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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.

Female breast cancer (BC) is the leading cause of cancer-related deaths worldwide with higher mortality rates and early onset in developing countries. The molecular basis of early disease onset is still elusive. We recruited 472 female breast cancer from two sub-Saharan African countries (Cameroon and Congo) between 2007 and 2018 and collected clinical data from these patients. To investigate the molecular drivers of early disease onset, we analyzed publicly available breast cancer molecular data from the cancer genome atlas (TCGA) and the gene expression omnibus (GEO) for copy number alteration, mutation and gene expression. Early BC onset (EOBRCA) (diagnosis before 45 years) was higher in African women compared with the TCGA cohort (51.7% vs 15.6%). The tumor grade, mitotic index, HER2 + phenotype, basal-like phenotype and ki67 were higher in EOBRCA for all cohorts. BC risk factors such as parity, breastfeeding early onset of menarche and use of hormonal contraceptives were significantly associated with EOBRCA (p CDH6 and FOXM1 and tumor suppressor including TGM3 and DMBT1 as well as higher TP53 mutation rates (OR: 2.93, p TGM3 deletions, which was associated with high expression of all SMAD transcription factors as well as WNT ligands. The Frizzled receptors FZD1, FZD4 and FZD6 were significantly upregulated in EOBRCA, suggesting activation of non-canonical WNT signaling. Our data, suggest the implication of TGM3 deletion in early breast cancer onset. Further molecular investigations are warranted in African patients.

BackgroundInflammation is undoubtedly a hallmark of cancer development. Its maintenance within tumors and subsequent consequences on disease aggressiveness is less understood.MethodsMulti-omic analyses of 27 (~ 5000 samples) entities from the TCGA, GEO and in-house data was performed to investigate the molecular determinant of tumor aggressiveness. Using molecular loss of function data, the mechanistic underpinnings of inflammation-induced tumor aggressiveness was addressed.ResultsThe data revealed a significant association between somatic copy number alterations (sCNA) and tumor aggressiveness, with amplification of the transcription factorSOX2being the most important feature among novel and known aggressiveness-associated genes such asZIC5andMYEOV.Mechanistically,SOX2regulates a group of aggressiveness-related genes including theAP1transcription factorFOSL2to sustain pro-inflammatory pathway such asIL6-JAK-STAT3, TNFAandIL17signaling pathways. Prolonged inflammation induces immunosuppression and further leads to activation of cytidine deamination and consequential DNA damage evidenced by enrichment in cytidine deamination mutational signatures in aggressive tumors.The resulting DNA damage affects tumor suppressor genes such asTP53,which was the most mutated gene in aggressive tumors compared with less aggressive tumors (38% vs 14%), thereby releasing cell cycle control. This was exemplified in Glioblastoma multiform, whereTP53andIDH1mutations are predominant.IDH1mutations were almost only seen in younger patients (>45 years, > 90%) and may explain the previously reported favorable prognosis.ConclusionTaken together, our data demonstrate the implication ofSOX2in promoting DNA damage and genome instability by sustaining inflammation viaFOSL2/IL6,resulting in tumor aggressiveness.

BackgroundFemale breast cancer (BC) is the leading cause of cancer-related deaths among women worldwide and higher mortality rates are observed in developing countries. Data from African Americans women are often used to draw inference in African BC and little is currently known about BC traits in indigenous African (IA) women.MethodsThis study prospectively enrolled 472 female patients with BC from Cameroon and Congo Brazzaville between 2007 and 2018. Patient demographics and clinical phenotypes were documented. Additional BC molecular data was downloaded from the cancer genome atlas (TCGA) and the gene expression omnibus (GEO). Demographic and clinical data from the different IA patient cohorts were analyzed to identify unique BC traits in African women. Molecular data from the TCGA was then analyzed to understand key BC traits identified in IA women.ResultsThe average age at BC diagnosis in IA women was more than 10 years earlier (46.5 ± 12.9) than within the TCGA samples (58.5 ± 13.2). Early onset of BC was observed more in IA women than in the TCGS (51.7% vs 15.6%). There was no difference in mean age at diagnosis between African American and white women. IA women had about 60% of T4 stage tumors compared with less than 5% in the TCGA cohort. Using CNA, mutation and gene expression data from the TCGA, we identified somatic copy number amplifications and upregulation of oncogenes such as CDH6, FOXM1, NAAA and CXCL10 in patients with early onset of BC. Deletion of tumor suppressor with corresponding downregulation was equally observed for TGM3, DMBT1 and MYO18B. TP53 mutations were associated with early BC onset (OR: 2.93, p < 0.01). Amplification and upregulation of the oncogene CDH6, PPP1R9B and SLC1A3 were associated with survival (HR: 0.51, 95% CI; 0.23-1.13, p = 0.096 and HR: 0.36, 95% CI: 0.18-0.68, p = 0.002, and HR: 0.14, 95% CI: 0.03-0.6, p = 0.008, respectively).ConclusionEarly onset of BC in IA women in this study might reflect yet unidentified genomic alterations. Dedicated studies are required to establish the genetic underpinning of BC in IA women to support tailored management strategies

adequate community perception of malaria is crucial to improving prevention, diagnosis, and treatment. This study aimed to determine the caregiver´s perception of the home-based management of childhood malaria in Baneghang and Fombap health areas, Cameroon.a cross-sectional study was carried out to assess the knowledge, attitudes, and practices of home caregivers (HCGs) in Baneghang, a health area under Community Directed Intervention (CDI), and Fombap, a CDI-free health area. Community health workers (CHWs) conducted a census to identify households with under-five children that constituted a sample frame, and then a systematic random sampling was used to select HCGs. Analysis of variance was used to compare the mean score perceptions of HCGs in the management of childhood malaria at the two sites.out of 420 HCGs interviewed, 161 (38.3%), 226 (53.8%), and 271 (64.5%) displayed correct knowledge, positive attitude, and good practice, respectively, towards childhood malaria prevention, diagnosis, and treatment in both study sites. The mean score knowledge of HCGs in Baneghang was significantly higher than that of those in Fombap (7.33 versus 6.94, p0.001). The mean score of attitudes of HCGs towards childhood malaria was also higher in Baneghang than in Fombap (6.59 versus 6.29, p=0.013). However, the mean scores of good practices of HCGs on childhood malaria in both health areas were similar (5.94 versus 5.92, p=0.921).home-based management of childhood malaria seems to have contributed to good knowledge and positive attitudes of HCGs on malaria prevention, diagnosis, and treatment.

Background Antibiotic resistance has contributed to the burden of infectious diseases both in the hospital and community setting, and represents a great threat to public health. Previous studies have revealed the role of reactive oxygen species as intermediate mediators of tissue damage, following antibiotherapies, indicating the need of associating antioxidants to these treatments. Therefore, the present work was designed to study the antibacterial, antifungal and antioxidant activities of extracts and compounds from Rumex abyssinicus Jacq. (Polygonaceae), as well as to investigate the antibacterial mechanisms of action of the most effective agents. Methods The plant extracts were prepared by maceration in organic solvents followed by column chromatography of the EtOAc fraction and purification of different fractions which led to the isolation and characterization of pure compounds. The antimicrobial activities of the extracts/compounds and their combinations with ciprofloxacin and fluconazole were evaluated using the broth microdilution method by determining the minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC). The effects of the extracts on the bacterial cell membrane and microbial respiratory chain dehydrogenase enzyme activity were determined by spectrophotometric methods. Antioxidant activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and gallic acid equivalent antioxidant capacity (GAEAC) assays. Results Chrysophanol (1), physcion (2), Ergosta-6,22-diene-3,5,8-triol (3), emodin (4), 6-hydroxyemodin (citreorosein) (5), chrysophanein (6) and physcionin (7) were isolated from EtOAc fraction of R. abyssinicus and displayed different degrees of antimicrobial activities (MIC = 8–256 μg/mL). The MeOH extract and compounds 2 and 4 exhibited synergistic effects with ciprofloxacin and fluconazole. Compounds 1, 2 and the combined mixture of 6 + 7 displayed the highest antioxidant activity (GAEAC = 83.38–106.03 μg/mL). Conclusion R. abyssinicus is a potential source of antibacterial, antifungal and antioxidant agents. The antibacterial mechanisms of action of the MeOH extract and compound 2 are due to disruption of the cytoplasmic membrane and inhibition of the microbial respiratory chain dehydrogenase enzyme activity. To the best of our knowledge, this is the first report of test samples and ciprofloxacin / fluconazole association against MDR strains. The observed activity of the isolated compounds against bacteria and fungi including MDR strains deserves further exploration.

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., Graphical abstract Image 1, Highlights • There is a need of developing safe macrofilaricide for human onchocerciasis using cattle Onchocerca ochengi. • Skin from naturally infected cattle have been used as a reliable parasite source for development of murine pre-clinical screening models. • The role of source-related factors on O. ochengi production and the success of pre-clinical screening models is assessed. • Worms from positive microfilaridermic cattle are successfully recovered after their implantation into rodents.

Introduction:la pandémie à COVID-19 pose des problèmes de diagnostic biologique qui restent d´actualité dans les pays à faible revenu en général et au Cameroun en particulier. Les tests rapides détectant l´antigène du virus SARS-CoV-2 fiables se présentent comme une alternative importante. L´objectif de notre étude était d´évaluer les performances diagnostiques des tests de diagnostic rapideBIOSYNEX®COVID-19 Ag BSSetBIOSYNEX®COVID-19 Ag+ BSScomparés entre eux, puis comparés au test PCR AmpliQuick®SARS-CoV-2. Méthodes:une étude transversale et comparative a été menée du 27 avril au 29 mai 2021 dans la ville de Douala au Cameroun. Les échantillons étaient constitués de prélèvements nasopharyngés reçus au laboratoire de biologie moléculaire de l'Hôpital Gynéco-Obstétrique et Pédiatrique de Douala. Les paramètres sociodémographiques (âge, profession (footballeurs, voyageurs, autres), statut matrimonial, nationalité), comorbidité et statut connu du COVID-19, ont été enregistrés sur les sites de collecte. Les principaux sites de collecte étaient le District de santé de Deïdo et l'Hôpital Gynéco-Obstétrique et Pédiatrique de Douala. Nous avons effectué le diagnostic du COVID-19 en utilisant les tests de diagnostic rapideBIOSYNEX®COVID-19 Ag BSSetBIOSYNEX®COVID-19 Ag+ BSSque nous avons d´abord comparé entre eux, puis nous les avons comparés au test PCR AmpliQuick®SARS-CoV-2 sur chaque échantillon. L'analyse statistique des données a été réalisée à l'aide des logiciels Microsoft Excel et SPSS version 17. Pour déterminer la sensibilité des deux tests de diagnostic rapide, le modèle bayésien de classe latente a été réalisé sur la médiane avec un intervalle de confiance à 95 %, et p

Introduction:COVID-19 equation in Cameroon is yet to be resolved. There is an urgent need for a rapid response strategy to the increasing demand of polymerase chain reaction (PCR) test results for both patients, travelers and competitors to various games. We assessed the diagnostic performance of the AmpliQuick® SARS-CoV-2 against the classic Reverse transcription polymerase chain reaction (RT-PCR). Methods:a cross-sectional and comparative study was conducted from April 27thto May 29th, 2021 in the city of Douala, Cameroon. The samples consisted of any nasopharyngeal sample received at the Douala Gynaeco-Obstetrics and Pediatric Hospital molecular biology laboratory, regardless of its origin. Sociodemographic parameters (age, profession (footballers, travelers, other), matrimonial status, nationality), comorbidity and known status of COVID-19, were recorded at collection sites. The main collection sites were the Deido Health District and the Douala Gynaeco-Obstetric and Pediatric Hospital. We performed testing using AmpliQuick® SARS-CoV-2 and the classic RT-PCR (Da An Gene Co.Ltd) on each sample during the one month period. Analytical performance parameters were determined. To determine the sensitivity of both methods, the Bayesian latent class model was performed on the median with 95% confidence interval, with p≤0.05 as significant level, as well as Kappa (κ) agreement between tests. An ethical clearance was sought and obtained from the University of Douala Institutional Ethics Committee. Results:a total of 1813 participants were enrolled, with the predominance of male (68.68%) and the age group 31 to 40 years old (31.33%). Most participants were married (53.46%) with only few with known COVID-19 status (5.47%). One thousand eight hundred and ten (1810) tests were performed by AMPLIQUICK® SARS-CoV-2 while only 1107 could be achieved with the classic RT-PCR. Over the study period, it was noted a drastic reduction in the time necessary to render results with the AMPLIQUICK® SARS-CoV-2 from 24 hours to 3 hours. The AMPLIQUICK® SARS-CoV-2 reduced technician hands-on time and its practicability was noticed based on the prefilled and ready-to-use microplates. A prevalence of 1.93% and 1.45% were obtained for AMPLIQUICK® SARS-CoV-2 and the classic RT-PCR respectively. This difference in the prevalence showed that AMPLIQUICK® SARS-CoV-2 (Sensitivity 83.5% [CI=64.6-95.2]) was more accurate than the classic RT-PCR (67.8% [CI=46.6-84.9]). Conclusion:it is time for a change of attitude to scale up the COVID-19 testing ability in Cameroon and the AMPLIQUICK® SARS-CoV-2 is an alternative diagnosis strategy which should help resolve the situation of timely and reliable results.

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., 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 affects millions of people in Sub-Saharan Africa. The current control strategy is the annual mass administration of ivermectin which has accumulated several drawbacks over time, especially the action of the drug is solely microfilaricidal, very long treatment period (15–17 years) and reports of ivermectin losing its efficacy; thus, raising the urgent need for new adulticidal compounds. Our study has established an in vitro platform capable of supporting the growth and development of Onchocerca volvulus for up to 315 days, enabling the observation of parasite developmental processes: moulting (from the infective L3 stage to adults), increase in morphometry, the attachment of adult male and female worms and the potential initiation of nodulogenesis. Moreover, the platform might provide more insight into O. volvulus adult worms behavioural pattern in vitro. Also, 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 developmental stages of this filarial parasite within 315 days.

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.

Background: The Mass Drug Administration of ivermectin for onchocerciasis control has contributed to a significant drop in Loa loa microfilaria loads in humans that led to reduction of the levels of infection in Chrysops vectors. Accurate parasite detection is essential for assessing loiasis transmission; as such a potential alternative or an indirect strategy to address the problem co-endemic loiasis presents to the LF and onchocerciasis elimination programme and also, further reflecting 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, its sensitivity is compromised when the intensity of infection is low. The Loop-mediated isothermal amplification (LAMP) of parasite DNA is an alternative method for detecting infection which offers operational simplicity, rapidity and versatility of visual readout options. This study aimed at validating 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 (30,000mf/ml) microfilarial load. The fed flies were maintained under laboratory conditions for 14 days and then analysed with LAMP for the detection of L. loa infection. In addition, a total of 9,270 flies were collected from the North West, East, and South West regions of Cameroon using sweep nets and subjected to microscopy (7,841 flies) and LAMP (1,291 flies plus 138 nulliparous flies) analyses. Results: LAMP assay successfully detected parasites in Chrysops fed on both low and high microfilariaemic volunteers. Field validation and surveillance studies phase revealed LAMP-based infection rates ranging from 0.5 % to 31.6 %, with the lowest levels in the South West 2 and highest infection rates in South West 1. LAMP assay detected significantly higher infection rates than microscopy in 4 of 5 study sites. Conclusion: This study demonstrated the potential of LAMP as a simple surveillance tool, and is more sensitive than microscopy for the detection of experimental and natural L. loa infections in Chrysops vectors.

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., Author summary The Neglected Tropical Disease, loiasis (tropical eye worm disease) affects 13–15 million individuals in Central Africa. Loiasis has recently been identified as a cause of premature mortality and is a barrier to ivermectin-based elimination of onchocerciasis or lymphatic filariasis where co-infections occur, due to the risk of serious adverse events. Loiasis lacks laboratory preclinical models for drug development, biomarker discovery and inflammatory pathology research. Here we detail the successful development of an experimental technique for the laboratory production of Loa loa infective larvae via injection of purified microfilariae into the thorax of wild-caught tabanid flies that are the natural transmission vector. The high yielding infective larvae produced in the laboratory were validated as biologically viable in culture and in a new mouse infection model whereby adult-stage parasites could be produced. The evidence reported herein is an important step to establishing a full laboratory life cycle of L. loa by passage between animal models and experimental injections of the wild-caught vector, Chrysops.

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.

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 ( 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. Graphical Abstract

BackgroundInfections 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 FindingsPrior 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-MK2) 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-MK2 (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-MK2 co-culture system. Early nodulogenesis was observed in both DMEM–10% FBS–LLC-MK2 and DMEM– 10%NCS–LLC-MK2 systems.Conclusions/SignificanceThe 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 summaryRiver 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 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.

Traditional medicine has remained the most affordable and easily accessible source of treatment in the primary healthcare system of resource-poor communities in Cameroon. Despite the increasing acceptance of ethnoveterinary medicine (EVM) in Cameroon, this rich indigenous knowledge is not adequately documented. The aim of this review is to summarize information on ethnoveterinary medicine practices and medicinal plants used in the treatment of livestock diseases in Cameroon. This study is based on a review of the literature published in scientific journals, books, reports from national, regional and international organizations, theses and conference papers. A review of the literature shows that livestock production in Cameroon is carried out mainly by Fulani herd pastoralists. Parasitic and infectious diseases are the most commonly found diseases. Herbs are the most popular ingredients in the preparation of ethnoveterinary medicines. Modes of administration of ethnoveterinary drugs involve predominantly the oral route, followed by the topical and nasal route. We recorded 138 plants belonging to 69 families and 110 genera that are used in Cameroon to manage livestock diseases. In Cameroon, an overwhelming majority of animal owners throughout the country rely on traditional healthcare practices to keep their animals healthy and to treat them when they are sick. Ethnoveterinary practices play significantly greater roles in livestock health care as an alternative or integral part of modern veterinary practices. Data from this study can be preserved and the utilized plants recorded for sustainable use.

The availability of a safe macrofilaricidal drug would help to accelerate onchocerciasis elimination. A trial was conducted in Cameroon to evaluate the effects of a subcutaneous injectable long-acting formulation of ivermectin (LAFI) on the microfilariae (mf) and adult stages of Onchocerca ochengi. Ten zebu cattle naturally infected with the parasite were injected subcutaneously with either 500 mg (group A, N = 4), or 1000 mg long-acting ivermectin (group B, N = 4) or the vehicle (group C, N = 2). Skin samples were collected from each animal before, and 6, 12, and 24 months after treatment to measure microfilarial densities (MFDs). Nodules excised before, and 6 and 12 months after treatment were examined histologically to assess the adult worms' viability and reproductive status. Blood samples were collected at pre-determined time-points to obtain pharmacokinetic data. Before treatment, the average O. ochengi MFDs were similar in the three groups. Six months after treatment, all animals in groups A and B were free of skin mf, whereas those in group C still showed high MFDs (mean = 324.5 mf/g). Only one ivermectin-treated animal (belonging to group A) had skin mf 12 months after treatment (0.9 mf/g). At 24 months, another animal in group A showed skin mf (10.0 mf/g). The histologic examination of nodules at 6 and 12 months showed that LAFI was not macrofilaricidal but had a strong effect on embryogenesis. The new LAFI regimen might be an additional tool to accelerate the elimination of human onchocerciasis in specific settings.Effets d’une formulation injectable d’ivermectine à activité prolongée sur Onchocerca ochengi chez les bovins zébu.La disponibilité d’un médicament macrofilaricide et sans danger permettrait d’accélérer l’élimination de l’onchocercose. Un essai a été mené au Cameroun pour évaluer les effets d’une formulation injectable en sous-cutané d’ivermectine à activité prolongée (FIAP) sur les microfilaires (mf) et les stades adultes d’Onchocerca ochengi. Dix vaches zébu infectées naturellement par le parasite ont reçu une injection sous-cutanée de 500 mg (groupe A, N = 4) ou de 1000 mg d’ivermectine à activité prolongée (groupe B, N = 4) ou le véhicule (groupe C, N = 2). Des échantillons de peau ont été collectés de chaque animal avant, puis 6, 12 et 24 mois après traitement pour mesurer les densités microfilariennes (DMF). Des nodules prélevés avant et 6 et 12 mois après traitement ont été examinés histologiquement pour évaluer la viabilité et le statut reproductif des vers adultes. Des échantillons de sang ont été prélevés pour obtenir des données de pharmacocinétique. Avant traitement, les DMF à O. ochengi étaient similaires dans les 3 groupes. Six mois après traitement, aucun des animaux des groupes A et B ne présentait de mf dermiques, alors que ceux du groupe C présentaient encore des DMF élevées (moyenne : 324,5 mf/g). Parmi les animaux traités par ivermectine, un seul (du groupe A) avait des mf dermiques 12 mois après traitement (0,9 mf/g). A 24 mois, un autre animal du groupe A avait des mf (10,0 mf/g). L’examen histologique des nodules collectés à 6 et 12 mois montrait que la FIAP n’était pas macrofilaricide mais avait un effet marqué sur l’embryogénèse. La nouvelle FIAP pourrait représenter un outil pour accélérer l’élimination de l’onchocercose dans certaines circonstances spécifiques.

Background: Onchocerciasis is targeted for elimination with annual community-directed treatment with ivermectin (CDTi). High infection levels were reported in South-West Cameroon, despite ≥ 15 years of CDTi. 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 to evaluate factors associated with continued transmission and skin disease. 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 9,456 participants. 5·9% of adults participated in ≥ 75% of CDTi rounds and 17·5% were systematic non-adherers. 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 negatively influenced by fear of adverse events linked with economic loss and concern of lethal adverse events, and positively influenced by perceived health benefits. Interpretation: High onchocerciasis prevalence and morbidity still occur in South-West Cameroon, including among children born in the CDTi era. Because non-adherence is mostly due to community perception of ivermectin-related adverse events, including death and lack of perceived health benefit, CDTi alone is unlikely to achieve elimination in this area of high transmission. Alternative strategies are needed for onchocerciasis elimination where negative perception of ivermectin is an entrenched barrier to community participation in programmes. Funding Statement: Department for International Development, UK. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: This protocol was reviewed and approved by the Liverpool School of Tropical Medicine Research Ethics Committee (reference: 16-027), the Cameroonian National Ethics Committee for Research on Human Health (approval no. 2016/11/838/CE/CNERSH/SP), and the Division of Health Operations Research within the Cameroonian Ministry of Public Health (approval no. 631-03·17). All censused individuals were explained the objectives and procedures of the intervention study. Informed assent was obtained from children and adolescents aged under 18 years with parental consent, and consent was provided by all adult participants.

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 Mansonellosis arises from infections with threadlike filarial nematodes in millions of individuals, especially in sub-Saharan Africa. Since infections present no overt clinical symptoms but attenuate immune responses that might lead to increased susceptibility and worsened disease course of concomitant infections, it is truly a neglected tropical disease. Nevertheless, only few studies focus on identifying suitable safe drugs for its control and little is known about the requirements for in vitro maintenance of the Mansonella perstans transmission stage. This study, therefore, evaluated the survival of M. perstans microfilariae (mf) using in vitro conditions that have been shown to promote survival of Loa loa, a closely related filarial nematode. Furthermore, the in vitro microfilaricidal effect of 15 agents was assessed on this helminth. Methods The ability of two basic culture media; Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) and a monkey kidney epithelial cell line (LLC-MK2) to support the survival of M. perstans microfilariae was investigated. Subsequently, 6 anti-helminthics, 5 anti-malarials, 1 anti-microbacterial, 2 trypanocidals and 1 anti-cancer agent were tested in vitro against mf. The suitability of the culture media as well as the effect of the anti-infective agents on mf survival was assessed by scoring their motility. Results FBS supplement and additional LLC-MK2 cells significantly improved the survival of mf in DMEM and RPMI-1640 culture. In detail, RPMI-1640 supplemented with 10% FBS and LLC-MK2 cells sustained the maintenance of mf for at least 20 days (100.00 ± 0.00% survival). In co-cultures with LLC-MK2 cells without serum, M. perstans mf were maintained in DMEM and RPMI-1640 medium with a motility above 99% by day 5. Mefloquine displayed the highest microfilaricidal effect in vitro followed by artesunate. Conclusion Both RPMI and DMEM in the presence of LLC-MK2 cells are suitable for the maintenance of M. perstans mf in vitro. In absence of the feeder cells, the addition of 10% FBS to RPMI-1640 medium improved the parasite survival rate and motility. The microfilaricidal activity of mefloquine and artesunate on M. perstans mf was documented for the first time in this study and can therefore be considered as reference for further screening of agents against this parasite stage.

Evidence is emerging that shows elevated mental distress and disorder amongst people with several neglected tropical diseases (NTDs). This study aimed to establish the prevalence of depressive symptoms amongst people with podoconiosis and lower limb lymphoedema of other cause in Cameroon. The study was part of a larger research piece that mapped the geographical distribution of podoconiosis in Cameroon. The Patient Health Questionnaire (PHQ-9, mean) was employed to determine the prevalence of depressive symptoms amongst people with lower limb lymphoedema. Linear regression was used to assess the association between socio-demographic characteristics of participants and depressive symptoms. Internal consistency of the PHQ-9 was estimated through Cronbach&rsquo, s alpha (&alpha, = 0.651). The mean PHQ-9 score among people with lower limb lymphoedema was 3.48 (SD &plusmn, 3.25). Using a PHQ-9 score of 5 or above as the cut-off score, 32 participants (38.6%) displayed at least mild depressive symptoms. Unemployment was the only factor that was significantly associated with more depressive symptoms overall. This study shows that depressive symptoms are common amongst people with lower limb lymphoedema in Cameroon. The findings provide support for the integration of psychosocial interventions into packages of care for the management of lower limb lymphoedema.

BackgroundOnchocerciasis 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/designWe 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.ConclusionsUsing 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.