Tridibes Adak

Plasmodium vivax Polymorphism in a Clinical Drug Trial

ABSTRACT Data from a double-blind randomized clinical drug trial were analyzed to find the comparative responses of two antirelapse drugs, bulaquine and primaquine, against different relapsing forms ofPlasmodium vivax infection. A 1-year follow-up study strongly suggests that the duration of preerythrocytic development ofP. vivax is a polymorphic characteristic, exhibited by two strains of hypnozoites responsible for early and late manifestations after primary infection. Short-term relapses were significantly higher in the first half year than long-term relapses, and the reverse was true in the second half year. Clinical drug response data showed that the hypnozoites characterized for short-term relapse were not susceptible to either of the antirelapse drugs in the currently administered dose, whereas hypnozoites characterized for long incubation were significantly susceptible.

Presence of two alternative kdr -like mutations, L1014F and L1014S, and a novel mutation, V1010L, in the voltage gated Na + channel of Anopheles culicifacies from Orissa, India

BackgroundKnockdown 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.MethodsAnopheles 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.ResultsDNA 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.ConclusionsThis 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.

Pyrethroid-Resistance and Presence of Two Knockdown Resistance (kdr) Mutations, F1534C and a Novel Mutation T1520I, in Indian Aedes aegypti

Background Control of Aedes aegypti, the mosquito vector of dengue, chikungunya and yellow fever, is a challenging task. Pyrethroid insecticides have emerged as a preferred choice for vector control but are threatened by the emergence of resistance. The present study reports a focus of pyrethroid resistance and presence of two kdr mutations—F1534C and a novel mutation T1520I, in Ae. aegypti from Delhi, India. Methodology/Principal Findings Insecticide susceptibility status of adult-female Ae. aegypti against DDT (4%), deltamethrin (0.05%) and permethrin (0.75%) was determined using WHOs standard insecticide susceptibility kit, which revealed resistance to DDT, deltamethrin and permethrin with corrected mortalities of 35%, 72% and 76% respectively. Mosquitoes were screened for the presence of kdr mutations including those reported earlier (I1011V/M, V1016G/I, F1534C, D1794Y and S989P), which revealed the presence of F1534C and a novel mutation T1520I. Highly specific PCR-RFLP assays were developed for genotyping of these two mutations. Genotyping using allele specific PCR and new PCR-RFLP assays revealed a high frequency of F1534C (0.41–0.79) and low frequency of novel mutation T1520I (0.13). The latter was observed to be tightly linked with F1534C and possibly serve as a compensatory mutation. A positive association of F1534C mutation with DDT and deltamethrin resistance in Ae. aegypti was established. However, F1534C-kdr did not show significant protection against permethrin. Conclusions/Significance The Aedes aegypti population of Delhi is resistant to DDT, deltamethrin and permethrin. Two kdr mutations, F1534C and a novel mutation T1520I, were identified in this population. This is the first report of kdr mutations being present in the Indian Ae. aegypti population. Highly specific PCR-RFLP assays were developed for discrimination of alleles at both kdr loci. A positive association of F1534C mutation with DDT and deltamethrin resistance was confirmed.

Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection

BackgroundKnockdown resistance (kdr) in insects, resulting from mutation(s) in the voltage-gated sodium channel (vgsc) gene is one of the mechanisms of resistance against DDT and pyrethroid-group of insecticides. The most common mutation(s) associated with knockdown resistance in insects, including anophelines, has been reported to be present at residue Leu1014 in the IIS6 transmembrane segment of the vgsc gene. This study reports the presence of two alternative kdr-like mutations, L1014S and L1014F, at this residue in a major malaria vector Anopheles stephensi and describes new PCR assays for their detection.MethodsPart of the vgsc (IIS4-S5 linker-to-IIS6 transmembrane segment) of An. stephensi collected from Alwar (Rajasthan, India) was PCR-amplified from genomic DNA, sequenced and analysed for the presence of deduced amino acid substitution(s).ResultsAnalysis of DNA sequences revealed the presence of two alternative non-synonymous point mutations at L1014 residue in the IIS6 transmembrane segment of vgsc, i.e., T>C mutation on the second position and A>T mutation on the third position of the codon, leading to Leu (TTA)-to-Ser (TC A) and -Phe (TTT) amino acid substitutions, respectively. Polymerase chain reaction (PCR) assays were developed for identification of each of these two point mutations. Genotyping of An. stephensi mosquitoes from Alwar by PCR assays revealed the presence of both mutations, with a high frequency of L1014S. The PCR assays developed for detection of the kdr mutations were specific as confirmed by DNA sequencing of PCR-genotyped samples.ConclusionsTwo alternative kdr- like mutations, L1014S and L1014F, were detected in An. stephensi with a high allelic frequency of L1014S. The occurrence of L1014S is being reported for the first time in An. stephensi. Two specific PCR assays were developed for detection of two kdr-like mutations in An. stephensi.

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

BackgroundAnopheles 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.MethodsThe 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.ResultsThe 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.ConclusionThe 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.

Molecular evidence of misidentification of Anopheles minimus as Anopheles fluviatilis in Assam (India).

Anophelesminimus s.l. and Anophelesfluviatilis s.l., two closely related taxa, are reported vectors of malaria in Assam state of India. We determined the DNA sequences of morphologically identified A. minimus s.l. and A. fluviatilis s.l. collected from the Kamrup district in Assam, for two rDNA loci-internal transcribed spacer 2 (ITS2) and D3 domain of 28S rDNA (28S-D3). Analysis of rDNA data revealed that the sequences of both the morphologically identified A. minimus s.l. and A. fluviatilis s.l. from Assam are identical, homologous to the sequences of A. minimus s.s. (former species A) and different from that of all the reported members of the Fluviatilis Complex (species S, T and U). This indicates that A. fluviatilis s.l. being reported in Kamrup district, Assam, in low density, mostly during January to April, is actually a hypermelanic and seasonal variant of A. minimus. It was also found that the banding pattern on chromosome arm 2 (which bears species-diagnostic inversions for the Fluviatilis Complex) of A. minimus and of A. fluviatilis s.l. from Assam is homosequential with A. fluviatilis species U suggesting that probably previously described A. fluviatilis U from Assam were also A. minimus.

Parasite killing in malaria non-vector mosquito Anopheles culicifacies species B: implication of nitric oxide synthase upregulation.

Background Anopheles culicifacies, the main vector of human malaria in rural India, is a complex of five sibling species. Despite being phylogenetically related, a naturally selected subgroup species B of this sibling species complex is found to be a poor vector of malaria. We have attempted to understand the differences between vector and non-vector Anopheles culicifacies mosquitoes in terms of transcriptionally activated nitric oxide synthase (AcNOS) physiologies to elucidate the mechanism of refractoriness. Identification of the differences between genes and gene products that may impart refractory phenotype can facilitate development of novel malaria transmission blocking strategies. Methodology/Principal Findings We conducted a study on phylogenetically related susceptible (species A) and refractory (species B) sibling species of An. culicifacies mosquitoes to characterize biochemical and molecular differences in AcNOS gene and gene elements and their ability to inhibit oocyst growth. We demonstrate that in species B, AcNOS specific activity and nitrite/nitrates in mid-guts and haemolymph were higher as compared to species A after invasion of the mid-gut by P. vivax at the beginning and during the course of blood feeding. Semiquantitative RT-PCR and real time PCR data of AcNOS concluded that this gene is more abundantly expressed in midgut of species B than in species A and is transcriptionally upregulated post blood meals. Dietary feeding of L-NAME along with blood meals significantly inhibited midgut AcNOS activity leading to an increase in oocyst production in An. culicifacies species B. Conclusions/Significance We hypothesize that upregulation of mosquito innate cytotoxicity due to NOS in refractory strain to Plasmodium vivax infection may contribute to natural refractoriness in An. culicifacies mosquito population. This innate capacity of refractory mosquitoes could represent the ancestral function of the mosquito immune system against the parasite and could be utilized to understand the molecular basis of refractoriness in planning effective vector control strategies.

Allelic dimorphism of Plasmodium vivax gam-1 in the Indian subcontinent

BackgroundGenetic polymorphism is an inevitable component of a complex organism especially in multistage infectious organisms such as malaria parasites. Understanding the population genetic structure of the parasites would provide valuable information for effective malaria control strategies. Recently, the development of molecular tools like PCR has made analysis of field samples possible and easier and research on Plasmodium vivax has also been strengthened. Not many reports are available on the genetic polymorphism of P. vivax from the Indian sub-continent. This study evaluates the extent of diversity in field isolates of India with respect to Pvgam-1.MethodsA study was designed to assess the diversity of Pvgam-1 among field isolates from India, using a nested PCR assay. Field isolates were collected from different regions of the country and the observed variability was confirmed by sequencing data.ResultsBoth Belem and Chesson type alleles were present either exclusively or in mixed form among isolates of all 10 study sites. The Belem type allele was predominant, occurring in 67% of isolates. The proportion of isolates showing the mixed form (both Belem and Chesson type alleles occurring together in the same isolate) was about 13 overall (up to 38.5% in some isolates). Sequencing of the PCR-amplified Belem and Chesson type alleles confirmed the PCR results. Among the 10 study sequences, 11 polymorphic sites and four singleton variations were observed. All the nucleotide substitutions were non-synonymous.ConclusionStudy shows limited diversity of Pvgam-1 marker in Indian isolates with well representation of both Belem and Chesson type alleles.

Isolation of a Plasmodium vivax refractory Anopheles culicifacies strain from India

Anopheles culicifacies sensu lato comprises five sibling species. We report the isolation of an An. culicifacies species B strain which is completely refractory to Plasmodium vivax sporogonic development and partially refractory to P. falciparum. Parasite development in this strain is arrested by a melanotic encapsulation mechanism in the mid‐gut. We compare the infectivity of this refractory strain and four other species B strains from different epidemiological zones of India with P. vivax in the laboratory.

Building small dams can decrease malaria: A comparative study from Sundargarh District, Orissa, India

The adverse health effect of environmental changes brought about with the construction of large and small dams has often been reported. Here, we present results of a 5-year (2001-2005) study documenting the positive effect of such developmental projects in reducing malaria in an area where malaria transmission is mainly due to the highly efficient anthropophagic vector Anopheles fluviatilis with some contribution from Anopheles culicifacies. The former breeds exclusively in the slow-flowing streams and the latter breeds in a variety of habitats. The study was conducted in San Dulakudar village and comparisons were made with two control villages situated near the stream with similar topography and malaria transmission pattern. Epidemiological data was collected through longitudinal weekly surveillance and cross-sectional surveys in all the study villages. The mean annual malaria incidence rates due to Plasmodium falciparum in children of 1-5 years age group during 2001 before construction of dam was 1304.3 and 785.7 cases/1000 population in dam site village and control villages, respectively. However, after construction of dam, there was gradual reduction in the malaria cases in dam site village and during 2005 the incidence was significantly reduced to 181.8 (P<0.01) whereas it was increased to 1000 in control villages without any significant change in comparison to baseline year (P>0.05). A significant reduction in malaria incidence and parasite rate was also recorded in all the age groups in dam site village without registering any significant change in control villages. The construction of a small dam in the study village altered the water flow above and below the dam thereby making it unfavourable for the breeding of A. fluviatilis which in turn brought about significant impact on malaria transmission.