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.

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. [...].

Diagnosis of Indigenous Non-Malarial Vector-Borne Infections from Malaria Negative Samples from Community and Rural Hospital Surveillance in Dhalai District, Tripura, North-East India.

The aetiology of non-malaria vector-borne diseases in malaria-endemic, forested, rural, and tribal-dominated areas of Dhalai, Tripura, in north-east India, was studied for the first time in the samples collected from malaria Rapid Diagnostic Kit negative febrile patients by door-to-door visits in the villages and primary health centres. Two hundred and sixty serum samples were tested for the Dengue NS1 antigen and the IgM antibodies of Dengue, Chikungunya, Scrub Typhus (ST), and Japanese Encephalitis (JE) during April 2019-March 2020. Fifteen Dengue, six JE, twelve Chikungunya, nine ST and three Leptospirosis, and mixed infections of three JE + Chikungunya, four Dengue + Chikungunya, three Dengue + JE + Chikungunya, one Dengue + Chikungunya + ST, and one Dengue + ST were found positive by IgM ELISA tests, and four for the Dengue NS1 antigen, all without any travel history. True prevalence values estimated for infections detected by Dengue IgM were 0.134 (95% CI: 0.08-0.2), Chikungunya were 0.084 (95% CI: 0.05-0.13), Scrub were 0.043 (95% CI: 0.01-0.09), and Japanese Encephalitis were 0.045 (95% CI: 0.02-0.09). Dengue and Chikungunya were associated significantly more with a younger age. There was a lack of a defined set of symptoms for any of the Dengue, Chikungunya, JE or ST infections, as indicated by the k-modes cluster analysis. Interestingly, most of these symptoms have an overlapping set with malaria; thereby, it becomes imperative that malaria and these non-malaria vector-borne disease diagnoses are made in a coordinated manner. Findings from this study call for advances in routine diagnostic procedures and the development of a protocol that can accommodate, currently, in practicing the rapid diagnosis of malaria and other vector-borne diseases, which is doable even in the resource-poor settings of rural hospitals and during community fever surveillance.

Validation of a Mobile Health Technology Platform (FeverTracker) for Malaria Surveillance in India: Development and Usability Study.

BACKGROUND: A surveillance system is the foundation for disease prevention and control. Malaria surveillance is crucial for tracking regional and temporal patterns in disease incidence, assisting in recorded details, timely reporting, and frequency of analysis. OBJECTIVE: In this study, we aim to develop an integrated surveillance graphical app called FeverTracker, which has been designed to assist the community and health care workers in digital surveillance and thereby contribute toward malaria control and elimination. METHODS: FeverTracker uses a geographic information system and is linked to a web app with automated data digitization, SMS text messaging, and advisory instructions, thereby allowing immediate notification of individual cases to district and state health authorities in real time. RESULTS: The use of FeverTracker for malaria surveillance is evident, given the archaic paper-based surveillance tools used currently. The use of the app in 19 tribal villages of the Dhalai district in Tripura, India, assisted in the surveillance of 1880 suspected malaria patients and confirmed malaria infection in 93.4% (114/122; Plasmodium falciparum), 4.9% (6/122; P vivax), and 1.6% (2/122; P falciparum/P vivax mixed infection) of cases. Digital tools such as FeverTracker will be critical in integrating disease surveillance, and they offer instant data digitization for downstream processing. CONCLUSIONS: The use of this technology in health care and research will strengthen the ongoing efforts to eliminate malaria. Moreover, FeverTracker provides a modifiable template for deployment in other disease systems.

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.

Pattern of forest recovery and carbon stock following shifting cultivation in Manipur, North-East India.

Shifting cultivation has resulted in large-scale deforestation and forest degradation in the tropics; however the abandoned fallows are known to have high potential for carbon capture. The paper is an attempt to determine the forest recovery patterns following shifting cultivation by evaluating the tree species composition, diversity and abundance with respect to topographical factors in Manipur, India. We also used ordination analysis to understand the change in species composition with regard to environmental variables. The living woody biomass carbon of each fallow was quantified, and the factors affecting the recovery of carbon stock along an increasing fallow gradient was assessed. Our results showed that the species richness and basal area recovered relatively with time since abandonment, and the north-facing lower elevation fallow sites displayed higher species richness and stem density than those in higher elevations. Environmental variables had no impact on the regeneration of Elaeocarpus floribundus Blume and Castanopsis hystrix Hook. f. & Thomson ex A. DC. which suggests that they may be capable of effective restoration of degraded forest areas. As these species appear naturally in the forests, it would facilitate quicker rehabilitation and reinstate the soil nutrients making the soil reusable in a short term. We also found that fallow age plays a vital role in recovering above-ground biomass carbon from living woody species followed by the aspect of the site. The total living woody biomass carbon ranged from 0.98 Mg ha-1 in 5 years fallow to 142.58 Mg ha-1 in 20 years fallow. The above-ground biomass carbon recovery of the oldest fallow was 39% to 40% of the reference undisturbed forest and the estimated time for the shifting cultivation fallows to reach that of the undisturbed forest level was approximately 39 years to 41 years.