P. K. Mohapatra

Role of the prevalent Anopheles species in the transmission of Plasmodium falciparum and P. vivax in Assam state, north-eastern India.

Abstract In north–eastern India, Anopheles minimus, An. dirus and An. fluviatilis are considered the three major vectors of the parasites causing human malaria. The role in transmission of the other Anopheles species present in this region is not, however, very clear. To examine the vectorial role of the more common anopheline mosquitoes, the heads and thoraces of 4126 female Anopheles belonging to 16 species (collected using miniature light traps set in human dwellings in a foothill village in the Jorhat district of Assam state) were tested, in ELISA, for the circumsporozoite proteins (CSP) of Plasmodium falciparum or the VK-210 and VK-247 polymorphs of P. vivax. Sixty-five pools of head–thorax homogenates, representing 10 different species of Anopheles , were found reactive, giving an overall minimum prevalence of infection (MPI) of 1.58%, with a 95% confidence interval (CI) of 1.21%–2.0%. Among the CSP-reactive pools of mosquitoes, 31% were positive only for P. falciparum, 45% only for P. vivax VK 247, 6% only for P. vivax VK 210, and 18% for both P. falciparum and P. vivax VK 247. The results indicate that not only the proven vector, An. minimus s.l. (MPI = 0.71%), but also several species of Anopheles previously considered unimportant in the epidemiology of malaria, especially An. aconitus (MPI = 3.95%), An. annularis (MPI = 5.8%), the An. hyrcanus group (MPI = 0.48%), An. kochi (MPI = 1.28%), the An. philippinensis-nivipes complex (MPI = 0.94%), and An. vagus (MPI = 3.87%), are important vectors in the foothills of Assam.

Molecular evidence of increased resistance to anti-folate drugs in Plasmodium falciparum in North-East India: a signal for potential failure of artemisinin plus sulphadoxine-pyrimethamine combination therapy.

North-east India, being a corridor to South-east Asia, is believed to play an important role in transmitting drug resistant Plasmodium falciparum malaria to India and South Asia. North-east India was the first place in India to record the emergence of drug resistance to chloroquine as well as sulphadoxine/pyrimethamine. Presently chloroquine resistance is widespread all over the North-east India and resistance to other anti-malarials is increasing. In this study both in vivo therapeutic efficacy and molecular assays were used to screen the spectrum of drug resistance to chloroquine and sulphadoxine/pyrimethamine in the circulating P. falciparum strains. A total of 220 P. falciparum positives subjects were enrolled in the study for therapeutic assessment of chloroquine and sulphadoxine/pyrimethamine and assessment of point mutations conferring resistances to these drugs were carried out by genotyping the isolates following standard methods. Overall clinical failures in sulphadoxine/pyrimethamine and chloroquine were found 12.6 and 69.5% respectively, while overall treatment failures recorded were 13.7 and 81.5% in the two arms. Nearly all (99.0%) the isolates had mutant pfcrt genotype (76T), while 68% had mutant pfmdr-1 genotype (86Y). Mutation in dhps 437 codon was the most prevalent one while dhfr codon 108 showed 100% mutation. A total of 23 unique haplotypes at the dhps locus and 7 at dhfr locus were found while dhps-dhfr combined loci revealed 49 unique haplotypes. Prevalence of double, triple and quadruple mutations were common while 1 haplotype was found with all five mutated codons (F/AGEGS/T) at dhps locus. Detection of quadruple mutants (51I/59R/108N/164L) in the present study, earlier recorded from Car Nicobar Island, India only, indicates the presence of high levels of resistance to sulphadoxine/pyrimethamine in north-east India. Associations between resistant haplotypes and the clinical outcomes and emerging resistance in sulphadoxine/pyrimethamine in relation to the efficacy of the currently used artemisinin combination therapy are discussed.

Evaluation of chloroquine (CQ) and sulphadoxine/pyrimethamine (SP) therapy in uncomplicated falciparum malaria in Indo‐Myanmar border areas

Chloroquine (CQ) and sulphadoxine/pyrimethamine (SP) are two first‐line antimalarials used under the existing Indian National Drug Policy in the north‐eastern region of India bordering several countries including Myanmar. Although widespread resistance to antimalarials in Plasmodium falciparum has been reported from western Myanmar, information from the Indian side of the border is scarce. We studied the therapeutic response to CQ and SP at four sites in Changlang and Lohit, two administrative districts of Arunachal Pradesh bordering Myanmar. We monitored uncomplicated falciparum malaria patients after treatment with standard regimens of CQ and SP for 28 days following the revised in‐vivo protocol of the World Health Organization. A total of 236 patients, 95 in the CQ group and 141 in the SP group, participated. We recorded 23.8% early treatment failures to CQ and 14.1% to SP; late clinical failures of 14.3 and 12.6%; late parasitological failures of 10.7 and 8.1% and adequate clinical and parasitological responses of 51.2 and 65.2%, respectively. The significantly different treatment failure rates seen in Chowkham (furthest from Indo‐Myanmar border) and Jairampur/Nampong (nearest to Indo‐Myanmar border) for chloroquine (Cox proportion hazard ratio 9.1, P < 0.0001) and SP (Cox proportion hazard ratio 7.35, P = 0.001) denote a non‐response gradient to the two antimalarials extending from the international border. The gradient is probably indicative of the direction of movement of the drug‐resistant P. falciparum parasite. The utility of chloroquine as the first‐line drug under the present National Drug Policy in these areas needs reconsideration.

Estimation of Vectorial Capacity of Anopheles dirus (Diptera: Culicidae) in a Forest-Fringed Village of Assam (India)

To estimate the vectorial capacity of Anopheles dirus, the main vector of forest malaria in the northeastern region of India, in order to gain an understanding of entomological factors related to malaria transmission in forest-fringe areas of Assam, India, an isolated village in the tropical rain forest-fringed area in the district of Dibrugarh, Assam, under the influence of An. dirus alone was studied. Data on various entomological variables required for computation of the vectorial capacity were generated in each month from June 1999 to May 2000 in the field using standard techniques. Malaria prevalence was also studied during the same period in the study village and correlated with the estimated vectorial capacity of An. dirus. Vectorial capacity of An. dirus was highest, 0.779 for Plasmodium vivax (Pv) and 0.649 for Plasmodium falciparum (Pf), during the hot-monsoon season (June-September) and decreased to 0.08 (Pv) and 0.07(Pf) in the temperate postmonsoon season (October-November) before attaining zero values in the cool-dry season (December-February). With increasing temperature in the temperate premonsoon season (March-May), vectorial capacity recorded was 0.119 and 0.82 for Pv and Pf, respectively. Significant positive correlation was seen between the estimated vectorial capacity of An. dirus and the number of new Pf (r = 0.86, p < 0.001) and Pv (r = 0.69, p < 0.02) cases in the study village in different months. Thus, this study highlights the pattern of malaria transmission by An. dirus in a forest-fringe area of Assam that begins in March, peaks in July/August, subsides by November, and remains interrupted between December and February. Measures for controlling malaria in forest-fringe areas should be scheduled accordingly.

Malaria transmission risk by the mosquito Anopheles baimaii (formerly known as An. dirus species D) at different hours of the night in North-east India.

Abstract.  The risk of acquiring malaria transmitted by Anopheles baimaii Sallum & Peyton, 2005, formerly known as An. dirus species D ( Sallum et al., 2005 ) (Diptera: Culicidae), at different hours of the night in a forest‐fringed village of Assam, North‐east India was assessed through all‐night mosquito landing catches during 1995–2000. An estimated overall mean biting rate of 36.1 bites/person/night (95% CI = 26.2–45.8), a sporozoite rate of 1.9% (95% CI = 1.1–2.9%) and a parous rate of 58.7% (95% CI = 55.3–62.0%) were recorded. Parous and sporozoite‐positive females tended to be caught mainly before midnight. The effective entomological inoculation rate was the highest (0.249 positive bites/person/night) from 21.00 to 24.00 hours, suggesting that the second quartile of the night is the most risky period for malaria transmission by An. baimaii. Considering that ∼21% of mean inoculations take place before 21.00 hours, it appears that there is a need for appropriate protective measures during the pre‐bed time period to supplement the impact of insecticide‐treated nets against An. baimaii in north‐east India.

Spatial distribution and r-DNA second internal transcribed spacer characterization of Anopheles dirus (Diptera: Culicidae) complex species in north-east India.

The identity and distribution of the prevalent member species of the Anopheles dirus complex mosquitoes in the north-eastern region of India was investigated in a cross-sectional study. We altogether collected 267 individuals of An. dirus s.l. from 27 forested/forest fringed locations spread across the seven north-eastern states, identified the species using a ribosomal DNA (r-DNA) second internal transcribed spacer (ITS2) based allele specific polymerase chain reaction (ASPCR) method and sequenced the ITS2 locus in a sub set of mosquitoes. An. baimaii was identified as the main, almost exclusive (266/267), species of the Dirus complex throughout the north-east India with no intraspecific variation in the 479 base pair long ITS2 sequences in 59 of the 60 individuals sequenced. Ribosomal DNA of one individual from Assam state did not amplify in the ASPCR, possessed 786 base pair long ITS2 sequence and showed 99.7% similarity with the sequence of An. dirus species D (An. baimaii) from Yunnan province of China, later referred to as species X of the Dirus complex. These observations suggest the presence of another, possibly the new, species of the Dirus complex, sympatric with An. baimaii, in Assam warranting investigations on its distribution, biology and role in human malaria transmission in north-east India.

Spatial distribution and molecular characterization of Anopheles nivipes and Anopheles philippinensis (Diptera: Culicidae) in north-east India.

Anopheles philippinensis-nivipes species of mosquitoes are considered secondary malaria vectors in the north-east India. However, difficulty in separating An. philippinensis and An. nivipes accurately on the basis of morphological identification, due to the overlapping female wing characters, has led to the paucity of information on their spatial distribution and vectorial importance, essential for understanding malaria disease epidemiology in areas under their influence. In view of this a study was undertaken to generate authentic information on distribution of An. philippinensis and An. nivipes in the north-east India using molecular tools. Between the two, An. nivipes was recorded as the dominant species in Assam, and Nagaland states whereas An. philippinensis was found predominant in the state of Mizoram and Arunachal Pradesh. Species differentiation based on female wing characters proved wrong in about 74% specimens of An. philippinensis but found accurate in case of An. nivipes. No intra-specific variation in second internal transcribed spacer (ITS2) region of ribosomal DNA(r-DNA) in either An. nivipes or An. philippinensis from different geographical areas of north-east India was noted.

Anopheles (Cellia) maculatus group: Its spatial distribution and molecular characterization of member species in north-east India

Anopheles (Cellia) maculatus is considered a group of at least nine formally named species. Faced with the difficulty of correct morphological identification due to overlapping characters, several member species of the An. maculatus group are known to play important role in malaria transmission in the Oriental region. Current assemblage, distribution and vectorial importance of the member species within the Maculatus group is far from clear in the north-eastern region of India. Our study encompassing 410 individuals, collected from 67 geo-referenced spots across the eight north-east Indian states, identified the presence of 6 member species of the Maculatus group using the molecular tools. Anopheles dravidicus and Anopheles rampae were documented for the first time in this part of India with latter forming the new country record. While Anopheles pseudowillmori (59.5%) and An. maculatus (32%) were widely available species in most of the north-eastern states, restricted distribution of Anopheles willmori to Nagaland and that of Anopheles sawadwongporni and An. rampae to Mizoram state was noted. None of the species was found positive for human malaria parasite. While no intraspecific differences existed in the sequences of second internal transcribed spacer (ITS2) region of ribosomal DNA (r-DNA) of the member species of the Maculatus group within north-east India, few differences were detected in the sequences of An. dravidicus, An. maculatus and An. pseudowillmori from north-east India with species from the neighbouring countries.

Population attributable risk estimates for risk factors associated with hypertension in an Indian population.

Background: Little evidence exists regarding the magnitude of contribution of risk factors associated with hypertension in India. Determination of potentially modifiable risk factors is necessary to focus prevention strategies. Design: Age-matched case–control study. Methods: A total of 350 hypertensive cases and 350 controls of both sexes in the age group 20–65 years. Hypertension was defined according to JNC VII criteria. Adjusted odds ratio (OR) and population attributable risk percentage (PAR %) for hypertension were calculated. Results: In multivariate analysis, tobacco users (either tobacco chewing/smoking or both) (adjusted OR 5.1, 95% CI 3.6–7.3), tobacco chewing (adjusted OR 3.2, 95% CI 2.2–4.6), smoking (adjusted OR 2.9, 95% CI 1.9–4.4), and alcohol consumption (adjusted OR 1.5, 95% CI 1.1–2.2) was the strongest determinants of hypertension. A dose–response relation was found between the number of cigarettes smoked per day (χ2 for trend = 26.07; p < 0.0001) and the amount of alcohol consumption per day (χ2 for trend = 24.26; p < 0.0001) and the risk of hypertension. PARs were 70.3% (95% CI 63.0–77.5) for tobacco use, 45.3% (95% CI 37.1–53.4) for tobacco chewing, 31.5% (95% CI 21.3–40.9) for smoking, and 33.6% (95% CI 22.9–44.4) for alcohol consumption. Conclusion: Our results indicate that incident hypertension cases are largely attributable to the habit of tobacco use and alcohol consumption. Therefore, changing these selected lifestyle factors needs to be prioritized as a major strategy for reducing incidence of hypertension in our population.

Genetic diversity and antibody responses against Plasmodium falciparum vaccine candidate genes from Chhattisgarh, Central India: Implication for vaccine development

The genetic diversity in Plasmodium falciparum antigens is a major hurdle in developing an effective malaria vaccine. Protective efficacy of the vaccine is dependent on the polymorphic alleles of the vaccine candidate antigens. Therefore, we investigated the genetic diversity of the potential vaccine candidate antigens i.e. msp-1, msp-2, glurp, csp and pfs25 from field isolates of P.falciparum and determined the natural immune response against the synthetic peptide of these antigens. Genotyping was performed using Sanger method and size of alleles, multiplicity of infection, heterogeneity and recombination rate were analyzed. Asexual stage antigens were highly polymorphic with 55 and 50 unique alleles in msp-1 and msp-2 genes, respectively. The MOI for msp-1 and msp-2 were 1.67 and 1.28 respectively. A total 59 genotype was found in glurp gene with 8 types of amino acid repeats in the conserved part of RII repeat region. The number of NANP repeats from 40 to 44 was found among 55% samples in csp gene while pfs25 was found almost conserved with only two amino acid substitution site. The level of genetic diversity in the present study population was very similar to that from Asian countries. A higher IgG response was found in the B-cell epitopes of msp-1 and csp antigens and higher level of antibodies against csp B-cell epitope and glurp antigen were recorded with increasing age groups. Significantly, higher positive responses were observed in the csp antigen among the samples with ≥42 NANP repeats. The present finding showed extensive diversity in the asexual stage antigens.