Epidemiology, Chemotherapy of Onchocerciasis and Development of Onchocerca-Specific Polymerase Chain Reaction Based on A P-Glycoprotein Gene.

Abstract

Onchocerciasis is a disease caused by a filarial nematode Onchocerca volvulus, which is transmitted to man through the bite of infected black flies of the Simulium damnosum complex. In Achi community (South Eastern Nigeria) - ivermectin treatments started since 1990 on a yearly basis under the auspices of a WHO/UNDP/WORLD BANK ONCHO. PROJECT. During this study in Achi and Amansea, the participants medical history, onchocercomata and skin snips were obtained between January, 2009 to December, 2012 from the studied groups. The studied population of 240 subjects was randomly selected thus: Achi (57), Amansea (52) and UNTH (131) (health seeking patients at Oncho. Project, University of Nigeria Teaching Hospital). The studied groups were out patients of the WHO/UNDP/World Bank Oncho Project in the various villages. The patients were grouped into age, sex, occupation, residence, dosage levels, skin microfilariae and degrees of onchodermatitis. The uterine contents of the female worms after ivermectin treatment of patients were qualitatively and quantitatively assessed using embryogram techniques. Sodium Dodecyle Sulphate Poly Acrylamide Gel Electrophoresis (SDS PAGE) and Immuno blotting techniques were used to separate the protein antigens of the female worms. Chemiluminescence was used to visualize the blots. Genomic DNAs were isolated from various female worms of different dosage levels for the investigations into the presence of mdr genes. Multidrug resistant (mdr) degenerate primers were used with the genomic DNA in polymerase chain reaction (PCR) experiments. The PCR product was isolated, cloned and sequenced and an Onchocerca- specific mdr primer designed from homologous and conserved regions of available mdr genes. The O. volvulus mdr primer was used to check for amplification on other Onchocerca samples using PCR. A laser densitometer was used to read the positive products. A statistical evaluation of the densitometer values was done using t-test analysis. In Achi, 53(93.0%) of the patients were infected thus confirming Achi – as an onchocerciasis endemic community, in Amansea 30(58.0%) had onchocerciasis thus confirming it as a meso-endemic region, while 34(25.0%) of the patients from UNTH had onchocerciasis. All prevalence for onchocerciasis were significantly different (p<0.05). Out of 57 patients sampled at Achi, 53((93.0%) had nodules which when digested, contained viable adult worms. Similarly, in Amansea, 30(58.0%) patients had nodules that contained little or no worms but rather unidentifiable contents or already calcified worms. The prevalence of onchocercal noodles was significantly different (p<0.05) in all the studied groups, suggesting that the presence of nodules might be a good sign for the rapid assessment of onchocerciasis. The embryograms of the uteri of the O. volvulus females from ivermectin treated patients revealed that the contents were significantly (p<0.05) more deformed than those from non-treated patients. Using C 219 polyclonal antibody, the presence of P-glycoprotein was established in O. volvulus. Furthermore, using mdr –degenerate primers (P3 – P4) and DNA sequencing, mdr genes has been found in O. volvulus for the first time. There was a 53% homology with the available mdr genes. Additionally, an Onchocerca-specific mdr primer has been designed from this study. The primers P11 – P12 showed specificity towards Onchocerca species and not to other nematodes. The laser densitometer values when analyzed with the t-test revealed that there was no significant difference (p > F = 0.8247) between the two major groups, treated and untreated.