Dynamics of the malaria-vector populations in coastal Lagos, south-western Nigeria.

An entomological study was carried out in coastal Lagos, south-western Nigeria, to explore the role of Anopheles gambiae s.l. and An. moucheti in the transmission of Plasmodium falciparum in this holo-endemic area. Mosquitoes were caught, on human bait at night and by pyrethrum-spray catches during the day, twice a month throughout 2000. Of the 1812 Anopheles mosquitoes collected, An. gambiae s.l., the predominant vector species, represented 78.7% while the other 21.3% were An. moucheti. The results of a PCR-based test identified 56.8% of the mosquitoes of the An. gambiae complex collected as An. gambiae s.s., 36.9% as An. melas and 6.3% as An. arabiensis. Anopheles gambiae s.s. was predominantly recorded in the wet season, biting females being collected from May to October, with a peak in July. Anopheles melas and An. moucheti were present throughout the year-long study whereas An. arabiensis was mainly found in the dry season. The results of ELISA-based analyses of bloodmeals indicated that An. gambiae s.s., An. melas and An. moucheti were predominantly anthropophagic whereas An. arabiensis was largely zoophagic. Among all of the females investigated, 3.6% of the An. gambiae s.s., 1.9% of the An. melas, 1.8% of the An. moucheti and 0% of the An. arabiensis were found to be infected with P. falciparum (i.e. carrying the parasite's circumsporozoite antigen). The corresponding proportions for the females collected during the dry season were 1.3%, 2.3%, 2.7% and 0%. The entomological inoculation rates for An. melas and An. moucheti were significantly higher during the dry season than at other times of the year. Taken together, these results indicate that An. melas and An. moucheti maintain transmission of P. falciparum during the dry season, while the biting population ofAn. gambiae s.s. is relatively small.

Knowledge and beliefs about causes, transmission, treatment and control of human onchocerciasis in rural communities in south western Nigeria.

Descriptive and quantitative information on onchocerciasis transmission and control were collected using focus group discussions and structured questionnaire to determine what changes if introduced, could upset the established human-vector-parasite relationship in limiting transmission. People's knowledge of cause of infection and transmission was very poor. Of the 1012 people interviewed, only 3% related the clinical manifestations of onchocerciasis to Simulium bites. Thirty six percent had no idea of the cause of infection while the rest attributed the clinical symptoms of the disease to many other causes. People's knowledge of the current treatment and control measures was also poor. However an impressive knowledge of the daily and seasonal distribution of Simulium flies was observed. The study identifies the need for health education campaigns aimed at relating the clinical manifestations of onchocerciasis to Simulium bites. This could help people in taking personal protective measures and seeking appropriate treatment.

Longitudinal studies of skin microfilaria and antibody conversion rates in children living in an endemic focus of onchocerciasis in Nigeria.

We monitored skin microfilarial conversion and antibody (serum) conversion rates to Onchocerca volvulus recombinant antigens (OC3.6 and OC9.3) over a 2-year period, in a cohort of 208 children aged 9-11 years and living in a mesoendemic focus of savannah-type onchocerciasis in Nigeria in order to detect prepatent onchocerciasis in children. Between time point A and time point B (9 months apart) the seroconversion rate was 19.4 per cent, and between time points B and C (15 months apart), a further 31.4 per cent of seronegative children had seroconverted. However, only a third of the seroconverted children became microfilaria-positive. From the time-lag between antibody conversion and skin snip conversion, we estimated that a 9-12 month interval was required for the detection of O. volvulus microfilariae following infection with viable third stage larva (L3s). The predictive value of antibody detection as a measure of skin microfilarial conversion was >90 per cent when the optical density (OD) at 490 nm was >0.80. We therefore propose a strategy for monitoring changes in transmission of onchocerciasis following vector control or chemotherapy through an evaluation of annual antibody seroconversion rates in a standardized sentinel population of children.

Prevalence and seasonal changes in the gastro-intestinal helminths of Nigerian goats.

A total of 120 gastro-intestinal tracts and 960 faecal samples were examined to assess the prevalence and seasonal changes in the gastro-intestinal helminth parasites of Red Sokoto (maradi) goats slaughtered at Ibadan between May 1991 and April 1992. Egg types of strongyles, Strongyloides, Trichuris, Skrjabinema, Dicrocoelium and Moniezia were encountered in 93%, 83%, 44%, 0.9%, 2.3% and 31% of the faecal samples respectively. However, only strongyle, Strongyloides and Trichuris eggs occurred in large numbers and were more common during the rainy season than in the dry season. The parasites recorded and their prevalences were Haemonchus contortus (90.0%), H. ovis (5.0%), Strongyloides papillosus (80.8%), Trichostrongylus colubriformis (78.3%), T. axei (69.2%), Trichuris ovis (72.5%), T. globulosa (38.3%), Oesophagostomum columbianum (67.5%), Cooperia curticei (58.3%) Gaigeria pachyscelis (40.8%), Skrjabinema ovis (5.0%), Nematodirus battus (5.8%), Moniezia expansa (29.2%), M. benedeni (10.0%), Paramphistomum spp. (5.0%) and Cysticercus tenuicollis (33.3%). Haemonchus ovis is reported for the first time in Nigeria. Mixed infections were most prevalent. Young goats were more commonly infected and had higher worm counts than adult goats. Only Haemonchus, Trichostrongylus, Strongyloides and Cooperia spp. occurred in large numbers. Irrespective of the age of the goats, higher worm counts were generally encountered during the rainy season than in the dry season. The results are discussed in relation to the control of helminthiasis in grazing animals in Nigeria.

Use of recombinant Onchocerca volvulus antigens for diagnosis and surveillance of human onchocerciasis.

The diagnostic value of ELISAs based on recombinant Onchocerca volvulus antigens OC 3.6 and OC 9.3 was evaluated with sera from endemic areas in West Africa, Guatemala and Ecuador. IgG assays were slightly more sensitive than those that detected IgG4, and the antigen combination was significantly more sensitive than either antigen alone (OC 3.6, 93%; OC 9.3, 84%, combined 98%). These assays were also evaluated with sera from 2 villages in the Onchocerciasis Control Programme area of West Africa including one village (Pendie) with recent recrudescence of infection and one (Niarba) where transmission had been interrupted for 15 years by vector control. The OC 3.6 IgG antibody assay was sensitive for new infections and exposure in Pendie; 24/24 (100%) of people with positive skin snips and 15/74 (20%) of sera from MF negative people had IgG antibodies to this antigen. In addition, antibodies to OC 3.6 often preceded the onset of skin snip positivity in Pendie. In contrast, IgG antibodies to OC 3.6 and OC 9.3 were rarely seen in children born during the 15 years since transmission was interrupted by vector control in Niarba. These encouraging results suggest that antibody assays based on OC 3.6 and OC 9.3 may be valuable tools for surveillance of onchocerciasis and also for monitoring the efficacy of control programmes.

Onchocerca volvulus: monoclonal antibodies to immune complex-associated parasite antigens.

Improved methods for diagnosis of onchocerciasis are needed. We have recently identified immune complex-associated parasite antigens in sera from onchocerciasis patients. The goal of this study was to produce monoclonal antibodies to these antigens that might be used in antigen detection assays. Two monoclonal antibodies (OV-1 and OV-5) that bind to parasite antigens in immunoblots of PEG-precipitated immune complexes from human onchocerciasis sera and to corresponding antigens in adult worm extracts and excretory-secretory products were produced. The target epitopes of the monoclonals are heat stable, resistant to trypsin, and destroyed by Pronase. The two monoclonals produce similar but not identical patterns of binding to immunoblots of Onchocerca volvulus adult worm antigen with major bands at 43-47, 58-63, and 70 kDa. OV-1 and OV-5 appear to bind to two distinct but closely related epitopes, neither of which is phosphorylcholine. Immunoelectron microscopy showed that the epitopes recognized by these monoclonals are widely distributed in adult female worms, but concentrated in the uterus and intestine. Antigen assays based on these antibodies detected parasite antigen in 9 of 14 sera from onchocerciasis patients, but significant background signal was detected in some nonendemic human sera. Thus, although this study has provided new information on parasite antigens in sera from onchocerciasis patients, additional work will be needed to achieve the goal of producing a sensitive and specific antigen diagnostic test for onchocerciasis.

Preliminary evaluation of recombinant Onchocerca volvulus antigens for serodiagnosis of onchocerciasis.

Serodiagnostic assays for onchocerciasis based on native antigens are hampered by the scarcity of antigen, and they suffer from poor specificity. The present study was designed to evaluate the diagnostic utility of recently described recombinant Onchocerca volvulus antigens OC 3.6 and OC 9.3 in enzyme immunoassays. The recombinant proteins were expressed as glutathione S-transferase fusions and were tested in several enzyme immunoassay formats to measure immunoglobulin G (IgG) and IgG4 antibodies with sera from patients with onchocerciasis in Nigeria and with various types of control sera. The best results were obtained by measuring IgG4 antibodies to the fusion proteins. Forty of 42 (95%) serum specimens from patients with onchocerciasis were reactive with OC 3.6; the reactivity with OC 9.3 was 81%. Results obtained with sera from experimentally infected chimpanzees suggest that OC 3.6 might be especially useful for detecting prepatent infections in humans, while OC 9.3 mainly detects mature, patent infections. Sera from individuals in Nigeria and the United States residing in areas nonendemic for onchocerciasis were uniformly nonreactive with these antigens in IgG and IgG4 assays, as were sera from patients with bancroftian filariasis, brugian filariasis, loiasis, ascariasis, schistomiasis, and dracunculiasis. These results suggest that enzyme immunoassays based on the recombinant antigens OC 3.6 and OC 9.3 are useful for the diagnosis of onchocerciasis.

Use of a recombinant antigen (OC 3.6 cDNA) for the serological diagnosis of onchocerciasis in exposed Nigerian children.

We evaluated a recombinant antigen (OC 3.6 cDNA) expressed in pMAL TM vector for the diagnosis of onchocerciasis in children living in an endemic focus of forest-type onchocerciasis in Nigeria. Using the Western blot with the maltose-binding fusion protein as antigen, 91 per cent of mf-positive children and 24 per cent of endemic normal children were positive. Furthermore, age was not a limitation to the assay. The practical limitations of the assay for field diagnosis of onchocerciasis is discussed.

Field evaluation of IgG4 serology for the diagnosis of onchocerciasis in children.

IgG4 serology was evaluated for early diagnosis of onchocerciasis in children living within an endemic focus of forest-type onchocerciasis in Nigeria. IgG4 serology proved to be more sensitive than conventional skin snip examination being able to detect 42% of infected children as opposed to 24% by snip examination. Furthermore, age was not a limitation to the technique within the 8-12 y spectrum. We conclude that IgG4 serology in children is a reliable indicator of community microfilarial rate and ongoing transmission of onchocerciasis.

Molecular cloning and characterization of recombinant parasite antigens for immunodiagnosis of onchocerciasis.

Immunological cross-reactivity among nematodes has hampered the development of specific serodiagnostic assays for onchocerciasis. In the present study, an Onchocerca volvulus adult worm complementary DNA expression library was differentially screened with human sera from patients infected with O. volvulus and with an omnibus anti-nematode serum pool comprised of sera from patients infected with Brugia malayi, Loa loa, Wuchereria bancrofti, Mansonella perstans, Strongyloides stercoralis, Ancylostoma duodenale, Ascaris lumbricoides, and Dracunculus medinensis. Seven Onchocerca-specific clones were identified and screened with individual onchocerciasis patient sera. Additional studies were performed to characterize the most immunoreactive clones, OC 3.6 and OC 9.3. OC 3.6 produced a 152-kD beta-galactosidase fusion protein that was recognized in dot-immunoblots by 54 of 55 sera from onchocerciasis patients (98%). The OC 3.6 DNA insert is 996 bp long with an open reading frame of 627 bp and a 369-bp untranslated 3' end. OC 3.6 is closely related to a previously reported clone (OV 33-3), but it differs from that clone at both the 5' and 3' ends. OC 9.3 contained a novel 565-bp insert and produced a 138-kD fusion protein that was recognized by 46 of 55 sera from onchocerciasis patients (83%). Additional studies are in progress to develop and evaluate immunodiagnostic tests for onchocerciasis based on measurement of antibodies to these promising recombinant antigens.

Circulating immune complex-associated parasite antigens in human onchocerciasis.

This study identified and characterized parasite antigens in sera from humans infected with Onchocerca volvulus. Immune complexes were precipitated from human sera with polyethylene glycol and analyzed by immunoblot with rabbit antibodies to O. volvulus. A 23-kDa parasite antigen was detected in sera from 17 of 23 Nigerian onchocerciasis patients and 5 of 10 endemic controls. Other parasite antigens with apparent molecular masses of 62, 66, and 70 kDa were less consistently observed. These antigens were not present in Nigerian or US nonendemic control sera, in sera from patients with various other parasitic infections, or in sera from US patients with autoimmune diseases. Biochemical studies indicated that these antigens are nonglycosylated acidic proteins that do not contain phosphorylcholine. These antigens may be useful as targets for improved diagnostic tests for onchocerciasis based on parasite antigen detection.

IgG4 subclass antibody serology for onchocerciasis.

Antibodies to phosphorylcholine and carbohydrate determinants responsible for much of the cross-reactivity among nematodes are subclass restricted in humans and absent in the IgG4 subclass. Total IgG and IgG4 antibody responses to Onchocerca volvulus were examined by enzyme immunoassay and by immunoblot. Significant background IgG reactivity was detected in both assays in US control sera and sera from patients with intestinal nematode infections, but background reactivity was negligible in the IgG4 assays. IgG4 antibodies were detected by enzyme immunoassay in 17 of 18 Nigerian onchocerciasis serum samples and in 2 of 9 endemic control serum samples. Cross-reactive IgG4 antibodies were present in serum pools from patients infected with other filariae. IgG4 antibodies recognized a restricted subset of O. volvulus antigens in immunoblots relative to IgG. These results confirm the previously reported enhanced specificity of IgG4 antibody assays for filariasis and extend the observation to include onchocerciasis. Whereas IgG4 subclass antibody serology is more specific than measurement of total IgG antibodies for onchocerciasis, cross-reactivity among filariae limits the utility of this approach.

A comparison of two proteolytic techniques for hatching Hymenolepis diminuta eggs.

Two techniques for hatching taeniid eggs, based on enzymic proteolysis and disaggregation of the embryophore, were evaluated using Hymenolepis diminuta. Both techniques proved ineffective on H. diminuta in their original form, achieving only a hatching rate of 1-2%. However, a gradual increase in pepsin concentration up to 3 mg ml-1 in the pretreatment solution enhanced hatching significantly (P less than 0.05). Normal or immune rat serum had no appreciable effect on the hatching process.

Malaria and loaisis among blood donors at Ibadan, Nigeria.

115 blood donors were screened for the presence of malaria and other blood parasites at the Blood Bank, University College Hospital, Ibadan, between January and July, 1984. 11.3% of these had blood parasites; 7.8% had Plasmodium falciparum with parasitaemias from 0.03-0.2%, and 3.5% had Loa loa microfilaraemia. No other blood parasites were observed. Serological examination by the indirect fluorescent antibody test revealed that 86% of the donors had malaria antibody with reciprocal titres ranging from 16 to 512. There was no significant association between ABO blood group and malaria parasitaemia or antibody titre.