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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.)

Background: Antimalarial drug resistance surveillance and containment are crucial for countries aiming to eliminate malaria. Monitoring resistance evolution through studies before and after treatment policy changes is essential., Methods: A total of 939 Plasmodium falciparum-positive blood samples were collected between 2014 and 2015 across 10 sites in India, categorized into 4 geographic clusters. Polymerase chain reaction-amplified products were sequenced to identify point mutations at drug resistance-conferring genes (Pfdhfr, Pfdhps, Pfmdr1, and Pfk13)., Results: Triple Pfdhfr mutants were found only in northeast India bordering Myanmar, while the wild type (WT) was dominant in central India. Pfdhps WTs were prevalent in all areas, and no double mutants were found. Except in northwest India, Pfmdr1 WT was dominant in all clusters. Nonsynonymous double mutations were only found in northwest India. Only synonymous mutations occurred in Pfk13. These were found in central India at a low frequency. The pattern of linkage disequilibrium and principal component analysis reflects low pressure for drug resistance and heterogeneity between the geographic clusters., Conclusions: Resistance levels were highest in northeast India, close to the Myanmar border, where resistance is common. Primaquine has been widely used as a gametocidal and schizonticidal drug, likely contributing to maintaining low drug resistance levels and preventing strong selection for resistance., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Theileria orientalis, the causal agent of oriental theileriosis, is known to cause mild disease in cattle and buffalo across the world. Recently, different genotypes of T. orientalis have emerged as pathogenic, causing high reported morbidity in cattle. This study focuses on investigating three suspected outbreaks of oriental theileriosis that resulted in fatalities among crossbred and indigenous bulls in Karnataka, India. Examination of blood smears revealed the presence of T. orientalis piroplasms within erythrocytes. The genetic characterization of T. orientalis was conducted by targeting specific markers, including the mpsp gene, p23 gene, and ribosomal DNA markers (18S rRNA gene, ITS-1, and ITS-2). Analysis based on the 18S rRNA gene unveiled the presence of both Type A and Type E genotypes of T. orientalis in the outbreaks. The mpsp gene-based analysis identified genotype 7 of T. orientalis in crossbred cows, whereas genotype 1 (Chitose B) was found to be present in indigenous bulls. Haplotype network analysis based on the mpsp gene revealed the presence of 39 distinct haplotypes within the 12 defined genotypes of T. orientalis with a high haplotype diversity of 0.9545 ± 0.017. Hematological and biochemical analysis revealed a decrease in calcium, hemoglobin levels, red blood cell counts, and phosphorus. This study constitutes the initial documentation of a clinical outbreak of oriental theileriosis in indigenous bulls with genotype 1 (Chitose 1B). Substantial epidemiological investigations are imperative to gain a comprehensive understanding of the geographical distribution of distinct genotypes and the diverse clinical manifestations of the disease across various hosts., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Genetically diverse parasites enhances resistance against antimalarials, vaccines and host immune responses. The present study was designed to evaluate the role played by Plasmodium falciparum genetic diversity in predicting the real world malarial population., Methods: Initially, the incidence pattern of all four northern Indian malarial species was examined using 18S rRNA gene and performed principal component analysis (PCA) based on frequencies of Plasmodium species. Consequently, genetic variance of Plasmodium falciparum histidine-rich protein-2 (Pfhrp2) gene among different malarial populations were compared using phylogenetic analysis. Multi-dimensional scaling was performed to assess genetic similarities and distances among studied populations., Results: Of total 2168 patients screened, 561 patients with fever of unknown origin were included. 18S rRNA and Pfhrp2 genes were amplified in 78 and 45 samples, respectively. Among them 13.9%(78/561) patients had Plasmodium infection. Infections by P. falciparum, P. vivax and mixed infections were diagnosed among 47(60.2%) and 28(35.9%) and 3(3.8%) patients, respectively. We found eight types of Pfhrp2 amino acid sequence repeats among northern Indian population. The PCA findings were in line with genetic diversity and phylogenetic data. Temporal analysis showed the proportion of total diversity present in total subpopulation (ΔS/ΔT) was maximum for P. falciparum., Conclusions: Higher incidence of Pfhrp2 sequence variation through genetic recombination among multiple strains during sexual reproduction is potentially correlated with high transmission activity. This sequence variation might alter RDT detection sensitivities for different parasites by modulating the structure and frequency of antigenic epitopes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Ujjala Ghoshal reports financial support was provided by UP Council of Science & Technology Lucknow Uttar Pradesh., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

The bulk of malaria rapid diagnostic tests (RDTs) target histidine-rich protein 2 of Plasmodium falciparum, the deadliest malaria species. The WHO considers pfhrp2/3 deletions as one of the main threats to successful malaria control and/or elimination; as such, parasites that lack part or all of the pfhrp2 gene are missed by pfHRP2-targeting RDTs. Such deletions have been reported in several African and Asian countries, but little is known in Cameroon and India. Blood samples were collected from individuals living in four areas of Cameroon (Douala, Maroua, Mayo-Oulo, Pette) and India (Mewat, Raipur, Ranchi, Rourkela). Deletions in pfhrp2/3 genes were confirmed if samples 1) had ≥100 parasites/µL by quantitative polymerase chain reaction (PCR), 2) PCR negative for pfhrp2/3, and 3) PCR positive for at least two single-copy genes. The overall proportion of pfhrp2 and pfhrp3 deletions in Cameroon was 13.5% and 3.1%. In India, the overall proportion was 8% for pfhrp2 and 4% for pfhrp3. The overall proportions of samples with both gene deletions (pfhrp2-/3-) were 3.1% in Cameroon and 1.3% in India. In Cameroon, pfhrp2-/3+ and pfhrp2-/3- deletions were common in Maroua (P = 0.02), in asymptomatic parasitemia (P = 0.006) and submicroscopic parasitemia (P <0.0001). In both countries, pfhrp2/3 deletions, including pfhrp2-/3- deletions, were mainly seen in monoclonal infections. This study outlines that double deletions that result in false negative RDTs are uncommon in our settings, and highlights the importance of active molecular surveillance for pfhrp2/3 deletions in Cameroon and India.

Surveillance for genetic markers of resistance can provide valuable information on the likely efficacy of antimalarials but needs to be targeted to ensure optimal use of resources. We conducted a systematic search and review of publications in seven databases to compile resistance marker data from studies in India. The sample collection from the studies identified from this search was conducted between 1994 and 2020, and these studies were published between 1994 and 2022. In all, Plasmodium falciparum Kelch13 (PfK13), P. falciparum dihydropteroate synthase, and P. falciparum dihydrofolate reductase (PfDHPS) genotype data from 2,953, 4,148, and 4,222 blood samples from patients with laboratory-confirmed malaria, respectively, were extracted from these publications and uploaded onto the WorldWide Antimalarial Resistance Network molecular surveyors. These data were fed into hierarchical geostatistical models to produce maps with a predicted prevalence of the PfK13 and PfDHPS markers, and of the associated uncertainty. Zones with a predicted PfDHPS 540E prevalence of >15% were identified in central, eastern, and northeastern India. The predicted prevalence of PfK13 mutants was nonzero at only a few locations, but were within or adjacent to the zones with >15% prevalence of PfDHPS 540E. There may be a greater probability of artesunate-sulfadoxine-pyrimethamine failures in these regions, but these predictions need confirmation. This work can be applied in India and elsewhere to help identify the treatments most likely to be effective for malaria elimination.

Background: Resistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine-pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum., Methods: Dried blood spots were collected during the active fever survey and mass screening and treatment activities as part of the Malaria Elimination Demonstration Project (MEDP) from 2019 to 2020. Isolated DNA samples were used to amplify the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes using nested PCR and sequenced for mutation analysis using the Sanger sequencing method., Results: A total of 393 samples were subjected to PCR amplification, sequencing and sequence analysis; 199, 215, 235, and 141 samples were successfully sequenced for Pfdhfr, Pfdhps, Pfk13, Pfmdr1, respectively. Analysis revealed that the 53.3% double mutation (C59R, S108N) in Pfdhfr, 89.3% single mutation (G437A) in Pfdhps, 13.5% single mutants (N86Y), and 51.1% synonymous mutations in Pfmdr1 in the study area. Five different non-synonymous and two synonymous point mutations found in Pfk13, which were not associated to artemisinin resistance., Conclusion: The study has found that mutations linked to SP resistance are increasing in frequency, which may reduce the effectiveness of this drug as a future partner in artemisinin-based combinations. No evidence of mutations linked to artemisinin resistance in Pfk13 was found, suggesting that parasites are sensitive to artemisinin derivatives in the study area. These findings are a baseline for routine molecular surveillance to proactively identify the emergence and spread of artemisinin-resistant parasites., (© 2023. The Author(s).)

Background: Drug resistance is a serious impediment to efficient control and elimination of malaria in endemic areas., Methods: This study aimed at analysing the genetic profile of molecular drug resistance in Plasmodium falciparum and Plasmodium vivax parasites from India over a ~ 30-year period (1993-2019). Blood samples of P. falciparum and/or P. vivax-infected patients were collected from 14 regions across India. Plasmodial genome was extracted and used for PCR amplification and sequencing of drug resistance genes in P. falciparum (crt, dhps, dhfr, mdr1, k13) and P. vivax (crt-o, dhps, dhfr, mdr1, k12) field isolates., Results: The double mutant pfcrt SVMNT was highly predominant across the country over three decades, with restricted presence of triple mutant CVIET from Maharashtra in 2012. High rates of pfdhfr-pfdhps quadruple mutants were observed with marginal presence of "fully resistant" quintuple mutant ACIRNI-ISGEAA. Also, resistant pfdhfr and pfdhps haplotype has significantly increased in Delhi between 1994 and 2010. For pfmdr1, only 86Y and 184F mutations were present while no pfk13 mutations associated with artemisinin resistance were observed. Regarding P. vivax isolates, the pvcrt-o K10 "AAG" insertion was absent in all samples collected from Delhi in 2017. Pvdhps double mutant SGNAV was found only in Goa samples of year 2008 for the first time. The pvmdr1 908L, 958M and 1076L mutations were highly prevalent in Delhi and Haryana between 2015 and 2019 at complete fixation. One nonsynonymous novel pvk12 polymorphism was identified (K264R) in Goa., Conclusions: These findings support continuous surveillance and characterization of P. falciparum and P. vivax populations as proxy for effectiveness of anti-malarial drugs in India, especially for independent emergence of artemisinin drug resistance as recently seen in Africa., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The global malaria control and elimination program faces major threats due to the emergence and transmission of the anti-malarial drug-resistant strain of Plasmodium falciparum. Monitoring of artemisinin (ART) resistance marker Kelch-13 in the malaria-endemic region is essential in mitigating the disease's morbidity and mortality. The current study aimed to generate baseline information for further surveillance in the future. The current research was designed and conducted from July 2019 to June 2021 to monitor Pfkelch13 mutation at the molecular level in the eastern region of India. We also conducted an in silico study to understand the drug-protein interactions between ART and the protein crystal of PfKelch13 (KELCH) with PDB id:4ZGC. The kelch-13 gene was amplified by nested polymerase chain reaction (PCR) and sequenced through the Sanger sequencing method. Reference 3D7 clone (PF3D7&#95;1343700) was used to align and probe all the sequences. The sequence analysis showed the absence of validated or associated mutation in the Kelch-13 propeller domain. The absence of natural selection in drug resistance was confirmed by the Tajima test. Further, in silico interaction studies between the drug ART and the Kelch propeller domain of P. falciparum were evaluated by structure predictions, molecular docking, molecular dynamics (MD) simulations, and estimations of binding free energies for the KELCH-ART complex. The results were compared with the apoprotein (KELCH-APO). The study confirmed the favorable binding of ART with the Kelch-13 propeller domain., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Recent studies have documented Pfhrp2/3 gene deletion globally as one of the biological threats in the fight against malaria. For malaria diagnosis, PfHRP2 based RDTs are most widely used in India, and performance of these RDTs are affected by deleted Pfhrp2/3 gene in Plasmodium falciparum. This study was planned to confirm Pfhrp2/3 gene deletion incidences and genetic variation in PfHRP2-based RDT positive with P.falciparum malaria cases from India., Methodology: Confirmed positive samples by PfHRP2-based RDTs as P. falciparum (n = 240) from six different endemic regions of India were validated by PCR to assure the actual infection. Two hundred forty samples qualified for DNA intactness by single-copy genes were subjected to amplification for the Pfhrp2/3 gene and its neighbouring gene (downstream and upstream) by PCR genotyping. Genetic variation in samples was analysed post-sequencing using Mega X software. Statistical analysis was performed to validate the genetic variation using Mann-Whitney Test., Results: RDT target region of Pfhrp2 gene (exon2) was found deleted in a single sample with presence of the Pfhrp3 exon2. Complete gene deletion of 4.2% was observed in the Pfhrp3 gene. Partial gene deletion was recorded for both pfhrp2 gene (exon2-0.4%, upstream 25.8% and downstream -9.1%) and Pfhrp3 gene (exon2-18.75%, upstream - 22.08% and downstream 13.3%). Eleven new unique types of amino acid repeat sequence and earlier reported amino acid repeat type was found in the Pfhrp2 gene, prompting high genetic variation., Conclusions: This study suggests that parasites lacking Pfhrp2/3 gene and its neighbouring gene (downstream and upstream) are present in malaria endemic areas of India, resulting in false positive results by RDT. Systematic countrywide monitoring for malaria control and elimination of malaria is warranted in this regard., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Malaria continues to be a major public health problem in the Northeastern part of India despite the implementation of vector control measures and changes in drug policies. To develop successful vaccines against malaria, it is important to assess the diversity of vaccine candidate antigens in field isolates. This study was done to assess the diversity of Plasmodium falciparum AMA-1 vaccine candidate antigen in a malaria-endemic region of Tripura in Northeast India and compare it with previously reported global isolates with a view to assess the feasibility of developing a universal vaccine based on this antigen., Methods: Patients with fever and malaria-like illness were screened for malaria and P. falciparum positive cases were recruited for the current study. The diversity of PfAMA-1 vaccine candidate antigen was evaluated by nested PCR and RFLP. A selected number of samples were sequenced using the Sanger technique., Results: Among 56 P. falciparum positive isolates, Pfama-1 was successfully amplified in 75% (n = 42) isolates. Allele frequencies of PfAMA-1 antigen were 16.6% (n = 7) for 3D7 allele and 33.3% (n = 14) in both K1 and HB3 alleles. DNA sequencing revealed 13 haplotypes in the Pfama-1 gene including three unique haplotypes not reported earlier. No unique amino-acid substitutions were found. Global analysis with 2761 sequences revealed 435 haplotypes with a very complex network composition and few clusters. Nucleotide diversity for Tripura (0.02582 ± 0.00160) showed concordance with South-East Asian isolates while recombination parameter (Rm = 8) was lower than previous reports from India. Population genetic structure showed moderate differentiation., Conclusions: Besides documenting all previously reported allelic forms of the vaccine candidate PfAMA-1 antigen of P. falciparum, new haplotypes not reported earlier, were found in Tripura. Neutrality tests indicate that the Pfama-1 population in Tripura is under balancing selection. This is consistent with global patterns. However, the high haplotype diversity observed in the global Pfama-1 network analysis indicates that designing a universal vaccine based on this antigen may be difficult. This information adds to the existing database of genetic diversity of field isolates of P. falciparum and may be helpful in the development of more effective vaccines against the parasite., (© 2022. The Author(s).)

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.

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 &objectives: The diagnosis of Plasmodium falciparum malaria is widely dependent on the P. falciparum histidine rich protein 2 (PfHRP2) antigens based rapid diagnostic tests. There are few possible factors like Pfhrp2 polymorphism, Pfhrp2 deletion and density of malaria parasite which can affect the sensitivity of the Pf-HRP2-based RDT. The primary objective of the investigation was to check whether the Pfhrp2 gene deletion is the primary cause of RDT false negative cases., Methods: Febrile patients from three districts of Chhattisgarh, India were screened for malaria during 2016-2017 by microscopy and RDT. All microscopy P. falciparum positive samples were validated by PCR. Microscopy positive and RDT negative samples were analyzed for the presence of Exon 2, across Exon 1-2, upstream and downstream of both the Pfhrp2 and Pfhrp3 genes fragment by PCR., Results: Out of 203 screened samples, 85 were detected positive for P. falciparum malaria based on microscopy and PCR. Among these 85 P. falciparum positive samples, 4 samples were observed Pf-HRP2 RDT negative. Although, it signified that the RDTs used were reliable with sensitivity of 95.3% (81/85). 3/4 PfHRP2-RDT negative samples of the P. falciparum isolates exhibited complete deletion of Pfhrp2 and Pfhrp3 genes and one sample was found RDT false negative due to high parasite density., Interpretation & Conclusion: Pfhrp2 and Pfhrp3 deletions that result in false negative RDTs were uncommon in our setting. The continued monitoring of RDTS which results in false negative tests due to Pfhrp2/3 gene deletion is the need of the hour for an effective malaria elimination strategy., Competing Interests: None

Artemisinin is the frontline fast-acting anti-malarial against P. falciparum. Emergence and spread of resistant parasite in eastern-India poses a threat to national malaria control programs. Therefore, the objective of our study is to evaluate the artesunate-sulfadoxine-pyrimethamine efficacy in Central India. 180 monoclonal P. falciparum-infected patients received standard ASSP therapy during August 2015-January 2017, soon after diagnosis and monitored over next 42-days. Artemisinin-resistance was assessed through in-vivo parasite clearance half-life (PC 1/2 ), ex-vivo ring-stage survivability (RSA), and genome analysis of kelch13 and other candidate gene (pfcrt, pfmdr1, pfatpase 6, pfdhfr and pfdhps). Of 180 P. falciparum positive patients, 9.5% showed increased PC 1/2 (> 5.5 h), among them eleven isolates (6.1%) showed reduced sensitivity to RSA. In 4.4% of cases, parasites were not cleared by 72 h and showed prolonged PC 1/2 (5.6 h) (P < 0.005) along with significantly higher RSA (2.2%) than cured patients (0.4%). None of day-3 positive isolates contained the pfkelch13 mutation implicated in artemisinin resistance. Parasite recrudescence was observed in 5.6% patients, which was associated with triple dhfr-dhps (A 16 I 51 R 59 N 108 I 164 -S 436 G 437 K 540 G 581 T 613 ) combination mutation. Emergence of reduced sensitivity to artesunate-sulfadoxine-pyrimethamine, in central India highlighted the risk toward spread of resistant parasite across different parts of India. Day-3 positive parasite, featuring the phenotype of artemisinin-resistance without pfkelch13 mutation, suggested kelch13-independent artemisinin-resistance.

Background: Plasmodium falciparum erythrocyte membrane protein is encoded by a highly variable multicopy var gene family known to play a key role in malaria pathogenicity. Therefore, we investigated sequence variation, expression profile and immune response of the Duffy binding-like domain (DBLα) region of the var gene., Methods: Blood samples were collected from patients with cerebral, severe and mild malaria in Chhattisgarh, India, a region with endemic malaria. Polymerase chain reaction amplicons were cloned and sequenced to determine sequence variation. The expression level was analyzed targeting the upstream region of var gene using the Delta-Delta-Ct method. Immunoglobulin G (IgG) level was determined against the 6 synthetic peptides of the DBLα region., Results: The study identified that group 1 and group 5 sequences (cysteine/position of limited variability (cys/PoLV) classification) along with cys2/cys4 and MFK*/REY motifs and short amino acid length were significantly associated with malaria severity. The specific PoLV (MFKS, LREA, PTNL) were restricted to cerebral malaria. The expression level of var group A was higher than var groups B and C, demonstrating its prognostic characteristic. All peptides showed high-quality IgG response, while VAR P5 appeared to be a good marker for severity., Conclusions: The present study illustrates the presence of specific sequences of DBLα tags involved in severe malaria that could be targeted in future interventions for malaria control and elimination., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cryptosporidium felis is the major etiologic agent of cryptosporidiosis in felines and has been reported in numerous human cryptosporidiosis cases. Sequence analysis of the 60-kDa glycoprotein ( gp60 ) gene has been developed for subtyping C. felis recently. In this study, 66 C. felis isolates from the United States, Jamaica, Peru, Portugal, Slovakia, Nigeria, Ethiopia, Kenya, China, India and Australia were subtyped using the newly established tool. Forty-four specimens yielded gp60 sequences, generating 23 subtypes clustered in 4 subtype families (XIXa, XIXc, XIXd and XIXe) with high bootstrap support in a phylogenetic analysis of sequence data. Among them, XIXa showed high genetic diversity at the nucleotide level, with the formation of 18 subtypes from both cats and humans with different geographic distribution. In contrast, all 11 XIXd isolates derived from humans from various countries had identical sequences. Results of this study improve our understanding of the genetic diversity, host specificity and transmission dynamics of C. felis .

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.)

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: Artesunate plus sulfadoxine-pyrimethamine (ASP) is first-line treatment for uncomplicated Plasmodium falciparum malaria in most of India, except for six North-eastern provinces where treatment failure rates were high. In Ujjain, central India, the frequency of mutations associated with increased drug tolerance, but not overt resistance to sulfadoxine and pyrimethamine were 9% and > 80%, respectively, in 2009 and 2010, just prior to the introduction of ASP. The frequency of drug resistance associated mutations in Ujjain in 2015-2016 after 3-4 years of ASP use, are reported., Methods: Blood samples from patients with P. falciparum mono-infection verified by microscopy were collected on filter-paper at all nine major pathology laboratories in Ujjain city. Codons pfdhfr 16-185, pfdhps 436-632 and K13 407-689 were identified by sequencing. Pfcrt K76T and pfmdr1 N86Y were identified by restriction fragment length polymorphism., Results: Sulfadoxine-pyrimethamine resistance-associated pfdhfr 108 N and 59R alleles were found in 100/104 (96%) and 87/91 (96%) samples, respectively. Pfdhps 437G was found in 10/105 (10%) samples. Double mutant pfdhfr 59R + 108 N were found in 75/81 (93%) samples. Triple mutant pfdhfr 59R + 108 N and pfdhps 437G were found in 6/78 (8%) samples. Chloroquine-resistance-associated pfcrt 76T was found in 102/102 (100%). Pfmdr1 N86 and 86Y were identified in 83/115 (72%) and 32/115 (28%) samples, respectively., Conclusion: The frequency of P. falciparum with reduced susceptibility to sulfadoxine-pyrimethamine remained high, but did not appear to have increased significantly since the introduction of ASP. No polymorphisms in K13 associated with decreased artemisinin susceptibility were found. ASP probably remained effective, supporting continued ASP use.

Background: Chloroquine (CQ) is generally prescribed as the front-line antimalarial drug of choice to treat Plasmodium vivax infections; however, some clinical CQ-resistant P. vivax isolates have been indigenously reported around the world during the last decade., Methods: In this study, P. vivax isolates (n=52) were obtained from autochthonous samples in southeast Iran during 2015-2017. The genomic DNA of samples was extracted, amplified (nested PCR) and sequenced by targeting the multidrug-resistance 1 gene. To verify the global genetic diversity of CQ-resistant P. vivax strains, the sequences of Pvmdr1 originating from Asia and the Americas were retrieved., Results: A total of 46 haplotypes were grouped into three distinct geographical haplogroups. The haplotype diversity and occurrence rates of Pvmdr1 976F/1076L mutations indicate that the efficacy of CQ is being compromised in Mexico, China, Nicaragua, Thailand, Brazil (2016), Ethiopia, Mauritania (2012) and southwest India in the near future. The cladistic phylogenetic tree showed that Pvmdr1 sequences isolated from the southeast Asian clade has a partial sister relationship with the American clade., Conclusions: The current findings will serve as a basis to develop appropriate malaria control strategies and public health policies in symptomatic imported malaria cases or plausible CQ-resistant P. vivax strains., (© The Author(s) 2020. 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.)

Estimation of the spread and advancement of Plasmodium falciparum artemisinin-resistant parasites can be done by probing polymorphisms in the kelch ( Pfk13 ) domain (a validated molecular marker). This study aimed to provide baseline information for future artemisinin surveillance by analyzing the k13 -propeller domain in P. falciparum field isolates collected from 24 study areas in 14 malaria hot spots of Odisha (previously Orissa) during July 2018-January 2019. A total of 178 P. falciparum mono infections were assessed. An 849-base pair fragment encoding the Pfk13 propeller was amplified by nested polymerase chain reaction and sequenced in both directions (PCR). After DNA alignment with the 3D7 reference sequence, all samples were found to be wild type. It can be anticipated that malaria public health is not under direct threat in Odisha relating to ART resistance., Competing Interests: The authors declare that they have no conflicts of interest regarding the publication of this manuscript., (Copyright © 2020 Ramakanta Rana et al.)

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.

Background: In 2017, nearly 80% of malaria morbidity and mortality occurred in sub-Saharan African (SSA) countries and India. Rapid diagnostic tests (RDTs), especially those targeting histidine-rich protein 2 (PfHRP2) of Plasmodium falciparum, have become an important diagnostic tool in these malaria-endemic areas. However, the chances of RDT-oriented successful treatment are increasingly jeopardized by the appearance of mutants with deletions in pfhrp2 and pfhrp3 genes. This systematic review and meta-analysis determines the prevalence of field P. falciparum isolates with deletion in pfhrp2 and/or pfhrp3 genes and their proportion among false-negative results in the PfHRP2-based RDTs in SSA and India., Methods: Eight electronic databases were used for searching potentially relevant publications for the systematic review analysis, wherein the main methodological aspects of included studies were analysed and some missing links in the included studies were identified., Results: A total of 19 studies were included, 16 from SSA and 3 from India. The pooled prevalence of pfhrp2 deletions was 8 and 5% while 16 and 4% for pfhrp3 gene deletions in Africa and India, respectively. The pooled proportion of pfhrp2 gene deletions found among false negative PfHRP2-based RDTs results was about 27.0 and 69.0% in Africa and India, respectively., Conclusions: This review study indicates a relatively high proportion of both pfhrp2/3 genes deletions in P. falciparum isolates and among false-negative malaria cases using PfHRP2-based RDT results in SSA and India. Recently the deletions in pfhrp2/3 genes have also been reported from two African countries (Nigeria and Sudan). This review emphasizes the importance of more extensive studies and standardization of studies addressing the pfhrp2/3 gene deletions in malarious areas.

Background: Toxoplasma gondii, an important food borne zoonotic parasite, infects almost all warm-blooded animals including pigs. People primarily become infected with T. gondii via consuming meat of infected animals. Status of T. gondii is largely unknown in pigs in India including northern regions. We, therefore, determined the prevalence of T. gondii infection in pigs from North India., Results: DNA of T. gondii was detected in 6.7% (54/810) of the tested slaughter pigs. Highest prevalence was observed in pigs from Punjab (8.2%) followed by Chandigarh (5.3%) and Uttarakhand (4.8%). Phylogenetic analysis revealed that the isolates from pigs had 96-100% nucleotide identity with Type I RH strain (AF179871), 96-99.7% with VEG type III strain (LN714499) and 67-72% with type II ME 49 strain (XM002370240). However, low level of polymorphism in the targeted B1 gene did not allow the determination of the clonal lineages of the isolates. Antibodies against T. gondii was reported in 48.3% (73/151) of the sera obtained from pigs slaughtered at Chandigarh abattoir, and scavenging by pigs was a significant risk factor., Conclusion: Prevalence of T. gondii DNA was low in pigs in North India, however, presence of the parasite warrants food safety concerns. Further studies are required to identify the clonal lineage of T. gondii circulating in pigs reared in North India. Pig farmers should be educated about the hygienic management practices.

Artemisinin-based combination therapy (ACT) has been used to treat uncomplicated Plasmodium falciparum infections in India since 2004. Since 2008, a decrease in artemisinin effectiveness has been seen throughout the Greater Mekong Subregion. The geographic proximity and ecological similarities of northeastern India to Southeast Asia may differentially affect the long-term management and sustainability of ACT in India. In order to collect baseline data on variations in ACT sensitivity in Indian parasites, 12 P. falciparum isolates from northeast India and 10 isolates from southwest India were studied in vitro Ring-stage survival assay (RSA) showed reduced sensitivity to dihydroartemisinin in 50% of the samples collected in northeast India in 2014 and 2015. Two of the 10 assayed samples from the southwest region of India from as far back as 2012 also showed decreased sensitivity to artemisinin. In both these regions, kelch gene sequences were not predictive of reduced artemisinin sensitivity, as measured by RSA. The present data justify future investments in integrated approaches involving clinical follow-up studies, in vitro survival assays, and molecular markers for tracking potential changes in the effectiveness of artemisinin against P. falciparum throughout India., (Copyright © 2019 Chakrabarti et al.)

Background: Artesunate-sulfadoxine-pyrimethamine (ASSP) is the frontline artemisinin combination therapy (ACT) in India. Random, irrational, subtherapeutic artemisinin doses and self-medication with ACT along with predominance of sulfadoxine-pyrimethamine resistance parasite invoked a strong possibility of emerging artemisinin-resistant malaria parasites., Methods: This study involved 226 patients with uncomplicated Plasmodium falciparum infection who had successfully completed the 42 days follow-up after ASSP combination therapy from April 2014 to January 2016. We assessed the ASSP treatment efficacy by evaluating parasite clearance half-life, pfkelch13, and other (pfdhfr, pfdhps, pfmdr1, pfcrt) gene mutations and survival of parasites as detected by an ex vivo ring-stage survival assay (RSA)., Findings: Slow-clearing infections with longer parasite clearance half-lives (>5 hours) were observed in 12% isolates. Cure rate after ASSP treatment was declining to about 84.1%. ASSP failure was recorded in 15.9% (early treatment failure, 7.9%; late treatment failure, 7.9%) of isolates. In sum, 24 patients (10.6%) had parasite clearance half-lives greater than 5 hours with pfkelch13 polymorphism after 441 codon; in 15 of those patients (6.6%), parasites had not cleared by 72 hours after initiation of therapy. Median ex vivo ring-stage survival rate of these isolates was very high (12.2%; 95% confidence interval [CI], 10.9-13.8) from that of cured patients (0.9%; 95% CI, 0.09-1.07). Of these 15 patients, 13 patients had pfkelch13 G625R polymorphism, whereas 2 patients contained R539T polymorphism. As per the World Health Organization guideline, these 15 isolates were true artemisinin-resistant isolates., Interpretation: Identification of artemisinin-resistant isolates in India together with new mutations and increasing combination therapy failures blow alarms for urgent malaria control., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Malaria is one of the important vector-borne diseases with high fatality rates in tropical countries. The pattern of emergence and spread of novel antigenic variants, leading to escape of vaccine-induced immunity might be factors responsible for severe malaria. A high level of polymorphism has been reported among malarial antigens which are under selection pressure imposed by host immunity. There are limited reports available on comparative stage-specific genetic diversity among Plasmodium vivax candidate genes in complicated vivax malaria. The present study was planned to study genetic diversity (Pvcsp and Pvs25) among complicated and uncomplicated P. vivax isolates., Methods: Pvcsp and Pvs2-specific PCRs and DNA sequencing were performed on P. vivax PCR positive samples. Genetic diversity was analysed using appropriate software., Results: The present study was carried out on 143 P. vivax clinical isolates, collected from Postgraduate Institute of Medical Education and Research, Chandigarh. Among the classic and variant types of Pvcsp, the VK210 (99%; 115/116) was found to be predominant in both complicated and uncomplicated group isolates. Out of the various peptide repeat motifs (PRMs) observed, GDRADGQPA (PRM1) and GDRAAGQPA (PRM2) was the most widely distributed among the P. vivax isolates. Whereas among the Pvs25 isolates, 100% of double mutants (E97Q/I130T) in both the complicated (45/45) as well as in the uncomplicated (81/81) group was observed., Conclusion: An analysis of genetic variability enables an understanding of the role of genetic variants in severe vivax malaria.

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.)

Accurate and timely diagnosis is very critical for management, control and elimination of the malaria. Malaria rapid diagnostic tests (RDTs) have improved the diagnosis and management of malaria in remote areas, community and places where microscopy is not available for diagnosis. According to WHO report 2018, Plasmodium falciparum malaria constitutes more than 50% of malaria cases in India. Most of the RDTs used for diagnosis of falciparum malaria today employ HRP2 as a target antigen. However, low density parasitemia and deletion of hrp-2 gene in P. falciparum leads to false negative results and necessitates the development of alternative/ new or improved RDT for malaria diagnosis. We have analysed the genetic diversity and homology modelling of Pfgdh (glutamate dehydrogenase), ldh (lactate dehydrogenase) and aldolase genes in P. falciparum isolates from the eight endemic states of India to assess their potential as antigen for RDT development. We observed negligible sequence diversity in Pfgdh in comparison to the low level of diversity in ldh and aldolase gene. No structural or functional changes were observed in modelling studies and all three genes were under negative purifying selection pressure. The highly conserved nature of pfgdh gene suggests that GDH could be a potential target molecule for Pan/Pf diagnostic test for malaria., Competing Interests: The authors have declared that no competing interests exist.

Malaria is a sterning public health concern in India and contribute to a major part of malaria burden in Southeast Asia. Being more populated and diverse geographic conditions makes more suitable place for sustaining malaria parasite in India. Anti-malarial resistance is a major concern in the battle against malaria, and the identified molecular markers will aid us to monitor the drug resistance in endemic areas. The aim of the current study is to determine the genotype of drug resistance associated genes pvmdr-1 and pvcrt-o from four different regions of India. Especially from Puducherry and Jodhpur, there were no prior studies focused on screening of drug resistance genes in P. vivax parasite. A total of 240 positive P. vivax infected patient samples were collected from four tertiary care hospitals from four different regions of India, namely, Puducherry (PDY), Mangaluru (MAQ), Cuttack (CTC), Jodhpur (JDH). All samples were screened by microscopy, RDT, QBC, and further DNA was extracted and vivax mono-infection was confirmed by nested PCR. Randomly selected amplicons were further subjected to nucleotide sequencing. The prevalence of K10 insertion in pvcrt-o gene was detected with 18.8% in PDY, 12.5% in MAQ and 6.3% in CTC P. vivax isolates, whereas no change in nucleotide was identified in P. vivax isolates collected from JDH region. Based on the F1076L mutation in pvmdr-1 gene, resistant P. vivax isolates was highly predominant in both the regions, JDH and CTC, with 100%, followed by MAQ with 93.3% and PDY with 73.3%. This study showed less frequency of pvcrt-o and high frequency of pvmdr-1 gene variants associated with CQ resistance, which act as an indicator and the onset of P. vivax drug resistance trend in four different regions of India. Due to the poor phenotypic studies available for P. vivax parasite, the present study data for CQ resistance based on pvcrt-o and pvmdr-1 markers should assist by providing base-line data for future monitoring of drug resistance., (Copyright © 2019. Published by Elsevier B.V.)

The clinical presentation of severe Plasmodium falciparum malaria differs between children and adults, but the mechanistic basis for this remains unclear. Contributing factors to disease severity include total parasite biomass and the diverse cytoadhesive properties mediated by the polymorphic var gene parasite ligand family displayed on infected erythrocytes. To explore these factors, we performed a multicohort analysis of the contribution of var expression and parasite biomass to severe malaria in two previously published pediatric cohorts in Tanzania and Malawi and an adult cohort in India. Machine learning analysis revealed independent and complementary roles for var adhesion types and parasite biomass in adult and pediatric severe malaria and showed that similar var profiles, including upregulation of group A and DC8 var , predict severe malaria in adults and children. Among adults, patients with multiorgan complications presented infections with significantly higher parasite biomass without significant differences in var adhesion types. Conversely, pediatric patients with specific complications showed distinct var signatures. Cerebral malaria patients showed broadly increased expression of var genes, in particular group A and DC8 var , while children with severe malaria anemia were classified based on high transcription of DC8 var only. This study represents the first large multisite meta-analysis of var expression, and it demonstrates the presence of common var profiles in severe malaria patients of different ages across distant geographical sites, as well as syndrome-specific disease signatures. The complex associations between parasite biomass, var adhesion type, and clinical presentation revealed here represent the most comprehensive picture so far of the relationship between cytoadhesion, parasite load, and clinical syndrome. IMPORTANCE P. falciparum malaria can cause multiple disease complications that differ by patient age. Previous studies have attempted to address the roles of parasite adhesion and biomass in disease severity; however, these studies have been limited to single geographical sites, and there is limited understanding of how parasite adhesion and biomass interact to influence disease manifestations. In this meta-analysis, we compared parasite disease determinants in African children and Indian adults. This study demonstrates that parasite biomass and specific subsets of var genes are independently associated with detrimental outcomes in both childhood and adult malaria. We also explored how parasite var adhesion types and biomass play different roles in the development of specific severe malaria pathologies, including childhood cerebral malaria and multiorgan complications in adults. This work represents the largest study to date of the role of both var adhesion types and biomass in severe malaria., (Copyright © 2019 Duffy et al.)

Background: A large number of recombinant proteins are tested over a period of time for diagnosis of toxoplasmosis throughout the world. However, such literature is very much limited from an Indian perspective., Purpose: The aim of the study was to assess the sero prevalence of Toxoplasma gondii in adult cattle from Kerala, India, using recombinant surface antigen 2 (recSAG2) protein., Methods: An antibody detection recombinant ELISA specific for T gondii was laboratory-standardized using recSAG2 protein. The optimum antigen concentration, serum concentration, and conjugate dilutions were determined by the initial checkerboard titrations. Subsequently, the diagnostic potential of the recombinant protein was assessed with 258 field sera samples cattle and compared with indirect fluorescent antibody test (IFAT)., Results: Among the cattle sera tested, 61.5% showed sero positivity of T. gondii-speciic IgG. When compared to IFAT, the sensitivity of the recSAG2 ELISA was found to be 80.00% with 88.57% specificity at 95% confidence interval with substantial agreement between the tests., Conclusion: The results of present study have revealed the presence of high seroprevalence of the parasite and, hence, immense public health significance.

In most of India, sulfadoxine-pyrimethamine (SP) plus artesunate serves as first-line treatment for uncomplicated falciparum malaria. In 112 clinical Plasmodium falciparum isolates from Mangaluru, southwestern India, we sequenced molecular markers associated with resistance to SP, lumefantrine, and artemisinin ( pfdhfr , pfdhps , pfmdr1 , and K13 ). The pfdhfr double mutation 59R-108N combined with the dhps 437G mutation occurred in 39.3% and the pfdhfr double mutation plus the pfdhps double mutation 437G-540E in additional 24.1%. As for pfmdr1 , the allele combination N86-184F-D1246 dominated (98.2%). K13 variants were absent. No evidence for artemisinin resistance was seen. However, the antifolate resistance alleles compromise the current first-line antimalarial sulfadoxine-pyrimethamine plus artesunate, which may facilitate the emergence of artemisinin resistance. Artemether-lumefantrine, introduced in northeastern parts of the country, in the study area faces the predominant pfmdr1 NFD genotype, known to impair lumefantrine efficacy. Further monitoring of resistance alleles and treatment trials on alternative artemisinin-based combination therapies are required.

Background & Objectives: Plasmodium parasite harbours unique methylerythritol phosphate (MEP) pathway which is obligatory for the biosynthesis of isoprenoids. In malaria parasites, the isoprenoids are indispensable during hepatic, erythrocytic and gametocytic stages. Owing to the criticality of MEP pathway and the potential of its enzymes to act as antimalarial drug target, this study comprehensively investigated the genetic diversity and structural composition of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE), fourth enzyme of MEP pathway in Indian Plasmodium falciparum (PfIspE)., Methods: The study employed sequencing, modeling and bioinformatics approaches to examine the genetic diversity and associated structural polymorphism in the PfIspE gene amplified from the clinical blood samples collected from seven malaria endemic geographical regions of India., Results: The sequence analysis showed that PfIspE gene is highly conserved with 100% sequence identity among all the P. falciparum Indian isolates as well as with the PfIspE gene of reference strain 3D7. Phylogenetic analysis suggested that PfIspE is highly evolved and differ sufficiently from human orthologue mevalonate kinase gene. Structural modeling studies revealed that PfIspE has conserved ATP and CDPME-binding domains. The active site was observed to be relatively rigid in architecture with >60% β-pleated sheets., Interpretation & Conclusion: The results of genetic, phylogeny and modeling studies strengthen the potential of PfIspE enzyme as a promising antimalarial drug target., Competing Interests: None

Malaria parasites utilize Methylerythritol phosphate (MEP) pathway for synthesis of isoprenoid precursors which are essential for maturation and survival of parasites during erythrocytic and gametocytic stages. The absence of MEP pathway in the human host establishes MEP pathway enzymes as a repertoire of essential drug targets. The fourth enzyme, 4-diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE) has been proved essential in pathogenic bacteria, however; it has not yet been studied in any Plasmodium species. This study was undertaken to investigate genetic polymorphism and concomitant structural implications of the Plasmodium vivax IspE (PvIspE) by employing sequencing, modeling and bioinformatics approach. We report that PvIspE gene displayed six non-synonymous mutations which were restricted to non-conserved regions within the gene from seven topographically distinct malaria-endemic regions of India. Phylogenetic studies reflected that PvIspE occupies unique status within Plasmodia genus and reflects that Plasmodium vivax IspE gene has a distant and non-conserved relation with human ortholog Mevalonate Kinase (MAVK). Structural modeling analysis revealed that all PvIspE Indian isolates have critically conserved canonical galacto-homoserine-mevalonate-phosphomevalonate kinase (GHMP) domain within the active site lying in a deep cleft sandwiched between ATP and CDPME-binding domains. The active core region was highly conserved among all clinical isolates, may be due to >60% β-pleated rigid architecture. The mapped structural analysis revealed the critically conserved active site of PvIspE, both sequence, and spacially among all Indian isolates; showing no significant changes in the active site. Our study strengthens the candidature of Plasmodium vivax IspE enzyme as a future target for novel antimalarials., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Antimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because of Leishmania donovani resistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations of L. donovani : the core group (CG) in the Gangetic plains and ISC1 in the Nepalese highlands. Sb resistance was only encountered within the CG, and a series of potential markers were identified. Here, we analyzed the development of resistance to trivalent antimonials (Sb III ) upon experimental selection in ISC1 and CG strains. We observed that (i) baseline Sb III susceptibility of parasites was higher in ISC1 than in the CG, (ii) time to Sb III resistance was higher for ISC1 parasites than for CG strains, and (iii) untargeted genomic and metabolomic analyses revealed molecular changes along the selection process: these were more numerous in ISC1 than in the CG. Altogether these observations led to the hypothesis that CG parasites are preadapted to Sb III resistance. This hypothesis was experimentally confirmed by showing that only wild-type CG strains could survive a direct exposure to the maximal concentration of Sb III The main driver of this preadaptation was shown to be MRPA , a gene involved in Sb III sequestration and amplified in an intrachromosomal amplicon in all CG strains characterized so far. This amplicon emerged around 1850 in the CG, well before the implementation of antimonials for VL chemotherapy, and we discuss here several hypotheses of selective pressure that could have accompanied its emergence. IMPORTANCE The "antibiotic resistance crisis" is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti- Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious disease with limited therapeutic options. Therefore, understanding how parasites became resistant to antimonials is of commanding importance. In this study, we experimentally characterized the dynamics of this resistance acquisition and show for the first time that some Leishmania populations of the ISC were preadapted to antimony resistance, likely driven by environmental factors or by drugs used in the 19th century., (Copyright © 2018 Dumetz et al.)

Background: Genes encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) are the targets of sulfadoxine-pyrimethamine (SP) present in artemisinin based combination therapy (ACT; artesunate + sulfadoxine pyrimethamine) for Plasmodium falciparum. Although SP is generally not used to treat vivax infection, mutations in dhfr and dhps that confer antifolate resistance in Plasmodium vivax are common; which may be attributed to its sympatric existence with P. falciparum. Current study was aimed to determine the pattern of mutations in dhfr and dhps in P. vivax isolates from Mangaluru region., Methods: A total of 140 blood samples were collected from P. vivax-infected people attending Wenlock Hospital Mangaluru during July 2014 to January 2016. Out of 140 isolates, 25 (18%) and 50 (36%) isolates were selected randomly for sequence analysis of pvdhfr and pvdhps genes respectively. Fragment of pvdhps and full length pvdhfr were amplified, sequenced and analysed for single nucleotide polymorphisms. dhps was analysed by PCR-RFLP also, to detect the two specific mutations (A383G and A553G)., Results: Analysis of pvdhps sequences from 50 isolates revealed single and double mutants at 38 and 46% respectively. Three non-synonymous mutations (K55R, S58R and S117N) were identified for pvdhfr. Among these, K55R was detected for the first time., Conclusions: The current study indicates that P. vivax dhps and dhfr mutant alleles are prevalent in this area, suggesting significant SP pressure.

The human MN blood group antigens are isoforms of glycophorin A (GPA) encoded by the gene, GYPA, and are the most abundant erythrocyte sialoglycoproteins. The distribution of MN antigens has been widely studied in human populations yet the evolutionary and/or demographic factors affecting population variation remain elusive. While the primary function of GPA is yet to be discovered, it serves as the major binding site for the 175-kD erythrocyte-binding antigen (EB-175) of the malarial parasite, Plasmodium falciparum, a major selective pressure in recent human history. More specifically, exon two of GYPA encodes the receptor-binding ligand to which P. falciparum binds. Accordingly, there has been keen interest in understanding what impact, if any, natural selection has had on the distribution of variation in GYPA and exon two in particular. To this end, we resequenced GYPA in individuals sampled from both P. falciparum endemic (sub-Saharan Africa and South India) and non-endemic (Europe and East Asia) regions of the world. Observed patterns of variation suggest that GYPA has been subject to balancing selection in populations living in malaria endemic areas and in Europeans, but no such evidence was found in samples from East Asia, Oceania, and the Americas. These results are consistent with malaria acting as a selective pressure on GYPA, but also suggest that another selective force has resulted in a similar pattern of variation in Europeans. Accordingly, GYPA has perhaps a more complex evolutionary history, wherein on a global scale, spatially varying selective pressures have governed its natural history.

Background: Malaria is highly prevalent in many parts of India and is mostly caused by the parasite species Plasmodium vivax followed by Plasmodium falciparum. Chloroquine (CQ) is the first-line treatment for blood stage P. vivax parasites, but cases of drug resistance to CQ have been reported from India. One of the surveillance strategies which is used to monitor CQ drug resistance, is the analysis of single nucleotide polymorphisms (SNPs) of the associated gene markers. Susceptibility to CQ can also be determined by copy number assessment of multidrug resistant gene (mdr-1). The current study has examined the prevalence of SNPs in P. vivax orthologs of P. falciparum chloroquine resistant and multi-drug resistant genes (pvcrt-o and pvmdr-1, respectively) and pvmdr-1 copy number variations in isolates from the highly endemic Mangaluru city near the South Western Coastal region of India., Methods: A total of 140 blood samples were collected from P. vivax infected patients attending Wenlock Hospital Mangaluru during July 2014 to January 2016. Out of these 140 samples, sequencing was carried out for 54 (38.5%) and 85 (60.7%) isolates for pvcrt-o and pvmdr-1, respectively. Single nucleotide polymorphisms (SNPs) in the pvcrt-o and pvmdr-1 genes were analysed by direct sequencing method, while copy number variations of 60 isolates (42. 8%) were determined by real time PCR., Results: Out of 54 clinical isolates analysed for pvcrt-o, three (5.6%) showed K10 insertion and the rest had wild type sequence. This is the first report to show K10 insertion in P. vivax isolates from India. Further, out of 85 clinical isolates of P. vivax analysed for mutations in pvmdr-1 gene, only one isolate had wild type sequence (~ 1%) while the remaining (99%) carried mutant alleles. Seven non-synonymous mutations with two novel mutations (I946V and Y1028C) were observed. Of all the observed mutations in pvmdr-1 gene, T958M was most highly prevalent (present in 90% of samples) followed by F1076L (76%), and Y976F (7%). Amplification of pvmdr-1 gene was observed in 31.6% of the isolates, out of 60 amplified., Conclusion: The observed variations both in pvmdr-1 and pvcrt-o genes indicate a trend towards parasite acquiring CQ resistance in this endemic area.

Malaria is a major public health concern in Northeast India with a preponderance of drug-resistant strains. Until recently the partner drug for artemisinin combination therapy (ACT) was sulphadoxine pyrimethamine (SP). Antifolate drug resistance has been associated with the mutations at dihydropteroate synthase (dhps) and dihydrofolatereductase (dhfr) genes. This study investigated antifolate drug resistance at the molecular level. A total of 249 fever cases from Arunachal Pradesh, NE India, were screened for malaria, and of these, 75 were found to be positive for Plasmodium falciparum . Samples were sequenced and analysed with the help of BioEdit and ClustalW. Three novel point mutations were found in the dhps gene with 10 haplotypes along with the already reported mutations. A single haplotype having quadruple mutation was found in the dhfr gene. The study reports higher degree of antifolate drug resistance as evidenced by the presence of multiple point mutations in dhps and dhfr genes. The findings of this study strongly discourage the use SP as a partner drug in ACT.

Background: In India, sulphadoxine-pyrimethamine (SP) is now in use as a partner drug of ACT (AS+SP) to treat uncomplicated falciparum malaria since 2010. Declined trend of AS+SP efficacy has been reported from north-eastern states of the country. It is not possible to determine the efficacy of SP alone from any study with ACT. So, this work was designed to study the pattern of polymorphisms in pfdhfr and pfdhps genes to predict the SP resistance status among parasite population of urban Kolkata after five years of ACT implementation., Methods: A total of 125 P. falciparum positive patients were enrolled in the study during December 2014 to July 2016 and treated with AS+SP. Parasitic DNA was isolated and subjected to sequencing of pfdhfr and pfdhps genes directly from purified PCR products., Results: Genotyping of both the genes was successfully done in 113 isolates. In pfdhfr, 94.69% (107/113) isolates showed mutations at codon 59 and 108. A double mutant genotype ANRNI was mostly prevalent (107/113, 94.69%), while wild-type genotype ANCSI was found only in 5.3% (6/113) isolates. In pfdhps, mutations were recorded at codon 436 and 437 in 65.49% (74/113) and 23.01% (26/113) isolates, respectively. In combined pfdhfr-pfdhps genes, triple mutant ANRNI-FAKAA was most prevalent (45/113, 39.82%) followed by double mutant ANRNI-SAKAA (37/113, 32.74%) and quadruple mutant ANRNI-FGKAA (24/113, 21.24%)., Conclusion: SP resistance hallmark mutations i.e., quadruple (AIRNI-SAEAA) or quintuple (AIRNI-SGEAA) genotype in pfdhfr and pfdhps was absent which indicates that SP components of used ACT is still effective in the study area. It is also evident by the clinical response of AS+SP. Monitoring the efficacy of this combination (both by therapeutic and molecular marker study) at a regular interval is highly suggested to record any development of SP resistance in near future., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Commercial malaria rapid diagnostic tests (RDTs) detect P. falciparum histidine rich protein 2 (PfHRP2) and cross react with PfHRP3, a structural homologue. Here, we analysed natural variations in PfHRP2 and PfHRP3 sequences from Indian isolates and correlated these variations with RDT reactivity. A total 1392 P. falciparum positive samples collected from eight endemic states were PCR amplified for Pfhrp2 and Pfhrp3 genes and were sequenced. The deduced protein sequences were analysed for repeat variations and correlated with RDT reactivity. Out of 1392 PCR amplified samples, a single sample was Pfhrp2 negative and two samples were Pfhrp3 negative. Complete Pfhrp2 and Pfhrp3 sequences were obtained for 769 samples and 750 samples, respectively. A total of 16 distinct repeat motifs were observed for Pfhrp2 and 11 for Pfhrp3, including some new repeat types. No correlation was found between variations in the size of Pfhrp2 repeat types 2 and 7, nor between any combinations of repeat motifs, and performance of a commercial RDT at low parasite densities. The findings suggest that sequence diversity in Pfhrp2 and Pfhrp3 genes in Indian isolates is not likely to negatively influence performance of currently used PfHRP2 RDTs.

Background: The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38 and Pfs12 and three other merozoite surface proteins: Glutamate-rich protein (GLURP), SERA5 and MSP-1 were identified on the Plasmodium falciparum merozoite surface., Methods: Recombinant 6-cys proteins i.e. Pfs38, Pfs12, Pfs41 as well as PfMSP-1 65 were expressed and purified using Escherichia coli expression system and antibodies were raised against each of these proteins. These antibodies were used to immunoprecipitate the native proteins and their associated partners from parasite lysate. ELISA, Far western, surface plasmon resonance and glycerol density gradient fractionation were carried out to confirm the respective interactions. Furthermore, erythrocyte binding assay with 6-cys proteins were undertaken to find out their possible role in host-parasite infection and seropositivity was assessed using Indian and Liberian sera., Results: Immunoprecipitation of parasite-derived polypeptides, followed by LC-MS/MS analysis, identified a large Pfs38 complex comprising of 6-cys proteins: Pfs41, Pfs38, Pfs12 and other merozoite surface proteins: GLURP, SERA5 and MSP-1. The existence of such a complex was further corroborated by several protein-protein interaction tools, co-localization and co-sedimentation analysis. Pfs38 protein of Pfs38 complex binds to host red blood cells (RBCs) directly via glycophorin A as a receptor. Seroprevalence analysis showed that of the six antigens, prevalence varied from 40 to 99%, being generally highest for MSP-1 65 and GLURP proteins., Conclusions: Together the data show the presence of a large Pfs38 protein-associated complex on the parasite surface which is involved in RBC binding. These results highlight the complex molecular interactions among the P. falciparum merozoite surface proteins and advocate the development of a multi-sub-unit malaria vaccine based on some of these protein complexes on merozoite surface.

Genotyping the sulfadoxine-pyrimethamine (SP) genes will help in identifying the genes under drug selection and the emergence of resistance in dhfr and dhps genes. India is an important hotspot for studying malaria due to the immense climatic diversity prevalent in the country. The central and eastern parts of the country are most vulnerable sites where malaria cases are reported throughout the year. From different regions of the country 173 field isolates were genotyped at various loci in dhfr and dhps genes collected between 1994 and 2013. This encompasses the period before antimalarial resistance emerged and the period after the use of combination therapy was made mandatory in the country. We observed the rise of resistant SP alleles from very low frequencies (in the year 1994) to steadily rising (in the year 2000) and maintaining this increasing trend subsequently (in the year 2013) as shown by the sequence analysis of dhfr and dhps genes. This study assessed the prevalence of mutations in dhfr and dhps genes associated with SP resistance in samples indicative of increase in resistance levels of Plasmodium falciparum to SP even after the change in malaria treatment policy in the country., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bovine theileriosis is a serious threat to livestock worldwide. Uncertainty around species prevalence, antigenic diversity and genotypes of strains make it difficult to assess the impact of this parasite and to provide necessary treatment. We aimed to characterize genotypic diversity, phylogeny and prevalence of Theileria orientalis parasites from the states of Telangana and Andhra Pradesh, India by collecting bovine blood samples from the major districts of the two states. Bioinformatic analysis identified antigenic diversity among the prevalent parasite strains using major piroplasm surface protein (MPSP) gene. Our study revealed a prevalence rate of 4.8% (n=41/862) of T. orientalis parasites in bovine animals and a new genotype of T. orientalis parasite which was not previously reported in India. The emergence of these new genotypes could be an explanation for the frequent outbreaks of bovine theileriosis. Further, whole genome sequencing of T. orientalis strains will help to elucidate the genetic factors relevant for transmissibility and virulence as well as vaccine and new drug development., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cytoadherence of Plasmodium falciparum-infected red blood cells (RBCs) in host microvasculature and complex regulation of the immune response are important contributors to the clinical outcome of disease. We tested the association of 23 single nucleotide polymorphisms (SNPs) and a microsatellite repeat in adhesion molecule genes THBS1 and ESEL, and immune regulatory molecule genes NOSII, CRP, and MBL2 with falciparum malaria in populations residing in a malaria-endemic and a non-endemic region of India. The THBS1 haplotype CCCCA (rs1478604, rs7170682, rs2664141, rs12912082, rs3743125) was a risk factor in the endemic region (relative risk = 3.78) and an ESEL SNP (rs5368, His468Tyr) associated with cerebral malaria (CM) [CM vs. non-cerebral malaria (NCM), odds ratio (OR) = 2.23, p = 0.03]. In the non-endemic region, an ESEL 3'UTR SNP (rs5359) associated with enhanced risk of disease (OR = 3.62, p = 1 × 10(-4)) and the CT genotype of the CRP promoter SNP (C/T/A) strongly associated with protection (severe vs. control, OR = 0.29, p = 6 × 10(-5)). Long repeat alleles of the NOSII promoter microsatellite (CCTTT)n exhibited strong association with protection and the NOSII ATG haplotype (rs3729508, rs2297520, rs9282801) was strongly protective against severe malaria in both regions (endemic, severe vs. control, OR = 0.05, p = 0.0001; non-endemic, severe vs. control, OR = 0.3, p = 1 × 10(-5)). Our results suggest differential contribution of variants of the investigated genes in determining the outcome of malaria in Indian populations.