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Abstract Aedes aegypti and Aedes albopictus mosquitoes spread major vector-borne viral diseases in tropical and sub-tropical regions of the globe. In this study, we sequenced the genome of Indian Ae. aegypti and Ae. albopictus and mapped to their reference genomes. Comparative genomics were performed between our strain and the reference strains. A total of 14,416,484 single nucleotide polymorphisms (SNPs) and 156,487 insertions and deletions (InDels) were found in Ae. aegypti, and 28,940,433 SNPs and 188,987 InDels in Ae. albopictus. Particular emphasis was given to gene families involved in mosquito digestion, development, and innate immunity, which could be putative candidates for vector control. Serine protease cascades and their inhibitors called serpins, play a central role in these processes. We extracted high-impact variants in genes associated with serine proteases and serpins. This study reports for the first time a high coverage genome sequence data of an Indian Ae. albopictus mosquito. The results from this study will provide insights into Indian Aedes specific polymorphisms and the evolution of immune related genes in mosquitoes, which can serve as a resource for future comparative genomics and those pursuing the development of targeted biopesticides for effective mosquito control strategies.

Abstract 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.

Since the first reported case of COVID-19 in December 2019, several SARS-CoV-2 variants have evolved, and some of them have shown higher transmissibility, becoming the prevalent strains. Genomic epidemiological investigations into strains from different time points, including the early stages of the pandemic, are very crucial for understanding the evolution and transmission patterns. Using whole-genome sequences, our study describes the early landscape of SARS-CoV-2 variants in central India retrospectively (including the first known occurrence of SARS-CoV-2 in Madhya Pradesh). We performed amplicon-based whole-genome sequencing of randomly selected SARS-CoV-2 isolates (n = 38) collected between 2020 and 2022 at state level VRDL, ICMR-NIRTH, Jabalpur, from 11899 RT-qPCR-positive samples. We observed the presence of five lineages, namely B.1, B.1.1, B.1.36.8, B.1.195, and B.6, in 19 genomes from the first wave cases and variants of concern (VOCs) lineages, i.e., B.1.617.2 (Delta) and BA.2.10 (Omicron) in the second wave cases. There was a shift in mutational pattern in the spike protein coding region of SRAS-CoV-2 strains from the second wave in contrast to the first wave. In the first wave of infections, we observed variations in the ORF1Ab region, and with the emergence of Delta lineages, the D614G mutation associated with an increase in infectivity became a prominent change. We have identified five immune escape variants in the S gene, P681R, P681H, L452R, Q57H, and N501Y, in the isolates collected during the second wave. Furthermore, these genomes were compared with 2160 complete genome sequences reported from central India that encompass 109 different SARS-CoV-2 lineages. Among them, VOC lineages Delta (28.93%) and Omicron (56.11%) were circulating predominantly in this region. This study provides useful insights into the genetic diversity of SARS-CoV-2 strains over the initial course of the COVID-19 pandemic in central India.

Tuberculosis (TB) is a significant public health problem among the Saharia community, an underprivileged tribal group in the west-central part of India. There are several challenges for India’s TB control program to curtail TB in the Saharia tribe. Malnutrition, poor health sector facilities, lower socio-economic status, and substance abuse are interconnected and synergistic factors contributing to a high burden of TB in the Saharia tribe. In this review, efforts are made to collate the findings of previous studies discussing the causes of high burden of TB in the Saharia tribe, social gaps for mitigating these preventable risk factors of TB in the Saharia tribe, and the plausible solutions for closing these gaps. The concept of Health in All Policies and intersectoral co-ordination is needed for the reduction of TB in the Saharia tribe and to make India TB-free by the year 2025.

Abstract Background Global malaria cases rose by 14 million, and deaths by 69,000, in 2020. In India, a 46% decline has been reported between 2020 and 2019. In 2017, the Malaria Elimination Demonstration Project conducted a needs-assessment of the Accredited Social Health Activists (ASHAs) of Mandla district. This survey revealed the inadequate level of knowledge in malaria diagnosis and treatment. Subsequently, a training programme was launched for enhancing malaria-related knowledge of ASHAs. The present study was conducted in 2021 to evaluate the impact of training on malaria-related knowledge and practices of ASHAs in Mandla. This assessment was also done in two adjoining districts: Balaghat and Dindori. Methods A cross-sectional survey using a structured questionnaire was administered to ASHAs to measure their knowledge and practices related to malaria etiology, prevention, diagnosis, and treatment. A comparison of information collected from these three districts was performed using simple descriptive statistics, comparison of means and multivariate logistic regression analysis. Results Significant improvement was noted amongst ASHAs of district Mandla between 2017 (baseline) and 2021 (endline) in knowledge related to malaria transmission, preventive measures, adherence to the national drug policy, diagnosis using rapid diagnostic tests, and identification of age group-specific, colour-coded artemisinin combination therapy blister packs (p

Abstract Background Compared to 2017, India achieved a significant reduction in malaria cases in 2020. Madhya Pradesh (MP) is a tribal dominated state of India with history of high malaria burden in some districts. District Mandla of MP state showed a considerable decline in malaria cases between 2000 and 2013, except in 2007. Subsequently, a resurgence of malaria cases was observed during 2014 and 2015. The Malaria Elimination Demonstration Project (MEDP) was launched in 2017 in Mandla with the goal to achieve zero indigenous malaria cases. This project used: (1) active surveillance and case management using T4 (Track fever, Test fever, Treat patient, and Track patient); (2) vector control using indoor residual sprays and long-lasting insecticidal nets; (3) information education communication and behaviour change communication; and (4) regular monitoring and evaluation with an emphasis on operational and management accountability. This study has investigated malaria prevalence trends from 2008 to 2020, and has predicted trends for the next 5 years for Mandla and its bordering districts. Methods The malaria prevalence data of the district Mandla for the period of January 2008 to August 2017 was obtained from District Malaria Office (DMO) Mandla and data for the period of September 2017 to December 2020 was taken from MEDP data repository. Further, the malaria prevalence data for the period of January 2008 to December 2020 was collected from DMOs of the neighbouring districts of Mandla. A univariate time series and forecast analysis was performed using seasonal autoregressive integrated moving average model. Findings Malaria prevalence in Mandla showed a sharp decline [− 87% (95% CI − 90%, − 84%)] from 2017 to 2020. The malaria forecast for Mandla predicts zero cases in the next 5 years (2021–2025), provided current interventions are sustained. By contrast, the model has forecasted a risk of resurgence of malaria in other districts in MP (Balaghat, Dindori, Jabalpur, Seoni, and Kawardha) that were not the part of MEDP. Conclusion The interventions deployed as part of MEDP have resulted in a sustainable zero indigenous malaria cases in Mandla. Use of similar strategies in neighbouring and other malaria-endemic districts in India could achieve similar results. However, without adding extra cost to the existing intervention, sincere efforts are needed to sustain these interventions and their impact using accountability framework, data transparency, and programme ownership from state to district level.

Abstract Background Mass screening and treatment (MSaT) aims at reducing the spread of malaria in communities by identifying and treating infected persons regardless of the symptoms. This study was conducted to identify and treat asymptomatic cases using MSaT approaches in the community. Methods Three rounds of MSaT using cluster combination approaches were carried out during September 2018 to December 2019 to identify and treat asymptomatic malaria cases in the community. All individuals who were present in the household were screened using RDT irrespective of malaria related symptoms. Simultaneously thick and thin blood smear and blood spot were collected for further analysis using microscopy and diagnostic PCR done in a subset of the samples. Results Logistic regression analysis revealed that asymptomatic malaria cases significantly less among the older age groups compared with

Abstract Background Despite significant progress in eliminating malaria from the state of Odisha, India, the disease is still considered endemic. Artesunate plus sulfadoxine-pyrimethamine (AS + SP) has been introduced since 2010 as first-line treatment for uncomplicated Plasmodium falciparum malaria. This study aimed to investigate the prevalence of mutations associated with resistance to chloroquine (CQ), sulfadoxine-pyrimethamine (SP), and artesunate (ART) in P. falciparum parasites circulating in the state. Methods A total of 239 isolates of P. falciparum mono infection were collected during July 2018-November 2020 from the four different geographical regions of the state. Genomic DNA was extracted from 200 µL of venous blood and amplified using nested polymerase chain reaction. Mutations on gene associated with CQ (Pfcrt and Pfmdr1) were assessed by PCR amplification and restriction fragment length polymorphism, artemisinin (Pfk13) gene by DNA sequencing and SP (Pfdhfr and Pfdhps) genes by allele-specific polymerase chain reaction (AsPCR). Results The point mutation in Pfcrt (K76T) was detected 2.1%, in Pfmdr1 (N86Y) 3.4%, and no mutations were found in Pfkelch13 propeller domain. Prevalence of Pfdhfr, Pfdhps and Pfhdfr-Pfdhps (two locus) gene mutations were 50.43%, 47.05% and 49.79% respectively. The single, double, triple and quadruple point mutations in Pfdhfr gene was 11.2%, 8.2%, 17.2% and 3.4% while, in Pfdhps gene was 10.9%,19.5%, 9.5% and 2.7% respectively. Of the total 13 haplotypes found in Pfdhfr, 8 were detected for the first time in the state and of the total 26 haplotypes found in Pfdhps, 7 were detected for the fisrt time in the state. The linked quintuple mutation Pfdhfr (N51I-C59R-S108N)-Pfdhps (A437G-K540E) responsible for clinical failure (RIII level of resistance) of SP resistance and A16V-S108T mutation in Pfdhfr responsible for cycloguanil was absent. Conclusion The study has demonstrated a low prevalence of CQ resistance alleles in the study area. Despite the absence of the Pfkelch13 mutations, high prevalence of Pfdhfr and Pfdhps point mutations undermine the efficacy of SP partner drug, thereby threatening the P. falciparum malaria treatment policy. Therefore, continuous molecular and in vivo monitoring of ACT efficacy is warranted in Odisha.

Abstract Background The utilization and impact of the healthcare services depend on the perceived quality, appropriateness, ease of availability, and cost of the services. This study aimed to understand the community's perception of the quality of healthcare services delivered as part of the Malaria Elimination Demonstration Project (MEDP), Mandla, Madhya Pradesh, India. Methods The study used qualitative techniques to analyze the community perceptions that emerged from the participants’ narratives during the Focus Group Discussions (FGDs) and in-depth Interviews with Key Informants (IKIs) on the promptness and quality of healthcare service delivery, the behaviour of MEDP staff, Information, Education and Communication, and Behavioural Change Communication activities, coordination with community members and other health personnel, and capacity building of healthcare workers and the community. Results 36 FGDs and 63 IKIs with 419 respondents were conducted in nine blocks of district Mandla. Overall, 97% to 100% of beneficiaries associated MEDP with regularity and prompt service delivery, availability of diagnostics and drugs, friendly behaviour, good coordination, and community mobilization to enhance treatment-seeking behaviour. Conclusions The study's findings highlighted the importance of building and maintaining the community's participation and promoting the demand for optimal utilization of healthcare services inside the village to promptly achieve the malaria elimination goal.

Abstract Background Low-density malaria infections (LDMI) are defined as infections that are missed by the rapid diagnostic test (RDT) and/or microscopy which can lead to continued transmission and poses a challenge in malaria elimination efforts. This study was conducted to investigate the prevalence of LDMI in febrile cases using species-specific nested Polymerase Chain Reaction (PCR) tests in the Malaria Elimination Demonstration Project, where routine diagnosis was conducted using RDT. Methods Every 10th fever case from a cross-sectional community based fever surveillance was tested with RDT, microscopy and nested PCR. Parasite DNA was isolated from the filter paper using Chelex based method. Molecular diagnosis by nested PCR was performed targeting 18SrRNA gene for Plasmodium species. Results The prevalence of malaria was 2.50% (436/17405) diagnosed by PCR, 1.13% (196/17405) by RDT, and 0.68% (118/ 17,405) by microscopy. Amongst 17,405 febrile samples, the prevalence of LDMI was 1.51% (263/17405) (95% CI 1.33–1.70), which were missed by conventional methods. Logistic regression analysis revealed that illness during summer season [OR = 1.90 (p

Scrub typhus is a seriously neglected life threatening disease in Asia and the islands of the Pacific and Indian oceans. The causative agent is Orientia tsutsugamushi, an obligate intra-cytosolic bacterium transmitted to vertebrate animals such as human or rodent by larval trombiculid mites (chiggers). Here, we conducted an outbreak investigation in Madhya Pradesh state in central India after sudden upsurge in scrub typhus cases and mortality in humans. Sixty-five live traps were placed for capturing the rodents. The captured rodent species were identified and the samples were subjected to nested PCR targeting 56kDa TSA gene. Analysis revealed for the first-time predominant circulation of Karp genotype in this region. The vector species involved in transmission is Leptotrobidium deliense. In conclusion, we reported the circulation of Karp strain in outbreak regions of Madhya Pradesh and suggest that public health monitoring and surveillance needs to be improved. Studies on antigenic diversity, association of strains with clinical spectrum and pathogenicity in the local setup will be essential for development of region-specific diagnostics and vaccines.

Abstract Background The capacity of the field staff to conduct activities related to disease surveillance, case management, and vector control has been one of the key components for successfully achieving malaria elimination. India has committed to eliminate malaria by 2030, and it has placed significance on monitoring and evaluation at the district level as one of the key strategies in its national framework. To support and guide the country’s malaria elimination objectives, the Malaria Elimination Demonstration Project was conducted in the tribal district of Mandla, Madhya Pradesh. Robust monitoring of human resources received special attention to help the national programme formulate a strategy to plug the gaps in its supply chain and monitoring and evaluation systems. Methods A monitoring tool was developed to test the capabilities of field workers to conduct activities related to malaria elimination work. Between November 2018 to February 2021, twenty-five Malaria Field Coordinators (MFCs) of the project utilized this tool everyday during the supervisory visits for their respective Village Malaria Workers (VMWs). The data was analysed and the scores were tested for variations against different blocks, educational status, duration of monitoring, and post-training scores. Results During the study period, the VMWs were monitored a total of 8974 times using the monitoring tool. Each VMW was supervised an average of 1.8 times each month. The critical monitoring indicators scored well in all seven quarters of the study as monitored by the MFCs. Monitoring by MFCs remained stable at 97.3% in all quarters. Contrary to expectations, the study observed longer diagnosis to treatment initiation time in urban areas of the district. Conclusion This study demonstrated the significance of a robust monitoring tool as an instrument to determine the capacity of the field workers in conducting surveillance, case management, and vector control related work for the malaria elimination programme. Similar tools can be replicated not only for malaria elimination, but other public health interventions as well.

Background and objectivesExploring the disease profile in a defined area helps policymakers to understand local health issues. It is essential to collect such information in countries, like India, which have a wide range of socioeconomic, geographic, and cultural diversity. Madhya Pradesh is the second largest state of India and has diversified populations living in urban, rural, and tribal areas. In this study, we performed a hospital record-based analysis to find out the status of different diseases in various outpatient departments (OPDs) of the District Hospital where patients from both rural and urban areas are treated.Materials and methodsThe retrospective data was collected from medical records available for the period September 2018 to September 2020. These datasets were analyzed to determine the most common presentations among patients.Results and conclusionsA total of 138,756 records of patients were available for this study, whose department-wise analysis revealed that most records were related to respiratory tract infection, superficial dermatophytosis, anemia among women, suppurative otitis media, and pneumonia. This study provides a broad overview of the overall health issues of people living in rural and urban areas of Datia. However, a major limitation of the study was that other correlations with age and gender were not established due to the absence of such information. Nonetheless, these findings will help policymakers and researchers to set the agenda for interventions and set goals for achieving better health for all people including those living in rural and tribal regions.

Abstract Background Changes in social, belief, and behavioural practices are essential for the success of any public health delivery programme. In the planning stages of the Malaria Elimination Demonstration Project (MEDP), priority was given to communication with a goal to develop capacity of health workers and to improve the knowledge, attitude and practices (KAP) of the people of Mandla. This paper describes the level of community knowledge on malaria, including its prevention, diagnosis, treatment-seeking behaviour, and the level of satisfaction with the services provided by the project. Methods A cross sectional survey was undertaken in 1233 villages of Mandla to study the KAP and self-assessed improvement in knowledge and satisfaction level of the community. The goal of the study was to understand whether there is need for strengthening communication strategy of MEDP for better impact. The survey was conducted amongst the head/eligible members of the 733 households located in the nine blocks of the district using clustered random sampling. Results Though four-fifths of the respondents were able to correlate the transmission of malaria with mosquitoes, misconceptions existed among them. The types of malaria were not known to everyone. Only 39% were aware of the Indoor Residual Spray (IRS) and 41% understood the value of Long-Lasting Insecticidal Nets (LLIN). Around 71% of subjects surveyed were aware of the proper diagnostic tests for malaria. A total of 87% of the respondents knew about the MEDP staff working in their respective villages. Conclusion The study reported gaps in knowledge on malaria at community level. The self-assessment of the community revealed that the communication strategy established by MEDP in Mandla district has been useful to them as they are becoming better informed about the prevention and treatment aspects of disease. The lessons learned as revealed in the KAP survey will improve malaria elimination outcomes in a timely manner.

Background: Earlier serosurveys in India revealed seroprevalence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) of 0.73% in May–June 2020 and 7.1% in August–September 2020. A third serosurvey was conducted between December 2020 and January 2021 to estimate the seroprevalence of SARS-CoV-2 infection among the general population and healthcare workers (HCWs) in India. Methods: The third serosurvey was conducted in the same 70 districts as the first and second serosurveys. For each district, at least 400 individuals aged ≥10 years from the general population and 100 HCWs from subdistrict-level health facilities were enrolled. Serum samples from the general population were tested for the presence of immunoglobulin G (IgG) antibodies against the nucleocapsid (N) and spike (S1-RBD) proteins of SARS-CoV-2, whereas serum samples from HCWs were tested for anti-S1-RBD. Weighted seroprevalence adjusted for assay characteristics was estimated. Results: Of the 28,598 serum samples from the general population, 4585 (16%) had IgG antibodies against the N protein, 6647 (23.2%) had IgG antibodies against the S1-RBD protein, and 7436 (26%) had IgG antibodies against either the N protein or the S1-RBD protein. Weighted and assay-characteristic-adjusted seroprevalence against either of the antibodies was 24.1% [95% confidence interval (CI) 23.0–25.3%]. Among 7385 HCWs, the seroprevalence of anti-S1-RBD IgG antibodies was 25.6% (95% CI 23.5–27.8%). Conclusions: Nearly one in four individuals aged ≥10 years from the general population as well as HCWs in India had been exposed to SARS-CoV-2 by December 2020.

Abstract Background Malaria is a major public health problem in India and accounts for about 88% of malaria burden in South-East Asia. India alone accounted for 2% of total malaria cases globally. Anti-malarial drug resistance is one of the major problems for malaria control and elimination programme. Artemether-lumefantrine (AL) is the first-line treatment of uncomplicated Plasmodium falciparum in north eastern states of India since 2013 after confirming the resistance against sulfadoxine-pyrimethamine. In the present study, therapeutic efficacy of artemether-lumefantrine and k13 polymorphism was assessed in uncomplicated P. falciparum malaria. Methods This study was conducted at four community health centres located in Koraput district of Odisha, Bastar district of Chhattisgarh, Balaghat district of Madhya Pradesh and Gondia district of Maharashtra state. Patients with uncomplicated P. falciparum malaria were administered with fixed dose combination (6 doses) of artemether-lumefantrine for 3 days and clinical and parasitological response was recorded up to 28 days as per World Health Organization protocol. Nucleotide sequencing of msp1 and msp2 gene was performed to differentiate between recrudescence and reinfection. Amplification and sequencing of k13 propeller gene region covering codon 450–680 was also carried out to identify the polymorphism. Results A total 376 malaria patients who fulfilled the enrolment criteria as well as consented for the study were enrolled. Total 356 patients were followed up successfully up to 28 days. Overall, the adequate clinical and parasitological response was 98.9% and 99.4% with and without PCR correction respectively. No case of early treatment failure was observed. However, four cases (1.1%) of late parasitological failure were found from the Bastar district of Chhattisgarh. Genotyping of msp1 and msp2 confirmed 2 cases each of recrudescence and reinfection, respectively. Mutation analysis of k13 propeller gene showed one non-synonymous mutation Q613H in one isolate from Bastar. Conclusions The study results showed that artemether-lumefantrine is highly effective in the treatment of uncomplicated P. falciparum malaria among all age groups. No functional mutation in k13 was found in the study area. The data from this study will be helpful in implementation of artemether-lumefantrine in case of treatment failure by artesunate plus sulfadoxine-pyrimethamine.

Abstract 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

Abstract Background The role of Accredited Social Health Activist (ASHA) in the health care delivery services at the periphery level is crucial for achieving disease prevention, control and elimination goals. The objective of the study was to assess the knowledge, attitude, practices, priorities and capability of ASHA related to malaria diagnosis and treatment as part of the Malaria Elimination Demonstration Project in 1233 villages of district Mandla, Madhya Pradesh. Methods A cross sectional study was conducted using a fully structured, pre-tested interview schedule during June and July 2017 (before the field operations of MEDP were started). Two hundred twenty (17%) of the total ASHAs were selected for the interview from the 9 developmental blocks of Mandla district. Results Knowledge, Attitude and Practices (KAP) study revealed that most ASHAs knew that mosquitoes are the main agent for spread of malaria (97.7%). They mostly used Rapid Diagnostic Test (RDT) for diagnosis (91.8%). The majority (87.3%) correctly identified negative RDT result while only 15% and 10.5%, respectively, identified Plasmodium vivax and Plasmodium falciparum positive cases correctly. Further analysis showed that 85% ASHAs used chloroquine, 44.5% used artemisinin-based combination therapy (ACT), and 55.5% used primaquine for treatment of malaria. It was also found that only 38.2% ASHA gave PQ for 14 days in cases of P. vivax. At the time of the interview, 19.1% ASHAs did not have any RDTs for diagnosis and 47.7% reported not having ACT for treatment of P. falciparum malaria. Conclusions This study has revealed that ASHAs in the test district were not adequately trained or stocked for malaria parasite species identification and treatment, which are the major components of malaria elimination programme. This study has, therefore, revealed a need for training ASHAs on testing by RDT and proper treatment regimen for P. vivax and P. falciparum.

Abstract Background Malaria Elimination Demonstration Project (MEDP) was started as a Public-Private-Partnership between the Indian Council of Medical Research through National Institute of Research in Tribal Health, Govt. of Madhya Pradesh and Foundation of Disease Elimination and Control of India, which is a Corporate Social Responsibility (CSR) initiative of the Sun Pharmaceutical Industries Limited. The project’s goal was to demonstrate that malaria can be eliminated from a high malaria endemic district along with prevention of re-establishment of malaria and to develop a model for malaria elimination using the lessons learned and knowledge acquired from the demonstration project. Methods The project employed tested protocols of robust surveillance, case management, vector control, and capacity building through continuous evaluation and training. The model was developed using the learnings from the operational plan, surveillance and case management, monitoring and feedback, entomological investigations and vector control, IEC and capacity building, supply chain management, mobile application (SOCH), and independent reviews of MEDP. Results The MEDP has been operational since April 2017 with field operations from August 2017, and has observed: (1) reduction in indigenous cases of malaria by about 91 %; (2) need for training and capacity building of field staff for diagnosis and treatment of malaria; (3) need for improvement insecticide spraying and for distribution and usage of bed-nets; (4) need for robust surveillance system that captures and documents information on febrile cases, RDT positive individuals, and treatments provided; (5) need for effective supervision of field staff based on advance tour plan; (6) accountability and controls from the highest level to field workers; and (7) need for context-specific IEC. Conclusions Malaria elimination is a high-priority public health goal of the Indian Government with a committed deadline of 2030. In order to achieve this goal, built-in systems of accountability, ownership, effective management, operational, technical, and financial controls will be crucial components for malaria elimination in India. This manuscript presents a model for malaria elimination with district as an operational unit, which may be considered for malaria elimination in India and other countries with similar geography, topography, climate, endemicity, health infrastructure, and socio-economic characteristics.

Abstract Background The Government of Madhya Pradesh employed Indoor Residual Spraying (IRS) with alpha-cypermethrin synthetic pyrethroids in sub-centres with Annual Parasite Incidence (API) from 2 to 4.99. In sub-centres with API more than 5, Long-Lasting Insecticidal Nets (LLINs) were distributed. At the request of the State Government, the Malaria Elimination Demonstration Project (MEDP) staff observed and provided support to both IRS and LLINs campaigns. In the year 2017, the study team monitored only the IRS campaigns, however, in the year 2018, the supportive supervision was provided to the IRS campaign teams along with post-distribution monitoring of the LLINs. Methods The study was carried out during IRS spraying using a pre-tested, closed-ended monitoring checklist which consisted of two parts- observations of spraying team and observation of sprayed houses. For LLINs, a sample of the households that received the bed nets was taken for the study. For IRS, the spraying teams were monitored for quality and technique for a total of 159 times in 2017 and 183 times in the year 2018, respectively. For post spraying observations, a total of 1261 and 1791 households were observed in the years 2017 and 2018, respectively. The use of LLINs was observed in 5 % of the households in 2018 and 2020, which is about 2,000 houses in each survey where each house received about 2.5 LLINs per household. The results of surveys were compared to assess impact of supportive supervision and monitoring. Results Significant improvement was noted after supportive supervision in year 2018 in various aspects of spraying. Preparedness of spraying, such as advance information to villagers, presence of equipment and records improved by up to 70 %. The methodology of spraying preparation improved from 50 to 90 %, spraying technique improved from 54 to 80 %, and proper use equipment during spraying improved from 51 to 92 %. After eight months post distribution of the LLINs in 2019, improvement was seen in regular usage of LLINs by 28 %. It was found that on-spot demonstrations during distribution and carrying of LLINs when sleeping outside homes increased by 56 %. Results of IEC campaigns revealed the reduction in adverse effects by 64 % and increase in awareness by 97 %. Conclusions Effective supervision improved the quality of IRS and usage of LLINs in the study area. Based on these results, continued training and monitoring of staff that is deployed to spraying houses and distribute bed nets was suggested. The study also revealed that proper IEC/BCC drives help increase community acceptance of vector control measures and their rational usage.

Abstract Background Malaria is known as a disease of poverty because of its dominance in poverty-stricken areas. Madhya Pradesh state in central India is one of the most vulnerable states for malaria morbidity and mortality. Socio-economic, environmental and demographic factors present challenges in malaria control and elimination. As part of the Malaria Elimination Demonstration Project in the tribal district of Mandla in Madhya Pradesh, this study was undertaken to assess the role of different social-economic factors contributing to malaria incidence. Methods The study was conducted in the 1233 villages of district Mandla, where 87% population resides in rural areas. The data was collected using the android based mobile application—SOCH for a period of 2 years (September 2017 to August 2019). A wealth index was computed along with analysis of the socio-economic characteristics of houses with malaria cases. Variables with significant variation in malaria cases were used in logistic regression. Results More than 70% of houses in Mandla are Kuccha (made of thatched roof or mud), 20% do not have any toilet facilities, and only 11% had an annual income of more than 50,000 INR, which converts to about $700 per year. Households with younger heads, male heads, more number of family members were more likely to have malaria cases. Kuccha construction, improper water supply, low household income houses were also more likely to have a malaria case and the odds doubled in houses with no toilet facilities. Conclusion Based on the results of the study, it has been found that there is an association between the odds of having malaria cases and different household variables such as age, gender, number of members, number of rooms, caste, type of house, toilet facilities, water supply, cattle sheds, agricultural land, income, and vector control interventions. Therefore, a better understanding of the association of various risk factors that influence the incidence of malaria is required to design and/or deploy effective policies and strategies for malaria elimination. The results of this study suggest that appropriate economic and environmental interventions even in low-income and poverty-stricken tribal areas could have huge impact on the success of the national malaria elimination goals.

Abstract Background The Malaria Elimination Demonstration Project (MEDP) maintained a workforce of 235 Village Malaria Workers (VMWs) and 25 Malaria Field Coordinators (MFCs) to conduct disease surveillance, case management, IEC/BCC activities, capacity building, and monitoring of vector control activities in 1233 villages of Mandla, a high malaria endemic district of Madhya Pradesh in central India. Methods The induction training was conducted for 3 days on malaria diagnosis, treatment, prevention, and ethics. All trainings were assessed using a pre and post-training assessment questionnaire, with 70% marks as qualifying threshold. The questionnaire was divided into three thematic areas viz. general knowledge related to malaria (KAP), diagnosis and treatment (DXRX), and vector control (PVC). Results In 2017, the project trained 330 candidates, followed by 243 and 247 candidates in 2018 and 2019, respectively. 94.3% candidates passed after a single training session. Almost all (95%) candidates showed improvement in knowledge after the training with 4% showing no effect and 1% showing deterioration. Progressive improvement in scores of 2017 cohort was seen along with significant improvement in performance of candidates in 2019 after the introduction of systematic monitoring and ‘shadowing’ training exercises. Conclusion The project has successfully demonstrated the value of recruitment of workers from the study area, outcome of training, and performance evaluation of field staff in malaria elimination programme. This careful strategy of recruitment and training resulted in a work-force that was capable of independently conducting surveillance, case management, vector control, and Information Education Communication/Behaviour Change Communication (IEC/BCC). The learnings of this study, including the training modules and monitoring processes, can be used to train the health delivery staff for achieving national goal for malaria elimination by 2030. Similar training and monitoring programmes could also be used for other public health delivery programmes.

Serology remains the mainstay for diagnosis of scrub typhus. In central India, diagnosis of this neglected emerging zoonotic disease suffers due to lack of standardized region-specific cutoff value and diagnostic test. Henceforth, standardized region-specific cutoff value for diagnostic ELISA play a significant role in initial treatment of patients and to differentiate other febrile diseases in endemic setup. A total of 144 patients of all age groups with acute undifferentiated febrile illness patients, forty healthy controls, dengue and chikungunya positive thirty-five samples in each category, respectively were enrolled in the study and subjected to IgM ELISA (InBioS, International, Inc.). Samples showing OD value more than 0.5 in IgM ELISA, were subjected to nested PCR. Both, receiver operating characteristic (ROC) curve and healthy volunteer samples mean with +3 SD were considered to generate region specific cutoff OD value. A total of 48 patients were diagnosed as cases of scrub typhus through IgM ELISA. Out of 48 samples, 30 were positive by nested PCR. The ROC curve analysis revealed a diagnostic ELISA cutoff value of 0.73 with sensitivity and specificity of 95% and 100%, respectively. The cut off arrived from healthy volunteer is mean OD + 3 SD is 0.72. Considering the significance of scrub typhus diagnosis for treatment and to understand disease dynamics in region wise, the cutoff value of >0.72 for diagnostic ELISA for Madhya Pradesh in central India can be used.

Abstract Background Understanding of malaria vector density, distribution, insecticide resistance, vector incrimination, infection status, and identification of sibling species are some of the essential components of vector control measures for achieving malaria elimination goals. Methods As part of the malaria elimination demonstration project, entomological surveillance was carried out from October 2017 to October 2019 by collecting indoor resting mosquitoes using hand catch method. Susceptibility test was done for determining the insecticide resistance status of vector mosquito Anopheles culicifacies using standard protocols by the World Health Organization. The cone bioassay method was used for determining the efficacy and quality of insecticide sprayed. Mosquitoes collected from different ecotypes were identified and processed for parasite identification, vector incrimination and sibling species determination. Results The two known malaria vector species (Anopheles culicifacies and Anopheles fluviatilis) were found in the study area, which have been previously reported in this and adjoining areas of the State of Madhya Pradesh. The prevalence of An. culicifacies was significantly higher in all study villages with peak in July while lowest number was recorded in May. Proportion of vector density was observed to be low in foothill terrains. The other anopheline species viz, Anopheles subpictus, Anopheles annularis, Anopheles vagus, Anopheles splendidus, Anopheles pallidus, Anopheles nigerrimus and Anopheles barbirostris were also recorded in the study area, although their prevalence was significantly less compared to the An. culicifacies. In 2017, An. culicifacies was found to be resistant to dichloro-diphenyl-trichloroethane (DDT) and malathion, with possible resistance to alphacypermethrin and susceptible to deltamethrin. However, in 2019, the species was found to be resistant to alphacypermethrin, DDT, malathion, with possible resistance to deltamethrin. The bioassays revealed 82 to > 98% corrected % mortality of An. culicifacies on day-one post-spraying and 35 to 62% on follow-up day-30. Anopheles culicifacies sibling species C was most prevalent (38.5%) followed by A/D and E while B was least pre-dominant (11.9%). Anopheles fluviatilis sibling species T was most prevalent (74.6%) followed by U (25.4%) while species S was not recorded. One An.culicifacies (sibling species C) was found positive for Plasmodium falciparum by PCR tests in the mosquitoes sampled from the test areas. Conclusion Based on the nine entomologic investigations conducted between 2017–2019, it was concluded that An. culicifacies was present throughout the year while An. fluviatilis had seasonal presence in the study areas. Anopheles culicifacies was resistant to alphacypermethrin and emerging resistance to deltamethrin was observed in this area. Anopheles culicifacies was confirmed as the malaria vector. This type of information on indigenous malaria vectors and insecticide resistance is important in implementation of vector control through indoor residual spraying (IRS) and use of insecticide-impregnated bed nets for achieving the malaria elimination goals.

Abstract Background In the past decade substantial reduction in malaria morbidity and mortality has been observed through well-implemented case management and vector control strategies. India has also achieved a significant reduction in malaria burden in 2018 and has committed to eliminate malaria by 2030. The Mandla Malaria Elimination Demonstration Project (MEDP) was started in 2017 in 1233 villages of District Mandla to demonstrate malaria elimination in a tribal district with hard-to-reach areas was possible using active and passive surveillance, case management, vector control, and targeted information, education and communication campaigns. An operational plan was developed to strengthen the existing surveillance and malaria elimination systems, through fortnightly active case detection to ensure that all cases including those that are introduced into the communities are rapidly identified and treated promptly. The plan also focused on the reduction of human-mosquito contact through the use of Long-Lasting Insecticial Nets (LLINs) and Indoor Residual Spray (IRS). The operational plan was modified in view of the present COVID-19 pandemic by creating systems of assistance for the local administration for COVID-related work while ensuring the operational integrity of malaria elimination efforts. Results The use of MEDP study design and operational plan, with its built-in management control systems, has yielded significant (91%) reduction of indigenous cases of malaria during the period from June 2017 to May 2020. The malaria positivity rate was 0.33% in 2017–18, 0.13% in 2018–19, and 0.06% in 2019–20. Mass screening revealed 0.18% malaria positivity in September–October 2018, followed by 0.06% in June 2019, and 0.03% in December 2019, and these were mostly asymptomatic cases in the community. The project has been able to sustain the gains of the past three years during the ongoing COVID-19 pandemic. Conclusion This paper provides the study design and the operational plan for malaria elimination in a high-burden district of Central India, which presented difficulties of hard to reach areas, forest malaria, and complex epidemiology of urban and rural malaria. The lessons learned could be used for malaria elimination efforts in rest of the country and other parts of South Asia with comparable demography and epidemiology.

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

Abstract Background Malaria in sub-Saharan Africa (sSA) is thought to be mostly caused by Plasmodium falciparum. Recently, growing reports of cases due to Plasmodium ovale, Plasmodium malariae, and Plasmodium vivax have been increasingly observed to play a role in malaria epidemiology in sSA. This in fact is due to the usage of very sensitive diagnostic tools (e.g. PCR), which have highlighted the underestimation of non-falciparum malaria in this sub-region. Plasmodium vivax was historically thought to be absent in sSA due to the high prevalence of the Duffy negativity in individuals residing in this sub-continent. Recent studies reporting detection of vivax malaria in Duffy-negative individuals from Mali, Mauritania, Cameroon challenge this notion. Methods Following previous report of P. vivax in Duffy-negative individuals in Nigeria, samples were further collected and assessed RDT and/or microscopy. Thereafter, malaria positive samples were subjected to conventional PCR method and DNA sequencing to confirm both single/mixed infections as well as the Duffy status of the individuals. Results Amplification of Plasmodium gDNA was successful in 59.9% (145/242) of the evaluated isolates and as expected P. falciparum was the most predominant (91.7%) species identified. Interestingly, four P. vivax isolates were identified either as single (3) or mixed (one P. falciparum/P. vivax) infection. Sequencing results confirmed all vivax isolates as truly vivax malaria and the patient were of Duffy-negative genotype. Conclusion Identification of additional vivax isolates among Duffy-negative individuals from Nigeria, substantiate the expanding body of evidence on the ability of P. vivax to infect RBCs that do not express the DARC gene. Hence, such genetic-epidemiological study should be conducted at the country level in order to evaluate the true burden of P. vivax in Nigeria.

Background. Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are malaria vector control measures used in India, but the development of insecticide resistance poses major impediments for effective vector control strategies. As per the guidelines of the National Vector Borne Disease Control Programme (NVBDCP), the study was conducted in 12 districts of Madhya Pradesh to generate data on insecticide resistance in malaria vectors. Methods. The susceptibility tests were conducted on adult An. culicifacies as per the WHO standard technique with wild-caught mosquitoes. The blood-fed female mosquitoes were exposed in 3 to 4 replicates on each occasion to the impregnated papers with specified discriminating dosages of the insecticides (DDT: 4%, malathion: 5%, deltamethrin: 0.05%, and alphacypermethrin: 0.05%), for one hour, and mortality was recorded after 24-hour holding. Results. An. culicifacies was found resistant to DDT 4% in all the 12 districts and malathion in 11 districts. The resistance to alphacypermethrin was also observed in two districts, and possible resistance was found to alphacypermethrin in seven districts and to deltamethrin in eight districts, while the vector was found susceptible to both deltamethrin and alphacypermethrin in only 3 districts. Conclusion. An. culicifacies is resistant to DDT and malathion and has emerging resistance to pyrethroids, alphacypermethrin, and deltamethrin. Therefore, regular monitoring of insecticide susceptibility in malaria vectors is needed for implementing effective vector management strategies. However, studies to verify the impact of IRS with good coverage on the transmission of disease are required before deciding on the change of insecticide in conjunction with epidemiological data.

Background. The major malaria vector in India is Anopheles culicifacies, and indoor residual spraying (IRS) and distribution of long-lasting insecticidal nets (LLINs) are the two main vector control measures in the national program. This species has shown resistance to dichlorodiphenyltrichloroethane (DDT), malathion, and synthetic pyrethroids (SP). The study was carried out in three districts, that is, Dindori of Madhya Pradesh and Kanker and Bastar from Chhattisgarh state to know the range of resistance phenotypes and to assess the strength of resistance in An. culicifacies. Methods. An. culicifacies collected from the field was tested to determine the susceptibility status to the discriminating concentration (DC) of different insecticides, that is, DDT 4.0%, malathion 5.0%, alphacypermethrin 0.05%, and deltamethrin 0.05% following the World Health Organization (WHO) procedures. Further, intensity bioassays of the resistant An. culicifacies to 1X discriminating concentration (DC) of alphacypermethrin and deltamethrin were conducted by exposing 5X and 10X concentrations of 1X DC. Results are interpreted as per the WHO criterion. Results. The overall result of susceptibility status in An. culicifacies in districts Dindori, Kanker, and Bastar revealed confirmed resistance to DDT, malathion, alphacypermethrin, and deltamethrin registering mortality of 15% (10–20%), 70% (65–75%), 78.6% (77–82.5%), and 87% (84.8–91.3%), respectively. Further, in district Dindori and Baster, the intensity bioassay test at 5X DC of alphacypermethrin and deltamethrin revealed 99% and 100%, respectively, while in district Kanker, the species registered moderate resistance with 92.5% and 95% mortality, respectively, in 5X DC of alphacypermethrin and deltamethrin. However, in 10X DC, the An. culicifacies was susceptible to both pyrethroids (100%) in district Kanker. Conclusion. The results of the intensity bioassay tests with SP-resistant An. culicifacies suggested no change of insecticide is required in the ongoing intervention. However, regular monitoring of insecticide susceptibility and intensity bioassays in malaria vectors in view of continued use of these interventions may increase resistance and for implementing effective vector management strategies.

Sickle cell disease is a major public health problem in India. Lack of rapid and reliable diagnostic methods result in many avoidable deaths in affected population. Current diagnostic tools are laboratory based, expensive and need trained manpower. Here, we evaluated the performance of a microchip-based cellulose acetate electrophoresis test, “Gazelle” in the tribal-dominated Indian states of Chhattisgarh and Madhya Pradesh. A total of 1,050 patients were screened by sickle cell solubility, hemoglobin (cellulose acetate) electrophoresis, high-performance liquid chromatography (HPLC) and Gazelle. Of the total 1,027 test results obtained, 960 tests were “Valid” (93.5%) and included in the analysis. Gazelle identified all patients with disease (HbSS and Thalassemia Major) with 100% accuracy. Gazelle demonstrated 100% sensitivity when comparing sickle cell disease (SCD) vs. sickle cell trait and SCD vs. normal. Specificity was 98.9% and 99.5% when comparing SCD vs. trait and trait vs. normal, respectively. Specificity was 99.8% when comparing SCD vs. normal and sensitivity was 99.3% when comparing trait vs. normal. Overall, Gazelle yielded a high accuracy (99.0%) compared to reference standard tests (hemoglobin electrophoresis and HPLC). Gazelle is a low-cost, rapid diagnostic test with high accuracy for detecting SCD both quantitatively and qualitatively. Gazelle can be a potential screening tool for the rapid diagnosis in resource limited settings and developing countries with high burden of hemoglobin disorders.

Malaria causes significant morbidity and mortality worldwide. Since 2005, malaria cases have been declining globally with many countries having eliminated malaria and several other countries heading towards malaria elimination. The World Health Organization’s Global Technical Strategy for malaria targets at least 90% reduction in case incidences and mortality rates, and elimination in 35 countries by 2030. India along with other Asia-Pacific countries has pledged to eliminate malaria by 2030. Sustainable vector control and case management interventions have played a pivotal role in malaria control leading to elimination. Malaria is complex in India due to the presence of multiple parasites and vectors species, asymptomatic cases, resistance against antimalarials and insecticides, social, demographic, cultural and behavioural beliefs. Therefore, maintaining zero indigenous malaria transmission and preventing malaria through importation of cases requires well-planned multi-pronged intervention strategies. This article provides insights into the past and present malaria control and elimination efforts that may be useful for the national programme for eliminating malaria from India by 2030.

Background: Early and accurate diagnosis of malaria is critical to the success of malaria elimination. However, the current mainstay of malaria diagnosis in the field, such as light microscopy and rapid diagnostic tests (RDTs), have limitations due to low parasite density or mutation in diagnostic markers. Methods: We evaluated an inexpensive, robust, rapid, malaria diagnostic device, called Gazelle, that employs magneto-optical detection to identify haemozoin crystals (Hz) produced by all species of human malaria parasites in infected individuals. A beam of polarised light is passed through the lysed diluted blood sample under the influence of high (~.55T) and low magnetic fields. The difference in light transmission through the sample between the high and low magnetic fields indicates presence of Hz, suggesting possible malarial infection. A total of 300 febrile patients were screened at the malaria clinic of Indian Council of Medical Research-National Institute of Research in Tribal Health (ICMR-NIRTH), Jabalpur, India, from August 2018 to November 2018. Malaria diagnosis was done using four diagnostic methods: Gazelle, light microscopy, RDT, and malaria specific Polymerase Chain Reaction (PCR). Measures of diagnostic accuracy were compared. Findings: Out of 300 febrile patients enroled and tested for the presence of malaria parasites, 262 patient samples were included in the final analysis. The sensitivity and specificity of Gazelle was 98% and 97% in comparison to light microscopy, 82% and 99% to PCR and 78% and 99% to RDT, respectively. The results of the four diagnostic methods were comparable and statistically no significant differences in sensitivity or specificity was observed between these methods. Enhanced diagnostic accuracy of Gazelle in malaria patients with no prior history of malaria treatment was observed in this study. Interpretation: The diagnostic ability of Gazelle was comparable to light microscopy and better than RDTs even in low parasitemia and in presence of pfhrp2/3 deletion mutant parasites. Gazelle may be a novel valuable diagnostic tool in resource poor settings where (i) microscopy is not feasible and (ii) pfhrp2/3gene deleted parasite are present. Its speed, cost-efficiency, and alternative to lack of microscopists makes it an important adjunct in field settings. Funding: HemexDx, India.

Malaria is a vector-borne infectious disease, caused by five different species of the genus Plasmodium, and is endemic to many tropical and sub-tropical countries of the globe. At present, malaria diagnosis at the primary health care level in India is conducted by either microscopy or rapid diagnostic test (RDT). In recent years, molecular diagnosis (by PCR assay), has emerged as the most sensitive method for malaria diagnosis. India is highly endemic to malaria and shoulders the burden of two major malaria parasites, Plasmodium falciparum and P. vivax. Previous studies using PCR diagnostic assay had unraveled several interesting facts on distribution of malaria parasites in India. However, these studies had several limitations from small sample size to limited geographical areas of sampling. In order to mitigate these limitations, we have collected finger-prick blood samples from 2,333 malaria symptomatic individuals in nine states from 11 geographic locations, covering almost the entire malaria endemic regions of India and performed all the three diagnostic tests (microscopy, RDT and PCR assay) and also have conducted comparative assessment on the performance of the three diagnostic tests. Since PCR assay turned out to be highly sensitive (827 malaria positive cases) among the three types of tests, we have utilized data from PCR diagnostic assay for analyses and inferences. The results indicate varied distributional prevalence of P. vivax and P. falciparum according to locations in India, and also the mixed species infection due to these two species. The proportion of P. falciparum to P. vivax was found to be 49:51, and percentage of mixed species infections due to these two parasites was found to be 13% of total infections. Considering India is set for malaria elimination by 2030, the present malaria epidemiological information is of high importance.

We have analysed the whole mitochondrial (mt) genome sequences (each ~6 kilo nucleotide base pairs in length) of four field isolates of the malaria parasite Plasmodium falciparum collected from different locations in India. Comparative genomic analyses of mt genome sequences revealed three novel India-specific single nucleotide polymorphisms. In general, high mt genome diversity was found in Indian P. falciparum, at a level comparable to African isolates. A population phylogenetic tree placed the presently sequenced Indian P. falciparum with the global isolates, while a previously sequenced Indian isolate was an outlier. Although this preliminary study is limited to a few numbers of isolates, the data have provided fundamental evidence of the mt genome diversity and evolutionary relationships of Indian P. falciparum with that of global isolates.

The development and rapid spread of chloroquine resistance (CQR) in Plasmodium falciparum have triggered the identification of several genetic target(s) in the P. falciparum genome. In particular, mutations in the Pfcrt gene, specifically, K76T and mutations in three other amino acids in the region adjoining K76 (residues 72, 74, 75 and 76), are considered to be highly related to CQR. These various mutations form several different haplotypes and Pfcrt gene polymorphisms and the global distribution of the different CQR- Pfcrt haplotypes in endemic and non-endemic regions of P. falciparum malaria have been the subject of extensive study. Despite the fact that the Pfcrt gene is considered to be the primary CQR gene in P. falciparum , several studies have suggested that this may not be the case. Furthermore, there is a poor correlation between the evolutionary implications of the Pfcrt haplotypes and the inferred migration of CQR P. falciparum based on CQR epidemiological surveillance data. The present paper aims to clarify the existing knowledge on the genetic basis of the different CQR- Pfcrt haplotypes that are prevalent in worldwide populations based on the published literature and to analyse the data to generate hypotheses on the genetics and evolution of CQR malaria.

Mutations in the Pfcrt gene that change the resulting amino acids and form different haplotypes are common and correlate with the prevalence of chloroquine resistant (CQR) field isolates of the malaria parasite, Plasmodium falciparum. This correlation provides opportunities to infer the global evolutionary history of CQ resistance by analysing CQR Pfcrt haplotype data. We collated data on the Pfcrt haplotypes from different global studies and performed evolutionary genetic analysis to present comprehensive and comparative information on the global distribution of five major CQR-Pfcrt haplotypes and evolutionary inter-relationships among 38 different countries. Using the haplotype diversity data, inter-continental genetic differentiation was also ascertained.

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.

Evolutionary genetic studies in genomes offer excellent opportunities to infer population structure and demographic history of species populations. However, such kinds of studies are very limited in malaria. Malaria is highly endemic in India and both of the causative agents of malaria, Plasmodium vivax and Plasmodium falciparum occur in almost equal proportion. The widespread distribution of P. vivax is attributing to socio-economic loss, and thereby increasing the public health concern. Therefore, it is important to understand the genetic features of P. vivax population in India. Comparative genomics of P. falciparum and P. vivax has revealed several syntenic chromosomal segments. One such 200 kb segment has been utilized to design several small DNA fragments from non-coding regions, and tested for ‘putatively neutral’ marker for inference of population structure and demography of P. vivax. Utilizing 126 P. vivax isolates collected from 10 different widespread geographic locations in India, it was found that two neutral DNA fragments (P10 and P17) showed fairly less nucleotide diversity in all the population samples of P. vivax. A sudden drop in diversity in putatively neutral genetic fragments indicates the role of positive natural selection under the hitchhiking model of molecular evolution. Evolutionary genetic studies in the regions surrounding P10 and P17 with functional validation might provide meaningful insights and help identify targets in P. vivax in India. The study can further extended to P.falciparum as it is syntenic to P.vivax. It was found that the neutral fragment P17 is flanked by putative circumsporozoite protein (PvCSP) gene. Since, CSP is a major surface protein of the infective stage of malaria parasite; it is believed that the PvCSP gene might be under a certain kind of selective pressure. The objective of this study is to obtain new DNA sequence information of the putative PvCSP gene in isolates from India and compare it with the estimated diversity of the non-coding DNA fragments located in-and around this gene for inference of natural selection. Effectiveness of putative PvCSP gene as a suitable vaccine candidate on the basis of genetic diversity in parasite populations can be evaluated.

The malaria parasite Plasmodium vivax is known to be majorly endemic to Asian and Latin American countries with no or very few reports of Africans infected with this parasite. Since the human Duffy antigens act as receptors for P. vivax to invade human RBCs and Africans are generally Duffy-negative, non-endemicity of P. vivax in Africa has been attributed to this fact. However, recent reports describing P. vivax infections in Duffy-negative Africans from West and Central parts of Africa have been surfaced including a recent report on P. vivax infection in native Cameroonians. In order to know if Cameroonians living in the southern regions are also susceptible to P. vivax infection, we collected finger-prick blood samples from 485 malarial symptomatic patients in five locations and followed PCR diagnostic assays with DNA sequencing of the 18S ribosomal RNA gene. Out of the 201 malaria positive cases detected, 193 were pure P. falciparum, six pure P. vivax and two mixed parasite infections (P. falciparum + P. vivax). The eight P. vivax infected samples (six single + two mixed) were further subjected to DNA sequencing of the P. vivax multidrug resistance 1 (pvmdr1) and the P.vivax circumsporozoite (pvcsp) genes. Alignment of the eight Cameroonian pvmdr1 sequences with the reference sequence showed high sequence similarities, reconfirming P. vivax infection in all the eight patients. DNA sequencing of the pvcsp gene indicated all the eight P. vivax to be of VK247 type. Interestingly, DNA sequencing of a part of the human Duffy gene covering the promoter region in the eight P. vivax-infected Cameroonians to identify the T-33C mutation revealed all these patients as Duffy-negative. The results provide evidence of single P. vivax as well as mixed malaria parasite infection in native Cameroonians and add knowledge to the growing evidences of P. vivax infection in Duffy-negative Africans.

The Duffy (Fy) antigens act as receptors for chemokines as well as for Plasmodium vivax to invade human RBCs. A recent study has correlated the occurrence of the FY*A allele of Duffy gene with decreased susceptibility to vivax malaria, but no epidemiological correlation between the distribution of FY*A allele and incidences of vivax malaria has been established so far. Furthermore, if such correlations exist, whether natural selection has mediated the association, is an important question. Since India is highly endemic to P. vivax malaria with variable eco-climatic and varying vivax malaria epidemiology across different regions, such a question could well be answered in Indians. For this, we have genotyped the FY gene at the -33(rd) and the 125(th) nucleotide positions in 250 Indians sampled from six different zonal plus one tribal population covering the whole of India and studied possible correlations with eco-climatic and vivax malaria incidences. No FY*O allele was found, however, both the FY*A and FY*B alleles forming FY*A/FY*A, FY*A/FY*B and FY*B/FY*B genotypes were widely distributed among Indians. Five out of seven population samples significantly deviated from the Hardy-Weinberg equilibrium expectation, and two alleles (FY*A and FY*B) and the homozygote genotype, FY*B/FY*B were clinically distributed over the population coordinates. Furthermore, vivax malaria incidences over the past five years were significantly negatively and positively associated with the frequencies of the FY*A and FY*B alleles, respectively. The Northern Indians were highly differentiated from the other zonal population samples at the FY gene, as evidenced from the reconstructed Neighbor-Joining phylogenetic tree. The results specify the role of natural selection in the distribution of FY gene polymorphism in India. Furthermore, the hypotheses on the part of the FY*A allele in conferring protection to vivax malaria could be validated following population genetic studies in a vivax malaria epidemiological setting, such as India.

TNF-α is an important human cytokine that imparts dualism in malaria pathogenicity. At high dosages, TNF-α is believed to provoke pathogenicity in cerebral malaria; while at lower dosages TNF-α is protective against severe human malaria. In order to understand the human TNF-α gene and to ascertain evolutionary aspects of its dualistic nature for malaria pathogenicity, we characterized this gene in detail in six different mammalian taxa. The avian taxon, Gallus gallus was included in our study, as TNF-α is not present in birds; therefore, a tandemly placed duplicate of TNF-α (LT-α or TNF-β) was included. A comparative study was made of nucleotide length variations, intron and exon sizes and number variations, differential compositions of coding to non-coding bases, etc., to look for similarities/dissimilarities in the TNF-α gene across all seven taxa. A phylogenetic analysis revealed the pattern found in other genes, as humans, chimpanzees and rhesus monkeys were placed in a single clade, and rats and mice in another; the chicken was in a clearly separate branch. We further focused on these three taxa and aligned the amino acid sequences; there were small differences between humans and chimpanzees; both were more different from the rhesus monkey. Further, comparison of coding and non-coding nucleotide length variations and coding to non-coding nucleotide ratio between TNF-α and TNF-β among these three mammalian taxa provided a first-hand indication of the role of the TNF-α gene, but not of TNF-β in the dualistic nature of TNF-α in malaria pathogenicity.