Malaria, a protozoan parasitic disease, is one of the major public health concerns with limited data in Pakistan . As Pakistan shares border with malaria endemic countries like Iran, India and Afghanistan so human migration across the border is inevitable which may facilitate substantial cross-border transmission of malaria. Human migration, immune responses exhibited by the human host, chemotherapy, genetic mutation and recombination lead to genetic diversity and affects the frequency of new alleles in the parasite population [33, 34]. The study of genetic diversity can give significant indications about parasite response to different drugs and vaccines as positive selection will favor the fixation of important alleles in the population and can lead to reduction of genetic diversity . Despite P. vivax contributing to 88% of malaria burden in Pakistan [4–6], data regarding genetic diversity of this key circulating species is lacking. There are limited studies from Pakistan which analyzed the diversity of local P. vivax in detail [4, 5, 23, 33]. So, the need for the detailed understanding of the extent and nature of P. vivax genetic diversity is obvious. PvCSP and PvMSP-1 are among the other important genetic markers used by the researchers to understand population structure and evolutionary dynamics from different geographical regions [4, 25]. In the present study, genetic diversity of both PvCSP and PvMSP-1 and PvMSP-1 genes were estimated among clinical isolates collected from different hospitals of Federal and Punjab region of Pakistan.
The results of PvCSP uphold the findings of the previously published results showing that VK210 strain is predominant type with the prevalence rate ranging between 81–100% [9, 12, 14, 36]. There are few malaria endemic areas where VK247 isolates are commonly present [37, 38]. In the present study, the translated nucleotide sequences suggested that there were peptide repetitive motifs in which GDRADGQPA (PRM1) and GDRAAGQPA (PRM2) which found to be the two major PRMs. Earlier studies also pointed out the dominance of these two prime PRMs (GDRADGQPA, GDRAAGQPA) in the clinical isolates [7, 8 12, 39]. All the isolates collected were composed of similar pre-repeat sequence (KLKQP) region and conserved post-repeat sequence GGNAANK (PRM4). At the end of the sequence the conserved post-repeat sequence is present as a last section in all of the VK210 isolates, consistent with the findings of aforementioned studies from India, Iran and Sri Lanka [8, 14, 20]. Another peptide repeat motif GNGAGGQAA (PRM3) was found at lower frequency (0.6%) in the isolates. The essential behind the development of genetic diversity found in the PvCSP gene, across various spatial locations, is the number of PRMs. Difference in the amino acid and nucleotide sequences of the Plasmodium antigens due to variations exist in the repeat unit numbers depicted the pressure of natural selection displayed by the host immune system [14, 40]. The arrangement of the main PRM1 and PRM2 factors lead to 15 different haplotypes in PvCSP. The pragmatic neutrality tests also accepted a neutral model of polymorphism which indicated that events of positive selection occurred in the complex group P. vivax isolate .
PvMSP-1 is one of the most promising vaccine candidates and is available for antigenic and genetic variation studies of P. vivax populations . In this study, partial sequence (~ 400 bp) at N-terminal of PvMSP-1 gene depicted a high-level of diversity. Such high level of genetic diversity is in concordance with what has already observed in neighboring country Iran  and in previous study from Pakistan . In the northwestern region of Thailand, a high degree of mutational variety was observed in PvMSP-1 gens of P. vivax isolates [28, 42]. The natural selection was determined by the neutrality tests on the PvMSP-1 N-terminal fragment of P. vivax. Overflow of transitional frequency alleles were observed because of significantly positive values which may be the result of balancing the selection and population bottlenecks. The sequence diversity of the population is best studied by the intragenic recombination of PvMSP-1 gene where the allelic recombination frequencies may aid as a character reference for understanding the parasitic population structure . Kibria et al.  also indicated that the given variations suggest high genetic diversity in all areas under study, furthermore, that the PvMSP-1 gene undergoes selective pressure for the existence and spread of the parasite. The PvMSP-1 gene sequences were instructive in distinguishing the two central localities of sample origin in terms of geography as well as helping them to group in two different clades. These clades are further sub divided into different clusters and group these sequences on these sub clusters according to its geographic origin .
Other studies suggested that the mode of evolution in PvCSP gene can lead to cohort of variants that can elude the host immune response under the effect of both mitotic recombinantion and positive selection of P. vivax new variants . It is safe to assume, therefore, that the wide variety of P. vivax is perhaps interrelated with multiple other variables including, but not limited to, genetic and biological characteristics, immunity of the host and the displacement of individuals within the boundaries of the endemic areas. Furthermore, the spread of P. vivax infections is also buttressed by relapse and early gametocytaemia, which in turn sustains local diversity, paving way for a more efficient transmission to the vector mosquitoes [12, 44].
In the research undertaken here, a broad range of genetic variety of PvCSP and PvMSP-1 genes in P. vivax population was noticed. The localities of the study areas, inhabited by various ethnicities, suggest that the migration of people may carry diverse parasite entities that increase the variety of the gene-pool. Furthermore, individuals infected by the disease might carry various clones with varying degree of PvCSP derivatives, which may recombine during the sexual stage of the mosquito leading to the production of offspring with new PvCSP genotypes. This prevalent phenomenon could strengthen the introduction of new strains of P. vivax into the regions where conditions for malaria transmission are conducive [12, 25].