An analysis of genetic diversity and inbreeding in Wuchereria bancrofti: implications for the spread and detection of drug resistance

Abstract

Estimates of genetic diversity in helminth infections of humans often have to rely on genotyping (immature) parasite transmission stages instead of adult worms. Here we analyse the results of one such study investigating a single polymorphic locus (a change at position 200 of the beta-tubulin gene) in microfilariae of the lymphatic filarial parasite Wuchereria bancrofti. The presence of this genetic change has been implicated in benzimidazole resistance in parasitic nematodes of farmed ruminants. Microfilariae were obtained from patients of three West African villages, two of which were sampled prior to the introduction of mass drug administration. An individual-based stochastic model was developed showing that a wide range of allele frequencies in the adult worm populations could have generated the observed microfilarial genetic diversity. This suggests that appropriate theoretical null models are required in order to interpret studies that genotype transmission stages. Wright's hierarchical F-statistic was used to investigate the population structure in W. bancrofti microfilariae and showed significant deficiency of heterozygotes compared to the Hardy-Weinberg equilibrium; this may be partially caused by a high degree of parasite genetic differentiation between hosts. Studies seeking to quantify accurately the genetic diversity of helminth populations by analysing transmission stages should increase their sample size to account for the variability in allele frequency between different parasite life-stages. Helminth genetic differentiation between hosts and non-random mating will also increase the number of hosts (and the number of samples per host) that need to be genotyped, and could enhance the rate of spread of anthelmintic resistance.

Figure 1. Estimates of Wright's F-statistics in Wuchereria bancrofti for the pre-treatment villages of Tangonko (black diamonds), Badongo (grey open circles) and for the treated village of Perigban (black squares), which received one round of chemotherapy (albendazole+ivermectin).

The error bars are the 95% confidence intervals. F_IT estimates the total degree of parasite inbreeding; F_IS describes the level of non-random mating within the infrapopulation; and shows the variation in microfilarial allele frequency within the host subpopulation (village).

Figure 2. The impact of inbreeding on the relationship between the sample microfilarial allele frequencies, , and the (inferred) underlying adult worm allele frequency, q^W, for the substitution at codon 200 of the β-tubulin gene in W. bancrofti.

The figure shows 95% confidence intervals for a population with no excess inbreeding (the null model, dark grey shaded area), and a population with the observed levels of inbreeding (F_IS = 0.28, , light grey shaded area). Simulations are based on the same sampling scheme used in Burkina Faso . The thick black solid line indicates the mean result for both models. The observed pre-treatment microfilarial allele frequency (; black thin, horizontal dotted line) was compared to simulation results to indicate the possible range of adult worm allele frequencies which could have given rise to the West African data. The null model (black vertical dotted-dashed lines) indicated values of q^W ranging from 0.21 to 0.32 compared to the inbred model (F_IS = 0.28, , black vertical dashed lines), which gave values of q^W between 0.18 and 0.37.

Figure 3. De Finetti diagram showing the genotype distribution of W. bancrofti microfilariae generated from a given underlying adult worm allele frequency, q^W, taken from villages prior to the introduction of chemotherapy.

A full explanation of the De Finetti diagram is given in . The black diamond represents the value originating from the observed data (with , and F_IT = 0.44), and the error bars indicate the uncertainty in genotype distribution stemming from the values of q^W (0.21, 0.32) that were estimated from the null (random) model (Figure 2). Y indicates the allele coding for tyrosine at position 200 of β-tubulin that is associated with benzimidazole (BZ) resistance in nematodes of livestock, and F denotes the allele (coding for phenylalanine) indicative of BZ susceptibility. The solid-black curve represents the Hardy-Weinberg equilibrium (HWE). The null model generating microfilarial allele frequencies (see text) was used to investigate the range of sample microfilarial genotype distributions that could be obtained from a population exhibiting no excess inbreeding (i.e. assuming that the underlying adult parasite population would have values of ). Simulations mimic the same sampling scheme described in Schwab et al. The observed microfilarial genotype distribution falls outside the 95% confidence interval range (grey shaded area surrounding the HWE curve) generated by the null model, despite the uncertainty in the underlying q^W estimates, indicating strong parasite inbreeding even before introduction of antifilarial combination therapy.

Figure 4. The impact of inbreeding on the relationship between the mean proportion of hosts harbouring microfilariae with one or two copies of allele Y and the (assumed) underlying adult worm allele frequency, q^W.

The figure compares the proportion of hosts exhibiting microfilariae with allele Y (i.e. both heterozygous and homozygous YY microfilariae, solid lines) with that of hosts which have only microfilariae with the homozygous YY genotype (broken lines). Model outcomes are compared for two hypothetical parasite populations; the former (thin grey lines) without excess inbreeding (generated by the null model), and the latter (thick black lines) with the levels of inbreeding (F_IS = 0.28, ) observed in the Burkina Faso data. Simulations used the same sampling scheme described in Schwab et al. and assume an overall microfilarial prevalence of ∼25% (see text).

Figure 5. The impact of helminth inbreeding on the minimum number of microfilaria-positive hosts who should be sampled and the minimum number of microfilariae that should be genotyped to be 95% confident of detecting at least one rare allele.

A randomly mating population (, grey open squares) is compared to an inbred population (F_IS = 0.28 and , black diamonds). The underlying adult worm allele frequency of both populations is set at q^W = 0.05. Each data point represents 100,000 runs of the stochastic model generating microfilarial allele frequencies. The number of microfilariae analysed per host is proportional to host microfilaraemia.

Figure 6. The impact of the observed level of parasite inbreeding on the production of resistant microfilariae.

The graph gives the relative change in the number of resistant genotypes in an inbred parasite population compared to that in a population at HWE. Results are shown for different resistance allele frequencies. The graph assumes that a known resistance allele is either recessive (A), black lines, or dominant (B), grey lines. The inbreeding coefficients are those reported in Figure 1: mean result (F_IT = 0.44, solid line); upper 95% confidence limit (F_IT = 0.68, dashed line); lower 95% confidence limit (F_IT = 0.17, dotted line). The relative change in the number of resistant genotypes caused by parasite inbreeding is estimated as in (A) and in (B).