Peter Enyong

Biting midges belonging to the genus Culicoides are tiny stout-shaped hematophagous insects and are thought to transmit the filarial nematode Mansonella perstans. Little is known about the Culicoides fauna in the rain forest belt of the Littoral Region of Cameroon. This study was designed to investigate the diversity, abundance and distribution of Culicoides spp. and their role as the purported vector(s) of M. perstans. Overnight light trap collections and human landing catches (HLCs) revealed eight species of Culicoides with C. grahamii being the most abundant species followed by C. milnei. Four anthropophilic species (C. inornatipennis, C. grahamii, C. fulvithorax and C. milnei) were determined by the HLCs with a higher abundance in the 4–6 p.m. collections. The drop trap technique and Mp419 LAMP assay confirmed C. milnei to be the most efficient vector in enabling the development of the microfilarial stage to the infective larval form of M. perstans. The LAMP assay also revealed that natural transmission of this nematode is fostered by C. milnei and C. grahamii in the wild. In conclusion, C. milnei was shown to be the main vector of M. perstans in the rain forest belt of the Littoral Region of Cameroon.

Background: Despite decades of community-directed treatment with ivermectin (CDTI), onchocerciasis transmission persists in Cameroon and has been associated with increased risk for epilepsy in endemic communities. We investigated the onchocerciasis situation in the Ntui Health District (a known onchocerciasis focus in Cameroon where the Sanaga River constitutes the main source of black fly vectors) using parasitological, entomological and serological parameters. Methods: In July 2021, community-based surveys were conducted in four villages (Essougli, Nachtigal, Ndjame and Ndowe). Onchocerciasis was diagnosed via microscopic examination of skin snips. Using rapid diagnostic tests, we screened children aged 3–6 years for Ov16 antibodies as a proxy for recent onchocerciasis transmission. Monthly black fly biting rates were obtained from the two riverside villages (Nachtigal and Essougli) for 12 consecutive months (July 2021 to June 2022) using the human landing catch technique. Some black flies were dissected each month to check for infection. Results: Overall, 460 participants were recruited; mean age was 32.1 (range: 3–85) years with 248 (53.9%) being males. Among skin snipped participants (n = 425), onchocerciasis prevalence was 14.6%. Participants with epilepsy (n = 25) were more often skin snip positive (45.8% vs 12.7%; P < 0.001) and had higher microfilarial loads (9.2 ± 22.0 vs 0.7 ± 3.5 microfilariae/skin snip; P < 0.001) compared to their peers without epilepsy. Eight (6.5%) of the 123 tested children were Ov16 seropositive. The breeding sites we investigated along the Sanaga River during the current study harbored fewer vectors (annual biting rates reaching 530,322 vs 606,370 in the Mbam River) and exhibited lower black fly infection rates (annual transmission potentials reaching 1479 vs 4488 in the Mbam River) when compared to recent entomological reports in Cameroon. Conclusion: Despite substantial biting rates, black fly infection rates (by microscopy) in the Ntui Health District were rather low resulting in overall low transmission potentials in study villages. Thanks to CDTI, O. volvulus infection in both humans and insects is on the decrease. However, there is evidence that O. volvulus is still endemic in these communities. Reducing the vector population will further accelerate onchocerciasis elimination prospects. Graphical

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. Graphical

Background: The onchocerciasis focus surrounding the lower Mbam and Sanaga rivers, where Onchocerca volvulus is transmitted by Simulium damnosum s.l. (Diptera: Simuliidae), was historically the largest in the southern regions of Cameroon. Annual community-directed treatment with ivermectin (CDTI) has been taking place since 2000, but recent studies have shown that new infections are occurring in children. We aimed to investigate blackfly biting and O. volvulus transmission rates along the lower Mbam river 16 years after the formal onset of annual CDTI. Methods: Black flies were collected for three consecutive days each month between July 2016 and June 2017 at two riverside villages and two inland sites situated 4.9 km and 7.9 km from the riverside. Specimens collected at each site were dissected on one of the three collection days each month to estimate parity rates and O. volvulus infection rates, while the remaining samples were preserved for pool screening. Results: In total, 93,573 S. damnosum s.l. black flies were recorded biting humans and 9281 were dissected. Annual biting rates of up to 606,370 were estimated at the riverside, decreasing to 20,540 at 7.9 km, while, based on dissections, annual transmission potentials of up to 4488 were estimated at the riverside, decreasing to 102 and 0 at 4.9 km and 7.9 km, respectively. However, pool screening showed evidence of infection in black flies at the furthest distance from the river. Results of both methods demonstrated the percentage of infective flies to be relatively low (0.10–0.36%), but above the WHO threshold for interruption of transmission. In addition, a small number of larvae collected during the dry season revealed the presence of Simulium squamosum E. This is the first time S. squamosum E has been found east of Lake Volta in Ghana, but our material was chromosomally distinctive, and we call it S. squamosum E2. Conclusions: Relatively low O. volvulus infection rates appear to be offset by extremely high densities of biting black flies which are sustaining transmission along the banks of the lower Mbam river. Graphical

Background: Infections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after two decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology, especially mating behaviour and nodule formation, as well as development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite’s reproductive and behavioural pattern. Methodology/Principal Findings: Prior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium were purified by centrifugation using a 30% Percoll solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both cell-free and cell-based co-culture systems, and monitored daily by microscopic visual inspection. Exhausted culture medium was replenished every 2–3 days. The cell-free culture system supported the viability and motility of O. volvulus larvae for up to 84 days (DMEM–10%NCS), while the co-culture system (DMEM–10%FBS–LLC-MK ₂ ) extended the worm survival period to 315 days. Co-culture systems alone promoted the two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates observed in DMEM–10%FBS–LLC-MK ₂ (69.2±30 %), while no moult was observed in DMEM–10%NCS–LEC condition. O. volvulus adult worms mating and even mating competitions were observed in DMEM–10% FBS –LLC-MK ₂ co-culture system. Early nodulogenesis was observed in both DMEM–10% FBS–LLC-MK ₂ and DMEM– 10%NCS–LLC-MK ₂ systems. Conclusions/Significance: The present study describes an in vitro system in which O. volvulus L3 larvae can be maintained in culture leading to the development of reproductive adult stages. Thus, this platform gives potential for the investigation of mating, mating competition and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for onchocercacidal drug screening. Author summary: River blindness affects people living in mostly remote and underserved rural communities in some of the poorest areas of the world. Although significant efforts have been achieved towards the reduction of disease morbidity, onchocerciasis still affect million of people in Sub-Saharan Africa. The current control strategy is the annual mass administration of ivermectin which have accumulated several drawbacks overtime: as the sole microfilaricidal action of the drug, very long treatment period (15-17 years) and reports of ivermectin losing its efficacy; Therefore, raising the urgent need for new onchocercacidal molecules. Our study has established an in vitro platform capable of supporting the growth and development of all developmental stages of O. volvulus (L3 infective stage, L4, L5 and adult worms), moreover the platform provided more insight on O. volvulus adult worms reproductive and behavioural pattern. Our findings provide more avenues for mass production of different parasite stages, the investigation of parasite developmental biology and the identification of targets for drug discovery against different phases of development of this filaria parasite

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 ofLoa loaco-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 isO. volvulusprevalence 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 andLoa loainfection 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/m³) 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.

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. 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. 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.

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. 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. 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. 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.