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Diverse nature of P. falciparum isolates is observed in all 13 studied regions of India in respect of length polymorphism. The prevalence of all the allelic families of msp-1 and msp-2 is observed throughout India. The high level of diversity in surface antigens at high transmission areas (HTA) concurs the earlier reports on Indian isolates (Joshi, 2003; Raj et al., 2004; Ranjit, Sharma, 1999). Observed high proportion of multiclonal isolates in HTA was in accordance with the reports of other workers (Raj et al., 2004; Ranjit et al., 2005) on Indian isolates. A comparatively higher proportion of multiclonal isolates as well as multiplicity of infection (MOI) was observed among isolates of Assam, Orissa, Jharkhand and Maharastra. All these area are highly endemic for P. falciparum malaria and its proportion being above 80%. The extent of diversity and multiplicity of infection in a region was correlated to level of malaria endemicity in earlier studies (Babiker et al., 1997; Ranjit, Sharma, 1999; Zwetyenga et al., 1998). However, in our study, Madhya Pradesh from LTA shows higher proportion of multiclonal isolates as well as higher MOI. A recent study from Iran reports emergence of drug resistant P. falciparum as a potential cause of high level of genetic diversity in msp-1 and msp-2 gene along with high proportion of multiclonal isolates (87%) and MOI (3.06) (Zakeri et al., 2005). This study for the first time revealed that an identical population structure of P. falciparum isolates is existing in various high transmission regions (Assam, Orissa, Jharkhand, West Bengal and Maharastra) of India, which is revealed by presence of common allelic composition in all these states as well as high level of identity among allelic sequences of isolates from two states Assam and Orissa was reported earlier (Joshi et al., 2007). A higher MOI based on msp-1 in some areas and higher MOI based on msp-2 in other areas suggests that local factors such as type of vector population, immunity status of human host and drug susceptibility pattern of the parasites in a region may be playing a role in defining the population structure of the field isolates. Prevalence of identical allelic composition in different study areas of these 13 states suggests a considerable amount of gene flow between the P. falciparum populations of different states. In India, one potentially important factor in the spread of P. falciparum is migrant workers. The human movement can influence local malaria epidemiology, including transmission and its seasonality (Kumar. A et al.) . This to and fro movement from native place to work place may be one of the major facilitating factors in the gene flow and can promote transmission of drug-resistant parasite strains. Distribution of allelic families of msp-1 and msp-2 and their variations were similar to that reported from other countries with low or meso-endemicity of malaria i.e. Southeast Asia, Latin America and Papua New Guinea (Ariey et al., 1999; Gomez et al., 2002; Montoya et al., 2003). Monomorphic nature of RO33 family of msp-1 has been reported earlier in isolates of other regions of India and the observed 150/160 bp was the most commonly reported allele in other continents also (Aubouy et al., 2003; Konate et al., 1999; Peyerl-Hoffmann et al., 2001). The presence of both alleles of RO33 family in msp-1gene at areas such as Assam, West Bengal, Maharastra, Madhya Pradesh and Goa indicates allelic identity among Indian isolates and with data of other regions suggests that Indian P. falciparum population could be a mixture of different strains interacting due to geneflow among parasite from Northeast to Southwest India. Longitudinal studies on the genetic diversity of P. falciparum isolates from regions with varied malaria epidemiology are required to understand association between the clonal fluctuations and transmission intensity in the country. A cohort study on genetic diversity of malaria parasite from Indian subcontinent, which is a composed of countries with varied malaria paradigm would probably quantum the understanding in spread of malaria in this part of world. Thus, present study shows that field isolates of eastern and north-eastern regions of India are highly diverse in respect of msp-1 (block 2) and msp-2 (central repeat region, block3) with identical population structure and exhibit a level of diversity similar to that in Papua New Guinea, Southeast Asia and South and Central America, regions with low to meso-endemicity of malaria.
7.2 DISTRIBUTION OF MUTATIONS CONFERRING CHLOROQUINE RESISTANCE AMONG INDIAN P.FALCIPARUM ISOLATES
7.2.1 Analysis of mutations in in vivo chloroquine efficacy outcomes
In this study, the distribution of pfcrt haplotypes and the pfmdr-1 mutation (N86Y) has been described across 13 sites in India. We also evaluated for a correlation between the prevalence of mutations and the clinical outcome of CQ treatment. The distribution of pfcrt haplotypes in association with the clinical outcome of treatment supports that the K76T mutation is the most predictive marker of CQR in the field (Djimde et al., 2001a; Djimde et al., 2001b). The wild type CVMNK-A haplotype was found to be exclusively restricted to the ACPR outcome group (P < 0.001, Fisher's exact test). However, the wild type CVMNK-A haplotype was not the predominant haplotype in the ACPR outcome group; in fact, 82.07% of the isolates identified as ACPR carried a mutant haplotype. From this data, it is proposed that the typing of molecular markers for CQ resistance may infer an intrinsic characteristic of the parasite, but may not necessarily be sufficient to predict treatment outcome. Treatment outcome may depend on other factors, such as host-parasite or host-drug interaction(Pillai et al., 2001). Exposure-related host immunity may play an essential role in naturally clearing the parasite infection, irrespective of their response to any drug. Some studies have shown that host immunity is also associated with clearance of resistant genotypes (Djimde et al., 2003; Dorsey et al., 2001).
7.2.2 Regional distribution of mutations among Indian P.falciparum isolates
The mutant haplotype SVMNT-S (characteristic of chloroquine resistant South American or PNG isolates) was observed in all sites across India, regardless of the clinical outcome. Observation made in this study support earlier reports regarding the prevalence of SVMNT-S haplotype among the chloroquine resistant isolates in India (Keen et al., 2007; Mittra et al., 2006; Vathsala et al., 2004). Perhaps the most striking observation in the study is that the SVMNT-S haplotype is endemic across all the sites of low P. falciparum malaria transmission, while in high transmission regions there are multiple mutant pfcrt haplotypes observed. These different haplotype combinations observed in regions of high transmission might be indicative of random mating patterns among parasites within these regions, which could be influenced by the local rate of malaria transmission. The role of transmission in the evolution and spread of drug resistance remains a matter of debate, as it has been hypothesized by some that low transmission may decrease circulating drug resistance, while others hypothesize that low transmission may increase drug resistance (Hastings, Mackinnon, 1998; Schmidt, 1995). These studies signify the effect of transmission on altering the spread of drug resistance. In this study, the distribution of mutations hints that the spread of chloroquine resistant parasite might be under the influence of the transmission intensity and the distribution of anti-malarial drugs. The complexity of infection (minimum number of clones within an infection) was commonly observe to bear higher proportion of multiclonal infections in regions of high transmission, compared to regions of low transmission (Baruah et al., 2009; Joshi et al., 2007; Talisuna et al., 2003; Urdaneta et al., 2001), which in turn lead to high genetic diversity in parasite. The ubiquitous nature of mutant SVMNT-S pfcrt haplotype in the low transmission regions in this study may be a direct consequence of higher inbreeding potential of resistant parasites due to low frequencies of complex and multiclonal infections. An inbreeding population, consisting mostly of resistant genotypes could spread parasite drug resistance expeditiously(Paul et al., 1995; Schmidt, 1995). This expansion of mutant SVMNT-S is likely to be enhanced by extensive exposure of CQ in these P. vivax predominated areas, as chloroquine is the first-line drug for vivax malaria in India. A similar expansion has been observed for the pfmdr-1 wild type N86 in these P. vivax prevalent areas. Again inbreeding might be the cause for this expansion in low transmission regions.
The significant presence of wild type CVMNK-A in high P. falciparum prevalent areas (Orissa, Jharkhand, Chhattisgarh) supports earlier reports on acquisition of immunity in high endemic areas, which creates a natural ecological refuge for drug sensitive parasites (Djimde et al., 2003; Klein et al., 2008). This could possibly explain the occurrence of the wild type haplotype in high P. falciparum prevalent areas. Further, appearance of the mutant haplotype CVIET-S (characteristic of Southeast Asian CQR parasite) is expected to be observed within eastern and northeastern parts of the country, because this region shares international borders with Bangladesh, Nepal, Bhutan, Myanmar and China and harbor migrant populations. The CVIET-S haplotype might be indicative of high drug pressure in regions of high transmission, as earlier observations have shown that the CVIET-S haplotype have higher IC50 for CQ (Mittra et al., 2006; Sa. J. M et al., 2009). Similarly, the prevalence of pfmdr-1 mutant N86Y found in these areas may be involved in modulating the CQ response in Indian isolates. Isolates with the two loci mutation (pfcrt-pfmdr-1) were limited to high P. falciparum transmission regions, where N86Y appeared with both the pfcrt mutant haplotype SVMNT-S and CVIET-S. These two mutant haplotypes were observed in high numbers at Assam, Orissa, and Jharkhand, whereas Chhattisgarh shows a higher frequency of the wild type for both loci. This study has not evaluated IC50 values for particular pfcrt haplotypes, but the observed prevalence of the mutant SVMNT-S, even in the high transmission regions may be an indication of increased transmission potential of these resistant parasites (Sa. J. M et al., 2009). The use of amodiaquine as monotherapy or in combination has been associated with the prevalence of SVMNT haplotype in chloroquine resistant areas (Alifrangis M et al., 2006; Dittrich et al., 2005). In India, amodiaquine was used as presumptive anti-malarial drug for CQR parasite in 1980's in these high transmission regions (Barkakaty et al., 1980; Ghosh et al., 1992; Pandya et al., 1994) and this may be a possible explanation for the introduction and spread of SVMNT-S haplotype. On the other hand, a recent investigation from central India, using multilocus microsatellites, compared the evolutionary proximity of Indian SVMNT-S haplotype with other parts of the world and reveals its close relation to the SVMNT genotype found in PNG (Mixson-Hayden T et al., 2010) . So, overall the spread of SVMNT is intriguing and require further investigation about its origin and spread in India.
The observed prevalence of the wild type pfmdr-1 N86 allele could be a cause for concern, because a recent study reported that the selection of this allele is associated with resistance to one of the artemisinin-based combination therapy (ACT-Coartem®) desseminated in East Africa (Sisowath et al., 2005). This allele has also been associated with a decreased sensitivity to lumefantrine in vitro (Duraisingh et al., 2000; Sisowath et al., 2007). Furthermore, the significant prevalence (P < 0.001) of wild type N86 in low P. falciparum prevalent areas seems to be fixed and may result in an easy escape for parasites exposed to this drug combination. Ultimately, this fixation could lead to the rapid spread of resistant parasites. However, a recent investigation of therapeutic efficacy of Coartem® in the high P. falciparum transmission regions marks successful outcome without any selection of N86 (Valecha et al., 2009).
This study observed a striking pattern in fixation of mutant SVMNT pfcrt haplotype at low P. falciparum prevalent areas, which raise concerns about faster spread of anti-malarial resistance in these areas. The absence of wild type pfcrt haplotype in most part of the country may lead to a situation where no reversal of wild type would happen even in the absence of CQ pressure. This study leads towards understanding the role of malaria transmission intensity in spread of anti-malarial resistant parasite in India, and it will be useful in designing anti-malarial treatment policy.
7.3 TREND OF STRENGTH OF SELECTION AROUND pfcrt GENE AMONG INDIAN P.FALCIPARUM ISOLATES
7.3.1 Parasite diversity in microsatellite loci across Indian P. falciparum populations
P. falciparum populations from high transmission areas (HTA) generally report higher genetic variation than those from low transmission areas (LTA) (Anderson et al., 2000) and maintains multiclonal infections (i.e., multiple genotypes per infected person) in such areas (Paul et al., 1998). The genetic diversity within both neutral microsatellite loci and pfcrt-flanking microsatellite loci varies according to transmission intensity. The extensive microsatellite diversity within HTA corroborates earlier studies by (Garg et al., 2007; Joshi, 2003), which demonstrated extensive polymorphism in surface antigens akin to high level of multiple infection in HTA. Higher values for many measures of genetic diversity (AR, PR and He) within HTA indicate high rate of recombination and less inbreeding in parasite population (Conway et al., 1999). Furthermore, a reduction in all measures of genetic variation was observed at pfcrt-flanking loci of resistant pfcrt haplotypes, when compared with the neutral loci, suggests selection around the pfcrt gene.
7.3.2 Trend of chloroquine selection around pfcrt gene
The significant reduction in extent of heterozygosity observed within the pfcrt-flanking regions of the mutant pfcrt alleles relative to wildtype alleles, indicate chloroquine selective pressure on the pfcrt gene. Reduced width of selective sweep (pfcrt-flanking region with reduced variation) around resistant CVIET allele in comparison to resistant SVMNT allele is possibly due to predominance of resistant CVIET allele and higher recombination rate within HTA. Approximately two fold higher He values around SVMNT alleles in HTA reiterates higher recombination events in populations at HTA and also a varied strength of selection at different geographic region cannot be ignored. The next possible reason may be varied time points of colonization of different resistant allele at different regions. The CVIET was possibly introduced earlier than SVMNT in India and may have evolved multiple times due to recombination events adding new alleles to population under HTA epidemiology. When reduction of heterozygosity was assessed at different locations, it provides a selective sweep around -20kb to +6kb in all site under high transmission, whereas locations under low transmission epidemiology showed wider selective sweep around ±24kb.The exception in LTA was MP, where considerably higher amount of He was observed. The fitness of resistant alleles may also play a role in hitchhiking, as the data set show fixation of resistant SVMNT at LTA in comparison to HTA where resistant alleles had still not reached fixation. This difference in relative fitness of pfcrt alleles at different epidemiology may contribute to the observed differential hitchhiking and to the difference in transit time (spread to fixation) of resistant allele. The data revealed that the molecular basis of resistance differs with change in transmission intensities, which concurs a recent report (Lumb et al., 2012) and also likely to translate the varied CQR level at different locations. In a selective sweep, reduction of heterozygosity was also accompanied with large regions of elevated linkage disequilibrium around resistant gene. The observed LD value supports above narrower selection valley as it got reduced with addition of a distant loci at upstream (+106kb) onwards. The decline in LD is more rapid around CVIET (IAS =0.08) in HTA than around SVMNT (IAS =0.10) in LTA, consistent with weaker selection event at HTA as discussed above. Hence, we clearly visualize a pattern of different genotypes occurred at different regions (broadly HTA and LTA), which in turn raises a possibility of detecting geographical structure in relation to chloroquine resistance among Indian P.falciparum.
7.4 ANALYSIS OF MICROSATELLITE IN THE INTRONS OF pfcrt GENE
The most effective way to understand the evolutionary history of a gene is to study genetic variation in and around that gene because positive natural selection would leave its footprint on the level of genetic variation. Our result of reduced genetic variation around pfcrt gene and increase in genetic variation as the recombinational distance from the site of selection increases i.e., at +106kb supports a strong directional selection at this site of genome. This observation concurs with prior reports of selective sweep (genetic hitchhiking) model of molecular evolution in global chloroquine resistant isolates (Wootton J. C et al., 2002). However, a recent report of high genetic variability of pfcrt in India (Vinayak S et al., 2006), deviates from the globally accepted evolutionary history of CQ resistance in P. falciparum. Observations of high genetic variations of pfcrt do not fit with either the selective-sweep model of molecular evolution or with the assumptions of origin and spread of drug resistance caused by drug pressure in the field. This implies that the pfcrt gene in parasites of India may be in the process of a massive genetic reconstruction, whereas the global isolates seem to have an almost fixed pfcrt gene, which in turn raises a need to insight the genetic variation inside the pfcrt gene. Thus we have evaluated the genetic variation in microsatellites in different introns of pfcrt gene. Collectively the data seems to have enormous amount of variation, as all the microsatellite was polymorphic at each intron except intron 5. However the frequency of alleles reveals that each intron has two predominant alleles and was moreover fixed with the mutant pfcrt haplotype (Table 6.11). This distinguished pattern of genotype supports the differential genetic background of two predominant mutant pfcrt haplotype found in Indian isolates. This observation continues with the evaluation of expected heterozygosity at different intron microsatellite. The significant reduction in extent of heterozygosity observed within mutant pfcrt alleles relative to wildtype alleles, indicate chloroquine selective pressure on the mutant pfcrt gene. Infact, like pfcrt-flanking loci, similar reduction in genetic variation was observed in mutant SVMNT haplotype in comparison of mutant CVIET. Interestingly, we also observed higher He values in intron microsatellites of SVMNT alleles from HTA in comparison of LTA (Table 6.12), which again reiterates higher recombination events in populations at HTA and confirms a varied strength of selection at different geographic region that cannot be ignored. Equivalent level of expected heterozygosity was observed in both intron microsatellite loci and the pfcrt-flanking microsatellite of all the wild and mutant pfcrt gene i.e., if reduction in variation happens in flanking region then similar reduction occurs in introns. Hence, the level of reduced genetic variation in mutant pfcrt haplotypes observed here confirms the selective sweep model of natural selection in and around pfcrt gene among Indian P.falciparum isolates. In turn we do not support the point of contradiction between global and Indian scenario of chloroquine resistance (Das A, Dash, 2007). The six locus microsatellite genotype was not shared between the two mutant pfcrt haplotype, which again indicates the varied genetic background associated with them. Not six but at five alleles of the intron microsatellite of Southeast Asian isolates (Dd2 and INDO) were similar to HAP6 Indian isolates bearing mutant CVIET haplotype (Table 6.13), hints about the probable migration of chloroquine resistant parasite from Southeast Asia region and may have common origin. However, mutant SVMNT isolates from Brazil (7G8) show similar allele at intron 2, 6 and 9 only. A recent study reported similar predominant microsatellite alleles at intron 2 and 3 between India and Papua New Guinea (DaRe J. T et al., 2007). This might indicate the multiple origins for chloroquine resistant parasites with mutant SVMNT haplotype.
7.5 GENE FLOW AMONG CHLOROQUINE RESISTANT P.falciparum ISOLATES ACROSS INDIA.
7.5.1 Inference of Population structure among Indian P.falciparum isolates
Three neutral loci located on three different chromosomes were used to identify the population structure of Indian P. falciparum. After STRUCTURE analysis, two discrete clusters associated with transmission intensity were identified, where Neutral cluster1 was significantly comprised of infections from HTA and Neutral cluster2 was significantly comprised of infections from LTA. Furthermore, there was minimal admixture between HTA and LTA, which supports earlier reports of the association between the genetic structure of P. falciparum and patterns of transmission (Anderson et al., 2000). Thus, the STRUCTURE results corroborate the differential measurements of genetic diversity at HTA and LTA. Further pairwise FST analysis confirmed the population differentiation provided by STRUCTURE, estimating substantial levels of genetic differentiation between populations from HTA and LTA (Table 6.15). This genetic differentiation could be due to genetic drift; however, the observed value of Nm â‰¥ 1 between all of the subpopulations does not support genetic drift (Slatkin, 1987). Low population differentiation was detected between falciparum subpopulations within HTA except WBG, which has moderate differentiation when compared with other regions. Corroborating earlier reports of genetically similar surface antigens (msp-1 and msp-2 ) between Assam and Eastern India (Joshi et al., 2007), indicate a pattern of current or historical migration due to their geographic proximity. CN and Eastern India were also found to be genetically similar, indicating that there is considerable parasite gene flow between mainland and island parasite populations, which supports previously reported data based on surface antigen (AMA1) (Garg et al., 2007). In LTA, MP show low genetic differentiation with RAJ, GOA and TN (FST = 0.05, P > 0.094). (RAJ-GUJ-UP) and (GOA&TN) showed high genetic differentiation between them. Thus, Southwest (GOA&TN) part of India have most genetically isolated population from rest of India except with MP. The three-level hierarchical analysis of genetic differentiation (AMOVA) supported the division of Indian P.falciparum population to four geographical groups, as maximum variations observed were within the subpopulation rather than among subpopulations of a particular geographical group. A significant positive correlation between genetic variation and geographic distance was obtained by Mantel test on all the populations. This indicates a genetic isolation by geographic distance in falciparum population of India. Geographical distance, alone, does not adequately explain the divergent parasite populations across India, especially given populations like ASM-MAH, MAH-CN, and ORS-CN are greater than 1200 km apart and showed no genetic differentiation. However, the extend analysis at LTA and HTA revealed a significant isolation by distance (IBD) at LTA only, and no such correlation between genetic distances and geographic distances in HTA. This again supports our above division of Indian P.falciparum population to four geographical groups. Further mantel test of all populations excluding the Southwest group revealed no correlation between genetic distances and geographic distances both in HTA and LTA. Probably the genetic differentiation of Southwest region with all other group is core responsible for IBD observation in whole dataset and even in LTA alone.
7.5.2 Spread of hitchhiking in Indian P. falciparum population
The results (STRUCTURE, FST, AMOVA and IBD) collectively show that groups of subpopulations have become differentiated from one another. This deviation from single random mating population may effect the genetic hitchhiking around a beneficial mutation accrued in population. We observed interesting pattern while comparing pairwise FST between neutral loci and pfcrt-flanking loci (Table 6.17A,B) at the four patches of population inferred above; Southwest and Northeast-East-Island show highly differentiated populations at both neutral and pfcrt-flanking loci indicating heterogenous population and low gene flow; Central and Northeast-East-Island or Northwest show low genetic differentiation at both neutral and pfcrt-flanking loci indicating homogenous population and continuous gene flow; Central and Southwest show moderate differentiation at both neutral and pfcrt-flanking loci indicating a limited gene flow between populations; Southwest and Northwest show high genetic differentiation at neutral loci but low genetic differentiation at pfcrt-flanking loci indicating that evolutionary process (i.e., strength of selection) is faster than rate of migration. This could be due to population subdivision which causes several important modifications in strength and pattern of a selective sweep. Studies (Bierne, 2010; Slatkin. M, Wiehe. T, 1998) showed that in a subdivided population, genetic hitchhiking can introduce population differentiation (large FST) in an initially homogeneous population (small FST) and the similar pattern is displayed between Northwest and Northeast-East-Island group. The gradient of heterozygosity may be used along the pathway of a sweep to infer the geographical movement of the beneficial mutation as it leaves weaker signature of selection while spreading across distant subpopulations after its introduction in first population i.e., heterozygosity is lowest in subpopulation where beneficial mutation is introduced and subsequently increased with the spread across neighboring demes (Kim, Maruki, 2011). Eventually, our data also show a gradient of heterozygosity in pfcrt-flanking loci from Southwest to Northeast-East-Island part of India and this raises thought about probable introduction of SVMNT allele in southern India and then progress to other parts of India.
7.6 Dispersal/Probable migratory route of chloroquine resistant haplotype
The STRUCTURE analysis of pfcrt-flanking loci showed 3 discrete clusters associated with the different pfcrt haplotypes (CVIET, SVMNT, and CVMNK). This in turn reflects the observation of varied level of selective sweep associated to different pfcrt haplotypes could be due to different chloroquine pressure at various regions. The pairwise FST estimate show high genetic differentiation between East-Island (ORS, JHK, CHG, CN), with that of Northwest (UP, RAJ, GUJ) and strong differentiation between East-Island and Southwest (GOA&TN), indicating a limited migration of resistant alleles between East-Island with that of Northwest and Southwest. In turn, the Northeast region may have indeed, been a major migratory route of the resistant alleles (CVIET& SVMNT) likely originating from the Southeast Asia region and then spreading into Eastern India and consequently to other parts of India. While moderate genetic differentiation at pfcrt-flanking loci was observed between Central (MP, MAH) group, Northeast-East-Island, Northwest group and Southwest group.
Admittedly, more neutral loci may be needed to make concrete conclusions about geographic structure in Indian P.falciparum, but even with these three neutral loci, there is evidence of a genetic structure that is strongly linked with the patterns of malaria transmission. Additionally, resistant alleles are also differentially structured; probably due to above found geographic structure or varied chloroquine usage in these locations. While determining F-statistics estimations, a small amount of genetic exchange between populations is enough to prevent the accumulation of large genetic differences between them. Thus, the significant strong genetic differentiation found between Southwest and Northeast-East-Island part of India implies limitations in direct dispersal of resistant alleles and supports the movement of resistant alleles via Central India.
Human migration between malaria endemic regions plays an important role in the movement of resistant alleles in distant populations (Hume et al., 2003; Lqbal et al., 2002). For example, two recent studies based on pfcrt-flanking microsatellite markers (Mixson-Hayden et al., 2010; Rawasia et al., 2012) reported that the resistant SVMNT found in India and Pakistan migrated from PNG. Evidence for this was also found by clusters of neutral microsatellite markers that were earlier reported between PNG (Pacific region) and Thailand (Southeast Asia) (Anderson et al., 2000). In this study, the microsatellite profile of pfcrt-flanking loci associated with the resistant allele SVMNT and CVIET were similar to that of microsatellite profile reported from PNG and SEA, respectively (Mixson-Hayden et al., 2010; Wootton et al., 2002). A recent study (Awasthi et al., 2011) examining the allele frequency of pfcrt haplotypes (CVMNK, CVIET and SVMNT) postulates a probable route of migration for different pfcrt haplotypes in India and reported that the mutant SVMNT allele arrived India from PNG via SEA. In presence of population differentiation and gene flow data inferring a geographic structure in Indian P.falciparum, we postulate another probable route for dispersal of mutant SVMNT across India through Sri Lanka or southern India (Figure 6.7).
Human migration related to labour and tourisim had been associated with prevalence of chloroquine resistance in India, particularly labourers travelling from eastern part of India to the western states (Sethi et al., 1990; Sharma, Sharma, 1988; Sharma, 2000). The study site in TN (Rameswarum Island) has been associated with continuous human migration with Sri Lanka and being a holy place the study site also receives a large number of pilgrims from all over India. In addition a large number of tourists stay here before or after visiting Sri Lanka. These human migrations within this region have likely helped to facilitate the spread of chloroquine resistant parasites between both country and throughout India (Rajagopalan et al., 1986). This, in turn, may have given chance for immigration of SVMNT from Sri Lanka to India as a recent study reported ubiquitous appearance of SVMNT in Sri Lanka (Zhang et al., 2011) and we also observed similar frequency of SVMNT from this study site also. The predominance of similar mosquito vector (Anopheles culicifacies; sibling B,E) in both locations (Surendran et al., 2000) provide a basis for this postulation. It is well known about human migration between southern India, Sri Lanka and Indonesia for trading and the cultural contacts was much greater than hitherto been imagined (Karafet et al., 2005). Similarly, Pakistan shares international borders with Rajasthan and Gujarat, which reports the fixation of SVMNT allele with similar pfcrt-flanking loci in both countries. Where as the countries sharing border with northeast India like Nepal, Bangladesh and Myanmar reports higher frequency of CVIET (Banjara et al., 2011; Kawai et al., 2011; Mohapatra et al., 2005). It is worth putting the South Asia-Southeast Asia migration corridor in perspective. Thus, we here, infer immigration of SVMNT allele from PNG to India via Sri Lanka or south India. At last the results of median joining NETWORK construction reveal two distinct cluster for resistant alleles CVIET and SVMNT, indicating accumulation of resistant alleles on a distinct genetic background. However, clustering of multilocus haplotype in SVMNT supports a recent origin than that of CVIET cluster which seems to undergone large number of mutational steps over time. The maintenance of diversity over time in HTA suggests a large population size. Thus, we observed a strong geographic structure governed by transmission intensity and it could be possible that different selective sweep must have occurred independently in different geographic locations due to local adaptation.
7.6 TREND OF SELECTION IN FLANKING OF pfmdr-1 GENE
Mutations in the multi drug resistance gene (pfmdr-1; chromosome 5) play a significant role in the parasite's resistance to various antimalarials and also modulate resistance against chloroquine (Dorsey et al., 2001).Out of all mutation observed in worldwide isolates (Foote et al., 1990), the N86Y mutation was detected to modulate the chloroquine resistance in Indian isolates (Mittra et al., 2006; Vathsala et al., 2004).However, the mutation was not well correlated with both in vitro and clinical outcome in all these reported studies in Indian isolates. In the above sections of experiment we observed a significant reduction of variation around the mutant pfcrt gene; whose action of maintenance of chloroquine resistance was complimented by mutant pfmdr-1 gene. Thus, we investigated the pattern of genetic variation at flanking of pfmdr-1 gene to compare that with the above reduced genetic variation in flanking of pfcrt gene. The data show enormous amount of variation at all the polymorphic microsatellite loci. Reduction in genetic variation was observed in the flanking microsatellite loci of mutant 86Y allele in comparison to the wild N86. The striking observations were; first, the chloroquine resistant pfcrt loci exhibiting reduced variation and chloroquine resistant pfmdr-1 loci exhibiting high variation. However this reduction in variation at flanking loci of mutant pfmdr-1 86Y allele was observed in ~5kb only. Second, the collective data of expected heterozygosity show higher genetic variation in proximity i.e., ~5kb loci in comparison to extended ~10kb loci which is not according to selective sweep model of molecular evolution observed in case pfcrt gene of Indian isolates. Third, the isolate carrying wild type allele at both the genes(CVMNK-N) exhibit higher genetic variation than that of isolates bearing wild type allele at pfmdr-1gene only, which could be associated with any kind of allele at pfcrt gene i.e., wild or mutant. Fourth, the isolates carrying mutant allele at both the genes, CVIET-Y exhibited an unsymmetrical reduction in genetic variation at these flanking loci (reduced variation at -400bp and +4.3kb). These observations support a varied mechanism responsible for generation or maintenance of genetic variation in pfcrt and pfmdr-1gene (Mehlotra et al., 2008). Both genes has different genomic characteristic; pfcrt is a single-copy locus (Fidock et al., 2000) and pfmdr-1 can occur as a single or multiple-copy locus (Triglia et al., 1991). However, we did not evaluate the copy number of pfmdr-1 gene; the possibility of different local recombination rate at both the genes can not be ignored. The difference in drug selection history and selection strength may alter patterns of genetic variation. Probably, strength of chloroquine selection pressure for both the gene is different in Indian isolates. The pfcrt gene is subjected under stronger selection pressure in comparison to pfmdr-1 gene in India. Our observation of rapid spread of mutant K76T in comparison of mutant N86 (Mallick et al., 2012) mark the differential selection pressure on both gene. Finally, the polymorphisms in four microsatellite loci flanking ~ ±5kb of pfmdr-1 gene displayed 94 different haplotypes in comparison to 54 haplotypes in approx. ±24kb of pfcrt gene. These moderately polymorphic wild and mutant-type alleles of pfmdr-1 gene could be evolving repeatedly on different genetic backgrounds. Which in turn will diminish the difference in diversity associated with resistant versus sensitive alleles and the association of individual haplotypes with resistant mutation became less clear in Indian isolates.
Thus, the reduced variation at pfcrt is result of strong chloroquine selection together with low local recombination or microsatellite mutation rates, whereas high level of variation at pfmdr-1 gene is result of weak chloroquine selection pressure together with high local recombination or microsatellite mutation rates.