Development of loop mediated isothermal amplification assay for rapid detection and its comparison with PCR techniques for Ancylostoma duodenale

Hookworm infections, primarily affecting children and pregnant women are major health concern. As part of global efforts to eradicate neglected tropical diseases (NTDs) through mass chemotherapy, reliable diagnostic methods are urgently needed as regions work towards elimination. In this study, we have developed a LAMP-based method to detect hookworm infection in faecal samples. The primers, tailored for the LAMP assay, were designed targeting ITS-1 region of Ancylostoma duodenale using Primer Explorer version 5. Five hundred (500) human stool samples were subjected to microscopy and DNA extraction simultaneously, followed by conventional PCR, real-time PCR and LAMP amplification. The specificity, sensitivity and inter-rater agreement of all assays were evaluated using MedCalc software taking real-time PCR as gold standard. The newly devised LAMP method successfully identified A. duodenale DNA in fecal samples in less than 40 min. It demonstrated perfect specificity (100%), with no instances of cross-reactivity. The LAMP assay achieved a sensitivity of 87.8%, 83.7% for conventional PCR and 59% for microscopy with 100% specificity for all methods. The newly developed LAMP assay represents a suitable diagnostic approach for identifying A. duodenale DNA in human stool samples due to its simplicity, affordability, sensitivity, specificity and potential for field applicability.