Serosurveillance of dengue infection and correlation with mosquito pools for dengue virus positivity during the COVID-19 pandemic in Tamil Nadu, India - A state-wide cross-sectional cluster randomized community-based study.

Background: Dengue is a vector-borne viral disease impacting millions across the globe. Nevertheless, akin to many other diseases, reports indicated a decline in dengue incidence and seroprevalence during the COVID-19 pandemic (2020-22). This presumably could be attributed to reduced treatment-seeking rates, under-reporting, misdiagnosis, disrupted health services and reduced exposure to vectors due to lockdowns. Scientific evidence on dengue virus (DENV) disease during the COVID-19 pandemic is limited globally. Methods: A cross-sectional, randomized cluster sampling community-based survey was carried out to assess anti-dengue IgM and IgG and SARS-CoV-2 IgG seroprevalence across all 38 districts of Tamil Nadu, India. The prevalence of DENV in the Aedes mosquito pools during 2021 was analyzed and compared with previous and following years of vector surveillance for DENV by real-time PCR. Findings: Results implicate that both DENV-IgM and IgG seroprevalence and mosquito viral positivity were reduced across all the districts. A total of 13464 mosquito pools and 5577 human serum samples from 186 clusters were collected. Of these, 3·76% of mosquito pools were positive for DENV. In the human sera, 4·12% were positive for DENV IgM and 6·4% were positive for DENV IgG. The anti-SARS-CoV-2 antibody titres correlated with dengue seropositivity with a significant association whereas vaccination status significantly correlated with dengue IgM levels. Interpretation: Continuous monitoring of DENV seroprevalence, especially with the evolving variants of the SARS-CoV-2 virus and surge in COVID-19 cases will shed light on the transmission and therapeutic attributes of dengue infection.

The Impact, Emerging Needs, and New Research Questions Arising from 12 Years of the Center for the Study of Complex Malaria in India.

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

Learnings from two independent malaria elimination demonstration projects in India.

PROBLEM: India and sub-Saharan Africa contributes about 85% of the global malaria burden, and India is committed to eliminating malaria by 2030. APPROACH: Two novel initiatives-the Malaria Elimination Demonstration Project (MEDP) in Madhya Pradesh and Durgama Anchalare Malaria Nirakaran (DAMaN) in Odisha-were initiated independently to demonstrate that indigenous malaria can be eliminated in a short period of time. LOCAL SETTING: These initiatives focused on rural, tribal areas where there is a high malaria burden and complex epidemiology. RELEVANT CHANGES: The case management and vector control strategies used in these programmes were based on the national guidelines, with context-specific changes and introduction of accountability at management, operational, technical and financial levels. The MEDP achieved a 91% reduction in malaria cases and recorded zero transmission for 6 consecutive and a total of 9 mo. The DAMaN project brought about an 88% reduction in malaria cases. LESSONS LEARNED: Malaria elimination will require robust surveillance and case management, monitoring of vector control interventions, community-centric information education communication and behaviour change communication initiatives and management controls, as well as regular internal and external reviews.

A model for malaria elimination based on learnings from the Malaria Elimination Demonstration Project, Mandla district, Madhya Pradesh.

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.

Increase of Plasma TNF-α Is Associated with Decreased Levels of Blood Platelets in Clinical Dengue Infection.

Dengue virus (DENV) infection has become an increasingly common concern in tropical and subtropical regions. It has protean manifestations ranging from febrile phase to severe life-threatening illness. In this study, we estimated Th1 and Th2 cytokines and correlated the levels with dengue severity along with certain hematological and biochemical parameters. We also studied the seroprevalence of dengue between October and December 2017 at the Government Theni Medical College, India. Individuals with dengue fever (DF) were positive for either IgM or IgG, or both. The biochemical and hematological parameters along with plasma tumor necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), granulocyte monocyte-colony stimulating factor (GM-CSF), interleukin (IL)-13, IL-12p70, IL-10, IL-5, IL-4, and IL-2 cytokines were estimated. The prevalence of DF was 42.9% during the study period. IL-2, TNF-α, IL-4, and IL-10 levels were significantly elevated (p < 0.005) in patients with secondary DENV infection, whereas the level of IL-13 remained unaltered during both primary and secondary infections. No statistically significant difference was noticed with IL-12p70, IL-5, IFN-γ, and GM-CSF between the healthy controls and the primary and secondary DENV-infected groups. Increase of 1 unit of TNF-α was associated with a decrease of 160 units of blood platelets. Together, the study suggests that TNF-α could play a key role in the pathogenesis of dengue, and despite the decrease in platelet levels, it remains to be seen whether any other inflammatory cells regulate the levels of TNF-α in DENV infection.

Microsatellite analysis of chloroquine resistance associated alleles and neutral loci reveal genetic structure of Indian Plasmodium falciparum.

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

Population cytogenetic and molecular evidence for existence of a new species in Anopheles fluviatilis complex (Diptera: Culicidae).

Anopheles fluviatilis James, an important malaria vector in the Oriental region has been established as a complex of at least three cryptic species which vary in their biological characteristics and malaria transmission potential. The sibling species S, T and U of Fluviatilis Complex can be identified by examination of species-specific fixed inversions in the polytene chromosomes and can also be differentiated by an allele-specific PCR assay based on differences in the D3 region of 28S ribosomal DNA (rDNA) of these species. Here we report a new An. fluviatilis population from villages under Laksar Community Health Centre, District Haridwar (Uttarakhand state), India which differs from the three sibling species of Fluviatilis Complex by two fixed paracentric inversions, s(1) and S in polytene chromosome arms 2 and 3 respectively. Longitudinal study carried out in study villages showed that the new cytotype was sympatric with species T and U in all the collections and no inversion heterozygotes were observed between them. Thus presence of two fixed paracentric inversions in polytene chromosomes with total absence of inversion heterozygotes demonstrates reproductive isolation which unequivocally establishes this cytological variant as a new species, provisionally designated as species V in the Fluviatilis Complex. Analysis of DNA sequences of D3 domain of 28S rDNA and ITS 2 region has also shown that species V is distinctly different from species S, T and U. With the discovery of new species in the Fluviatilis Complex, in-depth studies are required to know its distribution pattern and biological characteristics and to ascertain its role in malaria transmission.

Changing epidemiology of dengue in South-East Asia.

The burden of dengue and its potential threat to global health are now globally recognized, with 2.5 billion people at risk worldwide. The pathogenesis of severe dengue is particularly intriguing with the involvement of different immune factors. Also, the epidemiology of dengue in South-East Asia is undergoing a change in the human host, the dengue virus and the vector bionomics. Shift in affected age groups, sex differences and expansion to rural areas are evident, while the virulence and genotype of the virus determine the severity and time interval between sequential infections. The Aedes mosquito, a potent and adaptive vector, has evolved in longevity and survival, affected by seasonality and climate variability, socio-cultural and economic factors of human habitation and development. This review provides insights into the changing epidemiology and its factors in South-East Asia, one of the most important epicentres of dengue in the world, highlighting the major factors influencing these rapid changes. Addressing the changes may help mitigate the challenges in the current dengue control and prevention efforts.

Vector control interventions for visceral leishmaniasis elimination initiative in South Asia, 2005-2010.

The Visceral Leishmaniasis (VL) Elimination Initiative in the Indian subcontinent was launched in 2005 as a joint effort between the governments in the Region (India, Nepal and Bangladesh) and the World Health Organization (WHO). The objective is to reduce the annual VL incidence below 1/10,000 inhabitants by 2015 based on detection and treatment of VL cases and vector control. We present here a review of studies published in the period 2005-2010 on the efficacy of different tools to control Phlebotomus argentipes. The review indicates that the current indoor residual spraying (IRS) and novel vector control methods mainly insecticide treated nets (ITN) have low effectiveness for several reasons. Efforts to improve quality of IRS operations and further research on alternative and integrated vector control methods need to be promoted to reach the VL elimination target by 2015.

The malaria parasite Plasmodium vivax exhibits greater genetic diversity than Plasmodium falciparum.

We sequenced and annotated the genomes of four P. vivax strains collected from disparate geographic locations, tripling the number of genome sequences available for this understudied parasite and providing the first genome-wide perspective of global variability in this species. We observe approximately twice as much SNP diversity among these isolates as we do among a comparable collection of isolates of P. falciparum, a malaria-causing parasite that results in higher mortality. This indicates a distinct history of global colonization and/or a more stable demographic history for P. vivax relative to P. falciparum, which is thought to have undergone a recent population bottleneck. The SNP diversity, as well as additional microsatellite and gene family variability, suggests a capacity for greater functional variation in the global population of P. vivax. These findings warrant a deeper survey of variation in P. vivax to equip disease interventions targeting the distinctive biology of this neglected but major pathogen.

Effectiveness and durability of Interceptor® long-lasting insecticidal nets in a malaria endemic area of central India.

BACKGROUND: In the present study, Interceptor®, long-lasting polyester net, 75 denier and bursting strength of minimum 250 kPa coated with alpha-cypermethrin @ 200 mg/m² was evaluated for its efficacy in reducing the mosquito density, blood feeding inhibition and malaria incidence in a tribal dominated malaria endemic area in Chhattisgarh state, central India. Its durability, washing practices and usage pattern by the community was also assessed up to a period of three years. METHODS: The study was carried out in two phases. In the first phase (September 2006 to August 2007), 16 malaria endemic villages in district Kanker were randomized into three groups, viz. Interceptor net (LN), untreated polyester net (100 denier) and without net. Malaria cases were detected by undertaking fortnightly surveillance by home visits and treated as per the national drug policy. Mosquito collections were made by hand catch and pyrethrum space spray methods from human dwellings once every month. Slide positivity rate (SPR) and malaria incidence per 1000 population (PI) were compared between the three study arms to assess the impact of use of Interceptor nets. Simultaneously, wash resistance studies were carried out in the laboratory by doing cone bioassays on Interceptor LNs washed up to 20 times. Activities undertaken in second Phase (April 2008 to October 2009) after an interval of about 18 months post-net distribution included questionnaire based surveys at every six months, i.e. 18, 24, 30 and 36 months to observe durability, usage pattern of LNs and washing practices by the community. After 36 months of field use, 30 nets were retrieved and sampled destructively for chemical analysis. RESULTS: Interceptor nets were found effective in reducing the density, parity rate and blood feeding success rate of main malaria vector Anopheles culicifacies as compared to that in untreated net and no net villages. SPR in LN villages was 3.7% as compared to 6.5% in untreated and 11% in no net villages. PI in LN villages was 16.4 in comparison to 24.8 and 44.2 in untreated polyester net and no net villages respectively. In surveys carried out after three years of initial distribution, 78.7% (737/936) nets were still in possession with the households, of which 68% were used every night. An. culicifacies mortality was >80% in cone bioassays done on LNs washed up to 20 times in laboratory. Mean alpha-cypermethrin content was 43.5 ± 31.7 mg/m² on Interceptor LNs withdrawn after three years of household use against the baseline specification of 200 mg/m². A gradual increase in the proportion of holed nets was observed with the increased period of usage. CONCLUSION: Interceptor nets were highly effective in reducing vector densities as well as malaria incidence in the study villages. Availability of 78% nets with the households in usable condition clearly indicated durability of Interceptor LNs up to three years in the rural setting of India. The nets were found to contain an effective concentration of alpha-cypermethrin against malaria vector after three years of household use.

Mosquito biting activity on humans & detection of Plasmodium falciparum infection in Anopheles stephensi in Goa, India.

BACKGROUND & OBJECTIVES: Knowledge of the bionomics of mosquitoes, especially of disease vectors, is essential to plan appropriate vector avoidance and control strategies. Information on biting activity of vectors during the night hours in different seasons is important for choosing personal protection measures. This study was carried out to find out the composition of mosquito fauna biting on humans and seasonal biting trends in Goa, India. METHODS: Biting activities of all mosquitoes including vectors were studied from 1800 to 0600 h during 85 nights using human volunteers in 14 different localities of three distinct ecotypes in Goa. Seasonal biting trends of vector species were analysed and compared. Seasonal biting periodicity during different phases of night was also studied using William's mean. RESULTS: A total of 4,191 mosquitoes of five genera and 23 species were collected. Ten species belonged to Anopheles, eight to Culex, three to Aedes and one each to Mansonia and Armigeres. Eleven vector species had human hosts, including malaria vectors Anopheles stephensi (1.3%), An. fluviatilis (1.8%), and An. culicifacies (0.76%); filariasis vectors Culex quinquefasciatus (40.8%) and Mansonia uniformis (1.8%); Japanese encephalitis vectors Cx. tritaeniorhynchus (17.4%), Cx. vishnui (7.7%), Cx. pseudovishnui (0.1%), and Cx. gelidus (2.4%); and dengue and chikungunya vectors Aedes albopictus (0.9%) and Ae. aegypti (0.6%). Two An. stephensi of the total 831 female anophelines, were found positive for P. falciparum sporozoites. The entomological inoculation rate (EIR) of P. falciparum was 18.1 and 2.35 for Panaji city and Goa, respectively. INTERPRETATION & CONCLUSIONS: Most of the mosquito vector species were collected in all seasons and throughout the scotophase. Biting rates of different vector species differed during different phases of night and seasons. Personal protection methods could be used to stop vector-host contact.

Artesunate-amodiaquine fixed dose combination for the treatment of Plasmodium falciparum malaria in India.

BACKGROUND: Artemisinin-based combination therapy (ACT) has been recommended for the treatment of falciparum malaria by the World Health Organization. Though India has already switched to ACT for treating falciparum malaria, there is need to have multiple options of alternative forms of ACT. A randomized trial was conducted to assess the safety and efficacy of the fixed dose combination of artesunate-amodiaquine (ASAQ) and amodiaquine (AQ) for the treatment of uncomplicated falciparum malaria for the first time in India. The study sites are located in malaria-endemic, chloroquine-resistant areas. METHODS: This was an open label, randomized trial conducted at two sites in India from January 2007 to January 2008. Patients between six months and 60 years of age having Plasmodium falciparum mono-infection were randomly allocated to ASAQ and AQ arms. The primary endpoint was 28-day PCR-corrected parasitological cure rate. RESULTS: Three hundred patients were enrolled at two participating centres, Ranchi, Jharkhand and Rourkela, Odisha. Two patients in AQ arm had early treatment failure while there was no early treatment failure in ASAQ arm. Late treatment failures were seen in 13 and 12 patients in ASAQ and AQ arms, respectively. The PCR-corrected cure rates in intent-to-treat population were 97.51% (94.6-99.1%) in ASAQ and 88.65% (81.3-93.9%) in AQ arms. In per-protocol population, they were 97.47% (94.2-99.2%) and 88.30% (80-94%) in ASAQ and AQ arms respectively. Seven serious adverse events (SAEs) were reported in five patients, of which two were reported as related to the treatment. All SAEs resolved without sequel. CONCLUSION: The fixed dose combination of ASAQ was found to be efficacious and safe treatment for P. falciparum malaria. Amodiaquine also showed acceptable efficacy, making it a suitable partner of artesunate. The combination could prove to be a viable option in case India opts for fixed dose combination ACT. CLINICAL TRIAL REGISTRY: ISRCTN84408319.

Mutant pfcrt "SVMNT" haplotype and wild type pfmdr1 "N86" are endemic in Plasmodium vivax dominated areas of India under high chloroquine exposure.

BACKGROUND: Chloroquine resistance (CQR) phenotype in Plasmodium falciparum is associated with mutations in pfcrt and pfmdr-1 genes. Mutations at amino acid position 72-76 of pfcrt gene, here defined as pfcrt haplotype are associated with the geographic origin of chloroquine resistant parasite. Here, mutations at 72-76 and codon 220 of pfcrt gene and N86Y pfmdr-1 mutation were studied in blood samples collected across 11 field sites, inclusive of high and low P. falciparum prevalent areas in India. Any probable correlation between these mutations and clinical outcome of CQ treatment was also investigated. METHODS: Finger pricked blood spotted on Whatman No.3 papers were collected from falciparum malaria patients of high and low P. falciparum prevalent areas. For pfcrt haplotype investigation, the parasite DNA was extracted from blood samples and used for PCR amplification, followed by partial sequencing of the pfcrt gene. For pfmdr-1 N86Y mutation, the PCR product was subjected to restriction digestion with AflIII endonuclease enzyme. RESULTS: In 240 P. falciparum isolates with reported in vivo CQ therapeutic efficacy, the analysis of mutations in pfcrt gene shows that mutant SVMNT-S (67.50%) and CVIET-S (23.75%) occurred irrespective of clinical outcome and wild type CVMNK-A (7.91%) occurred only in adequate clinical and parasitological response samples. Of 287 P. falciparum isolates, SVMNTS 192 (66.89%) prevailed in all study sites and showed almost monomorphic existence (98.42% isolates) in low P. falciparum prevalent areas. However, CVIETS-S (19.51%) and CVMNK-A (11.84%) occurrence was limited to high P. falciparum prevalent areas. Investigation of pfmdr-1 N86Y mutation shows no correlation with clinical outcomes. The wild type N86 was prevalent in all the low P. falciparum prevalent areas (94.48%). However, mutant N86Y was comparably higher in numbers at the high P. falciparum prevalent areas (42.76%). CONCLUSIONS: The wild type pfcrt gene is linked to chloroquine sensitivity; however, presence of mutation cannot explain the therapeutic efficacy of CQ in the current scenario of chloroquine resistance. The monomorphic existence of mutant SVMNT haplotype, infer inbreeding and faster spread of CQR parasite in areas with higher P. vivax prevalance and chloroquine exposure, whereas, diversity is maintained in pfcrt gene at high P. falciparum prevalent areas.

Plasmodium vivax lineages: geographical distribution, tandem repeat polymorphism, and phylogenetic relationship.

BACKGROUND: Multi-drug resistance and severe/complicated cases are the emerging phenotypes of vivax malaria, which may deteriorate current anti-malarial control measures. The emergence of these phenotypes could be associated with either of the two Plasmodium vivax lineages. The two lineages had been categorized as Old World and New World, based on geographical sub-division and genetic and phenotypical markers. This study revisited the lineage hypothesis of P. vivax by typing the distribution of lineages among global isolates and evaluated their genetic relatedness using a panel of new mini-satellite markers. METHODS: 18S SSU rRNA S-type gene was amplified from 420 Plasmodium vivax field isolates collected from different geographical regions of India, Thailand and Colombia as well as four strains each of P. vivax originating from Nicaragua, Panama, Thailand (Pak Chang), and Vietnam (ONG). A mini-satellite marker panel was then developed to understand the population genetic parameters and tested on a sample subset of both lineages. RESULTS: 18S SSU rRNA S-type gene typing revealed the distribution of both lineages (Old World and New World) in all geographical regions. However, distribution of Plasmodium vivax lineages was highly variable in every geographical region. The lack of geographical sub-division between lineages suggests that both lineages are globally distributed. Ten mini-satellites were scanned from the P. vivax genome sequence; these tandem repeats were located in eight of the chromosomes. Mini-satellites revealed substantial allelic diversity (7-21, AE = 14.6 ± 2.0) and heterozygosity (He = 0.697-0.924, AE = 0.857 ± 0.033) per locus. Mini-satellite comparison between the two lineages revealed high but similar pattern of genetic diversity, allele frequency, and high degree of allele sharing. A Neighbour-Joining phylogenetic tree derived from genetic distance data obtained from ten mini-satellites also placed both lineages together in every cluster. CONCLUSIONS: The global lineage distribution, lack of genetic distance, similar pattern of genetic diversity, and allele sharing strongly suggested that both lineages are a single species and thus new emerging phenotypes associated with vivax malaria could not be clearly classified as belonging to a particular lineage on basis of their geographical origin.

Comparative efficacy of two poeciliid fish in indoor cement tanks against chikungunya vector Aedes aegypti in villages in Karnataka, India.

BACKGROUND: In 2006, severe outbreaks of Aedes aegypti-transmitted chikungunya occurred in villages in Karnataka, South India. We evaluated the effectiveness of combined information, education and communication (IEC) campaigns using two potential poeciliid larvivorous fish guppy (Poecilia reticulata) and mosquitofish (Gambusia affinis), in indoor cement tanks for Aedes larval control. METHODS: Trials were conducted in two villages (Domatmari and Srinivaspura) in Tumkur District from March to May 2006 for Poecilia and one village (Balmanda) in Kolar District from July to October 2006 for Gambusia. A survey on knowledge, attitude and practice (KAP) on chikungunya was initially conducted and IEC campaigns were performed before and after fish release in Domatmari (IEC alone, followed by IEC + Poecilia) and Balmanda (IEC + Gambusia). In Srinivaspura, IEC was not conducted. Larval surveys were conducted at the baseline followed by one-week and one-month post-intervention periods. The impact of fish on Aedes larvae and disease was assessed based on baseline and post-intervention observations. RESULTS: Only 18% of respondents knew of the role of mosquitoes in fever outbreaks, while almost all (n = 50 each) gained new knowledge from the IEC campaigns. In Domatmari, IEC alone was not effective (OR 0.54; p = 0.067). Indoor cement tanks were the most preferred Ae. aegypti breeding habitat (86.9%), and had a significant impact on Aedes breeding (Breteau Index) in all villages in the one-week period (p < 0.001). In the one-month period, the impact was most sustained in Domatmari (OR 1.58, p < 0.001) then Srinivaspura (OR 0.45, p = 0.063) and Balmanda (OR 0.51, p = 0.067). After fish introductions, chikungunya cases were reduced by 99.87% in Domatmari, 65.48% in Srinivaspura and 68.51% in Balmanda. CONCLUSIONS: Poecilia exhibited greater survival rates than Gambusia (86.04 vs.16.03%) in cement tanks. Neither IEC nor Poecilia alone was effective against Aedes (p > 0.05). We conclude that Poecilia + IEC is an effective intervention strategy. The operational cost was 0.50 (US$ 0.011, 1 US$= 47) per capita per application. Proper water storage practices, focused IEC with Poecilia introductions and vector sanitation involving the local administration and community, is suggested as the best strategy for Aedes control.

A proteogenomic analysis of Anopheles gambiae using high-resolution Fourier transform mass spectrometry.

Anopheles gambiae is a major mosquito vector responsible for malaria transmission, whose genome sequence was reported in 2002. Genome annotation is a continuing effort, and many of the approximately 13,000 genes listed in VectorBase for Anopheles gambiae are predictions that have still not been validated by any other method. To identify protein-coding genes of An. gambiae based on its genomic sequence, we carried out a deep proteomic analysis using high-resolution Fourier transform mass spectrometry for both precursor and fragment ions. Based on peptide evidence, we were able to support or correct more than 6000 gene annotations including 80 novel gene structures and about 500 translational start sites. An additional validation by RT-PCR and cDNA sequencing was successfully performed for 105 selected genes. Our proteogenomic analysis led to the identification of 2682 genome search-specific peptides. Numerous cases of encoded proteins were documented in regions annotated as intergenic, introns, or untranslated regions. Using a database created to contain potential splice sites, we also identified 35 novel splice junctions. This is a first report to annotate the An. gambiae genome using high-accuracy mass spectrometry data as a complementary technology for genome annotation.

Andrographolide: A Novel Antimalarial Diterpene Lactone Compound from Andrographis paniculata and Its Interaction with Curcumin and Artesunate.

Andrographolide (AND), the diterpene lactone compound, was purified by HPLC from the methanolic fraction of the plant Andrographis paniculata. The compound was found to have potent antiplasmodial activity when tested in isolation and in combination with curcumin and artesunate against the erythrocytic stages of Plasmodium falciparum in vitro and Plasmodium berghei ANKA in vivo. IC(50)s for artesunate (AS), andrographolide (AND), and curcumin (CUR) were found to be 0.05, 9.1 and 17.4 µM, respectively. The compound (AND) was found synergistic with curcumin (CUR) and addictively interactive with artesunate (AS). In vivo, andrographolide-curcumin exhibited better antimalarial activity, not only by reducing parasitemia (29%), compared to the control (81%), but also by extending the life span by 2-3 folds. Being nontoxic to the in vivo system this agent can be used as template molecule for designing new derivatives with improved antimalarial properties.

Status of pesticide management in the practice of vector control: a global survey in countries at risk of malaria or other major vector-borne diseases.

BACKGROUND: It is critical that vector control pesticides are used for their acceptable purpose without causing adverse effects on health and the environment. This paper provides a global overview of the current status of pesticides management in the practice of vector control. METHODS: A questionnaire was distributed to WHO member states and completed either by the director of the vector-borne disease control programme or by the national manager for vector control. In all, 113 countries responded to the questionnaire (80% response rate), representing 94% of the total population of the countries targeted. RESULTS: Major gaps were evident in countries in pesticide procurement practices, training on vector control decision making, certification and quality control of pesticide application, monitoring of worker safety, public awareness programmes, and safe disposal of pesticide-related waste. Nevertheless, basic conditions of policy and coordination have been established in many countries through which the management of vector control pesticides could potentially be improved. Most countries responded that they have adopted relevant recommendations by the WHO. CONCLUSIONS: Given the deficiencies identified in this first global survey on public health pesticide management and the recent rise in pesticide use for malaria control, the effectiveness and safety of pesticide use are being compromised. This highlights the urgent need for countries to strengthen their capacity on pesticide management and evidence-based decision making within the context of an integrated vector management approach.

CXCL4 and CXCL10 predict risk of fatal cerebral malaria.

Plasmodium falciparum in a subset of patients can lead to a diffuse encephalopathy known as cerebral malaria (CM). Despite treatment, mortality caused by CM can be as high as 30% while 10% of survivors of the disease may experience short- and long-term neurological complications. The pathogenesis of CM involves alterations in cytokine and chemokine expression, local inflammation, vascular injury and repair processes. These diverse factors have limited the rate of discovery of prognostic predictors of fatal CM. Identification of reliable early predictors of CM severity will enable clinicians to adjust this risk with appropriate management of CM. Recent studies revealed that elevated levels of CXCL10 expression in cerebrospinal fluid and peripheral blood plasma independently predicted severe and fatal CM. CXCR3, a promiscuous receptor of CXCL10, plays an important role in pathogenesis of mouse model of CM. In this study the role of corresponding CXCR3 ligands (CXCL11, CXCL10, CXCL9 & CXCL4) in fatal or severe CM was evaluated by comparing their levels in 16 healthy control (HC), 26 mild malaria (MM), 26 cerebral malaria survivors (CMS) and 12 non-survivors (CMNS) using enzyme linked immunosorbent assay (ELISA). Levels of CXCL4 and CXCL10 were significantly elevated in CMNS patients (p < 0.05) when compared with HC, MM and CMS. Elevated plasma levels of CXCL10 and CXCL4 were tightly associated with CM mortality. Receiver Operating Characteristic (ROC) curve analysis revealed that CXCL4 and CXCL10 can discriminate CMNS from MM (p < 0.0001) and CMS (p <0.0001) with an area under the curve (AUC)=1. These results suggest that CXCL4 and CXCL10 play a prominent role in pathogenesis of CM associated death and may be used as functional or surrogate biomarkers for predicting CM severity.