W09　JSPS malaria research project in Vietnam (in English)

Malaria eradication in Vietnam shows its success in the last decades as malaria has been reduced in the whole country and eliminated in the several provinces. Nevertheless, the disease still remains in the forested-mountainous areas and long-terms threat to the population living in other regions in Viet nam. Since 1987, Malaria has been resurgent with high peak in 1991 when 144 outbreaks with more than one million malaria cases and 4 646 deaths were reported. So the strategy of National Malaria Control Program in the 1991-2006 period aims to reduce malaria morbidity and malaria mortality and develop sustainable factors for malaria control. By 2000, mortality and morbidity had reduced by 97% and 77% respectively, as compared to those in 1991. The number of malaria outbreaks was decreased by 98% compared to 1991. In 2005, mortality and morbidity had reduced by 98% and 68% respectively, as compared to those in 2000. But the number of malaria outbreaks was increased surprisingly. Thus, the challenges for the National Malaria Control Program are still much remain to be done to ensure the sustainable results and success. The problem is big populations as 46% of the country's population living in the malaria endemic areas. The knowledge-behavior, living condition and habit of local people is main factors related directly to malaria disease. In the next five years, the objective of the National Malaria Control Program is to reduce morbidity and mortality by 30% and 25% respectively, and no malaria outbreak occurred.

An endemic state is an existence of parasites. To know the causes that parasites remain there, we first estimate the size and distribution of a parasite pool and explore a possible way of tracing malaria infection. We hypothesized that there should be a group of people highly infected with parasites within an endemic area and that they are a major source providing parasites to mosquitoes with or without any malaria symptoms. We chose villages in Binh Phuoc Province as a research site because we expected to observe the reduction of prevalence. People are presumed to quickly become rich by selling rubber, nuts, pepper and others to the world. We noticed that prevalence was different among communes in our research site. Further, we found there lived extremely highly infected families. Among them, we report two families. One family consisted of six people, examined in a rainy season in 2001. Five out of six were diagnosed falciparum malaria by microscopy. The remaining one was slide negative. By PCR analysis, all the five had a mixed infection. Even in falciparum infection, they showed several genotypes for MSP-1, MSP-2, GLURP and two types for Pfmdr1. The other family consists of 11 people, examined in a dry season in 2006. Seven out of 11 were diagnosed falciparum malaria by microscopy. It was confirmed by PCR. All the seven showed the same type of pfg377, but two types were detected for Pfmdr1.

We investigated distribution of the main malaria vector, Anopheles dirus in eight villages in Binh Phuoc Province, southern Vietnam. The density of An. dirus varied greatly among the villages. An. dirus is generally considered as a forest-dependent species. However, the density in each village was not correlated with the distance from the natural forest and the highest density was observed in a village located more than 20 km from the forest. It appeared that when suitable breeding sites such as small streams covered with bush are available, An. dirus can maintain its population after deforestation. Within the village of the highest density, An. dirus showed aggregated distribution to some houses, i.e. the very high density in a few houses and relatively low density in many houses. Regression analysis indicated the significant negative relationship between the An. dirus abundance in each house and the number of houses in the surrounding area. Regression models fit well to the observed pattern when the surrounding area was set within a radius of 350-550 meters. The result suggests that the An. dirus females are attracted to the houses in a range of several hundred meters and thus they are concentrated where houses are sparse. We discuss the significance of spatially heterogeneous distribution of houses in malaria transmission.

We conducted epidemiological studies in a village in Binh Phuoc Province, southern Vietnam where malaria prevalence has been high. The village consists of 170 households and 90 % of the residents are cashew nuts farmers of an ethnic minority, Stieng. They rarely stay outside in the night except for the harvest season (Feb. to Mar.), and this suggests that malaria infection occur mainly in and around their houses by nocturnal mosquitoes. 87% of households have bednets although the number of bednets is insufficient in many households. Insecticide treatment is done by limited households. Majority of people consult private clinic in the nearest town when they get fever. However, they often stop taking anti-malarial drugs before completing treatment. We will present the results of analysis which aimed to explore human-side factors influencing malaria infection, such as the location, type of house, number of family members, and use of bednets.

The detection of gametocytes in the peripheral human blood is one of the most important measurements of malaria transmission in an epidemiological survey. We attempted to detect gametocytes of Plasmodium falciparum by RT-PCR in a dried blood on filter paper and to evaluate the relevancy between a gametocyte carriage and environmental factors. Specific RT-PCR product for the region 3 of Pfg377 mRNA could be detected as few as one gametocyte/μl in the dried blood obtained from parasite culture. In the field samples analysis, the RT-PCR product for Pfg377 mRNA was observed in 67 of 131 specimens but it was not detected in any blood samples from healthy Vietnamese and Japanese controls. In the RT-PCR analysis, four types of allele of Pfg377 were found among Pfg377 mRNA-positive patients. Patients with a single Pfg377 allele were more frequent than patients with the mixed alleles. On the relevancy between a gametocyte carriage and environmental factors, the higher positivity of gametocytes carriages was found in the single P. falciparum infection compared with mixed Plasmodia infection. In this study, 47 asymptomatic patients were found. Although 18 of 47 asymptomatic patients showed Pfg377 mRNA expression, the positive rate of those in the asymptomatic patients was significantly lower than that in the symptomatic patients. Patients with Pfg377 mRNA expression in both symptomatic single P. falciparum infection and asymptomatic infection groups showed that the level of Th1-related cytokine expression was higher than that of Th2-related cytokine expression.

A mutation from Lys to Thr in the codon 76 of Plasmodium falciparum chloroquine resistant transporter protein (PfCRT) is associated with CQ resistance, and the genotype is used as a molecular marker to monitor the distribution and frequency of the CQ resistant malaria. In the present study, we analyzed the frequency of the mutation in codon 72-76 of the pfcrt gene and 3 microsatellite DNA markers (B5M77, 2E10, PE14F) flanking the pfcrt, using 39 P. falciparum isolates from southern part of Vietnam. Twenty-seven of 39 (69%) isolates showed CQ susceptible genotype coding CVMNK, while other 10 (26%) isolates showed CQ resistance type (CVIET, CVIDT, CVMDT). The other 2 (5%) isolates were proved to be mixed with CQ susceptible/resistant isolates whose genotypes were coding CVMNK/CVIDT. The microsatellite DNA markers of the CQ susceptible isolates were revealed to be highly polymorphic, while those of CQ resistant isolates were less polymorphic. One of the CQ resistant isolates (CVIET) had a different microsatellite DNA pattern from K1 (CQ resistant strain; CVIET) from Thailand. The other CQ resistant isolate types (CVIDT, CVMDT) shared the same microsatellite DNA patterns and these patterns were different from those of the CQ susceptible isolates nor the K1. These results showed that the origin of the CQ resistant isolates in the southern part of Vietnam might not be due to an introduction of the isolates from Thailand. Instead, it is suggested that the CQ resistant mutations possibly occurred in the endemic areas in Vietnam.