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Background: Device-associated infections (DAIs) such as ventilator associated pneumonia (VAP), central line-associated blood stream infection (CLABSI), and catheter-related urinary tract infection (CAUTI) are principal contributors to health hazard and a major preventable threat to patient safety. Robust surveillance of DAI delineates infections, pathogens, resistograms, and facilitates antimicrobial therapy, infection-control, antimicrobial stewardship, and improvement in quality of care., Methods: This prospective outcome surveillance study was conducted amongst 2067 ICU patients in a 1000-bedded teaching hospital. Clinical, laboratory, and environmental surveillance, as well as screening of health care professionals (HCPs) were conducted using the modified US Centers for Disease Control and Prevention-National Healthcare Safety Network definitions and methods. Morbidity, mortality, and health-care indices were analyzed and two-tier infection prevention and control was promulgated., Results: Mean occupancy was 95.34% for 2061 patients of 7381 patients/bed/ICU days. One hundred seventeen episodes of DAI occurred in 1258 patients of 12,882 device-days with mean device utilization ratio of 1.79. Mean rate of DAI was 7.40 per 1000 device days. Multiresistant Pseudomonas aeruginosa was most commonly followed by Acinetobacter. Mean all-cause mortality in ICU was 24.85%, whereas all-cause mortality after DAI was 9.79%. Methicillin-resistant Staphylococcus aureus prevalence was 38.46% amongst health-care professionals., Conclusion: Mean rates of VAP, CLABSI, and CAUTI were 20.69, 2.53, and 2.23 per 1000 device days comparable with Indian and global ICUs. Resolute conviction and sustained momentum in infection prevention and control is an essential step toward patient safety., Competing Interests: The authors have none to declare., (© 2022 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd.)

Background: Hepatitis B (HepB) is an important vaccine preventable infection among healthcare workers (HCWs). Vaccination against Hep B virus, remains the foremost preventive approach. This study aims to measure the antibody titres to Hep B surface antigen (anti-HBs) in a mixed cohort of HCWs. It also aims to study the association between time since vaccination and the anti-HBs titres thus evaluating the duration of seroprotection., Methods: A total of 200 HCWs, including nursing students (n = 112), nursing staff (n = 49), laboratory technicians (n = 30) and doctors (n = 9) who had received all three doses of the Hep B vaccine and met the inclusion criteria of having taken all three doses of vaccine were included in this study. Anti-HBs titres were estimated by bioMérieux mini VIDAS® automated immunoassay based on the principle of enzyme-linked fluorescence assay., Results: Two hundred subjects aged 19 to 52 years were included in the study; mean age was 27.29 ± 0.568 years. Duration since vaccination in the study cohort was ≤ 5 years in 149 (74.5.0%), 6-10 years in 20 (10.0%) and >10 years in 31 (15.5%) subjects. Postvaccination antibody titres were > 100 mIU/ml in 85.0%, 10-100 mIU/ml in 11.0% and ≤ 10 mIU/ml in 3.5%. There was a decline noted in antibody titres as duration after vaccination increased. Increasing age was associated with falling protective titres., Conclusion: The study revealed that majority of the HCWs had adequate anti-HBs titres and were protected after vaccination., Competing Interests: The authors have none to declare., (© 2022 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd.)

Enlargement or aneurysm of the aorta predisposes to dissection, an important cause of sudden death. We trained a deep learning model to evaluate the dimensions of the ascending and descending thoracic aorta in 4.6 million cardiac magnetic resonance images from the UK Biobank. We then conducted genome-wide association studies in 39,688 individuals, identifying 82 loci associated with ascending and 47 with descending thoracic aortic diameter, of which 14 loci overlapped. Transcriptome-wide analyses, rare-variant burden tests and human aortic single nucleus RNA sequencing prioritized genes including SVIL, which was strongly associated with descending aortic diameter. A polygenic score for ascending aortic diameter was associated with thoracic aortic aneurysm in 385,621 UK Biobank participants (hazard ratio = 1.43 per s.d., confidence interval 1.32-1.54, P = 3.3 × 10 -20 ). Our results illustrate the potential for rapidly defining quantitative traits with deep learning, an approach that can be broadly applied to biomedical images., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Genome editing of primary human cells with CRISPR-Cas9 is a powerful tool to study gene function. For many cell types, there are efficient protocols for editing with optimized plasmids for Cas9 and sgRNA expression. Vascular cells, however, remain refractory to plasmid-based delivery of CRISPR machinery for in vitro genome editing due to low transfection efficiency, poor expression of the Cas9 machinery, and toxic effects of the selection antibiotics. Here, we describe a method for high-efficiency editing of primary human vascular cells in vitro using nucleofection for direct delivery of sgRNA:Cas9-NLS ribonucleoprotein complexes. This method is more rapid and its high editing efficiency eliminates the need for additional selection steps. The edited cells can be employed in diverse applications, such as gene expression measurement or functional assays to assess various genetic perturbation effects in vitro. This method proves effective in vascular cells that are refractory to standard genome manipulation techniques using viral plasmid delivery. We anticipate that this technique will be applied to other non-vascular cell types that face similar barriers to efficient genome editing. © 2021 Wiley Periodicals LLC. Basic Protocol: CRISPR-Cas9 genome editing of primary human vascular cells in vitro., (© 2021 Wiley Periodicals LLC.)

Background: Corin is a protease expressed in cardiomyocytes that plays a key role in salt handling and intravascular volume homeostasis via activation of natriuretic peptides. It is unknown if Corin loss-of-function (LOF) is causally associated with risk of coronary artery disease (CAD)., Methods: We analyzed all coding CORIN variants in an Italian case-control study of CAD. We functionally tested all 64 rare missense mutations in Western Blot and Mass Spectroscopy assays for proatrial natriuretic peptide cleavage. An expanded rare variant association analysis for Corin LOF mutations was conducted in whole exome sequencing data from 37 799 CAD cases and 212 184 controls., Results: We observed LOF variants in CORIN in 8 of 1803 (0.4%) CAD cases versus 0 of 1725 controls ( P , 0.007). Of 64 rare missense variants profiled, 21 (33%) demonstrated <30% of wild-type activity and were deemed damaging in the 2 functional assays for Corin activity. In a rare variant association study that aggregated rare LOF and functionally validated damaging missense variants from the Italian study, we observed no association with CAD-21 of 1803 CAD cases versus 12 of 1725 controls with adjusted odds ratio of 1.61 ([95% CI, 0.79-3.29]; P =0.17). In the expanded sequencing dataset, there was no relationship between rare LOF variants with CAD was also observed (odds ratio, 1.15 [95% CI, 0.89-1.49]; P =0.30). Consistent with the genetic analysis, we observed no relationship between circulating Corin concentrations with incident CAD events among 4744 participants of a prospective cohort study-sex-stratified hazard ratio per SD increment of 0.96 ([95% CI, 0.87-1.07], P =0.48)., Conclusions: Functional testing of missense mutations improved the accuracy of rare variant association analysis. Despite compelling pathophysiology and a preliminary observation suggesting association, we observed no relationship between rare damaging variants in CORIN or circulating Corin concentrations with risk of CAD.

Introduction and Background: Both human papillomavirus (HPV) and the human immunodeficiency virus (HIV) are sexually transmitted. High-risk (HR) HPV types are a causal factor in cervical cancer. Persistent HPV infection in this subset of immunocompromised women results in faster disease progression. The study determined the prevalence of HPV genotypes in cervicovaginal secretions of HIV seropositive women and the correlation with CD4 counts and cytology. Method: One hundred, non-pregnant, HIV-positive women of 18 years of age and above were enrolled in this cross-sectional study following approval by the institutional ethical committee. A written consent, questionnaire, followed by sample collection including a Papanicolaou (Pap) smear for cytology was undertaken. Cervicovaginal secretion samples were collected in the Digene ® specimen transport medium (STM) (Qiagen Gaithersburg Inc., MD, USA). HPV genotyping was carried out with PCR amplification of a 65-base pair (bp) fragment in the L1 region of the HPV genome using the short PCR fragment (SPF10) primers followed by reverse hybridization by line probe assay (LPA) using the INNOLiPA HPV Genotyping Extra kit (Fujirebio, Belgium). Quantitation of HPV-16 and-18 viral loads (VLs) was done by real-time PCR. Results of Pap smear cytology were correlated with CD4 counts and HPV-16 and-18 VLs. Results: Mean age of the subjects was 34.9 years ± 7.2 years (median 33.0 years, range 24-60 years). HPV was detected in 62 of 93 (66.6%) samples. Twenty (32.25%) of these 62 samples harbored a single HPV genotype. Multiple genotypes (more than two) were detected in 38 (61.3%) samples. HPV-16 was the commonest genotype detected in 26 (27.9%) of all samples and 41.9% of HPV positive samples. Pap smear cytology was reported for 93 women included in the study. Women who had normal cytology were reported as negative for intraepithelial malignancy or lesion (NILM; n = 62; 71.36%), two women had a high-grade squamous intraepithelial lesion (HSIL), low-grade squamous intraepithelial lesion (LSIL; n = 11), atypical squamous cells of undetermined significance (ASCUS; n = 12). Those smears with inadequate material were reported as scant ( n = 6). The median CD4 count was 363/cu.mm (range 39-787) in HPV-positive women compared to 423/cu.mm (range 141-996) in those HPV-negative women. Quantitation of HPV-16 and-18 VL was done in duplicate for samples positive by PCR reverse hybridization (INNOLiPA). Of these 20 samples (65%), 12 samples were positive by real-time PCR. The normalized HPV-16 VL ranged between 18 and 240,000 copies/cell. The normalized HPV-18 VL in cervical samples ranged between ~24 and 60,000 copies/cell. Conclusion: HIV-positive women may be infected with multiple genotypes other than HPV-16 and-18. This may have implications on the vaccines available currently which target few specific genotypes only. Studies are required to determine the predictive role of HR HPV genotypes, in significant copy numbers especially in HIV seropositive women. It would be clinically relevant if the HPV VLs, cervical cytology, and CD4 counts are considered into cervical cancer screening programs for triage and follow-up of these women., 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 © 2021 Lall, Dar, Bhatla, Kumar, Choudhary, Mathur and Gupta.)

Background: Antibody response to SARS-CoV may be estimated to give trends and patterns emerging in a population during an evolving epidemic. The novel coronavirus has opened a new chapter in the history of pandemics and understanding the disease epidemiology., Methods: The study was a cross-sectional descriptive study. Institutional Ethical clearance and informed consent were taken for participation in the study. The study population included all personnel reporting to the institute for training courses, permanent posting or joining back from leave during the study period of 2 months (16 June to 16 August 2020). The sample size was calculated assuming the prevalence of COVID-19 to be 1% with the absolute precision of 0.5% and 5% level of significance, and finite correction for population size of 500, and the calculated sample size was 377. Inclusion criteria were all personnel reporting to the institute from different states and districts. Exclusion criteria-Any personnel reported for a short visit of lesser than 14 days. Demographic details and details of any likely exposure to a confirmed COVID-19 case were noted. A blood sample was collected, and serological tests were done using ErbaLisa COVID-19 IgG kit by Calbiotech, as per the manufacturer's instructions., Results: Overall seropositivity of IgG COVID-19 antibodies was 7.5% (31/413) (95% CI: 5.3-10.4%). Study population (n = 413) comprised of an adult population in the age range of 21 years-53 years, and the mean age was 31.4 years (SD = 6.2 years)., Conclusion: As the personnel joining the institute have come from various parts of the country the study provides an estimation of antibodies against COVID-19., Competing Interests: The authors have none to declare., (© 2021 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd.)

Competing Interests: The authors have none to declare.

Background: With virtually dried out new antibiotic discovery pipeline, emergence and spread of antimicrobial resistance is a cause for global concern. Colistin, a cyclic polypeptide antibiotic, often regarded as last resort for multi drug resistance gram-negative bacteria, is also rendered ineffective by horizontal transfer of resistance genes. Surveillance of colistin resistance in GNB is essential to ascertain molecular epidemiology., Methods: Whole genome sequencing (WGS) of an unusual colistin resistant urinary isolate of Escherichia coli was performed using Illumina MiSeq platform using 2x250bp V2 chemistry by following the manufactures protocol (Illumina Inc. USA). Multiple web-based bio-informatic tools were utilized to ascertain antibiotic resistant genes., Results: An approximate 5.4 Mb of genome of the urinary isolate AFMC&#95;UC19 was sequenced successfully. Mobile colistin resistance gene (mcr) on the plasmid responsible for horizontal spread was absent in the isolate., Conclusion: Colistin resistance has been reported previously in Klebsiella pneumoniae and it is a rare occurrence in Escherichia coli in Indian setting. Although the isolate lack mcr mediated colistin resistance, emergence and spread of colistin resistant in gram-negative bacteria pose a threat., Competing Interests: The authors have none to declare., (© 2020 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd.)

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure 1-4 , but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63 + intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.

Background: Currently CD4+ T lymphocyte counts and HIV-1 RNA levels are being utilized to predict outcome of human immunodeficiency virus (HIV) disease. Recently, the role of immune activation in HIV disease progression and response to treatment is being investigated. This study focused on the expression of CD38 and HLA-DR on lymphocyte subsets in various groups of HIV-infected individuals and to determine their association with HIV-1 disease progression., Methods: Ninety-eight cases of patients with HIV/AIDS in different disease stages and twenty-four healthy HIV-negative individuals were included in the cross-sectional study. Their immune function and abnormal immune activation markers (CD38 & HLA-DR) were detected using a flowcytometer, and HIV-1 RNA levels in individuals receiving antiretroviral drugs were estimated., Results: The immune activation marker levels were significantly different between patients with different disease stages (P < 0.001). A significant negative correlation was observed between peripheral blood CD4+ T cell counts and immune activation markers. Also, a significant positive correlation was observed between HIV-1 RNA levels and CD38+CD8+ T lymphocyte., Conclusion: Immune activation markers (CD38 & HLA-DR) increase with disease progression. CD38+ on CD8+ T lymphocyte correlates well with HIV1 RNA levels in individuals failing on antiretroviral therapy., (© 2019 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd.)

The world is currently face to face with a pandemic which is spreading rapidly across the globe caused by SARS-CoV-2, a strain of Coronaviruses (CoVs) belonging to subgenus Sarbecovirus of genus Betacoronavirus. World Health Organisation (WHO) on 11 Feb 20 named this disease caused by SARS-CoV-2 as Covid-19. This pandemic is spreading rapidly and more than 20,00,000 cases have occurred globally. The human Coronaviruses discovered in 1960s were considered potentially harmless endemic viruses with seasonal distribution before late 2002. The CoVs are found in a large number of domestic and wild animals and birds. The first pandemic caused by Coronavirus caused by SARS-CoV was recognized in the late 2002 in Guangdong Province and resulted in widespread morbidity and mortality. This was followed by MERS-CoV which began in 2012 in the Arabian peninsula with multiple outbreaks related to it in various parts of the globe. Various studies have suggested how these viruses made their entry from their natural reservoir bats via intermediate host like civets and camels in case of SARS-CoV and MERS-CoV respectively. The intermediate host of the SARS-CoV-2 still needs to be established. The SARS-CoV-2 has 96.2% similarity to the bat Severe Acute Respiratory Syndrome related-Coronavirus (SARSr-CoV RaTG13). SARS-CoV-2 has been found to be more distant in relation to SARS-CoV (79%) and MERS-CoV (50%). At the whole genome sequence level pangolin CoV and SARSr-CoV RaTG13 show 91.02% and 96.2% similarity with SARS-CoV-2 but the S1 subunit of spike protein of pangolin CoV is more closely related to SARS-CoV-2 than SARSr-CoV RaTG13. The genetic analysis of the currently circulating strains of the pandemic have shown 99.98-100% similarity in their genomes implying a recent shift to humans. The animal source of SARS-CoV-2 needs to be identified to implement control measures in the present pandemic. Also, how the virus moves interspecies will help predict and prevent future pandemics., Competing Interests: The authors have none to declare., (© 2020 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd.)

Background: Timely initiation of appropriate antimicrobial can improve the outcome in terms of reduced morbidity and mortality in addition to reduced health-care costs. Availability of early preliminary Antimicrobial Susceptibility Test (AST) report will be useful in directing antimicrobial therapy. The aim of the study was to correlate AST by disc diffusion method, directly from positively flagged blood culture bottles, with the AST by automated method., Methods: A total of 144 aerobic blood culture bottles flagged positive by the automated blood culture system were processed. The bacteria were pelleted by two-step centrifugation of the broth from the bottle and used to make a smear for Gram stain as well as an inoculum for antimicrobial sensitivity testing by Kirby Bauer disc diffusion method. Automated identification and AST were also carried out., Results: On direct staining, 94 samples showed gram-negative bacilli, 39 showed gram-positive cocci, and 11 showed yeasts or polymicrobial growth. In the case of gram-negative bacteria, there was 99% categorical agreement between direct sensitivity testing and automated sensitivity testing with 1% disagreement. Among the gram-positive cocci, there was 96% categorical agreement with 4% disagreement between the two methods., Conclusion: High degree of agreement between the two methods is promising and applicable to situations where automated sensitivity testing is not available. Even if the systems are available, this method would prove useful as an adjunct to standard AST reporting. This sensitivity report can be generated earlier than the conventional AST, enabling choice of appropriate antimicrobial., (© 2018 Armed Forces Medical Services (AFMS).)

Background: The cells that form the arterial wall contribute to multiple vascular diseases. The extent of cellular heterogeneity within these populations has not been fully characterized. Recent advances in single-cell RNA-sequencing make it possible to identify and characterize cellular subpopulations., Methods: We validate a method for generating a droplet-based single-cell atlas of gene expression in a normal blood vessel. Enzymatic dissociation of 4 whole mouse aortas was followed by single-cell sequencing of >10 000 cells., Results: Clustering analysis of gene expression from aortic cells identified 10 populations of cells representing each of the main arterial cell types: fibroblasts, vascular smooth muscle cells, endothelial cells (ECs), and immune cells, including monocytes, macrophages, and lymphocytes. The most significant cellular heterogeneity was seen in the 3 distinct EC populations. Gene set enrichment analysis of these EC subpopulations identified a lymphatic EC cluster and 2 other populations more specialized in lipoprotein handling, angiogenesis, and extracellular matrix production. These subpopulations persist and exhibit similar changes in gene expression in response to a Western diet. Immunofluorescence for Vcam1 and Cd36 demonstrates regional heterogeneity in EC populations throughout the aorta., Conclusions: We present a comprehensive single-cell atlas of all cells in the aorta. By integrating expression from >1900 genes per cell, we are better able to characterize cellular heterogeneity compared with conventional approaches. Gene expression signatures identify cell subpopulations with vascular disease-relevant functions.

Background: Chikungunya virus is an alpha virus with high similarity to Dengue and Zika viruses, both in transmission cycle and in clinical presentation. Chikungunya is a re-emerging mosquito-borne infection known to cause small to very large outbreaks/epidemics at frequent intervals. In 2016, India witnessed a large outbreak of Chikungunya infection affecting more than 58,000 people. This study was undertaken to look at the genotypic phylogeny to know the relatedness with previously reported strains., Methods: During the 2016 outbreak, samples from all patients clinically suspected to have Chikungunya were collected and subjected to testing for IgM antibody by ELISA and viral RNA detection by RT-PCR. Sequencing of the E1 gene segment was done to create a phylogenetic tree comparison with other strains., Results: Serum samples were collected from 142 patients of clinically suspected Chikungunya infection. Majority of the patients were in the age group of 31-50 years accounting for more than 35% of the total cases. Twenty eight samples were positive for IgM antibody. Thirty seven samples were positive for viral RNA by RT-PCR. Only 06 cases were positive by both tests. Mutations in the amino acids K211E, M269V and D284E in the E1 gene segment of the Chikungunya virus were observed in the seven strains that were sequenced. On phylogeny tree, all the strains were found to belong to the ECSA genotype., Conclusion: Actively searching for the potential epidemic causing mutations and reporting of novel mutations may help in better understanding and probably forecasting of future CHIKV outbreaks and its nature.

Background: Worldwide prevalence of Hepatitis C virus (HCV) infection hemodialysis (HD) ranges from 1 to 84.6% with serious complications. Assessment of prevalence, risk factors, and genotyping of HCV infection in patient on HD was carried out at Pune, India., Methods: A total of 250 patients on HD from five HD centers were recruited and tested for anti-HCV antibody using third-generation enzyme-linked immunosorbent assay (ELISA). Qualitative HCV RNA detection was carried out by nested reverse transcriptase polymerase chain reaction (RT-PCR). Genotyping and sequencing were carried out using the BigDye Terminator cycle sequencing ready reaction kit., Results: Mean age of patients was 47.3 years. Forty-seven cases out of a total of 250 were reactor for HCV antibody. Overall prevalence rate was 18.8% ranging from 6.7% to 35.6% in the five centers. Of total, 44.1% of females and 13.5% of males were HCV infected. The mean duration of HD in HCV-infected patients was 6.03 years. Prevalence was higher in patients aged > 5 years on HD with higher number of blood transfusions. Thirty-six cases were positive for HCV RNA. Only one HCV RNA was detected among the 203 anti-HCV negative samples. Discordance between antibody and HCV RNA positivity was noted. Seventeen infected cases had changed dialysis centers four times. Thirteen cases were HBsAg positive, of which six cases were coinfected with HCV. Thirty-seven samples were genotyped., Conclusion: The predominant genotype was 1a (54.1%) followed by 1b (43.2%) and 3a (2.7%). Highest prevalence of HCV (35.6%) and intracenter PNI of 99.3% of genotype 1b (84.6%) in center 3 indicates a possible nosocomial transmission.

Background: Prototheca is an emerging, opportunistic, pathogenic, zoonotic achlorophyllous green alga, expanding in pathogenicity and host range, causing localized and disseminated infections. This outbreak of Prototheca wickerhamii algaemia and sepsis in a tertiary care 30-bedded chemotherapy oncology unit is the first human outbreak to the best of our knowledge., Methods: P. wickerhamii algaemia was confirmed on consecutive isolation. Person to person transmission was hypothesized considering all patients in the unit at risk. Clinico-demographic, diagnostic and treatment profile were correlated. Both manual and automated systems were used for blood culture, isolation, identification and susceptibility of Prototheca . Liposomal amphotericin B was given. Outbreak surveillance of faeces, fingertips and environmental reservoirs, retrospective surveillance during past 15 years and prospective surveillance was continued for two years., Results: The outbreak affected 12 neutropenic patients over 50 days. No specific clinical features were noted. The hypothesis could not be substantiated. P. wickerhamii was isolated as yeast-like colonies revealing Gram positive yeast-like cells without budding and pseudohyphae which were confirmed by automated system. Post amphotericin B blood cultures were negative for Prototheca . Surveillance studies were not contributory., Conclusion: P. wickerhamii has no documented reservoirs or transmission. Endogenous colonization in the gut followed by translocation during chemotherapy induced immunosuppression is likely to cause algaemia and sepsis. Outbreaks are difficult to detect and control as incubation period is variable and clinical presentation is muted, emphasizing the need to strengthen hospital and laboratory based surveillance systems to ensure adequate preparedness, rapid detection and response to outbreaks.

Background: Carbapenems are considered "drugs of last resort" in many life-threatening infections. Advent of carbapenemases like KPC, OXA-48, VIM, IMP, and NDM have greatly affected the efficacy of these drugs, posing serious threat to global health and infection control. NDM bears special significance to the India subcontinent, labeled as place of origin and reservoir. NDM tends to escape detection by routine phenotypic methods, requiring molecular confirmation. This study utilizes nested, multiplex polymerase chain reaction (PCR) for reliable detection of bla NDM-1 in nosocomial Enterobacteriaceae isolates., Methods: This study was conducted to detect prevalence of bla NDM-1 , bla IMP , bla VIM and bla KPC genes by multiplex PCR among multidrug/carbapenem-resistant nosocomial Enterobacteriaceae isolates. From March 2013 to April 2014, 100 consecutive non-repeat isolates of Enterobacteriaceae from various inpatient clinical samples were analyzed. Imipenem-resistant isolates identified by Kirby Bauer disk diffusion method with Clinical and Laboratory Standards Institute guidelines were further subjected to nested, multiplex PCR to simultaneously detect bla NDM-1 , bla IMP , bla VIM and bla KPC genes., Results: Out of 100 isolates, 17 (17%) were found to be imipenem-resistant. bla NDM-1 was detected in all 17 isolates by nested, multiplex PCR. bla VIM was co-carried in 4 isolates while one isolate co-harbored bla IMP with bla NDM-1 . Imipenem resistance and NDM-1 carriage was predominant amongst Klebsiella isolates. Maximum NDM-1 producers were isolated from the intensive care unit (70.6%)., Conclusion: NDM-1 prevalence in nosocomial Enterobacteriaceae isolates in our hospital was found to be 17%. A nested, multiplex PCR was used for rapid detection of various carbapenemase genes with high sensitivity and specificity which is essential not only for favorable patient outcome but also for timely implementation of appropriate infection control practices to prevent further spread of such organisms.

Four antigenically different dengue virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) are known to cause infections in humans. Some of these are known to cause more severe disease than the others. Chances for developing Dengue hemorrhagic fever-dengue shock syndrome (DHF-DSS) increases significantly with history of previous infection with one of the four serotypes. Therefore, early diagnosis, serotyping and providing early warning of dengue fever epidemics to concerned authorities becomes very important for better patient outcome and to curb the rapid spread in the community. During the 2014 outbreak, a total of 100 samples from suspected cases of dengue were collected. NS1 antigen based rapid test was used for serological diagnosis. Dengue complex one step reverse transcription-polymerase chain reaction was performed to look for presence of viral RNA. Single tube multiplex RT-PCR was also performed to look for infecting serotype. CDC Dengue Multiplex Real Time PCR assay was performed for rapid diagnosis and simultaneous serotyping of the dengue virus. Out of the 100 samples screened, 69 were found to be positive by NS1Ag Rapid test. 34 samples were found positive by dengue consensus RT-PCR assay. 22 samples were found to be positive by single tube Dengue multiplex RT-PCR assay. Serotype DEN-2 was present in maximum numbers followed by DEN-3. 44 samples were found positive by DENV CDC Multiplex Real time PCR assay. DEN-2 was found in maximum numbers followed by DEN-1. Dengue remains to be an important health problem in India and across the globe. Few serotypes of dengue are more dangerous than the others. Rapid diagnosis and serotyping remains the key for better patient management and prevention of disease spreading in the community. Highly sensitive, specific and rapid CDC real time RT-PCR assay was found to be most promising tool among all available molecular diagnostic methods. This will serve a rapid and reliable simultaneous dengue virus detection as well serotyping assay in near future for rapid identification of dengue suspected sample screening.

Introduction: Enteric-fever is a major public-health problem in developing countries emerging as multidrug-resistant, Nalidixic-acid resistant and extremely drug-resistant Salmonella (Pakistan, 2016), has intensified the use of WHO watch/reserve group antimicrobials such as azithromycin and meropenem., Methods: This ambispective-study was conducted on 782 non-repeat blood-culture isolates of S. Typhi, S. Paratyphi A and S. Paratyphi B obtained from 29,184 blood cultures received at a 1000-bedded tertiary-care hospital of North-India from 2011-2017. Identification and antibiograms were obtained by Vitek-2 compact and Kirby-Bauer's disc diffusion with resistance to ampicillin, chloramphenicol and cotrimoxazole being labeled as multidrug-resistant. Decreased ciprofloxacin-susceptibility and ciprofloxacin-resistance were defined as MIC 0.125-0.5 and >1 μg/ml., Results: S. Typhi and S. Paratyphi A in a ratio of 3.9:1 were seen between July-September predominantly distributed between 6-45 year age group. Resistance to co-trimoxazole, chloramphenicol, ceftriaxone and azithromycin was 6.1%, 13.8%, 16.1 and 5.78% respectively. Multidrug-resistant S. typhi and S. paratyphi A were 2.73% and 1.91% respectively., Conclusion: Enteric-fever is a major public-health problem in India. Emergence of multidrug-resistant, Nalidixic-acid resistant and extremely-drug resistant Salmonella mandates ongoing surveillance for targeted empirical therapy and containment of spread. Repeated epidemics call for water, sanitation, hygiene and vaccination strategies to sustain herd-immunity.

Induced pluripotent stem cells (iPSCs) offer an unprece-dented opportunity to study human physiology and disease at the cellular level. They also have the potential to be leveraged in the practice of precision medicine, for example, personalized drug testing. This statement comprehensively describes the provenance of iPSC lines, their use for cardiovascular disease modeling, their use for precision medicine, and strategies through which to promote their wider use for biomedical applications. Human iPSCs exhibit properties that render them uniquely qualified as model systems for studying human diseases: they are of human origin, which means they carry human genomes; they are pluripotent, which means that in principle, they can be differentiated into any of the human body's somatic cell types; and they are stem cells, which means they can be expanded from a single cell into millions or even billions of cell progeny. iPSCs offer the opportunity to study cells that are genetically matched to individual patients, and genome-editing tools allow introduction or correction of genetic variants. Initial progress has been made in using iPSCs to better understand cardiomyopathies, rhythm disorders, valvular and vascular disorders, and metabolic risk factors for ischemic heart disease. This promising work is still in its infancy. Similarly, iPSCs are only just starting to be used to identify the optimal medications to be used in patients from whom the cells were derived. This statement is intended to (1) summarize the state of the science with respect to the use of iPSCs for modeling of cardiovascular traits and disorders and for therapeutic screening; (2) identify opportunities and challenges in the use of iPSCs for disease modeling and precision medicine; and (3) outline strategies that will facilitate the use of iPSCs for biomedical applications. This statement is not intended to address the use of stem cells as regenerative therapy, such as transplantation into the body to treat ischemic heart disease or heart failure., (© 2018 American Heart Association, Inc.)

Introduction: Many patients with asthma use inhalers incorrectly. Better inhaler technique is associated with better asthma control. We tested the effectiveness of a computer-based video training solution versus traditional written instructions, both which may be used in a resource-limited setting, for teaching inhaler technique. We hypothesized that computer based training will provide a higher quality of instruction which will improve technique more effectively than written training., Methods: 50 asthma patients were recruited from pulmonary clinic at the Junta De Beneficencia Hospital, Ecuador (average age 48.2 years, 58% female). Inhaler technique was taught using written instructions in 20 and video in 30 patients. Inhaler technique was analyzed by video recording pre and post training inhaler use. Inhaler technique score was calculated for each video recording., Results: Baseline performance was equivalent in each group, achieving an average of around 5 of 11 of the inhaler steps. Video training was significantly more effective than written instructions (change of 3.6 points vs. change of 0.4 points, p < 0.001), and improved inhaler technique by 70% (8.6 vs 5.03, p < 0.001); written training did not result in a significant increase in inhaler competency (5.9 vs. 5.5, p = 0.11)., Conclusions: We conclude that written instruction appears to be inadequate to achieve safe and effective administration of inhaled medicine. In contrast, video-based education can effectively create adequate inhaler technique without additional provider time. REGISTRATION NUMBER (CLINICALTRIALS.GOV IDENTIFIER): NCT02660879., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Genome-wide association studies (GWASs) implicate the PHACTR1 locus (6p24) in risk for five vascular diseases, including coronary artery disease, migraine headache, cervical artery dissection, fibromuscular dysplasia, and hypertension. Through genetic fine mapping, we prioritized rs9349379, a common SNP in the third intron of the PHACTR1 gene, as the putative causal variant. Epigenomic data from human tissue revealed an enhancer signature at rs9349379 exclusively in aorta, suggesting a regulatory function for this SNP in the vasculature. CRISPR-edited stem cell-derived endothelial cells demonstrate rs9349379 regulates expression of endothelin 1 (EDN1), a gene located 600 kb upstream of PHACTR1. The known physiologic effects of EDN1 on the vasculature may explain the pattern of risk for the five associated diseases. Overall, these data illustrate the integration of genetic, phenotypic, and epigenetic analysis to identify the biologic mechanism by which a common, non-coding variant can distally regulate a gene and contribute to the pathogenesis of multiple vascular diseases., (Copyright © 2017 Elsevier Inc. All rights reserved.)

AMP-activated protein kinase (AMPK) is a metabolic enzyme that can be activated by nutrient stress or genetic mutations. Missense mutations in the regulatory subunit, PRKAG2, activate AMPK and cause left ventricular hypertrophy, glycogen accumulation, and ventricular pre-excitation. Using human iPS cell models combined with three-dimensional cardiac microtissues, we show that activating PRKAG2 mutations increase microtissue twitch force by enhancing myocyte survival. Integrating RNA sequencing with metabolomics, PRKAG2 mutations that activate AMPK remodeled global metabolism by regulating RNA transcripts to favor glycogen storage and oxidative metabolism instead of glycolysis. As in patients with PRKAG2 cardiomyopathy, iPS cell and mouse models are protected from cardiac fibrosis, and we define a crosstalk between AMPK and post-transcriptional regulation of TGFβ isoform signaling that has implications in fibrotic forms of cardiomyopathy. Our results establish critical connections among metabolic sensing, myocyte survival, and TGFβ signaling., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Advances in genomics are enhancing our understanding of the genetic basis of cardiovascular diseases, both congenital and acquired, and stroke. These advances include finding genes that cause or increase the risk for childhood and adult-onset diseases, finding genes that influence how patients respond to medications, and the development of genetics-guided therapies for diseases. However, the ability of cardiovascular and stroke clinicians to fully understand and apply this knowledge to the care of their patients has lagged. This statement addresses what the specialist caring for patients with cardiovascular diseases and stroke should know about genetics; how they can gain this knowledge; how they can keep up-to-date with advances in genetics, genomics, and pharmacogenetics; and how they can apply this knowledge to improve the care of patients and families with cardiovascular diseases and stroke., (© 2016 American Heart Association, Inc.)

Objective: To create isogenic human pluripotent stem cell-derived macrophages with and without ABCA1 expression as a model for reverse cholesterol transport., Approach and Results: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) genome-editing system was used to introduce frameshift mutations into the coding sequence of ATP-binding cassette, subfamily A, member 1. Individual human pluripotent stem cell clones with deleterious mutations were identified, expanded, and differentiated into mature macrophages with a cytokine-based, feeder-free differentiation protocol. Wild-type cells demonstrated effective cholesterol efflux to apoAI acceptor, whereas ABCA1(-/-) cells displayed significantly reduced efflux ability and increased expression of proinflammatory cytokines., Conclusions: Human pluripotent stem cell-derived macrophages capable of reverse cholesterol transport can be rapidly generated and genetically edited with CRISPR/Cas9. Introduction of homozygous frameshift mutations results in loss of ABCA1 expression in differentiated macrophages and subsequent reduction of cholesterol efflux capability. This facile genome-editing approach and differentiation protocol pave the way for future studies of the molecular determinants of reverse cholesterol transport and other macrophage properties., (© 2015 American Heart Association, Inc.)

Objective: Coronary artery disease (CAD), including myocardial infarction (MI), is the main cause of death in the world. Genome-wide association studies have identified dozens of single nucleotide polymorphisms (SNPs) associated with CAD/MI. One of the most robust CAD/MI genetic associations is with intronic SNPs in the gene PHACTR1 on chromosome 6p24. How these PHACTR1 SNPs influence CAD/MI risk, and whether PHACTR1 itself is the causal gene at the locus, is currently unknown., Approach and Results: Using genetic fine-mapping and DNA resequencing experiments, we prioritized an intronic SNP (rs9349379) in PHACTR1 as causal variant. We showed that this variant is an expression quantitative trait locus for PHACTR1 expression in human coronary arteries. Experiments in endothelial cell extracts confirmed that alleles at rs9349379 are differentially bound by the transcription factors myocyte enhancer factor-2. We engineered a deletion of this myocyte enhancer factor-2-binding site using CRISPR/Cas9 genome-editing methodology. Heterozygous endothelial cells carrying this deletion express 35% less PHACTR1. Finally, we found no evidence that PHACTR1 expression levels are induced when stimulating human endothelial cells with vascular endothelial growth factor, tumor necrosis factor-α, or shear stress., Conclusions: Our results establish a link between intronic SNPs in PHACTR1, myocyte enhancer factor-2 binding, and transcriptional functions at the locus, PHACTR1 expression levels in coronary arteries and CAD/MI risk. Because PHACTR1 SNPs are not associated with the traditional risk factors for CAD/MI (eg, blood lipids or pressure, diabetes mellitus), our results suggest that PHACTR1 may influence CAD/MI risk through as yet unknown mechanisms in the vascular endothelium., (© 2015 American Heart Association, Inc.)

Background: Prevention of parent to child transmission (PPTCT) program was initiated in Armed Forces to reduce the vertical transmission of HIV by instituting single dose Nevirapine (sdNVP) in untreated HIV positive mothers in labour. The aim of this study was to evaluate the role of sdNVP to decrease viral load of HIV infected mother during labour and its efficacy in prevention of mother to child transmission of HIV., Methods: Thirty antenatal women tested positive for HIV at our PPTCT centre and delivered between Jan 2006 and May 2008 were evaluated. During labour these women were given sdNVP. Newborns were given syrup Nevirapine. The babies were tested for HIV infection at 48 h and six weeks after delivery., Results: Thirty HIV positive women delivered at our centre and four newborns were found positive for HIV infection at 48 h. After six weeks interval three neonates were detected for HIV infection as one infant at six weeks was found to be negative for HIV infection., Conclusion: The protection rate of Nevirapine in untreated HIV positive women is not ideal. It is recommended that all HIV positive women should be offered Highly Active Antiretroviral therapy as primary mode for PPTCT.

The past decade has been one of rapid innovation in genome-editing technology. The opportunity now exists for investigators to manipulate virtually any gene in a diverse range of cell types and organisms with targeted nucleases designed with sequence-specific DNA-binding domains. The rapid development of the field has allowed for highly efficient, precise, and now cost-effective means by which to generate human and animal models of disease using these technologies. This review will outline the recent development of genome-editing technology, culminating with the use of CRISPR-Cas9 to generate novel mammalian models of disease. While the road to using this same technology for treatment of human disease is long, the pace of innovation over the past five years and early successes in model systems build anticipation for this prospect.

Background: Various Serosurveys and studies provide ample evidence of differing perspectives regarding epidemiology of HAV and HEV in India. This study was conducted to assess the seroprevalence of HAV and HEV and its associated factors with an aim to provide inputs to planners regarding requirement of HAV vaccine., Methods: A multi-centric cross sectional survey amongst 4175 healthy trainees (young adults) was carried out in training centres, selected by multistage random sampling, giving equal representation to all regions of India. Sample size was calculated by taking prevalence of HAV seropositivity amongst adults as 60% and alpha 0.05., Results: Seroprevalence for HAV and HEV was 92.68% (95% CI 91.82, 93.47) and 17.05% (15.90, 18.26), respectively. Logistic regression showed that hand washing without soap, regular close contact with domestic animals, consumption of unpasteurized milk and regular consumption of food outside home were risk factors for HAV (p < 0.05). For HEV, irregular hand washing, consumption of unpasteurized milk and irregular consumption of freshly prepared food were risk factors (p < 0.05)., Conclusion: High level of immunity against HAV among the healthy young adults clearly demonstrates that vaccination against HAV is not required at present in our country. The large proportion being susceptible to HEV points towards the requirement of preventive strategies in the form of safe drinking water supply, hygiene, sanitation, increasing awareness and behaviour change with respect to personal hygiene especially hand and food hygiene.

Introduction. In India, 4,86,173 HIV infected patients are on first line antiretroviral therapy (ART) as of January 2012. HIV drug resistance (HIVDR) is drug and regimen-specific and should be balanced against the benefits of providing a given ART regimen. Material & Methods. The emergence of HIVDR mutations in a cohort of 100 consecutive HIV-1 infected individuals attending ART centre, on first line ART for 12 months, was studied. CD4(+) T-cell counts and plasma HIV-1 RNA level were determined. Result. Out of the 100 HIV-1 infected individuals, 81 showed HIVDR prevention (HIV-1 RNA level < 1000/mL), while the remaining 19 had HIV-1 viral RNA level > 1000/mL. HIVDR genotyping was carried out for individuals with evidence of virologic failure (HIV-1 RNA level > 1000/mL). The most frequent NRTI-associated mutation observed was M184V, while K103N/S was the commonest mutation at NNRTI resistance position. Conclusion. Our study has revealed the emergence of HIVDR in HIV-1 infected patients at the end of 12 months of first line ART initiation. For NRTIs, the prevalence of HIVDR mutations was 9% and 10% for NNRTIs. Our findings will contribute information in evidence-based decision making with reference to first and second line ART delivery and prevention of HIVDR emergence.

Background: The progressive decline in the CD4 count in HIV patients leads to a more general decline in immune functioning. The study has been carried out to determine the decline in CD4 count in HIV patients., Methods: The study was conducted in a medical college hospital at Maharashtra. The information on baseline CD4 count was gathered from positive patient records registered in the central disease registry. The baseline CD4 count was the first count of CD4 obtained when the patient is diagnosed as HIV positive and further two subsequent readings. The time from baseline (t1) till the last CD4 count (t2) was divided into the different quartiles and the median decline in CD4 count in each quartile was determined. As the time between the two CD4 count measurements was not uniform the rate of change in CD4 was measured with respect to time as [X (t2) - X (t1)/(t2 - t1)]. Correlation was assessed using correlation coefficient., Results: As the CD4 counts were following skewed distribution, the normality was achieved by cuberoot transformation. The overall rate of decline in CD4 count was estimated to be 35 cells/μL per year with 95% confidence interval (CI) as (17.01, 85.04). The correlation coefficient between decline in CD4 and the initial CD4 count in the four time quartiles was (r = -0.51; p = 0.001, r = -0.79; p = 0.000, r = -0.48; p = 0.015 and r = -0.80; p = 0.000) respectively. The median decline in the CD4 count in 0-6 months was 3 cells/μL, in (6-11) months was approximately 26 cells/μL, in (11-21.5) months was 30 cells/μL and in more than 21.5 months the median decline was 52 cells/μL., Conclusions: There was a progressive decline in the CD4 count following HIV infection. An understanding of the influence of decline in CD4 count in HIV patients not on ART is important for clinical management of HIV disease.

Background: Human parvovirus B19 is an emerging transfusion transmitted infection. Although parvovirus B19 infection is connected with severe complications in some recipients, donor screening is not yet mandatory. To reduce the risk of contamination, plasma-pool screening and exclusion of highly viraemic donations are recommended. In this study the prevalence of parvovirus B19 in healthy blood donors was detected by ELISA., Methods: A total of 1633 samples were screened for IgM and IgG antibodies against parvovirus B19 by ELISA. The initial 540 samples were screened for both IgM and IgG class antibodies and remaining 1093 samples were screened for only IgM class antibodies by ELISA., Results: Net prevalence of IgM antibodies to human parvovirus B19 in our study was 7.53% and prevalence of IgG antibodies was 27.96%. Dual positivity (IgG and IgM) was 2.40%., Conclusion: The seroprevalence of human parvovirus B19 among blood donor population in our study is high, and poses an adverse transfusion risk especially in high-risk group of patients who have no detectable antibodies to B19. Studies with large sample size are needed to validate these results.