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One of the major challenges for malaria control and elimination is the spread and emergence of antimalarial drug resistance. Mutations in Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) field isolates for five drug resistance genes viz. crt, mdr1, dhps, dhfr and kelch known to confer resistance to choloroquine (CQ), sulfadoxine-pyrimethamine (SP) and artemisinin (ART) and its derivatives were analyzed. A total of 342 symptomatic isolates of P. falciparum (Pf) and P. vivax (Pv) from 1993 to 2014 were retrieved from malaria parasite repository at National Institute of Malaria Research (NIMR). Sample DNA was extracted from dried blood spots and various targeted single nucleotide polymorphisms (SNPs) associated with antimalarial drug resistance were analysed for these isolates. 72S (67.7%) and 76T (83.8%) mutations along with SVMNT haplotype (67.7%) predominated the study population for Pfcrt. The most prevalent SNPs were 108N (73.2%) and 437G (24.8%) and the most prevalent haplotypes were ACNRNI (51.9%) and SAKAA (74.5%) in Pfdhfr and Pfdhps respectively. Only two mutations in Pfmdr1, 86Y (26.31%) and 184F (56.26%), were seen frequently in our study population. No mutations associated with Pfk13 were observed. For Pv, all the studied isolates showed two Pvdhps mutations, 383G and 553G, and two Pfdhfr mutations, 58R and 117N. Similarly, three mutations, viz. 958M, 908L and 1076L were found in Pvmdr1. No variations were observed in Pvcrt-o and Pvk12 genes. Overall, our study demonstrates an increase in mutations associated with SP resistance in both Pf and Pv, however, no single nucleotide polymorphisms (SNPs) associated with ART resistance have been observed for either species. Various SNPs associated with CQ resistance were seen in Pf; whereas only Pvmdr1 associated resistant SNPs were observed in Pv. Therefore, molecular characterization of drug resistance genes is essential for timely monitoring and prevention of malaria by identifying the circulating drug resistant parasites in the country., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Malaria elimination is one of the top health care priorities in India, necessitating accessible and accurate diagnosis for effective treatment. A malaria slide bank in India is a collection of quality-controlled malaria-positive and -negative slides and is considered a vital asset for quality diagnosis. The collection of blood samples, preparation of blood smears, staining, quality control, molecular characterizations, and slide validation were carried out according to standard operating procedures in accordance with the WHO reference laboratory. The true count and parasite density per microliter were computed in accordance with WHO guidelines. Over 27 months, 48 batches (8,196 slides) were prepared. Overall, the majority of slide batches were Plasmodium vivax (45.9%; 22/48), followed by Plasmodium falciparum (25%; 12/48), malaria-negative infections (25%; 12/48), and mixed infections (4.1%; 2/48). All 48 batches passed internal validation by WHO-certified level-1 microscopists. For a batch, the true count was the median of the validators' counts (range, 111-280,795 parasites/µL). Except for mixed infections, the PCR results agreed with the verified microscopy results. Malaria slide bank slides would be a valuable tool for quality control, assurance, and microscopist training.

Background: A detailed analysis of household and individual level Plasmodium infection patterns in two low-endemic districts of Meghalaya was undertaken to better understand the epidemiology of malaria in northeast India., Methods: Socio-demographic and behavioural information from residents (aged 1-69 years) of households were collected through pre-tested, questionnaire conducted in 2018 and 2019. Blood samples collected from participants were tested for Plasmodium falciparum and/or Plasmodium vivax infection using rapid diagnostic test, microscopy and PCR. Plasma samples from a subset of participants were analysed for antibodies against thirteen P. falciparum and four P. vivax antigens. Associations between household and individual level risk factors, and Plasmodium infections were evaluated using multilevel logistic regression models., Results: A total of 2753 individuals from 827 households were enrolled in 2018, and 834 individuals from 222 households were enrolled in 2019. Of them, 33 (1.2%) were positive by PCR for P. falciparum in 2018 and none were positive for P. vivax. In 2019, no PCR-positive individuals were detected. All, but one, infections were asymptomatic; all 33 infections were sub-microscopic. Reported history of malaria in the past 12 months (OR = 8.84) and history of travel in the past 14 days (OR = 10.06) were significantly associated with Plasmodium infection. A significant trend of increased seropositivity with age was noted for all 17 antigens. Although adults (≥ 18 years) consistently had the highest seropositivity rates, a sizeable proportion of under-five children were also found to be seropositive. Almost all individuals (99.4%) reported sleeping under an insecticide-treated bed-net, and household indoor residual spray coverage in the 12 months preceding the survey was low (23%). Most participants correctly identified common signs and symptoms of malaria, i.e., fever (96.4%), headache (71.2%), chills (83.2%) and body-ache (61.8%). Almost all participants (94.3%) used government-provided services for treatment of malaria., Conclusion: This study explored the epidemiology of malaria in two communities in Meghalaya, India, in the context of declining transmission. The presence of widespread asymptomatic infections and seropositivity among under-five children suggest that low-level Plasmodium transmission persists in this region. Implications of the study findings for malaria elimination efforts in low-transmission settings are discussed., (© 2021. The Author(s).)

Plasmodium vivax, a major contributor to the malaria burden in India, has the broadest geographic distribution and shows higher genetic diversity than P. falciparum. Here, we investigated the genetic diversity of two leading P. vivax vaccine candidate antigens, at three geographically diverse malaria-endemic regions in India. Pvama1 and Pvmsp119 partial coding sequences were generated from one hundred P. vivax isolates in India (Chennai n = 28, Nadiad n = 50 and Rourkela n = 22) and ~1100 published sequences from Asia, South America, North America, and Oceania regions included. These data were used to assess the genetic diversity and potential for vaccine candidacy of both antigens on a global scale. A total of 44 single nucleotide polymorphism (SNPs) were identified among 100 Indian Pvama1 sequences, including 10 synonymous and 34 nonsynonymous mutations. Nucleotide diversity was higher in Rourkela and Nadiad as compared to Chennai. Nucleotide diversity measures showed a strong balancing selection in Indian and global population for domain I of Pvama1, which suggests that it is a dominant target of the protective immune response. In contrast, the Pvmsp119 region showed highly conserved sequences in India and across the Oceania, South America, North America and Asia, demonstrating low genetic diversity in the global population when compared to Pvama1. Results suggest the possibility of including Pvmsp119 in a multivalent vaccine formulation against P. vivax infections. However, the high genetic diversity seen in Pvama1 would be more challenging for vaccine development., Competing Interests: The authors have declared that no competing interests exist.

Background: Automated detection of malaria and dengue infection has been actively researched for more than two decades. Although many improvements have been achieved, these solutions remain too expensive for most laboratories and clinics in developing countries. The low range HORIBA Medical Haematology Analyzer, Yumizen H550, now provides dedicated flags 'vivax malaria' and 'dengue fever' in routine blood testing, developed through machine learning methods, to be used as a screening tool for malaria and dengue fever in endemic areas. This study sought to evaluate the effectiveness of these flags under real clinical conditions., Methods: A total of 1420 samples were tested using the Yumizen H550 Haematology Analyzer, including 1339 samples from febrile patients among whom 202 were infected with malaria parasites (Plasmodium vivax only: 182, Plasmodium falciparum only: 18, both: 2), 210 were from febrile dengue infected patients, 3 were from afebrile dengue infected patients and 78 were samples from healthy controls, in an outpatient laboratory clinic in Mumbai, India. Microscopic examination was carried out as the confirmatory reference method for detection of malarial parasite, species identification and assessing parasitaemia based on different stages of parasite life cycle. Rapid diagnostic malarial antigen tests were used for additional confirmation. For dengue infection, NS1 antigen detection by ELISA was used as a diagnostic marker., Results: For the automated vivax malaria flag, the original manufacturer's cut off yielded a sensitivity and specificity of 65.2% and 98.9% respectively with the ROC AUC of 0.9. After optimization of cut-off value, flag performance improved to 72% for sensitivity and 97.9% specificity. Additionally it demonstrated a positive correlation with increasing levels of parasitaemia. For the automated dengue fever flag it yielded a ROC AUC of 0.82 with 79.3% sensitivity and 71.5% specificity., Conclusions: The results demonstrate a possibility of the effective use of automated infectious flags for screening vivax malaria and dengue infection in a clinical setting.

Background: Many malaria endemic countries are heading towards malaria elimination through the use of case management and vector control strategies, which employ surveillance, improving access to early diagnosis, prompt treatment., and integrated vector control measures. There is a consensus that elimination of malaria is feasible when rapid detection and prompt treatment is combined with mosquito-human contact interruption in an efficient and sustainable manner at community levels. This paper describes results of an integrated case management and vector control strategy for reducing malaria cases in 1233 villages over 3 years in district Mandla, Madhya Pradesh, India., Methods: The project enrolled the entire population (1,143,126) of Mandla district for fever surveillance followed by testing of febrile cases and treatment of positive subjects using T4 strategy, which is Track (by fever), Test (by RDTs), Treat (by ACT) and Track (for completion of treatment). In addition to the active and passive surveillance for detection and treatment of febrile cases, the project conducted mass screening and treatment to clear the asymptomatic reservoirs of infection. Febrile cases were also tested in the out-patient department of the District Hospital from June 2018 to September, 2018 and in a community-based medical camp from November 7 to 14, 2019. The project also used vector control measures for interrupting human-mosquito contact, and information, education and communication (IEC) campaigns to increase demand for malaria services at community level., Results: This project has revealed about 91% reduction of indigenous cases of malaria during the period from June 2017 to May 2020, through case management and vector control strategies. A total 357,143 febrile cases were screened, out of which 0.19% were found positive for the presence of malaria parasites, with Plasmodium falciparum and Plasmodium vivax ratio of 62:38. The prevalence of malaria was higher in individuals > 15 years of age (69% cases). The positivity rate was 0.33% in 2017-18, 0.13% in 2018-19, and 0.06% in 2019-20. In all of the 3 years of the project, the peak transmission correlated with rains. Mass screening revealed 0.18% positivity in Sep-Oct 2018, followed by 0.06% in June 2019, and 0.03% in December 2019, and these were mostly asymptomatic cases in the community. Imported cases into the district were mostly contributed by the distant state of Telangana (51.13%). Fever patients tested for malaria parasites in the District Hospital and medical camp revealed zero cases., Conclusion: Using the current intervention and prevention tools along with optimum utilization of human resources, a 91% reduction in indigenous cases of malaria was seen in the district in 3 years. The reduction was similar in the three high prevalence blocks of the district. These results reveal that malaria elimination is achievable in India within a stipulated time frame. The reduction of malaria at the community level was further validated when zero malaria cases were diagnosed during hospital and community-based studies in Mandla. Prompt detection and treatment of imported/migratory cases may have prevented outbreaks in the district. This project has demonstrated that field programmes backed by adequate technical, management, operational, and financial controls with robust monitoring are needed for achieving malaria elimination.

Background: The increasing antimalarial drug resistance is a significant hindrance to malaria control and elimination programs. For the last six decades, chloroquine (CQ) plus pyrimethamine remains the first-line treatment for P. vivax malaria. Regions where both P. falciparum and P. vivax co-exist, P. vivax is exposed to antifolate drugs due to either misdiagnosis or improper treatment that causes selective drug pressure to evolve. Therefore, the present study aims to estimate antimalarial drug resistance among the complicated and uncomplicated P. vivax patients., Methods: A total of 143 P. vivax malaria positive patients were enrolled in this study, and DNA was isolated from their blood samples. Pvcrt-o, Pvmdr-1, Pvdhps, and Pvdhfr genes were PCRs amplified, and drug resistance-associated gene mutations were analyzed. Statistical analysis of the drug resistance genes and population diversity was performed using MEGA vs. 7.0.21 and DnaSP v software., Results: Among the CQ resistance marker gene Pvcrt-o, the prevalence of K10 insertion was 17.5% (7/40) and 9.5% (7/73) of complicated and uncomplicated P vivax group isolates respectively. In Pvmdr-1, double mutant haplotype (M 958 /L 1076 ) was found in 99% of the clinical isolates. Among the pyrimethamine resistance-associated gene Pvdhfr, the double mutant haplotype I 13 P 33 F 57 R 58 T 61 N 117 I 173 was detected in 23% (11/48) in complicated and 20% (17/85) in uncomplicated group isolates. In the sulphadoxine resistance-associated Pvdhps gene, limited polymorphism was observed with the presence of a single mutant (D459A) among 16 and 5% of the clinical isolates in the complicated and uncomplicated group respectively., Conclusion: The study presents the situations of polymorphism in the antimalarial drug resistance-associated genes and emphasizes the need for regular surveillance. It is imperative for the development of suitable antimalarial drug policy in India.

Background: Malaria is a major public health problem in India. Data from surveys totaling 3031 participants at three sites revealed a high proportion of asymptomatic infections, complicating diagnosis. The aim of this study was to identify differences in complaints and symptoms between sites, and factors associated with asymptomatic Plasmodium infections., Methods: Published data from community-based cross-sectional studies conducted between 2012 and 2015 in Nadiad (Gujarat), Chennai (Tamil Nadu), and Rourkela (Odisha) as part of the Center for the Study of Complex Malaria in India were analysed. Complaints and symptoms were systematically recorded, and Plasmodium infections confirmed using microscopy, rapid diagnostic tests (RDTs), and polymerase chain reaction (PCR). Multivariate analyses were conducted to determine the association between general symptoms and age, season, or gender, and factors associated with asymptomatic Plasmodium infections were assessed., Results: Complaints of any illness were lowest in Chennai (17.7%), 30.6% in Rourkela and 42.7% in Nadiad. Complaints were more often reported for children; gender differences were noted in Rourkela only. In Nadiad, 7.0% of 796 participants were positive for malaria by PCR (32% Plasmodium falciparum); 78.6% had a history of fever or documented fever, 14.3% had other symptoms, and 7.1% were "truly asymptomatic". For Chennai this was 29.2%, 4.2% and 66.7% respectively, with a malaria prevalence of 2.6% by PCR of 928 participants (29% P. falciparum). In Rourkela, with 7.7% of 1307 participants positive for malaria by PCR (82% P. falciparum), the percentages were 35.6%, 24.8% and 39.6%, respectively. In Rourkela, asymptomatic infections were associated with young age and male gender (microscopy or RDT), and with rainy season (PCR). In the same site, participants with Plasmodium vivax were more likely to be asymptomatic (11/18 or 61.1%) than persons with P. falciparum mono-infections (27/78 or 34.6%); gametocytes for P. falciparum were evenly distributed between symptomatic and asymptomatic infections (2/53 vs. 2/49, respectively). The addition of the symptoms "headache", "aches" and "chills" to fever improved the case-definition of symptomatic malaria., Conclusion: There were considerable differences in complaints at the three sites in India. Malaria and asymptomatic infections differ by region, indicating that malaria elimination will require localized approaches.

Background: Vivax malaria is associated with significant morbidity and economic loss, and constitutes the bulk of malaria cases in large parts of Asia and South America as well as recent case reports in Africa. The widespread prevalence of vivax is a challenge to global malaria elimination programmes. Vivax malaria control is particularly challenged by existence of dormant liver stage forms that are difficult to treat and are responsible for multiple relapses, growing drug resistance to the asexual blood stages and host-genetic factors that preclude use of specific drugs like primaquine capable of targeting Plasmodium vivax liver stages. Despite an obligatory liver-stage in the Plasmodium life cycle, both the difficulty in obtaining P. vivax sporozoites and the limited availability of robust host cell models permissive to P. vivax infection are responsible for the limited knowledge of hypnozoite formation biology and relapse mechanisms, as well as the limited capability to do drug screening. Although India accounts for about half of vivax malaria cases world-wide, very little is known about the vivax liver stage forms in the context of Indian clinical isolates., Methods: To address this, methods were established to obtain infective P. vivax sporozoites from an endemic region in India and multiple assay platforms set up to detect and characterize vivax liver stage forms. Different hepatoma cell lines, including the widely used HCO4 cells, primary human hepatocytes as well as hepatocytes obtained from iPSC's generated from vivax patients and healthy donors were tested for infectivity with P. vivax sporozoites., Results: Both large and small forms of vivax liver stage are detected in these assays, although the infectivity obtained in these platforms are low., Conclusions: This study provides a proof of concept for detecting liver stage P. vivax and provide the first characterization of P. vivax liver stage forms from an endemic region in India.

Background: India has launched the malaria elimination initiative in February 2016. Studies suggest that estimates of malaria are useful to rationalize interventions and track their impact. Hence, a national study was launched to estimate burden of malaria in India in 2015., Methods: For sampling, all 624 districts of India were grouped in three Annual Parasite Incidence (cases per thousand population) categories, < two (low); two-five (moderate) and > five (high) API. Using probability proportional to size (PPS) method, two districts from each stratum were selected covering randomly 200,000 persons per district. Active surveillance was strengthened with 40 trained workers per study district. Data on malaria cases and deaths was collated from all health care providers i.e. pathological laboratories, private practitioners and hospitals in private and public health sectors and was used for analysis and burden estimation., Results: Out of 1215,114 population under surveillance, 198,612 (16.3%) tests were performed and 19,386 (9.7%) malaria cases were detected. The malaria cases estimated in India were 3875,078 (95% confidence interval 3792,018-3958,137) with API of 3.05 (2.99-3.12) including 2789,483 (2740,577-2838,389) Plasmodium falciparum with Annual Falciparum Incidence of 2.2 (2.16-2.24). Out of 8025 deaths investigated, 102 (1.27%) were attributed to malaria. The estimated deaths in India were 29,341 (23,354-35,327) including 19,067 (13,665-24,470) confirmed and 10,274 (7694-12,853) suspected deaths in 2015-2016., Conclusions: Estimated malaria incidence was about four folds greater than one million reported by the national programme, but three folds lesser than thirteen million estimated by the World Health Organization (WHO). However, the estimated deaths were 93 folds more than average 313 deaths reported by the national malaria programme in 2015-2016. The 29,341 deaths were comparable with 24,000 deaths in 2015 and 22,786 deaths in 2016 estimated by the WHO for India. These malaria estimates can serve as a benchmark for tracking the success of malaria elimination campaign in India.

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&#95;003545, PVX&#95;003555 and PVX&#95;121935), a hypothetical protein (PVX&#95;083555) and Pvstp1 (subtelomeric transmembrane protein 1, PVX&#95;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.

The aim of this study was to assess the clinical profile, severity and complications of patients suffering from malaria in Mangaluru, a southwestern coastal city in India. A total of 579 patients, who were treated at the District Wenlock Hospital, Mangaluru, and 168 healthy controls were recruited in this study. The clinical profile, haematological and biochemical parameters, and disease complications were assessed. The majority of patients were treated as outpatients and patients who had severe clinical conditions were admitted to the hospital for treatment and supportive care. Among the total 579 patients recruited in this study, the distribution of P. vivax, P. falciparum and mixed infections were 364 (62.9%), 150 (25.9%) and 65 (11.2%), respectively. Among these, 506 (87.4%) had mild malaria, whereas 73 (12.6%) had severe malaria. Overall, the clinical features and severity of malaria in P. vivax and mixed infection patients were comparable to P. falciparum patients, albeit with some significant differences. The clinical complications in severe malaria cases included thrombocytopenia (50.7%), metabolic acidosis (30.1%), severe anaemia (26.0%), jaundice (21.9%), hepatic dysfunction (15.1%), acute renal failure (6.8%), haematuria (8.2%), hypotension (9.6%), cerebral malaria (1.4%) and acute respiratory distress syndrome (1.4%). All the patients with severe malaria recruited in our study were successfully treated and discharged. Majority of patients had mild malaria, likely due to seeking treatment soon after experiencing symptoms and/or having preexisting immune protection. However, a significant number of patients had severe malaria and required hospital admission indicating that there is a substantial need for creating awareness among vulnerable immigrant population. Implementing effective surveillance and vector control measures in malaria hotspot locations in the city and educating people about preventive measures are likely to reduce the malaria burden in this endemic region.

Objective: Acute kidney injury (AKI) is a frequent presentation in malaria infections. Several cases of AKI that are accompanied by clinical symptoms of malaria infection, such as fever, nausea, respiratory distress, and anemia remain undiagnosed due to challenges in accurate diagnosis using peripheral blood microscopy and rapid diagnostic tests that are currently used in clinical settings. This is particularly true for P. vivax and P. knowlesi infections. As a result, these patients are not able to receive anti-malarial therapy in a timely manner. The objective of the present study was to investigate if patients presenting with AKI harbored any of the five human Plasmodium species (P. falciparum, P. vivax, P. knowlesi, P. malariae, and P. ovale) within their renal tissues., Results: We found that renal biopsies from malaria associated AKI patients harbor the human malaria parasites P. falciparum, P. vivax and P. knowlesi as mono- and mixed species infections. Presence of microvascular injury in a majority of the malaria associated AKI cases suggested vascular involvement of P. vivax and P. knowlesi. This research note also highlights P. knowlesi as an emerging pathogen in the Indian subcontinent.

India accounts for approximately half of the global Plasmodium vivax cases, but information as to the presence of chloroquine (CQ) resistance is scarce. In an observational study in Mangaluru, south-western India, of 116 vivax malaria patients analyzed, 89.5% (102/114) had cleared parasitemia on days two or three of CQ treatment. Two remaining patients presented on days four and five without parasitemia. One hundred eight isolates of these 116 patients were successfully sequenced for pvmdr1 polymorphisms. Eight non-synonymous polymorphisms but no wild-type isolate were detected. Ten pvmdr1 haplotypes were observed with mutations T958M and F1076L occurring in all isolates, whereas the candidate CQ resistance marker Y976F was present in one isolate only. Pvmdr1 polymorphisms were not associated with early parasite clearance. The high proportion of early parasite clearance and the virtual absence of pvmdr1 Y976F and of sextuple pvmdr1 mutants suggest that CQ in the study area is still sufficiently effective. However, the abundance of pvmdr1 mutations in the local parasite population warrants monitoring.

Background: Plasmodium vivax malaria accounts for 80% of the malaria cases in Delhi, India. The gene merozoite surface protein 3 alpha (MSP3α) is highly polymorphic and has been used as marker in many P. vivax population studies., Methods: MSP3α has been used to assess the genetic diversity of P. vivax samples from Delhi (India) having more than one malaria episode (s) i.e. clinically identified relapse cases using PCR-RFLP and sequencing., Results: Three major genotypes 2.0 kb (A), 1.4 kb (B) and 1.2 kb (C) were amplified from 72 isolates with frequencies of 72.2%, 19.44% and 9.72% respectively. One sample out of 72 showed mixed infection having both A and B type genotypes. 82.05% patients showed same genotype while only 17.94% patients showed different genotypes after subsequent malaria episodes. 18 different genotypes with Alu I and 35 with Hha I were identified among 72 samples analyzed by restriction fragment length polymorphism (RFLP). 18 Pvmsp3α nucleotide sequences were analyzed and it did not reveal any distinct intragenic differences within sequences of the same type, however, allelic diversity among the three types (Π = 0.029703) was observed. Phylogenetic analysis showed allelic family types A, B and C were not clustered but distributed in different branches. The results indicate that the P. vivax parasite population is highly diverse in Delhi, India. A large number of amino acid substitutions were found at the locus of the isolates when compared with the Belem Strain (Π  = 0.030528). The substantial sequence diversity is largely restricted to certain domains of encoded protein. Analysis of synonymous and nonsynonymous substitutions suggested that different selection forces were operating on different regions of the protein molecule., Conclusion: We propose that genotyping of the PvMSP-3α gene as one of the molecular tools for differentiating relapse from new infection in epidemiological settings. The analyses of sequence polymorphism in PvMSP-3α gene enable it as potential candidate for inclusion in a P.vivax vaccine research., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Autoimmune haemolytic anaemia due to malaria or following its treatment with artesunate is rare. A child presented with severe anaemia after being treated with artesunate for P. vivax malaria. Blood transfusion was difficult as cross-matching showed major incompatibility; group O negative blood under the cover of steroids was transfused. Oral steroids were given for six weeks. The patient made a complete recovery.

Plasmodium vivax is the most geographically widespread species responsible for malaria in humans. Our study focused on identifying highly expressed parasite proteins using a shotgun proteomics approach. Parasites (P. vivax) are isolated from seven patient samples using saponin lysis. Protein extracts from these parasites are processed and subjected to LC-MS/MS analysis. An overall proteome coverage of 605 P. vivax proteins along with 1670 human host proteins are obtained upon combining the data from LC-MS/MS runs. While a major proportion of the P. vivax proteins are either hypothetical or involved in basic cellular activities, few proteins such as tryptophan-rich antigen (Pv-fam-a; PVX&#95;090265), Pv-fam-d protein (PVX&#95;101520), Plasmodium exported protein (PVX&#95;003545), Pvstp1 (PVX&#95;094303) and hypothetical protein (PVX&#95;083555) are detected in more than 80% of the clinical isolates and found to be unique to P. vivax without orthologs in P. falciparum. Our proteomics study on individual parasite isolates reveals highly expressed P. vivax proteins, few of which may be good candidates for vivax malaria diagnosis due to their abundance and absence in P. falciparum. This study represents the first step towards the identification of biomarkers for P. vivax malaria. In future, their clinical diagnostic values must be explored and validated on large patient cohorts., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Malaria control program in the Arabian Peninsula, backed by adequate logistical support, has interrupted transmission with exception of limited sites in Saudi Arabia and sporadic outbreaks in Oman. However, sustained influx of imported malaria represents a direct threat to the above success. Here we examined the extent of genetic diversity among imported P. vivax in Qatar, and its ability to produce gametocytes, compared to parasites in main sites of imported cases, the Indian subcontinent (india) and East Africa (Sudan and Ethiopia). High diversity was seen among imported P. vivax in Qatar, comparable to parasites in the Indian subcontinent and East Africa. Limited genetic differentiation was seen among imported P. vivax, which overlapped with parasites in India, but differentiated from that in Sudan and Ethiopia. Parasite density among imported cases, ranged widely between 26.25-7985934.1 Pv18S rRNA copies/µl blood, with a high prevalence of infections carried gametocytes detectable by qRT-PCR. Parasitaemia was a stronger predictor for P. vivax gametocytes density (r = 0.211, P = 0.04). The extensive diversity of imported P. vivax and its ability to produce gametocytes represent a major threat for re-introduction of malaria in Qatar. The genetic relatedness between P. vivax reported in Qatar and those in India suggest that elimination strategy should target flow and dispersal of imported malaria into the region.

Background: As much as 80% of global Plasmodium vivax infections occur in South Asia and there is a shortage of direct studies on infectivity of P. vivax in Anopheles stephensi, the most common urban mosquito carrying human malaria. In this quest, the possible effects of laboratory colonization of mosquitoes on infectivity and development of P. vivax is of interest given that colonized mosquitoes can be genetically less divergent than the field population from which they originated., Methods: Patient-derived P. vivax infected blood was fed to age-matched wild and colonized An. stephensi. Such a comparison requires coordinated availability of same-age wild and colonized mosquito populations. Here, P. vivax infection are studied in colonized An. stephensi in their 66th-86th generation and fresh field-caught An. stephensi. Wild mosquitoes were caught as larvae and pupae and allowed to develop into adult mosquitoes in the insectary. Parasite development to oocyst and sporozoite stages were assessed on days 7/8 and 12/13, respectively., Results: While there were batch to batch variations in infectivity of individual patient-derived P. vivax samples, both wild and colonized An. stephensi were roughly equally susceptible to oocyst stage Plasmodium infection. At the level of sporozoite development, significantly more mosquitoes with sporozoite load of 4+ were seen in wild than in colonized populations.

Malaria is a major public health problem in India and in the Chhattisgarh state. The diagnosis of malaria presents a major challenge in remote areas The prevalence of malaria in Darbha and Kilepal Community Health Centers (CHCs) of the Jagdalpur district, Chhattisgarh affected by conflict was determined using microscopy and polymerase chain reaction (PCR). In the year 2015, 29.4% and 21.5% cases were found to be positive for malaria at the Darbha and Kilepal CHCs, respectively, by microscopy, and 7.4% and 1.6% of cases had mixed infections, respectively. Among the suspected cases of mixed infections and doubtful diagnoses, 21% had mixed infections with two or more species at the Darbha CHC, and 17% from the Kilepal CHC, as determined by PCR. Both the P. vivax subspecies Pv210 (56%) and Pv247 (44%) and the P. ovale curtisi subspecies were found in this area. The high proportion of mixed malaria parasitic infections detected in this study indicate the need to adequately train health staff involved in diagnosing malaria. This study showed that there is a need for site-specific data to understand the epidemiological picture and to develop appropriate intervention strategies and management guidelines for controlling and eliminating malaria in India.

The World Health Organization estimates that nearly 500 million malaria tests are performed annually. While microscopy and rapid diagnostic tests (RDTs) are the main diagnostic approaches, no single method is inexpensive, rapid, and highly accurate. Two recent studies from our group have demonstrated a prototype computer vision platform that meets those needs. Here we present the results from two clinical studies on the commercially available version of this technology, the Sight Diagnostics Parasight platform, which provides malaria diagnosis, species identification, and parasite quantification. We conducted a multisite trial in Chennai, India (Apollo Hospital [ n = 205]), and Nairobi, Kenya (Aga Khan University Hospital [ n = 263]), in which we compared the device to microscopy, RDTs, and PCR. For identification of malaria, the device performed similarly well in both contexts (sensitivity of 99% and specificity of 100% at the Indian site and sensitivity of 99.3% and specificity of 98.9% at the Kenyan site, compared to PCR). For species identification, the device correctly identified 100% of samples with Plasmodium vivax and 100% of samples with Plasmodium falciparum in India and 100% of samples with P. vivax and 96.1% of samples with P. falciparum in Kenya, compared to PCR. Lastly, comparisons of the device parasite counts with those of trained microscopists produced average Pearson correlation coefficients of 0.84 at the Indian site and 0.85 at the Kenyan site., (Copyright © 2017 American Society for Microbiology.)

Background: We report the first evidence of sympatric distribution of Plasmodium ovale curtisi and P. ovale wallikeri from India., Methods: Fingerprick blood samples were collected from fever cases in district Bastar, Chhattisgarh State for malaria screening by microscopy and PCR., Results: Two cases of mono infection of P. ovale, and a fatal case of cerebral malaria with a mixed infection of P. vivax, P. falciparum and P. ovale were confirmed by PCR. Sequencing analysis revealed the presence of P. ovale curtisi and P. ovale wallikeri., Conclusions: This study highlights the need of molecular diagnosis of malaria cases in forested areas for treatment and control., (© The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: For the calculation of parasite index (PI) by microscopy method, an assumed total leucocyte count (TLC) of 8,000/μL is used conventionally. However, due to obvious variation in the population and individual TLCs, use of 8,000/μL may result in either over/underestimation of the PI., Methods: This study was aimed at ascertaining the utility of 8,000/μL TLC, as well as other assumed TLCs, with respect to measured TLC for the calculation of PI. A tertiary care hospital and five primary health centres were the base for the prospective study conducted among microscopically proven, symptomatic Plasmodium vivax mono-infection patients aged ≥18 years. PIs calculated by assumed TLCs ranging from 4,000-11,000/μL were compared with those calculated by measured TLCs. Geometric mean with 95% confidence interval, Bland-Altman plot and Wilcoxon signed rank test were used for statistical analysis., Results: A total of 284 P. vivax mono-infection patients, including 156 from a tertiary care hospital and 128 from five primary health centres, were recruited in the study. Assumed TLCs below 5,000 cell/μL and above 5,500 cell/μL in tertiary care setting resulted in significant (p <0.05) underestimation and overestimation, respectively. However, in primary health centres, it was an assumed TLC of 5,000 cell/μL, below and above which there was significant (p <0.05) underestimation and overestimation observed, respectively., Conclusions: Assumed TLC of 8,000/μL is not suitable for the calculation of PI. Either actual TLC of the patient should be measured or a representative TLC should be derived for the population under investigation for any study requiring calculated PI by microscopy.

Background & Objectives: Description of severe vivax malaria and mixed species infection requires good clinical study. The present study was undertaken to evalute the characteristics of severe malaria patients in Bikaner, northwest India., Methods: This prospective study included 539 admitted adult patients of severe malaria (Plasmodium falciparum 274, P. vivax 221, and mixed infection of Pv + Pf 44). The diagnosis was confirmed by polymerase chain reaction. The categorization of severe malaria was done strictly as per WHO criteria., Results: The distribution of severe manifestation was similar in severe vivax, falciparum and mixed infections except more cases of thrombocytopenia in P. vivax (p=0.030) and in mixed infection (p=0.004). The risk of developing severe malaria was greatest in patients of mixed infection [53.01% (44/83)] in comparison to Plasmodium falciparum malaria [49.37% (274/555), RR= 1.135; p=0.616] and P. vivax malaria [45.38% (221/ 487), RR = 1.299, p=0.243]. Hepatic dysfunction was the commonest pernicious syndrome [P. falciparum 50% (137/274), P. vivax 43.89% (97/221), and mixed infections 54.55% (24/44)]. Multiorgan dysfunction was present in 40.26% (217/539) patients, the risk was greatest in mixed infection [90.90% (40/44)] in comparison to P. falciparum monoinfection [37.59% (103/274), RR = 12.238; p=0.0001] or P. vivax monoinfection [33.48% (74/ 221), RR = 13.25; p=0.0001]. The risk of mortality in severe malaria was 6.31% (34/539) in which mixed infection had greater risk [9.09% (4/44)] in comparison to P. falciparum [7.30% (20/274); OR = 1.270 (CI 0.347-4.217); p=0.757] or P. vivax [4.52% (10/221); 0R 2.110 (CI 0.527-7.826); p=0.260]., Interpretation & Conclusion: Severe vivax or falciparum malaria had almost similar features and prognosis including mortality. Risk of developing severe malaria, multiorgan dysfunction and mortality was more in patients of mixed infection in comparison to P. falciparum or P. vivax monoinfection. A multicentric study on larger number of patients requires further confirmation.

The conventional molecular diagnosis of malaria uses 18S rRNA-based PCR assay employing blood samples. This assay presents limitation in terms of long turnaround time and increased chances of false-positive results. Here, we evaluated one-step singleplex or multiplex PCR assay based on high copy species-specific consensus repeat sequences (CRS) along with standard 18S rRNA nested PCR (18S n-PCR) assay to detect P. falciparum and P. vivax infection using blood and saliva samples from Indian febrile patients. Out of 327 patients, 187 were found to be positive for malaria parasites by microscopic examination of peripheral blood smears. Among these 130 were P. vivax and 57 were P. falciparum cases. The 18S n-PCR assay and CRS PCR assay identified 186 out of 187 cases (99.4 %). Multiplex CRS PCR assay detected Plasmodium in 176 out of 187 cases (94.1 %). Both singleplex and multiplex CRS PCR assay identified 6 mixed infection cases, while 18S n-PCR assay detected 10 mixed infection cases of P. vivax and P. falciparum, which were not recognized by microscopy. Non-invasive Plasmodium detection rate with DNA derived from saliva samples was highest for 18S n-PCR (87.36 %), followed by singleplex CRS (81 %) and multiplex CRS PCR assay (70.5 %). Specificity for P. vivax and P. falciparum detection for all assays was 98.48 % and 100 % respectively. Detection rate for P. vivax in saliva correlated with parasite density for CRS target-based assays. The species-specific CRS PCR, either as a singleplex or multiplex assay, can have an impact on diagnosis and epidemiological studies in malaria.

Control and eradication of malaria is hindered by the acquisition of drug resistance by Plasmodium species. This has necessitated a persistent search for novel drugs and more efficient targets. Plasmodium species specific lactate dehydrogenase is one of the potential therapeutic and diagnostic targets, because of its indispensable role in endoerythrocytic stage of the parasite. A target molecule that is highly conserved in the parasite population can be more effectively used in diagnostics and therapeutics, hence, in the present study polymorphism in PfLDH (Plasmodiumfalciparum specific LDH) and PvLDH (Plasmodiumvivax specific LDH) genes was analyzed using PCR-single strand confirmation polymorphism (PCR-SSCP) and sequencing. Forty-six P. falciparum and thirty-five P. vivax samples were screened from different states of India. Our findings have revealed presence of a single PfLDH genotype and six PvLDH genotypes among the studied samples. Interestingly, along with synonymous substitutions, nonsynonymous substitutions were reported to be present for the first time in the PvLDH genotypes. Further, through amino acid sequence alignment and homology modeling studies we observed that the catalytic residues were conserved in all PvLDH genotypes and the nonsynonymous substitutions have not altered the enzyme structure significantly. Evolutionary genetics studies have confirmed that PfLDH and PvLDH loci are under strong purifying selection. Phylogenetic analysis of the pLDH gene sequences revealed that P. falciparum compared to P. vivax, has recent origin. The study therefore supports PfLDH and PvLDH as suitable therapeutic and diagnostic targets as well as phylogenetic markers to understand the genealogy of malaria species., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Malaria is one of the most devastating diseases of tropical countries with clinical manifestations such as anaemia, splenomegaly, thrombocytopenia, hepatomegaly and acute renal failures. In this study, cases of thrombocytopenia and haemoglobinemia were more prominent in subjects infected with Plasmodium falciparum (Welch, 1897) than those with Plasmodium vivax (Grassi et Feletti, 1890). However, anaemia, jaundice, convulsions and acute renal failure were significantly high (3-4 times) in subjects infected with P. falciparum than those infected with P. vivax. The incidence of splenomegaly and neurological sequelae were 2 and 6 times higher in P. falciparum infections compared to the infections of P. vivax. Both in P. vivax and P. falciparum malaria, the cases of splenomegaly, jaundice and neurological sequelae were almost double in children (<10 years) compared to older patients. The liver enzymes were generally in normal range in cases of low and mild infections. However, the AST, ALT, ALP activities and serum bilirubin, creatinine, and the urea content were increased in P. falciparum and P. vivax malaria patients having high parasitaemia, confirming liver dysfunction and renal failures in few cases of severe malaria both in India and Saudi Arabia.

Light microscopy of blood smears for diagnosis of malaria in the field has several limitations, notably delays in diagnosis. This study in Ahmedabad in Gujarat State, India, evaluated the diagnostic performance of a rapid diagnostic test for malaria (SD Bioline Malaria Ag P.f/Pan) versus blood smear examination as the gold standard. All fever cases presenting at 13 urban health centres were subjected to rapid diagnostic testing and thick and thin blood smears. A total of 677 cases with fever were examined; 135 (20.0%) tested positive by rapid diagnostic test and 86 (12.7%) by blood smear. The sensitivity of the rapid diagnostic test for malaria was 98.8%, specificity was 91.5%, positive predictive value 63.0% and negative predictive value 99.8%. For detection of Plasmodium falciparum the sensitivity of rapid diagnostic test was 100% and specificity was 97.3%. The results show the acceptability of the rapid test as an alternative to light microscopy in the field setting.

Background & Objectives: Recently, vivax malaria is also presenting as severe malaria causing multiorgan dysfunction similar to falciparum malaria. The present study was undertaken to evaluate the involvement of cardiovascular system in severe malaria., Methods: This is a clinical prospective study conducted on the cases of severe malaria in S.P. Medical College and PBM Hospital, Bikaner, India. In total, 100 cases (45 males, 55 females; age range 13-75 yr) of severe malaria (P. vivax 60; P. falciparum 28; and mixed 12) diagnosed by peripheral blood smear examination, rapid card test and PCR were studied. Evaluation of cardiovascular system was done by clinical examination, chest Xray, ECG, high resolution transthoracic echocardiography and estimation of cardiac markers., Results: In all, 17% cases (9 P. falciparum, 5 P. vivax and 3 mixed) were found to be suffering from cardiovascular involvement (11% circulatory failure, 7% congestive cardiac failure and 2% pulmonary edema). ECG showed sinus tachycardia in all the 17 patients, one had atrial ectopic and eight had non-specific ST-T changes. Cardiomegaly was seen in eight cases and pulmonary edema in two on X-ray chest. Echocardiography was within normal range but cardiac dimensions were increased in all the 17 cases. Troponin-I and CPK-MB were increased in 14 cases. Cardiovascular involvement in P. falciparum and mixed infection was associated with high parasite density but P. vivax infection was associated with relatively low parasite density. Involvement of cardiovascular system was associated with increased hospital stay (7.67 ± 2.23 vs 6.59 ± 0.87 days; p <0.001) and high mortality (5 died out of 17 patients). Significant ECG changes and cardiac markers indicate myocardial involvement in severe malaria., Interpretation & Conclusion: The present study indicates involvement of cardiovascular system in severe malaria as evidenced by changes in ECG and cardiac markers (Trop 1 and CPK-MB). The present study also highlights that vivax malaria is no more benign and pathophysiology of vivax malaria should be re-evaluated.

Background & Objectives: The search for effective polymorphic markers in Plasmodium vivax is highly demanding to understand its transmission in a population. Due to the limited knowledge existing for P. vivax, the search for polymorphic markers for population studies is ongoing. The MSP gene family of Plasmodia has been linked with immune evasion. To study the circulating parasite population P. vivax merozoite surface protein 3β (PvMSP3β) polymorphic marker was used to investigate the genetic diversity of P. vivax in natural infections., Methods: Polymorphism of PvMSP3β gene was determined in 46 P. vivax blood samples from six different regions of India by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) with PstI., Results: Two different parasite genotypes, viz. type-A and type-B were detected among 46 samples that were positive for PCR, based on the size of the amplification. RFLP analysis with PstI showed 22 allelic groups and 15.2% samples revealed mixed infections on analysis., Conclusion: PvMSP3β was found to be an effective molecular marker for P. vivax as it shows high diversity in India and multiple genotypes easily distinguishable without the need for sequencing.

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., (Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

Acute renal failure is one of the serious complications of malaria with untoward consequences including increased risk of mortality. This study was conducted to evaluate the incidence, clinical features, course, outcome, and predictors of mortality of acute renal failure (ARF) in children with malaria in the pediatric general wards and intensive care unit of our medical center from 1 January 2009 to 31 December 2009. Data of all children from 1 month to 12 years of age with confirmed malaria either on peripheral smear and/or OptiMal test were reviewed. Those suffering from ARF were selected for further study. Twelve (eight Plasmodium falciparum; three Plasmodium vivax, and one mixed infection) out of 227 (5%) cases of confirmed malaria had ARF. In addition to ARF, most of the patients had at least one other manifestation of severe malaria. Nine (75%) patients recovered completely, while 3 (25%) died. Presence of associated cerebral malaria, hyperbilirubinemia, and disseminated intravascular coagulopathy (DIC) was a poor prognostic factor and predictor of mortality. In conclusion, ARF can complicate both P. falciparum and P. vivax malaria. Malarial ARF as an isolated complication has a good prognosis. The presence of multiorgan involvement and delayed diagnosis increases morbidity and mortality.

Background: Koraput, a predominantly tribe-inhabited and one of the highly endemic districts of Odisha State that contributes a substantial number of malaria cases to the India's total. Control of malaria in such districts would contribute to change the national scenario on malaria situation. Hence, a study was carried out to measure the magnitude of malaria prevalence in the district to strengthen the malaria control activities., Methods: Prevalence of malaria was assessed through a sample blood survey (SBS) in seven randomly selected community health centres (CHCs). Individuals of all age groups in the villages selected (one in each subcentre) were screened for malaria infection. Both thick and thin smears were prepared from blood samples collected by finger prick, stained and examined for malaria parasites searching 100 fields in each smear. The results of a blood survey (n = 10,733) carried out, as a part of another study, during 1986-87 covering a population of 17,722 spread in 37 villages of Koraput district were compared with the current survey results. Software SPSS version 16.0 was used for data analysis., Result: During the current study, blood survey was done in 135 villages screening 12,045 individuals (16.1% of the total population) and among them, 1,983 (16.5%) were found positive for malaria parasites. Plasmodium falciparum was the major malaria parasite species accounted for 89.1% (1,767) of the total positives; Plasmodium vivax and Plasmodium malariae accounted for 9.3% (184) and 0.2% (5), respectively. Gametocytes were found in 7.7% (n = 152) of the positive cases. The majority of parasite carriers (78.9%) were afebrile. The 1986-87 blood survey showed that of 10,733 people screened, 833 (7.8%) were positive for malaria parasites, 714 (85.7%) with P. falciparum, 86 (10.3%) with P. vivax, 12 (1.4%) with P. malariae and 21 (2.5%) with mixed infections., Conclusion: The results of the current study indicated a rising trend in transmission of malaria in Koraput district compared to the situation during 1986-87 and indicated the necessity for a focused and reinforced approach for the control of the disease by improving people's access to diagnosis and treatment and ensuring implementation of the intervention measures with adequate coverage and compliance.

Variant surface antigens (VSAs) present on the surface of parasitized erythrocytes facilitate many Plasmodium spp. to escape the host immune system during infection. Multigene families coding for VSAs exist in several Plasmodium spp. and are located on telomeric and subtelomeric regions of the chromosomes. P. vivax genome also contains a multigene superfamily vir (variant interspersed repeats), present in the subtelomeric region with a possible role in immune evasion like the var gene in P. falciparum. Blood samples from 148 symptomatic malaria cases were collected from five different regions of India, viz. Mangalore, Rourkela, Goa, Delhi and Jabalpur. P. vivax isolates (74 single infections) were sequenced for four vir genes (viz. vir 27, vir 4, vir 12 and vir 21) and analyzed for the genetic variability existing in different populations of India. The results indicate that vir genes in different P. vivax populations in India are highly divergent both within and between the isolates. High levels of single nucleotide polymorphisms (SNPs) were observed attributing to the existing polymorphism for all the four vir genes studied across the population. Detailed knowledge of the genetic variation among the vir genes will help in understanding the evolutionary aspects of vir genes and may also provide basis for understanding the disease chronicity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Plasmodium vivax reticulocyte binding protein-2 (PvRBP-2) is a promising candidate for development of vaccine against parasite. DNA sequence polymorphism in pvrbp-2 which may hamper the vaccine development program has been identified in laboratory strains. Therefore, unraveling genetic polymorphism in pvrbp-2 from field isolates is a prerequisite for success in vaccine development. This study was designed with a primary aim to uncover genetic polymorphism in pvrbp-2 among P. vivax field isolates., Results: Using virtual restriction mapping of pvrbp-2 sequences, two restriction enzymes (AluI and ApoI) were selected for the development of pvrbp-2 as a PCR-RFLP marker. Restriction fragment length polymorphism (RFLP) analysis revealed a high degree of genetic polymorphism in the pvrbp-2 gene among field isolates of P. vivax. ApoI-RFLP was found to be more efficient in identifying the extent of genetic polymorphism in pvrbp-2 compared to AluI-RFLP. Combined genotyping/haplotyping of RFLP pattern revealed a total of 36 distinct RFLP patterns among 83 P. vivax isolates analyzed. DNA sequence analysis also supports high degree of genetic polymorphism among field isolates of P. vivax. Pvrbp-2 PCR-RFLP method is able to distinguish multiple infection up to 16.86% and it revealed a low level of shared genetic pool between more than two populations., Conclusion: The study suggests that pvrbp-2 is highly polymorphic genetic marker which can be used for population genetic analyses. RFLP analysis suggests presence of nearly similar proportion of Sal-1 and Belem alleles in Indian P. vivax populations. The larger extent of genetic polymorphism identified from limited samples advocates to screen genetic polymorphism in pvrbp-2 from malaria endemic geographical regions and countries for designing pvrbp-2 based anti-malarial control measures.

The apicomplexan parasite Plasmodium vivax is responsible for causing more than 70% of human malaria cases in Central and South America, Southeastern Asia and the Indian subcontinent. The rising severity of the disease and the increasing incidences of resistance shown by this parasite towards usual therapeutic regimens have necessitated investigation of putative novel drug targets to combat this disease. The apicoplast, an organelle of procaryotic origin, and its circular genome carrying genes of possible functional importance, are being looked upon as potential drug targets. The genes on this circular genome are believed to be highly conserved among all Plasmodium species. Till date, the plastid genome of P. falciparum, P. berghei and P. chabaudi have been detailed while partial sequences of some genes from other parasites including P. vivax have been studied for identifying evolutionary positions of these parasites. The functional aspects and significance of most of these genes are still hypothetical. In one of our previous reports, we have detailed the complete sequence, as well as structural and functional characteristics of the Elongation factor encoding tufA gene from the plastid genome of P. vivax. We present here the sequences of large and small subunit rRNA (lsu and ssu rRNA) genes, sufB (ORF470) gene, RNA polymerase (rpo B, C) subunit genes and clpC (casienolytic protease) gene from the plastid genome of P. vivax. A comparative analysis of these genes between P. vivax and P. falciparum reveals approximately 5-16% differences. A codon usage analysis of major plastid genes has shown a high frequency of codons rich in A/T at any or all of the three positions in all the species. TTA, AAT, AAA, TAT, and ATA are the major preferred codons. The sequences, functional domains and structural analysis of respective proteins do not show any variations in the active sites. A comparative analysis of these Indian P. vivax plastid genome encoded genes has also been done to understand the evolutionary position of the Indian parasite in comparison to other Plasmodium species., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The human malaria parasite Plasmodium vivax is globally widespread, causing high malaria morbidity. As P. vivax is highly endemic to India, and previous reports indicate genetic homogeneity in population samples, we tested the hypothesis of no genetic structuring in Indian P. vivax. Further, based on the reports of increasing incidence of Plasmodium falciparum infection in comparison with P. vivax in recent years in India, it was important to understand whether reduction in population size has resulted in decrease in P. vivax infection rate in India. For this, we utilized recently developed putatively neutral markers from chromosome 13 of P. vivax to score single nucleotide polymorphisms in 126 P. vivax isolates collected from 10 different places in India. The overall results indicated that Indian P. vivax bears high nucleotide diversity within population samples but moderate amount of genetic differentiation between population samples. STRUCTURE analysis grouped 10 population samples into three clusters based on the proportion of the genetic ancestries in each population. However, the pattern of clustering does not correlate with sampling locations in India. Furthermore, analyses of past demographic events indicated reduction in population size in majority of population samples, but when isolates from all the 10 samples were considered as a single population, the data fit to the demographic equilibrium model. All these observations clearly indicate that Indian P. vivax presents complex evolutionary history but possesses several features of being a part of ancestral distribution range of this species., (© 2012 Blackwell Publishing Ltd.)

Plasmodium vivax is traditionally known to cause benign tertian malaria, although recent reports suggest that P. vivax can also cause severe life-threatening disease analogous to severe infection due to P. falciparum. There are limited published data on the clinical and epidemiological profiles of children suffering from 'severe malaria' in an urban setting of India. To assess the clinical and epidemiological profiles of children with severe malaria, a prospective study was carried out during June 2008-December 2008 in the Department of Pediatrics, Guru Teg Bahadur Hospital, a tertiary hospital located in East Delhi, India. Data on children aged < or = 12 years, diagnosed with severe malaria, were analyzed for their demographic, clinical and laboratory parameters. All patients were categorized and treated as per the guidelines of the World Health Organization. In total, 1,680 children were screened for malaria at the paediatric outpatient and casualty facilities of the hospital. Thirty-eight children tested positive for malaria on peripheral smear examination (2.26% slide positivity rate). Of these, 27 (71%) were admitted and categorized as severe malaria as per the definition of the WHO while another 11 (29%) received treatment on outpatient basis. Most (24/27; 88.8%) cases of severe malaria (n=27) were infected with P. vivax. Among the cases of severe malaria caused by Plasmodium vivax (n=24), 12 (50%) presented with altered sensorium (cerebral malaria), seven (29.1%) had severe anaemia (haemoglobin <5 g/dL), and 17 (70.8%) had thrombocytopaenia, of which two had spontaneous bleeding (epistaxis). Cases of severe vivax malaria are clinically indistinguishable from severe falciparum malaria. Our study demonstrated that majority (88.8%) of severe malaria cases in children from Delhi and adjoining districts of Uttar Pradesh were due to P. vivax-associated infection. P. vivax should, thus, be regarded as an important causative agent for severe malaria in children.

Background: Chloroquine resistance (CQR) phenotype in Plasmodium falciparum is associated with mutations in pfcrt and pfmdr-1 genes. Mutations at amino acid position 72-76 of pfcrt gene, here defined as pfcrt haplotype are associated with the geographic origin of chloroquine resistant parasite. Here, mutations at 72-76 and codon 220 of pfcrt gene and N86Y pfmdr-1 mutation were studied in blood samples collected across 11 field sites, inclusive of high and low P. falciparum prevalent areas in India. Any probable correlation between these mutations and clinical outcome of CQ treatment was also investigated., Methods: Finger pricked blood spotted on Whatman No.3 papers were collected from falciparum malaria patients of high and low P. falciparum prevalent areas. For pfcrt haplotype investigation, the parasite DNA was extracted from blood samples and used for PCR amplification, followed by partial sequencing of the pfcrt gene. For pfmdr-1 N86Y mutation, the PCR product was subjected to restriction digestion with AflIII endonuclease enzyme., Results: In 240 P. falciparum isolates with reported in vivo CQ therapeutic efficacy, the analysis of mutations in pfcrt gene shows that mutant SVMNT-S (67.50%) and CVIET-S (23.75%) occurred irrespective of clinical outcome and wild type CVMNK-A (7.91%) occurred only in adequate clinical and parasitological response samples. Of 287 P. falciparum isolates, SVMNTS 192 (66.89%) prevailed in all study sites and showed almost monomorphic existence (98.42% isolates) in low P. falciparum prevalent areas. However, CVIETS-S (19.51%) and CVMNK-A (11.84%) occurrence was limited to high P. falciparum prevalent areas. Investigation of pfmdr-1 N86Y mutation shows no correlation with clinical outcomes. The wild type N86 was prevalent in all the low P. falciparum prevalent areas (94.48%). However, mutant N86Y was comparably higher in numbers at the high P. falciparum prevalent areas (42.76%)., Conclusions: The wild type pfcrt gene is linked to chloroquine sensitivity; however, presence of mutation cannot explain the therapeutic efficacy of CQ in the current scenario of chloroquine resistance. The monomorphic existence of mutant SVMNT haplotype, infer inbreeding and faster spread of CQR parasite in areas with higher P. vivax prevalance and chloroquine exposure, whereas, diversity is maintained in pfcrt gene at high P. falciparum prevalent areas.

Background: Malaria due to both Plasmodium falciparum and Plasmodium vivax is a major public health problem in India. The quantification of malaria transmission for the classification of malaria risk has long been a concern for epidemiologists. Results are presented from 30 cross-sectional surveys which measured spleen rates (SR) and infant parasite rates (IPR) in the forested districts of Madhya Pradesh during malaria outbreaks to assess whether both IPR and SR can still be used as indicators of malaria endemicity as spleen examination has lost much of its value as an epidemiological indicator in areas where anti-malarials drugs are widely used., Methods: Rapid fever surveys were carried out from door to door and all suspected malaria cases in the entire population of a village were screened for malaria parasites on the basis of clinical symptoms such as fever, chill, rigor, headache and body ache etc. Children between 2 and 9 years were examined for enlarged spleen according to Hacketts method. Finger prick blood smears were collected from all children with enlarged spleen with or without fever after obtaining written informed consent following institutional ethical guidelines. Infants less than 1 year were also screened for malaria with or without fever., Results: Since malaria is local and focal, in some areas the outbreak waned quickly in few months and in some areas continued for 3 to 4 years. The analysis of trend revealed that when IPR decline over the years as a result of malaria intervention measures, SR also decline. In case splenomegaly continues without diminution in size, it is probably due to recrudescence or relapse, although it is not possible to separate malaria parasite species on the basis of SR., Conclusion: Both the tools are of immense value in evaluating and assessing the malaria situation especially in remote areas where sophisticated molecular and serological techniques are difficult to establish. Therefore, in forested areas malaria surveillance system will require adoption of multiple approaches that have proven effective now or in the past.

Background & Objectives: Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are responsible for most of the global burden of malaria. With changing spectrum of clinical presentation in malaria, pulmonary system involvement has always been under diagnosed. The present study was planned to estimate the pulmonary system involvement in patients with malaria from north- western India (Bikaner). STUDY DESIGN & DESCRIPTION OF THE PATIENTS: Our study was conducted during 2007 to 2009 in 200 cases of severe malaria [Pf , Pv, and mixed (Pf + Pv)] with pulmonary involvement. It included adult patients of both sexes (145 males and 55 females) belonging to all age groups. The diagnosis of Pf and Pv was confirmed by demonstrating asexual form of parasites in peripheral blood smear and OptiMal test., Main Outcome Measures: Pulmonary involvement was observed in 30% (60/200) patients among which cough in 24% , dyspnea in 12%, acute respiratory distress syndrome (ARDS) in 7% , bronchitis in 3% and pneumonia in 1.5% were the major clinical manifestations of malaria. Metabolic acidosis and low oxygen saturation was observed in 7% patients. Chest X- ray abnormality in 11.5% patients, 7% had bilateral infiltrates, 1.5% had inflammatory patch and 3% had findings suggestive of bronchitis. Spirometry findings showed 17% patients had early small airway obstruction. All the patients with ARDS had poor disease outcome., Results & Conclusion: Our results suggest that pulmonary system involvement was observed in patients infected with Pf and Pv. If these clinical presentations are ignored, it may lead to delay in diagnosis and can alter the outcome and prognosis of the disease. Therefore, early diagnosis of malaria induced ARDS can significantly affect the outcome.

Although malaria is endemic in India, neonatal disease is considered rare. We report a case of neonatal malaria in a 26-day-old neonate with fever and splenomegaly who was diagnosed after a long and unsuccessful battery of tests for splenomegaly. Routine screening for malaria is essential for all neonates with fever in endemic areas. Early diagnosis and treatment of malaria could effectively prevent infant mortality.

Background: Genetic polymorphism is an inevitable component of a multistage infectious organism, such as the malaria parasite. By means of genetic polymorphism, parasite opts particular polymorph and reveals survival advantage. Pvs25 and pvs28 are sexual stage antigen genes, expressed at the ookinete stage inside the mosquito gut, and considered as potential transmission-blocking vaccine candidates. This study presents sequence variations in two important transmission blocking antigen genes pvs25 and pvs28 in the field isolates of P. vivax from the Indian subcontinent., Methods: One hundred microscopically diagnosed P. vivax isolates were collected from five geographical regions of India. Pvs25 and pvs28 genes were PCR amplified and sequenced to assess sequence variation among field isolates., Results: A total of 26 amino acid substitutions were observed in Pvs25 (10) and Pvs28 (16) among field isolates of P. vivax. Tandem repeat polymorphism observed in pvs28 shows 3-6 tandem repeats in the field isolates. Seven and eight novel amino acid substitutions were observed in Pvs25 and Pvs28, respectively in Indian isolates. Comparison of amino acid substitutions suggests that majority of substitutions observed in global isolates were also present in Indian subcontinent. A single haplotype was observed to be major haplotype among isolates of Delhi, Nadiad, Chennai and Panna except in isolates of Kamrup. Further, population comparison analyses suggest that P. vivax isolates inhabiting in north-eastern region (Kamrup) were distantly related with the isolates from remaining parts of the country. Majority of the amino acid substitutions observed in Indian isolates were more identical to the substitutions reported from isolates of Thailand and Bangladesh., Conclusion: Study uncovered many new amino acid substitutions as well as a predominance of single haplotype in Indian subcontinent except in north-eastern region of the country. The amino acid substitutions data generated in this study from different geographical regions of the Indian subcontinent could be helpful in designing a more effective anti-malarial transmission-blocking vaccine.

Background: Sulphadoxine and pyrimethamine are anti-folate drugs that show synergistic anti-malarial effect. Point mutations in dihydrofolate reductase (dhfr) and dihydropteorate synthatase (dhps) cause anti-folate drug resistance phenotype in human malaria parasites. This study presents pattern of point mutations in dhfr/dhps genes among Indian sub-continent., Methods: Microscopically diagnosed one hundred Plasmodium vivax field isolates were collected from five widely separated geographical regions of India. Dhfr and dhps genes were PCR amplified and sequenced. Previously published mutations data were collected and analyzed using Chi square test to identify geographical cluster of mutant/wild type genotypes., Results: Sequence analysis revealed single (S58R), double (S58R/S117N) and quadruple (F57L/S58R/T61M/S117T/) point mutations at dhfr and single (A383G) to double (A383G/A553G) mutations at dhps in P. vivax field isolates. In addition, three new mutations were also observed at dhfr. Both, dhfr and dhps genes revealed tandem repeat variations in field isolates. Dhps revealed very low mutation frequency (14.0%) compared to dhfr (50.70%). Comparative analysis revealed a progressive increase in frequency of quadruple mutant dhfr genotype (p<0.001) within five years in north-eastern state (Kamrup, Assam). Frequency of dhfr genotypes revealed three distinct geographical clusters of wild (northern India), double mutant (southern India), and quadruple mutant (north-eastern and island regions of India) on the Indian sub-continent., Conclusion: Study suggests that SP may be susceptible to P. vivax in India, except Andaman and north-eastern state. The distinction of geographical regions with sensitive and resistant parasite phenotypes would be highly useful for designing and administering national anti-malarial drug policy.

To assess the impact of irrigation canals on malaria transmission, a study was conducted in Dhenkanal district of Orissa, India. The district is situated in the central part of Orissa and hyperendemic area for malaria. A canal system is being constructed for irrigation in the district, which passes through Parjang and Analabereni Primary Health Centres (PHC), endemic for malaria. The water has been released only up to Parjang (Canal with water -CWW) area during the end of 2004 and construction work is still going on in Analabereni PHC (Canal under construction-CUC). Retrospective clinical data (2001-2008) collected from health services from two study sites showed average Slide Positivity Rate (SPR) before release of water (2001-2004) was 9.25% and 18.04% in CWW and CUC areas, respectively. After release of water (2005-2008) the SPR was 5.77% and 10.19%, in CWW and CUC areas, respectively. The average Annual Parasite Incidence (API) was 7.66 and 22.67 in CWW and CUC areas before the release of water and 5.32 and 12.28 after release of water, respectively. A point fever survey was conducted in 2009 which revealed the presence of Plasmodium falciparum (Pf) and P. vivax (Pv) in both study areas. The survey found SPR of 18.82% and 24.54%, and Pf percentages of 75% and 85%, in CWW and CUC areas, respectively. The present study revealed the presence of two malaria vectors, Anopheles culicifacies and Anopheles annularis in the area. Vector Per Man Hour Density was 2.38 in CWW and 2.69 in CUC for An. culicifacies and 1.46 and 1.54 for An. annularis respectively. The sporozoites rates were found to be 3.6 and 3.8 for CWW and CUC, respectively. The present study reveals that, the construction of canal system did not increase the malaria prevalence during post water release period - implying that the malaria control programme was effective although still more intensive situation specific vectors control programme need to be continued simultaneously so that malaria transmission can be curtailed.

Introduction: despite the high prevalence of Plasmodium vivax (P vivax) malaria, research into its complications has lagged disproportionately as compared to Plasmodium falciparum (P falciparum) malaria., Material and Methods: the present retrospective observational study was conducted on cases with P vivax mono-infection presenting with severe malaria on the basis of one or more criteria as per the World Health Organization guidelines being used for severe falciparum malaria in children, as well as other manifestations been classified as complicated malaria, during an outbreak of malaria in a single tertiary referral hospital of north India., Results: seventy-four patients of acute malaria presented during the outbreak, of which 50 cases with P vivax mono-infection were included for the study. Complicated malaria was diagnosed in 41/50 cases, with thrombocytopenia being the commonest manifestation. Other presentations of severe malaria in our patients were liver dysfunction (with or without jaundice) 31/50 cases, respiratory involvement 14/50 cases, renal impairment 11/50 cases, circulatory collapse (Shock) 8/50 cases, severe anaemia 3/50 cases and central nervous system (CNS) involvement 2/50 cases., Conclusion: the term "benign tertian malaria" no longer holds true for P vivax mono-infection. The authors wish to open a new front for researches on the possible genotypic abnormalities that the parasite or its carrier might have acquired over decades and has transformed into a species with the malignant potential of P falciparum., (2010 Elsevier B.V. All rights reserved.)