title: Molecular surveillance of Plasmodium falciparum drug resistance markers reveals partial recovery of chloroquine susceptibility but sustained sulfadoxine-pyrimethamine resistance at two sites of different malaria transmission intensities in Rwanda.
abstract: Faced with intense levels of chloroquine (CQ) resistance in Plasmodium falciparum malaria, Rwanda replaced CQ with amodiaquine (AQ)+sulfadoxine-pyrimethamine (SP) in 2001, and subsequently with artemether-lumefantrine (AL) in 2006, as first-line treatments for uncomplicated malaria. Following years of discontinuation of CQ use, re-emergence of CQ-susceptible parasites has been reported in countries including Malawi, Kenya and Tanzania. In contrast, high levels of SP resistant mutant parasites continue to be reported even in countries of presumed reduced SP drug selection pressure. The prevalence and distributions of genetic polymorphisms linked with CQ and SP resistance at two sites of different malaria transmission intensities are described here to better understand drug-related genomic adaptations over time and exposure to varying drug pressures in Rwanda. Using filter paper blood isolates collected from P. falciparum infected patients, DNA was extracted and a nested PCR performed to identify resistance-mediating polymorphisms in the pfcrt, pfmdr1, pfdhps and pfdhfr genes. Amplicons from a total of 399 genotyped samples were analysed by ligase detection reaction fluorescent microsphere assay. CQ susceptible pfcrt 76K and pfmdr1 86N wild-type parasites were found in about 50% and 81% of isolates, respectively. Concurrently, SP susceptible pfdhps double (437G-540E), pfdhfr triple (108N-51I-59R), quintuple pfdhps 437G-540E/pfdhfr 51I-59R-108N and sextuple haplotypes were found in about 84%, 85%, 74% and 18% of isolates, respectively. High-level SP resistance associated pfdhfr 164L and pfdhps 581G mutant prevalences were noted to decline. Mutations pfcrt 76T, pfdhfr 59R and pfdhfr 164L were found differentially distributed between the two study sites with the pfdhfr 164L mutants found only at Ruhuha site, eastern Rwanda. Overall, sustained intense levels of SP resistance mutations and a recovery of CQ susceptible parasites were found in this study following 7 years and 14 years of drug withdrawal from use, respectively. Most likely, the sustained high prevalence of resistant parasites is due to the use of DHFR/DHPS inhibitors like trimethoprim-sulfamethoxazole (TS) for the treatment of and prophylaxis against bacterial infections among HIV infected individuals as well as the continued use of IPTp-SP within the East and Central African regions for malaria prevention among pregnant women. With regard to CQ, the slow recovery of CQ susceptible parasites may have been caused partly by the continued use of CQ and/or CQ mimicking antimalarial drugs like AQ in spite of policies to withdraw it from Rwanda and the neighbouring countries of Uganda and Tanzania. Continued surveillance of P. falciparum CQ and SP associated polymorphisms is recommended for guiding future rational drug policy-making and mitigation of future risk of anti-malaria drug resistance development.
authors.affiliation: Medical Research Centre Division, Rwanda Biomedical Centre, PO Box 7162, Kigali, Rwanda; Centre of Tropical Medicine and Travel Medicine, Department of Infectious Diseases, Division of Internal Medicine, Meibergdreef 9, 1100 DD, Amsterdam, The Netherlands. Electronic address: fkkateera@yahoo.com.
authors.forename: Fredrick
authors.initials: F
authors.lastname: Kateera
authors.affiliation: Molecular Research Laboratory, Infectious Disease Research Collaboration (IDRC), New Mulago Hospital Complex, PO Box 7051, Kampala, Uganda; School of Biomedical Science, College of Medicine, Makerere University, Uganda. Electronic address: snsobya@idrc-uganda.org.
authors.forename: Sam L
authors.initials: SL
authors.lastname: Nsobya
authors.affiliation: Molecular Research Laboratory, Infectious Disease Research Collaboration (IDRC), New Mulago Hospital Complex, PO Box 7051, Kampala, Uganda. Electronic address: stephentukwasibwe@yahoo.com.
authors.forename: Steven
authors.initials: S
authors.lastname: Tukwasibwe
authors.affiliation: Medical Research Centre Division, Rwanda Biomedical Centre, PO Box 7162, Kigali, Rwanda; Malaria & Other Parasitic Diseases Division, Rwanda Biomedical Centre, Kigali, Rwanda. Electronic address: ehakizimana@gmail.com.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Hakizimana
authors.affiliation: College of Medicine & Health Sciences, University of Rwanda, PO Box 3286, Kigali, Rwanda. Electronic address: lmutesa@gmail.com.
authors.forename: Leon
authors.initials: L
authors.lastname: Mutesa
authors.affiliation: Centre of Tropical Medicine and Travel Medicine, Department of Infectious Diseases, Division of Internal Medicine, Meibergdreef 9, 1100 DD, Amsterdam, The Netherlands; Royal Tropical Institute/Koninklijk Instituutvoor de Tropen, KIT Biomedical Research, Meibergdreef 39, 1105 AZ, Amsterdam, The Netherlands. Electronic address: p.mens@kit.nl.
authors.forename: Petra F
authors.initials: PF
authors.lastname: Mens
authors.affiliation: Centre of Tropical Medicine and Travel Medicine, Department of Infectious Diseases, Division of Internal Medicine, Meibergdreef 9, 1100 DD, Amsterdam, The Netherlands. Electronic address: m.p.grobusch@amc.uva.nl.
authors.forename: Martin P
authors.initials: MP
authors.lastname: Grobusch
authors.affiliation: Centre of Tropical Medicine and Travel Medicine, Department of Infectious Diseases, Division of Internal Medicine, Meibergdreef 9, 1100 DD, Amsterdam, The Netherlands. Electronic address: m.vanvugt@amc.uva.nl.
authors.forename: Michèle
authors.initials: M
authors.lastname: van Vugt
authors.affiliation: Department of Tropical Medicine, School of Public Health and Tropical Medicine, Vector-Borne Infectious Disease Research Centre, Tulane University, 333S Liberty Street, Mail code 8317, New Orleans, LA 70112, United States. Electronic address: nkumar@tulane.edu.
authors.forename: Nirbhay
authors.initials: N
authors.lastname: Kumar
mesh_terms: D000293:Adolescent; D000655:Amodiaquine / Q000627:therapeutic use; D000962:Antimalarials / Q000494:pharmacology*; D002648:Child; D002675:Child, Preschool; D002738:Chloroquine / Q000494:pharmacology*; D004338:Drug Combinations; D004351:Drug Resistance / Q000235:genetics*; D005260:Female; D005838:Genotype; D006239:Haplotypes; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007630:Kenya; D008288:Malaria / Q000188:drug therapy / Q000469:parasitology / Q000635:transmission; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000469:parasitology / Q000635:transmission; D008295:Malawi; D008297:Male; D010963:Plasmodium falciparum / Q000235:genetics*; D011110:Polymorphism, Genetic; D011739:Pyrimethamine / Q000494:pharmacology*; D012432:Rwanda; D013413:Sulfadoxine / Q000494:pharmacology*; D013636:Tanzania; D014454:Uganda
chemical_list: D000962:Antimalarials; D004338:Drug Combinations; D000655:Amodiaquine; C001205:fanasil, pyrimethamine drug combination; D013413:Sulfadoxine; D002738:Chloroquine; D011739:Pyrimethamine
keywords: Chloroquine; Plasmodium falciparum; Polymorphisms; Resistance; Sulfadoxine-pyrimethamine