INTRODUCTION
An estimated 247 million clinical cases and 619,000 malaria-related deaths were reported worldwide in 2021.1 Plasmodium falciparum is the most prevalent and virulent of the human malaria species.1 Antimalarial drug resistance in P. falciparum parasites poses one of the greatest threats to malaria control. As a result of the widespread resistance to chloroquine (CQ) and sulfadoxine–pyrimethamine (SP), artemisinin-based combination therapies have been recommended for P. falciparum malaria treatment since 2006.2
Sulfadoxine–pyrimethamine plus amodiaquine (AQ) was used as a first-line antimalarial treatment of uncomplicated malaria in Rwanda from 2001 to 2006, at which time artemether–lumefantrine (AL) became the recommended first-line treatment.2 Resistance to SP is associated with polymorphisms in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes.3,4 Antimalarial drug policy change may influence drug sensitivity as a result of the withdrawal of drug pressure on parasites. This was demonstrated in a 2003 study5 in Malawi where, 12 years after CQ withdrawal, no evidence of the P. falciparum CQ resistance transporter (pfcrt K76T) mutation was detected among isolates, compared with 85% reported in 1992, indicating that CQ efficacy may have been restored. In Rwanda, a slower recovery rate of CQ sensitivity has been reported after 14 years of cessation.2 However, despite 7 years of the presumed absence of SP pressure, a high level of SP resistance was reported in samples collected from Rwanda in 2015,2 which persisted in 2020.6 Sulfadoxine–pyrimethamine remains the only drug recommended by the WHO for intermittent preventive treatment during pregnancy (IPTp) to prevent the adverse consequences of malaria. Intermittent preventive treatment during pregnancy–SP is a recommended intervention in 35 sub-Saharan African countries.1,7
Although the quintuple mutant genotype, consisting of triple mutations of pfdhfr (N51I/S59R/S108N) and double mutations of pfdhps (A437G/K540E), is associated with clinical and parasitological SP treatment failure, IPTp-SP remains efficacious and confers some protection, particularly against low birthweight, even in areas with a high level of quintuple mutant genotypes,8 presumably because of the nonantimalarial effects of SP.9,10 However, IPTp-SP efficacy for preventing low birthweight is reduced when a high proportion of P. falciparum parasites acquire an additional pfdhps A581G mutation, leading to the sextuple mutant (pfdhfr N51I + C59R + S108N/pfdhps A437G + K540E + A581G).8,11 As a result of the documented high level of resistance, SP was withdrawn completely from all uses in Rwanda 2008, with cessation of the IPTp policy.2
Rwanda experienced a dramatic increase in malaria cases between 2012 and 2017, from 564,407 in 2012 to 4,746,985 in 2017—a more than an 8-fold increase.12 More than 79% of the malaria burden was reported from the eastern and southern provinces. Because it is not ethically appropriate to conduct an SP efficacy trial in regions with known high rates of treatment failure, resistance marker detection may be the only evidence to support the notion of reintroducing drugs after their withdrawal. We report SP resistance markers in P. falciparum isolates from pregnant women in a relatively high malaria transmission zone in Rwanda, collected during a cluster randomized controlled trial investigating intermittent malaria screening and treatment of positive patients during pregnancy, compared with routine antenatal care for reducing malaria prevalence at delivery. We also compared samples obtained from placental or peripheral blood to determine whether peripheral blood captured drug-resistant genotypes accurately.
MATERIALS AND METHODS
Study area and study participants.
The study was conducted in two districts (Huye and Kamonyi), both of which are in the Southern Province of Rwanda (Figure 1). The Southern Province encompasses an area of 5,963 km2 with a population of about 2.6 million. Although malaria cases have declined in recent years, 1.8 million cases were reported in Rwanda in 2020 compared with 4.7 million cases in 2017. The Southern Province has the highest incidence in the country, with more than 450 cases per 1,000 people, compared with the national average of 198 per 1,000 people.12 In our study, we used clinical samples collected from pregnant women attending an antenatal care clinic in Rwanda between 2016 and 2018, and participating in a cluster-level randomized study to estimate the effectiveness of intermittent screening and treatment of malaria in pregnancy on maternal and birth outcomes (ClinicalTrials.gov identifier, NCT03508349).13 A total of 1,441 pregnant women participated in the study, which included rapid diagnostic testing locally. At the time of delivery, placental and peripheral blood samples for polymerase chain reaction (PCR) testing at later time were collected using a filter paper card.13 All available 1,267 placental or 1,347 peripheral samples were processed for PCR nucleic acid testing, with the 192 placental and 140 peripheral positive P. falciparum samples processed for further drug-resistant testing.
Map of Rwanda showing the study sites of the Huye and Kamonyi districts in Southern Province, Rwanda.
Citation: The American Journal of Tropical Medicine and Hygiene 109, 5; 10.4269/ajtmh.23-0225
Map of Rwanda showing the study sites of the Huye and Kamonyi districts in Southern Province, Rwanda.
Citation: The American Journal of Tropical Medicine and Hygiene 109, 5; 10.4269/ajtmh.23-0225
Map of Rwanda showing the study sites of the Huye and Kamonyi districts in Southern Province, Rwanda.
Citation: The American Journal of Tropical Medicine and Hygiene 109, 5; 10.4269/ajtmh.23-0225
Genotyping drug-resistant genes.
We genotyped the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes that confer resistance to pyrimethamine and sulfadoxine, respectively, using placental samples. Peripheral samples were compared with placental samples in matched pairs of PCR amplification from both sample locations. These genes were amplified by nested PCR and then a ligase detection reaction–fluorescent microsphere assay was performed to identify single nucleotide polymorphisms (SNPs) in the pfdhfr and pfdhps genes as described previously.14 We targeted four SNPs within the pfdhfr gene (N51I, C59R, S108N, and I164L) and five SNPs within the pfdhps gene (S436A, A437G, K540E, A581G, and A613S).
Statistical analysis.
The prevalence of each SNP was calculated by dividing the number of samples carrying the SNP by the total number of samples genotyped successfully at that SNP. Each isolate was coded based on the presence or absence of a mutant allele, for which three categories were considered: wildtype, mutant, and mixed infection. In the final analysis, both mutant and mixed infection were coded as mutant alleles to generate the number of mutant alleles per codon. All statistical analyses were conducted using IBM SPSS Statistics 28.0 (SPSS Inc., Chicago, IL).
RESULTS
From the original study, placental samples were collected and analyzed successfully by reverse transcription–PCR from 1,267 women. The prevalence of placental infection at delivery by PCR from placental blood was 15% (192 of 1,267). whereas fingerstick peripheral prevalence was 10% (140 of 1,347). A total of 148 of 192 placental blood samples were genotyped successfully for pfdhfr and pfdhps genes, and 58 were matched to fully genotyped peripheral fingerstick blood.
pfdhps gene.
The prevalence of mutant alleles (pure mutant and mixed) in placental samples at codons S436A, A437G, K540E, A581G, and A613S was 71%, 90%, 90%, 38%, and 3%, respectively (Table 1). The distribution of mutant alleles pfdhps S436A, A437G, K540E, A581G, and A613S were comparable across the two districts (Table 2).
The prevalence of single nucleotide polymorphisms in the pfdhps and pfdhfr genes in Southern Province, Rwanda, (N = 148)
| Gene/Haplotype | Huye (n = 47) | Kamonyi (n = 101) | Total, n (%) | ||||
|---|---|---|---|---|---|---|---|
| Wildtype, n (%) | Mutant, n (%) | Mixed, n (%) | Wildtype, n (%) | Mutant, n (%) | Mixed, n (%) | ||
| pfdhfr | |||||||
| 51 | 2 (4) | 44 (96) | 0 (0) | 2 (2) | 97 (97) | 0 (0) | 141 (97) |
| 59 | 5 (11) | 39 (83) | 3 (6) | 6 (6) | 93 (92) | 2 (2) | 137 (93) |
| 108 | 1 (2) | 46 (98) | 0 (0) | 0 (0) | 101 (100) | 0 (0) | 147 (99) |
| 164 | 43 (100) | 0 (0) | 0 (0) | 88 (100) | 0 (0) | 0 (0) | 0 |
| pfdhps | |||||||
| 436 | 11 (26) | 0 (0) | 32 (74) | 29 (31) | 0 (0) | 64 (68) | 96 (71) |
| 437 | 4 (9) | 39 (91) | 0 (4) | 10 (11) | 80 (86) | 3 (3) | 122 (90) |
| 540 | 4 (9) | 39 (91) | 0 (4) | 10 (11) | 82 (87) | 2 (2) | 123 (90) |
| 581 | 27 (69) | 8 (21) | 4 (10) | 52 (59) | 27 (31) | 9 (10) | 48 (38) |
Percentage of mutant (mixed and mutant) alleles
| Gene/Haplotype | Codons | Huye, n (%) | Kamonyi, n (%) | Pearson’s χ2 test | P value |
|---|---|---|---|---|---|
| pfdhfr | 51I | 44 (96) | 97 (98) | 0.418 | 0.680 |
| 59R | 42 (89) | 95 (94) | 1.029 | 0.327 | |
| 108N | 46 (98) | 101 (100) | 2.164 | 0.318 | |
| 164 L | ND | ND | – | – | |
| 436A | 32 (74) | 64 (69) | 0.313 | 0.576 | |
| pfdhps | 437G | 39 (91) | 83 (89) | 0.014 | 0.905 |
| 540E | 39 (91) | 84 (89) | 0.001 | 0.977 | |
| 581G | 12 (31) | 36 (41) | 1.497 | 0.221 | |
| 613S | 2 (5) | 2 (2) | 0.631 | 0.592 | |
| Triple pfdhfr (51I + 59R + 108N) | – | 40 (85) | 91 (90) | 0.786 | 0.375 |
| Double pfdhps (437G + 540E) | – | 39 (83) | 83 (82) | 0.14 | 0.905 |
| Quintuple mutant | – | 35 (74) | 76 (75) | 0.10 | 0.919 |
| Sextuple mutant | – | 11 (23) | 30 (30) | 0.635 | 0.425 |
| pfdhfr | 51I | 44 (96) | 97 (98) | 0.418 | 0.680 |
ND = not detected.
pfdhfr gene.
The prevalence of mutant alleles (pure mutant and mixed) in placental samples for N51I, C59R, and S108N was 97%, 93%, and 99%, respectively (Table 1). All samples were found to carry the wildtype allele at pfdhfr I164L. The prevalence of the pfdhfr triple (51 + 59 + 108) haplotype was 89% in the Huye District and 90% in the Kamonyi District; however, the distribution of haplotype combinations was comparable across the two sites (Table 2).
Haplotype combinations.
The prevalence of quintuple (pfdhfr 51 + 59 + 108/pfdhps 437 + 540) and sextuple (pfdhfr 51 + 59 + 108/pfdhps 437 + 540 + 581) mutant genotypes in placental samples was 75% and 28%, respectively. The distribution of mutant alleles and haplotype combinations were comparable across both Huye and Kamonyi districts (Table 2). Among monoclonal infections, the prevalence of quintuple mutants and sextuple mutants was 78 of 113 (69%) and 29 of 113 (26%), respectively, whereas for polyclonal infections, the quintuple and sextuple mutant prevalence was 27 of 35 (77%) and 11 of 35 (31%), respectively.
Concordance of placental and peripheral blood samples for both pfdhfr and pfdhps drug-resistant genotypes.
Matched peripheral-to-placental blood detection of drug-resistant mutants were genotyped for each locus in the pfdhfr and pfdhps genes. The observed agreements for the four loci in pfdhfr (51 + 59 + 108 + 164) were 207 of 208 (99%), and for the five loci in pfdhps (436 + 437 + 540 + 581 + 613) were 239 of 260 (92%) (Table 3). The peripheral blood sample detected all pfdhfr and all pfdhps drug-resistant mutants correctly except at the S436 loci. Four SNPs (pfdhfr N51I, C59R, and I164L, and pfdhps A613S) exhibited perfect agreement. The kappa values for pfdhfr S108N and pfdhps S436, A437G, K540E, and A581G were 0.60, 0.38, 0.91, 0.81 and 0.80, respectively. The overall degree of agreement between placental and peripheral blood in classifying SNPs associated with sulfadoxine and pyrimethamine was strong (κ = 0.83).
Placental and peripheral blood agreement on drug resistance genotyping
| Polymorphism | Capillary | Placental, n | Observed agreement, n/N (%) | Kappa value | |||
|---|---|---|---|---|---|---|---|
| Mutant | Wildtype | Mixed | Total | ||||
| pfdhfr 51 | Mutant | 50 | 0 | 0 | 50 | – | – |
| Wildtype | 0 | 1 | 0 | 1 | – | – | |
| Mixed | 0 | 0 | 0 | 0 | – | – | |
| Total | 50 | 1 | 0 | 51 | 51/51(100) | 1 | |
| pfdhfr 59 | Mutant | 49 | 0 | 0 | 49 | – | – |
| Wildtype | 0 | 5 | 0 | 5 | – | – | |
| Mixed | 0 | 0 | 1 | 1 | – | – | |
| Total | 49 | 5 | 1 | 55 | 55/55 (100) | 1 | |
| pfdhfr 108 | Mutant | 53 | 0 | 0 | 53 | – | – |
| Wildtype | 0 | 1 | 0 | 1 | – | – | |
| Mixed | 1 | 0 | 0 | 1 | – | – | |
| Total | 54 | 1 | 0 | 55 | 54/55 (98) | 0.66 | |
| pfdhfr 164 | Mutant | 0 | 0 | 0 | 0 | – | – |
| Wildtype | 0 | 47 | 0 | 47 | – | – | |
| Mixed | 0 | 0 | 0 | 0 | – | – | |
| Total | 0 | 47 | 0 | 47 | 47/47 (100) | 1 | |
| pfdps 436 | Mutant | 0 | 0 | 0 | 0 | – | – |
| Wildtype | 0 | 8 | 7 | 15 | – | – | |
| Mixed | 0 | 6 | 31 | 37 | – | – | |
| Total | 0 | 14 | 38 | 52 | 39/52 (75) | 0.32 | |
| pfdps 437 | Mutant | 45 | 1 | 0 | 46 | – | – |
| Wildtype | 0 | 6 | 0 | 6 | – | – | |
| Mixed | 0 | 0 | 0 | 0 | – | – | |
| Total | 45 | 7 | 0 | 52 | 51/52 (98) | 0.91 | |
| pfdps 540 | Mutant | 47 | 1 | 0 | 48 | – | – |
| Wildtype | 0 | 5 | 0 | 5 | – | – | |
| Mixed | 0 | 1 | 0 | 1 | – | – | |
| Total | 47 | 7 | 0 | 54 | 52/54 (96) | 0.81 | |
| pfdps 581 | Mutant | 11 | 1 | 0 | 12 | – | – |
| Wildtype | 0 | 35 | 1 | 36 | – | – | |
| Mixed | 1 | 2 | 2 | 5 | – | – | |
| Total | 12 | 38 | 3 | 53 | 48/53 (91) | 0.75 | |
| pfdps 613 | Mutant | 0 | 0 | 0 | 0 | – | – |
| Wildtype | 0 | 49 | 0 | 49 | – | – | |
| Mixed | 0 | 0 | 0 | 0 | – | – | |
| Total | 0 | 49 | 0 | 49 | 49/49 (100) | 1 | |
DISCUSSION
Despite transitioning from SP + AQ to AL as the first-line treatment in 2006, and discontinuing IPTp-SP in 2008, there continued to be a relatively high proportion of both quintuple (75%) and sextuple (28%) mutants in samples collected in southern Rwanda from 2016 to 2018, despite the limitation of collection 5 to 7 years ago.2 Overall, the prevalence of pfdhps A581G was 38%. Data from neighboring Uganda highlight the focal nature of the A581G mutant, as prevalence varied from 5% to 46% in 2018 and 2019, depending on the province.15 Although the prevalence of the quintuple mutant in our study, conducted in 2016 to 2018, is similar to that reported from Rwanda’s Ruhuha (Eastern Province) and Mubuga (Western Province) in 2015 (74%), the prevalence of the sextuple mutant genotype in our sample was greater (28% compared with 18%), with a corresponding greater prevalence of pfdhps A581G (from 24% in the 2015 study to 38% in our sample).2 Because resistance markers may be highly focal, the geographic difference in study sites may account for the difference in the prevalence between our findings and the previous study from Rwanda.2 Thus, it is not possible to state that this is definitive evidence of increasing resistance over time. However, older data from the Democratic Republic of the Congo, which shares a border with Rwanda, demonstrated an increase in pfdhps K540E/A581G (not defined as a sextuple mutant) from 2% in 2007 to 18% in 2013, suggesting that the increase in Rwanda may be real.16 The sextuple mutant has been associated with increased treatment failure, as well as delivery of low-birthweight infants among women taking IPTp with SP.17,18 A recent, large clinical trial with nearly 1,500 participants in each arm compared SP to dihydroartemisinin–piperaquine alone or combined with a single dose of azithromycin in settings with 20% to 30% prevalence of the sextuple mutant. Despite the relatively high prevalence of the sextuple mutant, adverse pregnancy outcomes were reported more frequently in the groups receiving dihydroartemisinin–piperaquine.10
Recovery of wildtype (susceptible) alleles after withdrawal of antimalarial drugs has been reported in several countries, although timelines have varied.2,5 For instance, high rates of recovery of CQ susceptibility have been reported in Malawi, where parasites reverted to wildtype at the pfcrt K76T codon responsible for CQ resistance 12 years after cessation of CQ use.5 In Rwanda, a slower recovery rate has been reported, with the prevalence of mutant pfcrt K76T declining from 74% in 2010 to 49% in 2015, suggesting an approximate 25% recovery of wildtype pfcrt K76T after a decade of CQ withdrawal.2,19 For SP, we observed a high level (> 92%) of pfdhfr N51I, C59R, and S108N and more than 89% of pfdhps A437G and K540E, which is similar or greater than reported previously in Rwanda.2 This demonstrates the persistence of high-level SP resistance even years after SP withdrawal, similar to what has been reported in Uganda and Tanzania, although a decrease in pfdhfr and pfdhps mutant alleles were reported in Ethiopia after SP withdrawal.20–22 The persistence of high levels of SP resistance in Rwanda even in the absence of SP pressure for almost a decade may be a result of gene flow from neighboring countries or the continued use of SP or other antifolate inhibitors such trimethoprim and sulfamethoxazole, which may have cross-resistance with SP.23 Alternatively, the SP mutations may not have the same fitness cost as those for CQ, and there may thus be less pressure to revert.
We found a high level of agreement between placental and peripheral blood SP drug-resistant mutants. Importantly, testing of peripheral blood did not miss mutant SP P. falciparum parasites for pfdhfr and all but one of the pfdhps genes, suggesting that maternal peripheral blood would be sufficient and provide a good measurement for the prevalence of SP resistance markers in pregnant women. A small study in Gabon24 saw greater than 90% agreement in SP drug-resistant mutations from 22 matched samples collected in 2005 and 2006. A similar study in Ghana25 with 294 matched pairs collected in 2000 and 2001 indicated 83% agreement between peripheral and placental blood samples that were influenced by parasite density, number of mutations, and fever. The agreement increases inversely with the number of alleles, such that the multiallelic MSP2 indicated less agreement between placental and peripheral samples.26
CONCLUSION
Our study demonstrated a high level of focal SP resistance, with a 23% and 30% prevalence of the sextuple mutant in the Huye and Kamonyi districts, respectively, despite nearly a decade of a presumed absence of SP pressure. At this level of resistance, the efficacy of SP for prevention of low birthweight is likely compromised. As drug resistance can be quite focal, resistance testing would be indicated prior to implementing any SP-containing preventive strategy in Rwanda.
ACKNOWLEDGMENTS
We thank Worod Allak at the Becton Dickinson Immunology and Flow Cytometry Core at Johns Hopkins Bloomberg School of Public Health for training, support, and technical assistance using the MagPix. The authors confirm that all ongoing and related trials for this drug/intervention are registered (#NCT03508349). This trial is registered at ClinicalTrials.gov (#NCT03508349, https://clinicaltrials.gov/ct2/show/NCT03508349).
REFERENCES
- 1.↑
Global Malaria Programme , 2022. World Malaria Report 2022. Geneva, Switzerland: World Health Organization.
- 2.↑
Kateera F, Nsobya SL, Tukwasibwe S, Mens PF, Hakizimana E, Grobusch MP, Mutesa L, Kumar N, Van Vugt M, 2016. Malaria case clinical profiles and Plasmodium falciparum parasite genetic diversity: a cross sectional survey at two sites of different malaria transmission intensities in Rwanda. Malar J 15: 237.
- 3.↑
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.
- 4.↑
Triglia T, Cowman AF, 1999. The mechanism of resistance to sulfa drugs in Plasmodium falciparum. Drug Resist Updat 2: 15–19.
- 5.↑
Laufer MK, Thesing PC, Eddington ND, Masonga R, Dzinjalamala FK, Takala SL, Taylor TE, Plowe CV, 2006. Return of chloroquine antimalarial efficacy in Malawi. N Engl J Med 355: 1959–1966.
- 6.↑
Flegg JA, Humphreys GS, Montanez B, Strickland T, Jacome-Meza ZJ, Barnes KI, Raman J, Guerin PJ, Hopkins Sibley C, Dahlström Otienoburu S, 2022. Spatiotemporal spread of Plasmodium falciparum mutations for resistance to sulfadoxine–pyrimethamine across Africa, 1990–2020. PLOS Comput Biol 18: e1010317.
- 7.↑
Venkatesan M, Alifrangis M, Roper C, Plowe CV, 2013. Monitoring antifolate resistance in intermittent preventive therapy for malaria. Trends Parasitol 29: 497–504.
- 8.↑
Van Eijk AM et al., 2019. Effect of Plasmodium falciparum sulfadoxine–pyrimethamine resistance on the effectiveness of intermittent preventive therapy for malaria in pregnancy in Africa: a systematic review and meta-analysis. Lancet Infect Dis 19: 546–556.
- 9.↑
Roh ME et al., 2020. Overall, anti-malarial, and non-malarial effect of intermittent preventive treatment during pregnancy with sulfadoxine–pyrimethamine on birthweight: a mediation analysis. Lancet Glob Health 8: e942–e953.
- 10.↑
Madanitsa M et al., 2023. Effect of monthly intermittent preventive treatment with dihydroartemisinin–piperaquine with and without azithromycin versus monthly sulfadoxine–pyrimethamine on adverse pregnancy outcomes in Africa: a double-blind randomised, partly placebo-controlled trial. Lancet 401: 1020–1036.
- 11.↑
Gutman J, van Eijk A, Rodriguez E, Ahn J, ter Kuile F, 2022. Sulfadoxine–Pyrimethamine Resistance and Intermittent Preventive Treatment in Pregnancy (IPTp) for the Prevention of Malaria in Pregnancy: A Systematic Review and Meta-analysis. Available at: zenodo.org/record/6559908. Accessed August 23, 2023.
- 12.↑
U.S. President’s Malaria Initiative [Rwanda] , Malaria Operational Plan FY 2022. Available at: www.pmi.gov. Accessed August 30, 2023.
- 13.↑
Uwimana A et al., 2023. Effectiveness of intermittent screening and treatment of malaria in pregnancy on maternal and birth outcomes in selected districts in Rwanda: a cluster randomized controlled trial. Clin Infect Dis 77: 127–134.
- 14.↑
LeClair NP, Conrad MD, Baliraine FN, Nsanzabana C, Nsobya SL, Rosenthal PJ, 2013. Optimization of a ligase detection reaction–fluorescent microsphere assay for characterization of resistance-mediating polymorphisms in African samples of Plasmodium falciparum. J Clin Microbiol 51: 2564–2570.
- 15.↑
Asua V et al., 2021. Changing prevalence of potential mediators of aminoquinoline, antifolate, and artemisinin resistance across Uganda. J Infect Dis 223: 985–994.
- 16.↑
Deutsch-Feldman M et al., 2019. The changing landscape of Plasmodium falciparum drug resistance in the Democratic Republic of Congo. BMC Infect Dis 19: 872.
- 17.↑
Hansson H et al., 2021. Reduced birth weight caused by sextuple drug-resistant Plasmodium falciparum infection in early second trimester. J Infect Dis 224: 1605–1613.
- 18.↑
Karema C, Imwong M, Fanello CI, Stepniewska K, Uwimana A, Nakeesathit S, Dondorp A, Day NP, White NJ, 2010. Molecular correlates of high-level antifolate resistance in Rwandan children with Plasmodium falciparum malaria. Antimicrob Agents Chemother 54: 477–483.
- 19.↑
Zeile I, Gahutu J-B, Shyirambere C, Steininger C, Musemakweri A, Sebahungu F, Karema C, Harms T, Eggelte TA, Mockenhaupt FP, 2012. Molecular markers of Plasmodium falciparum drug resistance in southern highland Rwanda. Acta Trop 121: 50–54.
- 20.↑
Mbogo GW et al., 2014. Temporal changes in prevalence of molecular markers mediating antimalarial drug resistance in a high malaria transmission setting in Uganda. Am J Trop Med Hyg 91: 54–61.
- 21.↑
Matondo SI, Temba GS, Kavishe AA, Kauki JS, Kalinga A, Van Zwetselaar M, Reyburn H, Kavishe RA, 2014. High levels of sulphadoxine–pyrimethamine resistance Pfdhfr-Pfdhps quintuple mutations: a cross sectional survey of six regions in Tanzania. Malar J 13: 152.
- 22.↑
Hailemeskel E, Kassa M, Taddesse G, Mohammed H, Woyessa A, Tasew G, Sleshi M, Kebede A, Petros B, 2013. Prevalence of sulfadoxine–pyrimethamine resistance-associated mutations in dhfr and dhps genes of Plasmodium falciparum three years after SP withdrawal in Bahir Dar, Northwest Ethiopia. Acta Trop 128: 636–641.
- 23.↑
Iyer JK, Milhous WK, Cortese JF, Kublin JG, Plowe CV, 2001. Plasmodium falciparum cross-resistance between trimethoprim and pyrimethamine. Lancet 358: 1066–1067.
- 24.↑
Bouyou-Akotet MK, Mawili-Mboumba DP, Tchantchou TdeD, Kombila M, 2010. High prevalence of sulfadoxine/pyrimethamine-resistant alleles of Plasmodium falciparum isolates in pregnant women at the time of introduction of intermittent preventive treatment with sulfadoxine/pyrimethamine in Gabon. J Antimicrob Chemother 65: 438–441.
- 25.↑
Mockenhaupt FP, Bedu-Addo G, Junge C, Hommerich L, Eggelte TA, Bienzle U, 2007. Markers of sulfadoxine–pyrimethamine-resistant Plasmodium falciparum in placenta and circulation of pregnant women. Antimicrob Agents Chemother 51: 332–334.
- 26.↑
Kamwendo DD, Dzinjalamala FK, Snounou G, Kanjala MC, Mhango CG, Molyneux ME, Rogerson SJ, 2002. Plasmodium falciparum: PCR detection and genotyping of isolates from peripheral, placental, and cord blood of pregnant Malawian women and their infants. Trans R Soc Trop Med Hyg 96: 145–149.