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Background: Alcohol imposes a significant burden on health, social and economic systems in Sri Lanka. In the present economic crisis taxes on alcohol provides necessary revenue increases. Yet, the perception of the public on alcohol policies in Sri Lanka is not well explored., Objectives: This opinion survey was conducted with the aim to understand the public's awareness on alcohol harm, alcohol industry influences, barriers and facilitators for implementing alcohol control policies in Sri Lanka, and the level of public support for alcohol policies, particularly taxes on alcohol products., Methods: A street intercept survey among 997 participants (with a ratio of 2:1 for males and females) selected through a cluster sampling method responded to an interviewer administered questionnaire. Bivariate and multivariate analyses were conducted to determine associations and a p  < 0.05 was considered significant., Results: Among the respondents, 36.1% have consumed alcohol at least once in their lifetime and 29.1% have consumed alcohol during the past 12 months with a significant gender difference (females - 2.8%; males- 43.4%; p  < 0.001). Significant proportions of both men (81.4%) and women (71.8%); p  < 0.017 agreed that policy measures to reduce alcohol consumption would benefit the government including a significant proportion (73.8%, p  < 0.008) of alcohol users. The vast majority -72.8%- agreed that increasing alcohol prices would help address the alcohol consumption problem in Sri Lanka. Moreover, only 30.8% of men and 44.3% of women agreed that the government's alcohol laws are currently strong enough to protect people from alcohol harm. The regression analysis revealed that men are 2.43 times more in agreement with the statement that "policy measures aimed at reducing alcohol consumption can benefit the public" as compared to women. However, individuals aged 50-64 years are 40% less likely to agree with this statement as compared to 18-33 years., Conclusion: The majority of the public, including people who consume alcohol, are supportive of improving alcohol related policies, including taxes, and acknowledge negative impact of alcohol consumption on the country. This presents a clear opportunity for Sri Lanka to strengthen and enforce the alcohol related policies to protect and improve public health., Competing Interests: The 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 © 2024 Chandraratne, Negi, Siyambalapitiya, De Seram, Selladurai, Pieris, Rothenstein-Henry and Murukutla.)

Background: Positive portrayals of tobacco use in entertainment media can normalize and perpetuate use. In 2012, the Government of India implemented the Tobacco-Free Film and Television Rules, a first-of-its-kind comprehensive regulation to restrict tobacco depiction in films and television programs. Two complementary studies were undertaken to assess the implementation of the film rules on television and in movie theaters., Methods: In the first part, movie theater observations and exit surveys were conducted from Feb. 3 to March 24, 2015. In total, 308 movie theaters were selected for the observation of films. A total of 3080 exit surveys were conducted to assess moviegoers' reactions toward the film rule. The second part comprised the systematic observation of 424 prerecorded television programs that aired from Nov. 20 to Dec. 30, 2015., Results: Compliance with the Tobacco-Free Film and Television Rules policy was lower on television than in movie theaters. While 66% of television programs with tobacco scenes implemented at least 1 of the 3 elements of the film rule, not a single program executed all required elements correctly. In movie theaters, 99% of films that contained tobacco scenes implemented at least one element of the film rule. However, all elements of the film rules were implemented correctly during 27% of the films observed. Exit surveys showed that among moviegoers who recalled viewing at least one element of the film rule, there was increased concern about tobacco's harms and intentions to quit., Conclusion: Implementation of the film rules was higher in movie theaters than on television, though there were gaps in implementation for both. Despite inconsistent application, audience reactions to the anti-tobacco messages were favorable, with increased concern about tobacco's harms and intention to quit. Overall, the film rules offer a strong tool for countering tobacco promotion, reaching hundreds of millions with anti-tobacco messaging., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2023.)

Background: This study assesses the effectiveness of a campaign “Are We Drinking Ourselves Sick?” that ran nationally in Jamaica in four phases from 2017 to 2019 to increase knowledge about the harms of sugary drinks, shift attitudes, and build support for policy actions to address sugary drink consumption, including a tax and a ban in schools. Methods: Campaign impact was measured in representative cross-sectional household surveys of adults ages 18 to 55. A baseline survey was conducted before the launch of the campaign (n = 1430). Evaluation surveys were conducted mid-campaign (n = 1571) and post-campaign (n = 1500). Campaign impact was assessed by comparing changes across survey periods on key knowledge, attitudinal and policy support outcome indicators. The independent association between campaign awareness and outcomes was analyzed using logistic regression analyses. Results: The campaign was recalled by more than 80% of respondents and was well-received with 90% or more respondents describing it as believable and relevant. There was a decline in knowledge on the harms of sugary drinks from the baseline to post-campaign period, notably on risks of diabetes (adjusted odds ratio or AOR = 0.62, p < 0.001), overweight and obesity (AOR = 0.58, p < 0.001), and heart disease (AOR = 0.79, p < 0.003). However, post-campaign awareness was independently associated in logistic regression analysis with improved knowledge of the harms of sugary drinks, including risks of diabetes (AOR = 1.45, p = 0.019), overweight or obesity (AOR = 1.65, p = 0.001), and heart disease (AOR = 1.44, p = 0.011). Support for government action remained high across survey waves (≥90%), and campaign awareness was independently associated with increased policy support for sugary drinks taxes (Mid-campaign: AOR = 1.43, p = 0.019; post-campaign: AOR = 1.46, p = 0.01) and restrictions on sugary drinks in schools (AOR = 1.55, p = 0.01). Conclusion: This study demonstrates the role that media campaigns can play in maintaining knowledge and concern about the health harms of sugary drinks and increasing support for policy passage.

Despite the dwindling number of visceral leishmaniasis (VL) cases in India, there is an urgent need for early and unequivocal diagnostics for controlling and preventing the reemergence of VL. Post-kala-azar dermal leishmaniasis (PKDL), a dermal sequela of VL, serves as a reservoir of the parasite. Diagnosis of PKDL, especially the macular variant, is challenging and poses impediment toward attainment of VL elimination. In this study, a real-time fluorimetry loop-mediated isothermal amplification (RealAmp) assay has been established for the detection of different clinical manifestations of leishmaniasis. The study included 150 leishmaniasis patients (25 VL, 25 cutaneous leishmaniasis [CL], and 100-PKDL) along with 120 controls. The assay demonstrated sensitivity of 100% (95% CI: 86.68-100) for diagnosis of VL and PKDL (95% CI: 79.61-100) and 96% (95% CI: 86.68-100) for CL with 100% specificity. Moreover, considering the cardinal role of PKDL, diagnosis using minimally invasive slit aspirate was explored, which demonstrated remarkable sensitivity of 96% (95% CI: 87.64-98.47). As a test of cure for PKDL, RealAmp successfully detected parasite in two of posttreatment cases who later reported relapse on follow-up. Also, direct sample lysis using slit aspirate was attempted in a small group that yielded sensitivity of 89% (95% CI: 67.20-96.90). RealAmp depicted excellent diagnostic accuracy in the diagnosis of leishmaniasis in concordance with the established SYBR Green I-based (Molecular Probes, Eugene, OR) visual loop-mediated isothermal amplification (LAMP) and the reference comparator real-time PCR. The study endorsed the employment of LAMP either as visual-LAMP or RealAmp for an accurate and expeditious diagnosis of PKDL and as a tool for assessment of cure.

Background and Aims: Exposure to tobacco, alcohol and fast-food use in films is associated with initiation of these behaviours. India is the world's largest film producer, but the extent of such imagery in Bollywood (Hindi cinema) films is unclear. We therefore aimed to describe the extent of and trends in tobacco, alcohol and fast-food imagery in Bollywood films, between 1994-2013., Methods: For the 15 top-grossing films each year between 1994-2013, the number of five-minute intervals containing product images were determined separately for tobacco, alcohol and fast-food. Both the proportion of films containing at least one image occurrence, and occurrences per film, were described overall and by year. Negative binomial regression described associations between film rating and occurrences/film, and estimated time-trends in occurrences/film, adjusted for rating., Results: We analysed 93 U-rated (unrestricted), 150 U/A-rated (parental guidance for children aged <12 years) and 55 A-rated (restricted to adult audience) films, containing 9,226 five-minute intervals (mean intervals/film 30.8, SD 4.0). 70% (n = 210), 93% (n = 278) and 21% (n = 62) of films contained at least one tobacco, alcohol and fast-food occurrence, respectively. Corresponding total mean occurrences/film were 4.0 (SD 4.9), 7.0 (4.7) and 0.4 (0.9). Tobacco occurrences were more common in U/A films (incidence rate ratio 1.49, 95% confidence interval 1.06-2.09) and A films (2.95; 1.95-4.48) than U-rated films. Alcohol occurrences were also more common in A-rated films than U-rated films (1.48; 1.15-1.85). Tobacco occurrences/film became less common over the observed period (adjusted trend -4% per annum; -2 to -7%; p <0.001), while alcohol (+2%; 0-3%; p = 0.02), and fast food (+8%; 2-14%; p = 0.01) occurrences/film became more common., Conclusions: Although the extent of tobacco imagery in Bollywood films fell over 1994-2013, it is still frequently observed. Alcohol imagery is widespread, even in U-rated films, and trends in both alcohol and fast-food imagery are upwards., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Globally, stroke is one of the leading causes of death and disability-adjusted life-years (DALYs). The red cell distribution width (RDW) is a readily available and inexpensive test which is done routinely as a part of complete blood count in these patients., Objective: In this study, we tried to correlate the RDW with severity of acute ischemic stroke (AIS)., Methods: Patients presenting to emergency department (ED) within 24 hours of the onset of clinical signs and symptoms suggestive of AIS were assessed for Glasgow Coma Scale (GCS) and National Institutes of Health Stroke Scale (NIHSS) score followed by non-contrast computed tomography (NCCT) scan. RDW value for all the patients who were included in the study were co-related with the severity of the stroke., Results: The median (IQR) RDW in the patients with minor stroke on the basis of GCS was 13.5 (13.3-13.5), moderate stroke was 13.8 (13.5-14.4) and with severe stroke was 15.4 (15.1-15.6) (p < 0.001). The median (IQR) RDW in the patients with minor stroke on the basis of NIHSS score was 13.4 (13.2 - 13.6), moderate stroke was 13.8 (13.5-14.3), and moderate to severe stroke was 14.7 (14.5-15.3) and with severe stroke was 15.5 (15.1-15.7) (p < 0.001). The median RDW in patients who were alive was 13.8 (13.5-15.1) and in patients who expired was 15.5 (14.5-15.7) (p = 0.048)., Conclusion: Based on the findings of this study, RDW index has statistically significant correlation with the severity of AIS. So it can potentially be an important parameter to predict the prognosis of AIS patients., Competing Interests: Conflict of interest None declared., (© 2020 Tehran University of Medical Sciences.)

Background: The World Health Organization has targeted elimination of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) by 2020. Despite distinctive decline seen in the number of VL cases in ISC, there is still a quest for development of a diagnostic test which has the utility for detection of active infection and relapse cases and as a test of cure. The present study validated the sensitivity and specificity of SYBR Green I based closed tube LAMP assay reported by us for diagnosis of VL., Methodology: The validation study was carried out at two endemic sites in India, located at Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna and Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi. Standard operating protocols were provided at the two sites for applying LAMP assay on confirmed VL cases. The diagnostic accuracy of LAMP assay was evaluated by Receiver operator curve (ROC) analysis. Furthermore, a simplified LAMP assay based on direct blood lysis, DBL-LAMP, was developed and verified for its diagnostic accuracy., Principal Findings: A total of 267 eligible participants were included in the study which comprised of 179 VL cases and 88 controls. Sensitivity and specificity of the LAMP assay were 98.32% (95% C.I- 95.2-99.7%) and 96.59% (95% C.I.-90.4-99.3%), respectively. ROC curve analysis depicted no significant difference between area under curve (AUCROC) for LAMP assay and rK39 RDT, indicative of LAMP as an excellent diagnostic test. DBL-LAMP assay, performed on 67 VL and 100 control samples, yielded a sensitivity of 93.05% (95% C.I- 84.75-97%) and specificity of 100% (95% C.I.- 96.30-100%)., Conclusions/significance: The validated closed tube LAMP for diagnosis of VL will provide impetus to the ongoing VL elimination programme in ISC. The assay based on direct blood lysis promotes its scope for application in field settings by further reducing time and cost., Competing Interests: The authors have declared that no competing interests exist.

Background: Tobacco control mass media campaigns are cost-effective in reducing tobacco consumption in high-income countries, but similar evidence from low-income countries is limited. An evaluation of a 2009 smokeless tobacco control mass media campaign in India provided an opportunity to test its cost-effectiveness., Methods: Campaign evaluation data from a nationally representative household survey of 2898 smokeless tobacco users were compared with campaign costs in a standard cost-effectiveness methodology. Costs and effects of the Surgeon campaign were compared with the status quo to calculate the cost per campaign-attributable benefit, including quit attempts, permanent quits and tobacco-related deaths averted. Sensitivity analyses at varied CIs and tobacco-related mortality risk were conducted., Results: The Surgeon campaign was found to be highly cost-effective. It successfully generated 17 259 148 additional quit attempts, 431 479 permanent quits and 120 814 deaths averted. The cost per benefit was US$0.06 per quit attempt, US$2.6 per permanent quit and US$9.2 per death averted. The campaign continued to be cost-effective in sensitivity analyses., Conclusion: This study suggests that tobacco control mass media campaigns can be cost-effective and economically justified in low-income and middle-income countries. It holds significant policy implications, calling for sustained investment in evidence-based mass media campaigns as part of a comprehensive tobacco control strategy., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background Air pollution is of particular concern in India, which contains 11 of the 20 most polluted cities in the world. Media coverage of air pollution issues plays an important role in influencing public opinion and increasing citizen demand for action on clean air policy. Hence, this study was designed to assess news coverage of air pollution in India and its implications for policy advancement. Methods Articles published online between 1 January 2014 and 31 October 2015 that discussed air pollution in India were systematically content analysed. From 6435 articles in the national media and 271 articles in the international media, a random selection of 500 articles (400 from national and 100 from international media) were analysed and coded by two independent coders, after high inter-rater reliability (kappa statistic above 0.8) was established. Results There was an increase in the number of news stories on air pollution in India in the national media over the study period; 317 (63%) stories described the risk to health from air pollution as moderately to extremely severe, and 393 (79%) stories described the situation as needing urgent action. Limited information was provided on the kinds of illnesses that can result from exposure. Less than 30% of stories in either media specifically mentioned the common illnesses resulting from air pollution. Very few articles in either media mentioned the population groups most at risk from air pollution, such as children or older people. Vehicles were presented most often as the cause of air pollution in India (in over 50% of articles in both national and international media). Some of the most important sources of air pollution were mentioned less often: 6% of national and 18% of international media articles mentioned unclean sources of household energy; 3% of national and 9% of international media articles mentioned agricultural field burning. Finally, the majority of articles (405; 81%) did not mention any specific institution or organization - such as the government or industry groups - as the primary responsible stakeholder, thus leaving ambiguous the organizations whose leadership was necessary to mitigate air pollution. Conclusion Gaps exist in the current media discourse on air pollution, suggesting the need for strengthening engagement with the media as a means of creating citizen engagement and enabling policy action. Through greater elaboration of the health burdens and evidence-based policy actions, the media can play a critical role in galvanizing India's action on air quality. These data may suggest opportunities for media advocacy and greater public and policy engagement to address issues around air quality in India.

Background: Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application., Methods: We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL)., Results: The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse., Conclusions: The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid molecular diagnosis of VL and PKDL with potential for application in assessment of cure.

Currently no effective vaccine is available for human visceral leishmaniasis(VL) caused by Leishmania donovani. Previously, we showed that centrin1 and p27gene deleted live attenuated Leishmania parasites (LdCen1(-/-) and Ldp27(-/-)) are safe, immunogenic and protective in animal models. Here, to assess the correlates of protection, we evaluated immune responses induced by LdCen1(-/-) and Ldp27(-/-) in human blood samples obtained from healthy, healed VL (HVL), post kala-azar dermal leishmaniasis(PKDL) and VL subjects. Both parasites infected human macrophages, as effectively as the wild type parasites. Further, LdCen1(-/-) and Ldp27(-/-) strongly stimulated production of pro-inflammatory cytokines including, IL-12, IFN-γ, TNF-α, IL-2, IL-6 and IL-17 in the PBMCs obtained from individuals with a prior exposure to Leishmania (HVL and PKDL). There was no significant stimulation of anti-inflammatory cytokines (IL-4 and IL-10). Induction of Th1 biased immune responses was supported by a remarkable increase in IFN-γ secreting CD4(+) and CD8(+) T cells and IL-17 secreting CD4(+) cells in PBMCs from HVL cases with no increase in IL-10 secreting T cells. Hence, LdCen1(-/-) and Ldp27(-/-) are promising as live vaccine candidates against VL since they elicit strong protective immune response in human PBMCs from HVL, similar to the wild type parasite infection, mimicking a naturally acquired protection following cure.

Background: Majority of individuals with history of visceral leishmaniasis (VL) exhibit strong immunity to re-infection, however, the mechanism of resistance is poorly understood. It is unclear whether CD8(+) T cells contribute to protection against Leishmania donovani infection through cytotoxic activity. The present study aims to evaluate immunological mechanism associated with resistance to the disease in healed VL (HVL) individuals and further, the contribution of CD8(+) T cells in the protective immunity., Methods: Peripheral blood mononuclear cells (PBMCs) from VL, HVL and naive groups were exposed in vitro to total soluble Leishmania antigen (TSLA) from L. donovani. The proliferation index was determined by ELISA based lymphoproliferative assay. Cytokines and granzyme B levels were measured by CBA. Activated T-cell populations were estimated using flow cytometry., Results: We observed significantly higher lymphoproliferation, cytokines and granzyme B levels in HVL group compared to naive or VL group. More strikingly, we found a strong association (rs = 0.895, P < 0.0001) between proliferation index (PI) and granzyme B level, with a significant proportion of activated CD8(+) T cells in HVL group., Conclusions: Leishmania immune group (HVL) exhibited durable and strong cellular immune response to TSLA in terms of lymphoproliferation as well as production of Th1 cytokines and granzyme B. Additionally, the elevated level of activated CD8(+) T cells and stimulation of cytotoxic activity through granzyme B production, indicated a possible role of CD8(+) T cells in resistance to L. donovani infection in the HVL group.

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.

Resistance to sodium antimony gluconate (SAG) is a major cause of therapeutic failure in a large proportion of visceral leishmaniasis (VL) cases. Determinants of SAG resistance have been widely studied; however, the mechanism operating in clinical isolates is poorly understood. In the present study, expression of parasite surface antigen-2 (PSA-2) gene was studied in clinical isolates of Leishmania donovani comprising of antimony resistant (n=10) and sensitive (n=4) parasites. The expression of PSA-2 gene was found to be consistently high in SAG resistant clinical isolates (≥1.5-fold) at both transcript and protein level. Further, over-expression of PSA-2 in L. donovani isolates (LdPSA-2(++)) resulted in conversion of SAG sensitive phenotype to resistant. The LdPSA-2(++) parasites showed significantly decreased susceptibility towards SAG (>12-fold), amphotericin B (>4-fold) and miltefosine (>2.5-fold). Marked decrease in antimony accumulation and enhanced tolerance towards complement mediated lysis was evident in LdPSA-2(++) parasites. The study established the role of PSA-2 gene in SAG resistance and its potential as a biomarker to distinguish resistant and sensitive clinical isolates of L. donovani., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Loop-mediated isothermal amplification (LAMP) is at the forefront in the search for innovative diagnostics for rapid and specific amplification of target DNA under isothermal conditions. We have applied LAMP assay using SYBR Green for clear-cut naked eye detection of Leishmania (Leishmania) donovani in 200 clinical samples of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). The assay was positive in 53/55 VL blood samples (sensitivity, 96.4%; 95% confidence interval [CI], 87.7-99%), 15/15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI, 79.6-100%), 60/62 PKDL tissue biopsy samples (sensitivity, 96.8%; 95% CI, 88.9-99.1%), and 1/68 control samples (specificity, 98.5%; 95% CI, 92.1-99.7%). The assay was specific for L. (L.) donovani, the causative species for VL and negative for L. (L.) infantum, L. (L.) tropica, and L. (L.) major. This is the first comprehensive clinical study demonstrating the applicability of the LAMP assay for a rapid and reliable molecular diagnosis of VL and PKDL., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Resistance to antimony is a major cause of failure to therapy in a large proportion of visceral leishmaniasis cases. Methods to distinguish resistant and sensitive parasite are urgently needed as the standard in vitro intracellular drug susceptibility assays are cumbersome and time consuming. Differential expression profiling studies have led to the identification of several antimony resistance-associated genes; however, their efficacy as a potential biomarker for monitoring antimony resistance remains imprecise. We analysed the expression of eight genes [antimony metabolism-associated genes - multidrug resistance protein A (MRPA), γ-glutamylcysteine synthetase (γ-GCS) and aquaporin-1 (AQP1) - and genes identified by proteome/transcriptome profiling—heat shock protein 83, mitogen-activated protein kinase 1 and histones H1, H2A and H4) in antimony-resistant (n=10) and antimony-sensitive (n=4) clinical isolates of Leishmania donovani by quantitative real-time PCR, in comparison with a lab-generated resistant and a standard sensitive isolate. We observed a significant differential expression of MRPA, histone H1 (p<0.01), γ-GCS, HSP83 (p<0.005) and histone H2A and H4 (p<0.0001) in a group of sodium antimony gluconate-resistant isolates compared to sensitive isolates. Preferential AQP1 expression was observed in all the sensitive isolates (p<0.0001). Overall, expression profile in field isolates for all the genes studied showed altered expression in majority of isolates, while in some, the expression was static. All the isolates showed a mosaic of expression pattern of the genes analysed indicating constellation of genes contributes towards the drug susceptibility of parasite. As none of the genes exhibit an absolute correlation with phenotype, targeted expression analysis of a set of genes should be considered as biomarker for distinguishing the antimony-resistant and antimony-sensitive parasite.

Widespread antimonial resistance in anthroponotic visceral leishmaniasis (VL) makes it critical to monitor the susceptibility of prevailing field isolates to upcoming antileishmanials in order to frame the right treatment policies to protect these drugs against development of resistance. We aimed to generate the baseline data on natural in vitro susceptibility to paromomycin and sitamaquine in Leishmania donovani field isolates from VL patients (n = 20) coming from zones of varying sodium antimony gluconate (SAG) resistance. We further monitored nitric oxide (NO) release in infected macrophages treated with these drugs. Field isolates exhibited variable sensitivity to paromomycin and sitamaquine with respective mean 50% effective dose (ED₅₀) values ± standard error of the mean (SEM) of 3.9 ± 0.3 μM and 2.1 ± 0.2 μM at the intracellular amastigote stage and 29.8 ± 2.5 μM and 17.7 ± 1.0 μM at the promastigote stage. Susceptibilities at the two parasite stages did not correlate for either drug. Isolates from high SAG resistance zones exhibited significantly lower susceptibility to sitamaquine than those from low SAG resistance zones, while isolates from different zones showed similar susceptibilities to paromomycin. NO release was promoted in L. donovani-infected macrophages upon treatment with paromomycin/sitamaquine. NO inhibitors significantly compromised amastigote killing by sitamaquine, but not by paromomycin. In conclusion, SAG-resistant/sensitive VL isolates were susceptible to both paromomycin and sitamaquine. Paromomycin, exhibiting higher efficacy against SAG-resistant parasites and having a distinct mechanism of action, appears to be a promising drug for combination therapy.

A rapid and accurate method to detect and quantify Leishmania parasite is urgently needed to facilitate early diagnosis of leishmaniasis and monitoring of antileishmania therapy. In this study, real-time assay was applied to estimate parasite load in clinical samples of visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) patients. The mean parasite load in blood of VL patients (n = 31) was 8,372 parasites/ml, while the mean parasite load in bone marrow aspirate (BMA) was 194,962 parasites/million nucleated cells (n = 12). Parasite load was undetectable after treatment with amphotericin B (n = 9) in VL, while a residual parasite burden was detected in 2 of 6 patients following treatment with sodium antimony gluconate. Further, circulating levels of IFN-gamma, TNF-alpha, IL-10, IL-6, IL-4 and IL-2 were analysed in VL patients (n = 29) by Cytometric Bead Array to evaluate correlation with parasitic load. Interestingly, IL-10 levels correlated significantly with parasite load (r = 0.82, P<0.0001). The mean parasite load in dermal lesions of PKDL patients was 9,502 parasites/microg tissue DNA at pre-treatment stage (n = 25), with no detectable parasites after therapy (n = 5). Parasite burden was distinctly higher (P<0.0001) in nodular lesions (n = 12) (19,586 parasites/microg tissue DNA) compared to papular/macular lesions (n = 13, 193 parasites/microg tissue DNA). Further, chronic PKDL lesions showed significantly (P = 0.0166) higher parasite load in comparison with acute lesions. Results indicate that chronic, nodular cases constitute the major parasite reservoir for anthroponotic transmission. Our results establish that the high parasite load in VL is strongly correlated with a high level of IL-10, implicating IL-10 as a marker of disease severity. The assay is applicable for diagnosis as well as prognosis of both VL and PKDL, providing a simple molecular tool to monitor the efficacy of antileishmanial drugs or vaccines.

The diagnostic potential of recombinant Lepp12 (rLepp12) antigen cloned from Leishmania infantum was assessed in L. donovani infections by Western blotting. Ninety-two serum samples, including 30 patients with active kala-azar (KA), 17 post-treated KA patients (KA-PT), 20 post-kala-azar dermal leishmaniasis (PKDL) patients and 25 controls, were analysed for rLepp12, rK39 and DAT positivity. All KA samples taken at pre-treatment stage were positive for Lepp12 antibodies. Seventeen of these were evaluated post treatment (KA-PT), 10 of which were found to be negative. Nine of these 10 negative cases corresponded to clinically cured patients with regressed spleen. Seven post-treatment cases were rLepp12-positive; all of them corresponded to patients who were considered clinically cured but continued to have an enlarged spleen (> or =5 cm). The majority of PKDL patients (18/20) were found to be seronegative by immunoblot test using rLepp12 antigen. The rLepp12-based Western blot diagnosed 100% of patients with visceral disease, whilst none of the control cases were found to be reactive to rLepp12. rLepp12 protein provides a useful reagent for highly sensitive and specific diagnosis of KA. Additionally, rLepp12 appears to have potential as a prognostic marker for the infection.

Individuals with visceral leishmaniasis, or kala azar (KA) and individuals with post-KA dermal leishmaniasis (PKDL) are considered to be reservoirs of transmission of Leishmania donovani in India. When intracellular amastigotes were used to assess the natural susceptibility that PKDL isolates and KA isolates have to sodium antimony gluconate (SAG), the mean ED(50) was found to be 12.0+/-2.49 and 11.0+/-1.38 microg/mL, respectively; and there was a significant correlation with the clinical response (r rank=0.99). All KA isolates, as well as a significant proportion (55%) of PKDL isolates from high-endemicity zones, were resistant to SAG. The median ED(50) for SAG-resistant PKDL isolates (20.0 microg/mL) was significantly higher (P<.05) than that for SAG-resistant KA isolates (15.7 microg/mL). SAG-resistant PKDL isolates may contribute to KA's increased refractoriness to SAG, via anthroponotic transmission of SAG-resistant strains.

The arbitrary-primed PCR (AP-PCR) technique was employed with the twin goals of identifying genetic polymorphisms within the Indian isolates and to identify differentially expressed gene sequences. The parasite isolates from Indian Kala-azar patients could be differentiated from Leishmania donovani isolates from distinct geographic regions. Moreover, differences within the Indian isolates could also be identified. A majority (17/19) of the Indian isolates gave identical AP-PCR pattern, while two isolates gave consistently divergent pattern. The distinctive AP-PCR fragments obtained with Indian isolates were used as probes in Northern blot analysis. Three such fragments were found to represent transcribed sequences that were differentially expressed in the two stages of the parasite. These sequences led to cloning and characterization of Leishmania Centrin gene and a novel gene termed A-1 that is over-expressed in amastigote stage of the parasite. The study demonstrates the utility of random genome sampling methods in genomic fingerprinting and in identifying differentially transcribed sequences that could potentially contribute to parasite virulence.

Development of simple, economical and non-invasive tests for the early diagnosis of visceral leishmaniasis (VL) or kala-azar (KA) remains a challenge, and serological studies based on antigen prepared from the amastigote stage of Leishmania donovani, the stage that causes infection, are lacking. In the present study, circulating antibodies to total antigen isolated from the promastigote and amastigote stages of the parasite, as well as to recombinant K39 (rK39) antigen, are measured by enzyme-linked immunosorbent assay (ELISA) and the results compared with a polymerase chain reaction (PCR) test for KA diagnosis. In 116 samples of KA examined, the amastigote antigen gave significantly higher mean absorbance values in ELISA than did the promastigote antigen. The sensitivity for KA detection was significantly higher using the amastigote antigen (94%) than the promastigote antigen (90.5%). Analysis in 91 controls showed that specificity was higher with amastigote antigen (92.3%) than with promastigote antigen (86.8-89.0%). Reliability of ELISA diagnosis with amastigote antigen was only marginally lower than that with rK39 ELISA or with the PCR test. Easy availability and low cost of indigenous amastigote antigen, together with the simplicity of ELISA compared with PCR, make ELISA based on amastigote antigen a promising choice for the diagnosis of KA.

Development of simple, economical and non-invasive tests for the early diagnosis of visceral leishmaniasis (VL) or kala-azar (KA) remains a challenge, and serological studies based on antigen prepared from the amastigote stage of Leishmania donovani, the stage that causes infection, are lacking. In the present study, circulating antibodies to total antigen isolated from the promastigote and amastigote stages of the parasite, as well as to recombinant K39 (rK39) antigen, are measured by enzyme-linked immunosorbent assay (ELISA) and the results compared with a polymerase chain reaction (PCR) test for KA diagnosis. In 116 samples of KA examined, the amastigote antigen gave significantly higher mean absorbance values in ELISA than did the promastigote antigen. The sensitivity for KA detection was significantly higher using the amastigote antigen (94%) than the promastigote antigen (90.5%). Analysis in 91 controls showed that specificity was higher with amastigote antigen (92.3%) than with promastigote antigen (86.8-89.0%). Reliability of ELISA diagnosis with amastigote antigen was only marginally lower than that with rK39 ELISA or with the PCR test. Easy availability and low cost of indigenous amastigote antigen, together with the simplicity of ELISA compared with PCR, make ELISA based on amastigote antigen a promising choice for the diagnosis of KA.

Leishmania donovani, a protozoan parasite, causes visceral disease in humans. To identify genes that control growth, we have isolated for the first time in the order Kinetoplastida a gene encoding for centrin from L. donovani. Centrin is a calcium-binding cytoskeletal protein essential for centrosome duplication or segregation. Protein sequence similarity and immunoreactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Immunofluorescence analysis localized the protein in the basal body. Calcium binding analysis revealed that its C-terminal Ca(2+) binding domain binds 16-fold more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lacking a Ca(2+) binding site suggest that Ca(2+) binding to these regions may have a role in the protein conformation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal-deleted centrin in the parasite significantly reduces its growth rate, and it was found that significantly more cells are arrested in the G(2)/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.

When infected with Leishmania donovani, patients develop specific antibodies that constitute the basis of serodiagnosis. Using immunoblot analysis, we examined the antibody response to antigens of L. donovani in 35 kala-azar (KA) patients and 67 controls. Sera from KA patients recognised numerous antigens with molecular weights ranging from 14-110 kDa. Antigens of 40 kDa, 55 kDa, 65 kDa, 70 kDa and 82 kDa were recognised most frequently. All KA patients produce an antibody response to one or more of these antigens. The majority (83%) of KA cases recognised at least four of these five parasite antigens. The 70 kDa antigen showed the greatest sensitivity for Indian KA, and produced a positive reaction in 94% of patients. This antigen gave 10% false-positive reactions in controls comprising patients with related diseases (i.e. tuberculosis, leprosy and malaria) and in healthy controls. Data indicated that the 70 kDa antigen may include a member of the heat shock protein 70 family. Studies with four clinical isolates of L. donovani showed that the 70 kDa component was expressed in all the strains examined. Immunoblot assay (Western blotting) provided a sensitive diagnostic test for KA patients, and identified the 70 kDa parasite antigen that is promising as a potential target antigen for the development of less complex serodiagnostic assays for KA.

During a 15 months period, 97 patients with AMI were monitored continuously at hospital discharge for one hour. The VPCs or no VPC noted during this period of continuous monitoring were correlated with subsequent cardiac events during one year follow up. Patients with complicated VPCs had a significantly higher incidence of cardiac deaths and non-fatal re-infarctions during one year follow up (54.5%) as compared to patients with no VPCs (6.5%) (p less than .001). Patients with higher grades of VPCs had more complicated course (30.3% to 66.7% for Lown Grade II to IV) as compared to 6.5% and 20.8% for grade Lown O and I. Though, the results of this 1 hour continuous monitoring at hospital discharge are not as sensitive as with ambulatory Holter monitoring as is evident from this study where complicated VPCs were detected in 11.4% patients vs. 33% with Holter monitoring, it could be a useful tool in institutions where the facility of Holter monitoring does not exist.

Roots and shoots of corn seedlings (Zea mays L. var. Dixie 18) germinated in trifluralin (alpha,alpha,alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) solutions are characterized by radial enlargement of the cortical cells and by multinucleate cells in the meristematic regions. Trifluralin inhibits elongation of Avena coleoptile sections at concentrations of 0.1 mum to 10 mum. Synthesis of DNA, RNA, and protein is suppressed in the root tips while no significant effect is noticeable in the shoots of corn germinated in trifluralin. A (32)P time-course study of 48, 72, and 96 hours utilizing phenol extraction and MAK column separation of corn root and shoot nucleic acids showed suppression of (32)P incorporation in the treated roots; however, the 72 and 96 hour treated shoots incorporated a much greater amount than the control with most of the increased incorporation found in the sRNA and DNA fractions. The increased activity in the DNA may be due to a high G-C type DNA. No selective suppression or enhancement of any particular RNA species was noticed in the treated plants.