Vaccination coverage and breakthrough infections of COVID-19 during the second wave among staff of selected medical institutions in India.

India experienced the second wave of SARS-CoV-2 infection from April 3 to June 10, 2021. During the second wave, Delta variant B.1617.2 emerged as the predominant strain, spiking cases from 12.5 million to 29.3 million (cumulative) by the end of the surge in India. Vaccines against COVID-19 are a potent tool to control and end the pandemic in addition to other control measures. India rolled out its vaccination programme on January 16, 2021, initially with two vaccines that were given emergency authorization-Covaxin (BBV152) and Covishield (ChAdOx1 nCoV- 19). Vaccination was initially started for the elderly (60+) and front-line workers and then gradually opened to different age groups. The second wave hit when vaccination was picking up pace in India. There were instances of vaccinated people (fully and partially) getting infected, and reinfections were also reported. We undertook a survey of staff (front line health care workers and supporting) of 15 medical colleges and research institutes across India to assess the vaccination coverage, incidence of breakthrough infections, and reinfections among them from June 2 to July 10, 2021. A total of 1876 staff participated, and 1484 forms were selected for analysis after removing duplicates and erroneous entries (n = 392). We found that among the respondents at the time of response, 17.6% were unvaccinated, 19.8% were partially vaccinated (received the first dose), and 62.5% were fully vaccinated (received both doses). Incidence of breakthrough infections was 8.7% among the 801 individuals (70/801) tested at least 14 days after the 2nd dose of vaccine. Eight participants reported reinfection in the overall infected group and reinfection incidence rate was 5.1%. Out of (N = 349) infected individuals 243 (69.6%) were unvaccinated and 106 (30.3%) were vaccinated. Our findings reveal the protective effect of vaccination and its role as an essential tool in the struggle against this pandemic.

Plasmodium Iron-Sulfur [Fe-S] cluster assembly protein Dre2 as a plausible target of Artemisinin: Mechanistic insights derived in a prokaryotic heterologous system.

Iron-sulfur (Fe-S) cluster containing proteins have been assigned roles in various essential cellular processes, such as regulation of gene expression, electron transfer, sensing of oxygen and balancing free radical chemistry. However, their role as the drug target remains sparse. Recently the screening of protein alkylation targets for artemisinin in Plasmodium falciparum led to identification of Dre2, a protein involved in redox mechanism for the cytoplasmic Fe-S cluster assembly in different organisms. In the present study, to further explore the interaction between artemisinin and Dre2, we have expressed the Dre2 protein of both P. falciparum and P. vivax in E. coli. The opaque brown colour of the IPTG induced recombinant Plasmodium Dre2 bacterial pellet, suggested iron accumulation as confirmed by the ICP-OES analysis. In addition, overexpression of rPvDre2 in E. coli reduced its viability, growth and increased the ROS levels of bacterial cells, which in turn led to an increase in expression of stress response genes of E. coli such as recA, soxS, mazF. Moreover, the overexpression of rDre2 induced cell death could be rescued by treatment with Artemisinin derivatives suggesting their interaction. The interaction between DHA and PfDre2 was later demonstrated by CETSA and microscale thermophoresis. Overall, this study suggests that Dre2 is the probable target of Artemisinin and the antimalarial activity of DHA/Artemether could also be due to yet unidentified molecular mechanism altering the Dre2 activity in addition to inducing DNA and protein damage.

Study of COVID-19 Infection, its Severity and Outcome in COVID-19 Vaccinated People at Tertiary Health Care Center, North West Rajasthan.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of corona virus-induced disease 19 (COVID-19) that was declared as a global pandemic in March 2020 by the world health organization (WHO). Two vaccines were granted for emergency use by the Central Drugs Standard Control Organization (CDSCO) in India, Covishield® (AstraZeneca's vaccine manufactured by Serum Institute of India) and Covaxin® (manufactured by Bharat Biotech Limited). Sputnik - V has been granted EUA in the month of April 2021. The purpose of this study is to determine the association of COVID-19 infection, its severity and outcome in COVID-19 vaccinated people. METHODS: This was a hospital based prospective cohort study done between March to June 2021 at PBM Associated Group of Hospitals (AGH), Bikaner, Raj. Total 1028 COVID suspected cases consulted in COVID OPD or hospitalized under department of medicine, out of which 146 satisfied the inclusion and exclusion criteria, out of these 146, first 100 cases who gave consent for part of study were selected. RESULTS: Among 100 COVID-19 infected cases, 49 received first dose while rest got both doses. After first dose of vaccination 42.86% had mild and 32.65% had severe clinical infection while after both doses 80.39% had mild and 11.76% had severe clinical infection. On evaluation of HRCT Chest, after first dose 8.16% had normal 40.82% were in severe category while those who got both doses it was 52.82% 3.92% respectively. Among 49 who got first dose, 10.20% recovered on just home based treatment without any need of hospitalization, while 89.8% got admitted in dedicated COVID hospital out of which 73.47% got recovered and 16.33% died. Among 51 who got both the doses, 66.67% recovered on just home based treatment, while 33.33% required hospitalization out of which 25.49% got recovered and 7.84% died. CONCLUSION: After 2nd dose of vaccine there is a significant risk reduction in need of hospitalization and getting severe infection and mortality when compared with first dose only.

Cytokine signatures of Plasmodium vivax infection during pregnancy and delivery outcomes.

Plasmodium vivax malaria is a neglected disease, particularly during pregnancy. Severe vivax malaria is associated with inflammatory responses but in pregnancy immune alterations make it uncertain as to what cytokine signatures predominate, and how the type and quantity of blood immune mediators influence delivery outcomes. We measured the plasma concentrations of a set of thirty-one biomarkers, comprising cytokines, chemokines and growth factors, in 987 plasma samples from a cohort of 572 pregnant women from five malaria-endemic tropical countries and related these concentrations to delivery outcomes (birth weight and hemoglobin levels) and malaria infection. Samples were collected at recruitment (first antenatal visit) and at delivery (periphery, cord and placenta). At recruitment, we found that P. vivax-infected pregnant women had higher plasma concentrations of proinflammatory (IL-6, IL-1ß, CCL4, CCL2, CXCL10) and TH1-related cytokines (mainly IL-12) than uninfected women. This biomarker signature was essentially lost at delivery and was not associated with birth weight nor hemoglobin levels. Antiinflammatory cytokines (IL-10) were positively associated with infection and poor delivery outcomes. CCL11 was the only biomarker to show a negative association with P. vivax infection and its concentration at recruitment was positively associated with hemoglobin levels at delivery. Birth weight was negatively associated with peripheral IL-4 levels at delivery. Our multi-biomarker multicenter study is the first comprehensive one to characterize the immunological signature of P. vivax infection in pregnancy thus far. In conclusion, data show that while TH1 and pro-inflammatory responses are dominant during P. vivax infection in pregnancy, antiinflammatory cytokines may compensate excessive inflammation avoiding poor delivery outcomes, and skewness toward a TH2 response may trigger worse delivery outcomes. CCL11, a chemokine largely neglected in the field of malaria, emerges as an important marker of exposure or mediator in this condition.

Reduction of Sphingosine Kinase 1 Phosphorylation and Activity in Plasmodium-Infected Erythrocytes.

Sphingosine-1-phosphate (S1P), a bioactive lipid mediator is involved in an array of biological processes and linked to pathological manifestations. Erythrocyte is known as the major reservoir for S1P as they lack S1P-degrading enzymes (S1P lyase and S1P phosphohydrolase) and harbor sphingosine kinase-1 (SphK-1) essential for sphingosine conversion to S1P. Reduced S1P concentration in serum was correlated with disease severity in patients with Plasmodium falciparum and Plasmodium vivax infections. Herein, we aimed to identify the underlying mechanism and contribution of host erythrocytes toward depleted S1P levels in Plasmodium-infected patients vs. healthy individuals. The level and activity of SphK-1 were measured in vitro in both uninfected and cultured P. falciparum-infected erythrocytes. Infected erythrocytes demonstrated a significant decrease in SphK-1 level in a time-dependent manner. We found that 10-42 h post invasion (hpi), SphK1 level was predominantly reduced to ∼50% in rings, trophozoites, and schizonts compared to uninfected erythrocytes. We next analyzed the phosphorylation status of SphK-1, a modification responsible for its activity and S1P production, in both uninfected control and Plasmodium-infected erythrocytes. Almost ∼50% decrease in phosphorylation of SphK-1 was observed that could be corroborated with significant reduction in the production and release of S1P in infected erythrocytes. Serum S1P levels were studied in parallel in P. falciparum (N = 15), P. vivax (N = 36)-infected patients, and healthy controls (N = 6). The findings revealed that S1P concentration was significantly depleted in uncomplicated malaria cases and was found to be lowest in complicated malaria and thrombocytopenia in both P. falciparum and P. vivax-infected groups (∗∗ p < 0.01). The lower serum S1P level could be correlated with the reduced platelet count defining the role of S1P level in platelet formation. In conclusion, erythrocyte SphK-1 and S1P levels were studied in Plasmodium-infected individuals and erythrocytes that helped in characterizing the complications associated with malaria and thrombocytopenia, providing insights into the contribution of host erythrocyte biology in malaria pathogenesis. Finally, this study proposes the use of S1P and its analog as a novel adjunct therapy for malaria complications.

Functional analysis of iron-sulfur cluster biogenesis (SUF pathway) from Plasmodium vivax clinical isolates.

Iron-sulfur (Fe-S) clusters are critical metallo-cofactors required for cell function. Assembly of these cofactors is a carefully controlled process in cells to avoid toxicity from free iron and sulfide. In Plasmodium, two pathways for these Fe-S cluster biogenesis have been reported; ISC pathway in the mitochondria and SUF pathway functional in the apicoplast. Amongst these, SUF pathway is reported essential for the apicoplast maintenance and parasite survival. Many of its components have been studied from P. falciparum and P. berghei in recent years, still few queries remain to be addressed; one of them being the assembly and transfer of Fe-S clusters. In this study, using P. vivax clinical isolates, we have shown the in vitro interaction of SUF pathway proteins SufS and SufE responsible for sulfur mobilization in the apicoplast. The sulfur mobilized by the SufSE complex assembles on the scaffold protein PvSufA along with iron provided by the external source. Here, we demonstrate in vitro transfer of these labile Fe-S clusters from the scaffold protein on to an apo-protein, PvIspG (a protein involved in penultimate step of Isoprenoids biosynthesis pathway) in order to provide an insight into the interaction of different components for the biosynthesis and transfer of Fe-S clusters. Our analysis indicate that inspite of the presence of variations in pathway proteins, the overall pathway remains well conserved in the clinical isolates when compared to that reported in lab strains.

Deciphering the role of IspD (2­C­methyl­D­erythritol 4­phosphate cytidyltransferase) enzyme as a potential therapeutic drug target against Plasmodium vivax.

Biosynthesis of isoprenoids (MEP Pathway) in apicoplast has an important role during the erythrocytic stages of Plasmodium, as it is the sole pathway to provide the major isoprene units required as metabolic precursor for various housekeeping activities. With the intensifying need to identify a novel therapeutic drug target against Plasmodium, the MEP pathway and its components are considered as potential therapeutic targets, due to the difference in the isoprenoid synthesis route (MVA) functional in the host cells. While few major components have already been studied from this pathway for their potential as a drug target, IspD (2-C-methyl-D-erythritol-4-phosphate cytidyltransferase) enzyme, the enzyme catalyzing the third step of the pathway has only been tested against a synthetic compound from Malaria box called MMV008138, which also has not shown adequate inhibitory activity against P. vivax IspD. In the present study, to validate the potential of PvIspD as a drug target, various antimicrobial agents were screened for their inhibition possibilities, using in-vitro High Throughput Screening (HTS) technique. Shortlisted antimicrobial drug molecules like Cefepime, Tunicamycin and Rifampicin were further validated by in-vitro biochemical enzyme inhibition assays where they showed activity at nanomolar concentrations suggesting them or their derivatives as prospective future antimalarials. This study also confirmed the in-vivo expression of PvIspD protein during asexual stages by sub-cellular localization in apicoplast and explores the importance of the IspD enzyme in the development of new therapeutics.

A Proteogenomic Analysis of Haptoglobin in Malaria.

SCOPE: Haptoglobin (Hp), an acute phase inflammatory protein is associated with malaria pathogenesis in several proteomics and genomics studies. The Hp gene has two co-dominant alleles: Hp1 and Hp2 that produce three genotypes: Hp1/Hp1, Hp1/Hp2 and Hp2/Hp2. EXPERIMENTAL DESIGN: In this study, validation of the proteomics data with Multiple Reaction Monitoring Mass Spectroscopy (MRM-MS) is performed and the association of the Hp gene variants with severe, non-severe malaria and community (healthy) controls using genotyping PCRs and DNA sequencing is analysed. RESULTS: Highly significant values of Hp is observed in the MRM assay that show a correlation with severity of malaria and is clearly distinguished from another febrile disease, dengue. Moreover, the Hp2/Hp2 genotype is seen in high percentages in non-severe malaria patients (74%) and community controls (72%) whereas patients diagnosed with severe malaria show only (31%) of this genotype. Sequencing of the Hp promoter region reveals three SNPs along with 10 unique haplotypes, out of which five are associated with non-severe and three with severe malaria populations (χ2  = 130; df = 18; p < 0.0001). CONCLUSION AND CLINICAL RELEVANCE: This proteo-genomic study focuses on the correlation of the Hp protein and gene with malaria, thus highlighting the pivotal role of this acute phase immune gene in malaria pathogenesis.

Burden and impact of Plasmodium vivax in pregnancy: A multi-centre prospective observational study.

BACKGROUND: Despite that over 90 million pregnancies are at risk of Plasmodium vivax infection annually, little is known about the epidemiology and impact of the infection in pregnancy. METHODOLOGY AND PRINCIPAL FINDINGS: We undertook a health facility-based prospective observational study in pregnant women from Guatemala (GT), Colombia (CO), Brazil (BR), India (IN) and Papua New Guinea PNG). Malaria and anemia were determined during pregnancy and fetal outcomes assessed at delivery. A total of 9388 women were enrolled at antennal care (ANC), of whom 53% (4957) were followed until delivery. Prevalence of P. vivax monoinfection in maternal blood at delivery was 0.4% (20/4461) by microscopy [GT 0.1%, CO 0.5%, BR 0.1%, IN 0.2%, PNG 1.2%] and 7% (104/1488) by PCR. P. falciparum monoinfection was found in 0.5% (22/4463) of women by microscopy [GT 0%, CO 0.5%, BR 0%, IN 0%, PNG 2%]. P. vivax infection was observed in 0.4% (14/3725) of placentas examined by microscopy and in 3.7% (19/508) by PCR. P. vivax in newborn blood was detected in 0.02% (1/4302) of samples examined by microscopy [in cord blood; 0.05% (2/4040) by microscopy, and 2.6% (13/497) by PCR]. Clinical P. vivax infection was associated with increased risk of maternal anemia (Odds Ratio-OR, 5.48, [95% CI 1.83-16.41]; p = 0.009), while submicroscopic vivax infection was not associated with increased risk of moderate-severe anemia (Hb<8g/dL) (OR, 1.16, [95% CI 0.52-2.59]; p = 0.717), or low birth weight (<2500g) (OR, 0.52, [95% CI, 0.23-1.16]; p = 0.110). CONCLUSIONS: In this multicenter study, the prevalence of P. vivax infection in pregnancy by microscopy was overall low across all endemic study sites; however, molecular methods revealed a significant number of submicroscopic infections. Clinical vivax infection in pregnancy was associated with maternal anemia, which may be deleterious for infant's health. These results may help to guide maternal health programs in settings where vivax malaria is endemic; they also highlight the need of addressing a vulnerable population such as pregnant women while embracing malaria elimination in endemic countries.

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.

Design, construction and validation of a Plasmodium vivax microarray for the transcriptome profiling of clinical isolates.

High density oligonucleotide microarrays have been used on Plasmodium vivax field isolates to estimate whole genome expression. However, no microarray platform has been experimentally optimized for studying the transcriptome of field isolates. In the present study, we adopted both bioinformatics and experimental testing approaches to select best optimized probes suitable for detecting parasite transcripts from field samples and included them in designing a custom 15K P. vivax microarray. This microarray has long oligonucleotide probes (60mer) that were in-situ synthesized onto glass slides using Agilent SurePrint technology and has been developed into an 8X15K format (8 identical arrays on a single slide). Probes in this array were experimentally validated and represents 4180 P. vivax genes in sense orientation, of which 1219 genes have also probes in antisense orientation. Validation of the 15K array by using field samples (n=14) has shown 99% of parasite transcript detection from any of the samples. Correlation analysis between duplicate probes (n=85) present in the arrays showed perfect correlation (r2=0.98) indicating the reproducibility. Multiple probes representing the same gene exhibited similar kind of expression pattern across the samples (positive correlation, r≥0.6). Comparison of hybridization data with the previous studies and quantitative real-time PCR experiments were performed to highlight the microarray validation procedure. This array is unique in its design, and results indicate that the array is sensitive and reproducible. Hence, this microarray could be a valuable functional genomics tool to generate reliable expression data from P. vivax field isolates.

Retinopathy of vivax malaria in adults and its relation with severity parameters.

Malarial retinopathy is a set of retinal signs in severe malaria due to falciparum malaria. With increased recognition of severe manifestations of vivax malaria, a systematic study to evaluate retinal changes in vivax malaria could elaborate our knowledge about this neglected entity. This observational study included retinal examination of 104 adult patients (>14 years) with varying severity of vivax malaria admitted to a tertiary care center during peak seasons of 2012 and 2013. Thirty-eight percent of severe cases had a retinal sign as compared to 6% of non-severe cases (p < 0.01). No statistically significant effect of residence or age on the presence of retinopathy was noted. Females were found to be more prone to develop a retinal sign (p < 0.01). Presence of retinal signs was significantly associated with anemia and jaundice. No statistical association was noted for retinal signs to be present in either renal dysfunction or altered thrombocytes count. The most common signs were arteriovenous changes, present in eight cases (19%) of severe malaria and three cases (5%) of non-severe malaria. Retinal hemorrhage was present in five cases (12%) of severe malaria and no case of non-severe malaria. Both superficial and deep hemorrhages were seen including white-centered hemorrhages. Other signs included cotton wool spots, hard exudates, blurred disk margins with spontaneous venous pulsations and bilateral disk edema. A correlation between retinal signs and severity parameters was drawn from the study. This is the first systemic study to evaluate the retinal changes in vivax malaria. Larger prospective studies should be done for further knowledge regarding retinal changes in vivax malaria, especially severe disease. Apart from its clinical significance, it might lead to a better understanding of the pathogenesis of the systemic disease of vivax malaria.

Structural and functional characterization of an iron-sulfur cluster assembly scaffold protein-SufA from Plasmodium vivax.

Iron-sulfur (Fe-S) clusters are utilized as prosthetic groups in all living organisms for diverse range of cellular processes including electron transport in respiration and photosynthesis, sensing of ambient conditions, regulation of gene expression and catalysis. In Plasmodium, two Fe-S cluster biogenesis pathways are reported, of which the Suf pathway in the apicoplast has been shown essential for the erythrocytic stages of the parasite. While the initial components of this pathway detailing the sulfur mobilization have been elucidated, the components required for the assembly and transfer of Fe-S clusters are not reported from the parasite. In Escherichia coli, SufB acts as a scaffold protein and SufA traffics the assembled Fe-S cluster from SufB to target apo-proteins. However, in Plasmodium, the homologs of these proteins are yet to be characterized for their function. Here, we report a putative SufA protein from Plasmodium vivax with signature motifs of A-type scaffold proteins, which is evolutionarily conserved. The presence of the [Fe4S4](3+) cluster under reduced conditions was confirmed by UV-visible and EPR spectroscopy and the interaction of these clusters with the conserved cysteine residues of chains A and B of PvSufA, validates its existence as a dimer, similar to that in E. coli. The H-bond interactions at the PvSufA-SufB interface demonstrate SufA as a scaffold protein in conjunction with SufB for the pre-assembly of Fe-S clusters and their transfer to the target proteins. Co-localization of the protein to the apicoplast further provides an experimental evidence of a functional scaffold protein SufA for the biogenesis of Fe-S clusters in apicoplast of Plasmodium.

Comparison of the safety and efficacy of fixed-dose combination of arterolane maleate and piperaquine phosphate with chloroquine in acute, uncomplicated Plasmodium vivax malaria: a phase III, multicentric, open-label study.

BACKGROUND: Chloroquine has been the treatment of choice for acute vivax malaria for more than 60 years. Malaria caused by Plasmodium vivax has recently shown resistance to chloroquine in some places. This study compared the efficacy and safety of fixed dose combination (FDC) of arterolane maleate and piperaquine phosphate (PQP) with chloroquine in the treatment of uncomplicated vivax malaria. METHODS: Patients aged 13-65 years with confirmed mono-infection of P. vivax along with fever or fever in the previous 48 h were included. The 317 eligible patients were randomly assigned to receive FDC of arterolane maleate and PQP (n = 159) or chloroquine (n = 158) for 3 days. Primaquine was given as an anti-relapse measure on day 3 and continued for 14 consecutive days. Primary efficacy analysis included assessment of the proportion of aparasitaemic and afebrile patients at 72 h. Safety endpoints were analysis of adverse events, vital signs, laboratory data, and abnormalities on electrocardiograph. Patients participated in the study for at least 42 days. RESULTS: In per protocol population, the proportion of aparasitaemic and afebrile patients at 72 h was 100% (140/140) in the FDC of arterolane maleate and PQP group, and 99.3% (145/146) in the chloroquine group (Fisher, p > 0.9999). In intent to treat population, the corresponding value was reported to be 96.9% (154/159) in the FDC of arterolane maleate and PQP group and 98.7 % (156/158) in the chloroquine group (Fisher, p = 0.4479). The median parasite clearance time was 24 h in FDC of arterolane maleate and PQP group and 26 h in chloroquine group (Log-rank, p = 0.2264). Similarly, median fever clearance time was 24 h in both the groups (Log-rank, p = 0.7750). In PP population, day 28 cure rates were 100 % in both the groups (95% CI (96.52, 100.0 for FDC of arterolane maleate and PQP and 96.73, 100.0 in chloroquine group)). Incidence of adverse events was 82.4% in the FDC of arterolane maleate and PQP group and 85.4% in the chloroquine group. Most of the adverse events were mild to moderate in intensity. The commonly reported clinical adverse events in the FDC of arterolane maleate and PQP versus chloroquine group were vomiting (5.0 vs 5.1%), headache (1.3 vs 3.2%) and prolonged QT (1.9 vs 3.2%). No deaths were reported. The pharmacokinetic analysis indicates that arterolane maleate is well absorbed and has a relatively short t1/2 of 3.2 h. Piperaquine is also well absorbed after oral administration with a t1/2 of about 228.33 h. CONCLUSIONS: The study showed that FDC of arterolane maleate and PQP effectively cured vivax malaria and attained acceptable level of cure up to day 28. Both the groups showed similar safety profile. Trial Registration Clinical Trial Registry India: CTRI/2011/11/002129.

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.

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 × 10(3)/mm(3) 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 × 10(3)/mm(3)) 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 × 10(3)/mm(3) [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/mm(3), we advocate it to include as one of the severe malaria criteria.

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.