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Background: Culicoides (Diptera; Ceratoponidae) are tiny, stout, blood-sucking flies with a near worldwide distribution. When present, they are often considered a biting nuisance but in addition, they are involved in the transmission of pathogens to humans, domestic and wild animals. Data on Culicoides species in the South-West region of Cameroon dates back to the 1950s. Over the decades, ecological transformation due to agriculture and deforestation may have affected the population dynamics of Culicoides and therefore our study provides an update of their bio-ecology in the region. Furthermore, the role of various Culicoides species in the transmission of parasitic filariae of the genus Mansonella remains inconclusive in this region. This study was designed to address these unknown issues and expand on current scientific knowledge., Results: Eight species of Culicoides (C. bedfordi, C. inornatipennis, C. fulvithorax, C. grahamii, C. imicola, C. milnei, C. neavei and C. kumbaensis) were collected using light traps and human baits. Culicoides grahamii was the most abundant species, followed closely by C. milnei. Three species (C. milnei, C. grahamii and C. inornatipennis) were common in all observed larval development sites. Only four species (C. inornatipennis, C. fulvithorax, C. grahamii and C. milnei) were collected on humans. Anthropophilic species were more abundant (P < 0.001) in the evening (4-7 pm) when compared to the morning collections (6-9 am). After overnight fly collections using a drop trap with a human microfilaremic donor, C. milnei emerged as the potential host for transmitting Mansonella perstans. Substantial heterogeneity was observed between the trap visiting cycles of the various species (P < 0.001). The biting cycle of the main vector, C. milnei, showed two peaks (10-11 pm and 4-5 am), the highest being 10-11 pm., Conclusions: The Culicoides fauna of the South-West region of Cameroon has not changed significantly since the 1950s. Culicoides milnei was demonstrated to be the major vector of M. perstans in this part of Cameroon. It is essentially a nocturnal species which peaks in abundance between 10 and 11 pm.

Background: The epidemiological profiles of vector-borne diseases, such as malaria, are strongly associated with landscape components. The reduction of malaria burden in endemic and epidemic regions mainly depends on knowledge of the malaria-transmitting mosquito species, populations and behavioural characteristics, as well as malaria exposure risks. This work aimed at carrying out a holistic study in order to characterise Anopheles species in relation to human malaria in seven wetlands along the lower section of the volcanic chain of Cameroon., Results: Eight malaria vectors: Anopheles arabiensis, An. coluzzii, An. funestus (s.s.), An. gambiae, An. hancocki, An. melas, An. nili and An. ziemanni, were found biting humans. Anopheles gambiae was widespread; however, it played a secondary role in the Ndop plain where An. ziemmani was the primary vector species (79.2%). Anophelines were more exophagic (73.6%) than endophagic (26.4%), showing a marked nocturnal activity (22:00-4:00 h) for An. coluzzii and An. gambiae while An. funestus (s.s.) was mostly caught between 1:00 and 6:00 h and An. ziemanni having an early evening biting behaviour (18:00-00:00 h). Female Anopheles were mostly observed to have relative high parity rates (≥ 70%), with the exception of the Meanja site where species parity varies from 46 to 55%. Overall, the transmission level was low with entomological inoculation rates estimated to 0.7 infected bites per person per month (ib/p/mth) in Tiko and Ndop, 1.4 ib/p/mth in Mamfe and 2.24 ib/p/mth in Santchou., Conclusions: The present study represents detailed Anopheles vector characterisation from an understudied area along the volcanic chain of Cameroon with endemic malaria transmission. The significant differences in bionomics and Anopheles species distribution within the studied wetlands, highlights the importance of providing baseline data and an opportunity to assess the outcome of ongoing malaria control interventions in the country.

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 (T 90 = 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 (T 90 = 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 (T 90 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., 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.

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.

Background: The immunochromatographic test (ICT) for lymphatic filariasis is a serological test designed for unequivocal detection of circulating Wuchereria bancrofti antigen. It was validated and promoted by WHO as the primary diagnostic tool for mapping and impact monitoring for disease elimination following interventions. The initial tests for specificity and sensitivity were based on samples collected in areas free of loiasis and the results suggested a near 100% specificity for W. bancrofti. The possibility of cross-reactivity with non-Wuchereria bancrofti antigens was not investigated until recently, when false positive results were observed in three independent studies carried out in Central Africa. Associations were demonstrated between ICT positivity and Loa loa microfilaraemia, but it was not clearly established if these false positive results were due to L. loa or can be extended to other filarial nematodes. This study brought further evidences of the cross-reactivity of ICT card with L. loa and Onchocerca ochengi (related to O. volvulus parasite) using in vivo and in vitro systems., Methods: Two filarial/host experimental systems (L. loa-baboon and O. ochengi-cattle) and the in vitro maintenance of different stages (microfilariae, infective larvae and adult worm) of the two filariae were used in three experiments per filarial species. First, whole blood and sera samples were prepared from venous blood of patent baboons and cattle, and applied on ICT cards to detect circulating filarial antigens. Secondly, larval stages of L. loa and O. ochengi as well as O. ochengi adult males were maintained in vitro. Culture supernatants were collected and applied on ICT cards after 6, 12 and 24 h of in vitro maintenance. Finally, total worm extracts (TWE) were prepared using L. loa microfilariae (Mf) and O. ochengi microfilariae, infective larvae and adult male worms. TWE were also tested on ICT cards. For each experiment, control assays (whole blood and sera from uninfected babon/cattle, culture medium and extraction buffer) were performed., Results: Positive ICT results were obtained with whole blood and sera of L. loa microfilaremic baboons, culture supernatants of L. loa Mf and infective larvae as well as with L. loa Mf protein extracts. In contrast, negative ICT results were observed with whole blood and sera from the O. ochengi-cattle system. Surprisingly, culture supernatant of O. ochengi adult males and total worm extracts (Mf, infective larvae and adult worm) were positive to the test., Conclusions: This study has provided further evidence of L. loa cross-reactivity for the ICT card. All stages of L. loa seem capable of inducing the cross-reactivity. Onchocerca ochengi. can also induce cross-reactivity in vitro, but this is less likely in vivo due to the location of parasite. The availability of the parasite proteins in the blood stream determines the magnitude of the cross-reactivity. The cross-reactivity of the ICT card to these non-W. bancrofti filariae poses some doubts to the reliability and validity of the current map of LF of Central Africa that was generated using this diagnostic tool.

Background: The African Programme for Onchocerciasis Control (APOC) was created to control onchocerciasis as a public health problem in 20 African countries. Its main strategy is community directed treatment with ivermectin. In order to identify all high risk areas where ivermectin treatment was needed, APOC used Rapid Epidemiological Mapping of Onchocerciasis (REMO). REMO has now been virtually completed and we report the results in two articles. The present article reports the mapping of high risk areas where onchocerciasis was a public health problem. The companion article reports the results of a geostatistical analysis of the REMO data to map endemicity levels and estimate the number infected., Methods: REMO consists of three stages: exclusion of areas that are unsuitable for the vector, selection of sample villages to be surveyed in each river basin, and examination of 30 to 50 adults for the presence of palpable onchocercal nodules in each selected village. The survey results and other relevant information were processed in a geographical information system. A panel of experts interpreted the data taking the river-based sampling into account and delineated high risk areas where the prevalence of nodules is greater than 20%., Results: Unsuitable areas were identified in eight countries. In the remaining areas surveys were done in a total of 14,473 sample villages in which more than half a million people were examined. High-risk areas were identified in 18 APOC countries, ranging from small isolated foci to a vast contiguous endemic area of 2 million km2 running across seven countries. In five countries the high risk area covered more than 48% of the total surface area, and 31% to 48% of the population. It is estimated that 86 million people live in high risk areas in the APOC countries., Conclusions: The REMO maps have played a significant role in onchocerciasis control in the 20 APOC countries. All high-risk areas where onchocerciasis used to be a serious public health problem have been clearly delineated. This led to the creation of community-directed treatment projects that by 2012 were providing annual ivermectin treatment to over 80 million people.

Background: Anti-Wolbachia treatment with doxycycline is effective in sterilising and killing adult Onchocerca volvulus nematodes, proving superior to ivermectin and of great potential as an alternative approach for the treatment and control of onchocerciasis, particularly in areas of Loa loa co-endemicity. Nevertheless, the length of the required treatment poses potential logistical problems and risk of poor compliance, raising a barrier to the use of doxycycline in Mass Drug Administration (MDA) strategies. In 2007 and 2008 a feasibility trial of community-directed treatment with doxycycline was carried out in two health districts in Cameroon, co-endemic for O. volvulus and L. loa. With 17,519 eligible subjects, the therapeutic coverage was 73.8% with 97.5% compliance, encouraging the feasibility of using doxycycline community-directed delivery in restricted populations of this size. The current study evaluated the effectiveness of this community-directed delivery of doxycycline four years after delivery., Findings: Infection with O. volvulus was evaluated by skin biopsy and nodule palpation. Of the 507 subjects recruited, 375 had completed the treatment with doxycycline followed by one or two rounds of annual ivermectin MDA and 132 received one or two rounds of annual ivermectin MDA alone. Statistically significant lower microfilarial prevalence (17.0% [doxycycline plus ivermectin group], 27.0% [ivermectin only group], p = 0.014) and load (p = 0.012) were found in people that had received doxycycline followed by ivermectin compared to those who received ivermectin only., Conclusions: This study demonstrates the long-term effectiveness of doxycycline treatment delivered with a community-directed strategy even when evaluated four years after delivery in an area of ongoing transmission. This finding shows that a multi-week course of treatment is not a barrier to community-delivery of MDA in restricted populations of this size and supports its implementation to compliment existing control strategies for onchocerciasis, where needed.

Background: Severe side effects following ivermectin treatment of onchocerciasis in areas of co-endemicity with loaisis have been an impediment for the work of the African Programme for Onchocerciasis Control (APOC) in forested regions of several countries. Doxycycline has been shown to be effective in the treatment of onchocerciasis and has the added advantages of killing adult Onchocerca volvulus but neither adult Loa loa nor their microfilariae. This drug therefore offers great potential for the treatment of onchocerciasis in areas of co-endemicity with loiasis. The limitation of use of this drug is the duration of treatment that may pose a potential problem with therapeutic coverage and compliance with treatment. To benefit from the advantages that doxycycline offers in the treatment of onchocerciasis, it will be necessary to establish an effective distribution system that can access remote communities. This study assessed the feasibility of a large-scale distribution of doxycycline for the treatment of onchocerciasis in areas of co-endemicity with loiasis using a community-directed approach., Methods: The study was carried out in 5 health areas co-endemic for Onchocerca volvulus and Loa loa which had no prior experience of the Community Directed Treatment with Ivermectin (CDTI). The community-directed delivery process was introduced using a cascade mechanism from the central health system that passed through the regional health delegation, health district and the health areas. Community health implementers (CHIs) were trained to deliver doxycycline to community members and, under the supervision of the health system, to monitor and document drug intake and side effects., Results: The community members adhered massively to the process. Of the 21355 individuals counted, 17519 were eligible for treatment and 12936 were treated with doxycycline; giving a therapeutic coverage of eligible population of 73.8%. Of the 12936 who started the treatment, 97.5% complied by the end of six weeks. No serious side effect was registered during the six week treatment., Conclusion: This study indicates that when empowered the community health implementers can successfully deliver doxycycline for six weeks for the treatment of onchocerciasis in areas of co-endemicity with loiasis. The therapeutic coverage and the compliance treatment rate achieved in this study coupled to the known efficacy of doxycycline on O. volvulus, are indicators that the strategy involving the mass administration of doxycycline can be used to control onchocerciasis in those areas of co-endemicity with loiasis where ivermectin may be contraindicated.