Multiplexed recombinase polymerase amplification detection of intestinal protozoa using lateral flow strips
Zachary A. Crannell, Miguel M. Cabada, Alejandro Castellanos-Gonzalez, Ayesha Irani,
Mauricio Rebolledo, A. Clinton White, Jr, and Rebecca Richards-Kortum
When: November 2-6, 2014
Where: Sheraton New Orleans Hotel and New Orleans Marriott Hotel, New Orleans
Protozoan infections of Cryptosporidium, Giardia, and Entamoeba are increasingly being recognized as important causes of diarrheal episodes and are associated with growth and cognitive impairment. While each is treatable, the treatments differ. However, the clinical presentations are similar, underlying the importance of accurate diagnostics. Current diagnosis relies heavily on stool smear, which even in the best hands is neither sensitive nor specific. We have developed isothermal nucleic acid amplification assays that can detect low-level infections as well as PCR, but does not require expensive equipment that is often unavailable in low-resource settings. The recombinase polymerase amplification (RPA)-Cryptosporidium assay demonstrates a limit-of-detection (LOD) comparable to or better than PCR (100 parasites/ml stool). Similarly the RPA-Giardia assay has a LOD that compares well with established PCR assays (1,000 parasites/ml stool). The RPA-Giardia assay was field tested in the highlands of Peru using 111 stool specimens collected from rural communities. For specimens containing low and medium concentrations of DNA (90/111 specimens), the sensitivity was 93% and the specificity was 94%. The RPA-Entamoeba assay is currently undergoing bench-top testing and, like the other RPA assays, demonstrated a LOD equivalent to the gold standard PCR. We are currently working on integrating the three assays with our low-resource DNA extraction protocols to build a multiplex assay that can detect all three parasites on a single lateral flow strip.