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Introduction: Several reports indicate high prevalences of both onchocerciasis and epilepsy in some regions of Africa. This raises the question of whether these diseases are associated. We therefore investigated people with epilepsy and/or onchocerciasis living in an area in Tanzania endemic for Onchocerca volvulus (O. volvulus)., Methods: We collected clinical information, skin snips, and blood from 300 individuals, and cerebrospinal fluid (CSF) from 197. Participants were allocated to 4 groups consisting of people with epilepsy and onchocerciasis (n=135), those with either epilepsy (n=61) or onchocerciasis only (n=35), and healthy individuals (n=69). Samples were evaluated for microfilaria, IgG4 antibodies against O. volvulus, O. volvulus antibody index (CSF/serum), and CSF routine parameters. Polymerase chain reaction (PCR) was performed on skin snips and CSF., Results: No difference was found in microfilarial density between participants with and without epilepsy (P=0.498). The antibody index was raised in 2 participants. CSF PCR was negative in all samples tested., Discussion: Our results do not give evidence of a relationship between O. volvulus and epilepsy. Despite the fact that 2 participants had raised antibody index, the existence of cerebral onchocerciasis caused by migration of microfilariae into the CSF appears unlikely. However, to date unexplored reactions to the infestation with O. volvulus causing epilepsy cannot be excluded.

Up to 5% of untreated female Onchocerca volvulus filariae develop potentially fatal pleomorphic neoplasms, whose incidence is increased following ivermectin treatment. We studied the occurrence of 8 filarial proteins and of Wolbachia endobacteria in the tumor cells. Onchocercomas from patients, untreated and treated with antibiotics and anthelminthics, were examined by immunohistology. Neoplasms were diagnosed in 112 of 3587 female and in 2 of 1570 male O. volvulus. The following proteins and other compounds of O. volvulus were expressed in the cells of the neoplasms: glutathione S-transferase 1, lysosomal aspartic protease, cAMP-dependent protein kinase, alpha-enolase, aspartate aminotransferase, ankyrin E1, tropomyosin, heat shock protein 60, transforming growth factor-beta, and prostaglandin E(2). These findings prove the filarial origin of the neoplasms and confirm the pleomorphism of the tumor cells. Signs indicating malignancy of the neoplasms are described. Wolbachia were observed in the hypodermis, oocytes, and embryos of tumor-harbouring filariae using antibodies against Wolbachia surface protein, Wolbachia HtrA-type serine protease, and Wolbachia aspartate aminotransferase. In contrast, Wolbachia were not found in the cells of the neoplasms. Further, neoplasm-containing worms were not observed after more than 10 months after the start of sufficient treatment with doxycycline or doxycycline plus ivermectin.

Observations of low response of patients infected with Onchocerca volvulus to ivermectin suggest that the parasite may be under a selection process toward potential resistance. To limit the extension of this phenomenon, it is crucial to characterize the genes of O. volvulus that are involved. For this, O. volvulus adult worms collected before the introduction of ivermectin in an onchocerciasis endemic area of central Cameroon were genotyped for beta-tubulin. To derive a baseline to investigate the selective pressure of ivermectin, we analysed (1) the frequency distribution of the beta-tubulin alleles, and (2) the relationship between the different beta-tubulin related genotypes and the fertility status of the female worms. The frequency of allele b of the beta-tubulin gene was very low, as it was observed in West Africa. We observed a deficit of heterozygous female worms leading to Hardy Weinberg disequilibrium, which might be explained by a shorter life-span of these worms compared to the homozygous worms. Unexpectedly, our results also show that the heterozygous female worms were much less fertile than the homozygotes: more than two thirds of the homozygotes were fertile, whereas only 37% of the heterozygotes were fertile. These results will be further considered when analysing post-treatment data.

The standard assay for onchocerciasis diagnosis is microscopical detection of microfilariae in skin snips. Skin snipping is painful, requires appropriate sterilization of equipment, and may fail to diagnose light infections. Two alternatives are a polymerase chain reaction (PCR) test which detects parasite DNA in pieces or scrapings of skin and a test based on allergic reactions to topical application of diethylcarbamazine (DEC). We compared these 2 diagnostics with standard skin snip microscopy in 313 individuals from 2 villages in Guinea, with low prevalence after over 10 years of control by the Onchocerciasis Control Programme. Lower and upper bounds on sensitivities and specificities of these 3 tests were estimated. In addition, these parameters were estimated using 5 different statistical models. Where prevalence was low, PCR and the DEC patch test appeared to be more sensitive than skin snipping which has low sensitivity. As the DEC test is non-invasive, simple and cheap, it may provide a good alternative to skin snipping alone for surveillance in low prevalence areas.

A model is presented which describes the aggregation of female Onchocerca volvulus in nodules and their distribution in the human population. The basic model is based on a single parameter, the formation probability q, which represents the probability with which incoming larvae form a new nodule. This parameter describes parasite behaviour which cannot easily be recognized in available data without modelling. The estimate for the average formation probability of muq = 0.39 suggests an attraction of the invading infective larvae to already existing nodules or resident worms with probability 0.61. No significant difference in muq was found between the forest and savanna parasite strains. The model can be used inversely to estimate the worm burden of persons from palpation data. The observed variance in the number of nodules per person requires the assumption of a variance-increasing mechanism which was implemented by heterogeneity within the host population (extended model with 2 parameters). Possible reasons for this heterogeneity are presented and its implications concerning the reproductive biology of the parasite are discussed.

Detection of Onchocerca volvulus larvae in vector populations is of prime importance in the assessment of the effectiveness of onchocerciasis control programmes. Traditionally, detection of larvae is attained by the dissection of flies, but this time-consuming method cannot easily discriminate between species of Onchocerca. The genome of all Onchocerca species has a unique 150 bp repeat, which can be amplified by PCR, and O. volvulus-specific DNA probes can detect these products by Southern blot (SB). This study optimizes a PCR/SB assay, and compares it with fly dissection to estimate the prevalence (p) and intensity of infection (m) in the local vector population of a Mexican community that has become hypoendemic as a result of 7 years of treatment with ivermectin and nodulectomy. The PCR detected 1 infected fly in a pool of 99 uninfected flies, but the optimal pool size was 50 flies. At the community level, 1 out of 10,550 flies was positive (p = 0.0095%, 95% confidence intervals CI = 0.00024-0.05280%; m = 0.00027 larvae/parous fly, CI = -0.00026-0.00081) by PCR, and 4 out of 10,772 flies (p = 0.0371%, CI = 0.01012-0.09505%; m = 0.00107 larvae/parous fly, 95% CI = 0.00002-0.00212) by dissection (observed m = 0.0005). Both methods produce statistically similar estimates of the prevalence and intensity, indicating that pool screening is a viable alternative for entomological surveillance in areas where the intensity of transmission is becoming extremely low as a result of control interventions.

The filarial nematodes Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus represent major public health problems in the Tropics. Effective diagnosis of infection with these parasites is required both for administration of drugs to infected individuals and for monitoring of control programs. However parasitological diagnosis is associated with a number of problems including frequently inadequate sensitivity, long pre-patency of infection and inconvenience for patients. For these reasons there has been considerable effort expended in developing other forms of diagnosis, in particular immunoassays for measuring antibody and circulating parasite antigen as well as molecular-biology-based assays for detecting parasite DNA. This article reviews the progress and achievements obtained to date. The latter include the development of ELISAs employing recombinant antigen for detection of antibody to O. volvulus which have both high sensitivity and specificity, the commercial availability of immunoassays to measure circulating antigen in W. bancrofti infection and the generation of specific DNA-based detection systems for all three parasites.

Polymerase chain reaction (PCR) combined with non-radioactive DNA hybridization was applied for the detection and characterization of a 150 bp tandem repeat of Onchocerca volvulus. DNA of worms from western Uganda was amplified and then probed with a digoxygenin-labelled oligonucleotide, specific for the forest form of O. volvulus and compared to samples from various African countries. Hybridization was only observed with PCR products from the forest in Liberia, south-eastern Ghana, Benin and southern Cameroon, but not with worms from Uganda or the savannah in Burkina Faso and northern Ghana. A nested PCR using primers derived form the forest form-specific DNA sequence confirmed these results. Morphometric studies revealed length differences between the microfilariae of Ugandan O. volvulus to those of West Africa, especially to those of the savannah in Burkina Faso. It is concluded that the forest/savannah classification of O. volvulus from West Africa is not suitable for Simulium neavei-transmitted O. volvulus from Uganda.

Immunization of chimpanzees with radiation-attenuated infective 3rd-stage larvae (L3) of Onchocerca volvulus did not induce strong protective immunity against a subsequent challenge infection; only 1 out of 4 immunized animals remained non-patent (i.e. microfilariae-negative) after challenge, and may have been protected. However, during immunization and before challenge, a broad range of adult O. volvulus-derived antigens (OvAg) and also uterus-derived OvAg were recognized by circulating antibodies; moreover, the repertory of antigens recognized increased further in subsequently patent animals after challenge, particularly in the range of M(r) 12-42 kDa. In the immunized and non-patent chimpanzee, by contrast, serological recognition of uterus-derived OvAg with M(r) 14 kDa and 105 kDa disappeared by 19 months post-challenge (p.c.). During immunization, Acanthocheilonema viteae L3 antigens of M(r) 11-12 kDa were strongly recognized only by the non-patent animal, suggesting that recognition of these antigens may have supported resistance to the subsequent challenge infection. In immunized chimpanzees, a substantial increase in the cellular reactivity to OvAg was induced; this, however, declined by 19 months p.c. to levels similar to those seen prior to immunization. At that time, 3 out of 4 immunized animals were patently infected. The effect of exogenous cytokines on in vitro-reactivity of PBMC to OvAg was examined. Addition of exogenous IL-2 alone, IFN-gamma alone, and IFN-gamma in combination with IL-2, did not augment net cellular responses to OvAg by PBMC from infected and control chimpanzees. In the presence of IL-4 alone, IL-6 alone, IL-2 with IL-4, IL-2 with IL-4 and IFN-gamma, or IL-2 with IL-4 and IL-6, the net cellular reactivity to OvAg increased significantly in patent chimpanzees and reached levels similar to non-patent animals. Thus, non-patent chimpanzees maintain high cellular reactivity to OvAg and in vitro cellular unresponsiveness to OvAg on the part of patent chimpanzees is reversible after addition of several cytokines which act individually or synergistically.