Extracellular Vesicles in Triple-Negative Breast Cancer: Immune Regulation, Biomarkers, and Immunotherapeutic Potential.

Triple-negative breast cancer (TNBC) is an aggressive subtype accounting for ~10-20% of all human BC and is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) amplification. Owing to its unique molecular profile and limited targeted therapies, TNBC treatment poses significant challenges. Unlike other BC subtypes, TNBC lacks specific molecular targets, rendering endocrine therapies and HER2-targeted treatments ineffective. The chemotherapeutic regimen is the predominant systemic treatment modality for TNBC in current clinical practice. However, the efficacy of chemotherapy in TNBC is variable, with response rates varying between a wide range of patients, and the emerging resistance further adds to the difficulties. Furthermore, TNBC exhibits a higher mutational burden and is acknowledged as the most immunogenic of all BC subtypes. Consequently, the application of immune checkpoint inhibition has been investigated in TNBC, yielding promising outcomes. Recent evidence identified extracellular vesicles (EVs) as an important contributor in the context of TNBC immunotherapy. In view of the extraordinary ability of EVs to transfer bioactive molecules, such as proteins, lipids, DNA, mRNAs, and small miRNAs, between the cells, EVs are considered a promising diagnostic biomarker and novel drug delivery system among the prospects for immunotherapy. The present review provides an in-depth understanding of how EVs influence TNBC progression, its immune regulation, and their contribution as a predictive biomarker for TNBC. The final part of the review focuses on the recent key advances in immunotherapeutic strategies for better understanding the complex interplay between EVs and the immune system in TNBC and further developing EV-based targeted immunotherapies.

Meropenem-vaborbactam restoration of first-line drug efficacy and comparison of meropenem-vaborbactam-moxifloxacin versus BPaL MDR-TB regimen.

BACKGROUND: Meropenem in combination with ß-lactamase inhibitors (BLIs) and other drugs was tested to identify alternative treatment regimens for multidrug-resistant tuberculosis (MDR-TB). METHODS: The following were performed: (1) MIC experiments; (2) static time-kill studies (STKs) with different BLIs; and (3) a hollow fibre model system of TB (HFS-TB) studies with meropenem-vaborbactam combined with human equivalent daily doses of 20 mg/kg or 35 mg/kg rifampin, or moxifloxacin 400 mg, or linezolid 600 mg vs. bedaquiline-pretonamid-linezolid (BPaL) for MDR-TB. The studies were performed using Mycobacterium tuberculosis (M. tuberculosis) H37Rv and an MDR-TB clinical strain (named M. tuberculosis 16D) that underwent whole genome sequencing. Exponential decline models were used to calculate the kill rate constant (K) of different HFS-TB regimens. RESULTS: Whole genome sequencing revealed mutations associated with resistance to rifampin, isoniazid, and cephalosporins. The meropenem-vaborbactam MIC of M. tuberculosis was H37Rv 2 mg/L and > 128 mg/L for M. tuberculosis 16D. Relebactam and vaborbactam improved both the potency and efficacy of meropenem in STKs. Meropenem-vaborbactam alone failed to kill M. tuberculosis 16D but killed below day 0 burden when combined with isoniazid and rifampin, with the moxifloxacin combination being the most effective and outranking bedaquiline and pretomanid. In the HFS-TB, meropenem-vaborbactam-moxifloxacin and BPaL had the highest K (log10 cfu/mL/day) of 0.31 (95% CI 0.17-0.58) and 0.34 (95% CI 0.21-0.56), while meropenem-vaborbactam-rifampin (35 mg/kg) had a K of 0.18 (95% CI 0.12-0.25). The K for meropenem-vaborbactam-moxifloxacin-linezolid demonstrated antagonism. CONCLUSION: Adding meropenem-vaborbactam could potentially restore the efficacy of isoniazid and rifampin against MDR-TB. The meropenem-vaborbactam-moxifloxacin backbone regimen has implications for creating a new effective MDR-TB regimen.

Evaluation of Xpert MTB/RIF Assay on Stool Samples for the Diagnosis of Pulmonary Tuberculosis among the Pediatric Population.

Objective Microbiological confirmation of tuberculosis (TB) in pediatric cases is challenging due to its paucibacillary nature and difficulty in specimen collection. This study aimed to validate stool as an alternative sample for the diagnosis of pediatric pulmonary TB via Xpert MTB/RIF (Xpert) assay. Materials and Methods This cross-sectional study included 75 pediatric patients up to 10 years of age with signs and symptoms suggestive of TB. From each recruited patient, pulmonary and stool samples were collected in a sterile container. The collected samples were subjected to Ziehl-Neelsen staining, BACTEC MGIT 960 culture (MGIT), Xpert, and in-house multiplex polymerase chain reaction for TB diagnosis. Results About 13.33% (10/75) of the pulmonary samples and, of them, 50% (5/75) of the stool samples were positive by Xpert assay. The sensitivity and specificity of Xpert assay with stool and pulmonary samples were 50 (95% confidence interval [CI]: 18.71-81.29%) and 100% (95% CI: 94.48-100%), respectively. Conclusion The Xpert assay on stool samples showed limited sensitivity and good specificity in the diagnosis of pulmonary TB. Therefore, it can be proposed as an alternative screening sample to diagnose TB in pediatric cases for which getting a respiratory sample is extremely difficult. However, further studies with greater number of samples and multiple baseline variables are required to support our findings. Strategies to optimize stool Xpert assay should be performed to enhance the sensitivity of this method to detect Mycobacterium tuberculosis in children.

Microbiome based approaches for the degradation of polycyclic aromatic hydrocarbons (PAHs): A current perception.

Globally, polycyclic aromatic hydrocarbons (PAHs) pollution is primarily driven by their release into the air through various combustion processes, including burning fossil fuels such as coal, oil, and gas in motor vehicles, power plants, and industries, as well as burning organic matter like wood, tobacco, and food in fireplaces, cigarettes, and grills. Apart from anthropogenic pollution sources, PAHs also occur naturally in crude oil, and their potential release during oil extraction, refining processes, and combustion further contributes to contamination and pollution concerns. PAHs are resistant and persistent in the environment because of their inherent features, viz., heterocyclic aromatic ring configurations, hydrophobicity, and thermostability. A wide range of microorganisms have been found to be effective degraders of these recalcitrant contaminants. The presence of hydrocarbons as a result of numerous anthropogenic activities is one of the primary environmental concerns. PAHs are found in soil, water, and the air, making them ubiquitous in nature. The presence of PAHs in the environment creates a problem, as their presence has a detrimental effect on humans and animals. For a variety of life forms, PAH pollutants are reported to be toxic, carcinogenic, mutation-inducing, teratogenic, and immune toxicogenics. Degradation of PAHs via biological activity is an extensively used approach in which diverse microorganisms (fungal, algal, clitellate, and protozoan) and plant species and their derived composites are utilized as biocatalysts and biosurfactants. Some microbes have the ability to transform and degrade these PAHs, allowing them to be removed from the environment. The goal of this review is to provide a critical overview of the existing understanding of PAH biodegradation. It also examines current advances in diverse methodologies for PAH degradation in order to shed light on fundamental challenges and future potential.

Beyond Macromolecules: Extracellular Vesicles as Regulators of Inflammatory Diseases.

Inflammation is the defense mechanism of the immune system against harmful stimuli such as pathogens, toxic compounds, damaged cells, radiation, etc., and is characterized by tissue redness, swelling, heat generation, pain, and loss of tissue functions. Inflammation is essential in the recruitment of immune cells at the site of infection, which not only aids in the elimination of the cause, but also initiates the healing process. However, prolonged inflammation often brings about several chronic inflammatory disorders; hence, a balance between the pro- and anti-inflammatory responses is essential in order to eliminate the cause while producing the least damage to the host. A growing body of evidence indicates that extracellular vesicles (EVs) play a major role in cell-cell communication via the transfer of bioactive molecules in the form of proteins, lipids, DNA, RNAs, miRNAs, etc., between the cells. The present review provides a brief classification of the EVs followed by a detailed description of how EVs contribute to the pathogenesis of various inflammation-associated diseases and their implications as a therapeutic measure. The latter part of the review also highlights how EVs act as a bridging entity in blood coagulation disorders and associated inflammation. The findings illustrated in the present review may open a new therapeutic window to target EV-associated inflammatory responses, thereby minimizing the negative outcomes.

The Role of Extracellular Vesicles in the Pathogenesis of Hematological Malignancies: Interaction with Tumor Microenvironment; a Potential Biomarker and Targeted Therapy.

The tumor microenvironment (TME) plays an important role in the development and progression of hematological malignancies. In recent years, studies have focused on understanding how tumor cells communicate within the TME. In addition to several factors, such as growth factors, cytokines, extracellular matrix (ECM) molecules, etc., a growing body of evidence has indicated that extracellular vesicles (EVs) play a crucial role in the communication of tumor cells within the TME, thereby contributing to the pathogenesis of hematological malignancies. The present review focuses on how EVs derived from tumor cells interact with the cells in the TME, such as immune cells, stromal cells, endothelial cells, and ECM components, and vice versa, in the context of various hematological malignancies. EVs recovered from the body fluids of cancer patients often carry the bioactive molecules of the originating cells and hence can be considered new predictive biomarkers for specific types of cancer, thereby also acting as potential therapeutic targets. Here, we discuss how EVs influence hematological tumor progression via tumor-host crosstalk and their use as biomarkers for hematological malignancies, thereby benefiting the development of potential therapeutic targets.

Diagnosis of tuberculous pleural effusion in a tertiary care hospital of central India: The role of xpert Mycobacterium tuberculosis/rifampicin.

Background: In India, 15%-20% of tuberculosis (TB) cases are categorized as extra-pulmonary TB, and tuberculous pleural effusion (TPE) is the second-most common type after tuberculous lymphadenitis. However, the paucibacillary nature of TPE makes its diagnosis challenging. As a result, relying on empirical anti-TB treatment (ATT) based on clinical diagnosis becomes necessary for achieving the best possible diagnostic outcome. The study aims to determine the diagnostic utility of Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) for the detection of TB in TPE in high incidence setting of Central India. Methods: The study enrolled 321 patients who had exudative pleural effusion detected through radiological testing and were suspected of having TB. The medical procedure of thoracentesis was conducted to collect the pleural fluid, which was then subjected to both the Ziehl-Neelsen staining and Xpert MTB/RIF test. The patients who showed improvement after receiving anti-tuberculosis treatment (ATT) were considered the composite reference standard. Results: The sensitivity of smear microscopy was found to be 10.19%, while that of the Xpert MTB/RIF method was 25.93% when compared to the composite reference standard. The accuracy of clinical diagnosis was measured using receiver operating characteristics based on clinical symptoms, and it was found to be 0.858 (area under the curve). Conclusions: The study shows that Xpert MTB/RIF has significant value in diagnosing TPE, despite its low sensitivity of 25.93%. Clinical diagnosis based on symptoms was relatively accurate, but relying on symptoms alone is not enough. Using multiple diagnostic tools, including Xpert MTB/RIF, is crucial for accurate diagnosis. Xpert MTB/RIF has excellent specificity and can detect RIF resistance. Its quick results make it useful in situations where a rapid diagnosis is necessary. While it should not be the only diagnostic tool, it has a valuable role in diagnosing TPE.

Omadacycline pharmacokinetics/pharmacodynamics in the hollow fiber model and clinical validation of efficacy to treat pulmonary Mycobacterium abscessus disease.

BACKGROUND: Guideline-based therapy (GBT) for pulmonary Mycobacterium abscessus (Mab) disease achieves sustained sputum culture conversion (SSCC) rates of 30%; this is reflected by poor efficacy of GBT in the hollow fiber system model of Mab (HFS-Mab), which killed ∼1.22 log10 CFU/mL. This study was performed to determine which clinical dose of omadacycline, a tetracycline antibiotic, should be used in combination therapy to treat pulmonary Mab disease for relapse-free cure. METHODS: First, omadacycline intrapulmonary concentration-time profiles of seven daily doses were mimicked in the HFS-Mab model and exposures associated with optimal efficacy were identified. Second, 10,000 subject Monte-Carlo simulations were performed to determine whether oral omadacycline 300 mg/day achieved these optimal exposures. Third, a retrospective clinical study on omadacycline vs. primarily tigecycline-based salvage therapy was conducted to assess rates of SSCC and toxicity. Fourth, a single patient was recruited to validate the findings. RESULTS: Omadacycline efficacy in the HFS-Mab was 2.09 log10 CFU/mL at exposures achieved in >99% of patients on 300 mg/day omadacycline. In the retrospective study of omadacycline 300 mg/day-based combinations vs. comparators, SSCC was achieved in 8/10 vs. 1/9 (P=0.006), symptom improvement in 8/8 vs. 5/9 (P=0.033), toxicity in 0 vs. 9/9 (P<0.001), and therapy discontinuation due to toxicity in 0 vs. 3/9 (P<0.001) cases, respectively. In one prospectively recruited patient, omadacycline 300 mg/day salvage therapy achieved SSCC and symptom-resolution in 3 months. CONCLUSION: Based on the preclinical and clinical data, omadacycline 300 mg/day in combination regimens could be appropriate for testing in Phase III trials in patients with Mab pulmonary disease.

Isoniazid pharmacokinetics/pharmacodynamics as monotherapy and in combination regimen in the hollow fiber system model of Mycobacterium kansasii.

BACKGROUND: There is limited high quality evidence to guide the optimal doses of drugs for the treatment of Mycobacterium kansasii pulmonary disease (Mkn-PD). METHODS: We performed (1) minimum inhibitory concentration experiment, (2) isoniazid dose-response study using the hollow fiber system model (HFS-Mkn) to determine PK/PD optimized exposure, and (3) another HFS-Mkn study to determine the efficacy of high dose isoniazid (15 mg/kg/day) with standard dose rifampin (10 mg/kg/day) and ethambutol (15 mg/kg/day). Inhibitory sigmoid maximal effect model and linear regression was used for data analysis. RESULTS: MIC of the 20 clinical isolates ranged between 0.5 mg/L to 32 mg/L. In the HFS-Mkn, isoniazid monotherapy failed to control the bacterial growth beyond day 7. On day 7, when the maximal Mkn kill was observed, the optimal isoniazid exposure for Mkn kill was calculated as 24hr area under the concentration-time curve to the MIC of 12.41. Target attainment probability of 300 mg/day dose fell below 90% above the MIC of 1 mg/L. High dose isoniazid combination sterilized the HFS-Mkn in 30-days with a kill rate of -0.15 ± 0.02 log10 CFU/mL/day. CONCLUSION: Despite initial kill, isoniazid monotherapy failed due to resistance emergence. Our pre-clinical model derived results suggest that higher than currently recommended 300 mg/day isoniazid dose may achieve better clinical efficacy against Mkn-PD.

Challenges and a potential solution to perform drug susceptibility testing of omadacycline against nontuberculous mycobacteria.

BACKGROUND: Minimum inhibitory concentration (MIC) of slow growing mycobacteria (SGM) often do not correlate with the treatment response. Among the challenges is the identification of MIC of drugs that degrade in solution faster than the doubling time of the SGM. METHODS: First, we identified the rate of omadacycline degradation in solution, and its effect on the rapidly growing methicillin resistant Staphylococcus aureus (MRSA). We then identified doubling times versus MICs for Mycobacterium abscessus, M. avium, and M. kansasii, with and without supplementation for degraded drug. RESULTS: Omadacycline concentration in solution declined ∼50% over 24hr. In the MRSA experiments, omadacycline demonstrated 66.48 ± 19.38% loss in potency over 24hr, confirming the degradation rate in solution. M. abscessus had a doubling time of 8.75 ± 1.23hr and the omadacycline MIC after 24hr of incubation was 2mg/L with and without 50% daily drug supplementation, indicating that drug degradation had no effect on this rapid grower. The doubling time for M. avium was 29.52hr (95% confidence interval (CI): 23.18-33.89hr) and 31.15hr (95%CI: 19.45-38.49 hr) for M. kansasii. The M. avium MICs ±50% daily omadacycline supplementation were 1mg/L and 0.5mg/L on day 7, whereas the M. kansasii MICs ±50% daily supplementation were >128mg/L and 32mg/L on day 7. CONCLUSION: Omadacycline degradation in solution leads to falsely high MICs when SGM doubling time exceed the drug degradation rates in solution. The challenge could be overcome by daily drug supplementation to account for the loss of potency, which is laborious, or perhaps stabilizing the drug from degradation.

Omadacycline Pharmacokinetics/Pharmacodynamics in the Hollow Fiber System Model and Potential Combination Regimen for Short Course Treatment of Mycobacterium kansasii Pulmonary Disease.

The 12-month therapy duration for the treatment of Mycobacterium kansasii pulmonary disease calls for more efficacious drugs for better treatment outcomes and to shorten the therapy duration. We performed (i) omadacycline MIC with M. kansasii ATCC 12478 strain and 21 clinical isolates, (ii) dose-response study in the hollow fiber system model of M. kansasii (HFS-Mkn) with six human equivalent omadacycline daily doses to determine the optimal drug exposure for the maximal kill, and (iii) a second HFS-Mkn study to determine the efficacy of omadacycline (300 mg/day) plus moxifloxacin (600 mg/day) plus tedizolid (200 mg/day) combination regimen with standard regimen as comparator. GraphPad Prism was used for data analysis and graphing. MIC of the reference strain was 4 mg/L but ranged from 8 to 32 mg/L among the 21 clinical isolates. In the HFS-Mkn, the exposure required for 50% of the maximal effect (EC50) was an omadacycline area under the concentration-time curve to MIC (AUC0-24/MIC) ratio of 1.95. The optimal exposure was an AUC0-24/MIC of 3.05, which could be achieved with 300 mg/day clinical dose. The omadacycline-moxifloxacin-tedizolid combination sterilized the HFS-Mkn in 14 days with a linear-regression based kill rate of -0.309 ± 0.044 log10 CFU/mL/day compared to the kill rate of -0.084 ± 0.036log10 CFU/mL/day with the standard regimen or 3.7-times faster. Omadacycline has efficacy against M. kansasii and could be used at 300 mg/day in combination with moxifloxacin and tedizolid for the treatment of M. kansasii pulmonary diseases with the potential to shorten the currently recommended 12-month therapy duration.

Organ Involvement in COVID-19: A Molecular Investigation of Autopsied Patients.

Precise reasons for severe manifestation of SARS-CoV-2 remain unanswered, and efforts have been focused on respiratory system management. Demonstration of unequivocal presence of SARS-CoV-2 in vital body organs by cadaver autopsy was the only way to prove multi-organ involvement. Hence, the primary objective of the study was to determine presence of the SARS-CoV-2 in various organs of patients succumbing to SARS-CoV-2 infection. A total of 246 samples from different organs of 21 patients who died due to severe COVID-19 illness were investigated by qRT-PCR, and SARS-CoV-2 was detected in 181 (73.57%) samples and highest positivity of SARS-CoV-2 being (expectedly) found in nasopharynx (90.4%) followed by bilateral lungs (87.30%), peritoneal fluid (80%), pancreas (72.72%), bilateral kidneys (68.42%), liver (65%) and even in brain (47.2%). The deceased patients were categorized to three subgroups based upon the extent of organs in which SARS-CoV-2 was detected by qRT-PCR (high intensity ≥80%, intermediate intensity = 65-80% and low intensity ≤65% organs involvement). It was conclusively established that SARS-CoV-2 has the property of invasion beyond lungs and even crosses the blood-brain barrier, resulting in multi-system disease; this is probably the reason behind cytokine storm, though it is not clear whether organ damage is due to direct injury caused by the virus or result of inflammatory assault. Significant inverse correlation was found between the Ct value of lung samples and number of organs involved, implying that higher viral load in lungs is directly proportionate to involvement of extrapulmonary organs and patients with higher viral load in respiratory secretions should be monitored more closely for any warning signs and the treatment strategies should also address involvement of other organs for better outcome, because lungs, though the primary site of infection, are not the only organ system responsible for pathogenesis of systemic illness.

Clinical, Virological, and Pathological Profile of Patients Who Died of COVID-19: An Autopsy-Based Study From India.

Background and objective Ever since its emergence in December 2019, coronavirus disease 2019 (COVID-19) has affected more than 220 million people worldwide, resulting in more than 45 million deaths. The present autopsy-based study was undertaken to understand the pathophysiology of the disease and correlate the histopathological and virological findings with the antemortem clinical and biochemical determinants. Methods In this prospective observational study, autopsies were carried out on 21 reverse transcription-polymerase chain reaction (RT-PCR)-proven COVID-19 patients who had died of the disease. The histopathological findings of tissue samples from lungs, liver, and kidneys collected during the autopsy were graded based on their presence or absence; if present, they were graded as either focal or diffuse. The findings were correlated with antemortem clinical and biochemical findings. Postmortem tissue RT-PCR analysis was conducted, and findings were compared with postmortem histopathological findings. Results There was multisystem involvement with the COVID-19 cases. The involvement of lungs was observed in most of the cases (90.4%). The presence of viral RNA was observed in all the organs including the liver (57.1%) and kidney (66.6%). An association was observed between antemortem biochemical parameters [aspartate aminotransferase (AST), alanine aminotransferase (ALT)] and the histopathological features in the liver. No correlation between the Sequential Organ Failure Assessment (SOFA) score recorded clinically and lung histopathology was observed; nor was there any correlation between blood urea-creatinine levels and kidney histopathology. Conclusions Our study shows that COVID-19 is a multisystemic disease and the mortality associated with it is likely to be multifactorial. Despite the presence of amplifiable severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in various organs, no association could be established between the clinical and histopathology findings. Neither the duration of hospitalization nor the duration of mechanical ventilation showed any correlation with the severity of histopathological findings in the lungs at autopsy.

Health Seeking Behaviour and Healthcare Utilization in a Rural Cohort of North India.

BACKGROUND: The healthcare infrastructure of a country determines the health-seeking behaviour of the population. In developing countries such as India, there is a great disparity in the distribution of healthcare institutions across urban and rural areas with disadvantages for people living in rural areas. OBJECTIVES: Our objectives were to study the health-seeking behavior and factors associated with the use of formal healthcare among the Gorakhpur Health and Demographic Surveillance System (GHDSS) cohort of North India. METHODS: The study was conducted in 28 villages from two rural blocks in the Gorakhpur district of eastern Uttar Pradesh, North India. Structured questionnaires were used to collect the data with regard to demographics, health-seeking behaviour and healthcare utilization. An adjusted odds ratio with 95% confidence interval was used to report the factors associated with the utilization of healthcare. RESULTS: Out of 120,306 individuals surveyed, 19,240 (16%) individuals reported having any health problem in the last 15 days. Of them, 90% sought healthcare for their health needs. The formal healthcare utilization was 79%. The use of public health facilities was very low (37%) with most of the people seeking treatment from private healthcare (63%). Females, people with a higher level of education (graduate and above), and those belonging to rich and middle tercile were more likely to use formal healthcare services. Among different ailments, respiratory problems, gastrointestinal problems, and musculoskeletal problems were associated with decreased use of formal healthcare. CONCLUSION: About four in five individuals surveyed who had health problems sought treatments from formal healthcare with three in five preferring private institutions to public healthcare facilities due to a perceived higher level of treatment quality and nearby availability. There is an urgent need to re-establish community trust among public healthcare facilities with a focus on delivering on-site health care and enhancing the quality of services offered by public healthcare institutions.

Relative Consolidation of the Kappa Variant Pre-Dates the Massive Second Wave of COVID-19 in India.

India experienced a tragic second wave after the end of March 2021, which was far more massive than the first wave and was driven by the emergence of the novel delta variant (B.1.617.2) of the SARS-CoV-2 virus. In this study, we explored the local and national landscape of the viral variants in the period immediately preceding the second wave to gain insight into the mechanism of emergence of the delta variant and thus improve our understanding of the causation of the second wave. We randomly selected 20 SARS-CoV-2 positive samples diagnosed in our lab between 3 February and 8 March 2021 and subjected them to whole genome sequencing. Nine of the 20 sequenced genomes were classified as kappa variant (B.1.617.1). The phylogenetic analysis of pan-India SARS-CoV-2 genome sequences also suggested the gradual replacement of the α variant with the kappa variant during this period. This relative consolidation of the kappa variant was significant, since it shared 3 of the 4 signature mutations (L452R, E484Q and P681R) observed in the spike protein of delta variant and thus was likely to be the precursor in its evolution. This study demonstrates the predominance of the kappa variant in the period immediately prior to the second wave and underscores its role as the "bridging variant" between the α and delta variants that drove the first and second waves of COVID-19 in India, respectively.

Heterogeneous patterns of COVID-19 transmission in an Urban set up - sero-epidemiological survey data from Ujjain, Madhya Pradesh (a central Indian city).

In the wake of rising number of SARS-CoV-2 cases, the Government of India had placed mass-quarantine measures, termed as "lockdown" measures from end-March 2020. The subsequent phase-wise relaxation from July 2020 led to a surge in the number of cases. This necessitated an understanding of the true burden of SARS-CoV-2 in the community. Consequently, a sero-epidemiological survey was carried out in the central Indian city of Ujjain, Madhya Pradesh. This article details the processes of data acquisition, compilation, handling, and information derivation from the survey. Information on socio-demographic and serological variables were collected from 4,883 participants using a multi-stage stratified random sampling method. Appropriate weightage was calculated for each participant as sampling fraction derived from Primary Sampling Unit (PSU), Secondary Sampling Unit (SSU) and Tertiary Sampling Unit (TSU). The weightage was then applied to the data to adjust the findings at population level. The comprehensive and robust methodology employed here may act as a model for similar future endeavours. At the same time, the dataset can also be relevant for researchers in fields such as data science, epidemiology, virology and earth modelling.

Testing of four-sample pools offers resource optimization without compromising diagnostic performance of real time reverse transcriptase-PCR assay for COVID-19.

Quick identification and isolation of SARS-CoV-2 infected individuals is central to managing the COVID-19 pandemic. Real time reverse transcriptase PCR (rRT-PCR) is the gold standard for COVID-19 diagnosis. However, this resource-intensive and relatively lengthy technique is not ideally suited for mass testing. While pooled testing offers substantial savings in cost and time, the size of the optimum pool that offers complete concordance with results of individualized testing remains elusive. To determine the optimum pool size, we first evaluated the utility of pool testing using simulated 5-sample pools with varying proportions of positive and negative samples. We observed that 5-sample pool testing resulted in false negativity rate of 5% when the pools contained one positive sample. We then examined the diagnostic performance of 4-sample pools in the operational setting of a diagnostic laboratory using 500 consecutive samples in 125 pools. With background prevalence of 2.4%, this 4-sample pool testing showed 100% concordance with individualized testing and resulted in 66% and 59% reduction in resource and turnaround time, respectively. Since the negative predictive value of a diagnostic test varies inversely with prevalence, we re-tested the 4-sample pooling strategy using a fresh batch of 500 samples in 125 pools when the prevalence rose to 12.7% and recorded 100% concordance and reduction in cost and turnaround time by 36% and 30%, respectively. These observations led us to conclude that 4-sample pool testing offers the optimal blend of resource optimization and diagnostic performance across difference disease prevalence settings.

Genomic analysis of a novel species Halomonas shambharensis isolated from hypersaline lake in Northwest India.

Genome analysis of Halomonas shambharensis, a novel species, was performed to understand the osmoprotectant strategies used by the strain to overcome the salinity stress and to explore the prospective industrial uses. It will also help to better understand the ecological roles of Halomonas species in hypersaline habitats. Ultrastructure of the cell was determined by using transmission electron microscopy. Standard microbiological methods were used to find out growth parameters and heterotrophic mode of nutrition. For Genome analysis, complete bacterial genome sequencing was performed using the Oxford Nanopore MinION DNA Sequencer. Assembly, annotation and finishing of the obtained sequence were done by using a Prokaryotic Genome Annotation Pipeline (PGAP) (SPAdes v. 3.10.1). Predicted Coading sequences (CDSs) obtained through the PGAP were used for functional annotation using Clusters of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes (KEGG) platforms. The H. shambharensis was found to be a Gram-stain-negative, rod-shaped bacterium, motile with a peritrichous flagella. The H. shambharensis bacterium can grow in a wide range of temperature (from 25 to 65 °C), pH (pH 4 to pH 12.0) and salt concentration (5.0% NaCl to 30.0% NaCl). After annotation and assembly, the total genome size obtained was 1,533,947 bp, which revealed 146 subsystems, 3847 coding sequences, and 19RNAs with G+C content of 63.6%. Gene annotation identified the genes related to various metabolic pathways, including carbohydrate metabolism, fatty acid metabolism and stress tolerance. The genomic dataset of H. shambharensis will be useful for analysis of protein-coding gene families and how these coding genes are significant for the survival and metabolism among the different species of Halomonas. The complete genome sequence presented here will help to unravel the biotechnological potential of H. shambharensis for production of the high-value products such as betaine, or as a source of gene-mining for individual enzymes.

Genome Sequencing Reveals a Mixed Picture of SARS-CoV-2 Variant of Concern Circulation in Eastern Uttar Pradesh, India.

Uttar Pradesh is the densely populated state of India and is the sixth highest COVID-19 affected state with 22,904 deaths recorded on November 12, 2021. Whole-genome sequencing (WGS) is being used as a potential approach to investigate genomic evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. In this study, a total of 87 SARS-CoV-2 genomes-49 genomes from the first wave (March 2020 to February 2021) and 38 genomes from the second wave (March 2021 to July 2021) from Eastern Uttar Pradesh (E-UP) were sequenced and analyzed to understand its evolutionary pattern and variants against publicaly available sequences. The complete genome analysis of SARS-CoV-2 during the first wave in E-UP largely reported transmission of G, GR, and GH clades with specific mutations. In contrast, variants of concerns (VOCs) such as Delta (71.0%) followed by Delta AY.1 (21.05%) and Kappa (7.9%) lineages belong to G clade with prominent signature amino acids were introduced in the second wave. Signature substitution at positions S:L452R, S:P681R, and S:D614G were commonly detected in the Delta, Delta AY.1, and Kappa variants whereas S:T19R and S:T478K were confined to Delta and Delta AY.1 variants only. Vaccine breakthrough infections showed unique mutational changes at position S:D574Y in the case of the Delta variant, whereas position S:T95 was conserved among Kappa variants compared to the Wuhan isolate. During the transition from the first to second waves, a shift in the predominant clade from GH to G clade was observed. The identified spike protein mutations in the SARS-CoV-2 genome could be used as the potential target for vaccine and drug development to combat the effects of the COVID-19 disease.

Evaluation of pooled sample analysis strategy in expediting case detection in areas with emerging outbreaks of COVID-19: A pilot study.

Timely diagnosis of COVID-19 infected individuals and their prompt isolation are essential for controlling the transmission of SARS-CoV-2. Though quantitative reverse transcriptase PCR (qRT-PCR) is the method of choice for COVID-19 diagnostics, the resource-intensive and time-consuming nature of the technique impairs its wide applicability in resource-constrained settings and calls for novel strategies to meet the ever-growing demand for more testing. In this context, a pooled sample testing strategy was evaluated in the setting of emerging disease outbreak in 3 central Indian districts to assess if the cost of the test and turn-around time could be reduced without compromising its diagnostic characteristics and thus lead to early containment of the outbreak. From 545 nasopharyngeal and oropharyngeal samples received from the three emerging districts, a total of 109 pools were created with 5 consecutive samples in each pool. The diagnostic performance of qRT-PCR on pooled sample was compared with that of individual samples in a blinded manner. While pooling reduced the cost of diagnosis by 68% and the laboratory processing time by 66%, 5 of the 109 pools showed discordant results when compared with induvial samples. Four pools which tested negative contained 1 positive sample and 1 pool which was positive did not show any positive sample on deconvolution. Presence of a single infected sample with Ct value of 34 or higher, in a pool of 5, was likely to be missed in pooled sample analysis. At the reported point prevalence of 4.8% in this study, the negative predictive value of qRT-PCR on pooled samples was around 96% suggesting that the adoption of this strategy as an effective screening tool for COVID-19 needs to be carefully evaluated.