Shilpi Garg

Clinical Features of Children Hospitalized with Malaria—A Study from Bikaner, Northwest India

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.

Thrombocytopenia in Plasmodium falciparum, Plasmodium vivax and mixed infection malaria: A study from Bikaner (Northwestern India)

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.

A glimpse into the clinical proteome of human malaria parasites Plasmodium falciparum and Plasmodium vivax

Malaria causes a worldwide annual mortality of about a million people. Rapidly evolving drug‐resistant species of the parasite have created a pressing need for the identification of new drug targets and vaccine candidates. By developing fractionation protocols to enrich parasites from low‐parasitemia patient samples, we have carried out the first ever proteomics analysis of clinical isolates of early stages of Plasmodium falciparum (Pf) and P. vivax. Patient‐derived malarial parasites were directly processed and analyzed using shotgun proteomics approach using high‐sensitivity MS for protein identification. Our study revealed about 100 parasite‐coded gene products that included many known drug targets such as Pf hypoxanthine guanine phosphoribosyl transferase, Pf L‐lactate dehydrogenase, and Plasmepsins. In addition, our study reports the expression of several parasite proteins in clinical ring stages that have never been reported in the ring stages of the laboratory‐cultivated parasite strain. This proof‐of‐principle study represents a noteworthy step forward in our understanding of pathways elaborated by the parasite within the malaria patient and will pave the way towards identification of new drug and vaccine targets that can aid malaria therapy.

Clinical profiles of 13 children with Plasmodium vivax cerebral malaria

Abstract Background: 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. Aims: To describe the clinical features of PCR-confirmed cerebral malaria owing to P. vivax mono-infection and its clinico-laboratory profile in Bikaner, Northwest India. Methods: This observational prospective study was based on detailed clinical and laboratory investigation of children admitted with cerebral malaria owing to P. vivax between November 2008 and December 2010. Cerebral malaria was categorised according to the WHO (2000) criteria for P. falciparum and the diagnosis of P. vivax mono-infection was established by peripheral blood film and rapid diagnostic tests and confirmed by polymerase chain reaction. The possibility of other diseases/infections causing similar illness were investigated thoroughly. Results: Thirteen children with P. vivax cerebral malaria were studied, eight of whom (61·5%) had multi-organ (two or more organs) dysfunction. Other associated severe manifestations included severe anaemia (7), hepatic dysfunction (2), renal dysfunction (2), bleeding manifestation (2), respiratory distress (2), metabolic acidosis (2) and shock (one). Hypoglycaemia was not observed in any patient. There was no evidence of neurological sequelae. All the children were managed according to WHO guidelines using intravenous artisunate. Thrombocytopenia was detected in five and hyponatraemia in four children. Conclusion: P. vivax mono-infection can cause cerebral malaria and multi-organ dysfunction.

Thrombocytopenia in childhood malaria with special reference to P. vivax monoinfection: A study from Bikaner (Northwestern India)

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.

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

OBJECTIVE 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). METHODS In this study, a multiplex PCR (P. falciparum and P. vivax) was further developed with the incorporation of Plasmodium malariae (P. malariae) specific primer and also a positive control. The performance of microscopy, plasmodium lactate dehydrogenase (pLDH) based malaria rapid diagnostic test OptiMAL(®) and 18S rRNA gene based multiplex PCR for the diagnosis of P. falciparum and P. vivax was compared. RESULTS The three species multiplex PCR (P. falciparum, P. vivax and P. malariae) with an inbuilt positive control was developed and evaluated. In comparison with multiplex PCR, which showed the sensitivity and specificity of 99.36% (95%CI, 98.11%-100.00%) and 100.00% (95%CI, 100.00%-100.00%), the sensitivity and specificity of microscopy was 90.44% (95%CI, 88.84%-95.04%) and 99.22% (95%CI, 97.71%-100.00%), and OptiMAL(®) was 93.58% (95%CI, 89.75%-97.42%) and 97.69% (95%CI, 95.10%-100.00%). The efficiencies were 99.65%, 95.10% and 95.45% for multiplex PCR, microscopy and OptiMAL(®), respectively. CONCLUSIONS Our results raise concerns over the overall sensitivities of microscopy and OptiMAL(®), when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites. This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.

Development and evaluation of a 28S rRNA gene-based nested PCR assay for P. falciparum and P. vivax

Abstract 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.

Novel mutations in the antifolate drug resistance marker genes among Plasmodium vivax isolates exhibiting severe manifestations

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.

Plasmodium vivax apicoplast genome: A comparative analysis of major genes from Indian field isolates ☆

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.

Characterization of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG) from Plasmodium vivax and it’s potential as an antimalarial drug target

The prokaryotic type Methyl Erythritol phosphate (MEP) pathway functional in the apicoplast of Plasmodium is indispensable for the erythrocytic stages of the parasite. It is the sole process of isoprenoids biosynthesis in the parasite and is different from that in humans. Among the seven enzymes known to be functional in the MEP pathway in prokaryotes, most enzymes from Plasmodium are yet uncharacterized. The penultimate enzyme of this pathway 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG), has been shown to act as a key target molecule in prokaryotes, where its deletion results in impairment of many housekeeping functions. The present study is the first detailed report of IspG enzyme from any Plasmodium sp. We report here that the protein is highly conserved across apicomplexans and prokaryotes and it localizes to the apicoplast as evident from the immune-localization studies performed on P. vivax infected blood smears made from clinical patients. The biochemical reconstitution and in silico docking of [4Fe-4S] clusters on the protein indicate their importance for the activity of enzyme. In-silico screening of different drug entities suggested the inhibitory role of alkyne diphosphate analogues and fosmidomycin against the IspG enzyme, suggesting the potential role of this enzyme as an antimalarial target.