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.

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.

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.

Generation of Loa loa infective larvae by experimental infection of the vector, Chrysops silacea.

Basic and translational research on loiasis, a filarial nematode infection of medical importance, is impeded by a lack of suitable Loa loa infection models and techniques of obtaining and culturing life cycle stages. We describe the development of a new method for routine production of infective third-stage larvae (L3) of L. loa from the natural intermediate arthropod vector host, Chrysops silacea, following experimental infection with purified microfilariae. At 14-days post-infection of C. silacea, the fly survival rate was 43%. Survival was significantly higher in flies injected with 50 mf (55.2%) than those that received 100 mf (31.0%). However, yield per surviving fly and total yield of L3 was markedly higher in the group of flies inoculated with 100 mf (3474 vs 2462 L3 produced). The abdominal segment hosted the highest percentage recovery of L3 (47.7%) followed by head (34.5%) and thorax (17.9%). L. loa larval survival was higher than 90% after 30 days of in vitro culture. The in vitro moulting success rate to the L4 larval stage was 59.1%. After experimental infection of RAG2-/-IL-2γc-/-mice, the average L. loa juvenile adult worm recovery rate was 10.5% at 62 dpi. More than 87% of the worms were recovered from the muscles and subcutaneous tissues. Worms recovered measured an average 24.3 mm and 11.4 mm in length for females (n = 5) and males (n = 5), respectively. In conclusion, L. loa mf injected into C. silacea intrathoracically develop into infective larvae that remain viable and infective comparable to L3 obtained through natural feeding on the human host. This technique further advances the development of a full laboratory life cycle of L. loa where mf derived from experimentally-infected animals may be utilized to passage life cycle generations via intrathoracic injections of wild-caught vector hosts.

Complete Mitochondrial Genome Sequence of Mansonella perstans.

The 13,647-bp complete mitochondrial genome of Mansonella perstans was sequenced and is syntenic to the mitochondrial genome of Mansonella ozzardi Phylogenetic analysis of the mitochondrial genome is consistent with the known phylogeny of ONC5 group filarial nematodes.

Mapping lymphatic filariasis in Loa loa endemic health districts naïve for ivermectin mass administration and situated in the forested zone of Cameroon.

BACKGROUND: The control of lymphatic filariasis (LF) caused by Wuchereria bancrofti in the Central African Region has been hampered by the presence of Loa loa due to severe adverse events that arise in the treatment with ivermectin. The immunochromatographic test (ICT) cards used for mapping LF demonstrated cross-reactivity with L. loa and posed the problem of delineating the LF map. To verify LF endemicity in forest areas of Cameroon where mass drug administration (MDA) has not been ongoing, we used the recently developed strategy that combined serology, microscopy and molecular techniques. METHODS: This study was carried out in 124 communities in 31 health districts (HDs) where L. loa is present. At least 125 persons per site were screened. Diurnal blood samples were investigated for circulating filarial antigen (CFA) by FTS and for L. loa microfilariae (mf) using TBF. FTS positive individuals were further subjected to night blood collection for detecting W. bancrofti. qPCR was used to detect DNA of the parasites. RESULTS: Overall, 14,446 individuals took part in this study, 233 participants tested positive with FTS in 29 HDs, with positivity rates ranging from 0.0 to 8.2%. No W. bancrofti mf was found in the night blood of any individuals but L. loa mf were found in both day and night blood of participants who were FTS positive. Also, qPCR revealed that no W. bancrofti but L.loa DNA was found with dry bloodspot. Positive FTS results were strongly associated with high L. loa mf load. Similarly, a strong positive association was observed between FTS positivity and L loa prevalence. CONCLUSIONS: Using a combination of parasitological and molecular tools, we were unable to find evidence of W. bancrofti presence in the 31 HDs, but L. loa instead. Therefore, LF is not endemic and LF MDA is not required in these districts.

Comparison of immune responses to Loa loa stage-specific antigen extracts in Loa loa-exposed BALB/c mice upon clearance of infection.

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.

Implementation of test-and-treat with doxycycline and temephos ground larviciding as alternative strategies for accelerating onchocerciasis elimination in an area of loiasis co-endemicity: the COUNTDOWN consortium multi-disciplinary study protocol.

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/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.

Discovery of short-course antiwolbachial quinazolines for elimination of filarial worm infections.

Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targets Wolbachia, a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of Wolbachia in the in vivo Litomosoides sigmodontis filarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantify Wolbachia elimination in Brugia pahangi filarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed in L. sigmodontis, Brugia malayi, and Onchocerca ochengi in vivo preclinical models of filarial disease and in vitro selectivity against Loa loa (a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections.

Preclinical development of an oral anti-Wolbachia macrolide drug for the treatment of lymphatic filariasis and onchocerciasis.

There is an urgent global need for a safe macrofilaricide drug to accelerate elimination of the neglected tropical diseases onchocerciasis and lymphatic filariasis. From an anti-infective compound library, the macrolide veterinary antibiotic, tylosin A, was identified as a hit against Wolbachia This bacterial endosymbiont is required for filarial worm viability and fertility and is a validated target for macrofilaricidal drugs. Medicinal chemistry was undertaken to develop tylosin A analogs with improved oral bioavailability. Two analogs, A-1535469 and A-1574083, were selected. Their efficacy was tested against the gold-standard second-generation tetracycline antibiotics, doxycycline and minocycline, in mouse and gerbil infection models of lymphatic filariasis (Brugia malayi and Litomosoides sigmodontis) and onchocerciasis (Onchocerca ochengi). A 1- or 2-week course of oral A-1535469 or A-1574083 provided >90% Wolbachia depletion from nematodes in infected animals, resulting in a block in embryogenesis and depletion of microfilarial worm loads. The two analogs delivered comparative or superior efficacy compared to a 3- to 4-week course of doxycycline or minocycline. A-1574083 (now called ABBV-4083) was selected for further preclinical testing. Cardiovascular studies in dogs and toxicology studies in rats and dogs revealed no adverse effects at doses (50 mg/kg) that achieved plasma concentrations >10-fold above the efficacious concentration. A-1574083 (ABBV-4083) shows potential as an anti-Wolbachia macrolide with an efficacy, pharmacology, and safety profile that is compatible with a short-term oral drug course for treating lymphatic filariasis and onchocerciasis.

Mouse models of Loa loa.

Elimination of the helminth disease, river blindness, remains challenging due to ivermectin treatment-associated adverse reactions in loiasis co-infected patients. Here, we address a deficit in preclinical research tools for filarial translational research by developing Loa loa mouse infection models. We demonstrate that adult Loa loa worms in subcutaneous tissues, circulating microfilariae (mf) and presence of filarial biomarkers in sera occur following experimental infections of lymphopenic mice deficient in interleukin (IL)-2/7 gamma-chain signaling. A microfilaraemic infection model is also achievable, utilizing immune-competent or -deficient mice infused with purified Loa mf. Ivermectin but not benzimidazole treatments induce rapid decline (>90%) in parasitaemias in microfilaraemic mice. We identify up-regulation of inflammatory markers associated with allergic type-2 immune responses and eosinophilia post-ivermectin treatment. Thus, we provide validation of murine research models to identify loiasis biomarkers, to counter-screen candidate river blindness cures and to interrogate the inflammatory etiology of loiasis ivermectin-associated adverse reactions.

Short-course, oral flubendazole does not mediate significant efficacy against Onchocerca adult male worms or Brugia microfilariae in murine infection models.

The Onchocerca ochengi adult implant and Brugia malayi microfilariemic Severe-Combined Immunodeficient (SCID) mouse models are validated screens to measure macrofilaricidal and microfilaricidal activities of candidate onchocerciasis drugs. The purpose of this study was to assess whether 5 daily sub-cutaneous (s.c.) injections of standard flubendazole (FBZ) suspension (10mg/kg), a single s.c. injection (10mg/kg) or 5 daily repeated oral doses of FBZ amorphous solid dispersion (ASD) formulation (0.2, 1.5 or 15mg/kg) mediated macrofilaricidal efficacy against O. ochengi male worms implanted into SCID mice. The direct microfilaricidal activity against circulating B. malayi microfilariae of single dose FBZ ASD formulation (2 or 40 mg/kg) was also evaluated and compared against the standard microfilaricide, ivermectin (IVM). Systemic exposures of FBZ/FBZ metabolites achieved following dosing were measured by pharmacokinetic (PK) bioanalysis. At necropsy, five weeks following start of FBZ SC injections, there were significant reductions in burdens of motile O. ochengi worms following multiple injections (93%) or single injection (82%). Further, significant proportions of mice dosed following multiple injections (5/6; 83%) or single injection (6/10; 60%) were infection negative (drug-cured). In comparison, no significant reduction in recovery of motile adult O. ochengi adult worms was obtained in any multiple-oral dosage group. Single oral-dosed FBZ did not mediate any significant microfilaricidal activity against circulating B. malayi mf at 2 or 7 days compared with >80% efficacy of single dose IVM. In conclusion, multiple oral FBZ formulation doses, whilst achieving substantial bioavailability, do not emulate the efficacy delivered by the parenteral route in vivo against adult O. ochengi. PK analysis determined FBZ efficacy was related to sustained systemic drug levels rather than achievable Cmax. PK modelling predicted that oral FBZ would have to be given at low dose for up to 5 weeks in the mouse model to achieve a matching efficacious exposure profile.

Heterogeneity in the in vitro susceptibility of Loa loa microfilariae to drugs commonly used in parasitological infections.

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 (CR50 = 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 (CR50 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.

Antimicrobial and toxicological activities of five medicinal plant species from Cameroon traditional medicine.

BACKGROUND: Infectious diseases caused by multiresistant microbial strains are on the increase. Fighting these diseases with natural products may be more efficacious. The aim of this study was to investigate the in vitro antimicrobial activity of methanolic, ethylacetate (EtOAc) and hexanic fractions of five Cameroonian medicinal plants (Piptadeniastum africana, Cissus aralioides, Hileria latifolia, Phyllanthus muellerianus and Gladiolus gregasius) against 10 pathogenic microorganisms of the urogenital and gastrointestinal tracts. METHODS: The fractions were screened for their chemical composition and in vivo acute toxicity was carried out on the most active extracts in order to assess their inhibitory selectivity. The agar well-diffusion and the micro dilution methods were used for the determination of the inhibition diameters (ID) and Minimum inhibitory concentrations (MIC) respectively on 8 bacterial species including two Gram positive species (Staphylococcus aureus, Enterococcus faecalis), and six Gram negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Shigella flexneri, Salmonella typhi) and two fungal isolates (Candida albicans, Candida krusei). The chemical composition was done according to Harbone (1976), the acute toxicity evaluation according to WHO protocol and the hepatic as well as serum parameters measured to assess liver and kidney functions. RESULTS: The chemical components of each plant's extract varied according to the solvent used, and they were found to contain alkaloids, flavonoids, polyphenols, triterpens, sterols, tannins, coumarins, glycosides, cardiac glycosides and reducing sugars. The methanolic and ethylacetate extracts of Phyllanthus muellerianus and Piptadeniastum africana presented the highest antimicrobial activities against all tested microorganisms with ID varying from 8 to 26 mm and MIC from 2.5 to 0.31 mg/ml. The in vivo acute toxicity study carried out on the methanolic extracts of Phyllanthus muellerianus and Piptadeniastrum africana indicated that these two plants were not toxic. At the dose of 4 g/kg body weight, kidney and liver function tests indicated that these two medicinal plants induced no adverse effect on these organs. CONCLUSION: These results showed that, all these plant's extracts can be used as antimicrobial phytomedicines which can be therapeutically used against infections caused by multiresistant agents. Phyllanthus muellerianus, Piptadeniastum africana, antimicrobial, acute toxicity, kidney and liver function tests, Cameroon Traditional Medicine.