Bacteriology
A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens

https://doi.org/10.1016/j.diagmicrobio.2014.02.022Get rights and content

Abstract

Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens.

Introduction

Enteric pathogens that cause gastroenteritis remain a major global health concern and are 1 of the top infectious causes of morbidity and mortality worldwide (Cunningham et al., 2010, de Boer et al., 2010, Gomez-Duarte et al., 2009, Nguyen et al., 2005, O'Leary et al., 2009). Infectious gastroenteritis is responsible for 1.7–2.5 million deaths annually, mostly of children under 5 years of age in developing countries (Gomez-Duarte et al., 2009, Kosek et al., 2003, Kosek et al., 2009, Liu et al., 2011, UN, 2005, WHO, 2011). In light of major global infectious disease emergencies, such as the cholera epidemic in Haiti and the highly virulent Escherichia coli outbreak across Europe, enteric bacterial pathogens remain a significant public health threat in both the developed and developing parts of the world (Askar et al., 2011, Enserink, 2011, Walton and Ivers, 2011, Nature News, 2011). Conventional diagnostic procedures for routine detection of enteric bacterial pathogens entail enrichment steps, selective culture, biochemical identification, serotyping, and/or resistance profiling. The steps for pathogen identification are laborious and time consuming, often taking 3–5 days for a final result. Several pathogens are indistinguishable from normal flora by morphology and biochemical properties. Other pathogens are difficult to culture while some have limited viability outside the host (Cunningham et al., 2010, de Boer et al., 2010, Schuurman et al., 2007). Molecular methods, including TaqMan assays or antigen detection tests, offer more rapid and sensitive detection than conventional culture methods (Gomez-Duarte et al., 2009, Liu et al., 2011, O'Leary et al., 2009). However, these molecular methods are limited in scope and scale for both the number of samples processed annually and the broad and wide-ranging number of possible pathogens that can be detected.

We have developed, characterized, and applied a high-throughput multiplex molecular assay for the simultaneous detection of Campylobacter spp. (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter fetus, Campylobacter upsaliensis), Vibrio spp. (Vibrio cholerae, Vibrio mimicus, Vibrio parahaemolyticus, Vibrio fluvialis, Vibrio furnissii, Vibrio metschnikovii, Vibrio hollisae), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), diffusely adherent E. coli (DAEC), Shigella spp. (Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Shigella boydii), Salmonella spp. (Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi, Salmonella arizoniae, Salmonella cholerasuis), Listeria monocytogenes, and Yersinia enterocolitica. This assay can detect and identify over 29 species of enteropathogenic bacteria across 7 different genera in a single assay protocol. Additionally, we applied this assay to clinically significant culture-positive samples for V. cholerae obtained from our international study site, the Groupe Haitien d’Etude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) in Port-au-Prince, Haiti. Currently, this multiplex assay is the only comprehensive molecular test reported to date for the detection of all category B bacterial food and water-borne pathogens.

Section snippets

Bacterial isolates and clinical specimens

Clinical isolates from stool and other clinical specimens that had been previously phenotypically identified using conventional microbiological techniques were obtained from the clinical microbiology laboratory at New York-Presbyterian Hospital/Weill Cornell Medical Center (NYPH/WCMC) and the New York City Department of Health. Molecularly characterized isolates of diarrheagenic E. coli were kindly provided by Dr Lee Riley of the University of California Berkeley. Pure cultures of the following

Analytical sensitivity and specificity of the PCR/LDR assay with clinical isolates

The analytical specificity of the assay was 100% for pure cultures of all 97 clinical isolates. There were no false positives when testing each pure culture isolate with the PCR/LDR assay, indicating no cross-reaction between the 51 PCR primers and 157 LDR primers. We determined the limit of detection of our assay on seeded specimens to range from 101 CFU/mL to 105 CFU/mL. The concentration in which all pathogens were detected was 105 CFU/mL.

Diagnostic sensitivity and specificity of PCR/LDR with seeded specimens

The diagnostic sensitivity and specificity of the

Discussion

In this study, we describe the development and application of a multiplex PCR/LDR assay for the identification of all NIAID category B bacterial food and water-borne pathogens. The assay was designed to PCR amplify 17 genetic targets attributed to a panel of 29 pathogenic bacterial species. Species-specific single base variants within these PCR amplicons are then queried using LDR primer pairs that generate ligation products that are subsequently hybridized to their discrete individual VeraCode

Acknowledgments

We thank Stephen Jenkins, Audrey Schuetz, and the technical staff of the clinical microbiology laboratory at NYPH/WCMC for collecting and providing clinical isolates and specimens and Bill Pape at GHESKIO for his advice and support. We thank Jianmin Huang for providing the AK16D ligase enzyme. We acknowledge Lee Riley of the University of California Berkeley and the New York City Department of Health for providing us with clinical isolates.

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    Funding: This work was supported by Public Service grants UCI-AI062579 and U01-AI075470-01 from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health. This work was also supported by Ruth L. Kirschstein National Service Award (NRSA) T32 Training Grant 5T32A1007613 from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health.

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