In response to chloroquine (CQ) resistance, the policy for the first-line treatment of uncomplicated malaria in the Democratic Republic of East Timor (DRET) was changed in early 2000. The combination of sulphadoxine-pyrimethamine (SP) was then introduced for the treatment of uncomplicated falciparum malaria.
Methods
Blood samples were collected in two different periods (2003–2004 and 2004–2005) from individuals attending hospitals or clinics in six districts of the DRET and checked for Plasmodium falciparum infection. 112 PCR-positive samples were inspected for genetic polymorphisms in the pfcrt, pfmdr1, pfdhfr and pfdhps genes. Different alleles were interrogated for potential associations that could be indicative of non-random linkage.
Results
Overall prevalence of mutations associated with resistance to CQ and SP was extremely high. The mutant form of Pfcrt (76T) was found to be fixed even after five years of alleged CQ removal. There was a significant increase in the prevalence of the pfdhps 437G mutation (X2 = 31.1; p = 0.001) from the first to second survey periods. A non-random association was observed between pfdhfr 51/pfdhps 437 (p = 0.001) and pfdhfr 59/pfdhps 437 (p = 0.013) alleles.
Conclusion
Persistence of CQ-resistant mutants even after supposed drug withdrawal suggests one or all of the following: local P. falciparum may still be inadvertently exposed to the drug, that mutant parasites are being "imported" into the country, and/or reduced genetic diversity and low parasite transmission help maintain mutant haplotypes. The association between pfdhfr 51/pfdhps 437 and pfdhfr 59/pfdhps 437 alleles indicates that these are undergoing concomitant positive selection in the DRET.
Background
In the Democratic Republic of East Timor (DRET), chloroquine (CQ) resistance in Plasmodium falciparum was first reported in the 1980s [1, 2]. Despite this, until 1999, CQ plus primaquine continued to be used as the first-line treatment of uncomplicated malaria, with sulphadoxine-pyrimethamine (SP) as second-line, severe malaria being treated with quinine [3].
In 1992, high levels of resistance to chloroquine were reported [4]. In vitro and in vivo resistance to choroquine, amodiaquine and SP was documented in the district of Lospalos, where more than 67% treatment failure to CQ was reported between 1999 and 2000 [3, 5–8]. Consequently, in early 2000, the policy for the first-line treatment of uncomplicated malaria was changed. SP was introduced for the treatment of uncomplicated falciparum malaria and chloroquine was used for the treatment of Plasmodium vivax only. Currently, SP has been replaced by artemether-lumefantrine for treating P. falciparum, but chloroquine is still the recommended treatment for vivax malaria.
Genetic variation associated with both CQ and SP resistance can be monitored with specific molecular markers. The K76T mutation at the pfcrt is considered a reliable genetic marker for CQ resistance. Polymorphisms in pfmdr 1, which encodes the P. falciparum P glycoprotein homologue 1, modulate chloroquine resistance in mutant pfcrt-harboring parasites in vitro [9], although their role in vivo has not been sufficiently substantiated [10].
Molecular mechanisms of antifolate resistance in P. falciparum have been explored in detail [11]. Specific point mutations in the parasite's dihydrofolate reductase (dhfr and dihydropterate synthase (dhps) genes are associated resistance to pyrimethamine-sulphadoxine [12–14]. The first study of SP efficacy for the treatment of uncomplicated falciparum malaria, conducted in 2001, reported 81.6% single pfdhfr 108N or double C59R/S108N mutants, but none of the isolates harboured mutations in dhps, and the drug was confirmed to be efficacious [15].
The aim of the present study was to investigate the proportions and distribution of molecular polymorphisms in the parasite Plasmodium falciparum dihydrofolate reductase (pfdhfr), dihydropterate synthase (pfdhps), chloroquine resistance transporter (pfcrt) and multi-drug resistance (pfmdr 1) genes, from samples collected four to five years after the replacement of CQ by SP as the recommended first-line treatment.
Methods
Study area
The Democratic Republic of East Timor (DRET) is situated on the Eastern Part of the Island of Timor, the eastern most of the Lesser Sunda Island (Figure 1). The study was carried out in two different periods. The first period (2003/2004) was carried out in two districts: (Dili and Suai), and the second period (2004/2005) was carried out in the former and an additional four districts: (Liquiça, Same, Viqueque, Lospalos). These districts can be classified into three different zones, according to geographical location: North (Dili and Liquica), South (Suai, Same and Viqueque) and East (Lospalos).
Figure 1
Map of the Democratic Republic of East Timor showing locations of the districts surveyed.
Sample collection
Two-hundred and fifty blood samples were collected from 17 November 2003 to 7 January 2004 (period 1) and 650 from 15 December 2004 to 9 March 2005 (period 2). This study was approved by Ministry of Health (Ref.: MS-DG/PESQUISA-IHMT/XI/03/346 and MS/VM/PESQUISA/XII/04) and patients gave their consent to participate. Finger-prick blood samples were collected by passive case detection (PCD) from suspected malaria carriers presenting at hospital/clinic, after informed consent was obtained. No age restrictions were applied. Collected blood was used to make thin and thick Giemsa-stained smears, which were checked for malaria parasites by optical microscopy. A sub-sample of the collected blood was blotted onto filter paper for assessment of molecular markers.
Genotyping
Genomic DNA was extracted with phenol-chloroform [16] and confirmed to include P. falciparum DNA, by use of a species-specific PCR [17]. Each sample was then checked for polymorphisms in three codons (51, 59 and 108) of the pfdhfr gene and two codons (437 and 540) of the pfdhps gene, using a slight modification of the PCR-RFLP methodology described by [18]. Polymorphisms in two codons (75 and 76) of the pfcrt gene and two codons (86 and 1246) of the pfmdr 1 gene were detected as described previously [19]. Digested products were separated on 2–3% agarose gel, then stained with ethidium bromide and visualized under UV.
Statistical analysis
A Chi-square (χ2) Test was performed to compare differences in frequency of mutant alleles among codons of the pfcrt, pfmdr 1, pfdhfr and pfdhps genes and between periods of sampling, using SPSS 11.5 for windows, and P < 0.05 was considered significant. Association between alleles at any two loci was tested by Fisher's Exact Test. Data from isolates containing both alleles (polyclonal infections) were excluded from the analysis.
Results
A total of 900 samples were checked for presence of malaria. Mean age of sampled individuals was 21 ± 17.04 (SD) of which 51% and 49% were males and females, respectively. Prevalence of falciparum, vivax and mixed malaria infections as determined by optical microscopy (OM) was underestimated in all cases, with PCR allowing detection of a significantly higher number of cases (X2 = 15,02; p = 0,001). Thus, infection prevalence detected by OM vs PCR, for P. falciparum, P. vivax and mixed infections, was 7.5% vs 11.4%, 3.7% vs 6.6% and 0% vs 1.0%, respectively.
38 of the 250 and 74 of the 650 patients were infected by P. falciparum in period 1 and period 2, respectively, as determined by PCR. Results of Single Nucleotide Polymorphism (SNP) typing are summarized in Table 1. Over 97% of the samples inspected showed the CQ resistance pfcrt 76 core mutation and of these, 38 and 56% had the additional pfmdr 1 86Y mutation in the first and second periods respectively. The 75E polymorphism was also observed in 12.5% of isolates from period 2. No mutant alleles at codon pfmdr 1 1246 were detected.
Table 1
Frequency (%) of the Pfdhfr (codons 51, 59 and 108), Pfdhps (codons 437 and 540), Pfcrt (codons 76 and 75), Pdmdr 1 (codons 86 and 1246) genotypes and multiple mutations in first and second assay periods.
Gene
Codon
Period 1
(2003–2004)
Period 2
(2004–2005)
(N = 38)
(%)
(N = 74)
(%)
Pfcrt
75
(wild-type) N
16
100
27
56
(Mutant) E
0
0
6
13
N + E
0
0
15
31
Not determined
22
22
76
(wild-type) K
1
3
0
0
(Mutant) T
32
97
59
100
K + T
0
0
0
0
Not determined
5
15
Pfmdr1
86
(wild-type) N
8
24
18
27
(Mutant) Y
13
38
37
56
N + Y
13
38
11
17
Not determined
4
8
1246
(wild-type) D
32
100
64
97
(Mutant) Y
0
0
0
0
D + Y
0
0
2
3
Not determined
6
8
Pfdhfr
51
(wild-type) N
9
24
8
11
(Mutant) I
25
66
66
89
N + I
4
10
0
0
Not determined
0
0
59
(wild-type) C
11
29
9
14
(Mutant) R
24
63
53
84
C + R
3
8
1
2
Not determined
0
11
108
(wild-type) S
0
0
2
3
(Mutant) N
28
100
62
97
S + N
0
0
0
0
Not determined
10
10
Pfdhps
437
(wild-type) A
14
40
0
0
(Mutant) G
21
60
67
100
A + G
0
0
0
0
Not determined
3
7
540
(wild-type) K
34
97
67
100
(Mutant) E
1
3
0
0
K + E
0
0
0
0
Not determined
3
7
Multiple mutations
dhfr 51I+108N
19
68
60
97
dhfr 59R+108N
17
61
52
84
dhfr 51I +59R+108N
16
57
53
86
dhfr 51I +59R+108N+dhps 437G
13
46
51
82
More than 97% of the parasite population inspected exhibited the pfdhfr core mutation 108N, and an increase in mutant alleles from the first to second period in two others loci examined (pfdhfr 51 and pfdhfr 59) was detected. All isolates presented the Pfdhfr 108N mutation in combination with dhfr 51I and/or dhfr 59R. In particular, the pfdhfr triple mutation was seen in the great majority of samples (Table 1). There was a significant difference (X2 = 31.1; p = 0.001) in the prevalence of the pfdhps 437G mutation which increased from 60% to 100% from the first to second periods, respectively. The quadruple mutation [triple dhfr (51I, 59R and 108N) + dhps 437G ] was found in 82.3% of isolates. None of the samples contained quintuple mutations. There was no significant association between any particular genotype prevalence and patient age or sex.
Potential associations between alleles at different loci were evaluated, except for those whose prevalence was found to be fixed, or near fixation. Consequently, a non-random association was observed between the pfdhfr and pfdhps genes in the group of isolates collected during the first survey (2003/2004). Here, concomitant significant occurrence of wild-type/wild-type or mutant/mutant pfdhfr 51/pfdhps 437 alleles were observed among 29 out of 31 samples, whilst pfdhfr 59/pfdhps 437 occurred in 25 of the 33 cases successfully genotyped (Table 2).
Table 2
Significant associations between different alleles of the Pfdhfr e Pfdhps genes.
Codons
Genotype
Number observed
Fisher's Exact Test (2-sided)
(P- value)
51 × 437 (N = 31)
N-A (wild-type)
8
P = 0.001 < 0.05
I-G (mutant)
21
59 × 437 (N = 33)
C-A (wild-type)
7
P = 0.001 < 0.05
R-G (mutant)
18
Discussion
The present study evaluated mutation prevalence in the Pfcrt, Pfmdr 1, Pfdhfr and Pfdhps genes among natural parasite populations of East Timor. Overall, the frequency of mutant alleles progressively increased from the first to second period, including those suggested to be involved in chloroquine resistance. These data is in contrast with previous studies where the frequency of the Pfcrt 76T mutation decreased progressively with abolition of chloroquine for treatment for P. falciparum malaria [20, 21]. The explanation for the persistence of the K76T mutation in East Timor P. falciparum populations may entail several factors. First, despite the discontinuation of CQ, the drug was still recommended for vivax malaria infections. Vivax malaria accounts for 20 – 40% of all malaria cases [7]. Furthermore, there is the problem of underestimation of mixed infections, as the diagnosis of malaria species in East Timor is made by conventional microscopy alone. Second, anti-malarial drugs are still widely available commercially in East Timor and most people use drugs acquired from parallel markets. The indiscriminate or inappropriate use and unsupervised drugs use for malaria infections is likely to be implicated in the maintenance of CQ-resistant parasites. Third, widespread use of quinine and amodiaquine in East Timor, which have been associated to certain extent to pfcrt 76T and pfmdr 1 86Y mutations [22, 23], may impose positive selection, maintaining CQ resistance. Fourth, there has been extensive population movement between East Timor and West Timor-Indonesia, where malaria is highly endemic and CQ resistance levels are high. Thus, dissemination of resistant genotypes is likely to play an important role in maintaining CQ resistance in the region. Last, the reduced genetic diversity and lower recombination rates in south-east Asian parasites (when compared to Africa) may help maintain predominant genotypes even if mutations carry a fitness cost.
Point mutations in pfmdr 1 N86Y and D1246Y, occasionally cited as potential contributors to chloroquine resistance [24] were also inspected. The pfmdr 1 86Y allele was slightly predominant (56% prevalence) as previously observed both in Africa [25] and in some regions in Indonesia [26].
Since SP replaced CQ as recommended first line drug, the putative efficacy of SP was inferred by inspecting mutations in key codons of P. falciparum dhfr and dhps genes. Results indicated that mutation prevalence in those genes have increased steadily over a short period of time in contrast to an earlier study by [15]. Additionally, all isolates exhibited the pfdhfr 108N mutation in combination with dhfr 51I and/or dhfr 59R mutations, indicative of increased levels of pyrimethamine resistance [24, 27–29]. Also, triple mutants in the Pfdhfr gene (51I+59R+108N) accounted for 85.5% of all isolates further re-enforcing that high levels of pyrimethamine resistance are present [27, 30].
The most common mutations polymorphism in the pfdhps gene was the 437G; only one case presented a 540E mutation. These observations are consistent with pfdhps 437G being the first to emerge as result of pressure by sulpha drugs [11, 31, 32], which confers resistance to sulphadoxine in P. falciparum [17, 33, 34]. It was also verified that a high number isolates (82.3%) carried 4 mutations distributed among the dhfr and dhps genes {quadruple mutation: (51I + 59R + 108N + 437G)}, a pattern inferred in the presence of high levels of resistance to the pyrimethamine and in some cases, clinical resistance to SP [35].
Pfdhfr is present in chromosome 4 and pfdhps lies present in chromosome 8 of P. falciparum. Thus, both genes are physically distant and in conditions of normal transmission any particular haplotypes of each gene should be found randomly in human hosts. However, the present data highlights that resistance-associated alleles encoding both the pfdhfr 51 and 59 mutants are non-randomly associated with pfdhps 437 mutants, therefore indicating that both loci are under strong directional selection by sulphadoxine-pyrimethamine (SP), suggestive of selective advantage conferred by the presence of the two resistant alleles.
Conclusion
This work suggests that SP resistance may already exist in the East Timor, and that the continuous use of the drug will contribute to higher patterns of inefficacy in the treatment of falciparum malaria. Nevertheless, these findings could be complemented with in vivo data as to reflect more closely the therapeutic efficacy of CQ and SP patterns in the epidemiological scenario. Persistence of CQ-resistant mutants even after supposed drug withdrawal re-enforces the need of its use against P. vivax exclusively. The association between pfdhfr 51/pfdhps 437 and pfdhfr 59/pfdhps 437 alleles indicates that these are undergoing concomitant positive selection.
Declarations
Acknowledgements
The authors gratefully acknowledge the support of the Ministry of Health of The Democratic Republic of East Timor, laboratory staff and people of East Timor for their collaboration. Afonso de Almeida is funded by Fundação Calouste Gulbenkian (via Scholarship for PhD Course; E-18836, P-69095 (TL)).
Authors’ Affiliations
(1)
Universidade Nacional de Timor Leste, Avenida Cidade de Lisboa
(2)
Centro de Malária e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa
(3)
Unidade de Biologia Molecular, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa
References
WHO: Advances in malaria chemotherapy. WHO Technical Reports. 1984, World Health Organ Geneva, 711:Google Scholar
D'Alessandro U, Buttiens H: History and importance antimalarial drug resistance. Trop Med Int Health. 2001, 6: 845-848. 10.1046/j.1365-3156.2001.00819.x.View ArticlePubMedGoogle Scholar
Kolaczinski J, Webstar J: Malaria Control in Complex Emergencies: the example of East Timor. Trop Med Int Health. 2003, 8: 48-55. 10.1046/j.1365-3156.2003.00969.x.View ArticlePubMedGoogle Scholar
Morris K: Growing plains of East Timor: health of an infant nation. The Lancet. 2001, 357: 873-877. 10.1016/S0140-6736(00)04203-3.View ArticleGoogle Scholar
Arbani PR, Petersen G: Malaria Epidemiological Profile – East and West Timor. World Health Organ Geneva. 1999Google Scholar
Andjaparidze A, Marwah SK, Debnath C: East Timor Health Sector Situation report. World Health Organ East Timor. 2000Google Scholar
Webster J: Malaria Consortium, RBM Complex Emergency Malaria Data Base East Timor. World Organ Health Geneva. 2000Google Scholar
Ezard N, Burns M, Lynch C, Cheng Q, Edstein MD: Efficacy of cloroquine in the treatment of uncomplicated Plasmodium falciparum infection in East Timor, 2000. Acta Trop. 2003, 88: 87-90. 10.1016/S0001-706X(03)00161-X.View ArticlePubMedGoogle Scholar
Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF: Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature. 2000, 403: 906-909. 10.1038/35002615.View ArticlePubMedGoogle Scholar
Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA: A molecular marker for surveillance of chloroquine-resistant falciparum malaria. NEJM. 2001, 344: 257-263. 10.1056/NEJM200101253440403.View ArticlePubMedGoogle Scholar
Triglia T, Menting JGT, Wilson C, Cowman AF: Mutations in dihydropteroate synthase are responsible for sulfone and sufonamide resistance in Plasmodium falciparum. Proc Natl Acad Sci USA. 1997, 94: 13944-13949. 10.1073/pnas.94.25.13944.PubMed CentralView ArticlePubMedGoogle Scholar
Cowman AF, Morry MJ, Biggs BA, Cross GA, Foote SJ: Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. Proc Natl Acad Sci USA. 1988, 85: 9109-9113. 10.1073/pnas.85.23.9109.PubMed CentralView ArticlePubMedGoogle Scholar
Peterson DS, Walliker D, Wellems TE: Evidence that a point mutation in dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine in falciparum malaria. Proc Natl Acad Sci USA. 1988, 85: 9114-9118. 10.1073/pnas.85.23.9114.PubMed CentralView ArticlePubMedGoogle Scholar
Triglia T, Wang P, Sims PF, Hyde JE, Cowman AF: Allelicexchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria. EMBO J. 1998, 17: 3807-3815. 10.1093/emboj/17.14.3807.PubMed CentralView ArticlePubMedGoogle Scholar
Burns M, Joanne Baker, Alyson MA, Michellel G, Michael DE, Cheng Q: Efficacy of sulfadoxine-pyrimethamine in the treatment of uncomplicated P. falciparum malaria in East Timor. Am J Trop Med Hyg. 2006, 74: 361-366.PubMedGoogle Scholar
Snounou G: Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol. 1993, 58: 283-292. 10.1016/0166-6851(93)90050-8.View ArticlePubMedGoogle Scholar
Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, Rosário VE, Thaithong S, Brown KN: High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol. 1993, 1: 315-320. 10.1016/0166-6851(93)90077-B.View ArticleGoogle Scholar
Duraisingh MT, Curtis J, Warhurst DC: Plasmodium falciparum: Detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion. Exp Parasitol. 1998, 89: 1-8. 10.1006/expr.1998.4274.View ArticlePubMedGoogle Scholar
Lopes D, Rungsihirunrat K, Noqueira F, Seugorn A, Gil JP, do Rosário VE, Cravo P: Molecular characterisation of drug-resistant Plasmodium falciparum from Thailand. Malar J. 2002, 1: 1-11. 10.1186/1475-2875-1-12.View ArticleGoogle Scholar
Kublin JG, Cortese JF, Njunju EM, Mukadam RA, Wirima JJ, Kazembe PN, Djimde AA, Kouriba B, Taylor TE, Plowe CV: Reemergence of chloroquine-sensitive Plasmodium falciparum malaria after cessation of chloroquine use in Malawi. J Infect Dis. 2003, 187: 1870-5. 10.1086/375419.View ArticlePubMedGoogle Scholar
Wang X, Um J, Li Q, Chen P, Guo X, Fu L, Chen L, Su X, Wellems TE: Decreased prevalence of the Plasmodium falciparum chloroquine resistance transporter 76T marker associated with cessation of choroquine use against P. falciparum malaria in Hainan, people's Republic of China. Am J Trop Med Hyg. 2005, 72: 410-414.PubMedGoogle Scholar
Holmgren G, Gil GP, Ferreira PM, Veiga MI, Obonyo CO, Bjorkman A: Amodiaquine resistant Plasmodium falciparum malaria in vivo is associated with selection of Pfcrt 76T and Pfmdr 1 86Y. Infect Genet Evol. 2006, 6: 309-314. 10.1016/j.meegid.2005.09.001.View ArticlePubMedGoogle Scholar
Happi CT, Gbotosho GO, Folarin OA, Bolaji OM, Sowunmi A, Kyle DE, Milhous W, Wirth DF, Oduola AMJ: Association between mutation in Plsamodium falciparum chloroquine resistance transport and Plsamodium falciparum multidrug resistance 1 genes and in vivo amodiaquine resistance in Plsamodium falciparum malaria-infected children in Nigeria. Am J Trop Med Hyg. 2006, 75: 155-161.PubMedGoogle Scholar
Plowe CV: Monitoring antimalarial drug resistance: making the most of the tools at hand. J Exp Biol. 2003, 206: 3745-3752. 10.1242/jeb.00658.View ArticlePubMedGoogle Scholar
Mawili-Mboumba DP, Kun JFJ, Lell B, Kremsner PG, Ntoumi F: Pfmdr 1 alleles and response to ultralow-dose mefloquine treatment in Gabonese patients. Antimicrob Agents Chemother. 2002, 46: 166-170. 10.1128/AAC.46.1.166-170.2002.PubMed CentralView ArticlePubMedGoogle Scholar
Syarfruddin D, Asih PBS, Aggarwal SL, Shankar AH: Frequency distribution on antimalarial drug-resistant alleles among isolates of P. falciparum in Purworejo District, Central Java Province, Indonesia. Am J Trop Med Hyg. 2003, 69: 614-620.Google Scholar
Basco LK, Eldin de Pécoulas P, Wilson CM, Bras JL, Mazabraud A: Point mutations in the dihydrofolate reductase thymidylate synthase gene and pyrimethamine and cycloguanil resistance in Plasmodium falciparum. Mol Biochem Parasitol. 1995, 69: 135-138. 10.1016/0166-6851(94)00207-4.View ArticlePubMedGoogle Scholar
Plowe CV, Cortese JF, Djimde A, Nwanyanwu OC, Watkins WM, Winstanley PA, Franco JGE, Mollinedo RE, Avila JC, Cespedes JL, Carter D, Doumbo OK: Mutations in Plasmodium falciparum difydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance. J Infect Dis. 1997, 176: 1590-1596.View ArticlePubMedGoogle Scholar
Urdaneta L, Plowe C, Goldman I, Lal AA: Point mutations in dihydrofolate reductase and dihydopteroate synthase genes of Plasmodium falciparum isolates from Venezuela. Am J Trop Med Hyg. 1999, 61: 457-462.PubMedGoogle Scholar
Wang P, Lee CS, Bayoumi R, Djimde A, Doumbo O, Swedberg G, Dao LD, Mshinda H, Tanner M, Watkins WM, Sims PFG, Hyde JE: Resistance to antiolates in Plasmodium falciparum monitored by sequence analysis of dihydropteroate synthase and dihydrofolate reductase alleles in a large number of field samples of diverse origins. Mol Biochem Parasitol. 1997, 89: 161-177. 10.1016/S0166-6851(97)00114-X.View ArticlePubMedGoogle Scholar
Ngo Thanh, Durasingh M, Reed Michael, Hipgrave D, Biggs B, Cowman AF: Analysis Pfcrt, Pfmdr1, dhfr, and dhps mutations and drugs sensitivities in P. falciparum isolates from patients in Vietnam before and after treatment with artemisinin. Am Soc Trop Med Hyg. 2003, 68: 350-356.Google Scholar
Nzila AM, Mberu EK, Sulo J, Dayo H, Winstanley PA, Sibley CH, Watkins WM: Towards an understanding of the mechanism of pyrimethamine-sulfadoxine resistance in P. falciparum: genotyping of dihydrofolate reductase and dihydropteroate synthase of Kenyan parasites. Am Soc Microbiol. 2000, 44: 991-996.Google Scholar
Jelinek T, Kilian AHD, Kabagambe G, Sonnenberg FV: Plasmodium falciparum resistance to sulfadoxyne/pyrimethamine in Uganda: correlation with polymorphisms in the dehidrofolate reductase and dihydropterate synthetase genes. Am J Trop Med Hyg. 1999, 61: 463-466.PubMedGoogle Scholar
Syarfruddin D, Asih PBS, Casey GJ, Maguire J, Baird JK, Nagesha HS, Cowman AF, Reeder JC: Molecular epidemiology of Plasmodium falciparum resistance to antimalarial drugs in Indonesia. Am J Trop Med Hyg. 2005, 72: 174-181.Google Scholar
Ndounga M, Tahar R, Basco LK, Casimiro PN, Malonga DA, Ntoumi F: Therapeutic efficacy of sulfadoxine-pyrimethamine and the prevalence of molecular markers of resistance in under 5-years olds in Brazzaville, Congo. Trop Med Int Health. 2007, 12: 1164-1171.View ArticlePubMedGoogle Scholar
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