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Durgama Anchalare Malaria Nirakaran (DAMaN) is a multi-component malaria intervention for hard-to-reach villages in Odisha, India. The main component, malaria camps (MCs), consists of mass screening, treatment, education, and intensified vector control. We evaluated MC effectiveness using a quasi-experimental cluster-assigned stepped-wedge study with a pretest–posttest control group in 15 villages: six immediate (Arm A), six delayed (Arm B), and three previous interventions (Arm C). The primary outcome was PCR + Plasmodium infection prevalence. The time (i.e., baseline vs. follow-up 3) x study arm interaction term shows that there were statistically significant lower odds of PCR + Plasmodium infection in Arm A (AOR = 0.36, 95% CI = 0.17, 0.74) but not Arm C as compared to Arm B at the third follow-up. The cost per person ranged between US$3–8, the cost per tested US$4–9, and the cost per treated US$82–1,614, per camp round. These results suggest that the DAMaN intervention is a promising and financially feasible approach for malaria control. [ABSTRACT FROM AUTHOR]

Background: India has made considerable progress in malaria reduction over the past two decades, with government-sponsored indoor residual spraying (IRS) and insecticide-treated bed net (ITN) or long-lasting insecticidal nets (LLIN) distribution being the main vector-related prevention efforts. Few investigations have used non-participant observational methods to assess malaria control measures while they were being implemented, nor documented people's perceptions and acceptance of IRS or LLINs in India, and none have done so in the northeast region. This study evaluated household (HH)-level operation of IRS and distribution of LLINs by India's National Vector Borne Disease Control Programme (NVBDCP) in 50 villages of Meghalaya state, and documented their acceptance and use. Methods: Study field teams accompanied the government health system teams during August-October, 2019 and 2020 to observe deployment of LLINs, and record HH-level data on LLIN numbers and use. In addition, NVBDCP spray teams were followed during 2019–2021 to observe IRS preparation and administration. HH members were interviewed to better understand reasons for acceptance or refusal of spraying. Results: A total of 8386 LLINs were distributed to 2727 HHs in 24 villages from five Primary Health Centres, representing 99.5% of planned coverage. Interviews with 80 HH residents indicated that they appreciated the LLIN dissemination programme, and generally made regular and appropriate use of LLINs, except during overnight travel or when working in agricultural fields. However, HH-level IRS application, which was observed at 632 HHs, did not always follow standard insecticide preparation and safety protocols. Of 1,079 occupied HHs visited by the spray team, 632 (58.6%) refused to allow any spraying. Only 198 (18.4%) HHs agreed to be sprayed, comprising 152 (14.1%) that were only partly sprayed, and 46 (4.3%) that were fully sprayed. Reasons for refusal included: inadequate time to rearrange HH items, young children were present, annoying smell, staining of walls, and threat to bee-keeping or Eri silk moth cultivation. Conclusions: These findings are among the first in India that independently evaluate people's perceptions and acceptance of ongoing government-sponsored IRS and LLIN programmes for malaria prevention. They represent important insights for achieving India's goal of malaria elimination by 2030. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Author summary: Plasmodium vivax is the dominant malaria species in several countries, and a safe and effective vaccine against the species is needed if progress is to be made towards malaria control and elimination. The P. vivax Apical Membrane Antigen 1 (AMA1) ectodomain and the C-terminal region (19 kDa) of the Merozoite Surface Protein 1 (MSP1) are important malaria vaccine antigens. However, antigenic diversity remains a critical factor for acquisition of immunity and also a major barrier for development of an effective vaccine. Here, investigations on the genetic diversity of two blood stage vaccine candidate antigens, Merozoite surface protein 119 and Apical membrane antigen 1, of P. vivax from three malaria endemic regions of the India was attempted and compared with global population. High genetic variation was observed in Pvama1 domain I, which has been reported as a dominant target of host immune response. In contrast, Pvmsp119 showed highly conserved sequences from three geographically diverse populations of India and globally, which further suggests Pvmsp119 as an extremely conserved protein. Collectively, the diversity data generated in this study provides a baseline data towards development of a broadly efficacious Pvama1 and Pvmsp119 based vaccine. [ABSTRACT FROM AUTHOR]

Background: Despite declining incidence over the past decade, malaria remains an important health burden in India. This study aimed to assess the village-level temporal patterns of Plasmodium infection in two districts of the north-eastern state of Meghalaya and evaluate risk factors that might explain these patterns. Methods: Primary Health Centre passive malaria case data from 2014 to 2018 were analysed to characterize village-specific annual incidence and temporal trends. Active malaria case detection was undertaken in 2018 and 2019 to detect Plasmodium infections using PCR. A questionnaire collected socio-demographic, environmental, and behavioural data, and households were spatially mapped via GPS. Adult mosquitoes were sampled at a subset of subjects' houses, and Anopheles were identified by PCR and sequencing. Risk factors for Plasmodium infection were evaluated using bivariate and multivariate logistic regression analysis, and spatial cluster analysis was undertaken. Results: The annual malaria incidence from PHC-based passive surveillance datasets in 2014–2018 was heterogenous but declining across villages in both districts. Active surveillance in 2018 enrolled 1468 individuals from 468 households (West Jaintia Hills) and 1274 individuals from 359 households (West Khasi Hills). Plasmodium falciparum prevalence per 100 people varied from 0 to 4.1% in the nine villages of West Jaintia Hills, and from 0 to 10.6% in the 12 villages of West Khasi Hills. Significant clustering of P. falciparum infections [observed = 11, expected = 2.15, Relative Risk (RR) = 12.65; p < 0.001] was observed in West Khasi Hills. A total of 13 Anopheles species were found at 53 houses in five villages, with Anopheles jeyporiensis being the most abundant. Risk of infection increased with presence of mosquitoes and electricity in the households [Odds Ratio (OR) = 1.19 and 1.11], respectively. Households with reported animals had reduced infection risk (OR = 0.91). Conclusion: Malaria incidence during 2014–2018 declined in all study villages covered by the passive surveillance data, a period that includes the first widespread insecticide-treated net campaign. The survey data from 2018 revealed a significant association between Plasmodium infection and certain household characteristics. Since species of Plasmodium-competent mosquito vectors continue to be abundant, malaria resurgence remains a threat, and control efforts should continue. [ABSTRACT FROM AUTHOR]

The Indian state of Odisha has a longstanding battle with forest malaria. Many remote and rural villages have poor access to health care, a problem that is exacerbated during the rainy season when malaria transmission is at its peak. Approximately 62% of the rural population consists of tribal groups who are among the communities most negatively impacted by malaria. To address the persistently high rates of malaria in these remote regions, the Odisha State Malaria Control Program introduced 'malaria camps' in 2017 where teams of health workers visit villages to educate the population, enhance vector control methods, and perform village-wide screening and treatment. Malaria rates declined statewide, particularly in forested areas, following the introduction of the malaria camps, but the impact of the intervention is yet to be externally evaluated. This study protocol describes a cluster-assigned quasi-experimental stepped-wedge study with a pretest-posttest control group design that evaluates if malaria camps reduce the prevalence of malaria, compared to control villages which receive the usual malaria control interventions (e.g. IRS, ITNs), as detected by PCR. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The mounting evidence supporting the capacity of Plasmodium vivax to cause severe disease has prompted the need for a better characterization of the resulting clinical complications. India is making progress with reducing malaria, but epidemics of severe vivax malaria in Gujarat, one of the main contributors to the vivax malaria burden in the country, have been reported recently and may be the result of a decrease in transmission and immune development. Over a period of one year, we enrolled severe malaria patients admitted at the Civil Hospital in Ahmedabad, the largest city in Gujarat, to investigate the morbidity of severe vivax malaria compared to severe falciparum malaria. Patients were submitted to standard thorough clinical and laboratory investigations and only PCR-confirmed infections were selected for the present study. Severevivax malaria (30 patients) was more frequent than severe falciparum malaria (8 patients) in our setting, and it predominantly affected adults (median age 32 years, interquartile range 22.5 years). This suggests a potential age shift in anti-malarial immunity, likely to result from the recent decrease in transmission across India. The clinical presentation of severe vivax patients was in line with previous reports, with jaundice as the most common complication. Our findings further support the need for epidemiological studies combining clinical characterization of severe vivax malaria and serological evaluation of exposure markers to monitor the impact of elimination programmes. [ABSTRACT FROM AUTHOR]

Background: Identifying highly immunogenic blood stage antigens which can work as target for naturally acquired antibodies in different eco-epidemiological settings is an important step for designing malaria vaccine. Blood stage proteins of Plasmodium vivax, apical membrane antigen-1 (PvAMA-1) and 19 kDa fragment of merozoite surface protein (PvMSP-119) are such promising vaccine candidate antigens. This study determined the naturally-acquired antibody response to PvAMA-1 and PvMSP-119 antigens in individuals living in three geographically diverse malaria endemic regions of India. Methods: A total of 234 blood samples were collected from individuals living in three different eco-epidemiological settings, Chennai, Nadiad, and Rourkela of India. Indirect ELISA was performed to measure human IgG antibodies against recombinant PvAMA-1 and PvMSP-119 antigens. The difference in seroprevalence and factors associated with antibody responses at each site was statistically analysed. Results: The overall seroprevalence was 40.6% for PvAMA-1 and 62.4% for PvMSP-119. Seroprevalence to PvAMA-1 was higher in Chennai (47%) followed by Nadiad (46.7%) and Rourkela (27.6%). For PvMSP-119, seroprevalence was higher in Chennai (80.3%) as compared to Nadiad (53.3%) and Rourkela (57.9%). Seroprevalence for both the antigens were found to be higher in Chennai where P. vivax is the dominant malaria species. In addition, heterogeneous antibody response was observed for PvAMA-1 and PvMSP-119 antigens at each of the study sites. Two factors, age and malaria positivity were significantly associated with seropositivity for both the antigens PvAMA-1 and PvMSP-119. Conclusion: These data suggest that natural acquired antibody response is higher for PvMSP-119 antigen as compared to PvAMA-1 antigen in individuals living in three geographically diverse malaria endemic regions in India. PvMSP-119 appears to be highly immunogenic in Indian population and has great potential as a malaria vaccine candidate. The differences in immune response against vaccine candidate antigens in different endemic settings should be taken into account for development of asexual stage based P. vivax malaria vaccine, which in turn can enhance malaria control efforts. [ABSTRACT FROM AUTHOR]

Background: Repellents such as coils, vaporizers, mats and creams can be used to reduce the risk of malaria and other infectious diseases. Although evidence for their effectiveness is limited, they are advertised as providing an additional approach to mosquito control in combination with other strategies, e.g. insecticide-treated nets. We examined the use of repellents in India in an urban setting in Chennai (mainly Plasmodium vivax malaria), a peri-urban setting in Nadiad (both P. vivax and P. falciparum malaria), and a more rural setting in Raurkela (mainly P. falciparum malaria). Methods: The use of repellents was examined at the household level during a census, and at the individual level in cross-sectional surveys and among patients visiting a clinic with fever or other symptoms. Factors associated with their use were examined in a multivariate analysis, and the association between malaria and the use of repellents was assessed among survey- and clinic participants. Results: Characteristics of participants differed by region, with more people of higher education present in Chennai. Use of repellents varied between 56-77 % at the household level and between 32-78 % at the individual level. Vaporizers were the main repellents used in Chennai, whereas coils were more common in Nadiad and Raurkela. In Chennai and Nadiad, vaporizers were more likely to be used in households with young male children. Vaporizer use was associated with higher socio-economic status (SES) in households in Chennai and Nadiad, whereas use of coils was greater in the lower SES strata. In Raurkela, there was a higher use of coils among the higher SES strata. Education was associated with the use of a repellent among survey participants in Chennai and clinic study participants in Chennai and Nadiad. Repellent use was associated with less malaria in the clinic study in Chennai and Raurkela, but not in the surveys, with the exception of the use of coils in Nadiad. Conclusions: Repellents are widely used in India. Their use is influenced by the level of education and SES. Information on effectiveness and guidance on choices may improve rational use. [ABSTRACT FROM AUTHOR]

The evolutionary history and age of Plasmodium vivax has been inferred as both recent and ancient by several studies, mainly using mitochondrial genome diversity. Here we address the age of P. vivax on the Indian subcontinent using selectively neutral housekeeping genes and tandem repeat loci. Analysis of ten housekeeping genes revealed a substantial number of SNPs (n = 75) from 100 P. vivax isolates collected from five geographical regions of India. Neutrality tests showed a majority of the housekeeping genes were selectively neutral, confirming the suitability of housekeeping genes for inferring the evolutionary history of P. vivax. In addition, a genetic differentiation test using housekeeping gene polymorphism data showed a lack of geographical structuring between the five regions of India. The coalescence analysis of the time to the most recent common ancestor estimate yielded an ancient TMRCA (232,228 to 303,030 years) and long-term population history (79,235 to 104,008) of extant P. vivax on the Indian subcontinent. Analysis of 18 tandem repeat loci polymorphisms showed substantial allelic diversity and heterozygosity per locus, and analysis of potential bottlenecks revealed the signature of a stable P. vivax population, further corroborating our ancient age estimates. For the first time we report a comparable evolutionary history of P. vivax inferred by nuclear genetic markers (putative housekeeping genes) to that inferred from mitochondrial genome diversity. Author Summary: Plasmodium vivax is the most prevalent human malaria parasite outside Africa, responsible for significant morbidity, although it is commonly referred to as a benign malaria agent with respect to P. falciparum. Inferring the evolutionary history of an infectious agent provides important information for designing control measures. In this study, we developed a panel of novel molecular markers including housekeeping genes, minisatellites and microsatellites to infer the evolutionary history of P. vivax on the Indian subcontinent. The panel of markers uncovered an ancient and long evolutionary history and a stable population structure of P. vivax in India. [ABSTRACT FROM AUTHOR]

Background. Relapses originating from hypnozoites are characteristic of Plasmodium vivax infections. Thus, reappearance of parasitemia after treatment can result from relapse, recrudescence, or reinfection. It has been assumed that parasites causing relapse would be a subset of the parasites that caused the primary infection. Methods. Paired samples were collected before initiation of antimalarial treatment and at recurrence of parasitemia from 149 patients with vivax malaria in Thailand (n = 36), where reinfection could be excluded, and during field studies in Myanmar (n = 75) and India (n = 38). Results. Combined genetic data from 2 genotyping approaches showed that novel P. vivax populations were present in the majority of patients with recurrent infection (107 [72%] of 149 patients overall [78% of patients in Thailand, 75% of patients in Myanmar {Burma}, and 63% of patients in India]). In 61% of the Thai and Burmese patients and in 55% of the Indian patients, the recurrent infections contained none of the parasite genotypes that caused the acute infection. Conclusions. The P. vivax populations emerging from hypnozoites commonly differ from the populations that caused the acute episode. Activation of heterologous hypnozoite populations is the most common cause of first relapse in patients with vivax malaria. [ABSTRACT FROM AUTHOR]

Background: We conducted a diagnostic surveillance study to identify Plasmodium, dengue virus, chikungunya virus, and Orientia tsutsugamushi infections among febrile patients who underwent triage for malaria in the outpatient department at Ispat General Hospital, Rourkela, Odisha, India.Methods: Febrile patients were enrolled from January 2016-January 2017. Blood smears and small volumes or vacutainers of blood were collected from study participants to carry out diagnostic assays. Malaria was diagnosed using rapid diagnostic tests (RDT), microscopy, and PCR. Dengue, chikungunya, and scrub typhus infections were identified using rapid diagnostic test kits and ELISA.Results: Nine hundred and fifty-four patients were prospectively enrolled in our study. The majority of patients were male (58.4%) and more than 15 years of age (66.4%). All 954 enrollees underwent additional testing for malaria; a subset of enrollees (293/954) that had larger volumes of plasma available was also tested for dengue, chikungunya and scrub typhus by either RDT or ELISA or both tests. Fifty-four of 954 patients (5.7%) were positive for malaria by RDT, or microscopy, or PCR. Seventy-four of 293 patients (25.3%) tested positive for dengue by either RDT or ELISA, and 17 of 293 patients (5.8%) tested positive for chikungunya-specific IgM by either ELISA or RDT. Ten of 287 patients tested (3.5%) were positive for scrub typhus by ELISA specific for scrub typhus IgM. Seventeen patients among 290 (5.9%) with results for ≥3 infections tested positive for more than one infection. Patients with scrub typhus and chikungunya had high rates of co-infection: of the 10 patients positive for scrub typhus, six were positive for dengue (p = 0.009), and five of 17 patients positive for chikungunya (by RDT or ELISA) were also diagnosed with malaria (p < 0.001).Conclusions: Dengue, chikungunya and scrub typhus are important etiologies of non-malarial febrile illness in Rourkela, Odisha, and comorbidity should be considered. Routine febrile illness surveillance is required to accurately establish the prevalence of these infections in this region, to offer timely treatment, and to implement appropriate methods of control. [ABSTRACT FROM AUTHOR]

Malaria in India is declining, in part due to the use of long-lasting insecticide-treated nets (LLINs) and vector control. Historically, the north-eastern region of India has contributed ~10%–12% of the nation's malaria burden. The important mosquito vectors in northeast India have long been considered to be Anopheles baimaii and An. minimus , both associated with forest habitats. Local deforestation and increased rice cultivation, along with widespread LLIN use, may be changing vector species composition. Understanding if and how vector species composition is changing is critical to successful malaria control. In Meghalaya state, malaria is now at a low level of endemicity with occasional seasonal outbreaks. In a biodiverse setting like Meghalaya, where >24 Anopheles mosquito species have been recorded, accurate morphological identification of all species is logistically challenging. To accurately determine Anopheles species richness in the West Khasi Hills (WKH) and West Jaintia Hills (WJH) districts, adult and larval mosquitoes were collected and identified using molecular methods of allele-specific PCR and cytochrome oxidase I DNA barcoding. In 14 villages across both districts, we identified high species richness, 19 species in total. Molecular findings indicated that An. minimus and An. baimaii were rare, while four other species (An. maculatus , An. pseudowillmori, An. jeyporiensis and An. nitidus) were abundant. Anopheles maculatus was highly prevalent in WKH (39% of light trap collections) and An. pseudowillmori in WJH (45%). Larvae of these four species were found in rice fields, suggesting that land cover change is influencing species composition change. Our results suggest that rice fields might be contributing to the observed abundance of An. maculatus and An. pseudowillmori, which could be playing a role in malaria transmission, either independently due to their high abundance, or in combination with An. baimaii and/or An. minimus. • Accurate Anopheles species composition in Meghalaya, India using molecular methods. • New and previously unreported species inferred using molecular phylogeny approach. • Small numbers of primary vectors indicate a reduced role in malaria transmission. • Haplotype networks indicate demographic expansion in some species. • Species composition change may be due to changing land cover/use and LLINs. [ABSTRACT FROM AUTHOR]

Considering malaria as a local and focal disease, epidemiological understanding of different ecotypes of malaria can help in devising novel control measures. One of the major hurdles in malaria control lies on the evolution and dispersal of the drug-resistant malaria parasite, Plasmodium falciparum . We herewith present data on genetic variation at the Single Nucleotide Polymorphism (SNP) level in four different genes of P. falciparum ( Pfcrt , Pfmdr1 , Pfdhfr , and Pfdhps ) that confer resistance to different antimalarials in two different eco-epidemiological settings, i.e. Hilly-Forest (HF) and Riverine-Plain (RP), in a high malaria endemic district of Odisha state, India. Greater frequency of antimalarial resistance conferring SNPs and haplotypes was observed in all four genes in P. falciparum , and Pfdhps was the most variable gene among the four. No significant genetic differentiation could be observed in isolates from HF and RP ecotypes. Twelve novel, hitherto unreported nucleotide mutations could be observed in the Pfmdr1 and Pfdhps genes. While the Pfdhps gene presented highest haplotype diversity, the Pfcrt gene displayed the highest nucleotide diversity. When the data on all the four genes were complied, the isolates from HF ecotype were found to harbour higher average nucleotide diversity than those coming from RP ecotype. High and positive Tajima's D values were obtained for the Pfcrt and Pfdhfr genes in isolates from both the HF and RP ecotypes, with statistically significant deviation from neutrality in the RP ecotype. Different patterns of Linkage Disequilibrium (LD) among SNPs located in different drug-resistant genes were found in the isolates collected from HF and RP ecotypes. Whereas in the HF ecotype, SNPs in the Pfmdr1 and Pfdhfr were significantly associated, in the RP ecotype, SNPs located in Pfcrt were associated with Pfmdr1 , Pfdhfr and Pfdhps . These findings provide a baseline understanding on how different micro eco-epidemiological settings influence evolution and spread of different drug resistance alleles. Our findings further suggest that drug resistance to chloroquine and sulfadoxine–pyrimethamine is approaching fixation level, which requires urgent attention of malaria control programme in India. [ABSTRACT FROM AUTHOR]

Abstract: Efforts to control malignant malaria caused by Plasmodium falciparum are hampered by the parasite’s acquisition of resistance to antimalarial drugs, e.g., chloroquine. This necessitates evaluating the spread of chloroquine resistance in any malaria-endemic area. India displays highly variable malaria epidemiology and also shares porous international borders with malaria-endemic Southeast Asian countries having multi-drug resistant malaria. Malaria epidemiology in India is believed to be affected by two major factors: high genetic diversity and evolving drug resistance in P. falciparum. How transmission intensity of malaria can influence the genetic structure of chloroquine-resistant P. falciparum population in India is unknown. Here, genetic diversity within and among P. falciparum populations is analyzed with respect to their prevalence and chloroquine resistance observed in 13 different locations in India. Microsatellites developed for P. falciparum, including three putatively neutral and seven microsatellites thought to be under a hitchhiking effect due to chloroquine selection were used. Genetic hitchhiking is observed in five of seven microsatellites flanking the gene responsible for chloroquine resistance. Genetic admixture analysis and F-statistics detected genetically distinct groups in accordance with transmission intensity of different locations and the probable use of chloroquine. A large genetic break between the chloroquine-resistant parasite of the Northeast-East-Island group and Southwest group (FST =0.253, P <0.001) suggests a long period of isolation or a possibility of different origin between them. A pattern of significant isolation by distance was observed in low transmission areas (r =0.49, P =0.003, N =83, Mantel test). An unanticipated pattern of spread of hitchhiking suggests genetic structure for Indian P. falciparum population. Overall, the study suggests that transmission intensity can be an efficient driver for genetic differentiation at both neutral and adaptive loci across India. [Copyright &y& Elsevier]

Abstract: Malaria is a major public health problem in India and one which contributes significantly to the overall malaria burden in Southeast Asia. The National Vector Borne Disease Control Program of India reported ∼1.6 million cases and ∼1100 malaria deaths in 2009. Some experts argue that this is a serious underestimation and that the actual number of malaria cases per year is likely between 9 and 50 times greater, with an approximate 13-fold underestimation of malaria-related mortality. The difficulty in making these estimations is further exacerbated by (i) highly variable malaria eco-epidemiological profiles, (ii) the transmission and overlap of multiple Plasmodium species and Anopheles vectors, (iii) increasing antimalarial drug resistance and insecticide resistance, and (iv) the impact of climate change on each of these variables. Simply stated, the burden of malaria in India is complex. Here we describe plans for a Center for the Study of Complex Malaria in India (CSCMi), one of ten International Centers of Excellence in Malaria Research (ICEMRs) located in malarious regions of the world recently funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The CSCMi is a close partnership between Indian and United States scientists, and aims to address major gaps in our understanding of the complexity of malaria in India, including changing patterns of epidemiology, vector biology and control, drug resistance, and parasite genomics. We hope that such a multidisciplinary approach that integrates clinical and field studies with laboratory, molecular, and genomic methods will provide a powerful combination for malaria control and prevention in India. [Copyright &y& Elsevier]

Four Plasmodium species cause malaria in humans: Plasmodium vivax is the most widespread and results in pronounced morbidity. India (population >1 billion) is a major contributor to the burden of vivax malaria. With a resurgence in interest concerning the neglected burden of vivax malaria and the completion of the P. vivax genome, it is timely to review what is known concerning P. vivax in India. The P. vivax population is highly diverse in terms of relapse patterns, drug response and clinical profiles, and highly genetically variable according to studies of antigen genes, isoenzyme markers and microsatellites. The unique epidemiology of malaria in India, where P. vivax predominates over Plasmodium falciparum, renders this location ideal for studying the dynamics of co-infection. [Copyright &y& Elsevier]

• Previous studies identified rainy season and male gender as risk factors for malaria in India. • A subsequent behavioral survey investigated the behavioral aspects of malaria risk. • Men were more likely to be involved in outside activities in the evening and early morning. • The use of rmosquito epellents and creams was low. • Malaria is a localized disease in India, and requires local prevention plans whereby behavior-related factors taken into account. To characterize malaria and assist in prevention efforts, we conducted a series of epidemiological studies in Sundargarh district, India, as part of an NIH-funded International Center of Excellence for Malaria Research. In a published survey around Rourkela in 2013-2014 (N = 1307), malaria prevalence was found to be 8.3%. Using these data, villages were divided into low (<2%), medium (2-10%) and high (>10%) malaria prevalence, and risk factors assessed by type of village. In the six low malaria villages, four persons were positive by PCR; in the four medium malaria villages, prevalence was 7% (35 infections, 7 P. vivax); and in the three high malaria villages, prevalence was 21% (62 infections, 10 P. vivax and 5 mixed with P. vivax and P. falciparum). A total of 30.6% infections were submicroscopic and 40.6% were asymptomatic. Our analyses showed that the rainy season and male gender were risk factors for malaria; in high malaria villages, young age was an additional risk factor, and indoor and outdoor spraying was protective compared to no spraying. We undertook a subsequent behavioral survey in four of the medium and high malaria villages in 2017 to investigate the behavioral aspects of malaria risk. Among 500 participants in 237 households, adult men (15+ years) were more likely to be outside in the evening (34.5% vs. 7.9% among adult women 15+ years and 0.7% among children, p < 0.001), or to sleep outside (7.5% vs. 0.5% and 0%, respectively, p < 0.001). Although women were more likely to get up before 6 a.m. (86.6%, vs. 70.5% among men, 50.7% among children, p < 0.001), men were more likely to be outside in the early morning (77.6% among men, 11.2% among women, and 11.1% among children, p < 0.001). More children used insecticide treated nets the previous night (73.4%) than men (45.6%) or women (39.6%), and repellents were used by 29.5% of 234 households (insecticide creams were not used at all). Malaria control and elimination in India will need local approaches, and the promotion of repellent cream use by at-risk groups could be further explored in addition to mass-screen or treat programs in high-risk villages. [ABSTRACT FROM AUTHOR]

Background: Malaria remains a public health problem in regions of Northeastern India because of favourable bio-geographic transmission conditions, poor access to routine healthcare, and inadequate infrastructure for public health and disease prevention. This study was undertaken to better understand community members' and health workers' perceptions of malaria, as well as their knowledge, attitudes, and prevention practices related to the disease in Meghalaya state., Methods: The study included participants from three malaria endemic districts: West Khasi Hills, West Jaiñtia Hills, and South Garo Hills from 2019 to 2021. A total of 82 focus group discussions (FGD) involving 694 community members and 63 in-depth interviews (IDI) with health personnel and traditional healers residing within the three districts were conducted. A thematic content analysis approach was employed, using NVivo12 software for data management., Results: Most participants reported a perceived reduction in malaria during recent years, attributing this to changes in attitudes and behaviours in health seeking, and to more effective government interventions. Local availability of testing and treatment, and an improved, more responsive health system contributed to changing attitudes. Long-lasting insecticidal nets (LLINs) were largely preferred over indoor residual spraying (IRS), as LLINs were perceived to be effective and more durable. Community members also reported using personal protective measures such as applying repellents, burning neem tree leaves, straw/egg trays, wearing long sleeve clothes, and applying ointments or oils to protect themselves from mosquito bites. While most participants acknowledged the role of mosquitoes in malaria transmission, other conditions that are not mosquito-borne were also attributed to mosquitoes by some participants. The communities surveyed have largely shifted from seeking treatment for malaria from traditional healers to using public facilities, although some participants reported switching between the two or using both simultaneously. Improved understanding of cerebral malaria, which some participants previously attributed to mental illness due to 'bad spirits', is an example of how cultural and ritualistic practices have changed., Conclusion: The findings reveal diverse perceptions among community members regarding malaria, its prevention, practices to prevent mosquito-transmitted diseases, and their opinions about the healthcare system. A key finding was the shift in malaria treatment-seeking preferences of community members from traditional healers to the public sector. This shift highlights the changing dynamics and increasing acceptance of modern healthcare practices for malaria treatment and prevention within tribal and/or indigenous communities. By recognizing these evolving attitudes, policymakers and healthcare providers can better tailor their interventions and communication strategies to more effectively address ongoing needs and concerns as India faces the 'last mile' in malaria elimination., (© 2024. The Author(s).)

The Center for the Study of Complex Malaria in India (CSCMi) is one of 10 International Centers of Excellence in Malaria Research funded by the National Institutes of Health since 2010. The Center combines innovative research with capacity building and technology transfer to undertake studies with clinical and translational impact that will move malaria control in India toward the ultimate goal of malaria elimination/eradication. A key element of each research site in the four states of India (Tamil Nadu, Gujarat, Odisha, and Meghalaya) has been undertaking community- and clinic-based epidemiology projects to characterize the burden of malaria in the region. Demographic and clinical data and samples collected during these studies have been used in downstream projects on, for example, the widespread use of mosquito repellants, the population genomics of Plasmodium vivax, and the serological responses to P. vivax and Plasmodium falciparum antigens that reflect past or present exposure. A focus has been studying the pathogenesis of severe malaria caused by P. falciparum through magnetic resonance imaging of cerebral malaria patients. Here we provide a snapshot of some of the basic and applied research the CSCMi has undertaken over the past 12 years and indicate the further research and/or clinical and translational impact these studies have had.

The Center for the Study of Complex Malaria in India (CSCMi) was launched in 2010 with the overall goal of addressing major gaps in our understanding of "complex malaria" in India through projects on the epidemiology, transmission, and pathogenesis of the disease. The Center was mandated to adopt an integrated approach to malaria research, including building capacity, developing infrastructure, and nurturing future malaria leaders while conducting relevant and impactful studies to assist India as it moves from control to elimination. Here, we will outline some of the interactions and impacts the Center has had with malaria policy and control counterparts in India, as well as describe emerging needs and new research questions that have become apparent over the past 12 years.

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.

Malaria in India, while decreasing, remains a serious public health problem, and the contribution of submicroscopic and asymptomatic infections to its persistence is poorly understood. We conducted community surveys and clinic studies at three sites in India differing in their eco-epidemiologies: Chennai (Tamil Nadu), Nadiad (Gujarat), and Rourkela (Odisha), during 2012-2015. A total of 6,645 subject blood samples were collected for Plasmodium diagnosis by microscopy and PCR, and an extensive clinical questionnaire completed. Malaria prevalence ranged from 3-8% by PCR in community surveys (24 infections in Chennai, 56 in Nadiad, 101 in Rourkela), with Plasmodium vivax dominating in Chennai (70.8%) and Nadiad (67.9%), and Plasmodium falciparum in Rourkela (77.3%). A proportional high burden of asymptomatic and submicroscopic infections was detected in community surveys in Chennai (71% and 71%, respectively, 17 infections for both) and Rourkela (64% and 31%, 65 and 31 infections, respectively). In clinic studies, a proportional high burden of infections was identified as submicroscopic in Rourkela (45%, 42 infections) and Chennai (19%, 42 infections). In the community surveys, anemia and fever were significantly more common among microscopic than submicroscopic infections. Exploratory spatial analysis identified a number of potential malaria hotspots at all three sites. There is a considerable burden of submicroscopic and asymptomatic malaria in malarious regions in India, which may act as a reservoir with implications for malaria elimination strategies.

Background: Meghalaya, one of eight states in the northeastern region of India, has been reported to carry a high malaria burden. However, malaria surveillance, epidemiology, and vector studies are sparse, and no reviews combining these topics with malaria prevention and control strategies have been published in recent years. Furthermore, no analysis of surveillance data has been published documenting the changes in epidemiology following the first distribution of long-lasting insecticidal nets (LLINs) statewide in 2016., Methods: A hybrid approach was used to describe the status of malaria in Meghalaya. First, a literature search was performed using the terms 'malaria' and 'Meghalaya'. Second, data were obtained from the Meghalaya State Malaria Control Programme for 2006-2017 for analysis of trends. Data from 3 years 2015-2017 were analysed further by district and year to assess changes in malaria incidence and distribution following the introduction of LLINs., Results/conclusions: Like malaria in mainland India, malaria in Meghalaya is complex, with both Plasmodium falciparum and Plasmodium vivax parasites in circulation, multiple Anopheles vector species, and reports of both unusual and severe malaria syndromes across all age groups. Integrated statewide malaria epidemiology, vector, and prevention and control data for Meghalaya are not readily available, and published studies are largely focused on a single topic or a single district or region of the state. Although malaria prevention and control approaches are available, (e.g. spraying, LLINs, personal repellents), their use and effectiveness is also not well characterized in the literature. Analysis of state malaria control programme data indicates that case incidence and related fatalities in Meghalaya have declined over the last decade. This could be attributed to changes in treatment guidelines and/or statewide distribution of effective prevention methods such as LLINs. Since the distribution of more than 900,000 LLINs in 2016, the malaria caseload has declined significantly in most Meghalaya districts, excluding the remote and geographically isolated South Garo Hills. Additionally, the proportion of adult malaria cases (15+ years of age versus children 0-14 years) in most districts was significantly greater following LLIN distribution, which likely reflects common lifestyle practices in these areas (e.g. adults working during night hours; small children in the households receiving priority for bed net protection). While reduction in malaria case incidence and related deaths is clear, the changes in malaria transmission and clinical manifestation have not been characterized. Routine epidemiology and vector surveillance combined with real-time data reporting are essential for the continued reduction and eventual elimination of malaria in Meghalaya.

Background: Assessing the Plasmodium vivax burden in India is complicated by the potential threat of an emerging chloroquine (CQ) resistant parasite population from neighbouring countries in Southeast Asia. Chennai, the capital of Tamil Nadu and an urban setting for P. vivax in southern India, was selected as a sentinel site for investigating CQ efficacy and sensitivity in vivax malaria., Methods: CQ efficacy was evaluated with a 28-day in vivo therapeutic study, while CQ sensitivity was measured with an in vitro drug susceptibility assay. In both studies, isolates also underwent molecular genotyping to investigate correlations between parasite diversity and drug susceptibility to CQ. Molecular genotyping included sequencing a 604 base pair (bp) fragment of the P. vivax multidrug resistant gene-1 (Pvmdr1) for single nucleotide polymorphisms (SNPs) and also the amplification of eight microsatellite (MS) loci located across the genome on eight different chromosomes., Results: In the 28-day in vivo study (N=125), all subjects were aparasitaemic by Day 14. Passive case surveillance continuing beyond Day 28 in 22 subjects exposed 17 recurrent infections, which ranged from 44 to 148 days post-enrollment. Pvmdr1 sequencing of these recurrent infections revealed that 93.3% had identical mutant haplotypes (958M/Y976/1076L) to their baseline Day 0 infection. MS genotyping further revealed that nine infection pairs were related with ≥ 75% haplotype similarity (same allele at six or more loci). To test the impact of this mutation on CQ efficacy, an in vitro drug assay (N=68) was performed. No correlation between IC50 values and the percentage of ring-stage parasites prior to culture was observed (r(sadj): -0.00063, p = 0.3307) and the distribution of alleles among the Pvmdr1 SNPs and MS haplotypes showed no significant associations with IC50 values., Conclusions: Plasmodium vivax was found to be susceptible to CQ drug treatment in both the in vivo therapeutic drug study and the in vitro drug assay. Though the mutant 1076 L of Pvmdr1 was found in a majority of isolates tested, this single mutation did not associate with CQ resistance. MS haplotypes revealed strong heterogeneity in this population, indicating a low probability of reinfection with highly related haplotypes.