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Antimicrob Agents Chemother. 2011 January; 55(1): 155–164.
Published online 2010 October 18. doi:  10.1128/AAC.00691-10
PMCID: PMC3019650

Tracking Origins and Spread of Sulfadoxine-Resistant Plasmodium falciparum dhps Alleles in Thailand[down-pointing small open triangle]

Abstract

The emergence and spread of drug-resistant Plasmodium falciparum have been a major impediment for the control of malaria worldwide. Earlier studies have shown that similar to chloroquine (CQ) resistance, high levels of pyrimethamine resistance in P. falciparum originated independently 4 to 5 times globally, including one origin at the Thailand-Cambodia border. In this study we describe the origins and spread of sulfadoxine-resistance-conferring dihydropteroate synthase (dhps) alleles in Thailand. The dhps mutations and flanking microsatellite loci were genotyped for P. falciparum isolates collected from 11 Thai provinces along the Burma, Cambodia, and Malaysia borders. Results indicated that resistant dhps alleles were fixed in Thailand, predominantly being the SGEGA, AGEAA, and SGNGA triple mutants and the AGKAA double mutant (mutated codons are underlined). These alleles had different geographical distributions. The SGEGA alleles were found mostly at the Burma border, while the SGNGA alleles occurred mainly at the Cambodia border and nearby provinces. Microsatellite data suggested that there were two major genetic lineages of the triple mutants in Thailand, one common for SGEGA/SGNGA alleles and another one independent for AGEAA. Importantly, the newly reported SGNGA alleles possibly originated at the Thailand-Cambodia border. All parasites in the Yala province (Malaysia border) had AGKAA alleles with almost identical flanking microsatellites haplotypes. They were also identical at putatively neutral loci on chromosomes 2 and 3, suggesting a clonal nature of the parasite population in Yala. In summary, this study suggests multiple and independent origins of resistant dhps alleles in Thailand.

The Thailand-Cambodia border has been the epicenter of most antimalarial drug resistance, including chloroquine (CQ), sulfadoxine-pyrimethamine (SP), mefloquine (MQ), and, most recently, artemisinin (13, 14, 34, 50). CQ resistance was first reported in this region in the early 1960s (16, 20, 53). In 1973, the malaria control program of Thailand introduced SP as the first-line drug for the treatment of uncomplicated falciparum malaria (7, 11, 12, 30, 41). The efficacy of SP decreased sharply, and by 1980, the cure rate dropped to <25% in some Thai provinces (8, 10, 21, 37). The cure rates at the Thailand-Burma (Tak) (42%), Thailand-Cambodia (Chanthaburi) (32%) borders as well as in the northeastern province (Kalasin) (39%) were lower than those in the central (Phetchabun) (92%) and southern (Songkhla) (82%) provinces (37). Eventually, SP became virtually ineffective in Thailand (23, 30, 48). As a consequence, MQ was introduced in 1985 in a fixed-dose combination with SP (MSP). Clinical resistance to MQ emerged soon, with a significant drop in the cure rate by 1992 (51). In 1995, artemisinin-based combination therapy (ACT), consisting of artesunate (AS) plus MQ, was introduced as the first-line treatment against uncomplicated falciparum malaria in areas designated “high-MQ-resistance zones,” mainly around Mae Sot (Tak province) at the border of Thailand with Burma and around the southeastern border of Thailand (Trat and Chanthaburi provinces) with Cambodia. Subsequently, the combination of AS plus MQ was adopted across all of Thailand (49).

Resistance to pyrimethamine and sulfadoxine in Plasmodium falciparum is conferred mainly by key mutations in the genes encoding the dihydrofolate reductase (DHFR) (C50R, N51I, C59R, S108N/T, and I164L) and dihydropteroate synthase (DHPS) (S436A/F, A437G, K540E, A581G, and A613S/T) enzymes, respectively (4, 36, 39, 42-44). Evidence indicates that these mutations in the parasite appear in a stepwise manner, and the number of mutations is positively correlated with the level of both in vivo and in vitro SP resistance (19, 27, 43). The genotyping of these mutations and the microsatellite loci linked to them provides important insights into the evolutionary history of drug resistance in a given region where malaria is endemic. For example, analyses of pfcrt mutations and flanking microsatellites in P. falciparum populations from different continents revealed that CQ resistance originated only 4 to 5 times globally, including one origin at the Thailand-Cambodia border (18, 28, 52). Similarly, high levels of pyrimethamine resistance (dhfr alleles with ≥3 mutations) also have a limited number of origins (4 to 5 origins) worldwide (32, 40). Furthermore, it has been shown that resistant pfcrt and triple mutant dhfr alleles prevalent in Africa originally came from Southeast Asia (Thailand-Cambodia border), most likely through the Indian subcontinent. Therefore, population migration has played an important role in the intercontinental spread of both CQ and SP resistances (40, 52).

The evolutionary history of CQ and pyrimethamine resistance in Southeast Asia has been extensively investigated (1, 29). In Southeast Asia, dhfr alleles with a single mutation have multiple origins, whereas those with ≥2 mutations have a single common origin (32, 40). Recently, we demonstrated that there are three major triple mutant dhps alleles in Cambodia: AGEAA, SGEGA, and the novel SGNGA (dhps codons 436, 437, 540, 581, and 613 are sequentially represented, with mutated codons underlined), especially in the regions (Western Cambodia) bordering Thailand (46). The Eastern Cambodia regions bordering Vietnam had predominantly wild-type, single mutant, and double mutant dhps alleles. Unlike dhfr, we observed multiple origins of the dhps alleles carrying ≥2 mutations in Cambodia. We also showed that the SGEGA and SGNGA triple mutant dhps alleles in Cambodia originated from a common genetic background, while the AGEAA allele was derived from another independent background (46). There are only limited data available on dhps mutations from Thailand (3, 31). In a previous study of the Thailand-Burma border, only two triple mutant alleles (SGEGA and AGEAA) were described (31).

Due to significant interest in understanding the evolution of multidrug-resistant malaria on the Thailand-Burma and Thailand-Cambodia borders, it is valuable to systematically characterize how different sulfadoxine-resistant dhps alleles have originated and spread throughout this region. We addressed the following questions in this study. First, is there any difference in the prevalence of resistant dhps alleles among different provinces in Thailand? Second, are there any differences in the genetic backgrounds of the parasites among different Thai provinces? Third, are there multiple origins of resistant dhps alleles in Thailand? Fourth, is the novel SGNGA allele found in Cambodia also present in Thailand? Finally, do the Thailand and Cambodian resistant dhps alleles share an ancestry?

MATERIALS AND METHODS

Parasite isolates and study sites.

Plasmodium falciparum-infected blood samples (n = 444) used in this study were collected in 2007 to 2008 from 11 Thai provinces as part of a malaria surveillance study conducted by the Thailand Ministry of Public Health. Seven of these provinces (Mae Hong Son, Chiangmai, Tak, Kanchanaburi, Prachuap Khiri Khan, Chumphon, and Ranong) are close to the Burma border, and three (Trat, Chanthaburi, and Si Sa Ket) border Cambodia, while one (Yala) is close to Malaysia (Fig. (Fig.1).1). Finger prick blood samples were collected on filter paper with informed consent from each patient. The study was approved by the Ethical Review Committee for Research in Human Subjects of the Thailand Ministry of Public Health.

FIG. 1.
Region-wise distribution of dhps alleles in Thailand. The gray-shaded areas in the map indicate the 11 Thai provinces from where samples were collected (Mae Hong Son, n = 38, including 5 samples from Chiangmai; Tak, n = 151; Kanchanaburi, ...

Although numbers of annual malaria cases in Thailand have substantially declined over the past decade, it is still problematic in forested areas and along the Burma and Cambodia borders due to significant population movement (6, 9, 49, 51, 54). In recent years, there has been a dramatic increase in the number of malaria cases in Yala and other southern provinces near the Malaysia border, and this can be attributed to ongoing civil unrest, which is a major obstacle for the government malaria control staff to carry out their routine activities effectively. In 2007, the malaria incidence rate was higher in Yala (17.2 cases per 1,000 population), Mae Hong Son (9.2 cases per 1,000 population), Tak (8.5 cases per 1,000 population), and Ranong (8.7 cases per 1,000 population) than in other provinces (3.9 cases per 1,000 population in Chumphon, 2.9 cases per 1,000 population in Prachuap Khiri Khan, 1.7 cases per 1,000 population in Chanthaburi, and 1.2 cases per 1,000 population in Kanchanaburi) (49). Historically, the Trat and Chanthaburi provinces in Thailand and the Pailin province in Cambodia have been collectively known as the epicenter of drug-resistant malaria. However, continuous efforts to contain malaria on the Thailand-Cambodia border have led to a substantial decrease in the number of malaria cases in this region (51).

Genotyping of dhps codons.

DNA from all 444 P. falciparum-confirmed filter paper blood spots were extracted by using a QIAamp DNA minikit (Qiagen, Valencia, CA). Samples were genotyped for the five key codons (dhps codons 436, 437, 540, 581, and 613) implicated in sulfadoxine resistance using methods described previously (46). Briefly, a portion (647 bp) of the dhps gene spanning codons 436 to 613 was amplified by using a nested PCR strategy. The first round of amplification was performed with primers 5′-AACCTAAACGTGCTGTTCAA-3′ (F1) and 5′-AATTGTGTGATTTGTCCACAA-3′ (R1), and the second round was done with primers 5′-ATGATAAATGAAGGTGCTAG-3′ (F2) and 5′-TCATTTTGTTGTTCATCATGT-3′ (R2) (46). Sequencing of the nested PCR product was performed by using primers F2 and R2 (46). Samples with multiple peaks at genotyped codons (mixed genotype) were excluded from allele frequency calculations and microsatellite genotyping.

Genotyping of microsatellite loci.

A total of 18 microsatellite loci (4 each on chromosomes 2 and 3 and 10 on chromosome 8) were typed. The loci on chromosome 2 (GenBank UniSTS C2M27, C2M29, C2M34, and C2M33) and chromosome 3 (GenBank UniSTS C3M40, C3M88, C3M69, and C3M39) are putatively neutral and were typed to get an estimate of the baseline heterozygosity in the Thai P. falciparum population. Ten loci on chromosome 8 around the dhps gene (at −11 kb, −7.5 kb, −2.9 kb, −1.5 kb, and −0.13 kb in the upstream region and 0.03 kb, 0.5 kb, 1.4 kb, 6.4 kb, and 9 kb in the downstream region) were typed to study the selective sweeps and genetic lineages of resistant dhps alleles in Thailand. Samples with multiple peaks at any one of the 18 loci were considered multiply infected and not included for either constructing multilocus microsatellite haplotypes or determining lineages of dhps alleles. Primer details and PCR cycling conditions for all neutral and dhps microsatellite loci were reported previously (46). The amplified microsatellite products were resolved on an ABI 3130xl genetic analyzer and scored by using GeneMapper software v3.7 (Applied Biosystems, Foster City, CA).

Estimating genetic diversity and determining genetic lineages.

As a measure of genetic diversity, the expected heterozygosity (He) at all dhps and neutral microsatellite loci was estimated by using the Excel Microsatellite Toolkit, version 3.1.1 (35). He was calculated by using the formula (He) = [n/(n − 1)][1 − Σpi2], where n is the number of isolates genotyped for that locus and pi is the frequency of the ith allele. The sampling variance for He was calculated as 2(n − 1)/n3 {2(n − 2) [Σ(pi3 − (Σpi2)2]}. For He analysis, samples were grouped either allele-wise or region-wise.

For determining genetic lineages of dhps alleles, we constructed multilocus haplotypes using all 10 dhps microsatellite loci with eBURST, version 3 (17). We also compared the Thai dhps microsatellite haplotypes with previously reported Cambodian dhps microsatellite haplotypes (46) to determine the genetic relationships between Thai and Cambodian dhps alleles. For this comparison, we constructed a neighbor-joining (NJ) tree based on Cavalli-Sforza (CS) chord genetic distance (5) as implemented with Powermarker v3.25 software (26).

Statistical analysis.

The prevalences of triple mutant dhps alleles among different provinces in Thailand were compared by the chi-square (χ2) test. Differences in mean He values were compared by using a Mann-Whitney U test implemented in the statistical package Stata, version 8.1, for Windows (Stata Corporation, College Station, TX). A P value of ≤0.05 was considered statistically significant.

RESULTS

Determination of singly infected samples.

All 444 samples were subjected to sequencing of the five dhps codons. Twenty-seven samples showed multiple peaks (mixed genotype) in the sequencing electropherograms at any of the five codons and thus were not processed further. The remaining 417 samples presented a single genotype at all five dhps codons and were typed for 8 neutral microsatellite loci on chromosomes 2 and 3 and 10 microsatellite loci flanking dhps on chromosome 8. Of these 417 samples, 117 showed >1 peak (multiple allele) at any microsatellite locus, while 300 had a single peak at all 18 loci. Thus, we found ~32% (144/444) of samples to be multiply infected, which is in agreement with data from previous studies in Thailand (33, 38). We report here the dhps allele frequency for 417 samples (Fig. (Fig.11 and Table Table1),1), and only 300 singly infected samples were used for constructing multilocus microsatellite haplotypes and defining dhps lineages.

TABLE 1.
Distribution of P. falciparum dhps alleles in Thailand

Distribution of dhps alleles in Thailand.

All 417 samples had mutations in the dhps gene, mostly (353/417; 85%) triple mutants (SGEGA, 206/417 samples; SGNGA, 46/417; AGEAA, 98/417; AGNAA, 3/417). Other dhps alleles in the order of decreasing frequencies were AGKAA (47/417 samples), SGEAA (6/417), SGKGA (5/417), AGEGA (4/417), SGKAA (1/417), and FGEAT (1/417) (Fig. (Fig.11 and Table Table1).1). Overall, the proportion of SGEGA-bearing parasites was greater than that of AGEAA-bearing parasites, which in turn was greater than that of SGNGA-bearing parasites. There were some noticeable differences in the distributions of three major triple mutant alleles in different provinces of Thailand (Fig. (Fig.1,1, inset). The northern provinces had a high frequency of the SGEGA allele (32/38 in Mae Hong Son and 108/151 in Tak) and low frequencies of the AGEAA (3/38 in Mae Hong Son and 28/151 in Tak) and SGNGA (1/38 in Mae Hong Son and 9/151 in Tak) (P = 0) alleles. Ranong in the south had high frequencies of both the SGEGA (31/55) and AGEAA (21/55) alleles (P = 0.056) and only one isolate with the SGNGA allele. Interestingly, Yala, a southernmost province near the Malaysia border, had only AGKAA double mutants (Fig. (Fig.1).1). In other words, all but one of the AGKAA-bearing isolates were from Yala. The central provinces Kanchanaburi and Prachuap Khiri Khan had almost equal frequencies (P > 0.2 for all comparisons) of all three triple mutant alleles (Fig. (Fig.1).1). The southeastern province Trat at the Cambodia border had almost equal frequencies of the AGEAA and SGNGA alleles (P = 0.54). The SGEGA allele was totally absent in Trat, or, if present at a low frequency, the limited sample size may not have allowed us to detect it. We also analyzed isolates from the northeastern province Si Sa Ket, bordering northern Cambodia, where a majority of isolates had AGEAA alleles (P = 0.001) with a minor frequency of SGEGA (Fig. (Fig.11).

Broadly, the AGEAA alleles were substantially present in central, south, southeast (Thailand-Cambodia border), and northeastern provinces, while they were present in low numbers in the northern provinces bordering Burma (46). The SGEGA allele was predominantly present in northern provinces, followed by south and central provinces. The frequency of this allele was low in the regions closer to the Thailand-Cambodia border (46). Interestingly, the newly reported SGNGA triple mutant occurred mostly at the Thailand-Cambodia border (Trat) (7/24), in the provinces close to it in central Thailand (Kanchanaburi [19/52] and Prachuap Khiri Khan [9/37]), as well as in Cambodia (Pailin [13/29] and Chumkiri [12/49]) (Fig. (Fig.1)1) (46).

Selective sweeps and lineages of resistant dhps alleles.

All parasites (n = 30) from Yala harbored the AGKAA double mutants with identical or nearly identical flanking microsatellite haplotypes. These parasites also had identical microsatellite haplotypes at neutral loci on chromosomes 2 and 3. Thus, the Yala parasites were treated as a separate population different from the rest of the Thai isolates (n = 270) (Fig. (Fig.2A).2A). The mean He at 10 dhps loci for the remainder of Thailand was 0.42 ± 0.06, which was very low (P = 0.0002 by a Mann-Whitney U test) compared to the mean He at 8 neutral loci (0.82 ± 0.04), implying that the dhps gene has undergone strong selection in Thailand (the mean He values at dhps and neutral microsatellite loci for all provinces are given in Table Table2).2). On the other hand, the mean He at 10 dhps loci for the Yala parasites was 0.02 ± 0.01 and was not significantly different (P = 0.76 by a Mann-Whitney U test) from the mean He at neutral loci (0.05 ± 0.03) (Table (Table2).2). The extremely low diversity at both dhps (selected) as well as neutral (unselected) loci in Yala could be the result of a recent expansion of a few founder lineages of AGKAA-bearing parasites that are the vestiges of a bottlenecked population or a recent introduction. We also analyzed the three major triple mutant dhps alleles for selective sweeps (Fig. (Fig.2B).2B). The mean He values around these alleles were extremely low (SGEGA, 0.23 ± 0.06 [n = 148]; AGEAA, 0.27 ± 0.06 [n = 73]; SGNGA, 0.34 ± 0.03 [n = 34]) but not significantly different from each other (P > 0.05 by a Mann-Whitney U test). Nonetheless, it was significantly lower (P = 0.0001 by a Mann-Whitney U test) than those of neutral loci (SGEGA, 0.81 ± 0.04; AGEAA, 0.73 ± 0.03; SGNGA, 0.82 ± 0.03).

FIG. 2.
Selection valley around dhps alleles in Thailand. (A) The genetic diversity of the isolates from Yala (n = 30) were calculated separately from the remainder of the Thai isolates (n = 270). The diversity was extremely reduced at all 10 ...
TABLE 2.
Mean expected heterozygosity (He) at 10 dhps and 8 neutral microsatellite loci in different provinces of Thailand and Cambodiaa

To investigate lineages of resistant dhps alleles in Thailand, we constructed 10-loci dhps microsatellite haplotypes (haplotypes T1 to T98) for all 300 singly infected samples (Fig. (Fig.3).3). Among 73 isolates bearing the AGEAA allele, 30 were found to have identical 10-locus microsatellite haplotype T1, 15 were its single-locus variants (SLVs) (T2 to T12), 9 were double-locus variants (DLVs) (T13 to T15), and 5 were triple-locus variants (TLVs) (T16 and T17). The remaining 14 isolates (T18 to T29) differed at ≥4 loci. This suggests that there was one major lineage for the AGEAA allele in Thailand along with few minor variants, which could represent either independent lineages or recombinants. Among 148 isolates bearing SGEGA, 39 carried an identical haplotype, T37, 50 were SLVs (T38 to T45), 15 were DLVs (T46 to T55), and 27 were TLVs (T20 and T56 to T68). The remaining 17 isolates differed at ≥4 loci (T69 to T81). Thus, the SGEGA allele also had one major lineage with a few minor variants. Some of these SGEGA-bearing isolates (T65 to T79) had haplotypes closer to that of AGEAA, especially in the 5′ region, possibly due to recombination. The majority (30/34) of SGNGA-bearing isolates carried identical or very similar haplotypes, which were in turn identical (T37, T38, and T46) or very similar to the SGEGA haplotypes, suggesting a common lineage of these two alleles. Of the 31 isolates bearing AGKAA, 30 were from Yala (T30 to T35) and had identical or very similar 10-locus microsatellite haplotypes (Fig. (Fig.3).3). The one isolate from Si Sa Ket bearing the AGKAA allele had a distinct haplotype (T36) different from that of Yala but very similar to T1, a major haplotype for the AGEAA allele.

FIG. 3.
The 10-locus microsatellite haplotype profiles (T1 to T98) flanking dhps in Thailand. Each box represents the occurrence of the dhps allele (codons 436, 437, 540, 581, and 613 are sequentially represented, with mutated codons in red) and its flanking ...

Defining lineages for low-frequency dhps alleles was difficult. However, the microsatellite backgrounds suggest that the SGKGA and SGEAA haplotypes are identical or very similar to the SGEGA/SGNGA haplotypes, whereas AGEGA was identical to AGEAA (Fig. (Fig.3).3). The AGNAA alleles (T97 and T98) were also closer to one of the haplotypes (T28 and T29) of AGEAA. Although AGKAA alleles in Yala had a single lineage, their backgrounds were closely related to AGEAA haplotypes (Fig. (Fig.33).

Shared ancestry of resistant dhps alleles in Thailand and Cambodia.

We also investigated relationships between resistant dhps alleles from Thailand (n = 300) (this study) and Cambodia (n = 78) (46), as depicted in a neighbor-joining tree (Fig. (Fig.4).4). Interestingly, ~50% of the Thai isolates (n = 151) shared identical 10-locus haplotypes with ~70% (n = 54) of the Cambodian isolates (Table (Table3).3). The remaining 24 Cambodian isolates also had closely related haplotypes with the respective alleles in Thailand, as depicted (haplotypes C1 to C20) in Fig. Fig.4.4. Thus, results from this study provide evidence that resistant dhps alleles in the Thailand-Cambodia region have common shared origins.

FIG. 4.
NJ tree depicting relationships between dhps microsatellite haplotypes in Thailand and Cambodia. T1 to T98 represent haplotypes for 300 Thai P. falciparum isolates, as shown in Fig. Fig.3.3. Blue (AGEAA) and black (SGEGA/SGNGA) branches indicate ...
TABLE 3.
List of 10-locus dhps microsatellite haplotypes shared between Thai and Cambodian P. falciparum samples

DISCUSSION

The Thailand-Cambodia border has been the epicenter of drug-resistant malaria (45). During the past 2 decades, Mae Sot (an area in the Tak province of Thailand) on the Burma border has become an additional focus of antimalarial drug resistance (51). To our knowledge, this may be the first study to systematically analyze the dhps mutations and flanking microsatellite loci in parasites from 11 Thai provinces, which are among those with the highest incidence of malaria. Previously, only two studies of dhps mutations from Thailand have been reported, but neither study characterized the flanking microsatellites around dhps (3, 31). Biswas et al. (3) analyzed only 50 isolates (from the Kanchanaburi, Tak, Trat, and Nongkhai provinces during 1995 to 1996) and observed ~66% of them with 1 to 4 dhps mutations (S436F, 26%; A437G, 30%; A581G, 38%; A613S, 14%; A613T, 38%). Since they did not genotype dhps codon 540, it was difficult to compare data from their study with our current data. We could directly compare our data with data from only one other study, a study by Nair et al. (31), where all five dhps codons were genotyped for 157 parasite isolates collected during 1998 to 2001 from Mawker-Thai (Tak Province). All isolates in that study harbored mutant dhps (SGEGA, 68%; AGEAA, 20%; SGKGA, 8%; AGKAA, 2% SGEAA, 1%). We also found mutant dhps in all 151 samples from Tak, with frequencies of the SGEGA (72%), AGEAA (19%), and SGEAA (0.66%) alleles almost equal to those reported previously by Nair et al. (Table (Table1).1). However, unlike the study reported by Nair et al., we did not find any AGKAA-bearing parasites in Tak, and the frequency of the SGKGA allele was also low (0.7%). Interestingly, we report for the first time a new triple mutant, SGNGA, in Tak as well as in other provinces of Thailand (Fig. (Fig.11 and Table Table1).1). It is likely that this novel SGNGA allele may have spread to these Thai provinces recently from the Cambodia border.

One of the important observations from this study is that the prevalence of the mutant dhps alleles were geographically different, which provides interesting insights into the gene flow/parasite migration patterns in Thailand. The SGEGA allele was found in high frequencies in the northern, central, and southern provinces bordering Burma but in low frequencies in the provinces bordering Cambodia (Fig. (Fig.1).1). The Cambodian side of the border, especially Pailin and Chumkiri, also had low frequencies of the SGEGA allele (46). The frequency of the SGNGA allele was high in the provinces closer to the Thailand-Cambodia border (~45% in Pailin and ~25% in Chumkiri in Cambodia and 29% in Trat, 37% in Kanchanaburi, and 24% in Prachuap Khiri Khan in Thailand) compared to those in other provinces (Fig. (Fig.11 and Table Table1).1). The microsatellite haplotypes flanking the SGEGA and SGNGA alleles were identical or very similar, suggesting a common origin for these alleles (Fig. (Fig.3)3) (46). The AGEAA allele occurred at high frequencies at the Thailand-Cambodia border and central, northeastern, and southern (Ranong) Thai provinces and at low frequencies in the northern provinces. In Cambodia, Pailin and Chumkiri had large numbers of isolates harboring the AGEAA allele (46). Although AGEAA alleles were found along with the SGEGA/SGNGA alleles in almost all Thai provinces, their microsatellite haplotypes were different, consistent with independent origins for these alleles. Based on the distribution pattern of these resistant alleles, it is tempting to speculate that the SGNGA and AGEAA alleles originated at the Thailand-Cambodia border, whereas the SGEGA allele originated at the Thailand-Burma border (Fig. (Fig.1).1). However, based on the common origins of the SGEGA and SGNGA alleles, it is also possible that both of these alleles originated at the Thailand-Cambodia border and that only the SGEGA allele spread and became established in the provinces bordering Burma (Fig. (Fig.1).1). These hypotheses can be validated only if retrospective parasites from the Thailand-Cambodia and Thailand-Burma borders and other neighboring countries are analyzed. Nonetheless, there were two major (one for SGEGA/SGNGA and another for AGEAA) and several minor independent lineages of triple mutant dhps alleles in Thailand (Fig. (Fig.3),3), which were identical or very similar to the dhps lineages prevalent in Cambodia (Fig. (Fig.4)4) (46).

The high occurrence of triple mutant dhps alleles at the Thailand-Burma and Thailand-Cambodia borders may have resulted from the combination of several factors, including the extensive use of SP in these regions in the past. The frequent movement of people across these borders has immensely contributed to the dissemination of multidrug-resistant parasites from the Thailand-Cambodia border to other regions. In the 1970s and 1980s, a large number of gem miners from Burma and elsewhere in Thailand temporarily migrated to the southeastern border of Thailand and Cambodia and in Western Cambodia, where malaria transmission was intense. When these workers returned back to Thailand and Burma, they were often infected with drug-resistant parasites. In recent years, this inflow of Burmese migrants to the Thailand-Cambodia border has continued, but mainly for agricultural reasons, by migrants entering Thailand through Mae Sot (Tak province), Kanchanaburi, and Ranong. These migrants are known as a key factor in spreading drug-resistant malaria from its epicenter in southeast Thailand westward.

Interestingly, the Yala parasite population had only AGKAA double mutants with identical flanking microsatellite haplotypes (mean He at 10 dhps loci of 0.02 ± 0.01; n = 30) (Fig. (Fig.2A2A and and33 and Table Table2).2). Moreover, they were also identical at neutral microsatellite loci (mean He at 8 neutral loci of 0.05 ± 0.03; n = 30). This observation is consistent with results from recent studies that also indicated a population bottleneck in the Yala population, leading to a reduction in genetic diversity (24, 38). Pumpaibool et al. (38) previously analyzed the genetic diversity at 12 neutral microsatellite markers in parasites from seven Thai provinces (Mae Hong Son, Tak, Kanchanaburi, and Ranong on the Burma border and Trat, Ubonratchathani, and Yala on the Cambodia, Laos, and Malaysia borders, respectively) and found a significant level of genetic differentiation between the Yala population and the populations from Thailand-Burma (fixation index [FST] = 0.1321) and Thailand-Cambodia (FST = 0.1199) borders (38). The genetic differentiation between Thailand-Burma and Thailand-Cambodia populations was relatively low (FST = 0.0478). We also found high FST values (based on 8 neutral loci) for Yala versus Thailand-Burma (FST = 0.317) and Yala versus Thailand-Cambodia (FST = 0.348) populations, whereas we found low FST values for Thailand-Burma versus Thailand-Cambodia populations (FST = 0.045). This indicates that there is more parasite migration between the Thailand-Cambodia and Thailand-Burma borders, whereas Yala is isolated from the rest of Thailand.

Evidence indicates that in the absence of drug pressure, parasites with resistant mutations are relatively less fit than their ancestral wild-type counterparts (1, 2, 15, 47). Therefore, one might expect to note a gradual decline in the prevalence of resistant mutations in the population once the use of a drug has been discontinued. This has been observed for Malawi and other regions where malaria is endemic, where the prevalence of the CQ-resistant pfcrt (76T) allele has been found to decline after CQ was removed from the national treatment policy (1, 2, 25). Similarly, the prevalence of triple mutant dhfr and dhps alleles has declined in Peru 5 years after SP was removed (55). This scenario is in striking contrast to the fixation of sulfadoxine-resistant dhps alleles in Thailand despite SP treatment being officially discontinued more than 20 years ago. A similar fixation of resistant dhps alleles was also observed for parasites from Cambodia, especially in the Pailin and Chumkiri regions (46). There could be several reasons for this fixation. First, additional selective pressure from an antifolate drug other than sulfadoxine may be acting to maintain mutant dhps alleles (22). There is widespread use of cotrimoxazole (trimethoprim-sulfamethoxazole) for the treatment of bacterial infections and Pneumocystis infections in HIV-infected patients in Southeast Asia (22). However, there is no experimental or epidemiological evidence showing an association between cotrimoxazole use and the occurrence of dhps mutations in P. falciparum. Second, the parasites may have acquired additional compensatory mutations. Third, the fitness cost imposed by mutations in the dhps gene is too small to have a measurable deleterious impact on the parasite. Fourth, the parasite populations are fixed for resistance, and therefore, there are no competing sensitive parasites that would have greater fitness in the absence of sulfadoxine.

In summary, the results from this study indicate that (i) resistant dhps alleles were fixed in Thailand with substantial differences in their regional distributions; (ii) the recently reported SGNGA alleles were found at high frequencies in Thailand and most likely originated on the Thailand-Cambodia border; (iii) there were two major independent lineages of the triple mutant alleles, one common for SGEGA/SGNGA and another independent for AGEAA; and (iv) the Yala province had a recently expanded clonal population of AGKAA-bearing parasites. Finally, the resistant dhps alleles in Thailand originated on multiple, but limited, genetic backgrounds, which were identical or very similar to their counterparts in Cambodia. Given the historical importance of the Thailand-Cambodia and Thailand-Burma borders in the emergence and spread of multidrug-resistant parasites, this study could be helpful in understanding the movement of resistant parasites in the region. It is important to consider the current pattern of human migration in this region to devise a comprehensive strategy to contain the spread of drug-resistant parasites from the Thailand and Cambodia region to other areas.

Acknowledgments

M.T.A. is supported by an American Society for Microbiology (ASM) and Coordinating Center for Infectious Diseases (CCID) postdoctoral fellowship. We acknowledge support from the Atlanta Research and Education Foundation, VAMC, Atlanta, GA, and the CDC Antimicrobial Resistance Working Group. A.A.E. is supported by National Institutes of Health (NIH) grant R01GM084320. The funders had no role in study design, data collection and analysis, preparation of the manuscript, or decision to publish.

We thank the staff from the regional Offices of Disease Prevention and Control (ODPC) of the Thailand Ministry of Public Health. We are grateful to Ira Goldman and Sean Griffing for proofreading the manuscript.

The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Footnotes

[down-pointing small open triangle]Published ahead of print on 18 October 2010.

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