Malaria Vector Bionomics: Countrywide Surveillance Study on Implications for Malaria Elimination in India.

BACKGROUND: The biological characteristics of mosquito vectors vary, impacting their response to control measures. Thus, having up-to-date information on vector bionomics is essential to maintain the effectiveness of existing control strategies and tools, particularly as India aims for malaria elimination by 2030. OBJECTIVE: This study aims to assess the proportions of vector species resting indoors and outdoors, determine their preference for host biting/feeding, identify transmission sites, and evaluate the susceptibility of vectors to insecticides used in public health programs. METHODS: Mosquito collections were conducted in 13 districts across 8 Indian states from 2017 to 2020 using various methods to estimate their densities. Following morphological identification in the field, sibling species of Anopheles mosquitoes were identified molecularly using polymerase chain reaction (PCR)-specific alleles. Plasmodium falciparum and Plasmodium vivax infections in the vectors were detected using enzyme-linked immunosorbent assay (ELISA) and PCR assays. In addition, we assessed the insecticide susceptibility status of primary malaria vectors following the World Health Organization (WHO) protocol. RESULTS: Anopheles culicifacies, a primary malaria vector, was collected (with a man-hour density ranging from 3.1 to 15.9) from all states of India except those in the northeastern region. Anopheles fluviatilis, another primary vector, was collected from the states of Madhya Pradesh, Maharashtra, Karnataka, and Odisha. In Haryana and Karnataka, An. culicifacies sibling species A predominated, whereas species C and E were predominant in Madhya Pradesh and Maharashtra. An. culicifacies displayed mainly endophilic behavior across all states, except in Madhya Pradesh, where the proportion of semigravid and gravid mosquitoes was nearly half of that of unfed mosquitoes. The human blood index of An. culicifacies ranged from 0.001 to 0.220 across all study sites. The sporozoite rate of An. culicifacies ranged from 0.06 to 4.24, except in Madhya Pradesh, where none of the vector mosquitoes were found to be infected with the Plasmodium parasite. In the study area, An. culicifacies exhibited resistance to DDT (dichlorodiphenyltrichloroethane; with <39% mortality). Moreover, it showed resistance to malathion (with mortality rates ranging from 49% to 78%) in all districts except Angul in Odisha and Palwal in Haryana. In addition, resistance to deltamethrin was observed in districts of Maharashtra, Gujarat, Haryana, and Karnataka. CONCLUSIONS: Our study offers vital insights into the prevalence, resting behavior, and sibling species composition of malaria vectors in India. It is evident from our findings that resistance development in An. culicifacies, the primary vector, to synthetic pyrethroids is on the rise in the country. Furthermore, the results of our study suggest a potential change in the resting behavior of An. culicifacies in Madhya Pradesh, although further studies are required to confirm this shift definitively. These findings are essential for the development of effective vector control strategies in India, aligning with the goal of malaria elimination by 2030.

Monitoring of Leishmania transmission in the post-elimination phase: the potential of serological surveys.

OBJECTIVES: Monitoring of Leishmania transmission is considered a strategic priority for sustaining elimination of visceral leishmaniasis as a public health problem in the Indian subcontinent. The objective of this study was to evaluate whether serological surveys can distinguish between communities with and without Leishmania transmission, and to assess which serological marker performs best. METHODS: Seven villages were selected from Bihar and Uttar Pradesh state, India, and categorized as either currently endemic, previously endemic or non-endemic. Blood samples were analyzed with the rK39 RDT, DAT, and rK39 ELISA. RESULTS: Contrary to the rK39 RDT and DAT, the rK39 ELISA showed a significant difference between all three categories of endemicity, with a seroprevalence of 5.21% in currently endemic villages, 1.55% in previously endemic villages, and 0.13% in non-endemic villages. Even when only looking at the seroprevalence among children aged <10 years, the rK39 ELISA was still able to differentiate between villages with and without ongoing transmission. CONCLUSIONS: Our findings suggest the rK39 ELISA to be the most promising marker for monitoring of Leishmania transmission. Further validation is required, and practical, context-adapted recommendations need to be formulated in order to guide policy makers towards meaningful and sustainable surveillance strategies in the post-elimination phase.

Visceral Leishmaniasis-Human Immunodeficiency Virus-Coinfected Patients Are Highly Infectious to Sandflies in an Endemic Area in India.

In an area endemic with Indian visceral leishmaniasis (VL), we performed direct xenodiagnosis to evaluate the transmission of Leishmania donovani from patients with VL-human immunodeficiency virus (HIV) coinfection to the vector sandflies, Phlebotomus argentipes. Fourteen patients with confirmed VL-HIV coinfection, with a median parasitemia of 42 205 parasite genome/mL of blood, were exposed to 732 laboratory-reared pathogen-free female P argentipes sandflies on their lower arms and legs. Microscopy revealed that 16.66% (122/732) of blood-fed flies were xenodiagnosis positive. Notably, 93% (13/14) of the VL-HIV group infected the flies, as confirmed by quantitative polymerase chain reaction and/or microscopy, and were 3 times more infectious than those who had VL without HIV.

An experimental and prediction modeling study on water lettuce (Pistia stratiotes L.) assisted heavy metals removal from glass industry effluent.

The glass manufacturing industry produces hazardous effluent that is difficult to manage and causes numerous environmental problems when disposed of in the open. In this study, an attempt was made to study the phytoremediation feasibility of water lettuce (Pistia stratiotes L.), a free-floating aquatic macrophyte, for the removal of six heavy metals from glass industry effluent (GIE) at varying concentrations (0, 25, 50, 75, and 100%). After a 40-day experiment, the results showed that 25% GIE dilution showed maximum removal of heavy metals i.e., Cu (91.74%), Cr (95.29%), Fe (86.47%), Mn (92.95%), Pb (87.10%), and Zn (91.34%), respectively. The bioaccumulation, translocation, and Pearson correlation studies showed that the amount of heavy metals absorbed by vegetative parts of P. stratiotes was significantly correlated with concentrations. The highest biomass production, chlorophyll content, relative growth rate, and biomass productivity were also noted in the 25% GIE treatment. Moreover, the multiple linear regression models developed for the prediction of heavy metal uptake by P. stratiotes also showed good performance in determining the impact of GIE properties. The models showed a high coefficient of determination (R2 > 0.99), low mean average normalizing error (MANE = 0.01), and high model efficiency (ME > 0.99) supporting the robustness of the developed equations. This study outlined an efficient method for the biological treatment of GIE using P. stratiotes to reduce risks associated with its unsafe disposal.

Color Doppler Ultrasound Indices as Predictors of Propranolol Response in Infantile Hemangioma: A Prospective Study.

OBJECTIVE: To evaluate the utility of color Doppler ultrasonography in assessing infantile hemangioma response to treatment with oral propranolol. METHODS: A prospective study was conducted between January, 2016 and December, 2022, wherein children with symptomatic (ulceration, bleeding, pain and scarring) infantile hemangioma were given oral propranol (2 mg/kg per day in three divided doses) as outpatient therapy. The clinical response was assessed three months post-initiation of treatment (intermediate clinical response) and three months post-completion of treatment (final clinical response, FCR). The primary outcome measurement was a clinical and radiological response (resistivity index (RI), pulsatility index (PI) and peak systolic velocity) to treatment. The secondary outcomes assessed were the complications related to treatment. RESULTS: Out of 601 patients who were started on propranolol, 99 developed severe adverse effects and were excluded from analysis. At FCR assessment, out of 502 participants, 64.3% (n = 323) showed excellent response, 17.7% (n = 89) showed partial, and 17.9% (n = 90) were non-responders. A significant increase in RI and PI values was noted in all children following propranolol treatment for six months. An increase > 7.5% in RI could identify responders with 92% sensitivity, 91% specificity and area under the curve (AUC) of 0.963. An increase of > 11.5% in PI could identify responders with 86% sensitivity, 91% specificity and AUC of 0.896. Patients initially showing no response but later becoming excellent responders had significantly higher RI and PI values. CONCLUSIONS: Color Doppler ultrasonography is a valuable tool in predicting the treatment outcome of infantile hemangioma using propranolol.

Efficacy of Individualized Homeopathic Medicines in the Treatment of Hemorrhoids: Double-Blind, Randomized, Placebo-Controlled Trial.

Objectives: To investigate the efficacy and safety of individualized homeopathic medicines (IHMs) in treating hemorrhoids compared with placebo. Design: This is a double-blind, randomized (1:1), two parallel arms, placebo-controlled trial. Setting: The trial was conducted at the surgery outpatient department of the State National Homoeopathic Medical College and Hospital, Lucknow, Uttar Pradesh, India. Subjects: Patients were 140 women and men, aged between 18 and 65 years, with a diagnosis of primary hemorrhoids grades I-III for at least 3 months. Excluded were the patients with grade IV hemorrhoids, anal fissure, and fistula, hypertrophic anal papillae, inflammatory bowel disease, coagulation disorders, rectal malignancies, obstructed portal circulation, patients requiring immediate surgical intervention, and vulnerable samples. Interventions: Patients were randomized to Group 1 (n = 70; IHMs plus concomitant care; verum) and Group 2 (n = 70; placebos plus concomitant care; control). Outcome measures: Primary-the anorectal symptom severity and quality-of-life (ARSSQoL) questionnaire, and secondary-the EuroQol 5-dimensions 5-levels (EQ-5D-5L) questionnaire and EQ visual analogue scale (VAS); all of them were measured at baseline, and every month, up to 3 months. Results: Out of the 140 randomized patients, 122 were protocol compliant. Intention-to-treat sample (n = 140) was analyzed. The level of significance was set at p < 0.05 two tailed. Statistically significant between-group differences were elicited in the ARSSQoL total (Mann-Whitney U [MWU]: 1227.0, p < 0.001) and EQ-5D-5L VAS (MWU: 1228.0, p = 0.001) favoring homeopathy against placebos. Sulfur was the most frequently prescribed medicine. No harm or serious adverse events were reported from either of the groups. Conclusions: IHMs demonstrated superior results over placebo in the short-term treatment of hemorrhoids of grades I-III. The findings are promising, but need to be substantiated by further phase 3 trials. Clinical Trial Registration Number: CTRI/2020/03/024342.

Polyubiquitin protein of Aedes aegypti as an interacting partner of dengue virus envelope protein.

Dengue virus (DENV) is an arbovirus that comprises four antigenically different serotypes. Aedes aegypti (Diptera: Culicidae) acts as the principal vector for DENV transmission, and vector control is crucial for dengue fever epidemic management. To design effective vector control strategies, a comprehensive understanding of the insect vector and virus interaction is required. Female Ae. aegypti ingests DENV during the acquisition of a blood meal from an infected human. DENV enters the insect midgut, replicates inside it and reaches the salivary gland for transmitting DENV to healthy humans during the subsequent feeding cycles. DENV must interact with the proteins present in the midgut and salivary glands to gain entry and accomplish successful replication and transmission. Ae. aegypti midgut cDNA library was prepared, and yeast two-hybrid screening was performed against the envelope protein domain III (EDIII) protein of DENV-2. The polyubiquitin protein was selected from the various candidate proteins for subsequent analysis. Polyubiquitin gene was amplified, and the protein was purified in a heterologous expression system for in vitro interaction studies. In vitro pull-down assay presented a clear interaction between polyubiquitin protein and EDIII. To further confirm this interaction, a dot blot assay was employed, and polyubiquitin protein was found to interact with DENV particles. Our results enable us to suggest that polyubiquitin plays an important role in DENV infection within mosquitoes.

Evaluating Ayurvedic mouthwash and soda-salt mouthwash for oral mucositis in head and neck cancer: A randomized controlled trial.

BACKGROUND: Patients undergoing radiotherapy (RT) or concurrent chemo-radiation (CCRT) for head and neck squamous cell carcinoma (HNSCC) often suffer from side effects such as mucositis, xerostomia, pharyngitis, laryngitis, and pain, which are being managed symptomatically by alcohol-based mouthwashes. OBJECTIVES: To determine the effectiveness of Ayurvedic mouthwash "Draksha Guduchyadi Kashaya" in reducing the severity of oral side effects of chemo-radiation. MATERIAL AND METHODS: This concurrent parallel randomized controlled study was conducted at Sir Sunderlal Hospital, BHU, on 70 HNSCC patients scheduled to undergo RT/CCRT. Patients who met the inclusion-exclusion criteria were enrolled, and 35 were randomly assigned to either the intervention group (Ayurveda) or the control group using a simple random technique (lottery method). Blinding was not implemented in this study. Patients in the intervention group (Ayurveda) were instructed to perform kavala with 50 ml of "Draksha Guduchyadi Kashaya" for 2 min, ten times daily, while the control group performed 2-min gargling with soda-salt mouthwash ten times daily. RESULTS: Out of the 70 patients enrolled, data from 60 patients were analyzed, revealing statistically significant differences in the onset of mucositis (p = 0.049), pharyngitis (p = 0.034), laryngitis (p = 0.009) and intensity of variables such as mucositis (p = 0.000), xerostomia (p = 0.046), pharyngitis (p = 0.002), laryngitis (p = 0.035), and pain (p = 0.000). These findings indicate that Ayurvedic mouthwash may be beneficial in managing the oral side effects of chemo-radiation in HNSCC. CONCLUSION: This AYUSH financially supported trial (Reg No: CTRI/2020/04/024672) demonstrates promise as a safe and cost-effective alternative for managing oral complications of RT/CCRT, offering complementary treatment for comprehensive cancer care.

Wuchereria bancrofti infection is associated with progression to clinical visceral leishmaniasis in VL- endemic areas in Muzaffarpur, Bihar, India.

BACKGROUND: Co-endemicity of neglected tropical diseases (NTDs) necessitates that these diseases should be considered concomitantly to understand the relationship between pathology and to support disease management and control programs. The aims of the study were to assess the prevalence of filarial infection in asymptomatic Leishmania donovani infected individuals and the correlation of Wuchereria bancrofti infection with progression to clinical visceral leishmaniasis (VL) in Bihar, India. METHODOLOGY/PRINCIPAL FINDINGS: Within the Muzaffarpur-TMRC Health and Demographic Surveillance System (HDSS) area, a cohort of Leishmania seropositive (n = 476) or seronegative individuals (n = 1130) were sampled annually for three years for filarial infection and followed for progression to clinical VL. To corroborate the results from the cohort study, we also used a retrospective case-control study of 36 VL cases and 71 controls selected from a subset of the HDSS population to investigate the relationship between progression to clinical VL and the prevalence of filarial infection at baseline. Our findings suggest a higher probability of progression to clinical VL in individuals with a history of filarial infection: in both the cohort and case-control studies, progression to clinical VL was higher among filaria infected individuals (RR = 2.57, p = 0.056, and OR = 2.52, p = 0.046 respectively). CONCLUSION: This study describes that progression to clinical VL disease is associated with serological evidence of prior infection with W. bancrofti. The integration of disease programs for Leishmania and lymphatic filariasis extend beyond the relationship of sequential or co-infection with disease burden. To ensure elimination targets can be reached and sustained, we suggest areas of co-endemicity would benefit from overlapping vector control activities, health system networks and surveillance infrastructure.

Artemisinin resistance in P. falciparum: probing the interacting partners of Kelch13 protein in parasite.

OBJECTIVES: Artemisinin (ART) resistance in Plasmodium is threatening the artemisinin combination therapies-the first line of defence against malaria. ART resistance has been established to be mediated by the Plasmodium Kelch13 (PfK13) protein. For the crucial role of PfK13 in multiple pathways of the Plasmodium life cycle and ART resistance, it is imperative that we investigate its interacting partners. METHODS: We recombinantly expressed PfK13-p (Bric a brac/Poxvirus and zinc finger and propeller domains), generating anti-PfK13-p antibodies to perform co-immunoprecipitation assays and probed PfK13 interacting partners. Surface plasmon resonance and pull-down assays were performed to establish physical interactions of representative proteins with PfK13-p. RESULTS: The co-immunoprecipitation assays identified 17 proteins with distinct functions in the parasite life cycle- protein folding, cellular metabolism, and protein binding and invasion. In addition to the overlap with previously identified proteins, our study identified 10 unique proteins. Fructose-biphosphate aldolase and heat shock protein 70 demonstrated strong biophysical interaction with PfK13-p, with KD values of 6.6 µM and 7.6 µM, respectively. Additionally, Plasmodium merozoite surface protein 1 formed a complex with PfK13-p, which is evident from the pull-down assay. CONCLUSION: This study adds to our knowledge of the PfK13 protein in mediating ART resistance by identifying new PfK13 interacting partners. Three representative proteins-fructose-biphosphate aldolase, heat shock protein 70, and merozoite surface protein 1-demonstrated clear evidence of biophysical interactions with PfK13-p. However, elucidation of the functional relevance of these physical interactions are crucial in context of PfK13 role in ART resistance.

Correction: Assessing insecticide susceptibility, diagnostic dose and time for the sand fly Phlebotomus argentipes, the vector of visceral leishmaniasis in India, using the CDC bottle bioassay.

[This corrects the article DOI: 10.1371/journal.pntd.0010613.].

Genetic differentiation among Aedes aegypti populations from different eco-geographical zones of India.

The present study explicitly evaluated the genetic structure of Aedes aegypti Linn, the vector of dengue, chikungunya, and Zika viruses, across different geo-climatic zones of India and also elucidated the impact of ecological and topographic factors. After data quality checks and removal of samples with excess null alleles, the final analysis was performed on 589 individual samples using 10 microsatellite markers. Overall findings of this study suggested that, Ae. aegypti populations are highly diverse with moderate genetic differentiation between them. Around half of the populations (13 out of 22) formed two genetic clusters roughly associated with geographical regions. The remaining nine populations shared genetic ancestries with either one or both of the clusters. A significant relationship between genetic and geographic distance was observed, indicating isolation by distance. However, spatial autocorrelation analysis predicted the signs of long-distance admixture. Post-hoc environmental association analysis showed that 52.7% of genetic variations were explained by a combination of climatic and topographic factors, with latitude and temperature being the best predictors. This study indicated that though overall genetic differentiation among Ae. aegypti populations across India is moderate (Fst = 0.099), the differences between the populations are developing due to the factors associated with geographic locations. This study improves the understanding of the Ae. aegypti population structure in India that may assist in predicting mosquito movements across the geo-climatic zones, enabling effective control strategies and assessing the risk of disease transmission.

Assessing insecticide susceptibility, diagnostic dose and time for the sand fly Phlebotomus argentipes, the vector of visceral leishmaniasis in India, using the CDC bottle bioassay.

Visceral leishmaniasis (VL) is a vector-borne protozoan disease, which can be fatal if left untreated. Synthetic chemical insecticides are very effective tools for controlling of insect vectors, including the sand fly Phlebotomus argentipes, the vector of VL in the Indian subcontinent. However, repeated use of the same insecticide with increasing doses potentially can create high selection pressure and lead to tolerance and resistance development. The objective of this study was to determine the lethal concentrations and assess levels of susceptibility, diagnostic doses and times to death of laboratory-reared P. argentipes to five insecticides that are used worldwide to control vectors. Using the Center for Disease Control and Prevention (CDC) bottle bioassay, 20-30 sand flies were exposed in insecticide- coated 500-ml glass bottles. Flies were then observed for 24 hours and mortality was recorded. Dose-response survival curves were generated for each insecticide using QCal software and lethal concentrations causing 50%, 90% and 95% mortality were determined. A bioassay was also conducted to determine diagnostic doses and diagnostic times by exposing 20-30 flies in each bottle containing set concentrations of insecticide. Mortality was recorded at 10-minute intervals for 120 minutes to generate the survival curve. Phlebotomus argentipes are highly susceptible to alpha-cypermethrin, followed by deltamethrin, malathion, chlorpyrifos, and least susceptible to DDT. Also, the lowest diagnostic doses and diagnostic times were established for alpha-cypermethrin (3µg/ml for 40 minutes) to kill 100% of the flies. The susceptibility data, diagnostic doses and diagnostic times presented here will be useful as baseline reference points for future studies to assess insecticide susceptibility and resistance monitoring of field caught sand flies and to assist in surveillance as VL elimination is achieved in the region.

Antimalarial Drug Resistance Profiling of Plasmodium falciparum Infections in India Using Next-Generation Sequencing.

Background: Tracking the emergence and spread of antimalarial drug resistance has become critical to sustaining progress towards the control and eventual elimination of malaria in South Asia, especially India. Methods: An amplicon sequencing protocol was used for high-throughput molecular surveillance of antimalarial drug resistance in a total of 158 isolates at three sites in India: Chennai, Nadiad and Rourkela. Five genes of the Plasmodium falciparum implicated in antimalarial resistance were investigated here; Pfcrt for chloroquine resistance, Pfdhfr for pyrimethamine resistance, Pfdhps for sulfadoxine resistance, Pfk13 for artemisinin resistance and Pfmdr1 for resistance to multiple antimalarials. Results: Mutations in the propeller domain of PfK13 were observed in two samples only, however these mutations are not validated for artemisinin resistance. A high proportion of parasites from the P. falciparum dominant site Rourkela showed wild-type Pfcrt and Pfdhfr haplotypes, while mutant Pfcrt and Pfdhfr haplotypes were fixed at the P. vivax dominant sites Chennai and Nadiad. The wild-type PfDHPS haplotype was predominant across all study sites. Finally, we observed the largest proportion of suspected multi-clonal infections at Rourkela, which has the highest transmission of P. falciparum among our study sites. Conclusion: This is the first simultaneous high-throughput next generation sequencing of five complete P. falciparum genes from infected patients in India.