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Introduction Administration of ivermectin (IVM) as part of mass drug administration (MDA) campaigns for onchocerciasis and/or lymphatic filariasis (LF) has been suspended in areas co-endemic for Loa loa due to severe post-treatment adverse events (SAEs) associated with high-burden of infection (>30,000 mf/ml). One simple approach for preventing SAEs is to identify and exclude individuals at risk from MDA. Here, we describe a repurposed hand-held automated cell counter (Scepter 2.0; HHAC) as a rapid, point-of-care method for quantifying microfilariae (mf) in the blood of infected individuals. Methodology/Principal Findings The quantification of microfilarial levels in blood of naturally infected humans, experimentally infected baboons, or mf-spiked human blood was tested using a microfluidic-based automated counter and compared to traditional calibrated thick-smears. We demonstrate that mf can be quantified in 20 µl of whole blood following lysis with 10% saponin within a minute of obtaining blood. There was a highly significant concordance between the counts obtained by the HHAC and those by microscopy for mf densities of >5,000 (p30,000 per ml (p, Author Summary Mass drug administration (MDA) efforts with ivermectin-based regimens for onchocerciasis and for lymphatic filariasis in Africa have been suspended in certain areas that are co-endemic for Loa loa infection. This is due to the serious adverse events (encephalopathy and death) that can develop following ivermectin administration in individuals with very high circulating levels of Loa loa microfilariae (mf) (>30,000 mf/ml). Currently thick-blood smears are performed to quantify the mf levels that are neither rapid nor high throughput. To develop a point of care (POC) strategy for rapid and high throughput mf quantification, a repurposed handheld automated counter (HHAC) was assessed and shown to be an efficient POC tool to identify individuals with very high Loa loa microfilaraemia in a manner sufficient to be used as part of a Test and (not) Treat (TNT) program in an effort to restart the suspended MDA programs while preventing severe adverse neurologic events.

Determining the effectiveness of a product in repelling mosquitoes or other flying insects is a difficult task. One approach is to use a bioassay with textile fabric. We investigated the role of the textile substrate in a bioassay with a numerical model, and compared the outcome with known results for DEET. The model was then used to determine the effectiveness of textile slow-release formulations based on coatings, and results were compared with those of a field study in the Cameroon. Slow-release formulations are difficult to evaluate with standard tests, as the compound needs a build-up time not present in these tests.We found excellent correspondence between the model and the known DEET results without matching parameters. Slow-release approaches are deemed possible but have several drawbacks. Modelling can help in identifying optimal use conditions. The field test with a slow-release system performed better than anticipated by the model, with initially more than 90% repellency. DEET-coated textile was considered not to be marketable, however.We advise that bioassays characterise in more detail the type of textile fabric used so as to allow conclusions to be drawn by textile modelling. As regards coated-textile slow-release systems, more research is needed. We nevertheless advise usage mainly at entry points, e.g. as scrims.

Background For two decades, onchocerciasis control has been based on mass treatment with ivermectin (IVM), repeated annually or six-monthly. This drug kills Onchocerca volvulus microfilariae (mf) present in the skin and the eyes (microfilaricidal effect) and prevents for 3–4 months the release of new mf by adult female worms (embryostatic effect). In some Ghanaian communities, the long-term use of IVM was associated with a more rapid than expected skin repopulation by mf after treatment. Here, we assessed whether the embryostatic effect of IVM on O. volvulus has been altered following frequent treatment in Cameroonian patients. Methodology Onchocercal nodules were surgically removed just before (D0) and 80 days (D80) after a standard dose of IVM in two cohorts with different treatment histories: a group who had received repeated doses of IVM over 13 years, and a control group with no history of large-scale treatments. Excised nodules were digested with collagenase to isolate adult worms. Embryograms were prepared with females for the evaluation of their reproductive capacities. Principal Findings Oocyte production was not affected by IVM. The mean number of intermediate embryos (morulae and coiled mf) decreased similarly in the two groups between D0 and D80. In contrast, an accumulation of stretched mf, either viable or degenerating, was observed at D80. However, it was observed that the increase in number of degenerating mf between D0 and D80 was much lower in the frequently treated group than in the control one (Incidence Rate Ratio: 0.25; 95% CI: 0.10–0.63; p = 0.003), which may indicate a reduced sequestration of mf in the worms from the frequently treated group. Conclusion/Significance IVM still had an embryostatic effect on O. volvulus, but the effect was reduced in the frequently treated cohort compared with the control population., Author Summary Onchocerciasis, also known as river blindness, is a parasitic disease due to the filarial nematode Onchocerca volvulus. It affects more than 37 million people worldwide, most of them (99%) living in Africa. The control of river blindness is, up to now, based on annual or six-monthly mass treatment with ivermectin. This drug kills O. volvulus microfilariae (mf) present in the skin and the eyes and prevents for 3–4 months the release of new mf by female worms (embryostatic effect). In Ghana, after 10–19 years of repeated treatments, the emergence of adult parasite populations not responding as expected to ivermectin was postulated. In this study, the reproductive status of female worms was compared, just before and 80 days after ivermectin treatment, between frequently treated and ivermectin-naïve cohorts from Cameroon. In both groups, embryogenesis of O. volvulus was not affected by ivermectin. However, the accumulation of microfilariae (mf) in the females uteri expected after ivermectin was less marked in the frequently treated population, suggesting that the temporary sequestration of mf following treatment may have been weakened in this group. After 13 years of repeated annual treatments, the embryostatic effect of ivermectin on O. volvulus still occurs but the present findings, associated with observations of higher rates of skin repopulation by mf in the same individuals, suggest that this effect has been decreased.

Lymphatic Filariasis and Onchocerciasis (river blindness) constitute pressing public health issues in tropical regions. Global elimination programs, involving mass drug administration (MDA), have been launched by the World Health Organisation. Although the drugs used are generally well tolerated, individuals who are highly co-infected with Loa loa are at risk of experiencing serious adverse events. Highly infected individuals are more likely to be found in communities with high prevalence. An understanding of the relationship between individual infection and population-level prevalence can therefore inform decisions on whether MDA can be safely administered in an endemic community. Based on Loa loa infection intensity data from individuals in Cameroon, the Republic of the Congo and the Democratic Republic of the Congo we develop a statistical model for the distribution of infection levels in communities. We then use this model to make predictive inferences regarding the proportion of individuals whose parasite count exceeds policy-relevant levels. In particular we show how to exploit the positive correlation between community-level prevalence and intensity of infection in order to predict the proportion of highly infected individuals in a community given only prevalence data from the community in question. The resulting prediction intervals are not substantially wider, and in some cases narrower, than the corresponding binomial confidence intervals obtained from data that include measurements of individual infection levels. Therefore the model developed here facilitates the estimation of the proportion of individuals highly infected with Loa loa using only estimated community level prevalence. It can be used to assess the risk of rolling out MDA in a specific community, or to guide policy decisions., Author Summary Lymphatic Filariasis (LF) is caused by parasitic worms which live in the lymphatic system. Though several body parts may be affected, LF characteristically leads to the enlargement of limbs, causing pain, physical disability and social stigma. Onchocerciasis (river blindness) is caused by similar worms which inhabit the subcutaneous tissue and lead to disturbing skin lesions, visual impairment and blindness. Onchocerciasis and LF affect over 146 million people, and have been targeted for elimination as public health problems by the World Health Organisation, using mass drug administration (MDA). Although the drugs used in MDA are generally well tolerated, individuals who are highly co-infected with Loa loa are at risk of experiencing serious adverse events. In order to inform decisions on whether MDA can be safely administered in a community, we investigate the relationship between community level prevalence of Loa loa and individual infection levels through the development of a statistical model. We find a strong positive correlation and show how this can be exploited to estimate the proportion of individuals highly infected with Loa loa without the need for data on individual infection levels. The model therefore provides a method to assess the risk of rolling out MDA in a specific community, or to guide policy decisions.

Immunocytochemical examination of onchocercal nodule tissues containing adult Onchocerca volvulus using immuno-markers for blood and lymphatic vessels (vWF, D2-40, podoplanin, Prox-1, and Lyve1) shows a distinct pattern of distribution of these vessels within nodules. Blood vessels were commonly seen associated with organized lymphoid cellular aggregates in the both the outer and inner areas of the nodules. In contrast, the majority of the lymphatic vessel positivity was seen in the central zone in close apposition to the adult parasites, and the remainder usually associated with microfilariae in the outer areas of the nodule. These findings suggest an intimate relationship between adult O. volvulus and lymphatic vessels, including the likely proliferation of lymphatic endothelial cells (lymphangectasia) akin to that seen with other filariae. These findings indicate that adult O. volvulus may migrate via the lymphatic system, and that clinical manifestations of this disease that involve tissue edema may be the result of the location of these worms in the lymphatic system.

To assess individual compliance with annual ivermectin treatment in onchocerciasis-endemic villages.Multi-site study in eight APOC-sponsored projects in Cameroon, Nigeria and Uganda to identify the socio-demographic correlates of compliance with ivermectin treatment. A structured questionnaire was administered on 2305 persons aged 10 years and above. Two categories of respondents were purposively selected to obtain both high and low compliers: people who took ivermectin 6-8 times and 0-2 times previously. Simple descriptive statistics were employed in characterizing the respondents into high and low compliers, while some socio-demographic and key perceptual factors were employed in regression models constructed to explain levels of compliance among the respondents.Some demographic and perceptual factors associated with compliance were identified. Compliance was more common among men (54.4%) (P0.001). Adults (54.6%) had greater rates of high compliance (P0.001. The mean age of high compliers (41.5 years) was significantly older (35.8 years) (t = 8.46, P0.001). Perception of onchocerciasis and effectiveness of ivermectin influenced compliance. 81.4% of respondents saw benefits in annual ivermectin treatment, high compliance among those who saw benefits was 59.3% compared to 13.3% of those who did not (P0.001).Efforts to increase compliance with ivermectin treatment should focus on providing health education to youth and women. Health education should also highlight the benefits of taking ivermectin.

International audience; The present study analyzed the relationship between the genetic diversity of Plasmodium falciparum and parasitologic/entomologic indices in the Mount Cameroon region by using merozoite surface protein 1 as a genetic marker. Blood samples were collected from asymptomatic children from three altitude zones (high, intermediate, and low). Parasitologic and entomologic indices were determined by microscopy and landing catch mosquito collection/ circumsporozoite protein–enzyme-linked immunosorbent assay, respectively. A total of 142 randomly selected P. falciparum-positive blood samples were genotyped by using a nested polymerase chain reaction–based technique. K-1 polymerase chain reaction products were also sequenced. As opposed to high altitude, the highest malaria prevalence (70.65%) and entomologic inoculation rate (2.43 infective/bites/night) were recorded at a low altitude site. Seven (18.91%), 22 (36.66%), and 19 (42.22%) samples from high, intermediate, and low altitudes, respectively, contained multiclonal infections. A new K-1 polymorphism was identified. This study shows a positive non-linear association between low/intermediate altitude (high malaria transmission) and an increase in P. falciparum merozoite surface protein 1 block 2 polymorphisms.

Malaria and Soil-Transmitted Helminths (STHs) do co-infect hosts and it has been speculated that urbanization alters the frequency and transmission dynamics of both infections. This study was aimed at finding out the influence of urbanization on the prevalence and intensity of co-infections of asymptomatic malaria and STHs in schoolchildren in the Mount Cameroon Region. A total of 235 and 208 children from Ekona (rural) and Great Soppo (urban) respectively aged 4-14 years were enrolled into a cross-sectional study. Blood films were Giemsa-stained for the determination of malaria parasite density and speciation. Capillary blood was spun for the determination of packed cell volume. Stool samples were examined by the Kato-Katz technique for the presence and intensity of STHs. The prevalences of malaria in Ekona and Great Soppo were, 92.34 % and 82.70 % respectively (X2 = 9.60, p = 0.002). Malaria Geometric Mean Parasite Density (GMPD) in Ekona (277 ± 1988) was higher than that in Great Soppo (251±2400 parasites/ µl). Prevalence of helminths was higher in Ekona (43.82%) than Great Soppo (11.54%) (X2 = 60.12, p < 0.001). Coinfections were higher in Ekona (41.70 %) than Great Soppo (10.58%) (X2 = 45.83, p < 0.001). Ascaris lumbricoides was the most prevalent helminth and also had the highest Geometric Mean Egg Density (GMED) in Ekona, followed by Trichuris trichiura and hookworm. A similar pattern was observed in Great Soppo except for T. trichiura which was the most prevalent helminth. Prevalences of anemia in Ekona and Great Soppo were 6.00% and 1.00% respectively. The highest prevalence of anemia, GMPD and GMED occurred in co-infected participants when compared to those infected only with malaria or STHs. The prevalence of the infeections decreased with increase in level of urbanization. Schoolbased control of these infections especially in rural areas will reduce their related morbidities.

Background Anti-Wolbachia treatment with doxycycline is effective in sterilising and killing adult Onchocerca volvulus nematodes, proving superior to ivermectin and of great potential as an alternative approach for the treatment and control of onchocerciasis, particularly in areas of Loa loa co-endemicity. Nevertheless, the length of the required treatment poses potential logistical problems and risk of poor compliance, raising a barrier to the use of doxycycline in Mass Drug Administration (MDA) strategies. In 2007 and 2008 a feasibility trial of community-directed treatment with doxycycline was carried out in two health districts in Cameroon, co-endemic for O. volvulus and L. loa. With 17,519 eligible subjects, the therapeutic coverage was 73.8% with 97.5% compliance, encouraging the feasibility of using doxycycline community-directed delivery in restricted populations of this size. The current study evaluated the effectiveness of this community-directed delivery of doxycycline four years after delivery. Findings Infection with O. volvulus was evaluated by skin biopsy and nodule palpation. Of the 507 subjects recruited, 375 had completed the treatment with doxycycline followed by one or two rounds of annual ivermectin MDA and 132 received one or two rounds of annual ivermectin MDA alone. Statistically significant lower microfilarial prevalence (17.0% [doxycycline plus ivermectin group], 27.0% [ivermectin only group], p = 0.014) and load (p = 0.012) were found in people that had received doxycycline followed by ivermectin compared to those who received ivermectin only. Conclusions This study demonstrates the long-term effectiveness of doxycycline treatment delivered with a community-directed strategy even when evaluated four years after delivery in an area of ongoing transmission. This finding shows that a multi-week course of treatment is not a barrier to community-delivery of MDA in restricted populations of this size and supports its implementation to compliment existing control strategies for onchocerciasis, where needed.

Background Immunochromatographic card test (ICT) is a tool to map the distribution of Wuchereria bancrofti. In areas highly endemic for loaisis in DRC and Cameroon, a relationship has been envisaged between high L. loa microfilaria (Mf) loads and ICT positivity. However, similar associations have not been demonstrated from other areas with contrasting levels of L. loa endemicity. This study investigated the cross-reactivity of ICT when mapping lymphatic filariasis (LF) in areas with contrasting endemicity levels of loiasis and mansonellosis in Cameroon. Methodology/Principal Findings A cross-sectional study to assess the prevalence and intensity of W. bancrofti, L. loa and M. perstans was carried out in 42 villages across three regions (East, North-west and South-west) of the Cameroon rainforest domain. Diurnal blood was collected from participants for the detection of circulating filarial antigen (CFA) by ICT and assessment of Mf using a thick blood smear. Clinical manifestations of LF were also assessed. ICT positives and patients clinically diagnosed with lymphoedema were further subjected to night blood collection for the detection of W. bancrofti Mf. Overall, 2190 individuals took part in the study. Overall, 24 individuals residing in 14 communities were tested positive by ICT, with prevalence rates ranging from 0% in the South-west to 2.1% in the North-west. Lymphoedema were diagnosed in 20 individuals with the majority of cases found in the North-west (11/20), and none of them were tested positive by ICT. No Mf of W. bancrofti were found in the night blood of any individual with a positive ICT result or clinical lymphoedema. Positive ICT results were strongly associated with high L. loa Mf intensity with 21 subjects having more than 8,000 L. loa Mf ml/blood (Odds ratio = 15.4; 95%CI: 6.1–39.0; p < 0.001). Similarly, a strong positive association (Spearman’s rho = 0.900; p = 0.037) was observed between the prevalence of L. loa and ICT positivity by area: a rate of 1% or more of positive ICT results was found only in areas with an L. loa Mf prevalence above 15%. In contrast, there was no association between ICT positivity and M. perstans prevalence (Spearman’s rho = - 0.200; p = 0.747) and Mf density (Odds ratio = 1.8; 95%CI: 0.8–4.2; p = 0.192). Conclusions/Significance This study has confirmed the strong association between the ICT positivity and L. loa intensity (Mf/ml of blood) at the individual level. Furthermore, the study has demonstrated that ICT positivity is strongly associated with high L. loa prevalence. These results suggest that the main confounding factor for positive ICT test card results are high levels of L. loa. The findings may indicate that W. bancrofti is much less prevalent in the Central African region where L. loa is highly endemic than previously assumed and accurate re-mapping of the region would be very useful for shrinking of the map of LF distribution., Author Summary Mapping of lymphatic filariasis (LF) caused by W. brancrofti is usually done by employing a rapid diagnostic test that permits the detection of worm antigen in daytime blood. This is sometimes combined with a thick blood film (TBF) for microscopic examination, as confirmatory tool for detecting W. bancrofti Mf in peripheral night blood. During recent epidemiological surveys using immunochromatographic card test (ICT) to map LF in areas highly endemic for loiasis, positive card tests were observed in individuals’ amicrofilaremic for W. bancrofti during night TBF examination, as well as by parasite DNA detection. The possibility of ICT cross-reacting with L. loa antigen was envisaged, but so far associations between ICT positivity and L. loa endemicity levels and loads of Mf in day blood have not yet been established. Moreover, M. perstans another filaria with blood dwelling Mf, that is often sympatric with L. loa, could contribute to the observed ICT cross-reactivity. The authors investigated the cross-reactivity of ICT in areas with contrasting endemicity levels of L. loa and M. perstans in Cameroon. Results incriminated L. loa as the major confounder in ICT cross-reactivity, with significant association between ICT positivity and loiasis both at individual level (load of Mf/ml of blood) and endemicity level (Mf prevalence). M. perstans displayed no association with ICT positivity. The findings raised concerns about the specificity of the whole blood ICT used for LF mapping in loiasis co-endemic areas. The development of an algorithm for LF mapping in loiasis co-endemic areas will be important to validate the LF map obtained using ICT in Central Africa.

Background A simple method called RAPLOA, to rapidly assess what proportion of people in a community are infected with L. loa and hence which communities are at high risk of severe adverse reactions following ivermectin treatment, was developed in Cameroon and Nigeria. The method needed further validation in other geographical and cultural contexts before its application in all endemic countries. The present study was designed to validate RAPLOA in two regions in the North East and South West of the Democratic Republic of Congo. Methods In each study region, villages were selected from different bio-ecological zones in order to cover a wide range of loiasis endemicity. In each selected community, 80 people above the age of 15 years were interviewed for a history of eye worm (migration of adult L. loa under the conjunctiva of the eye) and parasitologically examined for the presence and intensity of L. loa infection. In total, 8100 individuals from 99 villages were enrolled into the study. Results The results confirmed the findings of the original RAPLOA study: i) the eye worm phenomenon was well-known in all endemic areas, ii) there was a clear relationship between the prevalence of eye worm history and the prevalence and intensity of L. loa microfilaraemia, and iii) using a threshold of 40%, the prevalence of eye worm history was a sensitive and specific indicator of high-risk communities. Conclusion Following this successful validation, RAPLOA was recommended for the assessment of loiasis endemicity in areas targeted for ivermectin treatment by lymphatic filariasis and onchocerciasis control programmes.

Background: Wolbachia are intriguing symbiotic endobacteria with a peculiar host range that includes arthropods and a single nematode family, the Onchocercidae encompassing agents of filariases. This raises the question of the origin of infection in filariae. Wolbachia infect the female germline and the hypodermis. Some evidences lead to the theory that Wolbachia act as mutualist and coevolved with filariae from one infection event: their removal sterilizes female filariae; all the specimens of a positive species are infected; Wolbachia are vertically inherited; a few species lost the symbiont. However, most data on Wolbachia and filaria relationships derive from studies on few species of Onchocercinae and Dirofilariinae, from mammals. Methodology/Principal Findings: We investigated the Wolbachia distribution testing 35 filarial species, including 28 species and 7 genera and/or subgenera newly screened, using PCR, immunohistochemical staining, whole mount fluorescent analysis, and cocladogenesis analysis. (i) Among the newly screened Onchocercinae from mammals eight species harbour Wolbachia but for some of them, bacteria are absent in the hypodermis, or in variable density. (ii) Wolbachia are not detected in the pathological model Monanema martini and in 8, upon 9, species of Cercopithifilaria. (iii) Supergroup F Wolbachia is identified in two newly screened Mansonella species and in Cercopithifilaria japonica. (iv) Type F Wolbachia infect the intestinal cells and somatic female genital tract. (v) Among Oswaldofilariinae, Waltonellinae and Splendidofilariinae, from saurian, anuran and bird respectively, Wolbachia are not detected. Conclusions/Significance: The absence of Wolbachia in 63% of onchocercids, notably in the ancestral Oswaldofilariinae estimated 140 mya old, the diverse tissues or specimens distribution, and a recent lateral transfer in supergroup F Wolbachia, modify the current view on the role and evolution of the endosymbiont and their hosts. Further genomic analyses on some of the newly sampled species are welcomed to decipher the open questions. © 2011 Ferri et al.

Background Loiasis is a major obstacle to ivermectin treatment for onchocerciasis control and lymphatic filariasis elimination in central Africa. In communities with a high level of loiasis endemicity, there is a significant risk of severe adverse reactions to ivermectin treatment. Information on the geographic distribution of loiasis in Africa is urgently needed but available information is limited. The African Programme for Onchocerciasis Control (APOC) undertook large scale mapping of loiasis in 11 potentially endemic countries using a rapid assessment procedure for loiasis (RAPLOA) that uses a simple questionnaire on the history of eye worm. Methodology/Principal Findings RAPLOA surveys were done in a spatial sample of 4798 villages covering an area of 2500×3000 km centred on the heartland of loiasis in Africa. The surveys showed high risk levels of loiasis in 10 countries where an estimated 14.4 million people live in high risk areas. There was a strong spatial correlation among RAPLOA data, and kriging was used to produce spatially smoothed contour maps of the interpolated prevalence of eye worm and the predictive probability that the prevalence exceeds 40%. Conclusion/Significance The contour map of eye worm prevalence provides the first global map of loiasis based on actual survey data. It shows a clear distribution with two zones of hyper endemicity, large areas that are free of loiasis and several borderline or intermediate zones. The surveys detected several previously unknown hyperendemic foci, clarified the distribution of loiasis in the Central African Republic and large parts of the Republic of Congo and the Democratic Republic of Congo for which hardly any information was available, and confirmed known loiasis foci. The new maps of the prevalence of eye worm and the probability that the prevalence exceeds the risk threshold of 40% provide critical information for ivermectin treatment programs among millions of people in Africa., Author Summary Loiasis is a neglected tropical disease caused by infection with the filarial parasite Loa loa, transmitted by Chrysops vectors. Loiasis has recently emerged as a disease of public health importance when neurologic serious adverse events (SAEs) were reported in individuals with high L. loa microfilaraemia after ivermectin treatment. This had a negative impact on the control of onchocerciasis and lymphatic filariasis in areas of co-endemicity with loiasis. Microfilarial prevalence of 20% has been suggested as the threshold above which there is an unacceptable risk of SAEs with ivermectin treatment.The African Programme for Onchocerciasis Control (APOC) undertook large scale mapping of loiasis in 11 potentially endemic countries using a rapid assessment procedure for loiasis (RAPLOA) that uses a simple questionnaire on the history of eye worm. A geostatistical analysis method called kriging applied to the results in 4798 sampled villages generated a contour map of eye worm prevalence, providing the first global map of loiasis based on actual survey data. This map showed high risk levels of loiasis in 10 countries where an estimated 14.4 million people live in high risk areas.

Forms of onchocerciasis-related stigmatisation, following over 10 years of implementation of community directed treatment with ivermectin (CDTI), were documented through a survey conducted between September and November 2009 to look at perceived changes in stigma over the past 7–10 years. We documented people's perceptions towards stigma before and after the introduction of CDTI from 1600 structured interviews with households selected from the community treatment registers; in-depth interviews with 57 community leaders, community directed distributors and health personnel; 33 focus group discussions with male and female community members in different age groups (18–24, 25–54 and ≥55 years) and 13 case studies. People with onchocercal skin disease (OSD) with rough skin, swellings and rashes were the most stigmatised. People still fear sexual intimacy with infected persons. In the past, people with OSD were considered unclean and stigmatised because of fear of OSD transmission and embarrassment. People who had lived in the community less than 5 years tended to stigmatise OSD patients more than those people who had lived in the community for longer than 5 years. The youth stigmatised the most. Although stigmatisation persists, avoidance of people with OSD decreased from 32.7% to 4.3%. It is notable that treatment availability has improved relationships between healthy people and those with OSD symptoms in endemic communities and this can be attributed to CDTI. Health education should be emphasised in the communities during distribution.

Description de quatre nouvelles especes parasites de lezards de diverses familles: Thamugadia wertheimae n. sp., parasite d'Acanthodactylus b. boskianus en Israel, Madathamugadia versterae n. sp., parasite de Mabuya capensis au Nord Transvaal, M. bissani n. sp., parasite de Mabuya quinquetaeniata au Mali, M. ineichi n. sp., parasite de Pseudocordylus microlepidotus melanotus dans l'Etat Libre d'Orange. Le cycle de cette derniere espece est realise; il s'effectue chez un phlebotome, comme les deux autres cycles connus dans le genre. La nouvelle combinaison Madathamugadia huambensis (Petit, Bain, Gomes & Touratier, 1983) est proposee pour la filaire de Mabuya aff. quinquetaeniata en Angola, qui etait placee dans le genre Piratuboides, Oswaldofilariinae. Redescription de Pseudothamugadia physignathi, parasite de Physignathus lesueurii en Australie; en Nouvelle Guinee, cette filaire (ou une espece proche?) est parasite de Chlamydosaurus kingii. La presence de Splendidofilariinae chez les lezards en Afrique du Sud et de l'Ouest est un element nouveau. Ces especes ethiopiennes presentent divers caracteres originaux mais elles ont des affinites avec les deux Madathamugadia malgaches. M. ineichi apparait comme une forme primitive (capsule buccale presente, papilles pre et postcloacales developpees); deux lignees en deriveraient, la premiere caracterisee par la regression des papilles postcloacales: ce sont les sept autres especes de Madathamugadia, en regions ethiopienne et mediterraneenne, la seconde par la disparition des papilles precloacales: ce sont les quatre especes de Thamugadia, en region mediterraneenne. Pseudothamugadia apparait proche de Madathamugadia.

Background The risk of severe adverse events following treatment of onchocerciasis with ivermectin in areas co-endemic with loiasis currently compromises the development of control programmes and the treatment of co-infected individuals. We therefore assessed whether doxycycline treatment could be used without subsequent ivermectin administration to effectively deliver sustained effects on Onchocerca volvulus microfilaridermia and adult viability. Furthermore we assessed the safety of doxycycline treatment prior to ivermectin administration in a subset of onchocerciasis individuals co-infected with low to moderate intensities of Loa loa microfilaraemia. Methods A double-blind, randomized, field trial was conducted of 6 weeks of doxycycline (200 mg/day) alone, doxycycline in combination with ivermectin (150 µg/kg) at +4 months or placebo matching doxycycline + ivermectin at +4 months in 150 individuals infected with Onchocerca volvulus. A further 22 individuals infected with O. volvulus and low to moderate intensities of Loa loa infection were administered with a course of 6 weeks doxycycline with ivermectin at +4 months. Treatment efficacy was determined at 4, 12 and 21 months after the start of doxycycline treatment together with the frequency and severity of adverse events. Results One hundred and four (60.5%) participants completed all treatment allocations and follow up assessments over the 21-month trial period. At 12 months, doxycycline/ivermectin treated individuals had lower levels of microfilaridermia and higher frequency of amicrofilaridermia compared with ivermectin or doxycycline only groups. At 21 months, microfilaridermia in doxycycline/ivermectin and doxycycline only groups was significantly reduced compared to the ivermectin only group. 89% of the doxycycline/ivermectin group and 67% of the doxycycline only group were amicrofilaridermic, compared with 21% in the ivermectin only group. O. volvulus from doxycycline groups were depleted of Wolbachia and all embryonic stages in utero. Notably, the viability of female adult worms was significantly reduced in doxycycline treated groups and the macrofilaricidal and sterilising activity was unaffected by the addition of ivermectin. Treatment with doxycycline was well tolerated and the incidence of adverse event to doxycycline or ivermectin did not significantly deviate between treatment groups. Conclusions A six-week course of doxycycline delivers macrofilaricidal and sterilizing activities, which is not dependent upon co-administration of ivermectin. Doxycycline is well tolerated in patients co-infected with moderate intensities of L. loa microfilariae. Therefore, further trials are warranted to assess the safety and efficacy of doxycycline-based interventions to treat onchocerciasis in individuals at risk of serious adverse reactions to standard treatments due to the co-occurrence of high intensities of L. loa parasitaemias. The development of an anti-wolbachial treatment regime compatible with MDA control programmes could offer an alternative to the control of onchocerciasis in areas of co-endemicity with loiasis and at risk of severe adverse reactions to ivermectin. Trial Registration Controlled-Trials.com ISRCTN48118452, Author Summary The control of onchocerciasis in Africa relies on the sustained delivery of ivermectin. In certain areas, annual treatments delivered with high population coverage for at least 15–17 years can break transmission. In other endemic settings this strategy alone is thought to be insufficient to eradicate the disease. One of the major limitations occurs in areas that are co-endemic with another filarial infection caused by Loa loa, due to the risk of a rare severe adverse event associated with the rapid killing of L. loa microfilariae in heavily parasitized individuals. There are also concerns over recent evidence of reduced efficacy of ivermectin and the possible development of resistance. An alternative approach is to target the Wolbachia bacterial endosymbionts of Onchocerca volvulus with the antibiotic, doxycycline. In an area of Cameroon co-endemic for onchocerciasis and loiasis we conducted a trial comparing doxycycline with or without ivermectin treatment to ivermectin treatment alone. A six-week course of doxycycline delivers macrofilaricidal and sterilizing activities, which is not dependent upon co-administration of ivermectin. Doxycycline is well tolerated in patients co-infected with moderate intensities of L. loa microfilariae. The trial indicates that anti-wolbachial therapy is a feasible alternative to ivermectin in communities co-endemic for onchocerciasis and loiasis.

Onchocerciasis can be effectively controlled by annual mass treatment with ivermectin in endemic communities. However, in communities that are endemic for loiasis there may be significant risk of severe adverse reactions after ivermectin treatment. Planning of control requires therefore mapping of these two infections using rapid assessment tools developed for each disease. These tools were initially implemented independently till the feasibility of combining them was demonstrated. This paper reports the results of integrated mapping in four epidemiological zones in the Democratic Republic of Congo and its implications on operational decision-making on ivermectin treatment.Rapid assessment surveys were conducted between 2004 and 2005 using both rapid epidemiological mapping of onchocerciasis (REMO) and rapid assessment procedure for loiasis (RAPLOA). The survey results were subjected to a spatial analysis in order to generate for each of the two diseases maps of the estimated prevalence of infection throughout the four zones.Surveys were undertaken in 788 villages where 25,754 males were examined for palpable onchocercal nodules and 62,407 people were interviewed for history of eye worm. The results showed major differences in the geographic distribution of the two diseases. Loiasis was highly endemic in some areas, where special precautions were required, but not in others where routine ivermectin treatment could proceed.Integrated rapid mapping of onchocerciasis and loiasis reduces both time and cost of surveys and greatly facilitates operational decision-making on ivermectin treatment in areas where loiasis might be co-endemic.

Onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is a parasitic disease leading to debilitating skin disease and blindness, with major economic and social consequences. The pathology of onchocerciasis is principally considered to be a consequence of long-standing host inflammatory responses. In onchocerciasis a subcutaneous nodule is formed around the female worms, the core of which is a dense infiltrate of inflammatory cells in which microfilariae are released. It has been established that the formation of nodules is associated with angiogenesis. In this study, we show using specific markers of endothelium (CD31) and lymphatic endothelial cells (Lyve-1, Podoplanin) that not only angiogenesis but also lymphangiogenesis occurs within the nodule. 7% of the microfilariae could be found within the lymphatics, but none within blood vessels in these nodules, suggesting a possible route of migration for the larvae. The neovascularisation was associated with a particular pattern of angio/lymphangiogenic factors in nodules of onchocerciasis patients, characterized by the expression of CXCL12, CXCR4, VEGF-C, Angiopoietin-1 and Angiopoietin-2. Interestingly, a proportion of macrophages were found to be positive for Lyve-1 and some were integrated into the endothelium of the lymphatic vessels, revealing their plasticity in the nodular micro-environment. These results indicate that lymphatic as well as blood vascularization is induced around O. volvulus worms, either by the parasite itself, e. g. by the release of angiogenic and lymphangiogenic factors, or by consecutive host immune responses.

Les nematodes pulmonaires parasites de cameleons du genre Rhabdias n'etaient connus qu'en Afrique orientale et a Madagascar. Deux especes sont decrites au Cameroun: il Rhabdias okuensis n. sp., hote-type Chamaeleo (Trioceros) quadricornis gracilior au Mont Oku est frequent; l'espece est proche de R. jarki, au Burundi, par la capsule buccale courte et l'oesophage long et mince, et en est distincte par la grande vesicule cervicale et des caracteres cephaliques (bouche, papilles). Les femelles parasites sont hermaphrodites (spermatozoides identifies); en outre, elles perforent la paroi pulmonaire et provoquent des lesions, comme R. jarki. C. (T.) w. wiedersheimi au Mont Oku heberge aussi R. okuensis, comme cela est demontre par les sequences des genes 12S rADN et coxl. ii) R. cristati n. sp., hote-type C. (T.) cristatus ou Mont Cameroun est decrit chez un specimen fortement parasite; l'espece est proche de R. chamaeleonis d'Afrique Orientale, et en est distinct par la grande taille de la capsule buccale et l'apex epais de l'intestin. La phase libre du cycle, etudiee chez R. okuensis, presente les caracteres des autres Rhobdias de cameleons: heterogonie, developpement des larves par endotokie matricide, stade infectant avec une extremite caudale epaisse et arrondie, exuvie transformee en gaine cuticulaire epaisse; chaque femelle libre produit une larve, comme chez les autres Rhabdias africains, alors que la femelle de R. gemellipara, parasite de cameleon malgache, en produit deux.

Since 1987 onchocerciasis control has relied on the donation of ivermectin (Mectizan®, Merck & Co., Inc.) through the Mectizan Donation Programme. Recently, concern has been raised over the appearance of suboptimal responses to ivermectin in Ghana – highlighting the potential threat of the development of resistance to ivermectin. This report summarises a meeting held in Ghana to set the research agenda for future onchocerciasis control. The aim of this workshop was to define the research priorities for alternative drug and treatment regimes and control strategies to treat populations with existing evidence of suboptimal responsiveness and define research priorities for future control strategies in the event of the development of widespread ivermectin resistance.

International audience; Abstract: The main effects of ivermectin (IVM) on Onchocerca volvulus are a microfilaricidal effect, leading to a rapid decrease in the microfilarial loads, and a temporary blockage of the release of microfilariae (mf) by the adult worms. The mf loads re-increase slowly from 3 months after treatment. While reports from Ghana suggest that this re-increase is more rapid in patients who have received many IVM doses, the long term impact of repeated IVM treatments on the dynamics of mf loads is still unclear. To evaluate this, mf loads have been monitored in 1994, 1997 and 2007 in a cohort of Cameroonian subjects. They all had received either 4 annual or 13 3-monthly IVM doses between 1994 and 1997 (during a closely monitored trial), and were proposed the drug during the subsequent years as part of annual community-directed treatments with IVM (CDTI). In 2007, information was collected from each individual about the date of their last dose. Mf loads measured in 1994 (before the 1st dose), 1997 (one year after the 4th dose), and 2007 (10 months after the last round of CDTI) in a group of 32 patients treated annually between 1994 and 1997 and who had actually taken IVM in 2006, were 137.7, 15.2 and 15.4 mf/mg, respectively. Thirty-eight other individuals had been treated either annually or three-monthly between 1994 and 1997, and had not participated in the CDTIs organised in 2006; in these patients, the mf loads measured in 1994 and 2007 were 99.5 and 47.3 mf/mg, respectively. When extending the non compliance period to more than 3 years, the mean mf load in 2007 was 109.2 mf/mg (14 subjects). 105 skin-snip positive individuals from the 1994 cohort were retreated in 2007 after the parasitological examination, and then followed up at 15, 80 and 180 days after treatment. Mf loads recorded on D15 showed that the microfilaricidal effect of IVM was similar to that classically reported. However, the mf prevalence observed at D80 (41%) and D180 (77%) and the significant mf loads observed at D180 (13% of individuals present with >10mf/mg) reflect the persisting reproductive capacities of some adult worms. These observations suggest that despite 13 years of intervening treatments halting IVM treatment could result in a rapid return to the initial hyperendemic state. Embryograms and genotyping of parasites collected as part of this study are underway to assess whether the repopulation rates of skin by mf observed in some individuals reflects an emerging resistance of O. volvulus to IVM.

Background Rapid and correct diagnosis of malaria is considered an important strategy in the control of the disease. However, it remains to be determined how well these tests can perform in those who harbour the parasite, but are asymptomatic, so that rapid diagnostic tests (RDTs) could be used in rapid mass surveillance in malaria control programmes. Methods Microscopic and immunochromatographic diagnosis of malaria were performed on blood samples from the hyperendemic Mount Cameroon region. Thin and thick blood films were stained with Giemsa and examined under light microscopy for malaria parasites. The RDT was performed on the blood samples for the detection of Plasmodium species. In addition, the performance characteristics of the test were determined using microscopy as gold standard. Results Results revealed 40.32% to be positive for microscopy and 34.41% to be positive for the RDT. Parasites were detected in a greater proportion of samples as the parasite density increase. Plasmodium falciparum was the predominant Plasmodium species detected in the study population either by microscopy or by the RDT. Overall, the test recorded a sensitivity and specificity of 85.33% and 95.05% respectively, and an accuracy of 91.40%. The sensitivity and specificity of the RDT increased as parasite densities increased. Conclusion The Hexagon Malaria Combi™ test showed a high sensitivity and specificity in diagnosing malaria in asymptomatic subjects and so could be suitable for use in mass surveillance programmes for the management and control of malaria.

BACKGROUND: Individuals with high microfilarial loads of Loa loa are at increased risk of neurologic serious adverse (SAE) events following ivermectin treatment against onchocerciasis. RAPLOA (Rapid Assessment Procedure for loiasis), a newly developed rapid assessment procedure for loiasis that relates the prevalence of key clinical manifestation of loiasis (history of eye worm) to the level of endemicity of the infection (prevalence of high intensity), is a very useful tool to identify areas at potential risk of L. loa post ivermectin treatment encephalopathy. In a perspective of treatment decision making in areas of co-endemicity of loiasis/onchocerciasis, it would be advantageous (both in time and cost savings) for national onchocerciasis control programmes to use RAPLOA and the Rapid epidemiologic assessment for onchocerciasis (REA), in combination in given surveys. Since each of the two rapid assessment tools have their own specificities, the workability of combining the two methods needed to be tested. METHODS: We worked in 10 communities of a forest area presumed co-endemic for loiasis and onchocerciasis in the North-West Province of Cameroon where the mass-treatment with ivermectin had not been carried out. A four-step approach was used and comprised: (i) generating data on the prevalence and intensity of loiasis and onchocerciasis in an area where such information is scarce; (ii) testing the relationship between the L. loa microfilaraemia prevalence and the RAPLOA prevalence, (iii) testing the relationship between the O. volvulus microfiladermia prevalence and the REA prevalence, (iv) testing the workability of combining RAPLOA/REA by study teams in which a single individual can perform the interview for RAPLOA and the nodule palpation for REA. RESULTS: The microfilaraemia prevalence of loiasis in communities ranged from 3.6% to 14.3%. 6 (0.61%) individuals had L. loa microfilarial loads above 8000 mf/ml but none of them attained 30,000 mf/ml, the threshold value above which the risk of developing neurologic SAE after ivermectin treatment is very high. None of the communities surveyed had RAPLOA prevalence above 40%. All the communities had microfiladermia prevalence above 60%. The microfiladermia results could be confirmed by the rapid epidemiologic method (nodule palpation), with all the 10 communities having REA prevalence above 20%. For the first time, this study has demonstrated that the two rapid assessment procedures for loiasis and onchocerciasis can be carried out simultaneously by a survey team, in which a single individual can administer the questionnaire for RAPLOA and perform the nodule palpation for REA. CONCLUSION: This study has: (i) Revealed that the Momo valley of the North West province of Cameroon is hyperendemic for onchocerciasis, but is of lower level of endemicity for L. loa. (ii) Confirmed the previous relationships established between RAPLOA and the L. loa microfilaraemia prevalence in one hand and between the REA and the O. volvulus microfiladermia prevalence in another hand (iii) Shown that RAPLOA and REA could be used simultaneously for the evaluation of loiasis and onchocerciasis endemicity in areas targeted by the African Programme for onchocerciasis Control for community-directed treatment with ivermectin (CDTI).

The prevalence and intensity of loiasis were studied in 4532 individuals selected from 42 villages from 5 contrasting bioecological zones in Cameroon from February to July 2001. The individuals were examined for Loa loo microfilaraemia using the thick blood film method. Loiasis was heterogeneously distributed from one bioecological zone to another. The occurrence of the infection was largely related to environmental factors favouring the breeding of Chrysops vectors. The mean prevalence of infection varied from 0% in the grassland savannah, 6.6% in the deciduous equatorial rainforest, 9.7% in the dense-humid equatorial rainforest, to 33.3% in the forested savannah. Loiasis was totally absent in the grassland savannah. The intensity of loiasis infection (L. loa microfilariae per millilitre [mf/mL] of blood) increased with an increase in prevalence, varying from 0 mf/mL in the grassland savannah, 325 mf/mL in the deciduous equatorial rainforest, 433 mf/mL in the dense-humid equatorial rainforest, to 3125 mf/mL in the forested savannah. The proportion of individuals with high (> 8000 mf/mL) and very high (> 30 000 mf/mL) L. loa microfilaraemia was greater in bioecological zones with a higher prevalence of L. loa. These results will be valuable for the prediction of loiasis endemicity in different ecological zones of Central Africa and for the refinement of the existing geographical model for the mapping of loiasis.

Summary There is a lack of data on the Anopheles fauna, its biology and the roles played by different vector species in the transmission of malaria in the mount Cameroon region. The biting habits, feeding behaviour and entomological inoculation rates of different Anopheles species during the dry and rainy season were investigated. A total of 2165 Anopheles was collected, 805 in the rainy season and 1360 in the dry season. Five Anopheles species were identified: Anopheles gambiae s.l., An. funestus, An. hancocki, An. moucheti and An. nili. An. gambiae, An. funestus and An. hancocki, recorded during both seasons, were the main vectors of malaria in the region. An. gambiae s.s. was the only member of the An. gambiae (Giles) complex. These three species had their peak activity between 1 and 2 am. A human blood index (HBI) of 98.29% was recorded for fed Anopheles. The sporozoite rate, for all vectors together, was significantly higher in the rainy season (9.4%) than in the dry season (4.2%) with all the species infected by Plasmodium falciparum. The average inoculation rate was 0.44 infective bites per man per night, which adds up to 161 infective bites per year in this study area. Analyses of relative abundance and infection rate of malaria vectors at different sites situated along a transect of 20 km during the dry season showed high heterogeneity in biting and sporozoite rates. No malaria vector was caught at 1200 m a.s.l. The mount Cameroon region should be considered an area of high malaria transmission intensity.

BACKGROUND: Many filarial nematodes harbour Wolbachia endobacteria. These endobacteria are transmitted vertically from one generation to the next. In several filarial species that have been studied to date they are obligatory symbionts of their hosts. Elimination of the endobacteria by antibiotics interrupts the embryogenesis and hence the production of microfilariae. The medical implication of this being that the use of doxycycline for the treatment of human onchocerciasis and bancroftian filariasis leads to elimination of the Wolbachia and hence sterilisation of the female worms. Wolbachia play a role in the immunopathology of patients and may contribute to side effects seen after antifilarial chemotherapy. In several studies Wolbachia were not observed in Loa loa. Since these results have been doubted, and because of the medical significance, several independent methods were applied to search for Wolbachia in L. loa. METHODS: Loa loa and Onchocerca volvulus were studied by electron microscopy, histology with silver staining, and immunohistology using antibodies against WSP, Wolbachia aspartate aminotransferase, and heat shock protein 60. The results achieved with L. loa and O. volvulus were compared. Searching for Wolbachia, genes were amplified by PCR coding for the bacterial 16S rDNA, the FTSZ cell division protein, and WSP. RESULTS: No Wolbachia endobacteria were discovered by immunohistology in 13 male and 14 female L. loa worms and in numerous L. loa microfilariae. In contrast, endobacteria were found in large numbers in O. volvulus and 14 other filaria species. No intracellular bacteria were seen in electron micrographs of oocytes and young morulae of L. loa in contrast to O. volvulus. In agreement with these results, Wolbachia DNA was not detected by PCR in three male and six female L. loa worms and in two microfilariae samples of L. loa. CONCLUSIONS: Loa loa do not harbour obligatory symbiotic Wolbachia endobacteria in essential numbers to enable their efficient vertical transmission or to play a role in production of microfilariae. Exclusively, the filariae cause the immunopathology of loiasis is patients and the adverse side effects after antifilarial chemotherapy. Doxycycline cannot be used to cure loiais but it probably does not represent a risk for L. loa patients when administered to patients with co-infections of onchocerciasis.

BACKGROUND: The majority of filarial nematode species are host to Wolbachia bacterial endosymbionts, although a few including Acanthocheilonema viteae, Onchocerca flexuosa and Setaria equina have been shown to be free of infection. Comparisons of species with and without symbionts can provide important information on the role of Wolbachia symbiosis in the biology of the nematode hosts and the contribution of the bacteria to the development of disease. Previous studies by electron microscopy and PCR have failed to detect intracellular bacterial infection in Loa loa. Here we use molecular and immunohistological techniques to confirm this finding. METHODS: We have used a combination of PCR amplification of bacterial genes (16S ribosomal DNA [rDNA], ftsZ and Wolbachia surface protein [WSP]) on samples of L. loa adults, third-stage larvae (L3) and microfilariae (mf) and immunohistology on L. loa adults and mf derived from human volunteers to determine the presence or absence of Wolbachia endosymbionts. Samples used in the PCR analysis included 5 adult female worms, 4 adult male worms, 5 mf samples and 2 samples of L3. The quality and purity of nematode DNA was tested by PCR amplification of nematode 5S rDNA and with diagnostic primers from the target species and used to confirm the absence of contamination from Onchocerca sp., Mansonella perstans, M. streptocerca and Wuchereria bancrofti. Immunohistology was carried out by light and electron microscopy on L. loa adults and mf and sections were probed with rabbit antibodies raised to recombinant Brugia malayi Wolbachia WSP. Samples from nematodes known to be infected with Wolbachia (O. volvulus, O. ochengi, Litomosoides sigmodontis and B. malayi) were used as positive controls and A. viteae as a negative control. RESULTS: Single PCR analysis using primer sets for the bacterial genes 16S rDNA, ftsZ, and WSP were negative for all DNA samples from L. loa. Positive PCR reactions were obtained from DNA samples derived from species known to be infected with Wolbachia, which confirmed the suitability of the primers and PCR conditions. The quality and purity of nematode DNA samples was verified by PCR amplification of 5S rDNA and with nematode diagnostic primers. Additional analysis by 'long PCR' failed to produce any further evidence for Wolbachia symbiosis. Immunohistology of L. loa adults and mf confirmed the results of the PCR with no evidence for Wolbachia symbiosis. CONCLUSION: DNA analysis and immunohistology provided no evidence for Wolbachia symbiosis in L. loa.

Encephalopathy has been reported in Cameroon in individuals heavily infected with Loa loa microfilariae who were treated with ivermectin against onchocerciasis. Prior to the initiation of the community-directed treatment of onchocerciasis with ivermectin (CDTI) in the South West Province of Cameroon, an epidemiological survey of loiasis was conducted (in July 1998-July 1999) to ascertain the intensity of the disease. Thick blood films were made from 1228 blood samples collected during the day. Rapid epidemiological assessment (REA) of onchocerciasis was conducted among 614 individuals in the Upper Bayang area. Sixteen percent of the population examined were carriers of L. loa microfilariae. More males (20.1%) than females (12.4%) were infected. The community mean microfilaraemias of the different villages were low (1100 mf/mL). However, 1 person was found harbouring 174,000 mf/mL of blood and 1% of the study population (12) had microfilaraemia8100 mf/mL. Results of the REA of onchocerciasis show that 31.3% of the population investigated in the Upper Bayang area have this disease. These findings show that loiasis and onchocerciasis are co-endemic in the area, but the risk of developing encephalopathy after taking ivermectin is small.

In the past 5 years, important progress in podoconiosis research and control has been made. The global distribution of this ascending, geochemical lymphoedema has been updated, advances have been made in disease assessment and treatment, and advocacy has successfully brought podoconiosis to the global stage. We highlight some of this progress here, and announce the launch of a new international initiative.

The filaria Monanema martini with skin-dwelling microfilariae induces in its natural murid hosts lesions similar to those in human onchocerciasis. This was demonstrated by histo-pathological studies but it appeared useful to evaluate the model by a clinical investigation. An ophthalmological analysis was performed on the two species of hosts, inoculated by one, two, or multiple doses of larvae, and with infections of at least one year duration. A total of 140 eyes was examined (anterior and posterior segments). We established a system for enumerating the different types and severities of lesions. We prepared a file for each eye and attempted to quantify our observations. The significant lesions were different in the two host species. In Arvicanthis niloticus, in which motile microfilariae were seen in the anterior segment, punctate keratitis was predominant. In Lemniscomys striatus, the posterior segment showed complete chorioretinal atrophy, similar to the final stage of onchocercal chorioretinitis in humans. M. martini represents in its natural hosts two complementary models for the study of the pathogenesis and treatment of human onchocerciasis.

River blindness is a seriously debilitating disease caused by the filarial parasite Onchocerca volvulus, which infects millions in Africa as well as in South and Central America. Research has been hampered by a lack of good animal models, as the parasite can only develop fully in humans and some primates. This review highlights the development of two animal model systems that have allowed significant advances in recent years and hold promise for the future. Experimental findings with Litomosoides sigmodontis in mice and Onchocerca ochengi in cattle are placed in the context of how these models can advance our ability to control the human disease.

Background Individuals with high microfilarial loads of Loa loa are at increased risk of neurologic serious adverse (SAE) events following ivermectin treatment against onchocerciasis. RAPLOA (Rapid Assessment Procedure for loiasis), a newly developed rapid assessment procedure for loiasis that relates the prevalence of key clinical manifestation of loiasis (history of eye worm) to the level of endemicity of the infection (prevalence of high intensity), is a very useful tool to identify areas at potential risk of L. loa post ivermectin treatment encephalopathy. In a perspective of treatment decision making in areas of co-endemicity of loiasis/onchocerciasis, it would be advantageous (both in time and cost savings) for national onchocerciasis control programmes to use RAPLOA and the Rapid epidemiologic assessment for onchocerciasis (REA), in combination in given surveys. Since each of the two rapid assessment tools have their own specificities, the workability of combining the two methods needed to be tested.

Background Loa loa has recently emerged as a filarial worm of significant public health importance as a consequence of its impact on the African Programme for Onchocerciasis Control (APOC). Severe, sometimes fatal, encephalopathic reactions to ivermectin (the drug of choice for onchocerciasis control) have occurred in some individuals with high Loa loa microfilarial counts. Since high density of Loa loa microfilariae is known to be associated with high prevalence rates, a distribution map of the latter may determine areas where severe reactions might occur. The aim of the study was to identify variables which were significantly associated with the presence of a Loa microfilaraemia in the subjects examined, and to develop a spatial model predicting the prevalence of the Loa microfilaraemia.

Background The majority of filarial nematode species are host to Wolbachia bacterial endosymbionts, although a few including Acanthocheilonema viteae, Onchocerca flexuosa and Setaria equina have been shown to be free of infection. Comparisons of species with and without symbionts can provide important information on the role of Wolbachia symbiosis in the biology of the nematode hosts and the contribution of the bacteria to the development of disease. Previous studies by electron microscopy and PCR have failed to detect intracellular bacterial infection in Loa loa. Here we use molecular and immunohistological techniques to confirm this finding.

Background Community-Directed Treatment with Ivermectin (CDTI) is the main strategy adopted by the African Programme for Onchocerciasis control (APOC). Recent reports from onchocerciasis endemic areas of savannah zones have demonstrated the feasibility of disease elimination through CDTI. Such information is lacking in rain forest zones. In this study, we investigated the parasitological and entomological indices of onchocerciasis transmission in three drainage basins in the rain forest area of Cameroon [after over a decade of CDTI]. River basins differed in terms of river number and their flow rates; and were characterized by high pre-control prevalence rates (60-98%). Methods Nodule palpation and skin snipping were carried out in the study communities to determine the nodule rates, microfilarial prevalences and intensity. Simulium flies were caught at capture points and dissected to determine the biting, parous, infection and infective rates and the transmission potential. Results The highest mean microfilaria (mf) prevalence was recorded in the Meme (52.7%), followed by Mungo (41.0%) and Manyu drainage basin (33.0%). The same trend was seen with nodule prevalence between the drainage basins. Twenty-three (23/39) communities (among which 13 in the Meme) still had mf prevalence above 40%. All the communities surveyed had community microfilarial loads (CMFL) below 10 mf/skin snip (ss). The infection was more intense in the Mungo and Meme. The intensity of infection was still high in younger individuals and children less than 10 years of age. Transmission potentials as high as 1211.7 infective larvae/person/month were found in some of the study communities. Entomological indices followed the same trend as the parasitological indices in the three river basins with the Meme having the highest values. Conclusion When compared with pre-control data, results of the present study show that after over a decade of CDTI, the burden of onchocerciasis has reduced. However, transmission is still going on in this study site where loiasis and onchocerciasis are co-endemic and where ecological factors strongly favour the onchocerciasis transmission. The possible reasons for this persistent and differential transmission despite over a decade of control efforts using ivermectin are discussed. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-0817-2) contains supplementary material, which is available to authorized users.

Background Immunological mechanisms involved in the survival and development of human filarial species in the vertebrate host are poorly known due to the lack of suitable experimental models. In order to understand the role of cytokines in the survival and development of filarial larvae in the vertebrate host, we infected different strains of BALB/c mice deficient in a number of cytokine or cytokine receptor genes with Loa loa. The survival and development of larvae were monitored. Methods BALB/c mice genetically deficient in IL-4R, IFN-γ, IFN-γ/IL-5, IL-5, and IL-4R/IL-5 cytokine or cytokine receptor genes were infected with a human strain of L. loa and necropsies were performed at different time intervals up to 70 days post infection to monitor the survival and development of L. loa larvae. The larvae were teased out of the skin, muscles, peritoneal and pleural cavities, heart and lung tissues. The length and width of the recovered larvae were measured to assess their growth. Results In mice deficient for IL-4R, IFN-γ, IFN-γ/IL-5, IL-5 and IL-4R/IL-5, the larvae survived up to 5, 20, 40, 50 and 70 days respectively. Worms recovered 70 days post infection in IL-4R/IL-5 DKO mice were young adults and measured 10.12 mm in length and 0.1 mm in width. Overall, 47% of larvae were recovered from subcutaneous tissues, 40% from muscles, 6% from the peritoneal cavity and 4% from the pleural cavity, lungs and heart. Conclusion L. loa exhibits a differential survival and development in different strains of cytokine or cytokine receptor gene knockout mice with IL-4R and IL-5 playing critical roles in the host resistance to L. loa infection. The knock out BALB/c mouse therefore represents a useful tool to explore the key effectors of adaptive immunity involved in the killing of the L. loa parasite in a mammal host.

Background As the African Programme for Onchocerciasis Control (APOC) matured into its 10th year of ensuring community involvement in mass annual treatment of onchocerciasis with ivermectin, there was recognition of a need to study not only annual coverage of ivermectin in villages but also the compliance of individual villagers with these annual treatments. This was based on the concern that while population coverage goals may be achieved each year, there might be segments of the population who systematically are not complying with the annual regimen, thus creating a reservoir of infection and threatening program gains. Methods A multi-site study in five APOC sponsored projects in Nigeria and Cameroon was undertaken to identify the socio-demographic correlates of compliance with ivermectin treatment. A total of 8,480 villagers above 9 years of age selected through a systematic random sampling from 101 communities were surveyed to ascertain their levels of compliance, by adapting APOC's standard household ivermectin survey form. Community leaders, community directed distributors (CDDs) of ivermectin and health workers were interviewed with in-depth interview guides, while focus group discussions were held with community members to help explain how socio-demographic factors might affect compliance. Results Eight-year compliance ranged from 0 to 8 times with 42.9% taking ivermectin between 6-8 times annually (high compliance). In bivariate analysis high compliance was positively associated with being male, over 24 years of age, having been married, not being Christian, having little or no formal education and being in the ethnic majority. These variables were also confirmed through regression analysis based on total times ivermectin was taken over the period. While these factors explained only 8% of the overall variation in compliance, ethnic status and education appeared to be the strongest factors. Those with higher education may be more mobile and harder to reach while neglect of ethnic minorities has also been documented in other programs. Conclusion These findings can help managers of CDTI programmes to ensure ivermectin reaches all segments of the population equally.

Background Control and elimination of filarial pathogens is a central focus of major global health efforts directed at parasitic diseases of developing countries. Accomplishment of these goals would be markedly enhanced by the enhanced destruction of the adult stage of filariae. The identification of new, more quantitative biomarkers that correlate with mortality or chemotherapeutic damage to adult filariae, would greatly facilitate, for example, the development of new macrofilaricides. Methods An immunocytochemical approach using an antibody against human Nras was used to identify and detect changes in the nematode homolog let-60 that is associated with cell growth and maintenance. Single Onchocerca volvulus nodules were removed from each of 13 patients treated with ivermectin (as part of a community-wide mass drug administration programme), and from each of 13 untreated individuals; these 26 nodules were stained with the anti-Nras antibody. The localization and degree of positivity of Nras/let-60 staining were assessed subjectively and compared between the two groups; the positivity of staining was also quantified, using image analysis, in a subgroup of these nodules. In addition, the specific morphological association between Nras/let-60 and the Wolbachia endosymbiont present in these parasites was also observed in 4 additional filarial species using an anti-Wolbachia surface protein (WSP) antibody under light and confocal microscopy. Results Nras/let-60 is present in many structures within the adult female worms. A statistically significant decrease in the general staining intensity of Nras/let-60 was observed in adult female O. volvulus treated with ivermectin when compared with parasites from untreated patients. Nras/let-60 staining was frequently observed to be co-localized with WSP in O.volvulus, Brugia malayi, Litomosoides sigmodontis and Dirofilaria immitis. Nras/let60 is also present in Onchocerca ochengi. Conclusion Nras/let-60, as detected by immunocytochemical staining, is decreased in ivermectin-treated adult female O. volvulus relative to untreated control specimens, suggesting a suppressive effect of ivermectin on the overall biochemical activity of these parasites. Co-localization of Nras/let-60 and WSP suggests the possibility that the endosymbiont utilizes this nematode protein as part of a mutualistic relationship. Nras/let60 appears to be a useful biomarker for assessing the health of filariae. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-0947-6) contains supplementary material, which is available to authorized users.

Background Severe side effects following ivermectin treatment of onchocerciasis in areas of co-endemicity with loaisis have been an impediment for the work of the African Programme for Onchocerciasis Control (APOC) in forested regions of several countries. Doxycycline has been shown to be effective in the treatment of onchocerciasis and has the added advantages of killing adult Onchocerca volvulus but neither adult Loa loa nor their microfilariae. This drug therefore offers great potential for the treatment of onchocerciasis in areas of co-endemicity with loiasis. The limitation of use of this drug is the duration of treatment that may pose a potential problem with therapeutic coverage and compliance with treatment. To benefit from the advantages that doxycycline offers in the treatment of onchocerciasis, it will be necessary to establish an effective distribution system that can access remote communities. This study assessed the feasibility of a large-scale distribution of doxycycline for the treatment of onchocerciasis in areas of co-endemicity with loiasis using a community-directed approach. Methods The study was carried out in 5 health areas co-endemic for Onchocerca volvulus and Loa loa which had no prior experience of the Community Directed Treatment with Ivermectin (CDTI). The community-directed delivery process was introduced using a cascade mechanism from the central health system that passed through the regional health delegation, health district and the health areas. Community health implementers (CHIs) were trained to deliver doxycycline to community members and, under the supervision of the health system, to monitor and document drug intake and side effects. Results The community members adhered massively to the process. Of the 21355 individuals counted, 17519 were eligible for treatment and 12936 were treated with doxycycline; giving a therapeutic coverage of eligible population of 73.8%. Of the 12936 who started the treatment, 97.5% complied by the end of six weeks. No serious side effect was registered during the six week treatment. Conclusion This study indicates that when empowered the community health implementers can successfully deliver doxycycline for six weeks for the treatment of onchocerciasis in areas of co-endemicity with loiasis. The therapeutic coverage and the compliance treatment rate achieved in this study coupled to the known efficacy of doxycycline on O. volvulus, are indicators that the strategy involving the mass administration of doxycycline can be used to control onchocerciasis in those areas of co-endemicity with loiasis where ivermectin may be contraindicated.

Background The human parasite Onchocerca volvulus harbours Wolbachia endosymbionts essential for worm embryogenesis, larval development and adult survival. In this study, the development of Wolbachia-depleted microfilariae (first stage larvae) to infective third stage larvae (L3) in the insect vector Simulium damnosum was analysed. Methods Infected volunteers in Cameroon were randomly and blindly allocated into doxycycline (200 mg/day for 6 weeks) or placebo treatment groups. After treatment, blackflies were allowed to take a blood meal on the volunteers, captured and dissected for larval counting and DNA extraction for quantitative real-time PCR analysis. Results PCR results showed a clear reduction in Wolbachia DNA after doxycycline treatment in microfilariae from human skin biopsies with > 50% reduction at one month post-treatment, eventually reaching a reduction of > 80%. Larval stages recovered from the insect vector had similar levels of reduction of endosymbiotic bacteria. Larval recoveries were analysed longitudinally after treatment to follow the kinetics of larval development. Beginning at three months post-treatment, significantly fewer L3 were seen in the blackflies that had fed on doxycycline treated volunteers. Concomitant with this, the proportion of second stage larvae (L2) was significantly increased in this group. Conclusions Doxycycline treatment and the resulting decline of Wolbachia endobacteria from the microfilaria resulted in retarded development of larvae in the insect vector. Thus, anti-wolbachial treatment could have an additive effect for interrupting transmission by reducing the number of L3 that can be transmitted by blackflies.