Assessing Onchocerca volvulus Intensity of Infection and Genetic Diversity Using Mitochondrial Genome Sequencing of Single Microfilariae Obtained before and after Ivermectin Treatment

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: drug studies; elimination; epidemiology; macrofilariae; microfilariae; monitoring; onchocerciasis; population genetics.

Figure 1

Rarefaction curve indicating how the number of Onchocerca volvulus microfilariae sampled from (a) five individual hosts from the DRC and (b) seven hosts from the South Sudan study areas affects the number of haplotypes likely to be observed. Solid line: rarefaction; dotted line: extrapolation; shaded area: confidence interval.

Figure 2

(a) Haplotype network using 143 mitochondrial single-nucleotide polymorphic variants from 225 microfilariae collected from 10 people from the DRC. (b) Haplotype network based on 228 genetic variants from 211 microfilariae collected from 10 people from South Sudan. Each circle represents a haplotype and is colored based on person; circle size indicates the number of microfilariae with that haplotype. Hatch marks along connecting lines indicate the number of sequence differences between haplotypes.

Figure 3

Analysis of genetic differentiation based on 320 mitochondrial DNA variant sites sequences of microfilariae collected from people in the DRC and South Sudan. (a) Principal components analysis (PCA) of microfilariae genotyped from 10 people from the DRC, colored by host (as in (b)); (b) discriminant analysis of principal components (DAPC) for microfilariae from DRC, maximizing differentiation between hosts; (c) PCA of microfilariae from 10 people from South Sudan, colored by host (as in (d)); (d) DAPC of microfilariae from South Sudan, maximizing differentiation between hosts.

Figure 4

Analysis of genetic differentiation based on the mitochondrial genotypes of microfilariae collected from four people in South Sudan with both pre- and post-treatment samples. (a) Discriminate analysis of principal components (DAPC) by time collected: pre-treatment (pre) or 5 months post-treatment (post). (b) Each column represents a mitochondrial haplotype found within a person and the shading the probability that haplotype would be assigned to time of collection based on the DAPC, with the actual day collected indicated above and columns arranged by individual person, as in (d); (c) DAPC of haplotypes maximizing differentiation between each individual host and by day collected; (d) each column represents a haplotype within a person (M204, M206, M224, or M238) and the shading the probability that it would be assigned to the pre- or post-treatment sample of one of the four individuals.

Figure 5

(a) Principal component analysis of Onchocerca volvulus worms from Benin, Cameroon, Côte d’Ivoire, Democratic Republic of Congo (DRC), Ghana, Guinea, Liberia, Mali, Sierra Leone, South Sudan, and Uganda based on mitochondrial genome sequencing. (b) Discriminant analysis of principal components (DAPC) of worms from Cameroon, Côte d’Ivoire, DRC, Mali, and South Sudan. (c) Percentage of worms from each country that were correctly assigned to their country of origin based on DAPC.