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. 2023 Sep 12;8(9):445.
doi: 10.3390/tropicalmed8090445.

Procedure for Handling and Storage of Onchocerca volvulus Microfilariae Obtained from Skin Snips for Downstream Genetic Work

Shannon M Hedtke  1 , Anusha Kode  1 , Tony O Ukety  2 , Jöel L Mande  2 , Germain M Abhafule  2 , Anuarite A Raciu  2 , Claude B Uvon  2 , Stephen R Jada  3 , An Hotterbeekx  4 , Joseph Nelson Siewe Fodjo  4 , Makedonka Mitreva  5 , Wilson Sebit  6 , Robert Colebunders  4 , Warwick N Grant  1 , Annette C Kuesel  7
Affiliations

Procedure for Handling and Storage of Onchocerca volvulus Microfilariae Obtained from Skin Snips for Downstream Genetic Work

Shannon M Hedtke et al. Trop Med Infect Dis. .

Abstract

WHO and endemic countries target elimination of transmission of Onchocerca volvulus, the parasite causing onchocerciasis. Population genetic analysis of O. volvulus may provide data to improve the evidence base for decisions on when, where, and for how long to deploy which interventions and post-intervention surveillance to achieve elimination. Development of necessary methods and tools requires parasites suitable for genetic analysis. Based on our experience with microfilariae obtained from different collaborators, we developed a microfilariae transfer procedure for large-scale studies in the Democratic Republic of Congo (DRC) comparing safety and efficacy of ivermectin, the mainstay of current onchocerciasis elimination strategies, and moxidectin, a new drug. This procedure is designed to increase the percentage of microfilariae in skin snips suitable for genetic analysis, improve assignment to metadata, and minimize time and materials needed by the researchers collecting the microfilariae. Among 664 microfilariae from South Sudan, 35.7% and 39.5% failed the mitochondrial and nuclear qPCR assay. Among the 576 microfilariae from DRC, 16.0% and 16.7% failed these assays, respectively. This difference may not only be related to the microfilariae transfer procedure but also to other factors, notably the ethanol concentration in the tubes in which microfilariae were stored (64% vs. ≥75%).

Keywords: drug trials; epidemiological studies; genetic analysis; microfilariae; onchocerciasis.

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Conflict of interest statement

A.C.K. was staff of WHO/TDR at the time of this work. TDR, via co-author A.C.K., had a role in the development of the procedure, the writing of the manuscript, and in the decision to publish the result. NIAID had no role in the design of this research; in the analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Schematic of 96-format microfilariae transfer procedure. (a) Sample Source Record Form with right (R) and left (L) iliac crest (IC) and calf skin snip weight (Wt), microfilariae count, geographic origin, and study time point (and information required for assessing compliance with the standard operating procedure for obtaining skin microfilariae counts). Zero counts have been highlighted in yellow for this schematic. (b) Aligned 96-well plate, 96-format rack with ethanol-filled tubes, and 96-format rack with pipette tips. (c) Aligned 96-well plate, 96-format rack with ethanol-filled tubes, and 96-format rack with pipette tips after removal of ethanol tubes and pipettes corresponding to positions of wells without microfilariae.
Figure 2
Figure 2
Comparison of positive qPCR of a 67 bp mitochondrial target (Cq < 30) and 66 bp nuclear target (Cq < 33) of Onchocerca volvulus from South Sudan (2018) and the Democratic Republic of Congo (2021). The total number of microfilariae picked and analyzed is compared to the number that successfully passed detection standards for mitochondrial (mt) DNA and nuclear (nuc) DNA.
See this image and copyright information in PMC

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