Mapping malaria vectors and insecticide resistance in a high-endemic district of Haryana, India: implications for vector control strategies.

BACKGROUND: Achieving effective control and elimination of malaria in endemic regions necessitates a comprehensive understanding of local mosquito species responsible for malaria transmission and their susceptibility to insecticides. METHODS: The study was conducted in the highly malaria prone Ujina Primary Health Center of Nuh (Mewat) district of Haryana state of India. Monthly entomological surveys were carried out for adult mosquito collections via indoor resting collections, light trap collections, and pyrethrum spray collections. Larvae were also collected from different breeding sites prevalent in the region. Insecticide resistance bioassay, vector incrimination, blood meal analysis was done with the collected vector mosquitoes. RESULTS: A total of 34,974 adult Anopheles mosquitoes were caught during the survey period, out of which Anopheles subpictus was predominant (54.7%). Among vectors, Anopheles stephensi was predominant (15.5%) followed by Anopheles culicifacies (10.1%). The Human Blood Index (HBI) in the case of An. culicifacies and An. stephensi was 6.66 and 9.09, respectively. Vector incrimination results revealed Plasmodium vivax positivity rate of 1.6% for An. culicifacies. Both the vector species were found resistant to DDT, malathion and deltamethrin. CONCLUSION: The emergence of insecticide resistance in both vector species, compromises the effectiveness of commonly used public health insecticides. Consequently, the implementation of robust insecticide resistance management strategies becomes imperative. To effectively tackle the malaria transmission, a significant shift in vector control strategies is warranted, with careful consideration and adaptation to address specific challenges encountered in malaria elimination efforts.

A randomised controlled trial to compare the efficacy, safety, and tolerability of low dose, short course primaquine in adults with uncomplicated P. vivax malaria in two hospitals in India.

BACKGROUND: Plasmodium vivax remains a major challenge for malaria control and elimination due to its ability to cause relapsing illness. To prevent relapses the Indian National Center for Vector Borne Diseases Control (NCVBDC) recommends treatment with primaquine at a dose of 0.25 mg/kg/day provided over 14 days. Shorter treatment courses may improve adherence and treatment effectiveness. METHODS: This is a hospital-based, randomised, controlled, open-label trial in two centres in India. Patients above the age of 16 years, with uncomplicated vivax malaria, G6PD activity of ≥ 30% of the adjusted male median (AMM) and haemoglobin levels ≥ 8 g/dL will be recruited into the study and randomised in a 1:1 ratio to receive standard schizonticidal treatment plus 7-day primaquine at 0.50 mg/kg/day or standard care with schizonticidal treatment plus 14-day primaquine at 0.25 mg/kg/day. Patients will be followed up for 6 months. The primary endpoint is the incidence risk of any P. vivax parasitaemia at 6 months. Safety outcomes include the incidence risk of severe anaemia (haemoglobin < 8 g/dL), the risk of blood transfusion, a > 25% fall in haemoglobin and an acute drop in haemoglobin of > 5 g/dL during primaquine treatment. DISCUSSION: This study will evaluate the efficacy and safety of a 7-day primaquine regimen compared to the standard 14-day regimen in India. Results from this trial are likely to directly inform national treatment guidelines. TRIAL REGISTRATION: Trial is registered on CTRI portal, Registration No: CTRI/2022/12/048283.

Prevalence and Correlates of Malnutrition in Nuh District, Haryana State, India.

India has a substantial burden of undernutrition coupled with overweight and obesity at the other end of the spectrum of malnutrition. Nuh district, in the Haryana State in northern India, is an impoverished district in India. With an aim to investigate the problem of malnutrition in the community, a cross-sectional study was conducted in four villages of the Nuh district. Height/length, weight, and age data of children under 5 years were used to calculate three indices: weight-for-age, height-for-age, and weight-for-height. The body mass index was calculated for individuals older than 6 years. Associations between malnutrition and other factors were assessed using simple and multiple logistic regression to get adjusted coefficients. The total surveyed population comprised 11,496 individuals. Over 51% were female, and 13.2% of the surveyed population were children under 5 years. Almost half of the population was illiterate and unemployed. The prevalences of underweight, stunting, and wasting in children under 5 years were 37%, 53%, and 21%, respectively. The prevalences of underweight and stunting in the 6- to 19-year-old age group were 29% and 38%, respectively. The prevalence of overweight was 36% in the 20- to 40-year-old and > 60-year-old age groups, and 44% in the 41- to 60-year-old age group. Our findings reveal a considerable burden of undernutrition among children under 5 years and a dual burden of undernutrition and overnutrition in adults, highlighting the need to map these areas and sharpen our responses to mitigate the overwhelming and long-term consequences of malnutrition in the Nuh district.

Vaccination coverage and breakthrough infections of COVID-19 during the second wave among staff of selected medical institutions in India.

India experienced the second wave of SARS-CoV-2 infection from April 3 to June 10, 2021. During the second wave, Delta variant B.1617.2 emerged as the predominant strain, spiking cases from 12.5 million to 29.3 million (cumulative) by the end of the surge in India. Vaccines against COVID-19 are a potent tool to control and end the pandemic in addition to other control measures. India rolled out its vaccination programme on January 16, 2021, initially with two vaccines that were given emergency authorization-Covaxin (BBV152) and Covishield (ChAdOx1 nCoV- 19). Vaccination was initially started for the elderly (60+) and front-line workers and then gradually opened to different age groups. The second wave hit when vaccination was picking up pace in India. There were instances of vaccinated people (fully and partially) getting infected, and reinfections were also reported. We undertook a survey of staff (front line health care workers and supporting) of 15 medical colleges and research institutes across India to assess the vaccination coverage, incidence of breakthrough infections, and reinfections among them from June 2 to July 10, 2021. A total of 1876 staff participated, and 1484 forms were selected for analysis after removing duplicates and erroneous entries (n = 392). We found that among the respondents at the time of response, 17.6% were unvaccinated, 19.8% were partially vaccinated (received the first dose), and 62.5% were fully vaccinated (received both doses). Incidence of breakthrough infections was 8.7% among the 801 individuals (70/801) tested at least 14 days after the 2nd dose of vaccine. Eight participants reported reinfection in the overall infected group and reinfection incidence rate was 5.1%. Out of (N = 349) infected individuals 243 (69.6%) were unvaccinated and 106 (30.3%) were vaccinated. Our findings reveal the protective effect of vaccination and its role as an essential tool in the struggle against this pandemic.

Age-specific malaria vulnerability and transmission reservoir among children.

Purpose: The pediatric population, especially under-five children, is highly susceptible to malaria and accounts for 76 % of global malaria deaths according to the World Malaria Report 2022. The purpose of this manuscript is to discuss the various factors involved in the susceptibility of the pediatric population to Malaria and the importance of this age group for malaria elimination. Methodology: Data on pediatric malaria epidemiology that includes prevalence, risk factors, immune factors, socioeconomic factors, control methods, etc. were extracted from published literature using PubMed and Google Scholar. This data was further correlated with malaria incidence data from the World Health Organization (WHO) and the National Center for Vector Borne Diseases Control (NCVBDC). Results: The younger age group is vulnerable to severe malaria due to an immature immune system. The risk of infection and clinical disease increases after the waning of maternal immunity. In the initial years of life, the developing brain is more susceptible to malaria infection and its after-effects. The pediatric population may act as a malaria transmission reservoir due to parasite density and asymptomatic infections. WHO recommended RTS,S/AS01 has limitations and may not be applicable in all settings to propel malaria elimination. Conclusion: The diagnosis of malaria is based on clinical suspicion and confirmed with microscopy and/or rapid diagnostic testing. The school-age pediatric population serves as a transmission reservoir in the form of asymptomatic malaria since they have acquired some immunity due to exposure in early childhood. Targeting the hidden reservoir in the pediatric population and protecting this vulnerable group will be essential for malaria elimination from the countries targeting elimination.

Economic burden estimation associated with dengue and chikungunya in Gujarat, India.

Background: Dengue and chikungunya have been emerging as major vector-borne diseases. The global burden of the diseases is rising as a public health problem. The complexity of disease is governed by multiple constraints including only symptomatic treatment and inflicts heavy social and economic burden on society. The present study is designed to assess the economic burden of dengue and chikungunya infection by calculating cost per patient in Gujarat, India. Methods: A total of 210 patients were enrolled in the study from Ahmedabad and Kheda district of Gujarat from May 2018 to December 2019 of which 150 had dengue and 60 chikungunya infections, subject to the willingness of participation in the survey. Information on wage loss days, cost associated with medicines, diagnosis, special food and travel cost, etc., for the calculation of the direct and indirect costs associated with dengue and chikungunya were collected from these participants using a structured questionnaire. Informed consent was taken before including any participant in the study. Results: In the dengue sample, 86 were males (57.3%) and the rest were females, and in the chikungunya sample, 31 were males (51.7%) and the rest females. The median age of the participants with dengue and chikungunya was 18 (p25 to p75: 8 to 26) and 30 (p25 to p75: 21 to 45) years respectively. Median family income was recorded as Rs 15,000 (p25 to p75: 9000 to 25500) and Rs 12,000 (p25 to p75: 9000 to 18500) for the dengue and chikungunya cases, respectively. The average duration of the illness was observed to be higher in chikungunya (median days (P25 to p75): 15 (7-45)) than dengue (median days (P25 to p75): 10 (5-15)). The median indirect cost in the case of dengue was Rs 1,931 (p25 to p75: 300 to 4500) while Rs 2,550 (p25 to p75: 0 to 5250) was observed for chikungunya cases. Two types of direct cost, namely, direct cost related to medical expenses and direct cost related to other expenses were calculated. Direct cost related to medical expenses was observed to be higher in dengue (Md (P25 to p75): Rs 2,450 (400-5000)) than chikungunya (Md (P25 to p75): Rs 1,500 (150-5200)) while indirect cost related to other expenses were comparable between dengue (Md (P25 to p75): Rs 1,575 (1300-2600)) and chikungunya (Md (P25 to p75): Rs 1500 (850-2850)). The average total cost for one dengue episode was estimated to be Rs 6,860 (3700-12525) whereas it was Rs 7,000 (2550-14000) for one episode of Chikungunya. Conclusions: Overall, patients have to bear high costs while suffering from dengue and chikungunya infections. Furthermore, the duration of illness while suffering from viral diseases also contributes to the substantial economic burden. Improved knowledge about the impact of the cost and the economic burden associated with dengue and chikungunya will help policymakers allocate and appropriate resources accordingly.

An investigation of the efficacy of different statistical models in malaria forecasting in the semi-arid regions of Gujarat, India.

BACKGROUND & OBJECTIVES: Robust forecasting of malaria cases is desirable as we are approaching towards malaria elimination in India. Methods enabling robust forecasting and timely case detection in unstable transmission areas are the need of the hour. METHODS: Forecasting efficacy of the eight most prominent statistical models that are based on three statistical methods: Generalized linear model (Model A and Model B), Smoothing method (Model C), and SARIMA (Model D to model H) were compared using last twelve years (2008-19) monthly malaria data of two districts (Kheda and Anand) of Gujarat state of India. RESULTS: The SARIMA Model F was found the most appropriate when forecasted for 2017 and 2018 using model-building data sets 1 and 2, respectively, for both the districts: Kheda and Anand. Model H followed by model C were the two models found appropriate in terms of point estimates for 2019. Still, we regretted these two because confidence intervals from these models are wider that they do not have any forecasting utility. Model F is the third one in terms of point prediction but gives a relatively better confidence interval. Therefore, model F was considered the most appropriate for the year 2019 for both districts. INTERPRETATION & CONCLUSION: Model F was found relatively more appropriate than others and can be used to forecast malaria cases in both districts.

Bionomics of Anopheles culicifacies Sensu Lato in two Malaria Endemic Districts of Central Gujarat, India.

Background: Gujarat State has been witnessing large scale urbanization, in last two decades, resulting changes in local environment and microclimate may have also influenced the resting, feeding habits and development of Anopheles culicifacies sensu 1ato. Therefore, a systematic longitudinal study was undertaken to know the bionomics of An. culicifacies s.l. in present study. Methods: The study was conducted in four sentinel villages in Kheda and Panchmahal Districts. The mosquitoes resting indoors and outdoors were collected in early morning hours, using mouth aspirator, pyrethrum space spray and light traps. Mosquito landing collections on human volunteers was carried out from dusk to dawn. Species composition, abundance, seasonal prevalence, resting behavior (Endophily and Exophily), sibling species composition, vector potential and insecticide susceptibility status of malaria vectors was studied. Results: Six Anopheles species were collected, An. subpictus s.l. was the predominant species followed by An. culicifacies s.l., a known malaria vector was resting indoor and zoophagic behaviour. Anopheles culicifacies, sibling species B (89%) was found. The sporozoite rate (%) and entomological inoculation rate in Kheda was 2.33%, 3.09 per bite/person/annum and they were 1.05% and 0.475 bite/person/annum in Panchmahal, respectively. Anopheles culicifacies s.l. was found possible resistance to alpha-cypermethrin. Conclusion: Anopheles culicifacies s.l. showed endophillic, zoophagic behaviour and found possible resistance to alpha-cypermethrin. Early biting behaviour of An. culicifacies s.l. in this area is a cause of concern. Therefore, there is need for frequent monitoring and evaluation of vector control measures in order to achieve the elimination target of malaria in this area.

Four decades of epidemiological data reveal trajectories towards malaria elimination in Kheda district (Gujarat), western part of India.

BACKGROUND: Malaria is a main public health problem in India and was so particularly in the state of Gujarat in the western part of the country. This study assesses the effects of various interventions on malaria cases using data from the last 33 years (1987-2019). METHODS: Here we have analysed 33 years of malaria epidemiological data from a malaria clinic in Kheda district in Gujarat. The data were digitised yearly and monthly, age-wise and gender-wise, and descriptive analysis was performed to assess the effects of several interventions on malaria burden. RESULTS: During 1987-2019, our clinic diagnosed 5466 Plasmodium vivax and 4732 P. falciparum malaria cases. Overall, there was a declining trend in malaria cases except for the years 1991, 1994, 2004 and 2005. The year 2004 especially witnessed an epidemic in Kheda as well as throughout Gujarat. Malaria infections were most common (40%) among the 21-40 years age group. Fever was the most common symptom in all age groups. INTERPRETATION: Introduction of revised drug policy and improved surveillance technique (rapid diagnosis kits) have strengthened the diagnosis and treatment of malaria in the district. Use of pyrethroid in indoor residual insecticide spray has also strengthened vector control. Among the various interventions used, long-lasting insecticide nets and introduction of artemisinin-based combination therapy have played significant roles in controlling malaria cases. A more drastic decline in P. falciparum cases versus P. vivax is evident, but the latter persists in high proportions and therefore new tools for malaria control will be needed for elimination.

Correction: Nucleosomal Histone Proteins of L. donovani: A Combination of Recombinant H2A, H2B, H3 and H4 Proteins Were Highly Immunogenic and Offered Optimum Prophylactic Efficacy against Leishmania Challenge in Hamsters.

[This corrects the article DOI: 10.1371/journal.pone.0097911.].

Small-scale field evaluation of the entomological efficacy and the residual activity of Fludora® Fusion WP-SB indoor residual spraying against Anopheles culicifacies s.l. in Gujarat, India.

OBJECTIVES: To evaluate the entomological efficacy and the residual activity of indoor residual spraying with Fludora® Fusion 562.5 WP-SB, a combination formulation containing clothianidin, a neonicotinoid and deltamethrin, a pyrethroid, against the main rural malaria vector, Anopheles culicifacies s.l., in India in a small-scale trial. METHODS: In three study villages, suitable households were randomly allocated to five treatments: Fludora® Fusion 562.5 WP-SB (target dose 225 mg active ingredient AI/m2 ); clothianidin 70 WG (target dose 200 mg AI/m2 ); K-Othrine 250 WG (deltamethrin, target dose 25 mg AI/m2 ); Ficam VC 80 WP-SB (bendiocarb, target dose 400 mg AI/m2 ) and unsprayed control. Insecticides were sprayed by hand compression sprayers with control flow valves and 8002E nozzles. Post-spray cone bioassays were done on insecticide-treated walls using a colonised, deltamethrin-resistant strain of An. culicifacies. Mosquitoes were collected from treated rooms by different methods. The insecticide content on filter papers collected from the sprayed walls was determined by chemical assay to assess the spray quality. RESULTS: The ratios of applied to target doses of insecticides were within 0.84 to 1.4, showing a good spray quality. The cone bioassays revealed residual action lasting 7 months for all insecticides without significant differences in mortality between different surfaces treated nor between the four treatment arms (P > 0.05). Considering all entomological parameters such as indoor resting density, excito-repellency, blood-feeding inhibition and delayed mortality, the overall efficacy of Fludora® Fusion WG-SB was equal or better compared with other insecticides. CONCLUSIONS: Fludora® Fusion showed overall equal or better efficacy than deltamethrin and bendiocarb alone against a pyrethroid-resistant malaria vector population and can be considered as an alternative product for management of pyrethroid resistance in malaria vectors.

Overlaying COVID-19 mitigation plans on malaria control infrastructures.

To counter the coronavirus disease 2019 (COVID-19) pandemic, each country must design sustainable control plans given the inherent disparities in wealth and healthcare systems. Most malaria-endemic countries run well-entrenched malaria control programs via their established frameworks for diagnosis, case management, treatment and overall surveillance. We propose that the malaria control infrastructures can be partially co-opted for launching sustainable COVID-19 mitigation plans.

In-silico Designing and Testing of Primers for Sanger Genome Sequencing of Dengue Virus Types of Asian Origin.

Rarity in reporting whole genome sequence of Dengue virus from dengue endemic countries leaves lacunae in understanding regional pattern of virus mutation and ultimately leading to non-understanding of transmission pattern and clinical outcomes emerging at regional levels. Due to inter-serotype genomic similarity and intra-serotype genomic diversity, appropriate designing of primer pairs appears as an exhaustive exercise. Present paper reports new Dengue virus type-specific primer which may help in characterizing virus specific to Asian origin. Genomes of dengue virus serotypes of Asian region were searched and using advanced bioinformatics tools, serotype specific primers were designed and tested for their targeted amplification efficiency. 19 primers sets for DENV-1, 18 primer sets for DENV-2, 17 for DENV-3 and 18 for DENV-4 were designed. In-silico and experimental testing of the designed primers were performed on virus isolated from both clinical isolates and passaged cultures. While all 17 and 18 primer sets of DENV-3 and DENV-2 respectively yielded good quality sequencing results; in case of DENV-4, 16 out of 18 primer sets and in DENV-1, 16 out of 19 primer sets yielded good results. Average sequencing read length was 382 bases and around 82% nucleotide bases were Phred quality QV20 bases (representing an accuracy of circa one miscall every 100 bases) or higher. Results also highlighted importance of use of primer development algorithm and identified genomic regions which are conservative, yet specific for developing primers to achieve efficiency and specificity during experiments.

Molecular, biochemical characterization and assessment of immunogenic potential of cofactor-independent phosphoglycerate mutase against Leishmania donovani: a step towards exploring novel vaccine candidate.

Despite immense efforts, vaccine against visceral leishmaniasis has yet not been developed. Earlier our proteomic study revealed a novel protein, cofactor-independent phoshoglycerate mutase (LdiPGAM), an important enzyme in glucose metabolism, in T helper cells type 1 (Th1) stimulatory region of soluble Leishmania donovani antigen. In this study, LdiPGAM was biochemically and molecularly characterized and evaluated for its immunogenicity and prophylactic efficacy against L. donovani. Immunogenicity of recombinant LdiPGAM (rLdiPGAM) was initially assessed in naïve hamsters immunized with it by analysing mRNA expression of inducible nitric oxide (NO) synthase (iNOS) and other Th1/T helper cells type 2 cytokines, which revealed an upregulation of Th1 cytokines along with iNOS. Immunogenicity of rLdiPGAM was further evaluated in lymphocytes of treated Leishmania-infected hamsters and peripheral blood mononuclear cells of Leishmania patients in clinical remission by various parameters, viz. lymphoproliferation assay and NO production (hamsters and patients) and levels of various cytokines (patients). rLdiPGAM induced remarkable Lymphoproliferative response and NO production in treated Leishmania-infected hamsters as well as in patients and increase in interferon gamma (IFN-γ), interleukin-12 (IL-12p40) responses in Leishmania patients in clinical remission. Vaccination with rLdiPGAM exerted considerable prophylactic efficacy (73%) supported by increase in mRNA expression of iNOS, IFN-γ and IL-12p40 with decrease in transforming growth factor beta and interleukin-10. Above results indicate the importance of rLdiPGAM protein as a potential vaccine candidate against visceral leishmaniasis.

Comparative Analysis of Cellular Immune Responses in Treated Leishmania Patients and Hamsters against Recombinant Th1 Stimulatory Proteins of Leishmania donovani.

Our prior studies demonstrated that cellular response of T helper 1 (Th1) type was generated by a soluble antigenic fraction (ranging from 89.9 to 97.1 kDa) of Leishmania donovani promastigote, in treated Leishmania patients as well as hamsters and showed significant prophylactic potential against experimental visceral leishmaniasis (VL). Eighteen Th1 stimulatory proteins were identified through proteomic analysis of this subfraction, out of which 15 were developed as recombinant proteins. In the present work, we have evaluated these 15 recombinant proteins simultaneously for their comparative cellular responses in treated Leishmania patients and hamsters. Six proteins viz. elongation factor-2, enolase, aldolase, triose phosphate isomerase, protein disulfide isomerase, and p45 emerged as most immunogenic as they produced a significant lymphoproliferative response, nitric oxide generation and Th1 cytokine response in PBMCs and lymphocytes of treated Leishmania patients and hamsters respectively. The results suggested that these proteins may be exploited for developing a successful poly-protein and/or poly-epitope vaccine against VL.

Correction: Recombinant NAD-dependent SIR-2 protein of Leishmania donovani: immunobiochemical characterization as a potential vaccine against visceral leishmaniasis.

[This corrects the article DOI: 10.1371/journal.pntd.0003557.].

Recombinant NAD-dependent SIR-2 protein of Leishmania donovani: immunobiochemical characterization as a potential vaccine against visceral leishmaniasis.

BACKGROUND: The development of a vaccine conferring long-lasting immunity remains a challenge against visceral leishmaniasis (VL). Immunoproteomic characterization of Leishmania donovani proteins led to the identification of a novel protein NAD+-dependent Silent Information regulatory-2 (SIR2 family or sirtuin) protein (LdSir2RP) as one of the potent immunostimulatory proteins. Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity. In the present study, an immunobiochemical characterization of LdSir2RP and further evaluation of its immunogenicity and prophylactic potential was done to assess for its possible involvement as a vaccine candidate against leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: LdSir2RP was successfully cloned, expressed and purified. The gene was present as a monomeric protein of ~45 kDa and further established by the crosslinking experiment. rLdSir2RP shown cytosolic localization in L. donovani and demonstrating NAD+-dependent deacetylase activity. Bioinformatic analysis also confirmed that LdSir2RP protein has NAD binding domain. The rLdSir2RP was further assessed for its cellular response by lymphoproliferative assay and cytokine ELISA in cured Leishmania patients and hamsters (Mesocricetus auratus) in comparison to soluble Leishmania antigen and it was observed to stimulate the production of IFN-γ, IL-12 and TNF-α significantly but not the IL-4 and IL-10. The naïve hamsters when vaccinated with rLdSir2RP alongwith BCG resisted the L. donovani challenge to the tune of ~75% and generated strong IL-12 and IFN-γ mediated Th1 type immune response thereof. The efficacy was further supported by remarkable increase in IgG2 antibody level which is indicative of Th1 type of protective response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of rLdSir2RP was done using computational approach. CONCLUSION/SIGNIFICANCE: The immunobiochemical characterization strongly suggest the potential of rLdSir2RP as vaccine candidate against VL and supports the concept of its being effective T-cell stimulatory antigen.

Nucleosomal histone proteins of L. donovani: a combination of recombinant H2A, H2B, H3 and H4 proteins were highly immunogenic and offered optimum prophylactic efficacy against Leishmania challenge in hamsters.

The present study includes cloning and expression of recombinant Leishmania donovani histone proteins (rLdH2B, rLdH3, rLdH2A and rLdH4), assessment of their immunogenicity in Leishmania infected cured patients/endemic contacts as well as in cured hamsters and finally evaluation of their prophylactic efficacy in hamsters against L. donovani challenge. All recombinant proteins were expressed and purified from the heterologous bacterial host system. Leishmania infected cured patients/endemic contacts as well as cured hamsters exhibited significantly higher proliferative responses to individual recombinant histones and their pooled combination (rLdH2B+rLdH3+rLdH2A+rLdH4) than those of L.donovani infected hosts. The L.donovani soluble antigens (SLD) stimulated PBMCs of cured/exposed and Leishmania patients to produce a mixed Thl/Th2-type cytokine profile, whereas rLdH2B, rLdH3, rLdH2A, rLdH4 and pooled combination (rLdH2-4) stimulated the production of Th1 cytokines IFN-γ, IL-12 and TNF-α but not Th2 cytokines IL-4 or IL-10. The immunogenicity of these histone proteins along with their combination was also checked in cured hamsters where they stimulated higher lymphoproliferation and Nitric oxide production in lymphocytes of cured hamsters than that of infected controls. Moreover, significantly increased IgG2 response, an indicative of cell mediated immunity, was observed in cured hamsters against these individual proteins and their combination as compared to infected hamsters. Further, it was demonstrated that rLdH2B, rLdH3, rLdH2A and rLdH4 and pooled combination were able to provide considerable protection for hamsters against L. donovani challenge. The efficacy was supported by the increased inducible Nitric Oxide Synthase (iNOS) mRNA transcripts and Th1-type cytokines--IFN-γ, IL-12 and TNF-α and down-regulation of IL-4, IL-10 and TGF-ß. Hence, it is inferred that pooled rLdH2-4 elicits Thl-type of immune responses exclusively and confer considerable protection against experimental Visceral Leishmaniasis.

Characterization of the proliferating cell nuclear antigen of Leishmania donovani clinical isolates and its association with antimony resistance.

Previously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate of Leishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance in L. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥ 3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ~5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate. L. donovani PCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate of L. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (Sb(V)) and trivalent antimonial (Sb(III)) drugs was assessed in vitro against promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of Sb(V) (from 41.2 ± 0.6 µg/ml to 66.5 ± 3.9 µg/ml) and Sb(III) (from 24.0 ± 0.3 µg/ml to 43.4 ± 1.8 µg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon Sb(III) treatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance in L. donovani clinical isolates, which warrants detailed investigations regarding its mechanism.

Over-expression of 60s ribosomal L23a is associated with cellular proliferation in SAG resistant clinical isolates of Leishmania donovani.

BACKGROUND: Sodium antimony gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism. METHODOLOGY AND PRINCIPAL FINDINGS: The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode. CONCLUSION/SIGNIFICANCE: This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism.