Citation: Hedtke, S.M.; Choi, Y.-J.; Kode, A.; Chalasani, G.C.; Sirwani, N.; Jada, S.R.; Hotterbeekx, A.; Mandro, M.; Siewe Fodjo, J.N.; Amambo, G.N.; et al. Assessing Onchocerca volvulus Intensity of Infection and Genetic Diversity Using Mitochondrial Genome Sequencing of Single Microfilariae Obtained before and after Ivermectin Treatment. Pathogens 2023, 12, 971. https://doi.org/10.3390/ pathogens12070971 Academic Editor: Fabrizio Bruschi Received: 24 May 2023 Revised: 16 July 2023 Accepted: 17 July 2023 Published: 24 July 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pathogens Article Assessing Onchocerca volvulus Intensity of Infection and Genetic Diversity Using Mitochondrial Genome Sequencing of Single Microfilariae Obtained before and after Ivermectin Treatment Shannon M. Hedtke 1, * , Young-Jun Choi 2 , Anusha Kode 1 , Gowtam C. Chalasani 1 , Neha Sirwani 1 , Stephen R. Jada 3 , An Hotterbeekx 4 , Michel Mandro 5 , Joseph N. Siewe Fodjo 4 , Glory Ngongeh Amambo 6 , Raphael A. Abong 6,7 , Samuel Wanji 6,7 , Annette C. Kuesel 8 , Robert Colebunders 4 , Makedonka Mitreva 2,† and Warwick N. Grant 1,† 1 Department of Environment and Genetics, School of Agriculture, Biomedicine and Environment, La Trobe University, Bundoora, VIC 3086, Australia; a.kode@latrobe.edu.au (A.K.); gchalasani@illumina.com (G.C.C.); n.sirwani@latrobe.edu.au (N.S.); w.grant@latrobe.edu.au (W.N.G.) 2 Department of Medicine, Washington University in St. Louis and McDonnell Genome Institute, St. Louis, MO 63108, USA; choi.y@wustl.edu (Y.-J.C.); mmitreva@wustl.edu (M.M.) 3 Amref South Sudan, Juba P.O. Box 30125, South Sudan; stephen.jada@amref.org 4 Global Health Institute, University of Antwerp, Doornstraat 331, 2610 Antwerp, Belgium; an.hotterbeekx@uantwerpen.be (A.H.); josephnelson.siewefodjo@uantwerpen.be (J.N.S.F.); robert.colebunders@uantwerpen.be (R.C.) 5 Provincial Health Division Ituri, Ministry of Health, Bunia P.O. Box 57, Democratic Republic of the Congo; michelmandro8@gmail.com 6 Parasites and Vectors Research Unit, Department of Microbiology and Parasitology, Faculty of Science, University of Buea, Buea P.O. Box 63, Cameroon; gloryngongeh@yahoo.com (G.N.A.); abongr@gmail.com (R.A.A.); samwandji@gmail.com (S.W.) 7 Research Foundation for Tropical Diseases and Environment (REFOTDE), Buea P.O. Box 474, Cameroon 8 UNICEF/UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases (TDR), World Health Organization, 1202 Geneva, Switzerland; kuesela@who.int * Correspondence: s.hedtke@latrobe.edu.au † These authors contributed equally to this work. Abstract: Onchocerciasis is a neglected tropical disease targeted for elimination using ivermectin mass administration. Ivermectin kills the microfilariae and temporarily arrests microfilariae production by the macrofilariae. We genotyped 436 microfilariae from 10 people each in Ituri, Democratic Republic of the Congo (DRC), and Maridi County, South Sudan, collected before and 4–5 months after ivermectin treatment. Population genetic analyses identified 52 and 103 mitochondrial DNA haplotypes among the microfilariae from DRC and South Sudan, respectively, with few haplotypes shared between people. The percentage of genotype-based correct assignment to person within DRC was ~88% and within South Sudan ~64%. Rarefaction and extrapolation analysis showed that the genetic diversity in DRC, and even more so in South Sudan, was captured incompletely. The results indicate that the per-person adult worm burden is likely higher in South Sudan than DRC. Analyses of haplotype data from a subsample (n = 4) did not discriminate genetically between pre- and post-treatment microfilariae, confirming that post-treatment microfilariae are not the result of new infections. With appropriate sampling, mitochondrial haplotype analysis could help monitor changes in the number of macrofilariae in a population as a result of treatment, identify cases of potential treatment failure, and detect new infections as an indicator of continuing transmission. Keywords: onchocerciasis; population genetics; microfilariae; epidemiology; macrofilariae; elimina- tion; monitoring; drug studies Pathogens 2023, 12, 971. https://doi.org/10.3390/pathogens12070971 https://www.mdpi.com/journal/pathogens