Molecular Interactions Leading to Advancements in the Techniques for COVID-19 Detection: A Review.

Since 2019 the world has been in a combat with the highly contagious disease COVID-19 which is caused by the rapid transmission of the SARS-CoV-2 virus (severe acute respiratory syndrome coronavirus 2). Detection of this disease in an early stage helps to control its spread and management. To combat this epidemic with one-time effective medication, improved quick analytical procedures must be developed and validated. The requirement for accurate and precise analytical methods for the diagnosis of the virus and antibodies in infected patients has been a matter of concern. The global impact of this virus has motivated scientists and researchers to investigate and develop various analytical diagnostic techniques. This review includes the study of standard methods which are reliable and accredited for the analytical recognition of the said virus. For early detection of SARS-CoV-2 RNA, RT-PCR (Real-time reverse transcriptase-polymerase chain reaction) is an accurate method among other methods and, thus, considered as the "gold standard" technique. Here, we outline the most extensively used analytical methods for diagnosing COVID-19, along with a brief description of each technique and its analytical aspects/perspective.

Single-cell transcriptomics reveal different maturation stages and sublineage commitment of human thymic invariant natural killer T cells.

Invariant natural killer T cells are a rare, heterogeneous T-cell subset with cytotoxic and immunomodulatory properties. During thymic development, murine invariant natural killer T cells go through different maturation stages differentiating into distinct sublineages, namely, invariant natural killer T1, 2, and 17 cells. Recent reports indicate that invariant natural killer T2 cells display immature properties and give rise to other subsets, whereas invariant natural killer T1 cells seem to be terminally differentiated. Whether human invariant natural killer T cells follow a similar differentiation model is still unknown. To define the maturation stages and assess the sublineage commitment of human invariant natural killer T cells during thymic development, in this study, we performed single-cell RNA sequencing analysis on human Vα24+Vß11+ invariant natural killer T cells isolated from thymocytes. We show that these invariant natural killer T cells displayed heterogeneity, and our unsupervised analysis identified 5 clusters representing different maturation stages, from an immature profile with high expression of genes important for invariant natural killer T cell development and proliferation to a mature, fully differentiated profile with high levels of cytotoxic effector molecules. Evaluation of expression of sublineage-defining gene sets revealed mainly cells with an invariant natural killer T2 signature in the most immature cluster, whereas the more differentiated ones displayed an invariant natural killer T1 signature. Combined analysis with a publicly available single-cell RNA sequencing data set of human invariant natural killer T cells from peripheral blood suggested that the 2 main subsets exist both in thymus and in the periphery, while a third more immature one was restricted to the thymus. Our data point to the existence of different maturation stages of human thymic invariant natural killer T cells and provide evidence for sublineage commitment of invariant natural killer T cells in the human thymus.

Timing of Exposure to ICU Diaries and Its Impact on Mental Health, Memories, and Quality of Life: A Double-Blind Randomized Control Trial.

Optimal time for ICU diary delivery and impact on mental health (MH), anxiety-depression, post-traumatic stress symptoms (PTSS), quality of life (QOL), and memories is unclear. We evaluated the effect of ICU diaries, dispatched at different time points, on outcomes in an Indian cohort. DESIGN: Double-blind randomized controlled trial. SETTING: A 1,000-bedded teaching hospital in East India. PATIENTS: Mechanically ventilated (>24 hr) adults were recruited, excluding those dead or incapable of meaningful-communication at discharge or follow-up. Eighty-three patients, aged 46.2 ± 17.2 years, Acute Physiology and Chronic Health Evaluation II scores 13.7 ± 4.9 were assessed. Length of ICU stay was 8.2 ± 7.1 days with 3.7 ± 3.2 ventilator days. INTERVENTION: Of 820 screened, 164 had diaries created. Including photographs, diaries were comaintained by healthcare workers and family members. Ninety patients were randomized at 1-month follow-up: diary sent to 45 at 1 month (group ID1) and to 45 at 3 months (ID3). MEASUREMENTS AND MAIN RESULTS: Anxiety-depression, memory, and QOL were assessed telephonically or home visits by a psychologist using the Hospital Anxiety-Depression Scale (HADS) and other tools at ICU discharge, 1-month (prerandomization), and 3 months of discharge. ID3 was reassessed after receiving diaries at 3.5 months. Primary outcome was anxiety-depression; secondary outcomes included PTSS, QOL, and memories. There was 100% follow-up. At 3 months, ID1 patients had a significant (p < 0.001) reduction in HADS from baseline when compared with ID3 that had not received diaries (4.16 ± 2.9 vs 2.15 ± 1.8; 95% CI, 2.8-1.2). PTSS scores were likewise better (p < 0.001). ID3 patients demonstrated significant improvement (p < 0.01) in QOL and memories along with HADS and PTSS when assessed at 3.5 months. CONCLUSIONS: ICU diaries improve MH but not QOL when delivered at 1 month and assessed 2 months thereafter. Assessed after 15 days, delayed exposure at 3 months significantly improved QOL and memories in addition to MH.

Immunoglobulin-G Antibodies against Severe Acute Respiratory Syndrome - Coronavirus-2 among Health-Care Workers: A Serosurveillance Study from India.

Background: Seropositivity among health-care workers (HCWs) may help in better understanding of the immune response after COVID-19 infection. Objectives: To estimate seropositivity among HCWs and to compare available variables with seropositivity to understand the factors affecting seropositivity. Materials and Methods: A serosurveillance among HCWs was carried out using population proportion sampling during the second half of October 2020 in the city of Ahmedabad using the Covid-Kavach (immunoglobulin G [IgG] ELISA Antibody testing kit). Simple proportions and appropriate statistical tests were used as needed. Results: As on October' 2020, HCWs in Ahmedabad demonstrated a seropositivity of 20.84% (95% confidence interval [CI] 19.00-22.81%). Seropositivity among HCWs was lower than that of the general population (24.20%) which was estimated as part of the same study. Female HCWs had higher seropositivity 22.14% (95% CI 19.74-24.74%) as compared to 18.82% (95% CI 16.06-21.93%) among male HCWs and the difference was statistically not significant (Z = 1.66, P = 0.097). Age groups with increasing age show increasing trend in the seropositivity among HCWs. Conclusion: As on October 2020, with 20.84% seropositivity among HCWs in Ahmedabad, one in every five HCW already demonstrate IgG antibodies against severe acute respiratory syndrome- coronavirus-2. Further scientific studies on seropositivity and the factors affecting the seropositivity may be carried out to uncover more details of immune reaction after COVID-19 infection.

ICU Memories and Patient Outcomes in a Low Middle-Income Country: A Longitudinal Cohort Study.

OBJECTIVES: To study memories of ICU following discharge, their associations, and impact on mental health and quality of life in a low- and middle-income country. DESIGN: Prospective observational cohort; data on memories (pain, fear, nightmare, factual), clinical and demographic variables, anxiety-depression, posttraumatic stress symptoms, and quality of life were collected 0, 7, 14, 30, 90, and 180 days post discharge. Home visits for assessment minimized loss to follow-up. Linear mixed-models and regression analyses were used to estimate adjusted effects of memories controlling for age, sex, time, and severity of illness. SETTING: Twenty-five bedded ICU of a tertiary care center in East India. PATIENTS: Adult ICU survivors between January 2017 and July 2018 able to communicate their memories. INTERVENTIONS: Not applicable. MEASUREMENTS AND MAIN RESULTS: Final sample consisted of 322 patients who completed 180 days follow-up. Pain, fear, factual, and nightmare memories dropped from 85%, 56%, 55%, and 45% at discharge to less than or equal to 5% at 180 days. Patients with gaps in ICU memory had worse anxiety-depression, posttraumatic stress symptoms, and quality of life at all follow-up points. Sedation (odds ratio, 0.54; CI, 0.4-0.7), steroids (odds ratio, 0.47; CI, 0.3-0.8), benzodiazepines (odds ratio, 1.74; CI, 1-3.04), and mechanical ventilation (odds ratio, 0.43; CI, 0.2-0.8) were independently associated with gaps in memory. Non-ICU factor such as substance addiction (odds ratio, 5.38; CI, 2-14) was associated with memories affecting mental health and quality of life. CONCLUSIONS: Gaps in memory and various memory types were common after ICU admission, whose prevalence waned over time. Compared with nightmares and fearful memories, gaps in memories were most strongly associated with poor mental health and quality of life. Identifying patients with gaps in memories might be an objective way of planning interventions to improve their long-term outcomes.

Prevalence and Psychosocial Correlations of COVID-19-Related Worries in People with Diabetes Mellitus Seeking Services from East Indian Tertiary Care Center: A Cross-Sectional Survey Results.

Context: Patients with diabetes are more prone to psychosocial problems which are known to adversely impact clinical outcomes of diabetes. COVID-19 is understood to further worsen the psychosocial problems of patients with diabetes. Aims: We carried out this cross-sectional telephonic survey of COVID-19-related worries in patients with diabetes mellitus to understand the prevalence and correlates of COVID-19-related worries. Settings and Design: This was a telephonic survey of patients seeking care from noncommunicable disease clinic of a tertiary care medical center. Subjects and Methods: We used a structured questionnaire to assess sociodemographic, clinical, psychological variables and COVID-19-related worries. Statistical Analysis Used: We used SPSS 20.0 for descriptive statistics keeping significance levels at 0.05. Between-group comparisons of continuous variables were made with independent t-test and two-way ANOVA; correlations were carried out with Pearson correlation test. Results: Two hundred and nine patients completed the telephonic survey conducted from September to November 2020. The prevalence of diabetes-related worries in our sample was 80%. Younger age (P < 0.001), unemployment (P = 0.029), and the presence of mental disorder (P < 0.001) were associated with higher diabetes-related worries. Poor glycemic control (0.008) and symptoms of COVID-19 (0.03) were associated with diabetes-related worries. Diabetes-related worries correlated with diabetes distress (ρ =0.441, P < 0.001), social isolation (ρ =0.401, P < 0.001), and perception of social support (ρ = -0.158, P < 0.001). Conclusions: A large proportion of our patients with diabetes are at high risk to experience COVID-19-related worries especially, younger people, unemployed and those with mental illness. Furthermore, the presence of diabetes distress and the perception of social isolation increase COVID-19 worries.

Altered chromatin landscape in circulating T follicular helper and regulatory cells following grass pollen subcutaneous and sublingual immunotherapy.

BACKGROUND: Allergen-specific immunotherapy is a disease-modifying treatment that induces long-term T-cell tolerance. OBJECTIVE: We sought to evaluate the role of circulating CXCR5+PD-1+ T follicular helper (cTFH) and T follicular regulatory (TFR) cells following grass pollen subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) and the accompanying changes in their chromatin landscape. METHODS: Phenotype and function of cTFH cells were initially evaluated in the grass pollen-allergic (GPA) group (n = 28) and nonatopic healthy controls (NAC, n = 13) by mathematical algorithms developed to manage high-dimensional data and cell culture, respectively. cTFH and TFR cells were further enumerated in NAC (n = 12), GPA (n = 14), SCIT- (n = 10), and SLIT- (n = 8) treated groups. Chromatin accessibility in cTFH and TFR cells was assessed by assay for transposase-accessible chromatin sequencing (ATAC-seq) to investigate epigenetic mechanisms underlying the differences between NAC, GPA, SCIT, and SLIT groups. RESULTS: cTFH cells were shown to be distinct from TH2- and TH2A-cell subsets, capable of secreting IL-4 and IL-21. Both cytokines synergistically promoted B-cell class switching to IgE and plasma cell differentiation. Grass pollen allergen induced cTFH-cell proliferation in the GPA group but not in the NAC group (P < .05). cTFH cells were higher in the GPA group compared with the NAC group and were lower in the SCIT and SLIT groups (P < .01). Time-dependent induction of IL-4, IL-21, and IL-6 was observed in nasal mucosa following intranasal allergen challenge in the GPA group but not in SCIT and SLIT groups. TFR and IL-10+ cTFH cells were induced in SCIT and SLIT groups (all, P < .01). ATAC-seq analyses revealed differentially accessible chromatin regions in all groups. CONCLUSIONS: For the first time, we showed dysregulation of cTFH cells in the GPA group compared to NAC, SCIT, and SLIT groups and induction of TFR and IL-10+ cTFH cells following SCIT and SLIT. Changes in the chromatin landscape were observed following allergen-specific immunotherapy in cTFH and TFR cells.

Intensive care unit (ICU) diaries and the experiences of patients' families: a grounded theory approach in a lower middle-income country (LMIC).

OBJECTIVE: An intensive care unit (ICU) diary is a relatively new concept in low middle-income countries (LMICs). Illiteracy and socio-cultural inhibitions may affect the use and utility of this intervention, which has proven beneficial to patients and their families in high income countries (HICs). We aimed to explore how families of ICU patients experienced ICU diaries in our set up by using the Grounded Theory (GT) approach. A relatively new research tool, this enables exploration of a phenomenon to build theories in areas hitherto uncharted. METHOD: A clinical psychologist did 29 in-depth interviews of relatives of 13 patients admitted in the ICU for > 24 h for whom an ICU diary was being maintained. We used a three-step coding process- open, axial, and selective coding, followed by the formulation of a theory embedded in the data. RESULTS: We found that the younger relatives of ICU patients accepted the idea better (age 30, SD 6) Half (48%) had education between 5th to 10th standards. Emergent themes suggested that for the family members, reading and writing the diary brought novelty, acted as a communication enabler, spiritual truss, and improved knowledge leading to change in perspective about the health care system. It also became a bridge to community bonding after patient discharge. Starting with appreciating the novelty of 'diary entries,' which was a new and exciting concept, family members used the diaries to communicate with health care workers (to gain information and understanding about the disease and treatment) and the patient to express their love and to maintain a connection. The diary acted as a confessional for hopes, fears, guilt, and faith for many members. As a tool, it enabled them to understand medical personnel as human beings and to appreciate their efforts, effectively improving confidence in the system. Finally, upon returning home, the diary was a crowd puller for extended family and neighbors encouraging discussions and enhancing bonding and information sharing. CONCLUSIONS: Our findings indicate a good acceptance of ICU diaries by family members in our ICU. With less literate, admitted 'shy 'members, in a society where 'diary writing' is not culturally rampant, the appreciation for the novel concept was universal. We see a place for these interventions not only at the patient/ family level but also as a means to 'correct' the image of health care workers in our society by humanizing ourselves to the end-user- the patient and his family.

Post traumatic stress symptoms, anxiety, and depression in patients after intensive care unit discharge - a longitudinal cohort study from a LMIC tertiary care centre.

BACKGROUND: Data on intensive care unit (ICU) related psychiatric morbidity from Low Middle-Income Countries are sparse. We studied the ICU related posttraumatic stress symptoms (PTSS), anxiety, and depression symptoms in a cohort of patients from Eastern India. METHODS: We included adults admitted more than 24 h to a mixed ICU. PTSS, anxiety, and depression symptoms were assessed by telephonic or face to face interviews by using the Impact of Events-r (IES-r) and Hospital anxiety and depression (HADS), respectively, at 0, 7,14, 30, 90 and 180 days from ICU discharge. The loss to follow up was minimal. Demographic, socioeconomic, quality of life (QOL), and critical care related variables were studied. RESULTS: Of 527 patients, 322 (59.4%) completed 6 months' follow up. The majority were male (60%), mechanically ventilated > 48 h (59.4%), mean age of 48 (+/- 16), mean acute physiology and chronic health evaluation II (APACHE II) at admission 9.4 (+/- 4.6), median length of stay 3 (2-28 days). The rates of ICU related clinical PTSS was < 1 and < 3% for anxiety/depression at any point of follow up. Data were analyzed by linear mixed (random effects) models. There was a significant drop in all scores and association with repeated measures over time. Poor QOL at discharge from the ICU showed significant association with PTSS, anxiety, and depression (ß = - 2.94, - 1.34, - 0.7 respectively) when corrected for gender and education levels. Younger age, greater severity of illness, and prior stressful life experiences predicted worse PTSS (ß = - 0.02, 0.08, 3.82, respectively). Benzodiazepines and lower sedation scores (better alertness) predicted lower depression symptoms. (ß = - 0.43, 0.37 respectively). CONCLUSION: ICU related psychiatric morbidity rates in our population are low compared with reported rates in the literature. Poor QOL at ICU discharge may predict worse long-term mental health outcomes. Further research on the impact of ICU and sociocultural factors on mental health outcomes in patients from different backgrounds is needed. The study was registered at CTRI/2017/07/008959.

Enhancing Data Reliability in TOMAHAQ for Large-Scale Protein Quantification.

The analytical scale of most mass-spectrometry-based targeted proteomics assays is usually limited by assay performance and instrument utilization. A recently introduced method, called triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantitation (TOMAHAQ), combines both peptide and sample multiplexing to simultaneously improve analytical scale and quantitative performance. In the present work, critical technical requirements and data analysis considerations for successful implementation of the TOMAHAQ technique based on the study of a total of 185 target peptides across over 200 clinical plasma samples are discussed. Importantly, it is observed that significant interference originate from the TMTzero reporter ion used for the synthetic trigger peptides. This interference is not expected because only TMT10plex reporter ions from the target peptides should be observed under typical TOMAHAQ conditions. In order to unlock the great promise of the technique for high throughput quantification, here a post-acquisition data correction strategy to deconvolute the reporter ion superposition and recover reliable data is proposed.

Laundry detergents and detergent residue after rinsing directly disrupt tight junction barrier integrity in human bronchial epithelial cells.

BACKGROUND: Defects in the epithelial barrier have recently been associated with asthma and other allergies. The influence of laundry detergents on human bronchial epithelial cells (HBECs) and their barrier function remain unknown. OBJECTIVE: We investigated the effects of laundry detergents on cytotoxicity, barrier function, the transcriptome, and the epigenome in HBECs. METHODS: Air-liquid interface cultures of primary HBECs from healthy control subjects, patients with asthma, and patients with chronic obstructive pulmonary disease were exposed to laundry detergents and detergent residue after rinsing. Cytotoxicity and epithelial barrier function were evaluated. RNA sequencing, Assay for Transposase Accessible Chromatin with high-throughput sequencing, and DNA methylation arrays were used for checking the transcriptome and epigenome. RESULTS: Laundry detergents and rinse residue showed dose-dependent toxic effects on HBECs, with irregular cell shape and leakage of lactate dehydrogenase after 24 hours of exposure. A disrupted epithelial barrier function was found with decreased transepithelial electrical resistance, increased paracellular flux, and stratified tight junction (TJ) immunostaining in HBECs exposed to laundry detergent at 1:25,000 dilutions or rinse residue at further 1:10 dilutions. RNA sequencing analysis showed that lipid metabolism, apoptosis progress, and epithelially derived alarmin-related gene expression were upregulated, whereas cell adhesion-related gene expression was downregulated by laundry detergent at 1:50,000 dilutions after 24 hours of exposure without substantially affecting chromatin accessibility and DNA methylation. CONCLUSION: Our data demonstrate that laundry detergents, even at a very high dilution, and rinse residue show significant cell-toxic and directly disruptive effects on the TJ barrier integrity of HBECs without affecting the epigenome and TJ gene expression.

The Use of Biomarkers to Predict Aero-Allergen and Food Immunotherapy Responses.

The incidence of allergic conditions has continued to rise over the past several decades, with a growing body of research dedicated toward the treatment of such conditions. By driving a complex range of changes in the underlying immune response, immunotherapy is the only therapy that modulates the immune system with long-term effects and is presently utilized for the treatment of several atopic conditions. Recent efforts have focused on identifying biomarkers associated with these changes that may be of use in predicting patients with the highest likelihood of positive clinical outcomes during allergen immunotherapy (AIT), providing guidance regarding AIT discontinuation, and predicting symptomatic relapse and the need for booster AIT after therapy. The identification of such biomarkers in food allergy has the additional benefit of replacing oral food challenges, which are presently the gold standard for diagnosing food allergies. While several markers have shown early promise, research has yet to identify a marker that can invariably predict clinical response to AIT. Skin prick testing (SPT) and specific IgE have commonly been used as inclusion criteria for the initiation of AIT and prediction of reactions during subsequent allergen challenge; however, existing data suggests that changes in these markers are not always associated with clinical improvement and can be widely variable, reducing their utility in predicting clinical response. Similar findings have been described for the use of allergen-specific functional IgG4 antibodies, basophil activation and histamine release, and type 2 innate lymphoid cells. There appears to be a promising association between changes in the expression of dendritic cell-associated markers, as well as the use of DNA promoter region methylation patterns in the prediction of allergy status following therapy. The cellular and molecular changes brought about by immunotherapy are still under investigation, but major strides in our understanding are being made.

Food allergy and omics.

Food allergy (FA) prevalence has been increasing over the last few decades and is now a global health concern. Current diagnostic methods for FA result in a high number of false-positive results, and the standard of care is either allergen avoidance or use of epinephrine on accidental exposure, although currently with no other approved treatments. The increasing prevalence of FA, lack of robust biomarkers, and inadequate treatments warrants further research into the mechanism underlying food allergies. Recent technological advances have made it possible to move beyond traditional biological techniques to more sophisticated high-throughput approaches. These technologies have created the burgeoning field of omics sciences, which permit a more systematic investigation of biological problems. Omics sciences, such as genomics, epigenomics, transcriptomics, proteomics, metabolomics, microbiomics, and exposomics, have enabled the construction of regulatory networks and biological pathway models. Parallel advances in bioinformatics and computational techniques have enabled the integration, analysis, and interpretation of these exponentially growing data sets and opens the possibility of personalized or precision medicine for FA.

Cancellous osteoma of maxilla: A rare case report.

Osteoma is a slow growing benign tumor result from either cancellous or compact bone proliferation. Osteoma of the jaws may arise from the surface of the bone as a sessile mass located either peripherally or endosteally. This paper presents a rare case report of peripheral cancellous osteoma, involving the buccal cortical plate and alveolar process of the right maxilla of a 37-year-old female patient.

Defective sphingosine 1-phosphate receptor 1 (S1P1) phosphorylation exacerbates TH17-mediated autoimmune neuroinflammation.

Sphingosine 1-phosphate (S1P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. The sphingosine phosphate receptor 1 (S1P1) agonist FTY-720 (Gilenya) arrests immune trafficking and prevents multiple sclerosis (MS) relapses. However, alternative mechanisms of S1P-S1P1 signaling have been reported. Phosphoproteomic analysis of MS brain lesions revealed S1P1 phosphorylation on S351, a residue crucial for receptor internalization. Mutant mice harboring an S1pr1 gene encoding phosphorylation-deficient receptors (S1P1(S5A)) developed severe experimental autoimmune encephalomyelitis (EAE) due to autoimmunity mediated by interleukin 17 (IL-17)-producing helper T cells (TH17 cells) in the peripheral immune and nervous system. S1P1 directly activated the Jak-STAT3 signal-transduction pathway via IL-6. Impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.

Human neonatal naive CD4+ T cells have enhanced activation-dependent signaling regulated by the microRNA miR-181a.

Compared with older children and adults, human neonates have reduced and delayed CD4(+) T cell immunity to certain pathogens, but the mechanisms for these developmental differences in immune function remain poorly understood. We investigated the hypothesis that impaired human neonatal CD4(+) T cell immunity was due to reduced signaling by naive CD4(+) T cells following engagement of the αß-TCR/CD3 complex and CD28. Surprisingly, calcium flux following engagement of CD3 was significantly higher in neonatal naive CD4(+) T cells from umbilical cord blood (CB) compared with naive CD4(+) T cells from adult peripheral blood. Enhanced calcium flux was also observed in adult CD4(+) recent thymic emigrants. Neonatal naive CD4(+) T cells also had higher activation-induced Erk phosphorylation. The microRNA miR-181a, which enhances activation-induced calcium flux in murine thymocytes, was expressed at significantly higher levels in CB naive CD4(+) T cells compared with adult cells. Overexpression of miR-181a in adult naive CD4(+) T cells increased activation-induced calcium flux, implying that the increased miR-181a levels of CB naive CD4(+) T cells contributed to their enhanced signaling. In contrast, AP-1-dependent transcription, which is downstream of Erk and required for full T cell activation, was decreased in CB naive CD4(+) T cells compared with adult cells. Thus, CB naive CD4(+) T cells have enhanced activation-dependent calcium flux, indicative of the retention of a thymocyte-like phenotype. Enhanced calcium signaling and Erk phosphorylation are decoupled from downstream AP-1-dependent transcription, which is reduced and likely contributes to limitations of human fetal and neonatal CD4(+) T cell immunity.