Research Article: Genetic Selection of Low Fertile Onchocerca volvulus by Ivermectin Treatment

Date Published: October 30, 2007

Publisher: Public Library of Science

Author(s): Catherine Bourguinat, Sébastien D. S. Pion, Joseph Kamgno, Jacques Gardon, Brian O. L. Duke, Michel Boussinesq, Roger K. Prichard, Sara Lustigman

Abstract: BackgroundOnchocerca volvulus is the causative agent of
onchocerciasis, or “river blindness”. Ivermectin has
been used for mass treatment of onchocerciasis for up to 18 years, and
recently there have been reports of poor parasitological responses to the
drug. Should ivermectin resistance be developing, it would have a genetic
basis. We monitored genetic changes in parasites obtained from the same
patients before use of ivermectin and following different levels of
ivermectin exposure.Methods and FindingsO. volvulus adult worms were obtained from 73 patients
before exposure to ivermectin and in the same patients following three years
of annual or three-monthly treatment at 150 µg/kg or 800
µg/kg. Genotype frequencies were determined in
β-tubulin, a gene previously found to be linked
to ivermectin selection and resistance in parasitic nematodes. Such
frequencies were also determined in two other genes, heat shock
protein 60 and acidic ribosomal protein, not
known to be linked to ivermectin effects. In addition, we investigated the
relationship between β-tubulin genotype and female
parasite fertility. We found a significant selection for
β-tubulin heterozygotes in female worms. There
was no significant selection for the two other genes. Quarterly ivermectin
treatment over three years reduced the frequency of the
β-tubulin “aa” homozygotes
from 68.6% to 25.6%, while the
“ab” heterozygotes increased from 20.9% to
69.2% in the female parasites. The female worms that were
homozygous at the β-tubulin locus were more fertile
than the heterozygous female worms before treatment (67% versus
37%; p = 0.003)
and twelve months after the last dose of ivermectin in the groups treated
annually (60% versus 17%;
p<0.001). Differences in fertility between heterozygous and homozygous worms were less apparent three months after the last treatment in the groups treated three-monthly.ConclusionsThe results indicate that ivermectin is causing genetic selection on O. volvulus. This genetic selection is associated with a lower reproductive rate in the female parasites. We hypothesize that this genetic selection indicates that a population of O. volvulus, which is more tolerant to ivermectin, is being selected. This selection could have implications for the development of ivermectin resistance in O. volvulus and for the ongoing onchocerciasis control programmes.

Partial Text: Onchocerca volvulus is the filarial nematode, transmitted by
Simulium flies, that causes human onchocerciasis, or
“river blindness”. It is estimated that 37 million people,
mostly in Africa, are infected with this worm [1]. At present, ivermectin
(IVM, Mectizan) is the only safe drug available for mass treatment of
onchocerciasis. IVM, administered at the standard dose of 150 µg/kg, has a
rapid effect on the embryonic stage of the parasite, the microfilariae (mf), which
cause most of the ocular and cutaneous manifestations of the disease. As a result of
this microfilaricidal effect, the skin microfilarial loads decrease by
95–99% within one month after treatment. The drug also blocks
the production of new mf by the adult female worms, who only resume mf release
3–6 months after treatment. This “embryostatic effect”
of IVM explains why the mf loads remain at very low levels for up to one year.
Furthermore, IVM treatments repeated at 1- to 3-monthly intervals have some, though
moderate, effect on the longevity of the adult worms (“macrofilaricidal
effect”) [2,3].

A total of 73 patients provided one nodule at the outset of the trial and one nodule
after treatment. A total of 367 worms (248 females, 119 males) were isolated from
the 73 nodules collected before treatment, and 224 worms (153 females, 71 males)
were extracted from the 73 nodules provided by the same hosts after three years of
repeated treatment. Details on the numbers of worms analyzed in the different
treatment groups are given in Tables
1 and 2.

With the implementation of the onchocerciasis control programmes, an increasing
proportion of people in endemic areas have received community-directed treatment
with IVM on a regular basis. Even though children under 5 years of age and pregnant
women are excluded from mass treatment, a high proportion of the parasite
population, in control areas, is under treatment. As a consequence, and because most
of the parasite population is in the human hosts rather than in the vector, only a
relatively small proportion of the O. volvulus population is likely
to be in refugia (not exposed to the drug) at the time of
treatment. Thus, selection pressure for any IVM resistance alleles is expected to be
high in O. volvulus[7].



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