Community and health worker perspectives on malaria in Meghalaya, India: covering the last mile of elimination by 2030.

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.

Implementation and acceptance of government-sponsored malaria control interventions in Meghalaya, India.

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.

A cornucopia of research resources for the fourth rodent malaria parasite species.

The study of human malaria caused by species of Plasmodium has undoubtedly been enriched by the use of model systems, such as the rodent malaria parasites originally isolated from African thicket rats. A significant gap in the arsenal of resources of the species that make up the rodent malaria parasites has been the lack of any such tools for the fourth of the species, Plasmodium vinckei. This has recently been rectified by Abhinay Ramaprasad and colleagues, whose pivotal paper published in BMC Biology describes a cornucopia of new P. vinckei 'omics datasets, mosquito transmission experiments, transfection protocols, and virulence phenotypes, to propel this species firmly into the twenty-first century.

Household and individual level risk factors associated with declining malaria incidence in Meghalaya, India: implications for malaria elimination in low-endemic settings.

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.

Spatial and temporal village-level prevalence of Plasmodium infection and associated risk factors in two districts of Meghalaya, India.

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.

Defining symptoms of malaria in India in an era of asymptomatic infections.

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.

Antibody responses within two leading Plasmodium vivax vaccine candidate antigens in three geographically diverse malaria-endemic regions of India.

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.

Dengue, chikungunya, and scrub typhus are important etiologies of non-malarial febrile illness in Rourkela, Odisha, India.

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.

Overlapping Community Compositions of Gut and Fecal Microbiomes in Lab-Reared and Field-Collected German Cockroaches.

German cockroaches, Blattella germanica (Blattodea: Ectobiidae), are human commensals that move freely between food and waste, disseminating bacteria, including potential pathogens, through their feces. However, the relationship between the microbial communities of the cockroach gut and feces is poorly understood. We analyzed the V4 region of the 16S rRNA gene and the V9 region of the 18S rRNA gene by next-generation sequencing (NGS) to compare the bacterial and protist diversities in guts versus feces and males versus females, as well as assess variation across cockroach populations. Cockroaches harbored a diverse array of bacteria, and 80 to 90% of the operational taxonomic units (OTUs) were shared between the feces and gut. Lab-reared and field-collected cockroaches had distinct microbiota, and whereas lab-reared cockroaches had relatively conserved communities, considerable variation was observed in the microbial community composition of cockroaches collected in different apartments. Nonetheless, cockroaches from all locations shared some core bacterial taxa. The eukaryotic community in the feces of field-collected cockroaches was found to be more diverse than that in lab-reared cockroaches. These results demonstrate that cockroaches disseminate their gut microbiome in their feces, and they underscore the important contribution of the cockroach fecal microbiome to the microbial diversity of cockroach-infested homes.IMPORTANCE The German cockroach infests diverse human-built structures, including homes and hospitals. It produces potent allergens that trigger asthma and disseminates opportunistic pathogens in its feces. A comprehensive understanding of gut and fecal microbial communities of cockroaches is essential not only to understand their contribution to the biology of the cockroach, but also for exploring their clinical relevance. In this study, we compare the diversity of bacteria and eukaryotes in the cockroach gut and feces and assess the variation in the gut microbiota across cockroach populations.

Malaria in Meghalaya: a systematic literature review and analysis of data from the National Vector-Borne Disease Control Programme.

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.

An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads.

Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological importance, our knowledge of protists in urban environments lags behind that of bacteria, largely due to a lack of experimentally validated high-throughput protocols that produce accurate estimates of protist diversity while minimizing non-protist DNA representation. We optimized protocols for detecting zoonotic protists in raw sewage samples, with a focus on trichomonad taxa. First, we investigated the utility of two commonly used variable regions of the 18S rRNA marker gene, V4 and V9, by amplifying and Sanger sequencing 23 different eukaryotic species, including 16 protist species such as Cryptosporidium parvum, Giardia intestinalis, Toxoplasma gondii, and species of trichomonad. Next, we optimized wet-lab methods for sample processing and Illumina sequencing of both regions from raw sewage collected from a private apartment building in New York City. Our results show that both regions are effective at identifying several zoonotic protists that may be present in sewage. A combination of small extractions (1 mL volumes) performed on the same day as sample collection, and the incorporation of a vertebrate blocking primer, is ideal to detect protist taxa of interest and combat the effects of metazoan DNA. We expect that the robust, standardized methods presented in our workflow will be applicable to investigations of protists in other environmental samples, and will help facilitate large-scale investigations of protistan diversity.

A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India.

A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.

What is the value of reactive case detection in malaria control? A case-study in India and a systematic review.

BACKGROUND: Reactive case detection (RCD) for malaria is a strategy to identify additional malaria infections in areas of low malaria transmission and can complement passive surveillance. This study describes experiences with RCD in two Indian sites, and aimed to synthesize experiences with RCD across endemic countries. METHODS: RCD programmes were piloted in two urban areas of India with a low prevalence of mainly Plasmodium vivax malaria in 2014. Cases were identified in a clinic by microscopy and contacts were screened within 2 weeks; PCR, in addition to microscopy, was used to detect Plasmodium parasites. A systematic review was conducted to identify RCD experiences in the literature. RESULTS: In Chennai, 868 contacts were enrolled for 18 index cases of clinical malaria; in Nadiad, 131 contacts were enrolled for 20 index cases. No new malaria infections were detected in Nadiad among contacts, and four new infections were detected in Chennai (three P. vivax and one Plasmodium falciparum), of which two were among household members of index cases. An additional five studies describing results from an RCD strategy were identified in the literature: four in Africa and one in Thailand. Including the results from India, the average number of contacts screened per index case in a total of seven studies ranged from four to 50, and 126 in a case study in Thailand with one index case. Malaria was detected in 0-45 % of the contacted persons. The average number of index cases needed to be traced to find one new case of malaria ranged from one to five, and could not be assessed in one study in India (no contacts positive for 20 cases). Sharing the household with an index case was associated with a five-fold increased risk of malaria compared to contacts from households without an index case (pooled risk ratio 5.29, 95 % CI 3.31-8.47, I(2) 0 %, four studies). CONCLUSIONS: RCD in areas of low malaria transmission is a labour-intensive strategy, and its benefit is not clear. Studies are needed to assess how RCD can be optimized or into alternatives where interventions are targeted to family members or hotspots.

The origin and diversification of the merozoite surface protein 3 (msp3) multi-gene family in Plasmodium vivax and related parasites.

The genus Plasmodium is a diversified group of parasites with more than 200 known species that includes those causing malaria in humans. These parasites use numerous proteins in a complex process that allows them to invade the red blood cells of their vertebrate hosts. Many of those proteins are part of multi-gene families; one of which is the merozoite surface protein-3 (msp3) family. The msp3 multi-gene family is considered important in the two main human parasites, Plasmodium vivax and Plasmodium falciparum, as its paralogs are simultaneously expressed in the blood stage (merozoite) and are immunogenic. There are large differences among Plasmodium species in the number of paralogs in this family. Such differences have been previously explained, in part, as adaptations that allow the different Plasmodium species to invade their hosts. To investigate this, we characterized the array containing msp3 genes among several Plasmodium species, including P. falciparum and P. vivax. We first found no evidence indicating that the msp3 family of P. falciparum was homologous to that of P. vivax. Subsequently, by focusing on the diverse clade of nonhuman primate parasites to which P. vivax is closely related, where homology was evident, we found no evidence indicating that the interspecies variation in the number of paralogs was an adaptation related to changes in host range or host switches. Overall, we hypothesize that the evolution of the msp3 family in P. vivax is consistent with a model of multi-allelic diversifying selection where the paralogs may have functionally redundant roles in terms of increasing antigenic diversity. Thus, we suggest that the expressed MSP3 proteins could serve as "decoys", via antigenic diversity, during the critical process of invading the host red blood cells.

Chloroquine efficacy studies confirm drug susceptibility of Plasmodium vivax in Chennai, India.

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.

Comparative genomics of the neglected human malaria parasite Plasmodium vivax.

The human malaria parasite Plasmodium vivax is responsible for 25-40% of the approximately 515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.

Linkage maps from multiple genetic crosses and loci linked to growth-related virulent phenotype in Plasmodium yoelii.

Plasmodium yoelii is an excellent model for studying malaria pathogenesis that is often intractable to investigate using human parasites; however, genetic studies of the parasite have been hindered by lack of genome-wide linkage resources. Here, we performed 14 genetic crosses between three pairs of P. yoelii clones/subspecies, isolated 75 independent recombinant progeny from the crosses, and constructed a high-resolution linkage map for this parasite. Microsatellite genotypes from the progeny formed 14 linkage groups belonging to the 14 parasite chromosomes, allowing assignment of sequence contigs to chromosomes. Growth-related virulent phenotypes from 25 progeny of one of the crosses were significantly associated with a major locus on chromosome 13 and with two secondary loci on chromosomes 7 and 10. The chromosome 10 and 13 loci are both linked to day 5 parasitemia, and their effects on parasite growth rate are independent but additive. The locus on chromosome 7 is associated with day 10 parasitemia. The chromosome 13 locus spans ~220 kb of DNA containing 51 predicted genes, including the P. yoelii erythrocyte binding ligand, in which a C741Y substitution in the R6 domain is implicated in the change of growth rate. Similarly, the chromosome 10 locus spans ~234 kb with 71 candidate genes, containing a member of the 235-kDa rhoptry proteins (Py235) that can bind to the erythrocyte surface membrane. Atypical virulent phenotypes among the progeny were also observed. This study provides critical tools and information for genetic investigations of virulence and biology of P. yoelii.

Zombie malaria parasites.

Natural infections are distinct from those of laboratory-or zombie-strains.

Exploiting integrative metabolomics to study host-parasite interactions in Plasmodium infections.

Despite years of research, malaria remains a significant global health burden, with poor diagnostic tests and increasing antimalarial drug resistance challenging diagnosis and treatment. While 'single-omics'-based approaches have been instrumental in gaining insight into the biology and pathogenicity of the Plasmodium parasite and its interaction with the human host, a more comprehensive understanding of malaria pathogenesis can be achieved through 'multi-omics' approaches. Integrative methods, which combine metabolomics, lipidomics, transcriptomics, and genomics datasets, offer a holistic systems biology approach to studying malaria. This review highlights recent advances, future directions, and challenges involved in using integrative metabolomics approaches to interrogate the interactions between Plasmodium and the human host, paving the way towards targeted antimalaria therapeutics and control intervention methods.