LONGITUDINAL EVALUATION OF MALARIA EPIDEMIOLOGY IN AN ISOLATED VILLAGE IN WESTERN THAILAND: I. STUDY SITE AND ADULT ANOPHELINE BIONOMICS.

This is the first in a series of papers describing the epidemiology of malaria in an isolated village in western Thailand. The study site was the village of Kong Mong Tha, located in Sangkhla Buri District, Kanchanaburi Province, Thailand. In this paper we present an overview of the study site and results from our adult anopheline mosquito surveillance conducted over 56 consecutive months from June 1999 until January 2004. The collection site, indoor/outdoor location, parity, biting activity and Plasmodiumfalciparum (Pf) and P. vivax (Pv) infection rates were used to calculate seasonal entomological inoculation rates for the predominant four Anopheles species. A total of 21,566 anophelines representing 28 distinct species and 2 groups that were not identified to species were collected using human bait, with almost 95% of the collection consisting of Anopheles minimus, An. maculatus, An. sawadwongporni and An. barbirostris/campestris. Mosquitoes generally peaked during the wet season, were collected throughout the night, and were collected most often outside (ca. 75%) versus inside (ca. 25%) of houses. Approximately 50% of collected mosquitoes were parous. Overall Plasmodium infection rates were 0.27%, with a total of 16 and 42 pools of Pf- and Pv-positive mosquitoes, respectively. Annual EIRs were 2.3 times higher for Pv than for Pf, resulting in approximately 5.5 and 2.6 infective bites per person per year, respectively. The results suggest An. minimus and An. maculatus are the primary and secondary vectors of Pf and Pv transmission in Kong Mong Tha, while An. sawadwongporni and An. barbirostris/campestris also appear to play a role based on the presence of circumsporozoite protein (CSP) in the head/thorax of the specimens tested.

Semi-Field Evaluation of Metofluthrin-Impregnated Nets on Host-Seeking Aedes aegypti and Anopheles dirus.

The efficacy of a metofluthrin-impregnated net (MIN) known as the "Mushikonazu" on the house entry behavior of female Aedes aegypti and Anopheles dirus mosquitoes was evaluated using a semi-field 50-m tunnel setup. While the MIN is labeled for the control of chironomids and moth flies, this study determined the feasibility of using the device, given its current construction and metofluthrin formulation, as a spatial repellent against mosquitoes. Sentinel and cone bioassays were used to determine the insecticidal effect of the MIN. A spatial activity index (SAI) was calculated to evaluate responses of the mosquitoes. For the spatial repellent evaluation against Ae. aegypti, the overall mean of SAI was slightly less than 0 at wk 1 after the MIN application and then decreased for the last 4 wk showing a preference to treatment tent. For An. dirus, the mean SAI at wk 1 was positive, indicating a presumed repellent effect of the MIN against An. dirus. For the subsequent 4 wk, the SAI was negative, indicating a preference for the MIN. Results suggested that the MIN may not be a promising approach to repel Ae. aegypti and An. dirus under field conditions in Thailand. However, it remains probable that the MIN may be effective as a spatial repellent if modifications are made to the metofluthrin concentration or formulation and/or the construction of the device.

Detection of Dengue Virus in Mosquito Extracts and Human Clinical Samples Using a Field Expedient Molecular Platform.

Dengue fever occurs in localized outbreaks and can significantly erode troop strength and mission readiness. Timely identification of dengue virus (DENV) provides for rapid and appropriate patient management decisions, such as medical evacuation and supportive therapies, as well as help to promote Force Health Protection through vector control and personal protective measures. The "Ruggedized" Advanced Pathogen Identification Device is a field-friendly PCR (Polymerase Chain Reaction) platform that can be used to facilitate early identification of DENV. We developed a dry-format PCR assay on this platform. The assay demonstrated 100% analytical specificity for detecting dengue using a cross-reactivity panel. We used a panel of 102 acute, DENV isolation positive serum samples and 25 DENV negative samples; the assay demonstrated a clinical sensitivity of 97.1% (95% C.I. 91.6-99.4%) and specificity of 96.0% (95% C.I. 79.7-99.9%) in identifying patients with dengue infection. We also used the assay to test mosquito homogenates from 28 adult female Aedes aegypti. A single DENV infected mosquito was identified using the PCR assay and confirmed using immunofluorescence as a reference method. Much of the testing was performed under austere field conditions. Together, our results demonstrate the utility of this assay for detecting DENV in vector and human samples in field environments.

Field detection of Tembusu virus in western Thailand by rt-PCR and vector competence determination of select culex mosquitoes for transmission of the virus.

Tembusu virus (TMUV; Ntaya serocomplex) was detected in two pools of mosquitoes captured near Sangkhlaburi, Thailand, as well as from sera from sentinel ducks from the same area. Although TMUV has been isolated from several mosquito species in Asia, no studies have ever shown competent vectors for this virus. Therefore, we allowed mosquitoes captured near Sangkhlaburi to feed on young chickens that had been infected with TMUV. These mosquitoes were tested approximately 2 weeks later to determine infection, dissemination, and transmission rates. Culex vishnui developed high viral titers after feeding on TMUV-infected chicks and readily transmitted virus to naïve chickens. In contrast, Cx. fuscocephala seemed less susceptible to infection, and more importantly, zero of five fuscocephala with a disseminated infection transmitted virus by bite, indicating a salivary gland barrier. These results provide evidence for the involvement of Culex mosquitoes in the transmission of TMUV in the environment.

Soil analysis around anopheline breeding habitats in north-western Thailand.

The epidemiology of malaria is largely dependent on its vector habitat. Each species of Anopheles larvae has a specific habitat requirement for its development. Anopheline mosquitoes are common throughout Thailand and utilize a wide variety of habitats. The dominant malaria vectors in Thailand are An. dirus, An. maculatus, and An. minimus. The relationship between soil chemical components and the particular species of anopheline in their specific aquatic habitats was studied from September 2002 to July 2003 at Ban Khun Huay, Ban Pa Dae, and Ban Tham Seau in the Mae Sot district, Tak Province, Thailand. Mapping of each habitat was performed using a Global Positioning System unit. A total count of 2,130 laboratory reared adult Anopheles were collected from 138 habitats categorized into 11 different types identified into 18 species from larval sampling in three villages. An. dirus, An. maculatus, and An. minimus were found 5.26%, 10.70%, and 55.31%, respectively, along with other minor species. Drainage and/or season seemed to be associated with the presence of An. dirus, An. maculatus, An. minimus, An. jamesii, An. sawadwongporni, and An. peditaeniatus. Chemical tests: pH, aluminum, magnesium, calcium, and ferric iron showed some associations with the presence of Anopheles. Only drainage was found to be a parameter associated with the presence of An. minimus.

Naturally occurring mixed infection of Plasmodium vivax VK210 and P. vivax VK247 in anopheles mosquitoes (Diptera: Culicidae) in western Thailand.

We report the natural co-infection of a single Anopheles mosquito with Plasmodium vivax Grassi & Feletti phenotypes VK210 and VK247. In total, 8,452 anopheline mosquitoes collected between June 1999 and July 2001 were tested by ELISA for the presence of circumsporozoite (CS) protein to VK210, VK247, and P. falciparum (Welch) (PF). A total of 29 species was represented; however, the predominant species tested were A. minimus Theobald (4,632), A. sawadwongporni Rattanarithikul & Green (1,248), A. maculatus Theobald (1,201), A. campestris Reid (478), and A. barbirostris Van der Wulp (391). A total of 17 positive mosquitoes was identified by ELISA, and included the following: A. minimus infected with VK210 (5), PF (3), and both VK210 and VK247 (1), A. maculatus infected with VK210 (1), VK247 (1), and both VK210 and VK247 (1), A. campestris infected with VK210 (2), A. sawadwongporni infected with VK247 (1) and PF (1), and A. hodgkini Reid infected with VK247 (1). This is the first report of a single mosquito naturally infected with both VK210 and VK247.