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 postinfection (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 munoglobulin 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 mmunoglobulins secretion patterns which might contribute to immunity and protection against reinfection

Study of lymphoedema of non-filarial origin in the north west region of Cameroon: spatial distribution, profiling of cases and socio-economic aspects of podoconiosis

Background: Although podoconiosis is endemic in Cameroon, little is known about its epidemiology and spatial distribution. Methods: In this cross-sectional, population-based study, we enrolled all adults (≥15 years) residing in the districts of North-West Region of Cameroon for more than 10 or more years. Participants were interviewed, had physical examination. The study outcomes were prevalence estimates lymphoedema and podoconiosis. House-to-house screening was conducted by Community Health Implementers (CHIs). CHIs registered all individuals with lymphoedema and collected additional individual and household-related information. A panel of experts re-examined and validated all lymphoedema cases registered by CHIs. Results: Of the 439,781 individuals registered, 214,195 were adults (≥15 years old) and had lived in the districts of the Region for more than 10 years. A total of 2,143 lymphoedema cases, were identified by CHIs, giving a prevalence of lymphoedema 1.0% (95% confidence interval [CI]; 0.96-1.04) (2,143/214,195). After review by experts, podoconiosis prevalence in the study area was 0.48% (1,049/214,195) (95% CI; 0.46-0.52). The prevalence of podoconiosis varied by health district, from 0.16% in Oku to 1.92% in Bafut (p < 0.05). A total of 374 patients were recruited by stratified random sampling from the validated CHIs’ register to assess the clinical features and socio-economic aspects of the disease. Patients reportedly said to have first noticed swelling at an average age of 41.9 ± 19.1 (range: 6-90 years). Most patients (86.1%) complained of their legs suddenly becoming hot, red and painful. The majority (309, 96.5%) of the interviewees said they had worn shoes occasionally at some point in their life. The reportedly mean age at first shoe wearing was 14.2 ± 10.1 (± Standard Deviation), range (1-77 years). A high proportion (82.8%) of the participants wore shoes at the time of interview. Of those wearing shoes, only 67 (21.7%) were wearing protective shoes. Conclusion: This study provides insight into the geographical distribution and epidemiology of podoconiosis in the North West region of Cameroon, yet management is limited. Evidence-informed targeted interventions are needed to manage people with lymphoedem

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

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

Detecting and staging podoconiosis cases in North West Cameroon: positive predictive value of clinical screening of patients by community health workers and researchers

Background The suitability of using clinical assessment to identify patients with podoconiosis in endemic communities has previously been demonstrated. In this study, we explored the feasibility and accuracy of using Community Health Implementers (CHIs) for the large scale clinical screening of the population for podoconiosis in North-west Cameroon. Methods Before a regional podoconiosis mapping, 193 CHIs and 50 health personnel selected from 6 health districts were trained in the clinical diagnosis of the disease. After training, CHIs undertook community screening for podoconiosis patients under health personnel supervision. Identified cases were later re-examined by a research team with experience in the clinical identification of podoconiosis. Results Cases were identified by CHIs with an overall positive predictive value (PPV) of 48.5% [34.1–70%]. They were more accurate in detecting advanced stages of the disease compared to early stages; OR 2.07, 95% CI = 1.15–3.73, p = 0.015 for all advanced stages). Accuracy of detecting cases showed statistically significant differences among health districts (χ2 = 25.30, p = 0.0001). Conclusion Podoconiosis being a stigmatized disease, the use of CHIs who are familiar to the community appears appropriate for identifying cases through clinical diagnosis. However, to improve their effectiveness and accuracy, more training, supervision and support are required. More emphasis must be given in identifying early clinical stages and in health districts with relatively lower PPVs

Antimicrobial and toxicological activities of five medicinal plant species from Cameroon Traditional Medicine

<p>Abstract</p> <p>Background</p> <p>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 <it>in vitro </it>antimicrobial activity of methanolic, ethylacetate (EtOAc) and hexanic fractions of five Cameroonian medicinal plants (<it>Piptadeniastum africana</it>, <it>Cissus aralioides, Hileria latifolia, Phyllanthus muellerianus </it>and <it>Gladiolus gregasius) </it>against 10 pathogenic microorganisms of the urogenital and gastrointestinal tracts.</p> <p>Methods</p> <p>The fractions were screened for their chemical composition and <it>in vivo </it>acute toxicity was carried out on the most active extracts in order to assess their inhibitory selectivity.</p> <p>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 (<it>Staphylococcus aureus, Enterococcus faecalis)</it>, and six Gram negative <it>(Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Shigella flexneri, Salmonella typhi) </it>and two fungal isolates (<it>Candida albicans, Candida krusei)</it>. 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.</p> <p>Results</p> <p>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 <it>Phyllanthus muellerianus </it>and <it>Piptadeniastum africana </it>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 <it>in vivo </it>acute toxicity study carried out on the methanolic extracts of <it>Phyllanthus muellerianus </it>and <it>Piptadeniastrum africana </it>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.</p> <p>Conclusion</p> <p>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.</p> <p>Phyllanthus muellerianus, Piptadeniastum africana, antimicrobial, acute toxicity, kidney and liver function tests, Cameroon Traditional Medicine</p

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

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

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

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). Human strain L. loa mf were isolated from baboon blood using iso-osmotic Percoll gradient, and cultured in RPMI 1640/10% FBS with antimalarial drugs (mefloquine, amodiaquine, artesunate, chloroquine and quinine), anthelmintics (ivermectin, praziquantel, flubendazole and its reduced and hydrolyzed metabolites), two potential trypanocidal agents (fexinidazole and Scynexis-7158) and the anticancer drug imatinib. The drug concentrations used varied between 0.156 μg/ml and 10 μg/ml. Mf motility (CR = 50% immotility) and a metabolic viability assay (MTT) were used to assess the effects of these drugs on the parasites. Mf in control cultures showed only a slight reduction in motility after 5 days of culture. Active inhibition of Loa loa motility was seen with mefloquine and amodiaquine (CR 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. 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

Identification and characterization of Loa loa antigens responsible for cross-reactivity with rapid diagnostic tests for lymphatic filariasis.

The Global Program to Eliminate Lymphatic Filariasis (LF) relies on rapid diagnostic tests (RDTs) to determine where annual mass drug administration for LF is required and when it can be stopped. These tests detect a Wuchereria bancrofti glycoprotein in the blood of infected persons via a carbohydrate moiety recognized by the monoclonal antibodies AD12 and DH6.5. Loiasis cross-reactivity with LF RDTs has recently been recognized as a serious obstacle to LF elimination in loiasis-endemic areas. To better understand the nature of this cross-reactivity, we used the DH6.5 antibody to immunoaffinity purify Loa loa antigens from the sera of individuals with a positive RDT due to loiasis. Immunoblot analysis revealed many circulating AD12/DH6.5-reactive antigens, and proteomic analysis identified multiple L. loa proteins in LF RDT-positive loiasis sera. These included both secreted and somatic proteins, suggesting that they may be released by dying L. loa adult worms and/or microfilariae. Unlike the single high molecular weight W. bancrofti circulating filarial antigen that is reliably present in the blood of persons with bancroftian filariasis, reactive L. loa antigens appeared to be only transiently present in the blood of a subset of persons with loiasis. These key differences between the circulating antigens of W. bancrofti and L. loa can be used to differentiate positive results generated by both species and may lead to improved diagnostic tests for LF and loiasis