Browsing by Author "Das, Ashis"

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Analysis of elongation factor Tu (tuf A) of apicoplast from Indian Plasmodium vivax isolates
(Elsiever, 2007-09) Garg, Shilpi; Saxena, Vishal; Das, Ashis
The Apicomplexan parasite Plasmodium vivax is responsible for causing greater than 50% of human malaria cases in Central and South America, Southeastern Asia and the Indian subcontinent. The rising severity of the disease and the resistance shown by the parasite towards usual therapeutic regimen has put forth a demand for a novel drug target to combat this disease. Apicoplast, an organelle of prokaryotic origin, and its circular genome are being looked upon as a potential drug target. The Apicoplast genome is known to carry various genes of functional importance, including the gene encoding for the protein Elongation factor Tu (tuf A) that participates in the translational process in prokaryotes. The tuf A gene is translationally active within the organelle and is believed to be one of the best functionally conserved protein throughout the species. Till date there are no reports of this gene from another major human malaria parasite P. vivax. This is the first report detailing any complete gene analysis from the Apicoplast genome of the Indian P. vivax isolates. The study predicts and evaluates the complete Apicoplast Elongation factor tuf A gene and EF–Tu protein at primary, secondary and tertiary structure level. In addition, a comparative phylogenetic analysis using this gene is done to understand the evolutionary status of Indian P. vivax isolates. Our study shows that although the Indian P. vivax EF–Tu is not showing any major difference at the structural and predicted functional level, it is diverging way ahead from the P. vivax clade.
Clinical Features of Children Hospitalized with Malaria—A Study from Bikaner, Northwest India
(ASTMH, 2010) Garg, Shilpi; Saxena, Vishal; Das, Ashis
Severe Plasmodium vivax malaria in adults has been reported from Bikaner (northwestern India) but the reports on children are scanty. This prospective study was done on 303 admitted children of malaria. The diagnosis was done by peripheral blood smear and rapid diagnostic test. Further confirmation of severe P. vivax monoinfection was done by polymerase chain reaction (PCR). The proportion of P. falciparum, P. vivax, and mixed (P. falciparum and P. vivax) infection was 61.01%, 33.99%, and 4.95%, respectively. Severe disease was present in 49.5% (150/303) children with malaria, with the risk greatest among P. vivax monoinfection (63.1% [65/103]) compared with P. falciparum, either alone (42.7% [79/185]; odds ratio [OR] = 2.3 [95% confidence interval (CI) = 1.40–3.76], P = 0.001) or mixed infections (40% [6/15]; OR = 2.57 [95% CI = 0.88–7.48]). In children < 5 years of age, the proportion of severe malaria attributable to P. vivax rose to 67.4% (31/46) compared with 30.4% (14/46) of P. falciparum (OR = 4.7 [95% CI = 2.6–8.6], P < 0.0001) and 2.2% (1/46) of mixed infection (OR = 92 [95% CI = 24.6–339.9], P < 0.0001). The proportion of patients having severe manifestations, which included severe anemia, thrombocytopenia, cerebral malaria, acute respiratory distress syndrome, hepatic dysfunction, renal dysfunction, abnormal bleeding was significantly high in association with P. vivax monoinfection in 0–5 year age group, while the same was significantly high in association with P. falciparum monoinfection in 5–10 year age group. Similarly P. vivax monoinfection had greatest propensity to cause multiorgan dysfunction in 0–5 year age group (34.1% [17/41], P < 0.0001) in comparison to P. falciparum monoinfection, which had similar propensity in 5–10 year age group (36.8% [35/95], P = 0.039). Plasmodium vivax monoinfection was almost equally serious to cause significant mortality in comparison to P. falciparum (case fatality rate of severe P. vivax was 3.9% versus 3.2% of severe P. falciparum malaria; P = 1.0). This study reaffirms the evidence of severe P. vivax malaria in children in Bikaner.
Clinical profiles of 13 children with Plasmodium vivax cerebral malaria
(Taylor & Francis, 2011) Garg, Shilpi; Das, Ashis
Bikaner region is endemic for both P. vivax and P. falciparum malaria. Usually, cerebral malaria is caused by P. falciparum but it has been reported recently also in P. vivax mono-infection. Epidemiologic studies and clinical descriptions of P. vivax cerebral malaria in children are rare.
Comparative evaluation of microscopy, OptiMAL® and 18S rRNA gene based multiplex PCR for detection of Plasmodium falciparum & Plasmodium vivax from field isolates of Bikaner, India
(Elsiever, 2013-05-13) Garg, Shilpi; Saxena, Vishal; Das, Ashis
To evaluate microscopy, OptiMAL® and multiplex PCR for the identification of Plasmodium falciparumm (P. falciparum) and Plasmodium vivax (P. vivax) from the field isolates of Bikaner, Rajasthan (Northwest India).
Dataset of natural antisense transcripts in P. vivax clinical isolates derived using custom designed strand-specific microarray
(Elsiever, 2014-12) Garg, Shilpi; Saxena, Vishal; Das, Ashis
Natural antisense transcripts (NATs) have been detected in many organisms and shown to regulate gene expression. Similarly, NATs have also been observed in malaria parasites with most studies focused on Plasmodium falciparum. There were no reports on the presence of NATs in Plasmodium vivax, which has also been shown to cause severe malaria like P. falciparum, until a recent study published by us. To identify in vivo prevalence of antisense transcripts in P. vivax clinical isolates, we performed whole genome expression profiling using a custom designed strand-specific microarray that contains probes for both sense and antisense strands. Here we describe the experimental methods and analysis of the microarray data available in Gene Expression Omnibus (GEO) under GSE45165. Our data provides a resource for exploring the presence of antisense transcripts in P. vivax isolated from patients showing varying clinical symptoms. Related information about the description and interpretation of the data can be found in a recent publication by Boopathi and colleagues in Infection, Genetics and Evolution 2013.
Development and evaluation of a 28S rRNA gene-based nested PCR assay for P. falciparum and P. vivax
(Taylor & Francis, 2013-04-12) Garg, Shilpi; Saxena, Vishal; Das, Ashis
The 28S rRNA gene was amplified and sequenced from P. falciparum and P. vivax isolates collected from northwest India. Based upon the sequence diversity of the Plasmodium 28SrRNA gene in comparison with its human counterpart, various nested polymerase chain reaction (PCR) primers were designed from the 3R region of the 28SrRNA gene and evaluated on field isolates. This is the first report demonstrating the utility of this gene for species-specific diagnosis of malaria for these two species, prevalent in India. The initial evaluation on 363 clinical isolates indicated that, in comparison with microscopy, which showed sensitivity and specificity of 85·39% and 100% respectively, the sensitivity and specificity of the nested PCR assay was found to be 99·08% and 100% respectively. This assay was also successful in detecting mixed infections that are undetected by microscopy. Our results demonstrate the utility of the 28S rRNA gene as a diagnostic target for the detection of the major plasmodial species infecting humans.
Genomic dynamics of clinical Plasmodium vivax: comparative genomic hybridization in severe malaria cases
(Frontiers Media, 2025-11) Das, Ashis
Copy number variations (CNVs) in the Plasmodium vivax genome can influence key parasite traits such as erythrocyte invasion, immune evasion, drug resistance, and survival in the human host. Their potential role in severe manifestations of P. vivax malaria, such as cerebral malaria (CM) remains underexplored. In regions like India, where P. vivax is endemic, understanding genomic factors that contribute to disease severity is crucial. Given the limited understanding of genomic factors contributing to disease severity in P. vivax, this study aims to investigate genome-wide CNVs in clinical isolates from patients with cerebral and uncomplicated malaria.
An in vivo transcriptome data set of natural antisense transcripts from Plasmodium falciparum clinical isolates
(Elsiever, 2014-12) Garg, Shilpi; Saxena, Vishal; Das, Ashis
Antisense transcription is pervasive among biological systems and one of the products of antisense transcription is natural antisense transcripts (NATs). Emerging evidences suggest that they are key regulators of gene expression. With the discovery of NATs in Plasmodium falciparum, it has been suggested that these might also be playing regulatory roles in this parasite. However, all the reports describing the diversity of NATs have come from parasites in culture condition except for a recent study published by us. In order to explore the in vivo diversity of NATs in P. falciparum clinical isolates, we performed a whole genome expression profiling using a strand-specific 244 K microarray that contains probes for both sense and antisense transcripts. In this report, we describe the experimental procedure and analysis thereof of the microarray data published recently in Gene Expression Omnibus (GEO) under accession number GSE44921. This published data provide a wealth of information about the prevalence of NATs in P. falciparum clinical isolates from patients with diverse malaria related disease conditions. Supplementary information about the description and interpretation of the data can be found in a recent publication by Subudhi et al. in Experimental Parasitology (2014).
Integrative transcriptomic and machine learning approaches to decipher mitochondrial gene regulation in severe Plasmodium vivax malaria
(Springer, 2025-12) Das, Ashis
Mitochondria in Plasmodium vivax are functionally vital despite possessing a highly reduced genome and differing substantially from the human organelle. Beyond their classical role in energy production, they dynamically coordinate processes like pyrimidine biosynthesis and heme metabolism, adapting their functions across the intra-erythrocytic development cycle (IDC). Their unique architecture and stage-specific roles enable the parasite to fine-tune mitochondrial gene expression, involving both protein-coding sense transcripts and long non-coding natural antisense transcripts (NATs). This study unveils an unprecedented regulatory complexity by integrating transcriptomic profiling with advanced machine learning to decode the role of mitochondrial sense and natural antisense transcripts (NATs) in severe P. vivax malaria. We reveal distinct, clinically relevant expression signatures, where NATs emerge not as transcriptional by-products but as potent regulators tightly linked to mitochondrial pathways and translational machinery. This dual-layered transcriptomic landscape reflects an intricate molecular strategy by which the parasite fine-tunes mitochondrial function to survive under severe disease conditions. Importantly, while these findings illuminate novel regulatory mechanisms and position mitochondrial NATs as promising targets for antimalarial drug development, they represent preliminary insights derived from a limited clinical cohort and should not be interpreted as definitive clinical indicators. Validation in larger and diverse patient populations is essential to confirm their broader biological and clinical relevance. However, these results serve as indicators for potential innovative therapeutic interventions aimed at disrupting parasite bioenergetics and regulatory networks.
Novel mutations in the antifolate drug resistance marker genes among Plasmodium vivax isolates exhibiting severe manifestations
(Elsiever, 2012-12) Garg, Shilpi; Saxena, Vishal; Chowdhury, Shibasish; Das, Ashis
Plasmodium vivax is the predominant species of the human malaria parasite present in the Indian subcontinent. There have been recent reports on Chloroquine (CQ) resistance and severe manifestations shown by P. vivax from different regions of the world including India. This study focuses on Bikaner, India where during the last few years there have been continuous reports of severe manifestations by both Plasmodium falciparum and P. vivax. This region has a widespread use of Chloroquine and Sulfadoxine–Pyrimethamine for the treatment of malaria, but the resistance profiles of these drugs are not available. We report here the profile of mutations in marker genes associated with Chloroquine and antifolate drug resistance among the P. vivax parasites obtained from patients with severe (n = 30) and non-severe (n = 48) manifestations from this region. Most isolates showed the wild type alleles for both the Chloroquine and antifolate resistance markers (P < 0.0005). Except for one isolate showing Y976F mutation in the Pvmdr-1 gene, no reported mutation was observed in the Pvmdr-1 or Pvcrt gene. This is in accordance with the fact that till date no Chloroquine resistance has been reported from this region. However, the single isolate with a mutation in Pvmdr-1 may suggest the beginning of the trend towards decreased susceptibility to Chloroquine. The frequency of PvDHFR–PvDHPS two locus mutations was higher among the patients showing severe manifestations than the patient group with non-severe (uncomplicated) malaria (P < 0.003). None of the parasites from patients with uncomplicated P. vivax malaria showed the mutant PvDHPS genotype. Novel mutations in PvDHFR (S117H) and PvDHPS (F365L, D459A and M601I) were observed only in the parasite population obtained from patients exhibiting severe complications. Preliminary homology modeling and molecular docking studies predicted that these mutations apparently do not have any effect on the binding of the drug molecule to the enzyme. However, the presence of novel mutations in the PvDHPS gene indicate a degree of polymorphism of this molecule which is in contrast to available published information.
Novel point mutations in sulfadoxine resistance genes of Plasmodium falciparum from India
(Elsiever, 2009-04) Garg, Shilpi; Saxena, Vishal; Das, Ashis
Point mutations in the dhfr and dhps genes of Plasmodium falciparum are associated with pyrimethamine and sulfadoxine resistance respectively. In this study we have analyzed these genes from Bikaner (situated in North-West region of India), where both uncomplicated and severe manifestations of P. falciparum malaria are seen. A majority of isolates showed double mutant allele for DHFR. In contrast, the only reported mutation present in DHPS was A437G in few isolates. In addition, three novel non-synonymous mutations were observed in the PfDHPS from this region viz., S587F, N666K and C668W. The mutations at the 666 and 668 codon seem to form a bend in the big loop region of the DHPS enzyme and may affect the binding of the drug to the enzyme. Molecular docking of sulfadoxine to this mutated structure indicates reduction in its binding affinity to this enzyme
Plasmodium falciparum: genetic diversity of C-terminal region of MSP-1 in isolates from Indian sub-continent
(Elsiever, 2005-08) Saxena, Vishal; Das, Ashis
Malaria parasites exhibit sequence diversity for a number of stage specific antigens. Several studies have proved that merozoite surface protein-1 (MSP-1) is an effective target eliciting a protective immune response. The MSP-142 region comprising two EGF-like domains is involved in generating protective immune response in humans and other experimental animals. Searching for point mutations in this region is essential in view of vaccine development. We have investigated the sequence variations in Plasmodium falciparum MSP-1 carboxy terminal region in field isolates from different regions in India. Our study reveals the presence of eight variant types of MSP-119 in the Indian sub-continent, which comprise of E-TSR-L, Q-TSR-L, E-TSG-L, Q-KNG-L, Q-KNG-F, E-KNG-L, E-KNG-F, and E-KYG-F. The last named allele is a novel variant being reported for the first time.
Plasmodium vivax apicoplast genome: A comparative analysis of major genes from Indian field isolates
(Elsiever, 2012-04) Garg, Shilpi; Saxena, Vishal; Das, Ashis
The apicomplexan parasite Plasmodium vivax is responsible for causing more than 70% of human malaria cases in Central and South America, Southeastern Asia and the Indian subcontinent. The rising severity of the disease and the increasing incidences of resistance shown by this parasite towards usual therapeutic regimens have necessitated investigation of putative novel drug targets to combat this disease. The apicoplast, an organelle of procaryotic origin, and its circular genome carrying genes of possible functional importance, are being looked upon as potential drug targets. The genes on this circular genome are believed to be highly conserved among all Plasmodium species. Till date, the plastid genome of P. falciparum, P. berghei and P. chabaudi have been detailed while partial sequences of some genes from other parasites including P. vivax have been studied for identifying evolutionary positions of these parasites. The functional aspects and significance of most of these genes are still hypothetical. In one of our previous reports, we have detailed the complete sequence, as well as structural and functional characteristics of the Elongation factor encoding tufA gene from the plastid genome of P. vivax. We present here the sequences of large and small subunit rRNA (lsu and ssu rRNA) genes, sufB (ORF470) gene, RNA polymerase (rpo B, C) subunit genes and clpC (casienolytic protease) gene from the plastid genome of P. vivax. A comparative analysis of these genes between P. vivax and P. falciparum reveals approximately 5–16% differences. A codon usage analysis of major plastid genes has shown a high frequency of codons rich in A/T at any or all of the three positions in all the species. TTA, AAT, AAA, TAT, and ATA are the major preferred codons. The sequences, functional domains and structural analysis of respective proteins do not show any variations in the active sites. A comparative analysis of these Indian P. vivax plastid genome encoded genes has also been done to understand the evolutionary position of the Indian parasite in comparison to other Plasmodium species.
A prospective study on adult patients of severe malaria caused by Plasmodium falciparum, Plasmodium vivax and mixed infection from Bikaner, northwest India
(JVBD, 2014-10) Garg, Shilpi; Das, Ashis
Description of severe vivax malaria and mixed species infection requires good clinical study. The present study was undertaken to evalute the characteristics of severe malaria patients in Bikaner, northwest India.
Thrombocytopenia in childhood malaria with special reference to P. vivax monoinfection: A study from Bikaner (Northwestern India)
(Taylor & Francis, 2011-08-24) Garg, Shilpi; Das, Ashis
Thrombocytopenia is commonly seen in Plasmodium vivax malaria, but its prognostic value has not been addressed in children. This prospective study included 676 admitted children of malaria [Plasmodium falciparum (Pf) monoinfection 262, Plasmodium vivax (Pv) monoinfection 380, and mixed (Pf + Pv) infection 34], in which thrombocytopenia (platelet count <150 × 103/mm3 on admission) was found in 442 (65.38%) children [Pf monoinfection 55.3% (145/262), Pv monoinfection 73.16% (278/380), and mixed infection 55.88% (19/34)]. The association of thrombocytopenia was statistically significant with Pv monoinfection [73.16% (278/380)] in comparison to either Pf monoinfection [55.34% (145/262); odds ratio (OR) = 2.199 (95% confidence interval (CI) 1.577–3.068), p < 0.0001] or mixed infection [55.88% (19/34); OR = 2.152 (95%CI 1.054–4.394), p = 0.032]. In Pv monoinfection, thrombocytopenia was highest in 0–5 years age group and subsequently decreased with advancing age, whereas in Pf monoinfection it was reverse. Severe thrombocytopenia (platelet count <20 × 103/mm3) was present in 16.52% (73/442) children [Pv monoinfection 21.58% (60/278) and Pf monoinfection 8.97% (13/145)]. The risk of developing severe thrombocytopenia was also highest in Pv monoinfection [15.79% (60/380)] in comparison to Pf monoinfection [10.59% (13/262); OR = 3.591 (95%CI 1.928–6.690), p < 0.0001]. Bleeding manifestations were associated in 21.27% (94/442) children [Pf monoinfection 9.92% (26/262), Pv monoinfection 16.58% (63/380), and mixed malaria 14.71% (5/34)]. All the children having bleeding manifestations had thrombocytopenia but low platelet counts were not always associated with abnormal bleeding. The association of severe malaria was significantly more among children having Pv monoinfection with platelet counts <20 × 103/mm3 [OR = 2.569 (95%CI 1.196–5.517), p < 0.014] with specificity of 88.3% and positive predictive value of 85%. Till today, thrombocytopenia is not included in severe malaria criterion described by WHO, but when platelet counts <20 × 103/mm3, we advocate it to include as one of the severe malaria criteria.
Thrombocytopenia in Plasmodium falciparum, Plasmodium vivax and mixed infection malaria: A study from Bikaner (Northwestern India)
(Taylor & Francis, 2010) Garg, Shilpi; Saxena, Vishal; Das, Ashis
The occurrence, relation and magnitude of thrombocytopenia in different species of malaria are not clearly defined. This study included 1,064 patients admitted with malaria to study thrombocytopenia (platelet count <150,000 /cumm) in Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) mono infection and mixed infection (Pf + Pv). The species diagnosis was done by peripheral blood film (PBF) and rapid diagnostic test (RDT). Validation by polymerase chain reaction (PCR) was done only in patients with severe thrombocytopenia (platelet count <20,000 /cumm). The breakup of patients was 525 (49.34%) Pf, 460 (43.23%) Pv and 79 (7.42%) mixed malaria (Pf + Pv). Thrombocytopenia was observed in 24.6% (262/1064) patients. The risk was greatest in the mixed infections in comparison to monoinfection individually (43.04% [34/79]; mixed vs Pv monoinfection: Odds Ratio [OR] = 1.675 [95% Confidence Interval (CI) 1.029–2.726], p < 0.0366; mixed vs Pf monoinfection: OR=3.911 [95% CI 2.367–6.463], p < 0.0001). Pv monoinfection (31.09% [143/460]) had greater risk compared to Pf monoinfection (16.19% [85/525]; OR = 2.335 [95% CI 1.722–3.167], p < 0.0001). The occurrence of severe thrombocytopenia was also higher in Pv monoinfection (18.18% [26/143]) in comparison to either Pf monoinfection (10.59% [9/85], OR = 1.877 (95% CI 0.834–4.223)) or mixed infection (11.76% [4/34]; OR = 1.667 (95% CI 0.540–5.142) but this association was statistically not significant. Six patients (3 Pv, 2 Pf and 1 mixed) developed severe epistaxis requiring platelet transfusion. There was no relation between parasite density and platelet count as many patients with severe thrombocytopenia had parasite density similar to patients without thrombocytopenia. We found that the association of thrombocytopenia was statistically more significant with P. vivax monoinfection as compared to P. falciparum.