title: Evidence of triple mutantPfdhps ISGNGA haplotype inPlasmodium falciparumisolates from North-east India: An analysis of sulfadoxine resistant haplotype selection.
abstract: BACKGROUND
  North-east region of India has consistent role in the spread of multi drug resistantPlasmodium (P.) falciparumto other parts of Southeast Asia. After rapid clinical treatment failure of Artemisinin based combination therapy-Sulphadoxine/Pyrimethamine (ACT-SP) chemoprophylaxis, Artemether-Lumefantrine (ACT-AL) combination therapy was introduced in the year 2012 in this region for the treatment of uncomplicatedP. falciparummalaria. In a DNA sequencing based polymorphism analysis, seven codons ofP. falciparumdihydropteroate synthetase (Pfdhps) gene were screened in a total of 127P. falciparumisolates collected from Assam, Arunachal Pradesh and Tripura of North-east India during the year 2014 and 2015 to document current sulfadoxine resistant haplotypes.
  
  
  METHODS
  Sequences were analyzed to rearrange both nucleotide and protein haplotypes. Molecular diversity indices were analyzed in DNA Sequence Polymorphism software (DnaSP) on the basis ofPfdhps gene sequences. Disappearance from selective neutrality was assessed based on the ratio of non-synonomous to synonomous nucleotide substitutions [dN/dS ratio]. Moreover, two-tailed Z test was performed in search of the significance for probability of rejecting null hypothesis of strict neutrality [dN = dS]. Presence of mutantP. falciparummultidrug resistance protein1 (Pfmdr1) was also checked in those isolates that were present with newPfdhps haplotypes. Phylogenetic relationship based onPfdhps gene was reconstructed in Molecular Evolutionary Genetics Analysis (MEGA).
  
  
  RESULTS
  Among eight different sulfadoxine resistant haplotypes found, ISGNGA haplotype was documented in a total of five isolates from Tripura with association of a new mutant M538Rallele. Sequence analysis ofPfmdr1 gene in these five isolates came to notice that not all but only one isolate was mutant at codon 86 (N86Y;YYSND) in the multidrug resistance protein. Molecular diversity based onPfdhps haplotypes revealed thatP. falciparumpopulations in Assam and Tripura were under balancing selection for sulfadoxine resistant haplotypes but population from Arunachal Pradesh was under positive selection with comparatively high haplotype diversity (h= 0.870). In reconstructed phylogenetic analysis, isolates having ISGNGA haplotype were grouped into two separate sub-clusters from the other isolates based on their genetic distances and diversities.
  
  
  CONCLUSIONS
  This study suggests that sulfadoxine resistant isolates are still migrating from its epicenter to the other parts of Southeast Asia and hence control and elimination of the drug resistant isolates have become impedimental. Moreover,P. falciparumpopulations in different areas may undergo selection of particular sulfadoxine resistant haplotypes either in the presence of drug or after its removal to maintain their plasticity.
authors.affiliation: Department of Bioengineering & Technology, Gauhati University Institute of Science and Technology (GUIST), Gauhati University, Gopinath Bordoloi Nagar, Assam 786014, India.
authors.forename: Manuj K
authors.initials: MK
authors.lastname: Das
authors.affiliation: Division of Entomology and Filariasis, Regional Medical Research Centre (RMRC), North East Region (ICMR), Post Box No.-105, Dibrugarh, Assam 786001, India.
authors.forename: Sumi
authors.initials: S
authors.lastname: Chetry
authors.affiliation: Department of Biotechnology, Gauhati University, Gopinath Bordoloi Nagar, Assam 786014, India.
authors.forename: Mohan C
authors.initials: MC
authors.lastname: Kalita
authors.affiliation: Division of Entomology and Filariasis, Regional Medical Research Centre (RMRC), North East Region (ICMR), Post Box No.-105, Dibrugarh, Assam 786001, India.
authors.forename: Prafulla
authors.initials: P
authors.lastname: Dutta
keywords: Drug resistance; ISGNGA haplotype; North-east India; Pfdhps; Plasmodium falciparum