Summary In Southeast Asia the biodiversity of Anopheles species in the domestic environment is very high. Only few species are considered major vectors throughout the region, whereas the vector status of other species varies from area to area. Often it is difficult to identify an Anopheles species as a malaria vector in areas with low malaria incidence. The behaviour of Anopheles species largely determines their vector status, and insights into their behaviour are essential to evaluate the appropriateness of vector control measures. This study was conducted in six ecologically different localities in Southeast Asia to rank the different Anopheles species in terms of anthropophily and endophagy in order to estimate their current epidemiological importance. Concurrently, the biting and resting behaviour of the vectors was analysed to evaluate the appropriateness of insecticide‐impregnated bed nets and residual house spraying in vector control. Anopheles dirus A was highly anthropophilic at all sites where it occurred. By contrast, the degree of anthropophily exhibited by An. minimus A depended on availability of cattle. Anopheles campestris, An. nimpe, An. sinensis, An. maculatus, An. aconitus showed a high degree of anthropophily in certain villages, indicating their potential of participating in malaria transmission, although the actual incidence of malaria in the study villages can be fully explained by transmission of the major vectors (An. dirus A, An. minimus A and An. sundaicus). Late biting of An. minimus A and biting activity throughout the night of An. sundaicus favour bed nets as a control method for these species, whilst exophilic and outdoor biting in combination with early feeding behaviour of An. dirus A will make both insecticide‐impregnated bed nets and indoor residual spraying less suitable for controlling this species. Spatial variation in biting and resting behaviour was observed within almost all Anopheles species. These heterogeneities may result in the differences in epidemiological importance and in response to vector control of Anopheles species in different areas. Moreover, environmental changes and changes in human practice are expected to influence the behaviour, hence the role of the different species in malaria transmission. The effect of environmental changes on vector behaviour should be followed up carefully.

BackgroundThe entomological inoculation rate (EIR) is an important indicator in estimating malaria transmission and the impact of vector control. To assess the EIR, the enzyme-linked immunosorbent assay (ELISA) to detect the circumsporozoite protein (CSP) is increasingly used. However, several studies have reported false positive results in this ELISA. The false positive results could lead to an overestimation of the EIR. The aim of present study was to estimate the level of false positivity among different anopheline species in Cambodia and Vietnam and to check for the presence of other parasites that might interact with the anti-CSP monoclonal antibodies.MethodsMosquitoes collected in Cambodia and Vietnam were identified and tested for the presence of sporozoites in head and thorax by using CSP-ELISA. ELISA positive samples were confirmed by a Plasmodium specific PCR. False positive mosquitoes were checked by PCR for the presence of parasites belonging to the Haemosporidia, Trypanosomatidae, Piroplasmida, and Haemogregarines. The heat-stability and the presence of the cross-reacting antigen in the abdomen of the mosquitoes were also checked.ResultsSpecimens (N = 16,160) of seven anopheline species were tested by CSP-ELISA for Plasmodium falciparum and Plasmodium vivax (Pv210 and Pv247). Two new vector species were identified for the region: Anopheles pampanai (P. vivax) and Anopheles barbirostris (Plasmodium malariae). In 88% (155/176) of the mosquitoes found positive with the P. falciparum CSP-ELISA, the presence of Plasmodium sporozoites could not be confirmed by PCR. This percentage was much lower (28% or 5/18) for P. vivax CSP-ELISAs. False positive CSP-ELISA results were associated with zoophilic mosquito species. None of the targeted parasites could be detected in these CSP-ELISA false positive mosquitoes. The ELISA reacting antigen of P. falciparum was heat-stable in CSP-ELISA true positive specimens, but not in the false positives. The heat-unstable cross-reacting antigen is mainly present in head and thorax and almost absent in the abdomens (4 out of 147) of the false positive specimens.ConclusionThe CSP-ELISA can considerably overestimate the EIR, particularly for P. falciparum and for zoophilic species. The heat-unstable cross-reacting antigen in false positives remains unknown. Therefore it is highly recommended to confirm all positive CSP-ELISA results, either by re-analysing the heated ELISA lysate (100°C, 10 min), or by performing Plasmodium specific PCR followed if possible by sequencing of the amplicons for Plasmodium species determination.

Summary During the last decade, major progress in malaria control has been achieved in Vietnam, Laos and Cambodia. However, malaria is still a potentially fatal disease in some hilly‐forested areas and continues to be endemic in a few coastal foci. To estimate the risk that stems from the major vectors after a decade of intensive malaria control, an entomological study based on human landing collections was conducted between April 1998 and November 2000 in six study villages (four in Vietnam, one in Cambodia and one in Laos) located in different physio‐geographical areas. Five villages were selected in places where new cases of malaria still occurred. In the sixth village, in the northern hilly area of Vietnam, no case of malaria was detected during the past 3 years. In three study villages of the hilly forested areas of Cambodia and central Vietnam, Anopheles dirus A still played an important role in malaria transmission and maintain perennial transmission inside the villages despite its low density. Anopheles minimus A was found in all study villages except in the southern coastal village of Vietnam. Its role in malaria transmission, however, varied between localities and surveys. In one study village of central Vietnam it was almost absent (one specimen collected over 480 man nights), and in another village sporozoite positive specimens (2.8%) were only observed during the first two surveys whereas this species disappeared from the collections from November 1998 onwards (six surveys: 360 man nights). In the northern study site An. minimus A and C were found in all collections, but no local malaria transmission occurred. However, the constant presence of these two species associated with a high longevity (parous rate up around 80% and 65%, respectively), suggests that transmission can occur at almost any time if parasite reservoirs are reintroduced in the area. The proper management of malaria cases and population movement is, therefore, important to prevent outbreaks and the reintroduction of malaria in northern Vietnam. In the study site of the Mekong delta, An. sundaicus occurred at high densities (up to 190 bites/man/night). The recent changes in land use from rice cultivation to shrimp farming probably explains the increase of this brackish water breeding species during the study period. However, none of the 11 002 specimens was positive for Plasmodium circumsporozoite protein (CSP). The relative low survival rate as estimated by the parous rate (around 47 %) may reflect its low vectorial status that could explain the very low malaria incidence (1.9 case/100 persons/year) in this study site. A calculated sporozoite rate of maximum 1/300 000 is enough to explain this low malaria incidence. Despite the successes in malaria control, the vector An. dirus A continues to play an important role in malaria transmission, whereas An. minimus A showed temporal and spatial variation in its role as vector. The role of An. sundaicus as vector could not be confirmed because of the low incidence in the coastal study villa...

BackgroundIn Vietnam, malaria is becoming progressively restricted to specific foci where human and vector characteristics alter the known malaria epidemiology, urging for alternative or adapted control strategies. Long-lasting insecticidal hammocks (LLIH) were designed and introduced in Ninh Thuan province, south-central Vietnam, to control malaria in the specific context of forest malaria. An entomological study in this specific forested environment was conducted to assess the behavioural patterns of forest and village vectors and to assess the spatio-temporal risk factors of malaria transmission in the province.MethodsFive entomological surveys were conducted in three villages in Ma Noi commune and in five villages in Phuoc Binh commune in Ninh Thuan Province, south-central Vietnam. Collections were made inside the village, at the plot near the slash-and-burn fields in the forest and on the way to the forest. All collected mosquito species were subjected to enzyme-linked immunosorbent assay (ELISA) to detect Plasmodium in the head-thoracic portion of individual mosquitoes after morphological identification. Collection data were analysed by use of correspondence and multivariate analyses.ResultsThe mosquito density in the study area was low with on average 3.7 anopheline bites per man-night and 17.4 culicine bites per man-night. Plasmodium-infected mosquitoes were only found in the forest and on the way to the forest. Malaria transmission in the forested malaria foci was spread over the entire night, from dusk to dawn, but was most intense in the early evening as nine of the 13 Plasmodium positive bites occurred before 21H. The annual entomological inoculation rate of Plasmodium falciparum was 2.2 infective bites per person-year to which Anopheles dirus s.s. and Anopheles minimus s.s. contributed. The Plasmodium vivax annual entomological inoculation rate was 2.5 infective bites per person-year with Anopheles sawadwongporni, Anopheles dirus s.s. and Anopheles pampanai as vectors.ConclusionThe vector behaviour and spatio-temporal patterns of malaria transmission in Southeast Asia impose new challenges when changing objectives from control to elimination of malaria and make it necessary to focus not only on the known main vector species. Moreover, effective tools to prevent malaria transmission in the early evening and in the early morning, when the treated bed net cannot be used, need to be developed.