Polymerase Chain Reaction Pool Screening Used To Compare Prevalence of Infective Black Flies in Two Onchocerciasis Foci in Northern Sudan

Abstract

Onchocerciasis remains an important debilitating disease in many areas of Africa, including Sudan. The status of infection transmission in 2007 was assessed in the vectors of two disease foci in Sudan: Abu Hamed in northern Sudan, which has received at least 10 years of annual treatment and Galabat focus in eastern Sudan, where only minor, largely undocumented treatment activity has occurred. Assessment of more than 30,000 black flies for Onchocerca volvulus infectious stage L3 larvae by using an O-150 polymerase chain reaction protocol showed that black fly infectivity rates were 0.84 (95% confidence interval = 0.0497–1.88) per 10,000 flies for Abu Hamed and 6.9 (95% confidence interval = 1.1–16.4) infective flies per 10,000 for Galabat. These results provide entomologic evidence for suppressed Onchocerca volvulus transmission in the Abu Hamed focus and a moderate transmission rate of the parasite in the Galabat focus.

Interruption of Onchocerca volvulus Transmission in the Abu Hamed Focus, Sudan

“…In addition, we reported low levels of transmission based on the presence of L3 larvae in black flies that same year. 18 These reports indicated that the annual treatment regimen had achieved a high degree of onchocerciasis control in the area, with a considerable reduction in prevalence of dermal mf from the 34% to 37% reported in the mid-1980s, 7,27 but had not yet interrupted transmission. It is important to note, however, that coverage during the years where annual treatment was offered was inadequate (less than 65%).…”

“…Collection sites in and around Kelsaikal village in Mograt island and Nady were originally used for baseline entomological studies in 2007. 18 The Hamdab collection site was selected to investigate whether black fly breeding and transmission had been established in the spillways of the newly built dam. Flies were collected hourly 5 days per month from 6 AM to 7 PM, and the number of flies collected per hour was recorded.…”

“…The flies were divided into pools of 100 or less by site and date of collection and maintained in isopropanol until analysis. Heads of each black fly pool were first separated from bodies, and DNA was then extracted from pools of fly heads according to published procedures, 18,21,22 including the use of a 96-well purification module (Qiagen Inc., Valencia, CA) to streamline the DNA extraction process. A sham extraction was included with every 11 extractions to serve as contamination control.…”

“…Purified DNA was used as a template in the Onchocerca-specific O-150 polymerase chain reaction (PCR) followed by PCR-based enzyme-linked immunosorbent assay (ELISA) using probes specific for the parasite as previously reported. 18,[21][22][23] A sample was considered putatively positive if it had an ELISA score at or above the cutoff (i.e., the mean of 10 internal negative controls plus 3 SD). Only samples that were positive after a second independent PCR and ELISA were reported as confirmed positive.…”

“…Entomological analysis revealed that low levels of disease transmission existed before the shift to semiannual treatment activities in the area. 18 A more comprehensive follow-up assessment of entomological, serological, and parasitological data from the Abu Hamed focus was conducted in 2011.…”