BacteriologyDetection of enteropathogens associated with travelers' diarrhea using a multiplex Luminex-based assay performed on stool samples smeared on Whatman FTA Elute cards
Introduction
Travelers' diarrhea is frequently experienced by civilians and deployed military personnel traveling to developing countries. Although the identification of enteropathogens in epidemiologic studies has been greatly facilitated by the use of polymerase chain reaction (PCR) assays (Al Amri et al., 2007, Aranda et al., 2004, Couturier et al., 2011), testing remains limited due to requirements for collection, storage, and transportation of diarrheal specimens. As a result, epidemiological data are largely derived from cohorts with on-site testing and storage facilities.
Self-collected stool smears obtained on a filter paper matrix is an appealing alternative for stool collection and storage in field conditions (Grimes et al., 2008, Orlandi and Lampel, 2000). However, the utility of filter paper and impact on PCR detection is unknown. We performed a pilot study to compare the limits of detection (LoDs) of a multiplex PCR assay between tool samples and smeared Whatman™ FTA Elute cards (GE Healthcare Bio-Sciences Pittsburgh, PA) and determine the impact of prolonged storage and environmental conditions on detection from cards. Enteropathogens tested included norovirus (GI and GII), Salmonella enteritidis, Salmonella typhimurium, enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), Shigella sonnei, Campylobacter jejuni, Giardia lamblia, and Cryptosporidium.
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Materials and methods
Reference strains from commercial entities (American Type Culture Collection, Waterborne, and Zeptometrix) or clinical isolates (enteroaggregative E. coli and Campylobacter) were used. Bacterial strains were inoculated into Mueller–Hinton (MH) broth, incubated overnight, and the CFU/g concentration was determined by measuring the turbidity at 600 nm. Known concentrations of parasite oocysts and viruses were serially diluted in phosphate-buffered saline and deionized water, respectively. Five
Results
Overall, the LoDs ranged from 102 to 105 CFU, PFU, or cysts/g for most enteropathogens. LoDs were comparable (within 1–2 logs) between stool samples and stool cards at 1 week (Table 1). Cryptosporidium was not detected in spiked stool and had a high LoD in the stool card, probably due to the lack of oocyst disruption (e.g., bead beating) during sample processing. No sustained increase in the LoD at 3 months was noted for most pathogens except Campylobacter, which increased at 1 month and could not
Discussion
Our results indicate that the FTA Elute Card may be an effective method of storing genomic material from most diarrheal pathogens. Comparable LoDs were observed between stool samples and stool cards, indicating effective storage of genomic material and sequestration of factors inhibiting PCR. The LoDs observed were comparable to those reported in the literature (Liu et al., 2012, Navidad et al., 2013) and within the range associated with symptomatic infection (Granato et al., 2010, Lampel, 2005
Acknowledgments
The authors thank their collaborators at Naval Medical Research Center, Bethesda, MD, and Naval Medical Research Unit-6, Peru, for providing clinical isolates needed for assay validation.
Supported by the Infectious Disease Clinical Research Program, a Department of Defense (DoD) program executed through the Uniformed Services University of the Health Sciences, the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), under the Inter-Agency Agreement
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