Performance Characteristics of the TaqMan Array PCR on Repository Stool Specimens and Smears on Whatman® FTA Elute 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 [49–75%]), Campylobacter spp. (66 [43–85%]) and Norovirus (37 [16–61%]). Use of the FTA card resulted in a further reduction of PPA. Our ndings 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.


Introduction
Polymerase Chain Reaction (PCR) assays have signi cantly improved the detection of travelers' diarrhea (TD) pathogens but interpreting results is often challenging due to asymptomatic shedding and multipathogen detection [1][2][3]. Studies evaluating the performance characteristics of PCR assays have largely been conducted in clinic or hospital settings using diarrheal samples tested within days of collection [4,5]. This sampling method is limited by the infrequency of TD pathogens such as enterotoxigenic Escherichia coli, Shigella spp. and Campylobacter spp. and the lack of appropriately matched control specimens (e.g. asymptomatic travelers) for attribution of TD to detected pathogens. PCR data from longitudinal pediatric cohorts in developing countries cannot be readily extrapolated to adult TD populations due to differences in the host and environment that impact pathogen load and multipathogen detection in stool samples [6].
Biorepositories of clinically characterized diarrheal and non-diarrheal specimens provide an alternative resource to investigate clinical interpretation of PCR assay results. Biorepository specimens could be used to determine the odds of TD associated with detection of speci c pathogens, and potentially re ne estimates using quanti cation cycle (Cq) thresholds for pathogens detected in cases and controls. The suitability of archival specimens for DNA/RNA ampli cation and impact on assay performance is unclear, due to potential degradation. There are also logistical challenges in transporting frozen fecal specimens from global biorepositories to a single testing site while maintaining the cold-chain, which can Results Two hundred and sixty-one samples for targeted pathogens were available: ETEC (n = 70), Shigella (n = 62), Campylobacter (n = 59), Norovirus (n = 27). Four samples were positive for more than 1 pathogen and 47 samples were negative for all targeted pathogens. All stool samples were thawed, vortexed and approximately 20 µL smeared onto an FTA card and shipped at room temperature to Naval Medical Center Portsmouth (NMCP) for testing. Due to logistic and regulatory constraints a limited number of stool specimens were shipped from NAMRU-6 and only smeared FTA Cards were shipped from AFRIMS. 109 out of 261 (41.7%) stool samples were aliquoted and shipped at -20 • F for testing (Fig. 1). Laboratory personnel performing TAC testing were blinded to the results from previous testing. FTA Cards were received approximately two years before stool samples due to delays in getting required approvals for be cost-prohibitive. The Whatman FTA Elute Card® (FTA card, GE Healthcare Life Sciences, Marlborough, MA, USA) is an attractive alternative to conventional storage and shipment methods, as they can be shipped by regular mail at room temperature. The impact of using FTA Cards for sample storage and transportation on PCR sensitivity has not been evaluated.

Methods
We conducted a pilot study using a customized TD TaqMan Array Card (TAC) to assess the feasibility of using archived diarrheal specimens, of varying time periods, focusing on four TD pathogens: enterotoxigenic E coli (ETEC), Shigella spp., Campylobacter spp., and Norovirus. Two Department of Defense (DoD) fecal repositories of adult TD cases, tested using standard microbiologic methods at the time of sample collection, were utilized for the study [7,8]: We evaluated the Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) of TAC for detecting TD pathogens in archived fecal specimens, using results from previous microbiologic testing as the 'benchmark', the impact of storage duration and use of FTA cards on TAC sensitivity. Approximately 65 positive stool specimens per pathogen for ETEC, Shigella spp., Campylobacter spp., and Norovirus (based on an estimated PPA and NPA of 80% for the TAC assay and precision of 10%) and 30 samples negative for all pathogens, were requested. The study was approved by the Institutional Review Boards shipment. To reduce between run variability, we included corresponding FTA Card and stool samples in the same PCR run. Thus, FTA cards were stored at room temperature until stool samples were received. Stool samples were tested within a month of receipt from NAMRU-6. Extraction and TAC testing was performed as previously described [9].
The PPA for TAC on stool vs. benchmark was lower than our a priori PPA estimate of 80% across all pathogens except Shigella (Table 1) increasing duration of storage (i.e. ≤ 3 years and 5-13 years) was noted for ETEC and Campylobacter positive FTA Cards, although the analysis was limited by small sample size and wide con dence intervals of PPA estimates between strata of storage duration and could not be assessed for Shigella or Norovirus (Table 1). To distinguish whether the sensitivity loss was due to RNA/DNA degradation or lower extraction/ampli cation e ciency (i.e. due to PCR inhibition or loss during extraction), we compared the difference in extrinsic control Cq between the sample and extraction blank in samples stored for ≤ 3 years and 5-13 years. A positive Cq difference of 3.3 was considered equivalent to a 1-log loss in PCR signal due to inhibition. The proportion of samples that met this criterion were similar in the two strata of storage duration, suggesting that FTA cards and stool samples stored for 5-13 years did not demonstrate lower extraction/ampli cation e ciency or inhibition and thus the decrease was likely due to RNA/DNA degradation.  Next, we compared Cq values of TAC on stool and FTA cards for 109 paired samples strati ed by pathogen and results from benchmark testing (Fig. 2). We hypothesized that Cq values would be lower in FTA card and stool samples that were positive on benchmark testing vs. negative samples. A wide range of Cq values were observed across all pathogens and a signi cant difference in the median Cq value between benchmark positive and negative samples was only observed in FTA Cards positive for Campylobacter (p = 0.036) and Shigella (p = 0.046).

Discussion
We tested archived stool specimens using TAC to determine the feasibility of using these specimens for clinical validation of TAC. The results show a lower than expected PPA of TAC on archival samples and signi cant 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 con dence 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. Published reports, largely in the realm of microbiome research, focus on relatively short storage durations of days to months [11,12]. Cannon [14]. In addition, careful documentation of processing, storage and sampling methods and an understanding of their impact on quantitative PCR data is needed to appropriately adjust detection estimates using archival samples.
We also evaluated smeared FTA Cards for storage and transportation of archival samples. 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 nding suggests that using archival samples that are freeze-thawed may negatively impact TAC performance on smeared FTA Cards compared to fresh stool. Additional studies are needed to evaluate strategies for increasing PCR yield from smeared FTA card such as reducing storage duration of FTA Cards, refrigeration or adding preservatives (e.g. RNAlater [Invitrogen, Carlsbad, CA]).
An important limitation of our study was the small sample size of bacterial targets. We could not evaluate the loss of PCR signal with increasing storage duration since TAC testing of stool samples and FTA cards occurred at a single time-point and not longitudinally at pre-speci ed time intervals.
Microbiologic testing performed at the time of collection (i.e. benchmark) varied by site and time period, confounding the association between storage duration and TAC sensitivity. Despite these limitations, our pilot study adds important insights into the use of repository samples to validate quantitative PCR assays. Furthermore, it highlights the importance of developing standard operating procedures at the inception of studies to optimize processing and preservation of fecal samples and enhance the comparability and reproducibility of data [15,16]. In summary, our ndings suggest that TAC sensitivity on archival specimens may be lower than previous estimates using specimens with a shorter duration of storage.

Limitations
Limitations of our study were the small sample size of bacterial targets and timing of microbiological testing at differing sites. Future efforts should focus on adequately powered studies of TD stool archival specimens are needed to understand the impact of storage duration on quantitative PCR data.

Availability of Data and Materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Research data derived from an approved Uniformed Services University of the Health Sciences IRB (IDCRP-076, USUID.2013.0001) protocol. Consent to participate is not applicable.

Consent for Publication
Not applicable.