Monitoring Aedes populations for arboviruses, Wolbachia, insecticide resistance and its mechanisms in various agroecosystems in Benin.

Aedes mosquitoes are the main vectors of arboviruses in Benin. Cases of dengue have been reported in Benin with all four serotypes of the virus actively circulating in this region. Some agricultural settings are known to harbor Aedes vectors responsible for the transmission of arboviruses. The massive use of certain insecticides in agricultural settings has probably contributed to insecticide resistance in these vectors. In Benin, the susceptibility of arbovirus vectors to insecticides is poorly studied. In addition, the distribution of Wolbachia spp., which is used against some arboviruses is unknown. Moreover, there is limited information regarding the vectors responsible for the transmission of arboviruses in Benin. This present study monitored the species composition, arboviruses, and Wolbachia symbiont status, as well as the phenotypic and molecular insecticide resistance profile of Aedes populations from three agroecosystems in Benin. Aedes species identification was performed morphologically and confirmed using qPCR. (RT)-qPCR assay was applied for monitoring the presence of DENV, CHIKV, ZIKV, and WNV pathogens as well as for naturally occurring Wolbachia symbionts. Insecticide resistance was assessed phenotypically, by permethrin (0.75%) exposure of Adults (F0) using World Health Organization (WHO) bioassay protocols, and at the molecular level, using TaqMan (RT)-qPCR assays for assessing knock-down resistance (kdr) mutations (F1534C, V1016G/I, and S989P) and the expression levels of eight detoxification genes (P450s from the CYP9 and CYP6 families, carboxylesterases and glutathione-S-transferases). Aedes aegypti (Ae. aegypti) mosquitoes were the most abundant (93.9%) in the three agroecosystems studied, followed by Aedes albopictus (Ae. albopictus) mosquitoes (6.1%). No arboviruses were detected in the study's mosquito populations. Naturally occurring Wolbachia symbionts were present in 7 pools out of 15 pools tested. This could influence the effectiveness of vector control strategies based on exogenously introduced Wolbachia, all present in the three agroecosystems. Full susceptibility to permethrin was observed in all tested populations of Ae. albopictus. On the contrary, Ae. aegypti were found to be resistant in all three agroecosystem sites except for banana plantation sites, where full susceptibility was observed. Molecular analysis revealed that individual target site resistance kdr mutations F1534C and V1016G/I were detected in most Ae. aegypti populations. Additionally, double mutant (F1534C + V1016G/I) mosquitoes were found in some populations, and in one case, triple mutant (F1534C + V1016G/I + S989P) mosquitoes were detected. Metabolic resistance, as reflected by overexpression of three P450 genes (CYP6BB2, CYP9J26, and CYP9J32), was also detected in Ae. aegypti mosquitoes. Our study provides information that could be used to strategize future vector control strategies and highlights the importance of continuing vector surveillance. Future studies should assess the effect of piperonyl butoxide (PBO) on metabolic resistance and identify the different strains of Wolbachia spp., to choose the best vector control strategies in Benin.

Linking soils and human health: geospatial analysis of ground-sampled soil data in relation to community-level podoconiosis data in North West Cameroon.

BACKGROUND: Podoconiosis is a form of leg swelling, which arises when individuals are exposed over time to red clay soil formed from alkaline volcanic rock. The exact causal agent of the disease is unknown. This study investigates associations between podoconiosis disease data and ground-sampled soil data from North West Cameroon. METHODS: The mineralogy and elemental concentrations were measured in the soil samples and the data were spatially interpolated. Mean soil values were calculated from a 3 km buffer region around the prevalence data points to perform statistical analysis. Analysis included Spearman's rho correlation, binary logistic regression and principal component analysis (PCA). RESULTS: Six elements, barium, beryllium, potassium, rubidium, strontium and thallium, as well as two minerals, potassium feldspar and quartz, were identified as statistically related to podoconiosis. PCA did not show distinct separation between the spatial locations with or without recorded cases of podoconiosis, indicating that other factors such as shoe-wearing behaviour and genetics may significantly influence podoconiosis occurrence and prevalence in North West Cameroon. CONCLUSION: Several soil variables were statistically significantly related to podoconiosis. To further the current study, future investigations will look at the inflammatory pathway response of cells after exposure to these variables.

In Silico Identification of Novel Biomarkers and Development of New Rapid Diagnostic Tests for the Filarial Parasites Mansonella perstans and Mansonella ozzardi.

Mansonelliasis is a widespread yet neglected tropical infection of humans in Africa and South America caused by the filarial nematodes, Mansonella perstans, M. ozzardi, M. rodhaini and M. streptocerca. Clinical symptoms are non-distinct and diagnosis mainly relies on the detection of microfilariae in skin or blood. Species-specific DNA repeat sequences have been used as highly sensitive biomarkers for filarial nematodes. We have developed a bioinformatic pipeline to mine Illumina reads obtained from sequencing M. perstans and M. ozzardi genomic DNA for new repeat biomarker candidates which were used to develop loop-mediated isothermal amplification (LAMP) diagnostic tests. The M. perstans assay based on the Mp419 repeat has a limit of detection of 0.1 pg, equivalent of 1/1000th of a microfilaria, while the M. ozzardi assay based on the Mo2 repeat can detect as little as 0.01 pg. Both LAMP tests possess remarkable species-specificity as they did not amplify non-target DNAs from closely related filarial species, human or vectors. We show that both assays perform successfully on infected human samples. Additionally, we demonstrate the suitability of Mp419 to detect M. perstans infection in Culicoides midges. These new tools are field deployable and suitable for the surveillance of these understudied filarial infections.

Climate change and altitudinal structuring of malaria vectors in south-western Cameroon: their relation to malaria transmission.

An entomological survey was conducted in Cameroon between October 2004 and September 2005, in nine localities targeted for malaria vector control based on adult productivity and variability. Mosquitoes were collected by human-landing catches (HLCs) and pyrethrum spray catches. A total of 12 500 anophelines were collected and dissected: Anopheles gambiae s.l. (56.86%), An. funestus s.l. (32.57%), An. hancocki (9.38%), and An. nili (1.18%). Applying PCR revealed that specimens of the An. funestus group were An. funestus s.s. and An. gambiae complex were mostly An. melas and An. gambiae s.s. of the M and S molecular forms with the M forms being the most predominant. The natural distribution patterns of Anopheles species were largely determined by altitude with some species having unique environmental tolerance limits. A human blood index (HBI) of 99.05% was recorded. Mean probability of daily survival of the malaria vectors was 0.92, with annual mean life expectancy of 21.9 days and the expectation of infective life was long with a mean of 7.4 days. The high survival rates suggest a high vector potential for the species. This information enhances the development of a more focused and informed vector control intervention.

The association of adult Onchocerca volvulus with lymphatic vessels.

Immunocytochemical examination of onchocercal nodule tissues containing adult Onchocerca volvulus using immuno-markers for blood and lymphatic vessels (vWF, D2-40, podoplanin, Prox-1, and Lyve1) shows a distinct pattern of distribution of these vessels within nodules. Blood vessels were commonly seen associated with organized lymphoid cellular aggregates in the both the outer and inner areas of the nodules. In contrast, the majority of the lymphatic vessel positivity was seen in the central zone in close apposition to the adult parasites, and the remainder usually associated with microfilariae in the outer areas of the nodule. These findings suggest an intimate relationship between adult O. volvulus and lymphatic vessels, including the likely proliferation of lymphatic endothelial cells (lymphangectasia) akin to that seen with other filariae. These findings indicate that adult O. volvulus may migrate via the lymphatic system, and that clinical manifestations of this disease that involve tissue edema may be the result of the location of these worms in the lymphatic system.

New insights into the biology of filarial infections.

Recent successes in the control of lymphatic filariasis and onchocerciasis need continuing research in order to sustain the achievements and to develop further tools to tackle the new questions that arise when only reduced infection prevalences prevail. In this regard, in a symposium held at the Xth European Multicolloquium of Parasitology (August 2008, Paris) questions such as the impact of filarial immunosuppression, and its lack following filarial control, on the outcome of co-infections were addressed, as were new approaches to treatment with promising drugs such as moxidectin or the antibiotic chemotherapy against Wolbachia endosymbionts in filariae. In particular, longer treatment courses of doxycycline could be carried out by community-directed treatment at high coverage, thus potentially allowing its use in restricted areas with suboptimal responses to ivermectin against onchocerciasis, or in areas with co-infection by loiasis where onchocerciasis or lymphatic filariasis need to be controlled. New, more potent drugs, or eventually vaccines, will be of importance because in many vector-filarial parasite relationships worldwide, transmission efficacy increases with low numbers of ingested microfilariae, and since ivermectin may render treated hosts more susceptible to new infection.

Elephantiasis of non-filarial origin (podoconiosis) in the highlands of north-western Cameroon.

Lymphoedema, a condition of localized fluid retention, results from a compromised lymphatic system. Although one common cause in the tropics is infection with filarial worms, non-filarial lymphoedema, also known as podoconiosis, has been reported among barefoot farmers in volcanic highland zones of Africa, Central and South America and north-western India. There are conflicting reports on the causes of lymphoedema in the highland regions of Cameroon, where the condition is of great public-health importance. To characterise the focus of lymphoedema in the highlands of the North West province of Cameroon and investigate its real causes, a cross-sectional study was carried out on the adults (aged > or =15 years) living in the communities that fall within the Ndop and Tubah health districts. The subjects, who had to have lived in the study area for at least 10 years, were interviewed, examined clinically, and, when possible, checked for microfilaraemia. The cases of lymphoedema confirmed by ultrasonography and a random sample of the other subjects were also tested for filarial antigenaemia. The interviews, which explored knowledge, attitudes and perceptions (KAP) relating to lymphoedema, revealed that the condition was well known, with each study community having a local name for it. Of the 834 individuals examined clinically, 66 (8.1%) had lymphoedema of the lower limb, with all the clinical stages of this condition represented. None of the 792 individuals examined parasitologically, however, had microfilariae of W. bancrofti (or any other filarial parasite) in their peripheral blood, and only one (0.25%) of the 399 individuals tested for the circulating antigens of W. bancrofti gave a positive result. In addition, none of the 504 mosquitoes caught landing on human bait in the study area and dissected was found to harbour any stage of W. bancrofti. These findings indicate that the elephantiasis seen in the North West province of Cameroon is of non-filarial origin.

First report of Rhabdias (Nematoda: Rhabdiasoidea) from lungs of montane chameleons in Cameroon: description of two new species and notes on biology.

The lung nematodes of the genus Rhabdias parasitic in chameleons were previously only known from east Africa and Madagascar. Two new species are described from Cameroon: i) Rhabdias okuensis n. sp., type-host Chamaeleo (Trioceros) quadricomis gracilior, from Mont Oku, is frequent; it resembles R. jarki from Burundi, with a short buccal capsule and a long, thin oesophagus, and is distinguished by its large cervical vesicle and cephalic characters (mouth aperture, papillae). The female parasites are hermaphroditic (spermatozoa identified) and they pierce the lung wall and induce lesions, as R. jarki. In the same locality, another chameleon, C. (T.) w. wiedersheimi also harbours R. okuensis, as demonstrated with the 12S rDNA and coxl gene sequences. ii) R. cristati n. sp., type-host C. (T.) cristatus, from Mount Cameroon, is described from one heavily infected specimen; it resembles R. chamaeleonis from East Africa, and is distinguished by the large buccal capsule and the thick apex of the intestine. The free-living phase, studied in R. okuensis, presents characters of other Rhabdias from chameleons: heterogony, development of larvae through matricidal endotoky, infective larval stages with a thick, rounded caudal extremity, exuvium transformed into a thick cuticular sheeth. Each free-living female produces one larva, as in other African Rhabdias, whereas the female of R. gemellipara, a parasite of a Malagasy chameleon, produces two larvae.

Spatial modelling and the prediction of Loa loa risk: decision making under uncertainty.

Health decision-makers working in Africa often need to act for millions of people over large geographical areas on little and uncertain information. Spatial statistical modelling and Bayesian inference have now been used to quantify the uncertainty in the predictions of a regional, environmental risk map for Loa loa (a map that is currently being used as an essential decision tool by the African Programme for Onchocerciasis Control). The methodology allows the expression of the probability that, given the data, a particular location does or does not exceed a predefined high-risk threshold for which a change in strategy for the delivery of the antihelmintic ivermectin is required.

Epidemiology of concomitant infections due to Loa loa, Mansonella perstans, and Onchocerca volvulus in rain forest villages of Cameroon.

An epidemiological survey was conducted in 16 remote villages of the rain forest of southern Cameroon to ascertain the prevalence and intensity of three species of filariae: Loa loa, Onchocerca volvulus, and Mansonella perstans. We examined 1458 individuals for blood-dwelling microfilariae and 1255 of these were also for the presence of palpable nodules. All the villages surveyed were found highly endemic for onchocerciasis and mansonellosis with prevalence ranging from 28.44% to 87.17% for O. volvulus and 52.48% to 100% for M. perstans. The intensities of infection were also found high for M. perstans with arithmetic means of microfilaremia ranging from 280.94 to 4947.57 mf/ml. The loiasis prevalence was relatively low with value from 2.22% to 19.23%. Males were found more infected than females for the three species of filariae, and the prevalence and intensities of microfilaremia vary differently in males and females at different ages. The three species of filariae displayed different degrees of association in the inhabitants with a low prevalence of co-occurrence between L. loa/O. volvulus and between L. loa/M. perstans. In contrast, there was a high prevalence of co-occurrence between M. perstans and O. volvulus. The implications of the co-occurrence of the three species of filariae in the populations of these remote villages on the intervention programs based on mass treatment with mectizan are discussed.

The migration and localization of Loa loa infective and fourth-stage larvae in normal and immunosuppressed rodents.

The migration and localization of the human filarial parasite Loa loa in laboratory mice (BALB/c and Swiss) and jirds (Meriones unguiculatus) was investigated. The rodents, either left immunocompetent or immunosuppressed with hydrocortisone, were each inoculated subcutaneously or intraperitoneally with 50 or 200 infective, third-stage larvae (L(3)) of L. loa. Groups of the rodents were killed at various times post-infection, up to day 40, to enable histological studies and permit developing larvae to be recovered. Larvae survived and developed for only 1 week in the immunocompetent rodents but for a mean of 3 weeks in the immunosuppressed. Most of the larvae were found in the subcutaneous tissues (81.9%), peritoneal cavity (14.9%), pleural cavity (1.8%) or the lungs and heart (1.3%) and none was detected in the spleen, kidney, intestine, liver or pancreas. Localization of the larvae appeared unaffected by the site of inoculation, the rodent species or strain, or the dose of L(3) used. The recovery of larvae (as a percentage of the number inoculated) was better among the rodents inoculated with 50 L(3) each than among those given four times as many L(3). The results of the histological studies not only confirmed the presence of larvae in the subcutaneous tissue (72.5%), muscles (11.7%) and peritoneal and pleural cavities (7.8%) of the infected rodents but also revealed worms in the lymphatic vessels of the mesentery and spinal cord (7.3%). These results indicate that most L. loa L(3) inoculated into a mammalian host localize in the cutaneous sites and that only a small proportion of them might migrate, using the lymphatic system, into the internal organs. The observation of migrating L. loa larvae in the lymphatic vessel of the meningeal envelope of the spinal cord, albeit in an experimental host, may explain why, in areas where human loiasis is endemic, neurological manifestations occasionally occur in those with L. loa infections.

Co-endemicity of loiasis and onchocerciasis in the South West Province of Cameroon: implications for mass treatment with ivermectin.

Encephalopathy has been reported in Cameroon in individuals heavily infected with Loa loa microfilariae who were treated with ivermectin against onchocerciasis. Prior to the initiation of the community-directed treatment of onchocerciasis with ivermectin (CDTI) in the South West Province of Cameroon, an epidemiological survey of loiasis was conducted (in July 1998-July 1999) to ascertain the intensity of the disease. Thick blood films were made from 1228 blood samples collected during the day. Rapid epidemiological assessment (REA) of onchocerciasis was conducted among 614 individuals in the Upper Bayang area. Sixteen percent of the population examined were carriers of L. loa microfilariae. More males (20.1%) than females (12.4%) were infected. The community mean microfilaraemias of the different villages were low (< 1100 mf/mL). However, 1 person was found harbouring 174,000 mf/mL of blood and 1% of the study population (12) had microfilaraemia > 8100 mf/mL. Results of the REA of onchocerciasis show that 31.3% of the population investigated in the Upper Bayang area have this disease. These findings show that loiasis and onchocerciasis are co-endemic in the area, but the risk of developing encephalopathy after taking ivermectin is small.

New features on the moults and morphogenesis of the human filaria Loa loa by using rodent hosts consequences.

The development of the human filaria Loa loa (Dirofilariinae, Onchocercidae), previously studied in monkeys, was studied using the non permissive hosts-mice and jirds. The development proved to be rapid: moult 3 occurred on day 8 post-inoculation, the adult stage was reached on day 25 and measured at that time 3-3.5 mm in length. As in the other filarioids, the female genital apparatus developed during the fourth stage. A critical analysis of the studies on the development of Onchocercid species was made. The optimal duration of the stages (i.g. the shortest time) was chosen for the comparison. It appeared that the duration of the stage 3 was a constant character in a given species whatever the experimental conditions, whereas moult 4 might be retarded in a non susceptible host. Comparison between the 18 developmental cycles of Onchocercidae in the vertebrate host was made. Two biological types could be distinguished: either the moult 3 occurred on day 2-3 and was followed apparently by a late moult 4 (> or = 50 days), or the moult 3 occurred after about one week of development and it was associated with a less long stage 4 (20-40 days). The first group includes Dirofilaria and Onchocerca, the second group brings together mainly Loa and the Onchocercinae of the Dipetalonema line and related genera (Acanthocheilonema, Brugia, Litomosoides, etc.). The groups thus formed suggest real relationships as they fit with the morphology of the infective stage and the results of a recent molecular analysis of the 5S DNA.

Ivermectin and moxidectin in two filarial systems: resistance of Monanema martini; inhibition of Litomosoides sigmodontis insemination.

Effects of ivermectin and moxidectin were compared on two filarial species: Monanema martini which presents dermal microfilariae and induces Onchocerca-like lesions in its natural murid host Lemniscomys striatus, and Litomosoides sigmodontis (= L. carinii). M. martini microfilariae showed an unusual resistance to ivermectin, in vitro and in vivo; moxidectin was no more efficient. However, the two drugs used at high concentrations deeply altered the uterine embryogenesis, but had no lethal effect on adult filariae. L. sigmodontis blood microfilariae showed a great susceptibility to moxidectin, similar to that previously described for ivermectin. The two drugs also induced a long term effect because they inhibited the insemination of the female filariae. This result reinforces the observations made by other authors on the human parasite, Onchocerca volvulus.

Larval biology of six filariae of the sub-family Onchocercinae in a vertebrate host.

The development of six filariae of the sub-family Onchocercinae-Litomosoides sigmodontis, Acanthocheilonema viteae, Molinema dessetae, Monanema martini, Brugia malayi, B. pahangi-was compared in rodents, following a single inoculation of a low or high dose of infective larvae. Analysis was done with 105 rodents dissected and 53 rodents fixed for histopathology. The percentage of larvae which developed corresponded to the proportion of those which were able to penetrate into the sub-cutaneous lymphatic vessels; this percentage was determined during the first day (phase 1) and was characteristic of the filaria-host pair, and independent of the number of larvae inoculated. It could remain stable for a long time, more than eight months with M. martini (phase 2); the phenomena of regulation appeared later (phase 3). The larvae migrated through the lymphatic system, which represents a medium less protected and thus less aggressive than the blood system. The coelomic cavities, almost devoid of inflammatory cells, represented an ultimate shelter, as well as the joint-cavities (colonized by some Dirofilariinae). Localizations in the cardio-pulmonary blood system were accidental and occurred when, during the migrations, some larvae penetrated into the thoracic channel and arrived in the superior vena cava, then the right ventricle and the pulmonary arteries (the biology of Dirofilaria immitis resulted in a secondary adaptation); such accidents may occur with adult filariae, especially, after drug treatment. One may expect similar events in human filariasis. These "occult" filariae, more frequent than it is usually thought, influence the immunological status and the pathology.