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BACKGROUND: Anopheles fluviatilis is a species-complex comprising of four cryptic species provisionally designated as species S, T, U and V. Earlier, a 28S-rDNA based allele-specific polymerase chain reaction (ASPCR) assay was developed for the differentiation of the then known three members of the An. fluviatilis complex, i.e., species S, T, and U. This assay was modified in consequence of the discovery of a new cryptic member, species V, in the Fluviatilis Complex to include identification of new species. METHODS: In the modified procedure, the ASPCR assay was performed first, followed by restriction digestion of PCR product with an enzyme BamH I, which cleaves specifically PCR amplicon of species V and the resultant PCR-RFLP products can differentiate all the four cryptic members of the complex. Morphologically identified An. fluviatilis samples were subjected to sibling species identification by modified PCR-based assay and standard cytotaxonomy. The result of PCR-based assay was validated through cytotaxonomy as well as DNA sequencing of some representative samples. RESULTS: The modified PCR-based assay differentiates all four sibling species. The result of modified PCR-based assay tested on field samples was in agreement with results of cytotaxonomy as well as DNA sequencing of representative samples. CONCLUSIONS: The modified PCR-based assay unambiguously differentiates all four known members of the An. fluviatilis species complex. This assay will be useful in studies related to bionomics of members of the Fluviatilis Complex in their role in malaria transmission.
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Anopheles/classificação , Mosquitos Vetores/classificação , Reação em Cadeia da Polimerase/métodos , Animais , Feminino , Malária , Masculino , RNA Ribossômico 28S/análiseRESUMO
India has committed to eliminate malaria by 2030. The national framework for malaria elimination released by the Government of India plans to achieve this goal through strategic planning in a phased manner. Since vector control is a major component of disease management and vector elimination, it requires a thorough understanding of the biology and bionomics of malaria vectors exhibiting definite distribution patterns in diverse ecosystems in the country. Although a wealth of information is available on these aspects, lesser-known data are on biting time and rhythm, and the magnitude of outdoor transmission by the vectors which are crucial for effective implementation of the key vector control interventions. Most of the data available for the vector species are at sensu lato level, while the major vectors are species complexes and their members distinctly differ in biological characters. Furthermore, the persistent use of insecticides in indoor residual spray and long-lasting insecticidal nets has resulted in widespread resistance in vectors and changes in their behaviour. In this document, challenges in vector control in the Indian context have been identified and possible solutions to overcome the problem are suggested. Adequate addressing of the issues raised would greatly help make a deep dent in malaria transmission and consequently result in disease elimination within the targeted time frame.
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Anopheles/fisiologia , Malária/prevenção & controle , Controle de Mosquitos/métodos , Mosquitos Vetores/fisiologia , Animais , Índia , Características de História de VidaRESUMO
BACKGROUND & OBJECTIVES: The interactions between HIV and malaria co-infection have been shown to influence each other in their clinical outcomes in Sub-Saharan Africa. This study was carried out in the two States of north east India endemic for both HIV and malaria infections, to study the interactions between the two diseases in the HIV-infected population. METHODS: In this prospective study, a total of 333 HIV-infected individuals were followed up for a period of 6-18 months in Mizoram and Manipur during 2010-2011. The study assessed the changes in viral load and also the therapeutic efficacy of artesunate plus sulphadoxine-pyrimethamine (AS+SP) combination therapy in HIV-infected and HIV-uninfected individuals with Plasmodium falciparum malaria. RESULTS: Viral load in HIV-infected malaria patients on day zero (D0) ranged from 1110 to 147,000 copies/ml. The log transformation of the geometric means of HIV viral loads revealed no significant difference on different days of follow up. There was 100 per cent adequate clinical and parasitological response (ACPR) after treating with artemisinin based combination therapy (ACT) both in HIV-infected and HIV-uninfected P. falciparum-positive individuals. Similarly, chloroquine showed 100 per cent ACPR in P. vivax HIV-infected individuals. INTERPRETATION & CONCLUSION: The study showed no significant increase in HIV viral load in malaria cases. All HIV-infected and HIV-uninfected P. falciparum malaria-positive cases responded to the treatment with 100 per cent ACPR.
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Coinfecção/epidemiologia , Infecções por HIV/epidemiologia , Malária Falciparum/epidemiologia , Malária Vivax/epidemiologia , Adolescente , Adulto , Antimaláricos/uso terapêutico , Pré-Escolar , Cloroquina/uso terapêutico , Coinfecção/tratamento farmacológico , Coinfecção/parasitologia , Coinfecção/virologia , Resistência a Medicamentos/efeitos dos fármacos , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/parasitologia , Infecções por HIV/virologia , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Malária Falciparum/virologia , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Malária Vivax/virologia , Masculino , Plasmodium falciparum/patogenicidade , Carga Viral/efeitos dos fármacosRESUMO
BACKGROUND: The biological characteristics of mosquito vectors vary, impacting their response to control measures. Thus, having up-to-date information on vector bionomics is essential to maintain the effectiveness of existing control strategies and tools, particularly as India aims for malaria elimination by 2030. OBJECTIVE: This study aims to assess the proportions of vector species resting indoors and outdoors, determine their preference for host biting/feeding, identify transmission sites, and evaluate the susceptibility of vectors to insecticides used in public health programs. METHODS: Mosquito collections were conducted in 13 districts across 8 Indian states from 2017 to 2020 using various methods to estimate their densities. Following morphological identification in the field, sibling species of Anopheles mosquitoes were identified molecularly using polymerase chain reaction (PCR)-specific alleles. Plasmodium falciparum and Plasmodium vivax infections in the vectors were detected using enzyme-linked immunosorbent assay (ELISA) and PCR assays. In addition, we assessed the insecticide susceptibility status of primary malaria vectors following the World Health Organization (WHO) protocol. RESULTS: Anopheles culicifacies, a primary malaria vector, was collected (with a man-hour density ranging from 3.1 to 15.9) from all states of India except those in the northeastern region. Anopheles fluviatilis, another primary vector, was collected from the states of Madhya Pradesh, Maharashtra, Karnataka, and Odisha. In Haryana and Karnataka, An. culicifacies sibling species A predominated, whereas species C and E were predominant in Madhya Pradesh and Maharashtra. An. culicifacies displayed mainly endophilic behavior across all states, except in Madhya Pradesh, where the proportion of semigravid and gravid mosquitoes was nearly half of that of unfed mosquitoes. The human blood index of An. culicifacies ranged from 0.001 to 0.220 across all study sites. The sporozoite rate of An. culicifacies ranged from 0.06 to 4.24, except in Madhya Pradesh, where none of the vector mosquitoes were found to be infected with the Plasmodium parasite. In the study area, An. culicifacies exhibited resistance to DDT (dichlorodiphenyltrichloroethane; with <39% mortality). Moreover, it showed resistance to malathion (with mortality rates ranging from 49% to 78%) in all districts except Angul in Odisha and Palwal in Haryana. In addition, resistance to deltamethrin was observed in districts of Maharashtra, Gujarat, Haryana, and Karnataka. CONCLUSIONS: Our study offers vital insights into the prevalence, resting behavior, and sibling species composition of malaria vectors in India. It is evident from our findings that resistance development in An. culicifacies, the primary vector, to synthetic pyrethroids is on the rise in the country. Furthermore, the results of our study suggest a potential change in the resting behavior of An. culicifacies in Madhya Pradesh, although further studies are required to confirm this shift definitively. These findings are essential for the development of effective vector control strategies in India, aligning with the goal of malaria elimination by 2030.
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Anopheles , Malária , Mosquitos Vetores , Índia/epidemiologia , Animais , Malária/prevenção & controle , Malária/epidemiologia , Anopheles/efeitos dos fármacos , Humanos , Erradicação de Doenças/métodos , Inseticidas , Resistência a Inseticidas , EcologiaRESUMO
A comprehensive entomological survey was undertaken in Alipurduar District, West Bengal, from 2018 to 2020 and in 2022. This study was prompted by reported malaria cases and conducted across nine villages, seven Sub-Centres, and three Primary Health Centres (PHCs). Mosquitoes were hand-collected with aspirators and flashlights from human dwellings and cattle sheds during the daytime. Both morphological and molecular techniques were used for species identification. Additionally, mosquitoes were tested for Plasmodium parasites and human blood presence. Mosquito species such as An. barbirostris s.l., An. hyrcanus s.l., An. splendidus, and An. vagus were morphologically identified. For species like An. annularis s.l., An. minimus s.s., An. culicifacies s.l., and An. maculatus s.s., a combination of morphological and molecular techniques was essential. The mitochondrial cytochrome c oxidase gene subunit 1 (CO1) was sequenced for An. annularis s.l., An. maculatus s.s., An. culicifacies s.l., An. vagus, and some damaged samples, revealing the presence of An. pseudowillmori and An. fluviatilis. The major Anopheles species were An. annularis s.l., An. culicifacies s.l., and An. maculatus s.s., especially in Kumargram and Turturi PHCs. Plasmodium positivity was notably high in An. annularis s.l. and An. maculatus s.s. with significant human blood meal positivity across most species. Morphological, molecular, and phylogenetic analyses are crucial, especially for archived samples, to accurately identify the mosquito fauna of a region. Notably, this study confirms the first occurrence of An. pseudowillmori and An. sawadwongporni in West Bengal and implicates An. maculatus s.s., An. culicifacies s.l., and An. annularis s.l. as significant vectors in the Alipurduar region.
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BACKGROUND: With the reports of indigenous cases of dengue and chikungunya in the forest-covered rural tribal malaria-endemic villages of Dhalai District, Tripura, India, an exploratory study was undertaken to identify the vector breeding sites. METHODS: From June 2021 to August 2022, mosquito larvae were collected from both natural and artificial sources in the villages, house premises, and their nearby forested areas outside of the houses. Other than morphological characterisation, Aedes species were confirmed by polymerase chain reaction targeting both nuclear (ITS2) and mitochondrial genes (COI) followed by bidirectional Sanger sequencing. RESULTS: Aedes albopictus was abundantly found in this area in both natural and artificial containers, whereas Ae. aegypti was absent. Among the breeding sources of molecularly confirmed Ae. albopictus species, rubber collection bowls were found to be a breeding source reported for the first time. Plastic and indigenously made bamboo-polythene containers for storing supply water and harvesting rainwater in the villages with a shortage of water were found to be other major breeding sources, which calls for specific vector control strategies. Natural sources like ponds and rainwater collected on Tectona grandis leaves and Colocasia axil were also found to harbour the breeding, along with other commonly found sources like bamboo stumps and tree holes. No artificial containers as a breeding source were found inside the houses. Mixed breeding was observed in many containers with other Aedes and other mosquito species, necessitating molecular identification. We report six haplotypes in this study, among which two are reported for the first time. However, Aedes aegypti was not found in the area. Additionally, rubber collection bowls, ponds, and water containers also showed the presence of Culex quinquefasciatus and Culex vishnui, known JE vectors from this area, and reported JE cases as well. Different Anopheles vector spp. from this known malaria-endemic area were also found, corroborating this area as a hotbed of several vectors and vector-borne diseases. CONCLUSIONS: This study, for the first time, reports the breeding sources of Aedes albopictus in the forested areas of Tripura, with rubber collection bowls and large water storage containers as major sources. Also, for the first time, this study reports the molecular characterisation of the Ae. albopictus species of Tripura, elucidating the limitations of morphological identification and highlighting the importance of molecular studies for designing appropriate vector control strategies. The study also reports the co-breeding of JE and malaria vectors for the first time in the area reporting these vector-borne diseases.
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Wolbachia, an intracellular maternally transmitted endosymbiont, has been shown to interfere with the replication of dengue virus in Aedes aegypti mosquitoes. The Wolbachia-transinfected Ae. aegypti has been currently released in many countries to test its effectiveness in preventing the transmission of dengue virus. ICMR-Vector Control Research Centre in collaboration with World Mosquito Program Monash University, Australia, has generated two new Wolbachia-introgressed Ae. aegypti Puducherry (Pud) lines via backcrossing Ae. aegypti females of Australian (Aus) strains, infected with wMel and wAlbB Wolbachia with wild-type Ae. aegypti Puducherry (Pud) males. Wolbachia infections are known to induce a fitness cost and confer benefit on the host mosquito populations that will influence spread of the Wolbachia into native wild mosquito populations during the field release. Hence, the induced fitness cost or benefit/advantage in the two newly generated Ae. aegypti (Pud) lines was assessed in the laboratory in comparison with the wild-type Ae. aegypti (Pud) strain. In addition, maternal transmission (MT) efficiency, induced cytoplasmic incompatibility (CI), and insecticide resistance status of the two (Pud) lines were determined to assess the likely frequency of wMel and wAlbB infections in the native wild population after field invasion. The study shows that wMel and wAlbB infections did not induce any fitness cost on the two newly generated (Pud) lines. Rather, in terms of wing length, fecundity, egg hatch rate, and adult survival, the Wolbachia introgression conferred fitness benefits on the (Pud) lines compared to uninfected Wolbachia free wild Ae. aegypti population. wMel and wAlbB exhibited a high maternal transmission (99-100%) and induced nearly complete (98-100%) cytoplasmic incompatibility. Both the (Pud) lines were resistant to deltamethrin, malathion, DDT, and temephos, and the level of resistance was almost the same between the two lines as in the wild type. Overall, the stable association of wMel and wAlbB established with Ae. aegypti and the reproductive advantages of the (Pud) lines encourage a pilot release in the field for population replacement potential.
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BACKGROUND: ICMR-Vector Control Research Centre, Puducherry, India, developed two colonies of Aedes aegypti infected with wMel and wAlbB Wolbacia strains called Ae. aegypti (Pud) lines for dengue control. The sensitivity of wMel and wAlbB strains in Ae. aegypti (Pud) lines to heat stress was studied. METHODS: wMel and wAlbB infected and uninfected Ae. aegypti larvae (first to fourth instars) were reared in the laboratory to adults at 26 °C, 30 °C, 36 °C and 40 °C constant temperatures and also 26-30 °C, 26-36 °C and 26-40 °C diurnal cyclic temperatures. The adults were tested for Wolbachia infection. Experiments were also carried out rearing the larvae under simulated field conditions in summer (April and June) under sunlight using fully open and half open bowls and also under sunlight and natural shade. RESULTS: At 36 °C and 40 °C constant temperatures, complete larval mortality was observed. At 30 °C and 26 °C, no larval mortality occurred, but Wolbachia density was relatively low in wMel infected males compared to control (maintained at 26 ± 1 °C). At diurnal cyclic temperature of 26-40 °C, Wolbachia density was reduced in males of both the (Pud) lines, but not in females. At 26-36 °C, reduction in Wolbachia density was observed in wMel males but not in wAlbB males. At 26-30 °C, no significant reduction in Wolbachia density was observed with wMel and wAlbB strains. In simulated field conditions (April), under sunlight, the daytime water temperature reached a maximum of 35.7 °C in both full and half open bowls. No larval mortality occurred. Wolbachia frequency and density was reduced in wMel-infected Ae. aegypti (Pud) males from both type of bowls and in females from full open bowls, and in wAlbB males from half open bowls. In June, rearing of larvae under sunlight, the first-instar larvae experienced a maximum daytime water temperature of > 38 °C that caused complete mortality. No larval mortality was observed in bowls kept under shade (< 32 °C). CONCLUSIONS: Exposure of larvae to higher rearing temperatures in the laboratory and simulated-field conditions reduced the densities of wMel and wAlbB strains particularly in males, but the impact was more pronounced for wMel strain. The actual effect of heat stress on the stability of these two Wolbachia strains needs to be tested under natural field conditions.
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Aedes , Wolbachia , Animais , Feminino , Resposta ao Choque Térmico , Larva , Masculino , Mosquitos Vetores , ÁguaRESUMO
We thank Deora et al. [...].
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The aetiology of non-malaria vector-borne diseases in malaria-endemic, forested, rural, and tribal-dominated areas of Dhalai, Tripura, in north-east India, was studied for the first time in the samples collected from malaria Rapid Diagnostic Kit negative febrile patients by door-to-door visits in the villages and primary health centres. Two hundred and sixty serum samples were tested for the Dengue NS1 antigen and the IgM antibodies of Dengue, Chikungunya, Scrub Typhus (ST), and Japanese Encephalitis (JE) during April 2019-March 2020. Fifteen Dengue, six JE, twelve Chikungunya, nine ST and three Leptospirosis, and mixed infections of three JE + Chikungunya, four Dengue + Chikungunya, three Dengue + JE + Chikungunya, one Dengue + Chikungunya + ST, and one Dengue + ST were found positive by IgM ELISA tests, and four for the Dengue NS1 antigen, all without any travel history. True prevalence values estimated for infections detected by Dengue IgM were 0.134 (95% CI: 0.08-0.2), Chikungunya were 0.084 (95% CI: 0.05-0.13), Scrub were 0.043 (95% CI: 0.01-0.09), and Japanese Encephalitis were 0.045 (95% CI: 0.02-0.09). Dengue and Chikungunya were associated significantly more with a younger age. There was a lack of a defined set of symptoms for any of the Dengue, Chikungunya, JE or ST infections, as indicated by the k-modes cluster analysis. Interestingly, most of these symptoms have an overlapping set with malaria; thereby, it becomes imperative that malaria and these non-malaria vector-borne disease diagnoses are made in a coordinated manner. Findings from this study call for advances in routine diagnostic procedures and the development of a protocol that can accommodate, currently, in practicing the rapid diagnosis of malaria and other vector-borne diseases, which is doable even in the resource-poor settings of rural hospitals and during community fever surveillance.
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With India aiming to achieve malaria elimination by 2030, several strategies have been put in place. With that aim, mass surveillance is now being conducted in some malaria-endemic pockets. As dry season mass surveillance has been shown to have its importance in targeting the reservoir, a study was undertaken to assess the parasite load by a sensitive molecular method during one of the mass surveys conducted in the dry winter period. It was executed in two malaria-endemic villages of Dhalai District, Tripura, in northeast India, also reported as P. falciparum predominated area. The present study found an enormous burden of Rapid Diagnostic Test negative malaria cases with P. vivax along with P. vivax and P. falciparum mixed infections during the mass surveillance from febrile and afebrile cases in dry winter months (February 2021-March 2021). Of the total 150 samples tested, 72 (48%) were positive and 78 (52%) negative for malaria by PCR. Out of the 72 positives, 6 (8.33%) were P. falciparum, 40 (55.55%) P. vivax, and 26 (36.11%) mixed infections. Out of 78 malaria negative samples, 6 (7.7%) were with symptoms, while among the total malaria positive, 72 cases 7 (9.8%) were with symptoms, and 65 (90.2%) were asymptomatic. Out of 114 samples tested by both microscopy and PCR, 42 samples turned out to be submicroscopic with 4 P. falciparum, 23 P. vivax, and 15 mixed infections. Although all P. vivax submicroscopic infections were asymptomatic, three P. falciparum cases were found to be febrile. Evidence of malaria transmission was also found in the vectors in the winter month. The study ascertained the use of molecular diagnostic techniques in detecting the actual burden of malaria, especially of P. vivax, in mass surveys. As Jhum cultivators in Tripura are at high risk, screening for the malarial reservoirs in pre-Jhum months can help with malaria control and elimination.
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BACKGROUND: Anopheles culicifacies s.l., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids-the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (kdr) is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of kdr mutation (L1014F) in a field population of An. culicifacies s.l. and three new PCR-based methods for kdr genotyping. METHODS: The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant An. culicifacies s.l. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F) in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR), an Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR) were developed and tested for kdr genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped. RESULTS: The genotyping of this An. culicifacies s.l. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the kdr allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium. CONCLUSION: The Leu-Phe mutation, which generates the kdr phenotype in many insects, was detected in a pyrethroid and DDT resistant An. culicifacies s.l. population. Three PCR-based methods were developed for kdr genotyping. All the three assays were specific. The ARMS method was refractory to non-specific amplification in non-stringent amplification conditions. The PIRA-PCR assay is able to detect both the codons for the phenylalanine mutation at kdr locus, i.e., TTT and TTC, in a single assay, although the latter codon was not found in the population genotyped.
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Anopheles/genética , Dipeptídeos/genética , Resistência a Inseticidas/genética , Mutação Puntual/efeitos dos fármacos , Reação em Cadeia da Polimerase/métodos , Canais de Sódio/genética , Alelos , Animais , Anopheles/efeitos dos fármacos , Sequência de Bases , DDT/farmacologia , Primers do DNA/genética , Genes de Insetos/efeitos dos fármacos , Genótipo , Índia , Dados de Sequência Molecular , Mutação/efeitos dos fármacos , Piretrinas/farmacologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
BACKGROUND & OBJECTIVE: Aqueous and organic solvent extracts of plants/plant parts were effective in killing the mosquito larvae. Comparative efficacy of the aqueous and hexane extracts of dried fruit of Solanum nigrum was tested against five laboratory colonized strains of mosquito species, namely Anopheles culicifacies species A, An. culicifacies species C, An. stephensi, Culex quinquefasciatus and Aedes aegypti to assess the possibility for use of these extracts for their control. METHODS: Concentrations of aqueous extract of dried fruit in the range of 62.5 to 2000 ppm and hexane extract of dried fruit in the range of 0.781 to 150 ppm were used in bioassays. The mortality data were subjected to log probit regression analysis to determine the median lethal concentrations (LC(50) and LC(90)) to kill 50 and 90 per cent of the treated larvae of the respective species. RESULTS: All the five species registered 100 per cent mortality in larval bioassays at 1000 ppm with aqueous extract and at 100 ppm with hexane extract of dried fruit. In bioassays with aqueous extract An. culicifacies species A registered the lowest LC(50) of 208.5 ppm (range-208.5-359 ppm for different mosquito species) while with hexane extract, An. stephensi registered the lowest LC(50) of 6.25 ppm (6.25- 17.63 ppm for different mosquito species). The LC(50) of aqueous extract was 13-39 fold higher than the values of hexane extract of dried fruit for different species. The calculated LC(90) for hexane extract of dried fruit for different species was in the range of 43.38-95.28 ppm. INTERPRETATION & CONCLUSION: Hexane extract showed good mosquito larvicidal efficacy than that of the aqueous extract. The calculated LC(90) for the extract for different species was below 100 ppm and could be effective for comprehensive control of disease vectors.
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Culicidae/efeitos dos fármacos , Frutas/química , Larva/efeitos dos fármacos , Extratos Vegetais/farmacologia , Solanum nigrum/química , Animais , Culicidae/embriologia , Hexanos/química , Inseticidas/química , Inseticidas/farmacologia , Controle de Mosquitos/métodos , Controle Biológico de Vetores/métodos , Extratos Vegetais/química , Solanum nigrum/anatomia & histologia , Solventes/química , Água/químicaRESUMO
BACKGROUND: Molecular techniques have facilitated the studies on genetic diversity of Plasmodium species particularly from field isolates collected directly from patients. The msp-1 and msp-2 are highly polymorphic markers and the large allelic polymorphism has been reported in the block 2 of the msp-1 gene and the central repetitive domain (block3) of the msp-2 gene. Families differing in nucleotide sequences and in number of repetitive sequences (length variation) were used for genotyping purposes. As limited reports are available on the genetic diversity existing among Plasmodium falciparum population of India, this report evaluates the extent of genetic diversity in the field isolates of P. falciparum in eastern and north-eastern regions of India. METHODS: A study was designed to assess the diversity of msp-1 and msp-2 among the field isolates from India using allele specific nested PCR assays and sequence analysis. Field isolates were collected from five sites distributed in three states namely, Assam, West Bengal and Orissa. RESULTS: P. falciparum isolates of the study sites are highly diverse in respect of length as well as sequence motifs with prevalence of all the reported allelic families of msp-1 and msp-2. Prevalence of identical allelic composition as well as high level of sequence identity of alleles suggest a considerable amount of gene flow between the P. falciparum populations of different states. A comparatively higher proportion of multiclonal isolates as well as multiplicity of infection (MOI) was observed among isolates of highly malarious districts Karbi Anglong (Assam) and Sundergarh (Orissa). In all the five sites, R033 family of msp-1 was observed to be monomorphic with an allele size of 150/160 bp. The observed 80-90% sequence identity of Indian isolates with data of other regions suggests that Indian P. falciparum population is a mixture of different strains. CONCLUSION: The present study shows that the field isolates of eastern and north-eastern regions of India are highly diverse in respect of msp-1 (block 2) and msp-2 (central repeat region, block 3). As expected Indian isolates present a picture of diversity closer to southeast Asia, Papua New Guinea and Latin American countries, regions with low to meso-endemicity of malaria in comparison to African regions of hyper- to holo-endemicity.
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Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Variação Genética , Genótipo , Humanos , Índia/epidemiologia , Malária Falciparum/epidemiologia , Proteína 1 de Superfície de Merozoito/química , Proteína 1 de Superfície de Merozoito/genética , Dados de Sequência Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/genéticaRESUMO
Anopheles culicifacies, a complex of five isomorphic sibling species, is a major vector of malaria in India and neighboring countries. The five species are provisionally designated as species A, B, C, D, and E. Polytene chromosome examination has been the only method available that differentiates four members of this complex in areas where species E is not prevalent. However, this technique requires the mosquitoes to be in the half-gravid stage and thus limits its application to only about one fourth to one third of the total adult collection and excludes immature stages completely. For species E, both polytene chromosome examination and mitotic chromosome examination of F1 males are required. A polymerase chain reaction (PCR) assay based on the D3 domain (D3-PCR) of 28S rDNA and a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay involving ITS2 of rDNA are available for the discrimination of the members of the An. culicifacies complex. However, both these can only differentiate species A and D from species B, C, and E. We report here two allele-specific PCR assays (AD-PCR and BCE-PCR) using sequence differences in the mitochondrial cytochrome oxidase II (CO II) subunit. The AD-PCR assay distinguishes species A and D, whereas the BCE-PCR assay distinguishes species B, C, and E. Thus, with a combination of two PCR assays, namely the D3-PCR/ITS2-RsaI assay, followed by either the AD-PCR or the BCE-PCR assay, it is possible to identify individual specimens of any of the species of this complex. This assay system is the first, and the best available at present to distinguish all sibling species and especially to discriminate non-vector, species B from all the vector species, A, C, D, and E, of the An. culicifacies complex. Until another DNA-based method involving fewer steps is developed, this assay system can be used in all malaria epidemiologic studies where An. culicifacies is prevalent.
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Alelos , Culicidae/classificação , Insetos Vetores , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Culicidae/genética , DNA Ribossômico , Feminino , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
A study of the epidemiology of malaria transmission was undertaken in 13 tribal villages located in forest and plain areas of Sundargarh District of Orissa state, India, from January 2001 to December 2003. In forest areas, intense transmission of malaria is attributed to the highly anthropophagic vector Anopheles fluviatilis sibling species S and is complemented by A. culicifacies sibling species C. In plain areas, A. culicifacies sibling species C is responsible for malaria transmission. The entomological inoculation rate in the forest and plain areas was 0.311 and 0.014 infective bites/person/night, respectively, during 2003. Malaria transmission is perennial both in forest and plain areas but is markedly low in the plain area compared with the forest area. Plasmodium falciparum accounted for 85.0% of the total malaria cases during the study period. In forest and plain areas, the number of P. falciparum cases per 1000 population per year was 284.1 and 31.2, respectively, whereas the parasite rate was 14.0% and 1.7%, respectively. In forest areas, clinical malaria occurs more frequently in children aged 0-5 years and declines gradually with increasing age. The study showed that villages in forest and plain areas separated by short geographical distances have distinct epidemiology of malaria transmission.
Assuntos
Anopheles/parasitologia , Malária Falciparum/epidemiologia , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Estudos Transversais , Humanos , Incidência , Índia/epidemiologia , Lactente , Recém-Nascido , Insetos Vetores/parasitologia , Estudos Longitudinais , Malária Falciparum/parasitologia , Malária Falciparum/transmissão , Pessoa de Meia-Idade , Prevalência , Saúde da População Rural , Baço/parasitologiaRESUMO
A village-scale trial on the efficacy of mosquito nets treated with a tablet formulation of deltamethrin (K-OTAB) against malaria in comparison to untreated nets or no net was conducted in Sundargarh District of Orissa, India, which is characterized by perennial transmission with Plasmodium falciparum accounting for more than 80% of malaria cases. Three villages with similar topographical and epidemiological situations were selected and randomly assigned to 3 arms of the study: treated net, untreated net, and no net. Distribution of nets, based on a sleeping pattern survey, was carried out to cover 100% of the population in treated-net and untreated-net villages. Longitudinal and cross-sectional surveys were conducted to measure malaria incidence, prevalence, and splenomegaly. Malaria incidence was reduced by 64.3% in the village with treated nets, 45.2% in the village with plain nets, and 21.4% in the control village without nets. Comparison of malaria incidence data after 1 year of intervention showed significant difference between villages with treated net vs. untreated net (P < 0.05) and treated net vs. no net (P < 0.005). The incidence of clinical attack rate due to P. falciparum was significantly lower in the population using treated nets than in those using untreated nets and no nets. However, no age-specific protective efficacy of treated nets or untreated nets was observed. A significant reduction occurred in spleen rate and parasite rate in children aged 2-9 years using treated nets or untreated nets. An overall significant reduction was found in parasite rate in the total population using treated and untreated nets as compared to nonusers.
Assuntos
Inseticidas , Malária Falciparum/prevenção & controle , Controle de Mosquitos/métodos , Nitrilas , Piretrinas , Adolescente , Adulto , Roupas de Cama, Mesa e Banho , Criança , Pré-Escolar , Humanos , Incidência , Índia , Malária Falciparum/epidemiologia , Prevalência , Esplenomegalia/epidemiologia , Esplenomegalia/parasitologia , ComprimidosRESUMO
Aedes aegypti and Aedes albopictus are principal vectors for the transmission of chikungunya virus (CHIKV). India is a hub for both dengue and chikungunya infections and there are several reports of co-infection of dengue and chikungunya virus in the clinical scenario. The present pilot entomological survey was conducted to evaluate vertical transmission of CHIKV in Aedes field populations. Aedes immature (larvae and pupae) collection was done in 2012, over a period of six months from selected sites in Delhi and Haryana, India. The immatures collected were reared for adult emergence and species identification was done. A. aegypti male and female mosquitoes were separated and pooled collection spot-wise, RNA extracted and RT PCR performed to test for the presence of CHIKV in the pools. Container index (CI) and minimum infection rate (MIR) were estimated. From study areas that tested positive for CHIKV, adult collections were made and females upon feeding on uninfected blood in laboratory were allowed to lay eggs. The progeny that emerged from these field-collected mothers were tested for CHIKV presence. Our pilot survey showed the existence of A. aegypti population even during peak summer season in a few foci which eventually helped the mosquitoes to tide over adverse environmental conditions and with the start of rainfall, the population exploded within a short period of time. Immatures collected from field and progeny of adults collected from the field were CHIKV positive demonstrating the presence of vertical transmission of chikungunya virus in field population of A. aegypti. The present study further demonstrates the importance of identifying permanent breeding sites for proper Aedes species control.
Assuntos
Aedes/virologia , Febre de Chikungunya/transmissão , Febre de Chikungunya/virologia , Vírus Chikungunya/classificação , Vírus Chikungunya/genética , Transmissão Vertical de Doenças Infecciosas , Insetos Vetores/virologia , Adulto , Aedes/classificação , Animais , Febre de Chikungunya/epidemiologia , Feminino , Humanos , Índia , Insetos Vetores/classificação , Larva/virologia , Masculino , Pupa/virologia , Estações do AnoRESUMO
Aspartic proteases of human malarial parasites are thought to play key roles in essential pathways of merozoite release, invasion and host cell hemoglobin degradation during the intraerythrocytic stages of their life cycle. Therefore, we have purified and characterized Plasmodium vivax aspartic protease, to determine if this enzyme can be used as potential drug target/immunogen, and its inhibitors as potential antimalarial drug. The P. vivax aspartic protease has been purified by a combination of ion exchange and size exclusion chromatographies and HPLC. Its properties were examined in order to define a role in the hemoglobin degradation process. The purified enzyme migrated as a single band on native PAGE and SDS/PAGE with a molecular mass of 40 kDa. Gelatin zymogram analyses revealed a clear zone of proteolytic activity corresponding to the band obtained on native PAGE and SDS/PAGE. The enzyme has an optimal pH of 4.0 and exhibits its highest activity at 37 degrees C. The enzyme is inhibited by pepstatin, but not by other inhibitors including o-phenanthroline, EDTA, PMSF or E-64, supporting its designation as an aspartic protease; its IC50 value was found to be 3.0 microM. A Lineweaver Burk double reciprocal plot with pepstatin shows that the inhibition is competitive with respect to the substrate. Ca2+ and Mg2+ ions enhance the protease activity, whereas Cu2+ and Hg2+ ions were found to be inhibitory. The pivotal role of aspartic protease in initiating hemoglobin degradation in P. vivax malaria parasite is also demonstrated.
Assuntos
Ácido Aspártico Endopeptidases/isolamento & purificação , Hemoglobinas/metabolismo , Plasmodium vivax/enzimologia , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/efeitos dos fármacos , Ácido Aspártico Endopeptidases/metabolismo , Quelantes/farmacologia , Cromatografia DEAE-Celulose , Cromatografia em Gel , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Gelatina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Metais Pesados/farmacologia , Pepstatinas/farmacologia , Inibidores de Proteases/farmacologia , Reagentes de Sulfidrila/farmacologiaRESUMO
Anopheles culicifacies Giles is a complex of five sibling species, provisionally designated as species A, B, C, D and E. Species A, C, D and E are vectors of malaria in India. Species A, B, C and D can be identified by polytene chromosome examination except in areas where species B and E are sympatric. Species B and E share the same configuration of the polytene chromosomes but can be differentiated by examining the mitotic chromosomes of F(1) progeny from field collection. Further, polytene chromosome examination method requires the mosquitoes to be at the semigravid stage, which limits on use of this method to a very small proportion of the population. The present study investigated whether the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method can be used to differentiate the members of this complex. Complete ITS2 region along with part of the 5.8S and 28S rDNA sequences (512 bp) and the mitochondrial cytochrome oxidase II (530 bp) were amplified and digested with different restriction endonucleases. The Alu I digest of the COII amplicon and Rsa I digest of the ITS2 amplicon could distinguish two categories: species A and D forming one category and species B, C and E forming another. Further, Dde I digestion of the COII amplicon could distinguish species E from species B and C within the latter category. The PCR-RFLP techniques developed in this study can be applied to areas where species A and B and species B and E are sympatric.