C-225. PCR-Bead-Based Detection of Protozoan Enteropathogens from Stool

L. Krna, S. Stroup, E. R. Houpt;
Univ. of Virginia, Charlottesville, VA.

Background: AIDS associated chronic diarrhea is a significant problem in developing countries. Protozoal etiologies are common, including Cryptosporidium parvum, Giardia lamblia, Isospora belli, and the microsporidium Enterocytozoon bieneusi. Methods: Microscopy is insensitive and antigen detection unavailable for several of these pathogens, therefore we sought to develop a multiplex PCR assay followed by bead detection on a Luminex instrument. All PCR assays were designed with specific primers for the same gene (18s rRNA gene), with similar amplicon size (100 to 363 bp), master mixes, and cycling conditions. Reverse primers were biotinylated and internal specific probes were covalently linked to Luminex beads. Hybridization of amplicon to probe occurred at 37-52°C and the reporter used was streptavidin-PE. Results: The protocol revealed specific detection of C. parvum, G. lamblia, I. belli, and E. bieneusi in PBS and in spiked stool samples. Mean fluorescence intensity was 2959 (+/-117), 1582 (+/-51), 1555 (+/-87), and 9803 (+/-87), respectively, for each appropriate bead in 105 oocysts spiked into stool for C. parvum and G. lamblia and 5ng of cloned DNA from I. belli and E. bieneusi (p<0.01 versus background of 201 +/-30). Conclusion: We are now deploying the assay in Tanzania and incorporating remaining protozoal and enteric bacterial etiologies towards the goal of a single assay for all major enteropathogens.