Urban malaria in sub-Saharan Africa: dynamic of the vectorial system and the entomological inoculation rate.

Sub-Saharan Africa is registering one of the highest urban population growth across the world. It is estimated that over 75% of the population in this region will be living in urban settings by 2050. However, it is not known how this rapid urbanization will affect vector populations and disease transmission. The present study summarizes findings from studies conducted in urban settings between the 1970s and 2020 to assess the effects of urbanization on the entomological inoculation rate pattern and anopheline species distribution. Different online databases such as PubMed, ResearchGate, Google Scholar, Google were screened. A total of 90 publications were selected out of 1527. Besides, over 200 additional publications were consulted to collate information on anopheline breeding habitats and species distribution in urban settings. The study confirms high malaria transmission in rural compared to urban settings. The study also suggests that there had been an increase in malaria transmission in most cities after 2003, which could also be associated with an increase in sampling, resources and reporting. Species of the Anopheles gambiae complex were the predominant vectors in most urban settings. Anopheline larvae were reported to have adapted to different aquatic habitats. The study provides updated information on the distribution of the vector population and the dynamic of malaria transmission in urban settings. The study also highlights the need for implementing integrated control strategies in urban settings.

[Preliminary evaluation of the insecticide susceptibility in Anopheles gambiae and Culex quinquefasciatus from Lobito (Angola), using WHO standard assay]. / Premiers tests OMS d'évaluation de la sensibilité aux insecticides chez Anopheles gambiae et Culex quinquefasciatus à Lobito, Angola.

Field collections of the most common urban mosquito vectors Anopheles gambiae and Culex quinquefasciatus were carried out in June 2003, March 2004 and November 2005 to gather preliminary data on the insecticide susceptibility in mosquitoes from Lobito (Angola) using the WHO standard bioassays. Bioassays were performed on F0 adults emerging from the field larval collections and on unfed adults from landing catches on volunteers. Batches of mosquitoes from three selected locations (Alto Liro, San Jao and Bela Vista) were exposed for 1 hour to several insecticides such as DDT 4%, carbosulfan 0.4%, permethrin 1%, deltamethrin 0.05% and cyfluthrin 0.15%, in order to estimate the immediate knockdown times (kdT50 and kdT95) and the mortality rate after exposure. The results revealed that mosquito susceptibility to insecticides varied depending on the insecticide, the site and the period of collection. The main local malaria vector A. gambiae (both M and S forms) was basically resistant to DDT and susceptible to all pyrethoids, regardless of the period and the site of collections. The overall mortality rate due to DDT was 73% in Alto Liro, 89% in San Jao and varied depending on the period in Bela Vista between 95% in March 2004 and 100% in November 2005. The mortality due to pyrethoids was 100% at all locations, with the kdT50 and KdT95 times ranging between 9 and 16 minutes and between 18 and 29 minutes, respectively. Concerning the C. quinquefasciatus, populations from Yard and Caponte were resistant to all insecticides tested; the mortality rate was 40% with deltamethrin and 70% with permethrin, while no lethal effect was observed with DDT or carbosulfan. In conclusion, despite its probable high resistance to DDT, the main local malaria vector A. gambiae remained fully susceptible to pyrethroids. This could forecast a good biological efficacy of the scheduled vector control interventions in Angola, based on a large-scale distribution of long-lasting, insecticide-treated nets and on the implementation of indoor residual spraying. The local vector control programme must include well-adapted IEC campaigns and full participation of the community for better management of the insecticide resistance in targeted mosquitoes and for better control of malaria vector populations.

Optimization of breeding output for larval stage of Anopheles gambiae (Diptera: Culicidae): prospects for the creation and maintenance of laboratory colony from wild isolates.

Domesticating anopheline species from wild isolates provides an important laboratory tool but requires detailed knowledge of their natural biology and ecology, especially the natural breeding habitats of immature stages. The aim of this study was to determine the optimal values of some parameters of Anopheles gambiae larval development, so as to design a standard rearing protocol of highland isolates, which would ensure: the biggest fourth instars, the highest pupae productivity, the shortest duration of the larval stage and the best synchronization of pupation. The density of larvae, the size of breeding water and the quantity of food supplied were tested for their effect on larval growth. Moreover, three cheap foodstuffs were selected and tested for their capability to improve the breeding yield versus TetraMin® as the standard control. The larval density was a very sensitive parameter. Its optimal value, which was found to be ≈1 cm-2 surface area, yielded a daily pupation peak of 38.7% on day 8 post-oviposition, and a global pupae productivity of 78.7% over a duration range of three days. Anopheles gambiae's larval growth, survival and developmental synchronization were density-dependent, and this species responded to overcrowding by producing smaller fourth instars and fewer pupae, over elongated immature lifetime and duration range of pupae occurrence, as a consequence of intraspecific competition. While shallow breeding waters (<3 cm) produced a higher number of pupae than deeper ones, no effect of the breeding habitat's absolute surface area on larval development was observed. Increasing the daily food supply improved the pupae productivity but also boosted the water pollution level (which was assessed by the biological oxygen demand (BOD) and the chemical oxygen demand (COD)) up to a limit depending on the food quality, above which a rapid increase in larval mortality was recorded. The food quality that could substitute the manufactured baby fish food was obtained with weighed mixture of 1 wheat+1 shrimp+2 fish. On establishing an anopheline mosquito colony in the laboratory, special care should be taken to design and maintain the appropriate optimal values of larval density, water depth, daily diet quantity and nutritional quality.

La presence des bovins comme hotes alternatifs peut-elle modifier le comportement trophique des vecteurs du paludisme en zone de foret?

Des enquetes entomologiques longitudinales ont ete effectuees de novembre 1998 a novembre 2000 en vue de determiner le comportement trophique des anopheles en presence de bovins comme hotes alternatifs a l`homme dans deux localites de la zone de foret du sud Cameroun; Obala et Nkolbisson. Les collectes des moustiques ont eu lieu a l`interieur des habitations par pulverisation intra domiciliaire et a l`exterieur a l`aide de captures sous moustiquaire piege avec comme appats l`homme et le veau. L`origine du repas de sang et l`infectivite des moustiques ont ete identifiees par la technique ELISA (Enzyme Linked Immunosorbent Asay). Au total; 992 anopheles appartenant a 8 especes ont ete collectees au cours de cette etude. An. funestus etait l`espece la plus abondante a Obala tandis qu`An. nili predominait a Nkolbisson. Le taux d`anthropophilie etait plus eleve chez les moustiques collectes a l`interieur des habitations que chez ceux collectes a l`exterieur sous moustiquaire piege. An. gambiae et An. funestus ont montre une nette preference pour l`homme tandis qu`An. nili a pique indifferemment l`homme et le boeuf. An. gambiae etait de loin le vecteur principal dans les deux sites d`etude avec 24 cas d`infections sur les 42 trouves. Ces resultats montrent que les vecteurs en zone de foret peuvent diversifier la source de leur repas de sang en presence d`hotes alternatifs tels que les bovins. Toutefois; il serait important de comprendre si l`introduction de l`elevage de bovins dans cette region pourrait influencer le cycle de transmission du paludisme

Epidemiological Importance of the Anopheles Nili Group of Malaria Vectors in Equatorial Villages of Cameroon; Central Africa

From March 1999 to March 2007; entomological surveys were conducted in eight riverside locations of the equatorial domain in Cameroon; to assess the contribution of Anopheles nili group to malaria transmission. Results showed that the anthropophilic Anopheles nili; Anopheles carnevalei and Anopheles ovengensis were involved in 5-68of the Plasmodium falciparum entomological inoculation rates (EIRs) in all the study sites. Alongside; vectors such as Anopheles Gambiae; Anopheles moucheti; Anopheles funestus; Anopheles marshallii and Anopheles hancocki also took a substantial part to these transmission rates in sympatric conditions. Anopheles nili s.s. was the major vector in villages situated near the Sanaga river; with more than 100 infective bites/person/year. Its true Plasmodium falciparum infections rates ranged from 1.4to 6.1. Anopheles carnevalei and Anopheles ovengensis were mainly distributed in the equatorial cross-bordering areas; where they contributed for less than 30of the Plasmodium falciparum EIRs in villages located along the Nten river and Congo river networks. These findings confirm Anopheles nili s.s. as a major malaria vector mosquito; and also provided more data regarding the epidemiological role of Anopheles carnevalei and Anopheles ovengensis througout the central African region

Molecular differentiation of three closely related members of the mosquito species complex, Anopheles moucheti, by mitochondrial and ribosomal DNA polymorphism.

Distinction between members of the equatorial Africa malaria vector Anopheles moucheti (Evans) s.l. (Diptera: Culicidae) has been based mainly on doubtful morphological features. To determine the level of genetic differentiation between the three morphological forms of this complex, we investigated molecular polymorphism in the gene encoding for mitochondrial cytochrome oxidase b (CytB) and in the ribosomal internal transcribed spacers (ITS1 and ITS2). The three genomic regions revealed sequence differences between the three morphological forms similar in degree to the differences shown previously for members of other anopheline species groups or complexes (genetic distance d = 0.047-0.05 for CytB, 0.084-0.166 for ITS1 and 0.03-0.05 for ITS2). Using sequence variation in the ITS1 region, we set up a diagnostic polymerase chain reaction (PCR) for rapid and reliable identification of each subspecies within the An. moucheti complex. Specimens of An. moucheti s.l. collected in Cameroon, the Democratic Republic of Congo (DRC), Uganda and Nigeria were successfully identified, demonstrating the general applicability of this technique.

Insertion polymorphism of transposable elements and population structure of Anopheles gambiae M and S molecular forms in Cameroon.

The insertion polymorphism of five transposable element (TE) families was studied by Southern blots in several populations of the M and S molecular forms of the mosquito Anopheles gambiae sensu stricto from southern Cameroon. We showed that the mean TE insertion site number and the within-population insertion site polymorphism globally differed between the M and S molecular forms. The comparison of the TE insertion profiles of the populations revealed a significant differentiation between these two molecular forms (0.163 < Phi(ST) < 0.371). We cloned several insertions of a non-LTR retrotransposon (Aara8) that were fixed in one form and absent in the other one. The only insertion that could be clearly located on a chromosome arm mapped to cytological division 6 of chromosome X, confirming the importance of this region in the ongoing speciation between the M and S molecular forms.

Description and bionomics of Anopheles (Cellia) ovengensis (Diptera: Culicidae), a new malaria vector species of the Anopheles nili group from south Cameroon.

Mosquito species of the Anopheles nili group (Diptera: Culicidae) transmit malaria to humans along rivers in Africa. To date, the An. nili group includes the species Anopheles nili s.s. and its pale-winged variant known as the "Congo form," Anopheles somalicus and Anopheles carnevalei. Larval and adult mosquito collections in the forest region of Campo, in southern Cameroon, uncovered an additional morphological variant provisionally called "Oveng form" that was subsequently found to be genetically distinct from the other members of the An. nili group. In this study, we provide further biological data that characterizes this new taxon and justifies elevation to specific rank. We propose calling this new species Anopheles ovengensis, after its geographical origin. We present a morphological description of the adult female and fourth instars and original data on the biology, ecology, and role as a human malaria vector of this new species in its type location. We provide dichotomous keys for identification of adult females and fourth instars that can be used at least in tropical areas of west and central Africa.

[Development of a PCR (polymerase chain reaction) specific for complex Anopheles nili (Theobald) 1904 species in Cameroon ]. / Développement d'une PCR (polymerase chain reaction) spécifique d'espèces du complexe Anopheles nili (Théobald) 1904 au Cameroun.

The objective of this study was to develop new molecular tools for the identification of members of An. nili group, a malaria vector in Africa. Our strategy was based on the sequence analysis of portions of the rDNA. The ITS2 fragment of An. nili collected in Cameroon was sequenced and compared. The analysis of these sequences has revealed a great variability of ITS2 sequence. Three molecular forms: An. nili typical form, An. nili Oveng form and An. carnevalei were observed within the six morphological types. Specific primers were selected on ITS2 sequence to develop an allele-specific PCR giving 3 size bands. 169 specimens of An. nili collected in Cameroon were successfully tested. This method has been validated on specimens collected in others localities of tropical Africa. The multiplex PCR developed was very sensitive practical and applicable on large scale.

Comparison of direct and membrane feeding methods to infect Anopheles arabiensis with Plasmodium falciparum.

Two standard methods are available to infect mosquitoes with malaria parasites: direct feeding through the skin of the gametocyte carrier, and membrane feeding. Anopheles arabiensis collected at larval stages and reared in an insectary were fed in parallel by feeding on Plasmodium falciparum gametocyte carriers and by membrane feeding on venous blood of the same gametocyte carriers. Infection of mosquitoes was assessed at Day 7 post bloodmeal by oocyst count of the mosquito midguts. The following parameters were not significantly different between the two methods: the percentage of gametocyte carriers infective for at least one mosquito (52.4% through the skin versus 57.1% through the membrane), the mean infection rate of mosquitoes (10.0% versus 11.3%), the geometric mean oocyst number per mosquito (2.51 versus 3.83). In conclusion, infection of mosquitoes by membrane feeding was similar to infection by direct feeding. Most of the volunteers preferred venipuncture to mosquito bites.

High heterogeneity in the number of Plasmodium falciparum gametocytes in the bloodmeal of mosquitoes fed on the same host.

To investigate the quantitative distribution of Plasmodiumfalciparum gametocytes into the vector bloodmeal, Anopheles arabiensis mosquitoes were fed on 3 volunteers naturally infected with gametocytes. The content of each mosquito midgut was smeared on a microscope slide and Giemsa stained. The distribution of gametocytes ingested by mosquitoes followed a negative binomial distribution, with apparently constant overdispersion (parameter k +/- S.E. = 3.105 +/- 0.392) for the 3 series. This aggregation of gametocytes in some midguts probably facilitates the conjunction of gametes and fertilization. This suggests that P. falciparum gametocytes in the peripheral blood flow of infected man do not follow an independent, homogeneous pattern but show a significant aggregation.

Gametocytemia and infectivity to mosquitoes of patients with uncomplicated Plasmodium falciparum malaria attacks treated with chloroquine or sulfadoxine plus pyrimethamine.

Plasmodium falciparum gametocytemia and its related infectivity for mosquitoes was studied in 115 patients (median age = 18 years, range = 4-45) with simple malaria attacks who lived in the hypoendemic area of Dakar, Senegal. Patients were included in a 28-day in vivo sensitivity test after treatment with chloroquine (CQ, n = 82) or sulfadoxine plus pyrimethamine (SP, n = 33). The prevalence of resistant infections was 58.5% in those treated with CQ and 0% in those treated with SP. The gametocytemia peaked at day 7 after treatment. The maximal gametocyte prevalence was 38.2% in the CQ-sensitive infection group, 89.6% in the CQ-resistant group, and 97.0% in those treated with SP The maximal geometric mean gametocytemia was 2.19/microl in the CQ-sensitive infection group, 29.12/microl in the CQ-resistant group and 85.55/microl in those treated with SP. The period between appearance of the first clinical symptom and treatment was positively related to gametocyte prevalence at days 0 and 2. Experimental infection of wild Anopheles arabiensis using membrane feeders was performed at days 0 and 7, and mosquito infectivity was measured by oocyst detection on the midgut. At day 0, 14.1% of the patients had infected at least 1 mosquito, and at day 7, this value was 38.5%. The mean percentage of infected mosquitoes was 3.2% at day 0 and 12.6% at day 7. At day 7 after treatment with CQ, the relative risk for patients with resistant infections of infecting anophelines was 4.07 higher than in those with sensitive infections. No difference was observed in infectivity for mosquitoes between RI-type resistance and the RII + RIII-type resistance. A sporonticidal effect of SP was observed at day 7 after treatment. These data show that P. falciparum gametocytes and their infectivity for mosquitoes were differentiated according to the drug used, its efficacy, and the duration of symptoms before treatment; they were not dependent on the density of asexual stages. Prompt treatment of malaria cases performed at the beginning of symptoms could limit the spread of resistant parasites.

Survival and emergence of immature Anopheles arabiensis mosquitoes in market-gardener wells in Dakar, Senegal.

Anopheles arabiensis is the unique species of the An. gambiae complex observed in the wells dug by market-gardeners in the Dakar area. In order to relate the numbers of immature stages and emerging adults mosquitoes, population measurements were performed in eight wells in which An. arabiensis was the only mosquito species. Mean density of immature stages was measured using two sampling methods, the dipping with a tray by giving 50 dips in each well, and the quadrat with a frame on 2 or 3 m2 in each well. The absolute number of emergent adults was obtained by collecting mosquitoes under net-trap covering entirely each wells. The dipping method was quicker and more operational than quadrat method. Density estimations of larvae at stage I to IV did not significantly differed using dipping or quadrat methods. On the contrary, pupal density was underestimated when measured by dipping. Mosquito nets placed over wells increased significantly emergence rate of adults, thus measurement of emerging mosquitoes was possible only the first day following the net putting up. The total number of immature stages in each well was significantly correlated with the number of emergent mosquitoes. The mean number of mosquitoes emerging daily from one well corresponded to 5% of the total number of immature stages. Stage distribution for larvae I to IV and pupae, estimated by quadrat, was respectively 29%, 28%, 22%, 16% et 5% (total = 100%). Taking account the mean duration of various immature stages and the number of emerging mosquitoes by day, the equation of the survivorship curve from larval hatch (excluded) to emergence included was: y = 427.2-136.8 Log x. Therefore the mean mortality at immature stages was 80% i.e. an emerging rate of 20%. The results of this study, associated with those of previous ones, permit to evaluate the average productivity of malaria vectors in market-gardener wells in the Dakar area.

Ecology of larval mosquitoes, with special reference to Anopheles arabiensis (Diptera: Culcidae) in market-garden wells in urban Dakar, Senegal.

The urban area of Dakar, Senegal, contains > 5,000 market-garden wells that provide permanent sites for mosquito larvae, in particular Anopheles arabiensis Patton, the major vector of malaria. A study of the bioecology of mosquito larvae was conducted over 1 yr with a monthly visit to 48 of these wells. Overall, 9,589 larvae were collected of which 80.1% were Culicinae and 11.9% Anophelinae. Larvae from stages III and IV (n = 853) were identified to 10 species. An. arabiensis represented 86% of the anophelines collected and An. ziemanni Grunberg 14%. The most common Culicinae species included Aedeomyia africana Neveu-Lemaire, Culex quinquefasciatus Say, and Mimomyia splendens Theobald. Maximum anopheline abundance was observed at the end of the dry season in June, whereas maximum Culicinae abundance was observed at the end of the rainy season in September. Most wells (67%) did not harbor any An. arabiensis larvae and in the remaining 33% the larval abundance was low, averaging 0.54 larvae in stages III-IV per tray sample. To identify factors that determine the abundance of larvae in these wells, a co-inertia (multivariate) analysis was carried out to account for physicochemical variables (depth, turbidity, temperature, pH, conductivity, Na+, Cl-, HCO3-, CO3--, and NO3- concentrations) and biological variables (abundance of mosquito species, predators [e.g., fish, Dytiscidae, Notonectidae, odonates], molluscs [Bulinus and Biomphalaria], and surface plants [water lettuce, Lemna, and filamentous algae]). The co-inertia analysis indicated that the abundance of An. arabiensis was associated with Cx. quinquefasciatus and Cx. decens for the physiochemical data but was not associated with other mosquito species for floro-faunistic data. The conditions associated with abundant An. arabiensis were warm temperature (28-30 degrees C), clear and not too deep water (< 0.5 m), elevated concentrations of HCO3- and CO3--, low concentrations of NO3- and NaCl, low populations of larvivorous fish and invertebrate predators (notably odonates), the presence of water lettuce, and an absence of Lemna. These results indicate that many contributing factors influence the ecology of the immature stages of An. arabiensis.