Design, in silico study, synthesis and evaluation of hybrid pyrazole substituted 1,3,5-triazine derivatives for antimalarial activity.

OBJECTIVES: Malaria is a significant global health challenge, particularly in Africa, Asia, and Latin America, necessitating immediate investigation into innovative and efficacious treatments. This work involves the development of pyrazole substituted 1,3,5-triazine derivatives as antimalarial agent. METHODS: In this study, ten compounds 7(a-j) were synthesized by using nucleophilic substitution reaction, screened for in silico study and their antimalarial activity were evaluated against 3D7 (chloroquine-sensitive) strain of P. falciparum. KEY FINDING: The present work involves the development of hybrid trimethoxy pyrazole 1,3,5-triazine derivatives 7 (a-j). Through in silico analysis, four compounds were identified with favorable binding energy and dock scores. The primary focus of the docking investigations was on the examination of hydrogen bonding and the associated interactions with certain amino acid residues, including Arg A122, Ser A108, Ser A111, Ile A164, Asp A54, and Cys A15. The IC50 values of the four compounds were measured in vitro to assess their antimalarial activity against the chloroquine sensitive 3D7 strain of P. falciparum. The IC50 values varied from 25.02 to 54.82 µg/mL. CONCLUSION: Among the ten derivatives, compound 7J has considerable potential as an antimalarial agent, making it a viable contender for further refinement in the realm of pharmaceutical exploration, with the aim of mitigating the global malaria load.

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.

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.

Development and On-Field Deployment of a Mobile-Based Application 'MoSQuIT' for Malaria Surveillance in International Border Districts of Northeast India-Challenges and Opportunities.

The conventional paper-based system for malaria surveillance is time-consuming, difficult to track and resource-intensive. Few digital platforms are in use but wide-scale deployment and acceptability remain to be seen. To address this issue, we created a malaria surveillance mobile app that offers real-time data to stakeholders and establishes a centralised data repository. The MoSQuIT app was designed to collect data from the field and was integrated with a web-based platform for data integration and analysis. The MoSQuIT app was deployed on mobile phones of accredited social health activists (ASHA) working in international border villages in the northeast (NE) Indian states of Assam, Tripura and Arunachal Pradesh for 20 months in a phased manner. This paper shares the challenges and opportunities associated with the use of MoSQuIT for malaria surveillance. MoSQuIT employs the same data entry formats as the NVBDCP's malaria surveillance programme. Using this app, a total of 8221 fever cases were recorded, which included 1192 (14.5%) cases of P. falciparum malaria, 280 (3.4%) cases of P. vivax malaria and 52 (0.6%) mixed infection cases. Depending on network availability, GPS coordinates of the fever cases were acquired by the app. The present study demonstrated that mobile-phone-based malaria surveillance facilitates the quick transmission of data from the field to decision makers. Geospatial tagging of cases helped with easy visualisation of the case distribution for the identification of malaria-prone areas and potential outbreaks, especially in hilly and remote regions of Northeast India. However, to achieve the full operational potential of the system, operational challenges have to be overcome.

Incrimination of Aedes aegypti and Aedes albopictus as vectors of dengue virus serotypes 1, 2 and 3 from four states of Northeast India.

Dengue is an important vector borne disease with a great public health concern worldwide. Northeast India has experienced dengue almost every year for a decade. As studies on dengue vectors from this region are limited, we undertook an investigation to detect natural infection of the dengue virus (DENV) in potential dengue vectors of this region. Adult Aedes mosquitoes which were collected were subjected to RT-PCR for detection of infecting dengue serotype. Minimum infection rate was also determined for each positive pool. Out of the total 6229 adult Aedes mosquitoes collected, Aedes aegypti (63.3 %) was abundant in comparison to Aedes albopictus (36.7 %). These specimens (515 mosquito pools) were subjected to RT-PCR for detection of DENV-1, 2, 3 and 4. RT-PCR revealed the existence of DENV in both male as well as female mosquito pools suggesting natural transovarial transmission of DENV in this region. A total of 54 pools tested were positive for DENV-1, 2, 3 serotypes. This study revealed the occurence of DENV in both the potential dengue vectors from this region along with evidence of transovarial transmission which helps in persistence of the virus in nature.

Malaria in North-East India: Importance and Implications in the Era of Elimination.

Worldwide and in India, malaria elimination efforts are being ramped up to eradicate the disease by 2030. Malaria elimination efforts in North-East (NE) India will have a great bearing on the overall efforts to eradicate malaria in the rest of India. The first cases of chloroquine and sulfadoxine-pyrimethamine resistance were reported in NE India, and the source of these drug resistant parasites are most likely from South East Asia (SEA). NE India is the only land route through which the parasites from SEA can enter the Indian mainland. India's malaria drug policy had to be constantly updated due to the emergence of drug resistant parasites in NE India. Malaria is highly endemic in many parts of NE India, and Plasmodium falciparum is responsible for the majority of the cases. Highly efficient primary vectors and emerging secondary vectors complicate malaria elimination efforts in NE India. Many of the high transmission zones in NE India are tribal belts, and are difficult to access. The review details the malaria epidemiology in seven NE Indian states from 2008 to 2018. In addition, the origin and evolution of resistance to major anti-malarials are discussed. Furthermore, the bionomics of primary vectors and emergence of secondary malaria vectors, and possible strategies to prevent and control malaria in NE are outlined.

In silico ADMET study, docking, synthesis and antimalarial evaluation of thiazole-1,3,5-triazine derivatives as Pf-DHFR inhibitor.

BACKGROUND: Plasmodium falciparum dihydrofolate reductase (Pf-DHFR) is an essential enzyme in the folate pathway and is an important target for antimalarial drug discovery. In this study a modern approach has been undertaken to identify new hits of thiazole-1,3,5-triazine derivatives as antimalarials targeting Pf-DHFR. METHODS: The library of 378 thiazole-1,3,5-triazines were designed and subjected to ADME analysis. The compounds having optimal ADME score, was then evaluated by docking against wild and mutant Pf-DHFR complex. The resultant compound after screening from above these two methods were synthesized, and assayed for in vitro antimalarial against chloroquine-sensitive (3D-7) and chloroquine resistant (Dd-2) strains of P. falciparum. RESULTS: Twenty compounds were identified from the dataset based on considerable AlogP98 vs. PSA_2D confidence ellipse, ADME filter and TOPKAT toxicity analysis. Majority of compounds showed interaction with Asp54, Arg59, Arg122 and Ile 164 in docking analysis. Entire set of tested derivatives exhibited considerable activity at the tested dose against sensitive strain with IC50 values varying from 10.03 to 54.58 µg/ml. Furthermore, against chloroquine resistant strain, eight compounds showed IC50 from 11.29 to 40.92 µg/ml. Compound A5 and H16 were found to be the most potent against both the strains of P. Falciparum. CONCLUSION: Results of the study suggested the possible utility of thiazole-1,3,5-triazines as new lead for identifying new class of Pf-DHFR inhibitor.

In vitro adaptability of Plasmodium falciparum to different fresh serum alternatives.

To reduce the dependency on fresh AB+ serum in continuous culture of Plasmodium falciparum, a comparative study was undertaken to assess the in vitro adaptability of P. falciparum to media supplemented with fresh AB+ serum from whole blood, AB+ plasma, serum derived from AB+ plasma, AB+ human serum from Sigma, Albumax II, fetal bovine serum and new born calf serum, independently and in different combinations. Combinations were used to analyze whether two different substitutes demonstrate any synergistic effect on the growth of the parasites. Our findings exhibited that the combination of fresh human serum and Albumax II showed good growth pattern in comparison to that of fresh serum and can thereby be instrumental in reducing the role of fresh human serum in continuous parasite maintenance. Culture maintained with Albumax II with or without hypoxanthine showed average growth.

Molecular characterization of Plasmodium falciparum in Arunachal Pradesh from Northeast India based on merozoite surface protein 1 & glutamate-rich protein.

BACKGROUND & OBJECTIVES: Northeast (NE) India is one of the high endemic regions for malaria with a preponderance of Plasmodium falciparum, resulting in high morbidity and mortality. The P. falciparum parasite of this region showed high polymorphism in drug-resistant molecular biomarkers. However, there is a paucity of information related to merozoite surface protein 1 (msp-1) and glutamate-rich protein (glurp) which have been extensively studied in various parts of the world. The present study was, therefore, aimed at investigating the genetic diversity of P. falciparum based on msp-1 and glurp in Arunachal Pradesh, a State in NE India. METHODS: Two hundred and forty nine patients with fever were screened for malaria, of whom 75 were positive for P. falciparum. Blood samples were collected from each microscopically confirmed patient. The DNA was extracted; nested polymerase chain reaction and sequencing were performed to study the genetic diversity of msp-1 (block 2) and glurp. RESULTS: The block 2 of msp-1 gene was found to be highly polymorphic, and overall allelic distribution showed that RO33 was the dominant allele (63%), followed by MAD20 (29%) and K1 (8%) alleles. However, an extensive diversity (9 alleles and 4 genotypes) and 6-10 repeat regions exclusively of R2 type were observed in glurp. INTERPRETATION & CONCLUSIONS: The P. falciparum population of NE India was diverse which might be responsible for higher plasticity leading to the survival of the parasite and in turn to the higher endemicity of falciparum malaria of this region.

Molecular docking, synthesis and in vitro antimalarial evaluation of certain novel curcumin analogues

ABSTRACT The receptor protein PfATP6 has been identified as the common target of artemisinin and curcumin. The work was initiated to assess the antimalarial activity of six curcumin derivatives based on their binding affinities and correlating the in silico docking outcome with in vitro antimalarial screening results. A ligand library of thirty two Knoevenagel condensates of curcumin were designed and docked against PfATP6 protein and six compounds with the best binding scores were synthesized and screened for their antimalarial activity against the sensitive 3D7 strain of Plasmodium falciparum. ADME/Tox, pharmacokinetic and pharmacodynamic profiles of the designed compounds were analyzed and reported. 4-FB was found to have similar binding energy to the standard artemisinin (-6.75 and -6.73 respectively) while 4-MB, 3-HB, 2-HB, B, 4-NB displayed better binding energy than curcumin (-5.95, -5.89, -5.68, -5.35, -5.29 and -5.25 respectively). At a dose of 50 µg/mL all the six compounds showed 100% schizont inhibition while at 5µg/ml, five showed more than 75% inhibition and better results than curcumin. 4-FB showed the best activity with 97.8% schizonticidal activity. The in vitro results superimpose the results obtained from the in silico study thereby encouraging development of promising curcumin leads in the battle against malaria.

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 (76 T), while 68% had mutant pfmdr-1 genotype (86 Y). 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 (51 I/59 R/108 N/164 L) 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.