Three Tier Screening Tool and Second Triage to Minimize the Spread of COVID-19 in Emergency Department of a Tertiary Hospital in India

Introduction: Since the outbreak of Coronavirus on December 31, 2019 in Wuhan, Hubei Province, People’s Republic of China, the number of cases from China that have been imported into more than 180 countries and regions around the world. Objective: The goal of this study is to flatten the curve of new infection, through nosocomial transmission by health care system along with early identification of asymptomatic COVID-19 cases. Methods: A Survey was conducted over a period of 35 days. A total of 1709 individuals were screened (647 patients and 1062 patient attendees) coming to emergency Department. The waiting area of Emergency Care was divided into 3 screening zones and a separate second triage is established. The individuals entering are ensured that they are screened at all the 3 zones. Individuals were divided into two Groups after screening: Group A (suspected COVID-19) and Group B (unsuspected COVID-19). In Acute emergencies, the patient was directly treated at second triage. Results: A total of 1709 individuals, 247 in Group A (Suspected COVID-19) and 1462 in Group B (Unsuspected COVID-19). Among 247 individuals, 141 were males and 106 were females. Age ranged from 14-72 years with a mean age of 46.7years. Among 247 individuals (Group A), 81 were patients, of which one case was found to be COVID-19 Positive. Two Health care workers (HCW’s) found to be positive. Conclusion: Challenges from the widespread pandemic underscores the importance of early implementation of a second triage and vigorous screening for all the individuals to minimize the spread of infection, failing which pandemic infection may turn into an epidemic

Progressive Host-Directed Strategies to Potentiate BCG Vaccination Against Tuberculosis

The pursuit to improve the TB control program comprising one approved vaccine, M. bovis Bacille Calmette-Guerin (BCG) has directed researchers to explore progressive approaches to halt the eternal TB pandemic. Mycobacterium tuberculosis (M.tb) was first identified as the causative agent of TB in 1882 by Dr. Robert Koch. However, TB has plagued living beings since ancient times and continues to endure as an eternal scourge ravaging even with existing chemoprophylaxis and preventive therapy. We have scientifically come a long way since then, but despite accessibility to the standard antimycobacterial antibiotics and prophylactic vaccine, almost one-fourth of humankind is infected latently with M.tb. Existing therapeutics fail to control TB, due to the upsurge of drug-resistant strains and increasing incidents of co-infections in immune-compromised individuals. Unresponsiveness to established antibiotics leaves patients with no therapeutic possibilities. Hence the search for an efficacious TB immunization strategy is a global health priority. Researchers are paving the course for efficient vaccination strategies with the radically advanced operation of core principles of protective immune responses against M.tb. In this review; we have reassessed the progression of the TB vaccination program comprising BCG immunization in children and potential stratagems to reinforce BCG-induced protection in adults

The uncharted territory of host-pathogen interaction in tuberculosis

Mycobacterium tuberculosis (M.tb) effectively manipulates the host processes to establish the deadly respiratory disease, Tuberculosis (TB). M.tb has developed key mechanisms to disrupt the host cell health to combat immune responses and replicate efficaciously. M.tb antigens such as ESAT-6, 19kDa lipoprotein, Hip1, and Hsp70 destroy the integrity of cell organelles (Mitochondria, Endoplasmic Reticulum, Nucleus, Phagosomes) or delay innate/adaptive cell responses. This is followed by the induction of cellular stress responses in the host. Such cells can either undergo various cell death processes such as apoptosis or necrosis, or mount effective immune responses to clear the invading pathogen. Further, to combat the infection progression, the host secretes extracellular vesicles such as exosomes to initiate immune signaling. The exosomes can contain M.tb as well as host cell-derived peptides that can act as a double-edged sword in the immune signaling event. The host-symbiont microbiota produces various metabolites that are beneficial for maintaining healthy tissue microenvironment. In juxtaposition to the above-mentioned mechanisms, M.tb dysregulates the gut and respiratory microbiome to support its replication and dissemination process. The above-mentioned interconnected host cellular processes of Immunometabolism, Cellular stress, Host Microbiome, and Extracellular vesicles are less explored in the realm of exploration of novel Host-directed therapies for TB. Therefore, this review highlights the intertwined host cellular processes to control M.tb survival and showcases the important factors that can be targeted for designing efficacious therapy

Expression of Measles Virus Nucleoprotein Induces Apoptosis and Modulates Diverse Functional Proteins in Cultured Mammalian Cells

Background: Measles virus nucleoprotein (N) encapsidates the viral RNA, protects it from endonucleases and forms a virus specific template for transcription and replication. It is the most abundant protein during viral infection. Its C-terminal domain is intrinsically disordered imparting it the flexibility to interact with several cellular and viral partners. Principal Findings: In this study, we demonstrate that expression of N within mammalian cells resulted in morphological transitions, nuclear condensation, DNA fragmentation and activation of Caspase 3 eventuating into apoptosis. The rapid generation of intracellular reactive oxygen species (ROS) was involved in the mechanism of cell death. Addition of ascorbic acid (AA) or inhibitor of caspase-3 in the extracellular medium partially reversed N induced apoptosis. We also studied the protein profile of cells expressing N protein. MS analysis revealed the differential expression of 25 proteins out of which 11 proteins were up regulated while 14 show signs of down regulation upon N expression. 2DE results were validated by real time and semi quantitative RT-PCR analysis. Conclusion: These results show the pro-apoptotic effects of N indicating its possible development as an apoptogenic tool. Our 2DE results present prima facie evidence that the MV nucleoprotein interacts with or causes differential expression of a wide range of cellular factors. At this stage it is not clear as to what the adaptive response of the host cell is and what reflects a strategic modulation exerted by the virus

List of oligonucleotides used in this study.

<p>List of oligonucleotides used in this study.</p

N induces ROS production and caspase 3 activation in MCF7 and 293T cells.

<p>(a) N and P expression in MCF-7 cells. 24 h after transfection, total RNA was extracted from the cells and RT-PCR was performed as described in methods. Gel pictures showing expression of N and P. (b) Analysis of ROS generation by DCF fluorescence as described in Methods. Representative fluorescent images and corresponding mean fluorescence intensity. (c) Determination of caspase 3 activation in MCF7 cells by FITC fluorescence as described in Methods. Results are expressed as the fold increase in fluorescence and are given as the mean ± SD for three experiments.</p

Inhibition of apoptosis by AA and Z-VAD-FMK.

<p>(a) Percentage of apoptosis in control N expressing cells analyzed by flow cytometry after PI staining. Apoptotic cells were estimated by the percentage of cells in the sub-G1 peak. Each bar represents the mean ± SD of three independent experiments. (b) Nuclear staining with hoechst 33258. N transfected MCF7 cells showed apoptotic morphology; DNA condensation and nuclear fragmentation whereas rest of the cells remained uniformly stained with round and unpunctuated nucleus. White arrows indicate nuclear condensation and fragmentation. The figures represent one of the three independent experiments. (c) Single Cell Gel Electrophoresis assessment of N toxicity in human breast cancer cells (MCF7). Cells were harvested for comet tail formation assays under alkaline conditions. Comet images were captured using fluorescence microscopy, and tail moment was analyzed in 50–60 randomly chosen comets using Comet assay IV software. Representative comet images observed are shown. Histograms represent changes in the comet tail moments between control and treated cells. N transfected cells show a long tail indicating DNA damage. Each experiment was done in triplicate. Data is represented as means ±SD.</p

Typical 2D gel analysis of N transfected cells compared with control cells.

<p>Cells were transfected with either pCA or pCA-N and 24 h post transfection total cellular protein was extracted. Protein extracts were subjected to separation by 2DE prior to staining and visualization as described under methods. (a) Representative 2D gels of control and treated samples. Encircled proteins were consistently seen to vary in intensity in multiple experiments. Those spots were picked for sequencing. (b) The MALDI-TOF-MS mass spectrum of a spot, identified as the Poly(rC) binding protein 1 according to the matched peaks is shown.</p

N induces cell death in MCF7 cells.

<p>(a) Immunoblot analysis of N expression in MCF7 cells. (b) Representative fluorescent images of cells co transfected with GFP and N expression vectors showing 70–80% transfection efficiency. (c) Morphological features of MCF7 cells expressing N as seen by light microscopy. (d) Flow cytometric profile of representative cell populations 24 h post transfection, fixed and PI stained for cell cycle analysis as described in methods. The data is representative of three independent experiments.</p

Validation of 2DE by real time and semi quantitative RT-PCR.

<p>24 h after transfection, total RNA was extracted from the cells and real time and semi quantitative RT-PCR was performed as described in methods. (a) Gel pictures showing differential expression of PARK7 and PHB. Fold change in expression was analyzed by software ImageJ. Data was normalized with beta actin as the control house keeping gene. (b) Relative quantification of GAPDH and MAP2K4 mRNA in N transfected and control MCF7 cells. The change in gene expression is expressed as fold change in relation to control. Results are presented as the mean ± SD from 3 different experiments.</p