TaqMan Array Card testing of participant-collected stool smears to determine the pathogen-specific epidemiology of travellers' diarrhoea†.

BACKGROUND: We assessed the compliance with self-collection of stool smears on Whatman® FTA® Elute Card (FTA Card) and detection of travellers' diarrhoea (TD)-associated pathogens by using a quantitative Polymerase Chain Reaction (PCR) assay [customized TaqMan® array card (TAC)] in a prospective, observational cohort of travellers. METHODS: Enrolled travellers documented symptoms on a travel diary and collected an FTA Card during a diarrhoeal episode, or at the end of travel if they remained asymptomatic. TAC testing was performed on FTA Cards from TD cases and 1:1 matched asymptomatic controls and 1:1 matched loose stool cases that did not meet TD criteria. Odds ratios were used to determine the association between detected pathogens and TD. RESULTS: Of 2456 travellers, 484 (19.7%) completed an illness diary and met TD criteria, and 257 (53.1%) collected an FTA Card during the TD episode. FTA Cards were stored for a median of 2 years at room temperature (IQR: 1-4 years) before extraction and testing. The overall TAC detection rate in TD cases was 58.8% (95% CI: 52.5-64.8). Enterotoxigenic Escherichia coli was the most common pathogen in TD cases (26.8%), and 3.5% of samples were positive for norovirus. The odds of detecting TD-associated pathogens in 231 matched cases and asymptomatic controls were 5.4 (95% CI: 3.6-8.1) and 2.0 (95% CI: 1.1-3.7) in 121 matched TD and loose stool cases (P < 0.05). Enteroaggregative E. coli was the most common pathogen detected in asymptomatic controls and loose stool cases. Detection of diarrhoeagenic E. coli, Shigella/enteroinvasive E. coli and Campylobacter spp. was significantly associated with TD. CONCLUSION: FTA Cards are a useful adjunct to traditional stool collection methods for evaluating the pathogen-specific epidemiology of TD in austere environments. Qualitative detection of pathogens was associated with TD. Measures to improve compliance and quality of FTA Card collection with decreased storage duration may further optimize detection.

Performance characteristics of a quantitative PCR assay on repository stool specimens and smeared filter-paper cards.

OBJECTIVE: Stool repositories are a valuable resource for retrospective analyses including quantitative PCR assays to distinguish between asymptomatic shedding and clinical disease. The suitability of archival specimens for this purpose is unclear and requires assessment. We conducted a pilot study to evaluate pathogen detection by TaqMan Array Card (TAC) in travelers' diarrhea (TD) stool specimens stored for 1-13 years, as well as the impact of transporting specimens on Whatman FTA Elute cards (FTA Cards) on detection. RESULTS: The positive percent agreement (PPA) for TAC on stool vs. microbiologic testing was lower than our a priori PPA estimate of 80% for most pathogens: Shigella spp. (100% [95%CI 69-100%]), enterotoxigenic E coli (ETEC) (63% [95%CI 49-75%]), Campylobacter spp. (66% [95%CI 43-85%]) and Norovirus (37% [95%CI 16-61%]). Use of the FTA card resulted in a further reduction of PPA. Our findings suggest that archival specimens may lead to insensitive detection on quantitative PCR assays due to degradation of nucleic acid with prolonged storage, although our limited sample size precluded us from evaluating the impact of storage duration on nucleic acid yield. Additional studies are needed to understand the impact of storage duration on quantitative PCR data.

A Comparison of Stool Enteropathogen Detection by Semiquantitative PCR in Adults With Acute Travelers' Diarrhea Before and 3 Weeks After Successful Antibiotic Treatment.

We evaluated stool enteropathogen detection by semiquantitative polymerase chain reaction (PCR) in 108 subjects with travelers' diarrhea before and 3 weeks after treatment. Stool samples from 21 subjects were positive for the same pathogen species at both visits. We discuss factors that should be considered when interpreting stool PCR data after treatment.

Comparison of stool collection and storage on Whatman FTA Elute cards versus frozen stool for enteropathogen detection using the TaqMan Array Card PCR assay.

The use of Polymerase Chain Reaction (PCR) assays for pathogen detection in travelers' diarrhea (TD) field studies is limited by the on-site processing and storage requirements for fecal specimens. The objectives of this investigation were to i) characterize the pathogen distribution in deployed military personnel with TD using the TaqMan® Array Card PCR (TAC) on frozen stool and diarrheal smears on Whatman FTA Elute cards (FTA cards), and to ii) compare TAC detection of enteropathogen targets using smeared FTA cards and frozen stool, using TAC on frozen stool as the 'reference standard'. Stool samples, obtained from active duty personnel with acute TD enrolled in a field trial, were smeared onto FTA cards and stored at room temperature. A corresponding aliquot of stool was frozen in a cryovial. FTA cards and frozen stool samples were tested at a central lab, using a customized TAC for detection of TD pathogens. 187 paired frozen stool samples and smeared FTA cards were stored for a median of 712 days (IQR 396-750) before testing. Overall detection rates were 78.6% for frozen stool and 73.2% for FTA cards. Diarrheagenic Escherichia coli were the most common bacteria identified. Using the TAC results on frozen stool as the reference, the overall sensitivity and specificity of TAC on FTA cards was 72.9% and 98.0% respectively. TAC on FTA cards demonstrated a decrease in sensitivity with increasing frozen stool quantification cycle (Cq) (90.0% in FTA cards with a corresponding frozen stool Cq < 30, and 72.9% in samples with a corresponding frozen stool Cq < 35). Our findings support the use and further development of FTA cards in combination with a quantitative PCR assay for enteropathogen detection in TD field studies.

Prevalence of Staphylococcus aureus Colonization and Risk Factors for Infection Among Military Personnel in a Shipboard Setting.

Staphylococcal skin and soft tissue infections (SSTIs), especially those due to methicillin-resistant Staphylococcus aureus (MRSA) are an important public health issue for the military. Limited data exist regarding the prevalence of S. aureus colonization in the shipboard setting. We conducted a cross-sectional, observational study to determine the point prevalence of S. aureus colonization among military personnel onboard a naval vessel. Asymptomatic active duty personnel completed a survey for risk factors associated with colonization and SSTIs. Culture specimens were obtained from the anterior nares, pharynx, groin, and perirectal regions. MRSA isolates underwent testing for antimicrobial resistance, virulence factors, and pulsed-field type. 400 individuals were enrolled, 198 (49.5%) of whom were colonized with S. aureus, with MRSA identified in 14 participants (3.5%). No significant risk factors were associated with MRSA colonization. USA800 was the most common colonizing MRSA strain in the cohort and was detected in 10 participants (71%). Two participants (14%) were colonized with USA300 MRSA. In this first report of S. aureus epidemiology in a shipboard setting, we observed high rates of S. aureus and MRSA colonization. Longitudinal studies are needed to document the incident rates of S. aureus colonization during shipboard deployment and its impact on SSTI risk.

Detection of enteropathogens associated with travelers' diarrhea using a multiplex Luminex-based assay performed on stool samples smeared on Whatman FTA Elute cards.

We evaluated the limits of detection (LoD) for an 11-plex PCR-Luminex assay performed on Whatman(™) FTA Elute cards smeared with stool containing pathogens associated with travelers' diarrhea. LoDs ranged from 10(2) to 10(5)CFU, PFU, or cysts/g for most pathogens except Cryptosporidium. Campylobacter and norovirus LoDs increased with prolonged storage of cards.