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

Abstract

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.

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

“…The results show a lower than expected PPA of TAC on archival samples and significant variability in PPA estimates by pathogen when compared to previously published estimates [ 4 , 5 ]. This is partly explained by the small sample size resulting in wide confidence intervals, an important limitation in our study as well as prior reports [ 7 , 9 ]. The results suggest that using TAC on archival samples stored for several years may lower sensitivity and underestimate pathogen burden.…”

“…FTA Cards from AFRIMS were batch shipped to NMCP and tested within a month of receipt. Extraction and TAC testing was performed as previously described [ 9 ].

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“…Unfortunately, delays in receiving the stool specimens resulted in FTA Cards being stored for 2–3 years at room temperature prior to extraction and testing and negatively impacted PPA estimates for bacterial targets especially at higher stool Cqs. The PPA of TAC on FTA Cards across bacterial targets was lower than estimates from a post hoc analysis using fresh stool samples smeared on to FTA Cards and stored for approximately 2 years prior to extraction and testing [ 9 ]. This finding suggests that using archival samples that are freeze–thawed may negatively impact TAC performance on smeared FTA Cards compared to fresh stool.…”

Performance Characteristics of the TaqMan Array PCR on Repository Stool Specimens and Smears on Whatman® FTA Elute Cards

“…Stool samples were tested within a month of receipt from NAMRU-6. Extraction and TAC testing was performed as previously described [9].…”

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

“…FTA Cards from AFRIMS were batch shipped to NMCP and tested within a month of receipt. Extraction and TAC testing was performed as previously described [9].…”