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Abstract Background Chhattisgarh state in central India is highly endemic for malaria and contributes about 13% of annually reported malaria cases in the country with predominance of P. falciparum. Entomological investigations were carried out in a tribal forested area of district Bastar located in the southern part of Chhattisgarh state to record the prevalence of sibling species of Anopheles fluviatilis and An. culicifacies complexes. The vector species complexes were investigated at sibling species level for their biology in terms of resting and feeding behavior and malaria transmission potential. Methods Indoor resting vector mosquitoes collected during 2010–2011 were identified to sibling species by cytotaxonomy and polymerase chain reaction (PCR) assay. The blood meal source analysis and incrimination studies were done at sibling species level by counter current immunoelectrophoresis and enzyme linked immunosorbent assay (ELISA) respectively. Results Analysis of sibling species composition revealed predominance of An. fluviatilis species S in the study area, which was found to be highly anthropophagic and rested in human dwellings whereas the sympatric species T was primarily zoophagic. Incrimination studies showed high sporozoite rate in species S, thereby confirming its vectorial efficiency. An. culicifacies was encountered in low numbers and comprised species B and C in almost equal proportion. Both these species were found to be exclusively zoophagic. Conclusion The observations made strongly suggest that species S of Fluviatilis Complex is the principal vector of malaria in certain forest areas of district Bastar, Chhattisgarh state and should be the target species for vector control operation. Vector control strategies based on biological characteristics of Fluviatilis S will lead to substantial decline in malaria incidence in such areas.

Abstract Background In the present study, Interceptor®, long-lasting polyester net, 75 denier and bursting strength of minimum 250 kPa coated with alpha-cypermethrin @ 200 mg/m2 was evaluated for its efficacy in reducing the mosquito density, blood feeding inhibition and malaria incidence in a tribal dominated malaria endemic area in Chhattisgarh state, central India. Its durability, washing practices and usage pattern by the community was also assessed up to a period of three years. Methods The study was carried out in two phases. In the first phase (September 2006 to August 2007), 16 malaria endemic villages in district Kanker were randomized into three groups, viz. Interceptor net (LN), untreated polyester net (100 denier) and without net. Malaria cases were detected by undertaking fortnightly surveillance by home visits and treated as per the national drug policy. Mosquito collections were made by hand catch and pyrethrum space spray methods from human dwellings once every month. Slide positivity rate (SPR) and malaria incidence per 1000 population (PI) were compared between the three study arms to assess the impact of use of Interceptor nets. Simultaneously, wash resistance studies were carried out in the laboratory by doing cone bioassays on Interceptor LNs washed up to 20 times. Activities undertaken in second Phase (April 2008 to October 2009) after an interval of about 18 months post-net distribution included questionnaire based surveys at every six months, i.e. 18, 24, 30 and 36 months to observe durability, usage pattern of LNs and washing practices by the community. After 36 months of field use, 30 nets were retrieved and sampled destructively for chemical analysis. Results Interceptor nets were found effective in reducing the density, parity rate and blood feeding success rate of main malaria vector Anopheles culicifacies as compared to that in untreated net and no net villages. SPR in LN villages was 3.7% as compared to 6.5% in untreated and 11% in no net villages. PI in LN villages was 16.4 in comparison to 24.8 and 44.2 in untreated polyester net and no net villages respectively. In surveys carried out after three years of initial distribution, 78.7% (737/936) nets were still in possession with the households, of which 68% were used every night. An. culicifacies mortality was >80% in cone bioassays done on LNs washed up to 20 times in laboratory. Mean alpha-cypermethrin content was 43.5 ± 31.7 mg/m2 on Interceptor LNs withdrawn after three years of household use against the baseline specification of 200 mg/m2. A gradual increase in the proportion of holed nets was observed with the increased period of usage. Conclusion Interceptor nets were highly effective in reducing vector densities as well as malaria incidence in the study villages. Availability of 78% nets with the households in usable condition clearly indicated durability of Interceptor LNs up to three years in the rural setting of India. The nets were found to contain an effective concentration of alpha-cypermethrin against malaria vector after three years of household use.

Abstract Background Chloroquine resistance (CQR) phenotype in Plasmodium falciparum is associated with mutations in pfcrt and pfmdr-1 genes. Mutations at amino acid position 72-76 of pfcrt gene, here defined as pfcrt haplotype are associated with the geographic origin of chloroquine resistant parasite. Here, mutations at 72-76 and codon 220 of pfcrt gene and N86Y pfmdr-1 mutation were studied in blood samples collected across 11 field sites, inclusive of high and low P. falciparum prevalent areas in India. Any probable correlation between these mutations and clinical outcome of CQ treatment was also investigated. Methods Finger pricked blood spotted on Whatman No.3 papers were collected from falciparum malaria patients of high and low P. falciparum prevalent areas. For pfcrt haplotype investigation, the parasite DNA was extracted from blood samples and used for PCR amplification, followed by partial sequencing of the pfcrt gene. For pfmdr-1 N86Y mutation, the PCR product was subjected to restriction digestion with AflIII endonuclease enzyme. Results In 240 P. falciparum isolates with reported in vivo CQ therapeutic efficacy, the analysis of mutations in pfcrt gene shows that mutant SVMNT-S (67.50%) and CVIET-S (23.75%) occurred irrespective of clinical outcome and wild type CVMNK-A (7.91%) occurred only in adequate clinical and parasitological response samples. Of 287 P. falciparum isolates, SVMNTS 192 (66.89%) prevailed in all study sites and showed almost monomorphic existence (98.42% isolates) in low P. falciparum prevalent areas. However, CVIETS-S (19.51%) and CVMNK-A (11.84%) occurrence was limited to high P. falciparum prevalent areas. Investigation of pfmdr-1 N86Y mutation shows no correlation with clinical outcomes. The wild type N86 was prevalent in all the low P. falciparum prevalent areas (94.48%). However, mutant N86Y was comparably higher in numbers at the high P. falciparum prevalent areas (42.76%). Conclusions The wild type pfcrt gene is linked to chloroquine sensitivity; however, presence of mutation cannot explain the therapeutic efficacy of CQ in the current scenario of chloroquine resistance. The monomorphic existence of mutant SVMNT haplotype, infer inbreeding and faster spread of CQR parasite in areas with higher P. vivax prevalance and chloroquine exposure, whereas, diversity is maintained in pfcrt gene at high P. falciparum prevalent areas.

Abstract Background Multi-drug resistance and severe/complicated cases are the emerging phenotypes of vivax malaria, which may deteriorate current anti-malarial control measures. The emergence of these phenotypes could be associated with either of the two Plasmodium vivax lineages. The two lineages had been categorized as Old World and New World, based on geographical sub-division and genetic and phenotypical markers. This study revisited the lineage hypothesis of P. vivax by typing the distribution of lineages among global isolates and evaluated their genetic relatedness using a panel of new mini-satellite markers. Methods 18S SSU rRNA S-type gene was amplified from 420 Plasmodium vivax field isolates collected from different geographical regions of India, Thailand and Colombia as well as four strains each of P. vivax originating from Nicaragua, Panama, Thailand (Pak Chang), and Vietnam (ONG). A mini-satellite marker panel was then developed to understand the population genetic parameters and tested on a sample subset of both lineages. Results 18S SSU rRNA S-type gene typing revealed the distribution of both lineages (Old World and New World) in all geographical regions. However, distribution of Plasmodium vivax lineages was highly variable in every geographical region. The lack of geographical sub-division between lineages suggests that both lineages are globally distributed. Ten mini-satellites were scanned from the P. vivax genome sequence; these tandem repeats were located in eight of the chromosomes. Mini-satellites revealed substantial allelic diversity (7-21, AE = 14.6 ± 2.0) and heterozygosity (He = 0.697-0.924, AE = 0.857 ± 0.033) per locus. Mini-satellite comparison between the two lineages revealed high but similar pattern of genetic diversity, allele frequency, and high degree of allele sharing. A Neighbour-Joining phylogenetic tree derived from genetic distance data obtained from ten mini-satellites also placed both lineages together in every cluster. Conclusions The global lineage distribution, lack of genetic distance, similar pattern of genetic diversity, and allele sharing strongly suggested that both lineages are a single species and thus new emerging phenotypes associated with vivax malaria could not be clearly classified as belonging to a particular lineage on basis of their geographical origin.

Abstract Background Malaria vectors have acquired widespread resistance to many of the currently used insecticides, including synthetic pyrethroids. Hence, there is an urgent need to develop alternative insecticides for effective management of insecticide resistance in malaria vectors. In the present study, chlorfenapyr was evaluated against Anopheles culicifacies and Anopheles stephensi for its possible use in vector control. Methods Efficacy of chlorfenapyr against An. culicifacies and An. stephensi was assessed using adult bioassay tests. In the laboratory, determination of diagnostic dose, assessment of residual activity on different substrates, cross-resistance pattern with different insecticides and potentiation studies using piperonyl butoxide were undertaken by following standard procedures. Potential cross-resistance patterns were assessed on field populations of An. culicifacies. Results A dose of 5.0% chlorfenapyr was determined as the diagnostic concentration for assessing susceptibility applying the WHO tube test method in anopheline mosquitoes with 2 h exposure and 48 h holding period. The DDT-resistant/malathion-deltamethrin-susceptible strain of An. culicifacies species C showed higher LD50 and LD99 (0.67 and 2.39% respectively) values than the DDT-malathion-deltamethrin susceptible An. culicifacies species A (0.41 and 2.0% respectively) and An. stephensi strains (0.43 and 2.13% respectively) and there was no statistically significant difference in mortalities among the three mosquito species tested (p > 0.05). Residual activity of chlorfenapyr a.i. of 400 mg/m2 on five fabricated substrates, namely wood, mud, mud+lime, cement and cement + distemper was found to be effective up to 24 weeks against An. culicifacies and up to 34 weeks against An. stephensi. No cross-resistance to DDT, malathion, bendiocarb and deltamethrin was observed with chlorfenapyr in laboratory-reared strains of An. stephensi and field-caught An. culicifacies. Potentiation studies demonstrated the antagonistic effect of PBO. Conclusion Laboratory studies with susceptible and resistant strains of An. culicifacies and An. stephensi, coupled with limited field studies with multiple insecticide-resistant An. culicifacies have shown that chlorfenapyr can be a suitable insecticide for malaria vector control, in multiple-insecticide-resistant mosquitoes especially in areas with pyrethroid resistant mosquitoes.

Abstract Background Knockdown resistance in insects resulting from mutation(s) in the voltage gated Na+ channel (VGSC) is one of the mechanisms of resistance against DDT and pyrethroids. Recently a point mutation leading to Leu-to-Phe substitution in the VGSC at residue 1014, a most common kdr mutation in insects, was reported in Anopheles culicifacies-a major malaria vector in the Indian subcontinent. This study reports the presence of two additional amino acid substitutions in the VGSC of an An. culicifacies population from Malkangiri district of Orissa, India. Methods Anopheles culicifacies sensu lato (s.l.) samples, collected from a population of Malkangiri district of Orissa (India), were sequenced for part of the second transmembrane segment of VGSC and analyzed for the presence of non-synonymous mutations. A new primer introduced restriction analysis-PCR (PIRA-PCR) was developed for the detection of the new mutation L1014S. The An. culicifacies population was genotyped for the presence of L1014F substitution by an amplification refractory mutation system (ARMS) and for L1014S substitutions by using a new PIRA-PCR developed in this study. The results were validated through DNA sequencing. Results DNA sequencing of An. culicifacies individuals collected from district Malkangiri revealed the presence of three amino acid substitutions in the IIS6 transmembrane segments of VGSC, each one resulting from a single point mutation. Two alternative point mutations, 3042A>T transversion or 3041T>C transition, were found at residue L1014 leading to Leu (TTA)-to-Phe (TTT) or -Ser (TCA) changes, respectively. A third and novel substitution, Val (GTG)-to-Leu (TTG or CTG), was identified at residue V1010 resulting from either of the two transversions–3028G>T or 3028G>C. The L1014S substitution co-existed with V1010L in all the samples analyzed irrespective of the type of point mutation associated with the latter. The PIRA-PCR strategy developed for the identification of the new mutation L1014S was found specific as evident from DNA sequencing results of respective samples. Since L1014S was found tightly linked to V1010L, no separate assay was developed for the latter mutation. Screening of population using PIRA-PCR assays for 1014S and ARMS for 1014F alleles revealed the presence of all the three amino acid substitutions in low frequency. Conclusions This is the first report of the presence of L1014S (homologous to the kdr-e in An. gambiae) and a novel mutation V1010L (resulting from G-to-T or -C transversions) in the VGSC of An. culicifacies in addition to the previously described mutation L1014F. The V1010L substitution was tightly linked to L1014S substitution. A new PIRA-PCR strategy was developed for the detection of L1014S mutation and the linked V1010L mutation.