Detection of Onchocerca volvulus DNA in pools of wild-caught Simulium ochraceum by use of the polymerase chain reaction.

The presence of Onchocerca volvulus DNA in experimentally infected flies can now be detected by use of the PCR, so that, for example, one infected Simulium damnosum can be detected in a pool of 100 uninfected flies or one S. ochraceum can be detected in pools of 20-40. As this PCR technique is specific for O. volvulus, the results are not confounded by the presence of other, unimportant, Onchocerca species, and the technique could replace time-consuming, manual dissection of flies. In 1996 and 1997, pools of 16-21 Simulium ochraceum were tested by the PCR technique. These flies had been collected biting man, between 1992 and 1994, from two hyperendemic coffee estates (fincas) in Guatemala, and stored in commercial (95%) ethanol. Collections at finca Buena Vista (869 flies in 52 pools) were made 1-2 weeks and 46 weeks after 45% of eligible subjects had been treated with ivermectin for the first time. At finca El Brote, collections (360 flies in 18 pools) were made 13 weeks before and 7 weeks after 97% of eligible subjects had received their first treatment. DNA was easily recovered from simuliids that had been stored in ethanol for up to 4 years. Of the nine pools of flies with visible blood collected at Buena Vista, each of 20 flies, eight tested positive for O. volvulus DNA. In flies without blood, 13 of 22 pools collected at Buena Vista just after treatment tested positive, whereas there were 14 positives in 22 pools taken 46 weeks later (P > 0.05). At El Brote, nine of 10 pre-treatment pools were positive, compared with three of eight taken 7 weeks post-treatment (P = 0.04), indicating that the treatments in this finca had reduced infection in the vector, and possibly transmission, by about 60%. A sub-sample of Buena Vista flies was divided into 19 sets of three separate sub-pools containing heads, thoraces and abdomens. Three pools of heads alone were positive, and had corresponding pools of positive abdomens. Three positive pools of thoraces had negative corresponding pools of heads and abdomens. These results show that PCR can be used to determine the prevalence of O. volvulus DNA in wild-caught S. ochraceum. As the infection rates observed were higher than expected from dissections reported by other workers, PCR-determined rates may not be directly comparable with traditional parameters based on the dissection of flies to reveal O. volvulus larvae.

Assessment of vector microfilarial uptake as a comparatively non-invasive technique for monitoring onchocerciasis treatment campaigns in the Americas.

Since 1992, efforts have been made to combat onchocerciasis in Guatemala through mass distribution of ivermectin. The impact of the campaign is assessed by taking skin-snips from sentinel groups within selected communities. This method gives an estimate of the prevalence and intensity of infection, and thus the efficacy of the treatment. In some communities people are becoming reluctant to volunteer for skin-snipping, and so there is a need for an alternative technique that will give quantitative results. In most hyperendemic communities in Guatemala, biting blackflies are so ubiquitous that few people object to allowing 10 to 20 flies to engorge upon them. We examined data on the quantitative uptake of microfilariae by Simulium ochraceum before and after ivermectin distribution to see whether results similar to skin-snip data could be obtained. Counts of microfilariae ingested by S. ochraceum are compared to the numbers found in skin-snips from the same volunteers. In a group of 31 untreated infected persons, a skin-snip survey detected 64.5% positive, while feeding flies (vector microfilarial uptake, VmfU) detected 96.8%. Post-treatment, in a sample of 58 of whom 52 (89.7%) had a history of infection, both skin-snips and VmfU detected 54.2%. Vector blood meals contained more microfilariae than a mg of skin before treatment, but both recorded about equal numbers after treatment. When the data set was subdivided to compare samples taken at 2-3, 6-8 and 14-17 months post-treatment, the effect of ivermectin was still apparent at 6-8 months, but had virtually disappeared by 14 months post-treatment. A surprising observation was that the flies ingested fewer microfilariae from treated persons than was expected from the skin densities as estimated by skin-snip. This effect lasted for over 8 months, and could indicate that ivermectin has a greater effect on transmission than previously suspected. We conclude that VmfU could be used as an alternative to skin-snipping, and discuss the ethical implications.