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    Figure 1.

    A, Geographic distribution of Plasmodium falciparum dihydrofolate reductase (dhfr) mutations in Laos. Provinces with sample sizes < 10 are indicated as smaller pie charts. B, Geographic distribution of P. falciparum dihydropteroate synthase (dhps) mutations in Laos. Provinces with sample sizes < 10 are indicated as smaller pie charts. C, Geographic distribution of P. falciparum chloroquine resistance transporter (pfcrt) mutations in Laos. Provinces with sample sizes < 10 are indicated as smaller pie charts. D, Comparison of the frequency of alleles carrying mutant codons in northern, central, and southern Laos. North = Bokeo, Huaphanh, Luangphrabang, Luangnamtha, Phongsaly, Oudomxay, Vientiane, Xiengkhuang and Xayabury; Central = Borikhamxay and Khammouane; South = Attapeu, Champassack, Saravane, Savannakhet and Sekong.

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Combined Molecular and Clinical Assessment of Plasmodium falciparum Antimalarial Drug Resistance in the Lao People’s Democratic Republic (Laos)

Mayfong MayxayWellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic; Southwest Foundation for Biomedical Research, San Antonio, Texas; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Savannakhet Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic; Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

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Shalini NairWellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic; Southwest Foundation for Biomedical Research, San Antonio, Texas; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Savannakhet Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic; Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

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Dan SudimackWellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic; Southwest Foundation for Biomedical Research, San Antonio, Texas; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Savannakhet Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic; Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

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Mallika ImwongWellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic; Southwest Foundation for Biomedical Research, San Antonio, Texas; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Savannakhet Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic; Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

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Naowarat TanomsingWellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic; Southwest Foundation for Biomedical Research, San Antonio, Texas; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Savannakhet Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic; Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

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Tiengkham PongvongsaWellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic; Southwest Foundation for Biomedical Research, San Antonio, Texas; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Savannakhet Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic; Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

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Samlane PhompidaWellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic; Southwest Foundation for Biomedical Research, San Antonio, Texas; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Savannakhet Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic; Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

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Rattanaxay PhetsouvanhWellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic; Southwest Foundation for Biomedical Research, San Antonio, Texas; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Savannakhet Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic; Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

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Nicholas J. WhiteWellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic; Southwest Foundation for Biomedical Research, San Antonio, Texas; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Savannakhet Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic; Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

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Tim J. C. AndersonWellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic; Southwest Foundation for Biomedical Research, San Antonio, Texas; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Savannakhet Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic; Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

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Paul N. NewtonWellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic; Southwest Foundation for Biomedical Research, San Antonio, Texas; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Savannakhet Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic; Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

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Molecular markers provide a rapid and relatively inexpensive approach for assessing antimalarial drug susceptibility. We collected 884 Plasmodium falciparum–infected blood samples from 17 Lao provinces. Each sample was genotyped for 11 codons in the chloroquine resistance transporter (pfcrt), dihydrofolate reductase (pfdhfr), and dihydropteroate synthase (pfdhps) genes. The samples included 227 collected from patients recruited to clinical trials. The pfcrt K76T mutation was an excellent predictor of treatment failure for both chloroquine and chloroquine plus sulfadoxine-pyrimethamine, and mutations in both pfdhfr and pfdhps were predictive of sulfadoxine-pyrimethamine treatment failure. In multivariate analysis, the presence of the pfdhfr triple mutation (51 + 59 + 108) was strongly and independently correlated with sulfadoxine-pyrimethamine failure (odds ratio = 9.1, 95% confidence interval = 1.4–60.2, P = 0.017). Considerable geographic heterogeneity in allele frequencies occurred at all three loci with lower frequencies of mutant alleles in southern than in northern Laos. These findings suggest that chloroquine and sulfadoxine-pyrimethamine are no longer viable therapy in this country.

INTRODUCTION

Plasmodium falciparum antimalarial drug resistance has become an increasingly difficult problem in malaria-endemic countries and usually necessitates changes in national malaria treatment policy.1 Comparative clinical trials have played a vital role in the assessment of antimalarial drug resistance.2 However, because such trials require long patient follow-up, sufficient sample size, and are expensive, it is difficult to perform multiple trials in one country. Therefore, there is limited information on the variability of antimalarial drug sensitivity within countries. Widespread in vitro drug sensitivity assays are impractical because of technical limitations, variable correlations with clinical outcomes,3 and problems of repeatability between laboratories. The correlation of specific P. falciparum mutations with treatment failure and the development of rapid and relatively inexpensive molecular techniques to detect these mutations4 suggest that these methods may be appropriate and practicable for large-scale monitoring of antimalarial drug resistance patterns5,6 when appropriately calibrated with clinical efficacy studies. For example, mutations in dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes confer resistance to pyrimethamine (P) and sulfadoxine (S), respectively, both in vitro and in vivo.713 Mutations in codon 76 of the P. falciparum chloroquine (CQ) resistance transporter (pfcrt) gene contributes to CQ resistance.1417

Laos is a land-locked country, with a population of approximately 5.6 million that has borders with China on the north, Burma (Myanmar) on the northwest, Thailand on the west, Cambodia on the south, and Vietnam on the north and east. Malaria occurs in all 17 provinces and although prevalence varies considerably, it is more prevalent in the southern part of the country. Plasmodium falciparum resistance to CQ and SP were first noted in this country in the late 1960s,1820 but these drugs remained the first-line and second-line nationally recommended treatments for uncomplicated P. falciparum malaria until 2005. Clinical trials of oral CQ and SP for the treatment of uncomplicated P. falciparum malaria in five different areas of Laos between 2000 and 2003 showed high rates of treatment failure (35–80% for CQ and 18–35% for SP).16,2124 Subsequent trials of artesunate-mefloquine and artemether-lumefantrine at two sites in Laos showed 42-day failure rates ≤ 6%.25,26 Therefore, artemisinin-based combination therapy (ACT) with artemether-lumefantrine was introduced as first-line antimalarial drug treatment of uncomplicated P. falciparum malaria in this country in 2005.

We measured the prevalence of molecular markers of antimalarial drug resistance in P. falciparum parasites throughout the country to examine geographic variation in the prevalence of mutations, and to provide a baseline for examining changes in drug resistance mutation frequency after the change in drug treatment policy to ACT. We also measured the prevalence of molecular markers in parasites from patients recruited to efficacy trials at two sites in Laos to examine the relationships between these molecular markers and therapeutic responses.

MATERIALS AND METHODS

Blood sample collection, clinical trials, and DNA preparation.

Malaria clinics and health facilities in every province in Laos (with the exception of Vientiane City where there is no malaria transmission) were identified, and patients coming to these clinics with fever were studied between 2001 and 2004. Province names are given in accordance with Sisouphanthong and Taillard.27 Thick and thin blood smears, stained with Giemsa, and three finger prick blood spots (approximately 50 μL) onto Whatman (Maidstone, United Kingdom) 3 MM filter paper were obtained from all patients with malaria coming to the malaria clinics, health centers, and hospitals. The filter papers were stored in plastic bags with silica gel at room temperature and brief patient information recorded. In addition, pre-treatment finger prick blood samples from patients enrolled in clinical trials of CQ versus SP (Vientiane and Savannakhet Provinces)22,23 and CQ plus SP versus artesunate plus mefloquine versus artemether-lumefantrine (Coartem®; Novartis, Basel Switzerland) (Savannakhet Province)25 were collected and treatment responses to antimalarial drugs for these patients were classified according to World Health Organization criteria.28 Some of these samples have been included in previous reported studies.29,30

Drug treatments in clinical trials were CQ sulfate (Government Pharmaceutical Organization, Bangkok, Thailand) 10 mg base/kg, followed by 10 mg base/kg 24 hours later, followed by 5 mg base/kg at 48 hours or a single dose of 1.25 mg/kg of pyrimethamine and 25 mg/kg of sulfadoxine (Fansidar®; F. Hoffmann La Roche, Basel, Switzerland) or the combination of CQ and SP with the same doses as mentioned above. Patients not included in the efficacy trials were treated according to the protocols of the local malaria clinics and hospitals at the time of the investigation (mostly with either CQ, SP, or quinine). DNA was prepared from blood spots using an extraction kit (Qiagen, Valencia, CA). DNA templates were stored at −20°C until use. The study was reviewed and approved by the Ethical Committee of the Faculty of Medical Sciences, National University of Laos and by the Institutional Review Board at the University of Texas Health Science Center in San Antonio.

Malaria parasite genotyping.

pfdhfr and pfdhps.

Five point mutations encoding five amino acid changes in the pfdhfr gene (A16I, N51I, C59R, S108N, and I164L) and in the pfdhps gene (S436A/F, A437G, L540E, A581G, and A613S/T) were genotyped using a rapid primer extension using the SnaPshot kit (Applied Biosystems, Foster City, CA).31 An advantage of this method is that all five codons at pfdhfr or pfdhps can be genotyped in one capillary in a sequencer, and scoring can be automated using fragment analysis software.

pfcrt.

The amino acid 76 mutation in the pfcrt gene (K76T) was genotyped using a simple polymerase chain reaction (PCR)–restriction digest assay and fluorescent detection of products on a ABI 3100 capillary sequencer (Applied Biosystems).32 Briefly, a 132-basepair fragment of pfcrt was amplified by fluorescent end-labeled primers using seminested-nested PCR. The fluorescent end-labeled products from the second PCR were then digested with Apo I and the digested products were loaded in the capillary sequencer. The pfcrt-resistant alleles were uncut, giving a peak at 132 basepairs and wild-type alleles were cut, giving a labeled fragment of 101 basepairs.

Data analysis.

Data were analyzed using SPSS version 11.0 (SPSS Inc., Chicago, IL). A Fisher’s exact test and chi-square test with Yates’ correction was used to assess the significance of the association between mutations and treatment failure in univariate analysis. Multiple logistic regression was used to identify the independent effects of mutations or amplifications predictive of treatment failures. Two-tailed significance was tested at P < 0.02 because of multiple comparisons, and all confidence levels were set at 95%. We analyzed geographic variation in allele frequencies using Wright’s F-statistics33 using Arlequin™.34 Significance of population differentiation was evaluated by comparing the observed FST values with the values found in 10,000 permuted data sets, in which genotypes were reshuffled among population samples. We measured linkage disequilibrium (LD) between the three loci within population samples using FSTAT35 and tested the significance of associations using randomization procedures.

RESULTS

A total of 884 blood samples were collected from Lao patients with P. falciparum malaria; 345 (39%) of 884 patients were females. Sample sizes varied considerably between locations (range = 2–480) depending on the prevalence of malaria infection and the location of the clinical trial team (54% of samples were from patients recruited to clinical trials in Savannakhet). The overall mean (95% confidence interval [CI]) age of the patients was 16.8 (15.7–17.8) years and 453 (58%) were children ≤ 15 years of age. The geometric mean (95% CI) parasitemia was 20,054 (17,935–22,418) parasites/μL. Clinical trial results were available for 227 (26%) patients (59, 52, and 116 in CQ, SP and CQ plus SP groups, respectively).

Frequency and geographic distribution of mutations.

Of the 884 samples collected, 734 (83%), 760 (86%), and 871 (98.5%) were successfully amplified and genotyped for pfdhfr, pfdhps, and pfcrt, respectively. Of the samples successfully genotyped, 74 (10%) of pfdhfr, 75 (10%) of pfdhps, and 35 (4%) of pfcrt were multiple clone infections. Because a genotype cannot be ascribed, these were excluded from the main analysis. The overall frequency of mutations was 77% (509 of 660) for pfdhfr, 21% (146 of 685) for pfdhps, and 85% (713 of 836) for pfcrt (Table 1). A novel allele containing a Lys→Tyr mutation at codon 540 in pfdhps was found in 12 samples from northern Laos. These results were confirmed by sequencing.

The proportion of infections with pfdhfr mutations was 2% with single mutations (at 108 position), 47% with double mutations, 24% with triple mutations, and 4% with quadruple mutations, and pfdhps mutation frequencies were 8% with single mutations, 6% with double mutations, 6% with triple mutations, and 0.5% with quadruple mutations. Of these samples, 21% (127 of 598) had pfdhfr and pfdhps mutations, and 21% (122 of 580) had mutations in all three loci.

The prevalence of the I164L mutation in pfdhfr, which confers high-level antifolate resistance, was 5.5% (36 of 660), mainly as part of the quadruple pfdhfr mutations (51 + 59 + 108 + 164). The most frequent double pfdhfr mutation was 59 + 108 (45%) and all (100%) triple pfdhfr mutations were 51 + 59 + 108.

We observed considerable geographic heterogeneity in allele frequencies at all three loci (Figure 1a–d), with highly significant (P < 10−4 for each locus) differentiation among provinces with FST values of 0.20, 0.22, and 0.13 for dhfr, dhps, and pfcrt, respectively. However, the patterns of genetic differentiation varied between loci and we observed distinctive patterns of differentiation at dhfr in the northern and southern regions of Laos. In northern Laos, dhfr alleles with two mutations predominated and no wild-type alleles were found. In contrast, both wild-type and alleles with three mutations were common in southern Laos. Wild-type alleles for dhps were common in all provinces. For pfcrt, the 76T allele was fixed in northern Laos, but high frequencies (up to 44% in Khammouane) of wild-type (76K) alleles were found in central and southern provinces. There was less variation at this locus than at the other two, which was reflected in the lower fixation indices for this locus. The proportion of resistance mutations at all three loci was lowest in Savannakhet and Khammouane provinces. Furthermore, we observed strong correlations between numbers of wild-type alleles at each of three loci across the different provinces (dhfr × dhps, r2 = 0.49, F = 9.7, P = 0.011; dhfr × pfcrt, r2 = 0.90, F = 93.4, P < 0.0001; dhps × pfcrt, r2 = 0.47, F = 8.9, P = 0.014). We also found strong correlations between the mean number of mutations at dhps and dhfr across populations (r2 = 0.747, F = 29.5, P = 0.0003). These analyses used data from only the 12 provinces with samples sizes > 10. The I164L mutation was more common on the northern and southern borders with Thailand.

We investigated non-random association (linkage disequilibrium) between the three loci within each of nine provinces where greater than 20 genotyped samples were available for each locus (Table 2). Because one of the loci examined was monomorphic in six of nine provinces, tests involving this locus could not be conducted. We conducted 15 tests. Results of four of seven tests between dhfr and dhps were significant (P < 0.05), and two of these remained significant after Bonferroni correction, which reduced the threshold value for significance at the 5% level to P < 0.0033. Results of two of four tests between dhfr and pfcrt had P values < 0.05, and none of the results of three tests between dhps and pfcrt were significant.

Molecular markers of drug resistance and treatment outcome in clinical trials.

A total of 227 samples in the survey were from patients recruited to clinical trials. We examined the association between molecular markers and treatment outcome after CQ, SP, or CQ plus SP treatments. Point mutations within pfdhfr, pfdhps, and pfcrt were good predictors of treatment failure with both antimalarial monotherapy and combinations (Table 3). The proportion of infections carrying one or more mutant codons at three loci was significantly higher in patients who had CQ, SP, and CQ plus SP treatment failures compared with those with treatment success (P < 0.05), except for the pfdhfr mutation in the CQ plus SP group. The frequency of parasites carrying the combination of double pfdhfr plus pfdhps and triple pfdhfr plus pfdhps plus pfcrt gene mutations was also significantly higher in the treatment failure than in the treatment success groups for all three treatment regimens (P < 0.05). The sensitivity and specificity of the pfcrt mutation in predicting treatment failure was 100% and 62% for CQ and 100% and 25% for CQ plus SP. Thus, all infections that failed CQ treatment had pfcrt mutant alleles. For the presence of any pfdhfr mutations, the sensitivity and specificity in predicting treatment outcome was 100% and 38% for SP and 89% and 28% for CQ plus SP. The sensitivity and specificity of pfdhps mutations at any points in predicting treatment outcome was 58% and 85% for SP and 55% and 85% for CQ plus SP. The pfdhfr triple mutation (51 + 59 + 108) had sensitivities of 36% and 18% and specificities of 94% and 71% in predicting SP and CQ plus SP treatment outcomes, respectively.

Different point mutations in pfdhfr and pfdhps and combinations of point mutations in both genes were analyzed to investigate the predictive value on SP and CQ plus SP treatment outcomes (Tables 4 and 5). The presence of a pfdhfr mutation at position 108 was significantly associated with SP treatment failure (relative risk [RR] = 0.62, 95% CI = 0.47–0.80, P = 0.003 and RR = 9.14, 95% CI = 1.39–60.16, P = 0.017, respectively). In a multivariate analysis including single, double, and triple pfdhfr mutations, the pfdhfr triple mutations were correlated independently with SP treatment failure (OR = 5.09, 95% CI = 0.99–26.20, P = 0.023).

The genotype failure index (GFI), defined as the ratio of prevalence of pfcrt-resistant genotypes to CQ failures, was calculated5 using sympatric CQ clinical trial treatment failure data obtained 2–3 years before this survey16,21 and concurrently.22,23,25 The overall GFI for CQ treatment in Laos was 1.5. The GFI was higher in the central (2.7) region compared with northern (1.5) and the southern (2.0) regions. The mean fever and parasite clearance times were longer in patients whose parasites carried mutant genes than in those with wild-type genes, but the difference was not statistically significant (Table 6).

DISCUSSION

Three features of the Lao resistance mutation distribution maps are of interest. First, we observed considerable variation in resistance allele frequencies across Laos. For P. falciparum dhfr, wild-type alleles and triple mutant alleles were common in the southern region, but were absent or at very low frequency in the northern region, and the dhfr-164L allele was found at a high frequency only in border areas of the southern and northern regions of Laos. Wild-type alleles for pfcrt were only observed in central and southern Laos. The patterns observed are likely to result from a variety of forces, including selection by antifolate drugs, selection against mutant alleles in the absence of drug pressure, and migration of parasites from neighboring countries with differing treatment regimens.

Second, frequencies of resistance mutations in the three different genes were correlated, with loci encoding resistance to unrelated drugs showed similar trends in allele frequencies across Laos. This heterogeneity in allele frequencies and correlation between loci suggest systematic differences in selection pressure by CQ and SP. The underlying reasons for this are not clear, but are probably related to variation in antimalarial availability and use in different provinces. Drug pressure on parasites is probably very variable across Laos because there are large variations in the incidence of malaria, the availability and the ability to purchase the different drugs, and the isolation of human communities in mountainous areas. In other regions of the malaria-affected world, spread of resistance has been remarkably rapid with single resistance alleles traversing continents.17 However, despite being surrounded by high levels of CQ resistance for more than 30 years, resistance alleles for pfcrt have not spread and become fixed in central Laos. Similarly, the relative rarity of resistance alleles for dhps across Laos contrasts starkly with the high frequencies of resistance alleles observed in neighboring countries.36

Third, we observed extensive LD between both dhfr and dhps and dhfr and pfcrt within population samples from different provinces. Strong LD between dhfr and dhps mutations have been observed in other countries and are expected when there is strong drug selection with SP. However in Laos, this is surprising because although SP was the official second-line drug for uncomplicated P. falciparum malaria until 2005, this drug was probably seldom used compared with CQ. Cotrimoxazole is and has been a commonly used antibiotic and this may also have contributed to selection pressure. The observation that two of four comparisons between dhfr and pfcrt suggested LD is also surprising, given that these loci are selected by different drugs, but their availabilities may be linked. However, because LD may also result from heterogeneity in allele frequencies within the sampled population, LD could result from sampling rather than epistasis or co-selection during drug treatment. The geographic distribution of molecular markers of antimalarial drug resistance has recently been described across Cambodia where pfdhfr and pfdhps mutations were more homogeneously distributed than in Laos, perhaps representing a higher malaria endemicity and great human population movement and mixing in Cambodia.37 On a larger scale, surveys across six countries in southeast Asia indicate that resistance alleles at all three loci are rarer in Laos compared with neighboring countries.36

This study suggests that known resistance mutations in P. falciparum genes associated with CQ and SP resistance can be used to predict malaria treatment outcomes in Laos. Although previous studies in central and southern Laos have not detected the pfdhfr mutation at position 164,38,39 a low frequency (approximately 6%) was found in this study. This is consistent with the rarity of high-level SP resistance in Laos compared with neighboring Cambodia and Thailand, where the prevalence of pfdhfr mutation at 164 is high (range = 42–80%).29,31,37,40 In Vietnam, the 164L pfdhfr mutation was found in 0–63% of infections.29,41

Consistent with the high CQ treatment failure rates in clinical trials in Laos,16,2124 the overall frequency of pfcrt K76T mutations was 85%, which is similar to that found in Cambodia (88%),42 but lower than that in Thailand (99%), Burma (Myanmar), and Vietnam (97%).43,44 In Cambodia, CQ sensitivity was found until recently in the northeastern part of the country adjacent to southern Laos, but recent studies in southern and northern Laos have demonstrated high percentages of pfcrt K76T mutations (range = 64–100%),16,30,39,45,46 which supports the interpretation of clinical trial results that CQ alone is no longer useful for the treatment of uncomplicated P. falciparum malaria in Laos. In Savannakhet Province, CQ plus SP had a relatively high clinical treatment success of 92%, which was not significantly lower than that for artemether-lumefantrine, but was significantly lower than that for artesunate + mefloquine.25 However, this treatment success frequency is close to the current World Health Organization guidelines for change in antimalarial treatment policy of < 90%,47 and relies strongly on the efficacy of SP, which is unlikely to be sustained. This trial was conducted in the part of the country with the most drug-sensitive alleles and may overestimate treatment efficacy in other regions of the country.

There is no simple relationship between carriage of resistance mutations and treatment failure. The prevalence of mutations conferring resistance is higher than the proportion of patients with treatment failure because other factors contribute to the therapeutic responses, notably host immunity. The overall GFI for CQ treatment in this study was 1.5, which is lower than that observed in Mali (GFI = 1.6–2.8)5 and probably reflects the lower transmission intensity and thus lower immunity to P. falciparum in Laos compared with Mali. The GFI was higher in the central provinces compared with the northern and the southern provinces of Laos. On the basis of the overall GFI in Laos, a failure rate of approximately 57% after treatment with CQ would be expected over most of Laos where pfcrt 76T is fixed. The lowest frequency of pfcrt 76T is in Khammouane province (56%), where we would expect a failure rate of approximately 37%.

Further surveys with larger sample sizes are needed to monitor the distribution of molecular markers of antimalarial drug resistance mutation frequencies after the radical change in drug treatment policy to ACT. The heterogeneity in prevalence of drug resistance mutation across Laos argue strongly for a combined approach to drug resistance monitoring, in which information from clinical trials are combined with broad geographic surveys of frequencies of known resistance mutations.

Table 1

Frequency of pfdhfr and pfdhps in Plasmodium falciparum from Laos*

pfdhfr (n = 660)
165159108164TypeHaplotype (%)
AlaAsnCysSerIleWild23
AlaAsnArgAsnIleMutant45
AlaIleArgAsnIleMutant24
AlaIleArgAsnLeuMutant4
AlaAsnCysAsnIleMutant2
AlaAsnArgAsnLeuMutant1.5
AlaIleCysAsnIleMutant0.2
pfdhps (n = 685)
436437540581613TypeHaplotype (%)
* pfdhfr = P. falciparum dihydrofolate reductase; pfdhps = P. falciparum dihydropteroate synthase. Polymorphic amino acid residue numbers are shown along the top of each section, and amino acids conferring resistance are shown in bold. Rare haplotypes (frequency < 1%) were not confirmed by sequencing; we cannot exclude the possibility that they result from genotyping of multiple clone infections.
† Tyr mutation at codon 540 confirmed by sequencing.
SerAlaLysAlaAlaWild79
SerGlyLysAlaAlaMutant8
AlaGlyGluAlaAlaMutant6
AlaGlyLysAlaAlaMutant4
SerGlyTyr†AlaAlaMutant2
SerGlyGluGlyAlaMutant0.4
PheAlaLysAlaSerMutant0.4
PheGlyGluAlaSerMutant0.4
SerGlyGluAlaAlaMutant0.3
AlaAlaLysAlaAlaMutant0.1
AlaGlyLysAlaThrMutant0.1
PheGlyLysAlaThrMutant0.1
Table 2

Significance of linkage disequilibrium within parasite populations from nine Lao provinces*

Provincedhfr/dhpsdhfr/pfcrtdhps/pfcrt
* ND indicates that one or both of the loci compared were monomorphic. P values in bold are significant after Bonferroni correction. pfcrt = Plasmodium falciparum chloroquine resistance transporter. For definitions of other abbreviations, see Table 1.
Luangphrabang0.028NDND
Luangnamtha0.01NDND
Vientiane0.055NDND
KhammouaneND0.251ND
Attapeu0.5881.0001.000
Champassack0.001NDND
Saravane0.4490.0080.816
Savannakhet< 0.0010.0070.984
Sekong0.306NDND
Table 3

Summary of drug resistance polymorphisms from patients in clinical trials in Laos*

Treatment
DrugGeneNo.FailureSuccessPχ2
* The proportions of primary infections carrying one or more mutant codons at the three loci are shown for patients who were treated successfully and for those who failed treatment. CQ = chloroquine; SP = sulfadoxine-pyrimethamine. For definitions of other abbreviations, see Tables 1 and 2.
CQpfcrt5925/25 (100%)13/34 (38%)< 0.00123.97
CQ + SPpfcrt11620/20 (100%)72/96 (75%)0.0126.30
SPpfdhfr4511/11 (100%)21/34 (62%)0.0196.85
CQ + SPpfdhfr10717/19 (89%)63/88 (72%)0.142.65
SPpfdhps527/12 (58%)6/40 (15%)0.0058.04
CQ + SPpfdhps1025/15 (33%)10/87 (11%)0.044.86
SPpfdhfr + pfdhps544/10 (40%)4/44 (9%)0.0316.17
CQ + SPpfdhfr + pfdhps855/12 (42%)7/73 (10%)0.0118.75
CQ + SPpfdhfr + pfdhps + pfcrt805/11 (45%)6/69 (9%)0.00710.23
Table 4

Relative risk of developing treatment failure in patients treated with SP in clinical trials, by pfdhfr and pfdhps mutations*

Treatment outcome
No. of patients (%)
MutationsFailureSuccessRR (95% CI)P
* SP = sulfadoxine-pyrimethamine; RR = relative risk; CI = confidence interval. For definitions of other abbreviations, see Table 1.
† The likelihood ratio test was used for comparison between two groups.
pfdhfr
    Single (108)11/11 (100)21/34 (62)0.62 (0.47–0.80)0.003
    Double (59 + 108)7/11 (64)16/34 (47)1.97 (4.48–8.00)0.34
    Triple (51 + 59 + 108)4/11 (36)2/34 (6)9.14 (1.39–60.16)0.017
    Triple (59 + 108 + 164)0/110/34
    Quadruple (51 + 59 + 108 + 164)0/110/34
pfdhps
    Single (437)0/75/39 (13)0.87 (0.77–0.98)0.18
    Double (436 + 437)1/7 (14)0/391.17 (0.86–1.58)0.048
    Double (437 + 540)1/7 (14)0/391.17 (0.86–1.58)0.048
    Triple (436 + 437 + 540)0/70/39
Table 5

Relative risk of developing treatment failure in patients treated with CQ + SP in clinical trials, by pfdhfr and pfdhps mutations*

Treatment outcome
No. of patients (%)
MutationsFailureSuccessRR (95% CI)P
* CQ = chloroquine; SP = sulfadoxine-pyrimethamine; RR = relative risk; CI = confidence interval. For definitions of other abbreviations, see Table 1.
† The likelihood ratio test was used for comparison between two groups.
pfdhfr
    Single (108)2/17 (12)2/78 (25)5.08 (0.66–38.84)0.13
    Double (59 + 108)9/17 (53)31/78 (40)1.71 (0.59–4.90)0.32
    Triple (51 + 59 + 108)3/17 (18)23/78 (29)0.51 (0.13–1.95)0.30
    Triple (59 + 108 + 164)0/170/78
    Quadruple (51 + 59 + 108 + 164)2/17 (12)0/78
pfdhps
    Single (437)1/15 (7)5/87 (6)1.17 (0.13–10.79)0.89
    Double (436 + 437)0/151/87 (1)0.99 (0.97–1.00)0.57
    Double (437 + 540)1/15 (7)1/87 (1)6.14 (0.36–103.9)0.23
    Triple (436 + 437 + 540)2/15 (13)2/87 (2)6.54 (0.84–50.54)0.08
Table 6

Comparison of fever and parasite clearance times between patients whose parasites carried mutant and wild genes*

pfcrtpfdhfrpfdhps
VariableMutantWildPMutantWildPMutantWildP
* Assessed for CQ treatment in pfcrt and for SP treatment in pfdhfr and pfdhps. Values are mean (95% confidence interval). For definitions of abbreviations, see Tables 1, 2, and 3.
† FCT = fever clearance time, time in hours from the start of treatment at which the axillary temperature first decreased below 37.5°C and remained below 37.5°C for 48 hours.
‡ PCT = parasite clearance time, time in days from first treatment dose to the first thick film negative for Plasmodium falciparum parasites after checking ≥ 200 oil immersion fields.
FCT (hours)†44.128.30.1161.861.90.9963.758.50.67
(23.9–64.4)(19.5–37.0)(47.6–76.0)(41.6–82.3)(52.1–75.2)(45.6–71.3)
n = 15n = 18n = 22n = 14n = 9n = 32
PCT (days)‡3.22.90.133.43.00.184.03.00.004
(2.7–3.6)(2.6–3.1)(3.0–3.9)(2.5–3.4)(3.2–4.7)(2.7–3.3)
n = 21n = 21n = 27n = 14n = 10n = 38
Figure 1.
Figure 1.

A, Geographic distribution of Plasmodium falciparum dihydrofolate reductase (dhfr) mutations in Laos. Provinces with sample sizes < 10 are indicated as smaller pie charts. B, Geographic distribution of P. falciparum dihydropteroate synthase (dhps) mutations in Laos. Provinces with sample sizes < 10 are indicated as smaller pie charts. C, Geographic distribution of P. falciparum chloroquine resistance transporter (pfcrt) mutations in Laos. Provinces with sample sizes < 10 are indicated as smaller pie charts. D, Comparison of the frequency of alleles carrying mutant codons in northern, central, and southern Laos. North = Bokeo, Huaphanh, Luangphrabang, Luangnamtha, Phongsaly, Oudomxay, Vientiane, Xiengkhuang and Xayabury; Central = Borikhamxay and Khammouane; South = Attapeu, Champassack, Saravane, Savannakhet and Sekong.

Citation: The American Journal of Tropical Medicine and Hygiene 77, 1; 10.4269/ajtmh.2007.77.36

*

Address correspondence to Paul N. Newton, Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic. E-mail: paul@tropmedres.ac

Authors’ addresses: Mayfong Mayxay, Wellcome Trust–Mahosot Hospital–Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Mahosot Road, Vientiane, Lao People’s Democratic Republic, Telephone: 85-621-250-752, Fax: 85-621-242-168. E-mail: mmayxay@yahoo.com and Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Vientiane, Lao People’s Democratic Republic. Shalini Nair, Dan Sudimack, and Tim J. C. Anderson, Southwest Foundation for Biomedical Research. San Antonio, TX, Telephone: 210-258-9596. Mallika Imwong and Naowarat Tanomsing, Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, 420/6 Rajvithi Road, Bangkok, 10400, Thailand, Telephone: 66-2-354-9172, Fax: 66-2-354-9169. Tiengkham Pongvongsa, Savannakhet Provincial Malaria Station, Savannakhet Province, Savannakhet, Lao People’s Democratic Republic. Samlane Phompida and Rattanaxay Phetsouvanh, Centre of Malariology, Parasitology and Entomology, Vientiane, Lao People’s Democratic Republic, Telephone: 85-621-214-040, Fax: 85-621-218-131. Nicholas J. White, Wellcome Trust–Mahosot Hospital–Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Mahosot Road, Vientiane, Lao People’s Democratic Republic, Telephone: 85-621-250-752, Fax: 85-621-242-168, E-mail: nickw@tropmedres.ac, Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford OX3 7LJ, United Kingdom, and Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, 420/6 Rajvithi Road, Bangkok, 10400, Thailand, Telephone: 66-2-354-9172, Fax: 66-2-354-9169. Paul N. Newton, Wellcome Trust–Mahosot Hospital–Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Mahosot Road, Vientiane, Lao People’s Democratic Republic, Telephone: 85-621-250-752, Fax: 85-621-242-168; E-mail: paul@tropmedres.ac and Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford OX3 7LJ, United Kingdom.

Acknowledgments: We are grateful to all the patients, the medical staff from malaria clinics and hospitals across Laos, and the directors of all Provincial Malaria Stations for participating in the study, and Maniphone Khanthavong, Bouakham Vannachone, Vonthalom Thongpraseuth, Siamphay Keola, Somphane Sengphinthong, Manisack Phommasansack, Bounpone Phimphalat, Pitta Sengkeomahavong, Bounmy Syphachanh, Ammala Phomsimone, Vilayphone Phan-Anon, Kaiamphone Phonkeopaseuth, Chanthala Vilaihong, and Julie Simpson for technical help. We are also grateful to the staff of the Mahidol Oxford Research Unit, Bangkok, for their vital logistic support, and to Professor Nick Day, Drs. Pranom Phongmany, Odai Xaysitthideth, and Phomma Phengvilaysouk for valuable advice, and the Minister of Health, Dr. Ponmek Dalaloy, the Directors of Hygiene and Preventive Medicine, Drs. Douangchanh Keo-Asa and Bounlay Phommasack, the Director of Mahosot Hospital, Professor Chanpheng Thammavong, and Professor Sasithon Pukrittayakamee (Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand) for support.

Financial support: This study was supported by the Wellcome Trust of Great Britain. Shalini Nair, Dan Sudimack, and Tim J. C. Anderson are supported by National Institutes of Health grant RO1 AI48071. The molecular work at the Southwest Foundation for Biomedical Research was conducted in facilities constructed with support from Research Facilities Improvement Program grant C06 RR013556 from the National Center for Research Resources, National Institutes of Health.

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