Onchocerciasis-associated epilepsy: Charting a path forward.

This narrative review intends to inform neurologists and public health professionals about Onchocerciasis-Associated Epilepsy (OAE), a neglected public health problem in many remote onchocerciasis-endemic areas. For epidemiological purposes, we define OAE as sudden-onset of convulsive and non-convulsive seizure types, including head nodding seizures (nodding syndrome) in a previously healthy child aged 3 to 18 years in the absence of any other obvious cause for epilepsy, all happening within an area with high ongoing Onchocerca volvulus transmission. Several OAE pathophysiological mechanisms have been proposed, but none has been proven yet. Recent population-based studies showed that strengthening onchocerciasis elimination programs was followed by a significant reduction in the incidence of OAE and nodding syndrome. Treating epilepsy in onchocerciasis-endemic regions is challenging. More advocacy is needed to provide uninterrupted, free access to anti-seizure medication to persons with epilepsy in these remote, impoverished areas. It is crucial todevelop policies and increase funding for the prevention and treatment of OAE to reduce the associated burden of disease, notably via the establishment of morbidity management and disability prevention programs (MMDP). Moreover, effective collaboration between onchocerciasis elimination and mental health programs is imperative to alleviate the burden of OAE. This synergy promises reciprocal advantages and underscores the need for a comprehensive approach to address this multifaceted challenge.

Use of RNAlater as a preservation method for parasitic coprology studies in wild-living chimpanzees.

We evaluated the use of an RNA stabilisation buffer, RNAlater (Ambion, Austin, Texas), as a preservation medium for parasitic coprology analysis of faecal samples collected from chimpanzees living in the wild (Pan troglodytes troglodytes). Thirty faecal samples collected in the forests of south-east Cameroon (Mambele area) from 2003 to 2011 were preserved in RNAlater at -80 °C and analysed for their parasite content. We identified and counted parasitic elements and assessed their shape, size and morphology in relation to the storage time of the samples. We found that parasite elements were identifiable in RNAlater preserved samples after as many as 7 years, showing that RNAlater could be an effective and reliable preservation medium for coprology. Thus, its use could be an interesting way to optimise sample collection for several types of studies (parasitology and bacteriology/virology) at once, especially considering the logistically challenging and time-consuming field campaigns needed to obtain these faecal samples.

Microfilarial distribution of Loa loa in the human host: population dynamics and epidemiological implications.

Severe adverse events (SAEs) following ivermectin treatment may occur in people harbouring high Loa loa microfilarial (mf) densities. In the context of mass ivermectin distribution for onchocerciasis control in Africa, it is crucial to define precisely the geographical distribution of L. loa in relation to that of Onchocerca volvulus and predict the prevalence of heavy infections. To this end, we analysed the distribution of mf loads in 4183 individuals living in 36 villages of central Cameroon. Mf loads were assessed quantitatively by calibrated blood smears, collected prior to ivermectin distribution. We explored the pattern of L. loa mf aggregation by fitting the (zero-truncated) negative binomial distribution and estimating its overdispersion parameter k by maximum likelihood. The value of k varied around 0.3 independently of mf intensity, host age, village and endemicity level. Based on these results, we developed a semi-empirical model to predict the prevalence of heavy L. loa mf loads in a community given its overall mf prevalence. If validated at the continental scale and linked to predictive spatial models of loiasis distribution, this approach would be particularly useful for optimizing the identification of areas at risk of SAEs and providing estimates of populations at risk in localities where L. loa and O. volvulus are co-endemic.

Co-infection with Onchocerca volvulus and Loa loa microfilariae in central Cameroon: are these two species interacting?

Ivermectin treatment may induce severe adverse reactions in some individuals heavily infected with Loa loa. This hampers the implementation of mass ivermectin treatment against onchocerciasis in areas where Onchocerca volvulus and L. loa are co-endemic. In order to identify factors, including co-infections, which may explain the presence of high L. loa microfilaraemia in some individuals, we analysed data collected in 19 villages of central Cameroon. Two standardized skin snips and 30 mul of blood were obtained from each of 3190 participants and the microfilarial (mf) loads of both O. volvulus and L. loa were quantified. The data were analysed using multivariate hierarchical models. Individual-level variables were: age, sex, mf presence, and mf load; village-related variables included the endemicity levels for each infection. The two species show a certain degree of ecological separation in the study area. However, for a given individual host, the presence of microfilariae of one species was positively associated with the presence of microfilariae of the other (OR=1.79, 95% CI [1.43-2.24]). Among individuals harbouring Loa microfilariae, there was a slight positive relationship between the L. loa and O. volvulus mf loads which corresponded to an 11% increase in L. loa mf load per 100 O. volvulus microfilariae. Co-infection with O. volvulus is not sufficient to explain the very high L. loa mf loads harboured by some individuals.

Genetic polymorphism of the beta-tubulin gene of Onchocerca volvulus in ivermectin naïve patients from Cameroon, and its relationship with fertility of the worms.

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.

Loiasis: the individual factors associated with the presence of microfilaraemia.

No microfilariae are detectable in a significant percentage of those infected with the filarial worm Loa loa. While the probability of an infected individual becoming microfilaraemic is known to increase with age, the mechanisms underlying this trend are not well understood. Epidemiological data from an endemic village in central Cameroon were therefore explored, in an attempt to determine if, after taking into account any history of filaricidal treatment, the presence of Loa microfilaraemia in an individual was related to his/her gender, age, and/or exposure to the human-infective larvae of the parasite. An index of exposure, based on the monthly transmission potentials of the Chrysops in each of the main types of vegetation in a village and on the activity schedule of each inhabitant of the village, was developed. The results of the data analysis confirm that the acquisition of microfilaraemia is gender-dependent (males generally being more likely to be microfilaraemic than females), and indicate that, in males, a high level of exposure to infective larvae determines the shift from amicrofilaraemic to microfilaraemic status. They also indicate that filaricidal treatments have a long-lasting suppressive effect on Loa microfilaraemia, an observation that may have important implications for any strategy to limit the risk of Loa-associated encephalopathy following ivermectin treatment.

Effects of vitamin C supplementation on plasma ascorbic acid and oxalate concentrations and meat quality in swine.

Two experiments were conducted to determine the effects of vitamin C supplementation 48 h before slaughter on plasma ascorbic acid and oxalate concentrations and its effect on pork quality. In Exp. 1, 16 pigs (87.8+/-2.13 kg BW) were blocked by sex and weight and assigned randomly within block to one of three vitamin C treatments: 1) control; 2) 1,000 mg/L; or 3) 2,000 mg/L supplemented in the drinking water for a 48-h period. This was then followed by an additional 48-h period without supplemental vitamin C. Vitamin C increased plasma ascorbic acid concentrations (11.6, 19.5, and 23.4 microg/mL for 0, 1,000, and 2,000 mg/L of vitamin C; P < 0.05) within 6 h of supplementation. Plasma ascorbic acid concentrations from treated pigs decreased and did not differ from those of control pigs (13.7, 18.2, and 18.6 microg/mL for 0, 1,000, and 2,000 mg/L of vitamin C; P = 0.30) within 2 h of ending supplementation. No differences in plasma ascorbic acid concentrations were found between the two levels of supplementation. Vitamin C did not affect plasma oxalate or cortisol; however, cortisol tended to increase quadratically (P = 0.077) with vitamin C after 96 h. In Exp. 2, 30 pigs (107.5+/-0.54 kg BW) were blocked by sex and weight and assigned randomly within block to one of three vitamin C treatments: 1) control; 2) 500 mg/L; or 3) 1,000 mg/L supplemented in the drinking water 48 h before slaughter. Pigs were slaughtered 4 to 5 h after vitamin C supplementation ended, and loin samples were collected for meat quality measurements. At the time of slaughter, no differences in plasma ascorbic acid or cortisol were observed, but oxalate tended (P = 0.074) to increase quadratically with increasing vitamin C. Muscle ascorbic acid at slaughter and lactic acid in muscle at 0 and 1.5 h after slaughter were not different; however, lactic acid increased (P = 0.048) quadratically at 24 h after slaughter. Vitamin C did not affect initial or ultimate pH. Initial fluid loss (P = 0.041), and fluid loss on d 4 (P = 0.014) and 8 (P = 0.076) of simulated retail display; L* on d 0 (P = 0.038), 4 (P = 0.010), and 8 (P = 0.051); a* on d 0 (P = 0.021); and b* on d 0 (P = 0.006), 4 (P = 0.035), and 8 (P = 0.017) were negatively affected in a quadratic manner when vitamin C was supplemented. Vitamin C tended (P = 0.086) to increase oxidation in chops on d 0, but not d 4 or 8. Results indicate that on-farm supplementation of vitamin C was generally not effective in improving pork quality, which may be related to timing relative to slaughter.

Impact of four years of large-scale ivermectin treatment with low therapeutic coverage on the transmission of Onchocerca volvulus in the Mbam valley focus, central Cameroon.

The extent to which ivermectin treatments have an impact on onchocerciasis transmission is a matter of some concern. We investigated this issue in the Mbam valley, a hyperendemic focus located in a forest-savannah mosaic area of Cameroon. Parasitological examinations of 5-9-year-old children, who had never received any antifilarial drug, were conducted before the first distribution of ivermectin in 1991-1993 and again in 2002, after four annual rounds of mass treatments. After matching for gender, age and village of residence, the prevalence and intensity of microfilaridermia corresponded respectively, in 2002, to 66.2 and 42.0% of the initial values. The decrease was more marked among the youngest children who, compared with the older ones, were submitted to the reduced force-of-infection earlier in their life. The results of the present study suggest that the specific vectorial competence of Simulium squamosum cytotype B, the vector of Onchocerca volvulus in the Mbam valley, allows a significant decrease in onchocerciasis transmission after several years of treatment, despite low therapeutic coverage. Though these results are encouraging, efforts should be made to improve the therapeutic coverage in the area.

Experimental studies on the transmission of Onchocerca volvulus by its vector in the Sanaga valley (Cameroon): Simulium squamosum B. Intake of microfilariae and their migration to the haemocoel of the vector.

As large-scale ivermectin distribution is becoming the mainstay of onchocerciasis control in Africa, the issue of its impact on local transmission is increasing in importance. The vector competence of Simulium squamosum B in the severe focus of the Sanaga valley, Cameroon, was therefore investigated, by feeding 1320 flies on 14 carriers of Onchocerca volvulus microfilariae (mff). The results enabled the relationships between skin microfilarial load, microfilarial intake by the flies, the proportion and mean number of ingested mff that succeed in reaching the fly's haemocoel, and the frequency distribution of the ingested mff to be described, as functions of time post-engorgement (p.e.) and parasite density (while taking account of possible measurement error in the predictor variable). The proportion of flies with haemocoelic mff and the mean number of mff/fly increased up to 3 h p.e. The proportion of flies with ingested mff was non-linearly related to mean intake, via the negative-binomial distribution, with the overdispersion parameter k best described as an increasing (power) function of the mean. Approximately one in every three ingested mff escaped imprisonment by the peritrophic matrix, irrespective of the skin microfilarial load or the intake of mff. The relationship between successful and input mff is nearly linear (indicating proportionality) in S. squamosum B. These results are compared with those from O. volvulus-S. damnosum s.l. combinations in other West African foci.

Neoplastic change in Onchocerca volvulus and its relation to ivermectin treatment.

A pleomorphic neoplasm (PN) is described from sections of Onchocerca volvulus worms in nodules excised from Cameroonian patients. PN is confined to older, non-fecund, female worms, and those classed as moribund/dead. It is mainly composed of small, roundish, basophilic cells of diverse sizes, often forming a 'rosette' pattern around amorphous eosinophilic centres. The cells have a high nuclear/cytoplasmic ratio and up to 2-3 mitoses/high-power field; some become grossly enlarged, highly polymorphic and contain large, irregular blocks of chromatin. The eukaryotic PN cells first appear posteriorly in the pseudocoelom, probably from ovarian cells; they spread anteriorly, invading or compressing the uteri. Ivermectin treatment increased the prevalence PN from 3.7% of 1422 female worms in 637 patients before treatment to 17.5% of 1134 worms in 511 patients after 3 years treatment. Ivermectin at 400-800 microg/kg annually, or at 150 microg/kg or 400-800 microg/kg 3-monthly, over 3 years, did not increase the PN prevalence significantly, as compared with standard doses of 150 microg/kg annually. In other small series of African patients, PN prevalence increased in worms 2, 4, 6 and 10 months after ivermectin treatment; but there was no increase after treatment with amocarzine, albendazole or diethylcarbamazine and suramin. PN may partly account for the increased macrofilaricidal action of ivermectin on female O. volvulus in patients treated for 3 years at 3-monthly intervals.

Relationship between onchocerciasis and epilepsy: a matched case-control study in the Mbam Valley, Republic of Cameroon.

Studies conducted during the past 10 years to investigate the possible relationship between onchocerciasis and epilepsy have led to contradictory results. In 1991-92 and 2001 we investigated 14 villages in central Cameroon to evaluate the relationship, at the community level, between the prevalence of epilepsy and the endemicity level of onchocerciasis. A case-control study compared the microfilarial loads of 72 epileptic and 72 non-epileptic individuals, matched according to sex, age, and village of residence. The prevalence of epilepsy and the community microfilarial load (CMFL) were closely related (P < 0.02), and the case-control study demonstrated that the microfilarial loads (microfilariae per snip) in the epileptic group (arithmetic mean = 288, median = 216) were significantly higher (P < 10(-4)) than in the control group (arithmetic mean = 141, median = 63). The results strongly support the existence of a link between onchocerciasis and epilepsy. The fact that such a relationship has not been found recently in some other West and Central African areas is probably due to the lowered endemicity of onchocerciasis following vector- and ivermectin-related control measures applied over the past 5-25 years. The socio-economic and demographic impact of onchocerciasis-related epilepsy should be evaluated, and taken into account as part of all onchocerciasis control programmes.

Excess mortality associated with blindness in the onchocerciasis focus of the Mbam Valley, Cameroon.

The association between blindness, mortality and nutritional status was investigated in a retrospective cohort study in villages of central Cameroon where onchocerciasis is hyper-endemic. Overall, 101 blind subjects and 101 non-blind controls matched with the blind for age, sex and (generally) village of residence were followed for 10 years. Blindness gave rise to a significant increase in mortality (relative risk = 2.3; P = 0.012), the life expectancy of the blind adults being reduced by 4 years compared with that of their controls. For a given age, excess mortality was found to be associated with a late onset of blindness. The causes of death were similar for the blind and the controls but blind subjects had relatively low body mass indices, which may lead to relatively early fatal disease outcomes. These results are similar to those obtained in other parts of Africa and emphasise, once more, the demographic impact of blindness in developing countries.

Effects of a 3-day regimen of albendazole (800 mg daily) on Loa loa microfilaraemia.

The encephalopathy that sometimes develops after ivermectin treatment in patients with high Loa microfilaraemias is probably related to a massive effect of the drug on the Loa microfilariae. A trial was therefore conducted to evaluate whether a course of albendazole would bring about a slower decrease in the Loa microfilaraemia, and thus could be used as a mass 'clearing' treatment, before the distribution of ivermectin in areas where onchocerciasis and loiasis are co-endemic. The Loa microfilarial loads were followed monthly for 9 months in two groups of subjects, one treated with albendazole (400 mg twice a day for 3 days), and the other with vitamin (B(1), B(6) and B(12)) tablets. There were no significant between-group differences in the microfilarial loads at any of the examination rounds. During the follow-up period, there was also no significant change in the overall loads among those treated with albendazole, although the counts in those with high initial microfilaraemias (>8000 microfilariae/ml) tended to decrease progressively during the first 3 months. Further trials should now be performed, to evaluate the effects on Loa loa of two courses of albendazole given 2-3 months apart.

Relationships between the prevalence and intensity of Loa loa infection in the Central province of Cameroon.

Ivermectin treatment may induce marked adverse effects in those harbouring > 8000 Loa microfilariae (mff)/ml of blood, individuals with > 30 000 Loa mff/ml being at risk of developing serious neurological reactions. It is thus necessary to delineate the geographical areas where such responses may occur. To determine if the simple measure of prevalence of Loa microfilaraemia would be appropriate to identify the communities at risk, the relationships between prevalence and intensity of Loa microfilaraemia were investigated in 67 villages in Cameroon. The prevalence recorded in the adult population was found to be closely related to each of the indicators of infection intensity investigated. For example, when the prevalences of Loa microfilaraemia in adults were 20%, 30% and 40%, approximately 5%, 9% and 16% of the adults had microfilarial loads exceeding 8000 mff/ml, respectively; the corresponding percentages of adults with > 30 000 mff/ml were about 1%, 3% and 5%-6%. Thus it seems that, in areas where loiasis is co-endemic, the monitoring procedure during large-scale ivermectin treatments for the control of onchocerciasis only needs to be strengthened in those communities where the prevalence of Loa microfilaraemia in adults exceeds 20%.

Studies on the macrofilarial population of Onchocerca volvulus in hyper-endemic villages of the Central province of Cameroon.

The population structure of Onchocerca volvulus macrofilariae was studied in villages of central Cameroon where onchocerciasis is hyper-endemic. One nodule selected at random was removed from each of 576 adult males, and examined by histology. The numbers of male and female worms/nodule, and the status of the female worms (fecund, non-fecund, or dead) were recorded. The observations were analysed to evaluate whether the mean numbers of worms of each category varied in relation to the patient's age, the level of endemicity in his village, the anatomical localization of the nodule, the weight of the nodule, and the total number of palpable nodules harboured by the patient. The results obtained were very similar to those reported from West Africa. The mean numbers of dead female worms/nodule were relatively high in the villages with the lowest levels of endemicity. The mean numbers of fecund females and of live males were significantly higher in the nodules located around the knees. These results provide information which might be useful in modelling the population dynamics of O. volvulus, and also in the context of trials of any potentially macrofilaricidal drugs.

[Entomologic study of loaiasis transmission in the Lekie area (Cameroon)]. / Etude entomologique sur la transmission de la loase dans de département de la Lékié (Cameroun).

A number of cases of Loa encephalopathy have been recorded after ivermectin treatment in the Lekie Division, an area of degraded forest located in central Cameroon. An entomological study was carried out in a village of this region between May 1999 and April 2000 to determine whether the high microfilarial loads of Loa found in the population, which can exceed 10,000 microfilariae per ml of blood, were related to high densities of vector populations. The Chrysops collected at 10 catching stations, using hand nets, by persons standing by a wood fire, were dissected to evaluate their level of infection with Loa. The vectorial densities were three-fold higher in the forest stations than in those located near the habitations (2307 and 725 bites per man per year, respectively). These values are lower than those reported from similar studies in Cameroon, Congo and Gabon. Measurement of Chrysops densities does not seem to be an appropriate tool to evaluate the level of endemicity of loiasis, and to delineate the areas where there is a risk of post-ivermectin Loa encephalopathies.

The in vivo fate of APCs displaying minor H antigen and/or MHC differences is regulated by CTLs specific for immunodominant class I-associated epitopes.

The goal of this work was to evaluate the fate of APCs following interactions with T cells in unprimed mice with a normal T cell repertoire. We elaborated a model in which male adherent peritoneal mononuclear cells were injected into the foreleg footpads of naive female recipients mismatched for either minor or major histocompatibility Ags. At various times after injection, APC numbers in the draining (axillary and brachial) lymph nodes were assessed using a Ube1y gene-specific PCR assay. Our experimental model was designed so that the number of APCs expressing the priming epitope was similar to what is observed under real life conditions. Thus, early after injection, the frequency of afferent lymph-derived APCs expressing the priming epitope was in the range of 101-102/106 lymph node cells. We found that APCs presenting some, but not all, nonself epitopes were killed rapidly after entrance into the lymph nodes. Rapid elimination of APCs occurred following interactions with MHC class I-restricted, but not class II-restricted, T cells and was observed when APCs presented an immunodominant (B6dom1/H7a), but not a nondominant (HY), epitope. Killing of APCs was mediated partly, but not exclusively, by perforin-dependent process. We propose that killing of APCs by CTLs specific for immunodominant MHC class I-restricted epitopes may be instrumental in regulating the intensity, duration, and diversity of T cell responses.

Shaping the repertoire of cytotoxic T-lymphocyte responses: explanation for the immunodominance effect whereby cytotoxic T lymphocytes specific for immunodominant antigens prevent recognition of nondominant antigens.

The immunodominance effect, whereby the presence of immunodominant epitopes prevents recognition of nondominant determinants presented on the same antigen-presenting cell (APC) considerably restricts the repertoire of cytotoxic T lymphocyte (CTL) responses. To elucidate the molecular basis of the immunodominance effect, we compared the interactions of a dominant (B6(dom1)) and a nondominant epitope (H-Y) with their restricting class I molecule (H2-Db), and their ability to trigger cognate CTLs. We found that B6(dom1)/Db complexes behaved as optimal T-cell receptor (TCR) ligands and triggered a more rapid in vivo expansion of cognate CTLs than H-Y/Db complexes. The superiority of the dominant epitope was explained by its high cell surface density (1,012 copies/cell for B6(dom1) v 10 copies/cell for H-Y) and its optimal affinity for cognate TCRs. Based on these results, we conclude that dominant class I-associated epitopes are those that have optimal ability to trigger TCR signals in CTLs. We propose that the rapid expansion of CTLs specific for dominant antigens should enable them to compete more successfully than other CTLs for occupancy of the APC surface.

On the mechanisms of immunodominance in cytotoxic T lymphocyte responses to minor histocompatibility antigens.

Although there are numerous minor histocompatibility antigens (MiHA), T cell responses leading to graft-versus-host (GVH) and graft-versus-tumor effects involve only a small number of immunodominant MiHA. The goal of the present study was to analyze at the cellular and molecular levels the mechanisms responsible for MiHA immunodominance. Cytotoxic T lymphocytes (CTL) generated in eight combinations of H2b strains of mice were tested against syngeneic targets sensitized with HPLC-fractionated peptides eluted from immunizing cells. The number of dominant MiHA was found to range from as little as two up to ten depending on the strain combination used. The nature of dominant MiHA was influenced by both the antigen profile of the antigen-presenting cells (APC) and the repertoire of responding CTL. When C57BL/6 dominant MiHA (B6dom) and H-Y were presented on separate APC, they showed similar immunogenicity. In contrast, when they were presented on the same APC, B6dom MiHA totally dominated H-Y. B6dom MiHA did not suppress anti-H-Y responses by acting as T cell receptor antagonists for anti-H-Y CTL, nor were anti-B6dom CTL precursors more abundant than anti-H-Y CTL precursors. Dominance resulted from competition for the APC surface between anti-B6dom and anti-H-Y CTL; the crucial difference between the dominant and the dominated MiHA appears to depend on the differential avidity of their respective CTL for APC. The only B6dom epitope thus far identified is the nonapeptide AAPDNRETF presented by H2-D(b). We found that compared with other known D(b)-binding peptides, AAPDNRETF is expressed at very high levels on the cell surface, binds to the D(b) molecule with very high affinity, and dissociates very slowly from its presenting class I molecule. These data indicate that one cannot predict which MiHA will be dominant or dominated based simply on their respective immunogenicity when presented on separate APC. Indeed, the avidity of T cell/APC interactions appears to determine which antigen(s) will trigger T cell responses when numerous epitopes are presented by the same APC.