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.

Laboratory evaluation of the response of Aedes aegypti and Aedes albopictus uninfected and infected with dengue virus to deet.

Laboratory studies were conducted to compare the response of Aedes aegypti (L.) and Aedes albopictus (Skuse) adults, uninfected and infected with four serotypes of dengue virus to a repellent containing 5% N, N-diethyl-3-methylbenzamide (deet). The results showed that mosquitoes infected with the four serotypes of dengue responded similarly to uninfected mosquitoes.

Spatial and temporal patterns in pupal and adult production of the dengue vector Aedes aegypti in Kamphaeng Phet, Thailand.

We investigated how temporal and spatial effects confound the functional relationship between pupal and adult populations of Aedes aegypti and thus the value of pupal numbers as predictors of dengue transmission risk in Kamphaeng Phet, Thailand. We found considerable seasonal shifts in productivity of key containers. Tires contained much less pupae in the dry season than in the wet season. Earthenware jars and cement tanks for washing purposes were consistent producers over the entire study period. Houses in the two villages, with approximately twice as many houses per unit area, were significantly more likely to have adults and pupae. No significant annual, seasonal, or spatial effects on the strength of correlations between pupal and adult populations were found. Except for 2 (of 16) occasions, pupal, and adult populations were correlated strongly in time and space. Our results are consistent with application of the pupal survey technique for assessing dengue transmission risk.

Age-dependent survival of the dengue vector Aedes aegypti (Diptera: Culicidae) demonstrated by simultaneous release-recapture of different age cohorts.

The assumption that mosquito survival remains constant and that it is independent of age was tested with free-ranging Aedes aegypti (L.) (Diptera: Culicidae) by using a "synthetic cohort" mark-release-recapture method. Mosquito age cohorts (1-2, 3-4, and 19-20 d) were released simultaneously in homes in rural Thai villages during dry and rainy seasons (2002). Significant age-dependent effects were detected during the dry and rainy seasons. More young than older mosquitoes were recaptured (1-4 versus 5-20 d). The best predictive fit for recaptures was obtained using a quadratic function of age. Our results provide the first field-based evidence for age-dependent Ae. aegypti mortality in which the death rate increases with advancing age and highlight the need for research on ecological and epidemiological aspects of this process.

ELISA as an alternative tool for epidemiological surveillance for dengue in mosquitoes: a report from Thailand.

BACKGROUND & OBJECTIVES: Dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are the re-emerging infectious diseases caused by the four serotypes of dengue (DEN) virus, type 1 to 4, belonging to the family Flaviviridae and genus Flavivirus. In the absence of a safe and effective mass immunisation, the prevention and control of dengue outbreaks depend upon the surveillance of cases and mosquito vector. The aim of this work is to test enzyme-linked immunosorbent assay (ELISA) tool for the virological surveillance of dengue. METHODS: Virus-infected Aedes mosquitoes were collected from the field in order to serve as an early warning monitoring tool for dengue outbreaks. In a prospective field study conducted from April to September 2000, female adult Aedes mosquitoes were caught from selected dengue-sensitive area in Chombung district, Ratchaburi province and assayed by ELISA. RESULT: Approximately 18.3% were found positive for dengue virus. CONCLUSION: This can imply that ELISA can be an alternative tool for epidemiological surveillance for dengue in mosquitoes.

Leptospirosis in Kamphaeng Phet, Thailand.

Leptospirosis, which is caused by pathogenic spirochetes of the genus Leptospira, is a zoonotic disease of global importance and an emerging health problem. We studied patients suspected of having leptospirosis in Kamphaeng Phet Province, Thailand. Of 106 patients with suspected leptospirosis evaluated at the provincial hospital, 69 (65%) were confirmed positive (titer > or = 1:800 or > or = 4-fold increase in titer) by microscopic agglutination testing. Seventy-seven percent (53) of the cases occurred during the rainy season (June through November). Sera reacted predominantly with Bratislava, Autumnalis, and Icterohaemorrhagiae serovars. The screening Leptospira Dip-S-Ticks test had poor sensitivity (32%) but a specificity of 100% compared with a Leptospira IgM enzyme-linked immunosorbent assay. Leptospirosis was found to be a frequently confirmed cause of morbidity in Kamphaeng Phet Province in those suspected of having the illness.

Population genetic structure of Anopheles maculatus in Thailand.

Anopheles (Cellia) maculatus Theobald is a major malaria vector in southern Thailand and peninsular Malaysia, and previous population genetic studies suggested that mountain ranges act as barriers to gene flow. In this study, we examine the genetic variance among 12 collections of natural populations in southern Thailand by analyzing 7 microsatellite loci. Based on analysis of molecular variance (AMOVA), three geographic populations of An. maculatus are suggested. The southern population exists in western Thailand north of 12 degrees north latitude. Mosquitoes to the south fall into two genetic populations: 1) the middle southern collections located on the west side of the Phuket mountain range between 8 degrees and 10 degrees north latitude, and 2) the southern collections located on the east of the Phuket mountain range located between approximately 6.5 degrees and 11.5 degrees north latitude. AMOVA revealed significant genetic differentiation between northern and middle southern and southern populations. The middle southern population was moderately differentiated from the southern population. Furthermore, gene flow was restricted between proximal collections located on different sides of the Phuket mountain range. Collections separated by 50 km exhibited restriction of gene flow when separated by geographic barriers, whereas greater gene flow was evident among collections 650 km apart but without geographic barriers.

Laboratory and semi-field evaluation of Mosquito Dunks against Aedes aegypti and Aedes albopictus larvae (Diptera: Culicidae).

Laboratory bioassays and semi-field studies were conducted on the efficacy and longevity of Mosquito Dunks (7,000 ITU/mg Bti) in order to determine the concentration-response relationship and the effectiveness on the potency of the Bti product against Aedes mosquito species based on the WHO protocol standard methods and to determine the longevity of release for this product against Ae. aegypti mosquito larvae in water storage containers. This bio-potency study with the late 3rd instar larvae of Ae. aegypti and Ae. albopictus was carried out according to WHO standard protocols. The six concentrations of the Bti product used in each test were replicated 4 times with 25 mosquito larvae. Probit analysis was then used to determine the LC50 and LC95 which was equated with dosages of 1.02 and 1.86 ppm for Ae. aegypti; and 0.39 and 0.84 ppm for Ae. albopictus, which reveals a potency of 382.95 and 303.74 ITU/mg, respectively. The semi-field evaluation of this product in 200-liter earthen jars against 3rd instar larvae of Ae. aegypti showed satisfactory control of greater than 80% at 11 weeks post-treatment.

Dengue knowledge and practices and their impact on Aedes aegypti populations in Kamphaeng Phet, Thailand.

A knowledge, attitude, and practice (KAP) survey and an extensive entomologic survey were conducted in two sub-districts of Kamphaeng Phet province, Thailand, to test the hypothesis that correct dengue knowledge and practice reduce dengue vector populations. We found a negative association between respondents' knowledge of preventive measures and the number of unprotected containers in and around their houses. Knowledge of development sites was positively associated with unprotected containers. No relationships existed between knowledge of dengue and adult mosquito reduction practices. A higher number of unprotected containers increased the likelihood of the house being infested with one or more adult Aedes aegypti. Surprisingly, houses of respondents that used mosquito coils or had screening on doors and windows were significantly more likely to be infested (odds ratio =2.0) with adult Ae. aegypti. We conclude that there is a direct link between knowledge on dengue prevention and container protection practices, whereas measures against adult mosquitoes are used only when people experience a mosquito nuisance problem.

Morphological variations of Anopheles minimus A in Tak Province, Thailand.

Anopheles minimus Theobald is one of the major vectors of malaria throughout the Oriental Region, and it's complex consists of at least 2 sibling species (A and C) in Thailand. This study aimed to determine the morphological variations of wings of An. minimus A and to clarify the specific status of An. minimus in Ban Khun Huay, Ban Pa Dae, and Ban Tham Seau, Mae Sot district, Tak Province, Thailand. Anopheline larvae were collected from the fields between October 2002 and September 2003, allowed to emerge into adults in the laboratory and identified by morphological and molecular characterization. About 1,715 of female An. minimus A were separated into 8 groups based on their wing scale patterns. Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) assay (ribosomal DNA ITS2) confirmed the identification of An. minimus A in all 8 groups.

Identification and characterization of larval and adult anopheline mosquito habitats in the Republic of Korea: potential use of remotely sensed data to estimate mosquito distributions.

BACKGROUND: Plasmodium vivax malaria reemerged in the Republic of Korea in 1993, with more than 2,000 cases reported in the northwestern part of the country over the last 10 years. To better assess the risk of malaria transmission we conducted a surveillance study to identify and characterize the habitats that produce potential Anopheles vector mosquitoes. Immature and adult mosquito collection data were incorporated into a Geographic Information System (GIS) along with remotely sensed satellite imagery, and imagery classified to land use to determine if remote sensing data could be used to estimate mosquito habitats. RESULTS: More than 2,100 anopheline larvae were collected and mapped from 186 immature habitats, which were categorized into 9 types. Anopheles sinensis was the most commonly collected species, representing more than 97% of the specimens, followed by Anopheles pullus 1.0%, Anopheles lesteri 1.0%, and Anopheles sineroides 0.8%. Anopheles sinensis, pullus, and lesteri were found most frequently in rice paddies followed by: ditches, flooded areas, ground pools, wheel tracks, swamps, irrigation canals, and stream margins. Anopheles sineroides was found most commonly in flooded areas. Supervised classification was conducted on a LANDSAT 7 ETM+ image to identify 5-6 land-use classes that were considered to be of interest. The spatial distribution of the larval mosquito collections was overlaid on the land-use image derived from the LANDSAT image, and the number of mosquitoes habitats in each class calculated. On Ganghwa Island 67% of the immature habitats containing Anopheles sinensis were in the Paddy land-use class, although the class only represented 17% of the land area. In Paju District 53% of the immature habitats containing Anopheles sinensis were in the Paddy land-use class which represented only 9 % of the area of the district. There was significant (p < 0.05) correlation between habitats containing all four species and land use on Ganghwa Island; however, only Anopheles sinensis was significantly correlated with land-use in Paju District. In Ganghwa Island almost 50% of the Anopheles sinensis adults were collected in the paddy land-use class, and there was a positive correlation between larval and adult distributions. However, in Paju District adult Anopheles sinensis adults were most commonly collected in the Bare land-use class and only 17% collected in the Paddy class. There was a negative correlation between larval and adult distributions. CONCLUSION: Immature habitats most commonly associated with Anopheles sinensis, Anopheles pullus, Anopheles sineroides and Anopheles lesteri were flooded rice paddies. Adult Anopheles sinensis, the most commonly collected anopheline, were found closely associated with rice habitats on Ganghwa Island but not in Paju District. Both QuickBird and LANDSAT satellite data were used to display spatial data in the form of geographic coverage and descriptive information in the form of relational databases associated with the mapped features. Supervised classification of LANDSAT imagery permitted good separation between Paddy, Forest, and Water land-use classes. The immature collections of Anopheles sinensis were significantly correlated with land-use as determined in the land-use classification in both Ganghwa Island and Paju District. These data suggest that classified remotely sensed data could potentially be used to estimate the distribution of immature and adult mosquito populations in the Republic of Korea.

Bottle and biochemical assays on temephos resistance in Aedes aegypti in Thailand.

The bottle bioassay measuring the time-mortality rate is a simplified procedure for detecting insecticide resistance. It can be used with a biochemical microplate assay to identify the mechanism involved. This integrated approach was used to detect temephos resistance in Aedes aegypti from Nonthaburi (lowest use) and Roi Et (highest use). Ae. aegypti BKK1 laboratory strain was used as the susceptible reference strain. The appropriate concentration of insecticide for bottle bioassay was determined empirically for Ae. aegypti BKK1 strain and found to be in the range of 800-1,050 microg/bottle. The time-mortality rate at 800 microg/bottle was 170 +/- 8.66 minutes, significantly different from the time-mortality rates in the 850, 900, 950, and 1,050 microg/bottle (p = 0.008) concentrations, which were 135 +/- 15.00, 140 +/- 8.66, 135 +/- 15.00, and 125 +/- 8.66 minutes, respectively. The cut-off concentration selected for resistance detection was 850 microg/bottle. The time-mortality rate for the Roi Et strain was 382 +/- 26.41 minutes, significantly higher than the Nonthaburi (150 +/- 25.10 minutes) and BKK1 strains (145 +/- 20.49 minutes) (p < 0.001). The temephos resistance ratio (RR100) for the Ae. aegypti Roi Et strain was 2.64-fold higher at lethal time (LT100) than for the reference Ae. aegypti BKK1 strain. The mean optical density (OD) value from the biochemical microplate assay for the non-specific esterase of the Roi Et strain was higher than the mean OD for the non-specific esterase of both the Nonthaburi and BKK1 strains. Insensitive acetylcholinesterase was not found to be responsible for the resistance in the field-collected mosquitos. This study suggests that esterase detoxification is the primary cause of resistance in the Ae. aegypti population from Roi Et. Both the bottle bioassay and the biochemical microplate assay were proven to be promising tools for initial detection and field surveillance for temephos resistance.

Ikonos-derived malaria transmission risk in northwestern Thailand.

We mapped overall malaria cases and located each field observed major malaria vector breeding habitat using Global Positioning System (GPS) instruments from September 2000 to October 2003 around the three malaria-endemic villages of Ban Khun Huay, Ban Pa Dae, and Ban Tham Seau, Mae Sod district, Tak Province, Thailand. The land-use/land-cover classifications of the three villages and surrounding areas were performed on IKONOS satellite images acquired on 12 November 2001 with a spatial resolution of 1 x 1 m. Stream network was delineated and displayed. Proximity analysis was performed on the locations of the houses with and without malaria cases within a 1.5 km buffer from An. minimus immature mosquito breeding habitats, mainly stream margins. The 1.5 km used in our proximity analysis was arbitrarily estimated based on the An. minimus flight range. A statistical t-test at 5% significance level was performed to evaluate whether houses with malaria cases have higher proximities to streams than houses without malaria cases. The result shows no significant difference between proximity to streams between houses with malaria cases and houses without malaria cases. We suspect that the actual flight range of An. minimus may be greater than 1.5 km. The An. minimus larval habitat deserves more detailed investigation. Further studies on human behavior contrary to that required for adequate malaria control among these three villages are also recommended.

Water quality and breeding habitats of anopheline mosquito in northwestern Thailand.

Malaria transmission is dependent upon many hydrology-driven ecological factors that directly affect the vectorial competence, including the presence of suitable habitats for the development of anopheline larvae. Larval habitats were identified and characterized at three malaria endemic villages (Ban Khun Huay, Ban Pa Dae, and Ban Tham Seau) in Mae Sot district, Tak Province, in northwestern Thailand between July 2002 and June 2003. The Global Positioning System (GPS) was used to provide precise locational data for the spatial distribution of anopheline mosquito larvae and their habitats. Ten habitat categories were identified. Eighteen adult Anopheles species were identified from larvae in all the surveyed habitats. An. minimus was the most common species throughout the year. The relationship between eight abiotic variables (temperature, hardness, carbon dioxide, dissolved oxygen, nitrate, phosphate, silica and pH) and the abundance of four major species of malaria vectors (An. (Cel.) dirus, An. (Cel.) minimus, An. (Cel.) maculatus, and An. (Cel.) sawadwongporni), and six species of non-vectors (An. (Cel.) kochi, An. (Cel.) jamesii, An. (Ano.) peditaeniatus, An. (Ano.) barbirostris, An. (Ano.) campestris, and An (Cel.) vagus) larvae was investigated. The results from the multiple regression models suggest that hardness, water temperature and carbon dioxide are the best predictor variables associated with the abundance of An. minimus larvae (p < 0.001); water pH for An. dirus larvae (p < 0.001); temperature and pH for An. kochi larvae (p < 0.01); temperature and silica concentration for An. jamesii larvae (p < 0.001); dissolved oxygen and silica concentration for An. campestris larvae (p < 0.001); and pH and silica concentration for An. vagus larvae (p < 0.001). We could not identify key environmental variables for An. maculatus, An. sawadwongporni, An. peditaeniatus, and An. barbirostris.

Genetic structure of Aedes aegypti populations in Thailand using mitochondrial DNA.

A hierarchical population genetic study was conducted among 19 Aedes aegypti populations in Thailand from Chiang Mai in the north to Songkhla province in the south. Single-strand conformation polymorphism analysis was used to examine variation in a 359-basepair region of the NADH dehydrogenase subunit 4 mitochondrial DNA gene (ND4). Seven haplotypes were detected in two lineages previously identified in ND4 haplotypes from North America. Gene flow estimates and highly significant variation among populations within 25 kilometers implicated genetic drift and vector control efforts as major factors in genetic structure. Mantel regression analysis demonstrated no isolation by distance. Urban areas were relatively panmictic, while suburban/rural sites exhibited more restricted gene flow. Significant genetic structure among groups of collections > 100 kilometers apart is consistent with recent (approximately 50 year) expansion of Ae. aegypti from highly populated areas accompanied by founder effects, but could also reflect the overall low genetic diversity in ND4 in Thailand.

Dispersal of the dengue vector Aedes aegypti within and between rural communities.

Knowledge of mosquito dispersal is critical for vector-borne disease control and prevention strategies and for understanding population structure and pathogen dissemination. We determined Aedes aegypti flight range and dispersal patterns from 21 mark-release-recapture experiments conducted over 11 years (1991-2002) in Puerto Rico and Thailand. Dispersal was compared by release location, sex, age, season, and village. For all experiments, the majority of mosquitoes were collected from their release house or adjacent house. Inter-village movement was detected rarely, with a few mosquitoes moving a maximum of 512 meters from one Thai village to the next. Average dispersal distances were similar for males and females and females released indoors versus outdoors. The movement of Ae. aegypti was not influenced by season or age, but differed by village. Results demonstrate that adult Ae. aegypti disperse relatively short distances, suggesting that people rather than mosquitoes are the primary mode of dengue virus dissemination within and among communities.

Geographical distribution of Anopheles minimus species A and C in western Thailand.

Elucidating vector distribution based on an accurate species identification is important to understanding the nature of the species complex in order to achieve vector control. Morphologically, An. minimus s.l. is difficult to distinguish from both its species complex and its closely related species. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and a single multiplex-allele specific PCR developed for species identification were applied in this study in comparison with morphological identification. Both methods were used, combining with geographical information systems to determine the distribution of An. minimus species A and C. The investigation on the breeding habitats was performed in the malarious area of western Thailand. Anopheles larvae were collected from 36 bodies of water among five districts (Sangkhaburi, Thong Pha Phum, Si Sawat, Muang, and Sai Yok) of Kanchanaburi Province, Thailand. In this study, An. minimus A larvae were present in all study districts but the association differed when focusing on study sites within each district. Although there were many reports of An. minimus A in Ban Phu Rat and Ban Phu Toei villages in Sai Yok District, we did not find the breeding sites of species A in those two areas. An. minimus A and C were found in Ban Phu Ong Ka village in Sai Yok District. The breeding habitats of An. minimus C were present covering 30-40 km of distance in northern part of Sai Yok and this species was also found in the central and southern parts of Si Sawat District.

Use of a remote sensing-based geographic information system in the characterizing spatial patterns for Anopheles minimus A and C breeding habitats in western Thailand.

A remote sensing (RS)-based Geographic Information System (GIS) was used to characterize the breeding habitats of Anopheles minimus species A and C in five different districts of Kanchanaburi Province in western Thailand. The GIS and RS were used to monitor the area for the presence and absence of An. minimus A and C in five major land areas, forest, agriculture, urban, water and bare land. The results show that An. minimus A survives both in dense canopy forest and in open fields where agriculture is dominant. A scatter plot of land-use/land-cover for An. minimus, considering proximities to the forest and proximities to agriculture, suggests that An. minimus A has a wider habitat preference, ranging from dense canopy forest to open agricultural fields. A scatter plot for An. minimus C, on the other hand, showed a narrow habitat preference. A scatter plot for proximities performed on separate populations of An. minimus species A, one in the north and the other in the south, showed that there was an association in the northern population with the forest and in the southern population with agricultural areas. There were no statistically significant differences in the scatter plot of proximities to urban areas and water bodies with the An. minimus A north, south, and An. minimus C. LANDSAT TM satellite data classification was used to identify larval habitats that produce An. minimus A and C and analyze proximities between land-use/land-cover classes and locations of larval habitats. An. minimus A has a wide habitat preference, from dense canopy forest to open agricultural fields, while An. minimus C has a narrow habitat preference.

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.