Malaria Vector Bionomics: Countrywide Surveillance Study on Implications for Malaria Elimination in India.

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.

First Report of Anopheles annularis s.l., An. maculatus s.s., and An. culicifacies s.l. as Malaria Vectors and a New Occurrence Record for An. pseudowillmori and An. sawadwongporni in Alipurduar District Villages, West Bengal, India.

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.

First Report of Rubber Collection Bowls & Plastic and Bamboo Water Containers as the Major Breeding Source of Ae. albopictus with the Indigenous Transmission of Dengue and Chikungunya in Rural Forested Malaria-Endemic Villages of Dhalai District, Tripura, India: The Importance of Molecular Identification.

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.

Studies on the fitness characteristics of wMel- and wAlbB-introgressed Aedes aegypti (Pud) lines in comparison with wMel- and wAlbB-transinfected Aedes aegypti (Aus) and wild-type Aedes aegypti (Pud) lines.

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.

Reply to Deora et al. Multiplexing for Plasmodium spp.? Think Again! Comment on "Bhowmick et al. Dry Post Wintertime Mass Surveillance Unearths a Huge Burden of P. vivax, and Mixed Infection with P. vivaxP. falciparum, a Threat to Malaria Elimination, in Dhalai, Tripura, India. Pathogens 2021, 10, 1259".

We thank Deora et al. [...].

Sensitivity of wMel and wAlbB Wolbachia infections in Aedes aegypti Puducherry (Indian) strains to heat stress during larval development.

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.

Diagnosis of Indigenous Non-Malarial Vector-Borne Infections from Malaria Negative Samples from Community and Rural Hospital Surveillance in Dhalai District, Tripura, North-East India.

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.

Dry Post Wintertime Mass Surveillance Unearths a Huge Burden of P. vivax, and Mixed Infection with P. vivax P. falciparum, a Threat to Malaria Elimination, in Dhalai, Tripura, India.

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.

Modified PCR-based assay for the differentiation of members of Anopheles fluviatilis complex in consequence of the discovery of a new cryptic species (species V).

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.

Biology and bionomics of malaria vectors in India: existing information and what more needs to be known for strategizing elimination of malaria.

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.

HIV-malaria interactions in North-East India: A prospective cohort study.

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.

Evidence for natural vertical transmission of chikungunya viruses in field populations of Aedes aegypti in Delhi and Haryana states in India-a preliminary report.

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.

Analysis of population genetic structure of Indian Anopheles culicifacies species A using microsatellite markers.

BACKGROUND: Anopheles culicifacies sensu lato is an important vector of malaria in Southeast Asia contributing to almost 70% of malaria cases in India. It exists as morphologically similar sibling species A, B, C, D and E with varied geographical distribution patterns. Vector control measures have been difficult for this important vector as the sibling species have developed varying levels of resistance to the currently used insecticides. In view of the importance of this vector, we developed and validated a set of microsatellite markers and the same were used to analyze the population genetic structure of five different geographical populations of An. culicifacies A. METHODS: Anopheles culicifacies A samples were collected from different localities across India, and genotyping was performed using eight microsatellite markers on ABI Prism 310 Genetic Analyzer. Several statistical analyses were performed to ascertain the genetic diversity that exists within and between the populations. RESULTS: The markers were found to be moderately polymorphic in the populations. Genetic analysis indicated significant genetic differentiation between the majority of the population pairs analyzed and was not found to be related to the geographical distances between populations. CONCLUSION: This is the first and successful attempt to test the microsatellite markers developed for population genetic analysis of An. culicifacies A. Host feeding and breeding habits of species A suggest that factors other than ecological and geographical barriers were responsible for the genetic differentiation that has been observed between the populations.

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.

Centenary celebrations article: Plasmodium knowlesi: from macaque monkeys to humans in South-east Asia and the risk of its spread in India.

The identification of a large focus of Plasmodium knowlesi in Malaysian Borneo and subsequent reports from several countries in South-east Asia has led its recognition as the fifth human malaria parasite. The natural preferred hosts of this species still continue to be macaque monkeys that live in broad-leaf rain forests. This review describes the distribution of macaque monkeys, the Anopheles species belonging to the Leucosphyrus Group that have been incriminated as vectors, morphological and clinical features of this parasite, and the transmission cycles that have been identified for this parasite. As the North-eastern states of India share their borders with P. knowlesi malaria endemic countries and because travelers from countries in South-east Asia visit India and vice versa, risks of this parasite entering India and its spread are also discussed.

Laboratory studies on mosquito larvicidal efficacy of aqueous & hexane extracts of dried fruit of Solanum nigrum Linn.

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.

PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato.

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.

Multiplex PCR assay and phylogenetic analysis of sequences derived from D2 domain of 28S rDNA distinguished members of the Anopheles culicifacies complex into two groups, A/D and B/C/E.

A multiplex PCR assay was developed using the sequences of the D2 region of 28S ribosomal DNA (rDNA) to discriminate the five members of the Anopheles culicifacies complex provisionally designated as species A, B, C, D and E. Two minus strand primers derived from sequence differences in the D2 variable region and a universal plus strand primer derived from the conserved 28S (rDNA) has delimited five members into species A and D (group 1) and species B, C and E (group 2) in a PCR diagnostic assay. The complete 28S rDNA-D2 region sequence of A. culicifacies sibling species is reported for the first time. Inter-specific sequence divergence was greater than the intra-specific divergence. The phylogenetic relationships inferred from maximum likelihood, maximum parsimony and the neighbor joining analysis confirmed the presence of two unambiguous monophyly clades one consisting of species A and D and the other of species B, C and E and that the A. culicifacies sibling species diverged relatively recently in evolutionary terms despite their considerable differences in bionomics.

Urine levels of rifampicin & isoniazid in asymptomatic HIV-positive individuals.

BACKGROUND & OBJECTIVE: AIDS and its associated gastrointestinal complications may impair the absorption of anti-tuberculosis (TB) drugs. Impaired absorption of anti-TB drugs could lead to low drug exposure, which might contribute to acquired drug resistance and reduced effectiveness of anti-TB treatment. The aim of this study was to obtain information on the status of absorption of rifampicin (RMP) and isoniazid (INH) in asymptomatic HIV- positive individuals, who are less immunocompromised. The D-xylose absorption test was also carried out to assess the absorptive capacity of intestive. METHODS: The absorption of RMP, INH and D-xylose was studied in 15 asymptomatic HIV-positive individuals with CD4 cell counts>350 cells/mm3 and 16 healthy volunteers, after oral administration of single doses of RMP (450 mg), INH (300 mg) and D-xylose (5 g). Urine was collected up to 8 h after drug administration. Percentage dose of the drugs and their metabolites and D-xylose excreted in urine were calculated. RESULTS: A significant reduction in the urinary excretion of INH and D-xylose in HIV-positive persons compared to healthy volunteers was observed. The per cent dose of RMP and its metabolite, desacetyl RMP was also lower in HIV-positive persons compared to healthy volunteers, but this difference was not statistically significant. INTERPRETATION & CONCLUSION: Decreased urinary excretion of D-xylose and INH are suggestive of intestinal malabsorption in HIV-positive individuals. HIV infection could cause malabsorption of anti-TB drugs even at an early stage of the disease. The clinical implications of these findings need to be confirmed in larger studies.