title: High-level Plasmodium falciparum sulfadoxine-pyrimethamine resistance with the concomitant occurrence of septuple haplotype in Tanzania.
abstract: BACKGROUND
  Tanzania abandoned sulfadoxine-pyrimethamine (SP) as the first-line treatment for uncomplicated malaria in 2006 due to high levels Plasmodium falciparum resistance. However, SP is still being used for intermittent preventive treatment during pregnancy (IPTp-SP). This study aimed to assess the pattern of P. falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) mutations and associated haplotypes in areas with different malaria transmission intensities in mainland Tanzania, 6 years after withdrawal of SP as a first-line treatment regimen for uncomplicated malaria.
  
  
  METHODS
  A total of 264 samples were collected during cross-sectional surveys in three districts of Muheza, Muleba and Nachingwea in Tanga, Kagera and Lindi regions, respectively. Parasite genomic DNA was extracted from P. falciparum positive samples. The Pfdhfr, Pfdhps single nucleotide polymorphisms (SNPs) were amplified using nested polymerase chain reaction and detected by sequence specific oligonucleotide probe-enzyme linked immunosorbent assay (SSOP-ELISA).
  
  
  RESULTS
  The prevalence of the mutant Pfdhfr-Pfdhps haplotypes was heterogenous and transmission dependent. The triple Pfdhfr mutant haplotypes (CIRNI) were predominant in all sites with significantly higher frequencies at Muheza (93.3 %) compared to Muleba (75.0 %) and Nachingwea districts (70.6 %), (p < 0.001). Overall, the prevalence of the wild-type Pfdhps (SAKAA) haplotype was lowest at Muheza (1.3 %), (p = 0.002). Double Pfdhps haplotype SGEAA was significantly high at Muheza (27.2 %) and Muleba (20.8 %) while none (0 %) was detected at Nachingwea (p < 0.001). The prevalence of triple Pfdhps SGEGA haplotype was significantly higher at Muheza compared to Muleba and Nachingwea (p < 0.001). In contrast, Nachingwea and Muleba had significantly higher prevalence of another triple Pfdhps AGEAA haplotype (χ(2) = 39.9, p < 0.001). Conversely, Pfdhfr-Pfdhps as quintuple and sextuple haplotypes were predominant including the emergence of a septuple mutant haplotype CIRNI-AGEGA (n = 11) observed at Muheza and Muleba.
  
  
  CONCLUSIONS
  These results ascertain the high prevalence and saturation of Pfdhfr and Pfdhps haplotypes conferring SP resistance in areas with changing malaria epidemiology; and this could undermine the use of IPTp-SP in improving pregnancy outcomes. In these settings where high level SP resistance is documented, additional control efforts are needed and evaluation of an alternative drug for IPTp is an urgent priority.
authors.affiliation: National Institute for Medical Research, Tanga Research Centre, P. O. Box 5004, Tanga, United Republic of Tanzania. vitobaraka@gmail.com.|International Health Unit, Department of Epidemiology, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, 2610, Wilrijk, Belgium. vitobaraka@gmail.com.
authors.forename: Vito
authors.initials: V
authors.lastname: Baraka
authors.affiliation: National Institute for Medical Research, Tanga Research Centre, P. O. Box 5004, Tanga, United Republic of Tanzania. deusishe@yahoo.com.
authors.forename: Deus S
authors.initials: DS
authors.lastname: Ishengoma
authors.affiliation: National Institute for Medical Research, Tanga Research Centre, P. O. Box 5004, Tanga, United Republic of Tanzania. ffrancis8@gmail.com.
authors.forename: Filbert
authors.initials: F
authors.lastname: Fransis
authors.affiliation: National Institute for Medical Research, Tanga Research Centre, P. O. Box 5004, Tanga, United Republic of Tanzania. minjartd@gmail.com.
authors.forename: Daniel T R
authors.initials: DT
authors.lastname: Minja
authors.affiliation: National Institute for Medical Research, Tanga Research Centre, P. O. Box 5004, Tanga, United Republic of Tanzania. ramadebe11@gmail.com.
authors.forename: Rashid A
authors.initials: RA
authors.lastname: Madebe
authors.affiliation: Tanzania Food and Drugs Authority, P: O. Box 77150, Dar es Salaam, United Republic of Tanzania. degrate79@gmail.com.
authors.forename: Deogratius
authors.initials: D
authors.lastname: Ngatunga
authors.affiliation: International Health Unit, Department of Epidemiology, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, 2610, Wilrijk, Belgium. jean-pierre.vangeertruyden@uantwerpen.be.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Van Geertruyden
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D015415:Biomarkers / Q000032:analysis; D003430:Cross-Sectional Studies; D004094:Dihydropteroate Synthase / Q000235:genetics* / Q000378:metabolism; D004338:Drug Combinations; D004351:Drug Resistance; D004797:Enzyme-Linked Immunosorbent Assay; D006239:Haplotypes; D009154:Mutation; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011739:Pyrimethamine / Q000494:pharmacology*; D013413:Sulfadoxine / Q000494:pharmacology*; D013636:Tanzania; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors
chemical_list: D000962:Antimalarials; D015415:Biomarkers; D004338:Drug Combinations; D015800:Protozoan Proteins; C001205:fanasil, pyrimethamine drug combination; D013413:Sulfadoxine; D013762:Tetrahydrofolate Dehydrogenase; D004094:Dihydropteroate Synthase; D011739:Pyrimethamine