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Cerebral malaria (CM) affects children and adults, but brain swelling is more severe in children. To investigate features associated with brain swelling in malaria, we performed blood profiling and brain MRI in a cohort of pediatric and adult patients with CM in Rourkela, India, and compared them with an African pediatric CM cohort in Malawi. We determined that higher plasma Plasmodium falciparum histidine rich protein 2 (PfHRP2) levels and elevated var transcripts that encode for binding to endothelial protein C receptor (EPCR) were linked to CM at both sites. Machine learning models trained on the African pediatric cohort could classify brain swelling in Indian children CM cases but had weaker performance for adult classification, due to overall lower parasite var transcript levels in this age group and more severe thrombocytopenia in Rourkela adults. Subgrouping of patients with CM revealed higher parasite biomass linked to severe thrombocytopenia and higher Group A-EPCR var transcripts in mild thrombocytopenia. Overall, these findings provide evidence that higher parasite biomass and a subset of Group A-EPCR binding variants are common features in children and adult CM cases, despite age differences in brain swelling.

Malaria is a global socio-economic burden of which Plasmodium vivax contributes for about 70-80 million cases on an annual basis worldwide and 60-65% cases in India. Diversity observed in highly polymorphic Merozoite Surface Protein-3α (msp-3α) encoded by MSP-3 gene family, has been used efficiently for genotyping of P. vivax infection. This study aims to correlate the severity of clinical symptoms with parasite load, genotype of P. vivax and multiplicity of infection. Based on clinical symptoms classification, 31 (67.9%) out of 46 cases were found to be severe while 15 (32.6%) were non-severe and correlation of the severity of vivax infection with parasite load was not observed. Analysis of msp3-α allele genotype showed that out of 31 severe cases, 19 (61.2%) were single-clone infection cases whereas 12 (38.7%) were multi-clone infections. Similarly, out of 15 non-severe cases, 9 (60%) were single clone and 6 (40%) were multi-clone infections indicating the absence of a correlation between the multiplicity of infection and disease severity. Allele frequency observed was 65.9%, 23.4%, 23.4%, and 28.2% for allele A, B, C and D, respectively. An important finding was the greater distribution of allele D than alleles B and C, which has been reported as a rare allele otherwise. Further, of 13 cases with allele D, 76.9% (10/13) cases were severe. This study showed the absence of a correlation between the severity of clinical symptoms with parasite load and multiplicity of infection but at the same time drives a possibility of severe vivax malarial symptoms to have an association with the persistence of allele D in the population. This upon exploration can lead to the development of a target in detection of severe cases of malaria., (Copyright © 2020. Published by Elsevier B.V.)

In recent years, Plasmodium falciparum histidine-rich protein 2 gene deletion has been reported in India. Such isolates are prone to selective transmission and thus form a challenge to case management. As most of the rapid malaria diagnostic tests are based on the detection of HRP2 protein in the blood, we attempted to use Glutamate Dehydrogenase (GDH) as a biomarker for the diagnosis of P. falciparum. Recombinant PfGDH was successfully cloned, expressed and purified using the Ni-NTA approach. Polyclonal antibodies were raised against full-length rPfGDH and its peptides. Antibodies for rPfGDH showed a strong immune response against the recombinant protein. However, antibody showed no affinity towards the peptides, which suggests they failed as antigen. Antibodies for rPfGDH significantly detected the GDH in human blood specimens. This is the first report where P. falciparum GDH was detected in malaria cases from various parts of India. The raised polyclonal antibodies had shown an affinity for PfGDH in quantitative ELISA and are capable to be exploited for RDTs. This research needs further statistical validation on a large number and different sample types from candidates infected with P. falciparum and other species.

Background: In recent times, Plasmodium vivax (P. vivax) has become a serious threat to public health due to its ability to cause severe infection with fatal outcomes. Its unique biology makes it resilient to control measures that are otherwise effective against P. falciparum. A deeper understanding of P. vivax biology and pathogenesis is, therefore, essential for developing the right control strategies. Proteomics of P. falciparum has been helpful in studying disease biology and elucidating molecular mechanisms involved in the development of disease. However, unlike P. falciparum, proteomics data for P. vivax infection is minimal due to the absence of a continuous culture system. The dependence on clinical samples and animal models has drastically limited P. vivax research, creating critical knowledge gaps in our understanding of the disease. This study describes an in-depth proteomics analysis of P. vivax-infected human plasma and parasite isolates, to understand parasite biology, pathogenesis, and to identify new diagnostic targets for P. vivax malaria., Methods: A mass-spectrometry- (MS) based proteomics approach (Q Exactive) was applied to analyze human plasma and parasite isolates from vivax malaria patients visiting a primary health centre in India. Additionally, a targeted proteomics assay was standardized for validating unique peptides of most recurring parasite proteins., Results: Thirty-eight P. vivax proteins were detected in human plasma with high confidence. Several glycolytic enzymes were found along with hypothetical, cytoskeletal, ribosomal, and nuclear proteins. Additionally, 103 highly abundant P. vivax proteins were detected in parasite isolates. This represents the highest number of parasite proteins to be reported from clinical samples so far. Interestingly, five of these; three Plasmodium exported proteins (PVX_003545, PVX_003555 and PVX_121935), a hypothetical protein (PVX_083555) and Pvstp1 (subtelomeric transmembrane protein 1, PVX_094303) were found in both plasma and parasite isolates., Conclusions: A parasite proteomics investigation is essential to understand disease pathobiology and design novel interventions. Control strategies against P. vivax also depend on early diagnosis. This work provides deeper insights into the biology of P. vivax by identifying proteins expressed by the parasite during its complex life-cycle within the human host. The study also reports antigens that may be explored as diagnostic candidates.

Background: Identifying highly immunogenic blood stage antigens which can work as target for naturally acquired antibodies in different eco-epidemiological settings is an important step for designing malaria vaccine. Blood stage proteins of Plasmodium vivax, apical membrane antigen-1 (PvAMA-1) and 19 kDa fragment of merozoite surface protein (PvMSP-1 19 ) are such promising vaccine candidate antigens. This study determined the naturally-acquired antibody response to PvAMA-1 and PvMSP-1 19 antigens in individuals living in three geographically diverse malaria endemic regions of India., Methods: A total of 234 blood samples were collected from individuals living in three different eco-epidemiological settings, Chennai, Nadiad, and Rourkela of India. Indirect ELISA was performed to measure human IgG antibodies against recombinant PvAMA-1 and PvMSP-1 19 antigens. The difference in seroprevalence and factors associated with antibody responses at each site was statistically analysed., Results: The overall seroprevalence was 40.6% for PvAMA-1 and 62.4% for PvMSP-1 19 . Seroprevalence to PvAMA-1 was higher in Chennai (47%) followed by Nadiad (46.7%) and Rourkela (27.6%). For PvMSP-1 19 , seroprevalence was higher in Chennai (80.3%) as compared to Nadiad (53.3%) and Rourkela (57.9%). Seroprevalence for both the antigens were found to be higher in Chennai where P. vivax is the dominant malaria species. In addition, heterogeneous antibody response was observed for PvAMA-1 and PvMSP-1 19 antigens at each of the study sites. Two factors, age and malaria positivity were significantly associated with seropositivity for both the antigens PvAMA-1 and PvMSP-1 19 ., Conclusion: These data suggest that natural acquired antibody response is higher for PvMSP-1 19 antigen as compared to PvAMA-1 antigen in individuals living in three geographically diverse malaria endemic regions in India. PvMSP-1 19 appears to be highly immunogenic in Indian population and has great potential as a malaria vaccine candidate. The differences in immune response against vaccine candidate antigens in different endemic settings should be taken into account for development of asexual stage based P. vivax malaria vaccine, which in turn can enhance malaria control efforts.

Background: The rapid diagnostic test (RDT) has been adopted in contemporary malaria control and management programmes around the world as it represents a fast and apt alternative for malaria diagnosis in a resource-limited setting. This study assessed the performance of a HRP-2/pLDH based RDT (Parascreen® Pan/Pf) in a laboratory setting utilizing clinical samples obtained from the field., Methods: Whole blood samples were obtained from febrile patients referred for malaria diagnosis by clinicians from two different Upazila Health Complexes (UHCs) located near the Bangladesh-India and Bangladesh-Myanmar border where malaria is endemic. RDT was performed on archived samples and sensitivity and specificity evaluated with expert microscopy (EM) and quantitative PCR (qPCR)., Results: A total of 327 clinical samples were made available for the study, of which 153 were Plasmodium falciparum-positive and 54 were Plasmodium vivax-positive. In comparison with EM, for P. falciparum malaria, the RDT had sensitivity: 96.0% (95% CI, 91.2-98.3) and specificity: 98.2% (95% CI, 94.6-99.5) and for P. vivax, sensitivity: 90.7% (95% CI, 78.9-96.5) and specificity: 98.9% (95% CI, 96.5-99.7). Comparison with qPCR showed, for P. falciparum malaria, sensitivity: 95.4% (95% CI, 90.5-98.0) and specificity: 98.8% (95% CI, 95.4-99.7) and for P. vivax malaria, sensitivity: 89.0% (95% CI,77.0-95.4) and specificity: 98.8% (95% CI, 96.5-99.7). Sensitivity varied according to different parasitaemia for falciparum and vivax malaria diagnosis., Conclusion: Parascreen® Pan/Pf Rapid test for malaria showed acceptable sensitivity and specificity in border belt endemic areas of Bangladesh when compared with EM and qPCR.

Antibody detection is a safely applied method at the wide scale in diagnosis of visceral Leishmaniasis (VL). In order to further advance serodiagnosis, the rK28 antigen has been recently introduced as a candidate for diagnosis of VL. We evaluated the sensitivity and specificity of the rK28 antigen in a micro-ELISA format in comparison to the rk39 antigen. The test was conducted on 252 parasitologically confirmed VL cases, 103 endemic healthy controls, 95 non-endemic healthy controls, 88 other infectious disease and 53 follow-up cases. Of 252 parasitologically confirmed VL cases, 251 cases were reported positive by rK28 antigen, yielding 99.6% sensitivity (95% CI, 0.97-0.99), which was similar to the sensitivity of rK39 ELISA (99.6%) (95% CI, 0.97-0.99). Specificity of the rK28 antigen in non-endemic and endemic healthy controls was 100% (95% CI 0.96-1) and 94.17% (95% CI, 0.88-0.97), respectively. In 88 different diseases, specificity was 95.45% (95% CI, 0.84-0.96). With the rK39 antigen, specificity of non-endemic and endemic controls and different diseases was 100% (95% CI 0.96-1), 92.23% (95% CI 0.85-0.96) and 96.59% (95% CI 0.90-0.98), respectively. Our results show that rK39 and rK28 antigens have similar sensitivity and specificity and rK28 can also be used as a serodiagnostic tool in the endemic population of Bihar., (© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.)

Malaria transmission by Anopheles subpictus Grassi, 1899 and Anopheles culicifacies Giles, 1901 was studied from March 2004 to February 2007 in Angul District, Orissa, India, which is highly endemic for malaria. Adult mosquitoes were collected from human dwellings using sucking tubes and a mechanical aspirator. After identification, some An. subpictus and An. culicifacies specimens were subjected to a precipitin test to determine their anthropophilic index and the remaining samples were preserved in isopropyl alcohol for sporozoite detection by nested PCR. An. subpictus was the most prevalent (29.0%) anopheline species detected, followed by An. culicifacies (11.6%). The anthropophilic index for the An. subpictus was higher than An. culicifacies and was highest during the summer season. Malaria sporozoite rates of 0.52% and 1.82% were detected for An. subpictus and An. culicifacies, respectively. Sporozoites were detected during the summer in An. subpictus and during the rainy season and winter in An. culicifacies. The slide positivity rate (SPR) was high during the summer. The high anthropophilic index and presence of sporozoites in An. subpictus during the summer indicate An. subpictus is a contributory factor for the high SPR during the summer, and An. culicifacies is a contributory factor for the high SPR during the rainy and winter seasons, along with other anophelines. In the present study An. subpictus has been incriminated as a vector of malaria for the first time in Orissa.

Background: The newly recognized endemic focus of leishmaniasis in Satluj river valley of Himachal Pradesh (India) has both localized cutaneous leishmaniasis (LCL) and visceral leishmaniasis (VL) predominantly caused by Leishmania donovani. Rapid rK39 immunochromatographic dipstick test detects circulating antibodies to recombinant K39 antigen of L. donovani-infantum complex and is highly specific/sensitive in diagnosing symptomatic or asymptomatic infection in humans and dogs., Methods: The sera from two VL patients and 13 LCL patients, and 31 dogs were subjected to rK39 immunochromatographic dipstick testing with an aim to identify possible animal reservoir for leishmaniasis in this endemic focus., Results and Conclusion: The positive rapid rK39 immunochromatographic dipstick test in 100% VL and 31.8% LCL patients, and 6.5% dogs suggests that both VL and LCL in this focus are apparently being caused by L. donovani-infantum and that reservoir infection is perhaps being chiefly maintained in asymptomatic dogs. However, it needs corroborative evidence in the form of in-vitro parasite cultivation and/or PCR studies for confirmation. A more elaborate study is recommended.

Background: Malaria presents a diagnostic challenge in tribal belt of central India where two Plasmodium species, Plasmodium falciparum and Plasmodium vivax, are prevalent. In these areas, rapid detection of the malaria parasites and early treatment of infection remain the most important goals of disease management. Therefore, the usefulness of a new rapid diagnostic (RDT), the First Response(R) Combo Malaria Ag (pLDH/HRP2) card test was assessed for differential diagnosis between P. falciparum with other Plasmodium species in remote villages of Jabalpur district., Methods: A finger prick blood sample was collected to prepare blood smear and for testing with the RDT after taking informed consent. The figures for sensitivity, specificity, accuracy and predictive values were calculated using microscopy as gold standard., Results: Analysis revealed that overall, the RDT was 93% sensitive, 85% specific with a positive predictive value (PPV) of 79%, and a negative predictive value (NPV) of 95%. The accuracy 88% and J-index was 0.74. For P. falciparum, the sensitivity and specificity of the test were 96% and 95% respectively, with a PPV of 85% and a NPV of 99%. The RDT accuracy 95% and J-index was 0.84. For non-falciparum malaria, the sensitivity, specificity and accuracy were 83%, 94% and 92% respectively with a PPV of 69% and a NPV of 97%., Conclusion: The RDTs are easy to use, reliable and simple to interpret. RDTs are more suited to health workers in situations where health services are deficient or absent. Therefore, the test can be used as an epidemiological tool for the rapid screening of malaria.

This study sought to determine whether dip stick strip test containing antibody for Plasmodium falciparum histidine rich protein-II (PfHRP-II) antigen could be used for identification of P. falciparum and P. vivax malaria in man. The results obtained were also compared with the results of standard microscopic examination. A total of 150 cases were included for the study. Fifty cases were non-febrile cases with no history of malaria acting as control group and the rest 100 cases were having fever and formed the test group. All the cases in the control group was found to be negative for both microscopic examination and strip test. In the test group, all samples that showed positive for P. falciparum by microscopy was also found to be positive for strip test. Whereas, all those samples that were positive for P. vivax in microscopic examination was found to be negative for strip test indicating species specificity of the strip test. In addition, two other cases that were negative for microscopic examination were found to be positive for the strip test. Statistical analysis was done to compare the validity of the results of strip test with that of the results of microscopic examination.

A rapid immunodiagnostic test (ICT Malaria PfTest) has been developed by ICT Diagnostics (Sydney, Australia) for the diagnosis of Plasmodium falciparum infection. The test is an antigen capture assay based on the detection of P. falciparum histidine-rich protein 2 in peripheral blood. This study was undertaken to assess the performance and usefulness of the test as a diagnostic method in highly malarious, inaccessible forested villages of Mandla district, central India. In all, 353 patients with fever were scanned by the test in parallel with thick blood film examination. The sensitivity and specificity were 100% and 84.5%, respectively. The whole test took about 5 min. The test results became negative in most cases (70%) within 7 d after initiation of curative chemotherapy. The test is simple, easy to learn and accurate, and may prove to be an important tool in the battle against falciparum malaria.