PCR-Luminex Based Detection of 28 Enteropathogens

Abstract
Background: Given the range of potential enteropathogens implicated in diarrhea, we developed a PCR-Luminex based assay to detect 28 most common organisms associated with diarrhea. The enteropathogens included were Cryptosporidium spp., Entameoba histolytica, Giardia, Microsporidia, Cystoisospora, Cyclospora, Ascaris, Hookworms, Strongyloides, Tricuris, EAEC, EHEC, EIEC, EPEC, ETEC, Astrovirus, Norovirus G1 and G2, Rotavirus, Sapovirus, Adenovirus, and an internal control. This panel was used in a prospective study to determine etiology of diarrhea in Bangladeshi children in the first year of life. Methods: Specific primers and probes were designed for the organisms using targets which are conserved. DNA or RNA purified from stool was amplified using biotinylated primers, followed by hybridization to amine-modified probes covalently linked to carboxylated spectrally-distinct microspheres, followed by addition of streptavidin PE to detect specifically-bound amplicon. Luminex results are reported as corrected mean fluorescence intensity (cMFI) normalized to background, where cMFI > 2.5 was utilized as a “present” call with the exception of Cryptosporidium, Strongyloides, all EC, and all viruses where cMFI values were 7.3, 9.0, 10.0, and 2.0, respectively. Results: Performance of the assays yielded 95% to 100% sensitivity and specificity versus the assays performed via real-time PCR. We then applied the tests in a prospective study of 147 children from Mirpur, Bangladesh followed monthly for the first year of life. 83% of the children had at least 1 episode of diarrhea in the first year of life and 33% had 4 or more. The number of pathogens that were detected increased as the number of diarrheal episodes increased (e.g., from 1.7 to 2.5 from first diarrheal episodes to 4th or later diarrheal episodes, P <0.05). Rotavirus, ETEC, EAEC, and were the leading pathogens detected during the first diarrheal episodes, while Adenovirus, Shigella, ETEC, EPEC, and Giardia emerged as common in the later episodes. Conclusion: This Luminex based assay for the major enteropathogens offers sensitive and specific detection similar to real time PCR. When applied to field studies in endemic areas, a singular etiology of diarrhea is difficult to determine due to the frequency of mixed infections, and multiple pathogens may be the norm. Pathogens appear to accumulate in children that develop recurrent diarrhea.