Distribution and Functional Analyses of Mutations in Spike Protein and Phylogenic Diversity of SARS-CoV-2 Variants Emerged during the Year 2021 in India.

Introduction: Prolonged COVID-19 pandemic accelerates the emergence and transmissibility of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants through the accumulation of adaptive mutations. Particularly, adaptive mutations in spike (S) protein of SARS-CoV-2 leads to increased viral infectivity, severe morbidity and mortality, and immune evasion. This study focuses on the phylodynamic distribution of SARS-CoV-2 variants during the year 2021 in India besides analyzing the functional significance of mutations in S-protein of SARS-CoV-2 variants. Methods: Whole genome of SARS-CoV-2 sequences (n = 87957) from the various parts of India over the period of January to December 2021 was retrieved from Global Initiative on Sharing All Influenza Data. All the S-protein sequences were subjected to clade analysis, variant calling, protein stability, immune escape potential, structural divergence, Furin cleavage efficiency, and phylogenetic analysis using various in silico tools. Results: Delta variant belonging to 21A, 21I, and 21J clades was found to be predominant throughout the year 2021 though many variants were also present. A total of 4639 amino acid mutations were found in S-protein. D614G was the most predominant mutation in the S-protein followed by P681R, L452R, T19R, T478K, and D950N. The highest number of mutations was found in the N-terminal domain of S-protein. Mutations in the crucial sites of S-protein impacting pathogenicity, immunogenicity, and fusogenicity were identified. Intralineage diversity analysis showed that certain variants of SARS-CoV-2 possess high diversification. Conclusions: The study has disclosed the distribution of various variants including the Delta, the predominant variant, in India throughout the year 2021. The study has identified mutations in S-protein of each SARS-CoV-2 variant that can significantly impact the virulence, immune evasion, increased transmissibility, high morbidity, and mortality. In addition, it is found that mutations acquired during each viral replication cycle introduce new sub-lineages as studied by intralineage diversity analysis.

Colonic Eosinophilia: Clinicopathologic Study of Paired Right and Left Colon Biopsies from 276 Patients.

OBJECTIVE: This study evaluated differences in eosinophil (Eos) count in the right colon (RC) and left colon (LC) relative to known clinical and pathologic features. METHODS: H&E slides from 276 subjects with biopsies taken from both RC and LC were reviewed. Eos/mm2 were counted in the area with highest concentration then correlated with clinical and pathologic findings for RC and LC. RESULTS: There were higher numbers of Eos/mm2 in RC than in LC (mean 177 vs 122, respectively p<0.0001), and there was significant positive correlation between Eos numbers in the two locations (r=0.57, p<0.001). In RC, the mean Eos/mm2 was 242 with active chronic colitis, 195 with inactive chronic colitis, 160 in microscopic colitis, 144 in quiescent IBD, and 142 with normal histology (p<0.001), and was higher in males (204 vs 164, p=0.022). In LC, mean Eos/mm2 was 186 with active chronic colitis, 168 with inactive chronic colitis, 154 in microscopic colitis, 82 in quiescent IBD, and 84 with normal histology (p<0.001), and was higher in males (154 vs 107, p<0.001). In biopsies with normal histology, RC showed higher mean Eos/mm2 in Asian patients (228 vs 139, p=0.019), and patients with history of UC (205 vs 136, p=0.004), but was not significantly different in patients with or without irritable bowel syndrome with diarrhea (IBS-D) or history of Crohn's disease (CD). In LC the mean Eos/mm2 was higher in males (102 vs 77, p=0.036), and history of CD (117 vs 78, p=0.007), but was not significantly different in patients with or without IBS-D or history of UC. The number of Eos/mm2 was greater in biopsies performed in the summer than during other seasons of the year. CONCLUSION: The mean number of Eos/mm2 in colorectal biopsies varies significantly by location, histopathologic changes, clinical diagnosis, season, gender and ethnicity. Of particular interest is the association between high Eos/mm2 in RC biopsies with otherwise normal histology and clinical history of UC, and in LC biopsies with clinical history of CD. Additional larger and prospective studies that include normal healthy volunteers are needed to establish a reliable cutoff for the histopathologic diagnosis of eosinophilic colitis, taking into consideration the biopsy site within the colon and rectum, as well as patient gender and ethnicity.Presented in part at the annual American College of Gastroenterology meeting, San Antonio, TX October 2019.

Practice patterns and adherence to society guidelines for suspected choledocholithiasis: A comparison of academic and community settings from a large US healthcare system.

Background: The American Society of Gastrointestinal Endoscopy (ASGE) has proposed practice guidelines for evaluating patients with suspected choledocholithiasis. This study aims to assess and compare practice patterns for following ASGE guidelines for choledocholithiasis in a large academic vs. community hospital setting. Methods: A total of one thousand ER indicated for choledocholithiasis were randomly selected. Patients' demographics, total bilirubin, imaging studies including magnetic resonance cholangiopancreatography (MRCP), intraoperative cholangiogram (IOC), endoscopic ultrasound (EUS), and ERCP results were retrospectively collected. Patients with prior sphincterotomy were excluded. We examined the following practice deviations from the current ASGE guidelines; (1) ERCP was potentially delayed in high probability cases while awaiting additional imaging studies, (2) ERCP was performed without additional imaging studies in cases of low/intermediate-risk, or (3) ERCP was performed in low/intermediate-risk cases when additional imaging studies were negative. Results: A total of 640 patients with native papilla who underwent ERCP were included in the final analysis. Overall, the management of 43% (275) of patients was deviated from the applicable ASGE guidelines. Academic and community provider rates of non-adherence were 32 vs. 45%, respectively (p-value: < 0.01). Of 381 high-risk cases, 54.1% had additional imaging before ERCP. (Academic vs. community; 11.7 vs. 88.3%, p-value: < 0.01). In 26.7% (69/258) of low/intermediate risk cases, ERCP was performed without additional studies; academic (14.5%) vs. community (85.5%) (p-value: < 0.01). Finally, in 11.2% (19/170) of patients, ERCP was performed despite intermediate/low probability and negative imaging; academic (26.3%) vs. community (73.7%) (p-value: 0.02). Conclusion: Our study results show that providers do not adhere to ASGE practice guidelines in 43% of suspected choledocholithiasis cases. The rate of non-adherence was significantly higher in community settings. It could be due to various reasons, including lack/delays for alternate studies (i.e., MRCP, EUS), concern regarding the length of stay, patient preference, or lack of awareness/understanding of the guidelines. Increased availability of alternate imaging and educational strategies may be needed to increase the adoption of practice guidelines across academic and community settings to improve patient outcomes and save healthcare dollars.

Epitope Identification and Designing a Potent Multi-epitope Vaccine Construct against SARS-CoV-2 Including the Emerging Variants.

Introduction: The emergence of a novel coronavirus in China has turned into a SARS-CoV-2 pandemic with high fatality. As vaccines are developed through various strategies, their immunogenic potential may drastically vary and thus pose several challenges in offering immune responses against the virus. Methods: In this study, we adopted an immunoinformatics-aided approach for developing a new multi-epitope vaccine construct (MEVC). In silico approach was taken for the identification of B-cell and T-cell epitopes in the Spike protein, for MEVC various cytotoxic T-lymphocyte, helper T-lymphocyte, and B-cell epitopes with the highest affinity for the respective HLA alleles were assembled and joined by linkers. Results: The computational data suggest that the MEVC is nontoxic, nonallergenic and thermostable and elicit both humoral and cell-mediated immune responses. Subsequently, the biological activity of MEVC was assessed by bioinformatic tools using the interaction between the vaccine candidate and the innate immune system receptors TLR3 and TLR4. The epitopes of the construct were analyzed with that of the strains belonging to various clades including the emerging variants having multiple unique mutations in S protein. Conclusions: Due to the advantageous features, the MEVC can be tested in vitro for more practical validation and the study offers immense scope for developing a potential vaccine candidate against SARS-CoV-2 in view of the public health emergency associated with COVID-19 disease caused by SARS-CoV-2.

Comparing diagnostic accuracy of current practice guidelines in predicting choledocholithiasis: outcomes from a large healthcare system comprising both academic and community settings.

BACKGROUND AND AIMS: The American Society for Gastrointestinal Endoscopy (ASGE) 2010 guidelines for suspected choledocholithiasis were recently updated by proposing more specific criteria for selection of high-risk patients to undergo direct ERCP while advocating the use of additional imaging studies for intermediate- and low-risk individuals. We aim to compare the performance and diagnostic accuracy of 2019 versus 2010 ASGE criteria for suspected choledocholithiasis. METHODS: We performed a retrospective chart review of a prospectively maintained database (2013-2019) of over 10,000 ERCPs performed by 70 gastroenterologists in our 14-hospital system. We randomly selected 744 ERCPs in which the primary indication was suspected choledocholithiasis. Patients with a history of cholecystectomy or prior sphincterotomy were excluded. The same patient cohort was assigned as low, intermediate, or high risk according to the 2010 and 2019 guideline criteria. Overall accuracy of both guidelines was compared against the presence of stones and/or sludge on ERCP. RESULTS: Of 744 patients who underwent ERCP, 544 patients (73.1%) had definite stones during ERCP and 696 patients (93.5%) had stones and/or sludge during ERCP. When classified according to the 2019 guidelines, fewer patients were high risk (274/744, 36.8%) compared with 2010 guidelines (449/744, 60.4%; P < .001). Within the high-risk group per both guidelines, definitive stone was found during ERCP more frequently in the 2019 guideline cohort (226/274, 82.5%) compared with the 2010 guideline cohort (342/449, 76.2%; P < .001). In our patient cohort, overall specificity of the 2010 guideline was 46.5%, which improved to 76.0% as per 2019 guideline criteria (P < .001). However, no significant change was noted for either positive predictive value or negative predictive value between 2019 and 2010 guidelines. CONCLUSIONS: The 2019 ASGE guidelines are more specific for detection of choledocholithiasis during ERCP when compared with the 2010 guidelines. However, a large number of patients are categorized as intermediate risk per 2019 guidelines and will require an additional confirmatory imaging study.

Prosthetic Mitral Valve Obstruction with Left Atrial Appendage Thrombus: A Therapeutic Dilemma.

Valvular obstruction is a rare but life-threatening complication of mechanical prosthetic valves that raises significant challenges in management. We describe a unique case of mechanical mitral valve obstruction with co-existing left atrial appendage (LAA) thrombus. A 48-year-old man with a past medical history of atrial fibrillation and mechanical mitral valve replacement 18 months prior, presented with symptoms of new onset heart failure for 10 days. INR on presentation was sub-therapeutic. Trans-thoracic and trans-esophageal echocardiography revealed prosthetic mitral valve obstruction with mobile, echogenic masses seen on the mechanical valve as well as LAA, suggestive of thrombus. His clinical course rapidly deteriorated and he developed cardiogenic shock. He was deemed to have prohibitive risk for emergent surgical intervention. He received trial of thrombolytic therapy, with partial improvement of hemodynamic parameters and a mild decrease in thrombus burden. He then underwent surgical intervention with a favorable outcome. Intra-operative visualization of the prosthetic valve revealed a combination of pannus and thrombus. Prosthetic valve function should be promptly assessed in patients presenting with heart failure symptoms, as clinical deterioration can be rapid. Acute presentation, history of inadequate anticoagulation and appearance of soft mass on an echocardiogram, are suggestive of thrombus as the etiology of valve obstruction. However, thrombus and pannus are known to frequently co-exist. Emergent surgery is the recommended management strategy in patients with left-sided prosthetic valve thrombosis with the New York Heart Association (NYHA) III or IV symptoms, due to a lower rate of thrombo-embolism, major bleeding, and recurrent prosthetic valve thrombosis when compared with thrombolytic therapy. Slow-infusion, low-dose thrombolytics were recently shown to have favorable outcomes and can be considered when surgery is not available or the patient is deemed to have prohibitive surgical risk.

Dedifferentiated Liposarcoma in a Giant Esophageal Polyp: A Case Report and Review of the Literature.

Soft tissue sarcomas represent an extremely rare cause of esophageal masses, and undifferentiated sarcomas are rarer. The proportion of dedifferentiated liposarcomas (DDL) is even lower. The case of a 58-year-old male who complained of dysphagia and was found to have an 18-centimeter long esophageal mass/polyp on esophagogastroduodenoscopy is presented. The lesion was resected endoscopically and a diagnosis of DDL was confirmed by fluorescence in situ hybridization. Due to its rarity, the treatment experience with esophageal DDLs is limited. However, based on our experience, endoscopic resection of the lesion can be considered as the treatment of choice when feasible. We performed a review of the literature to identify and analyze similar reported cases.

Comparative study on production of a-Amylase from Bacillus licheniformis strains

Alpha amylase (α-1, 4-glucan-glucanhydrolase, EC 3.2.1.1), an extracellular enzyme, degrades α, 1-4 glucosidic linkages of starch and related substrates in an endo-fashion producing oligosaccharides including maltose, glucose and alpha limit dextrin (7). The present study deals with the production and comparative study of production of α-amylase from two strains of Bacillus licheniformis, MTCC 2617 and 2618, by using four different substrates, starch, rice, wheat and ragi powder as carbon source by submerged fermentation. The effect of varying pH and incubation temperature, activator, inhibitor, and substrate concentration was investigated on the activity of α-amylase produced by MTCC strain 2618. The results shows that the production of the α-amylase by the B.licheniformis strain MTCC 2618, using four different substrates were found to be maximum (Starch 3.64 IU/ml/minutes, Rice powder 2.93 IU/ml/minutes, Wheat powder 2.67 IU/ml/minutes, Ragi powder 2.36 IU/ml/minutes) on comparing the enzyme production of two strains. It was also observed that the maximum production was found on the 3rd day (i.e. 72 hr) and characterization of crude enzyme revealed that optimum activity was at pH 7 and 37ºC.

Comparative study on production of a-Amylase from Bacillus licheniformis strains.

Alpha amylase (α-1, 4-glucan-glucanhydrolase, EC 3.2.1.1), an extracellular enzyme, degrades α, 1-4 glucosidic linkages of starch and related substrates in an endo-fashion producing oligosaccharides including maltose, glucose and alpha limit dextrin (7). The present study deals with the production and comparative study of production of α-amylase from two strains of Bacillus licheniformis, MTCC 2617 and 2618, by using four different substrates, starch, rice, wheat and ragi powder as carbon source by submerged fermentation. The effect of varying pH and incubation temperature, activator, inhibitor, and substrate concentration was investigated on the activity of α-amylase produced by MTCC strain 2618. The results shows that the production of the α-amylase by the B.licheniformis strain MTCC 2618, using four different substrates were found to be maximum (Starch 3.64 IU/ml/minutes, Rice powder 2.93 IU/ml/minutes, Wheat powder 2.67 IU/ml/minutes, Ragi powder 2.36 IU/ml/minutes) on comparing the enzyme production of two strains. It was also observed that the maximum production was found on the 3(rd) day (i.e. 72 hr) and characterization of crude enzyme revealed that optimum activity was at pH 7 and 37°C.