Abdel Jelil Njouendou

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

BackgroundInfectious 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.MethodsThe 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.ResultsThe chemical components of each plants 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.ConclusionThese results showed that, all these plants 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

Cross-reactivity of filariais ICT cards in areas of contrasting endemicity of Loa loa and Mansonella perstans in Cameroon: Implications for shrinking of the lymphatic filariasis map in the central African region

Background Immunochromatographic card test (ICT) is a tool to map the distribution of Wuchereria bancrofti. In areas highly endemic for loaisis in DRC and Cameroon, a relationship has been envisaged between high L. loa microfilaria (Mf) loads and ICT positivity. However, similar associations have not been demonstrated from other areas with contrasting levels of L. loa endemicity. This study investigated the cross-reactivity of ICT when mapping lymphatic filariasis (LF) in areas with contrasting endemicity levels of loiasis and mansonellosis in Cameroon. Methodology/Principal Findings A cross-sectional study to assess the prevalence and intensity of W. bancrofti, L. loa and M. perstans was carried out in 42 villages across three regions (East, North-west and South-west) of the Cameroon rainforest domain. Diurnal blood was collected from participants for the detection of circulating filarial antigen (CFA) by ICT and assessment of Mf using a thick blood smear. Clinical manifestations of LF were also assessed. ICT positives and patients clinically diagnosed with lymphoedema were further subjected to night blood collection for the detection of W. bancrofti Mf. Overall, 2190 individuals took part in the study. Overall, 24 individuals residing in 14 communities were tested positive by ICT, with prevalence rates ranging from 0% in the South-west to 2.1% in the North-west. Lymphoedema were diagnosed in 20 individuals with the majority of cases found in the North-west (11/20), and none of them were tested positive by ICT. No Mf of W. bancrofti were found in the night blood of any individual with a positive ICT result or clinical lymphoedema. Positive ICT results were strongly associated with high L. loa Mf intensity with 21 subjects having more than 8,000 L. loa Mf ml/blood (Odds ratio = 15.4; 95%CI: 6.1–39.0; p < 0.001). Similarly, a strong positive association (Spearman’s rho = 0.900; p = 0.037) was observed between the prevalence of L. loa and ICT positivity by area: a rate of 1% or more of positive ICT results was found only in areas with an L. loa Mf prevalence above 15%. In contrast, there was no association between ICT positivity and M. perstans prevalence (Spearman’s rho = - 0.200; p = 0.747) and Mf density (Odds ratio = 1.8; 95%CI: 0.8–4.2; p = 0.192). Conclusions/Significance This study has confirmed the strong association between the ICT positivity and L. loa intensity (Mf/ml of blood) at the individual level. Furthermore, the study has demonstrated that ICT positivity is strongly associated with high L. loa prevalence. These results suggest that the main confounding factor for positive ICT test card results are high levels of L. loa. The findings may indicate that W. bancrofti is much less prevalent in the Central African region where L. loa is highly endemic than previously assumed and accurate re-mapping of the region would be very useful for shrinking of the map of LF distribution.

Evaluation of the cytotoxic activity of extracts from medicinal plants used for the treatment of malaria in Kagera and Lindi regions, Tanzania

A number of medicinal plants used for treatment of malaria in Tanzania have been documented, but information on their safety and efficacy is still based on traditional knowledge accumulated over years and not on pre-clinical and clinical evaluation. The present study aimed to assess the cytotoxic activity of extracts of selected plant species used for treatment of malaria in Tanzania. Ethanol extracts were evaluated for cytoxicity by using MTT assay on LLC-MK2 cells and by brine shrimp lethality assay. Forty five (93.75%) out of 48 crude extracts assessed using LLC-MK2 cells were non-cytotoxic while three extracts (6.25%) were cytotoxic with CC50

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

BackgroundThe 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.MethodsBefore 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.ResultsCases 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).ConclusionPodoconiosis 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.

Evaluation of in vitro culture systems for the maintenance of microfilariae and infective larvae of Loa loa

BackgroundSuitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa.MethodsIn vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco’s modified Eagle’s medium (DMEM) and Iscove’s modified Dulbecco’s medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval.ResultsOf the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T90 of 16.4–19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (β = 0.490), both IMDM (β = 0.256) and DMEM (β = 0.198) media and the protein supplements NCS (β = 0.052) and FBS (β = 0.022); while for L. loa L3, in addition to feeder cells (β = 0.259) and both IMDM (β = 0.401) and DMEM (β = 0.385) media, the protein supplements BSA (β = 0.029) were found important in maintaining the worm motility.ConclusionsThe findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.

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

BackgroundCo-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).MethodsHuman 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.ResultsMf 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.ConclusionsThe 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.

Mansonella perstans microfilaremic individuals are characterized by enhanced type 2 helper T and regulatory T and B cell subsets and dampened systemic innate and adaptive immune responses

The filarial nematode Mansonella perstans is endemic throughout Africa, northern South America and the Caribbean. Interestingly, M. perstans-infected individuals present no distinct clinical picture associated with certain pathology. Due to its relatively silent nature, research on this tropical disease has been neglected, especially M. perstans-driven immune responses. A hindrance in obtaining data on M. perstans-specific responses has been the inability to obtain adult worms since their habitats in serous cavities are difficult to access. Thus, in this study, for the first time, we used Mansonella perstans worm antigen extract as stimulant to obtain filarial-specific recall and immunoglobulin responses from M. perstans microfilaremic individuals (Mp MF+) from Cameroon. Moreover, systemic immune profiles in sera and immune cell composition in peripheral blood from Mp MF+ and amicrofilaremic individuals (Mp MF-) were obtained. Our data reveal that Mp MF+ individuals showed significantly reduced cytokine (IL-4, IL-6 and IL-12p70) and chemokine levels (IL-8 and RANTES), but significantly higher MIP-1β as well as increased M. perstans-specific IgG4 levels compared to Mp MF- individuals. In contrast, upon re-stimulation with worm antigen extract, IFN-γ, IL-13, IL-10 and IL-17A secretion was enhanced in cell cultures from Mp MF+ individuals when compared to those from cultures of healthy European individuals. Moreover, analysis of immune cell composition in peripheral blood from Mp MF+ individuals revealed increased type 2 helper T (Th2), natural killer (NK), regulatory B and T cell (Breg and Treg) subsets but decreased type 1 regulatory T (Tr1) cells. In summary, this study deciphers for the first time, M. perstans-specific immune responses using worm antigen extract and shows that patent M. perstans infections have distinct Th2, Breg and Treg subsets accompanied with reduced systemic innate and adaptive immune responses and dominant filarial-specific IgG4 levels.

Impact of repeated annual community directed treatment with ivermectin on loiasis parasitological indicators in Cameroon: Implications for onchocerciasis and lymphatic filariasis elimination in areas co-endemic with Loa loa in Africa

Background Loiasis is a filarial infection endemic in the rainforest zone of west and central Africa particularly in Cameroon, Gabon, Republic of Congo, and Democratic Republic of the Congo. Repeated treatments with ivermectin have been delivered using the annual community directed treatment with ivermectin (CDTI) approach for several years to control onchocerciasis in some Loa loa-Onchocerca volvulus co-endemic areas. The impact of CDTI on loiasis parasitological indicators is not known. We, therefore, designed this cross sectional study to explore the effects of several rounds of CDTI on parasitological indicators of loiasis. Methodology/Principal findings The study was conducted in the East, Northwest and Southwest 2 CDTI projects of Cameroon. Individuals who consented to participate were interviewed for ivermectin treatment history and enrolled for parasitological screening using thick smears. Ivermectin treatment history was correlated with loiasis prevalence/intensity. A total of 3,684 individuals were recruited from 36 communities of the 3 CDTI projects and 900 individuals from 9 villages in a non-CDTI district. In the East, loiasis prevalence was 29.3% (range = 24.2%–34.6%) in the non-CDTI district but 16.0% (3.3%–26.6%) in the CDTI district with 10 ivermectin rounds (there were no baseline data for the latter). In the Northwest and Southwest 2 districts, reductions from 30.5% to 17.9% (after 9 ivermectin rounds) but from 8.1% to 7.8% (not significantly different after 14 rounds) were registered post CDTI, respectively. Similar trends in infection intensity were observed in all sites. There was a negative relationship between adherence to ivermectin treatment and prevalence/intensity of infection in all sites. None of the children (aged 10–14 years) examined in the East CDTI project harboured high (8,000–30,000 mf/ml) or very high (>30,000 mf/ml) microfilarial loads. Individuals who had taken >5 ivermectin treatments were 2.1 times more likely to present with no microfilaraemia than those with less treatments. Conclusion In areas where onchocerciasis and loiasis are co-endemic, CDTI reduces the number of, and microfilaraemia in L. loa-infected individuals, and this, in turn, will help to prevent non-neurological and neurological complications post-ivermectin treatment among CDTI adherents.

Predicted distribution and burden of podoconiosis in Cameroon

Introduction Understanding the number of cases of podoconiosis, its geographical distribution and the population at risk are crucial to estimating the burden of this disease in endemic countries. We assessed each of these using nationwide data on podoconiosis prevalence in Cameroon. Methods We analysed data arising from two cross-sectional surveys in Cameroon. The dataset was combined with a suite of environmental and climate data and analysed within a robust statistical framework, which included machine learning-based approaches and geostatistical modelling. The environmental limits, spatial variation of predicted prevalence, population at risk and number of cases of podoconiosis were each estimated. Results A total of 214 729 records of individuals screened for podoconiosis were gathered from 748 communities in all 10 regions of Cameroon. Of these screened individuals, 882 (0.41%; 95% CI 0.38 to 0.44) were living with podoconiosis. High environmental suitability for podoconiosis was predicted in three regions of Cameroon (Adamawa, North West and North). The national population living in areas environmentally suitable for podoconiosis was estimated at 5.2 (95% CI 4.7 to 5.8) million, which corresponds to 22.3% of Cameroon’s population in 2015. Countrywide, in 2015, the number of adults estimated to be suffering from podoconiosis was 41 556 (95% CI, 1170 to 240 993). Four regions (Central, Littoral, North and North West) contributed 61.2% of the cases. Conclusion In Cameroon, podoconiosis is more widely distributed geographically than was initially expected. The number of cases and the population at risk are considerable. Expanding morbidity management and follow-up of cases is of utmost necessity. Promotion of footwear use and regular foot hygiene should be at the forefront of any intervention plan.