Resistance of Plasmodium falciparum to commonly used antimalarial drugs is a growing problem in Africa.1 In general, resistance is most severe in east and southern, compared with west Africa. Resistance to aminoquinolines, especially chloroquine (CQ), is widespread, and CQ is no longer recommended to treat malaria in sub-Saharan Africa. Resistance to amodiaquine (AQ), a related aminoquinoline, is less common, although its antimalarial efficacy is unsatisfactory in many areas. Resistance to antifolates, notably sulfadoxine-pyrimethamine (SP), is increasing. With increasing resistance to older drugs, highly efficacious artemisinin-based combination therapies (ACTs) are now the recommended first-line antimalarials in nearly all countries in sub-Saharan Africa.2
Despite the move to newer regimens, it remains important to assess resistance to older antimalarial agents for a number of reasons. First, many episodes of malaria continue to be treated with CQ, AQ, or SP, because transition to ACTs has been slow. Second, AQ and SP are included in ACTs (in both cases combined with artesunate) currently recommended by the World Health Organization (WHO). Third, one older combination regimen, AQ/SP, is recommended by the WHO to treat malaria when ACTs are not available, and this regimen remains highly efficacious in some areas, particularly parts of west Africa.3 Fourth, use of CQ and related drugs may be indicated in the future if the prevalence of resistance diminishes after removal of selective pressure, as has occurred in Malawi.4 Fifth, antifolates are widely used in Africa to prevent infections, including intermittent doses of SP to prevent malaria in children and pregnant women5 and trimethoprim-sulfamethoxazole to prevent opportunistic infections, including malaria, in HIV-infected individuals.6
Mechanisms of resistance of P. falciparum to aminoquinolines and antifolates are fairly well understood. Resistance to CQ is mediated principally by the 76T mutation in pfcrt, which encodes a putative transporter.7 Mutations in pfmdr1, which encodes another putative transporter, may contribute to resistance to CQ and seem to play a greater role in resistance to AQ. 8,9 Resistance to SP is mediated by a series of mutations in pfdhfr and pfdhps, the genes encoding dihydrofolate reductase and dihydropteroate synthase, the two enzyme targets of this combination, with both stepwise progression of mutations and selective sweeps of resistant parasites contributing to drug resistance. 10
Recent standardization of methods for the characterization of antimalarial treatment outcomes and identification of resistance-mediating polymorphisms has streamlined the characterization of drug resistance in Africa. However, information on resistance is spotty, with some parts of Africa poorly represented. One such area is the western Democratic Republic of the Congo (DRC), including Kinshasa, the third largest city in Africa, with a population of 8 million, which is the third largest city in Africa. In addition to the obvious importance of characterizing resistance in a major city, an understanding of malaria in Kinshasa will help us to appreciate the geographical flux of resistance between the very high levels recorded across east Africa and much lower levels seen in some areas of west Africa. To this end, we evaluated molecular markers of resistance to aminoquinoline and antifolate antimalarials in Kinshasa.
We evaluated a convenience sample of children 1–10 years of age at five clinics in Kinshasa (Center Hospitalier de Mont Amba, Center de Santé de Kindele, Center Pédiatrique de Kalembelembe, Center Hospitalier de Kingasani and Clinique Riviera) in March–April 2008. Children presenting with acute febrile illnesses received standard evaluations including Giemsa-stained blood smears. When uncomplicated malaria was diagnosed, parents or guardians of patients were asked to participate in this study. Selection criteria for our study were diagnosis of microscopy-proven uncomplicated malaria by the health center and provision of informed consent by the parent or guardian. Exclusion criteria were evidence or clinical suspicion of complicated malaria, as defined by the WHO. 11 Patients were managed for malaria following standard clinic protocols. With enrollment, a short questionnaire concerning prior use of antimalarials was completed, and blood was collected by finger prick for thin and thick blood smear and collection of blood spots on filter paper (Whatman 3MM). Blood smears were stained with 10% Giemsa for 10 minutes and examined by a trained microscopist. Filter paper samples were labeled and stored with desiccant at room temperature. Subsequently, DNA was extracted from filter paper with chelex, 12 and P. falciparum polymorphisms of interest were assessed by nested amplification of genes of interest, sequence-specific restriction endonuclease digestion, separation of DNA fragments by agarose gel electrophoresis, and visual characterization of DNA digestion patterns, with minor modifications of methods that have previously been described. 13,14 The study was approved by the Ethics Committee at the Kinshasa School of Public Health and the Committee for Human Research of the University of California, San Francisco, CA.
A total of 142 children with a mean age of 3.5 years were referred for the study. For 55 of these subjects, prior use of antimalarial treatment was reported by caregivers. This treatment was reported to be quinine in 38 (27% of study children), artesunate plus quinine in 4 (2.8%), other standard therapies in 7 (4.9%), and iron or traditional remedies in 6 (4.2%). Thus, antimalarial treatment before clinic presentation was common, and the drug most frequently reported was quinine, which is a standard therapy for severe, but not uncomplicated, malaria. No subjects reported prior use of artesunate/AQ, the recommended therapy to treat uncomplicated malaria in DRC. However, it is important to note that our findings do not provide a reliable gauge of presumptive antimalarial therapy in Kinshasa, because caretaker reports may have been inaccurate and because those who received effective initial therapy would not be expected to present to clinics with fever. Nonetheless, the results highlight heavy reliance on quinine for the treatment of uncomplicated malaria, a strategy that will likely be limited by poor tolerance and poor compliance with the full 7-day regimen.
The diagnosis of falciparum malaria was confirmed by repeat microscopy and polymerase chain reaction (PCR) in 121 study children. For the other 21 children, in nearly all cases, both the follow-up blood smear and PCR were negative, indicating a false-positive initial smear reading. P. falciparum polymorphisms associated with altered responses to aminoquinolines (pfcrt and pfmdr1) and antifolates (pfdhfr and pfdhps) were assessed (Table 1). Less then 121 outcomes were available for each polymorphism because of occasional failure of PCR reactions (despite repeat assays with increased template) and because, for the pfdhfr 164 polymorphism, analysis was stopped after the first 87 samples were all wild type. These results fill a gap in our appreciation of the map of drug resistance in Africa. In general, the prevalence of multiple resistance-mediating polymorphisms is highest in much of east and southern Africa and lowest in parts of west and central Africa (Figure 1). Consideration of key resistance mediating polymorphisms in Africa can be simplified to four sets of mutations. First, the key marker of CQ resistance is pfcrt 76T. This mutation is now common throughout sub-Saharan Africa, except in regions (primarily Malawi) where elimination of CQ use has allowed wild-type parasites to replace resistant mutants.4 Mutant parasites were common in Kinshasa, although prevalence of the 76T mutation was below that seen in east Africa, where it is commonly 100%. Second, the 86Y mutation in pfmdr1 mediates decreased sensitivity to aminoquinolines, but interestingly, increased sensitivity to mefloquine, halofantrine, and quinine. 15 Other pfmdr1 polymorphisms, including 184F, 1034C, 1042D, and 1246Y, may contribute to altered drug sensitivity. 16 Parasites from Kinshasa showed intermediate prevalence of pfmdr1 86Y, 184F, and 1246Y compared with sites with higher prevalence in east Africa and generally lower prevalence in west Africa. Third, considering antifolate resistance, two pfdhfr mutations (108N and 51I) and one pfdhps mutation (437G) are common in most areas, but not predictive of SP treatment outcomes. The key mediators of resistance seem to be pfdhfr 59R and pfdhps 540E, with both of these mutations needed for significant loss of SP treatment efficacy. 10,17 This conclusion is supported by the poor efficacy of SP in recent years at many locations in east Africa, 18 where prevalence of all five relevant mutations (the quintuple mutation) is common, and by continued good efficacy of SP in parts of west Africa where one relevant mutation, pfdhps 540E, is generally absent. 19 In Kinshasa, four of the five relevant mutations are very common, but pfdhps 540E is uncommon, although more prevalent than in countries farther to the west. Fourth, high-level antifolate resistance is mediated in Asia and South America by an additional mutation, pfdhfr 164L. This mutation has generally been rare in Africa, although modest prevalence has been noted recently in a few areas. 20,21 The pfdhfr 164L mutation was not identified in any parasites from Kinshasa.
In summary, P. falciparum causing symptomatic malaria in Kinshasa commonly contained mutations that mediate resistance to aminoquinoline and antifolate antimalarials. The results predict an intermediate level of drug resistance between the very high levels seen in east Africa and lower levels in parts of west Africa. Specifically, the results suggest poor antimalarial activity of CQ, uncertain efficacy of AQ, but fairly good efficacy for SP. The moderate prevalence of key pfmdr1 polymorphisms might suggest concern regarding the efficacy of aminoquinoline-containing ACTs (artesunate/AQ and dihydroartemisinin/piperaquine), although it remains unclear if these polymorphisms will affect ACT treatment outcomes. These results further suggest that SP or trimethoprim-sulfamethoxazole will remain efficacious in Kinshasa to prevent malaria. However, the genetics of parasite populations can change quickly under heavy drug pressure, and therefore continued surveillance of resistance mediating polymorphisms and, ideally, drug efficacy results from clinical trials, will be needed to best assess the utility of different antimalarial treatment and preventive regimens in Kinshasa over time.
P. falciparum genetic polymorphisms identified in samples from Kinshasa
Map of prevalence of pfdhfr/pfdhps quintuple mutation in Africa. Results from representative evaluable studies performed since 2000 are shown. Results are from our study (10%) and for one or more sites in Burkina Faso,3 Cameroon,22 Republic of the Congo, 23 DRC, 24,25 Ethiopia,26 Gabon,27,28 Ghana,29 Guinea,30 Guinea-Bissau,31 Malawi, 32,33 Mozambique,34 Nigeria,35 Senegal,36 Sudan,37 Tanzania,38 Uganda, 39 and Zambia. 40
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 4; 10.4269/ajtmh.2009.80.555
Map of prevalence of pfdhfr/pfdhps quintuple mutation in Africa. Results from representative evaluable studies performed since 2000 are shown. Results are from our study (10%) and for one or more sites in Burkina Faso,3 Cameroon,22 Republic of the Congo, 23 DRC, 24,25 Ethiopia,26 Gabon,27,28 Ghana,29 Guinea,30 Guinea-Bissau,31 Malawi, 32,33 Mozambique,34 Nigeria,35 Senegal,36 Sudan,37 Tanzania,38 Uganda, 39 and Zambia. 40
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 4; 10.4269/ajtmh.2009.80.555
Map of prevalence of pfdhfr/pfdhps quintuple mutation in Africa. Results from representative evaluable studies performed since 2000 are shown. Results are from our study (10%) and for one or more sites in Burkina Faso,3 Cameroon,22 Republic of the Congo, 23 DRC, 24,25 Ethiopia,26 Gabon,27,28 Ghana,29 Guinea,30 Guinea-Bissau,31 Malawi, 32,33 Mozambique,34 Nigeria,35 Senegal,36 Sudan,37 Tanzania,38 Uganda, 39 and Zambia. 40
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 4; 10.4269/ajtmh.2009.80.555
Address correspondence to Philip J. Rosenthal, Department of Medicine, Box 0811, University of California, San Francisco, CA 94143. E-mail: prosenthal@medsfgh.ucsf.edu
Authors’ addresses: Linda Mobula, Bruce Lilley, and Philip J. Rosenthal, Department of Medicine, Box 0811, University of California, San Francisco, CA 94143. Antoinette K. Tshefu, Ecole de Santé Publique, Faculté de Medecine, Université de Kinshasa, B.P. 11850 KIN I, Kinshasa, République Democratique du Congo.
Acknowledgments: The authors thank Pius Mafuta, Dr. Edouard Mayimbi, Dr. Jack Kokolomami, Dr. Michel Kaya, Bicko Makubikwa Mamengi, Grec Kiloto Kakoma, and laboratory technicians at Clinic Riviera and at Kalembelembe. We also thank the children and their parents or guardians who participated in this study.
Financial support: This work was supported by the Doris Duke Charitable Foundation, of which PJR is a Distinguished Clinical Scientist.
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