Book, Malte; Lehmann, Lutz Eric; Zhang, XiangHong; Stüber, Frank
In patients with sepsis, diagnosis of blood stream infection (BSI) is a key concern to the therapist. Direct verification of pathogens in the blood stream executed by blood cultures (BC) still is regarded as the gold standard up to date. The quickest possible initiation of an appropriate antimicrobial therapy is a cornerstone of an effective therapy. Moreover, in this view BC can also serve to identify antimicrobial agents to target the pathogen. However, when employing BC the time needed until microbiological results are available ranges from 24 up to 72 h. Moreover, infections caused by multiple pathogens often remain undetected and concurrent antibiotic therapy may lower the overall sensitivity. Alternative pathogen characterization can be performed by polymerase chain reaction (PCR) based amplification methods. Results using PCR can be obtained within 6-8 h. Therefore, the time delay until an appropriate therapy can be reduced enormously. Moreover, these methods have the potential to enhance the sensitivity in the diagnosis of blood stream infections. Therefore, PCR based methods might be a valuable adjunct to present procedures of diagnosing bacteraemia.
Hekmatimoghaddam, Seyedhosssein; Sadeh, Maryam; Khalili, Mohammad Bagher; Mollaabedin, Mansour; Sazmand, Alireza
Brucellosis has long been prevalent in Iran, with considerable medical and economic importance. Timely diagnosis is needed for early management and effective prevention of its consequences in human beings and animals. Current diagnostic methods impose peculiar challenges in terms of analytical method performance. This study compares diagnostic sensitivity, specificity, predictive Value of Positive (PVP) and Predictive Value of Negative (PVN) for Polymerase Chain Reaction (PCR), Wright agglutination test and blood culture used for patients suspected of brucellosis. In 120 patients clinically suspected of brucellosis and referred by physicians to the Yazd central Medical Laboratory, some relevant demographic, occupational, nutritional and clinical data were collected. Also, venous blood samples were drawn for diagnosis of brucellosis using PCR, Wright agglutination test and blood culture techniques. The most frequent symptom of patients was arthralgia (82 cases, 68.3%). PCR was positive in 25 cases (20.8%), wright test in 21 patients (17.5%) and blood culture in 6 cases (5%). In 20 out of 21 wright-positive cases, PCR was positive and all of the culture-positive patients had positive PCR. Sensitivity, specificity, PVP and PVN of blood culture compared to PCR (as the gold standard test) were 24, 100, 100 and 86%, respectively, but the above parameters when PCR is compared with blood culture (as gold standard) were 100, 83, 24 and 95%, respectively. PCR has better analytical performances than blood culture for diagnosis of brucellosis and is suitable for confirmation of Wright-positive cases.
Full Text Available Background: Bloodstream infections (BSI have a high incidence and high mortality in the worldwide. The mortality rate is variable between 20-70%. Therefore, early and timely detection of BSI agent in clinical laboratories is necessary. The aim of this study was to determine an efficient diagnostic tool to septicemia in accompany of blood culture method by Real-time PCR (using panbacterial 23S rRNA gene. Methods: This cross-sectional study was conducted in two analytical and clinical stages in Hamadan University of Medical Sciences, Iran, from October 2014 to June 2015. In analytical stage, sensitivity (by serial dilution from 104 to 1 CFU/ml and specificity of the primer were evaluated with the Staphylococcus aureus (as Gram positive indicator bacteria and Escherichia coli (as Gram-negative indicator bacteria, human genome (from Hella cell culture, Candida albicans yeast and Aspergillus fumigatus fungus. In clinical stage, 121 blood samples were collected from patients suspected to sepsis in intensive care unit (ICU from Hamadan University Hospitals. Finally, the results of Real-time PCR and blood culture methods were compared. Results: The Real-time PCR showed a sensitivity ranging from 2 to 10 target copies per reaction to the whole blood for Escherichia coli and Staphylococcus aureus respectively. The specificity of this method was evaluated and no false positive amplification was identified. 57.85% (70 cases of the samples were positive by Real-time PCR and 13.22% (16 cases of the samples were positive by blood culture. However, none of the cases that were positive by blood culture were negative in Real-time PCR. As well as, 44.62% (54 cases of cases were positive by Real-time PCR but blood culture showed no bacteria in the samples, and 42.15% (51 cases were negative by both methods. Correlation or agreement of Kappa was 0.20, that indicating poor agreement between the two methods. Conclusion: Real-time PCR is more sensitive than blood
Baty, G; Lanotte, P; Hocqueloux, L; Prazuck, T; Bret, L; Romano, M; Mereghetti, L
We report the case of a 55 year-old man presenting with a double aortic and mitral endocarditis for which resected valve culture was repeatedly negative. Specific PCR made on valves because of highly positive blood tests for Bartonella henselae remained negative. A molecular approach was made with 16S rDNA PCR, followed by sequencing. Bartonella quintana was identified as the etiology of endocarditis. B. quintana, "fastidious" bacteria, even if hard to identify in a laboratory, is often reported as a blood culture negative endocarditis (BCNE) agent. Molecular biology methods have strongly improved the diagnosis of BCNE. We propose a review of the literature focusing on the interest of broad-spectrum PCR on valve for the etiological diagnosis of BCNE.
Full Text Available Abstract Background Pseudomonas aeruginosa is responsible for numerous bloodstream infections associated with severe adverse outcomes in case of inappropriate initial antimicrobial therapy. The present study was aimed to develop a novel quantitative PCR (qPCR assay, using ecfX as the specific target gene, for the rapid and accurate identification of P. aeruginosa from positive blood cultures (BCs. Methods Over the period August 2008 to June 2009, 100 BC bottles positive for gram-negative bacilli were tested in order to evaluate performances of the qPCR technique with conventional methods as gold standard (i.e. culture and phenotypic identification. Results Thirty-three strains of P. aeruginosa, 53 strains of Enterobactericaeae, nine strains of Stenotrophomonas maltophilia and two other gram-negative species were isolated while 3 BCs were polymicrobial including one mixture containing P. aeruginosa. All P. aeruginosa clinical isolates were detected by qPCR except a single strain in mixed culture. Performances of the qPCR technique were: specificity, 100%; positive predictive value, 100%; negative predictive value, 98.5%; and sensitivity, 97%. Conclusions This reliable technique may offer a rapid (
Zhu, Lingxiang; Shen, Ding-Xia; Zhou, Qiming; Liu, Chao-Jun; Li, Zexia; Fang, Xiangdong; Li, Quan-Zhen
A set of real-time PCR based assays using the locked nucleic acid probes from Roche Universal ProbeLibrary were developed for rapid detection of eight bacterial species from positive blood culture bottles. Four duplex real-time PCR reactions targeting to one Gram-positive bacterium and one Gram-negative bacterium were optimized for species identification according to Gram stain results. We also included mecA-specific primers and probes in the assays to indicate the presence of methicillin resistance in the bacterial species. The analytical sensitivity was in the range of 1-10 CFU per PCR reaction mixture. The specificity and cross reactivity of the assay was validated by 28 ATCC reference strains and 77 negative blood culture specimens. No cross-reactivity was observed in these samples thus demonstrating 100 % specificity. 72 previously characterized clinical isolates were tested by the real-time PCR assay and validated the accuracy and feasibility of the real-time PCR assay. Furthermore, 55 positive blood culture samples were tested using real-time PCR and 50 (90.9 %) of them were identified as the same species as judged by biochemical analysis. In total, real-time PCR showed 98.2 % consistent to that of traditional methods. Real-time PCR can be used as a supplement for early detection of the frequently-occurred pathogens from the positive blood cultures.
Full Text Available Abstract Background Salmonella Typhi causes an estimated 21 million new cases of typhoid fever and 216,000 deaths every year. Blood culture is currently the gold standard for diagnosis of typhoid fever, but it is time-consuming and takes several days for isolation and identification of causative organisms. It is then too late to initiate proper antibiotic therapy. Serological tests have very low sensitivity and specificity, and no practical value in endemic areas. As early diagnosis of the disease and prompt treatment are essential for optimal management, especially in children, a rapid sensitive detection method for typhoid fever is urgently needed. Although PCR is sensitive and rapid, initial research indicated similar sensitivity to blood culture and lower specificity. We developed a fast and highly sensitive blood culture PCR method for detection of Salmonella Typhi, allowing same-day initiation of treatment after accurate diagnosis of typhoid. Methods An ox bile tryptone soy broth was optimized for blood culture, which allows the complete lysis of blood cells to release intracellular bacteria without inhibiting the growth of Salmonella Typhi. Using the optimised broth Salmonella Typhi bacteria in artificial blood samples were enriched in blood culture and then detected by a PCR targeting the fliC-d gene of Salmonella Typhi. Results Tests demonstrated that 2.4% ox bile in blood culture not only lyzes blood cells completely within 1.5 hours so that the intracellular bacteria could be released, but also has no inhibiting effect on the growth of Salmonella Typhi. Three hour enrichment of Salmonella Typhi in tryptone soya broth containing 2.4% ox bile could increase the bacterial number from 0.75 CFU per millilitre of blood which is similar to clinical typhoid samples to the level which regular PCR can detect. The whole blood culture PCR assay takes less than 8 hours to complete rather than several days for conventional blood culture
Wang, Hye-Young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok; Uh, Young; Lee, Hyeyoung
Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 10(3) CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene.
Culture - blood ... A blood sample is needed . The site where blood will be drawn is first cleaned with an antiseptic such ... organism from the skin getting into (contaminating) the blood sample and causing a false-positive result (see ...
McCann, Chase D.; Moore, Miranda S.; May, Larissa S.; McCarroll, Matthew; Jordan, Jeanne A.
Several molecular platforms can identify bacteria associated with bloodstream infections, but require positive culture bottles as starting material. Here we describe results of screening 1140 blood cultures at 8 hours post-inoculation, from 918 eligible adults being evaluated for bloodstream infection. DNA was extracted and analyzed by 16S and/or 23S rRNA real-time PCR/Pyrosequencing. Compared to culture, PCR/Pyrosequencing displayed 90.9% sensitivity, 99.6% specificity, 95.7% PPV, and 99.1% NPV. Overall concordance rate was 98.9% (1127/1140). In four cases with molecular-positive/culture-negative results, medical chart reviews provided evidence of identical bacteria from subsequent blood or concomitant urine/sputum cultures. Nine culture-positive/molecular-negative cases were associated with either polymicrobial growth, grew only in the anaerobic bottle of the clinical pair, and/or were detected by PCR/Pyrosequencing after 8 hours. In summary, this approach accurately detected and identified bacteria in ~91% of culture-confirmed cases significantly sooner than the phenotypic identification was available, having the potential to improve antibiotic stewardship. PMID:25534615
McCann, Chase D; Moore, Miranda S; May, Larissa S; McCarroll, Matthew G; Jordan, Jeanne A
Several molecular platforms can identify bacteria associated with bloodstream infections but require positive culture bottles as starting material. Here, we describe results of screening 1140 blood cultures at 8h postinoculation, from 918 eligible adults being evaluated for bloodstream infection. DNA was extracted and analyzed by 16S and/or 23S rRNA real-time PCR/pyrosequencing. Compared to culture, PCR/pyrosequencing displayed 90.9% sensitivity, 99.6% specificity, 95.7% positive predictive value, and 99.1% negative predictive value. Overall concordance rate was 98.9% (1127/1140). In 4 cases with molecular-positive/culture-negative results, medical chart reviews provided evidence of identical bacteria from subsequent blood or concomitant urine/sputum cultures. Nine culture-positive/molecular-negative cases were associated with either polymicrobial growth, grew only in the anaerobic bottle of the clinical pair, and/or were detected by PCR/pyrosequencing after 8h. In summary, this approach accurately detected and identified bacteria in ~91% of culture-confirmed cases significantly sooner than the phenotypic identification was available, having the potential to improve antibiotic stewardship.
Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok; Uh, Young; Lee, Hyeyoung
Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylo...
Westh, H; Lisby, G; Breysse, F
Severe sepsis is increasingly a cause of death. Rapid and correct initial antimicrobial treatment reduces mortality. The aetiological agent(s) cannot always be found in blood cultures (BCs). A novel multiplex PCR test (SeptiFast (alpha version)) that allows identification of 20 bacterial and fungal...... species directly from blood was used, comparatively with BC, in a multicentre trial of patients with suspected bacterial or fungal sepsis. Five hundred and fifty-eight paired samples from 359 patients were evaluated. The rate of positivity was 17% for BC and 26% for SeptiFast. Ninety-six microorganisms...
Foongladda, Suporn; Mongkol, Nanthanida; Petlum, Pornphan; Chayakulkeeree, Methee
We developed a single-tube real-time polymerase chain reaction (PCR) assay with multiple hybridization probes for detecting Candida albicans, C. tropicalis, C. glabrata, and C. parapsilosis. Primers were designed to amplify 18S rRNA gene of the genus Candida, and DNA probes were designed to hybridize two areas of the amplicons. The amplification curves and specific melting peaks of the probes hybridized with PCR product were used for definite species identifications. The reaction specificity was 100 % when evaluating the assay using DNA samples from 21 isolates of fungal and bacterial species. The assay was further evaluated in 129 fungal blood culture broth samples which were culture positive for fungus. Of the 129 samples, 119 were positively identified as: C. albicans (39), C. tropicalis (30), C. parapsilosis (23), C. glabrata (20), Candida spp. (5), and two samples containing mixed C. glabrata/C. albicans and C. glabrata/C. tropicalis. The five Candida spp. were identified by sequencing analysis as C. krusei, C. dubliniensis, C. aquaetextoris, and two isolates of C. athensensis. Of the ten samples which showed negative PCR results, six were Cryptococcus neoformans, and the others were Trichosporon sp., Rhodotorula sp., Fusarium sp., and Penicillium marneffei. Our findings show that the assay was highly effective in identifying the four medically important Candida species. The results can be available within 3 h after positivity of a blood culture broth sample.
Blaschke, Anne J.; Heyrend, Caroline; Byington, Carrie L.; Fisher, Mark A.; Barker, Elizabeth; Garrone, Nicholas F.; Thatcher, Stephanie A.; Pavia, Andrew T.; Barney, Trenda; Alger, Garrison D.; Daly, Judy A.; Ririe, Kirk M.; Ota, Irene; Poritz, Mark A.
Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Inc., Salt Lake City, UT) Blood Culture (BC) panel can identify > 25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 hour. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 of 92 pathogens (91%) covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven MRSA and VRE. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture. PMID:22999332
Full Text Available Abstract Background Early onset bacterial sepsis is a feared complication of the newborn. A large proportion of infants admitted to the Neonatal Intensive Care Unit (NICU for suspected sepsis receive treatment with potent systemic antibiotics while a diagnostic workup is in progress. The gold standard for detecting bacterial sepsis is blood culture. However, as pathogens in blood cultures are only detected in approximately 25% of patients, the sensitivity of blood culture is suspected to be low. Therefore, the diagnosis of sepsis is often based on the development of clinical signs, in combination with laboratory tests such as a rise in C – reactive protein (CRP. Molecular assays for the detection of bacterial DNA in the blood represent possible new diagnostic tools for early identification of a bacterial cause. Methods A broad range 16S rDNA polymerase chain reaction (PCR without preincubation was compared to conventional diagnostic work up for clinical sepsis, including BACTEC blood culture, for early determination of bacterial sepsis in the newborn. In addition, the relationship between known risk factors, clinical signs, and laboratory parameters considered in clinical sepsis in the newborn were explored. Results Forty-eight infants with suspected sepsis were included in this study. Thirty-one patients were diagnosed with sepsis, only 6 of these had a positive blood culture. 16S rDNA PCR analysis of blinded blood samples from the 48 infants revealed 10 samples positive for the presence of bacterial DNA. PCR failed to be positive in 2 samples from blood culture positive infants, and was positive in 1 sample where a diagnosis of a non-septic condition was established. Compared to blood culture the diagnosis of bacterial proven sepsis by PCR revealed a 66.7% sensitivity, 87.5% specificity, 95.4% positive and 75% negative predictive value. PCR combined with blood culture revealed bacteria in 35.1% of the patients diagnosed with sepsis
Karumaa, Santra; Kärpänoja, Pauliina; Sarkkinen, Hannu
We have evaluated the GenoType blood culture assay (Hain Lifescience, Nehren, Germany) for the identification of bacteria in 233 positive blood cultures and assessed its suitability in the workflow of a routine microbiology laboratory. In 68/233 (29.2%) samples, the culture result could not be confirmed by the GenoType assay due to a lack of primers in the test, multiple organisms in the sample, or inconsistency with respect to the identification by culture. Although the GenoType blood culture assay gives satisfactory results for bacteria for which primers are available, there are difficulties in applying the test in the routine microbiology laboratory.
Full Text Available Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA-based quantitative real-time PCR (LNA-qPCR method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4% were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.
Kaleta, Erin J; Clark, Andrew E; Johnson, Desiree R; Gamage, Dulini C; Wysocki, Vicki H; Cherkaoui, Abdessalam; Schrenzel, Jacques; Wolk, Donna M
Sepsis is among the top 10 causes of mortality in the United States. Rapid administration of antibiotics is one of the most important contributors to patient survival, yet only a limited number of methods exist for rapid identification of microbes cultivated from bloodstream infections, which can lead to sepsis. While traditional single-target molecular methods have been shown to greatly improve survival for septic patients by enabling rapid deescalation of broad-spectrum antibiotics, multiplex methods offer even greater possibilities. A novel multiplex method, PCR coupled to electrospray ionization mass spectrometry (PCR/ESI-MS), was used to identify the genus and species of microorganisms found to cause human bloodstream infections. DNA was directly extracted from 234 BacT-Alert blood culture bottles, and results were compared to those obtained by clinical reference standard methods. The study results demonstrated 98.7% and 96.6% concordance at the genus and species levels, respectively. Mixtures of microbes were identified in 29 blood culture bottles, including mixed species of the same genus, as well as mixtures containing Gram-positive and Gram-negative organisms, exemplifying the PCR/ESI-MS capability to identify multiple organisms simultaneously without the need for cultivation. This study demonstrates high analytical accuracy in comparison to routine subculture of blood culture bottles and phenotypic identification of microbes. Without foreknowledge of the microorganisms potentially present, the PCR/ESI-MS methods can deliver accurate results in as little as 5 to 6 h after a positive alarm from the automated blood culture system; however, current batch mode testing limits the method's clinical utility at this time.
... Feeding Your 1- to 2-Year-Old Blood Culture KidsHealth > For Parents > Blood Culture Print A A ... adjust the treatment choice. Why Do a Blood Culture? During some illnesses, certain infection-causing bacteria and ...
Ohshiro, Takeya; Miyagi, Chihiro; Tamaki, Yoshikazu; Mizuno, Takuya; Ezaki, Takayuki
Blood culturing and the rapid reporting of results are essential for infectious disease clinics to obtain bacterial information that can affect patient prognosis. When gram-positive coccoid cells are observed in blood culture bottles, it is important to determine whether the strain is Staphylococcus aureus and whether the strain has resistance genes, such as mecA and blaZ, for proper antibiotic selection. Previous work led to the development of a PCR method that is useful for rapid identification of bacterial species and antimicrobial susceptibility. However, that method has not yet been adopted in community hospitals due to the high cost and methodological complexity. We report here the development of a quick PCR and DNA-chromatography test, based on single-tag hybridization chromatography, that permits detection of S. aureus and the mecA and blaZ genes; results can be obtained within 1 h for positive blood culture bottles. We evaluated this method using 42 clinical isolates. Detection of S. aureus and the resistance genes by the PCR-DNA-chromatography method was compared with that obtained via the conventional identification method and actual antimicrobial susceptibility testing. Our method had a sensitivity of 97.0% and a specificity of 100% for the identification of the bacterial species. For the detection of the mecA gene of S. aureus, the sensitivity was 100% and the specificity was 95.2%. For the detection of the blaZ gene of S. aureus, the sensitivity was 100% and the specificity was 88.9%. The speed and simplicity of this PCR-DNA-chromatography method suggest that our method will facilitate rapid diagnoses.
Fujita, Shin-ichi; Yosizaki, Kentaro; Ogushi, Thikako; Uechi, Kouhei; Takemori, Yukiko; Senda, Yasuko
We evaluated the usefulness of PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) and the CTX-M extended-spectrum β-lactamase (ESBL) followed by microchip gel electrophoresis (MGE) for direct identification and CTX-M detection of Gram-negative bacteria (GNB) from positive blood culture bottles. Of 251 GNB isolated from blood cultures containing a single bacterium, 225 (90%) were correctly identified at the species level directly from positive blood culture bottles by comparing the ITS-PCR patterns of the sample strain with those of the control strains. There were no cases of incorrect identification. Limitations encountered included the inability to detect mixed cultures (four bottles) as well as some species (Enterobacter species and Klebsiella oxytoca) demonstrating identical ITS-PCR patterns. A total of 109 ESBL-producing isolates from various clinical materials obtained between January 2005 and December 2008 were examined for bla(CTX-M), bla(SHV), and bla(TEM) genes by PCR and sequences of PCR products. CTX-M ESBL was detected in 105 isolates, and SHV ESBL was detected in two isolates. The remaining two isolates (K. oxytoca) were shown to harbor bla(OXY.) Twenty (19%) of 104 Escherichia coli isolates from blood cultures were suspected to produce ESBL by the combination disk method, and these isolates were shown to harbor CTX-M ESBL by PCR-MGE. The results were obtained within 1.5 h at a calculated cost of $6.50 per specimen. In conclusion, simultaneous detection of ITS length polymorphisms and bla(CTX)-(M) by single PCR followed by MGE is useful for rapid, cost-effective, and reliable species-level identification of CTX-M ESBL-producing GNB responsible for bloodstream infections.
Rapid differentiation of Staphylococcus aureus, Staphylococcus epidermidis and other coagulase-negative staphylococci and meticillin susceptibility testing directly from growth-positive blood cultures by multiplex real-time PCR.
Jukes, Leanne; Mikhail, Jane; Bome-Mannathoko, Naledi; Hadfield, Stephen J; Harris, Llinos G; El-Bouri, Khalid; Davies, Angharad P; Mack, Dietrich
This study evaluated a multiplex real-time PCR method specific for the mecA, femA-SA and femA-SE genes for rapid identification of Staphylococcus aureus, Staphylococcus epidermidis and non-S. epidermidis coagulase-negative staphylococci (CoNS), and meticillin susceptibility testing directly in positive blood cultures that grew Gram-positive cocci in clusters. A total of 100 positive blood cultures produced: 39 S. aureus [12 meticillin-resistant S. aureus (MRSA), 31% of all the S. aureus]; 30 S. epidermidis (56.6% of the CoNS), 8 Staphylococcus capitis (15.1%), 3 Staphylococcus saprophyticus (5.7%), 4 Staphylococcus hominis (7.5%), 3 Staphylococcus haemolyticus (5.7%), 2 Staphylococcus warneri (3.8%), 1 Staphylococcus cohnii (1.9%) and 2 unidentified Staphylococcus spp. (3.8%); and 1 Micrococcus luteus in pure culture. Two blood cultures had no growth on subculture and five blood cultures grew mixed CoNS. For the 95 blood cultures with pure growth or no growth on subculture, there was very good agreement between real-time PCR and the BD Phoenix identification system for staphylococcal species categorization in S. aureus, S. epidermidis and non-S. epidermidis CoNS and meticillin-resistance determination (Cohen's unweighted kappa coefficient κ=0.882). All MRSA and meticillin-susceptible S. aureus were correctly identified by mecA amplification. PCR amplification of mecA was more sensitive for direct detection of meticillin-resistant CoNS in positive blood cultures than testing with the BD Phoenix system. There were no major errors when identifying staphylococcal isolates and their meticillin susceptibility within 2.5 h. Further studies are needed to evaluate the clinical benefit of using such a rapid test on the consumption of glycopeptide antibiotics and the alteration of empiric therapy in the situation of positive blood cultures growing staphylococci, and the respective clinical outcomes.
Wang, M-Y; Geng, J-L; Chen, Y-J; Song, Y; Sun, M; Liu, H-Z; Hu, C-J
Methicillin-resistant staphylococci (MRS) and ESBL(Extended-Spectrum β-Lactamase)-producing bacteria are the most important resistant pathogens in sepsis. In this study, a new multiplex-touchdown PCR method (MT-PCR) was developed to detect rapidly and simultaneously the presence of mecA, blaSHV , blaCTX-M , blaTEM and blaOXA genes from positive blood culture bottles. The technique showed a sensitivity of 10(3 ) CFU ml(-1) for mecA detection and of 10(2) CFU ml(-1) for other genes, and 100% specificity in the detection of all genes. All genes were detected in the spiked blood culture bottles artificially contaminated with reference strains. Three methicillin-resistant S. aureus (MRSA), two methicillin-resistant S. epidermidis (MRSE) and 32 ESBL-producing bacteria, were isolated from the clinical blood culture specimens in 48 h by standard microbiological procedures. The corresponding genes were detected directly in the three MRSA, two MRSE and 29 ESBL-producing bacteria from the clinical blood culture specimens in 4 h by MT-PCR assay. None of the blaSHV , blaCTX-M , blaTEM and blaOXA genes were detected in three other bottles with ESBL-producing bacteria because of other ESBL genotypes in the pathogens. Likewise, all bottles proven negative by culture remained negative by PCR. The proposed method was rapid, sensitive and specific, and was able to directly detect the genes of MRS and ESBL-producing bacteria from the blood culture bottles.
Keid, L B; Soares, R M; Vasconcellos, S A; Salgado, V R; Megid, J; Richtzenhain, L J
The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.
Duncan, Ashlee W; Maggi, Ricardo G; Breitschwerdt, Edward B
Historically, direct plating, lysis centrifugation, or freeze-thaw approaches have proven to be highly insensitive methods for confirming Bartonella species infection in dogs. A prospective study was designed to compare diagnostic methods for the detection of Bartonella using samples submitted to the Vector-Borne Disease Diagnostic Laboratory at North Carolina State University. Methods included indirect immunofluorescence assay, PCR, direct inoculation of a blood agar plate (trypticase soy agar with 5% rabbit blood), and inoculation into a novel pre-enrichment liquid medium, Bartonella/alpha-Proteobacteria growth medium (BAPGM). Sequential research efforts resulted in the development of a combinational approach consisting of pre-enrichment culture of Bartonella species in BAPGM, sub-inoculation of the liquid culture onto agar plates, followed by DNA amplification using PCR. The multi-faceted approach resulted in substantial improvement in the microbiological detection and isolation of Bartonella when compared to direct inoculation of a blood agar plate. Importantly, this approach facilitated the detection and subsequent isolation of both single and co-infections with two Bartonella species in the blood of naturally infected dogs. The use of a combinational approach of pre-enrichment culture and PCR may assist in the diagnostic confirmation of bartonellosis in dogs and other animals.
Full Text Available Background and Aim: Culture and specific staining (including Zeil-Nelson and fluorescent methods are standard measures for the diagnosis of tuberculosis (TB.Because these methods are time-consuming and, sometimes, due to their low accuracy faster and more accurate methods are necessitated. Methods, which can substitute invasive procedures, when obtaining smear samples and culture is not possible and in addition to being simple and fast, they have an acceptable diagnostic accuracy. The aim of the present study was to verify the diagnostic value of blood PCR in pulmonary TB. Materials and Methods: This case-control study included 64 proven pulmonary TB cases (according to The National TB Protocol and 28 subjects who were completely healthy. 4.5ml of blood was derived from each participant and then mixed with 0.5ml EDTA. Finally, DNA extraction and PCR testing using SI 6110 primers was performed for all blood samples. Results: Mean age of the cases and controls was 49.8±18.6 and 48.2±18.5, respectively. 49.2% of the cases and 25% of controls were male. Blood PCR in 23 patients with TB was positive, but none of the controls had a positive PCR (thus, sensitivity of 35.7% and specificity of 100%. Conclusion: With regard to specificity of 100% in PCR method (despite its low sensitivity, in conditions where there is no access to an appropriate specimen, a positive blood PCR can obviate invasive procedures and rapid and definitive diagnosis of the disorder and timely treatment of the patient, his life is saved.
Double triplex real-time PCR assay for simultaneous detection of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus and determination of their methicillin resistance directly from positive blood culture bottles.
Kilic, Abdullah; Basustaoglu, A Celal
We developed and validated here a double triplex real-time PCR assay to simultaneously detect and identify Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and their methicillin resistance in a single reaction directly from Gram-positive cocci-in-clusters (GPCs)-positive blood culture bottles. From August 15, 2009 through February 15, 2010, 238 GPC-positive samples were collected and identified by conventional methods as 11 methicillin-resistant S. aureus (MRSA), 28 methicillin-susceptible S. aureus (MSSA), 176 MR coagulase-negative staphylococci (MRCoNS), 21 MSCoNS and two Enterococcus faecalis. The double triplex real-time PCR assay was targeted and detected tuf, nuc and mecA genes in the first tube and atlE, gap and mvaA genes in the second tube which could be run simultaneously. The detection limit of the assay was found at 10(3) CFU/ml for the atleE gene, 10(4) CFU/ml for the mva gene and 10(5) CFU/ml for gap, nuc, mecA and tuf genes based on seeding experiments. All Staphylococcus species except two S. epidermidis were correctly identified by the assay. The double triplex real-time PCR assay quickly and accurately detects S. aureus, S. epidermidis, S. hominis and S. haemolyticus and their methicillin resistance in a single reaction directly from positive blood culture bottles within 83 min.
Kang, Hyunseok Peter; Dunne, W Michael
To examine the stability of repetitive-sequence (rep) PCR profiles, six species of bacteria were subcultured to blood agar plates and DNA was extracted from the cultures after 24, 48, and 72 h of incubation at 35 degrees C. In addition, the same species were subcultured to fresh blood plates daily and DNA was extracted from the cultures after growth of 5, 10, and 15 subcultures, respectively. rep PCR analysis demonstrated that all rep PCR fingerprints from a single species were identical.
Tattevin, P; Watt, G; Revest, M; Arvieux, C; Fournier, P-E
Blood culture-negative endocarditis is often severe, and difficult to diagnose. The rate of non-documented infective endocarditis has decreased with the advent of molecular biology - improved performance for the diagnosis of bacterial endocarditis with blood cultures sterilized by previous antibacterial treatment - and cardiac surgery - access to the main infected focus, the endocardium, for half of the patients. Blood culture-negative endocarditis are classified in 3 main categories: (i) bacterial endocarditis with blood cultures sterilized by previous antibacterial treatment (usually due to usual endocarditis-causing bacteria, i.e. streptococci, more rarely staphylococci, or enterococci); (ii) endocarditis related to fastidious microorganisms (e.g. HACEK bacteria; defective streptococci - Gemella, Granulicatella, and Abiotrophia sp. - Propionibacterium acnes, Candida sp.): in these cases, prolonged incubation will allow identifying the causative pathogen in a few days; (iii) and the "true" blood culture-negative endocarditis, due to intra-cellular bacteria that cannot be routinely cultured in blood with currently available techniques: in France, these are most frequently Bartonella sp., Coxiella burnetti (both easily diagnosed by ad hoc serological tests), and Tropheryma whipplei (usually diagnosed by PCR on excised cardiac valve tissue). Non-infective endocarditis is rare, mostly limited to marantic endocarditis, and the rare endocarditis related to systemic diseases (lupus, Behçet).
Gradel, Kim Oren; Knudsen, Jenny Dahl; Arpi, Magnus
ABSTRACT: BACKGROUND: Information from blood cultures is utilized for infection control, public health surveillance, and clinical outcome research. This information can be enriched by physicians assessments of positive blood cultures, which are, however, often available from selected patient groups...... or pathogens only. The aim of this work was to determine whether patients with positive blood cultures can be classified effectively for outcome research in epidemiological studies by the use of administrative data and computer algorithms, taking physicians assessments as reference. METHODS: Physicians...... assessments of positive blood cultures were routinely recorded at two Danish hospitals from 2006 through 2008. The physicians assessments classified positive blood cultures as: a) contamination or bloodstream infection; b) bloodstream infection as mono- or polymicrobial; c) bloodstream infection as community...
Laupland Kevin B
Full Text Available Abstract Background Blood cultures are a gold standard specific test for diagnosing many infections. However, the low yield may limit their usefulness, particularly in low-risk populations. This study was conducted to assess the utility of blood cultures drawn from ambulatory outpatients. Methods Blood cultures drawn at community-based collection sites in the Calgary Health Region (population 1 million in 2001 and 2002 were included in this study. These patients were analyzed by linkages to acute care health care databases for utilization of acute care facilities within 2 weeks of blood culture draw. Results 3102 sets of cultures were drawn from 1732 ambulatory outpatients (annual rate = 89.4 per 100,000 population. Significant isolates were identified from 73 (2.4% sets of cultures from 51 patients, including Escherichia coli in 18 (35% and seven (14% each of Staphylococcus aureus and Streptococcus pneumoniae. Compared to patients with negative cultures, those with positive cultures were older (mean 49.6 vs. 40.1 years, p Conclusion Blood cultures drawn in outpatient settings are uncommonly positive, but may define patients for increased intensity of therapy. Strategies to reduce utilization without excluding patients with positive cultures need to be developed for this patient population.
Girault, Dominique; Soupé-Gilbert, Marie-Estelle; Geroult, Sophie; Colot, Julien; Goarant, Cyrille
With the increasing use of real-time PCR techniques, Leptospira isolation has mostly been abandoned for the diagnosis of human leptospirosis. However, there is a great value of collecting Leptospira isolates to better understand the epidemiology of this complex zoonosis and to provide the researchers with different isolates. In this study, we have successfully isolated different Leptospira strains from BacT/Alert aerobic blood culture bottles and suggest that this privileged biological material offers an opportunity to isolate leptospires.
目的：观察real time PCR在血流感染病原体检测中的敏感性和特异性，并与常规血培养对比，探讨其临床应用价值。方法以该院各临床科室收集的108份脓毒血症患者血液标本进行real time PCR检测，同时进行常规血培养，比较两种方法的特异性和敏感性。结果108份标本当中，两种方法检测出12种病原微生物。 Real time PCR共检测出阳性标本25份，阴性标本83份。其中与血培养共同阳性标本9份，共同阴性标本78份。两方法的一致性为80.6%。Real time PCR的阴性预测值是0.94，敏感性64%，特异性83%。16例标本real time PCR阳性而血培养阴性，5例标本血培养阳性而real time PCR阴性。同时，有2病标超出real time PCR的检测范围，而血培养阳性。此外，real time PCR无法检测光滑念珠菌。结论real time PCR虽然能快速检测血液感染中病原微生物，但依然不能完全替代血培养。%Objective To observe the sensitivity and specificity of real-time PCR in the detection of unknown pathogen in blood-stream infection, and compare that with conventional blood culture, and thus to investigate its clinic application value in pathogen detection. Methods A total of 108 blood samples of patients with sepsis from the clinic departments in our hospital were collected for real-time PCR detection and conventional blood culture. And the sensitivity and specificity of these two methods were compared. Results Of the 108 samples, 12 kinds of pathogens were detected. 25 positive and 83 negative samples were detected by real-time PCR. 9 samples were positive, and 78 samples were negative in both real-time PCR and blood culture assays. The agreement rate of blood culture system and real-time PCR was 80.6%. The negative predictive value of real-time PCR was 0.94, sensitivity was 64%, and specificity 83%. In 16 samples where a positive real-time PCR and a negative blood culture system result were obtained
A.J.M. Munting-van Deventer; W.H.F. Goessens (Wil); A.F. van Belkum (Alex); H.J. van Vliet; E.W.M. van Etten (Els); H.A. Verbrugh (Henri)
textabstractA PCR using primers aimed at the multicopy gene coding for the small subunit rRNA and resulting in the synthesis of a 180-bp fragment was evaluated for its use in diagnosing invasive candidiasis in comparison with blood culture. With the use of a C. albicans
Taylor, Brian. J.; Martin, Kimberly A; Arango, Eliana; Agudelo, Olga M; Maestre, Amanda; Yanow, Stephanie K.
Background Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products. Methods Reagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical s...
Teranishi, Hideto; Ohzono, Nanae; Inamura, Norikazu; Kato, Atsushi; Wakabayashi, Tokio; Akaike, Hiroto; Terada, Kihei; Ouchi, Kazunobu
Febrile neutropenia is the main treatment-related cause of mortality in cancer patients. During June 2012 to April 2014, 97 blood culture samples were collected from patients receiving chemotherapy for hematological malignancy and cancer with febrile neutropenia episodes (FNEs). The samples were examined for the presence of bacteria and fungi using real-time PCR amplification and sequencing of 16S and 18S rRNA genes. Bacteria were identified in 20 of 97 samples (20.6%) by the real-time PCR assay and in 10 of 97 (10.3%) samples by blood culture. In 6 blood culture-positive samples, the real-time PCR assay detected the same type of bacteria. No fungi were detected by the real-time PCR assay or blood culture. During antibiotic therapy, all samples were negative by blood culture, but the real-time PCR assay yielded a positive result in 2 cases of 2 (100%). The bacterial DNA copy number was not well correlated with the serum C-reactive protein titer of patients with FNEs. We conclude that a real-time PCR assay could provide better detection of causative microbes' in a shorter time, and with a smaller blood sample than blood culture. Using a real-time PCR assay in combination with blood culture could improve microbiological documentation of FNEs.
Juana del Valle Mendoza
Full Text Available Bartonella bacilliformis is the etiologic agent of Carrion's disease. This disease has two well established phases, the most relevant being the so called Oroya Fever, in which B. bacilliformis infect the erythrocytes resulting in severe anemia and transient immunosuppression, with a high lethality in the absence of adequate antibiotic treatment. The presence of B. bacilliformis was studied in 113 blood samples suspected of Carrion's disease based on clinical criteria, despite the absence of a positive thin blood smear, by two different PCR techniques (using Bartonella-specific and universal 16S rRNA gene primers, and by bacterial culture. The specific 16S rRNA gene primers revealed the presence of 21 B. bacilliformis and 1 Bartonella elizabethae, while universal primers showed both the presence of 3 coinfections in which a concomitant pathogen was detected plus Bartonella, in addition to the presence of infections by other microorganisms such as Agrobacterium or Bacillus firmus. These data support the need to implement molecular tools to diagnose Carrion's disease.
Tinco Valdez, Carmen; Pons, Maria J.; del Valle, Luis J.; Oré, Verónica Casabona; Michelena, Denisse Champin; Mayra, Jorge Bazán; Gavidea, Víctor Zavaleta; Vargas, Martha; Ruiz, Joaquim
Bartonella bacilliformis is the etiologic agent of Carrion's disease. This disease has two well established phases, the most relevant being the so called Oroya Fever, in which B. bacilliformis infect the erythrocytes resulting in severe anemia and transient immunosuppression, with a high lethality in the absence of adequate antibiotic treatment. The presence of B. bacilliformis was studied in 113 blood samples suspected of Carrion’s disease based on clinical criteria, despite the absence of a positive thin blood smear, by two different PCR techniques (using Bartonella-specific and universal 16S rRNA gene primers), and by bacterial culture. The specific 16S rRNA gene primers revealed the presence of 21 B. bacilliformis and 1 Bartonella elizabethae, while universal primers showed both the presence of 3 coinfections in which a concomitant pathogen was detected plus Bartonella, in addition to the presence of infections by other microorganisms such as Agrobacterium or Bacillus firmus. These data support the need to implement molecular tools to diagnose Carrion’s disease. PMID:24651298
Di Giovanni, George D.; LeChevallier, Mark W.
A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-Q...
The use of 10% oxgall and bile broth medium, both supplemented with freshly prepared 100 u/ml streptokinase, for isolating Salmonella typhi by clot...significantly better rate of isolation than the clot culture methods. Keywords: Cultures biology; Clot cultures; Salmonella typhi ; Isolation of S. typhi; Whole blood culture; Blood-clot culture; Reprints.
Kara, Ateş; Kanra, Güler; Cengiz, A Bülent; Apiş, Menekşe; Gür, Deniz
The aim of this study was to determine how long it takes blood culture to become positive using a blood culture system that can be monitored continuously in pediatric patients. Data were collected prospectively on 1,000 positive blood culture results from a tertiary pediatric university hospital from April 2000 to May 2002. The laboratory used the BACTEC 9120 fluorescent blood culture system. Patient's age ranged from less than a day to 20 years of age (mean 3 years). Five hundred and four cultures (50.4%) out of 1,000 yielded coagulase negative staphylococcus (CNS), 81 (8.1%) S. aureus, 53 (5.3%). Pseudomonas and 50 (5.0%) Klebsiella species. Of the 504 coagulase negative staphylococcal blood culture isolates, 314 (62.3% of CNS) were regarded as skin contaminants. Of the 1,000 cultures, 9.6% were reported as positive in the first day, 27.8% in the second day, 54.7% in the third day, 77.0% in the fourth and 89.4% in the fifth day. There was no association between previous antibiotic usage and the period required for isolate recovery. The clinician can expect to get results of positive blood cultures with susceptibility data, at a rate of 77.1% by day four and almost 90% by day five of sampling in the bacteriemic patient. Blood cultures yielding coagulase negative staphylococci in the first three days almost always show bacteremia with those microorganisms.
Uno, Naoki; Suzuki, Hiromichi; Yamakawa, Hiromi; Yamada, Maiko; Yaguchi, Yuji; Notake, Shigeyuki; Tamai, Kiyoko; Yanagisawa, Hideji; Misawa, Shigeki; Yanagihara, Katsunori
The Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN) is a microarray-based assay that enables rapid detection of 9 common Gram-negative bacteria and 6 resistance determinants directly from positive blood cultures. We compared the performance of BC-GN with currently used automated systems, testing 141 clinical blood cultures and 205 spiked blood cultures. For identification of BC-GN target organisms in clinical and spiked blood cultures, the BC-GN assay showed 98.5% (130/132) and 98.9% (182/184) concordance, respectively. Of 140 resistance genes positively detected in clinical and spiked blood cultures with the BC-GN test, 139 (99.3%) were confirmed by PCR, and the detection results were consistent with the resistance phenotypes observed. The BC-GN assay, thus, can potentially improve care for sepsis patients by enabling timely detection and targeted antimicrobial therapy.
Jacobsen, Jonas; Lydolph, Magnus; Lange, Lene
Degenerate primers were designed for use in a culture-independent PCR screening of DNA from composite fungal communities, inhabiting residues of corn stovers and leaves. According to similarity searches and alignments amplified clone sequences affiliated with glycosyl hydrolase family 7 and glyco...
Li Ling Lee
Full Text Available Gammaretroviruses related to murine leukemia virus (MLV have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME patients and healthy controls. Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences. We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences. We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.
Çiftci, Alper; İça, Tuba; Savaşan, Serap; Sareyyüpoğlu, Barış; Akan, Mehmet; Diker, Kadir Serdar
The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.
Inglis, Timothy J J; Bzdyl, Nicole; Chua, I-Ly Joanna; Urosevic, Nadezda M; Leung, Michael J; Geelhoed, Elizabeth
Rapid identification of bacteria isolated from blood cultures by direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now in wide spread use in major centres but is not yet feasible in smaller hospital laboratories. A FilmArray multiplex PCR panel for blood culture isolate identification (BCID) provides an alternative approach to near point-of-care microbial identification in regional hospitals. We assessed the accuracy and time to identification of the BCID FilmArray in a consecutive series of 149 blood cultures from 143 patients in a teaching hospital and smaller regional hospitals, currently identified by direct MALDI-TOF and proprietary molecular methods. The BCID FilmArray contained 18 of 34 species and 20 of 23 species isolated from teaching and regional hospital, respectively. Overall, 85 % of the teaching hospital and 100 % of the regional hospital monomicrobial blood cultures were identified, compared with 60 and 68 %, respectively, for direct MALDI-TOF on the same cultures. There were no incorrect results from blood cultures containing Staphylococcus aureus, streptococci, Pseudomonas aeruginosa or Enterobacteriaceae. The three discrepant results were all in mixed cultures. The mean reduction in time to identification of blood culture isolates was 53 h, which did not include the time required to transport cultures from regional centres to a central laboratory. The overall performance of the BCID FilmArray is stronger in blood cultures from smaller regional hospitals that encounter a narrower range of bacterial species dominated by the commonest species. This approach is more suited to smaller clinical laboratories than the MALDI-TOF direct method.
Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Shively, Dawn A.; Nevers, Meredith B.
Quantitative polymerase chain reaction (qPCR) measurement of enterococci has been proposed as a rapid technique for assessment of beach water quality, but the response of qPCR results to environmental conditions has not been fully explored. Culture-based E. coli and enterococci have been used in empirical predictive models to characterize their responses to environmental conditions and to increase monitoring frequency and efficiency. This approach has been attempted with qPCR results only in few studies. During the summer of 2006, water samples were collected from two southern Lake Michigan beaches and the nearby river outfall (Burns Ditch) and were analyzed for enterococci by culture-based and non-culture-based (i.e., qPCR) methods, as well as culture-based E. coli. Culturable enterococci densities (log CFU/100 ml) for the beaches were significantly correlated with enterococci qPCR cell equivalents (CE) (R = 0.650, P N = 32). Enterococci CE and CFU densities were highest in Burns Ditch relative to the beach sites; however, only CFUs were significantly higher (P R = 0.565, P N = 32). Culturable E. coli and enterococci densities were significantly correlated (R = 0.682, P N = 32). Regression analyses suggested that enterococci CFU could be predicted by lake turbidity, Burns Ditch discharge, and wind direction (adjusted R2 = 0.608); enterococci CE was best predicted by Burns Ditch discharge and log-transformed lake turbidity × wave height (adjusted R2 = 0.40). In summary, our results show that analytically, the qPCR method compares well to the non-culture-based method for measuring enterococci densities in beach water and that both these approaches can be predicted by hydrometeorological conditions. Selected predictors and model results highlight the differences between the environmental responses of the two method endpoints and the potentially high variance in qPCR results
Tuomisto, Sari; Karhunen, Pekka J; Vuento, Risto; Aittoniemi, Janne; Pessi, Tanja
Postmortem bacteriology can be a valuable tool for evaluating deaths due to bacterial infection or for researching the involvement of bacteria in various diseases. In this study, time-dependent postmortem bacterial migration into liver, mesenteric lymph node, pericardial fluid, portal, and peripheral vein was analyzed in 33 autopsy cases by bacterial culturing and real-time quantitative polymerase chain reaction (RT-qPCR). None suffered or died from bacterial infection. According to culturing, pericardial fluid and liver were the most sterile samples up to 5 days postmortem. In these samples, multigrowth and staphylococci were not or rarely detected. RT-qPCR was more sensitive and showed higher bacterial positivity in all samples. Relative amounts of intestinal bacterial DNA (bifidobacteria, bacteroides, enterobacter, clostridia) increased with time. Sterility of blood samples was low during the studied time periods (1-7 days). The best postmortem microbiological sampling sites were pericardial fluid and liver up to 5 days after death.
Lu, Sha; Li, Xiqing; Calderone, Richard; Zhang, Jing; Ma, Jianchi; Cai, Wenying; Xi, Liyan
Talaromyces marneffei is a dimorphic pathogenic fungus, which is a life-threatening invasive mycosis in the immunocompromised host. Prompt diagnosis of T. marneffei infection remains difficult although there has been progress in attempts to expedite the diagnosis of this infection. We previously demonstrated the value of nested polymerase chain reaction (PCR) to detect T. marneffei in paraffin embedded tissue samples with high sensitivity and specificity. In this study, this assay was used to detect the DNA of T. marneffei in whole blood samples. Real-time PCR assay was also evaluated to identify T. marneffei in the same samples. Twenty out of 30 whole blood samples (67%) collected from 23 patients were found positive by using the nested PCR assay, while 23/30 (77%) samples were found positive by using the real-time PCR assay. In order to express accurately the fungal loads, we used a normalized linearized plasmid as an internal control for real-time PCR. The assay results were correlated as the initial quantity (copies/μl) with fungal burden. These data indicate that combination of nested PCR and real-time PCR assay provides an attractive alternative for identification of T. marneffei DNA in whole blood samples of HIV-infected patients.
Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil.
Fornazari, Felipe; da Silva, Rodrigo Costa; Richini-Pereira, Virginia Bodelão; Beserra, Hugo Enrique Orsini; Luvizotto, Maria Cecília Rui; Langoni, Helio
Leptospirosis is an infectious disease of worldwide importance. The development of diagnostic techniques allows sick animals to be identified, reservoirs to be eliminated and the disease prevented and controlled. The present study aimed to compare different techniques for diagnosing leptospirosis in sheep. Samples of kidney, liver and blood were collected from 465 animals that originated from a slaughterhouse. The sera were analyzed by the Microscopic Agglutination Test (MAT), and kidney and liver samples of seropositive animals were analyzed using four techniques: bacteriological culture, the Warthin Starry (WS) technique, conventional PCR (cPCR), and quantitative PCR (qPCR). With the MAT, 21 animals were positive (4.5%) to serovars Hardjo (n=12), Hebdomadis (n=5), Sentot (n=2), Wolfii (n=1) and Shermani (n=1). Titers were 100 (n=10), 200 (n=2), 400 (n=6) and 1600 (n=3). No animal was positive by bacteriological culture; four animals were positive by the WS technique in kidney samples; six animals were positive by cPCR in kidney samples; and 11 animals were positive by qPCR, eight of which in kidney samples and three in liver. The bacterial quantification revealed a median of 4.3 bacteria/μL in liver samples and 36.6 bacteria/μL in kidney samples. qPCR presented the highest sensitivity among the techniques, followed by cPCR, the WS technique and bacteriological culture. These results indicate that sheep can carry leptospires of the Sejroe serogroup, and demonstrate the efficiency of quantitative PCR to detect Leptospira spp. in tissue samples.
Kim, Jae-Seok; Kang, Go-Eun; Kim, Han-Sung; Kim, Hyun Soo; Song, Wonkeun; Lee, Kyu Man
The performance of molecular tests using the Verigene Gram-Positive and Gram-Negative Blood Culture nucleic acid tests (BC-GP and BC-GN, resp.; Naosphere, Northbrook, IL, USA) was evaluated for the identification of microorganisms detected from blood cultures. Ninety-nine blood cultures containing Gram-positive bacteria and 150 containing Gram-negative bacteria were analyzed using the BC-GP and BC-GN assays, respectively. Blood cultures were performed using the Bactec blood culture system (BD Diagnostic Systems, Franklin Lakes, NJ, USA) and conventional identification and antibiotic-susceptibility tests were performed using a MicroScan system (Siemens, West Sacramento, CA, USA). When a single strain of bacteria was isolated from the blood culture, Verigene assays correctly identified 97.9% (94/96) of Gram-positive bacteria and 93.8% (137/146) of Gram-negative bacteria. Resistance genes mecA and vanA were correctly detected by the BC-GP assay, while the extended-spectrum β-lactamase CTX-M and the carbapenemase OXA resistance gene were detected from 30 cases cultures by the BC-GN assay. The BC-GP and BC-GN assays showed high agreement with conventional identification and susceptibility tests. These tests are useful for rapid identification of microorganisms and the detection of clinically important resistance genes from positive Bactec blood cultures.
Waters, Sinéad M; Murphy, Richard A; Power, Ronan F G
Undefined Nurmi-type cultures (NTCs) have been used successfully to prevent salmonella colonisation in poultry for decades. Such cultures are derived from the caecal contents of specific-pathogen-free birds and are administered via drinking water or spray application onto eggs in the hatchery. These cultures consist of many non-culturable and obligately anaerobic bacteria. Due to their undefined nature it is difficult to obtain approval from regulatory agencies to use these preparations as direct fed microbials for poultry. In this study, 10 batches of prototype NTCs were produced using an identical protocol over a period of 2 years. Traditional microbiological techniques and a molecular culture-independent methodology, polymerase chain reaction combined with denaturing gradient gel electrophoresis (PCR-DGGE), were applied to characterise these cultures and also to examine if the constituents of the NTCs were identical. Culture-dependent analysis of these cultures included plating on a variety of selective and semi-selective agars, examination of colony morphology, Gram-staining and a series of biochemical tests (API, BioMerieux, France). Two sets of PCR-DGGE studies were performed. These involved the amplification of universal and subsequently lactic acid bacteria (LAB)-specific hypervariable regions of a 16S rRNA gene by PCR. Resultant amplicons were subjected to DGGE. Sequence analysis was performed on subsequent bands present in resultant DGGE profiles using the Basic Local Alignment Search Tool (BLAST). Microbiological culturing techniques tended to isolate common probiotic bacterial species from the genera Lactobacillus, Lactococcus, Bifidobacterium, Enterococcus, Clostridium, Escherichia, Pediococcus and Enterobacterium as well as members of the genera, Actinomyces, Bacteroides, Propionibacterium, Capnocytophaga, Proteus, and Klebsiella. Bacteroides, Enterococcus, Escherichia, Brevibacterium, Klebsiella, Lactobacillus, Clostridium, Bacillus, Eubacterium
Fouad, Ali Adil; Kalkancı, Ayşe
Direct demonstration of bacterial and/or fungal nucleic acids in the clinical samples of patients with blood stream infections is crucial in terms of rapid diagnosis, early and accurate therapy and patient management. This study was aimed to determine the presence of bacteria and fungi by polymerase chain reaction (PCR) in the clinical samples of experimental sepsis induced animals, to compare the results with culture and to evaluate the efficiency of PCR in the discrimination of bacteremia and fungemia. A total of 12 rabbits experimentally infected with standard strains of Staphylococcus aureus, Escherichia coli, Aspergillus fumigatus and Candida albicans to generate bacteremia (n= 4), fungemia (n= 4) and polymicrobial blood stream infection (n= 4), were included in the study. A total of 63 specimens of which 27 were blood and 36 were tissue (12 spleen, 12 liver, 12 kidney) samples were collected at 24, 48, 72 and 96th hours of infection. Uninfected healthy rabbits (n= 4), colony suspensions of standard bacterial and fungal strains (n= 15) and human blood samples contaminated with standard bacterial and fungal strains (n= 10) were used as controls. Microbial DNAs were searched by using real-time PCR in all the samples, and quantitative cultures were performed simultaneously. Gram-positive and gram-negative PCR protocols were performed for the samples of bacteremic animals, whereas panfungal PCR, Aspergillus and Candida PCR protocols were performed for the samples of animals with fungemia. All of those PCR protocols were applied separately for the samples of polymicrobial blood stream infection cases. Culture positivity was detected in 8 (29.6%) of the blood samples and bacterial and/or fungal DNAs were demonstrated in 20 (74%) of the blood samples by PCR. Microbial DNAs were also detected in 32 (89%) of 36 tissue samples (11 spleen, 11 liver, 10 kidney). Sensitivity rates of culture method to detect bacteremia and fungemia were 30% and 21.7%, respectively, whereas
Full Text Available The opportunistic pathogen C.albicans is able to cause disseminated infections in immunocompromised patients. Microbiological methods for the diagnosis of invasive candidiasis have many problems including low sensitivity, requirement to invasive clinical sampling such as biopsies or multiple blood cultures and need to expertise laboratory stuff. Since PCR has proven to be a powerful tool in the early diagnosis of several infectious diseases, we applied this approach as a rapid and sensitive method in detection of C.albicans cells in blood samples, for establishment a clinically useful method in diagnosing systemic candidiasis. DNA were extracted from blood samples seeded by serially diluted C.albicans cells, by omitting WBC and RBC followed by enzymatic breaking of fungal cell wall and phenol – chlorophorm extraction and alcohol precipitation of DNA. A new primer pair was designed for PCR-amplification of a part of ribosomal RNA gene. The primer set was able to amplify all medically important Candida species. When PCR was performed for detection of purified DNA, the sensitivity of the method was about 1 picogram fungal DNA, whereas the sensitivity for detection of C.albicans blastospores inoculated in blood was as few as 10 cell per 0.1 ml of blood. This method could be sensitive and useful for early and rapid diagnosis of systemic Candida infections and to simultaneous detection and speciation of Candida species by PCR-RFLP method.
Peretz, A; Isakovich, N; Pastukh, N; Koifman, A; Glyatman, T; Brodsky, D
Blood is one of the most important specimens sent to a microbiology laboratory for culture. Most blood cultures are incubated for 5-7 days, except in cases where there is a suspicion of infection caused by microorganisms that proliferate slowly, or infections expressed by a small number of bacteria in the bloodstream. Therefore, at the end of incubation, misidentification of positive cultures and false-negative results are a real possibility. The aim of this work was to perform a confirmation by Gram staining of the lack of any microorganisms in blood cultures that were identified as negative by the BACTEC™ FX system at the end of incubation. All bottles defined as negative by the BACTEC FX system were Gram-stained using an automatic device and inoculated on solid growth media. In our work, 15 cultures that were defined as negative by the BACTEC FX system at the end of the incubation were found to contain microorganisms when Gram-stained. The main characteristic of most bacteria and fungi growing in the culture bottles that were defined as negative was slow growth. This finding raises a problematic issue concerning the need to perform Gram staining of all blood cultures, which could overload the routine laboratory work, especially laboratories serving large medical centers and receiving a large number of blood cultures.
Full Text Available A study was conducted to develop a Trypanosoma vivax (T. vivax specific PCR based on the T. vivax proline racemase (TvPRAC gene. Forward and reverse primers were designed that bind at 764-783 bp and 983-1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide.
Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T
Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.
Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T
Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells. PMID:25437051
Full Text Available Abstract Reverse transcription-polymerase chain reaction (RT-PCR has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV. One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs. The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses.
Pardo, Joe; Klinker, Kenneth P.; Borgert, Samuel J.; Butler, Brittany M.; Rand, Kenneth H.; Iovine, Nicole M.
The FilmArray blood culture identification (BCID) panel is a rapid molecular diagnostic test approved for use with positive blood culture material. We describe a fatal case of meningococcemia with central nervous system (CNS) involvement detected using the BCID test with culture-negative blood and cerebrospinal fluid.
Pardo, Joe; Klinker, Kenneth P; Borgert, Samuel J; Butler, Brittany M; Rand, Kenneth H; Iovine, Nicole M
The FilmArray blood culture identification (BCID) panel is a rapid molecular diagnostic test approved for use with positive blood culture material. We describe a fatal case of meningococcemia with central nervous system (CNS) involvement detected using the BCID test with culture-negative blood and cerebrospinal fluid.
Oshitani, Yohei; Ishikawa, Tomoyuki; Murata, Ken; Aoyagi, Yoshiki; Yabe, Yasuyo; Aoshima, Masahiro
Despite blood culture's usefulness in antimicrobial therapy, fewer blood cultures and the infrequency of more than 1 set in cultures appear to be problems in Japan. Since June 2007 infection control team (ICT) recommended more than 1 set in blood sampling and intervention in positive blood culture, coagulase negative Staphylococci (CNS) has frequently been isolated from blood culture and its clinical significance is often difficult to judge. To determine the effect of ICT intervention, we evaluated the number of blood culture specimens, the frequency of more than 1 set in all blood culture specimens, and decision-making on antimicrobial treatment for CNS isolated retrospectively from blood. The study was divided into term I in August 2007 to July 2008, term II in August 2008 to July 2009, and term III in August 2009 to February 2010. We also analyzed how physicians treated infection or its suspicion after CNS and its drug susceptibility. The monthly number of blood culture specimens increased from 40.3 to 51.6 between terms I and III. The frequency of more than 1 set in a single blood culture session rose significantly from 67% to 89% between these terms (p blood culture specimens, enable more than 1 set in blood sampling, make it easier to judge the presence of infection, and increase appropriate treatment by physicians. We thus believe that the quality of antimicrobial treatment could be improved through education such as ICT action.
Yong, P V C; Chong, P P; Lau, L Y; Yeoh, R S C; Jamal, F
The incidence of candidemia and invasive candidiasis have increased markedly due to the increasing number of immunocompromised patients. There are five major medically important species of Candida with their frequency of isolation in the diminishing order namely Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei. In addition, there are numerous other species of Candida which differ in their genetic makeup, virulence properties, drug susceptibilities and sugar assimilation capabilities. In this report, an unusual Candida species was isolated from the blood of two leukaemic patients. Conventional culture and biochemical tests identified the Candida species as C. parapsilosis. Using fungal-specific oligonucleotide primers ITS1 and ITS4, we managed to amplify the ribosomal RNA gene and its internal transcribed spacer region from the genomic DNA of these isolates. The PCR products were then purified and subjected to automated DNA sequencing using BLAST and CLUSTAL sequence analysis identified these isolates to be Candida orthopsilosis. Candida orthopsilosis is a new species recently identified in 2005, being morphologically indistinguishable from C. parapsilosis and was previously classified as a subspecies of C. parapsilosis. This report highlights the importance of complementing traditional culture and biochemical-based identification methods with DNA-based molecular assays such as PCR as the latter is more superior in terms of its discriminatory power and speed.
Reza Mirnejad; Mozafar mohamadi; Vahbeh Piranfar; Seied Mojtaba Mortazavi; Reza Kachuei
Objective: To design a duplex PCR for rapid and simultaneous detection of Brucella species. in human blood samples. Methods: Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis. Following DNA extraction, PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals. Results: Of the 52 peripheral bloods samples tested, 25 sample (48%) showed positive reactions in PCR. Twelve samples were positive for Brucella abortus (B. abortus) (23%), 13 for Brucella melitensis (B. melitensis) (25%) and 0 for Brucella ovis (B. ovis) (0%). Conclusions: This work de=monstrates that in case where specific primers were utilized, duplex PCR has proved to be a simple, fast, and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples.
Giese, Steen Bjørck; Ahrens, Peter
-negative on intestinal mucosa, but culture-positive in milk, and both faeces and milk were negative in culture and PCR from 2 cows. In conclusion the presence of M. a. paratuberculosis could be detected in raw milk by PCR but cultivation of milk was more sensitive in detecting the organism.......Milk and faecal samples from cows with clinical symptoms of paratuberculosis were examined for the presence of Mycobacterium avium subsp.paratuberculosis (M. a. paratuberculosis) by culture and PCR. M. a. paratuberculosis was isolated in varied numbers from faeces or intestinal mucosa in 8 of 11...... animals. In milk from 5 cows (all faecal culture-positive) we cultivated a few colonies of M. a. paratuberculosis (less than 100 CFU per mi). Milk samples from 2 cows were PCR-positive (both animals were faecal culture-positive, and 1 cow was milk culture positive). One cow was culture...
Fischer, T K; Prag, J; Kharazmi, A
The survival and function of human phagocytes in sterile aerobic and anaerobic blood culture media were investigated using neutrophil morphology, white blood cell count in a haemoanalyser, flow cytometry, oxidative burst response, and bactericidal effect in Colorbact and Septi-Chek blood culture...
Pui-Ying Iroh Tam
Full Text Available Nigeria has one of the highest burdens of pneumococcal disease in the world, but accurate surveillance is lacking. Molecular detection of infectious pathogens in dried blood spots (DBS is an ideal method for surveillance of infections in resource-limited settings because of its low cost, minimal blood volumes involved, and ease of storage at ambient temperature. Our study aim was to evaluate a Streptococcus pneumoniae real-time polymerase chain reaction (rt-PCR assay on DBS from febrile Nigerian children on Whatman 903 and FTA filter papers, compared to the gold standard of culture.Between September 2011 to May 2015, blood was collected from children 5 years of age or under who presented to six hospital study sites throughout northern and central Nigeria with febrile illness, and inoculated into blood culture bottles or spotted onto Whatman 903 or FTA filter paper. Culture and rt-PCR were performed on all samples.A total of 537 DBS specimens from 535 children were included in the study, of which 15 were culture-positive for S. pneumoniae. The rt-PCR assay detected S. pneumoniae in 12 DBS specimens (2.2%. One positive rt-PCR result was identified in a culture-negative specimen from a high-risk subject, and two positive rt-PCR results were negative on repeat testing. Six culture-confirmed cases of S. pneumoniae bacteremia were missed. Compared to culture, the overall sensitivities of Whatman 903 and FTA DBS for detection of S. pneumoniae were 57.1% (95% CI 18.4-90.1% and 62.5% (95% CI 24.5-91.5%, respectively. Nonspecific amplification was noted in an additional 22 DBS (4.1%. Among these, six were positive for a non-S. pneumoniae pathogen on culture.Rt-PCR was able to detect S. pneumoniae from clinical DBS specimens, including from a culture-negative specimen. Our findings show promise of this approach as a surveillance diagnostic, but also raise important cautionary questions. Several DBS specimens were detected as S. pneumoniae by rt-PCR despite
Kunze, M; Volkman, H; Köhler, W
The importance of the skin contamination for the results of blood cultures was emphasized by model examinations. In the method of blood taking without previous desinfection of the skin the quota of positive blood cultures increased by the twofold to threefold per culture and test person (5.7 to 18.8% and 11.3 to 26.3%, respectively). In large-volume blood takings the contamination rate becomes smaller with increasing blood volume. The rejecting of a first blood sample is to be recommended, when the possibility is given. With an increased quantity o blood per taking by blood bactericidia a decreased contamination rate is to be expected. By the results of the examinations the necessity of a consequent desinfection of the skin is to be emphasized, also when closed systems of blood cultures are used.
Full Text Available Introduction. ABO blood group genotyping is a new technology in hematology that helps prevent adverse transfusion reactions in patients. Identification of antigens on the surface of red blood cells is based on serology; however, genotyping employs a different strategy and is aimed directly at genes that determine the surface proteins. ABO blood group genotyping by real-time PCR has several crucial advantages over other PCR-based techniques, such as high rapidity and reliability of analysis. The purpose of this study was to examine nucleotide substitutions differences by blood types using a PCR-based method on Kazakh blood donors.Methods. The study was approved by the Ethics Committee of the National Center for Biotechnology. Venous blood samples from 369 healthy Kazakh blood donors, whose blood types had been determined by serological methods, were collected after obtaining informed consent. The phenotypes of the samples included blood group A (n = 99, B (n = 93, O (n = 132, and AB (n = 45. Genomic DNA was extracted using a salting-out method. PCR products of ABO gene were sequenced on an ABI 3730xl DNA analyzer (Applied Biosystems. The resulting nucleotide sequences were compared and aligned against reference sequence NM_020469.2. Real-time PCR analysis was performed on CFX96 Touch™ Real-Time PCR Detection System (BioRad.Results. Direct sequencing of ABO gene in 369 samples revealed that the vast majority of nucleotide substitutions that change the ABO phenotype were limited to exons 6 and 7 of the ABO gene at positions 261, 467, 657, 796, 803, 930 and 1,060. However, genotyping of only three of them (261, 796 and 803 resulted in identification of major ABO genotypes in the Kazakh population. As a result, TaqMan probe based real-time PCR assay for the specific detection of genotypes 261, 796 and 803 was developed. The assay did not take into account several other mutations that may affect the determination of blood group, because they have a
Arango-Sabogal, Juan C; Labrecque, Olivia; Paré, Julie; Fairbrother, Julie-Hélène; Roy, Jean-Philippe; Wellemans, Vincent; Fecteau, Gilles
Culture of Mycobacterium avium subsp. paratuberculosis (MAP) is the definitive antemortem test method for paratuberculosis. Microbial overgrowth is a challenge for MAP culture, as it complicates, delays, and increases the cost of the process. Additionally, herd status determination is impeded when noninterpretable (NI) results are obtained. The performance of PCR is comparable to fecal culture, thus it may be a complementary detection tool to classify NI samples. Our study aimed to determine if MAP DNA can be identified by PCR performed on NI environmental samples and to evaluate the performance of PCR before and after the culture of these samples in liquid media. A total of 154 environmental samples (62 NI, 62 negative, and 30 positive) were analyzed by PCR before being incubated in an automated system. Growth was confirmed by acid-fast bacilli stain and then the same PCR method was again applied on incubated samples, regardless of culture and stain results. Change in MAP DNA after incubation was assessed by converting the PCR quantification cycle (Cq) values into fold change using the 2(-ΔCq) method (ΔCq = Cq after culture - Cq before culture). A total of 1.6% (standard error [SE] = 1.6) of the NI environmental samples had detectable MAP DNA. The PCR had a significantly better performance when applied after culture than before culture (p = 0.004). After culture, a 66-fold change (SE = 17.1) in MAP DNA was observed on average. Performing a PCR on NI samples improves MAP culturing. The PCR method used in our study is a reliable and consistent method to classify NI environmental samples.
bacterial culture. The results of BMCs and BCs were compared. Results:Total 57 cases of FUO were included in the study. Male female ratio was 1.22:1. Age range was fi ve to 83 years (median 30. Duration of fever was 21 to 365 days. Bacterial growth was seen in nine cases (15.78% of BMCs and in three cases (5.26% of corresponding BCs. Fungal or myocbacterial growth was not seen. Salmonella typhi was the commonest organism isolated in BMCs (three cases followed by Staphylococcus aureus (two cases, Escherichia coli, Non fermenting Gram negative bacilli, Enterococcus species and Salmonella paratyphi–A (one case each. Two cases of Salmonella typhi and one case of Salmonella paratyphi–A were isolated in BCs. Conclusions:BMCs are more useful than BCs in evaluation of patients with FUO, especially in cases of salmonella infection and are particularly important when the patient has already taken antibiotics. In immuno-competent patients presenting with FUO, BMCs for mycobacteria or fungi is unlikely to yield any growth. Key Words: blood culture, bone marrow culture, fever of unknown origin
Grange, P A; Gressier, L; Dion, P L; Farhi, D; Benhaddou, N; Gerhardt, P; Morini, J P; Deleuze, J; Pantoja, C; Bianchi, A; Lassau, F; Avril, M F; Janier, M; Dupin, N
Syphilis diagnosis is based on clinical observation, serological analysis, and dark-field microscopy (DFM) detection of Treponema pallidum subsp. pallidum, the etiological agent of syphilis, in skin ulcers. We performed a nested PCR (nPCR) assay specifically amplifying the tpp47 gene of T. pallidum from swab and blood specimens. We studied a cohort of 294 patients with suspected syphilis and 35 healthy volunteers. Eighty-seven of the 294 patients had primary syphilis, 103 had secondary syphilis, 40 had latent syphilis, and 64 were found not to have syphilis. The T. pallidum nPCR results for swab specimens were highly concordant with syphilis diagnosis, with a sensitivity of 82% and a specificity of 95%. Reasonable agreement was observed between the results obtained with the nPCR and DFM methods (kappa = 0.53). No agreement was found between the nPCR detection of T. pallidum in blood and the diagnosis of syphilis, with sensitivities of 29, 18, 14.7, and 24% and specificities of 96, 92, 93, and 97% for peripheral blood mononuclear cell (PBMC), plasma, serum, and whole-blood fractions, respectively. HIV status did not affect the frequency of T. pallidum detection in any of the specimens tested. Swab specimens from mucosal or skin lesions seemed to be more useful than blood for the efficient detection of the T. pallidum genome and, thus, for the diagnosis of syphilis.
Giselle FMC Lima
Full Text Available Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR, nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.
Chen, H; Griffin, D K; Jestice, K; Hackett, G; Cooper, J; Ferguson-Smith, M A
Fetal erythroblasts circulating in maternal blood are important candidate cells for non-invasive prenatal diagnosis. We have cultured erythroblasts from 16 maternal blood samples, both with and without prior enrichment by magnetic activated cell sorting (MACS), in a semi-solid medium containing growth factors. Individual colonies were examined by PCR with sex chromosome-specific primers and microsatellite marker primers. No conclusive Y-chromosome specific amplification could be demonstrated in any of the 16 cases, even when the mother was confirmed to be carrying a male fetus. All colonies tested by microsatellite marker PCR were of maternal origin. Our results suggest that the probability of obtaining fetal colonies from fetal erythroblasts circulating in maternal blood is very low and that approaches for culturing fetal erythroblasts in vitro cannot yet be used reliably for prenatal diagnosis using current methods for fetal cell enrichment.
Altun, Osman; Almuhayawi, Mohammed; Ullberg, Måns; Ozenci, Volkan
The FilmArray platform (FA; BioFire, Salt Lake City, UT) is a closed diagnostic system allowing high-order multiplex PCR analysis with automated readout of results directly from positive blood cultures in 1 h. In the present study, we evaluated the clinical performance of the FilmArray blood culture identification (BCID) panel, which includes 19 bacteria, five yeasts, and three antibiotic resistance genes. In total, 206 blood culture bottles were included in the study. The FilmArray could identify microorganisms in 153/167 (91.6%) samples with monomicrobial growth. Thirteen of the 167 (7.8%) microorganisms were not covered by the FilmArray BCID panel. In 6/167 (3.6%) samples, the FilmArray detected an additional microorganism compared to blood culture. When polymicrobial growth was analyzed, the FilmArray could detect all target microorganisms in 17/24 (71%) samples. Twelve blood culture bottles that yielded a positive signal but showed no growth were also negative by FilmArray. In 3/206 (1.5%) bottles, the FilmArray results were invalid. The results of the FilmArray were reproducible, as demonstrated by the testing and retesting of five bottles in the same day and a longitudinal follow-up of five other blood cultures up to 4 weeks. The present study shows that the FilmArray is a rapid identification method with high performance in direct identification of bacteria and yeasts from positive blood culture bottles.
Smit, Pieter W.; Peeling, Rosanna W.; Garcia, Patricia J.; Torres, Lorena L.; Pérez-Lu, José E.; Moore, David; Mabey, David
Bartonella bacilliformis is the etiological agent of a life-threatening illness. Thin blood smear is the most common diagnostic method for acute infection in endemic areas of Peru but remains of limited value because of low sensitivity. The aim of this study was to adapt a B. bacilliformis-specific real-time polymerase chain reaction (PCR) assay for use with dried blood spots (DBS) as a sampling method and assess its performance and use for the diagnosis and surveillance of acute Bartonella infection. Only two of 65 children (3%) that participated in this study had positive blood smears for B. bacilliformis, whereas 16 (including these two) were positive by PCR performed on DBS samples (24.6%). The use of DBS in combination with B. bacilliformis-specific PCR could be a useful tool for public health in identifying and monitoring outbreaks of infection and designing control programs to reduce the burden of this life-threatening illness. PMID:24043691
Mito, Tsutomu; Hirota, Yusuke; Suzuki, Shingo; Noda, Kazutaka; Uehara, Takanori; Ohira, Yoshiyuki; Ikusaka, Masatomi
A 65-year-old Japanese man was admitted with a 4-month history of fatigue and exertional dyspnea. Transthoracic echocardiography revealed a vegetation on the aortic valve and severe aortic regurgitation. Accordingly, infective endocarditis and heart failure were diagnosed. Although a blood culture was negative on day 7 after admission, a prolonged blood culture with subculture was performed according to the patient's history of contact with cats. Consequently, Bartonella henselae was isolated. Bartonella species are fastidious bacteria that cause blood culture-negative infective endocarditis. This case demonstrates that B. henselae may be detected by prolonged incubation of blood cultures. PMID:27746451
Sting, Reinhard; Runge, Martin; Eisenberg, Tobias; Braune, Silke; Müller, Wolfgang; Otto, Peter
Detection of the zoonotic pathogen Francisella tularensis subsp. holarctica (EF tularensis) in wild animals with culture techniques as well as polymerase chain reaction were compared and discussed on the basis of the investigation of 60 animals. The samples originated from 55 European brown hares (Lepus europaeus), two red foxes (Vulpes vulpes) and one each from a wild rabbit (Oryctolagus cuniculus), a European beaver (Castor fiber), and a lemur (Lemur catta). When comparing the growth of 28 F. tularensis isolates on the cysteine blood agar and the modified Martin-Lewis-agar used in this study, cultivation was successful for 26 isolates on both media, but for two isolates only on the cysteine blood agar. Out of 43 carcasses 19 tested positive in bacteriological culture and PCR. Two culture positive samples of tonsils originating from foxes could not be confirmed by PCR, although PCR was positive in 22 samples that missed growth of F. tularensis. Comparative studies on cultural detection of E. tularensis were performed on samples of 16 hares from lung, spleen, liver and gut and in one case with a peritoneal swab. In at least one of these localizations cultivation of the pathogen was successful. Detection rate was reduced to 94% (15 of 16 hares) considering only the results of the cultures of the lungs and spleens. For a sensitive and rapid detection of F. tularensis subsp. holarctica, the PCR is a suitable method thereby avoiding hazardous multiplying of the pathogen. However, cultivation of F. tularensis is often a prerequisite for further studies on antibiotic resistance patterns of the pathogen, molecular epidemiological and pathological analyses of tularaemia.
R.P.A.J. Verkooyen (Roel); D. Willemse; S.C. Hiep-van Casteren; S.A. Joulandan; R.J. Snijder; H.P. van Helden; M.F. Peeters; H.A. Verbrugh (Henri); J.M. van den Bosch (Jules)
textabstractWe prospectively studied 156 patients with a diagnosis of community-acquired pneumonia requiring admission. Several respiratory specimens were obtained for the detection of Chlamydia pneumoniae by cell culture and PCR. Three serum samples were obtained from
Downing, J; Darke, C
The ABO blood group antigens are carbohydrate molecules synthesized by the glycosyltransferases encoded by the ABO gene on chromosome 9. Kidney transplantation across the ABO barrier generally leads to rapid humoral graft rejection due to the presence of naturally occurring antibodies to the A and B antigens. We have developed a method for ABO typing our cadaveric organ donors by the polymerase chain reaction using sequence-specific primers (PCR-SSP). The method uses 12 primers in eight PCR mixtures and is performed under the same conditions as our routine HLA-A, B, C PCR-SSP typing. The PCR-SSP-based types of 166 regular blood donors and 148 cadaveric organ donors all showed total concordance with their serologically assigned ABO groups. Six individuals possessing the ABO A subgroups (A3, Ax and Aend) all typed as A1 by PCR-SSP, as expected. PCR-SSP is an appropriate method for ABO typing of cadaveric organ donors and, importantly, enables both ABO and HLA typing to be performed on the same DNA material.
Full Text Available The aim of this study was to compare the MPN-PCR (Most Probable Number- Polymerase Chain Reaction and MPN-Culture methods in enumerating of Listeria monocytogenes in milk. In order to compare the accuracy of these methods, 103 cell/ml Listeria monocytogenes and different background bacteria which may be present in raw milk, were inoculated in sterilized milk. After preparing serial dilutions, three replicates per dilution were inoculated in tubes containing listeria enrichment broth. After 48 hours of incubation, for MPN-Culture three inoculated replicates were subcultured on Oxford agar and suspected colonies were confirmed by performing by biochemical tests. For MPN-PCR assay, the DNA extraction was performed from the three inoculated replicates which were already used for MPN-Culture and PCR assay was performed using primers specific for Listeria monocytogenes. The experiment was repeated three times and the average of enumerated bacteria was calculated by each method separately. Statistical analysis using one sample Wilcoxon signed rank test showed that enumeration by MPN-PCR method was more accurate than enumeration by MPN-Culture method. The result of this study showed that MPN-PCR method in comparision with MPN-Culture even in the presence of different background microorganisms is more rapid and reliable. It is concluded that MPN-PCR method facilitates the enumeration of Listeria monocytogenes without excessive work and could be considered as an alternative to MPN-Culture technique.
Wang, Hye-Young; Kim, Seoyong; Kim, Jungho; Park, Soon Deok; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung
The aim of this study was to evaluate the performance of a commercially available multiplex real-time PCR assay and a PCR-reverse blot hybridization assay (PCR-REBA) for the rapid detection of bacteria and identification of antibiotic resistance genes directly from blood culture bottles and to compare the results of these molecular assays with conventional culture methods. The molecular diagnostic methods were used to evaluate 593 blood culture bottles from patients with bloodstream infections. The detection positivity of multiplex real-time PCR assay for Gram-positive bacteria, Gram-negative bacteria and Candida spp. was equivalent to PCR-REBA as 99.6 %, 99.1 % and 100 %, respectively. Using conventional bacterial cultures as the gold standard, the sensitivity, specificity, positive predictive value and negative predictive value of these two molecular methods were 99.5 % [95 % confidence interval (CI), 0.980-1.000; PReal-methicillin-resistant Staphylococcusaureus multiplex real-time PCR assay targeting the mecA gene to detect methicillin resistance was lower than that of the PCR-REBA method, detecting an overall positivity of 98.4 % (n=182; 95 % CI, 0.964-1.000; P<0.009) and 99.5 % (n=184; 95 % CI, 0.985-1.000; P<0.0001), respectively. The entire two methods take about 3 h, while results from culture can take up to 48-72 h. Therefore, the use of these two molecular methods was rapid and reliable for the characterization of causative pathogens in bloodstream infections.
Luijt, D.S.; Bos, P.A.; van Zwet, A.A.; Voorst-Vader, P.C.; Schirm, J.
: A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased
Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J
A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased t
Angen, Øystein; Ahrens, Peter; Tegtmeier, Conny
a single colony of H. somnus in the presence of 10(9) CFU of P. multocida even after 2 days of incubation. In conclusion, the present PCR test has been shown to represent a specific test for identification of H. somnus both in pure and mixed cultures. It represents a quick, sensitive and reliable method...... rise to an amplicon in the PCR test. The performance of the test on mixed cultures was evaluated by adding P. multocida to serial dilutions of H. somnus and incubating the agarplates for 1 and 2 days. This showed that the PCR test applied to the harvest from an agarplate can be expected to detect...
Sakurada, Koichi; Akutsu, Tomoko; Watanabe, Ken; Yoshino, Mineo
A new approach for the identification of body fluid stains by comparing specific mRNA expression levels has been extensively studied in recent years. Here, we examine whether nasal blood, which is regarded as one of the most difficult types of blood to identify, can be identified by comparing mRNA expression levels of target genes specific to saliva, nasal secretion, and blood. The saliva-specific statherin gene (STATH) was found to be expressed at high levels in not only saliva (dCt value: 1.32±1.39, n=5), but also nasal secretions (dCt value: 0.90±1.14, n=5), while the histatin gene (HTN3) was only expressed at high levels in saliva (dCt value: 1.08±2.35, n=5). We also confirmed that the hemoglobin-beta gene (HBB) showed high expression levels in blood (dCt value: -9.51±0.40, n=5). Four nasal blood stains were found to highly express STATH (dCt value: 5.65±3.98) and HBB (dCt value: -8.79±1.67) but not HTN3, suggesting that the stain samples contained both nasal secretions and blood and can therefore be identified as nasal blood stains. Although menstrual blood showed the same expression pattern as nasal blood, the menstrual blood-specific protein matrix metallopeptidase 7 (MMP7) was not expressed in all nasal blood stain samples. Therefore, its expression levels could be used to discriminate between nasal and menstrual blood. In conclusion, real-time RT-PCR was able to identify nasal blood, although the stability of gene expression in nasal blood stains was low over time, suggesting that this assay may not be effective for older stains. Future work should examine the usefulness of this assay under various environmental conditions.
Full Text Available BACKGROUND: Isolation of Rickettsia species from skin biopsies may be replaced by PCR. We evaluated culture sensitivity compared to PCR based on sampling delay and previous antibiotic treatment. METHODOLOGY/PRINCIPAL FINDINGS: Skin biopsies and ticks from patients with suspected Rickettsia infection were screened for Rickettsia spp. using qPCR, and positive results were amplified and sequenced for the gltA and ompA genes. Immunofluorescence for spotted fever group rickettsial antigens was done for 79 patients. All skin biopsies and only ticks that tested positive using qPCR were cultured in human embryonic lung (HEL fibroblasts using the centrifugation-shell vial technique. Patients and ticks were classified as definitely having rickettsioses if there was direct evidence of infection with a Rickettsia sp. using culture or molecular assays or in patients if serology was positive. Data on previous antibiotic treatments were obtained for patients with rickettsiosis. Rickettsia spp. infection was diagnosed in 47 out of 145 patients (32%, 41 by PCR and 12 by culture, whereas 3 isolates were obtained from PCR negative biopsies. For 3 of the patients serology was positive although PCR and culture were negative. Rickettsia africae was the most common detected species (n = 25, [17.2%] and isolated bacterium (n = 5, [3.4%]. The probability of isolating Rickettsia spp. was 12 times higher in untreated patients and 5.4 times higher in patients from our hometown. Rickettsia spp. was amplified in 24 out of 95 ticks (25% and we isolated 7 R. slovaca and 1 R. raoultii from Dermacentor marginatus. CONCLUSIONS/SIGNIFICANCE: We found a positive correlation between the bacteria copies and the isolation success in skin biopsies and ticks. Culture remains critical for strain analysis but is less sensitive than serology and PCR for the diagnosis of a Rickettsia infection.
Full Text Available Yaws, caused by Treponema pallidum ssp. pertenue, is a neglected tropical disease closely related to venereal syphilis and is targeted for eradication by 2020. Latent yaws represents a diagnostic challenge, and current tools cannot adequately distinguish between individuals with true latent infection and individuals who are serofast following successful treatment. PCR on blood has previously been shown to detect T. pallidum DNA in patients with syphilis, suggesting that this approach may be of value in yaws. We performed real-time PCR for Treponema pallidum ssp. pertenue on blood samples from 140 children with positive T. pallidum Particle Agglutination (TPPA and Rapid Plasma Reagin (RPR tests and 7 controls (negative serology, all collected as part of a prospective study of yaws in the Solomon Islands. All samples were also tested by a nested PCR for T. pallidum. 12 patients had clinical evidence of active yaws whilst 128 were considered to have latent yaws. 43 children had high titre rapid plasma reagins (RPRs of ≥1:32. PCR testing with both assays gave negative results in all cases. It is possible that the failure to detect T. pallidum ssp. pertenue in blood reflects lower loads of organism in latent yaws compared to those in latent infection with T. pallidum ssp. pertenue, and/or a lower propensity for haematogenous dissemination in yaws than in syphilis. As the goal of the yaws control programme is eradication, a tool that can differentiate true latent infection from individuals who are serofast would be of value; however, PCR of blood is not that tool.
Marks, Michael; Katz, Samantha; Chi, Kai-Hua; Vahi, Ventis; Sun, Yongcheng; Mabey, David C; Solomon, Anthony W; Chen, Cheng Y; Pillay, Allan
Yaws, caused by Treponema pallidum ssp. pertenue, is a neglected tropical disease closely related to venereal syphilis and is targeted for eradication by 2020. Latent yaws represents a diagnostic challenge, and current tools cannot adequately distinguish between individuals with true latent infection and individuals who are serofast following successful treatment. PCR on blood has previously been shown to detect T. pallidum DNA in patients with syphilis, suggesting that this approach may be of value in yaws. We performed real-time PCR for Treponema pallidum ssp. pertenue on blood samples from 140 children with positive T. pallidum Particle Agglutination (TPPA) and Rapid Plasma Reagin (RPR) tests and 7 controls (negative serology), all collected as part of a prospective study of yaws in the Solomon Islands. All samples were also tested by a nested PCR for T. pallidum. 12 patients had clinical evidence of active yaws whilst 128 were considered to have latent yaws. 43 children had high titre rapid plasma reagins (RPRs) of ≥1:32. PCR testing with both assays gave negative results in all cases. It is possible that the failure to detect T. pallidum ssp. pertenue in blood reflects lower loads of organism in latent yaws compared to those in latent infection with T. pallidum ssp. pertenue, and/or a lower propensity for haematogenous dissemination in yaws than in syphilis. As the goal of the yaws control programme is eradication, a tool that can differentiate true latent infection from individuals who are serofast would be of value; however, PCR of blood is not that tool.
Whiley, Harriet; Taylor, Michael
Legionella spp. are the causative agent of Legionnaire's disease and an opportunistic pathogen of significant public health concern. Identification and quantification from environmental sources is crucial for identifying outbreak origins and providing sufficient information for risk assessment and disease prevention. Currently there are a range of methods for Legionella spp. quantification from environmental sources, but the two most widely used and accepted are culture and real-time polymerase chain reaction (qPCR). This paper provides a review of these two methods and outlines their advantages and limitations. Studies from the last 10 years which have concurrently used culture and qPCR to quantify Legionella spp. from environmental sources have been compiled. 26/28 studies detected Legionella at a higher rate using qPCR compared to culture, whilst only one study detected equivalent levels of Legionella spp. using both qPCR and culture. Aggregating the environmental samples from all 28 studies, 2856/3967 (72%) tested positive for the presence of Legionella spp. using qPCR and 1331/3967 (34%) using culture. The lack of correlation between methods highlights the need to develop an acceptable standardized method for quantification that is sufficient for risk assessment and management of this human pathogen.
Mohammadiha, A; Mohebali, M; Haghighi, A; Mahdian, R; Abadi, A R; Zarei, Z; Yeganeh, F; Kazemi, B; Taghipour, N; Akhoundi, B
Zoonotic visceral leishmaniasis (VL) is endemic in northwestern Iran. Real-time PCR, conventional PCR, and the direct agglutination test (DAT) were used to diagnose Leishmania infantum infection in blood samples from 100 domestic dogs and 100 humans. Based on clinical evaluation, 82 humans and 72 dogs from the endemic area were categorized as having asymptomatic infection, DAT positive with no clinical signs of VL, or symptomatic infection, DAT positive with at least one sign of VL. Eighteen human samples containing no Leishmania antibodies (DAT(-)) and 28 dog DAT(-) sera from non-endemic areas with no history of VL constituted negative controls. All 46 DAT(-) samples were also negative by Dipstick rK39. Bone marrow material was used for parasitological examinations in symptomatic VL, and peripheral blood samples were used for detection of L. infantum infection using conventional PCR and real-time PCR in non-symptomatic subjects. Two DNA targets (ITS1 kDNA) were used for conventional PCR. L. infantum antibodies in sera were detected by DAT. Parasitemia was measured by real-time PCR targeting kDNA using Taqman Assay. All 72 (100%) symptomatic (38/38) and asymptomatic (34/34) dog DAT(+)samples, 45 of 48 (93.8%) symptomatic human DAT(+) samples, and 32 of 34 (94.1%) human asymptomatic cases were identified by real-time PCR. The mean (59.19 vs 12.38 parasite equivalents/mL of blood) and median (16.15 vs 1 parasite equivalents/mL of blood) ranges of parasitemia were higher in dogs than in humans (Preal-time PCR and DAT (99% in dogs and 95% in humans). Sensitivity of 100% and 93.9%, specificity of 96.4% and 100%, positive predictive values of 98.6% and 100%, and negative predictive values of 100% and 78.3% were found by real-time PCR for dog and human samples, respectively.
Balıkçı, Ahmet; Belas, Zeliha; Eren Topkaya, Aynur
Blood culture is the gold standard for diagnosis of bloodstream infections. Many studies have shown that rapid isolation and identification of the microorganisms in blood culture and initiation of early antimicrobial therapy are critically important to reduce the mortality rate. It was found that the rate of contamination in blood cultures is increasing with automated systems developed to facilitate the growth of microorganism and tracking positivity. It is more difficult to interpret a positive blood culture result especially in the case of having only one sample bottle. In this study the effect of growth time observed in the automated blood culture systems was evaluated in terms of interpretation of blood culture results as being pathogens or contaminants. A total of 1201 blood cultures tested in BACTEC 9120 (Becton Dickinson, USA) system in Maltepe University Hospital Medical Microbiology Laboratory, Istanbul, Turkey during one-year period were included in the study and growth times were recorded for positive bottles. The decision about the growth as being a pathogen or contamination was made by considering the clinical condition of the patient, the number of positive blood cultures and the results of inflammation markers (white blood cell counts, procalsitonin and CRP levels). Of the blood cultures 290 (24%) yielded positive results and 73% (212/290) of them were evaluated as pathogens, while 27% (78/290) were identified as contaminants. The mean detection time for clinically significant isolates was 17.87 hours and for contaminants was 40.56 hours. The difference between the growth time of pathogens and contaminants was found statistically significant (ppositive results, it was detected that 66% of the bacteria grew within the first 24 hours. While 29.6% of the pathogens grew within 12 hours, none of the contaminants grew during that time. The evaluation of growth time among staphylococci in terms of methicillin resistance revealed that methicillin- resistant
Tatiana Reis do Rosário
Full Text Available The initial growth of mycobacteria from 49 samples of cattle and buffalo organs collected in commercial slaughterhouses was compared between modified Middlebrook 7H11 thin layer microcolony culture and Stonebrink medium used in the isolation of Mycobacterium bovis. Aliquots were decontaminated by Petroff's method, processed and cultured in both media. The identity of the acid-fast bacilli stained by Ziehl-Neelsen was confirmed by PCR. Optical microscopy showed that results of the early observation of Mycobacterium bovis colonies in thin layer culture were similar to those obtained in macroscopic observation of the colonies in Stonebrink medium. However, early observation of the colonies enabled early confirmation by PCR, given the shorter time to the visualization of colonies when thin layer culture was used (between the 12nd and 25th day of culture.
Kaya, Dilek; Demirezen, Şayeste; Hasçelik, Gülşen; Gülmez Kivanç, Dolunay; Beksaç, Mehmet Sinan
Members of the genus Actinomyces, Gram-positive, non-spore-forming anaerobic bacteria, are normal inhabitants of the mucosal surfaces of the oral, gastrointestinal and genital tracts. Identification of these bacteria using conventional methods is generally difficult because of their complex transport and growth requirements and their fastidious and slow-growing nature. However, in recent years, the advancement of molecular techniques has provided much improved identification and differentiation of closely related Actinomyces species. The aim of the present study was to evaluate the efficacy of the PCR technique in the diagnosis of genital Actinomyces in comparison with culturing and Papanicolaou (Pap) smear microscopy. Multiple sampling was conducted from 200 women using smear microscopy, culturing and PCR. Cyto-brushes were smeared on glass slides and stained using the routine Pap technique. Culturing was performed from a sterile swab, and Actinomyces were determined using the BBL Crystal ANR ID kit. PCR was performed from a second swab, and the Actinomyces type was determined using type-specific primers designed in our laboratory. Only one vaginal fluid sample (0.5%) revealed Actinomyces-like organisms on Pap smear examination. Actinomyces were detected in nine samples (4.5%) using the BBL Crystal ANR ID kit. Using PCR, eight samples (4%) were found positive for Actinomyces. No specimens that gave positive results by Pap smear microscopy and culturing could be confirmed by PCR. Pap smear microscopy and culturing were both found to have zero sensitivity for Actinomyces. PCR appears to be a sensitive and reliable diagnostic method for the detection of Actinomyces, which are difficult to cultivate from genital samples. PCR can be used for diagnostic confirmation in cases diagnosed by conventional methods, to prevent false-positive results.
Akoglu, Gulsen; Karaduman, Aysen; Boztepe, Gonca; Ozkaya, Ozay; Sahin, Sedef; Erkin, Gul; Kolemen, Fikret
A 14-year-old boy presented with a pink firm plaque with well-defined borders in the right infra-orbital skin area. On diascopy, the infiltrate exhibited a typical apple-jelly appearance. No acid-fast bacilli could be demonstrated. A polymerase chain reaction (PCR) assay did not reveal the presence of mycobacteria in a lesional biopsy sample. Culture of biopsied tissue on Loewenstein-Jensen medium was negative. Although the tuberculosis culture and PCR did not confirm tuberculosis, a diagnosis of lupus vulgaris was made considering the clinical and histopathological findings. After a 9-month antituberculous therapy, the lesion disappeared. We believe that a diagnosis of lupus vulgaris still depends more on clinical and histopathological findings than on tuberculosis culture or PCR.
Huang, Ruijie; Zhang, Junjie; Yang, X Frank; Gregory, Richard L
Changes of bacterial profiles in microbial communities are strongly associated with human health. There is an increasing need for multiple species research in vitro. To avoid high cost or measurement of a limited number of species, PCR-based multiple species cell counting (PCR-MSCC) has been conceived. Species-specific sequence is defined as a unique sequence of one species in a multiple species mixed culture. This sequence is identified by comparing a random 1000 bp genomic sequence of one species with the whole genome sequences of the other species in the same artificial mixed culture. If absent in the other genomes, it is the species-specific sequence. Species-specific primers were designed based on the species-specific sequences. In the present study, ten different oral bacterial species were mixed and grown in Brain Heart Infusion Yeast Extract with 1% sucrose for 24 hours. Biofilm was harvested and processed for DNA extraction and q-PCR amplification with the species-specific primers. By comparing the q-PCR data of each species in the unknown culture with reference cultures, in which the cell number of each species was determined by colony forming units on agar plate, the cell number of that strain in the unknown mixed culture was calculated. This technique is reliable to count microorganism numbers that are less than 100,000 fold different from other species within the same culture. Theoretically, it can be used in detecting a species in a mixed culture of over 200 species. Currently PCR-MSCC is one of the most economic methods for quantifying single species cell numbers, especially for the low abundant species, in a multiple artificial mixed culture in vitro.
TERANISHI, Hideto; OUCHI, Kazunobu
Febrile neutropenia is the main treatment-related cause of mortality in cancer patients. During June 2012 to April 2013, 76 blood culture samples from patients receiving chemotherapy for hematological malignancy and cancer with febrile neutropenia episodes (FNEs) were examined for the presence of bacteria and fungi based on 16S rRNA gene and 18S rRNA combined with real-time PCR amplification and sequencing. Furthermore, we used a loopmediatedisothermal amplification (LAMP) assay for the detec...
De Vries, Jutte J. C.; Van Assen, Sander; Mulder, André B.; Kampinga, Greetje A.
An adult traveler presented with fever and malaise after returning from Sierra Leone. Young trophozoites of Plasmodium falciparum were seen in a blood smear, with parasitemia being 10%. Moreover, blood cultures drawn on admission signaled as "positive" after 1 day of incubation, but no bacteria were
De Vries, Jutte J. C.; Van Assen, Sander; Mulder, André B.; Kampinga, Greetje A.
An adult traveler presented with fever and malaise after returning from Sierra Leone. Young trophozoites of Plasmodium falciparum were seen in a blood smear, with parasitemia being 10%. Moreover, blood cultures drawn on admission signaled as "positive" after 1 day of incubation, but no bacteria were
Croxatto, Antony; Prod'hom, Guy; Durussel, Christian; Greub, Gilbert
Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.
David E Lucero
Full Text Available In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps. Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens, five for chicken (Gallus gallus and unicolored blackbird (Agelasticus cyanopus, and one for opossum (Monodelphis domestica. Using the qPCR assay we detected chicken (13 vectors, and human (14 vectors blood meals as well as an additional blood meal source, Canis sp. (4 vectors.We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.
Just, Søren Andreas; Knudsen, John Bonde; Uldum, Søren Anker
Legionella bozemanae is a rare isolate in clinical specimens. We describe a case of joint infection due to L. bozemanae in an immunocompromised patient with dermatomyositis. Without the use of PCR screening or culture on specialized medium, the organism would not have been detected.......Legionella bozemanae is a rare isolate in clinical specimens. We describe a case of joint infection due to L. bozemanae in an immunocompromised patient with dermatomyositis. Without the use of PCR screening or culture on specialized medium, the organism would not have been detected....
Frosth, Sara; Slettemeås, Jannice S.; Jørgensen, Hannah J.
a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR. METHODS: A D. nodosus-specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay...... was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126...... is fundamental to diagnosis of footrot, but D. nodosus should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop...
Griffin, M. R; Miller, A D; Davis, A. C.
During a 9-day period in August 1980 in a New Jersey hospital, three pairs of consecutively numbered blood cultures from different patients were identified as positive for the same organism (two pairs of Klebsiella pneumoniae and one pair of group A Streptococcus), for each pair, both cultures were positive in the same atmosphere, both organisms had the same sensitivities, and the second of each pair grew at least 2 days after the first and was the only positive blood culture obtained from th...
Polanco, Wanda; Carter, Donna; Shulman, Stanford
The performance of the FilmArray blood culture identification (BCID) panel has been studied in adult patients. We describe here an evaluation of this assay for the rapid identification of pathogens in Bactec Peds Plus/F and Bactec standard anaerobic/F bottles that contained blood samples from pediatric patients at a tertiary care children's hospital. PMID:25274998
In vitro culture, PCR , and nested PCR for the detection of Theileria equi in horses submitted to exercise Cultivo in vitro, PCR e nested PCR na detecção de Theileria equi em eqüinos submetidos a exercícios
Full Text Available This study compared the usefulness of in vitro culture, PCR, and nested PCR for the diagnosis of Theileria equi in horses submitted to stress during exercise. Blood samples from 15 apparently healthy horses, previously conditioned to a high-speed equine treadmill, were taken prior to and after exercise. The animals were divided into two experimental groups: 30-day training schedule (G1 and 90-day training schedule (G2. Statistical analysis was performed using a chi-square test and kappa statistic was used in order to assess agreement. No significant difference was observed between samples collected at resting or after exercise. In G1, merozoites of T. equi were detected in the blood smears of four horses before in vitro culture, whereas 14 samples were positive, confirmed by culture. In G2, five and 11 horses were positive before and after culture, respectively. No PCR amplified product was observed in any of the tested animals although the PCR system based on the 16S rRNA gene of T. equi detected DNA in blood with an equivalent 8x10-5% parasitaemia. The nested PCR based on the T. equi merozoite antigen gene (EMA-1 allowed the visualization of amplified products in all the horses. Therefore, nested PCR should be considered as a means of detection of sub-clinical T. equi infections and in vitro culture could be used as a complement to other methods of diagnosis.Comparou-se a utilização do cultivo in vitro, PCR e nested PCR no diagnóstico de Theileria equi em eqüinos submetidos ao estresse induzido por exercícios. Amostras de sangue foram obtidas de 15 eqüinos submetidos a treinamento em esteira rolante de alto desempenho, sendo as amostras colhidas antes e após os exercícios. Os animais foram divididos em dois grupos experimentais: 30 dias de treinamento (G1 e 90 dias de treinamento (G2. O teste do qui-quadrado foi empregado para as análises estatísticas e o índice kappa utilizado para avaliar a concordância. Não houve diferen
Blackmer, F; Reynolds, K A; Gerba, C P; Pepper, I L
Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.
Moore, M S; McCarroll, M G; McCann, C D; May, L; Younes, N; Jordan, J A
Here we compared the results of PCR/pyrosequencing to those of culture for detecting bacteria directly from blood. DNA was extracted from 1,130 blood samples from 913 patients suspected of bacteremia (enrollment criteria were physician-ordered blood culture and complete blood count [CBC]), and 102 controls (healthy blood donors). Real-time PCR assays for beta-globin and Universal 16S rRNA gene targets were performed on all 1,232 extracts. Specimens identified by Universal 16S rRNA gene PCR/pyrosequencing as containing staphylococci, streptococci, or enteric Gram-negative rods had target-specific PCR/pyrosequencing performed. Amplifiable beta-globin (melting temperature [Tm], 87.2°C ± 0.2°C) occurred in 99.1% (1,120/1,130) of patient extracts and 100% (102/102) of controls. Concordance between PCR/pyrosequencing and culture was 96.9% (1,085/1,120) for Universal 16S rRNA gene targets, with positivity rates of 9.4% (105/1,120) and 11.3% (126/1,120), respectively. Bacteria cultured included staphylococci (59/126, 46.8%), Gram-negative rods (34/126, 27%), streptococci (32/126, 25.4%), and a Gram-positive rod (1/126, 0.8%). All controls screened negative by PCR/pyrosequencing. Clinical performance characteristics (95% confidence interval [CI]) for Universal 16S rRNA gene PCR/pyrosequencing included sensitivity of 77.8% (69.5 to 84.7), specificity of 99.3% (98.6 to 99.7), positive predictive value (PPV) of 93.3% (86.8 to 97.3), and negative predictive value (NPV) of 97.2% (96.0 to 98.2). Bacteria were accurately identified in 77.8% (98/126) of culture-confirmed sepsis samples with Universal 16S PCR/pyrosequencing and in 76.4% (96/126) with follow-up target-specific PCR/pyrosequencing. The initial PCR/pyrosequencing took ∼5.5 h to complete or ∼7.5 h when including target-specific PCR/pyrosequencing compared to 27.9 ± 13.6 h for Gram stain or 81.6 ± 24.0 h for phenotypic identification. In summary, this molecular approach detected the causative bacteria in over
Tereza Emmanuelle de Farias Rotondano
Full Text Available Ehrlichiosis and anaplasmosis are tick-borne diseases. Ehrlichia canis and Anaplasma platys infect mainly white cells and platelets, respectively. The main DNA source for PCR is peripheral blood, but the potential of blood cell fractions has not been extensively investigated. This study aims at assessment of whole blood (WB and blood fractions potential in nested PCR (nPCR to diagnose canine ehrlichiosis and anaplasmosis. The 16S rRNA gene was amplified in 71.4, 17.8, 31.57, and 30% of the WB, granulocyte (G, mononuclear cells (M, and buffy coat (BC samples. Compared to the WB, the sensitivity of the PCR was 42.86% for the M, and BC fractions, 21.43% for the G, and 33.33% for the blood clot (C. There was fair agreement between the WB and M, BC and C, and slight with the G. Fair agreement occurred between the nPCR and morulae in the blood smear. One animal was coinfected with A. platys and E. canis. This study provided the first evidence of A. platys infection in dogs in Paraíba, Brazil, and demonstrated that WB is a better DNA source than blood fractions to detect Ehrlichia and Anaplasma by nPCR, probably because of the plasma bacterial concentration following host cell lysis.
Boer, P. de; Rahaoui, H.; Leer, R.J.; Montijn, R.C.; Vossen, J.M.B.M. van der
The major disadvantage of the current gold standard for detection of the food pathogen Campylobacter, i.e. culturing, is the lengthy procedure. In this study we assessed the use of real-time PCR for detection of Campylobacter. To this end, 926 poultry samples, taken from transport containers and bro
Giese, Steen Bjørck; Ahrens, Peter
Milk and faeces samples from cows with clinical symptoms of paratuberculosis were examined for the presence of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by culture and PCR. M. paratuberculosis was cultivated in variable numbers from faeces or intestinal mucosa in eight of 11...
GE Ying; LIU Xiao-qing; XU Ying-chun; XU Shan; YU Min-hong; ZHANG Wei; DENG Guo-hua
Background Blood culture contamination is a significant adverse event.The aim of this project was to evaluate the efficacy of a strict blood collection procedure in reducing the blood culture contamination rate.Methods A prospectively controlled study was performed in two different medical areas in Peking Union Medical College Hospital (PUMCH) for 16 months (from May 2006 to September 2007).In test group,a strict blood collection procedure was carried out by trained nurses with the veinpuncture sites were scrupulously disinfected with 2.5％ tincture of iodine plus 70％ alcohol.In control group,commonly used procedure in PUMCH was performed with 0.45％ chlorhexidine acetate plus 0.2％ iodine.Blood culture positive results for 4 target organisms (Coagulase-negative staphylococci,Propionibacterium acnes,Corynebacterium species and Bacillus species) were further assessed by physicians from infectious department to determine whether a sample was true positive (pathogen) or false positive (contamination).Results Total 9321 blood culture collections were analyzed.The blood culture contamination rate in test group was significantly lower than that in control group (5/3177 (0.16％) vs.77/6144 (1.25％); x2=13.382,P ＜0.001).The most common contaminant was Coagulase-negative staphylococcus (76.83％).The average cultural time during which contaminated samples became positive was longer than that for true pathogen samples (42.0 hours vs.13.9 hours,P=0.041).Conclusion Using a strict blood collection procedure can significantly reduce blood culture contamination rate.
Cengiz, Ali Bülent; Yildirim, Inci; Ceyhan, Mehmet; Seçmeer, Gülten; Gür, Deniz; Kara, Ateş
This prospective study, which was designed to compare nasopharyngeal culture, polymerase chain reaction (PCR) and serology in the diagnosis of pertussis, covered 35 children aged between 0 and 16 who were admitted to Hacettepe University Ihsan Doğramaci Children's Hospital between 1 March 2005 and 31 August 2006 with coughing for 7 days or longer, paroxysmal cough of any duration, or cough with inspiratory whoop and/or vomiting (or apnea) after coughs. The demographic data and vaccination history of the patients were recorded. During the initial examination, samples were taken from the posterior nasopharynx for Bordetella pertussis (B. pertussis) culture and PCR analysis. In order to determine antibody positivity and antibody levels against B. pertussis antigens, serum samples were taken during the initial examination (acute phase) and two weeks later (convalescent phase). In the first serum sample, immunoglobulin M (IgM) was determined against pertussis toxin. In the first and second samples, IgA and IgG antibodies were evaluated against pertussis toxin and filamentous hemagglutinin. Culture yielded negative results in all of the patients. PCR was positive in two cases (5.7%). In the PCR-positive patients, IgM, IgA and IgG type anti-pertussis antibodies were found to be positive in the first serum samples, and IgA and IgG antibodies were found to be positive in the second serum samples. Therefore, it was considered that serology could be as sensitive as PCR when type IgM, IgA and IgG antibodies were found to be positive against a minimum of two antigens of B. pertussis. In conclusion, both PCR and serologic tests--if evaluating all types of antibodies to a minimum of two antigens of B. pertussis obtained in both acute and convalescent sera--could be more sensitive than culture in the diagnosis of pertussis.
Full Text Available Abstract Background Pseudomonas aeruginosa is the major respiratory pathogen causing severe lung infections among CF patients, leading to high morbidity and mortality. Once infection is established, early antibiotic treatment is able to postpone the transition to chronic lung infection. In order to optimize the early detection, we compared the sensitivity of microbiological culture and quantitative PCR (qPCR for the detection of P. aeruginosa in respiratory samples of not chronically infected CF patients. Results In this national study, we followed CF patients during periods between 1 to 15 months. For a total of 852 samples, 729 (86% remained P. aeruginosa negative by both culture and qPCR, whereas 89 samples (10% were positive by both culture and qPCR. Twenty-six samples were negative by culture but positive by qPCR, and 10 samples were positive by culture but remained negative by qPCR. Five of the 26 patients with a culture negative, qPCR positive sample became later P. aeruginosa positive both by culture and qPCR. Conclusion Based on the results of this study, it can be concluded that qPCR may have a predictive value for impending P. aeruginosa infection for only a limited number of patients.
Rintala, H; Hyvärinen, A; Paulin, L; Nevalainen, A
Streptomycetes are gram-positive, spore producing, filamentous bacteria common in soil, but also present in indoor environments. They are potent producers of secondary metabolites and inducers of inflammatory responses in vitro. Polymerase chain reaction (PCR)- and culture-based detection methods for streptomycetes in house dust samples were compared. A total of 47 dust samples were investigated, and the presence of streptomycetes was determined by cultivation on tryptone-yeast-extract-glucose agar and PCR. The 16S rRNA gene of actinomycete isolates from house dust was partially sequenced to investigate if they belong to the genus Streptomyces. Both PCR and culture showed more frequent occurrence of streptomycetes in moisture-damaged homes, although the results did not correlate well. The occurrence of streptomycetes in house dust was associated with moisture damage of the home. The amount of Streptomyces-specific PCR amplification product was significantly higher in dust from moisture-damaged homes than in homes with no moisture damage (P culture, Fisher's exact test). Altogether, the presence of streptomycetes in house dust seems to indicate the presence of moisture damage in the building.
Ghotaslou, Reza; Farajnia, Safar; Yeganeh, Fatemeh; Abdoli-Oskouei, Shahram; Ahangarzadeh Rezaee, Mohammad; Barzegar, Mohammad
Meningitis is one of the hazardous and life threatening infections and is associated with mortality and morbidity. The aim of this study was to determine etiological agents of childhood bacterial meningitis. The culture, Gram staining, agglutination and PCR assays were used to examine CSF specimens from 277 patients with presumed bacterial meningitis for the occurrence of 4 most common infectious agents consist of N. meningitis, H. influnsae, S. pneumoniae and S. agalactiae between 2008 and 2009 at different wards of the Children Hospital of Tabriz. The mean age of patients was 35 ± 2 (Mean ± SEM) month, (minimum 11 days maximum 14 years), of all cases 59.6% male and 40.4% female. Overall the diagnosis was confirmed with a CSF culture in 11/277 (3.97%), by agglutination test in 14/277 (5.05%). The isolated bacteria included S. pneumoniae 5 cases, H. influnsae 2 cases, N. meningitis 3 cases and P. aeroginusae 1 case. A positive PCR assay allowed us to diagnose bacterial meningitis in 19 patients (6.8%). In the present study, we found PCR to be a useful and sensitive method for the detection of bacterial DNA in the CSF samples from suspected meningitis patients. Furthermore, to maximize management of meningitis cases, a combination of culture and PCR is necessary.
Eser, Ozgen Köseoğlu; Alp, Sehnaz; Ergin, Alper; Ipçi, Kaan; Alp, Alpaslan; Gür, Deniz; Hasçelik, Gülşen
Streptococcus pneumoniae and Haemophilus influenzae are the major etiologic agents of acute otitis media. This study was aimed to compare the detection rate of S.pneumoniae and H.influenzae by culture and real-time polymerase chain reaction (Rt-PCR) in the middle ear effusions of patients diagnosed as acute otitis media. A total of 60 middle ear effusion samples collected from children with acute otitis media were included in the study. The samples were inoculated and incubated in BACTEC Ped Plus blood culture bottles and BACTEC 9120 system (BD Diagnostic Systems, MD), respectively, and the isolates were identified by conventional methods. For the molecular diagnosis of H.influenzae and S.pneumoniae, ply pneumolysin gene and HIB capsule region, respectively were amplified by Rt-PCR (LightCycler, Roche Diagnostics, Germany). H.influenzae and S.pneumoniae were isolated from 5 (8.3%) and 3 (5%) of the patient samples with conventional culture methods, respectively. In addition in 11.6% of the samples other microorganisms (Staphylococcus epidermidis, Streptococcus intermedius, Streptococcus sanguinis, Moraxella catarrhalis, Pseudomonas aeruginosa, Candida albicans) were also isolated. On the other hand H.influenzae and S.pneumoniae were detected in 38 (63.3%) and 24 (40%) of the samples with Rt-PCR, respectively. There was about eight fold increase in the detection frequency of H.influenzae and S.pneumoniae with Rt-PCR compared to culture methods. When culture was accepted as the gold standard method, the sensitivity, specificity and positive predictive value of Rt-PCR in the detection of H.influenzae and S.pneumoniae were estimated as 80%, 51% and 98.2%, respectively. As a result, Rt-PCR was shown to be a sensitive method and could be preferred for the rapid diagnosis of H.influenzae and S.pneumoniae in the etiological diagnosis of acute otitis media, especially in culture negative cases.
Kärkkäinen, Päivi M; Valkonen, Maria; Hyvärinen, Anne; Nevalainen, Aino; Rintala, Helena
In this study, we developed two novel qPCR-assays for the detection of bacteria in house dust; one that determines the total bacterial amount and another that detects Gram-positive and Gram-negative bacteria separately. The methods were tested in silico and in vitro with microbial strains and vacuum cleaner dust samples, and validated in relation to culture and chemical marker analysis. We also compared the results of these three types of methods (qPCR, culture and chemical marker analysis) in 211 house dust samples from farming and non-farming environments. Microbial concentrations determined by the new qPCR assays (median 7.2 x 10(5) cell equivalents mg(-1)) were about two orders of magnitude higher than concentrations obtained by culture (median 6.7 x 10(3) cfu mg(-1)). The median concentration of muramic acid was 25.67 ng mg(-1) and that of 3-hydroxy fatty acids, expressed as LPS(10-16) was 26.14 pg mg(-1). Correlations between qPCR and chemical markers were moderate, while correlations between culture and qPCR and chemical markers were low to moderate. All the methods used in this study showed that the microbial concentrations are statistically significantly higher (p < 0.001, Mann-Whitney) in farming than non-farming environments.As a conclusion, all tested methods can be used for determining the bacterial load in dust samples, but none of the methods was superior to the others. The results obtained with these methods represent different aspects of bacterial exposure and therefore the results are not expected to be identical with each other.
Dey, Shuvra Kanti; Nishimura, Shuichi; Okitsu, Shoko; Hayakawa, Satoshi; Mizuguchi, Masashi; Ushijima, Hiroshi
A total of 463 fecal specimens from human patients were tested for the presence of Camphylobacter by a commercial immunochromatography kit (ImmunoCard STAT! CAMPY), polymerase chain reaction (PCR) and conventional bacteriological (CB) methods. Using culture as the standard of reference, the ImmunoCard STAT! CAMPY assay had a sensitivity of 86% and a specificity of 100%. On the other hand, using PCR as the standard of reference, ImmunoCard STAT! CAMPY assay had a sensitivity of 90.5% and a specificity of 100%.
Hassanpour, Gholamreza; Mohebali, Mehdi; Raeisi, Ahmad; Abolghasemi, Hassan; Zeraati, Hojjat; Alipour, Mohsen; Azizi, Ebrahim; Keshavarz, Hossein
The transmission of malaria by blood transfusion was one of the first transfusion-transmitted infections recorded in the world. Transfusion-transmitted malaria may lead to serious problems because infection with Plasmodium falciparum may cause rapidly fatal death. This study aimed to compare real-time polymerase chain reaction (real-time PCR) with rapid diagnostic test (RDT) and light microscopy for the detection of Plasmodium spp. in blood transfusion, both in endemic and non-endemic areas of malaria disease in Iran. Two sets of 50 blood samples were randomly collected. One set was taken from blood samples donated in blood bank of Bandar Abbas, a city located in a malarious-endemic area, and the other set from Tehran, a non-endemic one. Light microscopic examination on both thin and thick smears, RDTs, and real-time PCR were performed on the blood samples and the results were compared. Thin and thick light microscopic examinations of all samples as well as RDT results were negative for Plasmodium spp. Two blood samples from endemic area were positive only with real-time PCR. It seems that real-time PCR as a highly sensitive method can be helpful for the confirmation of malaria infection in different units of blood transfusion organization especially in malaria-endemic areas where the majority of donors may be potentially infected with malaria parasites.
Hagen, Ralf Matthias; Hinz, Rebecca; Tannich, Egbert; Frickmann, Hagen
We compared the performance of an in-house and a commercial malaria polymerase chain reaction (PCR) assay using freeze-thawed hemolytic blood samples. A total of 116 freeze-thawed ethylenediamine tetraacetic acid (EDTA) blood samples of patients with suspicion of malaria were analyzed by an in-house as well as by a commercially available real-time PCR. Concordant malaria negative PCR results were reported for 39 samples and malaria-positive PCR results for 67 samples. The in-house assay further detected one case of Plasmodium falciparum infection, which was negative in the commercial assay as well as five cases of P. falciparum malaria and three cases of Plasmodium vivax malaria, which showed sample inhibition in the commercial assay. The commercial malaria assay was positive in spite of a negative in-house PCR result in one case. In all concordant results, cycle threshold values of P. falciparum-positive samples were lower in the commercial PCR than in the in-house assay. Although Ct values of the commercial PCR kit suggest higher sensitivity in case of concordant results, it is prone to inhibition if it is applied to hemolytic freeze-thawed blood samples. The number of misidentifications was, however, identical for both real-time PCR assays.
Patton, Richard G; Schmitt, Timothy
We hypothesized that diversion of the first milliliter of venipuncture blood-the initial specimen diversion technique (ISDT)-would eliminate incompletely sterilized fragments of skin from the culture specimen and significantly reduce our blood culture contamination rate (R). We studied our hypothesis prospectively beginning with our control culture (C) definition: one venipuncture with two sequentially obtained specimens, 10 ml each, the first specimen (M1) for aerobic and the second (M2) for anaerobic media. The test ISDT culture (D) was identical, with the exception that each was preceded by diverting a 1-ml sample (DS) from the same venipuncture. During the first of two sequential 9-month periods, we captured D versus C data (n=3,733), where DMXR and CMXR are R for D and C specimens. Our hypothesis predicted DS would divert soiled skin fragments from DM1, and therefore, CM1R would be significantly greater than DM1R. This was confirmed by CM1R (30/1,061 [2.8%]) less DM1R (37/2,672 [1.4%]; P=0.005), which equals 1.4%. For the second 9-month follow-up period, data were compiled for all cultures (n=4,143), where ADMXR is R for all (A) diversion specimens, enabling comparison to test ISDT. Our hypothesis predicted no significant differences for test ISDT versus all ISDT. This was confirmed by DM1R (37/2,672 [1.4%]) versus ADM1R (42/4,143 [1.0%]; P=0.17) and DM2R (21/2,672 [0.80%]) versus ADM2R (39/4,143 [0.94%]; P=0.50). We conclude that our hypothesis is valid: venipuncture needles soil blood culture specimens with unsterilized skin fragments and increase R, and ISDT significantly reduces R from venipuncture-obtained blood culture specimens.
Edagawa, Akiko; Kimura, Akio; Kawabuchi-Kurata, Takako; Adachi, Shinichi; Furuhata, Katsunori; Miyamoto, Hiroshi
We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.
Full Text Available We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR, and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%. Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%. In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8% compared with real-time qPCR alone (46/68, 67.6%. Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1% compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%. Legionella was not detected in the remaining six samples (6/68, 8.8%, irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.
Weber, D J; Saviteer, S M; Rutala, W A; Thomann, C A
To determine the clinical significance of blood isolates of Bacillus, we reviewed all blood cultures obtained at North Carolina Memorial Hospital between 1981 and 1985. Over the five-year study period the number of patients (incidence per 10,000 hospital admissions) from whom Bacillus was isolated increased from 4.97 in 1981 to 12.5 in 1985. The incidence per 1,000 blood cultures also increased from 1.12 in 1981 to 2.33 in 1985. Review of the medical records of 78 of the 95 patients (82%) with positive cultures allowed retrospective classification of five isolates (6.4%) as clinically significant, 33 isolates (42.3%) as possibly significant, and 40 isolates (51.3%) as nonsignificant. Underlying diseases in patients with clinically significant Bacillus bacteremia included burn trauma in two, leukemia in one, carcinoma in one, and gastrointestinal hemorrhage in one. All isolates judged to be clinically significant and the majority of possibly significant isolates were B cereus. We conclude that the isolation of Bacillus species from blood cultures is clinically significant in 5% to 10% of cases, that the incidence of Bacillus bacteremia is increasing, and that burn trauma should be added to the list of conditions known to predispose to clinically significant Bacillus bacteremia.
Kernéis, Solen; Matta, Matta; Hoï, Annie Buu; Podglajen, Isabelle; Gutmann, Laurent; Novara, Ana; Latremouille, Christian; Mainardi, Jean-Luc
We report a case of Finegoldia magna (formerly known as Peptostreptococcus magnus) mediastinitis following coronary artery bypass in a 50-year-old patient. Even if staphylococci remain the main causative organism of postoperative mediastinitis, the responsibility of anaerobic bacteria must be considered in cases of fever and sternal drainage with negative blood cultures.
Alby, Kevin; Daniels, Lindsay M.; Weber, David J; Miller, Melissa B.
Seventy-eight blood cultures with a Gram stain result of Gram-positive cocci in pairs and/or chains were evaluated with the Nanosphere Verigene Gram-positive blood culture (BC-GP) assay. The overall concordance of the assay with culture was 89.7% (70/78 cultures), allowing for the development of a targeted treatment algorithm.
Kottaridi, Christine; Nomikou, Kiki; Lelli, Rossella; Markoulatos, Panayotis; Mangana, Olga
A new polymerase chain reaction (PCR) assay for rapid diagnosis of contagious ecthyma was designed and applied to 21 clinical samples from Greece. This assay, which detects a highly conserved gene from the parapox genome, was evaluated for its sensitivity and specificity in order to be considered as a useful diagnostic tool. A comparative study with two published PCR protocols one using primers PPP1-PPP3, PPP1-PPP4 which targets putative virion envelope gene B2L and the other using VIR1-VIR2 primers which amplifies ORF virus interferon resistant (VIR) gene, as well as cell culture virus neutralization assay was carried out. All samples tested were amplified successfully with the PCR protocol established in the laboratory. The combination of primers PPP1-PPP3 and PPP1-PPP4 in a semi-nested PCR gave a positive result in 20 of 21 samples while primers VIR1-VIR2 failed to amplify successfully 7 of 21 samples. The diagnostic value of parapox viral DNA amplification was also compared with the results of virus isolation by cell culture and was positive in three samples that the virus isolation was obtained.
Full Text Available Aim: Trichomoniasis, a sexually transmitted infection (STI caused by Trichomonas vaginalis, affects 180 million people worldwide and causes significant morbidity. Infection with T. vaginalis has been associated with vaginitis, exocervicitis, and urethritis in women. Material and Method: In this study, we aim to investigate the presence of T. vaginalis by using three different methods for comparing the results. Two hundred T. vaginalis isolates taken from swap samples were collected in Medical Faculty, Department of Gynecology, Mustafa Kemal University Polyclinic, and examined genotypically and phenotypically to identify T. vaginalis in Parasitology Department. This research is unique in terms of its contribution to patient treatment, being the first molecular study in Turkey/Hatay to determine Trichomonas (TV genes stemming from Trichomonas vaginalis strains. Result: 56 out of 200 patients examined were identified as positive and 24 (42.8% of these were identified through microscopy, 18 (32,1% with culture and 24 (42,8% with PCR. The number of those identified through all these methods is 14 (25%. In this study, difference was calculated using three methods (p=0.022 with Cochran%u2019s Q test. When compared with McNemar two by two, no superiority in T. vaginalis diagnosis was found between microscopy and culture (p=0.5, microscopy and PCR (p=0.063, or culture and PCR (p=0.25 methods. Discussion: Culture method is not used in routine laboratory procedures and has contamination risk. PCR method shows directly the parasite of DNAs, and so it is thought to be more reliable compared to the other two methods.
Full Text Available Background and Aims: Periodontitis is one of the most common oral diseases with the various incidence rates in different populations. A number of bacteria are considered as the major etiologic agents of periodontitis. The aim of the present study was to determine the prevalence of periodontopathogen bacteria in patients using both PCR and culture techniques.Materials and Methods: In this study, one-hundred patients (including 62 females and 38 males with an average age of 49±11.5 years with adult periodontitis referred to periodontics department of School of Dentistry/Tehran University of Medical Sciences were investigated. The samples were taken and sent immediately to the laboratory for culture and molecular evaluation. The PCR was performed using specific primers and the statistical analysis of data was performed using SPSS statistic software and McNemar test.Results: The results demonstrated that the total detection rate in culture method was 64%. The rate of Aggregatibacter actinomycetemcomitans (Aa was 28% which was significantly higher than that of Porphyromonas gingivalis (Pg (6% and Prevotella intermedia (Pi (3%. 27% of cases showed mixed bacterial growth. 65% of patients were positive using molecular method. The rate of Aa (30% was significantly higher than that of Pg (7% and Pi (5%. The mixed PCR positive rate containing of Aa, Pg and Pi was (23%.Conclusion: In this study, it was found that most of the bacteria isolated using culture and molecular methods were Aa, Pg and Pi, respectively. Although the detection frequencies of both techniques were similar, the specificity, sensitivity and bacterial detection speed of the PCR technique is obviously higher. Therefore, the use of molecular techniques is strongly recommended. However, both techniques seem to be suitable for microbiological diagnostics.
Andergassen, Ulrich; Kölbl, Alexandra C.; Hutter, Stefan; Friese, Klaus; Jeschke, Udo, E-mail: firstname.lastname@example.org [Department of Obstetrics and Gynaecology, Ludwig Maximilians University of Munich, Munich, Maistrasse 11, D-80337 Munich (Germany)
Breast cancer is still the most frequent cause of cancer-related death in women worldwide. Often death is not caused only by the primary tumour itself, but also by metastatic lesions. Today it is largely accepted, that these remote metastases arise out of cells, which detach from the primary tumour, enter circulation, settle down at secondary sites in the body and are called Circulating Tumour Cells (CTCs). The occurrence of such minimal residual diseases in the blood of breast cancer patients is mostly linked to a worse prognosis for therapy outcome and overall survival. Due to their very low frequency, the detection of CTCs is, still a technical challenge. RT-qPCR as a highly sensitive method could be an approach for CTC-detection from peripheral blood of breast cancer patients. This assumption is based on the fact that CTCs are of epithelial origin and therefore express a different gene panel than surrounding blood cells. For the technical approach it is necessary to identify appropriate marker genes and to correlate their gene expression levels to the number of tumour cells within a sample in an in vitro approach. After that, samples from adjuvant and metastatic patients can be analysed. This approach may lead to new concepts in diagnosis and treatment.
Full Text Available Background: No evidence-based guidelines or protocols to treat the infection-related symptoms in cancer patients with terminal stages have been established. Materials and Methods: We retrospectively analyzed all the patients with terminal stage cancer who died between April 2009 and March 2010. The patients' background, the prevalence of infection and clinical outcomes, pathogens isolated, antibiotics used, and whether blood cultures and some of examinations were performed or not were evaluated. Results: A total of 62 (44 males and 18 females patients were included in this study. The median age was 73 years (35-98 years. The most common cancer was that of the lung (n =59, 95.2%. A total of 32 patients were diagnosed with the following infections: Infection of respiratory tract in 27 (84.4%, of urinary tract in 4 (12.5%, and cholangitis in 1 (3.1%. Two cases (6.3% had pneumonia complicated with urinary tract infection. Blood cultures and antibiotic therapies were performed in 28 and 30 cases, respectively. Four (14.3% positive cultures were isolated from the blood obtained from 28 individual patients. As for clinical course, 3 (10% of them experienced improved symptoms after antibiotic therapy. Twenty-seven (90% patients were not confirmed as having any symptom improvement. Conclusions: Blood cultures and antibiotic therapy were limited, and might not be effective in terminally ill cancer patients with lung cancer. We suggest that administering an antibiotic therapy without performing a blood culture would be one of choices in those with respiratory tract infections if patients' life expectancy is short.
Wang, Chong; Robles, Francisco; Ramirez, Saul
species of the families Pasteurellaceae, Enterobacteriaceae and Flavobacteriaceae. Regarding specificity none of non-G. anatis strains tested positive with the proposed assay. To test and compare the qPCR method's ability to detect G. anatis from field samples, the sensitivity was compared to a previously......-time quantitative PCR (qPCR) method allowing species-specific identification and quantification of G. anatis. A G. anatis specific DNA sequence was identified in the gyrase subunit B gene (gyrB) and used to design a TaqMan probe and corresponding primers. The specificity of the assay was tested on 52 bacterial...... published conventional PCR method and culture-based identification, respectively. The detection rates were 97%, 78% and 34% for the current qPCR, the conventional PCR and the culture-based identification method, respectively. The qPCR assay was able to detect the gene gyrB in serial dilutions of 10...
Simpson, Kathleen E; Storch, Gregory A; Lee, Caroline K; Ward, Kent E; Danon, Saar; Simon, Catherine M; Delaney, Jeffrey W; Tong, Alan; Canter, Charles E
Specific viruses are associated with pediatric myocarditis, but the prevalence of viral DNAemia detected by blood polymerase chain reaction (PCR) is unknown. We evaluated the prevalence of known cardiotropic viruses (enterovirus, adenovirus, human herpesvirus 6, and parvovirus B19) in children with clinical myocarditis (n = 21). Results were compared to pediatric controls with similar viral PCR testing. The majority of positive PCR (89 %) was noted in children ≤12 months of age at diagnosis compared to older children. Infant myocarditis patients (8/10) had increased the prevalence of PCR positivity compared to infant pediatric controls (4/114) (p myocarditis infants had clinical recovery at follow-up. Of the PCR-positive myocarditis infants, 4 had clinical recovery, 2 developed chronic cardiomyopathy, 1 underwent heart transplant, and 1 died. Infants with clinical myocarditis have a high rate of blood viral positivity, which is higher compared to older children with myocarditis and healthy infant controls. Age-related differences in PCR positivity may be due to differences in host and/or virus characteristics. Our findings suggest that viral blood PCR may be a useful diagnostic tool and identify patients who would potentially benefit from virus-specific therapy.
Wei, Bo; Chen, Lei; Kibukawa, Miho; Kang, John; Waskin, Hetty; Marton, Matthew
Chagas disease is caused by the parasitic infection of Trypanosoma cruzi (T. cruzi). The STOP CHAGAS clinical trial was initiated in 2011 to evaluate posaconazole in treating Chagas disease, with treatment success defined as negative qualitative PCR results of detecting the parasites in blood specimens collected post-treatment. PAXgene Blood DNA tubes were utilized as a simple procedure to collect and process blood specimens. However, the PAXgene blood specimens challenged published T. cruzi PCR methods, resulting in poor sensitivity and reproducibility. To accurately evaluate the treatment efficacy of the clinical study, we developed and validated a robust PCR assay for detecting low level T. cruzi in PAXgene blood specimens. The assay combines a new DNA extraction method with a custom designed qPCR assay, resulting in limit of detection of 0.005 and 0.01 fg/μl for K98 and CL Brener, two representative strains of two of T. cruzi’s discrete typing units. Reliable qPCR standard curves were established for both strains to measure parasite loads, with amplification efficiency ≥ 90% and the lower limit of linearity ≥ 0.05 fg/μl. The assay successfully analyzed the samples collected from the STOP CHAGAS study and may prove useful for future global clinical trials evaluating new therapies for asymptomatic chronic Chagas disease. PMID:27906977
Tan Xue Ting
Full Text Available Pertussis or whooping cough is a highly infectious respiratory disease caused by Bordetella pertussis. In vaccinating countries, infants, adolescents, and adults are relevant patients groups. A total of 707 clinical specimens were received from major hospitals in Malaysia in year 2011. These specimens were cultured on Regan-Lowe charcoal agar and subjected to end-point PCR, which amplified the repetitive insertion sequence IS481 and pertussis toxin promoter gene. Out of these specimens, 275 were positive: 4 by culture only, 6 by both end-point PCR and culture, and 265 by end-point PCR only. The majority of the positive cases were from ≤3 months old patients (77.1% (. There was no significant association between type of samples collected and end-point PCR results (. Our study showed that the end-point PCR technique was able to pick up more positive cases compared to culture method.
Full Text Available The quality of microbiological diagnostic procedures depends on pre-analytic conditions. We compared the results of 16S rRNA gene PCR and sequencing from automatically incubated blood culture materials from tropical Ghana with the results of cultural growth after automated incubation.Real-time 16S rRNA gene PCR and subsequent sequencing were applied to 1500 retained blood culture samples of Ghanaian patients admitted to a hospital with an unknown febrile illness after enrichment by automated culture.Out of all 1500 samples, 191 were culture-positive and 98 isolates were considered etiologically relevant. Out of the 191 culture-positive samples, 16S rRNA gene PCR and sequencing led to concordant results in 65 cases at species level and an additional 62 cases at genus level. PCR was positive in further 360 out of 1309 culture-negative samples, sequencing results of which suggested etiologically relevant pathogen detections in 62 instances, detections of uncertain relevance in 50 instances, and DNA contamination due to sample preparation in 248 instances. In two instances, PCR failed to detect contaminants from the skin flora that were culturally detectable. Pre-analytical errors caused many Enterobacteriaceae to be missed by culture.Potentially correctable pre-analytical conditions and not the fastidious nature of the bacteria caused most of the discrepancies. Although 16S rRNA gene PCR and sequencing in addition to culture led to an increase in detections of presumably etiologically relevant blood culture pathogens, the application of this procedure to samples from the tropics was hampered by a high contamination rate. Careful interpretation of diagnostic results is required.
Thuret, G; Carricajo, A.; Chiquet, C.; Vautrin, A C; Celle, N; Boureille, M; Acquart, S; Aubert, G.; Maugery, J; Gain, P.
Aims: To test the bactericidal activity of standard organ culture medium, and to compare the sensitivity and rapidity of blood culture bottles with conventional microbiological methods for detection of bacteria and fungi inoculated in a standard cornea organ culture medium.
Azzari, Chiara; Cortimiglia, Martina; Moriondo, Maria; Canessa, Clementina; Lippi, Francesca; Ghiori, Federica; Becciolini, Laura; de Martino, Maurizio; Resti, Massimo
The diagnosis of invasive pneumococcal disease (IPD) is currently based on culture methods, which lack sensitivity, especially after antibiotic therapy. Molecular methods have improved sensitivity and do not require viable bacteria; however, their use is complicated by reports of low specificity with some assays. The present study investigated the specificity of a real-time PCR targeting lytA for the detection of IPD. A group of 147 healthy children, aged 6 months to 16 years (mean 6.4 years, median 4.9 years, interquartile range 6.4 years), who were in hospital for routine examinations, were tested for pneumococcal carrier status and for the presence of detectable pneumococcal DNA in their blood by real-time PCR targeting the pneumococcal lytA gene. In addition, 35 culture-positive biological samples were analysed. Urine was examined for the presence of pneumococcal DNA and C-polysaccharide antigen. Carriage was detected in 77 of the 147 subjects (52.4 %); however, regardless of carrier status, none of the subjects had a positive result from blood. Analysis of the culture-positive biological samples yielded positive results in 100 % (15/15) of cerebrospinal fluid samples and 95 % (19/20) of blood samples. All urine samples from healthy carriers were negative for DNA, whilst antigenuria was detected in 44/77 carriers (57.1 %). In conclusion, real-time PCR is both sensitive and specific and can be a useful tool in the routine diagnosis of IPD. Its sensitivity, which surpasses that of other methods for this purpose, does not come at the cost of reduced specificity.
Pfeiffer, Christopher D; Samsa, Gregory P; Schell, Wiley A; Reller, L Barth; Perfect, John R; Alexander, Barbara D
One potential limitation of DNA-based molecular diagnostic tests for Candida bloodstream infection (BSI) is organism burden, which is not sufficiently characterized. We hypothesized that the number of CFU per milliliter (CFU/ml) present in an episode of Candida BSI is too low for reliable DNA-based diagnostics. In this study, we determined Candida burden in the first positive blood culture and explored factors that affect organism numbers and patient outcomes. We reviewed records of consecutive patients with a positive blood culture for Candida in the lysis-centrifugation blood culture system (Isolator, Wampole Laboratories, Cranbury, NJ) from 1987 to 1991. Descriptive statistics and logistic regression analyses were performed. One hundred fifty-two episodes of Candida BSI were analyzed. Patient characteristics included adult age (72%), indwelling central venous catheters (83%), recent surgery (29%), neutropenia (24%), transplant (14%), and other immune suppression (21%). Rates of treatment success and 30-day mortality for candidemia were each 51%. The median CFU/ml was 1 (mode 0.1, range 0.1 to >1,000). In the multivariate analysis, pediatric patients were more likely than adults to have high organism burdens (odds ratio [OR], 10.7; 95% confidence interval [95% CI], 4.3 to 26.5). Initial organism density did not affect patient outcome. Candida CFU/ml in the first positive blood culture of a BSI episode varies greatly; >50% of cultures had ≤1 CFU/ml, a concentration below the experimental yeast cell threshold for reliable DNA-based diagnostics. DNA-based diagnostics for Candida BSI will be challenged by low organism density and the need for sufficient specimen volume; future research on alternate targets is warranted.
R. Munro; Collignon, P J; Sorrell, T C; Tomlinson, P.
Two hundred and thirty-four blood cultures from 140 patients receiving antibiotics were processed using the antimicrobial removal device (ARD) in parallel with conventional blood cultures. One hundred and seventy cultures were obtained from patients suspected to have bacteraemia and 64 from patients known to have a positive conventional blood culture within the preceding three days. A total of 38 (16.2%) ARD-processed cultures were positive, compared with 21 (8.9%) conventional cultures (p le...
Guo, Yi; Yang, Jing-Xian; Liang, Guo-Wei
The prevalence of Candida in bloodstream infections (BSIs) has increased. To date, the identification of Candida in BSIs still mainly relies on blood culture and serological tests, but they have various limitations. Therefore, a real-time PCR assay for the detection of Candida from whole blood is presented. The unique primers/probe system was designed on 5.8S rRNA gene (5.8S rDNA) of Candida genus. The analytical sensitivity was determined by numbers of positive PCRs in 12 repetitions. At the concentration of 10(1) CFU/ml blood, positive PCR rates of 100 % were obtained for C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. The detection rate for C. glabrata was 75 % at 10(1) CFU/ml blood. The reaction specificity was 100 % when evaluating the assay using DNA samples from clinical isolates and human blood. The maximum CVs of intra-assay and inter-assay for the detection limit were 1.22 and 2.22 %, respectively. To assess the clinical applicability, 328 blood samples from 82 patients were prospectively tested and real-time PCR results were compared with results from blood culture. Diagnostic sensitivity of the PCR was 100 % using as gold standard blood culture, and specificity was 98.4 %. Our data suggest that the developed assay can be used in clinical laboratories as an accurate and rapid screening test for the Candida from whole blood. Although further evaluation is warranted, our assay holds promise for earlier diagnosis of candidemia.
Jia Zeng Xia; Hao Ran Yin; Zheng Gang Zhu; Min yan
AIM To study the detection of micrometastasis in peripheral blood of patients with gastric carcinomas andits clinical significance.METHODS A cytokeratin 19 (CK19)-specific nested reverse transcriptase-polimerase chain reaction (RT-PCR) assay was developed to detect CK19 expressing cancer cells, the sensitivity was determined by serialdilution method using CK19 expressing gastric cancer cells, the specificity was assessed by examining 12negative controls and 12 positive controls. Then pre-operative peripheral blood from 42 patients with gastriccancer was detected and the relationship between positive results and biological behavior was studied.RESULTS CK19mRNA was expressed in all the 12 gastric cancer tissues but not in peripheral blood from12 healthy individuals；sensitivity of nested RT-PCR amplification for CK19mRNA was confirmed to be 1/106 by serial dilution method using human gastric cancer line SGC-7901; micrometastases in pre-operativeperipheral blood were detected in 13 (30,9%) patients with gastric carcinomas, the frequency ofmicrometastasis in peripheral blood was significantly correlated with tumor size,depth of invasion and TNMstage (x2 test, P<0.05).CONCLUSION Nested RT-PCR amplification for CK19mRNA is a sensitive and specific method for thedetection of micrometastases in peripheral blood in gastric cancer patients; pre-operative detection ofmicrometastasis in peripheral blood may be helpful in the prediction of tumor progression.
Khatib, R; Labalo, V; Sharma, M; Johnson, L B; Riederer, K
We retrospectively evaluated adult cases with Enterococcus spp. in 1 blood culture (BC) (1/1/2010-12/31/2015; n=294) and stratified them into bacteremia or contamination. Contamination frequency was similar in community versus hospital-onset, E. faecalis versus E. faecium, and number of BC drawn per day. Contamination predictors were vancomycin-resistance, ampicillin-resistance, commensal organism copresence, and nonurinary/abdominal sources.
A Sobhani; H. Shodjai S. Javanbakht
Bacteremia is an important infectious disease which may lead to death. Common bacteria and pattern of antibiotic resistance in different communities are different and understanding these differences is important. In the present study, relative frequency and pattern of drug resistance have been examined in bacteria isolated from blood cultures in Razi Hospital laboratory. The method of the study was descriptive. Data collection was carried out retrospectively. Total sample consisted of 311 pos...
Full Text Available Reverse transcription quantitative PCR (RT-qPCR is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults.
Saveliev Sergei V.
Full Text Available The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 107 or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.
De Vos Daniel
Full Text Available Abstract Background Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients - to mimick as closely as possible the sputa sent to routine laboratories - to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols. Results In our hands, all three culture methods and the bioMérieux easyMAG Nuclisens protocol Generic 2.0.1, preceded by proteinase K pretreatment and followed by any of the 3 real-time PCR formats with probes were most sensitive and able to detect P. aeruginosa up to 50 cfu/ml, i.e. the theoretical minimum of one cell per PCR mixture, when taking into account the volumes used in this study of sample for DNA-extraction, of DNA-elution and of DNA-extract in the PCR mixture. Conclusion In this study, no difference in sensitivity could be found for the detection of P. aeruginosa from sputum between microbiological culture and optimized DNA-extraction and real-time PCR. The results also indicate the importance of the optimization of the DNA-extraction protocol and the PCR format.
Parker, Alysia M.; House, John K.; Hazelton, Mark S.; Bosward, Katrina L.; Sheehy, Paul A.
Mycoplasma spp. are a major cause of mastitis, arthritis and pneumonia in cattle, and have been associated with reproductive disorders in cows. While culture is the traditional method of identification the use of PCR has become more common. Several investigators have developed PCR protocols to detect M. bovis in milk, yet few studies have evaluated other sample types or other important Mycoplasma species. Therefore the objective of this study was to develop a multiplex PCR assay to detect M. bovis, M. californicum and M. bovigenitalium, and evaluate its analytical performance against traditional culture of bovine milk, semen and swab samples. The PCR specificity was determined and the limit of detection evaluated in spiked milk, semen and swabs. The PCR was then compared to culture on 474 field samples from individual milk, bulk tank milk (BTM), semen and swab (vaginal, preputial, nose and eye) samples. Specificity analysis produced appropriate amplification for all M. bovis, M. californicum and M. bovigenitalium isolates. Amplification was not seen for any of the other Mollicutes or eubacterial isolates. The limit of detection of the PCR was best in milk, followed by semen and swabs. When all three Mycoplasma species were present in a sample, the limit of detection increased. When comparing culture and PCR, overall there was no significant difference in the proportion of culture and PCR positive samples. Culture could detect significantly more positive swab samples. No significant differences were identified for semen, individual milk or BTM samples. PCR identified five samples with two species present. Culture followed by 16S-23S rRNA sequencing did not enable identification of more than one species. Therefore, the superior method for identification of M. bovis, M. californicum and M. bovigenitalium may be dependent on the sample type being analysed, and whether the identification of multiple target species is required. PMID:28264012
Moskwa, Bożena; Bień, Justyna; Cybulska, Aleksandra; Kornacka, Aleksandra; Krzysiak, Michał; Cencek, Tomasz; Cabaj, Władysław
A simple polymerase chain reaction (PCR) test was used to identify Ashworthius sidemi, a blood-sucking gastrointestinal nematode that commonly infects bison, red and roe deer, and moose in Poland. The present study uses this technique to confirm the possibility of transmission of A. sidemi infection from wildlife to domestic animals, such as cattle and sheep, grazing on the same natural pastures. A 406 bp fragment of genomic A. sidemi DNA was actually detected in DNA isolated from larval cultures derived from feces from cattle. A. sidemi DNA has been detected in cattle which represent a new host for this parasite. This is the first evidence of A. sidemi in cattle. The results reveal that a PCR test based on DNA from L3 larvae can be used for in vivo detection of A. sidemi invasions in breeding animals. In conclusion, the transfer of A. sidemi infection from wildlife to the farm animals sharing the same pastures appears possible.
McCann, Chase D; Jordan, Jeanne A
Bacterial bloodstream infections (BSI) and ensuing sepsis are important causes of morbidity and mortality. Early diagnosis and rapid treatment with appropriate antibiotics are vital for improving outcome. Nucleic acid amplification of bacteria directly from whole blood has the potential of providing a faster means of diagnosing BSI than automated blood culture. However, effective DNA extraction of commonly low levels of bacterial target from whole blood is critical for this approach to be successful. This study compared the Molzyme MolYsis™ Complete5 DNA extraction method to a previously described organic bead-based method for use with whole blood. A well-characterized Staphylococcus aureus-induced pneumonia model of sepsis in canines was used to provide clinically relevant whole blood samples. DNA extracts were assessed for purity and concentration and analyzed for bacterial rRNA gene targets using PCR and sequence-based identification. Both extraction methods yielded relatively pure DNA with median A260/280 absorbance ratios of 1.71 (MolYsis™) and 1.97 (bead-based). The organic bead-based extraction method yielded significantly higher average DNA concentrations (PDNA concentrations of the MolYsis™ extracts closely mirrored quantitative blood culture results. Overall, S. aureus DNA was detected from whole blood samples in 70.7% (58/82) of MolYsis™ DNA extracts, and in 59.8% (49/82) of organic bead-based extracts, with peak detection rates seen at 48h for both MolYsis™ (87.0%) and organic bead-based (82.6%) methods. In summary, the MolYsis™ Complete5 DNA extraction kit proved to be the more effective method for isolating bacterial DNA directly from extracts made from whole blood.
Wu, Bin; Liang, Weili; Kan, Biao
The well-known human pathogenic bacterium, Vibrio cholerae, can enter a physiologically viable but non-culturable (VBNC) state under stress conditions. The differentiation of VBNC cells and nonviable cells is essential for both disease prevention and basic research. Among all the methods for detecting viability, propidium monoazide (PMA) combined with real-time PCR is popular because of its specificity, sensitivity, and speed. However, the effect of PMA treatment is not consistent and varies among different species and conditions. In this study, with an initial cell concentration of 1×10(8) CFU/ml, time and dose-effect relationships of different PMA treatments were evaluated via quantitative real-time PCR using live cell suspensions, dead cell suspensions and VBNC cell suspensions of V. cholerae O1 El Tor strain C6706. The results suggested that a PMA treatment of 20 μM PMA for 20 min was optimal under our conditions. This treatment maximized the suppression of the PCR signal from membrane-compromised dead cells but had little effect on the signal from membrane-intact live cells. In addition to the characteristics of PMA treatment itself, the initial concentration of the targeted bacteria showed a significant negative influence on the stability of PMA-PCR assay in this study. We developed a strategy that mimicked a 1×10(8) CFU/ml cell concentration with dead bacteria of a different bacterial species, the DNA of which cannot be amplified using the real time PCR primers. With this strategy, our optimal approach successfully overcame the impact of low cell density and generated stable and reliable results for counting viable cells of V. cholerae in the VBNC state.
Nicole L Podnecky
Full Text Available Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1 assay. Crossing threshold (C T values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10(4 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.
Park, Ki-Ho; Cho, Oh-Hyun; Lee, Sang-Oh; Choi, Sang-Ho; Kim, Yang Soo; Woo, Jun Hee; Kim, Mi-Na; Kim, Dae-Young; Lee, Jung-Hee; Lee, Je-Hwan; Lee, Kyoo-Hyung; Lee, Dae Ho; Suh, Cheolwon; Kim, Sung-Han
There are limited data on the incidence of subsequent bloodstream infection (BSI) and the effect of systemic antibiotics in patients who had positive catheter-drawn blood cultures (CBC) and negative peripheral blood cultures (PBC). We retrospectively reviewed all paired blood cultures from patients with Hickman catheter in the hematology-oncology ward between January 1997 and December 2008. There were 112 episodes with positive CBC and negative PBC. Nine episodes (8.0%; 95% CI, 3.0-13.1%) led to subsequent BSI within 28 days. Subsequent BSI developed in 6 of 31 episodes (19%) where empiric antibiotics were inappropriate but in 3 of 81 episodes (4%) where empiric antibiotics were appropriate (P = 0.01). Subsequent candidemia (50%, 2 of 4) was more common than subsequent bacteremia (6%, 7 of 108) (P = 0.03). In conclusion, for patients with positive CBC and negative PBC, the overall incidence of subsequent BSI was 8.0%, and inappropriate empiric antibiotics was associated with subsequent BSI.
Trisha N. Peel
Full Text Available Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM in addition to applying the Infectious Diseases Society of America (IDSA criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014 at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32% met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively; this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003. The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001, with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster.
Rosadas, Carolina; Cabral-Castro, Mauro Jorge; Vicente, Ana Carolina Paulo; Peralta, José Mauro; Puccioni-Sohler, Marzia
The objective of this study was to validate a TaqMan real-time PCR assay for HTLV-1 proviral load detection in peripheral blood mononuclear cells. TARL-2 cells were used to generate a standard curve. Peripheral blood mononuclear cell gDNA from 27 seropositive and 23 seronegative samples was analyzed. The sensitivity, specificity, accuracy, precision, dynamic range of the standard curve and qPCR efficiency were evaluated. All of the positive samples amplified the target gene. All of the negative samples amplified only the control gene (β-actin). The assay presented 100% specificity and sensibility. The intra- and inter-assay variability was 2.4% and 2.2%, respectively. The qPCR efficiency, slope and correlation coefficients (r2) were all acceptable. The limit of detection was 1 copy/rxn. This assay can reliably quantify HTLV-1 proviral load.
Aly, Balegh H; Hamad, Mostafa S; Mohey, Mervat; Amen, Sameh
The aim of this study was to compare between polymerase chain reaction (PCR) and bacterial culture in detection of Streptococcus Pneumonia and M. Catarrhalis in otitis media with effusion (OME) in children. Fifty patients having OME were included in this study between 2003 and 2008. Myringotomy and tympanostomy tube insertion were done in every patient and the middle ear effusion samples were aspirated. The samples were subjected to bacteriological study in the form of culture and molecular study in the form of PCR using JM201/202-204 primer probe set for both S. pneumonia and M. catarrhalis. The results of Bacterial cultures are as follows: five cases (10%) were culture positive for S. pneumonia. Six cases (12%) were culture positive for M. catarrhalis. Only one case (2%) showed positively for both S. pneumonia and M. catarrhalis. Polymerase chain reaction test shows that 18 cases (36%) were positive for S. pneumonia, 22 cases (44%) were positive for M. catarrhalis, 6 cases (12%) were positive for both organism and 4 cases (8%) were negative. The difference between the proportion of culture positive and PCR positive specimens for both organisms individually and collectively was significant (P PCR is more accurate than bacterial culture in detection of organisms in middle ear fluid in OME and that M. catarrhalis plays a significant rule in OME as it is the sole organism identified more than the other one by PCR.
Full Text Available Sepsis is a serious clinical condition, associated with high mortality despite advanced modern medical treatment. Traditionally, the detection and identification of bacteria and fungi circulating in the blood-stream is based on blood cultures. A number of factors influence the yield of blood culture, most of them concerning the microbiologist skill and the laboratory organization. In order to collect information about the practices and procedures used for the detection of microrganisms in blood cultures in the italian laboratory (lab, an e-mail with the invitation to participate in the survey was sent to 2000 members of the Italian Association of Clinical Microbiology. Responses were received from 100 lab, located from all over the country (in 18/20 italian regions. The results presented hereby concern specimen collection, culture techniques, rapid identification and susceptibility testing, laboratory organization, relationships with physicians. In summary, most lab use automated systems (96%, the bottles are incubated immediately during public holidays in 72/96 lab (75% and in 49/97 lab at night (50.5%, the lenght of incubation was 5 or 7 days in 93% of the lab, although it is common to extend the incubation period when brucellosis (74 lab, endocarditis (49 lab, systemic mycosis (33 lab is suspected. A wide variety of media are employed for subcultures. All lab process the positive bottles at least once a day, while only in 42 of 81 (51.9% lab the positive blood are processed on holiday. Communication between clinicians and microbiologist include: distribution of specimen collection guidelines (96/100 lab, availability to microbiologist of patients’ clinical situation (77/96 lab, 80.2%, and adding to report the microbiologist’ suggestion (75/98 lab, 76.5%. The results, compared with those collected with a similar questionnaire in 2001, show a greater adherence to guidelines: the number of bottles examined by lab yearly is almost doubled
Full Text Available Introduction: Blood culture is still the gold standard for the detection of the causative agent of sepsis. Especially in intensive care patients and those with vascular catheters, the most common organisms isolated are coagulase-negative staphylococci (CoNS and Staphylococcus aureus, both characterized by multidrug resistance. Purposes of our work are the study of the incidence of markers of resistance in staphylococci and evaluation of potential changes over the years. Materials and methods: In the period January 2008-June 2011 5239 blood cultures were analyzed.They were mainly obtained from the departments of Intensive Care, Cardiology, Hematology, General Medicine, Emergency Medicine, Infectious Diseases, Oncology, Pulmonology and Pediatric Hematoncology. The vials containing the blood were incubated in the BACTEC 9120 automated tool of Becton Dickinson and susceptibility testing performed with the Phoenix instrument of the same company. Results:Within a total of 5239 blood cultures, 3967 (75.7% were negative and 1272 (24.3% positive. Fungi were isolated in 6.2% (79 of the positive ones, Gram-negative bacteria in 24.6% (313 and Gram-positive bacteria in 69.2% (880. Within the latter, 187 (21.2% were not staphylococcal isolates, 693 (78.8% were stafiloccocci mainly represented by S. epidermidis, S. aureus, S. hominis, S. haemolyticus and S. saprophyticus. Of the 693 staphylococcal isolates, 436 (62.9% were b lactamase producers, and between them 336 (77.1% were methicillin resistant, while only 3 of 436 (0.69% were S. aureus resistant to vancomycin as well.The incidence of markers of resistance was very high, especially in patients in intensive care and cardiac surgery, who are usually subjected to combined antibiotic therapy. In the three years studied there were no statistically significant differences in the resistance of staphylococci. Conclusions: The data show an alarming high number of multi-resistant staphylococci, which is often a
Full Text Available Abstract Background Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments. Results A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA treated root cultures than those treated with sodium acetate (NaOAc. Conclusions This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2 and a gene encoding a ribosomal protein (RPL8C. A
de Boer, P; Rahaoui, H; Leer, R J; Montijn, R C; van der Vossen, J M B M
The major disadvantage of the current gold standard for detection of the food pathogen Campylobacter, i.e. culturing, is the lengthy procedure. In this study we assessed the use of real-time PCR for detection of Campylobacter. To this end, 926 poultry samples, taken from transport containers and broiler caeca in The Netherlands in 2007, were subjected to three different real-time PCR detection methods: one targeting the Campylobacter jejuni hipO gene, one targeting the Campylobacter coli glyA gene, and one generically targeting Campylobacter spp. 16S rDNA sequence. The PCR results from the three different PCR protocols were compared to the work of Nauta et al. (2009) who analyzed the same set of samples collected from 62 broiler flocks by means of enrichment culturing. The results indicate that the generic 16S campylobacter PCR detection is equally reliable but much faster (4 h instead of ≥2 days) than detection by means of culturing. Moreover, PCR detection targeting the hipO and the glyA gene provide the possibility of C. jejuni and C. coli species discrimination. The generic Campylobacter spp. PCR analysis also confirmed the high incidence of Campylobacter spp. in poultry samples (∼90%) and the species specific PCR showed the simultaneous presence of C. jejuni and C. coli in ∼24% of the samples. Furthermore, the results from the three PCR analyses suggested the occurrence of alternative Campylobacter species in almost 10% of the samples. The campylobacter PCR detection methods reported here can replace traditional culturing because of being quicker and more reliable.
Fuursted, K.; Arpi, M.; Lindblad, B.E.
We aimed to evaluate broad-range PCR and subsequent sequencing compared to conventional culture in the diagnosis of spinal infection. The method was a prospective study of all patients admitted to Aarhus University Hospital for surgery during a 12-months period with a clinically diagnosed infection...... allowed for a microbiological diagnosis in 72% of patients (13/18). A positive culture was found only in patients treated compared to PCR. However, PCR and culture result were equally negatively affected by duration of treatment. The combination of culture and broad-range PCR...... (clinically diagnosed spinal infections=18; non-infectious diseases=20). The specificity was excellent for both culture and PCR (95% and 100%, respectively). A true culture positive result was obtained in 50% of patients (9/18) and 61% was positive (11/18) by broad-range PCR. When combined, culture and PCR...
Bentley, James; Thakore, Shobhan; Muir, L; Baird, Alastair; Lee, Jennifer
Blood cultures are an important investigation to help tailor effective management for patients with severe sepsis. Frequent contaminated samples increase laboratory workload and can delay or cause incorrect changes to patient management. This can prolong patient hospitalisation, increase the risk of harm and increase cost to health boards. Current guidelines advocate a contamination rate of 2-3%. From January 2013 to November 2014 inclusive, the contamination rate was 4.74% in our Emergency Department, responsible for initial management and investigation of over 40 cases of sepsis per month. A Quality Improvement team was created to try to reduce contamination rates to the recommended target. An initial baseline survey of local staff showed good understanding of when to obtain a blood culture but there was variability in the methods and equipment used. A project was then conducted which focused on rationalising and standardising equipment and technique for blood culture sampling along with staff education to support this change. A simple department target of 30 days free from a contaminated blood culture was created which, if achieved, would ensure a contamination rate of less than 3%. This was supported by ongoing surveillance of contamination rates and investigation of contaminated sample cases. We were able to then identify high risk patients and factors which increased the chance of blood culture contamination. This allowed us to formulate solutions to help reduce the risks of contamination. Department achievements and learning points to help prevent further contamination were fed back positively to all staff. This project operated for 12-months and successfully reduced local contamination rates to 2.0%.
Full Text Available The clinical case studied concerns a woman 81 years old, with a history of prosthetic valve and mitral insufficiency, admitted to internal medicine ward of NOCSAE hospital as a result of a recurrent fever. Due to the suspicion of endocarditis and with the aim to identify the presence of aerobic/anaerobic microorganisms, two set of blood cultures collected within 24 hours were sent to the Laboratory of microbiology. All the bottles were incubated into the Bact-Alert 3D System (bioMérieux. After an 19 hours incubation time, the samples were identified as positive by the automated system; consequently they cultured on a blood agar and selective media, according to our laboratory operational protocol. In the same time Gram stain of the cultural broth revealed the presence of Gram positive cocci arranged in chains different in length. Since there wasn’t an evident microbial growth on solid media after 24-48 hours of incubation, a new culture was carried out on blood and chocolate agar after the addition of Staphylococcus aureus ATCC 25923. After 24 hours of incubation it was possible appreciate the growth of tiny colonies around the S. aureus ones. These colonies were identified by Vitek2 and Api Rapid 32 Strep (bioMérieux as Granulicatella adiacens. The results were confirmed by PCR and sequencing of the groESL gene. MIC values obtained by the means of E-test (bioMérieux were: 0.016mg/L for penicillin, 0.125mg/L for cefotaxime, 1mg/L for both vancomicin and levofloxacin. Resistance was observed for cloramphenicol (MIC=16mg/L. The timely communication of these findings, supported by clinical data like the appearance of vegetation on mitral valve highlighted by trans-oesophageal echocardiography, allowed to establish an adequate antibiotic therapy, rapid resolution of fever and normalisation of inflammatory parameters.
Full Text Available Abstract Background Capilia TB is a simple immunochromatographic assay based on the detection of MPB64 antigen specifically secreted by the Mycobacterium tuberculosis complex (MTC. Capilia TB was evaluated for rapid identification of MTC from BACTEC MGIT 960 and BACTEC 9120 systems in Kampala, Uganda. Since most studies have mainly dealt with respiratory samples, the performance of Capilia TB on blood culture samples was also evaluated. Methods One thousand samples from pulmonary and disseminated tuberculosis (TB suspects admitted to the JCRC clinic and the TB wards at Old Mulago hospital in Kampala, Uganda, were cultured in automated BACTEC MGIT 960 and BACTEC 9120 blood culture systems. BACTEC-positive samples were screened for purity by sub-culturing on blood agar plates. Two hundred and fifty three (253 samples with Acid fast bacilli (AFB, 174 BACTEC MGIT 960 and 79 BACTEC 9120 blood cultures were analyzed for presence of MTC using Capilia TB and in-house PCR assays. Results The overall Sensitivity, Specificity, Positive and Negative Predictive values, and Kappa statistic for Capilia TB assay for identification of MTC were 98.4%, 97.6%, 97.7%, 98.4% and 0.96, respectively. Initially, the performance of in-house PCR on BACTEC 9120 blood cultures was poor (Sensitivity, Specificity, PPV, NPV and Kappa statistic of 100%, 29.3%,7%, 100% and 0.04, respectively but improved upon sub-culturing on solid medium (Middlebrook 7H10 to 100%, 95.6%, 98.2%, 100% and 0.98, respectively. In contrast, the Sensitivity and Specificity of Capilia TB assay was 98.4% and 97.9%, respectively, both with BACTEC blood cultures and Middlebrook 7H10 cultured samples, revealing that Capilia was better than in-house PCR for identification of MTC in blood cultures. Additionally, Capilia TB was cheaper than in-house PCR for individual samples ($2.03 vs. $12.59, respectively, and was easier to perform with a shorter turnaround time (20 min vs. 480 min, respectively
Full Text Available Culture of Mycobacterium ulcerans from Buruli ulcer patients has very low sensitivity. Thus confirmation of M. ulcerans infection is primarily based on PCR directed against IS2404. In this study we compare the genotypes obtained by variable number of tandem repeat analysis of DNA from IS2404-PCR positive cultures with that obtained from IS2404 positive, culture-negative tissue. A significantly greater genetic heterogeneity was found among culture-negative samples compared with that found in cultured strains but a single genotype is over-represented in both sample sets. This study provides evidence that both the focal location of bacteria in a lesion as well as differences in the ability to culture a particular genotype may underlie the low sensitivity of culture. Though preliminary, data from this work also suggests that mycobacteria previously associated with fish disease (M. pseudoshottsii may be pathogenic for humans.
Kenyon, Chris R; Fatti, Geoff; Schrueder, Neshaad; Bonorchis, Kim; Meintjes, Graeme
Knowledge of local antibiotic sensitivities is crucial to creating appropriate empiric antibiotic guidelines. The new National Health Laboratory Service (NHLS) Data Warehouse allows clinicians to access collated spreadsheets of culture isolates and antimicrobial susceptibility patterns for their facilities. We used this service to study the trends in blood culture (BC) results at GF Jooste Hospital from 2005 to 2010. We investigated the BC contamination rate and changes in the antibiotic sensitivity profiles of selected organisms, and estimated the proportion of infections that were hospital-acquired. Over 3000 BCs were performed per year in this period. A very high contamination rate was observed (7 - 9%) in 2005 - 2007, with a gratifying reduction by 2010. Ceftriaxone resistance increased from 16% to 62% in Klebsiella pneumoniae (p<0.0001), and from 33% to 100% in Enterobacter spp. (p=0.053).
Matheny, Michael E.; FitzHenry, Fern; Speroff, Theodore; Hathaway, Jacob; Murff, Harvey J.; Brown, Steven H.; Fielstein, Elliot M.; Dittus, Robert S.; Elkin, Peter L.
Microbiology results are reported in semi-structured formats and have a high content of useful patient information. We developed and validated a hybrid regular expression and natural language processing solution for processing blood culture microbiology reports. Multi-center Veterans Affairs training and testing data sets were randomly extracted and manually reviewed to determine the culture and sensitivity as well as contamination results. The tool was iteratively developed for both outcomes using a training dataset, and then evaluated on the test dataset to determine antibiotic susceptibility data extraction and contamination detection performance. Our algorithm had a sensitivity of 84.8% and a positive predictive value of 96.0% for mapping the antibiotics and bacteria with appropriate sensitivity findings in the test data. The bacterial contamination detection algorithm had a sensitivity of 83.3% and a positive predictive value of 81.8%. PMID:20351890
Matheny, Michael E; Fitzhenry, Fern; Speroff, Theodore; Hathaway, Jacob; Murff, Harvey J; Brown, Steven H; Fielstein, Elliot M; Dittus, Robert S; Elkin, Peter L
Microbiology results are reported in semi-structured formats and have a high content of useful patient information. We developed and validated a hybrid regular expression and natural language processing solution for processing blood culture microbiology reports. Multi-center Veterans Affairs training and testing data sets were randomly extracted and manually reviewed to determine the culture and sensitivity as well as contamination results. The tool was iteratively developed for both outcomes using a training dataset, and then evaluated on the test dataset to determine antibiotic susceptibility data extraction and contamination detection performance. Our algorithm had a sensitivity of 84.8% and a positive predictive value of 96.0% for mapping the antibiotics and bacteria with appropriate sensitivity findings in the test data. The bacterial contamination detection algorithm had a sensitivity of 83.3% and a positive predictive value of 81.8%.
Takeshima, Shin-Nosuke; Watanuki, Sonoko; Ishizaki, Hiroshi; Matoba, Kazuhiro; Aida, Yoko
Bovine leukemia virus (BLV), the etiologic agent of enzootic bovine leucosis, has caused pandemic outbreaks worldwide. Because transcription of the BLV is quickly blocked after infection, detecting integrated provirus at host genome is an important method of identifying whether an animal is infected. The aim of the present study was to develop a novel direct blood-based PCR system to detect the BLV provirus with high specificity and at low cost. The assay was based on the BLV-CoCoMo degenerate primers, which amplify all known BLV strains. Cattle blood samples (n = 182) were collected from the same BLV-positive farm and subjected to BLV-CoCoMo-direct-PCR to detect the BLV provirus. The proviral load was then estimated. This novel PCR method showed 100 % specificity. The BLV-CoCoMo-direct-PCR can be used in a variety of laboratory situations because it does not require expensive equipment/reagents, DNA purification, or a second round of PCR. Therefore, the method is extremely cost-effective and the risk of a false-positive result due to DNA contamination is very low.
de Godoy, Natalia Souza; Andrino, Marcos Luiz Alves; de Souza, Regina Maia; Gakiya, Erika; Amato, Valdir Sabbaga; Lindoso, José Ângelo Lauletta
The aim of this study was to evaluate whether the molecular (kDNA-PCR) and parasitological diagnosis in peripheral blood (PB) could replace the invasive and painful bone marrow collection (BM) in the diagnosis of visceral leishmaniasis (VL). PB from suspected VL patients was evaluated by parasitological and molecular techniques using as the gold standard (GS) a combination of clinical, epidemiological, and immunochromatographic test (PB-rK39) results and parasitological examination of BM. Based on the GS, 38 samples from 32 patients were grouped: Group 1, 20 samples of VL cases, and Group 2, 18 samples of non-VL cases. In order to evaluate the parasitological and molecular techniques in PB, the samples were examined. From Group 1, PB kDNA-PCR was positive in 20 samples and in 19 of 20 in BM kDNA-PCR examination. However, the parasitological examination of buffy coat was insensitive, being able to detect only 4 cases from Group 1. All samples from Group 2 were negative. We concluded that, for the diagnosis of visceral leishmaniasis, the parasitological examination of peripheral blood was not useful; however, molecular diagnosis by kDNA-PCR, performed in peripheral blood, could be useful to replace the parasitological examination of bone marrow. PMID:27597892
Full Text Available Dental implants offer new treatment options for edentulous either partially or completely, now represent a viable alternative to conventional fixed protheses. Dental implants are colonized by a flora dominated by Gram-positive facultative aerobic, while in patients with bone loss and formation of pockets peri-implant diseases was found a significant difference in the composition of microflora, bacteria, Gram-negative anaerobes in particular Fusobacterium spp., Treponema denticola (Spirochetes, Tannerella forsythensis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia as interim black-pigmented bacteria, Porphyromonas gingivalis, often in high concentrations. Aims. The purpose of this study was to identify those at risk of perimplantitis using 2 techniques: RT-PCR examination of trade and culture. The results were compared taking into consideration the advantages and disadvantages of both methods. Materials and methods.We studied 24 patients (14 women and 10 men, aged, women between 43 and 76 years, with an average of 63.8 + / - 10.9 years, men between 45 and 88 years with a average of 64.3 years + / - 12.5 years. Was performed a double levy of sub-gingival plaque at multiple sites that had an implant CAL (clinical attachment level> 4mm in order to assess the microbiological identification with the two techniques: Examining culture and Real-Time PCR of Commerce ( Gum-Sunstar that identifies 4 bacterial species: A. actinomycetemcomitans (A.a., P.gingivalis (P.g., T.forsythensis (T.f., and T.denticola (T.d.. Results. All patients studied were positive to both tests with charger high: the consideration of tenure, with CFU / ml > 105, was positive in 66.6% of samples by:T.f., and P.g., in 12.5% for A.a., while T.d. not been sought by examining culture, the RT-PCR was positive, with high loads, in 95.8% of samples for T.f., in 79.1% for P.g., in 12.5% for A.a. and 20.8% for T.d.The test crop showed the presence of even P.intermedia in 91
MAYanping; LIRongping; ZOUPing; XIAOJuan; HUANGShiang; QIAOZhenhua
Objective: To investigate the expression and significance of caspase-3 protein in CD34+ cells from cord blood (CB) during culture in vitro with different growth factors. Methods: RT-PCR, Western blot and flow cytometry techniques were used to detect the expression of caspase-3 in CD34+ CB cells during culture in vitro. Results: Caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34+ cells. The expression of caspase-3 mRNA and protein was upregulated when these cellswere first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3 proenzyme was detected in the freshly isolated CD34+ cells as well as during the first 3 days expansion with cytokines. With longer culture time in vitro, especially in the presence of the combination of IL-3, IL-6 and GM-CSF, caspase-3 was activated and a cleavage product of 20 kDa became detectable.Conclusion: Caspase-3 is involved in apoptosis of primitive CB CD34+ cells during expansion in vitro.
Full Text Available Abstract Background Macrolide resistance in Helicobacter pylori is the major risk factor for treatment failure when using a proton pump inhibitor-clarithromycin containing therapy. Macrolide resistance is due to a few mutations on the 23S ribomosal subunit encoded by the 23S rRNA gene. The present study aimed at investigating the performance of the dual priming oligonucleotide (DPO-PCR kit named Seeplex® ClaR-H. pylori ACE detection designed to detect H. pylori and two types of point mutations causing clarithromycin resistance in H. pylori. Methods The performance of Seeplex® ClaR-H. pylori ACE detection was evaluated on 127 gastric biopsies in comparison to conventional bacterial culture followed by the determination of susceptibility to clarithromycin by E-test, as well as by an in-house real-time PCR using a fluorescence resonance energy transfer (FRET technology. Results Considering culture as the reference test, the sensitivity of DPO-PCR and real-time FRET-PCR was 97.7% and 100% while specificity was 83.1% and 80.7%, respectively. However, both PCR were concordant in detecting 14 H. pylori positive cases which were negative by culture. Globally, E-test and DPO-PCR were concordant with regard to clarithromycin susceptibility in 95.3% of the cases (41/43, while real-time FRET-PCR and DPO-PCR were concordant in 95% (57/60. Conclusion The DPO-PCR is an interesting tool to detect H. pylori on gastric biopsies and to study its susceptibility to clarithromycin in laboratories that cannot perform real-time PCR assays.
Alejandro G Schijman
Full Text Available BACKGROUND: A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. METHODOLOGY/FINDINGS: An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU I, IV and VI (set A, human blood spiked with parasite cells (set B and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C. Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA, 13 satellite DNA (Sat-DNA and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05-0.5 parasites/mL whereas specific kDNA tests detected 5.10(-3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood. The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3-94.4%, specificity of 85
Comandini, U V; Sönnerborg, A; Vahlne, A; Yun, Z
A multiple competitor PCR (mcPCR) was developed to quantify HIV-1 proviral DNA from peripheral blood mononuclear cells (PBMC). DNA extracted from a mixture of HIV infected PBMC and four size-mutated DNA competitors were co-amplified. The Cy5-fluorescence labelled PCR products were denatured by heating, separated using an automated DNA sequencer and quantified by a fragment analysis computer software. An internal standard was generated by plotting the peak areas of the four competitors against their inputs. Based on the internal standard, HIV sample DNA was quantified by extrapolating the corresponding signal. The linear range of the mcPCR was three log wide and the quantitation limit was about 20 copies of HIV DNA/10(6) PBMC. Using the mcPCR, HIV DNA was quantified from 14 long-term non progressors (LTNP) and 14 patients with advanced disease. A significantly lower copy number of HIV DNA was obtained in the LTNP (p = 0.018). These data suggest that the mcPCR is sensitive, reliable and especially useful for HIV DNA quantification of a large number of clinical samples.
Full Text Available Bacteremia is an important infectious disease which may lead to death. Common bacteria and pattern of antibiotic resistance in different communities are different and understanding these differences is important. In the present study, relative frequency and pattern of drug resistance have been examined in bacteria isolated from blood cultures in Razi Hospital laboratory. The method of the study was descriptive. Data collection was carried out retrospectively. Total sample consisted of 311 positive blood cultures from 1999 to 2001. Variables under study were bacterial strains, antibiotics examined in antibiogram, microbial resistance, and patients' age and sex. The most common isolated bacteria were Salmonella typhi (22.2% and the least common ones were Citrobacter (1.6%. The highest antibiotic resistance was seen against amoxicillin (88.4%. The proportion of males to females was1: 1/1 and the most common age group was 15-44 (47.3%. Common bacteria and pattern of antibiotic resistance were different in some areas and this subject requires further studies in the future.
McMahon, Tanis C.; Blais, Burton W.; Wong, Alex; Carrillo, Catherine D.
Foodborne illness attributed to enterohemorrhagic E. coli (EHEC), a highly pathogenic subset of Shiga toxin-producing E. coli (STEC), is increasingly recognized as a significant public health issue. Current microbiological methods for identification of EHEC in foods often use PCR-based approaches to screen enrichment broth cultures for characteristic gene markers [i.e., Shiga toxin (stx) and intimin (eae)]. However, false positives arise when complex food matrices, such as beef, contain mixtures of eae-negative STEC and eae-positive E. coli, but no EHEC with both markers in a single cell. To reduce false-positive detection of EHEC in food enrichment samples, a Multiplexed, Single Intact Cell droplet digital PCR (MuSIC ddPCR) assay capable of detecting the co-occurrence of the stx and eae genes in a single bacterial cell was developed. This method requires: (1) dispersal of intact bacteria into droplets; (2) release of genomic DNA (gDNA) by heat lysis; and (3) amplification and detection of genetic targets (stx and eae) using standard TaqMan chemistries with ddPCR. Performance of the method was tested with panels of EHEC and non-target E. coli. By determining the linkage (i.e., the proportion of droplets in which stx and eae targets were both amplified), samples containing EHEC (typically greater than 20% linkage) could be distinguished from samples containing mixtures of eae-negative STEC and eae-positive E. coli (0–2% linkage). The use of intact cells was necessary as this linkage was not observed with gDNA extracts. EHEC could be accurately identified in enrichment broth cultures containing excess amounts of background E. coli and in enrichment cultures derived from ground beef/pork and leafy-green produce samples. To our knowledge, this is the first report of dual-target detection in single bacterial cells using ddPCR. The application of MuSIC ddPCR to enrichment-culture screening would reduce false-positives, thereby improving the cost, speed, and accuracy of
Keegan, Alexandra R; Fanok, Stella; Monis, Paul T; Saint, Christopher P
Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with disinfection systems for drinking water and recycled water.
Full Text Available Adenovirus (AdV respiratory infections are usually described as being associated with high mortality rates. Laboratory diagnosis is essential for the establishment of the appropriate therapy, and for guiding the implementation of preventive measures in order to prevent the spread of the infection. Aiming to analyze the sensitivity and specificity of the laboratorial diagnosis methods available, we compared antigen detection by indirect immunofluorescence assay (IF, and a specific nested polymerase chain reaction (PCR, to detect AdV in respiratory samples collected from patients admitted to hospital with acute respiratory disease. Positive samples were inoculated into a cell culture to confirm the results. We analyzed 381 samples from the nasopharyngeal aspirates collected during the year 2008; of these, 2.6% tested were positive for adenovirus through IF and 10% through PCR; positive isolation was obtained in 40% and 26% of these cases, respectively. Most infected patients were children under six months of age, and despite of the fact that a significant number of patients required intensive care, the mortality rate was low (5%. In conclusion, molecular methods were found to be useful for rapid diagnosis of adenovirus infections with higher sensitivity than antigen detection; their introduction permitted a significant increase in diagnoses of adenovirus infections.
Kang, Heekyoung; Seong, Gi-Sang; Sohn, Hae-Jin; Kim, Jong-Hyun; Lee, Sang-Eun; Park, Mi Yeoun; Lee, Won-Ja; Shin, Ho-Joon
Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6.
Mogasale, Vittal; Ramani, Enusa; Mogasale, Vijayalaxmi V; Park, JuYeon
Blood culture is often used in definitive diagnosis of typhoid fever while, bone marrow culture has a greater sensitivity and considered reference standard. The sensitivity of blood culture measured against bone marrow culture results in measurement bias because both tests are not fully sensitive. Here we propose a combination of the two cultures as a reference to define true positive S. Typhi cases. Based on a systematic literature review, we identified ten papers that had performed blood and bone marrow culture for S. Typhi in same subjects. We estimated the weighted mean of proportion of cases detected by culture measured against true S. Typhi positive cases using a random effects model. Of 529 true positive S. Typhi cases, 61 % (95 % CI 52-70 %) and 96 % (95 % CI 93-99 %) were detected by blood and bone marrow cultures respectively. Blood culture sensitivity was 66 % (95 % CI 56-75 %) when compared with bone marrow culture results. The use of blood culture sensitivity as a proxy measure to estimate the proportion of typhoid fever cases detected by blood culture is likely to be an underestimate. As blood culture sensitivity is used as a correction factor in estimating typhoid disease burden, epidemiologists and policy makers should account for the underestimation.
Horii, Toshinobu; Notake, Shigeyuki; Tamai, Kiyoko; Yanagisawa, Hideji
We characterized the profiles of virulence genes and antimicrobial susceptibility of Bacillus cereus isolates from blood cultures as well as the risk factors for blood stream infections (BSIs). The diversity of virulence gene patterns was found to be wide among 15 B. cereus isolates from BSIs and also among 11 isolates from contaminated blood cultures. The MicroScan broth microdilution method yielded results corresponding with those of the agar dilution (reference) method for levofloxacin, linezolid, and vancomycin, while the Etest results were consistent with the reference results for clindamycin, gentamicin, imipenem, levofloxacin, and linezolid. Compared with the reference values, however, some isolates showed marked differences of the minimum inhibitory concentrations (MICs) for ampicillin and clindamycin when determined using the MicroScan method, or the MICs for ampicillin, meropenem, and vancomycin when determined using the Etest method. Significantly more patients were treated with antimicrobials for more than 3 days during the 3-month period before isolation in the BSI group. Prior antimicrobial therapy may be a risk factor for BSIs due to B. cereus.
Hansen, Wendy L J; Beuving, Judith; Verbon, Annelies; Wolffs, Petra F G
Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock(1). Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours(2, 4). The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes(5). Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h. Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods(6). This assay was based on a study in which PCR was used to measure the growth of bacteria(7). Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of
Golchin, Neda; Kheirandish, Maryam; Sharifi, Zohreh; Samiee, Shahram; Kokhaei, Parviz; Pourpak, Zahra
Umbilical cord blood (UCB) is one of the most important sources of hematopoietic stem cells which can be used for transplantation. The transplanted CB stem cells might cause infections in recipients. The aim of this study is to evaluate Human Herpes Virus8 (HHV8) as a Rhadinovirus among CB samples in order to assess safety of cord blood stem cells transplantation. To assess this aim, we surveyed 800 cord blood specimens by Real Time PCR.The overall HHV8 incidence in cord blood mononuclear cells was 1.38% and none of them was in lytic phase of HHV8. The authors suggest further HHV8 study on CB samples for transplantation.
Boutaga, Khalil; van Winkelhoff, Arie Jan; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.
Porphyromonas gingivalis is a major pathogen in destructive periodontal disease in humans. Detection and quantification of this microorganism are relevant for diagnosis and treatment planning. The prevalence and quantity of P. gingivalis in subgingival plaque samples of periodontitis patients were determined by anaerobic culture and real-time PCR amplification of the 16S small-subunit rRNA gene. The PCR was performed with primers and a fluorescently labeled probe specific for the P. gingivali...
Eamens, Graeme J; Whittington, Richard J; Turner, Mark J; Austin, Susan L; Fell, Shayne A; Marsh, Ian B
Dilution rates for pooled faecal culture (PFC) and direct IS900 polymerase chain reaction (D-PCR) tests were evaluated on faecal samples from infected cows mixed with uninfected faeces in dilutions from 1 in 5 to 1 in 50. PFC was performed by radiometric culture, with confirmation by IS900 PCR and restriction endonuclease analysis (PCR/REA) on growth, and by mycobactin dependency testing on solid medium. Using 37 culture positive faecal samples from 12 subclinical cows, 83.8% and 94.6% of samples were confirmed positive in the PFC assay at dilutions of 1 in 50 and 1 in 30, respectively. Lower dilutions (1 in 5 to 1 in 20) provided only marginally better sensitivity, and confirmation of PFC growth by PCR/REA was significantly more sensitive than mycobactin dependency. D-PCR had significantly lower sensitivity than PFC confirmed by PCR/REA, with pools of 1 in 50, 30, 10 and 5 yielding positive results in 64.9%, 70.3%, 78.4% and 83.8% of samples, respectively. Cattle considered to be shedding 1.5 x 10(6) viable M. avium subsp. paratuberculosis (Map)/g faeces (on the basis of estimated losses in processing and growth rates in radiometric broth) were positive at dilutions up to 1 in 50 in the PFC and D-PCR. Those shedding 5 x 10(5) viable Map/g were positive in the PFC at dilutions up to 1 in 40, but required a 1 in 10 dilution or less for D-PCR. The results suggest that for cattle shedding relatively high concentrations of Map in faeces (>2 x 10(5) viable Map/g), maximal dilutions of 1 in 30 for PFC and 1 in 10 for D-PCR would be applicable.
Caldas, Sérgio; Caldas, Ivo Santana; Diniz, Lívia de Figueiredo; Lima, Wanderson Geraldo de; Oliveira, Riva de Paula; Cecílio, Alzira Batista; Ribeiro, Isabela; Talvani, André; Bahia, Maria Terezinha
The lack of an accurate diagnosis has been a serious obstacle to the advancement of the anti-Trypanosoma cruzi chemotherapy and long-term infection can result in different health risks to human. PCRs are alternative methods, more sensitive than conventional parasitological techniques, which due to their low sensitivities are considered unsuitable for these purposes. The aim of this study was to investigate a sensitive diagnostic strategy to quantify blood and cardiac tissues parasites based on real-time PCR tools during acute and chronic phases of murine Chagas disease, as well as to monitor the evolution of infection in those mice under specific treatment. In parallel, fresh blood examination, immunological analysis and quantification of cardiac inflammation were also performed to confront and improve real-time PCR data. Similar profiles of parasitemia curves were observed in both quantification techniques during the acute phase of the infection. In contrast, parasites could be quantified only by real-time PCR at 60 and 120 days of infection. In cardiac tissue, real-time PCR detected T. cruzi DNA in 100% of infected mice, and using this tool a significant Pearson correlation between parasite load in peripheral blood and in cardiac tissue during acute and chronic phases was observed. Levels of serum CCL2, CCL5 and nitric oxide were coincident with parasite load but focal and diffuse mononuclear infiltrates was observed, even with significant (pblood and cardiac muscle at the treatment period, but after the end of chemotherapy an increase of parasitism was detected. Interestingly, inflammatory mediators levels and heart inflammation intensity had similar evolution to the parasite load, in the group of animals treated. Taken together, our data show that real-time PCR strategy used was suitable for studies of murine T. cruzi infection and may prove useful in investigations involving experimental chemotherapy of the disease and the benefits of treatment in relation to
Amores, Joaquín; Corrales, Juan C; Martín, Angel Gómez; Sánchez, Antonio; Contreras, Antonio; de la Fe, Christian
This study was designed to evaluate the validity of PCR for the direct detection of Mycoplasma (M.) agalactiae and Mycoplasma mycoides subsp. capri (Mmc), as the two species most frequently causing contagious agalactia (CA) in goats. The PCR method was compared with the traditional culture technique to determine which method was most efficient at identifying all auricular carriers present in herds. The samples analyzed were 307 ear swabs taken from goats reared in a CA endemic area. We assessed the validity of each technique to detect each species and agreement between both methods. For each species, the result was taken as true-positive when at least one of the two tests was positive. Of the swabs tested, 246 were scored positive by PCR (235 and 11 for Mmc and M. agalactiae, respectively) and 117 showed a positive culture result (113 for Mmc and 4 for M. agalactiae). 133 of the PCR-positive samples (124 and 9 for Mmc and M. agalactiae, respectively) yielded negative culture results and 4 culture-positive samples tested negative using PCR (2 for each species). Sensitivity and negative predictive values for PCR were 84.62 and 99.32 (for M. agalactiae) and 99.16 and 97.22% (for Mmc) respectively, and for culture were 30.77 and 97.03 (for M. agalactiae) and 47.08 and 36.08% (for Mmc), respectively. PCR proved to be a rapid and sensitive method for the detection of mycoplasmas in the external ear of asymptomatic carriers. Tools such as this are needed to adopt efficient control measures against CA.
Struniawski, R; Szpechcinski, A; Poplawska, B; Skronski, M; Chorostowska-Wynimko, J
The dried blood spot (DBS) specimens have been successfully employed for the large-scale diagnostics of α1-antitrypsin (AAT) deficiency as an easy to collect and transport alternative to plasma/serum. In the present study we propose a fast, efficient, and cost effective protocol of DNA extraction from dried blood spot (DBS) samples that provides sufficient quantity and quality of DNA and effectively eliminates any natural PCR inhibitors, allowing for successful AAT genotyping by real-time PCR and direct sequencing. DNA extracted from 84 DBS samples from chronic obstructive pulmonary disease patients was genotyped for AAT deficiency variants by real-time PCR. The results of DBS AAT genotyping were validated by serum IEF phenotyping and AAT concentration measurement. The proposed protocol allowed successful DNA extraction from all analyzed DBS samples. Both quantity and quality of DNA were sufficient for further real-time PCR and, if necessary, for genetic sequence analysis. A 100% concordance between AAT DBS genotypes and serum phenotypes in positive detection of two major deficiency S- and Z- alleles was achieved. Both assays, DBS AAT genotyping by real-time PCR and serum AAT phenotyping by IEF, positively identified PI*S and PI*Z allele in 8 out of the 84 (9.5%) and 16 out of 84 (19.0%) patients, respectively. In conclusion, the proposed protocol noticeably reduces the costs and the hand-on-time of DBS samples preparation providing genomic DNA of sufficient quantity and quality for further real-time PCR or genetic sequence analysis. Consequently, it is ideally suited for large-scale AAT deficiency screening programs and should be method of choice.
Aslanzadeh, J; de la Viuda, M; Fille, M; Smith, W B; Namdari, H
The major drawback in effective use of polymerase chain reaction (PCR) for detecting Mycobacterium tuberculosis (MTB) in clinical samples is the presence of PCR inhibitors and unique cell components of the organism that complicate DNA extraction and subsequent PCR amplification. A PCR assay with a unique multistep DNA extraction method that minimizes these problems was compared in a prospective study to acid-fast bacilli stain (AFBS) and culture for detecting MTB in clinical samples. A total of 254 clinical specimens in two separate studies were processed for MTB by these techniques. While PCR and culture were 100% sensitive and specific, culture required up to 8 weeks of incubation and additional time to perform biochemical testing to identify the isolated micro-organism. Acid-fast bacilli stain had a specificity of about 87% and did not differentiate among Mycobacterial species. In contrast, the results from PCR were available within 48 h and did not require additional testing to attain a final result. Polymerase chain reaction was highly reliable for detection and confirmation and interpretation of positive AFBS results. The assay was easy to perform with a turn around time of about 2 days.
Full Text Available In the present study, the efficacy of polymerase chain reaction (PCR based on mapA gene of C. jejuni was tested for detection of Campylobacter jejuni in naturally infected as well as spiked faecal and food samples of human and animal origin. Simultaneously, all the samples were subjected to the cultural isolation of organism and biochemical characterization. The positive samples resulted in the amplification of a DNA fragment of size ~589 bp in PCR assay whereas the absence of such amplicon in DNA extracted from E. coli, Listeria, Salmonella and Staphylococcus confirmed the specificity of the primers. Of randomly collected 143 faecal samples comprising human diarrheic stools (43, cattle diarrheic faeces (48 and poultry faecal swabs (52 only 4, 3 and 8, respectively, could be detected by isolation whereas 6, 3 and 10, respectively, were found positive by PCR. However, among food samples viz. beef (30, milk (35, cheese (30, only one beef sample was detected both by culture as well as PCR. Additionally, PCR was found to be more sensitive for C. jejuni detection in spiked faecal and food samples (96.1% each as relative to culture isolation which could detect the organism in 86.7% and 80% samples, respectively. The results depicted the superior efficacy of PCR for rapid screening of samples owing to its high sensitivity, specificity and automation potential.
Spoorenberg, V.; Prins, J.M.; Opmeer, B.C.; Reijke, T.M. de; Hulscher, M.E.; Geerlings, S.E.
We evaluated 800 hospitalized patients with a complicated urinary tract infection, from whom both a blood and a urine culture were obtained on the first day of antibiotic treatment. Urine cultures were positive in 70% of patients, and blood cultures were positive in 29%. In 7% of patients, uropathog
Full Text Available Bloodstream infection (BSI and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample, amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS. We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis, and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours.The IRIDICA BAC BSI Assay is not available in the United States.
Laukemann, Svenja; Kasper, Nina; Kulkarni, Prasad; Steiner, Deborah; Rast, Anna Christina; Kutz, Alexander; Felder, Susan; Haubitz, Sebastian; Faessler, Lukas; Huber, Andreas; Fux, Christoph A; Mueller, Beat; Schuetz, Philipp
Only a small proportion of blood cultures routinely performed in emergency department (ED) patients is positive. Multiple clinical scores and biomarkers have previously been examined for their ability to predict bacteremia. Conclusive clinical validation of these scores and biomarkers is essential.This observational cohort study included patients with suspected infection who had blood culture sampling at ED admission. We assessed 5 clinical scores and admission concentrations of procalcitonin (PCT), C-reactive protein (CRP), lymphocyte and white blood cell counts, the neutrophil-lymphocyte count ratio (NLCR), and the red blood cell distribution width (RDW). Two independent physicians assessed true blood culture positivity. We used logistic regression models with area under the curve (AUC) analysis.Of 1083 patients, 104 (9.6%) had positive blood cultures. Of the clinical scores, the Shapiro score performed best (AUC 0.729). The best biomarkers were PCT (AUC 0.803) and NLCR (AUC 0.700). Combining the Shapiro score with PCT levels significantly increased the AUC to 0.827. Limiting blood cultures only to patients with either a Shapiro score of ≥4 or PCT > 0.1 μg/L would reduce negative sampling by 20.2% while still identifying 100% of positive cultures. Similarly, a Shapiro score ≥3 or PCT >0.25 μg/L would reduce cultures by 41.7% and still identify 96.1% of positive blood cultures.Combination of the Shapiro score with admission levels of PCT can help reduce unnecessary blood cultures with minimal false negative rates.The study was registered on January 9, 2013 at the 'ClinicalTrials.gov' registration web site (NCT01768494).
Laukemann, Svenja; Kasper, Nina; Kulkarni, Prasad; Steiner, Deborah; Rast, Anna Christina; Kutz, Alexander; Felder, Susan; Haubitz, Sebastian; Faessler, Lukas; Huber, Andreas; Fux, Christoph A.; Mueller, Beat; Schuetz, Philipp
Abstract Only a small proportion of blood cultures routinely performed in emergency department (ED) patients is positive. Multiple clinical scores and biomarkers have previously been examined for their ability to predict bacteremia. Conclusive clinical validation of these scores and biomarkers is essential. This observational cohort study included patients with suspected infection who had blood culture sampling at ED admission. We assessed 5 clinical scores and admission concentrations of procalcitonin (PCT), C-reactive protein (CRP), lymphocyte and white blood cell counts, the neutrophil-lymphocyte count ratio (NLCR), and the red blood cell distribution width (RDW). Two independent physicians assessed true blood culture positivity. We used logistic regression models with area under the curve (AUC) analysis. Of 1083 patients, 104 (9.6%) had positive blood cultures. Of the clinical scores, the Shapiro score performed best (AUC 0.729). The best biomarkers were PCT (AUC 0.803) and NLCR (AUC 0.700). Combining the Shapiro score with PCT levels significantly increased the AUC to 0.827. Limiting blood cultures only to patients with either a Shapiro score of ≥4 or PCT > 0.1 μg/L would reduce negative sampling by 20.2% while still identifying 100% of positive cultures. Similarly, a Shapiro score ≥3 or PCT >0.25 μg/L would reduce cultures by 41.7% and still identify 96.1% of positive blood cultures. Combination of the Shapiro score with admission levels of PCT can help reduce unnecessary blood cultures with minimal false negative rates. The study was registered on January 9, 2013 at the ‘ClinicalTrials.gov’ registration web site (NCT01768494). PMID:26656373
Kaevska, M; Lvoncik, S; Slana, I; Kulich, P; Kralik, P
The environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grown in vitro and under field conditions. The substrate into which plants were seeded was previously contaminated with different strains of Mycobacterium avium (10(8) to 10(10) cells/g of soil) and feces from animals with paratuberculosis. We detected M. avium subsp. avium, hominissuis, and paratuberculosis in the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 10(4) cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment.
Heggers, J P; Rutan, R L; Strock, L L; Desai, M H; Robson, M C; Herndon, D N
A blood culture is an essential laboratory procedure necessary to confirm a septic episode. However, it is important to collect the blood sample at the appropriate time with an acceptable technique. The standard method is to collect at least 5 to 10 ml blood per culture bottle from patients with fevers. However, this volume of blood is an unrealistic amount to take from the frequently febrile pediatric patient. Alternatively, the pediatric blood culture bottle allows the collection of 1 ml blood per bottle to perform the same evaluation. We evaluated the two techniques of blood-culture collection over a 9-month period and compared the results between adult and pediatric blood culture bottles. Seventy-six patients, from November 1988 through February 1989, had blood cultures performed with the adult culture bottles, which produced a total of 1314 samples. A total of 113 patients, from March through July 1989, had blood cultures performed with the pediatric culture bottles, which produced a total of 758 samples. Percent recovery for the adult bottles versus the pediatric bottles was 13.95% versus 22.8% (p less than 0.0001). Since the amount of blood necessary to isolate an infectious agent is critical not only for laboratory identification but also for the volume of blood of pediatric patients, these data clearly establish the efficacy of pediatric blood culture bottles and the utilization of smaller amounts of blood. Not only did this approach significantly enhance organism recovery rate, but it may well be more cost-effective because fewer cultures need to be performed to isolate the infectious organism.
Laurin, Emilie; McKenna, Shawn; Chaffer, Marcelo; Keefe, Greg
Mycobacterium avium ssp. paratuberculosis (MAP) can be shed in feces, milk, and colostrum. The goal of this study was to assess assays that detect MAP in these sample types, including effects of lactation stage or season. Understanding the performance of these assays could improve how they are used, limiting the risk of infection to calves. Forty-six previously confirmed MAP-positive cows from 7 Atlantic Canadian dairy farms were identified for colostrum sampling and monthly sampling of milk and feces over a 12-mo period. Samples were assayed for MAP using solid culture, broth culture, and direct real-time PCR (qPCR). Across assay types, test sensitivity when applied to milk samples averaged 25% of that when applied to fecal samples. For colostrum samples, sensitivity depended on assay type, with sensitivity of qPCR being approximately 46% of that in feces. Across sample types, sensitivity of qPCR was higher than that of the other assays. Sensitivity of qPCR, when applied to milk samples, was significantly higher in summer than in other seasons. Summer was also the season with highest agreement between milk and fecal samples collected within the same month. Our results suggest that qPCR would detect more cows shedding MAP in their milk and colostrum than solid or broth culture assays, particularly during the summer, thus providing better management information to limit exposure of calves to this infectious organism.
Full Text Available Objective:Early and accurate diagnosis of bacterial meningitis is of critical concern. Optimum and rapid laboratory facilities are not routinely available for detecting the etiologic agents of meningitis. The objective of this study was to compare polymerase chain reaction (PCR assay with culture for detection of bacteria in central nervous system (CNS samples from patients suspected to have meningitis. Methods: One-hundred CSF samples were obtained and divided into two parts. One part of samples was used for standard bacterial culture and gram staining. The remaining was used for DNA extraction. PCR assay was performed with universal primers for 16S rDNA gene of bacteria. Performance characteristics of the test were determined. Findings:The PCR method was able to detect bacteria in all 36 culture-positive and in 38 of 64 culture-negative cases showing sensitivity and specificity of 100% and 40.6% respectively. Positive predictive value was 48.6% and negative predictive value 100%, however, Kappa coefficient showed the correlation of the 2 methods to be at 0.33. Conclusion:There are advantages and disadvantages in performance characteristics of the conventional CSF culture and universal CSF 16S rDNA PCR. Therefore, it is recommended to use both methods in clinical practice, particularly in suspicious contaminated samples, with presumable presence of fastidious or slow growing bacteria because of antibiotic consumption.
Full Text Available "nBackground: The aim was to compare three different methods (direct examination, culture and PCR methods for the diagnosis of Acanthamoeba keratitis (AK in corneal scrapes."nMethods: Twenty eight corneal scrapes and contact lenses were collected from keratitis patients and referred to the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences. Corneal scrapes were divided in three parts for direct examination, culture on non-nutrient agar and PCR analysis. PCR analysis was also performed using a 18S rRNA gene primer pair (DF3 region. DF3 (Diagnostic fragment 3 is a region of the nuclear small subunit ribosomal RNA gene which is specific for detecting Acanthamoeba strains."nResults: Acanthamoeba was the causative agent of keratitis in 50% of the patients. Direct smear of all prepared corneal scrapes in AK patients was negative and culture was positive in only 14.3% of the isolates. PCR analysis was positive in 71.4% of AK patients. These three methods were negative in corneal scrapes of non-AK patients. The sensitivity and specificity of PCR technique for the detection of Acanthamoeba sp. were calculated as 71.4% and 100%, respectively."nConclusion: According to high sensitivity and specificity of PCR-based method, this study confirmed that PCR using 18S rRNA gene primers (DF3 region is more useful for detecting AK cases compare to culture and direct microscopy methods.
Gómez-Rojo, Erica M; Romero-Santacreu, L; Jaime, I; Rovira, J
Weissella viridescens has been identified as one of the lactic acid bacteria (LAB) responsible for the spoilage of "morcilla de Burgos". In order to identify and quantify this bacterium in "morcilla de Burgos", a new specific PCR procedure has been developed. The primers and Taqman probe were designed on the basis of a sequence from the gene recN. To confirm the specificity of the primers, 77 strains from the genera Carnobacterium, Enterococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Vagococcus and Weissella were tested by conventional PCR. The specificity of the primers and the correct functioning of the probe was confirmed by performing real-time PCR (qPCR) with 21 W. viridescens strains and 27 strains from other LAB genera. The levels of detection and quantification for the qPCR procedure proposed herein were determined for a pure culture of W. viridescens CECT 283(T) and for "morcilla de Burgos" artificially inoculated with this species. The primers were specific for W. viridescens, with only one product of 91 bp being observed for this species. Similarly, the qPCR reactions were found to be specific, amplifying at a mean CT of 15.0±0.4 only for W. viridescens strains. The limit of detection (LOD) and quantification (LOQ) for this procedure was established in 0.082 pg for genomic DNA from W. viridescens. With regard to the artificially inoculated "morcilla", the limit of quantification was established in 80 CFU/reaction and the limit of detection in 8 CFU/reaction. Consequently, the qPCR developed herein can be considered to be a good, fast, simple and accurate tool for the specific detection and quantification of W. viridescens in meat samples.
Full Text Available Background: Hepatitis B virus (HBV is one of the major causative agents of acute and chronic liver disease worldwide and is believed to be responsible for a million deaths annually. On the basis of a comparison of complete genomic sequences, HBV has been classified into eight genotypes A-H which show a geographical distribution. Some genotypes are associated with different clinical outcomes. Identification of HBV genotypes is important to begin and follow up the treatment."nMethods: In this cross-sectional study, the serum samples of blood donors were collected from Tehran Blood Transfusion Centers in period during "2005-2006". Sera of 55 blood donors who were positive for hepatitis B surface antigen were selected. DNA was extracted using commercial kit and the S gene sequence was amplified by nested-PCR. PCR products were then analyzed for restriction enzymes that would be genotype specific."nResults: Genotype D was found the only type in all HBV DNA positive serum samples, in Tehran."nConclusion: Genotype D is dominant among Tehran's blood donors, which is consistent with Iran and the Middle East dominant genotype.
Holmes, R. L.; Harada, W A
A rapid technique used for the identification of Streptococcus agalactiae, Lancefield group B, from the blood cultures of two neonatal infants is reported. The method utilized the Phadebact Streptococcus Test System (Pharmacia Diagnostics, Piscataway, N.J.) and the supernatant from 13- and 14-h blood cultures. Additional studies with simulated neonatal blood cultures revealed that this method was reproducible. Additional studies also revealed that some non-specific agglutination did occur, wh...
Lefterova, Martina I; Budvytiene, Indre; Sandlund, Johanna; Färnert, Anna; Banaei, Niaz
Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.
Koshy, Linda; Anju, A L; Harikrishnan, S; Kutty, V R; Jissa, V T; Kurikesu, Irin; Jayachandran, Parvathy; Jayakumaran Nair, A; Gangaprasad, A; Nair, G M; Sudhakaran, P R
The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol-chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A260/A280 and A260/A230), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol-chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.
Bhat, Sham S; Salman, Afreen; Hegde, Sundeep
Aim To determine polymerase chain reaction (PCR)-based blood group on tooth pulp obtained from teeth stored for 1 month, 6 months, and 1 year following extraction and to evaluate the stability of deoxyribonucleic acid (DNA) in primary tooth subjected to a temperature of 200°C ± 5°C for 15 minutes. Materials and methods Dental pulp tissue was collected from 40 exfoliated primary teeth stored for various time durations and temperature and preserved at 4°C till DNA extraction was carried out. Deoxyribonucleic acid was extracted using silica membrane-based spin-column procedure of QIAamp DNA minikit from BioRad. Deoxyribonucleic acid was subjected to PCR amplification and monoplex allele-specific PCR primers for ABO genotyping. Statistical analysis used The data were analyzed by comparison (based on percentage). Results In our study, overall, 85% samples showed a DNA yield. Cent percent results were obtained for samples studied at the end of 1 month followed by 90 and 80% for samples studied for 6 months and 1 year respectively. Heated samples showed 70% result. Conclusion Polymerase chain reaction was found to be an effective method for blood group determination for teeth stored at various time durations and temperatures. However, as the time interval increased, the number of positive results obtained decreased. How to cite this article Pai RK, Bhat SS, Salman A, Hegde S. Blood Group Determination using DNA extracted from Exfoliated Primary Teeth at Various Time Durations and Temperatures: A PCR Study. Int J Clin Pediatr Dent 2016;9(4):308-312. PMID:28127161
Giri, Sidhartha; Rajan, Anand K; Kumar, Nirmal; Dhanapal, Pavithra; Venkatesan, Jayalakshmi; Iturriza-Gomara, Miren; Taniuchi, Mami; John, Jacob; Abraham, Asha Mary; Kang, Gagandeep
Although, culture is considered the gold standard for poliovirus detection from stool samples, real-time PCR has emerged as a faster and more sensitive alternative. Detection of poliovirus from the stool of recently vaccinated children by culture, single and multiplex real-time PCR was compared. Of the 80 samples tested, 55 (68.75%) were positive by culture compared to 61 (76.25%) and 60 (75%) samples by the single and one step multiplex real-time PCR assays respectively. Real-time PCR (singleplex and multiplex) is more sensitive than culture for poliovirus detection in stool, although the difference was not statistically significant.
Keramas, Georgios; Bang, Dang Duong; Lund, Marianne
. detection obtained with DNA microarrays were compared to those obtained by conventional culture and gel electrophoresis. By conventional culture, 60% of the samples were positive for either Campylobacter jejuni or Campylobacter coli. By PCR and capillary electrophoresis, 95% of the samples were positive...... for Campylobacter spp., whereas with DNA microarrays all samples were positive for Campylobacter spp. By application of DNA microarray analysis, the isolates in 4 samples (6%) could not be identified to the species level, whereas by PCR-capillary electrophoresis, the isolates in 12 samples (19%) remained...... unidentified. Interestingly, PCR-capillary electrophoresis analysis revealed that two (3%) of the samples were positive for both C. jejuni and C. coli, while DNA microarray analysis revealed that nine (14%) of the samples were positive for both species. Of 65 samples, 2 samples were identified to contain C...
Löfström, Charlotta; Schelin, Jenny; Norling, Börje;
, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100 cm2) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant....... The method allowed accurate quantification from 4.4×102 to at least 2.2×107 CFU Salmonella per swab sample using qPCR (without preceding DNA extraction) or selective plating on xylose lysine deoxycholate agar. Samples with 50 CFU could be detected occasionally but fell outside the linear range...... of the standard curve. The swab samples showed a broad biological diversity; for seven samples not inoculated with Salmonella, the microbial background flora (BGF) was determined to 5.0 ± 2.2 log CFU/ml sample withdrawn after flotation. It was determined that the proceeding PCR step was inhibited by BGF...
Ong, Siong Gim; Chan, Yiong Huak; Heng, Chew Kiat
Introduction The human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. Methods Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant. Taqman assay real-time qPCR and ddPCR were conducted to quantify AMY1 gene copy numbers. Statistical analysis was carried out to determine the difference in AMY1 gene copy number between the different biological specimens and different assay methods. Results We found significant within-individual difference (p<0.01) in AMY1 gene copy number between different biological samples as determined by qPCR. However, there was no significant within-individual difference in AMY1 gene copy number between different biological samples as determined by ddPCR. We also found that AMY1 gene copy number of blood samples were comparable between qPCR and ddPCR, while there is a significant difference (p<0.01) between AMY1 gene copy numbers measured by qPCR and ddPCR for both buccal swab and saliva samples. Conclusions Despite buccal cells and saliva samples being possible sources of DNA, it is pertinent that ddPCR or a single biological sample, preferably blood sample, be used for determining highly polymorphic gene copy numbers like AMY1, due to the large within-individual variability between different biological samples if real time qPCR is employed. PMID:28125683
Papadimitriou-Olivgeri, I; Giormezis, N; Papadimitriou-Olivgeris, M; Zotou, A; Kolonitsiou, F; Koutsileou, K; Fligou, F; Marangos, M; Anastassiou, E D; Spiliopoulou, I
The significance of the number of coagulase-negative staphylococci (CNS)-positive blood cultures remains obscure in regards to determining true bacteremia versus contamination. The goal of this study was to determine the predictors of real CNS bloodstream infection among intensive care unit (ICU) patients. ICU patients with at least one CNS-positive blood culture were identified from the microbiology database. Biofilm formation was tested by glass tube and microtiter plate assay. mecA gene, ica operon genes (icaA, icaB, icaD), and adhesin genes (aap, bap, atlE, fbe, fnbA) were detected by polymerase chain reaction (PCR). CNS were recovered from 120 septic episodes, 20 of which were true CNS bacteremias, whereas from the remaining 100 episodes, the isolated CNS were characterized as contaminants. The number of positive blood cultures was significantly associated with true CNS bacteremia. Nineteen true bacteremic Staphylococcus epidermidis strains were compared to 38 contaminants. Biofilm synthesis was documented in 37 isolates associated with the presence of the ica operon (p = 0.048). There were 39, 26, 38, 21, and 10 strains positive for the presence of atlE, bap, fbe, aap, and fnbA genes, respectively. Rifampicin resistance, absence of severe sepsis, number of S. epidermidis-positive blood cultures, and absence of the bap gene were independently associated with true S. epidermidis bacteremia as compared to contaminant strains. The number of positive blood cultures is associated with true CNS bacteremia. The presence of adhesin genes may play a role in differentiating true infection from contamination, whereas absence of the bap gene is associated with true S. epidermidis bacteremia.
Background: The carcinogenesis of hepatocellular carcinoma (HCC) is a multi-factorial, multistep and complex process. Its prognosis is poor and early detection is of the utmost importance. Transforming growth factor β1 (TGF-β1) message RNA (mRNA) has been reported to be elevated in HCC patients using Northern blotting. However, little work has been done about the detection of TGF-β1 mRNA levels in peripheral blood of patients with HCC using the real-time polymerase chain reactions (PCR) method. Objective: To assess the prognostic value of quantitative levels of TGF-β1 mRNA in peripheral blood of patients with HCC, and to investigate the relationship between the expression of TGF-β1 mRNA in peripheral blood and many diagnostic and pathological factors. Methods: We developed an optimized Taqman real-time PCR to quantify TGF-β1 mRNA in peripheral blood of 53 patients with HCC and 44 healthy volunteers. In addition, blood was collected from patients with HCC for measuring levels of total bilirubin (TBil), prealbumin, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltranspeptidase (GGT), alpha-L-fucosidase (AFU), alpha fetoprotein (AFP), carcino-embryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), viral load and platelet counts. Statistical analysis was performed using the SPSS software system (SPSS 10.0). Results: In real-time PCR, fluorescence was detectable in all blood specimens from patients with HCC and healthy volunteers. The levels of TGF-β1 mRNA expression in patients with HCC were significantly higher compared to that in healthy volunteers (P<0.000 1), suggesting an association of the activated TGF-β1 gene transcription with hepatocarcinogenesis. Patients with HCC were divided into 2 groups according to their TGF-β1 mRNA above (group A, n=28) or below (group B, n=25) the mean level. Statistical results demonstrated that TGF-β1 mRNA expression level was correlated with patients age, serum levels of CEA
Armstrong-Briley, Danielle; Hozhabri, Neda S T; Armstrong, Kris; Puthottile, Jason; Benavides, Raul; Beal, Stacy
In the United States, sepsis is the leading cause of death in critically ill patients. The fatality rate for severe sepsis is about 40%, and treatment costs over $16 billion annually. It is critical to identify and treat the source of sepsis. While there are varying guidelines determining when to draw blood for culture, at Baylor University Medical Center at Dallas, blood cultures are ordered for patients with new onset of fever, immunosuppression, or a suspicion of an underlying infectious etiology. We conducted a retrospective study of patients who had blood cultures after hospital admission or in the emergency department in December 2013. We compared length of stay and outcomes of patients with positive versus negative blood cultures. There was no significant difference for length of stay or outcomes among patients with positive and negative blood cultures. For patients admitted from the emergency department, there was a longer length of stay for patients with positive cultures; however, the overall prognosis was not worse.
Estrada, Cecilia S M Lucero; Velázquez, Lidia Del Carmen; Favier, Gabriela Isabel; Genaro, María Silvia Di; Escudero, María Esther
The prevalence of Yersinia enterocolitica in meat products was assessed by four methods: cold enrichment in trypticase soy broth (A), enrichment in modified Rappaport broth at 25 °C (B), concentration by immunomagnetic separation (C) and yadA nested PCR (D). Furthermore, the pathogenic potentials of the isolates were established by phenotypic and genotypic tests, and their genomic relationships were determined by pulsed-field gel electrophoresis (PFGE). A total of 238 samples were collected at retail level in the city of San Luis, Argentina, during the period 2007-2008. The highest Yersinia prevalence in meat products was observed by method D (92 positive samples), followed by methods A (13 positive samples) and C (5 positive samples); however, no isolation was obtained by method B. Fourteen Y. enterocolitica and 4 Yersinia intermedia strains were recovered by culture. All Y. enterocolitica 2/O:9 strains gave results related to virulence by phenotypic tests and exhibited the genotype virF(+)myfA(+)ail(+)ystA(+). Two biotype 1A strains showed a genotype virF(-)myfA(-)ail(+)ystA(+)ystB(+). The 14 Y. enterocolitica strains isolated during this work plus one reference strain were separated into 11 genomic types by PFGE. This genomic heterogeneity of the isolates shows the diversity of Y. enterocolitica strains in our region. It is the first time that IMS was used to search Y. enterocolitica strains from naturally contaminated meat products.
Higgins, K Lindsay; Coley, Phyllis D; Kursar, Thomas A; Arnold, A Elizabeth
Most studies examining endophytic fungi associated with grasses (Poaceae) have focused on agronomically important species in managed ecosystems or on wild grasses in subtropical, temperate and boreal grasslands. However grasses first arose in tropical forests, where they remain a significant and diverse component of understory and forest-edge communities. To provide a broader context for understanding grass-endophyte associations we characterized fungal endophyte communities inhabiting foliage of 11 species of phylogenetically diverse C(3) grasses in the understory of a lowland tropical forest at Barro Colorado Island, Panama. Our sample included members of early-arising subfamilies of Poaceae that are endemic to forests, as well as more recently arising subfamilies that transitioned to open environments. Isolation on culture media and direct PCR and cloning revealed that these grasses harbor species-rich and phylogenetically diverse communities that lack the endophytic Clavicipitaceae known from diverse woodland and pasture grasses in the temperate zone. Both the incidence and diversity of endophytes was consistent among grass species regardless of subfamily, clade affiliation or ancestral habitat use. Genotype and phylogenetic analyses suggest that these endophytic fungi are predominantly host generalists, shared not only among distinctive lineages of Poaceae but also with non-grass plants at the same site.
Huang, Shih-Wei; Hsu, Bing-Mu
Naegleria is a free-living amoeba. Pathogenic Naegleria may pose a health risk to people who come in contact with recreational waters. Here, we used Naegleria culture enrichment with PCR to identify the Naegleria species and investigated the distribution of Naegleria spp. in recreational waters including spring water, stream water and raw domestic water in central and southern Taiwan. In this study, Naegleria spp. were detected in 19 (17.8%) of the water samples. The occurrence of Naegleria in raw domestic water was 28.6%, higher than in stream water (14.7%) and in spring water (6.5%). The most frequently identified species exhibiting the closest phylogenetic relationships to the isolates were N. australiensis (n=4) and N. canariensis (n=4), followed by N. clarki (n=3) and N. philippinensis (n=3); N. americana (n=2). N. lovaniensis, N. dobsoni, and N. gruberi were each detected once. The pathogenic species N. fowleri was not detected, probably due to the low incubation temperature; however, the isolates exhibiting the closest phylogenetic relationships to the pathogenic species in mice of PAM, N. australiensis and N. philippinensis, were found. Results of this survey suggest the distribution of Naegleria spp. excluding N. fowleri in recreational waters. It should be considered a potential threat for health associated with human activities in recreational waters.
Cao, Heping; Cao, Fangping; Roussel, Anne-Marie; Anderson, Richard A
Quantitative real-time PCR (qPCR) such as TaqMan and SYBR Green qPCR are widely used for gene expression analysis. The drawbacks of SYBR Green assay are that the dye binds to any double-stranded DNA which can generate false-positive signals and that the length of the amplicon affects the intensity of the amplification. Previous results demonstrate that TaqMan assay is more sensitive but generates lower calculated expression levels than SYBR Green assay in quantifying seven mRNAs in tung tree tissues. The objective of this study is to expand the analysis using animal cells. We compared both qPCR assays for quantifying 24 mRNAs including those coding for glucose transporter (Glut) and mRNA-binding protein tristetraprolin (TTP) in mouse 3T3-L1 adipocytes and RAW264.7 macrophages. The results showed that SYBR Green and TaqMan qPCR were reliable for quantitative gene expression in animal cells. This result was supported by validation analysis of Glut and TTP family gene expression. However, SYBR Green qPCR overestimated the expression levels in most of the genes tested. Finally, both qPCR instruments (Bio-Rad's CFX96 real-time system and Applied Biosystems' Prism 7700 real-time PCR instrument) generated similar gene expression profiles in the mouse cells. These results support the conclusion that both qPCR assays (TaqMan and SYBR Green qPCR) and both qPCR instruments (Bio-Rad's CFX96 real-time system and Applied Biosystems' Prism 7700 real-time PCR instrument) are reliable for quantitative gene expression analyses in animal cells but SYBR Green qPCR generally overestimates gene expression levels than TaqMan qPCR.
Ajaz, Saima; Czajka, Anna; Malik, Afshan
We describe a protocol to accurately measure the amount of human mitochondrial DNA (MtDNA) in peripheral blood samples which can be modified to quantify MtDNA from other body fluids, human cells, and tissues. This protocol is based on the use of real-time quantitative PCR (qPCR) to quantify the amount of MtDNA relative to nuclear DNA (designated the Mt/N ratio). In the last decade, there have been increasing numbers of studies describing altered MtDNA or Mt/N in circulation in common nongenetic diseases where mitochondrial dysfunction may play a role (for review see Malik and Czajka, Mitochondrion 13:481-492, 2013). These studies are distinct from those looking at genetic mitochondrial disease and are attempting to identify acquired changes in circulating MtDNA content as an indicator of mitochondrial function. However, the methodology being used is not always specific and reproducible. As more than 95 % of the human mitochondrial genome is duplicated in the human nuclear genome, it is important to avoid co-amplification of nuclear pseudogenes. Furthermore, template preparation protocols can also affect the results because of the size and structural differences between the mitochondrial and nuclear genomes. Here we describe how to (1) prepare DNA from blood samples; (2) pretreat the DNA to prevent dilution bias; (3) prepare dilution standards for absolute quantification using the unique primers human mitochondrial genome forward primer (hMitoF3) and human mitochondrial genome reverse primer(hMitoR3) for the mitochondrial genome, and human nuclear genome forward primer (hB2MF1) and human nuclear genome reverse primer (hB2MR1) primers for the human nuclear genome; (4) carry out qPCR for either relative or absolute quantification from test samples; (5) analyze qPCR data; and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use.
Löfström, Charlotta; Schelin, Jenny; Norling, Börje
To facilitate quantitative risk assessment in the meat production chain, there is a need for culture-independent quantification methods. The aim of this study was to evaluate the use of flotation, a non-destructive sample preparation method based on traditional buoyant density centrifugation......, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100 cm2) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant...... concentrations of ≥ 6.1 × 108 CFU/swab sample, but not by concentrations ≤ 6.1 × 106 CFU/swab sample. By using the gauze swabs directly in the flotation procedure, the homogenization step normally used for preparation of food-related samples could be excluded, which simplified the culture independent...
Levy, Haim; Diallo, Souleymane; Tennant, Sharon M.; Livio, Sofie; Sow, Samba O.; Tapia, Milagritos; Fields, Patricia I.; Mikoleit, Matthew; Tamboura, Boubou; Kotloff, Karen L.; Lagos, Rosanna; Nataro, James P.; Galen, James E.; Levine, Myron M.
PCR methodology was developed to identify Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B. One multiplex PCR identifies serogroup D, A, and B and Vi-positive strains; another confirms flagellar antigen “d,” “a,” or “b.” Blinded testing of 664 Malian and Chilean Salmonella blood isolates demonstrated 100% sensitivity and specificity. PMID:18367574
S. Fontél, Kristina; Bøtner, Anette; Belsham, Graham
In Europe, clinical signs indicative of foot-and-mouth disease (FMD), would immediately lead to collection of blood and relevant organ material for further laboratory examination for this vesicular disease virus. Today, the first line system for detection of virus in the sample material is real t...... time RT-PCR (RT-qPCR). The aim of this study was to investigate the diagnostic utility of stabilized blood for detection of FMDV RNA in this system....
Vollmer, T; Knabbe, C; Dreier, J
Hepatitis E virus (HEV) infection is recognized as an emerging and often undiagnosed disease in industrialized countries, with asymptomatic infections actually occurring in blood donors. Sensitive detection of HEV-RNA is crucial for diagnosis and monitoring of disease progression. We evaluated the analytical sensitivity and performance of three HEV RT-PCR assays (RealStar HEV reverse transcription-PCR [RT-PCR], hepatitis@ceeramTools, and ampliCube HEV RT-PCR) for screening of individuals for HEV infections (ID-nucleic acid amplification technology [ID-NAT]) and for blood donor pool screening (minipool-NAT [MP-NAT]). RNA was extracted using NucliSens easyMAG (ID-NAT) and a high-volume extraction protocol (4.8 ml, chemagic Viral 5K, MP-NAT). Three NAT assays were evaluated for ID-NAT but only two assays for MP-NAT due to inhibition of the ampliCube HEV RT-PCR kit using the corresponding RNA extract. Assays provided good analytical sensitivity, ranging from 37.8 to 180.1 IU/ml (ID-NAT) and from 4.7 to 91.2 IU/ml (MP-NAT). The applicability of HEV antigen (HEV-Ag) screening was compared to that of RT-PCR screening and detection of HEV-IgM antibodies using seroconversion panels of 10 HEV genotype 3-infected individuals. Four individuals revealed a positive HEV-Ag detection result, with corresponding viremias ranging from 1.92 E + 03 to 2.19 E + 05 IU/ml, while the progression of HEV-Ag followed that of HEV viremia. The other six individuals showed no presence of HEV-Ag although the corresponding viremias were also in the range of >1.0 E + 03. Anti-HEV-IgM antibodies were detectable in seven donors; one donor presented parallel positivities of HEV-Ag and anti-HEV IgM. The evaluated NAT methods present powerful tools providing sensitive HEV detection. Application of HEV-Ag or anti-HEV IgM screening is currently inferior for the early detection of HEV infection due to the decreased sensitivity compared to NAT methods.
Full Text Available BACKGROUND: The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR. With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. RESULTS: In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569 outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN. PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. CONCLUSIONS: Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection
Hansen, S.L.; Hetmanski, J
Blood subcultures repeated 3 days after the cultures were first identified as positives increased our detection of polymicrobic bacteremia in 9.1 to 27% of clinically significant patient episodes. Reincubation and repeated subculture of previously positive blood cultures had a direct impact on the therapeutic management of patients with polymicrobic bacteremia.
Velden, L.B. van der; Vos, F.J.; Mouton, J.W.; Sturm, P.D.J.
The effect of immediate incubation of blood cultures at 37 degrees C on the turnaround time and the impact of Gram stain results on antimicrobial management were investigated. During a 6-month period, blood cultures collected at the emergency department outside laboratory operating hours were preinc
M. A. Gordukova
Full Text Available Primary immunodeficiencies (PID such as severe combined immunodeficiency (SCID and X-linked agammaglobulinemia are characterized by the lack of functional Tand B-cells, respectively. Without early diagnosis and prompt treatment children with PID suffer from severe infectious diseases, leading to their death or disability. Our purpose was developing of simple, inexpensive, high throughput technique based on the quantitative determination of TREC and KREC molecules by real-time PCR, and its validation in a group of children with a verified diagnosis of SCID and X-linked agammaglobulinemia.In this study, we developed and validated multiplex real-time PCR for the TREC’s and KREC’s quantitative analysis. We have shown that linear range of Ct changes depending on the concentrations of targets with a correlation coefficient R2 not worse than 0.98 was observed at concentrations from 109 to 5 × 104 copies per ml. The lowest amount of targets reliably detected in a reaction volume was 10 TREC’s copies, 5 KREC ‘s copies and 5 copies of internal control (IL17RA. We determined the age-depended reference values of TRECs and KRECs in whole blood in 29 boys and 27 girls with normal immunological parameters. The normal cut-offs for TRECs and KRECs were defined in dry blood spots depending on the method of extraction.The proposed method showed 100% diagnostic sensitivity and specificity in the studied group. The method can be proposed as a screening tool for the diagnosis of SCID and X-linked agammaglobulinemia both in whole blood and in the dry blood spots. The further investigation is required with larger number of samples.
Full Text Available Septicaemia is a major contributor of mortality. Blood culture is the essential investigation for the management of sepsis. Due to lack of resources blood culture is an irregularly used investigation in India. A three-tier level of development is being proposed to develop the blood culture based national programme for early detection of sepsis. The plan envisages the establishment of manual blood culture based elementary system in the health centre and district hospital level (Level 1, direct Gram stain and direct antibiotic sensitivity testing from the "positive" blood culture broths at the medical college hospital level (Level 2 and development of automated methods, enhancement of quality control and safety measures, clinical liaison and re-orientation of microbiology training at the tertiary care centre level (Level 3.
Adham, Fatma K; El-Samie-Abd, Emtithal M; Gabre, Refaat M; El Hussein, Hala
The prevalence of Borrelia burgdorferi sensu lato (s.l.), the etiologic agent of Lyme borrelosis (LB), was determined for the first time in Egypt by using polymerase chain reaction (PCR). Questing 5243 hard and soft ticks were collected from animal farms throughout Giza Governorate. DNA from 500 individual tick species was extracted and PCR was performed. Primers verified from the sequence of German strain Pko of Borrelia afzelii were used. Fragments of 642 bp were generated and sequenced. The prevalence of B. burgdorferi sensu lato (s.l.) was 28% of examined soft and hard ticks. High infection rate (66%) of B. burgdorferi s.l. was observed in both nymph and adult soft ticks Ornithodoros savignyi. Beside, the role of hard ticks as potential vectors of Lyme disease in Egypt, where the infection rate was between 0.0-50.0%. Sequence analysis of PCR product of Borrelia burgdorferi sensu lato shares high degree of similarity in sequence compared to similar species in GenBank.
Tierney, B M; Henry, N K; Washington, J A
Staining 2,205 macroscopically negative blood cultures with acridine orange after 6 to 17 h of inoculation and incubation was as sensitive as an early subculture in detecting positive blood cultures. Of the 179 positive blood cultures, 30 (16.8%) were detected by acridine orange alone, 19 (10.6%) were detected by early subculture alone, 84 (46.9%) were detected by both techniques, and 46 (25.7%) were not detected by either method. The latter group includes cultures that became positive after ...
Willasch, Andre M; Kreyenberg, Hermann; Shayegi, Nona; Rettinger, Eva; Meyer, Vida; Zabel, Marion; Lang, Peter; Kremens, Bernhard; Meisel, Roland; Strahm, Brigitte; Rossig, Claudia; Gruhn, Bernd; Klingebiel, Thomas; Niemeyer, Charlotte M; Bader, Peter
Quantitative real-time PCR (qPCR) has been proposed as a highly sensitive method for monitoring hematopoietic chimerism and may serve as a surrogate marker for the detection of minimal residual disease minimal residual disease in myelodysplastic syndrome (MDS), until specific methods of detection become available. Because a systematic comparison of the clinical utility of qPCR with the gold standard short tandem repeat (STR)-PCR has not been reported, we retrospectively measured chimerism by qPCR in 54 children transplanted for MDS in a previous study. Results obtained by STR-PCR in the initial study served as comparison. Because the detection limit of qPCR was sufficiently low to detect an autologous background, we defined the sample as mixed chimera if the proportion of recipient-derived cells exceeded .5%. The true positive rates were 100% versus 80% (qPCR versus STR-PCR, not significant), and mixed chimerism in most cases was detected earlier by qPCR than by STR-PCR (median, 31 days) when chimerism was quantified concurrently in peripheral blood and bone marrow. Both methods revealed a substantial rate of false positives (22.7% versus 13.6%, not significant), indicating the importance of serial testing of chimerism to monitor its progression. Finally, we propose criteria for monitoring chimerism in pediatric MDS with regard to the subtypes, specimens, PCR method, and timing of sampling.
Brook, I; Gruenwald, L D
Modified Castaneda blood culture bottles were used to diagnose bacteraemia in children attending the out patient clinic. Bacterial growth was detected in twelve out of 147 patients (8%), in both the routine and Castaneda blood culture bottles. Streptococcus pneumoniae was recovered in nine patients (6%), and H. influenzae in three patients (2%). The average length of time required to identify the organisms utilizing the routine blood culture bottles was 2 days (range 1 to 4 days), while the average time utilizing Castaneda bottles was 3.5 days (range 1 to 6 days). Castaneda blood bottles were found in this work to be effective in the detection of bacteraemia in children, and because of their simplicity they may serve for the detection of bacteraemia by physicians in general practice.
Shipitsyna, Elena; Guschin, Alexander; Maximova, Anna; Tseslyuk, Mariya; Savicheva, Alevtina; Sokolovsky, Evgenij; Shipulin, German; Domeika, Marius; Unemo, Magnus
This study aimed to assess the laboratory diagnosis of Neisseria gonorrhoeae in St. Petersburg, Russia. In total, 334 consecutive symptomatic patients were enrolled. Cervical and urethral specimens from women (n=286) and urethral specimens from men (n=48) were analyzed by microscopy, culture and two in-house NAATs, i.e. polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA), developed in Russia. All N. gonorrhoeae-positive samples were confirmed using porA pseudogene and 16S rRNA gene sequencing. All methods displayed 100% specificity, i.e. positive predictive values of 100%. Compared to the PCR (most sensitive method in the present study), in women the sensitivity of both microscopy and culture was 31.8%, and that of NASBA was 90.9%. In men, microscopy, culture and NASBA displayed a sensitivity of 75%, 50% and 100%, respectively. The negative predictive values of microscopy, culture, and NASBA were 97.3%, 97.3%, and 99.6% in women, and 97.8%, 95.7%, and 100% in men, respectively. According to the PCR, the prevalences of N. gonorrhoeae were 4.5% (women) and 8.3% (men). In conclusion, both the investigated Russian NAATs displayed a high sensitivity and specificity. However, in general the diagnosis of gonorrhoea in Russia is suboptimal and crucially requires validation, improvements and quality assurance.
Jawaheer, G; Neal, T; Shaw, N.
A prospective, blind study was carried out to determine: the amount of blood submitted for culture from neonates; whether small blood volumes resulted in false negative results; and whether there was a temporal relation between volume of blood cultured and time to positivity. Seventy three bottles were evaluated. They contained a median of 0.63 ml of blood. Twenty nine bottles (39.7%) contained less than 0.5 ml of blood; 21 bottles (28.8%) were positive. There were three false negative cult...
Full Text Available Neonatal sepsis is an important cause of death and morbidity in newborns and is diagnosed by isolation of organism in blood culture. In several reports,reliablity of blood cultures were done from umbi lical catheters,have been demonstrated. The objective of the present study was to determine,wether an inde welling umbilical catheter, could be an alternative site for blood culture. In a prospective study over 6 months during 2006,141 paired blood cultures from 134 infant,were done simultaneously from peripheral site and umbilical catheter (mostly U. V. C,during the first four days of life. Majority of these infants were preterm and admitted to NICU for special care. these infants had indwelling umbilical line and had indication of sepsis workup. A total of 141 pairs of blood cultures were obtained from 134 infants. In 16 infants blood culture pairs were positive for one organism in both peripheral vein and umbilical site. 71. 6% of total cultures (n=11pairs were negative in boths site. A total of 22 pairs were positive in one site only,with 5 positive from peripheral vein only and the other 17 from umblical site. Two pairs were positve in boths site with two different organism. In over all 16 infant (11%of blood were considered to be contaminated. Contamination rate were 2. 4% and 9. 2% for peripheral and umbilical catheter site. Contamination rate increased after 48 hours of age in umbilical catheter. The result showed that after 2 days contamination rate for blood culture taken from catheter line increased and specifity decreased. We recommended that blood culture via umblical catheter in first 2 days in sick neonates with indwelling catheter can be a alternate site of blood culture sampelling.
This study evaluated the ability of ten commercial blood cultures to support the growth of a nutritional variant Streptococcus mitior (pyridoxal-dependent). The abilities of two established and two new agar formulations are also reported. The dependable isolation of a fastidious streptococcus can best be obtained with fastidious anaerobe broth (FAB) (Lab M Ltd, Ford Lane, Salford) for blood cultures in conjunction with one of the new media. FAB agar with the addition of heated blood was found...
Evaluation of combined high-efficiency DNA extraction and real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in subclinically infected dairy cattle: comparison with faecal culture, milk real-time PCR and milk ELISA
Full Text Available Abstract Background Johne’s disease is caused by Mycobacterium avium subsp. paratuberculosis (Map and it is one of the most important diseases in cattle worldwide. Several laboratory tests for Map detection are available; however, these are limited by inadequate sensitivity and specificity when used in subclinically infected populations. To identify Map shedders in subclinically infected cattle, we used a new, high-yield method for DNA-extraction from Map in faeces combined with quantitative real-time PCR (qPCR for amplification of the insertion sequence IS900 of Map (HYDEqPCR. Evaluation of HYDEqPCR was carried out in comparison with faecal culture, milk qPCR, and milk enzyme-linked immunosorbent assay (ELISA, on 141 faecal and 91 milk samples, from 141 subclinically infected dairy cattle. Results The qPCR proved to be highly sensitive, with a detection limit of 2 IS900 DNA copies/μl in 67 % of the reactions. It also showed 100 % specificity, as determined from 50 Map and non-Map strains, and by the sequencing of qPCR amplicons. The detection limit of HYDEqPCR was 90 Map/g Map-spiked faeces, which corresponds to 2.4 colony forming units/g Map-spiked faeces, with an estimated efficiency of 85 % (±21 %. When tested on the field samples, HYDEqPCR showed 89 % of the samples as positive for Map, whereas faecal culture, milk qPCR, and milk ELISA detected 19 %, 36 % and 1 %, respectively. Fisher’s exact tests only show statistical significance (p ≤0.05 for the correlation between HYDEqPCR and faecal culture. The agreement between HYDEqPCR and milk qPCR and milk ELISA was poor, slight, and non-significant. Conclusions This study highlights the advantages of HYDEqPCR for detection of Map in subclinically infected populations, in comparison with faecal culture, milk qPCR and milk ELISA. HYDEqPCR can detect low-level Map shedders that go undetected using these other methods, which will thus underestimate the proportions of Map
Angeletti, Silvia; Gherardi, Giovanni; De Florio, Lucia; Avola, Alessandra; Crea, Francesca; Riva, Elisabetta; Vitali, Massimiliano Andrea; Galluzzo, Sara; Dicuonzo, Giordano
A Real-time polymerase chain reaction (PCR) with melting analysis was devised to target bacterial and fungal genes together with the most prevalent antimicrobial resistance genes in 250 positive blood culture broths. This method allowed the blood culture cultivated pathogens to be classified into clinically relevant groups such as Enterobacteriaceae, oxidase-positive bacilli, oxidase-positive coccobacilli, S. aureus and yeast. Enterococci and streptococci could be distinguished from CoNS only by the Gram stain. Gram-positive bacilli were discriminated from Gram-positive cocci by Gram stain. Furthermore, the most important antimicrobial resistant genes such as mecA, vanA, bla TEM , bla SHV and bla CTX-M could be identified. All results were obtained with a turnaround time of three hours from the moment of blood culture positivity compared to 24-72 hours for phenotypic methods. In conclusion, the proposed approach can allow the clinician to implement proper early management of sepsis patients.
Full Text Available Abstract Background Acute schistosomiasis constitutes a rare but serious condition in individuals experiencing their first prepatent Schistosoma infection. To circumvent costly and time-consuming diagnostics, an early and rapid diagnosis is required. So far, classic diagnostic tools such as parasite microscopy or serology lack considerable sensitivity at this early stage of Schistosoma infection. To validate the use of a blood based real-time polymerase chain reaction (PCR test for the detection of Schistosoma DNA in patients with acute schistosomiasis who acquired their infection in various endemic regions we conducted a European-wide prospective study in 11 centres specialized in travel medicine and tropical medicine. Methods Patients with a history of recent travelling to schistosomiasis endemic regions and freshwater contacts, an episode of fever (body temperature ≥38.5°C and an absolute or relative eosinophil count of ≥700/μl or 10%, were eligible for participation. PCR testing with DNA extracted from serum was compared with results from serology and microscopy. Results Of the 38 patients with acute schistosomiasis included into the study, PCR detected Schistosoma DNA in 35 patients at initial presentation (sensitivity 92%. In contrast, sensitivity of serology (enzyme immunoassay and/or immunofluorescence assay or parasite microscopy was only 70% and 24%, respectively. Conclusion For the early diagnosis of acute schistosomiasis, real-time PCR for the detection of schistosoma DNA in serum is more sensitive than classic diagnostic tools such as serology or microscopy, irrespective of the region of infection. Generalization of the results to all Schistosoma species may be difficult as in the study presented here only eggs of S. mansoni were detected by microscopy. A minimum amount of two millilitre of serum is required for sufficient diagnostic accuracy.
Matsuda, Mari; Iguchi, Shigekazu; Mizutani, Tomonori; Hiramatsu, Keiichi; Tega-Ishii, Michiru; Sansaka, Kaori; Negishi, Kenta; Shimada, Kimie; Umemura, Jun; Notake, Shigeyuki; Yanagisawa, Hideji; Yabusaki, Reiko; Araoka, Hideki; Yoneyama, Akiko
Background. Early detection of Gram-positive bacteremia and timely appropriate antimicrobial therapy are required for decreasing patient mortality. The purpose of our study was to evaluate the performance of the Verigene Gram-positive blood culture assay (BC-GP) in two special healthcare settings and determine the potential impact of rapid blood culture testing for Gram-positive bacteremia within the Japanese healthcare delivery system. Furthermore, the study included simulated blood cultures, which included a library of well-characterized methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) isolates reflecting different geographical regions in Japan. Methods. A total 347 BC-GP assays were performed on clinical and simulated blood cultures. BC-GP results were compared to results obtained by reference methods for genus/species identification and detection of resistance genes using molecular and MALDI-TOF MS methodologies. Results. For identification and detection of resistance genes at two clinical sites and simulated blood cultures, overall concordance of BC-GP with reference methods was 327/347 (94%). The time for identification and antimicrobial resistance detection by BC-GP was significantly shorter compared to routine testing especially at the cardiology hospital, which does not offer clinical microbiology services on weekends and holidays. Conclusion. BC-GP generated accurate identification and detection of resistance markers compared with routine laboratory methods for Gram-positive organisms in specialized clinical settings providing more rapid results than current routine testing.
Full Text Available Background. Early detection of Gram-positive bacteremia and timely appropriate antimicrobial therapy are required for decreasing patient mortality. The purpose of our study was to evaluate the performance of the Verigene Gram-positive blood culture assay (BC-GP in two special healthcare settings and determine the potential impact of rapid blood culture testing for Gram-positive bacteremia within the Japanese healthcare delivery system. Furthermore, the study included simulated blood cultures, which included a library of well-characterized methicillin-resistant Staphylococcus aureus (MRSA and vancomycin-resistant enterococci (VRE isolates reflecting different geographical regions in Japan. Methods. A total 347 BC-GP assays were performed on clinical and simulated blood cultures. BC-GP results were compared to results obtained by reference methods for genus/species identification and detection of resistance genes using molecular and MALDI-TOF MS methodologies. Results. For identification and detection of resistance genes at two clinical sites and simulated blood cultures, overall concordance of BC-GP with reference methods was 327/347 (94%. The time for identification and antimicrobial resistance detection by BC-GP was significantly shorter compared to routine testing especially at the cardiology hospital, which does not offer clinical microbiology services on weekends and holidays. Conclusion. BC-GP generated accurate identification and detection of resistance markers compared with routine laboratory methods for Gram-positive organisms in specialized clinical settings providing more rapid results than current routine testing.
Results: From 2009 to 2011, 72 patients with hematologic malignancies and bone marrow transplant recipients were evaluated for IC. The female to male ratio was 27:45; the mean age was 32.1 years. The most common malignancy in this patient was acute myeloid leukemia (AML (27.8% and acute lymphoblastic leukemia (ALL (26.4%. Out of 72 patients, 11 patients (15.3% had positive real time PCR /probe results. Based on the melting temperature (Tm analysis, 5 (45.4% C. krusei, 3 (27.2% C. tropicalis, 2 (18.1% C. parapsilosis and 1 C. albicans (9% were identified. According to the revised EORTC / MSG, 1 patient (9% and 10 patients (91% were defined as proven and possible groups of IC, respectively. The mortality rate in proven and possible IC patient was found 54.5%. Conclusion: The established Real-time PCR/FRET probe assay is an appropriate diagnostic tool for the detection of Candida species DNA and the management of patients suffering from hematologic malignancies and bone marrow recipient are at risk for IC.
Jalal Mardaneh; Parvin Hasanzadeh; Mohammad Motamedifar; Khadijeh Ahmadi; Farhad Nikkhahi
Background: Neisseria gonorrhoeae is a human obligate pathogen and the etiological agent of gonorrhea. Health irreparable complications resulting from gonorrhea disease occur mainly in pregnant women and neonates. Aim of this study was diagnosis of Neisseria gonorrhoeae among pregnant women with using culture and molecular method by amplification of cppB gene with PCR. Material and Methods: In this cross-sectional study, two endocervical swab specimens were obtained from 1100 pregnant women w...
Full Text Available Background: Neisseria gonorrhoeae is a human obligate pathogen and the etiological agent of gonorrhea. Health irreparable complications resulting from gonorrhea disease occur mainly in pregnant women and neonates. Aim of this study was diagnosis of Neisseria gonorrhoeae among pregnant women with using culture and molecular method by amplification of cppB gene with PCR. Material and Methods: In this cross-sectional study, two endocervical swab specimens were obtained from 1100 pregnant women who referred to Shiraz Hospitals. Culture on nonselective and selective media and nucleic acid amplification test (NAAT were performed for detection of Neisseria gonorrhoeae cppB gene. Results: All endocervical swabs cultures on selective and nonselective media were negative for Neisseria gonorrhoeae. Among examined endocervical swabs, 13samples (1.18% were positive by nucleic acid amplification of Neisseria gonorrgoeae cppB gene. Conclusion: Negative results of culture and positive results of PCR in this study indicate that however culture is gold standard method for detection of Neisseria gonorrhoeae but due to bacterial autolysis, poor sampling techniques and improper specimen storage and transport, its value decline as compared with Nucleic acid amplification test (NAAT.
Muñoz-San Martín, Catalina; Apt, Werner; Zulantay, Inés
The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a major public health problem in Latin America. This parasite has a complex population structure comprised by six or seven major evolutionary lineages (discrete typing units or DTUs) TcI-TcVI and TcBat, some of which have apparently resulted from ancient hybridization events. Because of the existence of significant biological differences between these lineages, strain characterization methods have been essential to study T. cruzi in its different vectors and hosts. However, available methods can be laborious and costly, limited in resolution or sensitivity. In this study, a new genotyping strategy by real-time PCR to identify each of the six DTUs in clinical blood samples have been developed and evaluated. Two nuclear (SL-IR and 18S rDNA) and two mitochondrial genes (COII and ND1) were selected to develop original primers. The method was evaluated with eight genomic DNA of T. cruzi populations belonging to the six DTUs, one genomic DNA of Trypanosoma rangeli, and 53 blood samples from individuals with chronic Chagas disease. The assays had an analytical sensitivity of 1-25fg of DNA per reaction tube depending on the DTU analyzed. The selectivity of trials with 20fg/μL of genomic DNA identified each DTU, excluding non-targets DTUs in every test. The method was able to characterize 67.9% of the chronically infected clinical samples with high detection of TcII followed by TcI. With the proposed original genotyping methodology, each DTU was established with high sensitivity after a single real-time PCR assay. This novel protocol reduces carryover contamination, enables detection of each DTU independently and in the future, the quantification of each DTU in clinical blood samples.
Ozüberk, Osman Özüberk; Gökahmetoğlu, Selma; Ozçelik, Bülent; Ekmekçioğlu, Oğuz
Chlamydia trachomatis infection is considered the most prevalent bacterial sexually transmitted disease worldwide. C.trachomatis causes eye infections such as trachoma and newborn inclusion conjunctivitis, newborn pneumonia, genitourinary system infections and suppurative inguinal lymphadenitis namely lymphogranuloma venerum. The aim of this study was to investigate C.trachomatis by direct fluorescent antibody (DFA), polymerase chain reaction (PCR) and cell culture methods in the clinical samples sent to the microbiology laboratory with the prediagnosis of genital infections. A total of 50 swab samples obtained from adult patients (49 female, 1 male) who were admitted to Erciyes University Hospital, Kayseri, Turkey between February-March 2010, were included in the study. C.trachomatis antigens were investigated by a commercial DFA (PathoDx, Remel, USA) method. McCoy cell cultures prepared in microplate wells were used for the isolation of C.trachomatis. The growth of C.trachomatis in cell cultures was confirmed by DFA and iodine staining methods. C.trachomatis DNA was investigated by commercially available PCR (Chlamydia trachomatis 330/740 IC; Sacace, Italy) method. In our study, 4 (8%) of the 50 swab samples were found positive with DFA, 1 (2%) was positive with cell culture, and 1 (2%) was positive with PCR. The only sample that gave positive results with all of the three methods was an urethral swab. Three cervical swab samples that were found positive only with DFA method was evaluated as false positivity. When cell culture was considered as the reference method, the sensitivity and specificity of DFA method were estimated as 100% and 94%, respectively, while those rates for PCR were 100% and 100%, respectively. In conclusion, although cell culture is still the gold standard in the diagnosis of C.trachomatis. infections, since it is time consuming and difficult to apply, more rapid and reliable PCR methods may be applied in diagnosis. DFA method which is
Springer, Jan; White, P Lewis; Hamilton, Shanna; Michel, Denise; Barnes, Rosemary A; Einsele, Hermann; Löffler, Juergen
Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P = 0.0019; for serum, P = 0.0049; and for WBP, P = 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P = 0.0238) and plasma (53%, P = 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time
Klanicova, B; Slana, I; Roubal, P; Pavlik, I; Kralik, P
Mycobacterium avium paratuberculosis (MAP), etiological agent of paratuberculosis in ruminants, is able to survive extreme conditions like very low pH (stomach), high temperature (pasteurization) or low temperature (refrigerated storage). Cheese, infant powder milk, cream and other milk and dairy products might thus be considered as possible sources of MAP for humans. The aim of this study was to investigate the survival of two MAP field isolates during fermentation of three different types of soured milk products (SMP; yogurt, acidophilus milk and kefir) under laboratory conditions. Pasteurized MAP-free milk was artificially contaminated with 10(6)MAPcells/mL and survival and absolute numbers of MAP were monitored during fermentation (4 or 16 h) and after six weeks of storage at 4°C by culture and quantitative real time PCR (qPCR). Viability of MAP was determined by culture using Herrold's egg yolk medium and Middlebrook 7H10 with antibiotics, supplemented with Mycobactin J and incubated at 37°C for up to 12 weeks. The absolute numbers of MAP were quantified by previously published qPCR assays targeting F57 and IS900 loci in MAP genome. We herein confirm that MAP can survive pH reduction, however, longer exposure to pH below 4 in SMP seems to be critical because it inhibits growth. Therefore, it is suggested that probiotic cultures that can decrease pH below 4 during fermentation could provide better inactivation of MAP in SMP.
Wildi, K; Tschudin-Sutter, S; Dell-Kuster, S; Frei, R; Bucher, H C; Nüesch, R
Blood cultures are routinely taken in outpatients with fever and suspected bacterial infections. However, in the majority of cases, they are not informative and of limited value for clinical decision making. The aim of this study was therefore to investigate factors associated with positive blood cultures in outpatients presenting to an outpatient clinic and emergency room. This was a case-control study of all outpatients with positive blood cultures from January 1, 2006 to October 31, 2007 and matched control patients with negative blood cultures in the same time period. Microbiology results and medical charts were reviewed to determine factors associated with positive blood cultures. The presence of a systemic inflammation response syndrome (SIRS) (OR 2.7, 95% Cl 1.0-7.2) and increased C-reactive protein (CRP) (OR 1.1 per 10 mg/l, 95% Cl 1.0-1.2) were the most powerful predictive values for the development of positive blood cultures. In positive cases serum albumin was lower (35 mg/l versus 39 mg/l) than in controls. SIRS, increasing CRP and low albumin were associated with positive blood cultures in outpatients. With simple clinical assessment and few laboratory tests indicative of infection, it is possible to define a group at higher risk for bacteremia in outpatients.
Ouedraogo, David Eric; Bollore, Karine; Viljoen, Johannes; Foulongne, Vincent; Reynes, Jacques; Cartron, Guillaume; Vendrell, Jean-Pierre; Van de Perre, Philippe; Tuaillon, Edouard
Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios.
Stecklum, Maria; Wulf-Goldenberg, Annika; Purfürst, Bettina; Siegert, Antje; Keil, Marlen; Eckert, Klaus; Fichtner, Iduna
In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction.
Taguchi, Hirokazu; Matsumoto, Tetsuya; Ishikawa, Hiroki; Ohta, Shoichi; Yukioka, Tetsuo
We investigated active screening for colonization with methicillin-resistant Staphylococcus aureus (MRSA) on admission and weekly follow-up surveillance after admission to a tertiary care center (TCC) between June 2007 and 31 December 2007. Eleven percent (30/267) of patients were found to be positive for MRSA by polymerase chain reaction (PCR) and/or culture on admission; 5% (12/267) became positive during the TCC stay. The major primary diagnoses in MRSA-positive patients were pneumonia and cerebrovascular diseases. Twenty-two (52%) of 42 patients were found to be MRSA positive by both PCR and culture, compared with 19 (45%) of 42 who were PCR positive and culture negative. These findings suggest that active surveillance with PCR is highly sensitive and useful for the detection of MRSA colonization. To our knowledge, this is the first report of active surveillance of MRSA by PCR and bacterial culture in critically ill inpatients in Japan.
Full Text Available La brucelosis canina, producida por Brucella canis, es una enfermedad asociada a problemas reproductivos y de carácter zoonótico. Estas bacterias son excretadas en orina, leche, fetos o semen de los animales infectados y la transmisión ocurre por contacto vía sexual, oral, nasal o conjuntival. El diagnóstico de rutina se realiza por serología, pero la confirmación requiere aislamiento del cultivo bacterial, lo cual es costoso y requiere laboratorios con nivel 3 de bioseguridad. Las técnicas moleculares son una posibilidad reconocida para determinar el ADN bacterial, con alta especificidad y sensibilidad. Este reporte evaluó como prueba de aplicación clínica una técnica de PCR desarrollada para cultivos bacteriales. A una hembra canina asintomática, con historia previa de la enfermedad, amamantando una camada sana de 4 días de nacidos, se le realizó la prueba serológica rápida en placa con 2ß-mercaptoetanol, hemocultivo y PCR, de leche y de sangre. Todas las pruebas fueron positivas a Brucella canis. Este es el primer reporte de diagnóstico en leche por PCR, lo que corrobora que animales clínicamente asintomáticos eliminan la bacteria por esta vía, lo que constituye un riesgo de infección para los neonatos y el riesgo zoonótico para veterinarios, propietarios del animal o personas que intervengan en el parto si no se toman medidas higiénicas preventivas.Canine brucellosis is a disease caused by Brucella canis that is associated to reproductive problems in dogs, and it is also known as zoonosis. These bacteria are excreted in urine, milk, fetus or semen of infected animals, and the transmission occurs via sexual, oral, nasal or conjunctival contact. Diagnosis is usually done through serology but confirmation requires isolation of bacterial culture, a costly process that requires laboratory biosafety level 3. Molecular techniques are a valid method to determine the bacterial DNA, offering high specificity and sensitivity
The EntericBio system uses a multiplex PCR assay for the simultaneous detection of Campylobacter spp., Salmonella enterica, Shigella spp., and Escherichia coli O157 from feces. It combines overnight broth enrichment with PCR amplification and detection by hybridization. An evaluation of this system was conducted by comparing the results obtained with the system with those obtained by routine culture, supplemented with alternative PCR detection methods. In a study of 773 samples, routine culture and the EntericBio system yielded 94.6 and 92.4% negative results, respectively. Forty-two samples had positive results by culture, and all of these were positive with the EntericBio system. This system detected an additional 17 positive samples (Campylobacter spp., n = 12; Shigella spp., n = 1; E. coli O157, n = 4), but the results for 5 samples (Campylobacter spp., n = 2; Shigella spp., n = 1; E. coli O157, n = 2) could not be confirmed. The target for Shigella spp. detected by the EntericBio system is the ipaH gene, and the molecular indication of the presence of Shigella spp. was investigated by sequence analysis, which confirmed that the ipaH gene was present in a Klebsiella pneumoniae isolate from the patient. The sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 99.3%, 91.5%, and 100%, respectively. Turnaround times were significantly reduced with the EntericBio system, and a result was available between 24 and 32 h after receipt of the sample in the laboratory. In addition, the amount of laboratory waste was significantly reduced by use of this system. In summary, the EntericBio system proved convenient to use, more sensitive than the conventional culture used in this study, and highly specific; and it generated results significantly faster than routine culture for the pathogens tested.
Full Text Available Background. The rapid identification of the etiology and the evaluation of the antimicrobial susceptibility of the bacteria causing bacteremia is of outmost relevance to set up an adequate treatment of sepsis. In this study we evaluated the microarray based method, Verigene Gram-positive blood cultures (BC-GP nucleic acid test (Nanosphere Inc., Northbrook, IL, USA for the identification of Gram positive pathogens from positive blood cultures. The panel BC-GP is capable to identify 13 germs and 3 genes associated with antimicrobial resistance. Materials and Methods. In this study a total of 100 positive, non replicated and monomicrobic blood cultures have been evaluated. For testing on the Verigene platform using the BC-GP assay, 350 L of blood culture media from a positive the blood culture bottle.Results. A total of 100 positive blood cultures were tested by the Verigene BC-GP assay: out of these a total of 100 Gram-positive cocci were identified. The most frequent bacteria identified included staphylococci, streptococci and enterococci. Among staphylococci, Staphylococcus aureus accounted for 25% (15/60, with 38% of S. epidermidis 37% (23/60 and 37% (22/60 other CoNS. All the S. aureus isolates were correctly identified by BC-GP whereas in 2/45 cases (4% BC-GP misidentified CoNS. In the case of enterococci 7/10 were E. faecalis and 3 E. faecium, all of these were correctly identified.Conclusions. The overall agreement with the results obtained by standard procedure is quite elevated (88% and as a consequence the BC-GP panel could be used as a rapid diagnostic tool to give a faster response in the case of bacteremia associated with sepsis.
Haque, Rashidul; Kabir, Mamun; Noor, Zannatun; Rahman, S M Mazidur; Mondal, Dinesh; Alam, Faisal; Rahman, Intekhab; Al Mahmood, Abdullh; Ahmed, Nooruddin; Petri, William A
The noninvasive diagnosis of amebic liver abscess is challenging, as most patients at the time of diagnosis do not have a concurrent intestinal infection with Entamoeba histolytica. Fecal testing for E. histolytica parasite antigen or DNA is negative in most patients. A real-time PCR assay was evaluated for detection of E. histolytica DNA in blood, urine, and saliva samples from amebic liver abscess as well as amebic colitis patients in Bangladesh. A total of 98 amebic liver abscess and 28 amebic colitis patients and 43 control subjects were examined. The real-time PCR assay detected E. histolytica DNA in 49%, 77%, and 69% of blood, urine, and saliva specimens from the amebic liver abscess patients. For amebic colitis the sensitivity of the real-time PCR assay for detection of E. histolytica DNA in blood, urine, and saliva was 36%, 61%, and 64%, respectively. All blood, urine, and saliva samples from control subjects were negative by the real-time PCR assay for E. histolytica DNA. When the real-time PCR assay results of the urine and saliva specimens were taken together (positive either in urine or saliva), the real-time PCR assay was 97% and 89% sensitive for detection of E. histolytica DNA in liver abscess and intestinal infection, respectively. We conclude that the detection of E. histolytica DNA in saliva and urine could be used as a diagnostic tool for amebic liver abscess.
Full Text Available The traditional methodology used for the identification of microbes colonizing our cultural heritage was the application of cultivation methods and/or microscopy. This approach has many advantages, as living microorganisms may be obtained for physiological investigations. In addition, these techniques allow the quantitative and qualitative assessment of the investigated environment. Quantitative analyses are done by plate count and the determination of abundance by the colony forming unit (CFU. Nevertheless, these techniques have many drawbacks that lead to an underestimation of the cell numbers and do not provide a comprehensive overview of the composition of the inhabiting microbiota. In the last decades, several molecular techniques have been developed enabling many advantages over the cultivation approach. Mainly PCR-based, fingerprinting techniques allow a qualitative detection and identification of the microbiota. In this study, we developed a real time PCR method as a simple, rapid and reliable tool to detect and quantify fungal abundance using the β-actin gene, which is known to appear as a single-copy gene in fungi. To this end, five different indoor thermal insulation materials applied for historical buildings that were previously tested for their bio-susceptibility against various fungi were subjected to qPCR analyses. The obtained results were compared with those obtained from a previous study investigating the bio-susceptibility of the insulation materials using classical cultivation experiments. Both results correlated well, revealing that Perlite plaster was the most suitable insulation material, showing the lowest fungal CFU and qPCR values. In contrast, insulations made of wood showed to be not recommendable from the microbiological point of view. In addition, the potential of qPCR was tested in other materials of cultural heritage, as old parchments, showing to be a suitable method for measuring fungal abundance in these
Ettenauer, Jörg; Piñar, Guadalupe; Tafer, Hakim; Sterflinger, Katja
The traditional methodology used for the identification of microbes colonizing our cultural heritage was the application of cultivation methods and/or microscopy. This approach has many advantages, as living microorganisms may be obtained for physiological investigations. In addition, these techniques allow the quantitative and qualitative assessment of the investigated environment. Quantitative analyses are done by plate count and the determination of abundance by the colony forming unit (CFU). Nevertheless, these techniques have many drawbacks that lead to an underestimation of the cell numbers and do not provide a comprehensive overview of the composition of the inhabiting microbiota. In the last decades, several molecular techniques have been developed enabling many advantages over the cultivation approach. Mainly PCR-based, fingerprinting techniques allow a qualitative detection and identification of the microbiota. In this study, we developed a real time PCR method as a simple, rapid and reliable tool to detect and quantify fungal abundance using the β-actin gene, which is known to appear as a single-copy gene in fungi. To this end, five different indoor thermal insulation materials applied for historical buildings that were previously tested for their bio-susceptibility against various fungi were subjected to qPCR analyses. The obtained results were compared with those obtained from a previous study investigating the bio-susceptibility of the insulation materials using classical cultivation experiments. Both results correlated well, revealing that Perlite plaster was the most suitable insulation material, showing the lowest fungal CFU and qPCR values. In contrast, insulations made of wood showed to be not recommendable from the microbiological point of view. In addition, the potential of qPCR was tested in other materials of cultural heritage, as old parchments, showing to be a suitable method for measuring fungal abundance in these delicate materials.
Radic, Marina; Goic-Barisic, Ivana; Novak, Anita; Rubic, Zana; Tonkic, Marija
PNA FISH(®) (peptide nucleic acid fluorescent in situ hybridization) Yeast Traffic Light (PNA FISH(®) YTL) assay is a commercially avaliable method for rapid identification of Candida spp. directly from positive blood cultures. This report provides a one-year experience in identification of yeasts from 25 specimens (15 positive blood cultures and 10 other clinically significant specimens) using PNA FISH(®) YTL and comparing it to VITEK 2 System. Overall, assay identification compatibility with VITEK 2 System was found among 21/25 (84%) isolates tested. Only 3/25 (12%) of the isolates were not identified, and one isolate was misidentified by the PNA FISH(®) YTL assay. Our results show that the assay is a reliable method in identification of Candida spp. not only from blood cultures, but even from other clinically significant specimens (urine cultures, catheter tip cultures, peritoneal fluid cultures) when compared to automated method like VITEK 2 System. This novel application of the PNA FISH(®) YTL assay could therefore contribute to cost savings and significant benefit to patients, as rapid information about isolated yeast species is provided.
Fabio Felipe dos Santos
Full Text Available ABSTRACT Purpose: To evaluate the ability of real-time quantitative PCR (qPCR for detectingToxoplasma gondii DNA in the peripheral blood and aqueous humor of patients with toxoplasmic active focal necrotizing retinochoroiditis. Methods: Fifty-five patients with infectious uveitis seen from 2009 to 2013 at the Department of Ophthalmology and Visual Sciences of the Federal University of São Paulo were enrolled in this study. Forty-three patients had toxoplasmic active focal necrotizing retinochoroiditis, and the remaining 12 had non-toxoplasmic infectious uveitis and served as controls. qPCR analysis forT. gondii DNA was performed on the patients' peripheral blood and aqueous humor samples. Results: The qPCR was positive for T. gondii DNA in 37.21% (16/43 of the aqueous humor samples and 2.33% (1/43 of the peripheral blood samples; further, 16.27% (7/43 of the patients had positive results in both their blood and aqueous humor samples. Conclusion: qPCR was able to detect T. gondii DNA in patients with toxoplasmic active focal necrotizing retinochoroiditis in the blood as well as the aqueous humor and can help with the diagnosis of the disease.
Full Text Available Aim: To study the incidence of S.enteritidis in poultry and meat samples by cultural and PCR methods. Materials and Methods: A total of 130 samples (25 each of chicken, mutton, poultry faeces, cloacal samples and 10 each of liver, spleen and kidney collected from different sources were subjected to cultural and PCR methods for the presence of Salmonella and Salmonella enteritidis. Primers for invA and sefA gene were used for Salmonella and S.enteritidis respectively. Results: Out of 130 samples, 87 were positive for Salmonella spp. i.e. chicken-16(64%, mutton-12(48%, faeces-23(92%, cloacal swabs-23(92%, liver-5(50%, spleen and kidney samples-4(40% each by PCR methods, whereas 77 were positive by cultural method i.e. chicken-14(56%, mutton-10(40%, faeces-22(88%, cloacal swabs-21(84%, liver-4(40%, spleen and kidney-3(30% each. Out of 87 positive for Salmonella by PCR method, 59(chicken-12, mutton-7, faeces-17, cloacal swabs-15, liver-3, spleen-2, kidney-3 were positive for S.enteritidis. High incidence of S.enteritidis (68% in all the above samples are indicative of unhygienic conditions in poultry farms. Selective enrichment with Rappaport-Vassilidias (RV broths and Tetrathionate (TT broths were superior over Selenite-F (SF and Selenite cysteine (SC broths. Conclusions: High incidence of S.enteritidis was seen in most of poultry samples like chicken, kidney, liver and it's faeces than mutton, which was indicative of contamination of S.enteritidis is more prevalent in poultry farms. [Vet World 2012; 5(9.000: 541-545
Johansen, Pernille; Vindeløv, Jannik; Arneborg, Nils; Brockmann, Elke
Although the strain composition of mixed cultures may hugely affect production of various fermented foods, such as e.g. cheese, tools for investigating it have so far been limited. In this study, two new approaches for quantification of seven Lactococcus lactis subsp. cremoris strains (S1-S7) in a defined mixed-strain starter culture were developed and verified. By mapping NGS reads from 47 sequenced L. lactis strains to de novo assembly contigs of the seven strains, two strain-specific sequence regions (SEQ1 and SEQ2) were identified for each strain for qPCR primer design (A1 and A2). The qPCR assays amplified their strain-specific sequence region target efficiently. Additionally, high reproducibility was obtained in a validation sample containing equal amounts of the seven strains, and assay-to-assay coefficients of variance (CVs) for six (i.e. S1, S2, S4-S7) of the seven strains correlated to the inter-plate CVs. Hence, at least for six strains, the qPCR assay design approach was successful. The metagenomics-based approach quantified the seven strains based on average coverage of SEQ1 and SEQ2 by mapping sequencing reads from the validation sample to the strain-specific sequence regions. Average coverages of the SEQ1 and SEQ2 in the metagenomics data showed CVs of ≤17.3% for six strains (i.e. S1-S4, S6, S7). Thus, the metagenomics-based quantification approach was considered successful for six strains, regardless of the strain-specific sequence region used. When comparing qPCR- and metagenomics-based quantifications of the validation sample, the identified strain-specific sequence regions were considered suitable and applicable for quantification at a strain level of defined mixed-strain starter cultures.
Archibald, Lennox K; Pallangyo, Kisali; Kazembe, Peter; Reller, L Barth
We conducted retrospective, comparative analyses of contamination rates for cultures of blood obtained in the emergency rooms of Muhimbili National Hospital (MNH) in Dar es Salaam, Tanzania; Lilongwe Central Hospital (LCH) in central Malawi; and the Duke University Medical Center (DUMC) in the United States. None of the emergency room patients had indwelling intravascular devices at the time that the blood samples for cultures were obtained. In addition, we reviewed the contamination rates for a cohort of patients already hospitalized in the DUMC inpatient medical service, most of whom had indwelling intravascular devices. The bloodstream infection rates among the patients at MNH (n=513) and LCH (n=486) were similar (approximately 28%); the contamination rates at the two hospitals were 1.3% (7/513) and 0.8% (4/486), respectively. Of 54 microorganisms isolated from cultures of blood collected in the DUMC emergency room, 26 (48%) were identified as skin contaminants. Cultures of blood collected in the DUMC emergency room were significantly more likely to yield growth of contaminants than the cultures of blood collected in the emergency rooms at MNH and LCH combined (26/332 versus 11/1,003; Pblood cultures, lower contamination rates were observed when skin was disinfected with isopropyl alcohol plus tincture of iodine rather than isopropyl alcohol plus povidone-iodine. In conclusion, blood culture contamination was minimized in sub-Saharan African hospitals with substantially limited resources through scrupulous attention to aseptic skin cleansing and improved venipuncture techniques. Application of these principles when blood samples for culture are obtained in U.S. hospital emergency rooms should help mitigate blood culture contamination rates and the unnecessary microbiology workup of skin contaminants.
De Gregorio, Eliana; Roscetto, Emanuela; Iula, Vita Dora; Martinucci, Marianna; Zarrilli, Raffaele; Di Nocera, Pier Paolo; Catania, Maria Rosaria
Acinetobacter baumannii is a multidrug-resistant pathogen associated with severe infections in hospitalized patients, including pneumonia, urinary and bloodstream infections. Rapid detection of A. baumannii infection is crucial for timely treatment of septicemic patients. The aim of the present study was to develop a specific marker for a quantitative polymerase chain reaction (PCR) assay for the detection of A. baumannii. The target gene chosen is the biofilm-associated protein (bap) gene, encoding a cell surface protein involved in biofilm formation. The assay is specific for A. baumannii, allowing its discrimination from different species of Acinetobacter and other clinically relevant bacterial pathogens. The assay is able to detect one genomic copy of A. baumannii, corresponding to 4 fg of purified DNA, and 20 colony-forming units/ml using DNA extracted from spiked whole blood samples.
Kanno, Syu-ichi; Tomizawa, Ayako; Yomogida, Shin
Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. Drug-induced liver injury from agents such as APAP is known to vary between individuals within a species. To avoid liver injury and ensure the proper use of pharmaceutical products, it is important to be able to predict such risks using genetic information. This study evaluated the use of quantitative real-time polymerase chain reaction (RT-qPCR) to identify mRNAs (carried in the blood of male ddY mice) capable of predicting susceptibility to APAP-induced hepatotoxicity. Screening was performed on samples obtained at 18 h after treatment from mice that had been orally treated with 500 mg/kg APAP. APAP-induced hepatotoxicity was seen in 60% of the mice, and the mortality rate was 12%. Blood APAP concentration did not differ significantly between mice with and without APAP-induced hepatotoxicity. We compared blood mRNA expression levels between mice with (positive, serious or lethal injury) and without hepatotoxicity in the APAP-treated group. The transcript levels of interleukin-encoding loci Il1β, Il10, and tumor necrosis factor (Tnf) were increased in the lethal injury group. Transcripts of the loci encoding transthyretin (Ttr) and metallothionein 1 (Mt1) showed increases in the liver injury group, while those of the glutathione peroxidase 3-encoding locus (Gpx3) were decreased. APAP hepatotoxicity was potentiated in fasted animals, although fasting did not appear to affect the level of expression of these genes. These results indicate that mRNA expression of Il1β, Il10, Tnf, Ttr, Mt1, and Gpx3 in mouse blood may provide useful surrogate markers of APAP-induced hepatotoxicity.
Objective:To explore the clinical signiifcance of apoptotic inhibitor gene Survivin in peripheral blood of patients with esophageal cancer. Methods:Real-time lfuorescence quantitative PCR was used to detect the expression of Survivin mRNA in peripheral blood of 93 patients with benign and malignant esophageal lesions. The relationship of Survivin mRNA expression and clinicopathologic feature was observed. Results:The expression of Survivin mRNA in peripheral blood which was associated with differentiated degree and clinical staging was progressively increased from benign lesion to carcinoma in situ and invasive carcinoma. Conclusion:The expression of Survivin mRNA in peripheral blood is significantly related to the genesis and progression of esophageal carcinoma. Real-time fluorescence quantitative PCR used to detect the expression of Survivin m-RNA in peripheral blood may be more convenient for diagnosing and guiding the treatment of esophageal carcinoma.
目的 通过PCR-SSP基因技术检测胎儿羊水细胞ABO血型基因型,产前诊断胎儿ABO血型.方法 选取了6名孕16 W以上的孕妇,抽取羊水细胞并进行分离,提取羊水细胞DNA,运用PCR-SSP技术分析其ABO血型基因型,并通过出生后的脐带血的血型鉴定进行确认.结果 6例羊水标本均通过PCR-SSP方法检测出了ABO血型的基因型;该6名胎儿的脐带血的ABO血型与羊水细胞的血型一致.结论 PCR-SSP技术可以准确地检测胎儿羊水细胞的ABO血型.%Objective To identify fetal ABO blood groups by analyzing amniotic fluid cells via PCR-SSP. Methods Six pregnant women with gestational age between 16 weeks and 25 weeks were selected. DNA was taken in amniotic fluid cells from these pregnant women and then ABO genotypes of amniotic fluid cells were detected by PCR-SSP. And the ABO genotypes were conformed by detecting the cord blood . Results The ABO genotypes were identified from the 6 samples of amniotic fluid cells, and the results were in accordance with cord blood. Conclusion Fetal ABO blood group can be accurately detected out by amniotic fluid cells.
Jindai, K; Strerath, M S; Hess, T; Safdar, N
Data on the clinical outcomes of patients with a single compared with multiple positive blood cultures for Enterococcus species is limited. We undertook a retrospective cohort study in adults with at least one positive blood culture for Enterococcus species in a single institution. Clinical outcomes included death and elimination of infection. We included 471 positive blood cultures from 206 enterococcal positive blood culture episodes in 189 patients. Multiple positive blood cultures for Enterococcus species occurred in 110/206 (53.4 %) episodes; 31.6 % of patients had diabetes mellitus; 42.9 % of patients had solid or hematologic malignancy; 26.5 % of patients were solid organ transplant recipients; hospital-acquired and healthcare-associated acquisition represented 55.3 % and 33.0 % of episodes, respectively. Thirty-five patients died and 110 episodes of enterococcal bloodstream infection were successfully treated. In the multivariable analysis, multiple positive blood cultures were not statistically significantly associated with an increased likelihood of in-hospital death [odds ratio (OR) 1.00, 95 % confidence interval (CI) 0.42-2.40] or elimination (OR 1.41, 95 % CI 0.76-2.64) compared with single positive blood cultures. Hematologic malignancy and diabetes mellitus were independently associated with in-hospital death (OR 2.83, 95 % Cl 1.02-7.82; OR 2.79, 95 % Cl 1.16-6.70, respectively). Infectious disease consultation was associated with a greater likelihood of elimination (OR 2.50, 95 % Cl 1.32-4.72). The clinical outcomes of patients with single versus multiple positive blood cultures with Enterococcus species were similar in our institution. Further studies should examine efficient methods to detect contamination versus true infection.
Motoshima, Maiko; Yanagihara, Katsunori; Morinaga, Yoshitomo; Matsuda, Junichi; Hasegawa, Hiroo; Kohno, Shigeru; Kamihira, Shimeru
Rapid identification of the causative bacteria of sepsis in patients can contribute to the selection of appropriate antibiotics and improvement of patients' prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures. We evaluated a rapid protocol for bacterial identification based on PCR and pyrosequencing of the V1 and V3 regions of the 16S rRNA gene using DNA extracted directly from positive blood culture samples. One hundred and two positive blood culture bottles from 68 patients were randomly selected and the bacteria were identified by phenotyping and pyrosequencing. The results of pyrosequencing identification displayed 84.3 and 64.7 % concordance with the results of phenotypic identification at the genus and species levels, respectively. In the monomicrobial samples, the concordance between the results of pyrosequencing and phenotypic identification at the genus level was 87.0 %. Pyrosequencing identified one isolate in 60 % of polymicrobial samples, which were confirmed by culture analysis. Of the samples identified by pyrosequencing, 55.7 % showed consistent results in V1 and V3 targeted sequencing; other samples were identified based on the results of V1 (12.5 %) or V3 (31.8 %) sequencing alone. One isolate was erroneously identified by pyrosequencing due to high sequence similarity with another isolate. Pyrosequencing identified one isolate that was not detected by phenotypic identification. The process of pyrosequencing identification can be completed within ~4 h. The information provided by DNA-pyrosequencing for the identification of micro-organisms in positive blood culture bottles is accurate and could prove to be a rapid and useful tool in standard laboratory practice.
Background: The mainstay of diagnosis of relapsing fever (RF is demonstration of the spirochetes in Giemsa-stained thick blood smears, but during non fever periods the bacteria are very scanty and rarely detected in blood smears by microscopy. This study is aimed to evaluate the sensitivity of different methods developed for detection of low-grade spirochetemia. Methods: Animal blood samples with low degrees of spirochetemia were tested with two PCRs and a nested PCR targeting flaB, GlpQ, and rrs genes. Also, a centrifuged-based enrichment method and Giemsa staining were performed on blood samples with various degrees of spirochetemia. Results: The flaB-PCR and nested rrs-PCR turned positive with various degrees of spirochetemia including the blood samples that turned negative with dark-field microscopy. The GlpQ-PCR was positive as far as at least one spirochete was seen in 5-10 microscopic fields. The sensitivity of GlpQ-PCR increased when DNA from Buffy Coat Layer (BCL was used as template. The centrifuged-based enrichment method turned positive with as low concentration as 50 bacteria/ml blood, while Giemsa thick staining detected bacteria with concentrations ≥ 25000 bacteria/ml. Conclusion: Centrifuged-based enrichment method appeared as much as 500-fold more sensitive than thick smears, which makes it even superior to some PCR assays. Due to simplicity and minimal laboratory requirements, this method can be considered a valuable tool for diagnosis of RF in rural health centers.
Full Text Available Androgen receptor splice variant V7 (AR-V7 was recently identified as a valuable predictive biomarker in metastatic castrate-resistant prostate cancer. Here, we report a new, sensitive and accurate screen for AR-V7 mRNA expression directly from circulating tumor cells (CTCs: We combined EpCAM-based immunomagnetic CTC isolation using the IsoFlux microfluidic platform with droplet digital polymerase chain reaction (ddPCR to analyze total AR and AR-V7 expression from prostate cancer patients CTCs. We demonstrate that AR-V7 is reliably detectable in enriched CTC samples with as little as five CTCs, even considering tumor heterogeneity, and confirm detection of AR-V7 in CTC samples from advanced prostate cancer (PCa patients with AR-V7 detection limited to castrate resistant disease status in our sample set. Sensitive molecular analyses of circulating tumor cells (CTCs or circulating tumor nucleic acids present exciting strategies to detect biomarkers, such as AR-V7 from non-invasive blood samples, so-called blood biopsies.
Comparison of a quantitative real-time polymerase chain reaction (qPCR) with conventional PCR, bacterial culture and ELISA for detection of Mycobacterium avium subsp. paratuberculosis infection in sheep showing pathology of Johne's disease.
Sonawane, Ganesh G; Tripathi, Bhupendra N
A quantitative real-time PCR (qPCR) assay employing IS900 gene specific primers of Mycobacterium avium subsp. parartuberculosis (MAP) was compared with conventional PCR, bacterial culture and enzyme-linked immunosorbent assay in 38 sheep showing granulomatous enteritis and lymphadenitis with and without demonstration of acid-fast bacilli (AFB). The lesions were classified as multibacillary (MB) (n = 23), which had diffuse granulomatous lesions with abundant AFB, and paucibacillary (PB) (n = 15), which had focal or multifocal granulomatous lesions with few or no AFB. In the multibacillary group (MB), IS900 PCR detected 19 (82.6%), and qPCR detected all 23 (100%) sheep positive for MAP in the intestine and lymph node tissues. In the paucibacillary group (PB), IS900 PCR detected 2 (13.3%), and qPCR detected all 15 (100%) sheep positive for MAP in tissues. When results of both groups were taken together, IS900 PCR detected 21(55.2%), and qPCR detected all 38 (100%) animals positive for MAP genome either in the intestine or lymph node tissues. On Herrold egg yolk medium, tissues of 14 (60.9%) MB and 5 (33.3%) PB sheep were found to be positive for MAP. Out of 27 sheep (PB = 8, MB = 19) tested by an ELISA, 21 (77.7%) were found to be positive for MAP antibody, of which 25% (2/8) and 100% (19/19) sheep were from PB and MB sheep, respectively. Based on the results of the present study, it was concluded that qPCR was a highly sensitive test in comparison to conventional PCR, ELISA and bacterial culture for the diagnosis of paratuberculosis on infected tissues especially from paucibacillary sheep.
HAO Chun; WANG Huan; LIU Qinhua; LI Xudong
The anaerobic ammonium-oxidizing (ANAMMOX) bacteria were enriched from a sequencing batch biofilm reactor (SBBR) biofilm.We successfully developed a quantitative competitive polymerase chain reaction (QC-PCR) system to detect and quantify ANAMMOX bacteria in environmental samples.For QC-PCR system,PCR primer sets targeting 16S ribosomal RNA genes of ANAMMOX bacteria were designed and used.The quantification range of this system was 4 orders of magnitude,from 10~3 to 10~6 copies per PCR,corresponding to the detection limit of 300 target copies per mL.A 312-bp internal standard (IS) was constructed,which showed very similar amplification efficiency with the target amxC fragment (349 bp) over 4 orders of magnitude (10~3-10~6).The linear regressions were obtained with a R~2 of 0.9824 for 10~3 copies,R~2 of 0.9882 for 10~4 copies,0.9857 for 10~5 copies and 0.9899 for 10~6 copies.Using this method,we quantified ANAMMOX bacteria in a shortcut nitrification/denitrification-anammox system which is set for piggery wastewater treatment.
Larsen, Annette Burkhart
of the thesis involves the establishment and characterization of an in vitro BBB models based on primary cells isolated from the rat brain. Co-culture and triple culture models with astrocytes and pericytes were found to be the superior to mono cultured BCECs with respect to many important BBB characteristics...... obstacle for the treatment of central nervous system (CNS) diseases, as many potentially CNS active drugs are unable to reach their site of action within the brain. In vitro BBB models are, therefore, being developed to investigate the BBB permeability of a drug early in its development. The first part....... In the second part of the thesis, the ability of turning BCECs into protein factories is investigated using a non-viral gene carrier. Transfection and protein synthesis of BCECs cultured with confined BBB properties were found to be feasible without disrupting the BBB properties, although it was not possible...
Stewart, Diana; Shieh, Y-Carol; Tortorello, Mary; Kukreja, Ankush; Shazer, Arlette; Schlesser, Joseph
The obligate intracellular pathogen Coxiella burnetii has long been considered the most heat resistant pathogen in raw milk, making it the reference pathogen for determining pasteurisation conditions for milk products. New milk formulations and novel non-thermal processes require validation of effectiveness which requires a more practical method for analysis than using the currently used animal model for assessing Coxiella survival. Also, there is an interest in better characterising thermal inactivation of Coxiella in various milk formulations. To avoid the use of the guinea pig model for evaluating Coxiella survival, an Integrated Cell Culture-PCR (ICC-PCR) method was developed for determining Coxiella viability in milk. Vero cell cultures were directly infected from Coxiella-contaminated milk in duplicate 24-well plates. Viability of the Coxiella in milk was shown by a ≥ 0.5 log genome equivalent (ge)/ml increase in the quantity of IS111a gene from the baseline post-infection (day 0) level after 9-11 d propagation. Coxiella in skim, 2%, and whole milk, and half and half successfully infected Vero cells and increased in number by at least 2 logs using a 48-h infection period followed by 9-d propagation time. As few as 125 Coxiella ge/ml in whole milk was shown to infect and propagate at least 2 logs in the optimised ICC-PCR assay, though variable confirmation of propagation was shown for as low as 25 Coxiella ge/ml. Applicability of the ICC-PCR method was further proven in an MPN format to quantitate the number of viable Coxiella remaining in whole milk after 60 °C thermal treatment at 0, 20, 40, 60 and 90 min.
Background Helicobacter pylori is an important agent of gastroduodenal disease in Africa and throughout the world. We sought to determine an optimum method for genotyping H. pylori strains from children and adults in The Gambia, West Africa. Results Virulence genes were amplified in 127 of 190 cases tested (121 adults and 6 children); each of 60 bacterial cultures, and 116 from DNA extracted directly from biopsies. The proportion of biopsies that were cagA+, the ratio of vacAs1/s2, and vacAm1/m2, and the proportion of mixed strain populations in individual subjects changed with age. Strains lacking virulence cagA and vacA genes and with apparently homogeneous (one predominant strain) infections were more common among infants than adults. Conclusions In order to detect the range of bacterial genotypes harbored by individual patients, direct PCR proved slightly superior to isolation of H. pylori by biopsy culture, but the techniques were complementary, and the combination of both culture and direct PCR produced the most complete picture. The seemingly higher virulence of strains from adult than infant infections in The Gambia merits further analysis. PMID:21507253
Full Text Available Abstract Background Helicobacter pylori is an important agent of gastroduodenal disease in Africa and throughout the world. We sought to determine an optimum method for genotyping H. pylori strains from children and adults in The Gambia, West Africa. Results Virulence genes were amplified in 127 of 190 cases tested (121 adults and 6 children; each of 60 bacterial cultures, and 116 from DNA extracted directly from biopsies. The proportion of biopsies that were cagA+, the ratio of vacAs1/s2, and vacAm1/m2, and the proportion of mixed strain populations in individual subjects changed with age. Strains lacking virulence cagA and vacA genes and with apparently homogeneous (one predominant strain infections were more common among infants than adults. Conclusions In order to detect the range of bacterial genotypes harbored by individual patients, direct PCR proved slightly superior to isolation of H. pylori by biopsy culture, but the techniques were complementary, and the combination of both culture and direct PCR produced the most complete picture. The seemingly higher virulence of strains from adult than infant infections in The Gambia merits further analysis.
GYUCHEOL LEE; CHANHEE LEE; CHANSEUNG PARK; SANGGI JEONG
Objective To identify waterborne enteric viruses in Korean surface water. Methods Integrated cell culture(ICC)-multiplex reverse transcription-polymerase chain reaction (RT-PCR) was simultaneously designed to detect coxsackieviruses (CV), polioviruses (PV), and reoviruses (RV). ICC-multiplex RT-PCR and phylogenetic analysis were conducted using 21 total culturable virus assay (TCVA)-positive sample-inoculated cell cultures. Results CV and RV were detected in 9 samples each, and 3 samples were positive for both CV and RV. PV was not etected in any sample. Molecular phylogenetic analysis of the VPl gene sequences revealed that CV types B2 and B4 redominated in Korean surface water, and the nucleotide sequences of CV type B2 were clustered with those of CVs isolated from China and Japan. The results suggested that the evolution of these viruses occurred in a region-specific manner. Conclusion CV and RV are detectable in Korean surface water, with a predominance of CV type B2, and the evolution of CV type B2 occur in a region-specific manner.
Sabino, E.C.; Lee, T.H.; Montalvo, L.; Nguyen, M.L.; Leiby, D.A.; Carrick, D.M.; Otani, M.M.; Vinelli, E.; Wright, D.; Stramer, S.L.; Busch, M.
Background The clinical significance of anti-T. cruzi low-level reactive samples is incompletely understood. PCR-positive rates and antibody levels among seropositive blood donors in three countries are described. Methods Follow-up whole blood and plasma samples were collected from T. cruzi-seropositive donors from 2008-2010 in the US (n=195) and Honduras (n=58). Also 143 samples from Brazil in 1996-2002, originally positive by three serological assays, were available and paired with contemporary follow-up samples from these donors. All samples were retested with the FDA-approved Ortho ELISA. PCR assays were performed on coded sample panels by two laboratories (BSRI and ARC) that amplified kinetoplast minicircle DNA sequences of T. cruzi. Results PCR testing at BSRI yielded slightly higher overall sensitivity and specificity (33% and 98%) compared with the ARC lab (28% and 94%). Among seropositive donors, PCR-positive rates varied by country (p<0.0001) for the BSRI laboratory: Brazil (57%), Honduras (32%) and the US (14%). ELISA signal/cutoff (S/CO) ratios were significantly higher for PCR-positive compared to PCR-negative donors (p<0.05 for all comparisons). Additionally, PCR-negative Brazilian donors exhibited greater frequencies of antibody decline over time versus PCR-positive donors (p=0.003). Conclusion For all three countries, persistent DNA positivity correlated with higher ELISA S/CO values, suggesting that high-level seroreactivity reflects chronic parasitemia. The higher rate of PCR positivity for Brazilian donors was likely attributable to required reactivity on three assays available a decade ago. Significant S/CO declines in 10% of the PCR-negative Brazilian donors may indicate seroreversion following parasite clearance in the absence of treatment. PMID:23002996
Sudan, Vikrant; Shanker, Daya; Jaiswal, Amit; Singh, Amit; Pandey, Vijay
Bovine Tropical Theileriosis (BTT) is an important vector-borne protozoan disease that imposing serious constraints on the health and productivity of domestic cattle. It is matter of common fact that following recovery from primary infection, cattle become persistent carriers and act as reservoirs of infection thereby, playing a critical role in disease epidemiology. The present study describes the comparative diagnostic efficiency of simplex PCR, duplex PCR and RAPD assays for detection of Theileria annulata in cattle. An optimized simple PCR and duplex PCR assay were established using TAMS F/R as primer sets encoding for 721 bp amplicon alongside a RAPD with arbitrary primer coding for 963 bp product of T. annulata. The simple PCR and duplex PCR detected pathogen with almost same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA of another pathogen without nonspecific amplifications. RAPD failed to give comparable results and suffered from limitations of sensitivity as well as specificity. The developed assays may be seen as a good tool for epidemiological studies aiming at assessing the burden of chronic infections and improving control of the associated diseases in endemic regions.
Soetens, Oriane; Vauloup-Fellous, Christelle; Foulon, Ina; Dubreuil, Pascal; De Saeger, Ben; Grangeot-Keros, Liliane; Naessens, Anne
Two protocols for the extraction of cytomegalovirus (CMV) DNA and two methods for the amplification of CMV DNA in dried blood spots were evaluated for the retrospective diagnosis of congenital CMV infection. During the period from 1996 to 2006, a urine screening program detected 76 congenitally infected neonates. Stored Guthrie cards with blood from 55 cases and 12 controls were tested. Two spots of dried blood were cut from each card and evaluated in two centers. CMV DNA was extracted from a whole single spot. Center 1 used phenol-chloroform extraction and ethanol precipitation followed by a conventional PCR. Center 2 used the NucliSens easyMAG automated DNA/RNA extraction platform (bioMérieux) followed by a real-time PCR. For evaluation of the extraction method, DNA extracted from each blood spot was evaluated by the amplification method used by the collaborating center. The sensitivities were 66% for center 1 and 73% for center 2. None of the controls were positive. A sensitivity as high as 82% could be obtained by combining the most sensitive extraction method (the phenol-chloroform procedure) with the most sensitive PCR method (real-time PCR). The detection rate was not influenced by the duration of storage of the spots. The sensitivity was higher with blood from congenitally infected cases due to a primary maternal CMV infection, regardless of the protocol used. However, the difference reached significance only for the least-sensitive protocol (P = 0.036).
Perkins, S E; Yan, L L; Shen, Z; Hayward, A; Murphy, J C; Fox, J G
Helicobacter pylori causes gastritis and peptic ulcers and is linked to gastric cancer. Domestic cats from a commercial source were found to be naturally infected with H. pylori, and studies were undertaken to eradicate H. pylori from infected cats by using triple antimicrobial therapy. Eight cats infected with H. pylori were used in the study. Six cats received a 21-day course of oral amoxicillin, metronidazole, and omeprazole, and two cats served as controls. Two weeks and 4 weeks posttreatment (p.t.), all six treated cats were negative at several sites (saliva, gastric juice, and gastric mucosa) for H. pylori by culture. However, as determined by PCR with primers specific for the 26-kDa product, the majority of cats at 2 and 4 weeks p.t. had gastric fluid samples which were positive for H. pylori and three of three cats at 2 weeks p.t. had dental plaque which was positive for H. pylori. At 6 weeks p.t., all six cats had H. pylori-negative cultures for samples from several gastric sites taken at necropsy, and only one cat had H. pylori cultured from gastric juice. PCR analysis revealed that five of six cats had H. pylori DNA amplification products from plaque, saliva, and/or gastric fluid samples. Negative bacterial cultures for cats for which there was demonstrable PCR amplification of H. pylori DNA may reflect the inability of in vitro culture techniques to isolate small numbers of H. pylori organisms, focal colonization at sites not cultured, or a failure of the antibiotics to successfully eradicate H. pylori from extragastric sites which allowed subsequent recolonization of the stomach after cessation of therapy. Alternatively, the treatment strategy may have induced in vivo viable but nonculturable coccoid forms of H. pylori. The H. pylori cat model should allow further studies to test these hypotheses as well as the efficacies of other combined therapeutic regimens. Also, because 100% of these cats were naturally infected with H.pylori, this model should
Gutiérrez, Ricardo; Morick, Danny; Gross, Ifat; Winkler, Ronen; Abdeen, Ziad; Harrus, Shimon
To determine the occurrence of feline bartonellosis in Israel, blood samples were collected from 179 stray and 155 domestic cats from 18 cities or villages in central and northcentral Israel. Samples were screened for Bartonella infection by culture isolation and molecular detection using high-resolution melt (HRM) real-time PCR assay targeting the 16S-23S rRNA internal transcribed spacer (ITS). All positive samples were confirmed by two additional HRM real-time PCR assays targeting two fragments of the β-subunit of RNA polymerase (rpoB) and the 16S rRNA genes. The prevalence of Bartonella spp. infection in the general tested population was 25.1% (84/334). A higher prevalence was detected in the stray (30.7%; 55/179) than the domestic cats (18.7%; 29/155). Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae were highly prevalent in both cat populations, however their distribution among the two populations varied significantly (p=0.016). B. clarridgeiae and B. koehlerae were found to be more prevalent in stray than domestic cats, whereas B. henselae was evenly distributed. Co-infection with two or more different Bartonella spp. was determined in 2.1% (7) of the cats. The ITS HRM real-time PCR assay used in this study was shown to have a greater screening power than bacterial isolation, detecting 94.0% (79/84) compared to 35.7% (30/84), respectively, of all positive samples. The high prevalence of these zoonotic Bartonella species, coupled with the overpopulation of stray cats, and increased numbers of domestic cats in the major urban centers in Israel represent a significant threat for the public health in this country.
Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.
The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported. This sequence suggests that B. cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.
Feng, Lei; Li, Cai-Xia; Han, Jun-Ping; Xu, Cheng; Hu, Lan
To obtain single-source short tandem repeat (STR) profiles in trace female/male blood mixture samples, we combined florescence in situ hybridization (FISH), laser microdissection, and low volume PCR (LV-PCR) to isolate male/female cells and improve sensitivity. The results showed that isolation of as few as 10 leukocytes was sufficient to yield full STR profiles in fresh female or male blood samples for 32 independent tests with a low additional alleles rate (3.91%) and drop-out alleles rate (5.01%). Moreover, this procedure was tested in two fresh blood mixture series at three ratios (1:5, 1:10, and 1:20), two mock female/male blood mixture casework samples, and one practical casework sample. Male and female STR profiles were successfully detected in all of these samples, showing that this procedure could be used in forensic casework in the future.
Nde, Chantal W; Fakhr, Mohamed K; Doetkott, Curt; Logue, Catherine M
This study was aimed at comparing the ability of conventional culture, the iQ-Check real-time PCR kit, and invA PCR to detect Salmonella in naturally contaminated premarket and retail turkey parts. Premarket (n = 120) turkey parts collected from a commercial turkey processing plant, and retail turkey parts (n = 138) were examined. Both PCR methods detected a significantly greater (P invA PCR for Salmonella detection in the premarket and retail parts, the indices of total agreement were 75.8% (95% CI: 67.2, 83.2) and 84.1% (95% CI: 76.9, 89.7), respectively. The rates of false positives (premarket: 31.9%, retail: 9.7%) and false negatives (premarket: 5.9%, retail: 9.7%) were determined between the culture method and the iQ-Check kit. When invA PCR was compared with the culture method, the rates of false positives (premarket: 37.7%, retail: 11.1%) and false negatives (premarket: 5.9%, retail: 18.3%) were obtained. The higher total agreement and the lower rates of both false positives and false negatives for the iQ-Check kit compared with invA PCR for both premarket and retail turkey parts corroborates the use of the iQ-Check kit as a screening tool for Salmonella in poultry meat.
Hare, K M; Binks, M J; Grimwood, K; Chang, A B; Leach, A J; Smith-Vaughan, H
A PCR for protein D (hpd#3) was used to differentiate nontypeable Haemophilus influenzae (NTHI) from Haemophilus haemolyticus. While 90% of nasopharyngeal specimens and 100% of lower-airway specimens from 84 Indigenous Australian children with bronchiectasis had phenotypic NTHI isolates confirmed as H. influenzae, only 39% of oropharyngeal specimens with phenotypic NTHI had H. influenzae. The nasopharynx is therefore the preferred site for NTHI colonization studies, and NTHI is confirmed as an important lower-airway pathogen.
Abstract Myostatin propeptide (MYOPRO) and follistatin (FOLLI) are potent myostatin inhibitors. In this study we analysed effects of training and androgens on MYOPRO and FOLLI concentrations in blood and skeletal muscle using Immuno PCR. Young healthy males performed either a 3-month endurance-training or a strength-training. Blood and biopsy samples were analysed. Training did not significantly affect MYOPRO and FOLLI concentrations in serum and muscle. To investigate whether tota...
Despite the customization of growth index thresholds within individual laboratories, use of the BACTEC NR 660 automated blood culture system results in a number of false-positive cultures. The results of Gram staining, acridine orange staining, and subculturing to agar media were evaluated on 210 false-positive blood cultures over a 6-month period. Inclusion of acridine orange staining in the routine workup of false-positive blood cultures can eliminate the need for subculturing.
Iris González Morales
Full Text Available Background: community-acquired pneumonia is a major health problem worldwide, in Cuba and in the province of Cienfuegos. Objective: to report the results of blood cultures performed at hospital admission in patients with community-acquired pneumonia. Methods: a prospective descriptive study was conducted in the Gustavo Aldereguía Lima Hospital in Cienfuegos, during the second half of 2012. It included 52 patients with community-acquired pneumonia who underwent blood culture at their admission to the hospital. Results: only five patients (9.6% of the cases had a positive test result; Streptococcus pneumoniae was isolated from only one positive culture; Staphylococcus aureus and Klebsiella pneumoniae were isolated from the rest. Conclusions: the percentage obtained in this study confirms the low diagnostic yield of blood cultures performed at admission in patients with community-acquired pneumonia; the low isolation rate of S. pneumoniae in our study was also significant.
Balkus Jennifer E
Full Text Available Abstract Background The presence of hydrogen peroxide (H2O2 producing Lactobacillus in the vagina may play a role in controlling genital HIV-1 shedding. Sensitive molecular methods improve our ability to characterize the vaginal microbiota; however, they cannot characterize phenotype. We assessed the concordance of H2O2-producing Lactobacillus detected by culture with quantitative PCR (qPCR detection of Lactobacillus species commonly assumed to be H2O2-producers. Methods Samples were collected as part of a prospective cohort study of HIV-1 seropositive US women. Cervicovaginal lavage specimens were tested for L. crispatus and L. jensenii using 16S rRNA gene qPCR assays. Vaginal swabs were cultured for Lactobacillus and tested for H2O2-production. We calculated a kappa statistic to assess concordance between culture and qPCR. Results Culture and qPCR results were available for 376 visits from 57 women. Lactobacilli were detected by culture at 308 (82% visits, of which 233 of 308 (76% produced H2O2. L. crispatus and/or L. jensenii were detected at 215 (57% visits. Concordance between detection of L. crispatus and/or L. jensenii by qPCR and H2O2-producing Lactobacillus by culture was 75% (kappa = 0.45. Conclusions Among HIV-1 seropositive women, there was a moderate level of concordance between H2O2-producing Lactobacillus detected by culture and the presence of L. crispatus and/or L. jensenii by qPCR. However, one-quarter of samples with growth of H2O2-producing lactobacilli did not have L. crispatus or L. jensenii detected by qPCR. This discordance may be due to the presence of other H2O2-producing Lactobacillus species.
Insa, Rosario; Marín, Mercedes; Martín, Adoración; Martín-Rabadán, Pablo; Alcalá, Luís; Cercenado, Emilia; Calatayud, Laura; Liñares, Josefina; Bouza, Emilio
Conventional culture of pleural fluid samples frequently provides false-negative results. Universal polymerase chain reaction (PCR) of the 16S ribosomal ribonucleic acid (rRNA) gene (16S PCR) has proven useful in the diagnosis of various bacterial infections. We conducted a prospective study to assess the value of 16S PCR in the etiologic diagnosis of pleural effusion. All pleural fluid samples received for culture were also studied using 16S PCR. Positive samples were sequenced for identification. Clinical records and conventional culture results were analyzed to classify pleural fluid samples as infected or not infected. We studied 723 samples. We excluded 188 samples because they were obtained from a long-term chest tube, there was a diagnosis of mycobacterial infection, or there were insufficient data to classify the episode. Finally, 535 pleural fluid samples were analyzed. According to our criteria, 82 (15.3%) were infected and 453 (84.7%) were not infected. In the infected samples, 16S PCR was positive in 67 samples (81.7%) while conventional culture was positive in 45 (54.9%). There were 4 false positives with 16S PCR (0.9%) and 12 with culture (2.6%). The values for the etiologic diagnosis of bacterial pleural effusion of conventional culture compared with 16S PCR were as follows: sensitivity, 54.9%/81.7%; specificity, 97.4%/99.1%; positive predictive value, 76.3%/94.4%; negative predictive value, 92.6%/96.8%; and accuracy, 90.8%/96.5%.When compared with conventional culture, 16S PCR plus sequencing substantially improves the etiologic diagnosis of infectious pleural effusion. In our opinion, this technique should be added to the routine diagnostic armamentarium of clinical microbiology laboratories.
Full Text Available Abstract Background previous studies have established that bacterial blood concentration is related with clinical outcome. Time to positivity of blood cultures (TTP has relationship with bacterial blood concentration and could be related with prognosis. As there is scarce information about the usefulness of TTP, we study the relationship of TTP with clinical parameters in patients with Streptococcus pneumoniae bacteremia. Methods TTP of all cases of Streptococcus pneumoniae bacteremia, detected between January 1995 and December 2004 using the BacT/Alert automated blood culture system in a teaching community hospital was analyzed. When multiple cultures were positive only the shortest TTP was selected for the analysis. Results in the study period 105 patients with Streptococcus pneumoniae bacteremia were detected. Median TTP was 14.1 hours (range 1.2 h to 127 h. Immunosuppressed patients (n = 5, patients with confusion (n = 19, severe sepsis or shock at the time of blood culture extraction (n = 12, those with a diagnosis of meningitis (n = 7 and those admitted to the ICU (n = 14 had lower TTP. Patients with TTP in the first quartile were more frequently hospitalized, admitted to the ICU, had meningitis, a non-pneumonic origin of the bacteremia, and a higher number of positive blood cultures than patients with TTP in the fourth quartile. None of the patients with TTP in the 90th decile had any of these factors associated with shorter TTP, and eight out of ten patients with TTP in the 10th decile had at least one of these factors. The number of positive blood cultures had an inverse correlation with TTP, suggesting a relationship of TTP with bacterial blood concentration. Conclusion Our data support the relationship of TTP with several clinical parameters in patients with Streptococcus pneumoniae bacteremia, and its potential usefulness as a surrogate marker of outcome.
Martinez, Raquel M; Bauerle, Elizabeth R; Fang, Ferric C; Butler-Wu, Susan M
The identification of organisms from positive blood cultures generally takes several days. However, recently developed rapid diagnostic methods offer the potential for organism identification within only a few hours of blood culture positivity. In this study, we evaluated the performance of three commercial methods to rapidly identify organisms directly from positive blood cultures: QuickFISH (AdvanDx, Wolburn, MA), Verigene Gram-Positive Blood Culture (BC-GP; Nanosphere, Northbrook, IL), and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with Sepsityper processing (Bruker Daltonics, Billerica, MA). A total of 159 blood cultures (VersaTREK Trek Diagnostic Systems, Cleveland, OH) positive for Gram-positive and Gram-negative bacteria as well as yeast were analyzed with QuickFISH and MALDI-TOF MS. In all, 102 blood cultures were analyzed using the BC-GP assay. For monomicrobial cultures, we observed 98.0% concordance with routine methods for both QuickFISH (143/146) and the BC-GP assay (93/95). MALDI-TOF MS demonstrated 80.1% (117/146) and 87.7% (128/146) concordance with routine methods to the genus and species levels, respectively. None of the methods tested were capable of consistently identifying polymicrobial cultures in their entirety or reliably differentiating Streptococcus pneumoniae from viridans streptococci. Nevertheless, the methods evaluated in this study are convenient and accurate for the most commonly encountered pathogens and have the potential to dramatically reduce turnaround time for the provision of results to the treating physician.
Kongklieng, Amornmas; Thanchomnang, Tongjit; Intapan, Pewpan M; Boonmars, Thidarut; Janwan, Penchom; Sanpool, Oranuch; Lulitanond, Viraphong; Taweethavonsawat, Piyanan; Chungpivat, Sudchit; Morakote, Nimit; Maleewong, Wanchai
Ehrlichia canis is a small pleomorphic gram-negative, coccoid, obligatory intracellular bacterium and the cause of canine monocytic ehrlichiosis. A real-time fluorescence resonance energy transfer polymerase chain reaction (real-time FRET PCR) coupled with melting curve analysis was established for detection of E. canis infection in canine blood samples. The VirB9 gene was amplified using one pair of primers and the melting curve analysis was generated by heating the hybridizing probes and amplified products. Eight E. canis-infected dog blood samples were initially identified using the Giemsa staining/microscopic method followed by conventional PCR (cPCR)/Sanger sequencing for confirmation. The sensitivity and specificity of the real-time FRET PCR detection were 87.5% and 100%, respectively and the limit of detection was 6.6 x 10(3) copies of positive E. canis control plasmids. The real-time FRET PCR with melting curve analysis reported here is better than microscopic visualization or cPCR because the method is not affected by the false bias inherent in the microscopic method. Furthermore, many samples can be processed rapidly at the same time. This convenient tool is beneficial as an alternative assay for the epidemiologic study of canine ehrlichiosis as well as for eradication of these organisms in prevention and control programs in endemic areas.
马艳; 胡必杰; 周春妹; 高晓东; 谢红梅; 黄声雷; 周昭彦; 鲍容
目的 了解送检厌氧血培养瓶对病原菌检出率及阳性结果报告时间的影响.方法 对2011年1月-2012年3月送检的4018份疑似血流感染患者的血培养结果进行统计学分析.结果 同时送检需氧瓶和厌氧瓶的检出率达14.11％,高于仅送检需氧瓶的9.26％,差异有统计学意义(P＜0.05)；厌氧瓶中大肠埃希菌和肠球菌属的阳性结果报告时间(306、630min)明显短于需氧瓶(612、810 min)(P＜0.05)；同时送检需氧瓶和厌氧瓶的病份中,厌氧瓶阳性而需氧瓶阴性者占2.42％,厌氧瓶培养可增加血流感染病原菌检出率达17.11％.结论 增加厌氧瓶培养可以提高阳性率并缩短阳性结果报告时间,临床上要加强厌氧血培养瓶的送检.%OBJECTIVE To evaluate the clinical significance of anaerobic blood culture bottles in the detection rate of the pathogens and the time of positive reports. METHODS The blood culture was statistically analyzed for 4018 patients with suspected blood stream infections submitted from Jan 21011 to Mar 2012. RESULTS The detection rate of the aerobic bottles and anaerobic bottles submitted at the same period was 14. 11%. only higher than 9. 265-6 of the aerobic bottles, the difference was statistically significant (P<0. 05) ; on detecting Escherichia coli and Enterocaccus , anaerobic blood cultures bottles (306,630 min)needed significantly shorter time than did the aerobic blood botiles(612.810 min), (P<0. 05) ; 2. 42% of the cases only were determined positive in anaerobic blood cultures bottles while both aerobic and anaerobic bottles were obtained, the anaerobic blood cultures could increase 17. 11% of the isolation rate of the pathogens causing blood stream infections. CONCLUSION To increase the anaerobic bottle blood cultures may significantly increase the isolation rate and shorten the report time, it is necessary for the hospital to intensify the submission of the anaerobic blood culture bottles.
Brown, D F; Warner, M; Taylor, C E; Warren, R E
A total of 3347 blood cultures from patients in all hospital wards were examined on a Malthus microbiological growth analyser and by a conventional system. There was no significant difference in the total numbers of positive cultures of clinical importance between the two systems (p greater than 0.05). Staphylococcus aureus, however, was isolated more often by the conventional method (p less than 0.05). Failure of the automatic detection routine limited the potential of the Malthus system for earlier detection of positive cultures. Daily visual examination of Malthus curves and subculture of bottles not promptly attached to the apparatus were necessary to avoid missing some positive cultures. False positive rates were 13% for the Malthus system and 2% for the conventional system. The contamination rate was considerably lower in the Malthus system (p less than 0.001). Further development would be necessary for the apparatus to be acceptable for routine screening of blood cultures.
A. A. Al-Sulami
Full Text Available Helicobacter pylori is recognized by the World Health Organization to be the primary cause of peptic ulcers, chronic gastritis, and stomach cancer, though the source of human infection is not well understood. One of the problems in understanding the source of human contamination is the difficulty in isolating the organism from the environment. However, the combination of PCR results with those of culturing of 471 drinking water samples can provide a more accurate picture of H. pylori detection. In this method 78 presumptive H. pylori colonies out of 266 tap water samples were obtained in the preliminary detection on modified Columbia agar (MCUA slant relying on urease positivity with a rate of 29.3%. However, only 11 out of them were confirmed by Gram staining and biochemical tests reducing the rate to 4.13% whereas only 3 (1.46% from 205 reverse osmosis (RO water samples. Furthermore, only 6 (54.5% out of the 11 isolates from tap water and 1 (33.3% of the 3 RO isolates were confirmed by 16SrRNA PCR. Thus PCR confirmation reduced the rate to 2.2%. In addition, only 4 (4% of 100 tap water samples negative for H. pylori by culture method were H. pylori positive by 16SrRNA. Water samples were collected from 24 districts of Basrah Governorate from February–December 2009. The direct recovery of H. pylori from drinking water is both alarming and scientifically exciting in terms of the investigation of its epidemiology.
Segura, Ferran; Pons, Immaculada; Pla, Júlia; Nogueras, María-Mercedes
Murine typhus is a zoonosis transmitted by fleas, whose etiological agent is Rickettsia typhi. Rickettsia felis infection can produces similar symptoms. Both are intracellular microorganisms. Therefore, their diagnosis is difficult and their infections can be misdiagnosed. Early diagnosis prevents severity and inappropriate treatment regimens. Serology can't be applied during the early stages of infection because it requires seroconversion. Shell-vial (SV) culture assay is a powerful tool to detect Rickettsia. The aim of the study was to optimize SV using a real-time PCR as monitoring method. Moreover, the study analyzes which antibiotics are useful to isolate these microorganisms from fleas avoiding contamination by other bacteria. For the first purpose, SVs were inoculated with each microorganism. They were incubated at different temperatures and monitored by real-time PCR and classical methods (Gimenez staining and indirect immunofluorescence assay). R. typhi grew at all temperatures. R. felis grew at 28 and 32 °C. Real-time PCR was more sensitive than classical methods and it detected microorganisms much earlier. Besides, the assay sensitivity was improved by increasing the number of SV. For the second purpose, microorganisms and fleas were incubated and monitored in different concentrations of antibiotics. Gentamicin, sufamethoxazole, trimethoprim were useful for R. typhi isolation. Gentamicin, streptomycin, penicillin, and amphotericin B were useful for R. felis isolation. Finally, the optimized conditions were used to isolate R. felis from fleas collected at a veterinary clinic. R. felis was isolated at 28 and 32 °C. However, successful establishment of cultures were not possible probably due to sub-optimal conditions of samples.
Full Text Available BACKGROUND: Recently, a clone of MRSA with clonal complex 398 (CC398 has emerged that is related to an extensive reservoir in animals, especially pigs and veal calves. It has been reported previously that methicillin-susceptible variants of CC398 circulate among humans at low frequency, and these have been isolated in a few cases of bloodstream infections (BSI. The purpose of this study was to determine the prevalence of S. aureus CC398 in blood cultures taken from patients in a geographic area with a high density of pigs. METHODOLOGY/PRINCIPAL FINDINGS: In total, 612 consecutive episodes of S. aureus BSI diagnosed before and during the emergence of CC398 were included. Three strains (2 MSSA and 1 MRSA that were isolated from bacteremic patients between 2010-2011 were positive in a CC398 specific PCR. There was a marked increase in prevalence of S. aureus CC398 BSI isolated between 2010-2011 compared to the combined collections that were isolated between 1996-1998 and 2002-2005 (3/157, 1.9% vs. 0/455, 0.0%; p = 0.017. CONCLUSIONS/SIGNIFICANCE: In conclusion, in an area with a relative high density of pigs, S. aureus CC398 was found as a cause of BSI in humans only recently. This indicates that S. aureus CC398 is able to cause invasive infections in humans and that the prevalence is rising. Careful monitoring of the evolution and epidemiology of S. aureus CC398 in animals and humans is therefore important.
Huys, Geert; Coopman, Vera; Van Varenbergh, Dirk; Cordonnier, Jan
The detection of autochthonous aquatic bacteria in tissue samples from drowning cases is increasingly considered as an alternative approach to assist the medico-legal diagnosis of death by drowning. Bacteria belonging to the genus Aeromonas may be suitable candidates for this application as they are ubiquitous in natural aquatic environments but are generally not part of the human microbiota. The research aims of this study were (i) to develop a sensitive, specific and rapid screening and confirmation method for Aeromonas species in tissue samples and (ii) to evaluate aseptic sternal puncture as a post-mortem sample technique and bone marrow as an alternative matrix to provide evidence of death by drowning. The presence of Aeromonas in tissue samples was verified by cultivation using the selective media Ampicillin Dextrin Agar (ADA) and Ryan's Aeromonas Medium. The use of ADA medium was found most optimal for the sensitive, inexpensive and quick detection of aeromonads in human tissue samples. Positive culture plates were confirmed by harvesting all colonies for DNA extraction and subsequent PCR amplification using Aeromonas genus-specific primers. Aeromonads were detected in lung swab, blood and bone marrow of drowned bodies (n=3), but were negative in these three matrices for all negative controls (n=90) tested. Bone marrow proved to be a suitable alternative matrix and can be sampled post-mortem by an aseptic sternal puncture. In conclusion, this study confirms previous indications that aeromonads in cultures from blood of water bodies can be considered a potential marker for drowning. Given the fact that the number of immersed bodies (drowned and non-drowned) included in this study is statistically not significant, however, more tissue samples need to be investigated to confirm the validity of these methods to aid the diagnosis of death by wet drowning.
Full Text Available BACKGROUND: Bacteriological confirmation of tuberculous (TB meningitis is difficult. Culture is slow and microscopy has insufficient sensitivity. We evaluated real time PCR targeting insertion sequence IS6110 among 230 consecutive adult patients with subacute meningitis in a referral hospital in Indonesia. METHODS: Cerebrospinal fluid (CSF samples were examined using microscopy, solid and liquid culture, and real time IS6110-PCR with a fluorescence-labeled probe using DNA extracted from CSF. CSF samples from 40 non-infectious neurology patients were used as negative controls. IS6110-PCR results were linked with clinical and CSF characteristics. RESULTS: Most patients presented with subacute meningitis, after a median of 14 days of symptoms (range 7-30. After exclusion of cryptococcal and bacterial meningitis, 207 patients were classified as definite or probable TB meningitis; 17.9% with HIV infection. Among this group IS6110-PCR gave the highest positivity rate (68%, 95% CI 62-74% compared with microscopy of ZN-stained slides (11%, 95% CI 7-15%, and mycobacterial culture using solid (36%, 95% CI 29-42% and liquid (44%, 95% CI 37-51% media. IS6110-PCR was positive in 92% of patients with culture-positive and 42% of patients with culture-negative probable TB meningitis. Among culture-negative patients, a positive PCR was associated with a history of TB treatment, a longer duration of illness, a higher CSF cell count and protein, and a lower CSF glucose. IS6110-PCR was negative in all CSF samples from non-meningitis control patients. CONCLUSIONS: Real time IS6110-PCR is a quick, sensitive, and specific test for diagnosing of TB meningitis in this setting. Its performance in other (less-developed settings needs further study.
Prier Lindvig, Katrine; Nielsen, Stig Lønberg; Henriksen, Daniel P;
: This was a hospital-based cohort study including all adult (≥15 years old) blood-cultured patients at the MED at Odense University Hospital between 1 August 2009 and 31 August 2011. RESULTS: During the study period, 5499/11 988 (45.9%) patients had blood cultures performed within 72 h of arrival and were included.......6 (95% CI 3.6-6.0)], at least two organ failure [HR 3.6 (2.9-4.5)], bacteraemia [HR 1.4 (1.1-1.8)], Charlson Comorbidity Index of at least 2 h [HR 1.7 (1.3-2.0)], SIRS [HR 1.5 (1.2-1.7)], a history of alcohol dependency [HR 1.7 (1.3-2.3)] and late drawing of blood cultures 24-48 h after arrival [HR 1.......7 (1.3-2.2)] were found to be prognostic factors of mortality among blood-cultured patients in the MED. CONCLUSION: Among blood-cultured patients in the MED, we found an 11.0% overall 30-day mortality. Factors associated with 30-day mortality were age more than 80 years, at least two organ failure...
Man Updesh Singh Sachdeva
Full Text Available Background: The conventional cytogenetic approach to demonstrate Philadelphia (Ph chromosome at times does not yield enough number of metaphases or are of suboptimal quality. Further, the rapid molecular tests have completely pushed this simple technique into disrepute. Aims: This study aimed to evaluate usefulness of phytohemagglutinin (PHA-stimulated peripheral blood culture for detection of Ph chromosome in chronic myeloid leukemia (CML patients. Materials and Methods: Fifty-six patients, including 11 newly diagnosed cases of CML and 45 patients of CML on imatinib therapy showing the presence of Ph chromosome in unstimulated samples, were included in the study. Cytogenetic analysis was done on unstimulated samples, i.e. bone marrow aspirate, 24- and 48-h peripheral blood culture, and compared with PHA-stimulated 72-h peripheral blood culture. Results: The preparations from PHA-stimulated peripheral blood culture samples in all 56 patients yielded high number of good-quality metaphases. All the 11 (100% newly diagnosed patients and 39/45 (87% of the patients on imatinib therapy showed the presence of Ph chromosome in PHA-stimulated samples. Addition of PHA-stimulated 72-h peripheral blood culture preparation can be of use for increasing the diagnostic yield in cases of CML with suboptimal results on conventional cytogenetics from bone marrow aspirate sample.
Franklin, Heather A; Stapp, Paul; Cohen, Amybeth
Elucidating feeding relationships between hosts and parasites remains a significant challenge in studies of the ecology of infectious diseases, especially those involving small or cryptic vectors. Black-tailed prairie dogs (Cynomys ludovicianus) are a species of conservation importance in the North American Great Plains whose populations are extirpated by plague, a flea-vectored, bacterial disease. Using polymerase chain reaction (PCR) assays, we determined that fleas (Oropsylla hirsuta) associated with prairie dogs feed upon northern grasshopper mice (Onychomys leucogaster), a rodent that has been implicated in the transmission and maintenance of plague in prairie-dog colonies. Our results definitively show that grasshopper mice not only share fleas with prairie dogs during plague epizootics, but also provide them with blood meals, offering a mechanism by which the pathogen, Yersinia pestis, may be transmitted between host species and maintained between epizootics. The lack of identifiable host DNA in a significant fraction of engorged Oropsylla hirsuta collected from animals (47%) and prairie-dog burrows (100%) suggests a rapid rate of digestion and feeding that may facilitate disease transmission during epizootics but also complicate efforts to detect feeding on alternative hosts. Combined with other analytical approaches, e.g., stable isotope analysis, molecular genetic techniques can provide novel insights into host-parasite feeding relationships and improve our understanding of the role of alternative hosts in the transmission and maintenance of disease.
Javadi, Ahmad; Verduin, Esther P; Brand, Anneke; Schonewille, Henk
The rhesus D blood group, which is expressed on the red blood cells (RBC) of 85% of the Caucasian population, is one of the most immunogenic RBC antigens, inducing D antibody formation in up to 20-80% of D-negative transfusion recipients and about 10% of pregnancies at risk. Pregnancy-induced D-antibodies can persist for many years, but the mechanisms underlying this persistence are unclear. The LOTUS study, a long-term follow-up study of mothers from severely affected children with hemolytic disease of the fetus and newborn investigates, among other endpoints, whether persistent feto-maternal chimerism is associated with long-term maternal anti-D persistence. We questioned which blood sample processing method should be used to detect low levels of RHD chimerism with the highest sensitivity and specificity using qPCR. After optimization of primer and probe concentrations for singleplex RHD exon 5 and 7 qPCR, sensitivity, specificity and efficiency of RHD and DYS1 qPCR were investigated in artificial chimeric samples. Sensitivity of DYS1 was one log higher (0.0001%) in enriched mononuclear cell fractions as compared with whole blood. Comparable linear sensitivity (0.007%) and mean efficiency (84-99%) for RHD qPCR were observed in all samples regardless whether whole blood or pre- or post-mixing of cellular fractions had been used. We conclude that RHD chimerism using singleplex exon 5 and 7 qPCR is linearly detectable down to 1.0 GE, without an advantage of fraction enrichment.
Hall, Leslie; Le Febre, Kara M; Deml, Sharon M; Wohlfiel, Sherri L; Wengenack, Nancy L
The Yeast Traffic Light PNA FISH kit (YTL) correctly identified Candida spp. in 207/216 (96%) positive blood cultures. Discordant results were seen with known cross-reacting species and cultures containing Candida lambica and Rhodotorula mucilaginosa. The YTL provides rapid, reliable identification of the five common Candida species found in blood cultures.
Mærkedahl, Rasmus Baadsgaard; Frøkiær, Hanne; Lauritzen, Lotte
Abstract Background: In large clinical trials, where RNA cannot be extracted immediately after sampling, preserving RNA in whole blood is a crucial initial step in obtaining robust qPCR data. The current golden standard for RNA preservation is costly and designed for time-consuming column-based RNA......-extraction. We investigated the use of lysis buffer for long-term storage of blood samples for qPCR analysis. Methods: Blood was collected from 13 healthy adults and diluted in MagMAX lysis/binding solution or PAXgene Blood RNA tubes and stored at -20 °C for 0, 1, or 4 months before RNA extraction...... by the matching method. RNA integrity, yield and purity were evaluated and the methods were compared by subsequent analyses of the gene expression levels of 18S, ACTB, IL1B, IL1RN, IL1R2, and PGK1 using qPCR. Results: The MagMAX system extracted 2.3-2.8 times more RNA per mL blood, with better performance...
Segura, Ferran; Pons, Immaculada; Sanfeliu, Isabel; Nogueras, María-Mercedes
Rickettsia conorii and Rickettsia massiliae-Bar29 are related to Mediterranean spotted fever (MSF). They are intracellular microorganisms. The Shell-vial culture assay (SV) improved Rickettsia culture but it still has some limitations: blood usually contains low amount of microorganisms and the samples that contain the highest amount of them are non-sterile. The objectives of this study were to optimize SV culture conditions and monitoring methods and to establish antibiotic concentrations useful for non-sterile samples. 12 SVs were inoculated with each microorganism, incubated at different temperatures and monitored by classical methods and real-time PCR. R. conorii was detected by all methods at all temperatures since 7th day of incubation. R. massiliae-Bar29 was firstly observed at 28°C. Real-time PCR allowed to detected it 2-7 days earlier (depend on temperature) than classical methods. Antibiotics concentration needed for the isolation of these Rickettsia species from non-sterile samples was determined inoculating SV with R. conorii, R. massiliae-Bar29, biopsy or tick, incubating them with different dilutions of antibiotics and monitoring them weekly. To sum up, if a MSF diagnosis is suspected, SV should be incubated at both 28°C and 32°C for 1-3 weeks and monitored by a sensitive real-time PCR. If the sample is non-sterile the panel of antibiotics tested can be added.
Morii, Daiichi; Yokozawa, Takayuki; Ichinose, Naoki; Oda, Toshimi
The blood culture contamination rate is often used to validate specimen-collection procedures. CUMITECH has set its optimal target to be 2% to 3%. However, the term "contamination rate" has been defined in many ways, limiting its generalizability. The definitions used in earlier studies can be divided into two categories; definitions based on clinical judgements, and those based on preset rules. According to each principle, the equation must be composed of a defined numerator and denominator. The problem with clinical definitions is that the decision is inevitably subjective, and the process is too cumbersome. Also, if the number of positive cultures is used as the denominator, the value would be equivalent to the positive predictive value, given that contamination is regarded as a "positive case." Thus, the value would not be useful for validating a procedure. On the other hand, when the preset algorithm was adopted, true infection would, to some degree, inevitably be classified as contamination. Also, if the algorithm adopted the number of blood culture sets as the denominator and contamination was defined as the identification of 1 or more specified organisms in only 1 of multiple sets of blood cultures, its theoretical maximum value would not be 100%. This is a problem because the value is a mixture of several numbers with different scales. In other words, whether the blood cultures are collected once, twice, or thrice or more a day would affect the result. The study cited by CUMITECH aimed to evaluate the equivalence between the clinical definition and the laboratory definition with preset rules, rather than to establish a benchmark for the contamination rate. It is undesirable for the number to be perceived as a benchmark. "A Guide to Blood Culture" (2013) by the Japanese Society for Clinical Microbiology introduced a calculation for the contamination rate, but the definition of the term "number of specimens" in the formula is ambiguous. In addition, the
Loonen, A J M; Jansz, A R; Kreeftenberg, H; Bruggeman, C A; Wolffs, P F G; van den Brule, A J C
To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit M(Grade)), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay.
Saegeman, V; Verhaegen, J; Lismont, D; Verduyckt, B; De Rijdt, T; Ectors, N
Tissue banks provide tissues of human cadaver donors for transplantation. The maximal time limit for tissue retrieval has been set at 24 h postmortem. This study aimed at evaluating the evidence for this limit from a microbiological point of view. The delay of growth in postmortem blood cultures, the identification of the species isolated and clinical/environmental factors were investigated among 100 potential tissue donors. No significant difference was found in the rate of donors with grown blood cultures within (25/65=38%) compared with after (24/65=37%) 24 h of death. Coagulase-negative staphylococci and gastro-intestinal microorganisms were isolated within and after 24 h of death. Two factors--antimicrobial therapy and "delay before body cooling"--were significantly inversely related with donors' blood culture results. From a microbiological point of view, there is no evidence for avoiding tissue retrieval among donors after 24 h of death.
Schnell, David; Lécuyer, Hervé; Geeraerts, Thomas; Dumenil, Anne-Sylvie; Bille, Emmanuelle; Mercier, Frédéric J; Benhamou, Dan; Zahar, Jean-Ralph
Evaluating the clinical significance of blood cultures positive for coagulase-negative staphylococci (CoNS) is of critical importance since these microorganisms represent both the first contaminants of blood cultures and one of the leading causes of bloodstream infection (BSI). This prospective 2-centre study aimed to compare a previously reported algorithm to a clinical algorithm based on our experience. We identified 84 patients with CoNS-positive blood cultures. Twenty-seven (32%) were considered to have BSI according to our study algorithm. Thirty-seven (44%) patients were considered to have CoNS BSI according to the previously reported algorithm. The 2 algorithms isolated patients with similar rates of recurrences and hospital mortality. Our algorithm seemed to result in less diagnoses of CoNS BSI without harmful consequences compared to the previously reported algorithm. The impact on patient outcome and the inappropriate use of antibiotics deserves further investigation.
王海; 崔大伟; 杨悦
目的 比较直接测序法和实时聚合酶链反应-高分辨率融解曲线技术(qPCR-HRM)检测非小细胞肺癌(NSCLC)表皮生长因子受体(EGFR)基因突变的差异,探讨适用于临床的EGFR基因突变检测方法.方法 52例NSCLC患者外周血中EGFR突变采用qPCR-HRM法,其组织标本中EGFR突变采用直接测序法,采用卡方检验比较检测结果.结果 qPCR-HRM法检测外周血中EGFR突变率为34.6％ (18/52),直接测序法检测的组织标本中突变率为30.8％ (16/52),结果差异无统计学意义(x2=0.17,P＞0.05);2种方法的总符合率为96.2％ (50/52).结论 与直接测序法相比,qPCR-HRM法检测NSCLC患者外周血中EGFR突变更适用于临床筛选适合EGFR酪氨酸激酶抑制剂(EGFR-TKI)治疗的NSCLC患者.
Full Text Available Background: Bacteremia due to Enterococcus faecalis is usually caused by strains resistant to most antibiotics. Effective management of the disease is dependent on rapid detection and characterization of the bacteria, and determination its sensitivity pattern to antimicrobial drugs. The aim of this study was to investigate a more rapid and reliable assay for simultaneous diagnosis of enterococcal bacteremia and its sensitivity pattern to antimicrobial drugs. Methods: Several bacterial suspensions with different content of two standard strains of Enterococcus faecalis resistant to vancomycin were used for inoculation to defibrinated sheep blood samples. PCR and routine assay was performed on all blood samples with different bacterial content.Results: Routine assay and PCR for all inoculated blood samples with ≥5 cfu/ml was positive. Mean time for PCR and routine assays was 10 hours and 5 days, respectively. Conclusion: PCR is a more rapid and sensitive assay for simultaneous detection and characterization for Enterococcus faecalis, and determination of its sensitivity pattern to vancomycin.
Full Text Available The blood culture is a very important laboratory test: if bacteremia or sepsis are suspected, the diagnosis of the pathogen and antibiotic therapy may be achieved making use of it. Identification and antibiotic susceptibility test carried out directly from the bottle may give important information in a shorter time. The introduction of the automatic instrumentation has improved the discovering of pathogens in the blood, however the elapsing time between the positive detection and the microbiological report is still along.The aim of our work was to verify the validity of the direct use of blood culture broth in which growth of microorganisms has been detected, which could reduce the response time of the bacteremia diagnosis. During the period February - July 2009, a total of 150 blood cultures were analysed:we compared the results obtained both by direct method and by reference method. 20 Gram positive microrganisms and 13 Gram negative microrganisms were respectively isolated and identified. The identification of Gram-negative and Gram-positive microrganisms showed an agreement of 100% between the direct and the reference method. For antibiotic susceptibility tests, among the Gram positive has reported 1.3% very major error, 2.9% major error and 1.4% minor error, while the Gram negative, respectivety 0.3%, 1.4%, 0%. The use of direct identification and susceptibility testing from positive blood cultures, can improve the response time and better efficiency in diagnostic procedures.
Lund, Lars Christian; Sydenham, Thomas Vognbjerg; Høgh, Silje Vermedal;
"Terrisporobacter othiniensis" (proposed species) was isolated from a blood culture. Genomic DNA was sequenced using a MiSeq benchtop sequencer (Illumina) and assembled using the SPAdes genome assembler. This resulted in a draft genome sequence comprising 3,980,019 bp in 167 contigs containing 3,......,449 coding sequences, 7 rRNAs, and 58 tRNAs......."Terrisporobacter othiniensis" (proposed species) was isolated from a blood culture. Genomic DNA was sequenced using a MiSeq benchtop sequencer (Illumina) and assembled using the SPAdes genome assembler. This resulted in a draft genome sequence comprising 3,980,019 bp in 167 contigs containing 3...
During an 8-month period at Children's Hospital, Oakland, Calif., a 9% rate for positive blood culture for children with Neisseria meningitidis meningitis was identified. The blood culture system used in each case was the BACTEC NR 730. This rate seemed significantly lower than previous rates (33 to 55%) (P.R. Dodge and M.N. Swartz, N. Engl. J. Med. 272:1003-1010, 1965; A.L. Hoyne and R.H. Brown, Ann. Intern. Med. 28:248-259, 1948; S. Levin and M.B. Painter, Ann. Intern. Med. 64:1049-1057, 19...
Hoorieh Saderi; Ali akbar Karimi; Marzieh Loni
Introduction: Determining frequency of bacteria, isolated from blood culture and their antibiotic susceptibility patterns, has epidemiological significance and can help in selecting empirical therapy. This study was aimed to assess, the frequency of bacteria isolated from blood culture of patients suspected to bacteremia and their antibiotic susceptibility patterns. Methods: Culture of blood and determination of antibiotic susceptibility was done by standard methods. In this study, a variety ...
RT-PCR em pools de soros sangüíneos para o diagnóstico da infecção aguda e de animais persistentemente infectados pelo vírus da diarréia viral bovina RT-PCR in pools of bovine blood serum to detect acute infection and persistently infected animals with bovine viral diarrhea virus
of serum from groups D and H resulted in positive reactions in serum samples from 11 cows and 12 calves. For the identification of persistently infected (PI animals, three months after the first examination, blood serum samples from 23 positive animals were reevaluated by RT-PCR, resulting in five positive calves. In two of these calves the BVDV was isolated in MDBK cell culture. The specificity of RT-PCR amplicons from one cow with acute infection, one PI calf, and two wild type BVDV strains isolated in cell culture were confirmed by nucleotide sequencing. The use of RT-PCR in pools of blood sera proved to be a quick and low cost strategy for the etiological diagnosis of the acute infection as well as to detect PI animals thereby favoring the implementation of control and prophylaxis measures.
Birkenheuer, Adam J; Levy, Michael G; Breitschwerdt, Edward B
Canine babesiosis has recently been recognized as an emerging infectious disease of dogs in North America. We sought to develop a seminested PCR to detect and differentiate Babesia gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. canis, and B. canis subsp. rossi DNA in canine blood samples. An outer primer pair was designed to amplify an approximately 340-bp fragment of the 18S rRNA genes from B. gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. rossi, and B. canis subsp. canis but not mammalian DNA. Forward primers were designed that would specifically amplify a smaller fragment from each organism in a seminested PCR. The practical limit of detection was 50 organisms/ml of mock-infected EDTA anticoagulated whole blood. The primer pair also amplified an approximately 370-bp fragment of the B. gibsoni (USA/California genotype) 18S rRNA gene from the blood of an experimentally infected dog with a high percentage of parasitemia. Amplicons were not detected when DNA extracted from the blood of a dog that was naturally infected with Theileria annae at a low percentage of parasitemia was amplified. Due to limited sensitivity, this test is not recommended for the routine diagnosis of B. gibsoni (USA/California genotype) or T. annae. The PCR test did not amplify Toxoplasma gondii, Neospora caninum, Leishmania infantum, Cryptosporidium parvum, or canine DNA under any of the conditions tested. The seminested PCR test was able to detect and discriminate B. gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. canis, and B. canis subsp. rossi DNA in blood samples from infected dogs.
Heinz, Stefan; Braspenning, Joris
An important function of the liver is the synthesis and secretion of blood coagulation factors. Within the liver, hepatocytes are involved in the synthesis of most blood coagulation factors, such as fibrinogen, prothrombin, factor V, VII, IX, X, XI, XII, as well as protein C and S, and antithrombin, whereas liver sinusoidal endothelial cells produce factor VIII and von Willebrand factor. Here, we describe methods for the detection and quantification of most blood coagulation factors in hepatocytes in vitro. Hepatocyte cultures indeed provide a valuable tool to study blood coagulation factors. In addition, the generation and expansion of hepatocytes or hepatocyte-like cells may be used in future for cell-based therapies of liver diseases, including blood coagulation factor deficiencies.
Laura Audrey Hug
Full Text Available Reductive dehalogenases are the critical enzymes for anaerobic organohalide respiration, a microbial metabolic process that has been harnessed for bioremediation efforts to resolve chlorinated solvent contamination in groundwater and is implicated in the global halogen cycle. Reductive dehalogenase sequence diversity is informative for the dechlorination potential of the site or enrichment culture. A suite of degenerate PCR primers targeting a comprehensive curated set of reductive dehalogenase genes was designed and applied to twelve DNA samples extracted from contaminated and pristine sites, as well as six enrichment cultures capable of reducing chlorinated compounds to non-toxic end-products. The amplified gene products from four environmental sites and two enrichment cultures were sequenced using Illumina HiSeq, and the reductive dehalogenase complement of each sample determined. The results indicate that the diversity of the reductive dehalogenase gene family is much deeper than is currently accounted for: one-third of the translated proteins have less than 70% pairwise amino acid identity to database sequences. Approximately 60% of the sequenced reductive dehalogenase genes were broadly distributed, being identified in four or more samples, and often in previously sequenced genomes as well. In contrast, 17% of the sequenced reductive dehalogenases were unique, present in only a single sample and bearing less than 90% pairwise amino acid identity to any previously identified proteins. Many of the broadly distributed reductive dehalogenases are uncharacterized in terms of their substrate specificity, making these intriguing targets for further biochemical experimentation. Finally, comparison of samples from a contaminated site and an enrichment culture derived from the same site eight years prior allowed examination of the effect of the enrichment process.
Jung, Yu Jung; Kim, Ji-Youn; Song, Dong Joon; Koh, Won-Jung; Huh, Hee Jae; Ki, Chang-Seok; Lee, Nam Yong
We evaluated the analytical performance of M. tuberculosis complex (MTBC)/nontuberculous mycobacteria (NTM) PCR assays for differential identification of MTBC and NTM using culture-positive liquid media. Eighty-five type strains and 100 consecutive mycobacterial liquid media cultures (MGIT 960 system) were analyzed by a conventional PCR assay (MTB-ID(®) V3) and three real-time PCR assays (AdvanSure™ TB/NTM real-time PCR, AdvanSure; GENEDIA(®) MTB/NTM Detection Kit, Genedia; Real-Q MTB & NTM kit, Real-Q). The accuracy rates for reference strains were 89.4%, 100%, 98.8%, and 98.8% for the MTB-ID V3, AdvanSure, Genedia, and Real-Q assays, respectively. Cross-reactivity in the MTB-ID V3 assay was mainly attributable to non-mycobacterium Corynebacterineae species. The diagnostic performance was determined using clinical isolates grown in liquid media, and the overall sensitivities for all PCR assays were higher than 95%. In conclusion, the three real-time PCR assays showed better performance in discriminating mycobacterium species and non-mycobacterium Corynebacterineae species than the conventional PCR assay.
Jimenez, L; Scalici, C; Smalls, S; Bosko, Y; Ignar, R
The BAX system, a PCR-based assay, was evaluated for detecting Salmonella typhimurium in pharmaceutical raw materials and products contaminated with mixed bacterial cultures of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella typhimurium. Artificially contaminated samples were preenriched in lactose broth with and without Tween 20. After preenrichment, samples were analyzed by PCR and standard methods. Ten of 25 samples did not show presence of the specific Salmonella spp. 740-base pair DNA fragment. However, S. typhimurium was isolated and identified by standard methods from all 25 samples. To optimize S. typhimurium detection in PCR negative samples, lactose broth was replaced by buffered peptone water (BPW) as the preenrichment broth. When BPW was used, all 10 samples were PCR positive. BPW enrichments increased S. typhimurium growth resulting in rapid PCR detection. The presence of non-Salmonella bacteria influenced the performance of the PCR-based assay. Optimization of S. typhimurium PCR detection in mixed culture required the use of different preenrichment broths. However, the BAX system detected S. typhimurium within 27 hours while standard methods required 5-7 days.
Nishikawa, Hirokazu; Shirano, Michinori; Kasamatsu, Yu; Morimura, Ayumi; Iida, Ko; Kishi, Tomomi; Goto, Tetsushi; Okamoto, Saki; Ehara, Eiji
To assess relationships of inflammatory markers and 2 related clinical factors with blood culture results, we retrospectively investigated inpatients' blood culture and blood chemistry findings that were recorded from January to December 2014 using electronic medical records and analyzed the data of 852 subjects (426 culture-positive and 426 culture-negative). Results suggested that the risk of positive blood culture statistically increased as inflammatory marker levels and the number of related factors increased. Concerning the effectiveness of inflammatory markers, when the outcome definition was also changed for C-reactive protein (CRP), the odds ratio had a similar value, whereas when the outcome definition of blood culture positivity was used for procalcitonin (PCT), the greatest effectiveness of that was detected. Therefore, the current results suggest that PCT is more useful than CRP as an auxiliary indication of bacterial infection.
Kane, Peter E.
Akira Kurosawa, the most popular Asian film maker with audiences in the United States, has found in William Shakespeare's plays themes and plots that resonate within Japanese culture. While the translations of "Macbeth" into "Throne of Blood" and "King Lear" into "Ran" are quite direct and literal with only…
A total of 302 samples of healthy farm-cultured Rana catesbeiana specimens (9-21 months-old, 50- 350 g liveweight, 50% each sex) from the north-east of Argentina, were analyzed through spectrophotometry, electrophoresis, densitometry, refractometry and microscopy in order to obtain blood and urine normal values. Confidence intervals (p
Kane, Peter E.
Akira Kurosawa, the most popular Asian film maker with audiences in the United States, has found in William Shakespeare's plays themes and plots that resonate within Japanese culture. While the translations of "Macbeth" into "Throne of Blood" and "King Lear" into "Ran" are quite direct and literal with only minor changes in plot and emphasis, in…
J.J. Kerremans (Jos); A.K. van der Bij (Akke); W.H.F. Goessens (Wil); H.A. Verbrugh (Henri); M.C. Vos (Margreet)
textabstractThe aim of this prospective randomized controlled clinical trial was to assess the impact of immediate incubation of blood cultures delivered to the laboratory outside its hours of operation on turnaround times, antibiotic prescription practices, and patient outcomes. A continuously moni
Robinson, Andrew M; Ussher, James E
MALDI-ToF MS can be used to identify microorganisms directly from blood cultures. This study compared two methods of sample preparation. Similar levels of genus- (91% vs 90%) and species-level identifications (79% vs 74%) were obtained with differential centrifugation and SDS methods. The SDS method is faster and requires minimal handling.
Houe, Hans; Eriksen, L.; Jungersen, Gregers;
This study investigated the number of blood culture-positive cattle among 215 animals clinically suspected of having bacterial endocarditis. For animals that were necropsied, the sensitivity, specificity and predictive value of the diagnosis of endocarditis were calculated on the basis...
Full Text Available The differentiation between acute and latent forms of the Toxoplasma gondii (T. gondii infection is still considered as a complicated issue. This study was aimed to elucidate the status of infection in the blood donors and the probable importance of blood transfusion in the transmission of the infection through detecting both immunological and genetic markers of acute and latent infection.Totally 235 blood samples from blood donors were collected. The levels of anti-T. gondii IgG and IgM antibodies were examined by specific ELISA kits. cDNA were synthesized from total extracted mRNA molecules from the serum samples and SAG1 gene, specific for tachyzoite form, were amplified using Real-Time PCR technique. Demographic information of study subjects including their gender, age, job, and habitat were recorded.Out of 235 serum samples, 80 (34.04% and 4 (1.71% were positive regarding anti-T. gondii IgG and IgM antibodies, respectively. Real-Time PCR results showed that 14 out of 200 (6.97% of blood donor had mRNA molecules of SAG1 gene. The positive results of Real-Time PCR of SAG1 in female gender and housekeepers were significantly higher than those of male gender and other job categories.The prevalence of chronic and acute infection is high in Iranian blood donors. Additionally, evaluation of antibodies could not be reliable, because several donors negative for anti-T. gondii IgM antibodies had detectable SAG1 mRNA molecules. Hence, it seems that molecular diagnostic tests are essential to detect acute infections.
Yuan, Yuan; Kitamura-Muramatsu, Yuri; Saito, Susumu; Ishizaki, Hiroshi; Nakano, Miwa; Haga, Satoshi; Matoba, Kazuhiro; Ohno, Ayumu; Murakami, Hironobu; Takeshima, Shin-Nosuke; Aida, Yoko
Bovine leukemia virus (BLV) induces enzootic bovine leukosis, which is the most common neoplastic disease in cattle. Sero-epidemiological studies show that BLV infection occurs worldwide. Direct contact between infected and uninfected cattle is thought to be one of the risk factors for BLV transmission. Contact transmission occurs via a mixture of natural sources, blood, and exudates. To confirm that BLV provirus is detectable in these samples, matched blood, nasal secretion, and saliva samples were collected from 50 cattle, and genomic DNA was extracted. BLV-CoCoMo-qPCR-2, an assay developed for the highly sensitive detection of BLV, was then used to measure the proviral load in blood (n=50), nasal secretions (n=48), and saliva (n=47) samples. The results showed that 35 blood samples, 14 nasal secretion samples, and 6 saliva samples were positive for the BLV provirus. Matched blood samples from cattle that were positive for the BLV provirus (either in nasal secretion or saliva samples) were also positive in their blood. The proviral load in the positive blood samples was >14,000 (copies/1×10(5) cells). Thus, even though the proviral load in the nasal secretion and saliva samples was much lower (<380 copies/1×10(5) cells) than that in the peripheral blood, prolonged direct contact between infected and healthy cattle may be considered as a risk factor for BLV transmission.
Stangegaard, Michael; Frøslev, Tobias G; Frank-Hansen, Rune
We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, Männedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO...... 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained...... the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpFlSTR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI...
Skovgaard, Kerstin; Mortensen, Shila; Poulsen, K.T.;
The quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive and very efficient technique for quantification of gene expression. However, qRT-PCR relies on accurate normalization of gene expression data, as RNA recovery and cDNA synthesis efficiency might vary...... from sample to sample. In the present study, six putative reference genes were validated for normalization of gene expression in three different tissues and in white blood cells from pigs experimentally infected with the common respiratory pathogen Actinobacillus pleuropneumoniae. Two dedicated...... (GAPDH). IL-6 expression was quantified in white blood cells, liver, lymph nodes and tonsils from 10 infected pigs and 5 control pigs. After normalization using either geNorm or Normfinder IL-6 was shown to be significantly up-regulated (P
Büsch, J; Huber, P; Pflüger, E; Miltenyi, S; Holtz, J; Radbruch, A
For simple and effective isolation of fetal cells from peripheral maternal blood, we combined depletion of maternal cells and enrichment of fetal cells by high-gradient magnetic cell separation (MACS). First CD45+ and CD14+ cells were depleted from maternal peripheral blood mononuclear cells by MACS. From the depleted fraction, CD71+ erythroid cells were enriched up to 80 per cent by MACS. This double-MACS' procedure yielded an average depletion rate of 780-fold and an average enrichment rate of 500-fold, with approximate recovery rates of 40-55 per cent. For paternity testing, cells from unseparated blood and the various fractions were analysed for polymorphism of the HLA-DQ-A1 locus and D1S80 locus by the polymerase chain reaction (PCR). In CD45-/CD71+ sorted cells from maternal blood, but not in unfractionated cells from maternal blood or CD45-/CD14- cells, paternal alleles could be detected. In the CD45-/CD71+ fraction, the relative frequency of paternal alleles compared with maternal alleles ranged from 1 in 20 to 1 in 200 (determined by titration and depending on the quality of separation and biological variation). In 7 out of 11 cases, between weeks 12 and 25 of gestation, we could identify paternal alleles by PCR, either HLA-DQ-A1 or D1S80. This double-MACS procedure is simple, fast, efficient, and reliable for non-invasive prenatal diagnosis.
Full Text Available Background and objectives: Blood culture is a critical part of evaluationof Neonate suspected wath Septicaemia. This phenomenon is one of the mostimportant causes of neonates in Neonatal.Material and Methods: This study was carried out on 1470 somples ofneonates suspected with bacteraemia , using reutine microbiologicultechnique. The samples wene assessed in hazrate Masoomeh hospilal ofKermansha,Iran.Results and Conclusions: of all samples, 112 (7.62% ane pasitive.Most of the Positive cultures were obtained after 24 hours of incubation inbroth mediu. we Confirmed this result by using diseriminafiue culture mediathe Isolated bacteria are Coagulase-negative Staphylococus(28.6%,AlfahymolylicStreptococus(0.09%, staphylococusaureus(10.7%,Klebseilla(6.2%,pseudomonas(12.5%,moraxella(0.9%,acineto bacter(13.4%, Alcalingenes(13.4%,protenos(1.8% and salmonela(0.9%.Conclusion: the frequency of Coagulase-negative Staphylococus Isolatedfrom neonates Blood culture is more than the other micro organisms.Generally, the frequency of gram negative bacteria is higher than gramPositive.Key words: Septicaemia, Blood culture, Bacteraemia, Neonatal
Aim: Bloodstream infections in hospitalized patients are one of the most important causes of morbidity and mortality despite antimicrobial therapy. Early diagnosis and treatment of these infections is crucial. The aim of this study was to evaluate the distribution and antibiotic susceptibility of bacteria isolated from blood cultures in a children’s hospital in the Southeastern Anatolia during an 18-month period. Material and Methods: 7 040 blood cultures which were sent from hospitalized patients in Gaziantep Children’s Hospital between 01.07.2010 and 01.01.2012 were evaluated. Results: A total of 7 040 blood cultures were evaluated in this study. Microbial growth was detected in 2075 (29.47%) blood cultures. The most frequently isolated bacteria were coagulase-negative staphylococci (%45.97) which were followed by Salmonella spp. (%7.8). 12.12% of enterococcal isolates were resistant to glycopeptide antibiotics. The most frequently isolated gram negative bacterium was Salmonella spp. 15.43% of Salmonella spp. showed decreased susceptibility against quinolones. The ESBL positivity rate of E. coli and K. pneumoniae strains was found to be 35.08% and 57.14%, respectively. The imipenem resistance rate of P. aeruginosa was found to be 33.33%. The most common nonfermentative bacterium was S. maltophilia. Conclusions: The distribution of bacteria isolated from blood cultures and antibiotic resistance rates differ among different regions of Turkey. Different results obtained in our study may be related with regional tendencies to infections and patient population. Distribution of infectious agents and antibiotic resistance rates should be evaluated at regular intervals. This will lead to establishment of proper antibiotic usage policies in our country. PMID:26265894
Full Text Available Objectives: The aim of this study was to evaluate the distribution of Staphylococcus species in bloodstream infections and to assess their susceptibility to methicillin. Material and Methods: Between January 1st 2008 - December 31st 2010, 7424 blood culture sets were submitted to the Laboratory Department of the Hospital for Clinical Infectious Diseases in Cluj-Napoca, Romania. The blood cultures were performed using BacT/Alert until January 2010 and BacT/Alert 3D automated system (bioMérieux after that date. The blood culture bottles were incubated at 37°C in a continuously monitoring system for up to 7 days. The strain identifications were performed by conventional methods, ApiStaph galleries and Vitek 2 Compact system. Susceptibility to methicillin was determined by disk diffusion method with cefoxitin disk and by using Vitek 2 Compact system. Results: From the total number of performed blood cultures, 568 were positive with Staphylococcus species. From 168 bacteriemic episodes 103 were with Staphylococcus aureus. Among 65 coagulase-negative staphylococci isolates, Staphylococcus epidermidis was the most frequently isolated species (34, followed by Staphylococcus hominis (15, Staphylococcus haemolyticus (8, Staphylococcus saprophyticus (3, Staphylococcus cohnii (1, Staphylococcus auricularis (1, and 3 strains that were not identified at species level. Methicillin resistance was encountered in 53.40% of Staphylococcus aureus strains and in 80% of coagulase-negative staphylococci. Conclusions: An important percentage of blood cultures were contaminated with Staphylococcus species. The main species identified in true bacteriemia cases were Staphylococcus aureus and Staphylococcus epidermidis. The percentage of methicillin-resistance, proved to be high not only for coagulase-negative staphylococci but also for Staphylococcus aureus.
Chun-Gang Xie; Jin-Fu Wang; Ying Xiang; Li-Yan Qiu; Bing-Bing Jia; Li-Juan Wang; Guo-Zhong Wang; Guo-Ping Huang
AIM: To establish a novel coculture system for ex vivo expansion of umbilical cord blood(UCB) hematopoietic progenitors using thrombopoietin (TPO)/Flt-3 ligand(FL)-transduced human marrow-derived mesenchymal stem cells (tfhMSCs) as feeder.METHODS: UCB CD34+ cells were isolated and cultured using four culture systems in serum-containing or serumfree medium. Suitable aliquots of cultured cells were used to monitor cell production, clonogenic activity,and long-term culture-initiating culture (LTC-IC) output.Finally, the severe-combined immunodeficient (SCID)mouse-repopulating cell (SRC) assay was performed to confirm ability of the cultured cells to reconstitute longterm hematopoiesis.RESULTS: There were no significant differences in the number of total nucleated cells among different culture systems in serum-containing medium during 21-d culture. However, on d 14, the outputs of CD34+ cells,CFU-C and CFU-GEMM in tfhMSCs coculture system were significantly enhanced. LTC-IC assay demonstrated that the tfhMSCs coculture system had the most powerful activity. The severe-combined immunodeficient (SCID)mouse repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. Furthermore, PCR analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of NOD/SCID mice.CONCLUSION: The TPO/FL-transduced hMSCs, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro and the tfhMSCs coculture system may be a suitable system for ex vivo manipulation of primitive progenitor cells under contact culture conditions.
Metwally, L; Fairley, D J; Coyle, P V; Hay, R J; Hedderwick, S; McCloskey, B; O'Neill, H J; Webb, C H; Elbaz, W; McMullan, R
The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.
Park, Jong Eun; Kim, Ji Youn; Yun, Sun Ae; Lee, Myoung Keun; Huh, Hee Jae; Kim, Jong Won; Ki, Chang Seok
Standardized cytomegalovirus (CMV) DNA quantification is important for managing CMV disease. We evaluated the performance of the Real-Q CMV Quantification Kit (Real-Q assay; BioSewoom, Korea) using whole blood (WB), with nucleic acid extraction using MagNA Pure 96 (Roche Diagnostics, Germany). Real-time PCR was performed on two platforms: the 7500 Fast real-time PCR (7500 Fast; Applied Biosystems, USA) and CFX96 real-time PCR detection (CFX96; Bio-Rad, USA) systems. The WHO international standard, diluted with CMV-negative WB, was used to validate the analytical performance. We used 90 WB clinical samples for comparison with the artus CMV RG PCR kit (artus assay; Qiagen, Germany). Limits of detections (LODs) in 7500 Fast and CFX96 were 367 and 479 IU/mL, respectively. The assay was linear from the LOD to 10⁶ IU/mL (R² ≥0.9886). The conversion factors from copies to IU in 7500 Fast and CFX96 were 0.95 and 1.06, respectively. Compared with the artus assay, for values 1,000 copies/mL, 73.3% and 80.6% of samples in 7500 Fast and CFX96, respectively, had real-time PCR platforms.
Katrin Hoffmann; Christiane Kerner; Wolfgang Wilfert; Marc Mueller; Joachim Thiery; Johann Hauss; Helmut Witzigmann
AIM: To evaluate the diagnostic potential of cytokeratin-19 (CK-19) mRNA for the detection of disseminated tumor cells in blood, bone marrow and peritoneal lavage in patients with ductal adenocarcinoma of the pancreas.METHODS: Sixty-eight patients with pancreatic cancer (n = 37), chronic pancreatitis (n = 16), and non-pancreatic benign surgical diseases (n = 15, control group)were included in the study. Venous blood was taken preoperatively, intraoperatively and at postoperative d 1 and 10. Preoperative bone marrow aspirates and peritoneal lavage taken before mobilization of the tumor were analyzed. All samples were evaluated for disseminated tumor cells by CK-19-specific nested-PCR and quantitative fluorogenic RT-PCR.RESULTS: CK-19 mRNA expression was increased in 24 (64%) blood samples and 11 (30%) of the peritoneal lavage samples in the patients with pancreatic cancer.In 15 (40%) of the patients with pancreatic cancer,disseminated tumor cells were detected in venous blood and bone marrow and/or peritoneal lavage. In the peritoneal lavage, the detection rates were correlated with the tumor size and the tumor differentiation. CK-19 levels were increased in pT3/T4 and moderately/poorly differentiated tumors (G2/G3). Pancreatic cancer patients with at least one CK-19 mRNA-positive sample showed a trend towards shorter survival. Pancreatic cancer patients showed significantly increased detection rates of disseminated tumor cells in blood and peritoneal lavage compared to the controls and the patients with chronic pancreatitis.CONCLUSION: Disseminated tumor cells can be detected in patients with pancreatic ductal adenocarcinoma by CK-19 fluorogenic RT-PCR. In peritoneal lavage, detection rate is correlated with tumor stage and differentiation. In the clinical use, CK-19 is suitable for the distinction between malignant and benign pancreatic disease in combination with other tumor-specific markers.
Conclusion: A rare disease, Aggregatibacter aphrophilus infective endocarditis was successfully treated with surgical repair and appropriate antibiotic therapy. To avoid misdiagnosis, br-PCR testing should be performed in patients with blood culture-negative endocarditis.
Alan O Marron
Full Text Available BACKGROUND: Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell. PRINCIPAL FINDINGS: Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample. CONCLUSIONS: The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient
Sen, Keya; L Sinclair, James; Boczek, Laura; Rice, Eugene W
A sensitive and specific method that also demonstrates viability is of interest for detection of E. coli O157:H7 in drinking water. A combination of culture and qPCR was investigated. Two triplex qPCRs, one from a commercial source and another designed for this study were optimized from 5 different assays to be run on a single qPCR plate. The qPCR assays were specific for 33 E. coli O157:H7 strains tested and detected 500 cells spiked in a background of 10(8) nontarget bacterial cells. The qPCR detection was combined with an enrichment process using Presence Absence (P/A) broth to detect chlorine and starvation stressed cells. qPCR analysis performed post-enrichment allowed the detection of 3-4 cells/L as indicated by a sharp increase in fluorescence (lowering of Ct values) from pre-enrichment levels, demonstrating a 5-6 log increase in the number of cells. When six vulnerable untreated surface water samples were examined, only one was positive for viable E. coli O157:H7 cells. These results suggest that the culture-PCR procedure can be used for rapid detection of E. coli O157:H7 in drinking water.
Shanthalingam, Sudarvili; Goldy, Andrea; Bavananthasivam, Jegarubee; Subramaniam, Renuka; Batra, Sai Arun; Kugadas, Abirami; Raghavan, Bindu; Dassanayake, Rohana P; Jennings-Gaines, Jessica E; Killion, Halcyon J; Edwards, William H; Ramsey, Jennifer M; Anderson, Neil J; Wolff, Peregrine L; Mansfield, Kristin; Bruning, Darren; Srikumaran, Subramaniam
Mannheimia haemolytica consistently causes severe bronchopneumonia and rapid death of bighorn sheep (Ovis canadensis) under experimental conditions. However, Bibersteinia trehalosi and Pasteurella multocida have been isolated from pneumonic bighorn lung tissues more frequently than M. haemolytica by culture-based methods. We hypothesized that assays more sensitive than culture would detect M. haemolytica in pneumonic lung tissues more accurately. Therefore, our first objective was to develop a PCR assay specific for M. haemolytica and use it to determine if this organism was present in the pneumonic lungs of bighorns during the 2009-2010 outbreaks in Montana, Nevada, and Washington, USA. Mannheimia haemolytica was detected by the species-specific PCR assay in 77% of archived pneumonic lung tissues that were negative by culture. Leukotoxin-negative M. haemolytica does not cause fatal pneumonia in bighorns. Therefore, our second objective was to determine if the leukotoxin gene was also present in the lung tissues as a means of determining the leukotoxicity of M. haemolytica that were present in the lungs. The leukotoxin-specific PCR assay detected leukotoxin gene in 91% of lung tissues that were negative for M. haemolytica by culture. Mycoplasma ovipneumoniae, an organism associated with bighorn pneumonia, was detected in 65% of pneumonic bighorn lung tissues by PCR or culture. A PCR assessment of distribution of these pathogens in the nasopharynx of healthy bighorns from populations that did not experience an all-age die-off in the past 20 yr revealed that M. ovipneumoniae was present in 31% of the animals whereas leukotoxin-positive M. haemolytica was present in only 4%. Taken together, these results indicate that culture-based methods are not reliable for detection of M. haemolytica and that leukotoxin-positive M. haemolytica was a predominant etiologic agent of the pneumonia outbreaks of 2009-2010.
Delgado, Susana; Rachid, Caio T C C; Fernández, Elena; Rychlik, Tomasz; Alegría, Angel; Peixoto, Raquel S; Mayo, Baltasar
Thermophilic lactic acid bacteria (LAB) species, such as Streptococcus thermophilus, Lactobacillus delbrueckii and Lactobacillus helveticus, enjoy worldwide economic importance as dairy starters. To assess the diversity of thermophilic bacteria in milk, milk samples were enriched in thermophilic organisms through a stepwise procedure which included pasteurization of milk at 63 °C for 30 min (PM samples) and pasteurization followed by incubation at 42 °C for 24 h (IPM samples). The microbial composition of these samples was analyzed by culture-dependent (at 42 °C) and culture-independent (PCR-DGGE and pyrosequencing of 16S rRNA gene amplicons) microbial techniques. The results were then compared to those obtained for their corresponding starting raw milk counterparts (RM samples). Twenty different species were scored by culturing among 352 isolates purified from the counting plates and identified by molecular methods. Mesophilic LAB species (Lactococcus lactis, Lactococcus garvieae) were dominant (87% of the isolates) among the RM samples. However, S. thermophilus and Lb. delbrueckii were found to be the dominant recoverable organisms in both PM and IPM samples. The DGGE profiles of RM and PM samples were found to be very similar; the most prominent bands belonging to Lactococcus, Leuconostoc and Streptococcus species. In contrast, just three DGGE bands were obtained for IPM samples, two of which were assigned to S. thermophilus. The pyrosequencing results scored 95 operational taxonomic units (OTUs) at 3% sequence divergence in an RM sample, while only 13 were encountered in two IPM samples. This technique identified Leuconostoc citreum as the dominant microorganism in the RM sample, while S. thermophilus constituted more than 98% of the reads in the IPM samples. The procedure followed in this study allowed to estimate the bacterial diversity in milk and afford a suitable strategy for the isolation of new thermophilic LAB strains, among which adequate
Orcel, P.; Bielakoff, J.; De Vernejoul, M.C. (INSERM U18, Hopital Lariboisiere, Paris (France))
Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes.
Oliveira, Fernanda S; Pirmez, Claude; Pires, Marize Q; Brazil, Reginaldo P; Pacheco, Raquel S
The technique of polymerase chain reaction (PCR) associated to hybridization was used to screen 123 samples collected from wild and synanthropic rodents captured in a cutaneous and visceral leishmaniasis endemic area in the State of Minas Gerais, Brazil. The detection of Leishmania spp in naturally infected rodents is of fundamental importance for incriminating them as possible reservoir hosts of the diseases in Minas Gerais. A total of 62 specimens belonging to wild (Thrichomys apereoides, Oryzomys subflavus, Galea spixii, Bolomys lasiurus and Wiedomys pyrrhorhinos) and synanthropic (R. rattus) rodent species were captured in different ecotopes. Blood and skin samples were submitted for PCR analyses followed by molecular hybridization with specific probes for the three Leishmania-species complexes. Fifteen samples were found positive after PCR-hybridization and identified as follows: nine belonging to the L. mexicana complex, three to the L. braziliensis complex and three to the L. donovani complex. Positive PCR results were found in 11 out of the 61 (18%) blood samples and in four out of the 62 (6.4%) skin fragments screened. R. rattus and T. apereoides were the most abundant species in the area also presenting high prevalence of natural infection. The presence of parasite DNA belonging to L. braziliensis, L. mexicana and L. donovani complexes was confirmed in several individuals of a rodent species, R. rattus. This work is the first report of the detection of L. (L.) chagasi in a naturally infected T. apereoides. The utility of filter paper as a substrate for PCR analyses and the efficacy of the procedure associated to the hybridization is emphasized.
Hardy, D J; Hulbert, B B; Migneault, P C
Consecutive BacT/Alert blood cultures which were instrument negative following a 7-day incubation were subcultured. Eighteen (0.2%) of 11,476 bottles had growth on subculture. Eleven of these eighteen isolates were considered contaminants on the basis of the identity of the organism and lack of other positive blood cultures from the same patient. In addition, analysis of time to instrument detection for approximately 2,900 positive blood cultures indicates that 5 or 6 days of incubation is su...
Luteijn, J. M.; Hubben, G. A. A.; Pechlivanoglou, P.; Bonten, M. J.; Postma, M. J.
P>A systematic review and meta-analysis were performed to determine and compare the sensitivity and specificity of PCR-based and culture-based diagnostic tests for methicillin-resistant Staphylococcus aureus (MRSA). Our analysis included 74 accuracy measurements from 29 publications. Nine tests were
Dehghan fatemeh; Najarian Negin; Kasravi Alii Reza; Falahat Saeed; Zolfaghari Mohammad Reza; Arjomandzadegan Mohammad; Kalantari Salomeh; Ahmari Gholam Reza; Sarmadian Hossein; Sadrnia Maryam; Ahmadi Azam; Shojapoor Mana
Objective:To analyse molecular detection of coliforms and shorten the time of PCR. Methods:Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated strains from certificated reference material and standard bacteria. The PCR and electrophoresis parameters were changed for reducing the operation time. Results:Results of PCR for lacZ and uidA genes were similar in all of standard, operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR. PCR results were confirmed by MPN culture method by sensitivity 86%(95%CI:0.71-0.93). Also the total execution time, with a successful change of factors, was reduced to less than two and a half hour. Conclusions:Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It’s recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN.
Nyman, A-K; Persson Waller, K; Emanuelson, U; Frössling, J
Real-time PCR analysis of milk samples is a fast method to identify intramammary infections (IMI) in dairy cows, and has the potential to be used for routine analysis of test milking composite milk samples. However, the results of the PCR analysis can be difficult to interpret. The objective of this study was to compare the sensitivity (Se) and specificity (Sp) of PCR analysis of composite milk samples, and conventional bacteriological culturing (BC) of quarter milk samples, when used to identify cows with IMI. The comparisons were performed for IMI with four common udder pathogens; Staphylococcus aureus (S aureus), Streptococcus dysgalactiae (Str dysgalactiae), Str uberis and coagulase negative staphylococci (CoNS). The Se and Sp of real-time PCR (SePCR; SpPCR) and BC (SeBC; SpBC) was simultaneously estimated using latent class analysis (LCA), studying one pathogen at the time. Milk samples from 970 dairy cows from 25 herds were included. Aseptically collected quarter milk samples taken at the day before test milking (TM), at the day of TM, and at the day after TM, were analyzed using BC. Non-aseptically collected composite milk samples taken at the day of TM were analyzed using PCR. Moreover, the composite milk somatic cell count (SCC) was recorded and summarized by diagnostic test and bacterial finding. LCA was first performed using only test results from samples taken at the day of TM, but in a second analysis BC results from the three consecutive samplings, interpreted in parallel, were included. The SePCR was significantly higher than the SeBC for S aureus, Str dysgalactiae and CoNS in the first analysis, but only for CoNS in the second analysis. The SpPCR was significantly lower than the SpBC for Str dysgalactiae and CoNS. In conclusion, using PCR analysis of composite milk samples, as a diagnostic tool for identifying cows with IMI increased the Se for all the pathogens investigated (although not always significantly), while Sp in general remained on a
Full Text Available Abstract Background Chlamydia trachomatis (Ct and Chlamydia pneumoniae (Cp are medically significant infectious agents associated with various chronic human pathologies. Nevertheless, specific roles in disease progression or initiation are incompletely defined. Both pathogens infect established cell lines in vitro and polymerase chain reaction (PCR has detected Chlamydia DNA in various clinical specimens as well as in normal donor peripheral blood monocytes (PBMC. However, Chlamydia infection of other blood cell types, quantification of Chlamydia infected cells in peripheral blood and transmission of this infection in vitro have not been examined. Methods Cp specific titers were assessed for sera from 459 normal human donor blood (NBD samples. Isolated white blood cells (WBC were assayed by in vitro culture to evaluate infection transmission of blood cell borne chlamydiae. Smears of fresh blood samples (FB were dual immunostained for microscopic identification of Chlamydia-infected cell types and aliquots also assessed using Flow Cytometry (FC. Results ELISA demonstrated that 219 (47.7% of the NBD samples exhibit elevated anti-Cp antibody titers. Imunofluorescence microscopy of smears demonstrated 113 (24.6% of samples contained intracellular Chlamydia and monoclonals to specific CD markers showed that in vivo infection of neutrophil and eosinophil/basophil cells as well as monocytes occurs. In vitro culture established WBCs of 114 (24.8% of the NBD samples harbored infectious chlamydiae, clinically a potentially source of transmission, FC demonstrated both Chlamydia infected and uninfected cells can be readily identified and quantified. Conclusion NBD can harbor infected neutrophils, eosinophil/basophils and monocytes. The chlamydiae are infectious in vitro, and both total, and cell type specific Chlamydia carriage is quantifiable by FC.
Nilma Cintra Lea
Full Text Available Background: In Brazil, listeriosis is not a notifiable disease; thus, the incidence of Brazilian cases remains unknown. Listeria monocytogenes is not always included in automated systems, and its detection depends on the high skill level of microbiology laboratory professionals. This paper describes the characteristics of L. monocytogenes isolates fortuitously obtained from an endocarditis case in Recife, PE, Brazil. Methods: Six bacterial isolates obtained from six blood cultures from a 28-year-old male bearing a prosthetic mitral heart valve were analyzed by PCR using primers specific of L. monocytogenes to confirm a presumptive identification, determine the serotype and presence of the virulence genes (inlA, inlB, inlC, inlJ, hly, plcA, actA, prfA in an attempt to determine the Listeria genotype by PCR-ribotyping. Results: The samples were identified as L. monocytogenes 4b. All investigated virulence genes were amplified by PCR, and the identity of the amplified segments was confirmed by sequencing. A deletion of 105 base pairs was detected in the actA gene. All of the samples generated the same PCR-ribotype pattern, clustered into a single ribotype, and were considered a single strain. Conclusion: L. monocytogenes infection should be considered in endocarditis differential diagnoses, especially among high-risk groups, due to its high pathogenicity and the environmental ubiquity.
Lupetti, A; Barnini, S; Castagna, B; Capria, A-L; Nibbering, P H
Rapid identification and antimicrobial susceptibility profiling of the bacteria in blood cultures can result in clinical and financial benefits. Addition of saponin to the fluid from blood culture bottles promotes the recovery of the bacteria and thus may shorten the turnaround time of the microbiological analyses. In this study we compared the identification and susceptibility profiles of saponin-treated and untreated (standard method) blood cultures monomicrobial for Gram-positive cocci using Vitek 2. We concordantly identified 49 (89%) of 55 monobacterial cultures using the results with the standard method as reference. Complete categorical agreement between the susceptibility profiles with the new and the standard method was found for 26 (53%) of 49 isolates, while discrepancies were seen for 23 (47%) cultures. E-tests indicated that the new method resulted in a correct susceptibility profile for 8 (35%) of these 23 blood cultures. Therefore, 34 (69%) of 49 cultures showed a concordant/correct susceptibility profile for all antimicrobials with an overall error rate of 2.3%. Thus, addition of saponin to the fluid from blood culture bottles of the Bactec 9240 leads to the rapid (results available >or=12 hours earlier) and reliable identification and susceptibility profiling of Gram-positive cocci in blood cultures with Vitek 2.
Freriks, Kim; Timmers, Henri J L M; Netea-Maier, Romana T; Beerendonk, Catharina C M; Otten, Barto J; van Alfen-van der Velden, Janiëlle A E M; Traas, Maaike A F; Mieloo, Hanneke; van de Zande, Guillaume W H J F L; Hoefsloot, Lies H; Hermus, Ad R M M; Smeets, Dominique F C M
Turner syndrome (TS) is the result of (partial) X chromosome monosomy. In general, the diagnosis is based on karyotyping of 30 blood lymphocytes. This technique, however, does not rule out tissue mosaicism or low grade mosaicism in the blood. Because of the associated risk of gonadoblastoma, mosaicism is especially important in case this involves a Y chromosome. We investigated different approaches to improve the detection of mosaicisms in 162 adult women with TS (mean age 29.9 ± 10.3). Standard karyotyping identified 75 patients (46.3%) with a non-mosaic monosomy 45,X. Of these 75 patients, 63 underwent additional investigations including FISH on buccal cells with X- and Y-specific probes and PCR-Y on blood. FISH analysis of buccal cells revealed a mosaicism in 19 of the 63 patients (30.2%). In five patients the additional cell lines contained a (derivative) Y chromosome. With sensitive real-time PCR we confirmed the presence of this Y chromosome in blood in three of the five cases. Although Y chromosome material was established in ovarian tissue in two patients, no gonadoblastoma was found. Our results confirm the notion that TS patients with 45,X on conventional karyotyping often have tissue specific mosaicisms, some of which include a Y chromosome. Although further investigations are needed to estimate the risk of gonadoblastoma in patients with Y chromosome material in buccal cells, we conclude that FISH or real-time PCR on buccal cells should be considered in TS patients with 45,X on standard karyotyping.
Ciarloni, Laura; Hosseinian, Sahar; Monnier-Benoit, Sylvain; Imaizumi, Natsuko; Dorta, Gian; Ruegg, Curzio
Colorectal cancer (CRC) is the second leading cause of cancer-related death in developed countries. Early detection of CRC leads to decreased CRC mortality. A blood-based CRC screening test is highly desirable due to limited invasiveness and high acceptance rate among patients compared to currently used fecal occult blood testing and colonoscopy. Here we describe the discovery and validation of a 29-gene panel in peripheral blood mononuclear cells (PBMC) for the detection of CRC and adenomatous polyps (AP). Blood samples were prospectively collected from a multicenter, case-control clinical study. First, we profiled 93 samples with 667 candidate and 3 reference genes by high throughput real-time PCR (OpenArray system). After analysis, 160 genes were retained and tested again on 51 additional samples. Low expressed and unstable genes were discarded resulting in a final dataset of 144 samples profiled with 140 genes. To define which genes, alone or in combinations had the highest potential to discriminate AP and/or CRC from controls, data were analyzed by a combination of univariate and multivariate methods. A list of 29 potentially discriminant genes was compiled and evaluated for its predictive accuracy by penalized logistic regression and bootstrap. This method discriminated AP >1cm and CRC from controls with a sensitivity of 59% and 75%, respectively, with 91% specificity. The behavior of the 29-gene panel was validated with a LightCycler 480 real-time PCR platform, commonly adopted by clinical laboratories. In this work we identified a 29-gene panel expressed in PBMC that can be used for developing a novel minimally-invasive test for accurate detection of AP and CRC using a standard real-time PCR platform.
Russell J McCulloh
Full Text Available Blood cultures are often recommended for the evaluation of community-acquired pneumonia (CAP. However, institutions vary in their use of blood cultures, and blood cultures have unclear utility in CAP management in hospitalized children.To identify clinical factors associated with obtaining blood cultures in children hospitalized with CAP, and to estimate the association between blood culture obtainment and hospital length of stay (LOS.We performed a multicenter retrospective cohort study of children admitted with a diagnosis of CAP to any of four pediatric hospitals in the United States from January 1, 2011-December 31, 2012. Demographics, medical history, diagnostic testing, and clinical outcomes were abstracted via manual chart review. Multivariable logistic regression evaluated patient and clinical factors for associations with obtaining blood cultures. Propensity score-matched Kaplan-Meier analysis compared patients with and without blood cultures for hospital LOS.Six hundred fourteen charts met inclusion criteria; 390 children had blood cultures obtained. Of children with blood cultures, six (1.5% were positive for a pathogen and nine (2.3% grew a contaminant. Factors associated with blood culture obtainment included presenting with symptoms of systemic inflammatory response syndrome (OR 1.78, 95% CI 1.10-2.89, receiving intravenous hydration (OR 3.94, 95% CI 3.22-4.83, receiving antibiotics before admission (OR 1.49, 95% CI 1.17-1.89, hospital admission from the ED (OR 1.65, 95% CI 1.05-2.60, and having health insurance (OR 0.42, 95% CI 0.30-0.60. In propensity score-matched analysis, patients with blood cultures had median 0.8 days longer LOS (2.0 vs 1.2 days, P < .0001 without increased odds of readmission (OR 0.94, 95% CI 0.45-1.97 or death (P = .25.Obtaining blood cultures in children hospitalized with CAP rarely identifies a causative pathogen and is associated with increased LOS. Our results highlight the need to refine the role of
Self, Wesley H; Speroff, Theodore; McNaughton, Candace D; Wright, Patty W; Miller, Geraldine; Johnson, James G; Daniels, Titus L; Talbot, Thomas R
Five hundred five blood cultures collected through a peripheral intravenous catheter (PIV) in an emergency department were matched to cultures obtained by dedicated venipuncture from the same patient within 10 minutes. The relative risk of contamination for cultures collected through PIVs compared with dedicated venipuncture was 1.83 (95% confidence interval, 1.08-3.11).
Lajbner, Z; Kotlík, P
The tench Tinca tinca is a valued table fish native to Europe and Asia, but which is now widely distributed in many temperate freshwater regions of the world as the result of human-mediated translocations. Fish are currently being transplanted between watersheds without concern for genetic similarity to wild populations or local adaptation, and efficient phylogeographic markers are therefore urgently needed to rapidly distinguish genetically distinct geographical populations and to assess their contribution to the hatchery breeds and to the stocked wild populations. Here, we present a new method to distinguish recently discovered and morphologically undistinguishable Western and Eastern phylogroups of the tench. The method relies on PCR-RFLP assays of two independent nuclear-encoded exon-primed intron-crossing (EPIC) markers and of one mitochondrial DNA (mDNA) marker and allows the rapid identification of the Western and Eastern phylogroup and also of three geographical mtDNA clades within the Eastern phylogroup. Our method will enable researchers and fishery practitioners to rapidly distinguish genetically divergent geographical populations of the tench and will be useful for monitoring the introduction and human-mediated spread of the phylogroups in wild populations, for characterization of cultured strains and in breeding experiments.
Arezi, Bahram; McKinney, Nancy; Hansen, Connie; Cayouette, Michelle; Fox, Jeffrey; Chen, Keith; Lapira, Jennifer; Hamilton, Sarah; Hogrefe, Holly
Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd ) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.
Full Text Available Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd for primed template and a moderate (2-fold increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.
Kishii, Kozue; Kikuchi, Ken; Yoshida, Atsushi; Okuzumi, Katsuko; Uetera, Yushi; Yasuhara, Hiroshi; Moriya, Kyoji
Carbapenem-resistant Acinetobacter baumannii has rapidly spread worldwide. This study investigated antibiotic susceptibility and genotypic resistance of 123 consecutive blood culture isolates of Acinetobacter species collected between 2003 and 2011 in two Japanese hospitals. The isolates were assigned to 13 species. Carbapenem resistance was detected in four isolates. Only one A. baumannii isolate had blaOXA-23 together with ISAba1; the remaining three isolates had IMP-1 metallo-β-lactamase. Quinolone resistance was detected in five isolates that had point mutations in the quinolone resistance-determining region. The predominance of various non-A. baumannii species and low prevalence of carbapenem resistance among blood culture isolates of Acinetobacter species in two Japanese hospitals were confirmed.
Choi, Sang-Ho; Woo, Jun Hee; Jeong, Jin-Yong; Kim, Nam Joong; Kim, Mi-Na; Kim, Yang Soo; Ryu, Jiso
Staphylococcus saprophyticus is a well-known cause of acute uncomplicated urinary tract infection in young women. However, the clinical significance of this organism isolated from blood culture has not been determined. We assessed the clinical significance and characteristics of S. saprophyticus identified on blood culture. A total of 24 patients were identified, and 7 patients (29.2%) were considered to have clinically significant bacteremia. Of the 7 patients with clinically significant bacteremia, hematologic malignancy was the most common underlying illness (5 patients), and tunneled-central venous catheter was the most common portal of entry (4 patients). In no case did S. saprophyticus bacteremia originate from the urinary tract. One patient died during hospitalization. However, the death was not directly related to bacteremia. In conclusion, our data suggest that bacteremia caused by S. saprophyticus is most commonly associated with tunneled-central venous catheter in patients with hematologic malignancies and may be associated with a lower risk of mortality.
Carretto, E; Bardaro, M; Russello, G; Mirra, M; Zuelli, C; Barbarini, D
Many studies demonstrate that delayed proper therapy in bloodstream infections caused by Staphylococcus aureus increases the mortality rate, emphasizing the need to shorten the turnaround time for positive blood cultures. Different techniques are currently available, from phenotypic methods to more complex tests such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), real-time PCR (RT-PCR), and fluorescence in situ hybridization using peptide nucleic acid probes (PNA FISH). This study evaluated the performance of the Staphylococcus QuickFISH BC test (QFT), a novel FISH methodology, compared with the direct tube coagulase test (DTCT) on blood cultures exhibiting Gram-positive cocci in clusters. A total of 173 blood cultures collected from 128 different patients were analyzed using the DTCT, evaluated after both 4 and 24 h, and the QFT. A total of 179 isolates were identified using the Vitek2 system. Thirty-five out of 35 Staphylococcus aureus were correctly identified by the QFT (sensitivity = 100%), with a specificity of 100% (no green fluorescence was detected for strains different from S. aureus). The DTCT was positive after 4 h for 28 out of the 35 samples (sensitivity = 80%) and after 24 h for 31 out of the 35 samples (sensitivity = 88.57%). Among the remaining 144 isolates, one was then identified as Corynebacterium striatum and two as Micrococcus luteus. QFT identified 139 out of the 141 coagulase-negative staphylococci (CoNS) (sensitivity = 98.58%), showing again a specificity of 100% (no fluorescent red signals were detected for strains different from CoNS). We also discuss also the implementation process of this methodology in our setting, with particular emphasis on the workflow and the cost-effectiveness.
Full Text Available The positivities of two methods for the diagnosis of localised cutaneous leishmaniasis (CL were estimated in 280 patients enrolled in a clinical trial. The trial was conducted in an endemic area of Leishmania (Viannia braziliensis and trial participants were patients with skin ulcers and positive leishmanin skin tests. Patients underwent aspirative skin punctures of the ulcerated lesions and lymph nodes for in vitro cultures, which were processed under field conditions at the local health centre. Skin lesion biopsies were tested at a reference laboratory using kinetoplastid DNA (kDNA-PCR to detect DNA. The median time required to obtain a positive culture from the skin samples was seven days and the contamination rate of the samples was 1.8%. The positivities of the cultures from skin lesions, kDNA-PCR and the combination of the two methods were 78.2% (95% CI: 73-82.6%, 89.3% (95% CI: 85.1-92.4% and 97.1% (95% CI: 94.5-98.5%. We conclude that parasite culture is a feasible method for the detection of Leishmania in field conditions and that the combination of culture and PCR has a potential role for the diagnosis of CL in candidates for clinical trials.
Ampuero, Julia; Rios, Alexandre Pereira; Carranza-Tamayo, César Omar; Romero, Gustavo Adolfo Sierra
The positivities of two methods for the diagnosis of localised cutaneous leishmaniasis (CL) were estimated in 280 patients enrolled in a clinical trial. The trial was conducted in an endemic area of Leishmania (Viannia) braziliensis and trial participants were patients with skin ulcers and positive leishmanin skin tests. Patients underwent aspirative skin punctures of the ulcerated lesions and lymph nodes for in vitro cultures, which were processed under field conditions at the local health centre. Skin lesion biopsies were tested at a reference laboratory using kinetoplastid DNA (kDNA)-PCR to detect DNA. The median time required to obtain a positive culture from the skin samples was seven days and the contamination rate of the samples was 1.8%. The positivities of the cultures from skin lesions, kDNA-PCR and the combination of the two methods were 78.2% (95% CI: 73-82.6%), 89.3% (95% CI: 85.1-92.4%) and 97.1% (95% CI: 94.5-98.5%). We conclude that parasite culture is a feasible method for the detection of Leishmania in field conditions and that the combination of culture and PCR has a potential role for the diagnosis of CL in candidates for clinical trials.
Schwaiger, K; Wimmer, M; Huber-Schlenstedt, R; Fehlings, K; Hölzel, C S; Bauer, J
A large proportion of mastitis milk samples yield negative or nonspecific results (i.e., no mastitis pathogen can be identified) in bacterial culturing. Therefore, the culture-independent PCR-single strand conformation polymorphism method was applied to the investigation of bovine mastitis milk samples. In addition to the known mastitis pathogens, the method was suitable for the detection of fastidious bacteria such as Mycoplasma spp., which are often missed by conventional culturing methods. The detection of Helcococcus ovis in 4 samples might indicate an involvement of this species in pathogenesis of bovine mastitis. In conclusion, PCR-single-strand conformation polymorphism is a promising tool for gaining new insights into the bacteriological etiology of mastitis.
Hope, Kristine M; Casey, Rufina N; Groocock, Geoffrey H; Getchell, Rodman G; Bowser, Paul R; Casey, James W
Viral hemorrhagic septicemia virus (VHSV) is an important pathogen of cultured and wild fish in marine and freshwater environments. A new genotype, VHSV IVb, was isolated from a fish collected from the Great Lakes in 2003. Since the first isolation, VHSV IVb has been confirmed in 28 species, signaling the early invasion and continued spread of this Office International des Epizooties-reportable agent. For surveillance of this virus in both wild and experimental settings, we have developed a rapid and sensitive one-step quantitative real-time polymerase chain reaction (qRT-PCR) assay that amplifies a 100-base-pair conserved segment from both the genomic negative strand and the mRNA positive strand of the nucleoprotein (N) gene of VHSV IVb. This assay is linear over seven orders of magnitude, with an analytical capability of detecting a single copy of viral RNA and reproducibility at 100 copies. The assay is approximately linear with RNA input from 50 to 1000 ng per assay and works equally well with RNA prepared from a column-based or phenol-chloroform-based method. In wild-caught fish, 97% of the cases were found to be more than three orders of magnitude more sensitive using qRT-PCR than using cell culture. Of the 1,428 fish from the Great Lakes region tested in 2006 and 2007, 24% were positive by qRT-PCR whereas only 5% were positive by cell culture. All of the fish that were positive by cell culture were also positive by qRT-PCR. Importantly, qRT-PCR sensitivity is comparable to that of cell culture detection when comparing VHSV viral RNA levels with viral titer stocks, confirming that the high qRT-PCR signals obtained with diagnostic samples are due to the accumulation of N gene mRNA by transcriptional attenuation. The qRT-PCR assay is particularly valuable for rapid and high-throughput prescreening of fish before confirmatory testing by cell culture or sequencing tissue-derived amplicons and especially in detecting infection in fish that do not show clinical
Full Text Available Blood culture, although represents the gold standard in detecting the ethiological agent of sepsis, is rather rarely required in relation to the real diagnostic importance. The result of this test depends in fact on many factors (sample volume, time of collection, accuracy, antibiotic therapy, contamination, number of drawings, drawing site, interpretation difficulties, etc. that are often considered by many clinicians so limited as to doubt about their actual value. The disadvantages are therefore represented by the lack of standardization but also by the low sensitivity and above all by the technical times too long for the clinical needs. Blood culture begins with the drawing of samples from the “septic” patient followed incubation of the bottles in automatic thermostated systems. In case of positive result (36 hours, the culture is Gram stained and streaked on solid media in order to obtain isolated colonies for the identification and the susceptibility testing (48 hours from positive result. The long time required for pathogen identification and susceptibility testing involves empirical broad spectrum antibiotic therapy that can promote the increase of bacterial resistance but also patient management costs. A clinically useful report should be available on short notice in order to guide the clinician to choose the most appropriate antibiotic. The microbiologist has therefore the hard work of reviewing the organization and the management of the procedures.We have therefore started to consider the possibility of treating the blood as an biological liquid in order to quickly determine the susceptibility of bacteria to antibiotics.
Valeria Cataneli Pereira
Full Text Available Coagulase-negative staphylococci (CoNS are the microorganisms most frequently isolated from clinical samples and are commonly found in neonatal blood cultures. Oxacillin is an alternative treatment of choice for CoNS infections; however, resistance to oxacillin can have a substantial impact on healthcare by adversely affecting morbidity and mortality. The objective of this study was to detect and characterise oxacillin-resistant CoNS strains in blood cultures of newborns hospitalised at the neonatal ward of the University Hospital of the Faculty of Medicine of Botucatu. One hundred CoNS strains were isolated and the mecA gene was detected in 69 of the CoNS strains, including 73.2% of Staphylococcus epidermidis strains, 85.7% of Staphylococcus haemolyticus strains, 28.6% of Staphylococcus hominis strains and 50% of Staphylococcus lugdunensis strains. Among these oxacillin-resistant CoNS strains, staphylococcal cassette chromosome mec (SCCmec type I was identified in 24.6%, type II in 4.3%, type III in 56.5% and type IV in 14.5% of the strains. The data revealed an increase in the percentage of CoNS strains isolated from blood cultures from 1991-2009. Furthermore, a predominant SCCmec profile of the oxacillin-resistant CoNS strains isolated from neonatal intensive care units was identified with a prevalence of SCCmec types found in hospital-acquired strains.
Yamada, Tomoyuki; Suzuki, Kaoru; Ohi, Yukimasa; Kawanishi, Fumiko; Shibata, Yuriko; Hosomi, Makoto; Goto, Emi; Nishihara, Masami; Katsumata, Takahiro; Ukimura, Akira
Cancer patients at a high risk of acquiring infectious diseases should be maintained in a facility where good infection control practices are followed. At our hospital, the infection control team(ICT)provides expertise, education, and support to the staff, helping them maintain proper standards, thereby minimizing the risks of infection. The ICT(established in 2004)has implemented infection control programs by employing an appropriate number of staff members after the revision of medical treatment fees in 2011. Our intervention program includes 2 general policies, namely, ordering and collection of blood cultures and intervention for the medical care of patients with positive blood cultures. In this study, we evaluated the effectiveness of our intervention for cancer patients with a positive blood culture. During the surveillance period(April 2011 to July 2012), 42 positive cases were determined to be infectious. ICT intervention was required in 37 cases. Our suggestions were accepted in 92%(34/37)of the cases, and improved outcome was estimated in 65%(22/34)of the cases. The results of our study contribute to the scientific bases on which routine clinical practices could be promoted in the future.
Hammarström, Björn; Nilson, Bo; Laurell, Thomas; Nilsson, Johan; Ekström, Simon
Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently changing the clinical routine for identification of microbial pathogens. One important application is the rapid identification of bacteria for the diagnosis of bloodstream infections (BSI). A novel approach based on acoustic trapping and an integrated selective enrichment target (ISET) microchip that improves the sample preparation step for this type of analysis is presented. The method is evaluated on clinically relevant samples in the form of Escherichia coli infected blood cultures. It is shown that noncontact acoustic trapping enables capture, enrichment, and washing of bacteria directly from the complex background of crude blood cultures. The technology replaces centrifugation-based separation with a faster and highly automated sample preparation method that minimizes manual handling of hazardous pathogens. The presented method includes a solid phase extraction step that was optimized for enrichment of the bacterial proteins and peptides that are used for bacterial identification. The acoustic trapping-based assay provided correct identification in 12 out 12 cases of E. coli positive blood cultures with an average score of 2.19 ± 0.09 compared to 1.98 ± 0.08 when using the standard assay. This new technology opens up the possibility to automate and speed up an important and widely used diagnostic assay for bloodstream infections.
Full Text Available Staphylococcus epidermidis is an important cause of bloodstream infections in patients with hematological malignancies. Knowledge of the long-term epidemiology of these infections is limited. We surveyed all S. epidermidis blood culture isolates from patients treated for hematological malignancies at the University Hospital of Örebro, Sweden from 1980 to 2009. A total of 373 S. epidermidis isolates were identified and multilocus sequence typing, staphylococcal chromosome cassette mec (SCCmec typing and standard antibiotic susceptibility testing were employed to characterize these isolates. The majority of the isolates 361/373 (97% belonged to clonal complex 2, and the 373 isolates were divided into 45 sequence types (STs; Simpson's Diversity Index was 0.56. The most prevalent STs were ST2 (243/373, 65% and ST215 (28/373, 8%. Ninety three percent (226/243 of the ST2 isolates displayed either SCCmec type III or IV. ST2 and 215 were isolated during the entire study period, and together these STs caused temporal peaks in the number of positive blood cultures of S. epidermidis. Methicillin resistance was detected in 213/273 (78% of all isolates. In the two predominating STs, ST2 and ST215, methicillin resistance was detected in 256/271 isolates (95%, compared with 34/100 (34% in other STs (p<0.001. In conclusion, in this long-term study of patients with hematological malignancies, we demonstrate a predominance of methicillin-resistant ST2 among S. epidermidis blood culture isolates.
Damsgaard, Camilla T.; Lauritzen, Lotte; Calder, Philip C.
assessed the intra- and inter-individual variation in cytokine production. In 64 healthy men (age 19-40 years) IL-6, TNF and IL-10 were measured by enzyme-linked immunosorbent assay in supernatants from whole-blood, PBMC and monocytes cultured 24 h with lipopolysaccharide (LPS) or UV-killed L acidophilus......Whole-blood and peripheral blood mononuclear cell (PBMC) cultures are used as non-validated surrogate measures of monocytic cytokine production. The aim of this investigation was to compare ex vivo cytokine production from human whole-blood and PBMC with that from isolated monocytes. We also...
Full Text Available Background: Acinetobacter spp are associated with various nosocomial infections like as septicemia and are isolated form blood cultures in hospitalized patients. Methods: In this study, 45 Acinetobacter strains were isolated from blood samples in Yazd shahid sadoughi hospital from 21 March 2005 to 20 September 2006 and were identified by biochemical tests. Antibiotic susceptibility of the strains was tested by standard disk diffusion method. Results: In this research, 45 isolates identified as Acinetobacter and of isolated strains, 88.8% of them found sensitive to imipenem and 80% to ciprofloxacin. Also 51.5% to nalidixic Acid 24.5% to trimethoprim/sulphametoxazole, 11.1% to ceftazidim and ceftriaxone, 8.8% to cefotaxime and cefexime and also 6.6% to ceftizoxime. Conclusion: Because of increasing of drug resistance in Acinetobacter spp. Isolated from blood samples, it is necessary to perform susceptibility testing, also imipenem and ciprofloxacin recommended for drug therapy.
Full Text Available Introduction: Determining frequency of bacteria, isolated from blood culture and their antibiotic susceptibility patterns, has epidemiological significance and can help in selecting empirical therapy. This study was aimed to assess, the frequency of bacteria isolated from blood culture of patients suspected to bacteremia and their antibiotic susceptibility patterns. Methods: Culture of blood and determination of antibiotic susceptibility was done by standard methods. In this study, a variety of isolated bacteria types, antibiotic susceptibility, as well as age, sex and type of admission of patients were analyzed in a university hospital from 21 March, 2006 to 20 March, 2007. Results: During one year, blood culture was done for 5116 patients and bacteria were isolated in 912 cases (17.8%. Three most frequently groups of bacteria in blood cultures of patients were non-fermentative gram negative bacteria (Pseudomonas and Acintobacter spp, coliforms (Escherichia coli and enterobacter and klebsiella spp. and coagulase negative staphylococci, respectively, which were isolated in 63.4%, 17.0% and 12.8% of patients, and constituted 93.2% of positive blood cultures. Higher resistance was shown in bacteria isolated from inpatients compare to outpatients. Conclusion: This study showed the influence of age, sex and type of admission (outpatient or inpatient in a variety of isolated bacteria in blood culture. The result of this study were the same as the other studies in Iran and other countries in respect of the variety of isolated bacteria and antibiotic susceptibility and show increase of antibiotic resistance in these bacteria.
Wang, Hye-young; Yoo, Gilsung; Kim, Juwon; Uh, Young; Song, Wonkeun; Kim, Jong Bae; Lee, Hyeyoung
Rapid and accurate identification of the causative pathogens of bloodstream infections is crucial for the prompt initiation of appropriate antimicrobial therapy to decrease the related morbidity and mortality rates. The aim of this study was to evaluate the performance of a newly developed PCR-reverse blot hybridization assay (REBA) for the rapid detection of Gram-negative bacteria (GNB) and their extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, and carbapenemase resistance genes directly from the blood culture bottles. The REBA-EAC (ESBL, AmpC β-lactamase, carbapenemase) assay was performed on 327 isolates that were confirmed to have an ESBL producer phenotype, 200 positive blood culture (PBCs) specimens, and 200 negative blood culture specimens. The concordance rate between the results of REBA-EAC assay and ESBL phenotypic test was 94.2%. The sensitivity, specificity, positive predictive value, and negative predictive value of the REBA-EAC assay for GNB identification in blood culture specimens were 100% (95% CI 0.938–1.000, P < 0.001), 100% (95% CI 0.986–1.000, P < 0.001), 100% (95% CI 0.938–1.000, P < 0.001), and 100% (95% CI 0.986–1.000, P < 0.001), respectively. All 17 EAC-producing GNB isolates from the 73 PBCs were detected by the REBA-EAC assay. The REBA-EAC assay allowed easy differentiation between EAC and non-EAC genes in all isolates. Moreover, the REBA-EAC assay was a rapid and reliable method for identifying GNB and their β-lactamase resistance genes in PBCs. Thus, this assay may provide essential information for accelerating therapeutic decisions to achieve earlier appropriate antibiotic treatment during the acute phase of bloodstream infection. PMID:28232823
Wang, Hye-Young; Yoo, Gilsung; Kim, Juwon; Uh, Young; Song, Wonkeun; Kim, Jong Bae; Lee, Hyeyoung
Rapid and accurate identification of the causative pathogens of bloodstream infections is crucial for the prompt initiation of appropriate antimicrobial therapy to decrease the related morbidity and mortality rates. The aim of this study was to evaluate the performance of a newly developed PCR-reverse blot hybridization assay (REBA) for the rapid detection of Gram-negative bacteria (GNB) and their extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, and carbapenemase resistance genes directly from the blood culture bottles. The REBA-EAC (ESBL, AmpC β-lactamase, carbapenemase) assay was performed on 327 isolates that were confirmed to have an ESBL producer phenotype, 200 positive blood culture (PBCs) specimens, and 200 negative blood culture specimens. The concordance rate between the results of REBA-EAC assay and ESBL phenotypic test was 94.2%. The sensitivity, specificity, positive predictive value, and negative predictive value of the REBA-EAC assay for GNB identification in blood culture specimens were 100% (95% CI 0.938-1.000, P < 0.001), 100% (95% CI 0.986-1.000, P < 0.001), 100% (95% CI 0.938-1.000, P < 0.001), and 100% (95% CI 0.986-1.000, P < 0.001), respectively. All 17 EAC-producing GNB isolates from the 73 PBCs were detected by the REBA-EAC assay. The REBA-EAC assay allowed easy differentiation between EAC and non-EAC genes in all isolates. Moreover, the REBA-EAC assay was a rapid and reliable method for identifying GNB and their β-lactamase resistance genes in PBCs. Thus, this assay may provide essential information for accelerating therapeutic decisions to achieve earlier appropriate antibiotic treatment during the acute phase of bloodstream infection.
Full Text Available Brucellosis is one of the most common zoonotic diseases in Iran and human brucellosis is endemic in all parts of the country. Growth of Brucella is slow and blood culture of these bacteria by use of classical methods is time-consuming. Furthermore, in endemic area culture is required for definitive diagnosis. In the present study, direct urease test and acridine orange staining were tried on the BACTEC blood culture broths for early presumptive identification of Brucella growth. Blood cultures were attempted in 102 seropositive patients. In the forty one blood cultures positive for Brucella, coccobacilli were seen in broth smears stained with acridine orange stain, and also were urease test positive, thus providing presumptive identification of Brucella growth. Urease test was negative and bacteria were not seen in the broth smears of the remaining 61 broths negative for Brucella growth. Because of simplicity, reliability and reproducibility, these tests can be routinely incorporated in the laboratory for diagnosis of brucellosis.
Delibato, Elisabetta; Fiore, Alfonsina; Anniballi, Fabrizio; Auricchio, Bruna; Filetici, Emma; Orefice, Leucio; Losio, Marina Nadia; De Medici, Dario
Food-borne diseases caused by Salmonella represent a worldwide public health problem. Salmonella must be absent in an established amount depending on the kind of the product and usually cultural methods have to be applied to evaluate the compliance of the products. ISO 6579:2002 in Europe and FSIS MLG 4.04.:2008 in the USA have usually been employed to detect Salmonella in meat, poultry and egg products. A Real Time PCR method using probes has recently been validated against the NMKL (Nordic Committee on Food Analysis) standard method. This method has been modified using the less expensive Sybr Green Real Time PCR approach and applied directly in the 18 hours preenrichment broth for the purpose of detecting Salmonella in meat products in less than 24 hours. The purpose of this study was to: - compare the effectiveness of ISO and FSIS cultural methods; - develop a new 24 hour duplex Sybr Green Real Time PCR-melting curve analysis; - evaluate the performance of Salmonella, Standard Method, Rapid Method, SYBR Green Real Time PCR. The equivalence between ISO and FSIS methods was demonstrated and the use of SYBR Green Real Time PCR as a screening tool for negative results seems appealing especially to evaluate compliance with the HACCP systems.
Intra, J; Sala, M R; Falbo, R; Cappellini, F; Brambilla, P
Rapid and early identification of micro-organisms in blood has a key role in the diagnosis of a febrile patient, in particular, in guiding the clinician to define the correct antibiotic therapy. This study presents a simple and very fast method with high performances for identifying bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after only 4 h of incubation. We used early bacterial growth on PolyViteX chocolate agar plates inoculated with five drops of blood-broth medium deposited in the same point and spread with a sterile loop, followed by a direct transfer procedure on MALDI-TOF MS target slides without additional modification. Ninety-nine percentage of aerobic bacteria were correctly identified from 600 monomicrobial-positive blood cultures. This procedure allowed obtaining the correct identification of fastidious pathogens, such as Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae that need complex nutritional and environmental requirements in order to grow. Compared to the traditional pathogen identification from blood cultures that takes over 24 h, the reliability of results, rapid performance and suitability of this protocol allowed a more rapid administration of optimal antimicrobial treatment in the patients.
Full Text Available BACKGROUND: The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. METHODS/PRINCIPAL FINDINGS: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD of 0.70 parasite equivalents/mL and a limit of quantification (LOQ of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. CONCLUSIONS/SIGNIFICANCE: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.
Abate, Teresa; Cayo, Nelly M.; Parrado, Rudy; Bello, Zoraida Diaz; Velazquez, Elsa; Muñoz-Calderon, Arturo; Juiz, Natalia A.; Basile, Joaquín; Garcia, Lineth; Riarte, Adelina; Nasser, Julio R.; Ocampo, Susana B.; Yadon, Zaida E.; Torrico, Faustino; de Noya, Belkisyole Alarcón; Ribeiro, Isabela; Schijman, Alejandro G.
Background The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal Findings We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/Significance The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment. PMID:23350002
Mantur, Basappa G; Mangalgi, Smita S
We investigated the role of the lysis centrifugation blood culture technique over the conventional Castaneda technique for the diagnosis of human brucellosis. The lysis centrifugation technique has been found to be more sensitive in both acute (20% higher sensitivity; P < 0.00001) and chronic (40% higher sensitivity; P = 0.087) forms of brucellosis. The major advantage of lysis centrifugation was in the mean detection time, which was only 2.4 days in acute and 2.7 days in chronic cases, with 103 out of 110 (93.6%) and 17 out of 20 (85%) cultures from acute and chronic brucellosis, respectively, detected before the conventional culture was positive. Our results confirmed the potential usefulness of the lysis technique in diagnosis and institution of appropriate antibiotic therapy.
Dodémont, Magali; De Mendonça, Ricardo; Nonhoff, Claire; Roisin, Sandrine; Denis, Olivier
Nonduplicate blood cultures that were positive for Gram-negative bacilli (n = 125) were tested by the Verigene Gram-negative blood culture (BC-GN) assay; 117 (90.7%) isolates were members of the panel. For identification and resistance markers, the agreements with routine methods were 97.4% (114/117) and 92.3% (12/13). The BC-GN assay is a rapid and accurate tool for the detection of pathogens from blood cultures and could be integrated alongside conventional systems to enable faster patient management, but the clinical benefits should be further evaluated.
Human adenovirus is relatively resistant to UV radiation and has been used as a conservative testing microbe for evaluations of UV disinfection systems as components of water treatment processes. In this study, we attempted to validate the applicability of integrated cell culture...
Birkenheuer, Adam J.; Levy, Michael G.; Breitschwerdt, Edward B.
Canine babesiosis has recently been recognized as an emerging infectious disease of dogs in North America. We sought to develop a seminested PCR to detect and differentiate Babesia gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. canis, and B. canis subsp. rossi DNA in canine blood samples. An outer primer pair was designed to amplify an ∼340-bp fragment of the 18S rRNA genes from B. gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. rossi, and B. canis subsp. ...
Mercier-Delarue, Severine; Vray, Muriel; Plantier, Jean Christophe; Maillard, Theodora; Adjout, Zidan; de Olivera, Fabienne; Schnepf, Nathalie; Maylin, Sarah; Simon, Francois; Delaugerre, Constance
Dried blood spots (DBS) are widely proposed as a plasma surrogate for monitoring antiretroviral treatment efficacy based on the HIV-1 RNA level (viral load [VL]) in resource-limited settings. Interfering coamplification of cell-associated HIV-1 DNA during reverse transcription (RT)-PCR can be avoided by using nucleic acid sequence-based amplification (NASBA) technology, which is based on an RNA template and isothermic conditions. We analyzed VL values obtained with DBS and plasma samples by comparing isothermic NASBA (NucliSENS EasyQ HIV-1 V2.0; bioMérieux) with real-time RT-PCR (Cobas TaqMan HIV-1 V2.0; Roche). Samples from 197 HIV-1-infected patients were tested (non-B subtypes in 51% of the cases). Nucleic acid extractions were performed by use of NucliSENS EasyMAG (bioMérieux) and Cobas AmpliPrep (Roche) before the NASBA and RT-PCR quantifications, respectively. Both quantification assays have lower limits of detection of 20 (1.3) and 800 (2.9) log10 copies/ml (log) in plasma and DBS, respectively. The mean (DBS minus plasma) differences were -0.39 and -0.46 log, respectively, for RT-PCR and NASBA. RT-PCR on DBS identified virological failure in 122 of 126 patients (sensitivity, 97%) and viral suppression in 58 of 70 patients (specificity, 83%), yielding 12 false-positive results (median, 3.2 log). NASBA on DBS identified virological failure in 85 of 96 patients (sensitivity, 89%) and viral suppression in 95 of 97 patients (specificity, 98%) and yielded 2 false-positive results (3.0 log for both). Both technologies detected HIV-1 RNA in DBS at a threshold of 800 copies/ml. This higher specificity of NASBA technology could avoid overestimation of poor compliance or the emergence of resistance when monitoring antiretroviral efficacy with the DBS method.
FANG Feng-qin; ZHANG Yue; LU Ping; ZHANG Li; JI Yu-hua
Objective To quantify proline 4-hydroxylase, alpha polypeptide Ⅱ (P4HA2) mRNA of human embryo lung fibroblast (HELF) with SYBR green based reversed transcript PCR (RT-PCR) for correcting cytomegalovirus (CMV) inactivation or clearance efficiency in donor blood.Methods A pair of specific primers of exon 12a of P4HA2 was designed, and the related PCR-reaction system and condition were optimized. Then the recombinant plasmid containing the target fragment was constructed for making standard curve with SYBR green based real-time RT-PCR. Finally, the sensitivity, reproducibility, and specificity of this method were fully estimated.Results The sensitivity of the method was 1.5E+04 copies/mL of P4HA2 mRNA, corresponding to 103 fibroblasts. In addition, existence of 8.67E+06 leukocytes could not interfere with the accurate quantification of HELF in the large dynamic range. The intra-assay variability and inter-assay variability both varied in different concentrations, being higher in low concentrations and lower in high concentrations. But all of them were below 13.76% in variation, which showed acceptable stability of this method.Conclusion SYBR green and specific primer based real-time RT-PCR show up a good quality for quantifying HELF P4HA2 mRNA with good specificity, stability, and high sensitivity. Approximate 10 copies of P4HA2 mRNA per cell in average can be detected by the method. Therefore, this method can be used to deduct fibroblast-associated CMV for correcting CMV inactivation efficiency in leukocytes.
Gwida, Mayada M; Al-Ashmawy, Maha A M
A total of 200 samples of milk and dairy products as well as 120 samples of dairy handlers were randomly collected from different dairy farms and supermarkets in Dakahlia Governorate, Egypt. The conventional cultural and serotyping methods for detection of Salmonella in dairy products were applied and the results were compared with those obtained by molecular screening assay using (ttr sequence). The obtained results revealed that 21% of milk and dairy products (42/200) were positive for Salmonella species using enrichment culture-based PCR method, while 12% of different dairy samples (24/200) were found to be positive for Salmonella species by using the conventional culture methods. Two stool specimens out of 40 apparently healthy dairy handlers were positive by the PCR method. Serotyping of Salmonella isolates revealed that 58.3% (14/24) from different dairy products were contaminated with Salmonella Typhimurium. We conclude that the enrichment culture-based PCR assay has high sensitivity and specificity for detection of Salmonella species in dairy products and handlers. High incidence of Salmonella Typhimurium in the examined dairy samples highlights the important role played by milk and dairy products as a vehicle in disease prevalence. Great effort should be applied for reducing foodborne risk for consumers.
Plain, Karren M; Waldron, Anna M; Begg, Douglas J; de Silva, Kumudika; Purdie, Auriol C; Whittington, Richard J
Pathogenic mycobacteria are difficult to culture, requiring specialized media and a long incubation time, and have complex and exceedingly robust cell walls. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, a chronic wasting disease of ruminants, is a typical example. Culture of MAP from the feces and intestinal tissues is a commonly used test for confirmation of infection. Liquid medium offers greater sensitivity than solid medium for detection of MAP; however, support for the BD Bactec 460 system commonly used for this purpose has been discontinued. We previously developed a new liquid culture medium, M7H9C, to replace it, with confirmation of growth reliant on PCR. Here, we report an efficient DNA isolation and quantitative PCR methodology for the specific detection and confirmation of MAP growth in liquid culture media containing egg yolk. The analytical sensitivity was at least 10(4)-fold higher than a commonly used method involving ethanol precipitation of DNA and conventional PCR; this may be partly due to the addition of a bead-beating step to manually disrupt the cell wall of the mycobacteria. The limit of detection, determined using pure cultures of two different MAP strains, was 100 to 1,000 MAP organisms/ml. The diagnostic accuracy was confirmed using a panel of cattle fecal (n=54) and sheep fecal and tissue (n=90) culture samples. This technique is directly relevant for diagnostic laboratories that perform MAP cultures but may also be applicable to the detection of other species, including M. avium and M. tuberculosis.
Idelevich, Evgeny A; Schüle, Isabel; Grünastel, Barbara; Wüllenweber, Jörg; Peters, Georg; Becker, Karsten
Briefly incubated agar cultures from positive blood cultures were used for antimicrobial susceptibility testing (AST) by Vitek 2. The cultivation time until inoculation was 3.8 h for Gram-positive cocci and 2.4 h for Gram-negative rods. The error rates were low, providing early and reliable AST without additional time or cost expenditure.
Performance of the FilmArray® blood culture identification panel utilized by non-expert staff compared with conventional microbial identification and antimicrobial resistance gene detection from positive blood cultures.
McCoy, Morgan H; Relich, Ryan F; Davis, Thomas E; Schmitt, Bryan H
Utilization of commercially available rapid platforms for microbial identification from positive blood cultures is useful during periods of, or in laboratories with, limited expert staffing. We compared the results of the FilmArray® BCID Panel performed by non-expert technologists to those of conventional methods for organism identification performed by skilled microbiologists. Within 8 h of signalling positive by a continuous monitoring blood culture system, positive bottles were analysed by the FilmArray BCID Panel. Data from these analyses were compared to standard-of-care testing, which included conventional and automated methods. To gauge the ease of use of the BCID Panel by non-expert staff, technologists unfamiliar with diagnostic bacteriology performed the testing without prior knowledge of the Gram stain results, or even whether organisms were detected. Identifications of 172/200 (86 %) positive blood cultures using the BCID Panel were consistent with identifications provided by standard-of-care methods. Standard-of-care testing identified organisms in 20 positive blood cultures, which were not represented on the BCID Panel. Seven (3.5 %) blood cultures demonstrated a discrepancy between the methods, which could not be attributed to either a lack of representation on the panel or unclear separate detection of organisms in a mixed blood culture of a shared genus or grouping of organisms, e.g. Staphylococcus or Enterobacteriaceae . One (0.5 %) blood culture yielded invalid results on two separate panels, so it was eliminated from the study. The easy-to-use FilmArray® technology shows good correlation with blood culture identification and antibiotic resistance detection performed by conventional methods. This technology may be particularly useful in laboratories with limited staffing or limited technical expertise.
Handrup, Mette Møller; Møller, Jens Kjølseth; Rutkjaer, Cecilie
When an infection is suspected in a child with cancer and a central venous line (CVL), cultures are often only obtained from the CVL and not from a peripheral vein (PV). This study was undertaken to evaluate the importance of concomitant blood cultures from the CVL and a PV.......When an infection is suspected in a child with cancer and a central venous line (CVL), cultures are often only obtained from the CVL and not from a peripheral vein (PV). This study was undertaken to evaluate the importance of concomitant blood cultures from the CVL and a PV....
Full Text Available BACKGROUND: Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. FINDINGS: We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2, hepatitis B virus (HBV, hepatitis C virus (HCV, human T-cell leukemia virus-1 and -2 (HTLV-1 and -2, vaccinia virus (VACV and West Nile virus (WNV. One hundred twenty (120 primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B, HBV (genotype A and WNV. It detected 100-1,000 geq/ml of plasma of HIV-1 subtypes (A - G, group N and CRF (AE and AG isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction. CONCLUSIONS: The viral array described here demonstrated adequate performance in the testing of donors' clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development.
Arnold, T; Scholz, H C; Marg, H; Rösler, U; Hensel, A
This study evaluated the suitability of invA gene amplification by PCR as an effective means of detecting Salmonella species in pigs experimentally infected with S. Typhimurium DT104. A controlled infection study using 24 pigs was performed in order to compare efficacy, precision and detection rates of the invA-based PCR method originally described by Rahn, K. De Grandis, S.A., Clarke, R.C., McEwan, S.A., Galan, J.E., Ginocchio, C., Curtiss, R. 3rd, C.L. Gyles, (Mol. Cell. Probes 1992; 6: 271-279) as a new in-house invA-based PCR method for the specific detection of Salmonella spp. in pork and different tissue samples of slaughter pigs. Finally, PCR results were compared with culture detection rates obtained by isolation procedures following the ISO 6579:2000, the 'gold standard'. After slaughtering, 14 different tissue samples of each pig were investigated to verify the usefulness of the two invA-based PCR methods in different matrices of slaughter pigs. The results demonstrate that the application of the widely used invA-based primer pair (139 + 141) may result in questionable products if samples gained from selective enrichment in the Rappaport-Vassiliadis medium were investigated. These questionable products can lead to false-positive results, if no additional hybridization procedure is attached or if unspecialized persons use this method in routine laboratory practice. The newly developed in-house PCR method used is based on the 3'-prime region of invA, especially designed and harmonized for the detection of Salmonella in different matrices of slaughtered pigs after bacterial enriched broth culture. In this study, this PCR revealed no questionable products and, furthermore, the specificity of the amplificate could be tested by means of the restriction enzyme NdeI. In comparison with the culture detection procedure, the new PCR method has a sensitivity of 100% and a specificity of 96%. Thus, this method might be used as a meaningful tool in eliminating
Lim, Sung H; Mix, Samantha; Xu, Zeyu; Taba, Brian; Budvytiene, Indre; Berliner, Anders N; Queralto, Nuria; Churi, Yair S; Huang, Richard S; Eiden, Michael; Martino, Raymond A; Rhodes, Paul; Banaei, Niaz
Sepsis is a medical emergency demanding early diagnosis and tailored antimicrobial therapy. Every hour of delay in initiating effective therapy measurably increases patient mortality. Blood culture is currently the reference standard for detecting bloodstream infection, a multistep process which may take one to several days. Here, we report a novel paradigm for earlier detection and the simultaneous identification of pathogens in spiked blood cultures by means of a metabolomic "fingerprint" of the volatile mixture outgassed by the organisms. The colorimetric sensor array provided significantly faster detection of positive blood cultures than a conventional blood culture system (12.1 h versus 14.9 h, P detection. The colorimetric sensor array also allowed for discrimination between unrelated strains of methicillin-resistant Staphylococcus aureus, indicating that the metabolomic fingerprint has the potential to track nosocomial transmissions. Altogether, the colorimetric sensor array is a promising tool that offers a new paradigm for diagnosing bloodstream infections.
Hartmann, Sofie Bruun; Emnéus, Jenny; Wolff, Anders;
. However, there is no consensus whether to use whole blood cultures or purified PBMCs for the assay, and both cell populations are being used and results compared. Therefore the aim of this study was to compare different culture settings using immune cells from previously vaccinated calves, and to shed...... light on external factors that could influence the read out in terms of IFN-γ levels. It was found that optimal culture conditions varied between individual animals; when polyclonal activated, cells from whole blood cultures were most responsive, but when activated specifically, the optimal cell...... concentration/population varied with whole blood, 10 × 106 cells/ml PBMC and 5 × 106 cells/ml PBMC being the highest performing conditions. A further investigation of the distribution of cell populations in PBMCs compared to whole blood was conducted, and a significant (p
Çelikezen, Fatih Çağlar; Toğar, Başak; Özgeriş, Fatma Betül; İzgi, Mehmet Sait; Türkez, Hasan
Boron compounds have an ability of supporting antioxidant properties in human and animal tissues. Lithium metaborate dihydrate (LiBO2·2H2O; LMD) is commonly used in nonlinear optic materials, cellular phones and pagers. But, there are limited data on the genotoxic and antioxidant effects of LMD in cultured human whole blood cells. The aim of this study was to evaluate for the genotoxicity and antioxidant/oxidant activity of LMD on human whole blood lymphocytes (n = 5). LMD was applied at various concentrations (0-1,280 µg/ml) to cultured blood samples. Antioxidant/oxidant activity was evaluated by measuring the total oxidant status (TOS) and total antioxidant capacity levels. Micronuclei and chromosomal aberration tests were used in genotoxicity studies. Our results clearly revealed that all tested concentrations of LMD were found to be non-genotoxic when compared to that of the control group. In addition, LMD exhibited antioxidant activities at low concentrations. In addition the TOS levels were not changed at all concentrations of LMD. Consequently, our results clearly demonstrated that LMD is non-genotoxic and it has an important antioxidant potential in vitro.
Full Text Available Abstract Background: Imipenem is a betalactam antibioticthat has antibacterial activity against gram positive,gram negative and anerobic species. Antibiotic resistance is a problem in Iran. In this study, we asses imipenem resistance in blood cultures in Bushehr educational hospitals. Material and method:This cross sectional study was done in 2010. Blood cultures were taken from admitted patients in hospitals. For all samples, antibiogram with disk diffusion was done. And result of culture was categorized into three groups :resistance, sensitive and intermediate. Data was analyzed with SPSS Version13. Results: This study consisted of 200 patients. 48% Male, 37% female, 14% NeonateThe age of study group was ranged between 1 and 90 years (Mean 21, Standard deviation 28. . Psuedomonus aeroginosa and staphilococcus epidermis had higher perevalence. Resistancy to imipenem was 29/5%. Resistancy to imipenem was 41/4% in NICU. Concdlsion: Findings indicated that antibiotic resistancy is increasing in Bushehr. Therfore, it is necessary to modify antibiotic prescription and restrict using wide spectrum antibiotics such as imipenem.
Verroken, A; Defourny, L; Lechgar, L; Magnette, A; Delmée, M; Glupczynski, Y
Speeding up the turn-around time of positive blood culture identifications is essential in order to optimize the treatment of septic patients. Several sample preparation techniques have been developed allowing direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification of positive blood cultures. Yet, the hands-on time restrains their routine workflow. In this study, we evaluated an approach whereby MALDI-TOF MS identification without any additional steps was carried out on short subcultured colonies from positive blood bottles with the objective of allowing results reporting on the day of positivity detection. Over a 7-month period in 2012, positive blood cultures detected by 9 am with an automated system were inoculated onto a Columbia blood agar and processed after a 5-h incubation on a MALDI-TOF MicroFlex platform (Bruker Daltonik GmbH). Single-spotted colonies were covered with 1 μl formic acid and 1 μl matrix solution. The results were compared to the validated identification techniques. A total of 925 positive blood culture bottles (representing 470 bacteremic episodes) were included. Concordant identification was obtained in 727 (81.1 %) of the 896 monomicrobial blood cultures, with failure being mostly observed with anaerobes and yeasts. In 17 episodes of polymicrobic bacteremia, the identification of one of the two isolates was achieved in 24/29 (82.7 %) positive cultures. Routine implementation of MALDI-TOF MS identification on young positive blood subcultures provides correct results to the clinician in more than 80 % of the bacteremic episodes and allows access to identification results on the day of blood culture positivity detection, potentially accelerating the implementation of targeted clinical treatments.
Naseri, Zahra; Alikhani, Mohammad Yousef; Hashemi, Seyed Hamid; Kamarehei, Farideh; Arabestani, Mohammad Reza
Brucellosis is a widespread zoonotic disease causing considerable economic and public health problems. Despite animal vaccination, brucellosis remains endemic in some areas such as Iran, especially in the western Iranian province of Hamadan. We sought to detect some of the most common virulence-associated genes in Brucella isolated from human blood cultures to determine the prevalence of some virulence genes among Brucella isolates. Fifty-seven isolates were studied from patients with a clinical diagnosis of brucellosis who referred to the Infectious Diseases Ward of Sina Hospital in Hamadan Province, Iran, between April 2013 and July 2014. Blood samples were collected for the diagnosis of brucellosis using the BACTEC blood culture system. All of these isolates were confirmed by the bcsp31 Brucella-specific gene. We detected 11 virulence-associated genes of Brucella, namely cβg, virB, znuA, ure, bvfA, omp25, omp31, wbkA, mviN, manA, and manB, which are important for the pathogenesis of this bacterium in the intracellular environment by multiplex PCR. Totally, 149 patients with a clinical diagnosis of brucellosis were enrolled in this study. Fifty-seven (38.3%) patients had positive blood cultures. On biochemical and molecular testing, all of the isolates were Brucella melitensis. Ten of the virulence genes were detected among all of the 57 isolates, but the bvf gene was detected in 53 (93%) isolates. The high prevalence of virulence-associated genes among the Brucella isolates detected in Hamadan Province, Iran, underscores the pathogenicity of this bacterium in this region. PMID:27582592
Full Text Available Brucellosis is a widespread zoonotic disease causing considerable economic and public health problems. Despite animal vaccination, brucellosis remains endemic in some areas such as Iran, especially in the western Iranian province of Hamadan. We sought to detect some of the most common virulence-associated genes in Brucella isolated from human blood cultures to determine the prevalence of some virulence genes among Brucella isolates. Fifty-seven isolates were studied from patients with a clinical diagnosis of brucellosis who referred to the Infectious Diseases Ward of Sina Hospital in Hamadan Province, Iran, between April 2013 and July 2014. Blood samples were collected for the diagnosis of brucellosis using the BACTEC blood culture system. All of these isolates were confirmed by the bcsp31 Brucella-specific gene. We detected 11 virulence-associated genes of Brucella, namely cβg, virB, znuA, ure, bvfA, omp25, omp31, wbkA, mviN, manA, and manB, which are important for the pathogenesis of this bacterium in the intracellular environment by multiplex PCR. Totally, 149 patients with a clinical diagnosis of brucellosis were enrolled in this study. Fifty-seven (38.3% patients had positive blood cultures. On biochemical and molecular testing, all of the isolates were Brucella melitensis. Ten of the virulence genes were detected among all of the 57 isolates, but the bvf gene was detected in 53 (93% isolates. The high prevalence of virulence-associated genes among the Brucella isolates detected in Hamadan Province, Iran, underscores the pathogenicity of this bacterium in this region.
Claeys, Kimberly C; Zasowski, Evan J; Lagnf, Abdalhamid M; Rybak, Michael J
Enterococci represent one of the most common causative pathogens of bloodstream infections (BSIs). There is debate in the literature regarding the clinical importance of single versus multiple positive blood cultures for Enterococci. This single-center retrospective study found that patients with multiple positive blood cultures experienced increased inpatient mortality and a shorter median survival. Additionally, BSIs >6.7 days resulted in approximately 20% increased mortality. These results are preliminary and require further exploration.
March, Gabriel A; García-Loygorri, María C; Simarro, María; Gutiérrez, María P; Orduña, Antonio; Bratos, Miguel A
The rapid identification and antibiotic susceptibility test of bacteria causing bloodstream infections are given a very high priority by clinical laboratories. In an effort to reduce the time required for performing antibiotic susceptibility test (AST), we have developed a new method to be applied from positive blood culture bottles. The design of method was performed using blood culture bottles prepared artificially with five strains which have a known susceptibility. An aliquot of the blood culture was subcultured in the presence of specific antibiotics and bacterial counts were monitored using the Sysmex UF-1000i flow cytometer at different times up to 180min. Receiver operating curve (ROC) analysis allowed us to find out the cut-off point for differentiating between sensitive and resistant strains to the tested antibiotic. This procedure was then validated against standard commercial methods on a total of 100 positive blood culture bottles from patients. First, bacterial identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) directly from positive blood culture bottles as we have previously reported. Secondly, antibiotic susceptibility test was performed in the same way that was carried out in artificially prepared blood culture bottles. Our results indicate that antibiotic susceptibility test can be determined as early as 120min since a blood culture bottle is flagged as positive. The essential agreement between our susceptibility test and commercial methods (E-test, MicroScan and Vitek) was 99%. In summary, we conclude that reliable results on bacterial identification and antibiotic susceptibility test performed directly from positive blood culture bottles can be obtained within 3h.
Park, Eunyoung; Lee, Cheonghoon; Bisesi, Michael; Lee, Jiyoung
The disinfection efficiency of peracetic acid (PAA) was investigated on three microbial types using three different methods (filtration-based ATP (adenosine-triphosphate) bioluminescence, quantitative polymerase chain reaction (qPCR), culture-based method). Fecal indicator bacteria (Enterococcus faecium), virus indicator (male-specific (F(+)) coliphages (coliphages)), and protozoa disinfection surrogate (Bacillus subtilis spores (spores)) were tested. The mode of action for spore disinfection was visualized using scanning electron microscopy. The results indicated that PAA concentrations of 5 ppm (contact time: 5 min), 50 ppm (10 min), and 3,000 ppm (5 min) were needed to achieve 3-log reduction of E. faecium, coliphages, and spores, respectively. Scanning electron microscopy observation showed that PAA targets the external layers of spores. The lower reduction rates of tested microbes measured with qPCR suggest that qPCR may overestimate the surviving microbes. Collectively, PAA showed broad disinfection efficiency (susceptibility: E. faecium > coliphages > spores). For E. faecium and spores, ATP bioluminescence was substantially faster (∼5 min) than culture-based method (>24 h) and qPCR (2-3 h). This study suggests PAA as an effective alternative to inactivate broad types of microbial contaminants in water. Together with the use of rapid detection methods, this approach can be useful for urgent situations when timely response is needed for ensuring water quality.
Albonico, Francesca; Loiacono, Monica; Gioia, Gloria; Genchi, Claudio; Genchi, Marco; Mortarino, Michele
Dirofilaria immitis and D. repens are the principal causative agents of canine filariosis and, although the number of dogs subjected to specific prevention is increasing, the prevalence of these parasites remains high in many areas of the world. The discrimination between the two Dirofilaria species using the classical diagnostic methods can be difficult and may lead to misdiagnosis especially on samples from areas where both Dirofilaria are present. Over the last years, several molecular methods with higher sensitivity and specificity compared to classical microscopy and ELISA assays were designed. Nevertheless, a need for simple, rapid, and cost-effective molecular protocols to accurately discriminate between D. immitis and D. repens still remains. High resolution melting analysis coupled to real-time PCR (real-time PCR-HRMA) is a widely used technique to target sequence polymorphisms of the same gene in different species without the need to perform DNA sequencing or to use species-specific probes. In this work, a fast and cost-effective real-time PCR-HRMA protocol to detect and differentiate simultaneously and unequivocally D. immitis and D. repens microfilarial DNA extracted from peripheral dog blood samples is described. The present method is simpler to use than most other DNA-based methods and provides comparable discrimination between the two sibling species.
Full Text Available Triatoma dimidiata, currently the major Central American vector of Trypanosoma cruzi, the parasite that causes Chagas disease, inhabits caves throughout the region. This research investigates the possibility that cave dwelling T. dimidiata might transmit the parasite to humans and links the blood meal sources of cave vectors to cultural practices that differ among locations.We determined the blood meal sources of twenty-four T. dimidiata collected from two locations in Guatemala and one in Belize where human interactions with the caves differ. Blood meal sources were determined by cloning and sequencing PCR products amplified from DNA extracted from the vector abdomen using primers specific for the vertebrate 12S mitochondrial gene. The blood meal sources were inferred by ≥ 99% identity with published sequences. We found 70% of cave-collected T. dimidiata positive for human DNA. The vectors had fed on 10 additional vertebrates with a variety of relationships to humans, including companion animal (dog, food animals (pig, sheep/goat, wild animals (duck, two bat, two opossum species and commensal animals (mouse, rat. Vectors from all locations fed on humans and commensal animals. The blood meal sources differ among locations, as well as the likelihood of feeding on dog and food animals. Vectors from one location were tested for T. cruzi infection, and 30% (3/10 tested positive, including two positive for human blood meals.Cave dwelling Chagas disease vectors feed on humans and commensal animals as well as dog, food animals and wild animals. Blood meal sources were related to human uses of the caves. We caution that just as T. dimidiata in caves may pose an epidemiological risk, there may be other situations where risk is thought to be minimal, but is not.
Rosec, Jean-Philippe; Simon, Marie; Causse, Véronique; Boudjemaa, Mireille
PCR protocols directly applied to enrichment broth cultures were compared with a culture method based on the ISO reference for detection of Vibrio parahaemolyticus in 57 natural bivalve mollusc samples. Comparisons were made on different primer pairs specifically targeting the V. parahaemolyticus-specific toxR gene (Vp-toxR) and pR72H fragment, and also tdh and trh hemolysin genes. The PCR method using these different primer pairs and the culture method were also examined for their limits of detection (LOD). The LODs ranged from 7-24 pg of purified DNA per reaction tube (RT) for primer pair Vp-toxR, but for primer pair pR72H, varied greatly depending on the V. parahaemolyticus strains used (0.7 pg-10.6 ng/RT). The Vp-toxR and pR72H primers allowed the detection of V. parahaemolyticus in 25 and 8 out of the 57 samples, respectively, while only 3 V. parahaemolyticus-positive samples were obtained by the culture method. The effective presence of V. parahaemolyticus in the Vp-toxR-positive samples was confirmed by sequencing the PCR products. The trh and Vp-toxR genes were simultaneously detected in 14% of the samples, which were thus considered as presumptively contaminated with pathogenic V. parahaemolyticus. These results emphasize the need for an efficient survey of both the total and pathogenic V. parahaemolyticus present in seafood in France. The PCR protocol targeting Vp-toxR followed by tdh and trh genes is an efficient and reliable method for the detection of total and presumptively pathogenic V. parahaemolyticus in bivalve molluscs.
Taşkın, Hatıra; Baktemur, Gökhan; Kurul, Mehmet; Büyükalaca, Saadet
This study was performed for comparison of meristem culture technique with shoot tip culture technique for obtaining virus-free plant, comparison of micropropagation success of two different nutrient media, and determination of effectiveness of real-time PCR assay for the detection of viruses. Two different garlic species (Allium sativum and Allium tuncelianum) and two different nutrient media were used in this experiment. Results showed that Medium 2 was more successful compared to Medium 1 for both A. tuncelianum and A. sativum (Kastamonu garlic clone). In vitro plants obtained via meristem and shoot tip cultures were tested for determination of onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV) through real-time PCR assay. In garlic plants propagated via meristem culture, we could not detect any virus. OYDV and LYSV viruses were detected in plants obtained via shoot tip culture. OYDV virus was observed in amount of 80% and 73% of tested plants for A. tuncelianum and A. sativum, respectively. LYSV virus was found in amount of 67% of tested plants of A. tuncelianum and in amount of 87% of tested plants of A. sativum in this study.
Full Text Available This study was performed for comparison of meristem culture technique with shoot tip culture technique for obtaining virus-free plant, comparison of micropropagation success of two different nutrient media, and determination of effectiveness of real-time PCR assay for the detection of viruses. Two different garlic species (Allium sativum and Allium tuncelianum and two different nutrient media were used in this experiment. Results showed that Medium 2 was more successful compared to Medium 1 for both A. tuncelianum and A. sativum (Kastamonu garlic clone. In vitro plants obtained via meristem and shoot tip cultures were tested for determination of onion yellow dwarf virus (OYDV and leek yellow stripe virus (LYSV through real-time PCR assay. In garlic plants propagated via meristem culture, we could not detect any virus. OYDV and LYSV viruses were detected in plants obtained via shoot tip culture. OYDV virus was observed in amount of 80% and 73% of tested plants for A. tuncelianum and A. sativum, respectively. LYSV virus was found in amount of 67% of tested plants of A. tuncelianum and in amount of 87% of tested plants of A. sativum in this study.
A nested PCR method was developed and its performance evaluated by detection of Shigella flexneri in food. The nested PCR amplifies sequences within an invasion-associated locus (ial) on the invasion plasmid specific for Shigella and enteroinvasive Eschrichia coli (EIEC). The nested PCR detected Sh. flexneri in lettuce inoculated with 2, 20 and 200 cfu g-1 after 1, 7 and 18 d of storage, respectively. In comparison, a culture method (NMKL no. 151) detected 10(5) cfu g-1 after 1 but not after 7 d of storage. The presence of inhibitors in blue cheese and shrimps reduced the sensitivity of the PCR assay. To overcome this inhibition, a sample preparation step based on buoyant density centrifugation was developed. This treatment resulted in a successful recovery of Sh. flexneri in lettuce, milk, shrimp and blue cheese inoculated with 10 cfu g-1. The proposed method, which includes a combination of enrichment, buoyant density centrifugation and a nested PCR, can be completed in less than two working days.
Bocsik, Alexandra; Darula, Zsuzsanna; Tóth, Géza; Deli, Mária A; Wollemann, Mária
Opioid peptides are potent analgesics with therapeutic potential in the treatment of acute and chronic pain. Their efficacy is limited by peptidases (enkephalinases). Opiorphin pentapeptide (QRFSR) is the first characterized human endogenous inhibitor of enkephalinases. The peptide is able to increase the binding and affinity of endogenous opiates to mu opioid receptors; thus, the mechanism of opiorphin may provide a new therapeutic approach in pain management. The analgesic effect of opiorphin was proven in several earlier published in vitro and in vivo studies. Our aim was to test the transfer of opiorphin through a blood-brain barrier model for the first time. The flux of opiorphin was tested on a blood-brain barrier culture model consisting of rat brain endothelial, glial and pericyte cells. Brain endothelial cells in this triple co-culture model form tight monolayers characterized by transendothelial electrical resistance measurement. Relative quantity of the peptide was estimated by mass spectrometry. The transfer of opiorphin through the blood-brain barrier model was estimated to be ∼3%, whereas the permeability coefficient was 0.53 ± 1.36 × 10(-6) cm/s (n = 4). We also observed rapid conversion of N-terminal glutamine into pyroglutamic acid during the transfer experiments. Our results indicate that opiorphin crosses cultured brain endothelial cells in the absence of serum factors in a significant amount. This is in agreement with previous in vivo data showing potentiation of enkephalin-mediated antinociception. We suggest that opiorphin may have a potential as a centrally acting novel drug to treat pain.
Full Text Available To assess the clinical relevance of a semi-quantitative measurement of human cytomegalovirus (HCMV DNA in renal transplant recipients within the typical clinical context of a developing country where virtually 100% of both receptors and donors are seropositive for this virus, we have undertaken HCMV DNA quantification using a simple, semi-quantitative, limiting dilution polymerase chain reaction (PCR. We evaluated this assay prospectively in 52 renal transplant patients from whom a total of 495 serial blood samples were collected. The samples scored HCMV positive by qualitative PCR had the levels of HCMV DNA determined by end-point dilution-PCR. All patients were HCMV DNA positive during the monitoring period and a diagnosis of symptomatic infection was made for 4 of 52 patients. In symptomatic patients the geometric mean of the highest level of HCMV DNAemia was 152,000 copies per 106 leukocytes, while for the asymptomatic group this value was 12,050. Symptomatic patients showed high, protracted HCMV DNA levels, whereas asymptomatic patients demonstrated intermittent low or moderate levels. Using a cut-off value of 100,000 copies per 106 leukocytes, the limiting dilution assay had sensitivity of 100%, specificity of 92%, a positive predictive value of 43% and a negative predictive value of 100% for HCMV disease. In this patient group, there was universal HCMV infection but relatively infrequent symptomatic HCMV disease. The two patient groups were readily distinguished by monitoring with the limiting dilution assay, an extremely simple technology immediately applicable in any clinical laboratory with PCR capability.
Dong, Tuo; Qu, Zhangyi; Zhang, Lijuan
Human granulocytic anaplasmosis (HGA) and human monocytic ehrlichiosis (HME) are emerging, tick-borne, zoonotic infectious diseases caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis, respectively. Early diagnosis is essential for rapid clinical treatment to avoid misdiagnosis and severe patient outcomes. Simple, sensitive and reliable diagnostic methods are urgently needed. In this study, we developed a duplex real-time PCR assay targeting the A. phagocytophilum ankA gene and the E. chaffeensis TRP120 gene, respectively. The lowest limit of detection of the duplex real-time PCR assay was 100 copies of the targeted A. phagocytophilum ankA gene and the E. chaffeensis TRP120 gene per reaction, and the specificity was 100%. Detection in blood DNA samples from the acute stage of illness for 22 HGA cases and 8 HME cases indicated that the duplex real-time PCR assay was more sensitive than the nested PCR assay. The infection of Citellusundulatus Pallas with A. phagocytophilum and E. chaffeensis was first confirmed in Xinjiang Province and the positive rate was 3.1% for A. phagocytophilum, 6.3% for E. chaffeensis and 3.1% for co-infection with both pathogens. The rates of A. phagocytophilum and E. chaffeensis infection of D. silvarum ticks collected from Shanxi Province were 8.2% and 14.8%, respectively, and the co-infection rate was 3.3%. The rates of A. phagocytophilum and E. chaffeensis infection in H. longicornis ticks collected from Shandong Province were 1.6% and 6.3%, respectively, and the co-infection rate was 1.6%.
McMaster, J P; Barr, J G; Campbell, R R; Bennett, R B; Smyth, E T
The Malthus Microbiological Growth Analyser has proved to be sensitive in detecting conductivity changes due to anaerobic metabolism in a number of widely used blood culture media. Freshly prepared cooked meat media and Thiol medium yielded the greatest gross conductivity changes, and were more sensitive of anaerobic metabolism than other media. Failure of the instrument to detect anaerobic metabolism was a problem particularly associated with growth in the thioglycollate medium. False positive detections of growth were attributed to a number of factors including electrode instability (6.0%) and bacterial contamination (8.75%).
Neela K. Patel
Full Text Available Cardiovascular disease is a leading cause of morbidity and mortality in the United States, and its prevention and treatment remain a priority for the medical community. Ethnic variations account for some differences in the prevalence of hypertension and blood pressure (BP control rates among Hispanics, indicating the need for culturally appropriate management models. Aggressive treatment strategies are key to achieving optimal BP control in high-risk Hispanic patients. Hypertension in this ethnic group continues to be a major health concern. Of note, when provided access to comprehensive care, Hispanics demonstrate similar response rates to treatment as the majority of non-Hispanic whites. This highlights the importance of effective, culturally responsive hypertension management among high-risk Hispanic patients for achieving observable, positive health outcomes.
Identification of blood culture isolates directly from positive blood cultures by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry and a commercial extraction system: analysis of performance, cost, and turnaround time.
Lagacé-Wiens, Philippe R S; Adam, Heather J; Karlowsky, James A; Nichol, Kimberly A; Pang, Paulette F; Guenther, Jodi; Webb, Amanda A; Miller, Crystal; Alfa, Michelle J
Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry represents a revolution in the rapid identification of bacterial and fungal pathogens in the clinical microbiology laboratory. Recently, MALDI-TOF has been applied directly to positive blood culture bottles for the rapid identification of pathogens, leading to reductions in turnaround time and potentially beneficial patient impacts. The development of a commercially available extraction kit (Bruker Sepsityper) for use with the Bruker MALDI BioTyper has facilitated the processing required for identification of pathogens directly from positive from blood cultures. We report the results of an evaluation of the accuracy, cost, and turnaround time of this method for 61 positive monomicrobial and 2 polymicrobial cultures representing 26 species. The Bruker MALDI BioTyper with the Sepsityper gave a valid (score, >1.7) identification for 85.2% of positive blood cultures with no misidentifications. The mean reduction in turnaround time to identification was 34.3 h (P blood cultures and 26.5 h in a more practical setting where conventional identification or identification from subcultures was required for isolates that could not be directly identified by MALDI-TOF. Implementation of a MALDI-TOF-based identification system for direct identification of pathogens from blood cultures is expected to be associated with a marginal increase in operating costs for most laboratories. However, the use of MALDI-TOF for direct identification is accurate and should result in reduced turnaround time to identification.
Al-Hamad, Arif; Al-Ibrahim, Maha; Alhajhouj, Eman; Al-Alshaikh Jaffer, Waseelah; Altowaileb, Jaffar; Alfaraj, Hassan
Compared with truly negative cultures, false positive blood cultures (BCs) not only increase laboratory work but also prolong the lengths of patient stays, which are likely to increase patient morbidity and costs. The present study aimed to evaluate the effectiveness of a hospital-wide educational intervention on BC contamination rates. Nurses performed all phlebotomies; therefore, educational workshops were offered to all nurses twice a week over a 3-month period. The workshops consisted of a questionnaire, PowerPoint presentation, video show, demonstration of the different materials used to collect BCs, and question session. Data from the questionnaires and laboratory culture results were compared between the 6-month pre- and post-intervention periods. Of the 503 eligible nurses, 216 (42.9%) attended the workshops. The survey identified areas for improvement, which included time of disinfectant application, volume of blood to be cultured, and disinfection of BC bottle tops. Of the 9903 BC sets that were drawn from 3649 patients during the study period, 676 (6.8%) were contaminated. The monthly BC contamination rates for the 6-month pre- and post-intervention periods were 8.1% and 5.2%, respectively, representing a 36% reduction (P=0.008). Only three wards had an acceptable contamination rate of ≤3% before the intervention, compared with eight wards after the intervention. While contamination of BCs can never be completely eliminated, there is evidence that adherence to best practice BC collection techniques can minimize BC contamination, which might be best achieved with a dedicated phlebotomy team.
Morton, Ben; Nagaraja, Shankara; Collins, Andrea; Pennington, Shaun H.; Blakey, John D.
Background The “weekend effect” describes an increase in adverse outcomes for patients admitted at the weekend. Critical care units have moved to higher intensity working patterns to address this with some improved outcomes. However, support services have persisted with traditional working patterns. Blood cultures are an essential diagnostic tool for patients with sepsis but yield is dependent on sampling technique and processing. We therefore used blood culture yield as a surrogate for the quality of support service provision. We hypothesized that blood culture yields would be lower over the weekend as a consequence of reduced support services. Methods We performed a retrospective observational study examining 1575 blood culture samples in a university hospital critical care unit over a one-year period. Results Patients with positive cultures had, on average, higher APACHE II scores (p = 0.015), longer durations of stay (p = 0.03), required more renal replacement therapy (p<0.001) and had higher mortality (p = 0.024). Blood culture yield decreased with repeated sampling with an increased proportion of contaminants. Blood cultures were 26.7% less likely to be positive if taken at the weekend (p = 0.0402). This effect size is the equivalent to the impact of sampling before and after antibiotic administration. Conclusions Our study demonstrates that blood culture yield is lower at the weekend. This is likely caused by delays or errors in incubation and processing, reflecting the reduced provision of support services at the weekend. Reorganization of services to address the “weekend effect” should acknowledge the interdependent nature of healthcare service delivery. PMID:26492559
Full Text Available The "weekend effect" describes an increase in adverse outcomes for patients admitted at the weekend. Critical care units have moved to higher intensity working patterns to address this with some improved outcomes. However, support services have persisted with traditional working patterns. Blood cultures are an essential diagnostic tool for patients with sepsis but yield is dependent on sampling technique and processing. We therefore used blood culture yield as a surrogate for the quality of support service provision. We hypothesized that blood culture yields would be lower over the weekend as a consequence of reduced support services.We performed a retrospective observational study examining 1575 blood culture samples in a university hospital critical care unit over a one-year period.Patients with positive cultures had, on average, higher APACHE II scores (p = 0.015, longer durations of stay (p = 0.03, required more renal replacement therapy (p<0.001 and had higher mortality (p = 0.024. Blood culture yield decreased with repeated sampling with an increased proportion of contaminants. Blood cultures were 26.7% less likely to be positive if taken at the weekend (p = 0.0402. This effect size is the equivalent to the impact of sampling before and after antibiotic administration.Our study demonstrates that blood culture yield is lower at the weekend. This is likely caused by delays or errors in incubation and processing, reflecting the reduced provision of support services at the weekend. Reorganization of services to address the "weekend effect" should acknowledge the interdependent nature of healthcare service delivery.
de Vries, Jutte J C; Wessels, Els; Korver, Anna M H; van der Eijk, Annemiek A; Rusman, Lisette G; Kroes, Aloys C M; Vossen, Ann C T M
Genotyping of cytomegalovirus (CMV) is useful to examine potential differences in the pathogenicity of strains and to demonstrate coinfection with multiple strains involved in CMV disease in adults and congenitally infected newborns. Studies on genotyping of CMV in dried blood spots (DBS) are rare and have been hampered by the small amount of dried blood available. In this study, two multiplex real-time PCR assays for rapid gB and gH genotyping of CMV in DBS were developed. Validation of the assays with 39 CMV-positive plasma samples of transplant recipients and 21 urine specimens of congenitally infected newborns was successful in genotyping 100% of the samples, with gB1 and gB3 being the most prevalent genotypes. Multiple gB and gH genotypes were detected in 36% and 33% of the plasma samples, respectively. One urine sample from a newborn with symptomatic congenital CMV was positive for gB1 and gB2. DBS of congenitally infected newborns (n = 41) were tested using 9 μl of dried blood, and genotypes were detected in 81% (gB) and 73% (gH) of the samples, with gB3 being the most prevalent genotype. No clear association of specific genotypes with clinical outcome was observed. In conclusion, the CMV gB and gH PCR assays were found to be rapid, sensitive for detecting mixed infections, and suitable for direct usage on DBS. These assays are efficient tools for genotyping of CMV in DBS of congenitally infected newborns.
Kılıc, Ayşe; Kalender, Hakan; Eroksuz, Hatice; Muz, Adile; Tasdemir, Bülent
This study used cultures, polymerase chain reaction (PCR), and immunoperoxidase to examine samples from 216 lungs from sheep and lambs with macroscopic pneumonia lesions for the presence of Mycoplasma species. DNA was extracted from lung tissue samples and broth cultures with the help of a DNA extraction kit and replicated using genus-specific and species-specific primers for mycoplasma. The lung samples were examined by the immunoperoxidase method using hyperimmune Mycoplasma ovipneumoniae serum. The randomly amplified polymorphic DNA (RAPD) test was used for the molecular typing of M. ovipneumoniae isolates. Mycoplasma was isolated in the cultures of 80 (37.03 %) of a total of 216 lung samples. Genus-specific mycoplasma DNA was identified by PCR in 96 (44.44 %) samples in broth cultures and 36 (16.66 %) directly in the lung tissue. Of these 96 cases in which genus-specific identification was made, 57 (59.37 %) were positive for reaction with species-specific primers for M. ovipneumoniae and 31 (32.29 %) for Mycoplasma arginini. The DNA of neither of the latter two species could be identified in the remaining eight samples (8.33 %) where mycoplasma had been identified. As for the immunoperoxidase method, it identified M. ovipneumoniae in 61 of 216 lung samples (28 %). Positive staining was concentrated in the bronchial epithelium cell cytoplasm and cell surface. RAPD analysis resulted in 15 different profiles. Our results suggest that PCR methods could be successfully used in the diagnosis of mycoplasma infections as an alternative to culture method and identifying this agent at the species level.
Riley, J. A.; Heiter, B J; Bourbeau, P P
bioMerieux, Inc., has recently introduced plastic bottles to replace glass bottles for use in the BacT/ALERT blood culture system. We compared the performance of the plastic to the glass bottles in a large clinical evaluation. Two blood cultures were collected from each patient, one using glass FA (aerobic) and FN (anaerobic) bottles and one using plastic FA and FN bottles. Of the 4,040 sets of four bottles collected, 3,110 contained the recommended 8 to 12 ml of blood, yielding 524 microorga...
Full Text Available Abstract Background The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. Methods In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22 that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription – polymerase chain reaction (RT-PCR. Results Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. Conclusion The design of new approaches to identify such markers is warranted.
Waters, Sinéad M; Doyle, Sean; Murphy, Richard A; Power, Ronan F G
Nurmi-type cultures (NTCs), derived from the fermentation of caecal contents of specifically pathogen-free (SPF) birds, have been used successfully to control salmonella colonisation in chicks. These cultures are undefined in nature and, consequently, it is difficult to obtain approval from regulatory agencies for their use as direct fed microbials (DFMs) for poultry. Progress towards the generation of effective defined probiotics requires further knowledge of the composition of these cultures. As such, species-specific, culture-independent quantification methodologies need to be developed to elucidate the concentration of specific bacterial constituents of NTCs. Quantification of specific bacterial species in such ill-defined complex cultures using conventional culturing methods is inaccurate due to low levels of sensitivity and reproducibility, in addition to slow turnaround times. Furthermore, these methods lack selectivity due to the nature of the accompanying microflora. This study describes the development of a rapid, sensitive, reliable, reproducible, and species-specific culture-independent, solution phase hybridisation PCR-ELISA procedure for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in NTCs. In this technique, biotin-labelled primers were designed to amplify a species-specific fragment of a marker gene of known copy number, in both species. Resulting amplicons were hybridised with a dinitrophenol (DNP)-labelled oligonucleotide probe in solution and were subsequently captured on a streptavidin-coated microtitre plate. The degree of binding was determined by the addition of IgG (anti-DNP)-horseradish peroxidase conjugate, which was subsequently visualised using a chromogenic substrate, tetramethylbenzidine. This novel quantitative method was capable of detecting E. faecalis and P. pentosaceus at levels as low as 5 CFU per PCR reaction.
Bach, H-J; Tomanova, J; Schloter, M; Munch, J C
Real-time quantitative PCR assays were developed for the absolute quantification of different groups of bacteria in pure cultures and in environmental samples. 16S rRNA genes were used as markers for eubacteria, and genes for extracellular peptidases were used as markers for potentially proteolytic bacteria. For the designed 16S rDNA TaqMan assay, specificity of the designed primer-probe combination for eubacteria, a high amplification efficiency over a wide range of starting copy numbers and a high reproducibility is demonstrated. Cell concentrations of Bacillus cereus, B. subtilis and Pseudomonas fluorescens in liquid culture were monitored by TaqMan-PCR using the 16S rDNA target sequence of Escherichia coli as external standard for quantification. Results agree with plate counts and microscopic counts of DAPI stained cells. The significance of 16S rRNA operon multiplicity to the quantification of bacteria is discussed.Furthermore, three sets of primer pair together with probe previously designed for targeting different classes of bacterial extracellular peptidases were tested for their suitability for TaqMan-PCR based quantification of proteolytic bacteria. Since high degeneracy of the probes did not allow accurate quantification, SybrGreen was used instead of molecular probes to visualize and quantify PCR products during PCR. The correlation between fluorescence and starting copy number was of the same high quality as for the 16S rDNA TaqMan assay for all the three peptidase gene classes. The detected amount of genes for neutral metallopeptidase of B. cereus, for subtilisin of B. subtilis and for alkaline metallopeptidase of P. fluorescens corresponded exactly to the numbers of bacteria investigated by the 16S rDNA targeting assay. The developed assays were applied for the quantification of bacteria in soil samples.
Pardo, Joe; Klinker, Kenneth P; Borgert, Samuel J; Butler, Brittany M; Giglio, Patricia G; Rand, Kenneth H
The purpose of this study was to evaluate the impact of the FilmArray Blood Culture Identification (BCID) Panel on the management of patients with blood cultures growing gram positive cocci and Candida. We retrospectively compared clinical and economic outcomes between patients during the BCID testing period and a matched historical control group before BCID testing was introduced. A total of 84 BCID patients were matched to 252 historical controls. BCID identification of coagulase negative staphylococci contaminants resulted in shorter post-culture length of stay (P historical controls (P = 0.047). The BCID, coupled with antimicrobial stewardship intervention, was a cost effective tool to improve patient care.
Full Text Available The ability to rapidly differentiate coagulase-negative staphylococcus (CoNS from Staphylococcus aureus and to determine methicillin resistance is important as it affects the decision to treat empiric antibiotic selection. The objective of this study was to evaluate CHROMagar S. aureus and CHROMagar MRSA (Becton Dickinson for rapid identification of Staphylococcus spp. directly from blood cultures. Consecutive blood culture bottles (BacT Alert 3D SA and SN, bioMérieux growing gram-positive cocci in clusters were evaluated. An aliquot was plated onto CHROMagar MRSA (C-MRSA and CHROMagar S. aureus (C-SA plates, which were read at 12 to 16 hours. C-SA correctly identified 147/147 S. aureus (100% sensitivity; 2 CoNS were misidentified as S. aureus (98% specificity. C-MRSA correctly identified 74/77 MRSA (96% sensitivity. None of the MSSA isolates grew on C-MRSA (100% specificity. In conclusion, CHROMagar is a rapid and sensitive method to distinguish MRSA, MSSA, and coagulase-negative Staphylococcus and may decrease time of reporting positive results.
Sangare, S A; Maiga, A I; Guindo, I; Maiga, A; Camara, N; Savadogo, S; Diallo, S; Bougoudogo, F; Armand-Lefevre, L; Andremont, A; Maiga, I I
Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae have been isolated from many regions of the world. Epidemiological studies are being conducted in Europe, North America, and Asia. No study has however been conducted in Africa to determine the prevalence and distribution of ESBLs on the continent. This literature review aimed at describing the prevalence of ESBL-producing Enterobacteriaceae isolated from blood cultures, as well as the ESBL genes involved at the international level. Our focus was mainly on Africa. We conducted a literature review on PubMed. Articles related to our study field and published between 1996 and 2014 were reviewed and entirely read for most of them, while we only focused on the abstracts of some other articles. Relevant articles to our study were then carefully reviewed and included in the review. The prevalence of ESBL-producing Enterobacteriaceae differs from one country to another. The results of our literature review however indicate that class A ESBLs prevail over the other types. We took into consideration articles focusing on various types of samples to assess the prevalence of ESBL-producing Enterobacteriaceae, but information on isolates from blood cultures is limited. The worldwide prevalence of ESBL-producing Enterobacteriaceae has increased over time. Evidence of ESBL-producing Enterobacteriaceae can be found in all regions of the world. Studies conducted in Africa mainly focused on the Northern and Eastern parts of the continent, while only rare studies were carried out in the rest of the continent.
Wojewoda, Christina M; Sercia, Linda; Navas, Maria; Tuohy, Marion; Wilson, Deborah; Hall, Geraldine S; Procop, Gary W; Richter, Sandra S
Rapid identification of pathogens from blood cultures can decrease lengths of stay and improve patient outcomes. We evaluated the accuracy of the Verigene Gram-positive blood culture (BC-GP) nucleic acid test for investigational use only (Nanosphere, Inc., Northbrook, IL) for the identification of Gram-positive bacteria from blood cultures. The detection of resistance genes (mecA in Staphylococcus aureus and Staphylococcus epidermidis and vanA or vanB in Enterococcus faecium and Enterococcus faecalis) by the BC-GP assay also was assessed. A total of 186 positive blood cultures (in BacT/Alert FA bottles) with Gram-positive cocci observed with Gram staining were analyzed using the BC-GP assay. The BC-GP results were compared with the identification and susceptibility profiles obtained with routine methods in the clinical laboratory. Discordant results were arbitrated with additional biochemical, cefoxitin disk, and repeat BC-GP testing. The initial BC-GP organism identification was concordant with routine method results for 94.6% of the blood cultures. Only 40% of the Streptococcus pneumoniae identifications were correct. The detection of the mecA gene for 69 blood cultures with only S. aureus or S. epidermidis was concordant with susceptibility testing results. For 3 of 6 cultures with multiple Staphylococcus spp., mecA detection was reported but was correlated with oxacillin resistance in a species other than S. aureus or S. epidermidis. The detection of vanA agreed with susceptibility testing results for 45 of 46 cultures with E. faecalis or E. faecium. Comparison of the mean times to results for each organism group showed that BC-GP results were available 31 to 42 h earlier than phenotypic identifications and 41 to 50 h earlier than susceptibility results.
Full Text Available Introduction: Bloodstream infections are associated with significant patient morbidity and mortality. Antimicrobial susceptibility patterns should guide the choice of empiric antimicrobial regimens for patients with bacteremia. Methods: Blood sample received from the patient attending Nepal Medical College and Teaching Hospital from March 2013 – August, 2013 were subjected to for culture. Isolate identification and antimicrobial susceptibility testing was done by standard microbiological method Results: Out of the total 2,766 blood samples, 13.3% showed bacterial growth. The percentage of neonatal septicemia was 13.3%. Staphylococcus aureus (28% was the most common isolates followed by Salmonella enterica Serotype Typhi (22%, Coagulase negative Staphylococci (9.5%, Salmonella enterica Serotype Paratyphi ((7.6% and Klebsiella pneumoniae (7.6%. 26.3% of the isolates of Staphylococcus aureus were oxacillin resistant. Most of the gram positive organisms were susceptible to amikacin and vancomycin and showed high level resistance to cefuroxime and cotrimoxazole. Out of 109 isolates of typhoid bacilli, 95.3% were resistant to nalidixic acid ,79% to ciprofloxacin and 60.5% to ofloxacin. More than 50% of the isolates of Klebsiella pneumoniae and Escherichia coli showed resistance to cephalosporins and cotrimoxazole. Acinetobacter spp showed high resistance (more than 60% to ceftriaxone and ofloxacin. More than 20% of the isolates of Pseudomonas aeruginosa were resistant to ciprofloxacin and amikacin. Conclusions: Ongoing surveillance for antimicrobial susceptibility remains essential, and will enhance efforts to identify resistance and attempt to limit its spread. Keywords: antibiotic; bacteria; blood stream infections.
Halo, Tiffany L; McMahon, Kaylin M; Angeloni, Nicholas L; Xu, Yilin; Wang, Wei; Chinen, Alyssa B; Malin, Dmitry; Strekalova, Elena; Cryns, Vincent L; Cheng, Chonghui; Mirkin, Chad A; Thaxton, C Shad
Metastasis portends a poor prognosis for cancer patients. Primary tumor cells disseminate through the bloodstream before the appearance of detectable metastatic lesions. The analysis of cancer cells in blood—so-called circulating tumor cells (CTCs)—may provide unprecedented opportunities for metastatic risk assessment and investigation. NanoFlares are nanoconstructs that enable live-cell detection of intracellular mRNA. NanoFlares, when coupled with flow cytometry, can be used to fluorescently detect genetic markers of CTCs in the context of whole blood. They allow one to detect as few as 100 live cancer cells per mL of blood and subsequently culture those cells. This technique can also be used to detect CTCs in a murine model of metastatic breast cancer. As such, NanoFlares provide, to our knowledge, the first genetic-based approach for detecting, isolating, and characterizing live cancer cells from blood and may provide new opportunities for cancer diagnosis, prognosis, and personalized therapy.
Full Text Available Yuji Watanabe,1,2 Nozomi Oikawa,1,2 Maya Hariu,1,2 Ryota Fuke,1 Masafumi Seki1 1Division of Infectious Diseases and Infection Control, 2Laboratory for Clinical Microbiology, Tohoku Medical and Pharmaceutical University Hospital, Sendai City, Miyagi, Japan Abstract: Procalcitonin (PCT and C-reactive protein serve as biomarkers of infection in patients with sepsis/bacteremia. The present study assessed the clinical characteristics of 280 patients with suspected sepsis who were admitted to Tohoku Medical and Pharmaceutical University Hospital between January 2012 and December 2013. Among the patients, 133 and 147 were positive and negative for PCT, respectively. Patients who were PCT positive were older and more frequently male, had reduced levels of platelets and albumin, and increased levels of aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, creatinine, and C-reactive protein. Patients who were PCT positive had significantly higher blood culture positivity compared with those who were PCT negative, and the sensitivity and specificity of PCT for detecting positive blood cultures were 74.5% and 59.1%, respectively. Escherichia coli was detected in PCT-positive patients, whereas Staphylococcus epidermidis and Staphylococcus lugdunensis were frequently detected in PCT-negative patients. Levels of PCT were higher in the patients infected with gram-negative rods than those with gram-positive cocci. Furthermore, extended-spectrum β-lactamase (ESBL-producing bacteria cases showed higher levels of PCT than those of non-ESBL cases. These results suggest that PCT may be a useful biomarker of sepsis, and it might serve as a strong tool to detect patients with severe gram-negative rod bacteremia including ESBL-producing bacteria cases early due to its relative high sensitivity. Keywords: biomarker, sepsis, Escherichia coli, gram-negative rods, ESBL
Full Text Available Abstract Background PCR amplification for the detection of pathogens in biological material is generally considered a rapid and informative diagnostic technique. Invasive Salmonella serovars, which cause enteric fever, can be commonly cultured from the blood of infected patients. Yet, the isolation of invasive Salmonella serovars from blood is protracted and potentially insensitive. Methods We developed and optimised a novel multiplex three colour real-time PCR assay to detect specific target sequences in the genomes of Salmonella serovars Typhi and Paratyphi A. We performed the assay on DNA extracted from blood and bone marrow samples from culture positive and negative enteric fever patients. Results The assay was validated and demonstrated a high level of specificity and reproducibility under experimental conditions. All bone marrow samples tested positive for Salmonella, however, the sensitivity on blood samples was limited. The assay demonstrated an overall specificity of 100% (75/75 and sensitivity of 53.9% (69/128 on all biological samples. We then tested the PCR detection limit by performing bacterial counts after inoculation into blood culture bottles. Conclusions Our findings corroborate previous clinical findings, whereby the bacterial load of S. Typhi in peripheral blood is low, often below detection by culture and, consequently, below detection by PCR. Whilst the assay may be utilised for environmental sampling or on differing biological samples, our data suggest that PCR performed directly on blood samples may be an unsuitable methodology and a potentially unachievable target for the routine diagnosis of enteric fever.
Heuvelink, Annet; Hassan, Abdulwahed Ahmed; van Weering, Hilmar; van Engelen, Erik; Bülte, Michael; Akineden, Ömer
Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne's disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold's egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Löwenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne's) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.
... Also, blood is either Rh-positive or Rh-negative. So if you have type A blood, it's either A positive or A negative. Which type you are is important if you need a blood transfusion. And your Rh factor could be important ...
Objective To investigate the distribution of pathogens detected in blood cultures and the value of double blood cul‐ture .Methods There were 2 033 cases of blood cultures including 1 425 cases of single blood culture and 608 cases of doube blood cultures were in the study .Blood cultures were performed by using Bact/Alert3D automated detection system ,and the positive blood culture results were statistically analyzed .Results There were 251 blood culture positive samples ,the detection rate was 12 .3% (251/2 033) .Among them ,the positive rate of single blood culture was 11 .9% (169/1 425);positive rate of double blood culture was 13 .5% (82/608) .There were 216 strains of pathogenic bacteria(repeatedly culture of the same pathogen from the same patient only accounted for 1 strain) .The isolates of gram positive(G+ ) cocci accounted for 65 .7% (142/216) and the major bacteria were coagulase negative Staphy lococci .Escherichia coli and Acinetobacter baumannii were the dominant isolates among Enterobac‐teriaceae[accounted for 20 .8% (45/216)] and Non‐fermenting gram‐negative(G-) rods[accounted for 8 .8% (19/216)] ,respective‐ly .The isolating rate of doube blood cultures was significantly higher than the single one(P<0 .05) .Coagulase‐negative staphylo‐coccus were isolated primarily in single blood culture and double blood culture with only one test postive .Non‐fermenting gram‐neg‐ative rods and fungi were the most common strains isolated from double blood culture with two tests both postive .Conclusion The distributions of pathogens isolated from single blood culture ,double blood culture with only one test positive and two tests positive are different .Because collecting double sets of blood cultures can significantly increase the isolating rates and distinguish contamina‐tion from real bloodstream infection ,collecting double sets of blood cultures should be recommended .Double blood culture is helpful to improve the detection rate of
Thanchomnang, Tongjit; Intapan, Pewpan M; Tantrawatpan, Chairat; Lulitanond, Viraphong; Chungpivat, Sudchit; Taweethavonsawat, Piyanan; Kaewkong, Worasak; Sanpool, Oranuch; Janwan, Penchom; Choochote, Wej; Maleewong, Wanchai