Comparative development of human filariae Loa loa, Onchocerca volvulus and Mansonella perstans in immunocompromised mouse strains.

Introduction: Mouse models of human filarial infections are not only urgently needed to investigate the biology of the nematodes and their modulation of the host's immunity, but will also provide a platform to screen and test novel anti-filarial drugs. Recently, murine Loa loa infection models have been stablished using immunocompromised mouse strains, whereas murine Mansonella perstans infections have not been implemented until now. Methods: Therefore, we aim to establish experimental M. perstans infections using the immunocompromised mouse strains RAG2IL-2Rγ-/- (lack B, T and natural killer cells), IL-4Rα/IL-5-/- (impaired IL-4/5 signalling and eosinophil activation) and NOD.Cg-PrkdcscidIl2rgtm1Wj l/SzJ (NOD scid gamma, NSG) BALB/c mice (lack mature lymphocytes) through subcutaneous (s.c.) or intraperitoneal (i.p.) inoculation of infective stage 3 larvae (L3) isolated from engorged vectors. Results: In total, 145 immunocompromised mice have been inoculated with 3,250 M. perstans, 3,337 O. volvulus, and 2,720 Loa loa L3 to comparatively analyse which immunocompromised mouse strain is susceptible to human filarial infections. Whereas, no M. perstans and O. volvulus L3 could be recovered upon 2-63 days post-inoculation, a 62-66% Loa loa L3 recovery rate could be achieved in the different mouse strains. Gender of mice, type of inoculation (s.c. or i.p.) or time point of analysis (2-63 days post inoculation) did not interfere with the success of L3 recovery. In addition, administration of the immune suppressants hydrocortisone, prednisolone and cyclophosphamide did not restore M. perstans L3 recovery rates. Discussion: These findings show that RAG2IL-2Rg-/-BALB/c and C57BL/6, IL-4Rα/IL-5-/- BALB/c and NSG mice were not susceptible to M. perstans and O. volvulus L3 inoculation using the applied methods, whereas Loa loa infection could be maintained. Further studies should investigate if humanized immunocompromised mice might be susceptible to M. perstans. and O. volvulus.

Impact of repeated mass ivermectin administration using a community directed approach on L. loa infection in Chrysops silacea of the rain forest and forest savanna of Cameroon.

Background: Loiasis is an endemic filarial infection in the rainforest zone of West and Central Africa. Repeated annual community-directed treatment with ivermectin (CDTI) delivered for several years to control onchocerciasis has been shown to reduce the prevalence and intensity of Loiasis in some Loa loa-Onchocerca volvulus co-endemic areas. However, the impact of these multiple rounds of CDTI on entomological indicators of loiasis transmission is not known, and was therefore assessed in this study in areas with contrasting histories of CDTI. Methods: The study was conducted in the East, North-west and South-west 1 CDTI project sites of Cameroon. Two communities per CDTI project were selected for fly collection and dissection. Ivermectin treatment coverage was documented in these areas, and this was correlated to Chrysops infection and infective rates. A total of 7029 female Chrysops were collected from 6 communities of the 3 CDTI projects (East, North-west, and South-west 1) and from 2 communities in a non-CDTI district (East). Results: Chrysops biting densities and parous rates were significantly reduced in the North-west and South-west sites post-CDTI, while in the East, biting densities were similar in non-CDTI and CDTI sites, with higher parous rates observed in the non-CDTI site. Infection and infective rates in the East non-CDTI site were 4.4% and 1.8% respectively, as compared to 3.3% and 1.3% in the CDTI site after 10 ivermectin rounds (there were no baseline data for the latter). In the North-west site, significant reductions in Chrysops infection and infective rates from 10.2% and 4.2% respectively, to 3.5% and 1.2 (after 9 rounds of ivermectin treatment), were recorded following CDTI. In the South-west, infection rate significantly increased from 1.74% to 2.8% and infective rate remained statistically unchanged after 14 rounds of CDTI (0.45% - 0.40%). Similar trends in Mean Head L3 were observed except in the East site where this indicator was similar in both CDTI and control sites. Only in the North-west site did monthly transmission potentials decrease significantly. Conclusion: This study demonstrated that the impact of repeated annual treatment with ivermectin for the control of onchocerciasis using community directed delivery approach on the entomological indicators of loiasis varies with bioecological zones. Community directed treatment with ivermectin induced a significant reduction in the entomological indicators of loiasis in the North-West project site which lies in forest savanna area. A non-significant decrease was observed in the East project site and in contrast, a significant increase was observed in the South-West 1 project site which both lies in the rainforest zones.

Culicoides Species of the Rain Forest Belt of the Littoral Region of Cameroon: Their Incrimination in the Transmission of Mansonella perstans.

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.

Application of loop mediated isothermal amplification (LAMP) assays for the detection of Onchocerca volvulus, Loa loa and Mansonella perstans in humans and vectors.

Conventional diagnosis of filarial infections is based on morphological identification of microfilariae using light microscopy and requires considerable expertise, is time-consuming, and can be subjective. Loop-mediated isothermal amplification (LAMP) has advantages over microscopy or PCR because of its operational simplicity, rapidity and versatility of readout options. LAMP assays represent a major step forward in improved filarial diagnostic tools suitable for low resource settings and field applicability. The study goal was to retrospectively evaluate the performance and suitability of the O-150, RF4, and Mp419 LAMP assays for diagnosing Onchocerca volvulus, Loa loa and Mansonella perstans infections, respectively, in humans and vectors under experimental and natural field conditions. Surveys were conducted in four health districts of Cameroon using skin snip and thick blood film methods to detect skin (O. volvulus) and blood (L. loa and M. perstans) dwelling microfilaria in humans. Engorged vectors (Simulium spp., Chrysops spp., and Culicoides spp.) were evaluated by LAMP. Dissected, wild-caught vectors were also analyzed. LAMP showed a prevalence of 40.4% (O. volvulus), 17.8% (L. loa) and 36.6% (M. perstans) versus 20.6% (O. volvulus), 17.4% (L. loa) and 33.8% (M. perstans) with microscopy. Simulium spp. were dissected for microscopy and pooled for LAMP. The O-150 LAMP assay infection rate was 4.3% versus 4.1% by microscopy. Chrysops spp. were dissected and analyzed individually in the LAMP assay. The RF4 LAMP assay infection rate was 23.5% versus 3.3% with microscopy. The RF4 LAMP assay also detected parasites in Chrysops spp. fed on low microfilaremic volunteers. The Mp419 LAMP assay infection rate was 0.2% for C. milnei and 0.04% for C. grahamii, while three other species were LAMP-negative. The sensitivity, species specificity, rapidity and ease of its use of these filarial LAMP assays, and validation of their performance in the field support use as alternatives to microscopy as diagnostic and surveillance tools in global health programs aimed to eliminate onchocerciasis.

Onchocerciasis in the Ntui Health District of Cameroon: epidemiological, entomological and parasitological findings in relation to elimination prospects.

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.

An analysis of social dimensions of podoconiosis and leprosy on affected households in endemic health districts of the North West Region of Cameroon.

Background: Podoconiosis and leprosy are Neglected Tropical Diseases associated with low quality of life, social stigma and isolation of affected people and families. Despite the substantial social burden it imposes, podoconiosis has largely been ignored in the global health literature until recently unlike leprosy. This study assessed and compared the quality of life and social impact of podoconiosis with that of leprosy among affected households and neighborhoods in North West Cameroon. Methods: A comparative cross-sectional design was used. Eighty-six households: 43 podoconiosis and 43 leprosy, plus household neighbours were enrolled from July and August 2015 from three health districts. Podoconiosis patients living in households within Batibo and Ndop health districts were sequentially sampled using a list of confirmed podoconioisis cases from previous studies. Leprosy patients living within communities in Mejang Health Area were sequentially sampled using the Mbingo treatment center register. WHO BREF tool was used to assess quality of life. Franklin Stigma Scale was adapted to assess felt and enacted stigma. Mann-Whitney U test was used to compare differences in stigma and QoL. Results: Physical domain showed a significant difference in the distribution in quality of life between groups (p < 0.05, median:70; U:635, r = 0.2). Overall enacted stigma revealed significant differences with p < 0.05 and r = 0.4. Overall stigma from family members (median:17, U:627 and r = 0.3) and neighbours (median:67, U:336 and r = 0.5) showed significant differences with p < 0.05 in the distribution of scores for both diseases. Sex and age showed significant associations with QoL and stigma. Conclusion: This study reveals the quality of life and stigma associated with podoconiosis on affected households to be comparable to that experienced by households with a leprosy patient. There is need for intensified preventive, management and control schemes to fight podoconiosis in Cameroon, just like leprosy.

The preparatory phase for ground larviciding implementation for chocerciasis control in the Meme River Basin in South West Cameroon: the COUNTDOWN Consortium alternative strategy implementation trial.

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.

Onchocerca volvulus transmission in the Mbam valley of Cameroon following 16 years of annual community-directed treatment with ivermectin, and the description of a new cytotype of Simulium squamosum.

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.

'Slash and clear' vector control for onchocerciasis elimination and epilepsy prevention: a protocol of a cluster randomised trial in Cameroonian villages.

INTRODUCTION: Onchocerciasis, caused by the filarial nematode Onchocerca volvulus, remains endemic in Cameroon despite decades of community-directed treatment with ivermectin (CDTI). CDTI is often hampered by coendemicity with loiasis (another filariasis caused by Loa loa) in some areas. Strong epidemiological evidence suggests that O. volvulus infection increases the risk for onchocerciasis-associated epilepsy (OAE) among Cameroonian children. This highlights the urgent need to strengthen onchocerciasis elimination programmes in mesoendemic/hyperendemic areas. Novel alternative strategies, such as the 'slash and clear' (S&C) vector control method, may be required to complement ongoing CDTI to accelerate elimination of transmission. The short-term impact of S&C on the biting rates of the blackfly vectors has been demonstrated in other settings. However, its long-term effectiveness and impact on parasitological and serological markers of onchocerciasis transmission as well as on OAE are still unknown. METHODS AND ANALYSIS: We aim to assess the effectiveness of annual S&C interventions combined with CDTI in reducing onchocerciasis transmission and epilepsy incidence. Eight onchocerciasis-endemic villages located <5 km from the Mbam or Sanaga rivers will be randomised to two arms: four villages will receive yearly CDTI only for two consecutive years (Arm 1), while the other four villages will receive CDTI plus annual S&C for 2 years (Arm 2). Study outcomes (blackfly biting rates, infectivity rates and seroprevalence of onchocerciasis antibodies (Ov16 antibodies) in children, prevalence of microfilaridermia and epilepsy incidence) will be monitored prospectively and compared across study arms. We expect that S&C will have an added benefit over CDTI alone. ETHICS AND DISSEMINATION: The protocol has received ethical approval from the institutional review board of the Cameroon Baptist Convention Health Board (reference number: IRB2021-03) and has been registered with the Pan African Clinical Trials Registry. Findings will be disseminated at national and international levels via meetings and peer-reviewed publications. TRIAL REGISTRATION NUMBER: PACTR202101751275357.

Onchocerca ochengi male worms implanted in SCID mice and Gerbil: Relationship between microfilaridermia status of cows, nodular worm viability and fertility and worm survival in the rodents.

BACKGROUND: Current treatment options for onchocerciasis are sub-optimal, prompting research and development of a safe cure (macrofilaricide). Onchocerca ochengi, a parasite of cattle, is used as a close surrogate for the human parasite O. volvulus in a murine model for pre-clinical screening of macrofilaricides. Skin from naturally infected cattle have been used in previous studies as a reliable source of parasite material. However, there is limited knowledge on how source-related factors such as the microfilaridermia status of the cattle, the nodule load and nodular worm viability may affect survival of male O. ochengi worms implanted in the rodent hosts. Such relationships were investigated in this study. METHODS: Dermal tissue and nodules were obtained from Gudali cattle, dissected and cultured to obtain migrating microfilariae (mf) and male worms. Emerged male worms were implanted into SCID mice and Gerbils (Meriones unguiculatus) and recovery rates were determined upon 42 days post implantation. Finally, nodules were processed for histology and embryogram analyses to assess the nodular worm viability and fertility, respectively. RESULTS: Of the 69 cattle sampled, 24 (34.8%) were mf+ and 45 (65.2%) were mf-. The mean nodule loads were 180.5 ± 117.7 (mf+) and 110.6 ± 102.7 (mf-) (p = 0.0186). The mean male worm harvest from nodules were 76.8 ± 120.3 and 47.2 ± 33.4 (p = 0.2488) for mf+ and mf- cattle, respectively. The number of male worms per 100 nodules were 57/100 and 46/100 nodules for mf+ and mf- cows, respectively. Female worms from nodules of mf- cows had higher counts of both normal and abnormal embryos with higher proportions of dead nodular worms evinced by histology compared to those from mf+ cows. A total of 651 worms were implanted into mice and gerbils, out of which 129 (19.81%) were recovered. Logistic regression analysis indicated that the microfilaridermia status of the cattle (presence of mf) (OR = 4.3319; P = 0.001) is the single most important predictor of the success of male worm recovery after implantation into rodents. CONCLUSION: Microfilaridermic cattle provide a promising source of adult O. ochengi. Male worms from this group of cattle have a better success rate of survival in a murine implant model. Nevertheless, in the programmatic point of view, amicrofilaridermic Gudali cattle would still constitute an important source of O. ochengi male worms with relatively good viability after implantation into rodents.

In vivo antimalarial activity of a probiotic bacterium Lactobacillus sakei isolated from traditionally fermented milk in BALB/c mice infected with Plasmodium berghei ANKA.

ETHNOPHARMACOLOGICAL RELEVANCE: Milk production, processing and consumption are integral part of traditional practices in Fulani tribe of Cameroon. It has been observed that Fulani are resistant to malaria. Dairy products traditionally processed by Fulani are intensively used in the ritual treatment of malarial, inflammations and behavioural disorders. Many studies have demonstrated that fermented milk is a rich source of probiotic bacteria. However, the antimalarial activity of probiotics isolated from this natural source has not been experimentally tested. AIM OF THE STUDY: Hence, this study was therefore aimed at evaluating the antimalarial activity of a probiotic bacterium Lactobacillus sakei isolated from traditionally fermented milk in mice infected with chloroquine sensitive Plasmodium berghei ANKA. MATERIALS AND METHODS: The probiotic bacterium was isolated from the Cameroonian Mborro Fulani's traditionally fermented milk and identified using the 16S r RNA gene sequencing. The schizontocidal activity of Lactobacillus sakei on established malaria infection was evaluated. Eighty-four healthy young adult Balb/c mice infected with Plasmodium berghei parasite were randomly divided into two sets of seven group of six mice each, and were given three different doses of Lactobacillus sakei, chloroquine and sulfadoxine/pyrimethamine for seven and fourteen days respectively. The level of parasitaemia, body temperature, survival time and haematological parameters were evaluated. RESULTS: The parasite growth inhibition was observed to increase with increasing dose of probiotic bacterium with maximum suppression being 100 % at dose 3 on day 20. Also, the probiotic bacterium significantly prevented body weight loss and was associated with body temperature reduction and prevented (p<0.05) a decrease in haematological parameters compared to that untreated malaria infected mice. CONCLUSION: The results obtained suggest that Lactobacillus sakei is a probiotic bacterium with antimalarial activity in mice infected with chloroquine sensitive Plasmodium berghei.

Capture of high numbers of Simulium vectors can be achieved with Host Decoy Traps to support data acquisition in the onchocerciasis elimination endgame.

Onchocerciasis elimination is within reach in many countries but requires enhanced surveillance of the Simulium vectors of Onchocerca volvulus. Collection of sufficient numbers of adult Simulium to detect infective O. volvulus larvae is hindered by limited sampling tools for these flies. Here, we tested for the first time the Host Decoy Trap (HDT), an exposure free method previously developed for Anopheles vectors of malaria parasites, as a potential sampling tool for adult Simulium. In three replicates of a randomized Latin square experimental design, the HDT was compared to Human Landing Catches (HLC) and the Esperanza Window Trap (EWT). A total of 8,531 adult S. damnosum sensu lato blackflies (S. squamosum group) were found in catches from the three different trapping methods. The HDT (mean catch 533 ± 111) caught significantly more S. squamosum than the EWT (mean catch 9.1 ± 2.2), a nearly 60-fold difference. There was no significant difference between the HLC (mean catch 385.6 ± 80.9) and the HDT. Larvae indistinguishable from those of O. volvulus were dissected from 2.86% of HDT samples (n = 70) and 0.35% of HLC samples (n = 285); a single infective third-stage larvae (L3) was  found during dissection of a sample from the HDT. Owing to its very high capture rate, which was comparable to the HLC and significantly greater than EWT, alongside the presence of infected flies in its catch, the HDT represents a potentially valuable new tool for blackfly collection in elimination settings, where thousands of flies are needed for parasite screening.

Establishment of an in vitro culture system to study the developmental biology of Onchocerca volvulus with implications for anti-Onchocerca drug discovery and screening.

BACKGROUND: Infections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after three decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology especially the mating behaviour and nodule formation as well as the development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite reproduction and behaviour. METHODOLOGY/PRINCIPAL FINDINGS: Prior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium damnosum flies 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 (DMEM supplemented with 10% NCS) supported the viability and motility of O. volvulus larvae for up to 84 days, while the co-culture system (DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells) extended worm survival for up to 315 days. Co-culture systems alone promoted two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates (69.2±30%) observed in DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, while no moult was observed in DMEM supplemented with 10% NCS and seeded on LEC feeder cells. In DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, O. volvulus adult male worms attached to the vulva region of adult female worms and may have mated in vitro. Apparent early initiation of nodulogenesis was observed in both DMEM supplemented with 10% FBS and seeded on LLC-MK2 and DMEM supplemented with 10% NCS and seeded on LLC-MK2 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 adult stages. Thus, this in vitro system may provide a platform to investigate mating behaviour and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for macrofilaricidal drug screening.

The Mbam drainage system and onchocerciasis transmission post ivermectin mass drug administration (MDA) campaign, Cameroon.

BACKGROUND: The impact of large scale Mass Drug Adminstration (MDA) of ivermectin on active onchocerciasis transmission by Simulium damnosum, which transmits the parasite O. volvulus is of great importance for onchocerciasis control programmes. We investigated in the Mbam river system area, the impact of MDA of ivermectin on entomological indices and also verify if there are river system factors that could have favoured the transmission of onchocerciasis in this area and contribute to the persistence of disease. We compared three independent techniques to detect Onchocerca larvae in blackflies and also analyzed the river system within 9 months post-MDA of ivermectin. METHOD: Simulium flies were captured before and after 1, 3, 6 and 9months of ivermectin-MDA. The biting rate was determined and 41% of the flies dissected while the rest were grouped into pools of 100 flies for DNA extraction. The extracted DNA was then subjected to O-150 LAMP and real-time PCR for the detection of infection by Onchocerca species using pool screening. The river system was analysed and the water discharge compared between rainy and dry seasons. PRINCIPAL FINDINGS: We used human landing collection method (previously called human bait) to collect 22,274 adult female Simulium flies from Mbam River System. Of this number, 9,134 were dissected while 129 pools constituted for molecular screening. Overall biting and parous rates of 1113 flies/man/day and 24.7%, respectively, were observed. All diagnostic techniques detected similar rates of O. volvulus infection (P = 0.9252) and infectivity (P = 0.4825) at all monitoring time points. Onchocerca ochengi larvae were only detected in 2 of the 129 pools. Analysis of the river drainage revealed two hydroelectric dams constructed on the tributaries of the Mbam river were the key contributing factor to the high-water discharge during both rainy and dry seasons. CONCLUSION: Results from fly dissection (Microscopy), real-time PCR and LAMP revealed the same trends pre- and post-MDA. The infection rate with animal Onchocerca sp was exceptionally low. The dense river system generate important breeding sites that govern the abundance of Simulium during both dry and rainy seasons.

Validation of loop-mediated isothermal amplification for the detection of Loa loa infection in Chrysops spp in experimental and natural field conditions.

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.

Why onchocerciasis transmission persists after 15 annual ivermectin mass drug administrations in South-West Cameroon.

INTRODUCTION: Onchocerciasis is targeted for elimination mainly with annual community-directed treatment with ivermectin (CDTI). High infection levels have been reported in South-West Cameroon, despite ≥15 years of CDTI. The aim of this study was to assess factors associated with continued onchocerciasis transmission and skin disease. METHODS: A large-scale cross-sectional study was conducted in 2017 in 20 communities in a loiasis-risk area in South-West Cameroon. A mixed-methods approach was used. Associations between infection levels, skin disease and adherence to CDTI were assessed using mixed regression modelling. Different community members' perception and acceptability of the CDTI strategy was explored using semi-structured interviews. RESULTS: Onchocerciasis prevalence was 44.4% among 9456 participants. 17.5% of adults were systematic non-adherers and 5.9% participated in ≥75% of CDTI rounds. Skin disease affected 1/10 participants, including children. Increasing self-reported adherence to CDTI was associated with lower infection levels in participants aged ≥15 years but not in children. Adherence to CDTI was positively influenced by perceived health benefits, and negatively influenced by fear of adverse events linked with economic loss. Concern of lethal adverse events was a common reason for systematic non-adherence. CONCLUSION: CDTI alone is unlikely to achieve elimination in those high transmission areas where low participation is commonly associated with the fear of adverse events, despite the current quasi absence of high-risk levels of loiasis. Such persisting historical memories and fear of ivermectin might impact adherence to CDTI also in areas with historical presence but current absence of loiasis. Because such issues are unlikely to be tackled by CDTI adaptive measures, alternative strategies are needed for onchocerciasis elimination where negative perception of ivermectin is an entrenched barrier to community participation in programmes.

Advances in preclinical platforms of Loa loa for filarial neglected tropical disease drug and diagnostics research.

The tropical disease, loiasis, caused by the filarial parasite, Loa, has gained prominence in global public health as a cause of excess mortality and a barrier to the elimination of the related prioritized neglected tropical diseases (NTDs), lymphatic filariasis and onchocerciasis, within Central Africa. There are no effective drug cures or vaccines available to treat loiasis safely. Here we review recent advances in loiasis preclinical platform technologies, including novel in vitro culturing systems, animal models and innovations in experimental infections of the L. loa vector, Chrysops, that have facilitated access to all L. loa filarial life-cycle stages. We detail applications of these new model systems in anti-filarial drug screening, diagnostic development, immunology, and pathophysiology research. Finally, we provide an overview of how loiasis preclinical platforms may be further utilized in translational medicine applications to support the development of much needed new interventions against filarial NTDs.

Clinical, haematological and biochemical profiling of podoconiosis lymphoedema patients prior to their involvement in a clinical trial in the Northwest Region of Cameroon.

BACKGROUND: Prior to carrying out clinical trials, it is important to assess the health status of the study participants to be able to interpret subsequent changes that may be related to the effects of the treatments during the follow-up of patients. This study presents the clinical, haematological and biochemical profiles of podoconiosis patients prior to their involvement in the PodoLEDoxy clinical trial. METHODS: All lower limb lymphoedema patients visiting the centre were screened and a podoconiosis diagnosis was based on clinical manifestation and detailed medical history. Patients who satisfied the eligibility criteria were enrolled in the study and their demographic data, vital signs and medical history were collected followed by biochemical and haematological examinations. RESULTS: Of the 222 participants enrolled in the study, 55.4% and 41.4% had either stage 3 or 2 podoconiosis as their highest stages, respectively. On physical examination, gastritis (46%) and poor vision (2.7%) were the most prevalent health issues identified. The majority of haematological and biochemical values were within the normal range except for mean platelet volume (47.7%), plateletcrit (58.1%), platelet distribution width (66.2%), mean corpuscular volume (67.6%) and red cell distribution width-standard deviation (79.3%), where >40% of the study participants had values out of the normal. CONCLUSION: The clinical, haematological and biochemical profiles of the study participants were largely within the normal range except for certain haematological parameters that might be worth investigating.

Differential susceptibility of Onchocerca volvulus microfilaria to ivermectin in two areas of contrasting history of mass drug administration in Cameroon: relevance of microscopy and molecular techniques for the monitoring of skin microfilarial repopulation within six months of direct observed treatment.

BACKGROUND: Ivermectin is an excellent microfilaricide against Onchocerca volvulus. However, in some regions, long term use of ivermectin has resulted in sub-optimal responses to the treatment. More data to properly document the phenomenon in various contexts of ivermectin mass drug administration (IVM-MDA) is needed. Also, there is a need to accurately monitor a possible repopulation of skin by microfilariae following treatment. Skin snip microscopy is known to have a low sensitivity in individuals with light infections, which can be the case following treatment. This study was designed with two complementary objectives: (i) to assess the susceptibility of O. volvulus microfilariae to ivermectin in two areas undergoing IVM-MDA for different lengths of time, and (ii) to document the repopulation of skin by the O. volvulus microfilariae following treatment, using 3 independent diagnostic techniques. METHOD: Identified microfilaridermic individuals were treated with ivermectin and re-examined after 1, 3, and 6 months using microscopy, actin real-time PCR (actin-qPCR) and O-150 LAMP assays. Susceptibility to ivermectin and trends in detecting reappearance of skin microfilariae were determined using three techniques. Microscopy was used as an imperfect gold standard to determine the performance of actin-qPCR and LAMP. RESULTS: In Bafia with over 20 years of IVM-MDA, 11/51 (21.6%) direct observe treated microfilaridemic participants were still positive for skin microfilariae after 1 month. In Melong, with 10 years of IVM-MDA, 2/29 (6.9%) treated participants were still positive. The microfilarial density reduction per skin biopsy within one month following treatment was significantly lower in participants from Bafia. In both study sites, the molecular techniques detected higher proportions of infected individuals than microscopy at all monitoring time points. LAMP demonstrated the highest levels of sensitivity and real-time PCR was found to have the highest specificity. CONCLUSION: Patterns in skin mirofilariae clearance and repopulation were established. O. volvulus worms from Bafia with higher number of annual MDA displayed a lower clearance and higher repopulation rate after treatment with ivermectin. Molecular assays displayed higher sensitivity in monitoring O. volvulus microfilaridemia within six months following treatment.