Study of lymphoedema of non-filarial origin in the northwest 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: This cross-sectional, population-based study enrolled all adults (≥15 y) residing in the districts of the northwest region of Cameroon for 10 or more years. Participants were interviewed and had a physical examination. The study outcomes were prevalence estimates of 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 y old) and had lived in the districts of the region for more than 10 y. A total of 2143 lymphoedema cases were identified by CHIs, giving a prevalence of lymphoedema of 1.0% (2143/214 195; 95% CI, 0.96-1.04). After review by experts, podoconiosis prevalence in the study area was 0.48% (1049/214 195; 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 were said to have first noticed swelling at an average age of 41.9±19.1 (range: 6-90 y). Most patients (86.1%; 315/366) complained of their legs suddenly becoming hot, red and painful. The majority (96.5%; 361/374) of the interviewees said they had worn shoes occasionally at some point in their life. The reported mean age at first shoe wearing was 14.2±10.1 (±SD,range 1-77 y). A high proportion (82.8%; 309/374) of the participants wore shoes at the time of interview. Of those wearing shoes, only 21.7% (67/309) were wearing protective shoes. Conclusion: This study provides an 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 lymphoedema.

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

BACKGROUND: Suitable 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. METHODS: In 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. RESULTS: Of 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 (ß = 0.490), both IMDM (ß = 0.256) and DMEM (ß = 0.198) media and the protein supplements NCS (ß = 0.052) and FBS (ß = 0.022); while for L. loa L3, in addition to feeder cells (ß = 0.259) and both IMDM (ß = 0.401) and DMEM (ß = 0.385) media, the protein supplements BSA (ß = 0.029) were found important in maintaining the worm motility. CONCLUSIONS: The 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.

Ivermectin treatment of Loa loa hyper-microfilaraemic baboons (Papio anubis): Assessment of microfilarial load reduction, haematological and biochemical parameters and histopathological changes following treatment.

BACKGROUND: Individuals with high intensity of Loa loa are at risk of developing serious adverse events (SAEs) post treatment with ivermectin. These SAEs have remained unclear and a programmatic impediment to the advancement of community directed treatment with ivermectin. The pathogenesis of these SAEs following ivermectin has never been investigated experimentally. The Loa/baboon (Papio anubis) model can be used to investigate the pathogenesis of Loa-associated encephalopathy following ivermectin treatment in humans. METHODS: 12 baboons with microfilarial loads > 8,000mf/mL of blood were randomised into four groups: Group 1 (control group receiving no drug), Group 2 receiving ivermectin (IVM) alone, Group 3 receiving ivermectin plus aspirin (IVM + ASA), and Group 4 receiving ivermectin plus prednisone (IVM + PSE). Blood samples collected before treatment and at Day 5, 7 or 10 post treatment, were analysed for parasitological, hematological and biochemical parameters using standard techniques. Clinical monitoring of animals for side effects took place every 6 hours post treatment until autopsy. At autopsy free fluids and a large number of standard organs were collected, examined and tissues fixed in 10% buffered formalin and processed for standard haematoxylin-eosin staining and specific immunocytochemical staining. RESULTS: Mf counts dropped significantly (p<0.05) in all animals following ivermectin treatment with reductions as high as (89.9%) recorded; while no significant drop was observed in the control animals. Apart from haemoglobin (Hb) levels which recorded a significant (p = 0.028) drop post treatment, all other haematological and biochemical parameters did not show any significant changes (p>0.05). All animals became withdrawn 48 hours after IVM administration. All treated animals recorded clinical manifestations including rashes, itching, diarrhoea, conjunctival haemorrhages, lymph node enlargement, pinkish ears, swollen face and restlessness; one animal died 5 hours after IVM administration. Macroscopic changes in post-mortem tissues observed comprised haemorrhages in the brain, lungs, heart, which seen in all groups given ivermectin but not in the untreated animals. Microscopically, the major cellular changes seen, which were present in all the ivermectin treated animals included microfilariae in varying degrees of degeneration in small vessels. These were frequently associated with fibrin deposition, endothelial changes including damage to the integrity of the blood vessel and the presence of extravascular erythrocytes (haemorrhages). There was an increased presence of eosinophils and other chronic inflammatory types in certain tissues and organs, often in large numbers and associated with microfilarial destruction. Highly vascularized organs like the brain, heart, lungs and kidneys were observed to have more microfilariae in tissue sections. The number of mf seen in the brain and kidneys of animals administered IVM alone tripled that of control animals. Co-administration of IVM + PSE caused a greater increase in mf in the brain and kidneys while the reverse was noticed with the co-administration of IVM + ASA. CONCLUSIONS: The treatment of Loa hyper-microfilaraemic individuals with ivermectin produces a clinical spectrum that parallels that seen in Loa hyper-microfilaraemic humans treated with ivermectin. The utilization of this experimental model can contribute to the improved management of the adverse responses in humans.

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.

Update on the distribution of Mansonella perstans in the southern part of Cameroon: influence of ecological factors and mass drug administration with ivermectin.

BACKGROUND: Mansonellosis remains one of the most neglected of tropical diseases and its current distribution in the entire forest block of southern Cameroon is unknown. In order to address this issue, we have surveyed the distribution of Mansonella perstans in different bioecological zones and in addition, elucidated the influence of multiple rounds of ivermectin (IVM) based mass drug administration (MDA). METHODS: A mixed design was used. Between 2000 and 2014, both cross-sectional and longitudinal surveys were carried out in 137 communities selected from 12 health districts belonging to five main bioecological zones of the southern part of Cameroon. The zones comprised of grassland savanna (GS), mosaic forest savanna (MFS), forested savanna (FS), deciduous equatorial rainforest (DERF) and the dense humid equatorial rainforest (DHERF). The survey was carried out in some areas with no treatment history as well as those currently under IVM MDA. Individuals within the participatory communities were screened for the presence of M. perstans microfilariae (mf) in peripheral blood by the calibrated thick film method to determine both prevalence and geometric mean intensities at the community level. RESULTS: Apart from sporadic cases in savanna areas, distribution of M. perstans was strongly linked to the equatorial rainforest zones. Before CDTI, the highest mean prevalence (70.0 %) and intensity (17,382.2 mf/ml) were obtained in communities in Mamfes' DHERF areas followed by communities in the DHERF zone of Lolodorf (53.8 % and 7,814.8 mf/ml, respectively). A longitudinal survey in Mamfe further showed that M. perstans infections had reduced by 34.5 % in DERF (P < 0.001) but not DHERF zones after ten years of IVM MDA. Further data from the cross-sectional study revealed that there was a decrease in prevalence in DHERF zones only after ten years of MDA. In DERF zones however, the infection was relatively lower after four years of MDA. CONCLUSIONS: The distribution of M. perstans in the southern part of Cameroon varies with bioecological zones and IVM MDA history. The zones with high prevalence and intensities lie in forested areas while those with low endemicity are in the savanna areas. MDA with ivermectin induced significant reduction in the endemicity of mansonellosis in the decidious equatorial rainforest. In contrast, the prevalence and intensity remained relatively high and stable in the dense humid equatorial rainforest zones even after a decade of mass drug administration with ivermectin. Since it is known that M. perstans down-regulates host's immune system, the findings from this work would be useful in designing studies to understand the impact of M. perstans on host immune response to vaccination and co-infection with other pathogens such as Mycobacterium spp. and Plasmodium spp. in areas of contrasting endemicities.

Relationship between oral declaration on adherence to ivermectin treatment and parasitological indicators of onchocerciasis in an area of persistent transmission despite a decade of mass drug administration in Cameroon.

BACKGROUND: Onchocerciasis control for years has been based on mass drug administration (MDA) with ivermectin (IVM). Adherence to IVM repeated treatment has recently been shown to be a confounding factor for onchocerciasis elimination precisely in rain forest areas where transmission continues and Loa loa co-exists with Onchocerca volvulus. In this study, participants' oral declarations were used as proxy to determine the relationship between adherence to IVM treatment and parasitological indicators of onchocerciasis in the rain forest area of Cameroon with more than a decade of MDA. METHODS: Participants were recruited based on their IVM intake profile with the aid of a semi-structured questionnaire. Parasitological examinations (skin sniping and nodule palpation) were done on eligible candidates. Parasitological indicators were calculated and correlated to IVM intake profile. RESULTS: Of 2,364 people examined, 15.5% had never taken IVM. The majority (40.4%) had taken the drug 1-3 times while only 18% had taken ≥ 7 times. Mf and nodule prevalence rates were still high at 47%, 95% CI [44.9-49.0%] and 36.4%, 95% CI [34.4-38.3%] respectively. There was a treatment-dependent reduction in microfilaria prevalence (rs =-0.986, P = 0.01) and intensity (rs =-0.96, P = 0.01). The highest mf prevalence (59.7%) was found in the zero treatment group and the lowest (33.9%) in the ≥ 7 times treatment group (OR = 2.8; 95% CI [2.09-3.74]; P < 0.001). Adults with ≥ 7 times IVM intake were 2.99 times more likely to have individuals with no microfilaria compared to the zero treatment group (OR = 2.99; 95% CI [2.19-4.08], P < 0.0001). There was no clear correlation between treatment and nodule prevalence and intensity. CONCLUSION: Adherence to ivermectin treatment is not adequate in this rain forest area where L. loa co-exists with O. volvulus. The prevalence and intensity of onchocerciasis remained high in individuals with zero IVM intake after more than a decade of MDA. Our findings show that using parasitological indicators, reduction in prevalence is IVM intake-dependent and that participants' oral declaration of treatment adherence could be relied upon for impact studies. The findings are discussed in the context of challenges for the elimination of onchocerciasis in this rain forest area.

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.

Situation analysis of parasitological and entomological indices of onchocerciasis transmission in three drainage basins of the rain forest of South West Cameroon after a decade of ivermectin treatment.

BACKGROUND: Community-Directed Treatment with Ivermectin (CDTI) is the main strategy adopted by the African Programme for Onchocerciasis control (APOC). Recent reports from onchocerciasis endemic areas of savannah zones have demonstrated the feasibility of disease elimination through CDTI. Such information is lacking in rain forest zones. In this study, we investigated the parasitological and entomological indices of onchocerciasis transmission in three drainage basins in the rain forest area of Cameroon [after over a decade of CDTI]. River basins differed in terms of river number and their flow rates; and were characterized by high pre-control prevalence rates (60-98%). METHODS: Nodule palpation and skin snipping were carried out in the study communities to determine the nodule rates, microfilarial prevalences and intensity. Simulium flies were caught at capture points and dissected to determine the biting, parous, infection and infective rates and the transmission potential. RESULTS: The highest mean microfilaria (mf) prevalence was recorded in the Meme (52.7%), followed by Mungo (41.0%) and Manyu drainage basin (33.0%). The same trend was seen with nodule prevalence between the drainage basins. Twenty-three (23/39) communities (among which 13 in the Meme) still had mf prevalence above 40%. All the communities surveyed had community microfilarial loads (CMFL) below 10 mf/skin snip (ss). The infection was more intense in the Mungo and Meme. The intensity of infection was still high in younger individuals and children less than 10 years of age. Transmission potentials as high as 1211.7 infective larvae/person/month were found in some of the study communities. Entomological indices followed the same trend as the parasitological indices in the three river basins with the Meme having the highest values. CONCLUSION: When compared with pre-control data, results of the present study show that after over a decade of CDTI, the burden of onchocerciasis has reduced. However, transmission is still going on in this study site where loiasis and onchocerciasis are co-endemic and where ecological factors strongly favour the onchocerciasis transmission. The possible reasons for this persistent and differential transmission despite over a decade of control efforts using ivermectin are discussed.