Background: Diagnosis of gastrointestinal parasites has traditionally relied on stool microscopy that has low diagnostic sensitivity and specificity, is time consuming and labor intensive. To overcome these existing barriers, we have developed a novel rapid, high throughput molecular diagnostic platform.
Methods: Species-specific primers and probes were generated and used for the 8 most common gastrointestinal parasite pathogens -- Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale, Giardia lamblia, Cryptosporidium parvum, Entamoeba histolytica, Trichuris trichiuria and Strongyloides stercoralis – in a standardized and quantitative multi-parallel fast chemistry-based real time PCR (qPCR) high throughput assay. This was then used to analyze stool samples collected from 540 children in rural Ecuador and compared to standard stool microscopy.
Results: The qPCR improved the sensitivity for Ascaris by 21.9% and had 97.8% specificity and 96.8% negative predictive value. Moreover there was a direct relationship between the egg counts by stool examination with nanograms of Ascaris DNA (spearman r: 0.69, p=0.0001) (Figure 1). qPCR improved the sensitivity for Giardia by 81.7% (126/400 for qPCR vs. 23/400 for stool examination p=0.001) (Figure 2). In addition, the qPCR assays were able to detect infections for Ancylostoma, Cryptosporidium, Strongyloides not seen by microscopy. It further provided methods to distinguish the pathogenic Entamoeba histolytica from the non-pathogenic E. dispar. Finally, by using a novel DNA extraction protocol, we were able to detect Trichuris, with an improved sensitivity of 75% and a direct correlation to DNA (spearman r: 0.74, p=0.0001).
Conclusion: Thus, we have developed a high throughput, rapid, operator-independent molecular-based system to improve the diagnostic accuracy of stool based parasite pathogen detection. This approach has been field tested in rural Ecuador and will be useful to refine treatment options for affected populations and ultimately lead to better health outcomes in resource-limited settings.
Figure 1: qPCR has a direct correlation to egg count for Ascaris
Figure 2: Prevalence of GI Parasites
P. Cooper, None
T. Nutman, None