|Year : 2015 | Volume
| Issue : 3 | Page : 401-405
Population genetic study of Plasmodium falciparum parasites pertaining to dhps gene sequence in malaria endemic areas of Assam
J Sharma, P Dutta, SA Khan
Entomology and Filariasis Division, Regional Medical Research Centre (ICMR), Dibrugarh, Assam, India
|Date of Submission||04-Jun-2014|
|Date of Acceptance||12-Mar-2015|
|Date of Web Publication||12-Jun-2015|
Entomology and Filariasis Division, Regional Medical Research Centre (ICMR), Dibrugarh, Assam
Source of Support: None, Conflict of Interest: None
Plasmodium falciparum malaria parasite had developed resistance to almost all the currently used antimalarial drugs. The purpose of the study was to come across the genetic distances in P. falciparum dhps gene sequences circulating in Assam. A partial fragment of P. falciparum dhps gene containing major single nucleotide polymorphisms associated with sulphadoxine resistance were amplified and sequenced. Thereafter specific bioinformatics tools like BioEdit v7.0.9, ClustalW in Mega 5, DnaSP version v.5.10.01 etc were used for the analysis. A total of 100 P. falciparum positive cases in different malaria endemic areas of Assam were included for the study. Based upon the mutation analysis, a total of seven different P. falciparum dhps genotypes were observed with five variable sites. Maximum five haplotypes were found in the P. falciparum isolates from Jorhat district of Assam. Four polymorphic sites were observed in the P. falciparum dhps gene sequences in Karbi Anglong, NC Hills, Chirang and Jorhat whereas the isolates from other study areas had three polymorphic sites. A statistically significant positive value of Tajima's D were observed among the P. falciparum field isolates in Assam indicating that there is an excess of intermediate frequency alleles and can result from population bottlenecks, structure and/or balancing selection. Extensive gene flow took place among the P. falciparum population of Jorhat with Sivasagar, Chirang with Sivasagar and Chirang with Karbi Anglong. However, large genetic differentiation was observed among the P. falciparum isolates of NC Hills with Lakhimpur, Tinsukia, Dibrugarh and Golaghat and also the parasite population of Karbi Anglong with Lakhimpur and Tinsukia signifying little gene flow among the population. This finding has shown that mutant Pfdhps gene associated with sulphadoxine resistance is circulating in Assam. It is believed that, the parasite population may have undergone high level of breeding.
Keywords: Assam, dhps, gene flow, mutation, P. falciparum, polymorphic site
|How to cite this article:|
Sharma J, Dutta P, Khan S A. Population genetic study of Plasmodium falciparum parasites pertaining to dhps gene sequence in malaria endemic areas of Assam. Indian J Med Microbiol 2015;33:401-5
|How to cite this URL:|
Sharma J, Dutta P, Khan S A. Population genetic study of Plasmodium falciparum parasites pertaining to dhps gene sequence in malaria endemic areas of Assam. Indian J Med Microbiol [serial online] 2015 [cited 2018 Dec 10];33:401-5. Available from: http://www.ijmm.org/text.asp?2015/33/3/401/158565
| ~ Introduction|| |
Drug resistance malaria is a global problem. It is widespread in Indian states. The North-Eastern region is highly vulnerable. The malaria parasites having antimalarial resistance activity are carrying some mutations in their target gene and convey it from one population to another. However, due to the presence of certain barrier, the P. falciparum parasite populations in a locality are unable to drift from one place to another. These barriers prevent to breed among the parasite population in two different localities. As a result of which there might be a high genetic distance between parasite populations due to low level of gene flow. Genetic distance is useful in reconstructing the history of population. Smaller genetic distances indicate that the population has more similar genes. In other way, lower genetic distance between population means that more breeding between them and as a result of which the populations are less isolated from one another. This indicates that they are closely related and they have a recent common ancestor or we may describe in other way that recent interbreeding has taken place between the populations. On the contrary, greater genetic distance between populations means that less breeding between them and more isolated from one another.
It has been seen that Assam is one of the high malarious state in India. Few studies conducted in this part indicated high prevalence of genetic polymorphisms associated with currently used antimalarial drugs. These studies confirmed that in P. falciparum dihydropteroate synthase (dhps) gene, mutations at codon position 436, 437, 540, 581, 613 are associated with sulphadoxine resistance (SR). ,, However, in this aspect extensive study regarding the genetic differentiation and gene flow is very much essential. Such studies are putting important insights into population dynamics. Keeping in view, a study in this line has been taken up in Assam to find out the genetic distances among the P. falciparum parasite population associated with SR.
| ~ Materials and Methods|| |
Patients of all the age-groups and both sexes having symptoms of fever/suspected malaria were included in our study. Prior to starting the work, institutional ethical clearance was obtained from institutional ethical board, Regional Medical Research Centre (RMRC), ICMR, Dibrugarh, Assam. Two millilitre of blood samples were collected from microscopic confirmed P .falciparum malaria cases in different malaria endemic areas of Assam. DNA extraction was done from the serum samples. A 710 bp portion of P. falciparum dhps gene containing the major single nucleotide polymorphisms related to sulphadoxine drug resistance was amplified by using specific primers as described earlier.  The amplified products were sequenced in an automated sequencer. The forward and reverse sequences of each sample were edited manually using BioEdit v7.0.9 software.  Sequence similarity searches were performed using NCBI's BLAST. DNA sequences were aligned through ClustalW in Mega 5 software and SNPs of concerned genes were estimated by software DnaSP version v.5.10.01.
Extensive analysis of the gene polymorphism, average number of nucleotide differences, nucleotide diversity  were analysed in DnaSP version v. 5.10.01. Neutrality test (Tajima F., 1989; Fu YX., 1997) was done to see if the population had undergone purifying selection, population expansion, and population bottleneck. , Besides this, the genetic differentiation and gene flow in the P. falciparum parasite population were analysed in DnaSP version v. 5.10.01.
| ~ Results|| |
The P. falciparum dhps gene sequences from 1300-1878 bp portions of 100 field samples in Assam were analysed (GenBank accession numbers: KJ403950-KJ404049). This result indicated that A437G and S436A were key point mutations in P. falciparum dhps gene associated with SR. Based upon the number of mutations a total of five variable sites (S: 5) were found in analysed samples of Assam [Table 1]. Nucleotide diversity (per site), average number of nucleotide differences etc., are shown in [Table 1]. Four polymorphic sites were detected in the P. falciparum dhps gene sequences in Karbi Anglong, NC Hills Chirang and Jorhat whereas the isolates from other study areas had three polymorphic sites. The P. falciparum dhps gene sequences from the isolates in Karbi Anglong have shown utmost average number of nucleotide differences that was closely followed by the P. falciparum population in Chirang district of Assam. Overall analysis of neutrality test showed a positive statistical significant value of Tajima's D: 2.08882 (Statistical significance: P < 0.05). Also, a positive value of Fu and Li's D test statistic: 1.03676 and Fu and Li's F test statistic: 1.62804 was obtained but did not show any statistically significant result. However, the value of Tajima's D, Fu and Li's D test statistic, Fu and Li's F test statistic obtained from the Pfdhps gene sequences in each district did not show any statistically significant result.
|Table 1: District wise mutation analysis for Pfdhps gene among the isolates of Assam |
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The P. falciparum field isolates in Assam had shown the presence of seven haplotypes (h: 7) of Pfdhps gene with a value of haplotype (gene) diversity (hd: 0.803), variance of haplotype diversity: 0.00029 and standard deviation of haplotype diversity: 0.017 [Table 2]. A maximum of five dhps haplotypes were observed from Jorhat district of Assam closely followed by Karbi Anglong and Chirang. The P. falciparum dhps gene sequences from NC hills, Golaghat, Dibrugarh and Lakhimpur district had shown the value of mean standard deviation of haplotype diversity (SD ± 0.161) whereas the value of standard deviation of haplotype diversity obtained from the remaining study areas was ≤0.1.
|Table 2: District wise haplotype analysis for Pfdhps gene among the isolates of Assam |
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Very little gene flow was observed among the parasite population of NC Hills with Lakhimpur, Tinsukia, Dibrugarh and Golaghat and also the parasite population of Karbi Anglong with Lakhimpur and Tinsukia. Accordingly very high genetic differentiation was estimated in those areas [Table 3]. The parasite population of Karbi Anglong with Dibrugarh and Golaghat had also shown very large genetic differentiation. Conversely, very little genetic differentiation was found among the parasite population in Jorhat and Sivasagar. The parasite population of Chirang with Sivasagar had also shown slight genetic differentiation which was closely observed with Karbi Anglong, signifying extensive gene flow among the parasite population of these areas with Nm value >>4 [Table 3] and [Figure 1]. Gene flow level was high enough among the parasite population of Sivasagar with Karbi Anglong, Jorhat with Dibrugarh and Golaghat, Chirang with NC Hills, Jorhat with Tinsukia, Jorhat with Karbi Anglong and Sivasagar with NC Hills. A negative value of Fst and Nm was observed among the parasite population of Karbi Anglong with NC Hills [Table 3]. However, the negative F ST value calculated between the localities were interpreted as zero.
|Figure 1: P. falciparum dhps gene flow among the field isolates in Assam|
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|Table 3: Analysis of genetic differentiation and gene flow among the parasite population of Assam (Pfdhps gene) |
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| ~ Discussion|| |
In case of Pfdhps gene, A437G and S436A mutation were ascertained as the key mutation associated with SR in different parts of Assam which is in accordance with previous findings. , Our study has shown that there is an increase in Pfdhps gene mutations associated with SR. Therefore it is suggested that the Sulphadoxine-Pyrimethamine antimalarial should be replaced and used another combination with artemisinin for treatment of P. falciparum malaria. In the meantime under the National vector borne disease control programme artemisinin in combination with lumefantrine is recommended for North eastern region. The combination is believed to be beneficial for the people of North-Eastern region.
A positive statistically significant value of Tajima's D was observed among the P. falciparum parasite population of Assam. It may be due to over dominant selection and population bottleneck. Negative Tajima's D values were observed among the field isolates from Golaghat, Dibrugarh, Tinsukia and Lakhimpur district of Assam but the results have not shown any statistical significance. The P. falciparum population from the other study areas revealed positive value of Tajima's D however, it was also not statistically significant. A very limited study has been carried out in this aspect for P. falciparum drug resistance markers.  However no extensive studies have been done for estimating the above parameter by using specific bioinformatics tools.
Likewise in case of Pfdhps gene analysis, the value of fixation index obtained between the parasite population of NC Hills with Tinsukia, Lakhimpur, Golaghat, Dibrugarh, Jorhat indicates much isolation between population and most likely mean that the population are not currently breeding with one another [Table 3]. The parasite population of Sivasagar with Jorhat and Chirang as well as Karbi Anglong with Chirang had a value of F ST on the low end of the standard range (close to 0) which indicates that the populations are sharing their genetic material through high level of breeding [Table 3]. Previous studies conducted in other areas revealed a decrease in the genetic distances in drug resistance marker gene among parasite population. It is due to the presence of most similar types of point mutations in drug resistance markers. ,, However in Northeastern region of India, no such studies have been carried out in P .falciparum drug resistance markers. So, this study brings importance for the public health interest.
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[Table 1], [Table 2], [Table 3]