Pan filarial-nested PCR with gelified tubes: a new approach towards point-of-care testing for human filariasisFIRST REVIEW ROUND - REVIEWERS' COMMENTSREVIEWERS' COMMENTS.

backgroundIn recent years, traditional point-of-care tests (POCTs) for diagnosing human filariasis have demonstrated substantial limitations, raising concerns regarding their reliability in the current epidemiological context, in which numerous countries are approaching the elimination of human filariasis. Consequently, there is a need for new molecular tests that allow the simultaneous and reliable detection of multiple human filarial species, even in cases of very low microfilaremia.

objectivesThe objective of this study was to convert the Fn-PCR from its original manual format to the new gelified tube-filarial-nested polymerase chain reaction (Gel-Fn-PCR), which overcomes some of the disadvantages of traditional molecular techniques thanks to its gelified tube format.

methodsA total of 661 dried blood spots, along with their corresponding stained blood smears, were employed for the optimisation and validation processes. The results of the Gel-Fn-PCR were compared to the microscopic diagnosis.

findingsThe Gel-Fn-PCR demonstrated good sensitivity and specificity for single-species infections (91.6% and 83.0% for Loa loa, and 95.0% and 97.7% for Mansonella perstans, respectively). However, for mixed infections, the sensitivity was low (43.9%), while specificity remained high (99.0%). MAIN

conclusionsThe assay proved to be effective for diagnosing single-species infections, even in cases with very low microfilaremia; however, its performance in identifying mixed infections was reduced, particularly when one species was present in much higher abundance than the other. Although not yet feasible as a fully deployable POCT, this study demonstrates the potential of integrating gelification with PCR for human filariasis diagnosis.