• 1

    Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI, 2005. The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 434 :214–217.

    • Search Google Scholar
    • Export Citation
  • 2

    Jima D, 2004. The 2003 malaria situation in Ethiopia. Proceedings of the Panel Discussion on the 2003 Malaria Epidemics in Ethiopia, May 29, 2004. Addis Ababa: Ethiopian Public Health Association, 6–19.

  • 3

    Bwijo B, Kaneko A, Takechi M, Zungu IL, Moriyama Y, Lum JK, Tsukahara T, Mita T, Takahashi N, Bergqvist Y, Bjorkman A, Kobayakawa T, 2003. High prevalence of quintuple mutant dhps/dhfr genes in Plasmodium falciparum infections seven years after introduction of sulfadoxine and pyrimethamine as first line treatment in Malawi. Acta Trop 85 :363–373.

    • Search Google Scholar
    • Export Citation
  • 4

    Kean BH, 1979. Chloroquine-resistant falciparum malaria from Africa. JAMA 241 :395.

  • 5

    Triglia T, Menting JG, Wilson C, Cowman AF, 1997. Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum. Proc Natl Acad Sci U S A 94 :13944–13949.

    • Search Google Scholar
    • Export Citation
  • 6

    Cowman AF, Morry MJ, Biggs BA, Cross GA, Foote SJ, 1988. Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. Proc Natl Acad Sci USA 85 :9109–9113.

    • Search Google Scholar
    • Export Citation
  • 7

    Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese JF, Martino LM, Mukadam RA, Rogerson SJ, Lescano AG, Molyneux ME, Winstanley PA, Chimpeni P, Taylor TE, Plowe CV, 2002. Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis 185 :380–388.

    • Search Google Scholar
    • Export Citation
  • 8

    Biswas S, Escalante A, Chaiyaroj S, Angkasekwinai P, Lal AA, 2000. Prevalence of point mutations in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum isolates from India and Thailand: a molecular epidemiologic study. Trop Med Int Health 5 :737–743.

    • Search Google Scholar
    • Export Citation
  • 9

    Meyer CG, May J, Arez AP, Gil JP, Do RV, 2002. Genetic diversity of Plasmodium falciparum: asexual stages. Trop Med Int Health 7 :395–408.

    • Search Google Scholar
    • Export Citation
  • 10

    Mutabingwa T, Nzila A, Mberu E, Nduati E, Winstanley P, Hills E, Watkins W, 2001. Chlorproguanil-dapsone for treatment of drug-resistant falciparum malaria in Tanzania. Lancet 358 :1218–1223.

    • Search Google Scholar
    • Export Citation
  • 11

    Berens N, Schwoebel B, Jordan S, Vanisaveth V, Phetsouvanh R, Christophel EM, Phompida S, Jelinek T, 2003. Plasmodium falciparum: correlation of in vivo resistance to chloroquine and antifolates with genetic polymorphisms in isolates from the south of Lao PDR. Trop Med Int Health 8 :775–782.

    • Search Google Scholar
    • Export Citation
  • 12

    Kain KC, Lanar DE, 1991. Determination of genetic variation within Plasmodium falciparum by using enzymatically amplified DNA from filter paper disks impregnated with whole blood. J Clin Microbiol 29 :1171–1174.

    • Search Google Scholar
    • Export Citation
  • 13

    Rason MA, Ariey F, Rafidimanantsoa L, Andrianantenaina BH, Sahondra Harisoa JL, Randrianarivelojosia M, 2002. Monitoring the drug-sensitivity of Plasmodium falciparum in coastal towns in Madagascar by use of in vitro chemosensitivity and mutation detection tests. Parasite 9 :247–253.

    • Search Google Scholar
    • Export Citation
  • 14

    D’Alessandro U, 1998. Antimalarial drug resistance: surveillance and molecular methods for national malaria control programmes. Mem Inst Oswaldo Cruz 93 :627–630.

    • Search Google Scholar
    • Export Citation
  • 15

    Nzila AM, Mberu EK, Sulo J, Dayo H, Winstanley PA, Sibley CH, Watkins WM, 2000. Towards an understanding of the mechanism of pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: genotyping of dihydrofolate reductase and dihydropteroate synthase of Kenyan parasites. Antimicrob Agents Chemother 44 :991–996.

    • Search Google Scholar
    • Export Citation
  • 16

    Djimde AA, Dolo A, Ouattara A, Diakite S, Plowe CV, Doumbo OK, 2004. Molecular diagnosis of resistance to antimalarial drugs during epidemics and in war zones. J Infect Dis 190 :853–855.

    • Search Google Scholar
    • Export Citation
  • 17

    Amukoye E, Winstanley PA, Watkins WM, Snow RW, Hatcher J, Mosobo M, Ngumbao E, Lowe B, Ton M, Minyiri G, Marsh K, 1997. Chlorproguanil-dapsone: effective treatment for uncomplicated falciparum malaria. Antimicrob Agents Chemother 41 :2261–2264.

    • Search Google Scholar
    • Export Citation
  • 18

    Sulo J, Chimpeni P, Hatcher J, Kublin JG, Plowe CV, Molyneux ME, Marsh K, Taylor TE, Watkins WM, Winstanley PA, 2002. Chlorproguanil-dapsone versus sulfadoxine-pyrimethamine for sequential episodes of uncomplicated falciparum malaria in Kenya and Malawi: a randomised clinical trial. Lancet 360 :1136–1143.

    • Search Google Scholar
    • Export Citation
  • 19

    Alloueche A, Bailey W, Barton S, Bwika J, Chimpeni P, Falade CO, Fehintola FA, Horton J, Jaffar S, Kanyok T, Kremsner PG, Kublin JG, Lang T, Missinou MA, Mkandala C, Oduola AM, Premji Z, Robertson L, Sowunmi A, Ward SA, Winstanley PA, 2004. Comparison of chlorproguanil-dapsone with sulfadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria in young African children: double-blind randomised controlled trial. Lancet 363 :1843–1848.

    • Search Google Scholar
    • Export Citation
  • 20

    Kremsner PG, Krishna S, 2004. Antimalarial combinations. Lancet 364 :285–294.

 

 

 

 

MOLECULAR SURVEILLANCE OF MUTATIONS IN DIHYDROFOLATE REDUCTASE AND DIHYROPTEROATE SYNTHASE GENES OF PLASMODIUM FALCIPARUM IN ETHIOPIA

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  • 1 Faculty of Medicine, Department of Microbiology, Immunology and Parasitology, Addis Ababa University Addis Ababa, Ethiopia; Institute for Tropical Medicine, Department of Parasitology, University of Tübingen, Tübingen, Germany

Point mutations in the genes for dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) of Plasmodium falciparum isolates are associated with sulfadoxine/pyrimethamine (SP) treatment failure, respectively. This study was conducted to assess the prevalence of SP resistance in P. falciparum isolates collected at the Jimma Health Center in southwestern Ethiopia. In this study, the genetic profile of P. falciparum isolates with respect to DHFR and DHPS genes was assessed in 124 individuals. The prevalence of single, double, and multiple mutations in these genes was calculated. The sequence profile showed that all samples carried a double mutation at the positions 51 and 108 (I51N108) in the DHFR gene. Sixty-seven (54.03%) of the isolates had an additional third mutation at position 59, resulting in the triple mutant I51R59N108. All isolates carried mutations G437 and E540 in the DHPS gene. Two isolates (1.61%) had additional mutations at codon 581 (A581).

INTRODUCTION

There are approximately 300–500 million episodes of malarial illness each year globally, with more than a million deaths. Africa, especially south of the Sahara, is the most affected continent, bearing most of the global deaths due to malaria.1 In Ethiopia, malaria is a major public health problem and has been reported as the main cause of morbidity and mortality in 2002–2003.2

Seventy-five percent of Ethiopia is malarious, and 65% of its population lives in this area and is at risk for malaria. Five to six million clinical cases and more than 600,000 confirmed cases have been reported from health facilities in non-epidemic years. This indicates only a portion of the actual magnitude of this problem since there is a poor accessibility to and use of health services. Plasmodium falciparum is the predominant species that causes severe and complicated clinical manifestations and almost all malaria deaths.2

Effective treatment is essential for malaria control. However, drug-resistant malaria has become a challenge in malaria control programs in recent years. The emergence of multidrug-resistant strains of P. falciparum has compromised the effectiveness of routinely used anti-malaria drugs. This has threatened the use of the cheap and safe drugs in resource-poor countries where the mosquito control has been ineffective.

Sulfadoxine/pyrimethamine (SP) has been used as an affordable alternative treatment of uncomplicated malaria cases in chloroquine-resistant areas of Africa.3 However, in some east African countries, including Ethiopia, SP was adopted earlier and has been used extensively as a first-line treatment due to the high prevalence of chloroquine-resistant strains of P. falciparum. This antifolate drug was used intensively in these areas, which has led to the selection of resistant strains against the drug.4

Resistance to pyrimethamine is associated with mutations in the gene encoding the parasite enzyme dihydrofolate reductase (DHFR), and resistance to sulfadoxine is correlated with mutations in the parasite gene for dihydropteroate synthetase (DHAP).5,6 The level of resistance is associated with the number of mutations in the genes for these two enzymes. Therefore, multiple mutations in the two genes are considered to be responsible for SP treatment failure.7

Mutations in DHFR have been reported at codons 16, 51, 59, 108, and 164 in a number of geographic isolates.8 Mutations S108 to N108 or T108 in DHFR has been proposed as the main mechanism of resistance against pyrimethamine. All multiple mutations emerge from stepwise selection of a single mutant at position 108 of the DHFR gene. It has also been shown that the resistance level is significantly increased by additional sequence changes at positions 51 (N51 to I51) and 59 (C59 to R59) in DHFR.8,9 In addition, a point mutation at codon 164 has been suggested to be responsible for the development of resistance to chlorproguanil-dapsone.10

The major amino acid mutation in the DHPS gene is at residue 437 (A to G), which plays a major role in the development of clinical resistance against sulfadoxine. Mutations in the DHPS gene associated with resistance to sulfadoxine include a change of S436 to F436, A437 to G437, K540 to E540, A581 to G581, and A613 to S613 or T613. Normally, multiple DHPS mutations result in a synergistic effect on SP resistance.9

A molecular geographic survey for the occurrence of P. falciparum drug resistance is of paramount importance for quantifying antimalarial drug efficacy and for monitoring the emergence of drug-resistant malaria.11 Molecular methods have an important application in surveillance programs as attractive tools to detect drug-resistant mutants in epidemiologic surveys. Moreover, molecular tests have many advantages compared with in vitro testing, which requires a complex parasite cultivation technique and several days to perform.1214

Patients infected with parasites carrying the DHPS G437E540 double mutant and the DHFR S108I51R59 triple mutant had a specifically high relative risk of treatment failure compared with those infected with parasites carrying only the DHFR triple mutant.15 In a similar study, the quintuple mutant (3 DHFR and 2 DHPS mutations) was shown to be a relevant molecular marker of SP treatment failure among uncomplicated P. falciparum malaria patients.7

Since the molecular basis of resistance against the anti-folate drugs is well characterized, it is advisable to use molecular methods to detect drug-resistant malaria. Based on this principle, we have performed a molecular survey on P. falciparum isolates collected from Ethiopia to measure the level of antifolate resistance in this country.

METHODS

The genetic profile of the P. falciparum isolates with respect to DHFR and DHPS genes have been assessed using capillary blood collected on filter paper (Whatman 3 mm chromatography paper; Krackeler Scientific Inc., Albany, NY) from 124 uncomplicated malaria patients living in southwestern Ethiopia.

Patients with clinically suspected malaria attending the Jimma Health Center (Jimma, Ethiopia) were screened and included in the study. Only those patients with uncomplicated malaria who had P. falciparum infections and who gave informed consent were included in the study. Severely ill patients and those who had a febrile illness other than malaria were excluded from the study. The study was reviewed and approved by the Addis Ababa University Ethical Committee. Individual written informed consent was obtained from each patient.

Thin and thick blood films were prepared and stained with Giemsa for the detection of P. falciparum infection. For mutation analysis, 20 μL of blood was collected by finger prick, blotted in triplicate on a filter paper, and air-dried. The filter papers from patients with malaria were wrapped separately in a plastic bag and transported at room temperature. DNA was isolated using the QIA amp® DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instruction.

The samples were analyzed using a nested polymerase chain reaction (PCR) and DNA sequencing to detect variation in the DHFR and DHPS genes. The extracted DNA (2 μL) was used as a template in 50-μL PCR that contained 0.2 μM of each oligonucleotide primer, 1× PCR buffer (Qiagen), 2.5 mM MgCl2, 0.2 mM dNTPs, and 0.02 units/μL of Taq DNA polymerase. Accordingly, a fragment of the DHFR gene containing codons 16, 51, 59, 108, and 164 and a fragment of the DHPS gene containing codons 436, 437, 540, 581, and 613 was amplified by a nested PCR approach.

In the first reaction, a 665-basepair (bp) portion of the DHFR gene was amplified by using primers Amp1 (5′-TTT ATA TTT TCT CCT TTT TAT-3′) and Amp22 (5′-TTA CTA GTA TAT ACA TCG CTA ACA G-3′). Similarly, a 727-bp portion of the DHPS gene was amplified by using primers sulf5′ (5′-GGT ATT TTT GTT GAA CCT AAA CG-3′ and sulf3′ (5′-TCC AAT TGT GTG ATT TGT CCA C-3′).

For the second reaction of the DHFR gene, 3 μL of amplified material from the first PCR product was added to the second PCR mixture. Primers SP1 (5′-ATG ATG GAA CAA GTC TGC GAC-3′) and Amp22 were used to amplify a 646-bp fragment containing codons 16, 51, 59, 108, and 164.

The second round PCR of the DHPS gene was done using similar reaction conditions like those used for DHFR, but two different primer pairs were used to obtain two separate fragments. Primers sulf5′ and Leo2′ (5′-CTG GAT TAT TTG TAC AAG CAC-3′) were used to amplify a 319-bp fragment of the DHPS gene containing the sequence of codons 436 and 437. Similarly a 472-bp DHPS fragment that included codons 540, 581, and 613 was amplified using the primer pair DS-5F (5′-GAA TGT GTT GAT AAT GAT TTA G- 3′) and sulf3′.

The products from the nested PCR were subjected to electrophoresis on a 1% agarose gel. Gels were stained with CYBR® GREEN I nucleic acid gel stain (Cambrex Bioscience, East Rutherford, NJ) and visualized on a dark reader transilluminator (Clare Chemical Research, Dolores, CO).

The amplified DNA was purified by a PCR purification kit (EZNA.® Cycle Pure Kit; Peqlab, Erlangen, Germany), following the supplier’s instructions. The DNA was then sequenced by using Big Dye 1.1 (Applied Biosystems, Foster City, CA) and purified by DNA grade Sephadex™ (Amersham Biosciences AB, Uppsala, Sweden). A genetic analyzer 3100 (Applied Biosystems) was used for analysis of DNA. The DNA sequences were transferred to the Bio-edit sequence alignment program (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) for detection of point mutations and sequence comparison.

RESULTS

A total of 174 subjects with uncomplicated malaria were screened for the presence of a P. falciparum infection and included in the study. Based on the amount of parasite DNA in the extracted samples, 124 patients were selected for the molecular analysis of the DHFR and DHPS genes of P. falciparum isolates. Amino acid sequences were identified by the direct sequencing procedure of DHFR and DHPS genes of the selected isolates.

The prevalence rate of previously known mutations has been detected. The sequence profile in this study showed that all isolates had double mutations at residues 51 and 108, as shown in Table 1 (I51N108). Sixty-seven (54%) of the isolates had a triple mutation in the DHFR gene (I51R59N108).

With respect to the DHPS gene, all isolates had mutations at two codons (G437 and E540). Two isolates (1.6%) had additional mutations at codon 581 (G581). Codons 16 and 164 in the DHFR gene and codons 436 and 613 in the DHPS gene were wild type in all isolates. There was no wild type sequence in both genes in any of the isolates analyzed. Fifty-four percent of the samples had quintuple mutations (DHFR triple and DHPS double), which is considered an appropriate marker for indicating complete treatment failure against SP. None of the mutations was associated with the age of the patients.

Higher mutation rates in the DHFR and DHPS genes indicate a higher level of resistance against SP treatment in Ethiopia, similar to previous in vivo studies. Chlorproguanil-dapsone, another antifolate drug, is dependent on the presence of a point mutation at position 164 of the DHFR gene. In this study, as previously reported in other African countries, there is no point mutation at this position in any isolates.

DISCUSSION

Sulfadoxine-pyrimethamine was a promising drug against chloroquine-resistant strains of P. falciparum in Africa. However, the emergence of resistant strains against SP has compromised the use of this drug. Mutations against SP are more prevalent in eastern Africa than in central and western Africa.

In Ethiopia, the development of resistance to currently used anti-malaria drugs has decreased the effectiveness of early diagnosis and treatment of malaria. Sulfadoxine-pyrimethamine was introduced as a first-line drug for treatment of uncomplicated P. falciparum malaria in July 1998. When it was introduced, the treatment failure rate was approximately 5%. In a recent nation-wide study that was conducted from October to December 2003, the mean failure rate had increased to 36% at the 14-day follow-up and 72% on the 28-day follow-up.2

The occurrence of DHFR and DHPS mutations in our study is similar to findings from Malawi, the first African country to adopt SP as first-line treatment of uncomplicated malaria.3 We found that 54% of the samples had quintuple mutations; a higher (78%) prevalence was reported in Malawi. The prevalence of DHPS double mutations (100%) was higher in Ethiopia than that reported in Malawi.3

Quintuple mutations served as an indicator for the presence of SP-resistant strains in Mali, where no quintuple mutation was found.16 A similar analysis in Kenya, which is in the same geographic region as Ethiopia, reported that 10% of the samples had quintuple mutations.15 The result of our study showed a higher rate of resistance against SP. This should warrant that immediate measures have to be taken to identify the most appropriate treatment option. Based on the World Health Organization guideline for Africa, artemether-lumefantrine was adopted as a first-line treatment against uncomplicated P. falciparum malaria in Ethiopia; however, it is too costly and unavailable.

Based on the absence of L164 mutation in the DHFR gene of P. falciparum, chlorproguanil-dapsone was recommended for Africa as affordable, readily available, and effective treatment option against SP-resistant strains of P. falciparum. It is rapidly eliminated from the body, resulting in low selection pressure for drug resistance.16,17 Chlorproguanil-dapsone may be an affordable alternative, but it may cause problems in areas with a high deficiency of glucose-6-phosphate dehydrogenase. It is not known whether in Ethiopia this deficiency exists.18,19

Artemisinin-based combinations have a short shelf-life (two years), and emergency supplies have to be quantified and maintained in well-managed stocks.20 Moreover, WHO has indicated that the rapid increase in demand for ACTs may result in shortages in the production and delivery of the drug because artimisinin compounds are derived from the plant Artemisia annua. The cultivation of this plant, as well as the extraction and the manufacturing process, requires approximately three years. Therefore, agricultural production should be coordinated with the increased demand for pharmaceutical products. WHO forecasts that a massive scale-up of production is needed to meet the global requirements of the drug in 2005.

Additional combination therapies and monotherapies have been recommended as effective treatment alternatives against P. falciparum. However, the ideal (inexpensive, readily available, effective, and safe) regimen for Africa is still not known.

Table 1

Polymorphisms in DHFR and DHPS genes of Plasmodium falciparum isolates from Ethiopia*

GeneCodonResults, no. (%)
*Amino acids in bold indicate mutations. DHFR = dihydrofolate reductase, DHPS = dihydropteroate synthase.
DHFR16-Alanine124 (100)
51-Isoleucine124 (100)
59-Cysteine57 (46)
59-Arginine67 (54)
108-Asparagine124 (100)
164-Isoleucine124 (100)
DHPS436-Serine124 (100)
437-Glycine124 (100)
540-Glutamic acid124 (100)
581-Alanine122 (98.4)
581-Glycine2 (1.6)
613-Alanine124 (100)

*

Address correspondence to Jürgen F. J. Kun, Institute for Tropical Medicine, Department of Parasitology, University of Tübingen, Wilhelmstr. 27, 72074 Tübingen, Germany. E-mail: juergen.kun@uni-tuebingen.de

Authors’ addresses: Tamirat Gebru-Woldearegai, Institute for Tropical Medicine, Department of Parasitology, University of Tübingen, Wilhelmstr. 27, 72074 Tübingen, Germany, Telephone: 49-7071-298-2195, Fax: 49-7071-294-684, E-mail: tamiratgw2002@yahoo.com (present address: P.O. Box 70226, Addis Ababa, Ethiopia, Telephone: 251-9-645-665). Asrat Hailu, P.O. Box 28017/1000, Addis Ababa, Ethiopia, Telephone: 251-9-480-993, Fax: 251-1-513099, E-mail: hailu_a2004@yahoo.com. Martin P. Grobusch, Institute for Tropical Medicine, Department of Parasitology, University of Tübingen, Wilhelmstr. 27, 72074 Tübingen, Germany, Telephone: 49-7071-298-0234, Fax: 49-7071-294-684 (present address: Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2196, Johannesburg, South Africa, Telephone: 27-11-489-8537, Fax: 27-11-489-8511, E-mail: grobuschm@pathology.wits.ac.za). Jürgen F. J. Kun, Institute for Tropical Medicine, Department of Parasitology, University of Tübingen, Wilhelmstr. 27, 72074 Tübingen, Germany, Telephone: 49-7071-298-2191, Fax: 49-7071-294-684, E-mail: juergen.kun@uni-tuebingen.de.

Acknowledgments: We thank the Graduate School of Addis Ababa University for organizing the study, Almaz Demissei (Jimma Health Centre) for recruiting patients, and Andrea Weierich and Velia Grummes for technical help at the Sequencing Facility of the Institute for Tropical Medicine.

Financial support: This work was supported by grants to Tamirat Gebru-Woldearegai from the Deutscher Akademischer Austausch Dienst and by the Professor Josef und Erika Hesselbach Stiftung to Jürgen F. J. Kun.

REFERENCES

  • 1

    Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI, 2005. The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 434 :214–217.

    • Search Google Scholar
    • Export Citation
  • 2

    Jima D, 2004. The 2003 malaria situation in Ethiopia. Proceedings of the Panel Discussion on the 2003 Malaria Epidemics in Ethiopia, May 29, 2004. Addis Ababa: Ethiopian Public Health Association, 6–19.

  • 3

    Bwijo B, Kaneko A, Takechi M, Zungu IL, Moriyama Y, Lum JK, Tsukahara T, Mita T, Takahashi N, Bergqvist Y, Bjorkman A, Kobayakawa T, 2003. High prevalence of quintuple mutant dhps/dhfr genes in Plasmodium falciparum infections seven years after introduction of sulfadoxine and pyrimethamine as first line treatment in Malawi. Acta Trop 85 :363–373.

    • Search Google Scholar
    • Export Citation
  • 4

    Kean BH, 1979. Chloroquine-resistant falciparum malaria from Africa. JAMA 241 :395.

  • 5

    Triglia T, Menting JG, Wilson C, Cowman AF, 1997. Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum. Proc Natl Acad Sci U S A 94 :13944–13949.

    • Search Google Scholar
    • Export Citation
  • 6

    Cowman AF, Morry MJ, Biggs BA, Cross GA, Foote SJ, 1988. Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. Proc Natl Acad Sci USA 85 :9109–9113.

    • Search Google Scholar
    • Export Citation
  • 7

    Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese JF, Martino LM, Mukadam RA, Rogerson SJ, Lescano AG, Molyneux ME, Winstanley PA, Chimpeni P, Taylor TE, Plowe CV, 2002. Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis 185 :380–388.

    • Search Google Scholar
    • Export Citation
  • 8

    Biswas S, Escalante A, Chaiyaroj S, Angkasekwinai P, Lal AA, 2000. Prevalence of point mutations in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum isolates from India and Thailand: a molecular epidemiologic study. Trop Med Int Health 5 :737–743.

    • Search Google Scholar
    • Export Citation
  • 9

    Meyer CG, May J, Arez AP, Gil JP, Do RV, 2002. Genetic diversity of Plasmodium falciparum: asexual stages. Trop Med Int Health 7 :395–408.

    • Search Google Scholar
    • Export Citation
  • 10

    Mutabingwa T, Nzila A, Mberu E, Nduati E, Winstanley P, Hills E, Watkins W, 2001. Chlorproguanil-dapsone for treatment of drug-resistant falciparum malaria in Tanzania. Lancet 358 :1218–1223.

    • Search Google Scholar
    • Export Citation
  • 11

    Berens N, Schwoebel B, Jordan S, Vanisaveth V, Phetsouvanh R, Christophel EM, Phompida S, Jelinek T, 2003. Plasmodium falciparum: correlation of in vivo resistance to chloroquine and antifolates with genetic polymorphisms in isolates from the south of Lao PDR. Trop Med Int Health 8 :775–782.

    • Search Google Scholar
    • Export Citation
  • 12

    Kain KC, Lanar DE, 1991. Determination of genetic variation within Plasmodium falciparum by using enzymatically amplified DNA from filter paper disks impregnated with whole blood. J Clin Microbiol 29 :1171–1174.

    • Search Google Scholar
    • Export Citation
  • 13

    Rason MA, Ariey F, Rafidimanantsoa L, Andrianantenaina BH, Sahondra Harisoa JL, Randrianarivelojosia M, 2002. Monitoring the drug-sensitivity of Plasmodium falciparum in coastal towns in Madagascar by use of in vitro chemosensitivity and mutation detection tests. Parasite 9 :247–253.

    • Search Google Scholar
    • Export Citation
  • 14

    D’Alessandro U, 1998. Antimalarial drug resistance: surveillance and molecular methods for national malaria control programmes. Mem Inst Oswaldo Cruz 93 :627–630.

    • Search Google Scholar
    • Export Citation
  • 15

    Nzila AM, Mberu EK, Sulo J, Dayo H, Winstanley PA, Sibley CH, Watkins WM, 2000. Towards an understanding of the mechanism of pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: genotyping of dihydrofolate reductase and dihydropteroate synthase of Kenyan parasites. Antimicrob Agents Chemother 44 :991–996.

    • Search Google Scholar
    • Export Citation
  • 16

    Djimde AA, Dolo A, Ouattara A, Diakite S, Plowe CV, Doumbo OK, 2004. Molecular diagnosis of resistance to antimalarial drugs during epidemics and in war zones. J Infect Dis 190 :853–855.

    • Search Google Scholar
    • Export Citation
  • 17

    Amukoye E, Winstanley PA, Watkins WM, Snow RW, Hatcher J, Mosobo M, Ngumbao E, Lowe B, Ton M, Minyiri G, Marsh K, 1997. Chlorproguanil-dapsone: effective treatment for uncomplicated falciparum malaria. Antimicrob Agents Chemother 41 :2261–2264.

    • Search Google Scholar
    • Export Citation
  • 18

    Sulo J, Chimpeni P, Hatcher J, Kublin JG, Plowe CV, Molyneux ME, Marsh K, Taylor TE, Watkins WM, Winstanley PA, 2002. Chlorproguanil-dapsone versus sulfadoxine-pyrimethamine for sequential episodes of uncomplicated falciparum malaria in Kenya and Malawi: a randomised clinical trial. Lancet 360 :1136–1143.

    • Search Google Scholar
    • Export Citation
  • 19

    Alloueche A, Bailey W, Barton S, Bwika J, Chimpeni P, Falade CO, Fehintola FA, Horton J, Jaffar S, Kanyok T, Kremsner PG, Kublin JG, Lang T, Missinou MA, Mkandala C, Oduola AM, Premji Z, Robertson L, Sowunmi A, Ward SA, Winstanley PA, 2004. Comparison of chlorproguanil-dapsone with sulfadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria in young African children: double-blind randomised controlled trial. Lancet 363 :1843–1848.

    • Search Google Scholar
    • Export Citation
  • 20

    Kremsner PG, Krishna S, 2004. Antimalarial combinations. Lancet 364 :285–294.

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