Abstract
Mansonelliasis is a widespread yet neglected tropical infection of humans in Africa and South America caused by the filarial nematodes, Mansonella perstans, M. ozzardi, M. rodhaini and M. streptocerca. Clinical symptoms are non-distinct and diagnosis mainly relies on the detection of microfilariae in skin or blood. Species-specific DNA repeat sequences have been used as highly sensitive biomarkers for filarial nematodes. We have developed a bioinformatic pipeline to mine Illumina reads obtained from sequencing M. perstans and M. ozzardi genomic DNA for new repeat biomarker candidates which were used to develop loop-mediated isothermal amplification (LAMP) diagnostic tests. The M. perstans assay based on the Mp419 repeat has a limit of detection of 0.1 pg, equivalent of 1/1000th of a microfilaria, while the M. ozzardi assay based on the Mo2 repeat can detect as little as 0.01 pg. Both LAMP tests possess remarkable species-specificity as they did not amplify non-target DNAs from closely related filarial species, human or vectors. We show that both assays perform successfully on infected human samples. Additionally, we demonstrate the suitability of Mp419 to detect M. perstans infection in Culicoides midges. These new tools are field deployable and suitable for the surveillance of these understudied filarial infections.
Highlights
Infective third stage larvae (L3s) of Mansonella are transmitted to humans by biting midges of the genus Culicoides[2,6,7,8,9]
Contaminating human reads present in the M. perstans (10%) and M. ozzardi (94%) libraries were removed, and high frequency k-mer reads collected from each library (M. perstans, 2,077,887; M. ozzardi, 138,139) were assembled to generate 61,182 candidate repeat sequences for M. perstans and 47 for M. ozzardi
Using available genome sequence from several filarial parasites, we previously developed a multi-step bioinformatic pipeline which enabled the discovery of novel DNA biomarkers suitable for the development of a new diagnostic loop-mediated isothermal amplification (LAMP) assay for L. loa[47]
Summary
Infective third stage larvae (L3s) of Mansonella are transmitted to humans by biting midges of the genus Culicoides[2,6,7,8,9]. A PCR assay has been developed to differentiate M. perstans from M. ozzardi, this requires sequencing of the amplicon[30]. These molecular assays are more sensitive than the standard parasitological techniques for the identification of Mansonella spp.[1,10,31,32,33,34,38,39,40]. The candidate biomarkers discovered were validated experimentally and used to develop new rapid colorimetric LAMP assays These assays were evaluated on patient samples and infected midges and compared with microscopy and PCR-based methods
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