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Background Objectives: At present, use of synthetic pesticides to manage insects and other arthropods creates a number of issues that are related to the environment and public health. The goal of the present study is to find repellent activity of Vitex negundo and Curcuma longa herbal extracts against the wild species Simulium (blackfly) bite., Methods: We have studied Simulium biting behavior and tested the repellency of herbal extract obtained from Vitex negundo L. (Lamiales: Lamiaceae) and Curcuma longa L. (Zingiberal: Zingiberaceae) along with their essential oils in three locations in Arunachal Pradesh, India on human volunteers' resistance to Simulium (blackflies). The reported herbal extracts were incorporated to topical drug delivery vehicle as a cream and gel., Results: The methanolic extract of Vitex negundo cream and gel formulation showed >2 h safety at 5% concentration and >5 h safety at 10% concentration in all the testing sites followed by ethyl acetate extract. Whereas, chloroform extract of Curcuma longa cream and gel formulation provided >1 h safety at 5% concentration and >4 h safety at 10% concentration., Interpretation Conclusion: At an optimum concentration of 10%, the methanolic extract of Vitex negundo cream provided complete protection times (CPTs) 320.4, 358.6, and 346.4 min, respectively. These findings offer confirmation of the methanolic extract and chloroform extract potential for use in new blackfly repellents., (Copyright © 2024 Copyright: © 2024 Journal of Vector Borne Diseases.)

The upregulation of HDAC1 facilitate the induction of epigenetic repression of genes responsible for suppressing tumourigenesis, thereby triggering the development of cancer. HDAC1 inhibitors have thus emerged as possible therapeutic approaches against a variety of human malignancies, as they can inhibit the activity of certain HDACs, repair the overexpression of tumour suppressor genes, and induce cell differentiation, cell cycle arrest, and apoptosis. In this study, among 810 virtually screened compounds, Pinocembrin (PHUB000396) had a significant binding affinity (-7.99 kcal/mol). In molecular dynamics simulation (MD) studies for 200 ns time scale, the compound Pinocembrin effectively undergoes conformational optimization, thereby enabling its accommodation within the active site of the receptor. This outcome serves as a rational for the observed binding affinity. The optimal binding free energy calculations using the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) (-35.86 ± 7.52 kcal/mol) showed the significant role of van der Waals forces and Coulomb interactions in the stability of the respective complex. The pharmacokinetic study showed its potential as a lead compound. The in-silico cytotoxicity prediction also confirmed its potential as an active anticancer phytocompound in lung and brain cancer. Therefore, it can be predicted that Pinocembrin could be a useful bioactive compound as an HDAC1 inhibitor and could be used in developing epigenetic therapy in cancer such as brain cancer and lung cancer to regulate gene expression., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Overexpression of HDAC 2 promotes cell proliferation in ovarian cancer. HDAC 2 is involved in chromatin remodeling, transcriptional repression, and the formation of condensed chromatin structures. Targeting HDAC 2 presents a promising therapeutic approach for correcting cancer-associated epigenetic abnormalities. Consequently, HDAC 2 inhibitors have evolved as an attractive class of anti-cancer agents. This work intended to investigate the anti-cancer abilities and underlying molecular mechanisms of Rhamnetin in human epithelial ovarian carcinoma cells (SKOV3), which remain largely unexplored. We employed various in vitro methods, including MTT, apoptosis study, cell cycle analysis, fluorescence microscopy imaging, and in vitro enzymatic HDAC 2 protein inhibition, to examine the chemotherapeutic sensitivity of Rhamnetin in SKOV3 cells. Additionally, we conducted in silico studies using molecular docking, MD simulation, MM-GBSA, DFT, and pharmacokinetic analysis to investigate the binding interaction mechanism within Rhamnetin and HDAC 2, alongside the compound's prospective as a lead candidate. The in vitro assay confirmed the cytotoxic effects of Rhamnetin on SKOV3 cells, through its inhibition of HDAC 2 activity. Rhamnetin, a nutraceutical flavonoid, halted at the G1 phase of the cell cycle and triggered apoptosis in SKOV3 cells. Furthermore, computational studies provided additional evidence of its stable binding to the HDAC 2 protein's binding site cavity. Based on our findings, we conclude that Rhamnetin effectively promotes apoptosis and mitigates the proliferation of SKOV3 cells through HDAC 2 inhibition. These results highlight Rhamnetin as a potential lead compound, opening a new therapeutic strategy for human epithelial ovarian cancer.Communicated by Ramaswamy H. Sarma.

The seaweed industries generate considerable amounts of waste that must be appropriately managed. This biomass from marine waste is a rich source of high-value bioactive compounds. Thus, this waste can be adequately utilized by recovering the compounds for therapeutic purposes. Histone deacetylases (HDACs) are key epigenetic regulators established as one of the most promising targets for cancer chemotherapy. In the present study, our objective is to find the HDAC 2 inhibitor. We performed top-down in silico methodologies to identify potential HDAC 2 inhibitors by screening compounds from edible seaweed waste. One hundred ninety-three ( n = 193) compounds from edible seaweeds were initially screened and filtered with drug-likeness properties using SwissADME. After that, the filtered compounds were followed to further evaluate their binding potential with HDAC 2 protein by using Glide high throughput virtual screening (HTVS), standard precision (SP), extra precision (XP), and quantum polarized ligand docking (QPLD). One compound with higher negative binding energy was selected, and to validate the binding mode and stability of the complex, molecular dynamics (MD) simulations using Desmond were performed. The complex-binding free energy calculation was performed using molecular mechanics-generalized born surface area (MM-GBSA) calculation. Post-MD simulation analyses such as PCA, DCCM, and free energy landscape were also evaluated. The quantum mechanical and electronic properties of the potential bioactive compounds were assessed using the density functional theory (DFT) study. These findings support the use of marine resources like edible seaweed waste for cancer drug development by using its bioactive compounds. The obtained results encourage further in vitro and in vivo research. Our in silico findings show that the compound has a high binding affinity for the catalytic site of the HDAC 2 protein and has drug-likeness properties, and can be utilized in drug development against cancer., Competing Interests: SP is employed by NatNov Bioscience Private Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bharadwaj, Ahmad, Pati, Ghosh, Sarkar, Rabha, Patel, Baishya, Edinur, Abdul Kari, Ahmad Mohd Zain and Wan Rosli.)

Brain cancers, mainly high-grade gliomas/glioblastoma, are characterized by uncontrolled proliferation and recurrence with an extremely poor prognosis. Despite various conventional treatment strategies, viz., resection, chemotherapy, and radiotherapy, the outcomes are still inefficient against glioblastoma. The blood-brain barrier is one of the major issues that affect the effective delivery of drugs to the brain for glioblastoma therapy. Various studies have been undergone in order to find novel therapeutic strategies for effective glioblastoma treatment. The advent of nanodiagnostics, i.e., imaging combined with therapies termed as nanotheranostics, can improve the therapeutic efficacy by determining the extent of tumour distribution prior to surgery as well as the response to a treatment regimen after surgery. Polymer nanoparticles gain tremendous attention due to their versatile nature for modification that allows precise targeting, diagnosis, and drug delivery to the brain with minimal adverse side effects. This review addresses the advancements of polymer nanoparticles in drug delivery, diagnosis, and therapy against brain cancer. The mechanisms of drug delivery to the brain of these systems and their future directions are also briefly discussed.

Gold nanoparticles (AuNPs) have been widely explored and are well-known for their medical applications. Chemical and physical synthesis methods are a way to make AuNPs. In any case, the hunt for other more ecologically friendly and cost-effective large-scale technologies, such as environmentally friendly biological processes known as green synthesis, has been gaining interest by worldwide researchers. The international focus on green nanotechnology research has resulted in various nanomaterials being used in environmentally and physiologically acceptable applications. Several advantages over conventional physical and chemical synthesis (simple, one-step approach to synthesize, cost-effectiveness, energy efficiency, and biocompatibility) have drawn scientists' attention to exploring the green synthesis of AuNPs by exploiting plants' secondary metabolites. Biogenic approaches, mainly the plant-based synthesis of metal nanoparticles, have been chosen as the ideal strategy due to their environmental and in vivo safety, as well as their ease of synthesis. In this review, we reviewed the use of green synthesized AuNPs in the treatment of cancer by utilizing phytochemicals found in plant extracts. This article reviews plant-based methods for producing AuNPs, characterization methods of synthesized AuNPs, and discusses their physiochemical properties. This study also discusses recent breakthroughs and achievements in using green synthesized AuNPs in cancer treatment and different mechanisms of action, such as reactive oxygen species (ROS), mediated mitochondrial dysfunction and caspase activation, leading to apoptosis, etc., for their anticancer and cytotoxic effects. Understanding the mechanisms underlying AuNPs therapeutic efficacy will aid in developing personalized medicines and treatments for cancer as a potential cancer therapeutic strategy.

COVID-19 is a disease that puts most of the world on lockdown and the search for therapeutic drugs is still ongoing. Therefore, this study used in silico screening to identify natural bioactive compounds from fruits, herbaceous plants, and marine invertebrates that are able to inhibit protease activity in SARS-CoV-2 (PDB: 6LU7). We have used extensive screening strategies such as drug likeliness, antiviral activity value prediction, molecular docking, ADME, molecular dynamics (MD) simulation, and MM/GBSA. A total of 17 compounds were shortlisted using Lipinski's rule in which 5 compounds showed significant predicted antiviral activity values. Among these 5, only 2 compounds, Macrolactin A and Stachyflin, showed good binding energy of -9.22 and -8.00 kcal/mol, respectively, within the binding pocket of the M pro catalytic residues (HIS 41 and CYS 145). These two compounds were further analyzed to determine their ADME properties. The ADME evaluation of these 2 compounds suggested that they could be effective in developing therapeutic drugs to be used in clinical trials. MD simulations showed that protein-ligand complexes of Macrolactin A and Stachyflin with the target receptor (6LU7) were stable for 100 nanoseconds. The MM/GBSA calculations of M pro -Macrolactin A complex indicated higher binding free energy (-42.58 ± 6.35 kcal/mol). Dynamic cross-correlation matrix (DCCM) and principal component analysis (PCA) on the residual movement in the MD trajectories further confirmed the stability of Macrolactin A bound state with 6LU7. In conclusion, this study showed that marine natural compound Macrolactin A could be an effective therapeutic inhibitor against SARS-CoV-2 protease (6LU7). Additional in vitro and in vivo validations are strongly needed to determine the efficacy and therapeutic dose of Macrolactin A in biological systems., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The green synthesis of silver nanoparticles (AgNPs) has currently been gaining wide applications in the medical field of nanomedicine. Green synthesis is one of the most effective procedures for the production of AgNPs. The Diospyros malabarica tree grown throughout India has been reported to have antioxidant and various therapeutic applications. In the context of this, we have investigated the fruit of Diospyros malabarica for the potential of forming AgNPs and analyzed its antibacterial and anticancer activity. We have developed a rapid, single-step, cost-effective and eco-friendly method for the synthesis of AgNPs using Diospyros malabarica aqueous fruit extract at room temperature. The AgNPs began to form just after the reaction was initiated. The formation and characterization of AgNPs were confirmed by UV-Vis spectrophotometry, XRD, FTIR, DLS, Zeta potential, FESEM, EDX, TEM and photoluminescence (PL) methods. The average size of AgNPs, in accordance with TEM results, was found to be 17.4 nm. The antibacterial activity of the silver nanoparticles against pathogenic microorganism strains of Staphylococcus aureus and Escherichia coli was confirmed by the well diffusion method and was found to inhibit the growth of the bacteria with an average zone of inhibition size of (8.4 ± 0.3 mm and 12.1 ± 0.5 mm) and (6.1 ± 0.7 mm and 13.1 ± 0.5 mm) at 500 and 1000 µg/mL concentrations of AgNPs, respectively. The anticancer effect of the AgNPs was confirmed by MTT assay using the U87-MG (human primary glioblastoma) cell line. The IC 50 value was found to be 58.63 ± 5.74 μg/mL. The results showed that green synthesized AgNPs exhibited significant antimicrobial and anticancer potency. In addition, nitrophenols, which are regarded as priority pollutants by the United States Environmental Protection Agency (USEPA), can also be catalytically reduced to less toxic aminophenols by utilizing synthesized AgNPs. As a model reaction, AgNPs are employed as a catalyst in the reduction of 4-nitrophenol to 4-aminophenol, which is an intermediate for numerous analgesics and antipyretic drugs. Thus, the study is expected to help immensely in the pharmaceutical industries in developing antimicrobial drugs and/or as an anticancer drug, as well as in the cosmetic and food industries.

Diosgenin encapsulated PCL-Pluronic nanoparticles (PCL-F68-D-NPs) were developed using the nanoprecipitation method to improve performance in brain cancer (glioblastoma) therapy. The nanoparticles were characterized by dynamic light scattering (DLS)/Zeta potential, Fourier-transform infrared (FTIR) spectra, X-ray diffraction (XRD), Field Emission Scanning Electron Microscopy (FESEM), and Transmission electron microscopy (TEM). The encapsulation efficiency, loading efficiency, and yield were calculated. The in vitro release rate was determined, and the kinetic model of diosgenin release was plotted and ascertained. The cytotoxicity was checked by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)assay against U87-MG cells (glioblastoma cell lines). The obtained nanoparticles demonstrated good size distribution, stability, morphology, chemical, and mechanical properties. The nanoparticles also possessed high encapsulation efficiency, loading efficiency, and yield. The release rate of Diosgenin was shown in a sustained manner. The in vitro cytotoxicity of PCL-F68-D-NPs showed higher toxicity against U87-MG cells than free Diosgenin.

Background & Objectives: Anopheles stephensi is one of the most important urban malaria vectors in India and contribute about 12% of total malaria cases. An. stephensi has three ecological variants; type, intermediate and mysorensis that can be differentiated on the basis of differences in number of ridges on egg float and on the basis of spiracular indices. Because of its anthropophilic nature the 'type' form is an efficient malaria vector. In the present study, the egg surface morphometry and morphology of An. stephensi 'type' form was studied and detail distinguish- ing characters were recorded for its correct identification., Methods: Eggs of An. stephensi 'type' form were studied by scanning electron microscopy (SEM) after sputter- coating with gold. In total 23 egg characters were analysed morphologically and morphometrically, which included egg attributes, deck attributes, ventral tubercles, micropyle and float attributes., Results: The dorsal surface of the egg of 'type' form was curved while the ventral surface was concave and both anterior and posterior ends were blunt. The average length and width of egg was 473.94 + 11.18 and 154.69 + 2.66 μm respectively. The number of float ribs observed was 20.33 ± 0.33. The maximum length of float was found to be 246.57 + 15.27 μm, whereas maximum width was 87.16 + 3.83 μm., Interpretation & Conclusion: The present study has generated some important data which is specific to An. Stephensi 'type' form and provided significant morphological and morphometric standards for its correct identification. This information could be useful in differentiation of An. stephensi 'type' form from other ecological forms of the same species as well as other species of Anopheles.

Background: Orang Primary Health Centre (OPHC) and Balipara Primary Health Centre (BPHC) of Assam (India) report mosquito borne diseases annually. Current study was performed to ascertain the prevalence of known malaria and Japanese Encephalitis (JE) vectors and their possible role in disease transmission., Methods: Malaria epidemiological data for 2006-2010 and JE data for 2008-2013 of Assam, India were obtained from the health authority. Mosquitoes were collected using CDC light traps and identified morpho-taxonomically., Results: Plasmodium falciparum cases (81.5%, 95% CI= 72.0-91.1) were statistically higher in OPHC (P< 0.0001, t= 8.0) during the recent years. There was 4.4 folds rise in the confirmed acute encephalitis syndrome (AES) and 3.2 folds increase in the confirmed JE cases during 2013 as compared to 2008. Altogether 9,218 mosquito specimens (PTND= 153.6), comprising of 44.1% anophelines (PTND= 67.7), 42.3% culicines (PTND= 65.0) and 9.5% mansonia (PTND= 14.6) were recorded. In BPHC, Anopheles vagus was recorded in high density (P< 0.0001), whereas Culex quinquefasciatus was the predominant JE vector (P= 0.04). In OPHC, among the known malaria vectors, the density of Anopheles annularis was significantly high (P< 0.0001). However Culex bitaeniorhynchus was the predominant known JE vector (P< 0.0001) followed by Cx. quinquefasciatus., Conclusion: Even in the absence of known efficient vectors, many Anopheles species are still involved in malaria transmission. There was disappearance of Anopheles minimus and Anopheles dirus and establishment of An. annularis , An. vagus and An. philippinensis/nivipes mosquitoes in study area.

During the recent past, development of DDT resistance and reduction to pyrethroid susceptibility among the malaria vectors has posed a serious challenge in many Southeast Asian countries including India. Current study presents the insecticide susceptibility and knock-down data of field collected Anopheles annularis sensu lato and An. vagus mosquito species from endemic areas of Assam in northeast India. Anopheles annularis s.l. and An. vagus adult females were collected from four randomly selected sentinel sites in Orang primary health centre (OPHC) and Balipara primary health centre (BPHC) areas, and used for testing susceptibility to DDT, malathion, deltamethrin and lambda-cyhalothrin. After insecticide susceptibility tests, mosquitoes were subjected to VectorTest™ assay kits to detect the presence of malaria sporozoite in the mosquitoes. An. annularis s.l. was completely susceptible to deltamethrin, lambda-cyhalothrin and malathion in both the study areas. An. vagus was highly susceptible to deltamethrin in both the areas, but exhibited reduced susceptibility to lambda-cyhalothrin in BPHC. Both the species were resistant to DDT and showed very high KDT50 and KDT99 values for DDT. Probit model used to calculate the KDT50 and KDT99 values did not display normal distribution of percent knock-down with time for malathion in both the mosquito species in OPHC (p<0.05) and An. vagus in BPHC (χ2 = 25.3; p = 0.0), and also for deltamethrin to An. vagus in BPHC area (χ2 = 15.4; p = 0.004). Minimum infection rate (MIR) of Plasmodium sporozoite for An. vagus was 0.56 in OPHC and 0.13 in BPHC, while for An. annularis MIR was found to be 0.22 in OPHC. Resistance management strategies should be identified to delay the expansion of resistance. Testing of field caught Anopheles vectors from different endemic areas for the presence of malaria sporozoite may be useful to ensure their role in malaria transmission.

Background: Resistance development in human malaria parasites against commonly used antimalarial drugs has necessitated the scientific exploration of traditionally used antimalarial plants. Plant derivatives have been used for curing malaria historically. The present study involves in vitro evaluation of two medicinally important plants Aristolochia griffithii and Thalictrum foliolosum DC used in antimalarial chemotherapy by the tribes of northeast India., Method: Chloroform, ethyl acetate and n-butanol extracts of Aristolochia griffithii and Thalictrum foliolosum DC were evaluated in vitro against chloroquine sensitive (SS) and chloroquine resistance strains (RS) of P. falciparum. The tests were conducted following WHO standard method and the inhibition of parasite (IC50) was calculated., Results: In A. griffithii, the IC50 value for ethyl acetate extracts against SS was 6.2 ± 0.02 μg/ml and found to be lower than chloroform extracts, which exhibited an IC50 value of 14.1 ± 0.1 μg/ml (t = 191.1; p < 0.0001). The IC50 values of both chloroform and ethyl acetate extracts for RS were higher as compared to the SS (p < 0.0001). In T. foliolosum DC the IC50 concentration of chloroform extracts for SS and RS were 0.5 ± 0.0 and 1.1 ± 0.0 μg/ml respectively (t = 54.2; p < 0.0001)., Conclusion: The present findings, although preliminary, but scientifically demonstrate that identification and isolation of active compounds of these two plant materials and testing against different Plasmodium species could lead to the development of potential antimalarial drugs for future.

Background: Malaria in northeast India affects children and adults annually. The number of malaria cases reported has declined over the past few years. Nevertheless, it is not clear whether there is an actual decline in parasitaemia or whether asymptomatic malaria infections are on the rise, especially in forested and forest-fringed areas. Asymptomatic malaria forms a parasite reservoir that acts as an epicentre for malaria spread during high-transmission season. Therefore it is important to understand the quantum of asymptomatic malaria infections among the vulnerable population., Method: Four forest fringed historically malaria endemic villages were selected for the study. A total of 133 individuals without a fever history in the past four weeks were tested for malaria parasite using rapid diagnostic test (RDT), microscopy and polymerase chain reaction (PCR) assay during January - February 2014. Indoor resting Anopheles vectors were collected, identified and tested for sporozoite using VectorTest™ panel assay during October 2013 to March 2014, which is a low transmission season for malaria. Social and demographic data were recorded during the study., Results: Mean age (± SEM) of the participants was 16.1 ± 1.2 years (95 % CI: 13.8-18.4). All participants (100 %) reported to use mosquito nets. Altogether, 43.6 % of participants had education below primary level and only 9 % reported a travel history during the past four weeks. All RDT, microscopy and PCR assays were found negative indicating no asymptomatic malaria parasitaemia. Seven known malaria vector species namely, Anopheles nivipes, An. minimus, An. annularis, An. vagus, An. aconitus, An. philippinensis and An. culicifacies, were recorded in the present study. VectorTest™ sporozoite panel assay conducted on 45 pools (N = 224) of vector mosquitoes were found negative for Plasmodium sporozoite., Discussion: Northeastern states of India report asymptomatic malaria parasitemia along with high malaria transmission. An. minimus and An. dirus are recognised as efficient vectors, but An. culicifacies, An. philippinensis and An. annularis also play role in malaria transmission. Currently all participants were found negative for asymptomatic malaria, however the small sample size may restrict the scope of present results to the population living in more remote areas., Conclusion: No cases of asymptomatic malaria infections parasitaemia was found in the present study conducted during a low transmission season indicating that asymptomatic malaria parasitaemia may not be prevalent in the region. Mosquito specimens were tested negative for the malaria sporozoites. Study findings encourage the ongoing malaria intervention efforts and recommends similar investigations in different ecological areas involving large populations.

Essential oil based insect repellents are environment friendly and provide dependable personal protection against the bites of mosquitoes and other blood-sucking insects. In the present study, optimized mixture of three essential oils was embedded into the ethylcellulose (EC) and polyvinylpyrrolidone (PVP K-30) polymers to develop essential oils based patch type mosquito repellent formulation. The developed formulation was characterized for various physico-chemical properties, oil release efficiency and essential oil-polymer interaction. Repellent activity of the formulation was evaluated against Ae. (S) albopictus mosquitoes and compared with commercially available synthetic insecticide based mosquito repellent cream Odomos(®) in the laboratory. The developed patches were 100% flat and there was no interaction between oil components and the excipients. Patches were smooth, homogenous and provided excellent mosquito repellent activity comparable to Odomos(®) under laboratory condition. Morphological and physico-chemical characterization indicated that the formulation was stable and suitable with the polymeric combination. The patch formulation did not show any inhalation toxicity in experimental Wistar rat. The repellent patches developed and evaluated currently, may provide a suitable, eco-friendly, acceptable and safe alternative to the existing synthetic repellent formulations for achieving protection against mosquitoes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Mosquito repellents play an important role in preventing man-mosquito contact. In the present study, we evaluated the synergistic mosquito-repellent activity of Curcuma longa, Pogostemon heyneanus and Zanthoxylum limonella essential oils. The mosquito repellent efficacies of three essential oils were evaluated separately and in combination under laboratory and field conditions. N,N-Diethylphenylacetamide (DEPA) and dimethylphthalate (DMP) were used for comparison of the protection time of the mixture of essential oils. At an optimum concentration of 20%, the essential oils of C. longa, Z. limonella and P. heyneanus provided complete protection times (CPTs) of 96.2, 91.4 and 123.4 min, respectively, against Aedes albopictus mosquitoes in the laboratory. The 1:1:2 mixture of the essential oils provided 329.4 and 391.0 min of CPT in the laboratory and field trials, respectively. The percent increases in CPTs for the essential oil mixture were 30 for DMP and 55 for N,N-diethylphenylacetamide (DEPA). The synergistic repellent activity of the essential oils used in the present study might be useful for developing safer alternatives to synthetic repellents for personal protection against mosquitoes., (Copyright © 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.)

Background: Dengue is rapidly expanding mosquito-borne viral infection globally facing operational challenges due to insecticide resistance in dengue vectors. We have studied the susceptibility status of potential dengue vectors St. albopicta and St. aegypti to the commonly used insecticides., Methods: Stegomyia larval bioassays were carried out to determine LC10, LC50 and LC99 values and resistance ratios (RR50 and RR99) for temephos. Adult susceptibility bioassay to 4% DDT, 0.05% deltamethrin, 5% malathion was assessed following standard procedure to determine the corrected mortality. Knock-down times (KDT50 and KDT99) were estimated and the knock-down resistance ratios (KRR50 and KRR99) were calculated., Results: St. albopicta wild population (WP) of Sotia was resistant to temephos as the LC99 value was 0.12 mg/l and found to be 2.3 fold high than the reference population (RP). St. aegypti WP of Borgong, Kusumtola and Serajuli displayed a RR99 of 2.5, 5.4 and 4.5 respectively suggesting high level of resistance to temephos. Results suggested that both St. albopicta and St. aegypti WP were fully resistant to DDT (mortality < 90%) in all the study locations. Both the species were completely susceptible to deltamethrin and malathion (corrected mortality > 98%), except for St. albopicta at Sotia which displayed low level of resistance to malathion (corrected mortality =95.4%). The estimated KDT values for both the species indicated high level of knock-down resistance to DDT and susceptibility to deltamethrin., Conclusion: WP of both the dengue vectors showed varied response to temephos, while resistant to DDT and completely susceptible to deltamethrin. Both the species were susceptible to malathion at majority of the testing sites. Current results strongly advocate that DDT is no longer effective against dengue vectors, while thorough monitoring of malathion susceptibility in geographical area at dengue risk is inexorable to ascertain whether or not the resistance to malathion is focal. Information generated herein may be useful in better planning and implementing in dengue control strategy using insecticides.

Background: Malaria transmission is perennial in the Assam-Arunachal Pradesh interstate border areas in the Sonitpur district of Assam, India. A yearlong study was carried out on the incidence of symptomatic and asymptomatic malaria and the role of asymptomatic malaria carriers in persistent transmission of the disease. The relationships between malaria incidence and weather parameters were also investigated., Methods: Active and mass blood surveys were conducted on a monthly basis in Bengenajuli, Sapairaumari Pathar, and Nigam villages near the Assam-Arunachal Pradesh border. Epidemiological indices were estimated for malaria-positive cases. Multiple linear regression between monthly malaria incidence and monthly average temperature, and relative humidity along with monthly total rainfall was carried out. The known malaria vectors collected in CDC light traps were identified and recorded., Results: Slide positivity rate (SPR) and Plasmodium falciparum percent (Pf%) for symptomatic malaria were 26.1 and 79.8, respectively. Prevalence of malaria vectors was observed throughout the year with varying density. Anopheles philippinensis/nivipes and A. annularis were predominant among the seven known vector species recorded currently. Asymptomatic parasitemia was detected throughout the year with SPR ranging from 4.8 to 5.3. Monthly rainfall with 1-month lag had the highest correlation (r=0.92) with SPR. The relationship between SPR and weather factors was established as SPR=-114.22+0.58 Tmin+1.38 RH+0.03 RF (R2=0.89; p=0.00)., Conclusion: Low and relatively constant levels of asymptomatic parasitemia was present in the study area. High malaria vector density and presence of asymptomatic malaria parasite carriers were responsible for persistent malaria transmission in the region. This study concludes that passive detection and prompt treatment of asymptomatic carriers is essential for preventing persistent disease transmission. Rainfall along with some other weather variables may be used for predicting the malaria epidemics in the region. The predictive information could be useful to target resources more effectively.

Background: The R2 repeat region of GLURP has been reported as a good genetic marker for Plasmodium falciparum genotyping. Proper knowledge of the extent and nature of P. falciparum genetic diversity using highly immunogenic R2 repeat region in malaria-endemic areas is a crucial element to understand various aspects related to immunity acquisition and disease pathogenesis., Methods: Population diversity of P. falciparum GLURP and amino acid sequence repeats in GLURP R2 region was studied in malaria-endemic Assam state, northeast India and compared at an interval of five years during 2005 (Group-A) and 2011 (Group-B)., Results: Of the 66 samples, a total of 55 samples showed positive PCR bands for GLURP R2 region and altogether ten types of alleles with size ranging from 501 bp to 1,050 bp (50 bp bin) were observed and coded as genotypes I-X. In Group-A (n = 29), 24 samples were found infected with single, four with double and one with triple P. falciparum genotype, while in Group-B (n = 26), single genotype was found in 23 samples, double in two samples and triple in one sample. Genotype IV showed significant increase (p = 0.002) during 2011 (Group-B). Genotypes I to V were more common in Group-B (62%), however genotypes VI to X were more frequently distributed in Group-A. The expected heterozygosity was found slightly higher in Group-A (HE = 0.87) than Group-B (HE = 0.85), whereas multiplicity of infection (MOI) in Group-A (MOI = 1.21 ± 0.49) and Group-B (MOI = 1.12 ± 0.43) did not display significant variation. The amino acid repeat sequence unit (AAU) DKNEKGQHEIVEVEEILPE (called 'a') was more frequent in the well-conserved part of R2 repeat region., Conclusion: The present study is the first extensive study in India which has generated substantial data for understanding the type and distribution of naturally evolved genetic polymorphism at amino acid sequence level in GLURP R2 repeat region in P. falciparum. There was decrease in the PCR amplicon size as well as the number of AAU [amino acid repeat unit] in Group-B displaying the bottleneck effect. The present study described a new type of AAU 'd' which varied from the other previous known AAUs.

Present study reports the prevalence and distribution of pfcrt K76T and mdr1 N86Y mutations in malaria endemic areas of Sonitpur district of Assam. Out of 163 individuals tested for malaria, 67 (SPR = 41.1 %) were detected positive for malaria infection using rapid detection kit in the field and PCR assay in the laboratory. Nested PCR-RFLP assay was used to amplify pfcrt K76T and mdr1 N86Y genes flanking the K76T and N86Y mutations. P. falciparum was most abundant (91.04 %) among the three Plasmodium species reported and its prevalence was significantly higher as compared to P. vivax and P. malariae (χ(2) = 150.76; p ≤ 0.0001; df = 2). Malaria was equally distributed among all the age groups and both the sexes. Hemoglobin contents in severe anaemic patients had a significant linear decreasing trend among patients with the increase in age (χ(2) = 4.33; p = 0.03), whereas non severe anaemic patients exhibited significant linear increasing trend among the patients with the increase in age (χ(2) = 18.38; p ≤ 0.0001). Pfcrt K76T mutation was recorded in 44 (72.13 %) isolates, whereas mdr1 N86Y mutation could be detected in 28 (41.79 %) isolates only. Only 32.7 % of the samples had both pfcrt K76T and mdr N86Y mutations. Number of pfcrt K76T mutant isolates was significantly higher than the wild type. However no significant difference was observed among the number of isolates with mdr1 N86Y mutant and wild isolates.

Background: Dengue vector control programmes are facing operational challenges due to resistance against commonly used insecticides throughout the endemic countries. Recently, there has been appreciable increase in the dengue cases in India, however, no recent data are available on susceptible status of dengue vectors. We have studied the susceptibility level of St. albopicta to commonly used insecticides in India. Adult mosquitoes were tested for the presence of dengue virus., Methods: St. albopicta larval bioassays were carried out to determine the lethal concentrations (LC10, LC50 and LC99) and the resistance ratios (RR10, RR50 and RR99) for temephos. Susceptibility to 4% DDT, 0.05% deltamethrin and 5% malathion was assessed following standard procedure. Knock-down times (KDT10, KDT50 and KDT99) were estimated and knock-down resistance ratios (KRR10, KRR50 and KRR99) were calculated. VectorTest™ dengue antigen assay was used to detect the dengue virus in the field collected mosquitoes., Results: In larval bioassays, the RR ranged from 1.4 (for RR99) to 1.7 (for RR50), which suggested that the tested St. albopicta were susceptible to temephos. There was no deviation among the lethal concentration data from linearity (r2=0.61). Adult St. albopicta mosquitoes were resistant to DDT, while fully susceptible to deltamethrin and malathion. The knock-down values (KDT10, KDT50 and KDT99) obtained for DDT displayed straight line in log-dose-probit analysis and follow linear regression model. The KRR99 for DDT was 4.9, which indicated a 4.9 folds increase in knock-down resistance to DDT. However, for malathion and deltamethrin, the KRR99 values were 1.6 and 1.5 respectively suggesting that mosquitoes were knock-down sensitive. None of the mosquito pool was dengue virus positive., Conclusion: St. albopicta showed resistance to DDT and reduced sensitivity to deltamethrin and malathion. This data on insecticide resistance could help public health authorities in India to design more effective vector control measures. More dengue vector specimens need to be scanned to identify the potential dengue vector.

Background: Malaria is a major cause of morbidity and mortality in Northeast India. As there is limited information available on the potential influence of socio-economic variables on malaria risk, the present study was conducted to assess the influence of demographic factors, the socio-economic status, and knowledge, awareness and education on malaria occurrence., Methods: Demographics, malaria knowledge and socio-economic variables were collected in four randomly selected health sub-centres of the Orang primary health centre in the Udalguri district, Assam and the association of malaria occurrence with different variables were analysed. The trend of malaria occurrence for different income groups, proximity to health centres and number of mosquito bites per day was also determined using the chi-square test. Relative risk (RR) for gender, house type, knowledge and use of bed nets was determined using Katz approximation., Results: Out of the 71 household heads interviewed, 70.4% (50/71) were males. About half (54.9%, 39/71) of the participants had a history of malaria in the last two years, of which 64.1% (25/39) were males, while 35.9% (14/39) were females (χ(2) = 5.13; p = 0.02; RR = 1.79). Of the total population surveyed, 49.3% lived in bamboo houses and 35.2% lived at a distance of >3 km from the nearest health centre. The number of participants who had a history of malaria decreased with an increasing monthly income (p < 0.0001). Malaria occurrence was higher among the households living in bamboo houses (69.2%), as compared to Kucha houses (20.5%) and Pucca houses (10.3%). No significant association was observed between education level and malaria occurrence (p = 0.93). The participants who did not use bed nets regularly reported a high occurrence of malaria infection as compared to those who used bed nets everyday (p < 0.0001)., Conclusions: Lower income, house type, distance to health sub-centre, knowledge and awareness about malaria, number of mosquito bites per day and use of bed nets were positively associated with malaria occurrence. Increasing the number of health sub-centres close to rural areas, improving the economic status and increasing awareness about malaria prevention measures will thus help to reduce malaria-associated morbidities.

Introduction: Chloroquine resistance in Plasmodium falciparum is associated with mutations in pfcrt and pfmdr1 genes. The frequency distribution of pfcrt K76T and pfmdr1 N86Y mutations and their association with chloroquine susceptibility was studied in an endemic area along the Indo-Bangladesh border., Methodology: A single-arm prospective study of clinical and parasitological responses in P. falciparum malaria patients to chloroquine was conducted in vivo. PCR-RFLP assay was used to detect pfcrt K76T and pfmdr1 N86Y mutations in P. falciparum. The PCR products of pfcrt gene were sequenced,  translated and aligned for haplotyping., Results: Out of 63 cases, 44 (69.8%) responded adequately to chloroquine treatment. Pfcrt K76T mutation was recorded in 100% of the treatment failure cases, whereas pfmdr1 N86Y mutation was found in 52.6% of the cases only. Early treatment failure (84.2%) occurred more frequently than late treatment failure (15.8%). Kaplan-Meier survival analysis showed that the probability estimate for treatment success after 7 and 15 days was 0.84 (95% CI = 0.72-0.92) and 0.70 (95% CI = 0.57-0.80), respectively. Sequence analysis of 72 to 76 pfcrt gene codons revealed the presence of two mutant (CVMNT, CVIET) and two wild (CVMNK, CVIEK) haplotypes. The mutant CVIET haplotype was predominantly distributed (42.1%)., Conclusions: The presence of mutations in pfcrt K76T and pfmdr1 N86Y genes is not sufficient to explain the therapeutic efficacy of chloroquine to P. falciparum. Study suggests that pfcrt K76T mutant haplotypes are widely distributed and are spreading diligently, which needs to be taken into account in devising an antimalarial policy.

The genus Simulium, black fly (Diptera), comprises >1800 species worldwide, of which 67 species under six subgenera have been well studied in India. While at the extreme, black fly bites can cause onchocerciasis or river blindness, the majority of clinical observations indicate mainly severe pain and continuous itching at bite sites. This investigation experimentally observed that black fly salivary extract (BFSE) causes unique biologic effects including immunomodulation, anti-coagulation, and hypersensitivity reactions in Wistar rats. Salivary glands from black flies were isolated, extracted in saline, and then ≈800 ng extract (BFSE) subcutaneously injected into rats. To evaluate potential immunosuppressive activity of the BFSE, serum levels of interleukins [IL]-6 and -10 and tumor necrosis factor [TNF]-α were assayed. To assess the BFSE impact on coagulation, activated partial thromboplastin time (aPTT), prothrombin time (PT), and bleeding time, as well as generation of coagulation activated factors I, IX, and X were analyzed. Anaphylaxis induction was monitored via electrocardiogram (ECG) and measures of blood pressure and rectal temperature. The data showed that BFSE treatment resulted in a significantly prolonged aPTT, PT, and bleeding time and reversibly inhibited generation of coagulation activated factors I, IX, and X. The extract also led to a prolonged (up to 48 h) suppression of serum IL-6, IL-10, and TNFα production. While these results suggest that BFSE possesses anti-thrombotic, anti-coagulant, and immunomodulatory activities, importantly, they also indicate that the extract has a capacity to induce anaphylaxis and acute cardiotoxicity.

Pupal stages of Simulium were collected and identified from various breeding habitats of twelve locations in northeastern India. Simulium flies, while attempting to land on human were collected between 07:00-17:00 hrs to understand the biting pattern. Seven species belonging to three sub-genera, Eusimulium (1), Gomphostilbia (1) and Simulium (5) belonging genus Simulium were encountered. Out of total seven species recorded, S. (E) aureohirtum, S. (G) tenuistylum and S. (S) rufibasis were predominant and shared 30.3%, 29.9% and 27.6% of total collection. Stream breeding habitat contributed 47.3% of total catch and was found to be preferred breeding habitat (p < 0.0001). S. (S) christophersi and S. (G) tenuistylum were recorded for the first time from the northeastern region of India. Simuliids biting rhythm showed bimodal pattern and were more active during sunny day (p < 0.0001). Microscopic dissection of simuliids (n = 266) did not incriminate simuliids as vector of onchocerciasis.

Background & Objective: Japanese encephalitis (JE) outbreaks are common in Assam, northeastern State of India. Information on resistance in known JE vectors in the affected area is important for effective control measures. This study was undertaken to determine the species abundance of JE vectors endemic to Sibsagar district of Assam, and their susceptibility against DDT and deltamethrin., Methods: Adult mosquitoes were collected using CDC light trap and aspirators from human dwellings from 13 endemic villages falling under three Primary Health Centres. Collected mosquitoes were identified and unfed female mosquitoes were used for DDT and deltamethrin sensitivity bioassay. The bioassay was performed following WHO protocol using standard susceptibility test kit. Knockdown time (KDT) was monitored at every 10 minutes intervals, whereas mortalities were recorded 24 h post-exposure. Vector density and resistance status were mapped using geographic information system (GIS) technique., Results: A total of 7655 mosquitoes were sampled under three genera, i.e. Anopheles, Culex and Mansonia, and nine species, the JE vector Cx. vishnui group (31.78%) was the most predominant species, followed by Ma. uniformis (16.81%) and Ma. indiana (16.45%). All vector species were suspected to be resistant to DDT and sensitive to deltamethrin, except Ma. indiana, which was suspected to deltamethrin resistant. The KDT50 and KDT95 values of vector mosquitoes for DDT were significantly higher as compared to deltamethrin. The probit model used to estimate KDT50 and KDT95 values did not display normal distribution of percentage knockdown with time for all the vectors tested for DDT and deltamethrin, except for Ma. indiana for deltamethrin assay and Cx. gelidus for the DDT assay., Interpretation & Conclusion: Differences in insecticide resistance status were observed between insecticides and vector species. The results of this study provided baseline data on insecticide resistance in known JE vectors of Sibsagar, Assam. The maps generated may allow better communication in control operations and comparison of changes in susceptibility status of these vectors over time.

The present study evaluates the performance of OptiMAL-IT test and nested PCR assay in detection of malaria parasites. A total of 76 randomly selected blood samples collected from two malaria endemic areas were tested for malaria parasites using microscopy and OptiMAL-IT test in the field. PCR assays were performed in the laboratory using DNA extracted from blood spots of the same samples collected on the FTA classic cards. Of the total of 61 field confirmed malaria positive samples, only 58 (95%) were detected positive using microscopy in the laboratory. Sensitivity, specificity, positive predictive value, negative predictive value and false discovery rate of OptiMal-IT in comparison to the microscopy were 93%, 83%, 95%, 79% and 5%, respectively. On the other hand, the sensitivity and specificity of PCR assay were 97% and 100%, respectively, whereas positive predictive value, negative predictive value and false discovery rate were 100%, 90% and 0%, respectively. The overall performance of OptiMal-IT and PCR assays for malaria diagnosis was 76% and 97%, respectively. PCR assay enabled the identification of infection with Plasmodium malariae Laveran, 1881 in four samples misidentified by microscopy and Plasmodium-specific antigen (PAN) identified by the OptiMAL-IT test. In addition to the standard methods, such PCR assay could be useful to obtain the real incidence of each malaria parasite species for epidemiological perspectives.

Background: Mosquito repellents and emanators confer protection against mosquito bites through spatial action of emanated vapours which are released into the adjoining environment. Synthetic insecticides released into the environment in ultra low volume vapour phase deter the mosquitoes from biting humans in a protected space., Methods: Formulation patches were prepared using the solvent evaporation method over a backing membrane and using Dibutylphthalate (DBT) as a plasticizer. The effect of formulation variables on the deltamethrin release from the patch matrices were studied under accelerated conditions, whereas, HPLC was used for quantitative estimation of deltamethrin. The prepared patch formulations were subjected to physicochemical studies, such as, deltamethrin content, thickness, weight variation, percent moisture content, moisture uptake, surface area and surface pH determination. Deltamethrin-polymer interaction and compatibility was ascertained using DSC and FT-IR, while surface morphology and deltamethrin distribution in the patch were studied using SEM technique. Repellent activity of the patch formulations was evaluated against Ae. albopictus mosquitoes., Results: Blends of polymeric combinations of polyvinylpyrrolidone (PVP) and ethyl-cellulose (EC) with admixture of deltamethrin provided prolonged repellent activity against Ae. albopictus mosquitoes. Physicochemical characterisation indicated the suitability of deltamethrin patch formulation with the polymeric combinations of PVP and EC. Patches were very effective against laboratory reared Ae. albopictus mosquitoes. No significant difference was observed between the performance of test patches and commercially available repellent cream Mosqshield®., Conclusion: Deltamethrin loaded patches provided effective repellency against Ae. albopictus mosquitoes. The study emphasised that deltamethrin released to the environment in low concentration could be an excellent spatial repellent against hematophagous mosquitoes.

India reports the highest number of malaria cases in Southeast Asia, of which Plasmodium falciparum contribute more than half of the cases every year. North eastern states of India contribute only 3.96% of country's population but account for >10% of total reported malaria cases, 11% of Plasmodium falciparum cases and 20% of malaria related deaths annually. In India, chloroquine resistance was reported for the first time from northeast region and since then chloroquine treatment failure is being reported from many parts of the region. Increased chloroquine treatment failure has led to change of the drug policy to artemisinin combination therapy as first line of malaria treatment in the region. However, replacing chloroquine to artemisinin combination therapy has not shown significant difference in the overall malaria incidence in the region. The present review addresses the current malaria situation of northeastern region of India in the light of antimalarials drug resistance., (Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

Background & Objectives: Simuliids (Diptera) are one of the medically important biting insects group and have worldwide distribution. Their immature stages proliferate in fast flowing river or stream water and have been considered as important ecological indicator., Methods: Aquatic stages of simuliids were collected and speciated from 16 different fresh water rivers and streams. Water flow rate was determined and water samples were analysed for various water variables such as water temperature, pH, dissolved oxygen concentration (DOC), dissolved oxygen saturation (DOS), conductivity, total dissolved solute (TDS), turbidity, resistivity and salinity. Linear regression was used to determine relationship between simuliid density and water variables, whereas multiple regression was used to determine the fitness for the presence of simuliid species. Principal component analysis (PCA) was used to determine the water parameters association with simuliid distribution., Results: Total 565 specimens comprising of three species namely, Simulium (S) barraudi Puri, S. (S) striatum Brunetti, and S. (S) himalayense Puri were recorded in the present study. Simulium barraudi was the most abundant (56.8%) and its density was high (χ2 = 289.3; df = 2; p <0.0001) as compared to the others. The average population size of each species was 188.3, whereas Simpson and Shannon-Wiener diversity indices were 0.4466 and 1.306 respectively. Linear regression showed that simuliid density was associated with the water flow rate. Principal component analysis indicated that the water parameters accounted for 42.25% variation along D1 axis, while 24.1% variation along D2 axis. Atleast two principal components have eigenvalue >1 and accounted for 32.6% of variation., Interpretation & Conclusion: Our study provides new information on simuliid species association with breeding water parameters in a little studied region of high biological interest. Turbidity, water flow and pH are important water parameters affecting the simuliid species prevalence. Each simuliid species preferred different sets of physicochemical parameters of breeding habitat, which are specific to that particular species. Therefore, simuliid species community as a whole cannot be considered as a suitable indicator of the streams water quality. In addition to describing simuliids, the information provided herein will be useful for the conservation of aquatic ecology and environment in Meghalaya state of India.

Background: Oral squamous cell carcinoma (OSCC) is the sixth most common cancer globally. Tobacco consumption and HPV infection, both are the major risk factor for the development of oral cancer and causes mitochondrial dysfunction. Genetic polymorphisms in xenobiotic-metabolizing enzymes modify the effect of environmental exposures, thereby playing a significant role in gene-environment interactions and hence contributing to the individual susceptibility to cancer. Here, we have investigated the association of tobacco - betel quid chewing, HPV infection, GSTM1-GSTT1 null genotypes, and tumour stages with mitochondrial DNA (mtDNA) content variation in oral cancer patients., Methodology/principal Findings: The study comprised of 124 cases of OSCC and 140 control subjects to PCR based detection was done for high-risk HPV using a consensus primer and multiplex PCR was done for detection of GSTM1-GSTT1 polymorphism. A comparative ΔCt method was used for determination of mtDNA content. The risk of OSCC increased with the ceased mtDNA copy number (Ptrend  = 0.003). The association between mtDNA copy number and OSCC risk was evident among tobacco - betel quid chewers rather than tobacco - betel quid non chewers; the interaction between mtDNA copy number and tobacco - betel quid was significant (P = 0.0005). Significant difference was observed between GSTM1 - GSTT1 null genotypes (P = 0.04, P = 0.001 respectively) and HPV infection (P<0.001) with mtDNA content variation in cases and controls. Positive correlation was found with decrease in mtDNA content with the increase in tumour stages (P<0.001). We are reporting for the first time the association of HPV infection and GSTM1-GSTT1 null genotypes with mtDNA content in OSCC., Conclusion: Our results indicate that the mtDNA content in tumour tissues changes with tumour stage and tobacco-betel quid chewing habits while low levels of mtDNA content suggests invasive thereby serving as a biomarker in detection of OSCC.

This study was carried out to determine the human host preference and presence of Plasmodium parasite in field collected Anopheles mosquitoes among four villages around a military cantonment located in malaria endemic Sonitpur district of Assam, India. Encountered malaria vector mosquitoes were identified and tested for host preference and Plasmodium presence using PCR method. Human host preference was detected using simple PCR, whereas vectorial status for Plasmodium parasite was confirmed using first round PCR with genus specific primers and thereafter nested PCR with three Plasmodium species specific primers. Out of 1874 blood fed vector mosquitoes collected, 187 (10%) were processed for PCR, which revealed that 40·6% had fed on human blood; 9·2% of human blood fed mosquito were harbouring Plasmodium parasites, 71·4% of which were confirmed to Plasmodium falciparum. In addition to An. minimus, An. annularis and An. culicifacies were also found positive for malaria parasites. The present study exhibits the human feeding tendency of Anopheles vectors highlighting their malaria parasite transmission potential. The present study may serve as a model for understanding the human host preference of malaria vectors and detection of malaria parasite inside the anopheline vector mosquitoes in order to update their vectorial status for estimating the possible role of these mosquitoes in malaria transmission. The study has used PCR method and suggests that PCR-based method should be used in this entire malarious region to correctly report the vectorial position of different malaria vectors.