Evidence of triple mutant Pf dhps IS GNG A haplotype in Plasmodium falciparum isolates 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 resistant Plasmodium (P.) falciparum to 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 uncomplicated P. falciparum malaria. In a DNA sequencing based polymorphism analysis, seven codons of P. falciparum dihydropteroate synthetase (Pfdhps) gene were screened in a total of 127 P. falciparum isolates collected from Assam, Arunachal Pradesh and Tripura of North-east India during the year 2014 and 2015 to document current sulfadoxine resistant haplotypes.

Materials and 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 of Pfdhps 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 mutant P. falciparum multidrug resistance protein1 (Pfmdr1) was also checked in those isolates that were present with new Pfdhps haplotypes. Phylogenetic relationship based on Pfdhps gene was reconstructed in Molecular Evolutionary Genetics Analysis (MEGA).

Results: Among eight different sulfadoxine resistant haplotypes found, IS GNG A haplotype was documented in a total of five isolates from Tripura with association of a new mutant M538 R allele. Sequence analysis of Pfmdr1 gene in these five isolates came to notice that not all but only one isolate was mutant at codon 86 (N86 Y ; Y YSND) in the multidrug resistance protein. Molecular diversity based on Pfdhps haplotypes revealed that P. falciparum populations 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 IS GNG A haplotype were grouped into two separate sub-clusters from the other isolates based on their genetic distances and diversities.

Conclusion: 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. falciparum populations 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.

Keywords: Drug resistance; ISGNGA haplotype; North-east India; Pfdhps; Plasmodium falciparum.

Fig. 1

Different alleles of Pfdhps gene and their prevalence.

Fig. 2

Representation of Pfdhps polymorphisms in 127 Northeastern isolates from Assam, Arunachal Pradesh and Tripura. Nucleotide sequences and respective amino acid codons with alleles are given according to P. falciparum 3D7 isolate sequence (GenBank accession No. XM_001349382). Sample location (numbers are given in parentheses) are abbreviated as ASM-Assam; AP-Arunachal Pradesh; TRP-Tripura. * New allele found in Tripura and #Synonymous mutation found in Tripura.

Fig. 3

Pfdhps wild and mutant protein haplotypes and their prevalence.

Fig. 4

Reconstructed Neighbour-Joining tree based on Pfdhps sequences of parasite population. Each sequence was represented after 1000 replication (bootstrap value = 1000). GenBank accession numbers of submitted sequences are given after the parasite ID number.

Fig. 5

Demographic map representing three states of North-east India and number of isolates collected from each sites.