Epidemic of Mucormycosis in COVID-19 Pandemic: A Position Paper.

During the second wave of COVID-19 pandemic, there is a sudden increase in number of cases mucormycosis infection in India. This communication by the Tropical Neurology subsection expert group of the Indian Academy of Neurology (IAN) describes the clinical and diagnostic features, treatment of the disease and gives recommendations about the ways forward.

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.

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.

Naturally Acquired Binding-Inhibitory Antibodies to Plasmodium vivax Duffy Binding Protein in Pregnant Women Are Associated with Higher Birth Weight in a Multicenter Study.

A vaccine to eliminate malaria would need a multi-stage and multi-species composition to achieve robust protection, but the lack of knowledge about antigen targets and mechanisms of protection precludes the development of fully efficacious malaria vaccines, especially for Plasmodium vivax (Pv). Pregnant women constitute a risk population who would greatly benefit from a vaccine preventing the adverse events of Plasmodium infection during gestation. We hypothesized that functional immune responses against putative targets of naturally acquired immunity to malaria and vaccine candidates will be associated with protection against malaria infection and/or poor outcomes during pregnancy. We measured (i) IgG responses to a large panel of Pv and Plasmodium falciparum (Pf) antigens, (ii) the capacity of anti-Pv ligand Duffy binding protein (PvDBP) antibodies to inhibit binding to Duffy antigen, and (iii) cellular immune responses to two Pv antigens, in a subset of 1,056 pregnant women from Brazil, Colombia, Guatemala, India, and Papua New Guinea (PNG). There were significant intraspecies and interspecies correlations for most antibody responses (e.g., PfMSP119 versus PfAMA1, Spearman's rho = 0.81). Women from PNG and Colombia had the highest levels of IgG overall. Submicroscopic infections seemed sufficient to boost antibody responses in Guatemala but not antigen-specific cellular responses in PNG. Brazil had the highest percentage of Duffy binding inhibition (p-values versus Colombia: 0.040; Guatemala: 0.047; India: 0.003, and PNG: 0.153) despite having low anti-PvDBP IgG levels. Almost all antibodies had a positive association with present infection, and coinfection with the other species increased this association. Anti-PvDBP, anti-PfMSP1, and anti-PfAMA1 IgG levels at recruitment were positively associated with infection at delivery (p-values: 0.010, 0.003, and 0.023, respectively), suggesting that they are markers of malaria exposure. Peripheral blood mononuclear cells from Pv-infected women presented fewer CD8+IFN-γ+ T cells and secreted more G-CSF and IL-4 independently of the stimulus used in vitro. Functional anti-PvDBP levels at recruitment had a positive association with birth weight (difference per doubling antibody levels: 45 g, p-value: 0.046). Thus, naturally acquired binding-inhibitory antibodies to PvDBP might confer protection against poor outcomes of Pv malaria in pregnancy.

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.

Plasmodium vivax VIR Proteins Are Targets of Naturally-Acquired Antibody and T Cell Immune Responses to Malaria in Pregnant Women.

P. vivax infection during pregnancy has been associated with poor outcomes such as anemia, low birth weight and congenital malaria, thus representing an important global health problem. However, no vaccine is currently available for its prevention. Vir genes were the first putative virulent factors associated with P. vivax infections, yet very few studies have examined their potential role as targets of immunity. We investigated the immunogenic properties of five VIR proteins and two long synthetic peptides containing conserved VIR sequences (PvLP1 and PvLP2) in the context of the PregVax cohort study including women from five malaria endemic countries: Brazil, Colombia, Guatemala, India and Papua New Guinea (PNG) at different timepoints during and after pregnancy. Antibody responses against all antigens were detected in all populations, with PNG women presenting the highest levels overall. P. vivax infection at sample collection time was positively associated with antibody levels against PvLP1 (fold-increase: 1.60 at recruitment -first antenatal visit-) and PvLP2 (fold-increase: 1.63 at delivery), and P. falciparum co-infection was found to increase those responses (for PvLP1 at recruitment, fold-increase: 2.25). Levels of IgG against two VIR proteins at delivery were associated with higher birth weight (27 g increase per duplicating antibody levels, p<0.05). Peripheral blood mononuclear cells from PNG uninfected pregnant women had significantly higher antigen-specific IFN-γ TH1 responses (p=0.006) and secreted less pro-inflammatory cytokines TNF and IL-6 after PvLP2 stimulation than P. vivax-infected women (p<0.05). These data demonstrate that VIR antigens induce the natural acquisition of antibody and T cell memory responses that might be important in immunity to P. vivax during pregnancy in very diverse geographical settings.

A cross strain Plasmodium falciparum microarray optimized for the transcriptome analysis of Plasmodium falciparum patient derived isolates.

Malarial parasite P. falciparum, an apicomplexan protozoan has a 23.3 MB nuclear genome and encodes ~ 5600 transcripts. The genetic diversity of the parasite within and across geographical zones is a challenge to gene expression studies which are essential for understanding of disease process, outcome and developing markers for diagnostics and prognostics. Here, we describe the strategy involved in designing a custom P. falciparum 15K array using the Agilent platform and Genotypic's Right Design methodology to study the transcriptome of Indian field isolates for which genome sequence information is limited. The array contains probes representing genome sequences of two distinct geographical isolates (i.e. 3D7 and HB3) and sub-telomeric var gene sequences of a third isolate (IT4) known to adhere in culture condition. Probes in the array have been selected based on their efficiency to detect transcripts through a 244K array experimentation. Array performance for the 15K array, was evaluated and validated using RNA materials from P. falciparum clinical isolates. A large percentage (91%) of the represented transcripts was detected from Indian P. falciparum patient isolates. Replicated probes and multiple probes representing the same gene showed perfect correlation between them suggesting good probe performance. Additional transcripts could be detected due to inclusion of unique probes representing HB3 strain transcripts. Variant surface antigen (VSA) transcripts were detected by optimized probes representing the VSA genes of three geographically distinct strains. The 15K cross strain P. falciparum array has shown good efficiency in detecting transcripts from P. falciparum parasite samples isolated from patients. The low parasite loads and presence of host RNA makes arrays a preferred platform for gene expression studies over RNA-Seq.

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.

Clinicopathological Analysis and Multipronged Quantitative Proteomics Reveal Oxidative Stress and Cytoskeletal Proteins as Possible Markers for Severe Vivax Malaria.

In Plasmodium vivax malaria, mechanisms that trigger transition from uncomplicated to fatal severe infections are obscure. In this multi-disciplinary study we have performed a comprehensive analysis of clinicopathological parameters and serum proteome profiles of vivax malaria patients with different severity levels of infection to investigate pathogenesis of severe malaria and identify surrogate markers of severity. Clinicopathological analysis and proteomics profiling has provided evidences for the modulation of diverse physiological pathways including oxidative stress, cytoskeletal regulation, lipid metabolism and complement cascades in severe malaria. Strikingly, unlike severe falciparum malaria the blood coagulation cascade was not found to be affected adversely in acute P. vivax infection. To the best of our knowledge, this is the first comprehensive proteomics study, which identified some possible cues for severe P. vivax infection. Our results suggest that Superoxide dismutase, Vitronectin, Titin, Apolipoprotein E, Serum amyloid A, and Haptoglobin are potential predictive markers for malaria severity.

Sustained Release Formulation of Primaquine for Prevention of Relapse of Plasmodium vivax Malaria: A Randomized, Double-Blind, Comparative, Multicentric Study.

Background. Primaquine is used to eradicate latent Plasmodium vivax parasite from liver, with administration of standard dose daily up to 14 days. We studied efficacy, safety, and tolerability of sustained release (SR) formulation of primaquine in comparison with conventional primaquine in preventing relapse of P. vivax malaria. Methods. Microscopically confirmed cases of P. vivax malaria received chloroquine therapy for three days. Aparasitemic and asymptomatic patients were then randomized to receive either conventional primaquine 15 mg for 14 days or primaquine SR 15 mg for 14 days, or primaquine SR 30 mg for seven days. Results. Of the 360 patients, who received chloroquine therapy, 358 patients were randomized. Two-hundred eighty-eight patients completed six-month follow-up and four patients (three: conventional primaquine 15 mg (2.86%), one: primaquine SR 30 mg (0.93%)) showed relapse confirmed by PCR genotyping. Drug compliance was significantly better in primaquine SR 30 mg group (95.57%, p = 0.039) without any serious adverse events. Conclusion. Primaquine SR 15 mg and primaquine SR 30 mg could be an effective alternative to conventional primaquine 15 mg due to their comparable cure rates and safety profile. Shorter treatment duration with primaquine SR 30 mg may increase patient compliance and may further reduce relapse rates. Clinical Trial Registration. This trial is registered with CTRI/2010/091/000245.

Disease specific modules and hub genes for intervention strategies: A co-expression network based approach for Plasmodium falciparum clinical isolates.

Systems biology approaches that are based on gene expression and bioinformatics analysis have been successful in predicting the functions of many genes in Plasmodium falciparum, a protozoan parasite responsible for most of the deaths due to malaria. However, approaches that can provide information about the biological processes that are active in this parasite in vivo during complicated malaria conditions have been scarcely deployed. Here we report the analysis of a weighted gene co-expression based network for P. falciparum, from non-cerebral clinical complications. Gene expression profiles of 20 P. falciparum clinical isolates were utilized to construct the same. A total of 20 highly interacting modules were identified post network creation. In 12 of these modules, at least 10% of the member genes, were found to be differentially regulated in parasites from patient isolates showing complications, when compared with those from patients with uncomplicated disease. Enrichment analysis helped identify biological processes like oxidation-reduction, electron transport chain, protein synthesis, ubiquitin dependent catabolic processes, RNA binding and purine nucleotide metabolic processes as associated with these modules. Additionally, for each module, highly connected hub genes were identified. Detailed functional analysis of many of these, which have known annotated functions underline their importance in parasite development and survival. This suggests, that other hub genes with unknown functions may also be playing crucial roles in parasite biology, and, are potential candidates for intervention strategies.

Plasmodium falciparum complicated malaria: Modulation and connectivity between exportome and variant surface antigen gene families.

In temperate and sub-tropical regions of Asia and Latin America, complicated malaria manifested as hepatic dysfunction or renal dysfunction is seen in all age groups. There has been a concerted focus on understanding the patho-physiological and molecular basis of complicated malaria in children, much less is known about it in adults. We report here, the analysis of data from a custom, cross strain microarray (Agilent Platform) using material from adult patient samples, showing hepatic dysfunction or renal failure. These are the most common manifestations seen in adults along with cerebral malaria. The data has been analyzed with reference to variant surface antigens, encoded by the var, rifin and stevor gene families. The differential regulation profiles of key genes (comparison between Plasmodium falciparum complicated and uncomplicated isolates) have been observed. The exportome has been analyzed using similar parameters. Gene ontology term based functional enrichment of differentially regulated genes identified, up-regulated genes statistically enriched (P<0.05) to critical biological processes like generation of precursor metabolite and energy, chromosome organization and electron transport chain. Systems network based functional enrichment of overall differentially regulated genes yielded a similar result. We are reporting here, up-regulation of var group B and C genes whose proteins are predicted to interact with CD36 receptor in the host, the up-regulation of domain cassette 13 (DC13) containing var group A, as also the up-regulation of group A rifins and many of the stevors. This is contrary to most other reports from pediatric patients, with cerebral malaria where the up-regulation of mostly var A group genes have been seen. A protein-protein interaction based network has been created and analysis performed. This co-expression and text mining based network has shown overall connectivity between the variant surface antigens (VSA) and the exportome. The up-regulation of var group B and C genes encoding PfEMP1 with different domain architecture would be important for deciding strategies for disease prevention.

Characterization of Plasmodium vivax-associated admissions to reference hospitals in Brazil and India.

BACKGROUND: The benign character formerly attributed to Plasmodium vivax infection has been dismantled by the increasing number of reports of severe disease associated with infection with this parasite, prompting the need for more thorough and comprehensive characterization of the spectrum of resulting clinical complications. Endemic areas exhibit wide variations regarding severe disease frequency. This study, conducted simultaneously in Brazil and India, constitutes, to our knowledge, the first multisite study focused on clinical characterization of P. vivax severe disease. METHODS: Patients admitted with P. vivax mono-infection at reference centers in Manaus (Amazon - Brazil) and Bikaner (Rajasthan - India), where P. vivax predominates, were submitted to standard thorough clinical and laboratory evaluations in order to characterize clinical manifestations and identify concurrent co-morbidities. RESULTS: In total, 778 patients (88.0% above 12 years old) were hospitalized at clinical discretion with PCR-confirmed P. vivax mono-infection (316 in Manaus and 462 in Bikaner), of which 197 (25.3%) presented at least one severity criterion as defined by the World Health Organization (2010). Hyperlactatemia, respiratory distress, hypoglycemia, and disseminated intravascular coagulation were more frequent in Manaus. Noteworthy, pregnancy status was associated as a risk factor for severe disease (OR = 2.03; 95% CI = 1.2-3.4; P = 0.007). The overall case fatality rate was 0.3/1,000 cases in Manaus and 6.1/1,000 cases in Bikaner, with all deaths occurring among patients fulfilling at least one severity criterion. Within this subgroup, case fatality rates increased respectively to 7.5% in Manaus and 4.4% in Bikaner. CONCLUSION: P. vivax-associated severity is not negligible, and although lethality observed for complicated cases was similar, the overall fatality rate was about 20-fold higher in India compared to Brazil, highlighting the variability observed in different settings. Our observations highlight that pregnant women and patients with co-morbidities need special attention when infected by this parasite due to higher risk of complications.

Performance of BinaxNOW G6PD deficiency point-of-care diagnostic in P. vivax-infected subjects.

Accurate diagnosis of glucose-6-phosphate dehydrogenase (G6PD) deficiency is required to avoid the risk of acute hemolysis associated with 8-aminoquinoline treatment. The performance of the BinaxNOW G6PD test compared with the quantitative spectrophotometric analysis of G6PD activity was assessed in 356 Plasmodium vivax-infected subjects in Brazil, Peru, Thailand, and India. In the quantitative assay, the median G6PD activity was 8.81 U/g hemoglobin (range = 0.05-20.19), with 11 (3%) subjects identified as deficient. Sensitivity of the BinaxNOW G6PD to detect deficient subjects was 54.5% (6 of 11), and specificity was 100% (345 of 345). Room temperatures inadvertently falling outside the range required to perform the rapid test (18-25°C) together with subtlety of color change and insufficient training could partially explain the low sensitivity found. Ensuring safe use of 8-aminoquinolines depends on additional development of simple, highly sensitive G6PD deficiency diagnostic tests suitable for routine use in malaria-endemic areas.

Clinical and histopathological profile of acute renal failure caused by falciparum and vivax monoinfection: an observational study from Bikaner, northwest zone of Rajasthan, India.

BACKGROUND & OBJECTIVES: Acute renal failure (ARF) is a known manifestation of severe Plasmodium falciparum (Pf) malaria but recently it has also been observed with P. vivax (Pv) monoinfection. A clinical observational study has been conducted to evaluate the clinical and histopathological profile of ARF in malaria. METHODS: This study was conducted on 288 consecutive cases of malaria with monoinfection (Pf 191 and Pv 97) diagnosed by peripheral blood film examination and rapid card test. ARF was diagnosed as per WHO criterion (serum creatinine >3 mg%). The data were analysed by Standard t-test using ANOVA software. RESULTS: ARF was seen in 52 cases of Pf and 14 cases of Pv malaria. Mean age was 32.58 yr (ranging 15-65; Pf 33.37 and Pv 29.14) and male to female ratio was 2:1 (Pf 3:1 and Pv 1:1). Most of the cases developed ARF within 10 days of onset of the disease. Associated severe manifestations were jaundice (53 cases: Pf 44 and Pv 9), cerebral malaria (28 cases: Pf 25 and Pv 3), severe anemia (18 cases: Pf 17 and Pv 1), hypotension (16 cases: Pf 11 and Pv 5), bleeding manifestations (16 cases: Pf 14 and Pv 2), multiorgan failure (12 cases: Pf 9 and Pv 3) and ARDS (6 cases: Pf 5 and Pv 1). Kidney biopsy (16 Pf and 2 Pv) showed acute tubular necrosis (5 Pf and 1 Pv), mesangioproliferative glomerulonephritis (2 Pf) or both (9 Pf and 1 Pv). Haemodialysis was done in 7 (Pf 4 and Pv 3) cases, out of which four survived. Most of the cases (48.49%) recovered within two weeks (range 3-20 days). Total mortality was 27.27% (Pf 28.85% and Pv 21.43%). INTERPRETATION & CONCLUSION: ARF can also be caused by vivax monoinfection with similar clinical and histopathological features although outcome is less severe as compared to falciparum monoinfection.

Natural antisense transcripts in Plasmodium falciparum isolates from patients with complicated malaria.

Mechanisms regulating gene expression in malaria parasites are not well understood. Little is known about how the parasite regulates its gene expression during transition from one developmental stage to another and in response to various environmental conditions. Parasites in a diseased host face environments which differ from the static, well adapted in vitro conditions. Parasites thus need to adapt quickly and effectively to these conditions by establishing transcriptional states which are best suited for better survival. With the discovery of natural antisense transcripts (NATs) in this parasite and considering the various proposed mechanisms by which NATs might regulate gene expression, it has been speculated that these might be playing a critical role in gene regulation. We report here the diversity of NATs in this parasite, using isolates taken directly from patients with differing clinical symptoms caused by malaria infection. Using a custom designed strand specific whole genome microarray, a total of 797 NATs targeted against annotated loci have been detected. Out of these, 545 NATs are unique to this study. The majority of NATs were positively correlated with the expression pattern of the sense transcript. However, 96 genes showed a change in sense/antisense ratio on comparison between uncomplicated and complicated disease conditions. The antisense transcripts map to a broad range of biochemical/metabolic pathways, especially pathways pertaining to the central carbon metabolism and stress related pathways. Our data strongly suggests that a large group of NATs detected here are unannotated transcription units antisense to annotated gene models. The results reveal a previously unknown set of NATs that prevails in this parasite, their differential regulation in disease conditions and mapping to functionally well annotated genes. The results detailed here call for studies to deduce the possible mechanism of action of NATs, which would further help in understanding the in vivo pathological adaptations of these parasites.

Dataset of natural antisense transcripts in P. vivax clinical isolates derived using custom designed strand-specific microarray.

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.