First Report of Anopheles annularis s.l., An. maculatus s.s., and An. culicifacies s.l. as Malaria Vectors and a New Occurrence Record for An. pseudowillmori and An. sawadwongporni in Alipurduar District Villages, West Bengal, India.

A comprehensive entomological survey was undertaken in Alipurduar District, West Bengal, from 2018 to 2020 and in 2022. This study was prompted by reported malaria cases and conducted across nine villages, seven Sub-Centres, and three Primary Health Centres (PHCs). Mosquitoes were hand-collected with aspirators and flashlights from human dwellings and cattle sheds during the daytime. Both morphological and molecular techniques were used for species identification. Additionally, mosquitoes were tested for Plasmodium parasites and human blood presence. Mosquito species such as An. barbirostris s.l., An. hyrcanus s.l., An. splendidus, and An. vagus were morphologically identified. For species like An. annularis s.l., An. minimus s.s., An. culicifacies s.l., and An. maculatus s.s., a combination of morphological and molecular techniques was essential. The mitochondrial cytochrome c oxidase gene subunit 1 (CO1) was sequenced for An. annularis s.l., An. maculatus s.s., An. culicifacies s.l., An. vagus, and some damaged samples, revealing the presence of An. pseudowillmori and An. fluviatilis. The major Anopheles species were An. annularis s.l., An. culicifacies s.l., and An. maculatus s.s., especially in Kumargram and Turturi PHCs. Plasmodium positivity was notably high in An. annularis s.l. and An. maculatus s.s. with significant human blood meal positivity across most species. Morphological, molecular, and phylogenetic analyses are crucial, especially for archived samples, to accurately identify the mosquito fauna of a region. Notably, this study confirms the first occurrence of An. pseudowillmori and An. sawadwongporni in West Bengal and implicates An. maculatus s.s., An. culicifacies s.l., and An. annularis s.l. as significant vectors in the Alipurduar region.

Molecular Evidence of Wolbachia Species in Wild-Caught Aedes albopictus and Aedes aegypti Mosquitoes in Four States of Northeast India.

Wolbachia, a Gram-negative intracellular bacterium, naturally infects many arthropods, including mosquito vectors responsible for the spread of arboviral diseases such as Zika, chikungunya, and dengue fever. Certain Wolbachia strains are involved in inhibiting arbovirus replication in mosquitoes, and this phenomenon is currently being studied to combat disease vectors. A study was conducted in four states in north-eastern India to investigate the presence of natural Wolbachia infection in wild-caught Aedes albopictus and Aedes aegypti mosquitoes, the established vectors of dengue. The detection of a Wolbachia infection was confirmed by nested PCR and sequencing in the two mosquito species Ae. aegypti and Ae. albopictus. Positivity rates observed in Ae. aegypti and Ae. albopictus pools were 38% (44 of 115) and 85% (41 of 48), respectively, and the difference was significant (chi-square = 28.3174, p = 0.00000010). Sequencing revealed that all detected Wolbachia strains belonged to supergroup B. Although Wolbachia infection in Ae. aegypti has been previously reported from India, no such reports are available from north-eastern India. Data on naturally occurring Wolbachia strains are essential for selecting the optimal strain for the development of Wolbachia-based control measures. This information will be helpful for the future application of Wolbachia-based vector control measures in this part of the country.

First Report of Rubber Collection Bowls & Plastic and Bamboo Water Containers as the Major Breeding Source of Ae. albopictus with the Indigenous Transmission of Dengue and Chikungunya in Rural Forested Malaria-Endemic Villages of Dhalai District, Tripura, India: The Importance of Molecular Identification.

BACKGROUND: With the reports of indigenous cases of dengue and chikungunya in the forest-covered rural tribal malaria-endemic villages of Dhalai District, Tripura, India, an exploratory study was undertaken to identify the vector breeding sites. METHODS: From June 2021 to August 2022, mosquito larvae were collected from both natural and artificial sources in the villages, house premises, and their nearby forested areas outside of the houses. Other than morphological characterisation, Aedes species were confirmed by polymerase chain reaction targeting both nuclear (ITS2) and mitochondrial genes (COI) followed by bidirectional Sanger sequencing. RESULTS: Aedes albopictus was abundantly found in this area in both natural and artificial containers, whereas Ae. aegypti was absent. Among the breeding sources of molecularly confirmed Ae. albopictus species, rubber collection bowls were found to be a breeding source reported for the first time. Plastic and indigenously made bamboo-polythene containers for storing supply water and harvesting rainwater in the villages with a shortage of water were found to be other major breeding sources, which calls for specific vector control strategies. Natural sources like ponds and rainwater collected on Tectona grandis leaves and Colocasia axil were also found to harbour the breeding, along with other commonly found sources like bamboo stumps and tree holes. No artificial containers as a breeding source were found inside the houses. Mixed breeding was observed in many containers with other Aedes and other mosquito species, necessitating molecular identification. We report six haplotypes in this study, among which two are reported for the first time. However, Aedes aegypti was not found in the area. Additionally, rubber collection bowls, ponds, and water containers also showed the presence of Culex quinquefasciatus and Culex vishnui, known JE vectors from this area, and reported JE cases as well. Different Anopheles vector spp. from this known malaria-endemic area were also found, corroborating this area as a hotbed of several vectors and vector-borne diseases. CONCLUSIONS: This study, for the first time, reports the breeding sources of Aedes albopictus in the forested areas of Tripura, with rubber collection bowls and large water storage containers as major sources. Also, for the first time, this study reports the molecular characterisation of the Ae. albopictus species of Tripura, elucidating the limitations of morphological identification and highlighting the importance of molecular studies for designing appropriate vector control strategies. The study also reports the co-breeding of JE and malaria vectors for the first time in the area reporting these vector-borne diseases.

Reply to Deora et al. Multiplexing for Plasmodium spp.? Think Again! Comment on "Bhowmick et al. Dry Post Wintertime Mass Surveillance Unearths a Huge Burden of P. vivax, and Mixed Infection with P. vivaxP. falciparum, a Threat to Malaria Elimination, in Dhalai, Tripura, India. Pathogens 2021, 10, 1259".

We thank Deora et al. [...].

Genetic polymorphism of Plasmodium falciparum msp-1, msp-2 and glurp vaccine candidate genes in pre-artemisinin era clinical isolates from Lakhimpur district in Assam, Northeast India.

Background: Northeast India shares its international border with Southeast Asia and has a number of malaria endemic zones. Monitoring genetic diversity of malaria parasites is important in this area as drug resistance and increasing genetic diversity form a vicious cycle in which one favours the development of the other. This retrospective study was done to evaluate the genetic diversity patterns in Plasmodium falciparum strains circulating in North Lakhimpur area of Assam in the pre-artemisinin era and compare the findings with current diversity patterns. Methods: Genomic DNA extraction was done from archived blood spot samples collected in 2006 from malaria-positive cases in Lakhimpur district of Assam, Northeast India. Three antigenic markers of genetic diversity were studied - msp-1 (block-2), msp-2 (block-3) and the glurp RII region of P. falciparum using nested PCR. Results: Allelic diversity was examined in 71 isolates and high polymorphism was observed. In msp-1, eight genotypes were detected; K1 (single allele), MAD20 (six different alleles) and RO33 (single allele) allelic families were noted. Among msp-2 genotypes, 22 distinct alleles were observed out of which FC27 had six alleles and IC/3D7 had 16 alleles. In RII region of glurp, nine genotypes were obtained. Expected heterozygosity (H E) values of the three antigenic markers were 0.72, 0.81 and 0.88, respectively. Multiplicity of infection (MOI) values noted were 1.28, 1.84 and 1.04 for msp-1, msp-2 and glurp, respectively. Conclusion: Results suggest a high level of genetic diversity in P. falciparum msp (block-2 of msp-1 and block-3 of msp-2) and the glurp RII region in Northeast India in the pre-artemisinin era when chloroqunine was the primary drug used for uncomplicated falciparum malaria. Comparison with current studies have revealed that the genetic diversity in these genes is still high in this region, complicating malaria vaccine research.

Genetic diversity of Plasmodium falciparum AMA-1 antigen from the Northeast Indian state of Tripura and comparison with global sequences: implications for vaccine development.

BACKGROUND: Malaria continues to be a major public health problem in the Northeastern part of India despite the implementation of vector control measures and changes in drug policies. To develop successful vaccines against malaria, it is important to assess the diversity of vaccine candidate antigens in field isolates. This study was done to assess the diversity of Plasmodium falciparum AMA-1 vaccine candidate antigen in a malaria-endemic region of Tripura in Northeast India and compare it with previously reported global isolates with a view to assess the feasibility of developing a universal vaccine based on this antigen. METHODS: Patients with fever and malaria-like illness were screened for malaria and P. falciparum positive cases were recruited for the current study. The diversity of PfAMA-1 vaccine candidate antigen was evaluated by nested PCR and RFLP. A selected number of samples were sequenced using the Sanger technique. RESULTS: Among 56 P. falciparum positive isolates, Pfama-1 was successfully amplified in 75% (n = 42) isolates. Allele frequencies of PfAMA-1 antigen were 16.6% (n = 7) for 3D7 allele and 33.3% (n = 14) in both K1 and HB3 alleles. DNA sequencing revealed 13 haplotypes in the Pfama-1 gene including three unique haplotypes not reported earlier. No unique amino-acid substitutions were found. Global analysis with 2761 sequences revealed 435 haplotypes with a very complex network composition and few clusters. Nucleotide diversity for Tripura (0.02582 ± 0.00160) showed concordance with South-East Asian isolates while recombination parameter (Rm = 8) was lower than previous reports from India. Population genetic structure showed moderate differentiation. CONCLUSIONS: Besides documenting all previously reported allelic forms of the vaccine candidate PfAMA-1 antigen of P. falciparum, new haplotypes not reported earlier, were found in Tripura. Neutrality tests indicate that the Pfama-1 population in Tripura is under balancing selection. This is consistent with global patterns. However, the high haplotype diversity observed in the global Pfama-1 network analysis indicates that designing a universal vaccine based on this antigen may be difficult. This information adds to the existing database of genetic diversity of field isolates of P. falciparum and may be helpful in the development of more effective vaccines against the parasite.

Dry Post Wintertime Mass Surveillance Unearths a Huge Burden of P. vivax, and Mixed Infection with P. vivax P. falciparum, a Threat to Malaria Elimination, in Dhalai, Tripura, India.

With India aiming to achieve malaria elimination by 2030, several strategies have been put in place. With that aim, mass surveillance is now being conducted in some malaria-endemic pockets. As dry season mass surveillance has been shown to have its importance in targeting the reservoir, a study was undertaken to assess the parasite load by a sensitive molecular method during one of the mass surveys conducted in the dry winter period. It was executed in two malaria-endemic villages of Dhalai District, Tripura, in northeast India, also reported as P. falciparum predominated area. The present study found an enormous burden of Rapid Diagnostic Test negative malaria cases with P. vivax along with P. vivax and P. falciparum mixed infections during the mass surveillance from febrile and afebrile cases in dry winter months (February 2021-March 2021). Of the total 150 samples tested, 72 (48%) were positive and 78 (52%) negative for malaria by PCR. Out of the 72 positives, 6 (8.33%) were P. falciparum, 40 (55.55%) P. vivax, and 26 (36.11%) mixed infections. Out of 78 malaria negative samples, 6 (7.7%) were with symptoms, while among the total malaria positive, 72 cases 7 (9.8%) were with symptoms, and 65 (90.2%) were asymptomatic. Out of 114 samples tested by both microscopy and PCR, 42 samples turned out to be submicroscopic with 4 P. falciparum, 23 P. vivax, and 15 mixed infections. Although all P. vivax submicroscopic infections were asymptomatic, three P. falciparum cases were found to be febrile. Evidence of malaria transmission was also found in the vectors in the winter month. The study ascertained the use of molecular diagnostic techniques in detecting the actual burden of malaria, especially of P. vivax, in mass surveys. As Jhum cultivators in Tripura are at high risk, screening for the malarial reservoirs in pre-Jhum months can help with malaria control and elimination.

Denominators: An attempt to adjust for cancer incidence and mortality rates and its role in cancer registries.

Cancer is an important public health burden in India and around the Globe. Incident cases of cancer are reported continuously, and trends in incidence rates vary among five yearly age group. In India National census is carried out after every 10 years since 1951, the latest census is carried out in 2011. As Incident cases of cancer are reported continuously for 5 year age group and estimation of rates and trends for a given year of interest fall in-between two census period population estimation of five yearly age group, which serves as Denominator sense importance. Denominator serves as the most essential part of any cancer registry program as it used for calculation of various rates for five yearly age group such as age-standardized rate, crude rate, truncated rate, cumulative risk etc., Calculated by different registries and Individual Researchers, Government organizations, etc. Currently most of the registries used individual exponential growth rate and differential distribution method to estimate the growth rate. However, it is found that the calculated rates and risk be suffering from bias most of the time since some method over and underestimate the growth rate while some does not able to maintain consistency. Here, an attempt is made to adjust the world standard population with two census period to estimate the five yearly age group for a given year of interest of a geographical region falls in-between two census period. It is also shown here that how the proposed model "age-adjusted population growth model" maintain the overall growth as in exponential growth model and difference distribution method also maintains the estimated growth rate is as found in difference distribution method. Further, it is tried to highlight the points how these method overcomes the problems compared with other methods that are currently used by registries.

Robertsonian translocation T (21; 21) in a female born to normal parents: a case report.

Downs syndrome (DS) occurs due to an extra copy of chromosome 21. About 3% of cases of Downs syndrome occur due to Robertsonian translocation, most commonly t (14; 21), other types of translocations are very rare cause of the syndrome. A 10-year-old patient with mental retardation was admitted following road traffic accident. Patient had flabby muscles, had delayed mile stones, stunted growth for the age, slanting of eyes, flat nasal bridge, and ineligible speech. On cytogenetic analysis the patient had karyotype showing one normal chromosome 21 and one Robertsonian translocation t (21; 21). Parents and siblings of the patient were phenotypically normal. Robertsonian translocation t (21; 21), can occur by transmission from carrier parent, due to ovarian mosaicism for Robertsonian translocation or may appear de novo. In the present case as the parents had normal karyotype and siblings were phenotypically normal, Robertsonian translocation probably have arisen de novo. The present case was a case of Downs syndrome with Robertsonian translocation t (21;21) probably arising de novo.

Cancer: scenario and relationship of different geographical areas of the globe with special reference to North East-India.

BACKGROUND: Cancer is becoming the most important public health burden around the globe. As per the GLOBOCAN 2008 estimates, about 12.7 million cancer cases and 7.6 million cancer deaths were estimated to have occurred in 2008. The burden of cancer cases for India in the year 2020 is calculated to be 1,148,757 (male 534,353; female 614,404) compared to 979,786 in 2010. The pattern of cancer incidence is varying among geographical regions, esophageal cancer for example being high in China, lung cancer in USA, and gallbladder cancer in Chile. The question remains why? Is it due to the diversity in genome pool, food habits, risk factor association and role of genetic susceptibility or some other factors associated with it? In India, the North East (NE)-India region is seeing a marked increase in cancer incidence and deaths, with a very different cancer incidence pattern compared to mainland India. The genome pool of the region is also quite distinct from the rest of India. Northeastern tribes are quite distinct from other groups; they are more closely related to East Asians than to other Indians. In this paper an attempt was made to see whether there is any similarity among the pattern of cancer incidence cases for different sites of NE-India region to South or East-Asia. MATERIALS AND METHODS: Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA), Pearson Correlation coefficient test was assessed to evaluate the linkage of North-East India region to other regions. A p value <0.05 was considered as statistically significant. RESULTS: The results clearly shows that there are similarities in occurrence of cancer incidence patterns for various cancer sites of NE-India with South and East-Asian regions, which may lead to the conclusion that there might be a genetic linkage between these regions.

Presence of three distinct genotypes within the Paragonimus westermani complex in northeastern India.

The name Paragonimus westermani (Kerbert, 1878) is commonly applied to members of a species complex that includes the well-known Asian lung fluke of medical and veterinary importance. Unambiguous molecular and morphological evidence showing the presence of a member of the complex in India has recently been published. In the present study we report the occurrence of 2 more members of the P. westermani complex in northeastern (NE) India. Surveys of the freshwater crabs Maydelliatelphusa lugubris in NE India revealed 2 morphologically distinct types of lung fluke metacercariae. Phylogenetic analyses, using DNA sequences from ITS2, 28S and cox1 gene regions indicate that these lung metacercariae belong to P. westermani complex. Type 1 metacercariae have a more basal position within the complex whereas type 2 metacercariae are closely related to the relatively derived forms of P. westermani from NE Asia (Japan, Korea, China) and Vietnam. A third type of metacercaria (type 3), detected in another crab host, Sartoriana spinigera in Assam, was phylogenetically close to P. siamensis, also a member of the P. westermani group. Molecular evidence has demonstrated the existence of 3 genotypes of lung flukes within the Paragonimus westermani complex in NE India. Two of these were previously unknown.