Genetic diversity and allelic variation in MSP3α gene of paired clinical Plasmodium vivax isolates from Delhi, India.

BACKGROUND: Plasmodium vivax malaria accounts for 80% of the malaria cases in Delhi, India. The gene merozoite surface protein 3 alpha (MSP3α) is highly polymorphic and has been used as marker in many P. vivax population studies. METHODS: MSP3α has been used to assess the genetic diversity of P. vivax samples from Delhi (India) having more than one malaria episode (s) i.e. clinically identified relapse cases using PCR-RFLP and sequencing. RESULTS: Three major genotypes 2.0 kb (A), 1.4 kb (B) and 1.2 kb (C) were amplified from 72 isolates with frequencies of 72.2%, 19.44% and 9.72% respectively. One sample out of 72 showed mixed infection having both A and B type genotypes. 82.05% patients showed same genotype while only 17.94% patients showed different genotypes after subsequent malaria episodes. 18 different genotypes with Alu I and 35 with Hha I were identified among 72 samples analyzed by restriction fragment length polymorphism (RFLP). 18 Pvmsp3α nucleotide sequences were analyzed and it did not reveal any distinct intragenic differences within sequences of the same type, however, allelic diversity among the three types (Π = 0.029703) was observed. Phylogenetic analysis showed allelic family types A, B and C were not clustered but distributed in different branches. The results indicate that the P. vivax parasite population is highly diverse in Delhi, India. A large number of amino acid substitutions were found at the locus of the isolates when compared with the Belem Strain (Π = 0.030528). The substantial sequence diversity is largely restricted to certain domains of encoded protein. Analysis of synonymous and nonsynonymous substitutions suggested that different selection forces were operating on different regions of the protein molecule. CONCLUSION: We propose that genotyping of the PvMSP-3α gene as one of the molecular tools for differentiating relapse from new infection in epidemiological settings. The analyses of sequence polymorphism in PvMSP-3α gene enable it as potential candidate for inclusion in a P.vivax vaccine research.

Role of IL-1ß, IL-6 and TNF-α cytokines and TNF-α promoter variability in Plasmodium vivax infection during pregnancy in endemic population of Jharkhand, India.

BACKGROUND: The combinatorial effects of Plasmodium infection, perturbation of inflammatory responses and the dichotomic role of TNF promoter polymorphism has potential clinical and physiological relevance during pregnancy. OBJECTIVE AND METHODS: This coordinated orchestration instigated us to investigate the circulating level of inflammatory cytokines (IL-1ß, TNF-α and IL-6) employing ELISA in a stratified group of samples and the plausible genetic association of TNF-α -308 G/A using PCR-RFLP/sequencing during Plasmodium vivax infection in pregnancy. RESULTS: We observed significantly elevated concentrations of IL-1ß were observed, followed by IL-6 and TNF-α in women with malaria (WWM) and in malaria in pregnancy (MIP). Further, elevated IL-1ß, followed by TNF-α and IL-6 were detected in the non-infected pregnancy group. The differential dynamics of inflammatory cytokine concentration during each trimester of pregnancy with and without P. vivax infection were detected. For the first time, a high level of IL-6 was observed in the first trimester of MIP and high IL-1ß in healthy pregnancies. In the second trimester, however, we observed a high level of IL-1ß in the MIP group compared to a sustained high level of IL-1ß in the healthy pregnancy group. In the third trimester, high IL-1ß was sustained in the MIP group and healthy pregnancies acquired a high TNF-α level. The genotypic distribution for the TNF-α promoter -308 G/A position was observed to be nonsignificant and mildly associated during MIP (OR = 1.4) and in WWM (OR = 1.2). Moreover, based on genotypic distribution, we observed a well-correlated and significantly elevated TNF-α concentration in the mutant homozygote genotype (AA; p = 0.001) followed by heterozygotes (GA; p = 0.0001) and ancestral genotypes (GG; p = 0.0001) in both MIP and WWM subjects. CONCLUSION: The observation of elevated IL-1ß and IL-6 in MIP and TNF-α in WWM may be regarded as a prognostic inflammatory marker of infection and pregnancy. Most particularly, the TNF-α concentration and its polymorphic variability in the promoter region may indicate genetic susceptibility and mildly influence the risk for P. vivax infection during pregnancy and in women with malaria.

iNOS polymorphism modulates iNOS/NO expression via impaired antioxidant and ROS content in P. vivax and P. falciparum infection.

Nitric oxide (NO) has dicotomic influence on modulating host-parasite interplay, synchronizing physiological orchestrations and diagnostic potential; instigated us to investigate the plausible association and genetic regulation among NO level, components of oxidative stress, iNOS polymorphisms and risk of malaria. Here, we experimentally elucidate that iNOS promoter polymorphisms are associated with risk of malaria; employing mutation specific genotyping, functional interplay using western blot and RT-PCR, quantitative estimation of NO, total antioxidant content (TAC) and reactive oxygen species (ROS). Genotyping revealed significantly associated risk of P. vivax (adjusted OR = 1.92 and 1.72) and P. falciparum (adjusted OR = 1.68 and 1.75) infection with SNP at iNOS-954G/C and iNOS-1173C/T positions, respectively; though vivax showed higher risk of infection. Intriguingly, mutation and infection specific differential upregulation of iNOS expression/NO level was observed and found to be significantly associated with mutant genotypes. Moreover, P. vivax showed pronounced iNOS protein (2.4 fold) and mRNA (2.5 fold) expression relative to healthy subjects. Furthermore, TAC and ROS were significantly decreased in infection; and differentially decreased in mutant genotypes. Our findings endorse polymorphic regulation of iNOS expression, altered oxidant-antioxidant components and evidences of risk association as the hallmark of malaria pathogenesis. iNOS/NO may serve as potential diagnostic marker in assessing clinical malaria.

Polymorphism in glutathione S-transferase P1 is associated with susceptibility to Plasmodium vivax malaria compared to P. falciparum and upregulates the GST level during malarial infection.

Glutathione S-transferase P1 (GSTP1) is a member of the GST superfamily, which has well-established multiple roles in various infectious and parasitic diseases. The genetic regulation of GSTP1 has been extensively studied. Thus, its biological significance and disease association prompted us to investigate the role of GSTP1 polymorphisms in Plasmodium-mediated pathogenesis in infected humans. The genotypic distribution of Ile105Val in Plasmodium vivax infection was observed to be significant and strongly associated (OR=4.5) with the progression of pathology, whereas in P. falciparum infection no significant association was observed compared to healthy subjects. Interestingly, we observed significant elevation of GST in vivax infection, with both genotypes Ile105Val and Val105Val, compared to healthy subjects, whereas in P. falciparum infection we found marginally elevated GST levels of mutated genotypes but significantly depleted compared to healthy subjects. Further, during vivax and falciparum infection overall significant elevations of glutathione, glutathione peroxidase, and GST levels were observed. Expression of both GSTP1 mRNA and protein was significantly upregulated during vivax infection compared to falciparum infection and both were significantly upregulated compared to the levels in healthy subjects as well. These studies suggest that GSTP1 polymorphism is involved in the pathogenesis of malaria and it may serve as a valuable molecular marker, possessing a promising rationale for diagnostic potential in assessing disease progression during clinical malaria.

Alleles -308A and -1031C in the TNF-alpha gene promoter do not increase the risk but associated with circulating levels of TNF-alpha and clinical features of vivax malaria in Indian patients.

The biological significance of TNF promoter polymorphism and infectious disease association prompted us to investigate whether TNF-alpha -308 G/A and -1031 T/C promoter polymorphisms are associated with Plasmodium vivax infection, cellular TNF-alpha level and possibly with clinical symptoms by employing PCR-RFLP methods. An overall significant elevation of serum TNF-alpha, IL-6 content (p=0.0002, p=0.002, respectively), whereas highly significant depletion of IL-10 content (p=0.0001) was observed in vivax patients. In addition, TNF-alpha concentration in patients with and without fever were found to be significant (p=0.0001, p=0.0004, respectively). The genotypic distribution for -308 G/A and -1031 T/C positions were found non significant, but it was clinically potent to observe statistically significant distribution of genotypes (p=0.032) in patients with and without fever. Furthermore, the TNF-alpha level in TNF1 and TNF2 genotype for -308 position was significantly higher (p=0.010, p=0.006 respectively). In case of -1031 position TNF-alpha level was significant in ancestral (TT) genotype (p=0.0007) in patients compared to healthy subjects and significantly higher in rare (CC) genotype (p=0.021) as compared to ancestral genotype. In addition, the two polymorphisms 308G/A and -1031T/C were in highly significant LD (D'=0.7992, r(2)=0.6005, p=0.0001) in the patients as well as it is interesting to report that the distribution of novel 308A: 1031C alleles associated haplotypes are nearly the same in patients (0.2610) and in healthy subjects (0.2636). In view of present observation of promoter polymorphism with TNF-alpha level and other clinical parameters of vivax infection, we suggest that evaluation of TNF level and its polymorphisms in the promoter region may be considered to be reliable molecular and immunological markers, possess promising rational for diagnostic potential and immunotherapeutic interventions in clinical vivax malaria. Genetic variation in the promoter region is of biological significance and may play important roles in host defense mechanisms against vivax infection by enhancing cell-mediated immunity and stimulating the protective immunological cascade.

Genetic structure of Plasmodium falciparum field isolates in eastern and north-eastern India.

BACKGROUND: Molecular techniques have facilitated the studies on genetic diversity of Plasmodium species particularly from field isolates collected directly from patients. The msp-1 and msp-2 are highly polymorphic markers and the large allelic polymorphism has been reported in the block 2 of the msp-1 gene and the central repetitive domain (block3) of the msp-2 gene. Families differing in nucleotide sequences and in number of repetitive sequences (length variation) were used for genotyping purposes. As limited reports are available on the genetic diversity existing among Plasmodium falciparum population of India, this report evaluates the extent of genetic diversity in the field isolates of P. falciparum in eastern and north-eastern regions of India. METHODS: A study was designed to assess the diversity of msp-1 and msp-2 among the field isolates from India using allele specific nested PCR assays and sequence analysis. Field isolates were collected from five sites distributed in three states namely, Assam, West Bengal and Orissa. RESULTS: P. falciparum isolates of the study sites are highly diverse in respect of length as well as sequence motifs with prevalence of all the reported allelic families of msp-1 and msp-2. Prevalence of identical allelic composition as well as high level of sequence identity of alleles suggest a considerable amount of gene flow between the P. falciparum populations of different states. A comparatively higher proportion of multiclonal isolates as well as multiplicity of infection (MOI) was observed among isolates of highly malarious districts Karbi Anglong (Assam) and Sundergarh (Orissa). In all the five sites, R033 family of msp-1 was observed to be monomorphic with an allele size of 150/160 bp. The observed 80-90% sequence identity of Indian isolates with data of other regions suggests that Indian P. falciparum population is a mixture of different strains. CONCLUSION: The present study shows that the 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, block 3). As expected Indian isolates present a picture of diversity closer to southeast Asia, Papua New Guinea and Latin American countries, regions with low to meso-endemicity of malaria in comparison to African regions of hyper- to holo-endemicity.

Decreased glutathione-S-transferase activity: diagnostic and protective role in vivax malaria.

OBJECTIVES: The study was undertaken to establish data on the comparative status of antioxidant enzyme GST activity, levels of lipid peroxidation and catalase activity during pathology of Plasmodium vivax malaria in Indian population. We investigated whether serum and plasma glutathione-S-transferase activity in vivax patients are unique to the disease or act as one of the important antioxidant marker for diagnostic potential and candidate for chemoprevention. METHODS: We measured activity of antioxidant enzyme GST, levels of lipid peroxidation and catalase activity during vivax infection. RESULTS: Mean activity of antioxidant enzyme GST in patients serum and plasma were less (6.43 and 5.65 IU/L respectively) than healthy subjects (11.65 and 10.09 IU/L respectively). Lipid peroxidation level and catalase activity of patients (1.77 micromol/L and 29.64 U/mL) with vivax malaria were higher than those of healthy subjects (1.03 micromol/L and 10.87 U/mL). GST activity in serum and plasma was inversely correlated with age in case of vivax patient and were found significant (R2=0.1907 and 0.1605 and p<0.0007 and p<0.01). CONCLUSIONS: In view of the present findings we suggest that GST, lipid peroxidation and catalase evaluation may be considered to be reliable biochemical markers and possess promising rational for diagnostic and therapeutic potential in vivax malaria. Decreasing GST activity and elevated activity of lipid peroxidation and catalase may play important roles in host defence mechanisms against vivax infection by up-regulating oxidative defence mechanisms.