Background: Reliable and sensitive diagnostic tests have important implications; otherwise it limits the understanding of disease magnitude and epidemiology. The primate whipworm Trichuris trichiura, a soil-transmitted helminth (STH) is the principal whipworm in the genus Trichuris infecting human. Methodology and Principal Findings: In this study, we examined fecal samples from 337 school- age children (≥15) of Southern Assam, India for Trichuris infection using microscopy followed by species discrimination using species specific nested PCR assay and bi-directional Sanger DNA sequencing of the diagnostic fragment. We further evaluated the sensitivity and specificity of primer pairs specific for Enterocytozoon bieneusi and Cyclospora cayetanensis. Based on single fecal examination 49 were microscopically positive for characteristic Trichuris eggs with a mean length and width of 54.3 (±2.8) μm and 23.8 (±1.9) μm respectively, while species specific PCR assay classified that all the 49 microscopy positive samples were T. trichiura. In addition, PCR assay classified 9 more positive cases of T. trichiura infection with an overall prevalence rate of 17.2% (58/337; 95% CI: 13.4–21.8%). Sensitivity and specificity evaluation of the primer pairs specific for E. bieneusi and C. cayetanensis showed no cross-amplification with the common pathogenic gastrointestinal protozoan, bacterial DNA and were able to detect 20ng and 30ng of DNA respectively. Conclusions: The findings of our study suggest that the newer molecular diagnostic methods, particularly species specific PCR assay intensifies the detection of Trichuris infection at species level and thus help to figure out the burden of zoonotic species and its transmission in a particular geographical area. In addition, the minimum detection levels of the primer sets suggest that the designed primers are sensitive enough for accurate detection of the two emerging protozoan parasites in clinical samples with low DNA yield.
