Outdoor malaria transmission in forested villages of Cambodia.

BACKGROUND: Despite progress in malaria control, malaria remains an important public health concern in Cambodia, mostly linked to forested areas. Large-scale vector control interventions in Cambodia are based on the free distribution of long-lasting insecticidal nets (LLINs), targeting indoor- and late-biting malaria vectors only. The present study evaluated the vector density, early biting activity and malaria transmission of outdoor-biting malaria vectors in two forested regions in Cambodia. METHODS: In 2005 two entomological surveys were conducted in 12 villages and their related forest plots in the east and west of Cambodia. Mosquitoes were collected outdoors by human landing collections and subjected to enzyme-linked immunosorbent assay (ELISA) to detect Plasmodium sporozoites after morphological identification. Blood samples were collected in the same villages for serological analyses. Collected data were analysed by the classification and regression tree (CART) method and linear regression analysis. RESULTS: A total of 11,826 anophelines were recorded landing in 787 man-night collections. The majority (82.9%) were the known primary and secondary vectors. Most of the variability in vector densities and early biting rates was explained by geographical factors, mainly at village level. Vector densities were similar between forest and village sites. Based on ELISA results, 29% out of 17 Plasmodium-positive bites occurred before sleeping time, and 65% in the forest plots. The entomological inoculation rates of survey 1 were important predictors of the respective seroconversion rates in survey 2, whereas the mosquito densities were not. DISCUSSION: In Cambodia, outdoor malaria transmission in villages and forest plots is important. In this context, deforestation might result in lower densities of the primary vectors, but also in higher densities of secondary vectors invading deforested areas. Moreover, higher accessibility of the forest could result in a higher man-vector contact. Therefore, additional vector control measures should be developed to target outdoor- and early-biting vectors.

False positive circumsporozoite protein ELISA: a challenge for the estimation of the entomological inoculation rate of malaria and for vector incrimination.

BACKGROUND: The 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. METHODS: Mosquitoes 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. RESULTS: Specimens (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. CONCLUSION: The 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.

Malaria transmission and vector behaviour in a forested malaria focus in central Vietnam and the implications for vector control.

BACKGROUND: In 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. METHODS: Five 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. RESULTS: The 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. CONCLUSION: The 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.

Spatio-temporal patterns in kdr frequency in permethrin and DDT resistant Anopheles gambiae s.s. from Uganda.

The planned upscaling of vector control strategies requires insight into the epidemiological consequences of vector resistance. Therefore, the pyrethroid and DDT resistance status of Anopheles gambiae s.l. was assessed in Uganda from 2004 to 2006, and spatial and seasonal variations in knockdown resistance (kdr) frequencies were analyzed in terms of epidemiological significance. Anopheles gambiae s.l. was DDT and pyrethroid resistant in central and eastern Uganda. The L1014S kdr allele frequencies varied from 3% to 48% in An. gambiae s.s. Although the homozygous resistant genotype was the most prevalent genotype among survivors, the genotypes could not entirely explain the bioassay results. In the dry season, the kdr frequency was significantly higher in Plasmodium falciparum-infected mosquitoes, indicating that mosquitoes bearing a kdr mutation have a better adult survival, hence a higher likelihood of becoming infectious. This study showed that kdr might have an epidemiological impact that could jeopardize the vector control strategies.

A significant increase in kdr in Anopheles gambiae is associated with an intensive vector control intervention in Burundi highlands.

OBJECTIVES AND METHODS: In Burundi, the occurrence of the knock down resistance (kdr) mutation in Anopheles gambiae sensu lato (s.l.) was determined for six consecutive years within the framework of a vector control programme. Findings were also linked with the insecticide resistance status observed with bioassay in An. gambiae s.l. and An. funestus. RESULTS: The proportion of An. gambiae s.l. carrying the East Leu-Ser kdr mutation was 1% before the spraying intervention in 2002; by 2007 it was 86% in sprayed valleys and 67% in untreated valleys. Multivariate analysis showed that increased risk of carrying the kdr mutation is associated with spraying interventions, location and time. In bioassays conducted between 2005 and 2007 at five sites, An. funestus was susceptible to permethrin, deltamethrin and DDT. Anopheles gambiae s.l. remained susceptible or tolerant to deltamethrin and resistant to DDT and permethrin, but only when kdr allele carriers reached 90% of the population. CONCLUSIONS: The cross-resistance against DDT and permethrin in Karuzi suggests a possible kdr resistance mechanism. Nevertheless, the homozygous resistant genotype alone does not entirely explain the bioassay results, and other mechanisms conferring resistance cannot be ruled out. After exposure to all three insecticides, homozygote individuals for the kdr allele dominate among the surviving An. gambiae s.l. This confirms the potential selection pressure of pyrethroids on kdr mutation. However, the high occurrence of the kdr mutation, even at sites far from the sprayed areas, suggests a selection pressure other than that exerted by the vector control programme.

The insecticide resistance status of malaria vectors in the Mekong region.

BACKGROUND: Knowledge on insecticide resistance in target species is a basic requirement to guide insecticide use in malaria control programmes. Malaria transmission in the Mekong region is mainly concentrated in forested areas along the country borders, so that decisions on insecticide use should ideally be made at regional level. Consequently, cross-country monitoring of insecticide resistance is indispensable to acquire comparable baseline data on insecticide resistance. METHODS: A network for the monitoring of insecticide resistance, MALVECASIA, was set up in the Mekong region in order to assess the insecticide resistance status of the major malaria vectors in Cambodia, Laos, Thailand, and Vietnam. From 2003 till 2005, bioassays were performed on adult mosquitoes using the standard WHO susceptibility test with diagnostic concentrations of permethrin 0.75% and DDT 4%. Additional tests were done with pyrethroid insecticides applied by the different national malaria control programmes. RESULTS: Anopheles dirus s.s., the main vector in forested malaria foci, was susceptible to permethrin. However, in central Vietnam, it showed possible resistance to type II pyrethroids. In the Mekong delta, Anopheles epiroticus was highly resistant to all pyrethroid insecticides tested. It was susceptible to DDT, except near Ho Chi Minh City where it showed possible DDT resistance. In Vietnam, pyrethroid susceptible and tolerant Anopheles minimus s.l. populations were found, whereas An. minimus s.l. from Cambodia, Laos and Thailand were susceptible. Only two An. minimus s.l. populations showed DDT tolerance. Anopheles vagus was found resistant to DDT and to several pyrethroids in Vietnam and Cambodia. CONCLUSION: This is the first large scale, cross-country survey of insecticide resistance in Anopheles species in the Mekong Region. A unique baseline data on insecticide resistance for the Mekong region is now available, which enables the follow-up of trends in susceptibility status in the region and which will serve as the basis for further resistance management. Large differences in insecticide resistance status were observed among species and countries. In Vietnam, insecticide resistance was mainly observed in low or transmission-free areas, hence an immediate change of malaria vector control strategy is not required. Though, resistance management is important because the risk of migration of mosquitoes carrying resistance genes from non-endemic to endemic areas. Moreover, trends in resistance status should be carefully monitored and the impact of existing vector control tools on resistant populations should be assessed.

Variation in malaria transmission intensity in seven sites throughout Uganda.

Knowledge of the baseline malaria transmission in a given environment is important to guide malaria control interventions. However, in Uganda, recent information on malaria transmission intensity is lacking. Therefore, a 1-year entomological study was conducted in seven ecologically different sites throughout the country to assess spatial and temporal patterns in malaria transmission intensity. Anopheles gambiae sensu stricto was the main vector in five of the seven study sites, and An. funestus was the most important vector in the two other sites. In a peri-urban village, An. arabiensis contributed substantially to malaria transmission. Clear differences in annual entomological inoculation rates (AEIR) were observed between the study sites, ranging from 4 infective bites per person per year in the southwestern part of the country to >1,500 infective bites per person per year in a swampy area near the Nile River. Between villages with parasite prevalences of >or= 80% in children between 1 and 9 years old, a 4-fold difference in AEIR was observed. Based on the observed behavior of the vectors, insecticide-treated bed nets will be highly effective in controlling malaria. However, in the high transmission areas, additional measures will be needed to reduce the malaria burden to acceptable levels.

Comparison of preservation methods of Rhipicephalus appendiculatus (Acari: Ixodidae) for reliable DNA amplification by PCR.

Five differently preserved groups of adult Rhipicephalus appendiculatus specimens were compared for quality of DNA extracted. Three methods were used to extract DNA from specimens i.e. two simple mosquito validated DNA extraction methods and a tick validated method. Extraction of DNA from tick legs was attempted. The quality of DNA extracted was evaluated by the success of PCR amplification of the ITS2 gene and the mitochondrial COI gene fragment. Fresh specimens (i.e. killed just before extraction) had the highest success of DNA amplification followed by specimens killed in ethanol and subsequently stored in the refrigerator (4 degrees C). There was no significant difference in amplification success between cryopreserved and 70% ethanol preserved specimens. It was possible to amplify DNA from legs of ticks. Sequenced ITS2 amplicon of template obtained from legs of ticks was as legible as those from whole tick extract. The two mosquito validated DNA extraction methods showed a significantly lower amplification success than the tick validated protocol.

Detection of the East and West African kdr mutation in Anopheles gambiae and Anopheles arabiensis from Uganda using a new assay based on FRET/Melt Curve analysis.

BACKGROUND: Appropriate monitoring of vector resistance to insecticides is an integral component of planning and evaluation of insecticide use in malaria control programmes. The malaria vectors Anopheles gambiae s.s. and Anopheles arabiensis have developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, better known as knockdown resistance (kdr). In An. gambiae s.s. and An. arabiensis, two different substitutions in the para-type sodium channel, a L1014F substitution common in West Africa and a L1014S replacement found in Kenya, are linked with kdr. Two different allele-specific polymerase chain reactions (AS-PCR) are needed to detect these known kdr mutations. However, these AS-PCR assays rely on a single nucleotide polymorphism mismatch, which can result in unreliable results. METHODS: Here, a new assay for the detection of knockdown resistance in An. gambiae s.s. and An. arabiensis based on Fluorescence Resonance Energy Transfer/Melt Curve analysis (FRET/MCA) is presented and compared with the existing assays. RESULTS: The new FRET/MCA method has the important advantage of detecting both kdr alleles in one assay. Moreover, results show that the FRET/MCA is more reliable and more sensitive than the existing AS-PCR assays and is able to detect new genotypes. By using this technique, the presence of the East African kdr mutation (L1014S) is shown for the first time in An. arabiensis specimens from Uganda. In addition, a new kdr genotype is reported in An. gambiae s.s. from Uganda, where four An. gambiae s.s. mosquitoes possess both, the West (L1014F) and East (L1014S) African kdr allele, simultaneously. CONCLUSION: The presence of both kdr mutations in the same geographical region shows the necessity of a reliable assay that enables to detect both mutations in one single assay. Hence, this new assay based on FRET/MCA will improve the screening of the kdr frequencies in An. gambiae s.s. and An. arabiensis.

Eco-ethological heterogeneity of the members of the Anopheles minimus complex (Diptera: Culicidae) in Southeast Asia and its consequences for vector control.

The presence of cryptic species within Anopheles minimus s.l. Theobald, one of the most widespread malaria vectors in Southeast Asia, was suggested on the basis of behavioral heterogeneities observed within this taxon. Subsequently, two species, A and C, were recognized. However, the existence of these cryptic species did not explain all observed behavioral heterogeneities within this complex. Besides, data on the behavior of vectors are essential to understand the dynamics of disease transmission and thus evaluate the appropriateness of vector control measures. Different collection methods were used to collect Anopheles species from several localities in Southeast Asia to assess the inter- and intraspecific behavioral divergences of An. minimus A and C. Collection results were subjected to a correspondence analysis. The members of the An. minimus complex were identified by use of the octanol dehydrogenase allozyme profiles or the polymerase chain reaction-restriction fragment length polymorphism assay. Large intraspecific behavioral differences were observed among populations of An. minimus A. These populations belong to the same species on the basis of the applied genetic markers. In northern Vietnam, species A tended to be more zoophilic, whereas in the study sites of south central Vietnam, Cambodia, and Laos it showed marked antropophilic behavior when cattle were scarce. In the most northern study site, An. minimus A showed noteworthy endophilic behavior. An. minimus C was primarily zoophilic and based on this behavior, its role in malaria transmission is questionable. However, it was only found in one locality, so that intraspecific behavioral variation could not be assessed. An. minimus A is able to change its host preference in function of local situations in host availability. Hence, its role in malaria transmission can differ from region to region. Similarly, the impact of vector control on this species may differ between localities. In conclusion, intraspecific behavioral differences in Anopheles species can occur and these behavioral heterogeneities, albeit important for disease transmission and control, are not a priori indicative for the presence of cryptic species.