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

Abstract

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.

Keywords: Archives; Diarrhea; Polymerase chain reaction; Travel.

Fig. 1

Flow diagram of samples received from both repositories and duration of storage prior to testing

Fig. 2

FTA card and stool samples positive vs. samples negative on microbiologic testing (n = 109). ETEC detection by TAC includes multiple targets: LT, STh and STp. Norovirus detection by TAC on FTA cards and stool includes the Norovirus GI or Norovirus GII target. Shigella and Campylobacter detection was based on a single target. Significant difference in median Cq values observed for the following by pairwise comparison: Campylobacter: Benchmark (+)/FTA Card(+) vs. Benchmark(−)/FTAcard(+) p = 0.036. Shigella/EIEC: Benchmark (+)/FTA Card(+) vs. Benchmark (−)/FTAcard(+) p = 0.046. Norovirus: Benchmark (−)/Stool (+) N = 18 Benchmark (-)/FTAcard(+) p = 0.003. Benchmark (+)/Stool (+) N = 7 Benchmark (+)/FTAcard(+) p = 0.033. The correlation between corresponding stool and FTA card targets was poor (r² : ETEC 0.37, Campylobacter: 0.27, Shigella 0.40 and Norovirus 0.07; p < 0.01 for all)