Molecular surveillance of drug-resistance associated mutations of Plasmodium falciparum in south-west Tanzania
© Schönfeld et al; licensee BioMed Central Ltd. 2007
Received: 05 October 2006
Accepted: 15 January 2007
Published: 15 January 2007
In Tanzania, drug-resistant malaria parasites are an increasing public health concern. Because of widespread chloroquine (CQ) resistance Tanzania changed its first line treatment recommendations for uncomplicated malaria from CQ to sulfadoxine-pyrimethamine (SP) in 2001. Loss of SP sensitivity is progressing rapidly. SP resistance is associated with mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes.
In samples from 86 patients with uncomplicated Plasmodium falciparum malaria from Mbeya and Matema, Mbeya region, south-western Tanzania, the occurrence of mutations was investigated in the pfcrt and pfmdr1 genes which are associated with CQ resistance and in pfdhfr and pfdhps, conferring SP resistance, as well in cytb which is linked to resistance to atovaquone.
Pfcrt T76 occurs in 50% and pfmdr1 Y86 in 51.7%. Pfdhfr triple mutations coexisting with pfdhps double mutations were detected in 64.3% of the P. falciparum isolates. This quintuple mutation is seen as a possible predictive molecular marker for SP treatment failure. Mutations of the cytb gene were not detected.
These findings of a high prevalence of mutations conferring SP resistance correspond to data of in vivo SP efficacy studies in other regions of Tanzania and underline the recommendation of changing first-line treatment to artemisinin-based combination therapy.
In Sub-Saharan Africa malaria is a leading cause of morbidity and mortality, especially in children under five years . Despite intensive campaigns, over 10 Million malaria cases occurred in Tanzania in 2003, about 30% more compared to the previous year . Most of these cases were caused by Plasmodium falciparum. This may be partly due to better surveillance systems, but raising drug resistance is the most likely reason for this tremendous increase.
Chloroquine (CQ) was the antimalarial treatment of choice during the second half of the 20th century. But increasing rates of CQ resistance led Tanzania to change its first line treatment of uncomplicated malaria to sulfadoxine-pyrimethamine (SP) in 2001 . This antifolate combination seemed to be an effective and reasonable alternative, but resistance to SP was rapidly gaining ground, facilitated by the slow elimination from the body. New data show a high level (45%) of SP treatment failures in Muheza, northeast Tanzania . Other effective drugs, where resistance is as yet not a frequent problem, such as atovaquone-proguanil or mefloquine, are of limited value due to their high current costs . A useful alternative is artemisinin-based combination therapy (ACT), e.g., artemether-lumefantrine which is introduced as the new first line drug in Tanzania in mid 2006. At the moment the availability of ACT is limited; while artemisinin monotherapy formulations and SP are commonly used, especially in the private sector. Some drug shops still sell the relegated CQ.
CQ resistance is associated with an amino acid change from lysine to threonine in codon 76 of the P. falciparum chloroquine resistance transporter gene (pfcrt) , and a mutation from asparagine to tyrosine in codon 86 of the multidrug resistant gene (pfmdr) [7–10]. Besides there are indices that pfmdr1 N86 is associated with resistance to lumefantrine that is widely used in combination with artemether , and also with decreased sensitivity to artemisinins .
SP resistance is associated with mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes. Pyrimethamine is a selective, competitive inhibitor of dihydrofolate reductase and earlier in the folate pathway sulfa drugs inhibit dihydropteroate synthase [13, 14]. Several point mutations are connected to antifolate drug resistance. The quintuple mutation (triple pfdhfr: I51, R59, N108 and double pfdhps: G437, E540) is discussed as a relevant molecular marker of SP treatment failure [15, 16].
Atovaquone-proguanil is a relatively new antimalarial drug that inhibits mitochondrial electron transport. Point mutations in the cytb codon 268 are associated with resistance to this combination [17–19].
Overall 86 blood samples were collected from patients with clinically diagnosed uncomplicated P. falciparum malaria (fever ≥ 38.0°C, parasitaemia ≥ 2 000/μl) from August to October in 2004 and from June to July in 2005 in Mbeya region of South-western Tanzania. Patients were enrolled at two locations: Matema Health Care Center located in a poor rural area at the shores of Lake Malawi with holoendemic malaria transmission and the Mbeya Referral Hospital, a tertiary hospital at an altitude of 1 700 meters that admits complicated cases from surrounding mesoendemic areas or people that have travelled within Tanzania. Written informed consent was obtained from each patient or the parental guide. The study was reviewed and approved by the local IRB at the Mbeya Referral Hospital and the Tanzanian National Ethics Board of the National Institute for Medical Research, Dar es Salaam.
From each patient a finger prick blood sample was taken for thick and thin blood film and another for filter paper blood sample. Giemsa-stained blood films were examined for malaria parasites (per 200 white blood cells) and densities were assessed based on a assumed mean WBC count of 8 000/μl. DNA extraction from filter paper bloodspots was done using Chelex® (Bio-Rad, Germany) as described elsewhere  Nested PCR assays were used to verify the parasite species . The DNA was amplified by nested PCR and digested by the RFLP-method to detect the mutations of P. falciparum pfcrt 76, pfmdr1 86, dhfr 16, 51, 59, 108, 164, dhps 436, 437, 540, 581, 613 and cytb 268 [17, 22, 23]. Mixed alleles (wild type and mutant) were assessed as mutant. The median age of the 86 patients with P. falciparum infection (45 female, 41 male) was 21 years (range 8 month to 55 years). All 86 isolates showed P. falciparum mono-infection, there was no P. vivax, P. ovale, or P. malariae infection. The geometric mean parasite density was 29 992/μl (range 3 320/μl to 127 440/μl).
Prevalence of mutations conferring resistance to chloroquine, sulfadoxine-pyrimethamine and atovaquone-proguanil in Plasmodium falciparum isolates from Mbeya & Matema, southern Tanzania
Mixed type (%)
Prevalence of pfcrt, pfmdr 1, dhfr and dhps genotype combinations conferring chloroquine and sulfadoxine-pyrimethamine resistance in Mbeya & Matema, together and each seperated. The risk ratio is the prevalence ratio of the combinations between Mbeya and Matema.
Mbeya & Matema (%)
Dhfr/dhps quintuple (triple dhfr + double dhps) ***
Even so this study investigated a limited number of patients and therefore differences may not reach significance, differences in mutation rates might reflect differences of access to health care between the two locations. While individuals in the rural area receive their malaria treatment, currently SP, almost exclusively through the Matema Health Care Center, patients from Mbeya can choose upon a wide variety of health care facilities and antimalarial drugs. Ongoing usage of CQ may be assumed there.
Although considerably higher resistance rates before 2001 are probable, the still relatively high rate of pfcrt and pfmdr1 mutations is contrary to other reports that have demonstrated a complete regression of pfcrt mutations several years after leaving CQ as first – line antimalarial drug [27, 28].
In vivo selection of pfmdr1 86N allele by artemether-lumefantrine has been found in Tanzania  and pfmdr copy numbers seem to influence susceptibility to lumefantrine and artemisinin . There is no prediction possible due to our results but continuing surveillance would be interesting concerning pfmdr1 polymorphisms.
This study confirms the high prevalence of point mutations in the pfdhfr and pfdhps genes in Tanzania which are associated with SP treatment failure [25, 26]. The rate of quintuple pfdhfr/pfdhps mutations in the Mbeya region, south-western Tanzania, is in the upper range of frequencies reported in East Africa. Data from Malawi, Kenya, Tanzania and Ethiopia range from 10 to 78% [25, 30–32]. The absence of cytb codon 268 supports atovaquone-proguanil as a possible second- or third-line drug for treatment of uncomplicated malaria.
The study data might be used as a basis for surveillance of resistance markers after introduction of ACT and might later indicate the possibility for reintroduction of one of the other drugs.
Conflict of interest
The authors do not have a commercial or other association that might pose a conflict of interest.
We thank MMRP for making their facilities available for this project. We acknowledge the contributions of NIMR, Dr Eleuter Samky, Diretor of Mbeya Referal Hospital and Dr. Donnan Mmbando, Regional Medical Officer for Mbeya Region and Heike Schimanowski, Head of the Matema Health Care Center, for the permission to use their respective facilities and facilitating ethical clearance. Vera Kleinfeldt was responsible for training of all laboratory technicians that participated in the study. This study is part of the thesis work of Mirjam Schönfeld at the Ludwig-Maximilians-University (LMU) Munich, Germany.
- WHO: World Malaria Report 2005. Geneva. 2005Google Scholar
- WHO: Global Health Atlas. Geneva. 2005Google Scholar
- Ministry of Health Tanzania: Malaria Treatment Guidelines. Dar es Salam. 2000Google Scholar
- Mutabingwa T, Nzila A, Mberu E, Nduati E, Winstanley P, Hills E, Watkins W: Chlorproguanil-dapsone for treatment of drug-resistant falciparum malaria in Tanzania. Lancet. 2001, 358: 1218-1223. 10.1016/S0140-6736(01)06344-9.View ArticlePubMedGoogle Scholar
- Winstanley P: Modern chemotherapeutic options for malaria. Lancet Infect Dis. 2001, 1: 242-250. 10.1016/S1473-3099(01)00119-0.View ArticlePubMedGoogle Scholar
- Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV, Coulibaly D: A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med. 2001, 344: 257-263. 10.1056/NEJM200101253440403.View ArticlePubMedGoogle Scholar
- Warhurst DC: Drug resistance in Plasmodium falciparum malaria. Infection. 1999, 27 (Suppl 2): S55-58.View ArticlePubMedGoogle Scholar
- Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naude B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE: Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell. 2000, 6: 861-871. 10.1016/S1097-2765(05)00077-8.PubMed CentralView ArticlePubMedGoogle Scholar
- Ouellette M: Biochemical and molecular mechanisms of drug resistance in parasites. Trop Med Int Health. 2001, 6: 874-882. 10.1046/j.1365-3156.2001.00777.x.View ArticlePubMedGoogle Scholar
- Wellems TE, Plowe CV: Chloroquine-resistant malaria. J Infect Dis. 2001, 184: 770-776. 10.1086/322858.View ArticlePubMedGoogle Scholar
- Sisowath C, Stromberg J, Martensson A, Msellem M, Obondo C, Bjorkman A, Gil JP: In vivo selection of Plasmodium falciparum pfmdr1 86N coding alleles by artemether-lumefantrine (Coartem). J Infect Dis. 2005, 191 (6): 1014-1017. 10.1086/427997.View ArticlePubMedGoogle Scholar
- Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC: The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. Mol Biochem Parasitol. 2000, 108: 13-23. 10.1016/S0166-6851(00)00201-2.View ArticlePubMedGoogle Scholar
- Bzik DJ, Li WB, Horii T, Inselburg J: Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene. Proc Natl Acad Sci USA. 1987, 84: 8360-8364. 10.1073/pnas.84.23.8360.PubMed CentralView ArticlePubMedGoogle Scholar
- Brooks DR, Wang P, Read M, Watkins WM, Sims PF, Hyde JE: Sequence variation of the hydroxymethyldihydropterin pyrophosphokinase: dihydropteroate synthase gene in lines of the human malaria parasite, Plasmodium falciparum, with differing resistance to sulfadoxine. Eur J Biochem. 1994, 224: 397-405. 10.1111/j.1432-1033.1994.00397.x.View ArticlePubMedGoogle Scholar
- 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: Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis. 2002, 185: 380-388. 10.1086/338566.View ArticlePubMedGoogle Scholar
- Happi CT, Gbotosho GO, Folarin OA, Bolaji OM, Sowunmi A, Kyle DE, Milhous W, Wirth DF, Oduola AM: Association between mutations in Plasmodium falciparum chloroquine resistance transporter and P. falciparum multidrug resistance 1 genes and in vivo amodiaquine resistance in P. falciparum malaria-infected children in Nigeria. Am J Trop Med Hyg. 2006, 75: 155-161.PubMedGoogle Scholar
- Schwobel B, Alifrangis M, Salanti A, Jelinek T: Different mutation patterns of atovaquone resistance to Plasmodium falciparum in vitro and in vivo: rapid detection of codon 268 polymorphisms in the cytochrome b as potential in vivo resistance marker. Malar J. 2003, 2: 5-10.1186/1475-2875-2-5.PubMed CentralView ArticlePubMedGoogle Scholar
- Musset L, Bouchaud O, Matheron S, Massias L, Le Bras J: Clinical atovaquone-proguanil resistance of Plasmodium falciparum associated with cytochrome b codon 268 mutations. Microbes Infect. 2006,Google Scholar
- Wichmann O, Muehlberger N, Jelinek T, Alifrangis M, Peyerl-Hoffmann G, Muhlen M, Grobusch MP, Gascon J, Matteelli A, Laferl H, Bisoffi Z, Ehrhardt S, Cuadros J, Hatz C, Gjorup I, McWhinney P, Beran J, da Cunha S, Schulze M, Kollaritsch H, Kern P, Fry G, Richter J, European Network on Surveillance of Imported Infectious Diseases: Screening for mutations related to atovaquone/proguanil resistance in treatment failures and other imported isolates of Plasmodium falciparum in Europe. J Infect Dis. 2004, 190: 1541-1546. 10.1086/424469.View ArticlePubMedGoogle Scholar
- Kain KC, Lanar DE: Determination of genetic variation within Plasmodium falciparum by using enzymatically amplified DNA from filter paper disks impregnated with whole blood. J Clin Microbiol. 1991, 29: 1171-1174.PubMed CentralPubMedGoogle Scholar
- Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario 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, 61: 315-320. 10.1016/0166-6851(93)90077-B.View ArticlePubMedGoogle 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
- Flueck TP, Jelinek T, Kilian AH, Adagu IS, Kabagambe G, Sonnenburg F, Warhurst DC: Correlation of in vivo-resistance to chloroquine and allelic polymorphisms in Plasmodium falciparum isolates from Uganda. Trop Med Int Health. 2000, 5: 174-178. 10.1046/j.1365-3156.2000.00543.x.View ArticlePubMedGoogle Scholar
- Gil JP, Nogueira F, Stromberg-Norklit J, Lindberg J, Carrolo M, Casimiro C, Lopes D, Arez AP, Cravo PV, Rosario VE: Detection of atovaquone and Malarone resistance conferring mutations in Plasmodium falciparum cytochrome b gene (cytb). Mol Cell Probes. 2003, 17: 85-89. 10.1016/S0890-8508(03)00006-9.View ArticlePubMedGoogle Scholar
- Mugittu K, Ndejembi M, Malisa A, Lemnge M, Premji Z, Mwita A, Nkya W, Kataraihya J, Abdulla S, Beck HP, Mshinda H: Therapeutic efficacy of sulfadoxine-pyrimethamine and prevalence of resistance markers in Tanzania prior to revision of malaria treatment policy: Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase mutations in monitoring in vivo resistance. Am J Trop Med Hyg. 2004, 71: 696-702.PubMedGoogle Scholar
- Mugittu K, Abdulla S, Falk N, Masanja H, Felger I, Mshinda H, Beck HP, Genton B: Efficacy of sulfadoxine-pyrimethamine in Tanzania after two years as first-line drug for uncomplicated malaria: assessment protocol and implication for treatment policy strategies. Malar J. 2005, 4: 55-10.1186/1475-2875-4-55.PubMed CentralView ArticlePubMedGoogle 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: 1870-1875. 10.1086/375419.View ArticlePubMedGoogle Scholar
- Mita T, Kaneko A, Lum JK, Bwijo B, Takechi M, Zungu IL, Tsukahara T, Tanabe K, Kobayakawa T, Bjorkman A: Recovery of chloroquine sensitivity and low prevalence of the Plasmodium falciparum chloroquine resistance transporter gene mutation K76T following the discontinuance of chloroquine use in Malawi. Am J Trop Med Hyg. 2003, 68: 413-415.PubMedGoogle Scholar
- Sidhu AB, Uhlemann AC, Valderramos SG, Valderramos JC, Krishna S, Fidock DA: Decreasing pfmdr1 copy number in plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J Infect Dis. 2006, 194: 528-535. 10.1086/507115.PubMed CentralView ArticlePubMedGoogle Scholar
- Bwijo B, Kaneko A, Takechi M, Zungu IL, Moriyama Y, Lum JK, Tsukahara T, Mita T, Takahashi N, Bergqvist Y, Bjorkman A, Kobayakawa T: 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. 2003, 85: 363-373. 10.1016/S0001-706X(02)00264-4.View ArticlePubMedGoogle 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 Plasmodium falciparum : genotyping of dihydrofolate reductase and dihydropteroate synthase of Kenyan parasites. Antimicrob Agents Chemother. 2000, 44: 991-996. 10.1128/AAC.44.4.991-996.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Gebru-Woldearegai T, Hailu A, Grobusch MP, Kun JF: Molecular surveillance of mutations in dihydrofolate reductase and dihyropteroate synthase genes of Plasmodium falciparum in Ethiopia. Am J Trop Med Hyg. 2005, 73: 1131-1134.PubMedGoogle Scholar
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