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.

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.

Village-scale evaluation of mosquito nets treated with a tablet formulation of deltamethrin against malaria vectors.

A field trial was carried out in the Sundargarh district of Orissa, India on the efficacy of mosquito nets treated with a tablet formulation of deltamethrin (K-O TAB) against malaria vectors. Treated nets were used in one village, and in the two control villages, one used untreated nets and the other used indoor spraying with DDT, without nets. In this area the primary malaria vectors are Anopheles culicifacies Giles sensu lato (Diptera: Culicidae) and An. fluviatilis James s.l., which are both endophagic and endophilic, and fully susceptible to deltamethrin. Treatment of a 10-m(2) mosquito net with one of the tablets gave a deltamethrin deposit of 25 mg/m(2). Bioassays repeated on domestically used nets over 7 months showed persistence of almost 100% mortality of An. fluviatilis, whereas An. culicifacies showed a decline from 100% to 71% mortality over this period, after which the nets were re-treated and bioassays were not continued. The sum of collections of mosquitoes resting in village houses and those in exit traps and dead on floor sheets showed a reduction in the numbers of the two vector species due to the treated nets, compared with untreated or no nets, but no reduction in other anophelines or Culex species. Large proportions of the collections of the vector and non-vector anophelines were dead on the floor sheets, but among Culex, mortality was delayed. Treated and untreated nets reduced the proportion of anophelines that had blood-fed; the treated nets did so more effectively than the untreated in the case of An. culicifacies and of Culex mosquitoes. In rooms with treated nets a larger proportion of the total collections [dead + live] were in the exit traps, which can be attributed to the excito-repellent effect of deltamethrin. It is easier to pack and handle tablets of insecticide than liquid concentrate and the use of one tablet per net may be preferable to making up a large volume of diluted insecticide and dipping many nets at a time.

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.

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.

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).

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.

Malarial morbidity in tribal communities living in the forest and plain ecotypes of Orissa, India.

A detailed epidemiological study of malarial morbidity was carried out in 13 'tribal' villages in the forest or plain ecotypes of Sundargarh district, Orissa, India. Longitudinal and cross-sectional, parasitological surveys were conducted in all the villages, to determine the incidence of malaria and the prevalence of malarial infection. The annual numbers of malaria cases/1000 were much higher in the forest (347.9) than on the plain (61.0). In the forest clinical malaria occurred more frequently in children than in adults but on the plain all age-groups were equally affected. In cross-sectional surveys, 14.1% of the subjects from the forest but only 2.8% of those from the plain were found smear-positive for malarial infection. The prevalences of infection in the forest area were highest in the young children (aged 1-5 years) and gradually declined with increasing age. The highest incidence of Plasmodium falciparum malaria (0.90 episode/person-year) was also recorded in the subjects from the forest who were aged 1-5 years. In the forest and plain communities surveyed, 78.5%-81.5% and 36.0%-52.0% of the children aged 2-9 years had detectable splenomegaly, respectively, indicating that the forest was hyper-endemic and the plain meso-endemic for malaria. Malaria is clearly a major problem among the tribal communities of Sundargarh, causing great morbidity and, consequently, considerable economic losses.

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.

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.

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.

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.

The hospital- and field-based performances of the OptiMAL test, for malaria diagnosis and treatment monitoring in central India.

The performance of the OptiMAL test, to detect and differentiate Plasmodium falciparum and P. vivax, was evaluated in central India. The subjects were either symptomatic patients, who presented at a referral hospital in urban Jabalpur, or the inhabitants of remote, tribal, forested villages where malaria is a major public-health problem. In each setting, the results of conventional microscopy were used as the 'gold standard'. Under hospital conditions, the test had excellent sensitivity (100%), good specificity (97%), a high positive predictive value (98%) and a high negative predictive value (100%). The corresponding values in the field-based study in the tribal villages (100%, 67%, 84% and 100%, respectively) were almost as good. The results of OptiMAL testing reveal the decline in parasitaemias (of P. falciparum or P. vivax) after drug administration. For monitoring the effectiveness of treatment, the test could therefore be a useful alternative to microscopy, particularly (1) in places where the facilities for microscopy are poor or non-existent and (2) among hospitalized patients with severe, complicated malaria (in whom parasitaemia and drug response need to be followed very carefully). Follow-up (within 28 days of diagnosis) of the 58 malaria cases detected in the field revealed that the OptiMAL test can be used to detect re-infection with a different Plasmodium sp. (sensitivity = 100%; specificity = 100%; J-index = 1) or recrudescence/re-infection with the same Plasmodium sp. (sensitivity = 83%; specificity = 100%; J-index = 0.83) accurately. The ability to use the test to distinguish P. falciparum from P. vivax, and to identify mixed infections of these two species, is of great significance in areas where the preferred and effective therapy for P. falciparum malaria differs from that for P. vivax.

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.

Ahaptoglobinemia (HpO) and malaria in India.

Haptoglobin (Hp) polymorphism analysed among P. vivax and P. falciparum patients and malaria negative subjects from areas with different epidemiological situations had shown high incidence of ahaptoglobinemia (HpO) among malaria patients. A definite association of HpO with P. vivax as well as P. falciparum malaria in Indian subjects had been observed. However, low sensitivity and reliability of HpO index indicates that it can not be a good indicator for determination of malaria endemicity. About 75 per cent of HpO subjects with P. vivax infection when treated with chloroquine showed typable Hp polymorphs by 8-9 days of post-treatment.