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Background: Culicoides, also known as biting midges, carry pathogens which include Mansonella perstans. Mansonella perstans is a nematode parasite implicated in a number of disease outcomes. Even though a high prevalence of about 75% M. perstans infection has been recorded in some communities in the middle belt of Ghana, and a wide diversity of Culicoides species has been identified, the exact Culicoides species transmitting M. perstans in Ghana has not yet been deciphered. This study therefore aimed at assessing the species diversity of Culicoides and their role in the transmission of M. perstans in the middle belt of Ghana., Methods: Culicoides species were sampled from 11 communities in the Asante-Akim North and Sene West districts in the middle belt of Ghana. Centre for Disease Control (CDC) UV light traps, as well as human bait (i.e. human landing catch and engorged catch) methods were used to assess the species abundance and diversity of Culicoides in the study communities in the wet and dry season. A colorimetric Loop-Mediated Isothermal Amplification (LAMP) assay was performed to assess the vector competence of the various Culicoides species., Results: A total of 4810 Culicoides from 6 species were sampled. These included Culicoides inornatipennis, C. milnei, C. schultzei, C. grahamii, C. neavei, and C. imicola. Culicoides imicola was the most abundant species (56%) followed by C. grahamii (16%). Light traps sampled the most diverse species (6 species). Human landing catch and engorged catch methods identified three anthropophilic species, C. grahamii, C. milnei, and C. inornatipennis, with C. grahamii being the most anthropophilic with a peak biting time between the hours of 5 p.m. to 6 p.m. Generally, there was relatively higher species abundance in the wet than dry season. LAMP assay identified C. grahamii as the potential vector for M. perstans transmission in the middle belt of Ghana., Conclusions: For the first time, we have demonstrated that C. grahamii is the potential competent vector for M. perstans transmission in the middle belt of Ghana. It is more abundant in the rainy season and has a peak biting time between the hours of 5 and 6 p.m., (© 2024. The Author(s).)

Background: Onchocerciasis control using ivermectin alone has been achieved in some endemic savannah zones of Africa. In the forest regions, the co-endemicity with Loa loa has led to severe adverse events (SAEs) resulting in poor adherence of community members to ivermectin mass drug administration (MDA). This may jeopardize achieving the interruption of transmission of onchocerciasis. Therefore, to accelerate the elimination of onchocerciasis in L. loa co-endemic zones, alternative treatment strategies (ATS) including ground larviciding may be necessary. This study aimed at identifying Simulium breeding sites, cytospecies, transmission profile, susceptibility of Simulium larvae to insecticide (temephos) and identification of some non-target aquatic fauna prior to the implementation of the COUNTDOWN consortium ground larviciding alternative strategy in the Meme River Basin in South West Cameroon., Methods: A topographic map and entomological survey were used to determine breeding sites. Larvae and adults were identified using standard identification keys. Susceptibility tests were carried out on collected larvae by exposing them to decreasing concentrations of temephos and assessing survival rates while the cytospecies were identified using cytotaxonomy. Various entomological indicators were assessed from dissected flies. Fishing was used as proxy to traps to assess some aquatic fauna at different sites., Results: Twenty-two breeding sites were prospected in the Meme River Basin with eight productive for larvae. A concentration of 0.5-0.1 mg/l temephos induced 100% larval mortality. As the concentration of temephos decreased from 0.05 to 0.0025 mg/l, mortality of larvae also decreased from 98.7 to 12%. Nine cytospecies were observed in the Meme River Basin; 13,633 flies were collected and 4033 dissected. A total of 1455 flies were parous (36.1%), 224 flies were infected (5.5%), and 64 were infective (1.6%). Aquatic fauna observed included Cyprinus spp., Clarias spp., crabs, tadpoles, beetles and larvae of damsel fly., Conclusions: Onchocerciasis is being actively transmitted within the Meme River Basin. Simulium larvae are susceptible to temephos, and nine cytospecies are present. Non-target fauna observed included fishes, frogs, crabs and insects. Besides treatment with ivermectin, vector control through ground larviciding may be a complementary strategy to accelerate onchocerciasis elimination in the study area., (© 2022. The Author(s).)

Background: The practice of agriculture in urban settings contributes to the rapid expansion of insecticide resistance in malaria vectors. However, there is still not enough information on pesticide usage in most urban settings. The present study aims to assess the evolution of Anopheles gambiae (s.l.) population susceptibility to insecticides and patterns of pesticide usage in agriculture in the city of Yaoundé, Cameroon., Methods: WHO susceptibility tests and synergist PBO bioassays were conducted on adult An. gambiae (s.l.) mosquitoes aged 3 to 5 days emerging from larvae collected from the field. Seven insecticides (deltamethrin, permethrin, DDT, bendiocarb, propoxur, fenitrothion and malathion) were evaluated. The presence of target site mutation conferring knockdown (kdr) resistance was investigated using TaqMan assay, and mosquito species were identified using SINE-PCR. Surveys on 81 retailers and 232 farmers were conducted to assess general knowledge and practices regarding agricultural pesticide usage., Results: High resistance intensity to pyrethroids was observed with a high frequency of the kdr allele 1014F and low frequency of the kdr 1014S allele. The level of susceptibility of An. gambiae (s.l.) to pyrethroids and carbamates was found to decrease with time (from > 34% in 2017 to < 23% in 2019 for deltamethrin and permethrin and from 97% in 2017 to < 86% in 2019 for bendiocarb). Both An. gambiae (s.s.) and An. coluzzii were recorded. Over 150 pesticides and fertilizers were sold by retailers for agricultural purposes in the city of Yaoundé. Most farmers do not respect safety practices. Poor practices including extensive and inappropriate application of pesticides as well as poor management of perished pesticides and empty pesticide containers were also documented., Conclusions: The study indicated rapid evolution of insecticide resistance and uncontrolled usage of pesticides by farmers in agriculture. There is an urgent need to address these gaps to improve the management of insecticide resistance., (© 2022. The Author(s).)

Background: The tropical disease onchocerciasis (river blindness), caused by Onchocerca volvulus filarial nematodes, is targeted for elimination by mass treatment with nematocidal and antimicrobial drugs. Diagnosis of O. volvulus infections is based on counts of skin-borne microfilariae, but additional diagnostic tools, e.g. worm- or host-derived small RNAs, proteins or metabolites, are required for high-throughput screening. N-acetyltyramine-O,β-glucuronide (NATOG) was suggested as a biomarker for onchocerciasis but its viability as diagnostic tool has been challenged., Methods: We performed a screening program of urine samples from individuals from Cameroon infected with O. volvulus, Loa loa, Mansonella perstans or a combination thereof. Urine metabolites were measured by liquid chromatography-mass spectrometry (LC-MS). Principle component analysis (PCA) revealed that onchocerciasis causes complex changes of the urine metabolome., Results: The mean NATOG content was elevated in urine of O. volvulus-infected compared with non-infected individuals, but NATOG levels showed considerable variation. However, 13.8% of all O. volvulus-infected individuals had high NATOG levels never reached by individuals without filarial infections or only infected with L. loa or M. perstans. Therefore, the identification of individuals with high NATOG levels might be used to screen for the elimination of onchocerciasis after mass drug application. Additional metabolites, including a compound identified as cinnamoylglycine, had high PC1/PC2 loadings in the data set. Mean levels of cinnamoylglycine were increased in O. volvulus-infected individuals, and 17.2% of all O. volvulus individuals had elevated cinnamoylglycine levels not reached by the controls., Conclusions: On an individual level, NATOG alone had poor discriminative power distinguishing infected from non-infected individuals. However, 13.8% of all O. volvulus-infected individuals had NATOG levels never reached by individuals without filarial infections or infected with only L. loa or M. perstans. Discrimination of O. volvulus infections from controls or individuals suffering from multiple infections was improved by the measurement of additional metabolites, e.g. cinnamoylglycine. Thus, measuring a combination of urine metabolites may provide a way to assess onchocerciasis on the population level. This provides the possibility to design a strategy for large-scale onchocerciasis epidemiological screening programs based on urine rather than invasive techniques., (© 2021. The Author(s).)

Background: The mass drug administration of ivermectin for onchocerciasis control has contributed to a significant drop in Loa loa microfilaria loads in humans that has, in turn, led to reduction of infection levels in Chrysops vectors. Accurate parasite detection is essential for assessing loiasis transmission as it provides a potential alternative or indirect strategy for addressing the problem of co-endemic loiasis and lymphatic filariasis through the Onchocerciasis Elimination Programme and it further reflects the true magnitude of the loiasis problem as excess human mortality has been reported to be associated with the disease. Although microscopy is the gold standard for detecting the infection, the sensitivity of this method is compromised when the intensity of infection is low. The loop-mediated isothermal amplification (LAMP) assay of parasite DNA is an alternative method for detecting infection which offers operational simplicity, rapidity and versatility of visual readout options. The aim of this study was to validate the Loa loa LAMP assay for the detection of infected Chrysops spp. under experimental and natural field conditions., Methods: Two sets of 18 flies were fed on volunteers with either a low (< 10 mf/ml) or high (> 30,000mf/ml) microfilarial load. The fed flies were maintained under laboratory conditions for 14 days and then analysed using LAMP for the detection of L. loa infection. In addition, a total of 9270 flies were collected from the north-west, east, and south-west regions (SW 1 and 2) of Cameroon using sweep nets and subjected to microscopy (7841 flies) and LAMP (1291 flies plus 138 nulliparous flies) analyses., Results: The LAMP assay successfully detected parasites in Chrysops fed on volunteers with both low and high microfilariaemic loads. Field validation and surveillance studies revealed LAMP-based infection rates ranging from 0.5 to 31.6%, with the lowest levels in SW 2 and the highest infection rates in SW 1. The LAMP assay detected significantly higher infection rates than microscopy in four of the five study sites., Conclusion: This study demonstrated the potential of LAMP as a simple surveillance tool. It was found to be more sensitive than microscopy for the detection of experimental and natural L. loa infections in Chrysops vectors.

Background: As new lymphatic filariasis infections are eliminated through mass chemotherapy, previously affected individuals are left with the sequellae, especially chronic progressive lymphoedema. Currently this is managed by careful attention to limb hygiene to prevent infection. Studies over the past 15 years have suggested that the incorporation of doxycycline treatment may arrest or even reverse progression of lymphoedema. Most of this work has been observational or based on small studies, and if this intervention is effective, studies need to be conducted on a larger scale and under diverse geographical and social conditions before it can be incorporated into treatment policy., Methods/design: The double-blind, placebo-controlled study was designed to investigate the impact of six weeks treatment with doxycycline added to standard limb hygiene on early stage filarial lymphoedema in five sites in Africa and the Indian subcontinent. One site in Cameroon is selected for studying lymphoedema in podoconiosis. Each site was individually powered with the potential to undertake a meta-analysis on completion. Evaluation methods followed those used in Ghana in 2012 with additions resulting from advances in technology. The details of the core protocol and how it was varied to take account of differing situations at each of the sites are provided. The study will enrol up to 1800 patients and will complete in mid-2021., Conclusions: This paper provides details of what challenges were faced during its development and discusses the issues and how they were resolved. In particular, the reasons for inclusion of new technology and the problems encountered with the supply of drugs for the studies are described in detail. By making these details available, it is hoped that the study protocol will help others interested in improving treatment for filarial lymphoedema in the design of future studies. Trial registration India: Clintrials.gov. NCT02929121 registered 10 Oct 2016: https://clinicaltrials.gov/ct2/show/NCT02929121 Mali: Clintrials.gov. NCT02927496 registered 7 Oct 2016: https://clinicaltrials.gov/ct2/show/NCT0292749 Sri Lanka: Clintrials.gov. NCT02929134 registered 10 Oct 2016: https://clinicaltrials.gov/ct2/show/NCT02929134 Ghana: ISRCTN. 14042737 registered 10 July 2017: https://doi.org/10.1186/ISRCTN14042737 Tanzania: ISRCTN. 65756724 registered 21 July 2017: https://doi.org/10.1186/ISRCTN65756724 Cameroon: ISRCTN. 1181662 registered 25 July 2017: https://doi.org/10.1186/ISRCTN11881662.

Background: Different immune mechanisms are capable of killing developmental stages of filarial nematodes and these mechanisms are also likely to vary between the primary and a challenge infection. However, the lack of a detailed analysis of cytokine, chemokine and immunoglobulin levels in human loiasis is still evident. Therefore, detailed analysis of immune responses induced by the different developmental stages of Loa loa in immune-competent BALB/c mice will aid in the characterization of distinct immune responses that are important for the immunity against loiasis., Methods: Different developmental stages of L. loa were obtained from human peripheral blood (microfilariae, MF), the transmitting vector, Chrysops (larval stage 3, L3) and infected immune-deficient BALB/cRAG2γc -/- mice (L4, L5, adult worms). Groups of wildtype BALB/c mice were then injected with the isolated stages and after 42 days post-infection (pi), systemic cytokine, chemokine and immunoglobulin levels were determined. These were then compared to L. loa-specific responses from in vitro re-stimulated splenocytes from individual mice. All parameters were determined using Luminex technology., Results: In a pilot study, BALB/c mice cleared the different life stages of L. loa within 42 days pi and systemic cytokine, chemokine and immunoglobulin levels were equal between infected and naive mice. Nevertheless, L. loa-specific re-stimulation of splenocytes from mice infected with L5, MF or adult worms led to induction of Th2, Th17 and chemokine secretion patterns., Conclusions: This study shows that although host immunity remains comparable to naive mice, clearance of L. loa life-cycle development stages can induce immune cell memory leading to cytokine, chemokine and immunoglobulins secretion patterns which might contribute to immunity and protection against reinfection.

Background: Onchocerciasis is a priority neglected tropical disease targeted for elimination by 2025. The standard strategy to combat onchocerciasis is annual Community-Directed Treatment with ivermectin (CDTi). Yet, high prevalence rates and transmission persist following > 12 rounds in South-West Cameroon. Challenges include programme coverage, adherence to, and acceptability of ivermectin in an area of Loa loa co-endemicity. Loiasis patients harbouring heavy infections are at risk of potentially fatal serious adverse events following CDTi. Alternative strategies are therefore needed to achieve onchocerciasis elimination where CDTi effectiveness is suboptimal., Methods/design: We designed an implementation study to evaluate integrating World Health Organisation-endorsed alternative strategies for the elimination of onchocerciasis, namely test-and-treat with the macrofilaricide, doxycycline (TTd), and ground larviciding for suppression of blackfly vectors with the organophosphate temephos. A community-based controlled before-after intervention study will be conducted among > 2000 participants in 20 intervention (Meme River Basin) and 10 control (Indian River Basin) communities. The primary outcome measure is O. volvulus prevalence at follow-up 18-months post-treatment. The study involves four inter-disciplinary components: parasitology, entomology, applied social sciences and health economics. Onchocerciasis skin infection will be diagnosed by skin biopsy and Loa loa infection will be diagnosed by parasitological examination of finger-prick blood samples. A simultaneous clinical skin disease assessment will be made. Eligible skin-snip-positive individuals will be offered directly-observed treatment for 5 weeks with 100 mg/day doxycycline. Transmission assessments of onchocerciasis in the communities will be collected post-human landing catch of the local biting blackfly vector prior to ground larviciding with temephos every week (0.3 l/m 3 ) until biting rate falls below 5/person/day. Qualitative research, including in-depth interviews and focus-group discussions will be used to assess acceptability and feasibility of the implemented alternative strategies among intervention recipients and providers. Health economics will assess the cost-effectiveness of the implemented interventions., Conclusions: Using a multidisciplinary approach, we aim to assess the effectiveness of TTd, alone or in combination with ground larviciding, following a single intervention round and scrutinise the acceptability and feasibility of implementing at scale in similar hotspots of onchocerciasis infection, to accelerate onchocerciasis elimination.

Background: The scale-up in the distribution of long-lasting insecticidal nets (LLINs) and indoor residual spraying has significantly reduced malaria burden and mortality. However, insecticide resistance, among other factors, is responsible for a recent rebound in malaria transmission in 2015-2016, threatening the progress so far made. As a contribution towards understanding patterns of resistance and its mechanism in the field we characterized a population of Anopheles gambiae (s.l.) from Gounougou, a Guinea savanna of north/central Cameroon., Results: Indoor collection conducted in September 2017 identified Anopheles coluzzii and Anopheles arabiensis as the unique Anopheles vector species, with abundances of 83 and 17%, respectively. Analysis of infection with TaqMan assays using heads/thoraces of indoor collected females of An. coluzzii revealed a low Plasmodium falciparum parasite rate of 4.7%. Bioassays conducted with female An. coluzzii revealed extreme resistance, with low mortalities of only 3.75 ± 1.25%, 3.03 ± 1.59% and 1.45 ± 1.45%, respectively, for permethrin, deltamethrin and DDT. In contrast, high susceptibility was obtained with the organophosphates and carbamates, with mortalities in the range of 98-100%. Synergist assays with piperonyl butoxide (PBO) recovered some susceptibility with increased mortality for permethrin to 14.88 ± 8.74%, and for deltamethrin to 32.50 ± 10.51% (~27-fold increase compared to mortalities with deltamethrin alone, χ 2  = 29, df = 1, P < 0.0001). These correlated with the results of cone bioassays which revealed complete loss of efficacy of Olyset®Net (0% mortality) and PermaNet®2.0 (0% mortality), and the considerable loss of efficacy of Olyset®Plus (mortality of 2 ± 2%), PermaNet®3.0 side panel (mortality of 2 ± 2%) and PermaNet3.0® roof (mortality of 16 ± 5.1%). Time-course bioassays conducted with deltamethrin established a high intensity of resistance, with LT 50 of 309.09 (95% CI 253.07-393.71, Fiducial), and a resistance ratio of 93.09 compared with the fully susceptible Ngoussou laboratory colony. TaqMan genotyping revealed a high frequency of the 1014F allele (65.25%) in the An. coluzzii populations. Sequencing of a fragment of the voltage-gated sodium channel identified a single An. arabiensis female harbouring the 1014S kdr mutation., Conclusions: This finding of high pyrethroid and DDT resistance in An. coluzzii from north-central Cameroon is a major obstacle to malaria control using pyrethroid bednets and indoor residual spraying with DDT.

Background: Culicoides (Diptera; Ceratoponidae) are tiny, stout, blood-sucking flies with a near worldwide distribution. When present, they are often considered a biting nuisance but in addition, they are involved in the transmission of pathogens to humans, domestic and wild animals. Data on Culicoides species in the South-West region of Cameroon dates back to the 1950s. Over the decades, ecological transformation due to agriculture and deforestation may have affected the population dynamics of Culicoides and therefore our study provides an update of their bio-ecology in the region. Furthermore, the role of various Culicoides species in the transmission of parasitic filariae of the genus Mansonella remains inconclusive in this region. This study was designed to address these unknown issues and expand on current scientific knowledge., Results: Eight species of Culicoides (C. bedfordi, C. inornatipennis, C. fulvithorax, C. grahamii, C. imicola, C. milnei, C. neavei and C. kumbaensis) were collected using light traps and human baits. Culicoides grahamii was the most abundant species, followed closely by C. milnei. Three species (C. milnei, C. grahamii and C. inornatipennis) were common in all observed larval development sites. Only four species (C. inornatipennis, C. fulvithorax, C. grahamii and C. milnei) were collected on humans. Anthropophilic species were more abundant (P < 0.001) in the evening (4-7 pm) when compared to the morning collections (6-9 am). After overnight fly collections using a drop trap with a human microfilaremic donor, C. milnei emerged as the potential host for transmitting Mansonella perstans. Substantial heterogeneity was observed between the trap visiting cycles of the various species (P < 0.001). The biting cycle of the main vector, C. milnei, showed two peaks (10-11 pm and 4-5 am), the highest being 10-11 pm., Conclusions: The Culicoides fauna of the South-West region of Cameroon has not changed significantly since the 1950s. Culicoides milnei was demonstrated to be the major vector of M. perstans in this part of Cameroon. It is essentially a nocturnal species which peaks in abundance between 10 and 11 pm.

Background: Loiasis, an often-neglected tropical disease, is a threat to the success of lymphatic filariasis and onchocerciasis elimination programmes in rainforest areas of the central and western Africa. Its control and even its elimination might be possible through the use of a safe macrofilaricide, a prophylactic drug, or perhaps a vaccine. This present study evaluated the effect of flubendazole (FLBZ) on the development of Loa loa L3 in vitro and in vivo., Methods: Infective stages of L. loa were isolated and co-cultured in Dulbecco's Modified Eagle's Medium in the presence of monkey kidney epithelial cells (LLC-MK2) feeder cells. FLBZ and its principal metabolites, reduced flubendazole (RFLBZ) and hydrolyzed flubendazole (HFLBZ), were screened in vitro at concentrations 0.05, 0.1, 0.5, 1 and 10 μg/ml. The viability of the parasites was assessed microscopically daily for 15 days. For in vivo study, a total of 48 CcR3 KO mice were infected subcutaneously with 200 L. loa L3 and treated with 10 mg/kg FLBZ once daily for 5 consecutive days. Twenty-four animals were used as control and received L3 and vehicle. They were dissected at 5, 10, 15 and 20 days post-treatment for worm recovery., Results: The motility of L3 larvae in vitro was reduced from the second day of incubation with drugs at in vivo plasma concentration levels, with a strong correlation found between reduced motility and increased drug concentration (Spearman's rho = -0.9, P < 0.0001). Except for HFLBZ (0.05 μg/ml and 0.01 μg/ml), all concentrations of FLBZ, HFLBZ and RFLBZ interrupted the moulting of L. loa infective larvae to L4. In vivo, regardless of the experimental group, there was a decrease in parasite recovery with time. However, at each time point this reduction was more pronounced in the group of animals treated with FLBZ compared to equivalent control. Parasites were recovered from the flubendazole-treated groups only on day 5 post-inoculation at an average rate of 2.1%, a value significantly lower (Mann-Whitney U-test, U = 28, P = 0.0156) than the average of 31.1% recovered from the control group., Conclusions: This study reveals the ability of flubendazole to inhibit the development of L. loa L3 both in vitro and in vivo, and in addition validates the importance of in vitro and animal models of L. loa as tools for the development of drugs against loiasis.

Background: The epidemiological profiles of vector-borne diseases, such as malaria, are strongly associated with landscape components. The reduction of malaria burden in endemic and epidemic regions mainly depends on knowledge of the malaria-transmitting mosquito species, populations and behavioural characteristics, as well as malaria exposure risks. This work aimed at carrying out a holistic study in order to characterise Anopheles species in relation to human malaria in seven wetlands along the lower section of the volcanic chain of Cameroon., Results: Eight malaria vectors: Anopheles arabiensis, An. coluzzii, An. funestus (s.s.), An. gambiae, An. hancocki, An. melas, An. nili and An. ziemanni, were found biting humans. Anopheles gambiae was widespread; however, it played a secondary role in the Ndop plain where An. ziemmani was the primary vector species (79.2%). Anophelines were more exophagic (73.6%) than endophagic (26.4%), showing a marked nocturnal activity (22:00-4:00 h) for An. coluzzii and An. gambiae while An. funestus (s.s.) was mostly caught between 1:00 and 6:00 h and An. ziemanni having an early evening biting behaviour (18:00-00:00 h). Female Anopheles were mostly observed to have relative high parity rates (≥ 70%), with the exception of the Meanja site where species parity varies from 46 to 55%. Overall, the transmission level was low with entomological inoculation rates estimated to 0.7 infected bites per person per month (ib/p/mth) in Tiko and Ndop, 1.4 ib/p/mth in Mamfe and 2.24 ib/p/mth in Santchou., Conclusions: The present study represents detailed Anopheles vector characterisation from an understudied area along the volcanic chain of Cameroon with endemic malaria transmission. The significant differences in bionomics and Anopheles species distribution within the studied wetlands, highlights the importance of providing baseline data and an opportunity to assess the outcome of ongoing malaria control interventions in the country.

Background: Suitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa., Methods: In vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco's modified Eagle's medium (DMEM) and Iscove's modified Dulbecco's medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T 90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval., Results: Of the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T 90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T 90 of 16.4-19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (β = 0.490), both IMDM (β = 0.256) and DMEM (β = 0.198) media and the protein supplements NCS (β = 0.052) and FBS (β = 0.022); while for L. loa L3, in addition to feeder cells (β = 0.259) and both IMDM (β = 0.401) and DMEM (β = 0.385) media, the protein supplements BSA (β = 0.029) were found important in maintaining the worm motility., Conclusions: The findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.

Background: Co-infection with loiasis remains a potential problem in control programs targeting filarial infections. The effects of many anti-parasitic drugs often administered to Loa loa infected people are not well documented. This study compared the in vitro activity of several of these drugs on the viability of L. loa microfilariae (mf)., Methods: Human strain L. loa mf were isolated from baboon blood using iso-osmotic Percoll gradient, and cultured in RPMI 1640/10% FBS with antimalarial drugs (mefloquine, amodiaquine, artesunate, chloroquine and quinine), anthelmintics (ivermectin, praziquantel, flubendazole and its reduced and hydrolyzed metabolites), two potential trypanocidal agents (fexinidazole and Scynexis-7158) and the anticancer drug imatinib. The drug concentrations used varied between 0.156 μg/ml and 10 μg/ml. Mf motility (CR 50 = 50% immotility) and a metabolic viability assay (MTT) were used to assess the effects of these drugs on the parasites., Results: Mf in control cultures showed only a slight reduction in motility after 5 days of culture. Active inhibition of Loa loa motility was seen with mefloquine and amodiaquine (CR 50 values of 3.87 and 4.05 μg/ml, respectively), immobilizing > 90% mf within the first 24 hours: mefloquine killed the mf after 24 hours of culture at concentrations ≥ 5 μg/ml. SCYX-7158 also induced a concentration-dependent reduction in mf motility, with > 50% reduction in mf motility seen after 5 days at 10 μg/ml. The anticancer drug imatinib reduced mf motility at 10 μg/ml from the first day of incubation to 55% by day 5, and the reduction in motility was concentration-dependent. Praziquantel and fexinidazole were inactive, and FLBZ and its metabolites, as well as ivermectin at concentrations > 5 μg/ml, had very minimal effects on mf motility over the first 4 days of culture., Conclusions: The considerable action of the anti-malarial drugs mefloquine and amodiaquine on Loa mf in vitro highlights the possibility of repurposing the existing anti-infectious agents for the development of drugs against loiasis. The heterogeneity in the activity of anti-parasitic agents on Loa loa mf supports the need for further investigation using animal models of loiasis.

Background: Onchocerciasis and lymphatic filariasis (LF) are major filarial infections targeted for elimination in most endemic sub-Saharan Africa (SSA) countries by 2020/2025. The current control strategies are built upon community-directed mass administration of ivermectin (CDTI) for onchocerciasis, and ivermectin plus albendazole for LF, with evidence pointing towards the potential for novel drug regimens. When distributing microfilaricides however, considerable care is needed to minimise the risk of severe adverse events (SAEs) in areas that are co-endemic for onchocerciasis or LF and loiasis. This work aims to combine previously published predictive risk maps for onchocerciasis, LF and loiasis to (i) explore the scale of spatial heterogeneity in co-distributions, (ii) delineate target populations for different treatment strategies, and (iii) quantify populations at risk of SAEs across the continent., Methods: Geographical co-endemicity of filarial infections prior to the implementation of large-scale mass treatment interventions was analysed by combining a contemporary LF endemicity map with predictive prevalence maps of onchocerciasis and loiasis. Potential treatment strategies were geographically delineated according to the level of co-endemicity and estimated transmission intensity., Results: In total, an estimated 251 million people live in areas of LF and/or onchocerciasis transmission in SSA, based on 2015 population estimates. Of these, 96 million live in areas co-endemic for both LF and onchocerciasis, providing opportunities for integrated control programmes, and 83 million live in LF-monoendemic areas potentially targetable for the novel ivermectin-diethylcarbamazine-albendazole (IDA) triple therapy. Only 4% of the at-risk population live in areas co-endemic with high loiasis transmission, representing up to 1.2 million individuals at high risk of experiencing SAEs if treated with ivermectin. In these areas, alternative treatment strategies should be explored, including biannual albendazole monotherapy for LF (1.4 million individuals) and 'test-and-treat' strategies (8.7 million individuals) for onchocerciasis., Conclusions: These maps are intended to initiate discussion around the potential for tailored treatment strategies, and highlight populations at risk of SAEs. Further work is required to test and refine strategies in programmatic settings, providing the empirical evidence needed to guide efforts towards the 2020/2025 goals and beyond.

Background: Approximately 114 million people are infected with Mansonella perstans in large proportions of Africa. In contrast to other filariae that infect humans, M. perstans-infected individuals show no distinct pathology or specific clinical picture, indicating a well-tuned adaptation to the host. In addition, since M. perstans adult worms reside in serous cavities which are difficult to access, research has been hindered and there is a paucity of knowledge about the biology of M. perstans, especially the development of the different life stages as well as M. perstans-driven immune responses. Thus in this study, an in vitro culture system was developed which allows an in-depth analysis of M. perstans., Results: Culicoides species were caught in Ediki (Kumba), Southwest Region within Cameroon following a blood meal on a microfilaremic donor that had 1500 microfilariae/ml of peripheral blood and kept in captivity for 12 days at 23 °C. In a pilot experiment, 15 infective larvae were obtained from the midges and co-cultured with a confluent monolayer of monkey kidney epithelial cells (LLC-MK2) in DMEM medium supplemented with 10% FBS for up to 77 days. The resulting survival rates of 33% revealed that the cell-conditioned medium was suitable for long-term maintenance of M. perstans worms. To confirm these preliminary observations, 249 infective larvae were cultured for 50 days and their development was monitored daily and microscopically graded for motility. In total, 170 (68.3%) filariae survived and 124 (49.8%) larvae moulted between days 21-30 to become L5 stage larvae which were motile and showed continuous vigorous movement., Conclusion: We have established an in vitro culture system for the generation and long-term maintenance of viable M. perstans worms. This technique will be an important tool to study parasite biology and development, the role in host immunity, and might be helpful to discover novel treatment strategies against this filariae.

Background: Mansonellosis was first reported in Ghana by Awadzi in the 1990s. Co-infections of Mansonella perstans have also been reported in a small cohort of patients with Buruli ulcer and their contacts. However, no study has assessed the exact prevalence of the disease in a larger study population. This study therefore aimed to find out the prevalence of M. perstans infection in some districts in Ghana and to determine the diversity of Culicoides that could be potential vectors for transmission., Methods: From each participant screened in the Asante Akim North (Ashanti Region), Sene West and Atebubu Amantin (Brong Ahafo Region) districts, a total of 70 μl of finger prick blood was collected for assessment of M. perstans microfilariae. Centre for Disease Control (CDC) light traps as well as the Human Landing Catch (HLC) method were used to assess the species diversity of Culicoides present in the study communities., Results: From 2,247 participants, an overall prevalence of 32% was recorded although up to 75% prevalence was demonstrated in some of the communities. Culicoides inornatipennis was the only species of Culicoides caught with the HLC method. By contrast, C. imicola (47%), C. neavei (25%) and C. schultzei (15%) were caught by the CDC light trap method. A wide diversity of other Culicoides spp. was also identified but correlation was only found between the prevalence of C. inornatipennis and M. perstans during the dry season., Conclusions: Here we demonstrate for the first time that M. perstans is highly prevalent in three districts in Ghana. We found a wide spectrum of Culicoides spp. Culicoides inornatipennis was the most anthropophilic and is therefore likely to be the species responsible for transmission of infection but formal proof has yet to be obtained., Trial Registration: NCT02281643 . Registered October 26, 2014. 'Retrospectively registered'., Trial Registry: ClinicalTrials.gov.

Background: Mansonellosis remains one of the most neglected of tropical diseases and its current distribution in the entire forest block of southern Cameroon is unknown. In order to address this issue, we have surveyed the distribution of Mansonella perstans in different bioecological zones and in addition, elucidated the influence of multiple rounds of ivermectin (IVM) based mass drug administration (MDA)., Methods: A mixed design was used. Between 2000 and 2014, both cross-sectional and longitudinal surveys were carried out in 137 communities selected from 12 health districts belonging to five main bioecological zones of the southern part of Cameroon. The zones comprised of grassland savanna (GS), mosaic forest savanna (MFS), forested savanna (FS), deciduous equatorial rainforest (DERF) and the dense humid equatorial rainforest (DHERF). The survey was carried out in some areas with no treatment history as well as those currently under IVM MDA. Individuals within the participatory communities were screened for the presence of M. perstans microfilariae (mf) in peripheral blood by the calibrated thick film method to determine both prevalence and geometric mean intensities at the community level., Results: Apart from sporadic cases in savanna areas, distribution of M. perstans was strongly linked to the equatorial rainforest zones. Before CDTI, the highest mean prevalence (70.0 %) and intensity (17,382.2 mf/ml) were obtained in communities in Mamfes' DHERF areas followed by communities in the DHERF zone of Lolodorf (53.8 % and 7,814.8 mf/ml, respectively). A longitudinal survey in Mamfe further showed that M. perstans infections had reduced by 34.5 % in DERF (P < 0.001) but not DHERF zones after ten years of IVM MDA. Further data from the cross-sectional study revealed that there was a decrease in prevalence in DHERF zones only after ten years of MDA. In DERF zones however, the infection was relatively lower after four years of MDA., Conclusions: The distribution of M. perstans in the southern part of Cameroon varies with bioecological zones and IVM MDA history. The zones with high prevalence and intensities lie in forested areas while those with low endemicity are in the savanna areas. MDA with ivermectin induced significant reduction in the endemicity of mansonellosis in the decidious equatorial rainforest. In contrast, the prevalence and intensity remained relatively high and stable in the dense humid equatorial rainforest zones even after a decade of mass drug administration with ivermectin. Since it is known that M. perstans down-regulates host's immune system, the findings from this work would be useful in designing studies to understand the impact of M. perstans on host immune response to vaccination and co-infection with other pathogens such as Mycobacterium spp. and Plasmodium spp. in areas of contrasting endemicities.

Background: The immunochromatographic test (ICT) for lymphatic filariasis is a serological test designed for unequivocal detection of circulating Wuchereria bancrofti antigen. It was validated and promoted by WHO as the primary diagnostic tool for mapping and impact monitoring for disease elimination following interventions. The initial tests for specificity and sensitivity were based on samples collected in areas free of loiasis and the results suggested a near 100% specificity for W. bancrofti. The possibility of cross-reactivity with non-Wuchereria bancrofti antigens was not investigated until recently, when false positive results were observed in three independent studies carried out in Central Africa. Associations were demonstrated between ICT positivity and Loa loa microfilaraemia, but it was not clearly established if these false positive results were due to L. loa or can be extended to other filarial nematodes. This study brought further evidences of the cross-reactivity of ICT card with L. loa and Onchocerca ochengi (related to O. volvulus parasite) using in vivo and in vitro systems., Methods: Two filarial/host experimental systems (L. loa-baboon and O. ochengi-cattle) and the in vitro maintenance of different stages (microfilariae, infective larvae and adult worm) of the two filariae were used in three experiments per filarial species. First, whole blood and sera samples were prepared from venous blood of patent baboons and cattle, and applied on ICT cards to detect circulating filarial antigens. Secondly, larval stages of L. loa and O. ochengi as well as O. ochengi adult males were maintained in vitro. Culture supernatants were collected and applied on ICT cards after 6, 12 and 24 h of in vitro maintenance. Finally, total worm extracts (TWE) were prepared using L. loa microfilariae (Mf) and O. ochengi microfilariae, infective larvae and adult male worms. TWE were also tested on ICT cards. For each experiment, control assays (whole blood and sera from uninfected babon/cattle, culture medium and extraction buffer) were performed., Results: Positive ICT results were obtained with whole blood and sera of L. loa microfilaremic baboons, culture supernatants of L. loa Mf and infective larvae as well as with L. loa Mf protein extracts. In contrast, negative ICT results were observed with whole blood and sera from the O. ochengi-cattle system. Surprisingly, culture supernatant of O. ochengi adult males and total worm extracts (Mf, infective larvae and adult worm) were positive to the test., Conclusions: This study has provided further evidence of L. loa cross-reactivity for the ICT card. All stages of L. loa seem capable of inducing the cross-reactivity. Onchocerca ochengi. can also induce cross-reactivity in vitro, but this is less likely in vivo due to the location of parasite. The availability of the parasite proteins in the blood stream determines the magnitude of the cross-reactivity. The cross-reactivity of the ICT card to these non-W. bancrofti filariae poses some doubts to the reliability and validity of the current map of LF of Central Africa that was generated using this diagnostic tool.

Background: Onchocerciasis control for years has been based on mass drug administration (MDA) with ivermectin (IVM). Adherence to IVM repeated treatment has recently been shown to be a confounding factor for onchocerciasis elimination precisely in rain forest areas where transmission continues and Loa loa co-exists with Onchocerca volvulus. In this study, participants' oral declarations were used as proxy to determine the relationship between adherence to IVM treatment and parasitological indicators of onchocerciasis in the rain forest area of Cameroon with more than a decade of MDA., Methods: Participants were recruited based on their IVM intake profile with the aid of a semi-structured questionnaire. Parasitological examinations (skin sniping and nodule palpation) were done on eligible candidates. Parasitological indicators were calculated and correlated to IVM intake profile., Results: Of 2,364 people examined, 15.5% had never taken IVM. The majority (40.4%) had taken the drug 1-3 times while only 18% had taken ≥ 7 times. Mf and nodule prevalence rates were still high at 47%, 95% CI [44.9-49.0%] and 36.4%, 95% CI [34.4-38.3%] respectively. There was a treatment-dependent reduction in microfilaria prevalence (rs =-0.986, P = 0.01) and intensity (rs =-0.96, P = 0.01). The highest mf prevalence (59.7%) was found in the zero treatment group and the lowest (33.9%) in the ≥ 7 times treatment group (OR = 2.8; 95% CI [2.09-3.74]; P < 0.001). Adults with ≥ 7 times IVM intake were 2.99 times more likely to have individuals with no microfilaria compared to the zero treatment group (OR = 2.99; 95% CI [2.19-4.08], P < 0.0001). There was no clear correlation between treatment and nodule prevalence and intensity., Conclusion: Adherence to ivermectin treatment is not adequate in this rain forest area where L. loa co-exists with O. volvulus. The prevalence and intensity of onchocerciasis remained high in individuals with zero IVM intake after more than a decade of MDA. Our findings show that using parasitological indicators, reduction in prevalence is IVM intake-dependent and that participants' oral declaration of treatment adherence could be relied upon for impact studies. The findings are discussed in the context of challenges for the elimination of onchocerciasis in this rain forest area.

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.

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.

Background: microRNAs (miRNAs), a class of short, non-coding RNA can be found in a highly stable, cell-free form in mammalian body fluids. Specific miRNAs are secreted by parasitic nematodes in exosomes and have been detected in the serum of murine and dog hosts infected with the filarial nematodes Litomosoides sigmodontis and Dirofilaria immitis, respectively. Here we identify extracellular, parasite-derived small RNAs associated with Onchocerca species infecting cattle and humans., Methods: Small RNA libraries were prepared from total RNA extracted from the nodule fluid of cattle infected with Onchocerca ochengi as well as serum and plasma from humans infected with Onchocerca volvulus in Cameroon and Ghana. Parasite-derived miRNAs were identified based on the criteria that sequences unambiguously map to hairpin structures in Onchocerca genomes, do not align to the human genome and are not present in European control serum., Results: A total of 62 mature miRNAs from 52 distinct pre-miRNA candidates were identified in nodule fluid from cattle infected with O. ochengi of which 59 are identical in the genome of the human parasite O. volvulus. Six of the extracellular miRNAs were also identified in sequencing analyses of serum and plasma from humans infected with O. volvulus. Based on sequencing analysis the abundance levels of the parasite miRNAs in serum or plasma range from 5 to 127 reads/per million total host miRNA reads identified, comparable to our previous analyses of Schistosoma mansoni and L. sigmodontis miRNAs in serum. All six of the O. volvulus miRNAs identified have orthologs in other filarial nematodes and four were identified in the serum of mice infected with L. sigmodontis., Conclusions: We have identified parasite-derived miRNAs associated with onchocerciasis in cattle and humans. Our results confirm the conserved nature of RNA secretion by diverse nematodes. Additional species-specific small RNAs from O. volvulus may be present in serum based on the novel miRNA sequences identified in the nodule fluid. In our analyses comparison to European control serum illuminates the scope for false-positives, warranting caution in criteria that should be applied to identification of biomarkers of infection.

Background: New drugs effective against adult filariae (macrofilaricides) would accelerate the elimination of lymphatic filariasis and onchocerciasis. Anti-Onchocerca drug development is hampered by the lack of a facile model. We postulated that SCID mice could be developed as a fmacrofilaricide screening model., Methods: The filaricides: albendazole (ABZ), diethylcarbamazine (DEC), flubendazole (FBZ), ivermectin (IVM) and the anti-Wolbachia macrofilaricide, minocycline (MIN) were tested in Brugia malayi (Bm)-parasitized BALB/c SCID mice vs vehicle control (VC). Responses were compared to BALB/c wild type (WT). Onchocerca ochengi male worms or onchocercomata were surgically implanted into BALB/c SCID, CB.17 SCID, BALB/c WT mice or Meriones gerbils. Survival was evaluated at 7-15 days. BALB/c SCID were tested to evaluate the responsiveness of pre-clinical macrofilaricides FBZ and rifapentine (RIFAP) against male Onchocerca., Results: WT and SCID responded with >95% efficacy following ABZ or DEC treatments against Bm larvae (P < 0.0001). IVM was partially filaricidal against Bm larvae in WT and SCID (WT; 39.8%, P = 0.0356 and SCID; 56.7%, P = 0.026). SCID responded similarly to WT following IVM treatment of microfilaraemias (WT; 79%, P = 0.0194. SCID; 76%, P = 0.0473). FBZ induced a total macrofilaricidal response against adult Bm in WT and SCID (WT; P = 0.0067, SCID; P = 0.0071). MIN induced a >90% reduction in Bm Wolbachia burdens (P < 0.0001) and a blockade of microfilarial release (P = 0.0215) in SCID. Male Onchocerca survival was significantly higher in SCID vs WT mice, but not gerbils, after +15 days (60% vs 22% vs 39% P = 0.0475). Onchocercoma implants had engrafted into host tissues, with evidence of neovascularisation, after +7 days and yielded viable macro/microfilariae ex vivo. FBZ induced a macrofilaricidal effect in Onchocerca male implanted SCID at +5 weeks (FBZ; 1.67% vs VC; 43.81%, P = 0.0089). Wolbachia loads within male Onchocerca were reduced by 99% in implanted SCID receiving RIFAP for +2 weeks., Conclusions: We have developed a 'pan-filarial' small animal research model that is sufficiently robust, with adequate capacity and throughput, to screen existing and future pre-clinical candidate macrofilaricides. Pilot data suggests a murine onchocercoma xenograft model is achievable.

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: 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: 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: 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., (© 2012 Albers et al; licensee BioMed Central Ltd.)

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: 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.

Since 1987 onchocerciasis control has relied on the donation of ivermectin (Mectizan(R), 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.