Ashis Das

Plasmodium vivax Malaria

We report 11 cases of severe Plasmodium vivax malaria in Bikaner (western India). Patients exhibited cerebral malaria, renal failure, circulatory collapse, severe anemia, hemoglobinurea, abnormal bleeding, acute respiratory distress syndrome, and jaundice. Peripheral blood microscopy, parasite antigen–based assays, and parasite 18s rRNA gene–based polymerase chain reaction showed the presence of P. vivax and absence of P. falciparum.

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.

An unexpected cause of fever and seizures

In June, 2007, a 27-year-old man was brought to our emergency department by ambulance, having regained consciousness after a generalised tonic-clonic seizure. He had been having fever, chills, and rigors, on alternate days, for the previous 8 days. He had no past history of convulsions, head injury, febrile convulsions during infancy, birth trauma, meningitis, encephalitis, or psychiatric illness. There was no other past medical history of note. Until he fell ill, he had been working in a jewellery shop in Surat, Gujarat—a city where both falciparum and vivax malaria are endemic. On examination, nothing abnormal was found. The patient’s full blood count was normal; biochemistry tests, including a blood glucose measurement, also gave unremarkable results. Electrocardiography, ophthalmoscopy, examination of the cerebrospinal fl uid, and CT of the head all showed nothing of note. However, examination of the blood fi lm showed trophozoites of Plasmodium vivax, at a density of 16 200 per μL (fi gure). A rapid diagnostic test (FalciVax, Zephyr Biomedical Systems, Goa, India) indicated the presence of parasite lactate dehydrogenase, specifi c to P vivax, and the absence of histidine-rich protein 2, specifi c to P falciparum. 6 h after he arrived, the patient had another generalised seizure. He was immediately given intravenous quinine, as per the WHO guidelines for severe vivax malaria; in addition, anticonvulsant drugs were given. Over the next 12 h, the patient had a total of eight generalised seizures, with intervals of 30–120 min, without regaining full consciousness. 48 h after treatment began, the fever subsided, and the patient became fully conscious. Further blood tests—for dengue fever, leptospirosis, and HIV—gave negative results; repeat CT of the head, and electroencephalography, showed nothing remarkable. PCR, which was done as described by Kochar and colleagues, confi rmed that the patient had been infected by P vivax, but not P falciparum. The patient was discharged 8 days after his arrival. When last seen, in August, 2007, he was entirely well. P falciparum is known to cause cerebral malaria, which can manifest with seizures. The parasite multiplies in red blood cells, which adhere to the walls of small blood vessels, causing reduced cerebral blood fl ow. P vivax is less likely than P falciparum to cause severe illness— indeed, the typical 48 h interval between fevers, and benign course, have led to vivax malaria being termed “benign tertian malaria”. Classically, P vivax has not been thought to cause cerebral malaria. However, it is now known that severe P vivax infection can cause cerebral malaria—although, to our knowledge, this is the fi rst case in which the cause of seizures has been confi rmed as P vivax alone. How P vivax causes cerebral malaria is unclear, but recent studies indicate that the mechanism may be similar to that triggered by P falciparum. Other causes of seizures in malaria include hypoglycaemia, hyponatraemia, lactic acidosis—and other illnesses, such as epilepsy.

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 falciparum: Genetic polymorphism in apical membrane antigen-1 gene from Indian isolates ☆

A number of stage-specific antigens have been characterized for vaccine development against Plasmodium falciparum malaria. This study presents a comprehensive analysis of the sequence polymorphism in Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) in population samples from the eastern and western parts of India. This is the first study of its kind for the nearly full length PfAMA-1 gene from these regions in India. Our observations confirmed that sequence diversity of PfAMA-1 confines only to point mutations and shows 4-8% variation as compared to the prototypes. As opposed to the previous studies on PfAMA-1, our study revealed a greater degree of polymorphism in the Domain II region of PfAMA-1 protein, though signature for diversifying selection is seen throughout the gene. Our present investigation also indicates a very high degree of variation in the reported T- and B-cell epitopes of PfAMA-1. Few noteworthy and unique observations made in this study are the substitution of Cysteine residues responsible for the disulfide bond structure of the protein and the presence of premature termination after 595 amino acids in 3 of the 13 isolates under consideration. These crucial findings add new perspectives to the future of AMA-1 research and could have major implications in establishing AMA-1 as a vaccine candidate.