Spatiotemporal patterns of malaria incidence in northern Thailand.

We present a detailed analysis of long-term time series of malaria incidence in northern Thailand. Positive cases for Plasmodium falciparum and P. vivax have been recorded monthly from 1977-2002 at 13 provinces in the region. Time series statistical methods are used to examine the long-term trends and seasonal dynamics of malaria incidence at regional and provincial scales. Both malarial types are declining throughout the region, except in the two provinces that share a large border with Myanmar. The rate of decline in P. vivax has decreased across the region since the end of the 1980s, and this may be a signal of developing resistance or changing vector potential. Both species display a two-peak annual seasonality that may be attributed to patterns of vector occurrence, farming practice and migration of individuals across international borders. In a number of provinces, the importance of the first seasonal peak has grown in recent years, possibly owing to increases in vector densities. The medium-term fluctuations of both species exhibit a clear spatial organisation. There is some evidence of a subtle close to 4-year super annual cycle in P. falciparum, which we suggest is driven by extrinsic factors relating to the climate of the region.

Laboratory and field evaluations of the insect repellent 3535 (ethyl butylacetylaminopropionate) and deet against mosquito vectors in Thailand.

The insect repellents 3535 (ethyl butylacetylaminopropionate or IR3535) and deet (N,N-diethyl-3-methylbenzamide) were prepared as 20% solutions in absolute ethanol and evaluated for repellency against many mosquito species in Thailand under laboratory and field conditions using human subjects. In the laboratory, 0.1 ml was applied per 30-cm2 of exposed area on a volunteer's forearm (0.66-0.67 mg active ingredient [AI]/ cm2), whereas in the field, volunteers' legs (from knee to ankle, with a surface area of about 712-782 cm2) were treated with 3 ml per exposed area (0.76-0.84 mg AI/cm2). In the laboratory, both IR3535 and deet showed equal repellency (P > 0.05) for 9.8 and 9.7 h against Aedes aegypti, for 13.7 and 12.7 h against Culex quinquefasciatus, and for 14.8 and 14.5 h against Cx. tritaeniorhynchus, respectively. Anopheles dirus was significantly less sensitive to IR3535 than to deet (P < 0.05), with a mean protection time of 3.8 and 5.8 h, respectively. Under field conditions, both IR3535 and deet provided a high degree of protection against various mosquito vectors ranging from 94 to 100% during the test periods. Both repellents provided a high level of protection for at least 8 h against Ae. albopictus and for at least 5 h against Cx. gelidus, Cx. tritaeniorhynchus, Cx. quinquefasciatus, Mansonia dives, Ma. uniformis, Ma. annulata, Ma. annulifera, Anopheles minimus, and An. maculatus. This study clearly documents the potential of IR3535 for use as a topical treatment against a wide range of mosquito species belonging to several genera.

Heterogeneity in the host preference of Japanese encephalitis vectors in Chiang Mai, northern Thailand.

Experiments, using the capture-mark-release-recapture technique inside large nets, were carried out in Chiang Mai, northern Thailand, to examine heterogeneity in the host preference of Japanese encephalitis (JE) vectors. A significantly higher proportion of the vector species that were initially attracted to a cow fed when released into a net with a cow than when released into a net containing a pig. However, Culex vishnui individuals that had been attracted to a pig had a higher feeding rate in a net containing a pig rather than a cow. When mosquitoes were given a choice by being released into a net containing both animals, they exhibited a tendency to feed on the host to which they had originally been attracted. This feeding preference was, however, not shown by the offspring of pig-fed individuals. We have therefore shown evidence of physiological/behavioural conditioning in the host preference of JE vectors rather than genetic variability. Our results suggest that effective control of JE might be achieved by increasing the availability of cows (the dead-end hosts of JE virus) to deflect the vectors from pigs (the amplifying host). The behavioural imprinting which we have found would tend to re-inforce the initial tendency of the vectors to bite cows.

A mark-release-recapture study on the spatial distribution of host-seeking anophelines in northern Thailand.

A mark-release-recapture experiment was conducted at Mae Taeng, Chiangmai, Thailand, in November 1990 to examine the movement of released host-seeking mosquitoes in a heterogeneous environment. A total of 1,848 mosquitoes of nine anopheline species was field-collected, marked with fluorescent dye, and released. Adult collections were made for four nights after the release at five collection sites surrounding the release site. Three different attractants, dry ice (5 kg/site/night), human, and cow bait were used to collect a large number and a wide variety of adult mosquitoes. The recapture rate of released mosquitoes differed among species, ranging from 0 to 7.5%. The species composition was significantly different among collection sites and collection methods, and samples from dry ice collection showed intermediate species composition between those from human and cow bait collections. The spatial distribution of released mosquitoes was not significantly different from that of unmarked mosquitoes. Different behavioral responses to heterogeneous environments by different species of host-seeking mosquitoes was suggested as the underlying mechanism of species-specific spatial distribution of mosquitoes in the study area.

Correlation of glutathione S-transferase and DDT dehydrochlorinase activities with DDT susceptibility in Anopheles and Culex mosquitos from northern Thailand.

Comparative DDT-susceptibility status as well as glutathione S-transferase activity and DDTase activity of Anopheles minimus (A). An. annularis and Culex quinquefasciatus were investigated to ascertain the role of these enzymes in DDT-resistance. The standard WHO susceptibility test kits was used to discriminate between resistant and susceptible populations. GST activity was measured in microtiter plates whereas DDTase activity was determined by HPLC quantitation of DDT metabolites. This is the first report of DDT-resistance in the Thai malaria vector, An. minimus species A. A positive correlation of DDT-resistance and DDTase activity was observed in this species as well as in the suspected vector, An. annularis. However, GST activity was not correlated to DDT-resistance in either species. Statistical analysis and scatter plots demonstrated the non-correlation between DDTase and GST activity in An. annularis. Studies in Culex quinquefisciatus revealed difference in GST/ DDTase and the relationship to DDT-resistance compared to the anopheline species. The Culex GST activity is correlated to DDTase activity. These results suggested that a positive correlation of GST and DDTase activity might be species dependent.

Selection of Anopheles dirus for refractoriness and susceptibility to Plasmodium yoelii nigeriensis.

Two lines of the Oriental malaria vector mosquito Anopheles dirus species A (Diptera: Culicidae), one fully refractory and one fully susceptible to Plasmodium yoelii nigeriensis (an African rodent malaria parasite), were established after 17 generations of mass selection, followed by single female selection for one or two generations. Prior to selection, the stock colony of An. dirus was 17% refractory. Both lines of An. dirus produced abundant ookinetes that started to invade the midgut within 24h post-infection, as seen in histological sections. In most of the refractory mosquitoes, oocysts stopped development <12 h post-invasion, indicating a rapid defence mechanism. Dead P. y. nigeriensis parasites were apparently localized as small melanized spots (2-5 microm) seen in wet preparations of mosquito midguts dissected 5-7 days post infective bloodmeal. In some refractory An. dirus females, apart from the spots, a small number of totally encapsulated oocysts (c. 10 microm) were also present. These larger melanized parasites predominated in a few females: they appeared 2-3 days post-infection as a secondary delayed defence mechanism. The progeny of reciprocal matings between susceptible and refractory lines had approximately 50% susceptibility. Backcrosses of F1 hybrids with susceptible or refractory lines increased or decreased the susceptibility of backcross progeny accordingly. Overall, these results suggest polygenic control of susceptibility to P. y. nigeriensis infection. The refractory line of An. dirus showed normal susceptibility to natural infections of the human malarias P. falciparum and P. vivax from local patients.

Comparisons of rice field mosquito (Diptera: Culicidae) abundance among areas with different agricultural practices in northern Thailand.

Adult mosquitoes were collected from 3 areas in northern Thailand with different availabilities of rice fields for larval habitats by using blacklight, truck, and pig-baited traps. Culex tritaeniorhynchus Giles and Culex gelidus Theobald were dominant in all samples. Significant study-area differences were found in light and truck trap collections for Cx. tritaeniorhynchus and Cx. gelidus, but not for Culex vishnui Theobald + Culex pseudovishnui Colless. In pig-baited sample area differences were significant only for Cx. gelidus. Adults of Culex fuscocephala Theobald were relatively rare, comprising only 0.6-7.9% of the total mosquitoes collected. When compared with the results from previous studies in the Chiangmai area, we conclude that Cx. fuscocephala has become a minor species and that changes in the mosquito fauna have occurred in northern Thailand.

Comparative susceptibility of two forms of Anopheles sinensis Wiedemann 1828 (Diptera : Culicidae) to infection with Plasmodium falciparum, P. vivax, P. yoelii and the determination of misleading factor for sporozoite identification.

Two karyotypic forms of laboratory-raised Anopheles sinensis, ie Form A (XY1) and Form B (XY2), were experimentally infected with various indigenous strains of Plasmodium falciparum and P. vivax using an artificial membrane feeding technique, and a rodent malaria, P. yoelii, using a direct feeding technic and dissected 7-9 days and 10-15 days after feeding for oocyst and sporozoite rates, respectively. The results revealed that two forms of An. sinensis were refractory vectors for P. falciparum and P. yoelii since 0% of oocyst and sporozoite rates were obtained, but poor vectors for P. vivax since 0.00-85.71% and 0.00-5.88% of oocyst and sporozoite rates were recovered. The sporozoite-like crystal found in the median lobe of the salivary gland of An. sinensis which could be a misleading factor in identification of true sporozoites in the salivary glands is reported for the first time.

High prevalence of Plasmodium malariae and Plasmodium ovale in malaria patients along the Thai-Myanmar border, as revealed by acridine orange staining and PCR-based diagnoses.

The prevalence of the four human malaria parasites was investigated among malaria patients at northern, central and southern towns in Thailand along the border with Myanmar between September 1995 and May 1996. Thin smears obtained from 548 Thai and Burmese patients were reviewed by an acridine orange staining method, and many mixed infections with two to four species, including P. malariae and P. ovale, were detected. These diagnostic results were compared with those by two PCR-based diagnoses, microtitre plate hybridization (MPH) and a nested PCR method, both of which targets the same, species-specific regions in the 18S rRNA genes. In both PCR diagnoses, many P. malariae and P. ovale infections were also detected. Detection sensitivity of P. malariae infection was higher in nested PCR than MPH, and a total prevalence of P. malariae infection estimated by nested PCR reached 24.3% (133/548). In 16 of them, the size of PCR products amplified by the P. malariae-specific primer was about 20-bp shorter than the expected size of 115-bp. Four of 16 possessed two different bands with normal and shorter sizes, suggesting that P. malariae isolates may be separated into two types, and that those with shorter products may be new variant form (s) with a nucleotide deletion in the target region. On the other hand, 21 P. ovale infections (3.8%) were detected by nested PCR, but four of them were MPH-negative because of the sequence variation at the probe region. These results indicated that the prevalence of P. malariae and P. ovale along the Thai-Myanmar border may be substantially higher than previously reported.

Effects of rice culture practices on the abundance of Culex mosquitoes (Diptera:Culicidae) in northern Thailand.

In 1991-1993, the density (numbers per 10 dips) and abundance (density x flooded area) of mosquitoes were studied in 3 areas of northern Thailand with different environmental conditions. Culex tritaeniorhynchus Giles, Cx. vishnui Theobald, and Cx. gelidus Theobald were predominant among the 8 Culex species collected. Abundance of these 3 species varied significantly among the 3 areas. Type of habitat classified according to agricultural practices in rainy and dry season significantly influenced larval abundance of Cx. tritaeniorhynchus and Cx. vishnui. Seasonal variation in larval abundance was significant only in Cx. vishnui. The response of the 3 vector mosquitoes to environmental heterogeneity is discussed.

Mark-release-recapture experiments to estimate the efficiency of the light trap in collecting Japanese encephalitis vector mosquitoes.

Efficiency of the light trap in collecting females of Culex tritaeniorhynchus, the principal vector mosquito of Japanese encephalitis, was evaluated by mark-release-recapture experiments. Females collected with dry ice in the field were marked and released in a pigsty. The recapture rate of marked females by the light trap was so low as around 1% or lower. Owing to this low efficiency, it is not likely that the light trap can be a useful tool in the control of Japanese encephalitis.

Characterization of two Japanese encephalitis virus strains isolated in Thailand.

Two strains of Japanese encephalitis (JE) virus were isolated from a pool of Culex tritaeniorhynchus captured in 1992 and another pool of Cx. vishnui captured in 1993, in Chiang Mai Area, Northern Thailand. These two strains, ThCMAr44/92 and ThCMAr67/93, could not be identified either as Nakayama or JaGAr01 subtype by the hemagglutination-inhibition (HI) and the neutralization (N) tests using immune sera raised against these standard JE virus strains. Reverse transcription-polymerase chain reaction showed the presence of JE-specific conserved sequences in these strains. Sequencing of 240 nucleotides in their PrM gene region identified that these two strains belong to the genotype 1 of JE virus. Nucleotide and encoded amino acid sequences of their envelope glycoprotein gene revealed 98.8 and 99.8% identity, respectively. These two strains shared 77.8 to 87.7% homology in the nucleotide sequence and 90.0 to 98.8% homology in the amino acid sequence with other reported JE strains. Five strain-specific amino acid changes were noted in ThCMAr44/92 strain, while one in ThCMAr67/93. In addition, four common amino acid changes were found in both strains. Thus, the findings indicated that these two strains were structurally different from each other as well as different from all the reported strains which was in agreement with the serological tests by hemagglutination-inhibition and neutralization.

Susceptibility of Thai zoophilic Anophelines and suspected malaria vectors to local strains of human malaria parasites.

Wild caught zoophilic Anopheles and suspected malaria vector species collected in northwest Thailand were experimentally infected with local human malaria parasites using a membrane feeding. One week post-feeding a number of mosquitos were dissected for oocyst examination. The remainder were kept for another one week or more, and then the salivary glands were examined for the presence of sporozoites. The results revealed that An. vagus, An. kochi and An. annularis were susceptible to both Plasmodium falciparum and P. vivax whereas An. barbirostris and An. sinensis were susceptible to only P. vivax. The non-susceptibility to P. falciparum of these two mosquito species may indicate their poor vector status of this malaria species in the field.

Plasmodium falciparum and P. vivax: factors affecting sensitivity and specificity of PCR-based diagnosis of malaria.

We have previously reported on development of a simple PCR-based method to detect Plasmodium falciparum in which patient blood samples are lysed and then filtered onto paper. The present studies, conducted in Thailand, were designed to identify factors contributing to differences between results from microscopy and PCR. To analyze microscope-positive, PCR-negative results, we demonstrated that using the lysis/filtration sample preparation method, target DNA degradation is not a significant factor. Similarly, we showed that sensitivity of PCR among patient samples did not differ using the lysis centrifugation method or organically extracted DNA. We further demonstrated that 7/13 samples which were negative by PCR for P. falciparum were positive by PCR when P. vivax-specific primers were used. Microscope-negative, P. falciparum PCR-positive samples were analyzed in two ways: the true rate of false-positivity for PCR (2%) was established by analyzing 498 samples from patients living in areas without transmission. We further showed that when microscope-negative, PCR-positive samples were amplified using an independent P. falciparum-specific PCR target sequence, 42/47 were PCR-positive. We conclude that the accuracy and reduced limit of detection of microscopy are major confounders when comparing this method to PCR.

DNA probes as epidemiological tools for surveillance of Plasmodium falciparum malaria in Thailand.

We have previously reported on development of a DNA probe-based method for diagnosing Plasmodium falciparum infection directly from patient blood samples. In the present studies, we sought to examine applicability of the method to large epidemiological surveys, comparing sensitivity, specificity, time required to obtain results, and costs with those of conventional microscopic examination. Results of DNA probe hybridization were also compared between laboratories in the US and Thailand, to assess transferability of the DNA probe technology. Five separate surveys of approximately 5000 villagers each were performed between December 1987 and June 1989 (26,176 samples total). Sensitivity ranged from 61% to 92% for both US and Thai laboratories, while specificity ranged from 98.2% to 99.9%. Agreement between the US and Thai laboratories was good, with kappa coefficients between 0.62 and 0.78 for different surveys. Between 4 and 8 person-days were required to obtain results from each set of 5000 samples by DNA hybridization, whereas microscopic examination required 150 person-days. Approximate costs were US 0.17 per sample for DNA probe analysis, and US$0.36 for microscopic examination. We conclude that the DNA probe method offers significant advantages when large numbers of samples must be surveyed for P. falciparum.

A simple method to detect Plasmodium falciparum directly from blood samples using the polymerase chain reaction.

We have developed a simple method for treating blood samples permitting direct detection of Plasmodium falciparum parasites using the P. falciparum-specific DNA probe pPF14 after polymerase chain reaction (PCR) amplification of target DNA sequences, and have compared this method with microscopic examination of thick blood smears. For PCR amplification, blood samples were lysed, then filtered onto filter paper. After drying, a piece of the filter paper was added directly to the PCR mixture for amplification. The presence of PCR products was detected using nonisotopically labeled probe. This method permits detection of less than than 10 parasites in a 20-microliters sample, and minimizes the effects of PCR inhibitors generally found in blood. Samples were collected from patients presenting at malaria clinics in Mae Sod and Mae Ramat, Thailand, and 626 were analyzed both by the PCR method and by conventional microscopy. Of these, 157 were positive both by microscopy and by PCR, while 297 were negative by both methods. PCR detected 131 samples that were negative by microscopy, and failed to detect 41 samples identified as positive by microscopy. All discordant samples were re-analyzed by microscopy and by PCR. Upon re-examination at a higher sensitivity, microscopy identified five additional positive cases, while six previously positive cases were found to be negative. This method of treating blood samples for PCR may also be useful in other diagnostic assays.