A qPCR to quantify Wolbachia from few Onchocerca volvulus microfilariae as a surrogate for adult worm histology in clinical trials of antiwolbachial drugs

Abstract

The filarial nematode Onchocerca volvulus causes onchocerciasis (river blindness), a neglected tropical disease affecting 21 million people, mostly in Sub-Saharan Africa. Targeting the endosymbiont Wolbachia with antibiotics leads to permanent sterilization and killing of adult worms. The gold standard to assess Wolbachia depletion is the histological examination of adult worms in nodules beginning at 6 months post-treatment. However, nodules can only be used once, limiting the time points to monitor Wolbachia depletion. A diagnostic to longitudinally monitor Wolbachia depletion from microfilariae (MF) at more frequent intervals < 6 months post-treatment would accelerate clinical trials of antiwolbachials. We developed a TaqMan qPCR amplifying the single-copy gene wOvftsZ to quantify Wolbachia from as few as one MF that had migrated from skin biopsies and compared quantification using circular and linearized plasmids or synthetic dsDNA (gBlock®). qPCR for MF from the rodent nematode Litomosoides sigmodontis was used to support the reproducibility and validate the principle. The qPCR using as few as 2 MF from O. volvulus and L. sigmodontis reproducibly quantified Wolbachia. Use of a linearized plasmid standard or synthesized dsDNA resulted in numbers of Wolbachia/MF congruent with biologically plausible estimates in O. volvulus and L. sigmodontis MF. The qPCR assay yielded a median of 48.8 (range 1.5–280.5) Wolbachia/O. volvulus MF. The qPCR is a sensitive tool for quantifying Wolbachia in a few MF from skin biopsies and allows for establishing the qPCR as a surrogate parameter for monitoring Wolbachia depletion in adult worms of new antiwolbachial candidates.

Filariasis research – from basic research to drug development and novel diagnostics, over a decade of research at the Institute for Medical Microbiology, Immunology and Parasitology, Bonn, Germany

“…The ability to monitor the effect of antiwolbachial drug in few microfilariae is essential because: (i) the level of microfilaridermia is decreasing as MDA coverage is being attained, and (ii) effective anitwolbachial drugs lead to a reduction in skin microfilariae. The method allows the analysis of anti-wolbachial drug efficacy from as few as 2 microfilariae and can be used to predict the effect in adult nematodes at less than 6 months post treatment without excising onchocermata, which are limited resource per patient; and the removal of onchocercomata can directly affect end point readoutsremoval of onchocercomata also removes adult nematodes, resulting in a concomitant reduction in microfilariae independent from treatment (171). The method is being validated using prospective samples from the flubentylosin phase 2 trial.…”