Nation-wide vector surveillance on Zika and Dengue did not indicate transmission of the American lineage-pandemic Zika virus in India.

OBJECTIVES: Following the Public Health Emergency of International Concern declared on Zika by the World Health Organization during 2016, the Indian Council of Medical Research carried out nationwide vector surveillance for Zika and Dengue viruses (ZIKV and DENV) in India as a preparedness measure in 2016-19. METHODS: High-risk zones distributed to 49 Districts in 14 states/union territories were included in the study. Seven ICMR institutions participated, following a standard operating protocol. Aedes specimens sampled weekly were processed by multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) for ZIKV/DENV and random samples crosschecked with real-time RT-PCR for ZIKV. RESULTS: Altogether, 79 492 Aedes specimens in 6492 pools were processed; 3 (0.05%) and 63 (0.97%) pools, respectively, were found positive for ZIKV and DENV. ZIKV infections were recorded in Aedes aegypti sampled during the 2018 sporadic Zika outbreak in Jaipur, Rajasthan. However, these belonged to the Asian lineage of the virus, already circulating in the country. Both Ae. aegypti and Aedes albopictus distributed to 8 states/union territories were found to be infected with DENV. Both sexes of Ae. albopictus were infected, indicating transovarial transmission. CONCLUSION: This investigation evinced no active transmission of the American lineage-pandemic Zika virus in India during the pandemic period.

Characterization of drug resistance and genetic diversity of Plasmodium falciparum parasites from Tripura, Northeast India.

Monitoring of anti-malarial drug resistance is vital in Northeast India as this region shares its international border with Southeast Asia. Genetic diversity of Plasmodium parasites regulates transmission dynamics, disease severity and vaccine efficacy. P. falciparum chloroquine resistance transporter (Pfcrt), multidrug resistance-1 (Pfmdr-1) and kelch 13 propeller (PfK-13) genes which govern antimalarial drug resistance and three genetic diversity markers, merozoite surface protein 1 and 2 (Pfmsp-1, Pfmsp-2) and glutamate rich protein (Pfglurp) were evaluated from Tripura, Northeast India using molecular tools. In the Pfcrt gene, 87% isolates showed triple mutations at codons M74I, N75E and K76T. 12.5% isolates in Pfmdr-1 gene showed mutation at N86Y. No polymorphism in PfK-13 propeller was found. Polyclonal infections were observed in 53.85% isolates and more commonly in adults (p = 0.0494). In the Pfmsp-1 locus, the K1 allelic family was predominant (71.2%) followed by the 3D7/IC family (69.2%) in the Pfmsp-2 locus. RII region of Pfglurp exhibited nine alleles with expected heterozygosity of 0.85. The multiplicity of infection for Pfmsp-1, Pfmsp-2 and Pfglurp were 1.56, 1.31 and 1.06 respectively. Overall, the study demonstrated a high level of chloroquine resistance and extensive parasite diversity in the region, necessitating regular surveillance in this population group.

Antifolate drug resistance: Novel mutations and haplotype distribution in dhps and dhfr from Northeast India.

Malaria is a major public health concern in Northeast India with a preponderance of drug-resistant strains. Until recently the partner drug for artemisinin combination therapy (ACT) was sulphadoxine pyrimethamine (SP). Antifolate drug resistance has been associated with the mutations at dihydropteroate synthase (dhps) and dihydrofolatereductase (dhfr) genes. This study investigated antifolate drug resistance at the molecular level. A total of 249 fever cases from Arunachal Pradesh, NE India, were screened for malaria, and of these, 75 were found to be positive for Plasmodium falciparum. Samples were sequenced and analysed with the help of BioEdit and ClustalW. Three novel point mutations were found in the dhps gene with 10 haplotypes along with the already reported mutations. A single haplotype having quadruple mutation was found in the dhfr gene. The study reports higher degree of antifolate drug resistance as evidenced by the presence of multiple point mutations in dhps and dhfr genes. The findings of this study strongly discourage the use SP as a partner drug in ACT.

Mitochondrial DNA-based genetic diversity of Anopheles nivipes in North East India.

Malaria is a major public health problem in north-east India mainly transmitted by Anopheles baimai and An. minimus while Anopheles nivipes plays an important supportive role. The genetic diversity of An. nivipes in north-east India was investigated by employing two mitochondrial DNA markers namely NADH dehydrogenase 5 (ND5) and cytochrome oxidase sub unit II (COII). High genetic diversity in An. nivipes was observed by the detection of 16 haplotypes among 30 sequences of ND5 gene and 29 haplotypes among 29 COII sequences. Anopheles nivipes of north-east India was significantly differentiated genetically with that of neighboring South-east Asian An. nivipes as revealed by pairwise FST values of 0.127 (p < 0.01) and 0.044 (p < 0.001) for ND5 and COII genes, respectively, suggesting geographical barriers to gene flow in this species between the two geographical areas resulting in significant population structuring.

Larvicidal activity of few select indigenous plants of North East India against disease vector mosquitoes (Diptera: Culicidae).

Mosquitoes are the vectors of several life threatening diseases like dengue, malaria, Japanese encephalitis and lymphatic filariasis, which are widely present in the north-eastern states of India. Investigations on five local plants of north-east India, selected on the basis of their use by indigenous communities as fish poison, were carried out to study their mosquito larvicidal potential against Anopheles stephensi (malaria vector), Stegomyia aegypti (dengue vector) and Culex quinquefasciatus (lymphatic filariasis vector) mosquitoes. Crude Petroleum ether extracts of the roots of three plants viz. Derris elliptica, Linostoma decandrum and Croton tiglium were found to have remarkable larvicidal activity; D. elliptica extract was the most effective and with LC50 value of 0.307 µg/ml its activity was superior to propoxur, the standard synthetic larvicide. Half-life of larvicidal activity of D. elliptica and L. decandrum extracts ranged from 2-4 days.

Genotyping of chloroquine resistant Plasmodium falciparum in wild caught Anopheles minimus mosquitoes in a malaria endemic area of Assam, India.

We validated the feasibility of using Plasmodium falciparum, the human malaria parasite, DNA present in wild caught vector mosquitoes for the characterization of chloroquine resistance status. House frequenting mosquitoes belonging to Anopheles minimus complex were collected from human dwellings in a malaria endemic area of Assam, Northeast India and DNA was extracted from the head-thorax region of individual mosquitoes. Anopheles minimus complex mosquitoes were identified to species level and screened for the presence of Plasmodium sp. using molecular tools. Nested PCR-RFLP method was used for genotyping of P. falciparum based on K76T mutation in the chloroquine resistance transporter (pfcrt) gene. Three of the 27 wild caught An. minimus mosquitoes were harbouring P. falciparum sporozoites (positivity 11.1%) and all 3 were had 76T mutation in the pfcrt gene, indicating chloroquine resistance. The approach of characterizing antimalarial resistance of malaria parasite in vector mosquitoes can potentially be used as a surveillance tool for monitoring transmission of antimalarial drug resistant parasite strains in the community.

Genetic analyses of ribosomal loci of Anopheles minimus species from north east India.

Anopheles minimus is one of the major vectors for transmission of malaria disease in north eastern (NE) region of India. The minimus species complex of Minimus subgroup of Myzomyia series of anophelines were studied in malaria affected states--Assam and Arunachal Pradesh (AP) of NE India. Ribosomal DNA markers--second internal transcribed spacer (ITS2) and third domain (D3) of 28S gene were used to characterize An. minimus species. Sequence homogeneity was observed in D3 sequences of An.minimus specimens throughout both the states. However, a transversion in ITS2 sequence of single specimen collected from Assam-Meghalaya border areas illustrates possibility of intra population polymorphism in ITS2 sequence within the geographical region.

In vitro and in vivo antiplasmodial activity of the root extracts of Brucea mollis Wall. ex Kurz.

Malaria control is compromised worldwide by continuously evolving drug-resistant strains of the parasite demanding exploration of natural resources for developing newer antimalarials. The northeastern region of India is endemic for malaria characterized by high prevalence of drug-resistant Plasmodium falciparum strains. Many plants are used by the indigenous communities living in the northeast India in their traditional system of medicine for the treatment of malarial fever. Folklore claim of antimalarial property of one such plant Brucea mollis was evaluated in vitro and in vivo for antiplasmodial activity. Crude extracts from dried B. mollis root powder were prepared through soxhlet extraction using petroleum ether, methanol, and water sequentially. Methanol extract was further partitioned between chloroform and water. These extracts were tested in vitro against laboratory-adapted chloroquine-sensitive and chloroquine-resistant strains of P. falciparum. In in vitro evaluation, extracts were found more active on the chloroquine-sensitive strain. Methanolic-chloroform (IC(50) 5.1 µg ml(-1)) and methanolic-aqueous (IC(50) 13.9 µg ml(-1)) extracts recorded significant in vitro antiplasmodial activity which was also supported by their promising in vivo activity (ED(50) 72 and 30 mg kg(-1) bw day(-1), respectively) against chloroquine-resistant Plasmodium yoelli N-67 strain in Swiss albino mice. Methanolic-aqueous extract-treated mice survived on average for 14 days that was comparable to the reference drug chloroquine. This is the first report of antiplasmodial activity of B. mollis validating the traditional use of this plant as antimalarial in the northeast India and calls for further detailed investigations.

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.

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.

Molecular evidence for the involvement of Anopheles nivipes (Diptera: Culicidae) in the transmission of Plasmodium falciparum in north-eastern India.

The Anopheles philippinensis and An. nivipes mosquitoes that form part of the An. annularis species group are morphologically very similar and difficult to differentiate as adults. In consequence, researchers generally refer to all field-collected individuals of these species simply as the An. philippinensis-nivipes species complex. Although this species complex is understood to play a role in the transmission of parasites causing human malaria in north-eastern India, the identity of the exact species involved, and the relative importance of the local An. philippinensis and An. nivipes, are far from clear. To settle this issue, house-frequenting female adults of the An. philippinensis-nivipes species complex were collected from 23 localities in the six north-eastern states, identified to species (using an allele-specific PCR to explore the insects' ribosomal-DNA internal-transcribed-spacer-2 sequences) and checked for Plasmodium DNA (using a nested PCR based on the 18S subunit of the parasite's ribosomal DNA). Of the 337 females of the An. philippinensis-nivipes species complex that were investigated, 275 were identified as An. nivipes and 62 as An. philippinensis. Malarial infection was detected in the heads/thoraces of just two specimens, with P. falciparum DNA detected in one An. nivipes from Nagaland state and one An. nivipes from Assam state. These results provide unambiguous evidence of the presence of both An. philippinensis and An. nivipes in the north-eastern region of India and the involvement of An. nivipes in transmitting P. falciparum in this area.

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.

Mosquito biodiversity of Dibru-Saikhowa biosphere reserve in Assam, India.

Entomological surveys were conducted for three consecutive years in core and buffer zone of the Dibru-Saikhowa biosphere reserve in pre monsoon, monsoon and post-monsoon seasons. A total of 52 species of mosquitoes under eleven genera have been detected. The genus Anopheles (18 species) was the predominant followed by Culex, Aedes, Mansonia, Armigeres, Mimomyia, Ochlerostatus, Malaya, Toxorhynchites, Ficalbia and Aedeomyia. The buffer zone of the forest reserve where human habitations are there exhibited the presence of maximum number of species (49 species under 10 genera) in comparison to core zone (42 species under 10 genera). In buffer zone, maximum numbers of species (38) were recorded in monsoon season followed by post- monsoon (35 species) and Pre-monsoon season (34 species). Whereas in core zone, maximum number of species were collected in post monsoon season followed by pre monsoon season and monsoon season. In Core and buffer zone, the maximum species were recorded from the ground pool habitat and slow flowing stream respectively. Among the disease vectors, the potential Japanese encephalitis vectors incriminated in India were very much prevalent. This study provides the list of available mosquito species recorded for the first time in the Dibru-Saikhowa biosphere reserve.

Evaluation of PermaNet 2.0 mosquito bednets against mosquitoes, including Anopheles minimus s.l., in India.

Wash resistance and field bioefficacy of PermaNet 2.0 nets, long lasting insecticidal nets, against mosquitoes were evaluated in Assam, northeastern India. After repeated hand washings at 12-day intervals a decline in the mosquito killing ability of PermaNet nets was noted (trend chi2 38.9, p < 0.0001), yet these nets retained good insecticidal efficacy for up to 15 wash cycles, producing a 72.5% mean mortality of An. minimus 24 hours after a 3-minute exposure in World Health Organization cones after 15 washings. Significantly fewer (p < 0.001) mosquitoes were captured in self-baited landing collections in houses equipped with PermaNet nets than in houses using untreated nets in the case of culicines (p < 0.001) but not with anophelines, including An. minimus. The use of PermaNet nets resulted in noticeably fewer bites from Culex pseudovishnui (68.5%) and Cx. quinquefasciatus (70%). Blood-feeding inhibition of mosquitoes in deliberately torn PermaNet nets was seen, indicating a protective effect for those sleeping under these nets. No serious adverse effects of the PermaNet nets were reported by users.

Armigeres (Armigeres) mahantai, a new mosquito species from India.

Female and male adults of Armigeres (Armigeres) mahantai, a new mosquito species, are described from Meghalaya state of northeastern India.

Detection & molecular confirmation of a focus of Plasmodium malariae in Arunachal Pradesh, India.

BACKGROUND & OBJECTIVE: During a malaria epidemiological study in Arunachal Pradesh, Plasmodium malariae like human malaria parasites were seen in blood smears from fever cases. The study was undertaken to detect the presence of P. malariae and to confirm its identity through DNA based polymerase chain reaction approach. METHODS: Fever survey was carried out in 22 villages in Indo-Myanmar bordering district of Lohit, Arunachal Pradesh in 2005. Morphologically suspected P. malariae cases were confirmed using nested PCR based on 18S small subunit ribosomal DNA gene sequence. RESULTS: Screening of 1,995 fever cases resulted in 9 probable cases of P. malariae based on morphological identification in Chakma tribe people residing in 2 villages. Nested PCR confirmed the identity of all probable cases of P. malariae by producing diagnostic band of 144 bp. PCR method was able to detect mixed infection of P. malariae with P. vivax and with P. falciparum. INTERPRETATION & CONCLUSION: P. malariae may have been present in Arunachal Pradesh but most probably is being misdiagnosed due to its close resemblance with P. vivax, especially in ring forms. Estimation of actual case load of P. malariae in north-east India is, therefore, important with accurate species identification using molecular methods.

Community practices of using bed nets & acceptance & prospects of scaling up insecticide treated nets in north-east India.

BACKGROUND & OBJECTIVE: The National Vector Borne Disease Control Programme (NVBDCP), India has been promoting and scaling up the use of insecticide-treated nets (ITNs) in the tribal dominated malarious areas of north-east India. But, information on sleeping habits, bed net ownership and use practices, and feedback of communities in ITN--targeted areas is needed for formulating a strategic framework for upscaling the coverage of ITNs. We carried out a community-based cross-sectional survey in select areas of Nagaland and Mizoram (where ITNs were introduced) along with Assam (where ITNs were not introduced) to know the response of community. METHODS: Following large scale introduction of ITNs during 2001-2002 in the north-eastern States of Nagaland and Mizoram by NVBDCP, India, a cross-sectional community-based survey was undertaken in April-May, 2003 covering 435 households of Nagaland and 464 households in Mizoram, using a structured questionnaire, to assess the demographic variables relevant to bed net use, bed net washing practices and acceptability of ITNs etc., for upscaling the coverage of ITNs in the surveyed communities. A total of 448 households in 8 villages in a non-ITN PHC area of Assam were served as the control area. RESULTS: The average bet net usage per family (2.01 to 2.65). Bed net use was mostly seasonal in Nagaland where a majority (65.1-78.7%) used bed nets only during summers and monsoon as compared to the year round use in Mizoram (83.5%) and Assam (78.9%). Frequent washing of nets was most common in Assamese communities with 77 per cent households washing their nets at least once in a month. More than two third users favoured use of ITNs over the conventional indoor residual spray of DDT for malaria control. INTERPRETATION & CONCLUSION: ITNs are widely acceptable in the user communities and a demand for ITNs is evident in the non-user communities of north-east India. Approach of treating community owned nets through an efficient service delivery mechanism will be a viable option for upscaling the ITN coverage.

Addition of three species of Topomyia (Diptera: Culicidae) to the mosquito fauna of Northeastern India.

Topomyia (Topomyia) hirtusa, Topomyia (Topomyia) bifurcata, and Topomyia (Suaymyia) cristata are recorded for the first time in India from the northeastern state of Arunachal Pradesh.

Molecular characterization and species identification of the Anopheles dirus and An. minimus complexes in north-east India using r-DNA ITS-2.

The sibling species composition of the Anopheles minimus and Anopheles dirus complexes is poorly known in the highly malarious north-eastern region of India where these two vector taxa are accountable for most of the malaria transmission among 30.7 million inhabitants. Prevalent members of these two complexes in this part of India were identified using sequences for the second internal transcribed spacer (ITS2) of ribosomal DNA. Anopheles baimaii (species D) of the An. dirus complex and An. minimus s.s. (species A) of the An. minimus complex were detected in Arunachal Pradesh, Assam, Meghalaya and Nagaland states. No intraspecific variation was observed in the ITS2 sequence (479bp) of An. baimaii whereas a single substitution was detected in the ITS2 sequence (372bp) of An. minimus from Nagaland state.

Characterization of ITS2 rDNA of Anopheles philippinensis and Anopheles nivipes (Diptera: Culicidae) from north-east India.

Anopheles (Cellia) philippinensis Ludlow and Anopheles (Cellia) nivipes (Theobald) are two closely related, morphologically very similar, mosquito species in the Annularis group, which play a supportive role in malaria transmission in north-east India. We amplified and performed sequence analysis for the internal transcribed spacer 2 (ITS2) locus of ribosomal DNA (rDNA) gene of morphologically confirmed specimens of these two species from the states of Assam and Nagaland. An. philippinensis and An. nivipes shared 85.2% sequence similarity and no intra-species variation was found in the nucleotide sequences of the two species. Overall, sequence data of the ITS2 marker revealed that both these species from north-east India differed by as much as have been reported from specimens of eastern Thailand.