Malaria vector sibling species distribution in different endemic districts of Punjab, India.

BACKGROUND & OBJECTIVES: Malaria transmission in Punjab, India is mainly seasonal with variation in its endemicity that may be due to varying vector behaviour in different areas of the state, primarily attributed to the existence of sibling species complexes among the vector species. So far there is no report regarding the existence of malaria vectors sibling species in the state of Punjab, therefore, the present study was planned to investigate the status of sibling species of two main vectors of malaria viz. Anopheles culcifacies and Anopheles fluviatilis in different districts of Punjab. METHODS: Mosquito collections were made through hand catch in the morning hours. Malaria vector species An. culicifacies and An. fluviatilis were morphologically identified and man hour density was calculated. Both the vector species were subjected to molecular assays for sibling species identification through amplification of D3 domain of 28S ribosomal DNA by allele-specific PCR. RESULTS: Four sibling species of An. culicifacies, were identified viz. A, B, C and E. Species A was identified from Bhatinda district, species B, C and E from. S.A.S. Nagar and species C from Hoshiarpur. Two sibling species S and T of An. fluviatilis were identified from districts S.A.S. Nagar and Rupnagar. INTERPRETATION & CONCLUSION: Presence of four sibling species of An. culicifacies and two sibling species of An. fluviatilis in Punjab necessitates planning of longitudinal studies to ascertain their role in disease transmission so that appropriate interventions may be applied to achieve malaria elimination.

Impact of the malaria comprehensive case management programme in Odisha, India.

BACKGROUND: The Comprehensive Case Management Project (CCMP), was a collaborative implementation research initiative to strengthen malaria early detection and complete treatment in Odisha State, India. METHODS: A two-arm quasi-experimental design was deployed across four districts in Odisha, representing a range of malaria endemicity: Bolangir (low), Dhenkanal (moderate), Angul (high), and Kandhamal (hyper). In each district, a control block received routine malaria control measures, whereas a CCMP block received a range of interventions to intensify surveillance, diagnosis, and case management. Impact was evaluated by difference-in-difference (DID) analysis and interrupted time-series (ITS) analysis of monthly blood examination rate (MBER) and monthly parasite index (MPI) over three phases: phase 1 pre-CCMP (2009-2012) phase 2 CCMP intervention (2013-2015), and phase 3 post-CCMP (2016-2017). RESULTS: During CCMP implementation, adjusting for control blocks, DID and ITS analysis indicated a 25% increase in MBER and a 96% increase in MPI, followed by a -47% decline in MPI post-CCMP, though MBER was maintained. Level changes in MPI between phases 1 and 2 were most marked in Dhenkanal and Angul with increases of 976% and 287%, respectively, but declines in Bolangir (-57%) and Kandhamal (-22%). Between phase 2 and phase 3, despite the MBER remaining relatively constant, substantial decreases in MPI were observed in Dhenkanal (-78%), and Angul (-59%), with a more modest decline in Bolangir (-13%), and an increase in Kandhamal (14%). CONCLUSIONS: Overall, CCMP improved malaria early detection and treatment through the enhancement of the existing network of malaria services which positively impacted case incidence in three districts. In Kandhamal, which is hyperendemic, the impact was not evident. However, in Dhenkanal and Angul, areas of moderate-to-high malaria endemicity, CCMP interventions precipitated a dramatic increase in case detection and a subsequent decline in malaria incidence, particularly in previously difficult-to-reach communities.

Are members of the Anopheles fluviatilis complex conspecific?

Anopheles fluviatilis sensu lato, a primary malaria vector in India, has been identified to be comprised of four cryptic species, provisionally designated as species S, T, U and V. However, Kumar et al. (Mol Ecol Resour, 2013;13:354-61) considered all of the then known three members of this species complex (S, T and U) conspecific. The specific status of species S and T was refuted based on the lack of sufficient barcode gap in mitochondrial-CO1 and the perceived presence of heterozygotes in populations as detected through one of the two species-specific PCR assays employed for the cryptic species identification. The existence of species U was refuted claiming that earlier investigations have already refuted their existence. Here we discuss problems associated with the CO1-based barcode approach for delimitation of cryptic species, the perceived heterozygosity between species S and T based on a species-specific PCR assay, and interpretation of published reports. We demonstrated that fixed differences do exist in the ITS2-rDNA sequence of species S and T with no evidence of heterozygotes in sympatric populations and, that the observed heterozygosity by Kumar et al. in the ITS2-based species diagnostic PCR is due to the high mispriming tendency of the T-specific primer with species S. We infer that mitochondrial DNA-based 'barcoding gap', an arbitrary threshold recommended for species delimitation, alone, is inadequate to delimit the members of An. fluviatilis complex.

Surveillance-based estimation of the malaria disease burden in a low endemic state of Punjab, India, targeted for malaria elimination.

BACKGROUND: The state of Punjab in India qualifies for malaria elimination because the number of cases reported through routine surveillance is in decline. However, surveillance system prevalence mainly provides malaria trends. Therefore, a prospective epidemiological study was designed to estimate the malaria burden in the state. METHODS: District-wise annual parasite incidence (API) was used for identification of three strata, representing high, moderate and low API zones. A total of 0.9 million people from nine districts was under malaria surveillance for 1 y. The weighted estimates of API for the three regions was calculated and combined to give an estimate of API for the total population of the state. RESULTS: Based upon the primary data generated, malaria cases from high, moderate and low malaria-endemic areas were estimated to be 3727, 904 and 106, respectively. Further, the total number of malaria cases in the state was estimated to be 4737 (95% CI 4006 to 5469) cases per annum. CONCLUSION: Actual burden of malaria in the state of Punjab, India, is about seven to eight times higher than that reported by routine surveillance activities. However, the state still qualifies for malaria elimination but needs vigorous efforts to strengthen the active surveillance and reporting system along with implementation of effective control strategies to achieve malaria elimination.

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.

Prevalence of submicroscopic malaria in low transmission state of Punjab: A potential threat to malaria elimination.

BACKGROUND & OBJECTIVES: Submicroscopic malaria infections with low parasite density serve as a silent reservoir for maintaining residual transmission in the population. These infections should be identified and targeted to be eliminated for sustained malaria control. The conventional methods of diagnosis such as light microscopy and rapid diagnostic kits often fail to detect low density infections. Therefore, the more sensitive molecular techniques should be employed to detect low density infections. The objectives of the study was to explore the prevalence of sub-microscopic infections in low transmission areas of Punjab using highly sensitive molecular tool. METHODS: A total of 1114 finger prick blood samples were collected through active surveillance and tested for malaria diagnosis using light microscopy, RDT and PCR. Nested PCR amplification was performed using a pair of Plasmodium genus-specific primers from the 18S rRNA small subunit gene (18S rRNA). The amplified PCR products were analysed using a 2% agarose gel, stained with ethidium bromide and observed under transilluminator. RESULTS: Test positive rate (TPR) by microscopy, RDT and PCR was 4.4, 3.95 and 5.75%, respectively. Microscopy and RDT failed to detect mixed infections whereas 0.26% cases were found to be mixed infection in PCR. Compared to LM and RDT, PCR has detected 1.3% additional positive cases. However, of the total positive cases detected by PCR, 23.4% infections were found to be submicroscopic, which could not be detected by conventional methods of diagnosis. INTERPRETATION & CONCLUSIONS: The molecular study revealed the existence of submicroscopic malaria cases in the study population which would have remained undetected by conventional methods of diagnosis. This is particularly important because Punjab state is in malaria elimination phase and targeted to achieve elimination in 2021. However, such undetected parasite positive cases may pose bigger problem any time due to continued transmission. Therefore, application of more sensitive diagnostic tools like PCR and LAMP with conventional methods may be much more useful in case detection particularly in low transmission settings for malaria elimination.

Cytophilic Antibodies Against Key Plasmodium falciparum Blood Stage Antigens Contribute to Protection Against Clinical Malaria in a High Transmission Region of Eastern India.

Background: The collection of clinical data from a tribal population in a malaria-endemic area of India suggests the occurrence of naturally acquired immunity (NAI) against Plasmodium falciparum malaria. Methods: Quantity and functionality of immunoglobulin G (IgG) antibodies against intact merozoites and recombinant proteins were assessed in a 13-month longitudinal cohort study of 121 individuals, 3-60 years of age. Results: Opsonic phagocytosis of merozoites activity was strongly associated (hazard ratio [HR] = 0.34; 95% confidence interval [CI] = .18-.66; P = .0013) with protection against febrile malaria. Of the different IgG subclasses, only IgG3 antibodies against intact whole merozoites was significantly associated with protection against febrile malaria (HR = 0.47; 95% CI = .26-.86; P = .01). Furthermore, a combination of IgG3 antibody responses against Pf12, MSP3.7, MSP3.3, and MSP2FC27 was strongly associated with protection against febrile malaria (HR = 0.15; 95% CI, .06-.37; P = .0001). Conclusions: These data suggest that NAI may, at least in part, be explained by opsonic phagocytosis of merozoites and IgG3 responses against whole merozoites, and in particular to a combination of 4 antigens is critical in this population. These results may have implications in the development of a subunit malaria vaccine. Opsonic phagocytosis of Plasmodium falciparum merozoites was associated with protection against clinical malaria in an India population. Antibody profiling identified four merozoite antigens (Pf12, MSP3.7, MSP3.3, and MSP2) as targets of protective Immunoglobuline G3 antibodies.

Interferon-γ (IFNG) microsatellite repeat and single nucleotide polymorphism haplotypes of IFN-α receptor (IFNAR1) associated with enhanced malaria susceptibility in Indian populations.

Pro-inflammatory cytokines IFNγ and IFNα function through their cellular receptors IFNγR1 and IFNαR1, respectively to mediate immune processes during malaria infection. A total of 21 SNPs, 2 ins/del polymorphisms and a microsatellite repeat, selected on the basis of their reported association with infectious diseases including malaria in world populations, were analysed for association with Plasmodium falciparum malaria susceptibility in a case-control study with adult patients and ethnically-matched controls drawn from a disease meso- to hyperendemic and a nonendemic region of India. Among the five IFNG SNPs tested, an intron 3 and a 3'UTR SNP associated with disease in the endemic region. In addition, large (CA)n repeats of IFNG intron 1 associated with protection from severe malaria in the endemic region (severe vs. control, odds ratio=0.21, 95% CI=0.08-0.52, P=1.3 × 10(-4)). The TA11CAG haplotype (rs2069705 T/C, rs2430561 A/T, rs3138557 (CA)n, rs2069718 T/C, rs2069727 A/G, rs2069728 G/A) carrying a short CA11 repeat also exhibited very strong association with severe malaria, particularly in the endemic region (severe vs. control, OR=14.56, 95% CI=3.39-85.81, P=3 × 10(-5)). One SNP each from the IFNA8 and IFNA17 of IFNA gene cluster had a protective effect in the non-endemic region but not in the endemic region. A promoter and an intron 2 SNP of IFNAR1 were risk factors for disease and the IFNAR1 haplotype GCCAGG (rs2843710 C/G, rs2850015 C/T, +6993 C/T, rs2243594 A/G, rs1012335 G/C, rs2257167 G/C) carrying both the risk alleles strikingly associated with disease manifestation in the endemic region (severe vs. control, OR=27.14, 95% CI=3.12-1254, P=2 × 10(-5); non-severe vs. control, OR=61.87, 95% CI=10.08-2521, P=1 × 10(-8)). The data indicates dissimilar contribution of cytokine and cytokine receptor variants to disease in populations residing in areas of differential malaria endemicity.

A clinical and molecular study of artesunate + sulphadoxine-pyrimethamine in three districts of central and eastern India.

BACKGROUND: Artesunate + sulphadoxine-pyrimethamine (AS + SP) is recommended throughout India as the first-line treatment for uncomplicated falciparum malaria. Due to the presence of several eco-epidemiological zones of malaria and variable drug pressure, it is necessary to evaluate the efficacy of this combination in different regions of India. The objective of this study was to use clinical and molecular methods to monitor the efficacy of AS + SP in three diverse sites. METHODS: The study was undertaken in three high endemic sites of central and eastern India. Patients with uncomplicated falciparum malaria were enrolled and followed for 28 days. Molecular genotyping was conducted for merozoite surface protein (msp1 and msp2) to differentiate between re-infection and recrudescence and for the dhfr and dhps genes to monitor antifolate drug resistance. RESULTS: In all, 149 patients were enrolled at the three sites. The crude cure rates were 95.9%, 100%, and 100% in Ranchi, Keonjhar, and West Garo Hills respectively. PCR-corrected cure rates were 100% at all sites. In dhfr, 27% of isolates had triple mutations, while 46% isolates were double-mutants. The most prevalent mutation was S108N followed by C59R. 164 L mutation was observed in 43/126 (34%) isolates. In dhps, most (76%) of the isolates were wild-type. Only 2.5% (2/80) isolates showed double mutation. dhfr-dhps two locus mutation were observed in 16% (13/80) isolates. Parasite clearance time was not related with antifolate mutations. CONCLUSIONS: AS + SP combination therapy remained effective against falciparum malaria despite common mutations promoting resistance to antifolate drugs. Although the prevalence of double and triple mutations in dhfr was high, the prevalence of dhfr-dhps two locus mutations were low. Even isolates with dhfr triple and dhfr-dhps two locus mutations achieved adequate clinical and parasitological response.

The impact of artemisinin combination therapy and long-lasting insecticidal nets on forest malaria incidence in tribal villages of India, 2006-2011.

INTRODUCTION: New tools for malaria control, artemisinin combination therapy (ACT) and long-lasting insecticidal nets (LLINs) were recently introduced across India. We estimated the impact of universal coverage of ACT and ACT plus LLINs in a setting of hyperendemic, forest malaria transmission. METHODS: We reviewed data collected through active and passive case detection in a vaccine trial cohort of 2,204 tribal people residing in Sundargarh district, Odisha between 2006 and 2011. We compared measures of transmission at the village and individual level in 2006-2009 versus 2010-2011 after ACT (in all villages) and LLINs (in three villages) were implemented. RESULTS: During 2006-2009 malaria incidence per village ranged from 156-512 per 1000 persons per year and slide prevalence ranged from 28-53%. Routine indoor residual spray did not prevent seasonal peaks of malaria. Post-intervention impact in 2010-2011 was dramatic with ranges of 14-71 per 1000 persons per year and 6-16% respectively. When adjusted for village, ACT alone decreased the incidence of malaria by 83% (IRR 0.17, 95%CI: 0.10, 0.27) and areas using ACT and LLINs decreased the incidence of malaria by 86% (IRR 0.14, 95%CI: 0.05, 0.38). After intervention, the age of malaria cases, their parasite density, and proportion with fever at the time of screening increased. CONCLUSIONS: ACT, and LLINs along with ACT, effectively reduced malaria incidence in a closely monitored population living in a forest ecotype. It is unclear whether LLINs were impactful when prompt and quality antimalarial treatment was available. In spite of universal coverage, substantial malaria burden remained.

Impact of changing over of insecticide from synthetic pyrethroids to DDT for indoor residual spray in a malaria endemic area of Orissa, India.

BACKGROUND & OBJECTIVES: Development of insecticide resistance in malaria vectors has been a major problem for achieving effective vector control. Due to limited availability of insecticides, the only option is management of resistance by judiciously using the insecticides and rotating them to maintain their effectiveness. This study was carried out in a malaria endemic area of Sundergarh district in Orissa where synthetic pyrethroids (SP) were in use for the last couple of years. The change-over from SP to DDT was done in one arm of study, and the other two arms remained on SP and insecticide-treated nets (ITN). Entomological and parasitological monitoring was done to assess the impact. METHODS: The study design comprised of three arms (i) two rounds of indoor residual spraying (IRS) with DDT 1g/m(2) as a change-over insecticide in areas previously under synthetic pyrethroids; (ii) two rounds of IRS with synthetic pyrethroid (alphacypermethrin, ACM) @ 25 mg/m(2) ; and (iii) an unsprayed area under ITN/long lasting insecticide nets (LNs). Indoor residual spraying was undertaken under strict supervision to maintain quality and coverage. Contact bioassays were conducted to know the persistence of insecticide on sprayed surfaces and adult vector density was monitored in fixed and randomly selected houses. Malaria incidence was measured through fortnightly domiciliary surveillance under primary health care system in all the study villages. RESULTS: The insecticide susceptibility tests showed that An.culicifacies was resistant to DDT but susceptible to malathion and ACM. However, An. fluviatilis was susceptible to all the three insecticides. ACM was effective in killing An. culicifacies on mud and wooden sprayed surfaces and maintained effective bioefficacy ranging from 92 to 100 per cent up to five months, whereas DDT failed to achieve effective mortality in An.culicifacies. However, there was significant decline in the density of An.culicifacies in ACM and DDT areas in comparison to ITNs/LNs. There was 61 per cent reduction in the slide positivity rate in ACM area in comparison to 48 and 51 per cent in DDT and ITN/LNs areas, respectively. The adjusted incidence rate of malaria cases per 1000 population in three study areas also showed significant declines within each group. INTERPRETATION & CONCLUSIONS: The present findings show that the change-over of insecticide from synthetic pyrethroids to DDT brings about the same epidemiological impact as envisaged from continuing SP spray or distributing insecticide treated nets/long-lasting insecticidal nets provided there is a good quality spray and house coverage.

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.

Deletion of the APOBEC3B gene strongly impacts susceptibility to falciparum malaria.

APOBEC3B, a gene involved in innate response, exhibits insertion-deletion polymorphism across world populations. We observed the insertion allele to be nearly fixed in malaria endemic regions of sub-Saharan Africa as well as populations with high malaria incidence in the past. This prompted us to investigate the possible association of the polymorphism with falciparum malaria. We studied the distribution of APOBEC3B, in 25 diverse Indian populations comprising of 500 samples and 176 severe or non-severe Plasmodium falciparum patients and 174 ethnically-matched uninfected individuals from a P. falciparum endemic and a non-endemic region of India. The deletion frequencies ranged from 0% to 43% in the Indian populations. The frequency of the insertion allele strikingly correlated with the endemicity map of P. falciparum malaria in India. A strong association of the deletion allele with susceptibility to falciparum malaria in the endemic region (non-severe vs. control, Odds ratio=4.96, P value=9.5E(-06); severe vs. control, OR=4.36, P value=5.76E(-05)) was observed. Although the frequency of deletion allele was higher in the non-endemic region, there was a significant association of the homozygous deletion genotype with malaria (OR=3.17, 95% CI=1.10-10.32, P value=0.0177). Our study also presents a case for malaria as a positive selection force for the APOBEC3B insertion and suggests a major role for this gene in innate immunity against malaria.

Malaria in India: the center for the study of complex malaria in India.

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

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.

Distinct cytokine profiles define clinical immune response to falciparum malaria in regions of high or low disease transmission.

The immune effector response to Plasmodium falciparum infection involves a finely-tuned interplay between different cell types and cytokines. However, the processes by which they mediate the development of clinical immunity, in areas of different endemicity, are poorly understood. We analyzed circulating levels of pro-inflammatory (TNF, IFN-γ, IL-12, IL-16) and anti-inflammatory (IL-4, IL-10, IL-13) cytokines in control and patient groups drawn from a P. falciparum-endemic and a non-endemic region of India. The endemic region control population exhibited a lower pro- to anti-inflammatory cytokine ratio, indicating a shift towards a high basal Th2 response. Levels of IL-10 contributed most towards the region-specific difference in basal cytokine response. IL-10 was also the strongest predictor of disease in the endemic region, while IL-12, along with IL-10 and IL-6, contributed most to disease outcome in the non-endemic region. A low, mean IFN-γ/IL-10 ratio was associated with disease severity in the endemic region (p < 0.0001). In contrast, a low mean IL-12/IL-10 ratio correlated with disease outcome in the non-endemic region (p < 0.0001). In the endemic region, IL-13 correlated negatively with IFN-γ in severe patients (Spearman's ρ: -0.49; p : 0.013), while in the non-endemic region, IL-13 correlated negatively with IL-6 in severe malaria patients (Spearman's ρ: -0.485; p : 0.001). In conclusion, levels of pro- and anti-inflammatory cytokines and the relative balance between the Th1 and Th2 response, illustrates how populations residing in areas of varying disease endemicity may respond to P. falciparum-induced immune challenge.

Wash-resistance and field evaluation of alphacypermethrin treated long-lasting insecticidal net (Interceptor) against malaria vectors Anopheles culicifacies and Anopheles fluviatilis in a tribal area of Orissa, India.

A field trial was conducted on the efficacy of Interceptor nets-a long-lasting insecticidal net (LLN) factory treated with alphacypermethrin 0.667% (w/w) corresponding to 200mg/m(2), against malaria vectors Anopheles culicifacies and Anopheles fluviatilis in one of the highly endemic areas of Orissa. The study area comprised 19 villages which were randomized into three clusters and designated as Interceptor net cluster, untreated net cluster, and no net cluster. Baseline studies showed that both the vector species An. culicifacies and An. fluviatilis were 100% susceptible to alphacypermethrin. Results of wash-resistance and bio-efficacy of Interceptor nets showed 100% mortality in An. culicifacies and An. fluviatilis even after 20 washings. Bioassays on the Interceptor nets while in use in the field conditions showed a knockdown effect on 70-90% mosquitoes during different months of intervention after 3 min of exposure and 100% mortality was recorded after 24h of recovery period. The median knockdown time for these species ranged between 4.10-5.25 min and 4.00-5.00 min respectively during intervention period. In Interceptor net study area, there was a significant reduction of 88.9, 96.3 and 90.6% in the entry rate of An. culicifacies, An. fluviatilis and other anopheline species respectively with an over all reduction of 87.5% in total mosquitoes. The overall feeding success rate of mosquitoes in the trial villages was only 12.8% in comparison to 35.0 and 78.8% in villages with untreated nets and no nets respectively. A significant reduction was also recorded in parity rate and human blood index of vector species in Interceptor net area. The results of the study showed that Interceptor nets are effective against the malaria vectors and may be used as a suitable intervention strategy in high-risk areas.

Defining the relationship between Plasmodium falciparum parasite rate and clinical disease: statistical models for disease burden estimation.

BACKGROUND: Clinical malaria has proven an elusive burden to enumerate. Many cases go undetected by routine disease recording systems. Epidemiologists have, therefore, frequently defaulted to actively measuring malaria in population cohorts through time. Measuring the clinical incidence of malaria longitudinally is labour-intensive and impossible to undertake universally. There is a need, therefore, to define a relationship between clinical incidence and the easier and more commonly measured index of infection prevalence: the "parasite rate". This relationship can help provide an informed basis to define malaria burdens in areas where health statistics are inadequate. METHODS: Formal literature searches were conducted for Plasmodium falciparum malaria incidence surveys undertaken prospectively through active case detection at least every 14 days. The data were abstracted, standardized and geo-referenced. Incidence surveys were time-space matched with modelled estimates of infection prevalence derived from a larger database of parasite prevalence surveys and modelling procedures developed for a global malaria endemicity map. Several potential relationships between clinical incidence and infection prevalence were then specified in a non-parametric Gaussian process model with minimal, biologically informed, prior constraints. Bayesian inference was then used to choose between the candidate models. RESULTS: The suggested relationships with credible intervals are shown for the Africa and a combined America and Central and South East Asia regions. In both regions clinical incidence increased slowly and smoothly as a function of infection prevalence. In Africa, when infection prevalence exceeded 40%, clinical incidence reached a plateau of 500 cases per thousand of the population per annum. In the combined America and Central and South East Asia regions, this plateau was reached at 250 cases per thousand of the population per annum. A temporal volatility model was also incorporated to facilitate a closer description of the variance in the observed data. CONCLUSION: It was possible to model a relationship between clinical incidence and P. falciparum infection prevalence but the best-fit models were very noisy reflecting the large variance within the observed opportunistic data sample. This continuous quantification allows for estimates of the clinical burden of P. falciparum of known confidence from wherever an estimate of P. falciparum prevalence is available.

Efficacy of permethrin treated long-lasting insecticidal nets on malaria transmission and observations on the perceived side effects, collateral benefits and human safety in a hyperendemic tribal area of Orissa, India.

Studies were conducted on the efficacy of Olyset nets-a long-lasting insecticidal net (LLIN) factory treated with 2% (w/w) permethrin on malaria transmission in an area under the influence of pyrethroid susceptible vector species Anopheles culicifacies and A. fluviatilis in Sundargarh District, Orissa, India. The study area comprised 22 villages that were randomized into three clusters and designated as Olyset net, untreated net, and no net area. Malaria incidence in the study population was measured through longitudinal active surveillance at fortnightly intervals. There was a reduction of 65-70% in malaria incidence in Olyset net area as compared to the control areas. The attack rate of Plasmodium falciparum or number of episodes per person per year in different age groups also showed significant reduction in Olyset net area as compared to untreated net and no net areas. Cross-sectional point prevalence surveys showed 45.7% reduction of malaria prevalence in Olyset net users, whereas there was an increase of 33.3% and 51% in untreated net and no net villages respectively. The compliance rate of Olyset net usage in the study population was 80-98% during different months, whereas it was between 70% and 90% for untreated nets. There were minimal complains of skin irritation (4%), itching (8%) and eye irritation (1.2%). However, these effects were only transitory in nature lasting for few hours of the first usage. Olyset nets also provided collateral benefits in terms of relief not only from mosquitoes and malaria but also from other household pests such as head lice, bed bugs, cockroaches, ants and houseflies. The Olyset nets were found to be safe to humans as no adverse event was recorded in the net users that can be attributed to the use of net. The study showed that Olyset nets are effective personal protection tool that can be used in a community based intervention programme.