A Novel Filarial‐Multiplexed Probe‐Quantitative <scp>PCR</scp> for the Advance in the Diagnosis of Multiple Infections With Human Filariasis

Abstract

The routine diagnostic method used for clinical samples suspected of filarial infection in our laboratory is the Filaria-real time-PCR (F-RT-PCR). The drawback of this method is the need for melting temperature analysis and PCR products' electrophoresis to identify the filarial species. Therefore, the aim of this study was to design a quantitative real-time PCR (qPCR) assay using filarial-specific hydrolysis probes, targeting 18S rRNA and ITS1 genes, allowing the simultaneous diagnosis of loiasis, mansonellosis and other human filariasis without the need of electrophoresis or melting temperature analysis. To achieve this objective, three filarial probes (Fil-Hum-GT1, Loa-Hum-GT2 and Mp-Hum-GT2) were designed, optimised and validated for integration into a single qPCR assay, named filarial-multiplexed probe-quantitative PCR (F-mp-qPCR). For the optimisation and validation of the F-mp-qPCR method, a total of 304 clinical samples as dried blood spot were used with their corresponding thick blood smears stained by Giemsa 3%. The detection limit of the Fil-Hum-GT1, Loa-Hum-GT2 and Mp-Hum-GT2 probes was 0.05, 0.5 and 3 mF/mL, respectively. The most sensitive and specific probe was the general filarial probe Fil-Hum-GT1, with a sensitivity of 92.0% to detect L. loa, 88.6% to detect M. perstans and 85.7% to detect mixed infections, with 100% specificity. Agreement with microscopy was excellent for the Fil-Hum-GT1 probe. In contrast, the Mp-Hum-GT2 probe showed the lowest performance, with a sensitivity of 81.8% to detect M. perstans, decreasing to 42.9% for mixed infections. Although the developed method did not prove to be significantly more sensitive than microscopy, this novel method is faster and easier to perform compared to microscopy and is very useful for screening large population groups in a context of medium-to-low human filariasis transmission.