Spatiotemporal Epidemiology of Malaria in India from 2007 to 2022.

India is a major contributor to the global burden of malaria, especially Plasmodium vivax infection. Understanding the spatiotemporal trends of malaria across India over the last two decades may assist in targeted intervention. The population-normalized spatiotemporal trends of malaria epidemiology in India from 2007 to 2022 were analyzed using a geographic information system with the publicly available "malaria situation" report of the National Vector Borne Disease Control Program (NVBDCP). The NVBDCP data showed malaria cases to have steeply declined from 1.17 million in 2015 to 0.18 million cases in 2022; this is 10.1 and 18.7 fold lower than the WHO's estimate of 11.93 million and 3.38 million cases in 2015 and 2022, respectively. From 2007 to 2022, Mizoram, Meghalaya, Tripura, Odisha, Chhattisgarh, and Jharkhand consistently reported high caseloads of Plasmodium falciparum. In the same period, the P. vivax caseload was high in Arunachal Pradesh, Mizoram, Nagaland, Jharkhand, Odisha, Chhattisgarh, Goa, Daman and Diu, Dadra and Nagar Haveli, and Andaman and Nicobar Islands. The distribution of forest cover, annual rainfall, and proportion of the Scheduled Tribe population (the most underprivileged in Indian society) spatially correlated with malaria cases and deaths. Mizoram is the only state where cases were higher in 2022 than in 2007. Overall, India has made tremendous progress in controlling malaria and malaria-related deaths in the last decade. The decline could be attributed to the effective vector and parasite control strategies implemented across the country.

Evaluating the carnivorous efficacy of Utricularia aurea (Lamiales: Lentibulariaceae) on the larval stages of Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti (Diptera: Culicidae).

The emergence of insecticide resistance in mosquitoes necessitates the exploration and validation of sustainable biological strategies for controlling mosquitoes in their natural habitats. We assessed the predatory effect of Utricularia aurea Lour (Lamiales: Lentibulariaceae), an aquatic carnivorous plant found in the Indian subcontinent, Japan, and Australia, on 4 instars of Anopheles stephensi Liston, Culex quinquefasciatus Say, and Aedes aegypti Linn (Diptera: Culicidae), in the laboratory and field settings. In the laboratory setting, predation of larvae by U. aurea was highest during the first hour when it predated 45%, 61%, and 58% of first instars of An. stephensi, Cx. quinquefasciatus, and, Ae. aegypti, respectively, and, within 12 h, U. aurea preyed upon ~95% of the first, second, and third instars of the 3 mosquito species, ~80% of the fourth instars of An. stephensi and Ae. aegypti, and ~60% of fourth instars of Cx. quinquefasciatus. The predatory effect of U. aurea varied with mosquito species and instar. Broadly, predation risk declined with the increase of the instar size. In the field setting, at the end of 16 days, U. aurea predated 76% and 71% of the immature An. stephensi and Ae. aegypti, respectively. Our findings suggest U. aurea can be utilized as a potential biocontrol agent for controlling mosquito larvae in natural habitats; however, the current claim warrants additional investigations in a variety of natural habitats.

Spatio-temporal epidemiology and associated indicators of COVID-19 (wave-I and II) in India.

The spatio-temporal distribution of COVID-19 across India's states and union territories is not uniform, and the reasons for the heterogeneous spread are unclear. Identifying the space-time trends and underlying indicators influencing COVID-19 epidemiology at micro-administrative units (districts) will help guide public health strategies. The district-wise daily COVID-19 data of cases and deaths from February 2020 to August 2021 (COVID-19 waves-I and II) for the entire country were downloaded and curated from public databases. The COVID-19 data normalized with the projected population (2020) and used for space-time trend analysis shows the states/districts in southern India are the worst hit. Coastal districts and districts adjoining large urban regions of Mumbai, Chennai, Bengaluru, Goa, and New Delhi experienced > 50,001 cases per million population. Negative binomial regression analysis with 21 independent variables (identified through multicollinearity analysis, with VIF < 10) covering demography, socio-economic status, environment, and health was carried out for wave-I, wave-II, and total (wave-I and wave-II) cases and deaths. It shows wealth index, derived from household amenities datasets, has a high positive risk ratio (RR) with COVID-19 cases (RR: 3.577; 95% CI: 2.062-6.205) and deaths (RR: 2.477; 95% CI: 1.361-4.506) across the districts. Furthermore, socio-economic factors such as literacy rate, health services, other workers' rate, alcohol use in men, tobacco use in women, overweight/obese women, and rainfall have a positive RR and are significantly associated with COVID-19 cases/deaths at the district level. These positively associated variables are highly interconnected in COVID-19 hotspot districts. Among these, the wealth index, literacy rate, and health services, the key indices of socio-economic development within a state, are some of the significant indicators associated with COVID-19 epidemiology in India. The identification of district-level space-time trends and indicators associated with COVID-19 would help policymakers devise strategies and guidelines during public health emergencies.

Epidemiology of scrub typhus and other rickettsial infections (2018-22) in the hyper-endemic setting of Mizoram, North-East India.

BACKGROUND: In the past decade, scrub typhus cases have been reported across India, even in regions that had no previous history of the disease. In the North-East Indian state of Mizoram, scrub typhus cases were first recorded only in 2012. However, in the last five years, the state has seen a substantial increase in the scrub typhus and other rickettsial infections. As part of the public health response, the Mizoram Government has integrated screening and line listing of scrub typhus and other rickettsial infections across all its health settings, a first in India. Here we detail the epidemiology of scrub typhus and other rickettsial infections from 2018-2022, systematically recorded across the state of Mizoram. METHODOLOGY/PRINCIPAL FINDINGS: The line-listed data positive for scrub typhus and other rickettsial infections identified by rapid immunochromatographic test and/or Weil-Felix test from 2018-22 was used for the analysis. During this period, 22,914 cases of rickettsial infections were recorded, out of which 19,651 were scrub typhus cases. Aizawl is the worst affected, with 10,580 cases (46.17%). The average incidence of rickettsial infections is 3.54 cases per 1000 persons-year, and the case fatality rate is 0.35. Only ∼2% of the reported scrub typhus cases had eschar. Multivariate logistic regression analysis indicate patients with eschar (aOR = 2.5, p<0.05), occupational workers [farmers (aOR:3.9), businessmen (aOR:1.8), construction workers (aOR:17.9); p<0.05], and children (≤10 years) (aOR = 5.4, p<0.05) have higher odds of death due to rickettsial infections. CONCLUSION: The integration of systematic surveillance and recording of rickettsial diseases across Mizoram has shed important insights into their prevalence, morbidity, and mortality. This study underscores the importance of active surveillance of rickettsial infections across India, as the burden could be substantially higher, and is probably going undetected.

Malaria hotspots and climate change trends in the hyper-endemic malaria settings of Mizoram along the India-Bangladesh borders.

India has made tremendous progress in reducing malaria mortality and morbidity in the last decade. Mizoram State in North-East India is one of the few malaria-endemic regions where malaria transmission has continued to remain high. As Mizoram shares international borders with Bangladesh and Myanmar, malaria control in this region is critical for malaria elimination efforts in all the three countries. For identifying hotspots for targeted intervention, malaria data from 385 public health sub-centers across Mizoram were analyzed in the Geographic Information System. Almost all the sub-centers reporting high Annual Parasite Index (> 10) are located in Mizoram's districts that border Bangladesh. Getis-Ord Gi* statistic shows most of the sub-centers located along the Bangladesh border in the Lawngtlai and Lunglei districts to be the malaria hotspots. The hotspots also extended into the Mamit and Siaha districts, especially along the borders of Lawngtlai and Lunglei. Analysis of terrain, climatic, and land use/land cover datasets obtained from the Global Modelling and Assimilation Office and satellite images show Mizoram's western part (Lawngtlai, Lunglei, and Mamit districts) to experience similar topographic and climatic conditions as the bordering Rangamati district in the Chittagong division of Bangladesh. Climatic trends in this region from 1981 to 2021, estimated by the Mann-Kendall test and Sen's slope estimates, show an increasing trend in minimum temperature, relative humidity, rainfall, and the associated shift of climatic pattern (temperate to tropical monsoon) could facilitate malaria transmission. The quasi-Poisson regression model estimates a strong association (p < 0.001) between total malaria cases, temperature range, and elevation. The Kruskal-Wallis H test shows a statistically significant association between malaria cases and forest classes (p < 0.001). A regional coordination and strategic plan are required to eliminate malaria from this hyper-endemic malaria region of North-East India.

Seroprevalence of scrub typhus and other rickettsial diseases among the household rodents of Mizoram, North-East India.

In the last decade, scrub typhus, a zoonotic disease has emerged as a major health concern in Mizoram, a North-East Indian state that shares international borders with Myanmar and Bangladesh. Mizoram is a biodiversity hotspot and >85% of the state is under forest cover, which provides an ideal ecological niche for the rodents and mites to transmit scrub typhus and other rickettsial infections. Using the Weil-Felix test, a serosurvey of household rodents from 41 villages spread across all the 11 districts in Mizoram was undertaken to gather important insights on their role in disease transmission. Furthermore, the chigger and flea indexes were calculated from the captured rodents. The 163 rodents captured belonged to five species; the highest numbers were from Rattus tanezumi (87), followed by Rattus rattus (41), Mus musculus (17), Suncus murinus (16), and Bandicota bengalensis (2). The rickettsial seropositivity of the captured rodents was 66.26% (108 out of 163 were positive). Among the 163 rodents, sera of 75 (46.01%), 61 (37.42%), and 73 (44.78%) were reactive to OXK, OX19, and OX2 antigens, respectively. The chigger and flea index were 17.92 and 0.16, respectively. Overall, the study has given important insights into the risk of multiple rickettsial infections that household rodents could transmit in Mizoram. These findings indicate the need for the urgent implementation of effective rodent control strategies in Mizoram.

Spatial epidemiology of acute respiratory infections in children under 5 years and associated risk factors in India: District-level analysis of health, household, and environmental datasets.

Background: In India, acute respiratory infections (ARIs) are a leading cause of mortality in children under 5 years. Mapping the hotspots of ARIs and the associated risk factors can help understand their association at the district level across India. Methods: Data on ARIs in children under 5 years and household variables (unclean fuel, improved sanitation, mean maternal BMI, mean household size, mean number of children, median months of breastfeeding the children, percentage of poor households, diarrhea in children, low birth weight, tobacco use, and immunization status of children) were obtained from the National Family Health Survey-4. Surface and ground-monitored PM2.5 and PM10 datasets were collected from the Global Estimates and National Ambient Air Quality Monitoring Programme. Population density and illiteracy data were extracted from the Census of India. The geographic information system was used for mapping, and ARI hotspots were identified using the Getis-Ord Gi* spatial statistic. The quasi-Poisson regression model was used to estimate the association between ARI and household, children, maternal, environmental, and demographic factors. Results: Acute respiratory infections hotspots were predominantly seen in the north Indian states/UTs of Uttar Pradesh, Bihar, Delhi, Haryana, Punjab, and Chandigarh, and also in the border districts of Uttarakhand, Himachal Pradesh, and Jammu and Kashmir. There is a substantial overlap among PM2.5, PM10, population density, tobacco smoking, and unclean fuel use with hotspots of ARI. The quasi-Poisson regression analysis showed that PM2.5, illiteracy levels, diarrhea in children, and maternal body mass index were associated with ARI. Conclusion: To decrease ARI in children, urgent interventions are required to reduce the levels of PM2.5 and PM10 (major environmental pollutants) in the hotspot districts. Furthermore, improving sanitation, literacy levels, using clean cooking fuel, and curbing indoor smoking may minimize the risk of ARI in children.

An assessment of remotely sensed environmental variables on Dengue epidemiology in Central India.

In recent decades, dengue has been expanding rapidly in the tropical cities. Even though environmental factors and landscape features profoundly impact dengue vector abundance and disease epidemiology, significant gaps exist in understanding the role of local environmental heterogeneity on dengue epidemiology in India. In this study, we assessed the role of remotely sensed climatic factors (rainfall, temperature and humidity) and landscape variables (land use pattern, vegetation and built up density) on dengue incidence (2012-2019) in Bhopal city, Central India. Dengue hotspots in the city were assessed through geographical information system based spatial statistics. Dengue incidence increased from 0.59 cases in 2012 to 9.11 cases in 2019 per 10,000 inhabitants, and wards located in Southern Bhopal were found to be dengue hotspots. Distributed lag non-linear model combined with quasi Poisson regression was used to assess the exposure-response association, relative risk (RR), and delayed effects of environmental factors on dengue incidence. The analysis revealed a non-linear relationship between meteorological variables and dengue cases. The model shows that the risk of dengue cases increases with increasing mean temperature, rainfall and absolute humidity. The highest RR of dengue cases (~2.0) was observed for absolute humidity ≥60 g/m3 with a 5-15 week lag. Rapid urbanization assessed by an increase in the built-up area (a 9.1% increase in 2020 compared to 2014) could also be a key factor driving dengue incidence in Bhopal city. The study sheds important insight into the synergistic effects of both the landscape and climatic factors on the transmission dynamics of dengue. Furthermore, the study provides key baseline information on the climatic variables that can be used in the micro-level dengue prediction models in Bhopal and other cities with similar climatic conditions.

Social and housing indicators of dengue and chikungunya in Indian adults aged 45 and above: Analysis of a nationally representative survey (2017-18).

BACKGROUND: Dengue and chikungunya (CHIKV) are the two major vector-borne diseases of serious public health concern in India. Studies on socioeconomic and housing determinants of dengue and CHIKV at a pan-India level are lacking. Here, we took advantage of the recently carried out Longitudinal Ageing Study in India (LASI) carried out across all the states and Union Territories of India to study the social indicators of dengue and CHIKV in India. METHODS: LASI-1 (2017-2018) data on the self-reported period prevalence of dengue and CHIKV from 70,932 respondents aged ≥45 years were used for this analysis. The state-wise distribution of dengue and CHIKV was mapped. Prevalence was estimated for each study variable, and the difference was compared using the χ2 test. The adjusted odds ratios (AOR) of the socioeconomic and housing variables for dengue and CHIKV were estimated using the multiple logistic regression model. RESULTS: Urban residence is the major socio-economic indicator of dengue and CHIKV (dengue AOR: 1.57, 95% CI: 1.18-2.11; CHIKV AOR: 1.84, 95% CI: 1.36-2.49). The other notable indicator is wealth; rich respondents have higher odds of dengue and CHIKV. Adults older than 54 years and those with high school education and above are associated with a lower likelihood of dengue and CHIKV. In addition, CHIKV is associated with scheduled and forward castes, households with improper toilet facilities, open defecation, and kutcha house type. CONCLUSIONS: Despite the limitation that the data is only from adults ≥ 45, this analysis provides important insights into the socioeconomic and housing variables associated with higher odds of dengue and CHIKV in India. Understanding these determinants may assist in the national planning of prevention and control strategies for dengue and CHIKV.

Epidemiology, Hot Spots, and Sociodemographic Risk Factors of Alcohol Consumption in Indian Men and Women: Analysis of National Family Health Survey-4 (2015-16), a Nationally Representative Cross-Sectional Study.

Objectives: To map the alcohol hot spots and understand the Sociodemographic Indices (SDI) affecting alcohol consumption in Indian men and women. Methods: Data from National Family Health Survey-4 carried out from 2015 to 2016 with a sample size of 103,411 men and 699,686 women were used for Geographic Information System mapping, and hot spot identification by spatial statistics (Getis-Ord Gi*). Bivariate analyses and multiple logistic regressions were used to analyze SDI. Results: India has three major alcohol hot spots: (1) North-East (NE) states, (2) Eastern Peninsular states formed by Chhattisgarh, Odisha, Jharkhand, and Telangana, and (3) Southern states of Tamil Nadu and Kerala. Hot spot analysis strongly correlated with region-wise analysis of SDI. Respondents who consumed tobacco have higher odds (men adjusted odds ratio [aOR]: 5.42; women aOR: 4.30) of consuming alcohol. Except for religion and social category, other socioeconomic factors have a low to moderate effect on alcohol consumption. Conclusions: Hot spots and high-risk districts of alcohol consumption identified in this study can guide public health policies for targeted intervention. Alcohol use is at the discretion of individual states and union territories, and stringent anti-alcohol policies strictly enforced across India are the keys to control alcohol use.

Socio-economic and household determinants of malaria in adults aged 45 and above: analysis of longitudinal ageing survey in India, 2017-2018.

BACKGROUND: Even though malaria cases have drastically come down in the last decade, malaria remains a serious public health concern in many parts of India. National Framework for Malaria Elimination in India (2016-2030) has been launched with the goal to eliminate malaria by 2030. Understanding the socio-economic and household determinants of malaria at the national level will greatly aid India's malaria elimination efforts. METHODS: The data from Longitudinal Ageing Survey of India (LASI) Wave 1 (2017-2018) survey comprising 70,671 respondents ≥ 45 years across all the States and Union Territories were used for the analysis. Simple and multiple logistic regressions were used to obtain the unadjusted and adjusted odds ratio respectively of the socio-economic and household variables. RESULTS: The major socio-economic variables that increase the likelihood of malaria are caste ('scheduled tribes'), low education levels and rural residence. The scheduled tribes have 1.8 times higher odds of malaria than the scheduled castes (AOR: 1.8; 95% CI: 1.5-2.1). Respondents with high school education (6-12 grade) (AOR: 0.7; 95% CI: 0.6-0.8) and college education (AOR: 0.5; 95% CI: 0.4-0.6) had a very low risk of malaria than those with no school years. Rural residence and occupation (agriculture and allied jobs) also increases the odds of malaria. The major housing determinants are household size (≥ 6), housing type (kutcha), use of unclean fuel, outside water source, improper sanitation (toilet facilities) and damp wall/ceiling. CONCLUSIONS: The study has identified the major socio-economic and housing factors associated with malaria in adults aged 45 and above. In addition to vector and parasite control strategies in the tribal dominated regions of India, improving literacy and housing conditions may help India's malaria elimination efforts.

Seroprevalence and risk factors of brucellosis in livestock in the wildlife and livestock interface area of Similipal Biosphere Reserve, India.

BACKGROUND AND AIM: Brucellosis is an important zoonotic disease that affects fertility in farm animals. The risk factors of brucellosis have not been well studied. This study aimed to understand the seroprevalence and risk factors of brucellosis among livestock in Bangriposi block of Mayurbhanj district in Odisha, a region that borders Similipal wildlife reserve. MATERIALS AND METHODS: Rose Bengal plate test (RBPT) was carried out to estimate the seroprevalence of the livestock in this region. Bivariate analysis was carried out to analyze the association between the variables and brucellosis. Binary logistic regression was performed to assess the risk factors associated with brucellosis in the livestock. RESULTS: Based on RBPT, the seroprevalence of brucellosis among cattle and goats was estimated to be 1.1% and 11.2%, respectively. Binary logistic regression analysis indicates that study area, age, goats, animals with a history of abortion, and rearing practices were the major risk factors in this region. CONCLUSION: This is one of the first studies in India to shed light on risk factors of brucellosis, an important neglected disease that affects the health of animals and humans and nation's economy.

Epidemiology of malaria and chloroquine resistance in Mizoram, northeastern India, a malaria-endemic region bordering Myanmar.

BACKGROUND: Mizoram, a northeastern state in India, shares international borders with Myanmar and Bangladesh and is considered to be one of the key routes through which drug-resistant parasites of Southeast Asia enter mainland India. Despite its strategic location and importance, malaria epidemiology and molecular status of chloroquine resistance had not been well documented, and since chloroquine (CQ), as the first-line treatment in Plasmodium falciparum infection was discontinued since 2008, it was expected that CQ-sensitive haplotype would be more abundant. METHODS: Malaria epidemiology data for the period 2010 to 2018 was collected from the office of State Vector Disease Control Programme. Plasmodium falciparum-positive blood samples were collected from government district hospitals, community health centres, primary health centres, sub-centres, and diagnostic centres from six malaria-prone districts. The samples were processed and analysed using genes-P. falciparum chloroquine-resistant transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) via sequencing of PCR amplicon from 2015 to 2017. RESULTS: Malaria occurred throughout the year and P. falciparum accounted for > 89% of total malaria cases. During 2010-2018, the highest number of malaria incidence was recorded in Lawngtlai (36% of total malaria cases; average API2010-2018 of 34.8) while Champhai remained consistently low (0.4%; average API2010-2018 of 0.04). Males of ≥ 15 years old contributed maximum (35.7%) among gender and age malarial distribution recorded during 2014-2018. Death due to malaria gradually decreased over the years. A higher abundance of mutated pfcrt (58.5% of the total sample analysed) and a lower prevalence of mutated pfmdr1 (48.7%) were observed. All mutations identified for pfcrt belong to the Southeast Asian CVIET haplotype. Only a single point mutation was observed at 86 (N → Y) position in pfmdr1 (48.7%). The key N86Y mutation in pfmdr1 that had been shown to modulate CQR was found in 67.1% of the samples positive for the CVIET haplotype. CONCLUSIONS: This is the first report that details malaria epidemiology and also the molecular status of CQ-resistance in P. falciparum population of the region. The efforts of the State Vector Borne Disease Control Programme have proved to be quite effective in controlling the malaria burden in the state. Despite the discontinuation of CQ for a decade, local P. falciparum is observed with decreased CQ-sensitive haplotype. It is believed that the present findings will form a basis for further studies on genetic diversity in P. falciparum, which could confer better understanding of the complexity of the disease in Southeast Asia.

Malaria in North-East India: Importance and Implications in the Era of Elimination.

Worldwide and in India, malaria elimination efforts are being ramped up to eradicate the disease by 2030. Malaria elimination efforts in North-East (NE) India will have a great bearing on the overall efforts to eradicate malaria in the rest of India. The first cases of chloroquine and sulfadoxine-pyrimethamine resistance were reported in NE India, and the source of these drug resistant parasites are most likely from South East Asia (SEA). NE India is the only land route through which the parasites from SEA can enter the Indian mainland. India's malaria drug policy had to be constantly updated due to the emergence of drug resistant parasites in NE India. Malaria is highly endemic in many parts of NE India, and Plasmodium falciparum is responsible for the majority of the cases. Highly efficient primary vectors and emerging secondary vectors complicate malaria elimination efforts in NE India. Many of the high transmission zones in NE India are tribal belts, and are difficult to access. The review details the malaria epidemiology in seven NE Indian states from 2008 to 2018. In addition, the origin and evolution of resistance to major anti-malarials are discussed. Furthermore, the bionomics of primary vectors and emergence of secondary malaria vectors, and possible strategies to prevent and control malaria in NE are outlined.

Diverse target gene modifications in Plasmodium falciparum using Bxb1 integrase and an intronic attB.

Genetic manipulation of the human malaria parasite Plasmodium falciparum is needed to explore pathogen biology and evaluate antimalarial targets. It is, however, aggravated by a low transfection efficiency, a paucity of selectable markers and a biased A/T-rich genome. While various enabling technologies have been introduced over the past two decades, facile and broad-range modification of essential genes remains challenging. We recently devised a new application of the Bxb1 integrase strategy to meet this need through an intronic attB sequence within the gene of interest. Although this attB is silent and without effect on intron splicing or protein translation and function, it allows efficient gene modification with minimal risk of unwanted changes at other genomic sites. We describe the range of applications for this new method as well as specific cases where it is preferred over CRISPR-Cas9 and other technologies. The advantages and limitations of various strategies for endogenous gene editing are also discussed.

CLAG3 Self-Associates in Malaria Parasites and Quantitatively Determines Nutrient Uptake Channels at the Host Membrane.

Malaria parasites increase host erythrocyte permeability to ions and nutrients via a broad-selectivity channel known as the plasmodial surface anion channel (PSAC), linked to parasite-encoded CLAG3 and two associated proteins. These proteins lack the multiple transmembrane domains typically present in channel-forming proteins, raising doubts about their precise roles. Using the virulent human Plasmodium falciparum parasite, we report that CLAG3 undergoes self-association and that this protein's expression determines channel phenotype quantitatively. We overcame epigenetic silencing of clag3 paralogs and engineered parasites that express two CLAG3 isoforms simultaneously. Stoichiometric expression of these isoforms yielded intermediate channel phenotypes, in agreement with observed trafficking of both proteins to the host membrane. Coimmunoprecipitation and surface labeling revealed formation of CLAG3 oligomers. In vitro selections applied to these transfectant lines yielded distinct mutants with correlated changes in channel activity. These findings support involvement of the identified oligomers in PSAC formation and parasite nutrient acquisition.IMPORTANCE Malaria parasites are globally important pathogens that evade host immunity by replicating within circulating erythrocytes. To facilitate intracellular growth, these parasites increase erythrocyte nutrient uptake through an unusual ion channel. The parasite CLAG3 protein is a key determinant of this channel, but its lack of homology to known ion channels has raised questions about possible mechanisms. Using a new method that allows simultaneous expression of two different CLAG3 proteins, we identify self-association of CLAG3. The two expressed isoforms faithfully traffic to and insert in the host membrane, while remaining associated with two unrelated parasite proteins. Both the channel phenotypes and molecular changes produced upon selections with a highly specific channel inhibitor are consistent with a multiprotein complex that forms the nutrient pore. These studies support direct involvement of the CLAG3 protein in channel formation and are relevant to antimalarial drug discovery projects targeting parasite nutrient acquisition.

Erratum for Gupta et al., "CLAG3 Self-Associates in Malaria Parasites and Quantitatively Determines Nutrient Uptake Channels at the Host Membrane".

[This corrects the article DOI: 10.1128/mBio.02293-17.].

Dynamics of Plasmodium vivax sporogony in wild Anopheles stephensi in a malaria-endemic region of Western India.

BACKGROUND: In global efforts to track mosquito infectivity and parasite elimination, controlled mosquito-feeding experiments can help in understanding the dynamics of parasite development in vectors. Anopheles stephensi is often accepted as the major urban malaria vector that transmits Plasmodium in Goa and elsewhere in South Asia. However, much needs to be learned about the interactions of Plasmodium vivax with An. stephensi. As a component of the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA), a series of membrane-feeding experiments with wild An. stephensi and P. vivax were carried out to better understand this vector-parasite interaction. METHODS: Wild An. stephensi larvae and pupae were collected from curing water in construction sites in the city of Ponda, Goa, India. The larvae and pupae were reared at the MESA ICEMR insectary within the National Institute of Malaria Research (NIMR) field unit in Goa until they emerged into adult mosquitoes. Blood for membrane-feeding experiments was obtained from malaria patients at the local Goa Medical College and Hospital who volunteered for the study. Parasites were counted by Miller reticule technique and correlation between gametocytaemia/parasitaemia and successful mosquito infection was studied. RESULTS: A weak but significant correlation was found between patient blood gametocytaemia/parasitaemia and mosquito oocyst load. No correlation was observed between gametocytaemia/parasitaemia and oocyst infection rates, and between gametocyte sex ratio and oocyst load. When it came to development of the parasite in the mosquito, a strong positive correlation was observed between oocyst midgut levels and sporozoite infection rates, and between oocyst levels and salivary gland sporozoite loads. Kinetic studies showed that sporozoites appeared in the salivary gland as early as day 7, post-infection. CONCLUSIONS: This is the first study in India to carry out membrane-feeding experiments with wild An. stephensi and P. vivax. A wide range of mosquito infection loads and infection rates were observed, pointing to a strong interplay between parasite, vector and human factors. Most of the present observations are in agreement with feeding experiments conducted with P. vivax elsewhere in the world.

Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila.

The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o) sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a of the F(o) sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics.