India's current malaria elimination success relies on targeting Plasmodium falciparum and Plasmodium vivax. However, sustaining complete elimination requires expanding beyond this species-centric focus. Embracing a broader One Health paradigm is essential for integrating hidden parasite diversity, addressing zoonotic malaria threats, and implementing risk-based surveillance to detect emerging transmission pathways early.

BACKGROUND:Vector-borne diseases (VBDs) continue to pose major public health challenges, particularly in tropical and subtropical regions. Conventional vector control strategies, including insecticide spraying, insecticide-treated bed nets, and larvicidal interventions, have substantially reduced disease burden; however, the emergence of insecticide resistance has compromised the long-term effectiveness of these approaches. Consequently, environmentally sustainable biological and genetic vector control methods are increasingly being explored as alternative strategies. MATERIALS AND METHODS:This review comprehensively evaluates recent advances in biological and genetic approaches for mosquito vector control. The article synthesizes evidence from published laboratory, semi-field, and field studies on Wolbachia-mediated pathogen blocking, paratransgenesis using Asaia and Serratia, entomopathogenic fungi, and genetic control tools, including the sterile insect technique (SIT), incompatible insect technique (IIT), release of insects carrying a dominant lethal (RIDL), and CRISPR-Cas9-based gene drive systems. RESULTS:Multiple biological and genetic interventions demonstrated substantial reductions in mosquito populations and pathogen transmission under experimental and field conditions. Wolbachia-based systems, SIT, IIT, and RIDL approaches showed promising operational feasibility and field validation, whereas gene drive technologies exhibited strong transformative potential but remain largely experimental. However, significant challenges persist, including scalability, infrastructure requirements, ecological uncertainty, biosafety concerns, regulatory complexity, and ethical considerations. Comparative evaluation indicates that these approaches differ considerably in technological maturity, deployment readiness, and sustainability. CONCLUSIONS:Emerging biological and genetic vector control strategies represent promising alternatives to conventional insecticide-based approaches for sustainable VBD management. Rather than relying on a single universal intervention, context-specific integration of complementary approaches within existing integrated vector management frameworks, supported by community participation and effective governance, is likely to provide more durable and environmentally sustainable solutions for controlling vector-borne diseases.

Effective metabolization of exogenous compounds in humans is a key for health and drug efficacy albeit a complex process determined by one’s genetic background. The enzyme, N-Acetyltransferase-2 (NAT2), which metabolizes different xenobiotic compounds (including folate) and various therapeutic drugs, is encoded by the NAT2 gene. Based on acetylation capacity, an individual is either a fast or slow acetylator. Although the apportionment of NAT2 gene and its acetylator types are widely documented in human populations, the evolutionary genetic affinities and functional background of NAT2 variations with closely-related taxa are not well understood. Herein, we have analyzed 60 NAT2 gene coding sequences (873 bp) representing 59 different taxa belonging to mammalian and avian taxa and two most prevalent human NAT2 alleles ((NAT2*4 and NAT2*6A) to document the phylogenetics relationship among taxa; to characterize DNA methylation patterns in this gene and to evaluate species specific binding efficiency to folate using molecular docking approach. Phylogenetic analysis showed 3 broad clades representing where humans and other primates were found to be closely related. The CpG analysis revealed a one conserved region of same length (total 200 bp) across taxa as well as variable short regions of differing lengths (ranging from minimum 50 to maximum 100 bp length sequences) in some taxa. Molecular docking showed variable binding affinities of NAT2 protein models with human slow acetylator allele (NAT2*6A) showing highest binding affinity to folate, indicative of adaptive response to folate bioavailability. Through our analysis we highlight the evolutionary conservation and functional divergence in NAT2 gene across Mammals and Aves likely to be driven by ecological and dietary factors. Our findings provide new insights into evolutionary adaptations to processing folate across various taxa, which may be significant in studies focusing on evolutionary genetics of drug metabolism.

Background This research describes the acceptability and feasibility of using portable Handheld X-ray device in screening for pulmonary TB in high TB burden rural and tribal settings. Methods The mixed methods study was conducted before and after handheld X-ray use was conducted in three blocks of Sheopur district, Madhya Pradesh, India, twice between February and March 2022 and then November 2022 and December 2022. Three groups of respondents were interviewed regarding the acceptability and feasibility of the device: (i) Suspected pulmonary TB participants whose Chest X-rays were to be captured using Hand-held X-ray machine; (ii) Technicians who used the Hand-held X-ray machine for capturing chest X-rays and (iii) Community leaders such as Gram Panchayat members, and health care workers like Community Health Officers (CHO), Medical Officer, CHC (Mo CHC) Auxiliary Nursing Midwives (ANMs) and Accredited Social Health Activist (ASHAs). Thereafter, suspected Tuberculosis patients underwent X-ray using the handheld X-ray device. Results Overall, the hand-held X-ray was acceptable to health care providers, X-ray technicians, the community, and patients. The hand-held X-ray machine offered portability and led to sizable number of X-rays in a day using a single machine. Community members were happy because they would not have to travel long distances for X-rays. Technicians reported that the device was easy to handle and provided acceptable image quality, thus plugging operational gaps in the diagnosis of pulmonary Tuberculosis. Conclusion Hand-held X-ray Machines can be a highly useful tool in TB prevention and care, particularly in tribal/rural areas and scaling up its use can help National Programme in early diagnosis of pulmonary Tuberculosis

Tuberculosis (TB) continues to be a significant health challenge in India, which necessitates accurate and personalized therapeutic strategies for its successful treatment. Polymorphisms in the N-acetyltransferase-2 (NAT2) enzyme, involved in metabolism of a first-line drug, isoniazid (INH), for treatment of TB, have three acetylation phenotypes (slow, intermediate, or fast) that influence drug efficacy, toxicity and treatment outcomes. This article is presented as a narrative review of current research retrieved from PubMed, Scopus, and Google Scholar, focusing on studies related to NAT2 polymorphisms, pharmacogenomics, and tuberculosis therapy. The selected literature was reviewed to address the biological importance of the acetylation process and the development of DNA-based methods for genotyping of the NAT2 gene and discussion of their clinical applications, as well as the effect of NAT2 phenotypes on the treatment outcomes of TB. In addition, how highly genetic diversity in Indian populations necessitates the development of simplified and personalized medication therapy using population-based NAT2 phenotyping approaches is discussed. The need for nationwide mapping of NAT2 variants and the deployment of rapid, cost-effective genotyping platforms, especially in resource-limited endemic settings, are also emphasized. Moreover, how combining NAT2 profiling with additional pharmacogenetic markers may lead to a comprehensive framework for TB treatment optimization is also discussed. It is envisioned that integration of all of these approaches under NAT2-guided therapy in India's National TB Elimination Programme (NTEP) might change the dynamics of TB management in India.

Some species of the Leucosphyrus Group of Anopheles mosquitoes in Southeast Asia are highly anthropophilic and efficient vectors of human malaria parasites, while others primarily feed on non-human primates (NHP) and transmit NHP malaria parasites. The evolutionary history of this group, particularly the origin of anthropophily, was studied using phylogenomic analysis of 2,657 high-confidence nuclear single-copy orthologous genes and 13 mitochondrial protein coding genes from 40 individuals of 11 species. Molecular dating and ancestral state reconstruction revealed that monkey-feeding is ancestral with speciation of monkey-feeding species dating to the Pliocene within Sundaland (Malay peninsula, Borneo, Sumatra and Java) which was covered in tropical rain forests during this period. Although less parsimonious alternatives cannot be excluded, molecular dating, ancestral state reconstruction and reticulation analysis indicated that anthropophily most likely evolved once, involving adaptive introgression, in the early Pleistocene in Sundaland, giving rise to multiple descendent anthropophilic species. Such early origination of anthropophily must necessarily have been in response to the arrival of early hominins (Homo erectus) rather than anatomically modern humans, likely associated with loss and fragmentation of rainforests during the early Pleistocene. The early origination of anthropophily also provides independent non-archaeological evidence supporting the limited fossil record of early hominin colonization in Southeast Asia around 1.8 Mya.

Phenotypic plasticity is often seen as an alternative adaptive strategy to genetic polymorphism, especially in response to rapid environmental changes. Indeed, a link between plasticity and heterozygosity, i.e., the measure of polymorphism, has previously been dismissed. Here, we compare the thermal plasticity of abdominal pigmentation in eight Drosophila species, four belonging to the melanogaster species group and four to the montium group. Despite a conserved developmental pathway for melanin synthesis, the genetic architecture of its variation has significantly evolved, being polygenic in most species (such as D. melanogaster) and Mendelian or invariable in others. By investigating the thermal plasticity of this trait in species with distinct architectures, we show that the degree of plasticity strongly associates with heterozygosity. Plasticity was resurrected in hybrids between species with no plastic responses but with contrasting melanism, and was higher in heterozygotes in species with simple Mendelian polymorphism. In plastic cases, pigmentation dominance is reversed depending on the developmental temperature. We propose simple genetic models with empirical molecular support to explain this link between phenotypic plasticity and genetic polymorphism. The relationship between these two phenomena, and the impact of each on the evolution of the other, may be more relevant than it is currently appreciated.

BACKGROUND OBJECTIVES:Accurate discrimination of mosquito vectors is essential for entomological surveillance in India, where multiple Anopheles, Aedes and Culex species transmit malaria, lymphatic filariasis and arboviral diseases. However, routine species identifications are challenged by cryptic and morphologically similar taxa. Furthermore, population genetic information of major mosquito vectors remains elusive. METHODS:A total of 265 adult mosquitoes representing 21 species from four mosquito genera and one non-mosquito dipteran genus ( Chironomus ) used as an outgroup, were collected from indoor and outdoor resting habitats in selected districts of Madhya Pradesh and Chhattisgarh. Mosquitoes were identified morphologically using standard keys and a mobile identification application. Subsets of specimens representing major genera and putative vector and non-vector taxa were subjected to PCR amplification and sequencing of the cox1 and ITS2 genes that are known to be genetic barcodes for mosquitoes. DNA sequences of these two genes were analyzed for inter- and intra-specific divergence, and phylogenetic clustering and genetic diversity. RESULTS:The cox1 marker showed 100% amplification success and reliably separated most species and genera, although it failed to fully resolve certain closely related taxa, particularly within Anopheles and culicine groups. In contrast, ITS2 amplification success was lower (~50%) but provided complementary resolution for selected species complexes that clarified ambiguous morphological assignments and improved discrimination of selected species complexes in combination with cox1 . Phylogenetic analyses recovered largely congruent species-level clustering across both markers, highlighting both the strengths and limitations of each marker for operational surveillance. Furthermore, mitochondrial diversity analyses revealed high haplotype diversity in Anopheles species with limited genetic differentiation between populations, suggesting weak population structure. INTERPRETATION CONCLUSION:The combined use of morphology with cox1 and ITS2 sequencing enhances species identification and phylogenetic resolution in diverse mosquito vectors. While cox1 serves as a robust primary barcode for routine surveillance, ITS2 provides additional resolution for cryptic taxa., However, population genetic inferences based on mitochondrial data alone should be interpreted cautiously. These findings support the strengthening of molecularly informed vector surveillance pipelines and backup more precise, evidence-based vector control in endemic settings.

Aedes aegypti and Aedes albopictus mosquitoes spread major vector-borne viral diseases in tropical and sub-tropical regions of the globe. In this study, we sequenced the genome of Indian Ae. aegypti and Ae. albopictus and mapped to their reference genomes. Comparative genomics were performed between our strain and the reference strains. A total of 14,416,484 single nucleotide polymorphisms (SNPs) and 156,487 insertions and deletions (InDels) were found in Ae. aegypti, and 28,940,433 SNPs and 188,987 InDels in Ae. albopictus. Particular emphasis was given to gene families involved in mosquito digestion, development, and innate immunity, which could be putative candidates for vector control. Serine protease cascades and their inhibitors called serpins, play a central role in these processes. We extracted high-impact variants in genes associated with serine proteases and serpins. This study reports for the first time a high coverage genome sequence data of an Indian Ae. albopictus mosquito. The results from this study will provide insights into Indian Aedes specific polymorphisms and the evolution of immune related genes in mosquitoes, which can serve as a resource for future comparative genomics and those pursuing the development of targeted biopesticides for effective mosquito control strategies.

Haemoglobinopathies are the most common inherited disorder of Red Blood Cells across the globe and one of the major public health problems in many regions of India. Variation in ethnic and regional prevalence is observed in many parts of India. The prevalence and pattern of haemoglobinopathies in Datia region has not been reported limiting the knowledge of disease profiles in this region. The present work aimed to find the broad view of various haemoglobinopathies in the local area and population at risk. To identify prevalence and patterns of three blood disorders namely sickle cell anaemia, β-thalassemia major and G-6-P-D deficiency samples were collected from the patients attending various OPDs of District Hospital, Datia, Madhya Pradesh, India. All anaemic patients referred by Government District Hospital, Datia District, M.P. were screened for various haemoglobinopathies. A total of 605 cases were received from September 2017 to March 2020 at the Model Rural Health Research Unit (MRHRU), Datia, M.P. All 605 samples were tested for different haemoglobinopathies by solubility test and G6PD deficiency, cellulose acetate electrophoresis, and suspected cases were confirmed from HPLC. A total of 605 patients were screened for haemoglobinopathies from District Hospital, Datia. It was found that 13 cases (2.14%) were of β thalassemia trait and other rare variants followed by 4 cases (0.66%) of sickle cell trait. A total of 3 cases (0.5%) of β thalassemia major were observed in a total sample of 605. No previous studies have reported the status of haemoglobinopathies in this region, since this region is known for tribes it is essential to have study of blood genetic disorders. Novelty of this study is that it is the first report from this region to the best of our knowledge. Very low prevalence rate of sickle cell anaemia and thalassemia was observed. So, large-scale screening in the region is required for identifying the true burden. Chelation therapy, gene therapies in future could be great help to the patients of this region.

Plasmodium falciparum is the main cause of malaria in North-Eastern (NE) states of India. Artemether–lumefantrine (AL) was introduced as first-line therapy against uncomplicated P. falciparum cases in 2013 after the emergence of resistance to sulfadoxine–pyrimethamine. The aim of the study was to assess the therapeutic efficacy of AL and status of molecular markers in the circulating parasites. Therapeutic efficacy of AL was assessed in NE states as per World Health Organization guidelines. Patients with P. falciparum positive peripheral blood smear were enrolled and treated with AL and clinical and parasitological parameters were monitored over a 28-day follow-up period. Furthermore, the pfmdr1, pfdhfr, pfdhps and pfk13 genes were amplified and sequenced for mutation analysis. A total of 231 cases were enrolled and therapeutic efficacy was determined in 215 (93.1

Background The hitchhiking model of molecular evolution proposes that linked non-coding regions also undergo fixation, while fixing a beneficial allele in a population. This concept can be applied to identify loci with functional and evolutionary significance. Putative circumsporozoite protein in Plasmodium vivax (PvpuCSP) identified following the molecular hitchhiking model, holds evolutionary significance. Methods We investigated the extent of genetic polymorphism in PvpuCSP and the role of natural selection which shapes the genetic composition and maintains the diversity in P. vivax isolates from India. Results Sequencing the putative CSP of P. vivax (PvpuCSP) in 71 isolates collected from P. vivax endemic regions of India, revealed a well-conserved N- and C-terminal, constituting around 80 % of the gene. PCR amplification and sequencing validated extensive diversity in the repeat region, ranging from 1.8 to 2.2 kb towards the C-terminal, identifying 37 different alleles from 71 samples. Conclusions The recent and exclusive accumulation of repeats in the putative circumsporozoite protein within Indian P. vivax highlights its highly variable length polymorphism, making it a potential marker for estimating diversity and infection complexity. Episodic diversifying selection in the PvpuCSP repeat region, supported by statistically significant p-values and likelihood ratios, contributes to amino acid diversity at various phylogenetic levels. Our previous systematic computational study proposed PvpuCSP as E3-ubiquitin ligase, and the observed diversity may facilitate adaptation to different ubiquitination-related interactions, though this remains to be experimentally validated.

BACKGROUND:Rapid diagnostic tests (RDTs) are vital for malaria diagnosis, especially in resource-limited areas. RDTs targeting histidine-rich protein 2 (PfHRP2) and its structural homologue PfHRP3 are commonly used for detecting Plasmodium falciparum. However, genetic deletions in these proteins can affect test accuracy. This study aims to examine the gene deletions and sequence variation in the Pfhrp2 and Pfhrp3 genes in P. falciparum isolates from Chhattisgarh, India, and assess their correlation with RDT reactivity. METHODS:A total of 264 microscopically confirmed P. falciparumpositive samples from Chhattisgarh were analyzed for deletions in the Pfhrp2 and Pfhrp3 genes. Nucleotide sequences were obtained for the Pfhrp2 (n=101) and Pfhrp3 (n=95) genes. The sequence data were analyzed for repeat motifs and correlated with the RDT performance, especially at low parasite densities. RESULTS:The deletion rates for Pfhrp2 and Pfhrp3 were found to be 3.8% and 14%, respectively. The Pfhrp2 gene exhibited 15 distinct repeat motifs, while the Pfhrp3 gene showed 10 repeat motifs. No significant correlation was observed between variations in repeat types 2 and 7 of Pfhrp2 and the commercial RDT performance, particularly at low parasite densities. CONCLUSIONS:The results indicate that the deletion rates and sequence diversity of Pfhrp2 and Pfhrp3 in Chhattisgarh are below the WHO threshold of 5% for a policy change regarding Pfhrp2 gene deletion. Sequence diversity does not appear to compromise the performance of current PfHRP2-based RDTs. However, a larger-scale study encompassing other endemic regions of India is recommended for a more comprehensive understanding of the impact on RDT efficacy over time.

BACKGROUND:This study aims to identify the signatures of natural selection in the pyrazinamidase (pncA) gene to see if genetic adaptations by Darwinian natural selection have shaped genetic composition of Mycobacterium tuberculosis (Mtb). METHODS:The present analyses were based on 209 DNA sequences (561 bp) of the pncA gene of the bacterial pathogen, Mtb from seven different counties (Peru, Pakistan, South Africa, Mexico, India, China, and Kuwait) endemic to tuberculosis (TB). Before conducting tests for Darwinian natural selection in the pncA gene, we conducted several tests for neutrality in all the available DNA sequences after retrieval from public domains. Several statistical analyses under different algorithms were conducted and biological/evolutionary inferences were drawn. RESULTS:The 209 sequences of the pncA gene in Mtb belonging to seven different countries were found to be perfectly aligned with the reference sequence. Data analyses under different population genetic models revealed the highest genetic diversity in India, followed by Peru; the lowest was in China. Interestingly, four populations; Peru, Pakistan, India, and Kuwait were found to be deviated from neutral model of evolution based on Tajima'D (TD) values; two populations (India and Peru) based on Fu and Li's D and F (FLD and FLF) test values and five populations (India, Peru, Pakistan, South Africa, and Kuwait) based on Fay and Wu's H (FWH) test. Moreover, based on the statistically significant results of neutrality tests, evidence for positive selection in three populations (Peru [P < 0.02945], Pakistan [P < 0.01767], and Kuwait [P < 0.00301]) at P < 0.05 level of significance] was found. CONCLUSION:The present evolutionary genetic analysis of the pncA gene indicates different levels of genetic diversity in seven different country populations. As almost all the global populations showed deviation from neutral model and three populations showed signatures of natural selection, with no specific hotspot region identified for PZA resistance, this gene needs to be studied with larger population size covering countries with TB incidences to study the evolution of drug resistance in Mtb. This will help in the management of drug resistance and TB elimination plan.

Current understanding of genetic polymorphisms and natural selection in Plasmodium falciparum circumsporozoite (PfCSP), the leading malaria vaccine, is crucial for the development of next-generation vaccines, and such data is lacking in Africa. Blood samples were collected among Plasmodium-infected individuals living in four Cameroonian areas (Douala, Maroua, Mayo-Oulo, Pette). DNA samples were amplified using nested PCR protocols, sequenced, and BLASTed. Single nucleotide polymorphisms (SNPs) were analysed in each PfCSP region, and their impact on PfCSP function/structure was predicted in silico. The N-terminal region showed a limited polymorphism with four haplotypes, and three novel SNPs (N68Y, R87W, K93E) were found. Thirty-five haplotypes were identified in the central region, with several variants (e.g., NVNP and KANP). The C-terminal region was also highly diverse, with 25 haplotypes and eight novel SNPs (N290D, N308I, S312G, K317A, V344I, D356E, E357L, D359Y). Most polymorphic codon sites were mainly observed in the Th2R subregion in isolates from Douala and Pette. The codon site 321 was under episodic positive selection. One novel (E357L) and three known (K322I, G349D, D359Y) SNPs show an impact on function/structure. This study showed extensive genetic diversity with geographical patterns and evidence of the selection of Cameroonian PfCSP central and C-terminal regions.

Rapid diagnostic tests (RDTs) are crucial for diagnosing malaria in resource-limited settings. These tests, which detect the histidine-rich protein 2 (PfHRP2) and its structural homologue PfHRP3, are specifically designed to identify Plasmodium falciparum. Deletion of the Pfhrp2 gene in parasite has been reported in India and other malaria-endemic countries. Therefore, periodic surveillance of Pfhrp2 and Pfhrp3 genetic deletions is crucial. We conducted a study to examine these gene deletions in P. falciparum isolates from nine malaria-endemic states in India. In this study, we analyzed 1,558 samples that were microscopically confirmed to be P. falciparum positive. We isolated genomic DNA from all the aforementioned samples, followed by PCR amplification of the Pfhrp2/3 gene. The results showed that the deletion rates for Pfhrp2 and Pfhrp3 genes were 0.44% and 1.47%, respectively. These findings indicate that the gene deletions in all nine states are at low level. Despite these low deletion rates, continuous surveillance is crucial to monitor the efficiency of HRP2 based malaria RDTs. It is recommend that conducting large-scale studies which include other endemic states in India to gain a more comprehensive understanding of the prevalence and impact of these gene deletions over time. This ongoing surveillance will ensure that diagnostic strategies remain effective and that any emerging trends in gene deletions are promptly addressed to achieve the malaria control and elimination.

Understanding the evolution of anthropophily, the preference of mosquitoes to feed on humans offers insights into current and future human disease transmission. Some species of the Leucosphyrus Group of Anopheles mosquitoes in Southeast Asia are highly anthropophilic and efficient vectors of human malaria parasites, while others primarily feed on non-human primates and transmit non-human primate malaria parasites. Through phylogenomic analysis of 11 recognized species, we studied the biogeography and evolutionary history of anthropophily in this group. Molecular dating and ancestral state reconstruction revealed that anthropophily evolved during the late Pliocene/early Pleistocene in Sundaland, likely in response to early hominins. This finding provides independent non-archaeological evidence supporting the limited fossil record of early hominin colonization in Southeast Asia around 1.8 million years ago. ### Competing Interest Statement The authors have declared no competing interest.

In the October issue of The Lancet Infectious Diseases, the editorial titled Can We Control Dengue?1 takes into consideration only two aspects of dengue control: prevention by vaccination and vector control strategies. However, a very important aspect of dengue virus (DENV) control, therapeutics, was left unmentioned despite being a potential major method of dengue control. Worldwide, dengue vaccination approaches are struggling for success, and Wolbachia-based vector control strategies have resulted in failure in parts of the world.