Genetic diversity and multiple infections of Plasmodium vivax malaria in Western Thailand

Using two polymorphic genetic markers, the merozoite surface protein-3alpha (MSP-3alpha) and the circumsporozoite protein (CSP), we investigated the population diversity of Plasmodium vivax in Mae Sod, Thailand from April 2000 through June 2001. Genotyping the parasites isolated from 90 malaria patients attending two local clinics for the dimorphic CSP gene revealed that the majority of the parasites (77%) were the VK210 type. Genotyping the MSP3-alpha gene indicated that P. vivax populations exhibited an equally high level of polymorphism as those from Papua New Guinea, a hyperendemic region. Based on the length of polymerase chain reaction products, three major types of the MSP-3alpha locus were distinguished, with frequencies of 74.8%, 18.7%, and 6.5%, respectively. The 13 alleles distinguished by restriction fragment length polymorphism analysis did not show a significant seasonal variation in frequency. Genotyping the MSP-3alpha and CSP genes showed that 19.3% and 25.6% of the patients had multiple infections, respectively, and the combined rate was 35.6%. Comparisons of MSP-3alpha sequences from nine clones further confirmed the high level of genetic diversity of the parasite and also suggested that geographic isolation may exist. These results strongly indicate that P. vivax populations are highly diverse and multiple clonal infections are common in this malaria-hypoendemic region of Thailand.     

Chloroquine sensitivity of Plasmodium vivax in Thailand

Chloroquine has been the standard treatment for Plasmodium vivax malaria for more than 40 years in most regions of the world. Recently, however, chloroquine-resistant P. vivax has been reported from Oceania, several parts of Asia, and South America. In order to assess the situation in Thailand, 886 patients with vivax malaria who were admitted to the Bangkok Hospital for Tropical Diseases from 1992 to 1997 were followed prospectively. Most of the patients had been infected on the western border of Thailand and were experiencing their first malarial infection when admitted. All received oral chloroquine (approximately 25 mg base/kg body weight, administered over 3 days) and then were randomized to receive primaquine (15 mg daily for 14 days) or no further treatment. All the patients were initially responsive to chloroquine, clearing their parasitaemias within 7 days, and there were no significant differences in the clinical or parasitological responses between those treated with primaquine and those given no further treatment. Plasmodium vivax parasitaemias re-appeared within 28 days of chloroquine treatment in just four patients. In each of these four cases, re-treatment with the same regimen of chloroquine resulted in eradication of the parasitaemia, with no further appearance of parasitaemia during the next, 28-day, follow-up period. These data indicate that virtually all acute (i.e. blood-stage) P. vivax infections acquired in Thailand can still be successfully treated with chloroquine.     

Antigenic diversity amongst ten geographic isolates of Plasmodium falciparum defined by merozoite invasion inhibition assay.

The extent to which human antibodies involved in functional immunity react with antigenic determinants varying between different isolates or strains of human malaria parasite Plasmodium falciparum will influence the design of vaccine against malaria. In this study, in vitro inhibition of merozoite invasion in erythrocytes by an immune human serum was used to define the antigenic differences in 10 isolates of P. falciparum from three endemic areas, i.e. Africa, South America and Southeast Asia. The serum inhibited the invasion of merozoites of all the strains but the extent of inhibition varied from low to moderate to high degree indicating antigenic differences amongst isolates of P. falciparum. The antigenic differences could not be correlated to the geographic origin of the parasite isolate.     

Antigenic disparity of Plasmodium vivax causing initial symptoms and causing relapse.

Relapse infections are an important obstacle to the successful treatment and control of Plasmodium vivax malaria, but little is known about the nature of the relapse. To provide insight into the antigenic disparity of the parasites causing initial clinical symptoms and causing relapse, a panel of 58 monoclonal antibodies (MAbs) against erythrocytic stages of Plasmodium vivax was tested by indirect fluorescent antibody test in five relapse cases. The initial and relapse strains from three patients (R3, R4, and R5) exhibited similar IFA reactivity with all MAbs tested, whereas the isolates from two relapse cases (R1 and R2) showed different patterns of reactivity and were seen only with 15 MAbs In case R1, different IFA reactivities were observed with 12 MAbs, nine of which reacted with the initial (RPV261) but not the relapse (RPV393) isolates, whereas the other three MAbs reacted only with the relapse isolates. With regards to the second relapse case (R2) in whom two relapses occurred, different IFA reactivities were demonstrated with seven MAbs that reacted only with the initial isolate (RPV 182) and with the isolate from the first relapse (RPV 240) but not with the isolate from the second relapse (RPV 300). The antibody responses from patients who developed primary clinical symptoms and relapse were detected by Western immunoblotting. In cases R3, R4 and R5, there was no difference in the spectrum of antigens from initial and relapse sera recognized by the antibodies. In contrast, in cases R1 and R2, the molecules recognized by antibodies in initial and relapse sera were markedly altered. In case R1, the series of molecules of P. vivax antigens recognized by initial (RPV 261) and relapse (RPV 393) sera were 21, 25, 31, 39, 42, 61, 95, 115, 200, > 200 kDa and 21, 24, 31, 35, 57, 75, 200, > 200 kDa, respectively. In case R2, the initial serum (RPV 182) recognized P. vivax antigens with molecular weights of 23, 30, 52, 57, 68, 75, 85, 95, 115, and 195 kDa while the first relapse (RPV 240) and the second relapse sera recognized P. vivax antigens with molecular weights of 23, 30, 52, 85, 95,115 kDa and 30, 57, 68, 75, 85,195 kDa, respectively.     

Single-nucleotide polymorphisms and genome diversity in Plasmodium vivax

The study of genetic variation in malaria parasites has practical significance for developing strategies to control the disease. Vaccines based on highly polymorphic antigens may be confounded by allelic restriction of the host immune response. In response to drug pressure, a highly plastic genome may generate resistant mutants more easily than a monomorphic one. Additionally, the study of the distribution of genomic polymorphisms may provide information leading to the identification of genes associated with traits such as parasite development and drug resistance. Indeed, the age and diversity of the human malaria parasite Plasmodium falciparum has been the subject of recent debate, because an ancient parasite with a complex genome is expected to present greater challenges for drug and vaccine development. The genome diversity of the important human pathogen Plasmodium vivax, however, remains essentially unknown. Here we analyze an approximately 100-kb contiguous chromosome segment from five isolates, revealing 191 single-nucleotide polymorphisms (SNPs) and 44 size polymorphisms. The SNPs are not evenly distributed across the segment with blocks of high and low diversity. Whereas the majority (approximately 63%) of the SNPs are in intergenic regions, introns contain significantly less SNPs than intergenic sequences. Polymorphic tandem repeats are abundant and are more uniformly distributed at a frequency of about one polymorphic tandem repeat per 3 kb. These data show that P. vivax has a highly diverse genome, and provide useful information for further understanding the genome diversity of the parasite.     

Risk factors for Plasmodium vivax gametocyte carriage in Thailand

To study the risk factors for Plasmodium vivax gametocyte carriage, the presence or absence of gametocytes was determined in 2,125 patients with P. vivax malaria participating in clinical trials at the Hospital for Tropical Diseases in Bangkok, Thailand. Stepwise logistic regression models were used to determine which variables were significantly related to gametocyte carriage. On admission, 615 patients (29%) had detectable gametocytes (before treatment). After treatment had started, an additional 245 patients (11%) developed patent gametocytemia. The variables retained by multivariate analysis were highest observed temperature (adjusted odds ratio [AOR] per degrees C increase = 0.82, 95% confidence interval [CI] = 0.71-0.94, P = 0.006), asexual parasitemia > 9,200/muL (AOR = 2.8, 95% CI = 1.9-4.2, P < 0.0001), erythrocyte counts (AOR = 0.8/million/muL increase, 95% CI = 0.67-0.95, P = 0.01), monocyte percentage (AOR = 0.93 per % increase, 95% CI = 0.89-0.96, P < 0.0001), lymphocyte percentage (AOR = 0.98 per % increase, 95% CI = 0.97-0.99, P = 0.006), albumin (AOR = 0.67 per 10 g/mL increase, 95% CI = 0.5-0.9, P = 0.007), and anion gap (AOR = 1.1 per unit increase, 95% CI = 1.02-1.14, P = 0.009). The possible significance of these observations is discussed.     

Therapeutic efficacy of artesunate in Plasmodium vivax malaria in Thailand

Our previous study showed that in vitro susceptibility of Plasmodium vivax to chloroquine has significantly decreased in Thailand within the past two decades. Thus, the evaluation of alternative antimalarials for treatment of vivax malaria is needed. The aim of this study was to examine parasitological and clinical efficacy of an artemisinin derivative (artesunate) for the treatment of vivax malaria in patients who were admitted to the Bangkok Hospital for Tropical Diseases. We randomly allocated patients aged 12-56 years to receive 3.3mg/kg (adult dose 200 mg) on the first day, and for the next four days each patient was given 1.65 mg/kg orally (adult dose 100 mg), total dose = 600 mg. After the five-day course of artesunate, primaquine was given: a single oral dose of 15mg for 14 days. A total number of 42 patients received treatment. All participants were followed up for 28 days. In all the cases, both parasitemia and fever were resolved rapidly; the mean fever clearance time and parasite clearance time, 14.6 and 36.7 hours, respectively, showed that therapeutic response to artesunate was better than that of chloroquine. The 14-day cure rate was 100%, but reappearance of parasitemia was seen in two patients on days 21 and 25 following treatment, respectively. These two cases of failure rate should be considered as true relapse rather than recrudescence, since the relapse interval in Southeast Asian vivax malaria according to recent findings seems to be 3 weeks after start of treatment, if primaquine is not given or an inadequate amount is given. In conclusion, artesunate might be useful in treatment of vivax malaria, causing a good blood schizontocidal effect. However, to prevent emerging resistance it should never be used alone.     

Genetic diversity of Plasmodium vivax and Plasmodium falciparum in Kohat District, Pakistan

Malaria is one of the serious diseases threatening human health in Pakistan and contributes to a large proportion of the total malaria deaths in South Asia. However, little is known about the nature and extent of genetic diversity of the malarial parasites circulating in Pakistan. This study was designed to assess the infection status of Plasmodium and the genetic diversity of Plasmodium vivax and Plasmodium falciparum by analyzing msp-3α, msp-3β and msp-1, msp-2 genes respectively using allele specific nested PCR and RFLP assays. For this purpose, 130 field isolates were collected from the individuals who exhibited clinical symptoms associated with malaria in the Kohat region of Khyber Pakhtoonkhwa (KPK), Pakistan. Among 130 blood samples collected, P. vivax was detected in 105/130 (80.8%) and P. falciparum in 21/130 (16.2%). Mixed infections with both parasites were detected in 4/130 (3%) of the isolates. A large number of distinguishable alleles were found for msp genetic markers: 10 alleles for msp-3α and seven for msp-3β with one mixed infection in case of msp-3β. The genotyping of P. falciparum showed that K1+MAD20 mixed genotype was dominant in msp-1 and FC27 in msp-2. The results collectively suggest that P. vivax and P. falciparum populations in this region are highly polymorphic and mixed infections are prevalent.     

Seasonal fluctuations in the carriage of Plasmodium vivax gametocytes in Thailand

To study the influence of season on Plasmodium vivax gametocyte carriage, the relationship between monthly rainfall and the proportion of P. vivax patients with detectable gametocytaemia was analysed. Most of the data used came from 6807 aggregated observations collected, in a refugee camp on the Thai-Burmese border, between January 2000 and December 2002. There was a positive correlation between rainfall and the incidence of P. vivax infection (Spearman's rho=+0.42; P =0.01) but the prevalence of gametocyte carriage among those with P. vivax infection was negatively correlated with rainfall (Spearman's rho=-0.58; P <0.001). The latter, negative correlation remained significant after controlling for the proportion of visitors relative to camp residents (P =0.003). Migrations, changes in transmission patterns, seasonal haematological changes, and ultraviolet immunosuppression are discussed as potential explanations for these observations.     

Geographical distribution of amino acid mutations in Plasmodium vivax DHFR and DHPS from malaria endemic areas of Thailand

Both malaria treatment and prophylaxis target the parasite dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes. Specific point mutations in these genes confer resistance to sulfadoxine-pyrimethamine (SP) in both Plasmodium falciparum and P. vivax. We used direct sequencing to examine the prevalence of point mutations in pvdhps and pvdhfr in 160 P. vivax isolates collected from areas along the international borders of Thailand. Results show that the majority of the isolates harbored a quadruple mutant allele of pvdhfr and a double mutant allele of pvdhps, but the distribution was not uniform. The highly mutant allele combination was especially prevalent along the Thai-Myanmar border, whereas the majority of the isolates from areas along the Thai-Cambodian and Thai-Malaysian borders carried double mutant alleles of pvdhfr and single mutant alleles of pvdhps. Novel mutations that have not been identified previously at codon 512 of pvdhps (K512M, K512E, K512T) were also found.     

Dramatic difference in diversity between Plasmodium falciparum and Plasmodium vivax reticulocyte binding-like genes

Malaria parasite proteins involved in erythrocyte invasion are considered important vaccine targets. Members of the reticulocyte binding-like (RBL) family of Plasmodium merozoite proteins are found in human, simian, and rodent malaria parasites and function in the initial steps of erythrocyte selection and invasion. The RBL genes are large, ranging in size from 7.7 to 10 kb, and the extent of any sequence diversity in parasite populations is unknown. We present the first assessment of sequence diversity within RBL genes from the two major human malaria parasites: Plasmodium falciparum and P. vivax. Polymorphism within the RBL genes is generally limited, except for P. vivax reticulocyte binding protein 2 (PvRBP2), which has nucleotide diversity levels 25-fold higher than the other RBL genes. The PvRBP2 haplotypes appear to fall into two distinct classes of alleles, suggesting large-scale dimorphism in this gene. Polymorphisms were frequently clustered, suggesting that different RBL domains may be evolving under different selection and functional pressures.     

Efficacy of primaquine regimens for primaquine-resistant Plasmodium vivax malaria in Thailand

To define the current efficacy of Fansidar (F. Hoffmann-La Roche Ltd., Basel Switzerland) (pyrimethamine and sulfadoxine), primaquine in a high dose, and artesunate for treating acute Plasmodium vivax malaria, we conducted a comparative clinical trial of these 3 drugs in an open-label study. Patients (15-65 years old) were assigned to 1 of 4 treatments regimens in a serial order. Ninety percent of the patients were infected at Thailand-Myanmar border. Patients in group I (n = 23) received Fansidar (3 tablets, 75 mg of pyrimethamine and 1,500 mg of sulfadoxine, a single dose on the first day), group II (n = 23) received Fansidar (3 tablets, 75 mg of pyrimethamine and 1,500 mg of sulfadoxine, a single dose on the first day) and then received primaquine (30 mg a day for 14 days), group III (n = 23) received primaquine (30 mg a day for 14 days), and group IV (n = 23) received artesunate (200 mg once a day for 3 days) and then primaquine (30 mg a day for 14 days). Cure rates on day 28 of follow-up were 40%, 100%, 100%, and 100% in groups I, II, II, and IV, respectively. There were 4 and 5 patients in group I showing post-treatment reappearance of parasitemia at < or = 16 days and between 17 and 28 days, respectively. Patients in the other 3 groups showed negative parasitemias within 7 days after treatment. Artesunate plus primaquine (group IV) cleared parasitemia faster than the other 3 regimens. There is a high proportion of ineffectiveness of Fansidar for treatment of P. vivax malaria and it should be no longer used for treatment of P. vivax malaria acquired at the Thailand-Myanmar border. A high dose of primaquine is safe and effective in the treatment of P. vivax malaria during the 28-day follow-up period.     

Humoral immune responses to Plasmodium vivax subtelomeric transmembrane proteins in Thailand

Plasmodium vivax subtelomeric transmembrane protein (PvSTP) is a homolog of P. falciparum SURFIN4.2', a protein exposed on the parasite-infected erythrocyte (iE) surface, and is thus considered to be exposed on P. vivax-iE. Because antibodies targeting antigens located on the surface of P. falciparum-iE, such as P. falciparum erythrocyte membrane protein 1, play an important role in regulating the course of disease, we evaluated the presence of antibodies in P. vivax-infected patients against two PvSTP paralogs, PvSTP1 and PvSTP2. Recombinant proteins corresponding to cysteine-rich domain (CRD) of the PvSTP extracellular region and the cytoplasmic region (CYT) were generated and used for the enzyme-linked immunosorbent assay. Plasma samples (n = 70) reacted positively with recombinant PvSTP1-CRD (40%), PvSTP1-CYT (31%), PvSTP2-CRD (27%), and PvSTP2-CYT (56%), suggesting that PvSTP1 and -2 are naturally immunogenic. Specific response against either PvSTP1 or PvSTP2 indicates the existence of specific antibodies for either PvSTP1 or -2.     

Antigenic diversity and size diversity of Plasmodium falciparum antigens in isolates from Gambian patients. I. S-antigens

Ring-stage asexual parasites of P. falciparum were collected from six Gambian children and the S-antigens radiolabelled by 3H-glycine uptake during in vitro culture up to rupture of infected cells and merozoite release. Ouchterlony double diffusion of boiled culture supernatants against a panel of adult Gambian sera identified one S-antigen precipitin arc for five isolates and two precipitin arcs for one isolate. Five of the six isolates were serologically distinct. Analysis of S-antigens by comparison of SDS-polyacrylamide gel electrophoresis patterns of heat-treated soluble proteins revealed a more complex pattern of 3H-labelled S-antigens that was different for each isolate. There were between two and six different 3H-labelled bands for each isolate in the size range of molecular weight 137 000 to 285 000. This result confirms the large size range of S-antigens identified with culture adapted P. falciparum. Several bands were relatively weakly labelled with 3H-glycine, suggesting that natural isolates contain one or two predominant S-antigen phenotypes and several other S-antigen phenotypes expressed by minor parasite subpopulations. Immunoprecipitation was performed using a panel of sera from Gambian adults, or, acute and 3 week convalescent sera from the same patients used for S-antigen radiolabelling. Adult sera generally immunoprecipitated some of the S-antigens in each isolate, including antigens that must represent extremely minor parasite subpopulations since they could not be seen in the patterns of non-immunoprecipitated heat-stable proteins. Sera from convalescent children were generally negative on immunoprecipitation, even with the homologous isolate. In one case we observed the acquisition of specific immunoprecipitating antibody to one of the homologous S-antigens during the convalescent period. The antigenic and structural complexity of S-antigens in natural isolates that have not been submitted to the selection pressure of adaptation for in vitro culture is clearly greater than for culture adapted P. falciparum.     

The genetic polymorphism of Plasmodium vivax genes in endemic regions of Thailand

ObjectiveTo investigate the genetic polymorphism of Plasmodium vivax (P. vivax) PvCSP and PvMSP1 genes from field isolates at four endemic regions (North, East, West and South) of Thailand.MethodsThe 152 P. vivax infected cases from dried blood spots were DNA extracted and confirmed by species-specific primer sets using multiplex PCR method. PvMSP1 fragments F2 and F3; PvCSP were genotyped using RFLP-PCR method.ResultsTotally amplified DNA which was multiple genotypes for PvMSP1 F2 and PvMSP1 F3 were 12.50% and 8.55%, respectively while PvCSP was 3.95%. The overall frequency of multiple genotypes was 25%. There were 12 allele types of PvMSP1 F2 using AluI enzyme digestion and 8 size variations were found in PvMSP1 F3. The isolates from western region was highly genetic diverse when compare among all isolates. The predominant variant type of PvCSP gene was VK210 type.ConclusionsThe multiple genotypes are common found in Thailand and it might hide the real genotype. PvCSP does not have extensive genetic diversity in this study. However, PvMSP1 marker due to multiple genotypes is difficult to be analyzed. The multiple genotypes findings might stem from population migration and vector species findings.     

Sensitivity of Plasmodium vivax to chloroquine in Sa Kaeo Province,Thailand

Following a recent, abrupt local increase in the incidence of vivax malaria, a study was conducted in order to evaluate the efficacy of chloroquine for the treatment of 26 adult patients with acute vivax malaria in Sa Kaeo Province, Thailand. The chloroquine sensitivity of Plasmodium vivax has been assessed in parallel, using a growth inhibition method. Blood samples for the in vitro tests were taken prior to the administration of the standard treatment with chloroquine--in total 25 mg base/kg over 3 days--and primaquine 0.25 mg base/kg once daily for 14 days. The efficacy has been assessed according to the WHO standard in vivo test. The cure rate was 100%. No recrudescence was observed during the follow-up period of 28 days. The mean fever clearance time (FCT) was 40 h, the mean parasite clearance time (PCT) was 49 h. Mean IC(50) and IC(90) of the parasites were 28 and 171 nM, respectively. These results show that local P. vivax is still sensitive to chloroquine. The epidemic outbreak was therefore obviously not due to the presence of chloroquine-resistant P. vivax.     

Sensitivity to antifolates and genetic analysis of Plasmodium vivax isolates from Thailand

We investigated the association between the Plasmodium vivax dihydrofolate reductase (Pvdhfrtas) and the P. vivax dihydropteroate synthase (Pvdhps) genotype and in vitro sensitivity to the antifolates pyrimethamine, WR99210, chlorcycloguanil, sulfadoxine, and dapsone. Drug responses of 32 P. vivax isolates were assessed in two in vitro systems: schizont maturation inhibition and a yeast expression system. The geometric mean of 50% inhibition concentration (IC(50)) values for pyrimethamine, chlorcycloguanil, WR99210, sulfadoxine, and dapsone were 85 +/- 88, 784 +/- 662, 95 +/- 87, 2,424 +/- 2,784, and 1,625 +/- 1,801 nM, respectively, for the schizont maturation assay. Five different Pvdhfr alleles and four Pvdhps alleles were observed: 26 of 32 quadruple mutant alleles of Pvdhfr (F57I,L/S58R/T61M/S117T), four triple mutants (S58R/T61M/S117T, K49C/S58R/S117N), and two double mutant isolates (S58R/S117N). All isolates carried Pvdhps 585V. Twenty four isolates carried double mutant Pvdhps (A383G/A553G), six an additional mutation, S382A,C/A383G/A553G, and two a single mutation, A383G. Increasing geometric mean IC(50) values were observed with increased number of Pvdhfr mutations from double to quadruple. Results suggest that quadruple mutant alleles confer decreased sensitivity to pyrimethamine but retain sensitivity to WR99210.     

Frequency of pruritus in Plasmodium vivax malaria patients treated with chloroquine in Thailand

Chloroquine-induced itch in black-skinned African malaria patients is common and frequently leads to poor compliance or treatment defaulting.To assess the frequency and severity of chloroquine-induced pruritus in an Asian population, we reviewed case records of 1189 Plasmodium vivax malaria patients treated with chloroquine (25 mg/kg over 3 days) at the Bangkok Hospital for Tropical Diseases from 1992 through 1997.The majority of patients were Thais or ethnic Burmese (light brown skin), referred from the western border of Thailand. Overall, there were 23 patients (1.9%) with complaints of pruritus during chloroquine therapy. Of these, 12 (52%) had palm and sole involvement, eight (35%) had generalized pruritus including the palms and soles, and three (13%) had palm itching only. One patient developed pruritus on the palms and soles on two consecutive admissions.The pruritus did not interfere with daily activity, was reduced in intensity by anti-histamine therapy, and did not affect the patient's willingness to complete the chloroquine regimen. Therapeutic responses in the 23 patients with chloroquine itch was similar to those without itch. Among the itch patients, there was no association with gender or level of parasitaemias. Our findings indicate that the frequency of chloroquine-induced pruritus in Asian patients treated with chloroquine for P. vivax malaria is low in comparison with black-skinned Africans.This may be related to pharmacogenetic factors, the infective Plasmodium species, drug metabolism or drug-parasite interactions, or a lower affinity of chloroquine for less pigmented skin.     

Spatio-temporal distribution of Plasmodium falciparum and p. Vivax malaria in Thailand

Malaria incidence data at the district level from 1997 to 2002 and total malaria case data from 1965 to 2002 in Thailand were analyzed to determine the spatial and temporal dynamics of Plasmodium falciparum and P. vivax malaria incidence. Over the 37-year period, there was a 35-fold reduction in the incidence rates of P. falciparum malaria (11.86% in 1965 versus 0.34% in 2002) and a 7-fold reduction in P. vivax malaria (2.89% in 1965 versus 0.40% in 2002). The incidence ratio of P. falciparum to P. vivax malaria was reduced from 4.1 to 0.8 during this period. Malaria incidence rate exhibited the most rapid reduction between 1975 and 1985, coinciding with the introduction of a combination of antifolate drugs (sulfadoxine-pyrimethamine). The distribution maps of P. falciparum and P. vivax malaria incidence rates indicated a high spatial heterogeneity. The Thailand-Myanmar and Thailand-Cambodia border areas, where migration of foreign workers was pronounced, had the highest incidence rates for P. falciparum, P. vivax, and mixed-species infections. Transition probability analysis based on the malaria incidence rate among Thai residents indicated that there was an overall trend of decrease in the number of malaria cases and the number of high incidence districts between 1997 and 2002. High spatial variation in malaria incidence and local human migration patterns suggest that malaria control measures need to be adjusted according to local environmental and demographic settings.