Complex population history of two Anopheles dirus mosquito species in Southeast Asia suggests the influence of Pleistocene climate change rather than human-mediated effects.

Anopheles dirus and Anopheles baimaii are closely related species which feed on primates, particularly humans, and transmit malaria in the tropical forests of mainland Southeast Asia. Here, we report an in-depth phylogeographic picture based on 269 individuals from 21 populations from mainland Southeast Asia. Analysis of 1537 bp of mtDNA sequence revealed that the population history of A. baimaii is far more complex than previously thought. An old expansion (pre-300 kyr BP) was inferred in northern India/Bangladesh with a wave of south-eastwards expansion arriving at the Thai border (ca 135-173 kyr BP) followed by leptokurtic dispersal very recently (ca 16 kyr BP) into peninsular Thailand. The long and complex population history of these anthropophilic species suggests their expansions are not in response to the relatively recent (ca 40 kyr BP) human expansions in mainland Southeast Asia but, rather, fit well with our understanding of Pleistocene climatic change there.

A field study on malaria prevalence in southeastern Laos by polymerase chain reaction assay.

A detection survey for malaria infection by routine microscopy and polymerase chain reaction (PCR) assay was conducted on 336 inhabitants of two villages in Khammouane Province, Lao People's Democratic Republic (Lao PDR), in July 1997. Malaria infection was demonstrated in 58 (17.3%) subjects by microscopy and in 117 (34.8%) by PCR assay. Specimens positive by both methods were frequent in young villagers, suggesting the presence of many subclinical infections in older persons. The most common species of malaria parasite was Plasmodium falciparum (82.9%). Polymerase chain reaction assay detected mixed infections with 2-4 species in 27 specimens (23.1%). The results demonstrate that there are many subclinical malaria infections with low parasite level and infection with all four human malaria species in Lao PDR.

Preliminary studies of Anopheles mosquitos in eight provinces in Lao PDR.

Malaria vector surveys were carried out in 8 provinces in Lao PDR in 1999. The surveys were conducted in 4 provinces - Savannakhet, Champasak, Luang Perbang and Sayaboury in May and in another 4 provinces - Bolikhamsay, Sarvan, Sekong and Vientiane in December 1999. Bare leg collection were carried out indoors and outdoors from 6 pm to 5 am. All anopheline mosquitos were identified, dissected and the gut, gland and ovaries were examined. A total of 438 Anopheles mosquitos belonging to 19 species were obtained. Of these only 3 species were found to be infected with oocysts - An. maculatus, An. dirus and An. minimus. All these species were found biting both indoors and outdoors. An. aconitus was the predominant species obtained in the December collection but its vectorial status remains unknown.

Current status of malaria infection in a southeastern province of Lao PDR.

In Khammouane, a southeastern province in Lao PDR, active case detection (ACD) surveys for malaria infection on the villagers in three villages were conducted four times from December, 1995 to August, 1996. The malaria infection was demonstrated in 5.3-10.5% of the villagers throughout the year in 2 villages located in mountainous forest, but the infection was rarely observed in the other village located in the plain along the Mekong River, in which only 4 malaria cases were detected in August, 1996. Although the positive rate of malaria parasites among the villagers was not so significantly different in the rainy season compared with the dry season, the intensity of infections (ie parasitemia) was significantly higher in the rainy season. More than 90% of the positive villagers were children under 15 years old. Most infections were due to Plasmodium falciparum.

An epidemiological survey on intestinal parasite infection in Khammouane Province, Lao PDR, with special reference to Strongyloides infection.

To estimate the current prevalence of intestinal helminth infection in Lao PDR, a parasitological survey was conducted in three villages in Khammouane Province in December 1996, with special reference to Strongyloides infection. A total of 669 fecal samples were collected in the villages and examined by agar-plate culture method and Kato-Katz thick smear method. Intestinal helminths were demonstrated in 82% of the samples as follows: Ascaris lumbricoides (30.5%), Trichuris trichiura (23.7%), hookworm (28.8%), Strongyloides stercoralis (19.0%), Enterobius vermicuralis (3.4%), Opisthorchis viverrini (56.7%) and Taenia sp (3.0%). The infection rates of S. stercoralis were 27.5% and 18.4% in two rural villages, but only 9.4% in an urban village. The highest prevalence rate of Strongyloides infection was obtained in the age group from 20 to 29 years old, although the infection rate already reached 10% in the age group under 10 years old. The prevalence was consistently higher in male subject than females in almost all age groups.

Plasmodium ovale infections detected by PCR assay in Lao PDR.

Plasmodium ovate infection was demonstrated in 5 out of 143 inhabitants in a village in Lao PDR by blood microscopy and PCR assay. Although the specimen confirmed to be positive for P. ovale by microscopical examination was only one, the target sequences in the 18S rRNA genes of malaria parasite detected in all of the five cases were consisted with those of P. ovale by the PCR assay. This is the first report concerning the presence of so many cases with P. ovale infection in Lao PDR.

An epidemiological study on Opisthorchis viverrini infection in Lao villages.

The prevalence of liver fluke, Opisthorchis viverrini, infection in rural and urban communities was studied in Khammouane Province in Lao PDR. The infection was the commonest among the villagers examined, showing the positive rates of 52.9% and 55.0% in two rural communities and 60.7% in an urban community, respectively. The infection rate reached up to 20% or more within 4 years after birth and increased with age to a plateau in the age group over 20 years. The highest prevalence rate was over 80% in the age group of 35-54 years. The age-related patterns of infection in males and females were almost the same, although there were some sex-related differences by village and by age group. Infection with Opisthorchis viverrini appears to be a serious public health problem strongly associated with the frequent eating habit of raw fish in low land Laos.

Collection of anopheline mosquitos in three villages endemic for malaria in Khammouane, Lao PDR.

Anopheline mosquito collections were made during the months of May and June (dry season) and August (rainy season), 1996 at villages, Nakham, Namdik and Hinboon Neua in Khammouane Province. Larval collections were also made in 3 slow running streams around Nakham villages in May 1996. Altogether, 3,549 adult females consisting 19 Anopheles species were collected by 3 nights of human and 6 animal bait traps and 3 resting collections. Among them, Anopheles vagus (19.75%), An. philippinensis (15.02%), An. nivipes (11.55%), An. pallidus (11.27%), An. peditaeniatus (8.34%), An. kochi (8.00%) and An. barbirostris (7.27%) were predominant species. The immatures of An. barbirostris, An. minimus, An. maculatus sensu lato, and An. culicifacies were found in the streams of Nakham. It is noteworthy that An. minimus, An. dirus and An. maculatus sensu lato, which are well known malaria vectors in Thailand, were recorded for the first time in this area.

Malaria prevalence and a brief entomological survey in a village surrounded by rice fields in Khammouan province, Lao PDR.

We surveyed Nongceng, a village in a south-eastern province of Lao PDR, for malaria and its vectors. Nongceng is situated in a basin and surrounded by rice fields. In February 1998 (dry season), 28.6% of 126 villagers were infected with malaria, and in September 1998 (rainy season), 16.3% of 147 villagers. The prevalence of malaria infection was consistently high in children under 10, and the predominant malaria species was Plasmodium falciparum. In brief surveys of the mosquitoes performed on the same day as the malaria surveys, 2007 Anopheles females from 12 species were collected by means of human bait, animal bait and resting collections. Of the vector species known to be important in transmitting malaria in neighbouring Thailand - An. minimus, An. dirus, and An. maculatus groups - only An. minimus was found. Its density was, however, very low in both seasons and it was therefore unlikely to be the vector. In fact, An. nivipes accounted for more than 65% of all mosquitoes collected and was the most common species collected from human baits. The results of this study show that endemic areas of malaria in Lao PDR are not necessarily related to forest. Rather, An. nivipes is suspected to be the most important vector.