Improved diagnosis of gastrointestinal infections using a semi-automated multiplex real-time PCR for detection of enteropathogens

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis. Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination. Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures. Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013–2018) included stool culture, microscopy and immunochromatography. Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013–2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis. Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

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Evaluation of Luminex xTAG Gastrointestinal Pathogen Panel Assay for Detection of Multiple Diarrheal Pathogens in Fecal Samples in Vietnam

Journal of Clinical Microbiology ◽

10.1128/jcm.03321-15 ◽

2016 ◽

Vol 54 (4) ◽

pp. 1094-1100 ◽

Cited By ~ 31

Author(s):

Vu Thuy Duong ◽

Voong Vinh Phat ◽

Ha Thanh Tuyen ◽

Tran Thi Ngoc Dung ◽

Pham Duc Trung ◽

...

Keyword(s):

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Diarrheal Disease ◽

Enteric Pathogens ◽

Molecular Diagnostic ◽

Molecular Diagnostic Techniques ◽

Content Type ◽

Microbiological Culture ◽

Gastrointestinal Pathogen

Diarrheal disease is a complex syndrome that remains a leading cause of global childhood morbidity and mortality. The diagnosis of enteric pathogens in a timely and precise manner is important for making treatment decisions and informing public health policy, but accurate diagnosis is a major challenge in industrializing countries. Multiplex molecular diagnostic techniques may represent a significant improvement over classical approaches. We evaluated the Luminex xTAG gastrointestinal pathogen panel (GPP) assay for the detection of common enteric bacterial and viral pathogens in Vietnam. Microbiological culture and real-time PCR were used as gold standards. The tests were performed on 479 stool samples collected from people admitted to the hospital for diarrheal disease throughout Vietnam. Sensitivity and specificity were calculated for the xTAG GPP for the seven principal diarrheal etiologies. The sensitivity and specificity for the xTAG GPP were >88% forShigellaspp.,Campylobacterspp., rotavirus, norovirus genotype 1/2 (GI/GII), and adenovirus compared to those of microbiological culture and/or real-time PCR. However, the specificity was low (∼60%) forSalmonellaspecies. Additionally, a number of important pathogens that are not identified in routine hospital procedures in this setting, such asCryptosporidiumspp. andClostridium difficile, were detected with the GPP. The use of the Luminex xTAG GPP for the detection of enteric pathogens in settings, like Vietnam, would dramatically improve the diagnostic accuracy and capacity of hospital laboratories, allowing for timely and appropriate therapy decisions and a wider understanding of the epidemiology of pathogens associated with severe diarrheal disease in low-resource settings.

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A pentaplex real-time PCR assay for rapid identification of major beta-lactamase genes KPC, NDM, CTX, CMY, and OXA-48 directly from bacteria in blood

Journal of Medical Microbiology ◽

10.1099/jmm.0.001465 ◽

2021 ◽

Vol 70 (12) ◽

Author(s):

Taalin R. Hoj ◽

Bradley McNeely ◽

Kylie Webber ◽

Evelyn Welling ◽

William G. Pitt ◽

...

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Real Time ◽

Real Time Pcr ◽

Type Species ◽

New Delhi ◽

Beta Lactamase ◽

Content Type ◽

Link Type ◽

Carbapenem Resistant

Introduction. Antibiotic resistance, particularly in cases of sepsis, has emerged as a growing global public health concern and economic burden. Current methods of blood culture and antimicrobial susceptibility testing of agents involved in sepsis can take as long as 3–5 days. It is vital to rapidly identify which antimicrobials can be used to effectively treat sepsis cases on an individual basis. Here, we present a pentaplex, real-time PCR-based assay that can quickly identify the most common beta-lactamase genes ( Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX-M); cephamycin AmpC beta-lactamases (CMY); and Oxacillinase-48 (OXA-48)) from pathogens derived directly from the blood of patients presenting with bacterial septicemia. Aim. To develop an assay which can rapidly identify the most common beta-lactamase genes in Carbapenem-resistant Enterobacteriaceae bacteria (CREs) from the United States. Hypothesis/Gap Statement. Septicemia caused by carbapenem-resistant bacteria has a death rate of 40–60 %. Rapid diagnosis of antibiotic susceptibility directly from bacteria in blood by identification of beta-lactamase genes will greatly improve survival rates. In this work, we develop an assay capable of concurrently identifying the five most common beta-lactamase and carbapenemase genes. Methodology. Primers and probes were created which can identify all subtypes of Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX); cephamycin AmpC beta-lactamase (CMY); and oxacillinase-48 (OXA-48). The assay was validated using 13 isolates containing various PCR targets from the Centre for Disease Control Antimicrobial Resistance Isolate Bank Enterobacterales Carbapenemase Diversity Panel. Blood obtained from volunteers was spiked with CREs and bacteria were separated, lysed, and subjected to analysis via the pentaplex assay. Results. This pentaplex assay successfully identified beta-lactamase genes derived from bacteria separated from blood at concentrations of 4–8 c.f.u. ml−1. Conclusion. This assay will improve patient outcomes by supplying physicians with critical drug resistance information within 2 h of septicemia onset, allowing them to prescribe effective antimicrobials corresponding to the resistance gene(s) present in the pathogen. In addition, information supplied by this assay will lessen the inappropriate use of broad-spectrum antimicrobials and prevent the evolution of further antibiotic resistance.

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Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting thegroELGene

Applied and Environmental Microbiology ◽

10.1128/aem.07749-11 ◽

2012 ◽

Vol 78 (8) ◽

pp. 2613-2622 ◽

Cited By ~ 57

Author(s):

Jana Junick ◽

Michael Blaut

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Housekeeping Gene ◽

Cell Number ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Bifidobacterium Adolescentis ◽

Fecal Samples ◽

Content Type ◽

Pcr Assays

ABSTRACTQuantitative real-time PCR assays targeting thegroELgene for the specific enumeration of 12 human fecalBifidobacteriumspecies were developed. The housekeeping genegroEL(HSP60in eukaryotes) was used as a discriminative marker for the differentiation ofBifidobacterium adolescentis,B. angulatum,B. animalis,B. bifidum,B. breve,B. catenulatum,B. dentium,B. gallicum,B. longum,B. pseudocatenulatum,B. pseudolongum, andB. thermophilum. The bifidobacterial chromosome contains a single copy of thegroELgene, allowing the determination of the cell number by quantification of thegroELcopy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a givenBifidobacteriumspecies. Independent of theBifidobacteriumspecies tested, the proportion ofgroELcopies recovered from fecal samples spiked with 5 to 9 log10cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10groELcopies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3Bifidobacteriumspecies andB. longumfrequently detected. The predominant species in infant and adult fecal samples wereB. breveandB. adolescentis, respectively. It was possible to distinguishB. catenulatumandB. pseudocatenulatum. We conclude that thegroELgene is a suitable molecular marker for the specific and accurate quantification of human fecalBifidobacteriumspecies by real-time PCR.

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Evaluation of a Multiplex Real-Time PCR Assay for Detecting Major Bacterial Enteric Pathogens in Fecal Specimens: Intestinal Inflammation and Bacterial Load Are Correlated in Campylobacter Infections

Journal of Clinical Microbiology ◽

10.1128/jcm.00558-16 ◽

2016 ◽

Vol 54 (9) ◽

pp. 2262-2266 ◽

Cited By ~ 12

Author(s):

Nadia Wohlwend ◽

Sacha Tiermann ◽

Lorenz Risch ◽

Martin Risch ◽

Thomas Bodmer

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Bacterial Load ◽

Positive Culture ◽

Fecal Calprotectin ◽

Enteric Pathogens ◽

Significant Linear Trend ◽

Content Type ◽

Culture Positive ◽

Culture Negative

A total of 1,056 native or Cary-Blair-preserved stool specimens were simultaneously tested by conventional stool culturing and by enteric bacterial panel (EBP) multiplex real-time PCR forCampylobacter jejuni,Campylobacter coli,Salmonellaspp., and shigellosis disease-causing agents (Shigellaspp. and enteroinvasiveEscherichia coli[EIEC]). Overall, 143 (13.5%) specimens tested positive by PCR for the targets named above; 3 coinfections and 109 (10.4%)Campylobacterspp., 17 (1.6%)Salmonellaspp., and 20 (1.9%)Shigellaspp./EIEC infections were detected. The respective positive stool culture rates were 75 (7.1%), 14 (1.3%), and 7 (0.7%). The median threshold cycle (CT) values of culture-positive specimens were significantly lower than those of culture-negative ones (CTvalues, 24.3 versus 28.7;P< 0.001), indicating that the relative bacterial load per fecal specimen was significantly associated with the culture results. InCampylobacterinfections, the respective median fecal calprotectin concentrations in PCR-negative/culture-negative (n =40), PCR-positive/culture-negative (n =14), and PCR-positive/culture-positive (n =15) specimens were 134 mg/kg (interquartile range [IQR], 30 to 1,374 mg/kg), 1,913 mg/kg (IQR, 165 to 3,813 mg/kg), and 5,327 mg/kg (IQR, 1,836 to 18,213 mg/kg). Significant differences were observed among the three groups (P< 0.001), and a significant linear trend was identified (P< 0.001). Furthermore, the fecal calprotectin concentrations andCTvalues were found to be correlated (r= −0.658). Our results demonstrate that molecular screening ofCampylobacterspp.,Salmonellaspp., andShigellaspp./EIEC using the BD Max EBP assay will result in timely diagnosis and improved sensitivity. The determination of inflammatory markers, such as calprotectin, in fecal specimens may aid in the interpretation of PCR results, particularly for enteric pathogens associated with mucosal damage and colonic inflammation.

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MicroSEQ® Salmonella spp. Detection Kit Using the Pathatrix® 10-Pooling Salmonella spp. Kit Linked Protocol Method Modification

Journal of AOAC International ◽

10.5740/jaoacint.13-251 ◽

2014 ◽

Vol 97 (2) ◽

pp. 484-491 ◽

Cited By ~ 3

Author(s):

Jason Wall ◽

Rick Conrad ◽

Kathy Latham ◽

Eric Liu

Keyword(s):

Real Time ◽

Study Design ◽

Foodborne Pathogens ◽

Real Time Pcr ◽

Reference Method ◽

Immunomagnetic Separation ◽

Pcr Analysis ◽

Salmonella Spp ◽

Culture Methods ◽

Design For All

Abstract Real-time PCR methods for detecting foodborne pathogens offer the advantages of simplicity and quick time to results compared to traditional culture methods. The addition of a recirculating pooled immunomagnetic separation method prior to real-time PCR analysis increases processing output while reducing bothcost and labor. This AOAC Research Institute method modification study validates the MicroSEQ®Salmonella spp. Detection Kit [AOAC Performance Tested Method(PTM) 031001] linked with the Pathatrix® 10-Pooling Salmonella spp. Kit (AOAC PTM 090203C) in diced tomatoes, chocolate, and deli ham. The Pathatrix 10-Pooling protocol represents a method modification of the enrichment portion of the MicroSEQ Salmonella spp. protocol. The results of the method modification were compared to standard cultural reference methods for diced tomatoes, chocolate, and deli ham. All threematrixes were analyzed in a paired study design. An additional set of chocolate test portions was analyzed using an alternative enrichment medium in an unpaired study design. For all matrixes tested, there were no statistically significant differences in the number of positive test portions detected by the modified candidate method compared to the appropriate reference method. The MicroSEQ Salmonella spp. protocol linked with the Pathatrix individual or 10-Pooling procedure demonstrated reliabilityas a rapid, simplified, method for the preparation of samples and subsequent detection of Salmonella in diced tomatoes, chocolate, and deliham.

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Detection and Characterization of Diphtheria Toxin Gene-Bearing Corynebacterium Species through a New Real-Time PCR Assay

Journal of Clinical Microbiology ◽

10.1128/jcm.00639-20 ◽

2020 ◽

Vol 58 (10) ◽

Cited By ~ 1

Author(s):

Margaret M. Williams ◽

Jessica L. Waller ◽

Janessa S. Aneke ◽

Michael R. Weigand ◽

Maureen H. Diaz ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Toxin Production ◽

Toxin Gene ◽

Analytical Sensitivity ◽

Rt Pcr ◽

Pcr Assay ◽

Culture Methods ◽

Content Type ◽

Corynebacterium Ulcerans

ABSTRACT Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of Corynebacterium diphtheriae, with similar illness produced occasionally by toxigenic Corynebacterium ulcerans or, rarely, Corynebacterium pseudotuberculosis. While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene (tox). Nontoxigenic tox-bearing (NTTB) Corynebacterium isolates have been described, but impact of these isolates on the accuracy of molecular diagnostics is not well characterized. Here, we describe a new triplex RT-PCR assay to detect tox and distinguish C. diphtheriae from the closely related species C. ulcerans and C. pseudotuberculosis. Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized Corynebacterium isolates accurately, with 100% analytical sensitivity for all targets. Analytical specificity with isolates was 94.1%, 100%, and 99.5% for tox, Diph_rpoB, and CUP_rpoB targets, respectively. Twenty-nine NTTB Corynebacterium isolates, representing 5.9% of 494 nontoxigenic isolates tested, were detected by RT-PCR. Whole-genome sequencing of NTTB isolates revealed varied mutations putatively underlying their lack of toxin production, as well as eight isolates with no mutation in tox or the promoter region. This new Corynebacterium RT-PCR method provides a rapid tool to screen isolates and identify probable diphtheria cases directly from specimens. However, the sporadic occurrence of NTTB isolates reinforces the viewpoint that diphtheria culture diagnostics continue to provide the most accurate case confirmation.

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Low Relative Abundances of the Mucolytic Bacterium Akkermansia muciniphila and Bifidobacterium spp. in Feces of Children with Autism

Applied and Environmental Microbiology ◽

10.1128/aem.05212-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6718-6721 ◽

Cited By ~ 169

Author(s):

Lv Wang ◽

Claus T. Christophersen ◽

Michael J. Sorich ◽

Jacobus P. Gerber ◽

Manya T. Angley ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Children With Autism ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Content Type ◽

Akkermansia Muciniphila ◽

Gastrointestinal Health ◽

Relative Abundances ◽

Mucus Barrier

ABSTRACTGastrointestinal disturbance is frequently reported for individuals with autism. We used quantitative real-time PCR analysis to quantify fecal bacteria that could influence gastrointestinal health in children with and without autism. Lower relative abundances ofBifidobacteriaspecies and the mucolytic bacteriumAkkermansia muciniphilawere found in children with autism, the latter suggesting mucus barrier changes.

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Real-time PCR analysis of enteric pathogens from fecal samples of irritable bowel syndrome subjects

Gut Pathogens ◽

10.1186/1757-4749-3-6 ◽

2011 ◽

Vol 3 (1) ◽

pp. 6 ◽

Cited By ~ 30

Author(s):

Teemu Rinttilä ◽

Anna Lyra ◽

Lotta Krogius-Kurikka ◽

Airi Palva

Keyword(s):

Irritable Bowel Syndrome ◽

Real Time ◽

Real Time Pcr ◽

Enteric Pathogens ◽

Pcr Analysis ◽

Fecal Samples ◽

Irritable Bowel

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Duplex real-time PCR assay for the simultaneous detection of Achromobacter xylosoxidans and Achromobacter spp.

Microbial Genomics ◽

10.1099/mgen.0.000406 ◽

2020 ◽

Vol 6 (7) ◽

Author(s):

Erin P. Price ◽

Valentina Soler Arango ◽

Timothy J. Kidd ◽

Tamieka A. Fraser ◽

Thuy-Khanh Nguyen ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Type Species ◽

Simultaneous Detection ◽

Diagnostic Methods ◽

Achromobacter Xylosoxidans ◽

Resistant Bacteria ◽

Pcr Assay ◽

Content Type ◽

Link Type

Several members of the Gram-negative environmental bacterial genus Achromobacter are associated with serious infections, with Achromobacter xylosoxidans being the most common. Despite their pathogenic potential, little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management. Here, we performed comparative genomics for 158 Achromobacter spp. genomes to robustly identify species boundaries, reassign several incorrectly speciated taxa and identify genetic sequences specific for the genus Achromobacter and for A. xylosoxidans . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans , with five Achromobacter isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ~110 genome equivalents and detected down to ~12 and ~1 genome equivalent(s), respectively. Extensive in silico analysis, and laboratory testing of 34 non- Achromobacter isolates and 38 adult cystic fibrosis sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans and will facilitate the rapid and accurate diagnosis of this important group of pathogens.

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Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples

Applied and Environmental Microbiology ◽

10.1128/aem.00623-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6570-6578 ◽

Cited By ~ 22

Author(s):

Sonia E. Létant ◽

Gloria A. Murphy ◽

Teneile M. Alfaro ◽

Julie R. Avila ◽

Staci R. Kane ◽

...

Keyword(s):

Bacillus Anthracis ◽

Real Time ◽

Incubation Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Limit Of Detection ◽

Turnaround Time ◽

Pcr Analysis ◽

Content Type ◽

Pcr Method

ABSTRACTIn the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulentBacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for theB. anthracischromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulentB. anthracisin environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.

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