期刊
PARASITOLOGY RESEARCH
卷 121, 期 4, 页码 1199-1206出版社
SPRINGER
DOI: 10.1007/s00436-021-07411-5
关键词
qPCR; Wolbachia; Onchocerca volvulus; Litomosoides sigmodontis
类别
资金
- German Center for Infection Research (DZIF) [TI 07.001_Schlabe_00, TI 07.001_Schlabe_01]
- German Center for Infection Research [TI 03.907]
- Bill and Melinda Gates Foundation (DOLF) [ISRCTN50035143]
- BONFOR Research Commission of the Medical Faculty at the University of Bonn
- Commission for Clinical Trials of the University Hospital of Bonn, Germany (MoRiOn) [ISRCTN43697583]
- Liverpool School of Tropical Medicine, A-WOL Consortium - Bill and Melinda Gates Foundation
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy [EXC2151 - 390873048]
The study developed a qPCR-based method to quantify Wolbachia from a small number of microfilariae obtained from skin biopsies. This method can be used as a surrogate parameter for monitoring Wolbachia depletion in adult worms of new anti-wolbachial candidates.
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 (R)). 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.
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