|Year : 2016 | Volume
| Issue : 2 | Page : 193-197
Mutational prevalence of chloroquine resistance transporter gene among Plasmodium falciparum field isolates in Assam and Arunachal Pradesh, India
J Sharma, M Soni, P Dutta, SA Khan, J Mahanta
Entomology and Filariasis Division, Regional Medical Research Centre (Indian Council of Medical Research), North East Region, Dibrugarh, Assam, India
|Date of Submission||26-May-2015|
|Date of Acceptance||19-Jan-2016|
|Date of Web Publication||14-Apr-2016|
Entomology and Filariasis Division, Regional Medical Research Centre (Indian Council of Medical Research), North East Region, Dibrugarh, Assam
Source of Support: None, Conflict of Interest: None
Objective: The present study aims to find out the mutational prevalence of Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene in Assam and Arunachal Pradesh, India. Methods: To fulfil the objective of the study, a total of 54 P. falciparum malaria positive samples were attempted for genotyping of Pfcrt gene using polymerase chain reaction (PCR) and direct DNA sequencing method. Results: The K76T mutation was observed in 77.78% cases followed by M74I (61.11%), N75E (61.11%) and C72S (16.67%). Triple mutant allele M74I+N75E+K76T was found in 61.11% P. falciparum field isolates. Double mutant allele C72S+K76T was seen among 16.67% samples. Mutation studies have shown existence of three different haplotypes of which two were associated with chloroquine resistance. The haplotype CVIET was found preponderance and circulated in all four districts. The other haplotype SVMNT was observed only in Karbi Anglong district of Assam. Maximum haplotype diversity was also recorded from Karbi Anglong district of Assam. Conclusion: Our study has confirmed high prevalence of mutant Pfcrt genotypes associated with chloroquine resistance in Assam and Arunachal Pradesh, India.
Keywords: Arunachal Pradesh, Assam, chloroquine, haplotype, Plasmodium falciparum, polymerase chain reaction
|How to cite this article:|
Sharma J, Soni M, Dutta P, Khan S A, Mahanta J. Mutational prevalence of chloroquine resistance transporter gene among Plasmodium falciparum field isolates in Assam and Arunachal Pradesh, India. Indian J Med Microbiol 2016;34:193-7
|How to cite this URL:|
Sharma J, Soni M, Dutta P, Khan S A, Mahanta J. Mutational prevalence of chloroquine resistance transporter gene among Plasmodium falciparum field isolates in Assam and Arunachal Pradesh, India. Indian J Med Microbiol [serial online] 2016 [cited 2018 Jan 17];34:193-7. Available from: http://www.ijmm.org/text.asp?2016/34/2/193/180298
| ~ Introduction|| |
At present, lots of preventative and treatment strategies are developed for reducing the incidence of malaria. However, malaria is still considered as one of the major health problem in developing countries. The emergence of parasite resistance to newly introduced drugs severely limits the armory of available drugs against protozoal pathogens. Several molecular markers are found to be associated with antimalarial resistance. Analyses of these molecular markers can provide significant information about resistance levels of the different antimalarial drugs. The K76T amino acid substitution in the Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) has been shown to be associated with chloroquine (CQ) and amodiaquine resistance. , Mutations at other amino acid codon such as 72, 74, 75 in Pfcrt gene was also detected by many studies in CQ resistance parasite lines. ,,
In India, the CQ resistant P. falciparum malaria was first reported in 1973 from Diphu area of the Karbi Anglong district in Assam.  Gradually it spread to the various parts of the country. ,, Only limited molecular studies in these aspects are available from North Eastern (NE) region of India, especially from Assam and Arunachal Pradesh. Therefore, the endeavour will certainly bring some outcomes that are very much helpful for understanding the changing pattern of drug resistance, distributions of different haplotypes and their diversity, parasite metabolism and their adaptation in regards to drug pressure. Keeping in mind, the study was conducted in Assam and Arunachal Pradesh, India to find out the mutational prevalence of Pfcrt gene among P. falciparum isolates.
| ~ Materials and Methods|| |
Study sites, blood samples and DNA extraction
The study was undertaken from December 2011 to December 2013 in some malaria-endemic areas of Assam and Arunachal Pradesh, the NE region of India. Clinically, suspected malaria cases were eligible for enrolment irrespective of age and sex. Patients who already took antimalarial drugs were not included in our study. Informed consent was obtained from patients or in the case of children from their guardians. Institutional ethical permission was taken from Institutional Ethical Board, Regional Medical Research Centre (Indian Council of Medical Research), Dibrugarh, Assam. Two millilitre of blood samples were collected from suspected malaria patients attended in different primary health centres. Thick and thin blood smears were prepared on a glass slide. Blood films were stained with 10% Giemsa and examined microscopically. Parasite density was assessed by counting asexual parasites in 200 white blood cells on the thick film and then quantified.  Parasite genomic DNA was extracted from blood samples using a QIAamp DNA blood kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions.
A polymerase chain reaction (PCR) method was done to amplify a partial 134 bp portion DNA sequence of Pfcrt gene containing the major single nucleotide polymorphisms related to CQ resistance [Figure 1]. The amplification of Pfcrt gene fragment was performed with 5 μl of DNA into a 45 μl of master mix containing 2.5 mM of MgCl2, 250 μM of each deoxyribonucleoside triphosphate (dATP, dTTP, dGTP and dCTP), 1.2 unit of taq DNA polymerase and 0.3 μM of each primer CRTD1 (TGT GCT CAT GTG TTT AAA CTT) and CRTD2 (CAA AAC TAT AGT TAC CAA TTT TG).  The reaction was settled with an initial hold (95°C/10 min), 40 cycles (94°C/30 s, 48°C/30 s, 72°C/45 s and final extension 65°C/3 min). Products were electrophoresed on 2% agarose gels and visualised under Gel documentation system (Kodak). Amplified products were further purified by using PCR Mini spin columns purification kit (Millipore Corporation). The quantity of purified PCR products was quantified in NanoDrop and depending upon the concentration of PCR products; samples were sent for sequencing in both directions (South Korea through Anshul Biotechnologies, Hyderabad, India). Sequencing products were further analysed by software DnaSP v. 5.10.01 for detection of point mutation, haplotype diversity (Hd), neutrality test and other parameters.
|Figure 1: Amplification of 134 bp portion of Pfcrt gene (Lane M represents 50 bp ladder, S1, S2, S3…. represents sample nos.)|
Click here to view
| ~ Results|| |
During the study period, a total 164 clinically suspected malaria patients were enroled from malaria-prone areas of Assam and Arunachal Pradesh. P. falciparum malaria parasite was detected among 35.37% (58/164) cases. The mean age groups among the malaria P. falciparum positive cases was found 25.21 (ranging from 1.6 year to 56 years). Mean parasite count level was found as 4.38% (standard deviation [SD] ±3.60 with a median value of 1.00). The two-tailed P < 0.0001 considered extremely significant having t = 6.209 with 25° of freedom. Low haemoglobin concentration was recorded in 86.21% (50/58) P. falciparum positive cases.
Mutations at the amino acid position from 72 to 76 of Pfcrt gene encoding protein were analysed in 54 samples to find out the drug resistance competency among the P. falciparum isolates in Assam and Arunachal Pradesh. Pfcrt double mutant allele C72S+K76T was found in 16.67% (9/54) samples and triple mutant allele M74I+N75E+K76T was noticed in 61.11% (33/54) P. falciparum field isolates from Assam and Arunachal Pradesh. The prevalence of Pfcrt triple mutant allele M74I+N75E+K76T revealed a high level of CQ resistance in NE region. Depending upon the mutation at amino acid position, atotal of three different Pfcrt haplotypes (CVMNK, SVMNT and CVIET) were identified, of which SVMNT and CVIET were found to be associated with CQ resistance. The haplotype (gene) diversity was (Hd: 0.560), the variance of Hd (0.00314) and SD of Hd (0.056). Maximum Hd was found in the P. falciparum field isolates of Karbi Anglong district in Assam (Hd: 0.692) with a value of SD of Hd 0.027. A total of 5 numbers of polymorphic (segregating) sites, (S: 5) and total number of mutations, (Eta: 5) were observed among the analysed samples of Pfcrt gene [Table 1].
|Table 1: Haplotype analysis for Plasmodium falciparum chloroquine resistance transporter gene among the isolates of Assam and Arunachal Pradesh at district level |
Click here to view
Mutation analysis at nucleotide level showed that at nucleotide position 214-216, TGT-AGT (C72S), at location 220-222, ATG-ATT (M74I), at 223-225 nucleotide positions, AAT-GAA (N75E) and at nucleotide position from 226 to 228, AAA-ACA (K76T) mutation leads to the occurrence of different haplotypes associated with CQ resistance. Nucleotide diversity and average number of nucleotide differences among the Pfcrt genome was found little beat high in the field isolates of Karbi Anglong district of Assam as compared to other study areas. The P. falciparum field isolated in Changlang district had shown no genetic differentiation [Table 2]. This is because all the analysed samples in Changlang district of Arunachal Pradesh had shown M74I + N75E + K76T mutation. A positive significant value of Tajima's D and Fu and Li's F (FLF) was observed among the field isolates of Assam whereas the analysed samples from Arunachal Pradesh had showed the negative significant value of Tajima's D, Fu and Li's D and FLF [Table 2].
|Table 2: Mutation analysis and neutrality test for Plasmodium falciparum chloroquine resistance transporter gene among the isolates of Assam and Arunachal Pradesh at district level |
Click here to view
| ~ Discussion|| |
In the past, CQ had been considered as an effective antimalarial drug in India against malaria. ,, However, after the occurrence of CQ resistance P. falciparum malaria parasite in Assam, the drug policy was changed and Sulphadoxine-Pyrimethamine (SP) combination was introduced by Government of India in 1982. ,, Again, SP resistance P. falciparum malaria cases were detected in Delhi and Assam (Karbi Anglong district) in 1987. ,,,, Accordingly in 2010, Government of India had introduced artemisinin-based combination therapy (ACT) for the treatment of all uncomplicated P. falciparum malaria cases. , However, nowadays, the NE region is facing the problem of reduced susceptibility to the ACT regime also. Keeping this in mind, the National Vector Borne Disease Control Programme (NVBDCP), Government of India has recommended artemether-lumefantrine combination for treatment of P. falciparum malaria cases in NE region in 2013.  This combination therapy is believed to be beneficial for the people of NE states.
Our study has provided significant information on mutational prevalence of Pfcrt gene among P. falciparum isolates in Assam and Arunachal Pradesh, India. In our result, Pfcrt 76T mutation was found in 77.78% cases. This data indicated high prevalence of CQ resistance P. falciparum malaria parasites in NE region of India. It has also shown resemblance with other studies conducted in different part of India as well as in neighboring countries. ,,,,,,,,
Double mutant allele C72S+K76T was found in 16.67% samples. The double mutant haplotype SVMNT was reported only in Karbi Anglong district of Assam [Table 3]. However, such haplotype was not observed from other study areas. According to previous studies, the SVMNT alleles were reported from various parts of the country. ,,,, Triple mutant allele K76T + N75E + M74I was found among 61.11% (33/54) P. falciparum field isolates, whereas no isolates with quadruple mutant C72S + M74I + N75E + K76T were observed during the study period. Triple mutant haplotype CVIET was found to be circulated in Karbi Anglong and Tinsukia district of Assam and Lohit and Changlang district of Arunachal Pradesh [Table 3]. This finding is in accordance with earlier studies conducted in East and South India. ,,,,,, Isolates with single mutant allele CVMNT were detected in many regions in previous consequence, but not reported among the isolates of Assam and Arunachal. 
|Table 3: Distribution of Plasmodium falciparum chloroquine resistance transporter haplotypes in Assam and Arunachal Pradesh |
Click here to view
Negative statistical significant values of Tajima's D were observed among the P. falciparum parasite population of Arunachal Pradesh. It has been suggested that there is an excess of low-frequency polymorphisms relative to an expectation among the parasite population of Arunachal Pradesh. It is an indication of population size expansion and purifying selection. In contrast, the positive statistical significant Tajima's D values observed among the parasite population of Assam have shown to be associated with low level of both low and high-frequency polymorphisms. The population may have suffered a recent bottleneck (or be decreasing) or we may have evidence for overdominant selection at this locus. The P. falciparum parasite population from Changlang district had shown only one type of haplotypes with a value of Hd and nucleotide diversity equal to zero. It may suggest that there is no evidence for any particular pattern of selection at this locus.
During the study period, wild type gene or mutant gene with a lower number of mutations were observed in very less numbers of cases. On the contrary, large numbers of parasite population were observed having higher numbers of mutations in the respective target gene and associated with increasing drug resistance capability. This phenomenon is due to the continuous exposure to drug pressure. Still today, some clinicians in government/private hospitals have provided CQ for the treatment of P. falciparum malaria cases in certain parts of the country. It needs to be released and should be focus on newly introduced ACT for the treatment of P. falciparum malaria cases.
| ~ Conclusion|| |
Our study has shown high prevalence of CQ resistance P. falciparum malaria cases in NE states of India. Continuous molecular surveillance and regular monitoring will help the policy makers for better management of malaria in near future.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, et al.
Mutations in the P. falciparum
digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 2000;6:861-71.
Djimdé A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourté Y, et al.
A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 2001;344:257-63.
Vinayak S, Biswas S, Dev V, Kumar A, Ansari MA, Sharma YD. Prevalence of the K76T mutation in the pfcrt gene of Plasmodium falciparu
m among chloroquine responders in India. Acta Trop 2003;87:287-93.
Sisowath C, Petersen I, Veiga MI, Mårtensson A, Premji Z, Björkman A, et al. In vivo
selection of Plasmodium falciparum
parasites carrying the chloroquine-susceptible pfcrt K76 allele after treatment with artemether-lumefantrine in Africa. J Infect Dis 2009;199:750-7.
Mwai L, Kiara SM, Abdirahman A, Pole L, Rippert A, Diriye A, et al. In vitro
activities of piperaquine, lumefantrine, and dihydroartemisinin in Kenyan Plasmodium falciparum
isolates and polymorphisms in pfcrt and pfmdr1. Antimicrob Agents Chemother 2009;53:5069-73.
Sehgal PN, Sharma MI, Sharma SI, Gopal S. Resistance to chloroquine in falciparum malaria in Assam state, India. J Commun Dis 1973;5:175-80.
Sharma VP. Current scenario of malaria in India. Parassitologia 1999;41:349-53.
Sharma YD, Biswas S, Pillai CR, Ansari MA, Adak T, Devi CU. High prevalence of chloroquine resistant Plasmodium falciparum
infection in Rajasthan epidemic. Acta Trop 1996;62:135-41.
Moody A. Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev 2002;15:66-78.
Gama BE, de Oliveira NK, Zalis MG, de Souza JM, Santos F, Daniel-Ribeiro CT, et al.
Chloroquine and sulphadoxine-pyrimethamine sensitivity of Plasmodium falciparum
parasites in a Brazilian endemic area. Malar J 2009;8:156.
Sharma YD. Molecular surveillance of drug-resistant malaria in India. Curr Sci 2012;102:696-703.
Sharma VP. Battling the malaria iceberg with chloroquine in India. Malar J 2007;6:105.
Saha P, Guha SK, Das S, Mullick S, Ganguly S, Biswas A, et al.
Comparative efficacies of artemisinin combination therapies in Plasmodium falciparum
malaria and polymorphism of pfATPase6, pfcrt, pfdhfr, and pfdhps genes in tea gardens of Jalpaiguri District, India. Antimicrob Agents Chemother 2012;56:2511-7.
National Vector Borne Disease Control Programme. National Drug Policy on Malaria. Directorate of National Vector Borne Disease Control Programme, Directorate General of Health Services, Ministry of Health and Family Welfare, Government of India; 2013. Available from: http://www.nvbdcp.gov.in/Doc/National-Drug-Policy-2013.pdf
. [Last accessed on 2014 Jun 12].
Sharma YD. Genetic alteration in drug resistance markers of Plasmodium falciparum
. Indian J Med Res 2005;121:13-22.
Das MK, Lumb V, Mittra P, Singh SS, Dash AP, Sharma YD. High chloroquine treatment failure rates and predominance of mutant genotypes associated with chloroquine and antifolate resistance among falciparum malaria patients from the island of Car Nicobar, India. J Antimicrob Chemother 2010;65:1258-61.
Awasthi G, Prasad GB, Das A. Population genetic analyses of Plasmodium falciparum
chloroquine receptor transporter gene haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 2011;41:705-9.
Ghanchi NK, Ursing J, Beg MA, Veiga MI, Jafri S, Mårtensson A. Prevalence of resistance associated polymorphisms in Plasmodium falciparum
field isolates from Southern Pakistan. Malar J 2011;10:18.
Bharti PK, Alam MT, Boxer R, Shukla MM, Gautam SP, Sharma YD, et al.
Therapeutic efficacy of chloroquine and sequence variation in pfcrt gene among patients with falciparum malaria in central India. Trop Med Int Health 2010;15:33-40.
Biswas S, Escalante A, Chaiyaroj S, Angkasekwinai P, Lal AA. Prevalence of point mutations in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum
isolates from India and Thailand: A molecular epidemiologic study. Trop Med Int Health 2000;5:737-43.
Lumb V, Das MK, Mittra P, Ahmed A, Kumar M, Kaur P, et al
. Emergence of an unusual sulphadoxine pyrimethamine resistance pattern and a novel K540N mutation in dihydropteroate synthetase in Plasmodium falciparum
isolates obtained from Car Nicobar Island, India, after the 2004 Tsunami. J Infect Dis 2009;199:1064-73.
Mittra P, Vinayak S, Chandawat H, Das MK, Singh N, Biswas S, et al.
Progressive increase in point mutations associated with chloroquine resistance in Plasmodium falciparum
isolates from India. J Infect Dis 2006;193:1304-12.
Sutar SK, Gupta B, Ranjit M, Kar SK, Das A. Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum
isolates from Odisha, India. Mem Inst Oswaldo Cruz 2011;106:78-84.
Vathsala PG, Pramanik A, Dhanasekaran S, Devi CU, Pillai CR, Subbarao SK, et al
. Widespread occurrence of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt
) gene haplotype SVMNT in P. falciparum malaria in India. Am J Trop Med Hyg 2004;70:256-9.
[Table 1], [Table 2], [Table 3]