Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani

Pentavalent antimonials have long been the first line of defence against leishmaniasis, but resistance has been reported in different parts of the world. Pentavalent antimony is reduced into trivalent form in the cells and is a potential inducer of HSP70 in L. donovani. Expression profile of HSP70 in antimony susceptible and resistant L. donovani isolates were characterized by Southern blot, Northern blot and western blot analysis. HSP70 gene copy number, gene expression and HSP70 protein expression was found uniform in both antimony sensitive and resistant clinical isolates. In laboratory condition, Leishmania cells respond to antimonial drug stress by three fold over expression of the HSP70 protein. The observed results indicated that HSP70 play important role in stress tolerance against antimonial drug without differential expression in antimony sensitive and resistance clinical isolates of L. donovani.Nepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1: 22-2

Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India

Failure of antimonial drugs, the mainstay therapy for leishmaniasis has become an escalating problem in the treatment of Indian leishmaniasis. Using 14 clinical isolates from both visceral (VL) and post-kala-azar dermal leismaniasis (PKDL) patients, we have examined the role of ATP-binding cassette transporter (ABC transporter) gene, multidrug resistant protein A (MRPA) and two building blocks of the major thiol, trypanothione namely, ornithine decarboxylase gene (ODC) (a rate limiting enzyme in the polyamine biosynthesis) and γ-glutamylcysteine synthetase (γ-GCS) (a rate limiting enzyme in glutathione biosynthesis) in antimony resistance. Amplification of these three genes was observed in some but not all clinical isolates. Increased expression of the three RNAs as determined by real-time PCR was observed in all SAG-R clinical isolates. Significant increase in cysteine and glutathione levels was observed in the resistant isolates. Our studies report the underlying mechanism of antimony resistance in the clinical isolates

Role of aquaglyceroporin (AQP1) gene and drug uptake in antimony-resistant clinical isolates of Leishmania donovani

Antimonial-containing drugs are the first line of treatment against Leishmaniasis. Resistance to antimonials in Leishmania is proposed to be due to reduced uptake of trivalent antimony (SbIII) through the aquaglyceroporin (AQP1). We investigated the uptake of SbIII and involvement of aquaglyceroporin in developing antimony resistance phenotype in Leishmania donovani clinical isolates. SbIII accumulation, copy number of AQP1 gene, and transcript levels were compared in antimony-sensitive versus -resistant isolates. Antimony-resistant field isolates showed reduced uptake of SbIII. The copy number of AQP1 gene showed higher copy number in the antimony-resistant isolates when compared with the sensitive isolates and did not correlate to the reduced uptake of SbIII. Downregulation of AQP1 RNA levels was not consistently found in the antimony-resistant isolates. Our studies indicate that while downregulation of AQP1 may be one of the mechanisms of antimony resistance, it is however not an invariable feature

Assessing aquaglyceroporin gene status and expression profile in antimony-susceptible and -resistant clinical isolates of Leishmania donovani from India

Objectives: Clinical resistance to pentavalent antimonials results from an interplay between uptake, efflux and sequestration in Leishmania. Aquaglyceroporins (AQPs) have been shown to facilitate uptake of trivalent metalloids. Down-regulation of AQP1 in Leishmania results in resistance to trivalent antimony, whereas overexpression of AQP1 in drug-resistant parasites can reverse the resistance. The present work investigates the role of AQP1 in monitoring antimonial resistance in Indian leishmaniasis. Methods and results: Susceptibility to trivalent antimony as determined in vitro with intracellular amastigotes from both visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients correlated well with the clinical response. Higher accumulation of trivalent antimony (SbIII) was observed in all susceptible isolates compared with resistant isolates. Reduced accumulation of SbIII correlated, with a few exceptions, with down-regulation of AQP1 RNA as determined by real-time PCR. Cloning and sequencing of the AQP1 gene from both VL and PKDL isolates showed sequence variation in four of the clinical isolates. None of the isolates had an alteration of Glu152 and Arg230, which have been previously shown to affect metalloid transport. Transfection of the AQP1 gene in a sodium antimony gluconate-resistant field isolate conferred susceptibility to the resistant isolate. Conclusions: Our studies indicate genetic variation in VL and PKDL isolates. Down-regulation of AQP1 correlates well with clinical drug resistance in a majority of Indian VL and PKDL isolates. AQP1 gene expression at both the genetic and transcriptional level showed positive correlation with SbIII accumulation, with some exceptions

Global age-sex-specific mortality, life expectancy, and population estimates in 204 countries and territories and 811 subnational locations, 1950–2021, and the impact of the COVID-19 pandemic: a comprehensive demographic analysis for the Global Burden of Disease Study 2021

Background: Estimates of demographic metrics are crucial to assess levels and trends of population health outcomes. The profound impact of the COVID-19 pandemic on populations worldwide has underscored the need for timely estimates to understand this unprecedented event within the context of long-term population health trends. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 provides new demographic estimates for 204 countries and territories and 811 additional subnational locations from 1950 to 2021, with a particular emphasis on changes in mortality and life expectancy that occurred during the 2020–21 COVID-19 pandemic period. Methods: 22 223 data sources from vital registration, sample registration, surveys, censuses, and other sources were used to estimate mortality, with a subset of these sources used exclusively to estimate excess mortality due to the COVID-19 pandemic. 2026 data sources were used for population estimation. Additional sources were used to estimate migration; the effects of the HIV epidemic; and demographic discontinuities due to conflicts, famines, natural disasters, and pandemics, which are used as inputs for estimating mortality and population. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate under-5 mortality rates, which synthesised 30 763 location-years of vital registration and sample registration data, 1365 surveys and censuses, and 80 other sources. ST-GPR was also used to estimate adult mortality (between ages 15 and 59 years) based on information from 31 642 location-years of vital registration and sample registration data, 355 surveys and censuses, and 24 other sources. Estimates of child and adult mortality rates were then used to generate life tables with a relational model life table system. For countries with large HIV epidemics, life tables were adjusted using independent estimates of HIV-specific mortality generated via an epidemiological analysis of HIV prevalence surveys, antenatal clinic serosurveillance, and other data sources. Excess mortality due to the COVID-19 pandemic in 2020 and 2021 was determined by subtracting observed all-cause mortality (adjusted for late registration and mortality anomalies) from the mortality expected in the absence of the pandemic. Expected mortality was calculated based on historical trends using an ensemble of models. In location-years where all-cause mortality data were unavailable, we estimated excess mortality rates using a regression model with covariates pertaining to the pandemic. Population size was computed using a Bayesian hierarchical cohort component model. Life expectancy was calculated using age-specific mortality rates and standard demographic methods. Uncertainty intervals (UIs) were calculated for every metric using the 25th and 975th ordered values from a 1000-draw posterior distribution. Findings: Global all-cause mortality followed two distinct patterns over the study period: age-standardised mortality rates declined between 1950 and 2019 (a 62·8% [95% UI 60·5–65·1] decline), and increased during the COVID-19 pandemic period (2020–21; 5·1% [0·9–9·6] increase). In contrast with the overall reverse in mortality trends during the pandemic period, child mortality continued to decline, with 4·66 million (3·98–5·50) global deaths in children younger than 5 years in 2021 compared with 5·21 million (4·50–6·01) in 2019. An estimated 131 million (126–137) people died globally from all causes in 2020 and 2021 combined, of which 15·9 million (14·7–17·2) were due to the COVID-19 pandemic (measured by excess mortality, which includes deaths directly due to SARS-CoV-2 infection and those indirectly due to other social, economic, or behavioural changes associated with the pandemic). Excess mortality rates exceeded 150 deaths per 100 000 population during at least one year of the pandemic in 80 countries and territories, whereas 20 nations had a negative excess mortality rate in 2020 or 2021, indicating that all-cause mortality in these countries was lower during the pandemic than expected based on historical trends. Between 1950 and 2021, global life expectancy at birth increased by 22·7 years (20·8–24·8), from 49·0 years (46·7–51·3) to 71·7 years (70·9–72·5). Global life expectancy at birth declined by 1·6 years (1·0–2·2) between 2019 and 2021, reversing historical trends. An increase in life expectancy was only observed in 32 (15·7%) of 204 countries and territories between 2019 and 2021. The global population reached 7·89 billion (7·67–8·13) people in 2021, by which time 56 of 204 countries and territories had peaked and subsequently populations have declined. The largest proportion of population growth between 2020 and 2021 was in sub-Saharan Africa (39·5% [28·4–52·7]) and south Asia (26·3% [9·0–44·7]). From 2000 to 2021, the ratio of the population aged 65 years and older to the population aged younger than 15 years increased in 188 (92·2%) of 204 nations. Interpretation: Global adult mortality rates markedly increased during the COVID-19 pandemic in 2020 and 2021, reversing past decreasing trends, while child mortality rates continued to decline, albeit more slowly than in earlier years. Although COVID-19 had a substantial impact on many demographic indicators during the first 2 years of the pandemic, overall global health progress over the 72 years evaluated has been profound, with considerable improvements in mortality and life expectancy. Additionally, we observed a deceleration of global population growth since 2017, despite steady or increasing growth in lower-income countries, combined with a continued global shift of population age structures towards older ages. These demographic changes will likely present future challenges to health systems, economies, and societies. The comprehensive demographic estimates reported here will enable researchers, policy makers, health practitioners, and other key stakeholders to better understand and address the profound changes that have occurred in the global health landscape following the first 2 years of the COVID-19 pandemic, and longer-term trends beyond the pandemic

Global incidence, prevalence, years lived with disability (YLDs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

Background: Detailed, comprehensive, and timely reporting on population health by underlying causes of disability and premature death is crucial to understanding and responding to complex patterns of disease and injury burden over time and across age groups, sexes, and locations. The availability of disease burden estimates can promote evidence-based interventions that enable public health researchers, policy makers, and other professionals to implement strategies that can mitigate diseases. It can also facilitate more rigorous monitoring of progress towards national and international health targets, such as the Sustainable Development Goals. For three decades, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) has filled that need. A global network of collaborators contributed to the production of GBD 2021 by providing, reviewing, and analysing all available data. GBD estimates are updated routinely with additional data and refined analytical methods. GBD 2021 presents, for the first time, estimates of health loss due to the COVID-19 pandemic. Methods: The GBD 2021 disease and injury burden analysis estimated years lived with disability (YLDs), years of life lost (YLLs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries using 100 983 data sources. Data were extracted from vital registration systems, verbal autopsies, censuses, household surveys, disease-specific registries, health service contact data, and other sources. YLDs were calculated by multiplying cause-age-sex-location-year-specific prevalence of sequelae by their respective disability weights, for each disease and injury. YLLs were calculated by multiplying cause-age-sex-location-year-specific deaths by the standard life expectancy at the age that death occurred. DALYs were calculated by summing YLDs and YLLs. HALE estimates were produced using YLDs per capita and age-specific mortality rates by location, age, sex, year, and cause. 95% uncertainty intervals (UIs) were generated for all final estimates as the 2·5th and 97·5th percentiles values of 500 draws. Uncertainty was propagated at each step of the estimation process. Counts and age-standardised rates were calculated globally, for seven super-regions, 21 regions, 204 countries and territories (including 21 countries with subnational locations), and 811 subnational locations, from 1990 to 2021. Here we report data for 2010 to 2021 to highlight trends in disease burden over the past decade and through the first 2 years of the COVID-19 pandemic. Findings: Global DALYs increased from 2·63 billion (95% UI 2·44–2·85) in 2010 to 2·88 billion (2·64–3·15) in 2021 for all causes combined. Much of this increase in the number of DALYs was due to population growth and ageing, as indicated by a decrease in global age-standardised all-cause DALY rates of 14·2% (95% UI 10·7–17·3) between 2010 and 2019. Notably, however, this decrease in rates reversed during the first 2 years of the COVID-19 pandemic, with increases in global age-standardised all-cause DALY rates since 2019 of 4·1% (1·8–6·3) in 2020 and 7·2% (4·7–10·0) in 2021. In 2021, COVID-19 was the leading cause of DALYs globally (212·0 million [198·0–234·5] DALYs), followed by ischaemic heart disease (188·3 million [176·7–198·3]), neonatal disorders (186·3 million [162·3–214·9]), and stroke (160·4 million [148·0–171·7]). However, notable health gains were seen among other leading communicable, maternal, neonatal, and nutritional (CMNN) diseases. Globally between 2010 and 2021, the age-standardised DALY rates for HIV/AIDS decreased by 47·8% (43·3–51·7) and for diarrhoeal diseases decreased by 47·0% (39·9–52·9). Non-communicable diseases contributed 1·73 billion (95% UI 1·54–1·94) DALYs in 2021, with a decrease in age-standardised DALY rates since 2010 of 6·4% (95% UI 3·5–9·5). Between 2010 and 2021, among the 25 leading Level 3 causes, age-standardised DALY rates increased most substantially for anxiety disorders (16·7% [14·0–19·8]), depressive disorders (16·4% [11·9–21·3]), and diabetes (14·0% [10·0–17·4]). Age-standardised DALY rates due to injuries decreased globally by 24·0% (20·7–27·2) between 2010 and 2021, although improvements were not uniform across locations, ages, and sexes. Globally, HALE at birth improved slightly, from 61·3 years (58·6–63·6) in 2010 to 62·2 years (59·4–64·7) in 2021. However, despite this overall increase, HALE decreased by 2·2% (1·6–2·9) between 2019 and 2021. Interpretation: Putting the COVID-19 pandemic in the context of a mutually exclusive and collectively exhaustive list of causes of health loss is crucial to understanding its impact and ensuring that health funding and policy address needs at both local and global levels through cost-effective and evidence-based interventions. A global epidemiological transition remains underway. Our findings suggest that prioritising non-communicable disease prevention and treatment policies, as well as strengthening health systems, continues to be crucially important. The progress on reducing the burden of CMNN diseases must not stall; although global trends are improving, the burden of CMNN diseases remains unacceptably high. Evidence-based interventions will help save the lives of young children and mothers and improve the overall health and economic conditions of societies across the world. Governments and multilateral organisations should prioritise pandemic preparedness planning alongside efforts to reduce the burden of diseases and injuries that will strain resources in the coming decades. Funding: Bill & Melinda Gates Foundation

Assessment of antigenemia among children in four hotspots of filarial endemic districts of Nepal during post-MDA surveillance

Abstract Background Sixty-three out of 77 districts reported lymphatic filariasis (LF) endemic in Nepal. Mass drug administration (MDA) with diethylcarbamazine (DEC) and albendazole (ALB) treatment program was continued for 6 to 11 rounds in these districts. Nepal government has stopped the MDA program based on the transmission assessment survey (TAS) report of 2014 and 2018 indicating Wuchereria bancrofti antigenemia prevalence < 2%. But the persistence of low levels of the circulating filarial antigen (CFA) in some foci of four endemic districts of Central Nepal, i.e., 0.4% in Dhading, 0.7% in Mahottari, 0.21% in Lalitpur and 1.2% in Bara district could responsible for enhancing the risk of infection resurgence. Hence the present study was designed to assess antigenic prevalence using Filariasis Test Strip (Alere, Scarborough ME) in children born after MDA in hotspot areas of four endemic districts of Central Nepal. Results The present study covers 70% children of the eligible population. The result revealed significantly high CFA prevalence in hotspots of Mahottari district belonging to the Terai region and Dhading district belonging to the hilly region, i.e., 13% and 10%, respectively, compared to baseline prevalence and TAS report. While in Lalitpur district and Bara district CFA prevalence was still found to be less than 2%. A higher number of MDA rounds covered in hotspots were found significantly associated with the low antigenic prevalence of W. bancrofti. Whereas median treatment coverage and inter-quartile range (IQR) in study districts were not found significantly associated with CFA prevalence. Although the clinical manifestation of hydrocele (1%) was found in all four study districts, it was not due to the W. bancrofti infection. Conclusions Two hotspot regions, one each from the Terai (Mahottari) and hilly (Dhading) districts were found highly prevalent with CFA and significantly associated with the number of MDA rounds but were not associated with treatment coverage and IQR. Higher CFA prevalence was observed in hotspots where baseline prevalence was high together indicating that rounds of MDA program need to be extended further in these hotspot regions of endemic districts

Entomological assessment in ‘hotspots’ of four lymphatic filariasis endemic districts, Central Nepal during post-MDA surveillance

Background & objectives: Annual mass drug administration (MDA) is the main strategy to interrupt the transmission of lymphatic filariasis (LF) in the community. The main aim of monitoring the MDA program, for its effectiveness and interruption of LF is the post-MDA surveillance using antigen survey in children born after MDA. The latest technique of new research suggests that xenomonitoring is an effective tool for monitoring LF intervention. The objective of this study was to assess the W. bancrofti infection/or infectivity in vector mosquitoes by xenomonitoring during post-MDA surveillance. Methods: A descriptive cross-sectional study was conducted in the hotspots of selected four districts of Central Nepal. A gravid trap technique was used for sampling mosquitoes. Infection/or infectivity was determined via the dissection of vector mosquitoes. Anopheles, Aedes, Armigerus and Culex species were collected from hotspots of four endemic districts, two from the hilly region (Lalitpur and Dhading) and two from Terai region (Bara and Mahottari) of Central Nepal. Results: A total of 4450 mosquitoes belonging to four genera, Anopheles, Culex, Armigeres, and Aedes were collected from four hotspots. The distribution of Culex quinquefasciatus was found to be the highest, 88.9% (n=3955/4450) followed by Cx. vishnui (4.5%), Armigeres sp (5.8%), An. culicifascies (0.2%), Aedes spp (0.8%). The proportion of female mosquitoes trapped is significantly higher. A total of 3344 parous Cx. quinquefasciatus mosquitoes were dissected for any larval stage of W. bancrofti. We could not find any filarial infection in dissected mosquito samples. Interpretation & conclusion: We conclude that the gravid trap is an efficient tool for the collection of gravid Cx. quinquefasciatus mosquitoes for xenomonitoring studies of filariasis endemic regions. Vector composition indicated a maximum number of vector mosquitoes of lymphatic filariasis were trapped compared with the other three species. Distribution and density of Cx. quinquefasciatus was found highest in four hotspots of endemic districts. None of the Cx. quinquefasciatus dissected were found to be infected by larval forms of filaria. Since the low levels of infection persistence in the human population in these hot spots, vector infection and infectivity can’t be ignored. Microscopic xenomonitoring at a low level of infection persistent is less likely to be efficient so molecular xenomonitoring along with a large sample should be required in each of the hot spots of the districts. Additionally, area is receptive so further vector control intervention should be required to reduce the risk of resurgence of infection

Assessment of microfilaremia in 'hotspots' of four lymphatic filariasis endemic districts of Nepal during post-MDA surveillance.

BackgroundThe lymphatic filariasis (LF) elimination program in all sixty-three endemic districts of Nepal was based on annual mass drug administration (MDA) using a combination of diethylcarbamazine (DEC) and albendazole for at least 5 years. The MDA program was started in the Parsa district of the Terai region and at least six rounds of MDA were completed between 2003 and 2017 in all filariasis endemic districts of Central Nepal. Transmission Assessment Survey (TAS) report indicated that circulating filarial antigen (CFA) prevalence was below the critical value i.e., ≤ 2% in selected LF endemic districts of Central Nepal. Based on the TAS report, antigen-positive cases were found clustered in the foci of those districts which were considered as "hotspots". Hence the present study was designed to assess microfilaremia in hotspots of four endemic districts of Central Nepal after the MDA program.Methodology and principal findingsThe present study assessed microfilaremia in hotspots of four endemic districts i.e. Lalitpur and Dhading from the hilly region and Bara and Mahottari from the Terai region of Central Nepal. Night blood samples (n = 1722) were collected by finger prick method from the eligible sample population irrespective of age and sex. Community people's participation in the MDA program was ensured using a structured questionnaire and chronic clinical manifestation of LF was assessed using standard case definition. Two districts one each from the hilly region (Lalitpur district) and Terai region (Bara district) showed improved microfilaria (MF) prevalence i.e. below the critical level (1%). Chronic clinical manifestation of LF showed significant association with the older age group (≥41 years) but not with sex.ConclusionsThe study revealed LF transmission improved in hotspots of two districts while continued in others but the risk of LF resurgence cannot be ignored since the upper confidence level of MF prevalence is over 1% in all the hotspots studied districts. High MF prevalence is well correlated with the number of MDA rounds but not with the MDA coverage. Community people involved in MDA drug uptake in any previous and last rounds have significantly less MF infection. Hence it is recommended that before deciding to stop the MDA rounds it is essential to conduct the MF survey at the hotspots of the sentinel sites