On the conspecificity of Anopheles fluviatilis species S with Anopheles minimus species C.

Anopheles fluviatilis and An. minimus complexes,each comprising of at least three sibling species, are closely related and important malaria vectors in Oriental Region. Recently An. fluviatilis species S, which is a highly efficient malaria vector in India, has been made conspecific with An. minimus species C (senior synonym) on the basis of homology in 335 base pair nucleotide sequence of D3 domain of 28S ribosomal DNA (rDNA). We examined the conspecificity of these two nominal species by obtaining and analysing the DNA sequences of nuclear ribosomal loci internal transcribed spacer 2 (ITS2) and D2-D3 domain of 28S rDNA (28S-D2/D3) from those of An. fluviatilis S and An. minimus C. We found that the sequences of An. fluviatilis S are appreciably different from those of An. minimus C with pair-wise distance (Kimura-2-parametre model)of 3.6 and 0.7%for loci ITS2 and 28S-D2/D3, respectively. Pair-wise distance and phylogenetic analyses using ITS2 sequences of members of Minimus and Fluviatilis Complexes revealed that An. fluviatilis S is distantly related to An. minimus C as compared to any other members of the Fluviatilis Complex. These findings suggest that the two nominal species, An. fluviatilis S and An. minimus C, do not merit synonymy. The study also confirms that the reported species An. fluviatilis X is synonym with species S.

Efficacy of Agnique MMF monomolecular surface film against Anopheles stephensi breeding in urban habitats in India.

Efficacy of Agnique MMF, a monomolecular film formulation, was tested against immatures of Anopheles stephensi, an urban malaria vector in India, in simulated and natural habitats. Simulated field trials carried out in cement tanks showed 100% inhibition of adult emergence for up to 1 wk at 0.4 ml/m2 and up to 3 wk at 1 ml/m2. A small-scale field trial in tanks and wells at 1 and 2 ml/m2 produced more than 75% reduction of late instars and 100% reduction of pupae on day 1. The reduction in pupae at 1 and 2 ml/m2 lasted up to 2 wk in tanks and 5 wk in wells. These results suggest that Agnique MMF could be used as one of the choices in an urban malaria control program.

Segregation of cytoplasmic incompatibility properties in Culex pipiens fatigans.

Maternally inherited variants, which arose within a laboratory colony of Culex pipiens fatigans, have been studied by rearing cultures from single egg rafts. Segregation, i.e, variation of cytoplasmic incompatibility properties between the male progeny of individual females, was demonstrated. Also, from the daughters of individual females, sub-lines were derived within which all the males showed the same incompatibility or compatibility properties. Among the descendants of tetracycline-treated individuals were lines which superficially simulated these phenomena, but theses lines ultimately reverted to the cytoplasmic compatibility type of the strain which was submitted to the treatment. The types of variation s in cytoplasmic incompatibility properties that have been studied are discussed.

Larvivorous fish in wells target the malaria vector sibling species of the Anopheles culicifacies complex in villages in Karnataka, India.

Malaria was a major problem in a sericulture area of Karnataka, south India, where Anopheles culicifacies s.l. and A. fluviatilis s.l. were considered to be the main vectors. Sibling species complexes of these two species were analysed in three ecologically different villages. Among A. culicifacies, only sibling species A and B were found. In Puram, a village with 22 wells, species A predominated; species B predominated in a village with four wells and a stream, and in a village with a stream and no wells. Poecilia reticulata fish were introduced into all wells and streams in the villages, and after one year no vectors were found in Puram, and all, or nearly all, A. culicifacies were species B in the other two villages. All A. fluviatilis belonged to the sibling species T. Blood meal analysis indicated that a few of the A. culicifacies collected had fed on humans while all the A. fluviatilis had fed on bovines. Before the introduction of fish, the annual parasite incidence for malaria was high in Puram, but much lower in the other two villages. From 1998 (over one year after release of fish) until 2003, no malaria cases were detected in the three villages.

Evaluation of bifenthrin treated mosquito nets against anopheline and culicine mosquitoes.

BACKGROUND AND OBJECTIVES: The main rural malaria vector Anopheles culicifacies has developed resistance to dichloro diphenyl trichloroethane (DDT), hexachloro cyclo hexane (HCH) and malathion in the state of Haryana in northern India. An alternative synthetic pyrethroid insecticide bifenthrin was therefore evaluated on mosquito nets against anopheline and culicine mosquitoes, in two villages Jagdishpur and Garh Mirakpur of Community Health Center (CHC) Badhkhalsa in district Sonipat, Haryana state. METHODS: Two formulations of bifenthrin, suspension concentrate (SC) and micro-emulsion (ME) were compared with micro-capsule suspension (CS) of lambdacyhalothrin. The impact of three doses of bifenthrin (10, 25 and 50 mg/m(2)) impregnated on mosquito nets was compared with lambdacyhalothrin (25 mg/m(2)) and untreated control. Quality assessment of treatment on treated nets was carried out by residue analysis and the persistence of the insecticide on nets was determined by contact bioassays. Efficacy of treated nets on mosquito density was assessed by calculating mosquito entry rate, immediate mortality, delayed mortality and excito-repellency to the insecticides. RESULTS: In susceptibility tests An. culicifacies was susceptible to bifenthrin (0.1% test papers) and to lambdacyhalothrin (0.05% test papers). Bioassays on treated nets against A. culicifacies recorded 100 per cent mortality up to tenth fortnight for all the doses of impregnation with bifenthrin (SC and ME) and lambdacyhalothrin (CS). Ring-net bioassays against An. culicifacies showed median knock-down time between 3.1 to 11.4 min. Behavioural indices were also studied for anopheline and culicine mosquitoes. The reduction in entry rates of anopheline and culicine mosquitoes into the rooms with treated nets compared to control indicated good efficacy with all the formulations and doses of the insecticides. INTERPRETAION AND CONCLUSION: Indoor (immediate) mortality of mosquitoes with bifenthrin ME formulation was relatively lower compared to SC fomulation of bifenthrin and based on delayed mortility and continued susceptibility in bioassays, bifenthrin ME at the rate of 10 mg/m(2) dose was found suitable for the impregnation of mosquito nets for phase III trial.

Multi-drug resistant Plasmodium falciparum malaria in Assam, India: timing of recurrence and anti-malarial drug concentrations in whole blood.

The susceptibility of 23 cases of Plasmodium falciparum malaria from the Sonapur primary health center in the Kamrup district of Assam, India to different antimalarials was investigated using the 28-day World Health Organization in vivo test. Whole blood concentrations of chloroquine, sulfadoxine, and quinine were determined at different intervals and at the time of parasites recrudescence after completion of treatment with the respective drugs to confirm the status of drug sensitivity. A case of multi-drug resistant P. falciparum malaria was found where recrudescence occurred, despite standard oral treatment with chloroquine, sulfadoxine/pyrimethamine, and quinine sequentially. Whole blood concentrations of chloroquine, sulfadoxine, and quinine at the time of recrudescence were 0.35 microg/ml (day 7), 18 microg/ml (day 14), and 0.009 microg/ml (day 14), respectively. Therefore, monitoring of drug-resistant P. falciparum malaria and its proper treatment should be intensified to check the spread of multi-drug resistant strains in other parts of the country.

Widespread occurrence of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene haplotype SVMNT in P. falciparum malaria in India.

The Plasmodium falciparum chloroquine resistance transporter (Pfcrt) K76T mutation and haplotype (amino acids 72-76) and the P. falciparum multidrug resistance 1 (Pfmdr1) mutation (N86Y) were analyzed as markers of chloroquine resistance in the DNAs of 73 blood samples from patients with P. falciparum malaria in India. Seventy of the 73 DNAs had the Pfcrt K76T mutation. Of these, 66 had the SVMNT haplotype and four had CVIET, the African/Southeast Asian haplotype. Only 20 of 69 DNAs had the Pfmdr1 N86Y mutation. It is surprising that the Pfcrt haplotype in India is predominantly SVMNT, rather than that seen in Southeast Asia. The widespread prevalence of the Pfcrt K76T mutation is a cause for concern.

Plasmodium vivax: enzyme polymorphism in isolates of Indian origin.

185 isolates of Plasmodium vivax were collected from patients visiting the malaria clinic run by the National Malaria Eradication Programme, Delhi, India. Percoll gradient centrifugation was used to concentrate P. vivax parasites from 0.4 to 0.5 ml of blood collected by finger prick. The parasite concentrate from each isolate was electrophoretically analysed for lactate dehydrogenase (LDH), NADP-dependent glutamate dehydrogenase (GDH), glucose phosphate isomerase (GPI) and adenosine deaminase (ADA). Variations were observed in GPI, GDH and ADA systems. Four electrophoretic forms of GPI and 5 each of GDH and ADA were observed. Electrophoretic mobilities of the different isoenzymic forms in P. vivax were identical to those reported for P. falciparum, indicating that the 2 species cannot be differentiated on the basis of electrophoretic patterns of the 4 enzyme systems studied.

Susceptibility of Anopheles culicifacies species A and B to Plasmodium vivax and Plasmodium falciparum as determined by immunoradiometric assay.

We have used a two-site immunoradiometric assay and species-specific antisporozoite monoclonal antibodies to determine the relative roles that sibling species A and B of the Anopheles culicifacies complex play in malaria transmission in western Uttar Pradesh, India. The results unequivocally establish species A as the primary vector of both Plasmodium vivax and P. falciparum in this area. Our results indicate active transmission of P. vivax from May to October and of P. falciparum from August to December. The identification of species A as the primary malaria vector in northern India will now allow suitable malaria control strategies to be designed.

Genetic structure of Plasmodium vivax isolates in India.

Variations in the allelic composition of glucose phosphate isomerase (GPI), NADP-dependent glutamate dehydrogenase (GDH) and adenosine deaminase (ADA) enzyme systems of Plasmodium vivax were observed in isolates of Indian origin in 1985-1993. No significant difference was observed in allelic frequencies in different years. The data indicated random distribution of GPI, GDH and ADA alleles among the isolates, suggesting that loci for these enzymes were not linked. A high proportion of the isolates comprised at least 2 genetically distinct clones, the mean number of clones per isolate being 1.4. There was no significant difference in the number of oocysts in Anopheles stephensi fed on uniclonal and multiclonal isolates. No difference was observed in the proportions of uniclonal and multiclonal isolates during low and high transmission periods.

Liquid chromatographic determination of amodiaquine in human plasma.

A normal-phase high-performance liquid chromatographic method using dichloromethane- methanol-1M perchloric acid (100:10:0.9, v/v/v) at a flow rate of 1.0 ml min(-1) on a LiChrospher Si column with UV (254 nm) detection has been developed for the determination of amodiaquine and its metabolites desethyl amodiaquine and bisdesethyl amodiaquine in plasma. The limit of quantification was 5 ng ml(-1). Mean within-day and day-to-day coefficients of variation (CV) were 4.10 and 6.27% for amodiaquine, 3.43 and 4.80% for desethyl amodiaquine and 3.53 and 5.23% for bisdesethyl amodiaquine, respectively. Mean extraction recovery of amodiaquine, desethyl amodiaquine and bisdesethyl amodiaquine from plasma were 82.48, 74.50 and 69.65%, respectively. Chloroquine and its metabolite desethyl chloroquine, quinine, sulfadoxine and primaquine do not interfere in the detection of amodiaquine, desethyl amodiaquine and bisdesethyl amodiaquine in plasma.

Spiracular indices in Anopheles stephensi: a taxonomic tool to identify ecological variants.

Thoracic spiracle length and its index was examined for their ability to discriminate two ecological variants, type form and mysorensis, of Anopheles stephensi in the adult stage. The type form is exclusively domestic in all seasons, whereas the mysorensis variant occupies the outdoor niche during monsoon and postmonsoon seasons, with spillover into domestic sites during summer ecological stress periods. A statistically significant co-relation was established between the ridge count of the egg and two adult measurements, the thoracic spiracle length, and the spiracular index. In An. stephensi type form, average spiracle length was 0.11-0.12 mm and average spiracular index was 8.09-9.23, whereas in mysorensis, the corresponding figures were 0.09-0.10 mm and 6.82-7.60. These parameters showed consistent variations in population of mosquitoes that emerged during monsoon and summer season. The thoracic lengths in both variants remained constant, and only spiracular lengths showed fluctuations in three seasonal populations. These measures provide discrimination of adult variants--identifications that are essential in malaria control programs.

Differential selection of malathion resistance in Anopheles culicifacies A and B (Diptera: Culicidae) in Haryana State, India.

In November 1989, for the first time after the introduction of malathion spray in 1982, increased densities of Anopheles culicifacies Giles s.l. were observed in Chhatera and Barota, two villages in Halalpur block in Sonepat District, Haryana State, India. In bioassay tests against the diagnostic dose of malathion (5% for 1 h), 41-47% of mosquitoes survived. An culicifacies populations in this area comprise two sibling species, A and B. Differential malathion resistance was observed; in species A it was 2-9% and in species B 62-66%. Likewise, species A was found to be more susceptible to DDT than was species B. The possible use of DDT spray to delay the onset of malathion resistance is discussed.

Evidence for a new malaria vector species, species E, within the Anopheles culicifacies complex (Diptera: Culicidae).

Female Anopheles culicifacies Giles from Ramanathapuram district, Tamil Nadu state, India, were examined for oocysts and sporozoites and their larval progeny for mitotic karyotype. Collections were made from Mandapam and Uchipuli on the mainland, and Thangachimadam and Pamban on Rameshwaram Island. Of the 451 An. culicifacies females that were collected and dissected, 24 were found positive for Plasmodia (21 for sporozoites and 3 for oocysts). Both acrocentric and submetacentric Y-chromosome karyotypes were observed among the progeny of females from all villages. All 11 iso-female lines whose parental females were positive for sporozoites or oocysts had progeny with submetacentric Y-chromosomes. Total absence of sporozoite-positives among mothers of acrocentric males was evidence of assortative mating between these 2 sympatric populations (i.e., 2 species). We propose that the nonvector population with acrocentric Y-chromosome sons retain the original designation of species B and that the vector population with the submetacentric Y-chromosome sons be designated as species E, a new species.

An allele-specific polymerase chain reaction assay for the differentiation of members of the Anopheles culicifacies complex.

Anopheles culicifacies, the principal vector of malaria in India, is a complex of five cryptic species which are morphologically indistinguishable at any stage of life. In view of the practical difficulties associated with classical cytotaxonomic method for the identification of members of the complex, an allele-specific polymerase chain reaction (ASPCR) assay targeted to the D3 domain of 28S ribosomal DNA was developed. The assay discriminates An. culicifacies species A and D from species B, C and E. The assay was validated using chromosomally identified specimens of An. culicifacies from different geographical regions of India representing different sympatric associations. The assay correctly differentiates species A and D from species B, C and E. The possible use of this diagnostic assay in disease vector control programmes is discussed.

Anopheline species complexes & malaria control.

Species complexes comprising morphologically indistinguishable biological species that are reproductively isolated, are of common occurrence among anophelines. A list of anopheline species complexes identified so far in the world has been given. To demonstrate the importance of species complexes in malaria control, we report the Anopheles culicifacies complex as a case study. An. culicifacies is a major vector of malaria in India and neighbouring countries. This complex comprises four sibling species, A, B, C and D. Stratification of U.P. state and district Allahabad has been shown taking into consideration the biological differences among sibling species, viz., sibling species composition and vectorial potential- species B is a non-vector while others are vectors. To achieve cost effective vector control, microlevel stratification at least at the block level has been suggested. Implications of differential responses of sibling species to DDT and malathion in field operations have been discussed. To achieve selective and sustainable control, and to reduce the unnecessary selection pressure of insecticides, an insecticide spray strategy to control An. culicifacies has been provided.

Investigation of malaria outbreak in Bahraich district, Uttar Pradesh.

BACKGROUND & OBJECTIVES: Based on the reports of 139 fever related deaths in Jarwal primary health centre (PHC) of Bahraich district, Uttar Pradesh (UP) during April to September 1999, a study was undertaken to explore the possibility of outbreak of Plasmodium falciparum malaria in the area and reasons of outbreak. METHODS: The study was undertaken during September-October 1999 in Bahraich district, UP. The study included a parasitological and an entomological survey. Blood slides from fever cases were collected and examined following standard procedures for detection of species and stage of parasite. The resting adult mosquitoes were collected from human dwellings and cattle sheds from selected villages. Susceptibility status of Anopheles culicifacies to 4 per cent DDT and 0.05 per cent deltamethrin was determined under laboratory conditions following the WHO procedure. In vitro drug sensitivity of P. falciparum to chloroquine was also estimated. RESULTS: Overall slide positivity rate (SPR) was found to be 33.8 with a preponderance of P. falciparum (88.4%). There was an outbreak of Pf malaria in Jarwal and surrounding areas as well. Foci of P. falciparum malaria were found in Jarwal, Fakharpur and Hazoorpur PHCs around Kaisarganj PHC. In addition, P. falciparum cases, were also reported from Motipur and Tejwapur. INTERPRETATION & CONCLUSION: Poor surveillance of affected areas resulting in low annual parasite incidence (API), lack of insecticidal spray in the currently affected PHCs as the API was less than 2 and development of resistance in P. falciparum to chloroquine were found as the possible reasons for the outbreak. It is recommended that surveillance be strengthened in all PHCs of Bahraich district to contain further extension of malaria in northeastern UP.

Anti-malarial activity of some xanthones isolated from the roots of Andrographis paniculata.

Four xanthones were isolated from the roots of Andrographis paniculata using a combination of column and thin-layer chromatographic methods. They were characterized as (i) 1,8-di-hydroxy-3,7-dimethoxy-xanthone, (ii) 4,8-dihydroxy-2,7-dimethoxy-xanthone, (iii) 1,2-dihydroxy-6,8-dimethoxy-xanthone and (iv) 3,7,8-trimethoxy-1-hydroxy xanthone by IR, MS and NMR spectroscopic methods. In vitro study revealed that compound 1,2-dihydroxy-6,8-dimethoxy-xanthone possessed substantial anti-plasmodial activity against Plasmodium falciparum with its IC(50) value of 4 microg ml(-1). Xanthones bearing hydroxyl group at 2 position demonstrated most potent activity while xanthones with hydroxyl group at 1,4 or 8 position possessed very low activity. In vivo anti-malarial sensitivity test of this compound on Swiss Albino mice with Plasmodium berghei infection using Peters' 4-day test gave substantial reduction (62%) in parasitaemia after treating the mice with 30 mg kg(-1) dose. In vitro cytotoxicity against mammalian cells revealed that 1,2-dihydroxy-6,8-dimethoxy-xanthone is non-cytotoxic with its IC(50) > 32 microg ml(-1).

GIS based malaria information management system for urban malaria scheme in India.

A GIS based information management system has been developed to help Urban Malaria Control in India. The basic objective is to develop a model to assist planning and implementation of a suitable control measure. The system can help in: (i) identifying high receptive areas in time and space domain; (ii) identifying risk factors for high receptivity; (iii) monitoring and evaluating control measures. To demonstrate this system, information on 33 parameters and malaria cases has been attached to a digitised map of Dindigul, an urban town in Tamil Nadu. Functionalities of the system and its utility are described in this paper. A GIS based information management system ensures that if a localised spurt of the disease occurs, it can be associated rapidly with a likely cause, a specific vector, and a probable human source, so that appropriate preventive action can be taken to arrest any rising trend.

Inheritance and linkage of malic enzyme in Anopheles stephensi.

Genetics and linkage analysis of malic enzyme (Me) have been worked out in Anopheles stephensi. Genetic analysis revealed the 2 variants to be codominant alleles at a locus Me, which is sex-linked. Linkage studies with another X-linked mutant red-eye (r), indicated that the map distance between 2 loci was 44.60 +/- 1.07.