Reverse Transcriptase-polymerase Chain Reaction Assay Research Articles

Omicron variants of SARS-CoV-2: Epidemiological and clinical insights from a tertiary care center in Saudi Arabia

BackgroundThe actual burden of the Omicron variants remains unclear. Therefore, this study aims to analyze the epidemiological and clinical features of Omicron-infected patients and investigate factors influencing hospital admission. MethodsThis retrospective single-center study included individuals with positive SARS-CoV-2 infection, specifically the Omicron variants (XBB, EG or JN), identified through real-time reverse-transcriptase polymerase chain reaction assays from January 2022 to December 2023. ResultsA total of 305 Omicron-infected patients were included; (53.11 %) were females and (46.89 %) were males, with a median age of 39 years [interquartile range (IQR): 30, 53]. Underlying diseases, including endocrine/metabolic disorders (22.30 %), hypertension (12.79 %), chronic respiratory disease (10.49 %), and malignancy (9.18 %) were prevalent, while (40.98 %) were medically free. The XBB variant was predominant (73.11 %), followed by JN (20.33 %), and EG variant (6.56 %). The seasonality analysis demonstrates XBB variants' domination in 2022, with a surge to 40 cases in December. The trend continued in 2023, peaking at 76 XBB cases in March. May 2023 reported 38 XBB cases and the emergence of 17 EG instances. Notably, in December, only one XBB case was reported, and 62 instances emerged with the JN variant. Overall, 233 out of 305 cases were reported during flu season (September to March) (76.39 %). Moreover, hospitalization occurred in (16.39 %), with a (1.31 %) mortality rate (all deaths in the JN variant). Multivariable analysis confirmed renal disease, chronic respiratory disease, endocrine/metabolism issues, and polymicrobial infection as positive predictors of hospitalization (p < 0.05). While COVID-19 vaccination significantly reduced hospitalization odds (Odds Ratio: 0.20, p = 0.001). ConclusionsThese findings contribute valuable insights into Omicron epidemiology and factors influencing hospitalization. The dynamic fluctuations in Omicron variants, particularly XBB, EG, and JN, over 2022 and 2023, with JN emerging as the dominant circulating variant globally, underscore the need for continuous vigilance and urgency for updated vaccine formulations.

Evolving Epidemiology of Pediatric Respiratory Syncytial Virus (RSV) Cases Around COVID-19 Pandemic: Impact and Clinical Insights, Retrospective Cohort Study.

The burden of respiratory syncytial virus (RSV) in high-risk pediatric patients remains unclear. Therefore, this study aims to characterize pediatric RSV cases from January 2019 to December 2022 and assess the impact of the COVID-19 pandemic on RSV burden and RSV-related outcomes. In addition, examining factors influencing RSV-related hospitalization. This is a retrospective study that included pediatric patients (aged 14 and below) who presented at King Faisal Specialist Hospital and Research Centre (KFSHRC) in Riyadh, Saudi Arabia with RSV infection identified using real-time reverse-transcriptase polymerase chain reaction assays. Statistical analyses were performed using STATA. A total of 885 RSV cases were reported; (56.05%) were males and (43.95%) were females with a median age of 24 months [interquartile range (IQR): 11-60]. 534 (60.34%) required hospitalization. As for RSV seasonality, there was a significant increase in RSV prevalence following the COVID-19 pandemic, escalating from 205 cases in 2019 to 425 cases in 2022. The increase in 2022 was evident in January and persisted from September to December, reaching its peak during the months of October (20.70% - 88 cases) and November (32.00% - 136 cases). About (27.12%) of RSV infected children were medically free patients. Symptomatic patients exhibited various clinical manifestations, with ventilation necessary in (13.11%) of cases. Further analysis revealed significant changes in RSV-related outcomes post-COVID-19, including a decrease in hospitalization rates, an increase in medically free patients, and a lower need for ventilation (p < 0.05). Notably, a significant proportion of RSV admissions occurred within the first 6 months of life, with (77.69%) in the age group of 0 to 5 months. In addition, previous RSV infection, prematurity, low birth weight, renal disease, congenital heart disease, endocrine/metabolic disease, neuro/neuromuscular diseases, and genetic disorders were positively associated with hospitalization (P < 0.05). Interestingly, asthma and bone marrow transplantation were negatively associated with hospitalization (P < 0.05). The mortality rate in this study is (2.37%) (21/885). This study provides a comprehensive understanding of the demographic and clinical factors influencing RSV outcomes, highlighting the impact of the COVID-19 pandemic and shedding light on potential risk factors for RSV-related hospitalization. The highest prevalence of RSV during (September to January), aligning with global patterns and emphasizing the importance of timing in preventive strategies.

Nationwide data from comprehensive genomic profiling assays for detecting driver oncogenes in non-small cell lung cancer.

Driver oncogenes are investigated upfront at diagnosis using multi-CDx systems with next-generation sequencing techniques or multiplex reverse-transcriptase polymerase chain reaction assays. Additionally, from 2019, comprehensive genomic profiling (CGP) assays have been available in Japan for patients with advanced solid tumors who had completed or were expected to complete standard chemotherapy. These assays are expected to comprehensively detect the driver oncogenes, especially for patients with non-small cell lung cancer (NSCLC). However, there are no reports of nationwide research on the detection of driver oncogenes in patients with advanced NSCLC who undergo CGP assays, especially in those with undetected driver oncogenes at diagnosis. In this study, we investigated the proportion of driver oncogenes detected in patients with advanced NSCLC with undetectable driver oncogenes at initial diagnosis and in all patients with advanced NSCLC who underwent CGP assays. We retrospectively analyzed data from 986 patients with advanced NSCLC who underwent CGP assays between August 2019 and March 2022, using the Center for Cancer Genomics and Advanced Therapeutics database. The proportion of driver oncogenes newly detected in patients with NSCLC who tested negative for driver oncogenes at diagnosis and in all patients with NSCLC were investigated. Driver oncogenes were detected in 451 patients (45.7%). EGFR was the most common (16.5%), followed by KRAS (14.5%). Among the 330 patients with undetected EGFR, ALK, ROS1, and BRAF V600E mutations at diagnosis, 81 patients (24.5%) had newly identified driver oncogenes. CGP assays could be useful to identify driver oncogenes in patients with advanced NSCLC, including those initially undetected, facilitating personalized treatment.

RET Fusion Testing in Patients With NSCLC: The RETING Study

IntroductionRET inhibitors with impressive overall response rates are now available for patients with NSCLC, yet the identification of RET fusions remains a difficult challenge. Most guidelines encourage the upfront use of next-generation sequencing (NGS), or alternatively, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) when NGS is not possible or available. Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in patients with advanced NSCLC. MethodsThis situation prompted us to evaluate several RET assays in a large multicenter cohort of RET fusion–positive NSCLC (n = 38) to obtain real-world data. In addition to RNA-based NGS (the criterion standard method), all positive specimens underwent break-apart RET FISH with two different assays and were also tested by an RT-PCR assay. ResultsThe most common RET partners were KIF5B (78.9%), followed by CCDC6 (15.8%). The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET(12) fusion. The three RET fusions not identified with RT-PCR were AKAP13(35)-RET(12), KIF5B(24)-RET(9) and KIF5B(24)-RET(11). All three false-negative RT-PCR cases were FISH-positive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays. Signet ring cells, psammoma bodies, and pleomorphic features were frequently observed (in 34.2%, 39.5%, and 39.5% of tumors, respectively). ConclusionsIn-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for the rapid and effective detection of RET fusions in patients with NSCLC. The likelihood of RET false-negative results with both FISH and RT-PCR reinforces the need for upfront NGS in patients with NSCLC.

Kilohertz high-frequency electrical stimulation ameliorate hyperalgesia by modulating transient receptor potential vanilloid-1 and N-methyl-D-aspartate receptor-2B signaling pathways in chronic constriction injury of sciatic nerve mice.

The number of patients with neuropathic pain is increasing in recent years, but drug treatments for neuropathic pain have a low success rate and often come with significant side effects. Consequently, the development of innovative therapeutic strategies has become an urgent necessity. Kilohertz High Frequency Electrical Stimulation (KHES) offers pain relief without inducing paresthesia. However, the specific therapeutic effects of KHES on neuropathic pain and its underlying mechanisms remain ambiguous, warranting further investigation. In our previous study, we utilized the Gene Expression Omnibus (GEO) database to identify datasets related to neuropathic pain mice. The majority of the identified pathways were found to be associated with inflammatory responses. From these pathways, we selected the transient receptor potential vanilloid-1 (TRPV1) and N-methyl-D-aspartate receptor-2B (NMDAR2B) pathway for further exploration. Mice were randomly divided into four groups: a Sham group, a Sham/KHES group, a chronic constriction injury of the sciatic nerve (CCI) group, and a CCI/KHES stimulation group. KHES administered 30 min every day for 1week. We evaluated the paw withdrawal threshold (PWT) and thermal withdrawal latency (TWL). The expression of TRPV1 and NMDAR2B in the spinal cord were analyzed using quantitative reverse-transcriptase polymerase chain reaction, Western blot, and immunofluorescence assay. KHES significantly alleviated the mechanical and thermal allodynia in neuropathic pain mice. KHES effectively suppressed the expression of TRPV1 and NMDAR2B, consequently inhibiting the activation of glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule 1 (IBA1) in the spinal cord. The administration of the TRPV1 pathway activator partially reversed the antinociceptive effects of KHES, while the TRPV1 pathway inhibitor achieved analgesic effects similar to KHES. KHES inhibited the activation of spinal dorsal horn glial cells, especially astrocytes and microglia, by inhibiting the activation of the TRPV1/NMDAR2B signaling pathway, ultimately alleviating neuropathic pain.

A Sanger approach based on overlapping fragments to screen SARS-CoV-2 variants

Since the beginning of the COVID-19 pandemic, the SARS-CoV-2 virus has undergone various genetic mutations which have led to the emergence of variants. The World Health Organization (WHO) defines Variants of Concern (VOCs) and Variants of Interest (VOIs) according to several criteria. These include significant changes in the transmissibility and pathogenicity of the virus characterized by mutations in the spike gene coding the spike glycoprotein. In this study, we designed ten Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) assays in order to identify mutations of SARS-CoV-2 in overlapping fragments. Each assay contained mutations on the fragments sequenced by a Sanger method. The genomic analysis of the fragments allowed to identify the variant according to the position of the mutations. The assembly of the 10 fragments refined the analysis, highlighting all the mutations present in the S gene. Finally, a comparison of methods using a Next-Generation Sequencing (NGS) approches for samples enabled the method to be validated. By this method we have highlighted a characteristic mutation of the lineage B of SARS-CoV-2. We showed the circulation of SARS-CoV-2 belonging to lineage A and B in the beginning of the pandemic in Gabon. We have identified the Alpha, Delta and Omicron variants. This method would allow laboratories with limited financial means or without NGS instrument to obtain sequences of the S gene. This method wase very effective to highlight the circulation of variants, in particular VOCs and VOIs, in this developing country, Gabon, during the COVID-19 pandemic.

A study to assess the impact of the cobas point-of-care RT-PCR assay (SARS-CoV-2 and Influenza A/B) on patient clinical management in the emergency department of the University of California at Davis Medical Center.

Rapid detection of SARS-CoV-2 is crucial for reduction of transmission and clinical decision-making. Several rapid (<30min) molecular point-of-care (POC) tests based on nucleic acid amplification exist for diagnosis of SARS-CoV-2 & Influenza A/B infections. This unblinded, pre-post study enrolled consecutive patients with symptoms/signs consistent with SARS-CoV-2 infection presenting to the University of California, Davis emergency department (ED). Outcomes following implementation of the cobas® SARS-CoV-2 & Influenza A/B test for use on the cobas Liat System (intervention: December 2020-May 2021) were compared with previous standard-of-care using centralized laboratory reverse transcriptase polymerase chain reaction (RT-PCR) methods (control: April 2020-October 2020). Electronic health records of 8879 symptomatic patient visits were analyzed, comprising 4339 and 4540 visits and 538 and 638 positive SARS-CoV-2 PCR test results in the control and intervention periods, respectively. Compared with the control period, turnaround time (TAT) was shorter in the intervention period (median 0.98vs 12.30h; p<0.0001). ED length of stay (LOS) was generally longer in the intervention period compared with the control period, but for those SARS-CoV-2-negative who were admitted, ED LOS was shorter (median 12.53vs 17.93h; p<0.0001). The rate of antibiotic prescribing was lower in the intervention than in the control period (42.86% vs 49.16%; p<0.0001) and antiviral prescribing was higher (7.64% vs 5.49%; p<0.0001). This real-world study confirms faster TAT with a POC RT-PCR method in an emergency care setting and highlights the importance of rapid SARS-CoV-2 detection to aid patient management and inform treatment decisions.

A-259 Detecting Human RNase P Gene Prevents False-Negative of SARS-CoV-2

Abstract Background Real-time Reverse transcriptase—polymerase chain reaction (RT-PCR) is a regular technique for detecting SARS-CoV-2 infection. Human RNase P sequence is using as internal control for PCR assay usually. This article discusses the correlation between SARS-CoV-2 PCR result and human RNase P gene result. Methods Nasopharyngeal swabs were collected from two patients within three consecutive days. We detected for SARS-CoV-2 target gene and human RNase P gene by using real-time RT-PCR assay, which SARS-CoV-2 target gene includes E gene, Rarp gene and N gene . And the threshold cycle values (Ct) were determined by cobas® z 480. Evaluate the results of SARS-CoV-2 and RNase P gene detection. All samples were also analysis for SARS-CoV-2 infection in Taiwan Centers for Disease Control. Results Results of SARS-CoV-2 PCR assay were consistent in both laboratories. SARS-CoV-2 target genes were detected from specimens of both patients on day1. Ct values of E gene were 29.63 and 29.50. Ct values of Rdrp gene were 32.23 and 32.50. Ct values of N gene were 33.09 and 32.68. Ct values of RNase P gene were 21.21 and 21.22. On day 2 and 3, all specimens were negative detected for SARS-CoV-2 PCR. Ct values of RNase P gene were 25.73 and 26.16 on day 2 and 29.82 and 30.28 on day 3. Conclusion The Ct values of human RNase P gene detection can be correlated with the numbers of collected cells. Comparing the results of two patients for three consecutive days, the Ct value of human RNase P gene related to SARS-CoV-2 PCR result. Collection technique can be a factor to affect Ct value of human RNase P gene detection. Develop acceptance range for Ct value of RNase P gene detection can be a quality standard for sample collection to prevent false-negative of SARS-CoV-2.

Expression Kinetics of Vitellogenin Gene in Estradiol-17β Induced &lt;i&gt;Catla catla&lt;/i&gt;

Vitellogenin (Vtg) is the yolk precursor protein in fish and is synthesized in the liver under estradiol-17β (E2) stimulation. In all oviparous animals, Vtg is synthesized as a response to endogenous estrogen in females in the breeding season. It also could be induced exogenously by E2 in males and juveniles. In the current study the inducibility of Vtg gene in Indian major carp, Catla catla was tested. A semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for kinetic study of Vtg mRNA level in C. catla after primary and secondary inductions by E2. It was found that the level of Vtg-specific mRNA was highest following 12 h post treatment in primary induction, and after 1 d post treatment in secondary induction. There was a sharp decrease in Vtg mRNA level on day one and day two in primary and secondary induction, respectively. Thus the ‘memory effect’ of vitellogenin induction by E2 in catla could not be confirmed.

Identification of hub genes for glaucoma: a study based on bioinformatics analysis and experimental verification.

To explore hub genes for glaucoma based on bioinformatics analysis and an experimental model verification. In the Gene Expression Omnibus (GEO) database, the GSE25812 and GSE26299 datasets were selected to analyze differentially expressed genes (DEGs) by the GEO2R tool. Through bioinformatics analysis, 9 hub genes were identified. Receiver operating characteristic (ROC) curves and principal component analysis (PCA) were performed to verify whether the hub gene can distinguish glaucoma from normal eyes. The mouse model of glaucoma was constructed, and the real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) assay was performed to detect the expression levels of hub genes in glaucoma. There were 128 overlapping DEGs in the GSE25812 and GSE26299 datasets, mainly involved in intracellular signalling, cell adhesion molecules and the Ras signalling pathway. A total of 9 hub genes were screened out, including GNAL, BGN, ETS2, FCGP4, MAPK10, MMP15, STAT1, TSPAN8, and VCAM1. The area under the curve (AUC) values of 9 hub genes were greater than 0.8. The PC1 axle could provide a 70.5% interpretation rate to distinguish glaucoma from normal eyes. In the ocular tissues of glaucoma in the mice model, the expression of BGN, ETS2, FCGR4, STAT1, TSPAN8, and VCAM1 was increased, while the expression of GNAL, MAPK10, and MMP15 was decreased. Nine hub genes in glaucoma are identified, which may provide new biomarkers and therapeutic targets for glaucoma.

Detection of infectious bronchitis virus in chicken in Kerala by real time Taqman RT-PCR assay

Chickens with signs of respiratory infection were screened for the presence of infectious bronchitis virus (IBV). Tissue samples were collected from dead and ailing chickens and were propagated intra-allantoically in embryonated chicken eggs and the allantoic fluid was harvested. The tissue samples and harvested allantoic fluid were probed with reverse transcriptase polymerase chain reaction (RT-PCR) with primers targeting 5’UTR of IBV. A total of 95 samples were tested, by RT-PCR. A Taqman probe labelled real time PCR assay targeting the 5’ UTR and Nucleocapsid (N) gene of IBV was standardised for the detection of IBV in all the 95 tissue and allantoic fluid samples to assess its efficiency. The real time PCR could detect IBV in all the 95 allantoic fluid and tissue samples including the samples, which were negative in preliminary detection. Hence, in the present study, the real time PCR assay was found to have equal efficacy on clinical samples and allantoic fluid and with a sensitivity of 100% and specificity of 90.90% in comparison with the conventional RT-PCR assay.

Prognostic role of intermountain risk score (IMRS) in intensive care unit patients with a diagnosis of COVID-19.

In this study, we aimed to assess the predictive value of Intermountain Risk Score (IMRS) in intensive care unit (ICU) patients with COVID-19. Our retrospective study included the data of 194 patients who were admitted to the COVID-19 ICU of a tertiary care center. COVID-19 diagnoses were made by a positive result from a real-time reverse-transcriptase (RT) polymerase chain reaction (PCR) assay of nasal and pharyngeal swab specimens. Patients who had negative RT-PCR results or who were not admitted to ICU and patients under 18 years old were excluded from the study. Complete blood count, biochemistry panel, and blood gas analysis results were gathered and compiled. 194 ICU patients with COVID-19 (PCR positive) were included in the study. The patients were divided into two groups according to IMRS (if IMRS was <15 in women and <17 in men, patients were included in the non-high-risk group, while patients with IMRS ≥15 in women and ≥17 in men were defined as a high-risk group). Multivariate regression analysis was performed to predict in-hospital mortality. The IMRS [OR: 1.17 (1.08-1.27) p<0.001)] was found to predict in-hospital mortality. In this study, we showed that the IMRS score at admission can predict in-hospital mortality in intensive care unit patients with a diagnosis of COVID-19.

The first study on clinicopathological changes in cats with feline infectious peritonitis with and without retrovirus coinfection.

Feline infectious peritonitis (FIP) is an infectious, immune-mediated, and fatal disease in cats caused by a mutant feline coronavirus (FCoV) infection. Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are two common retroviruses that play a role in reducing feline immune function with opportunistic retrovirus infection being a predisposing factor for the development of FIP. This study aimed to evaluate the clinicopathological parameters of FIP in cats with and without retrovirus coinfection. In total, 62 cats presenting with pleural and/or peritoneal effusion at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand, were selected for the study. Effusion samples were collected and a reverse transcriptase-polymerase chain reaction (RT-PCR) assay was performed on all samples using the 3' untranslated region primer. All FCoV-positive cats were tested for retrovirus infection using a commercial kit (Witness FeLV-FIV [Zoetis]; United States). Clinical signs, hematological, and biochemical parameters of these cats were investigated and grouped. Of the 62 cats with pleural and/or peritoneal effusion, FCoV was detected in 32, of which 21 were highly suspicious for FIP. The cats suspected of FIP were divided into three subgroups following viral detection. A total of 14 had only FCoV infection (Group A), four had FCoV and FeLV infection (Group B), and three had FCoV, FeLV, and FIV infection (Group C). Of the rest, 11 had definitive diagnoses, which included three being FCoV and FeLV-positive (Group D), and eight were retrovirus-negative (Group E). Mild anemia and lymphopenia were found in cats infected with these three viruses. An albumin-to-globulin ratio lower than 0.5 was found in FIP cats with only FCoV infection. Typically, cats with clinical effusion and FIP, with and without retrovirus coinfection, had similar hematological findings. Clinical signs, blood parameters, fluid analysis with cytological assessment, and RT-PCR assays could identify better criteria to diagnose FIP with and without retrovirus coinfection.

Molecular Epidemiology and Phylogenetic Analysis of Peste des Petits Ruminants Virus Circulating in Sheep in Bangladesh

Peste des petits ruminants (PPR) is a viral disease of small ruminants that is highly contagious, severe, reportable, and economically important. The present study was conducted to detect the PPR virus (PPRV) circulating in sheep in Bangladesh to determine its association with epidemiological risk factors and the degree of relationship between the F and H genes of the PPRV of sheep with those of other sheep and goat isolates. A cross-sectional study was conducted in five selected districts of Bangladesh to collect data on locations, ecological zones, breeds, age, sex, sources, time period, and farming systems using a structured questionnaire accompanied by face-to-face interviews. During sampling, 250 nasal swab samples were collected from live sheep with the typical clinical signs of PPR. Thereafter, a reverse-transcriptase polymerase chain reaction (RT-PCR) assay was employed to detect PPRV using the F and H genes. Risk factors were determined using bivariable and multivariable logistic regression analyses. Phylogenetic analysis of the detected PPRV was performed using MEGA software after sequencing both F and H genes. Using RT-PCR, 35.6% (89/250, 95% CI: 29.6%–41.6%) of the samples were found to be positive for PPRV. Locations, breeds, sources, and feeding systems were identified as potential molecular epidemiological risk factors for PPRV infection in a multivariate logistic regression model. Nucleotide sequencing and phylogenetic analysis showed that the PPRV strain was genetically related to the lineage IV virus isolates. For the F gene, the sequence divergence of our gene and other selected genes ranged from 0.01% to 0.018% within lineage IV, and the similarity ranged from 98.2% to 99.0%. In the case of the H gene, similar results were also observed in divergence, ranging from 0.017% to 0.083% among lineage IV and others, and similarity varied from 91.7% to 98.3%. To the best of our knowledge, this is the first study in Bangladesh conducted to determine the RT-PCR-based molecular epidemiology of PPRV in sheep. This study highlights the importance of establishing successful interventions for managing PPRV infections in small ruminants in Bangladesh.

Health-related quality of life of the adult COVID-19 patients following one-month illness experience since diagnosis: Findings of a cross-sectional study in Bangladesh.

The coronavirus disease 2019 (COVID-19) stances an incredible impact on the quality of life and denigrates the physical and mental health of the patients. This cross-sectional study aimed to assess the health-related quality of life (HRQOL) of COVID-19 patients. We conducted this study at the National Institute of Preventive and Social Medicine (NIPSOM) of Bangladesh for the period of June to November 2020. All the COVID-19 patients diagnosed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay in July 2020 formed the sampling frame. The study enrolled 1204 adult (aged >18 years) COVID-19 patients who completed a one-month duration of illness after being RT-PCR positive. The patients were interviewed with the CDC HRQOL-14 questionnaire to assess HRQOL. Data were collected by telephone interview on the 31st day of being diagnosed and by reviewing medical records using a semi-structured questionnaire and checklist. Around two-thirds (72.3%) of the COVID-19 patients were males and a half (50.2%) were urban residents. In 29.8% of patients, the general health condition was not good. The mean (±SD) duration of physical illness and mental illness was 9.83(±7.09) and 7.97(±8.12) days, respectively. Most of the patients (87.0%) required help with personal care, and 47.8% required assistance with routine needs. The mean duration of 'healthy days' and 'feeling very healthy' was significantly lower in patients with increasing age, symptoms, and comorbidity. The mean duration of 'usual activity limitation', 'health-related limited activity', 'feeling pain/worried', and 'not getting enough rest' were significantly higher among patients' having symptoms and comorbidity. 'Not so good' health condition was significantly higher in females (OR = 1.565, CI = 1.01-2.42) and those having a symptom (OR = 32.871, CI = 8.06-134.0) of COVID-19 and comorbidity (OR = 1.700, CI = 1.26-2.29). Mental distress was significantly higher among females (OR = 1.593, CI = 1.03-2.46) and those having a symptom (OR = 4.887, CI = 2.58-9.24). Special attention should be given to COVID-19 patients having symptoms and comorbidity to restore their general health, quality of life, and daily activities.

Low concentrations of tumor necrosis factor-alpha promote human periodontal ligament stem cells osteogenic differentiation by activation of autophagy via inhibition of AKT/mTOR pathway.

Tumor necrosis factor-alpha (TNF-α) is one of the crucial inflammatory factors in alveolar bone metabolism during the process of periodontitis. Autophagy is indispensable for proper osteoblast function. However, the effects of autophagy on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in inflammatory microenvironment and the underlying mechanisms remain to be clarified. The aim of the present study was to investigate whether autophagy participates in hPDLSCs differentiation after treated with TNF-α and explore the underlying mechanisms. Characterizations of hPDLSCs were evaluated by Alizarin-red S staining, Oil red staining and flow cytometry. hPDLSCs were treated with various concentrations of TNF-α. Rapamycin or 3MA was used to achieve or inhibit autophagy activation. AKT signaling was inhibited using ARQ092. Cell proliferation was evaluated using Cell Counting Kit-8 (CCK8) assay. Real-time reverse transcriptase-polymerase chain reaction assay (RT-PCR), western blot, alkaline phosphatase (ALP) staining and Alizarin Red S staining were applied to evaluate levels of osteogenic differentiation and autophagy. CCK8 showed that low concentrations of TNF-α had no influence on cell proliferation, while high concentrations of TNF-α inhibited proliferation. Low concentrations of TNF-α promoted osteogenic differentiation and autophagy, while high concentrations of TNF-α inhibited osteogenic differentiation and autophagy in hPDLSCs. The levels of osteogenic differentiation in hPDLSCs were partly effected after co-incubation with 0.1 ng/mL TNF-α with 3MA or Rapamycin. ARQ092 enhanced 0.1 ng/mL TNF-α-induced ALP expression and mineral nodule formation. Low concentrations of TNF-α promote hPDLSCs osteogenic differentiation by activation of autophagy via inhibition of AKT/mTOR signaling.

A novel missense ALMS1 variant causes aberrant splicing identified in a cohort of patients with Alström syndrome.

Purpose: Alström syndrome (AS) is a rare autosomal recessive disorder caused by variants of ALMS1. The objectives of this study were to describe the clinical and genetic characteristics of 19 Chinese patients with biallelic variants in ALMS1. Methods: We recruited 19 probands with biallelic disease-causing ALMS1 variants. All patients underwent ophthalmic and systematic evaluations and comprehensive molecular genetic analysis. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays were performed to observe the effect of a novel missense variant on ALMS1 pre-mRNA splicing. Results: We identified 33 causative variants in ALMS1, including 15 frameshift small indels, 14 non-sense variants, two gross deletions, one splicing variant, and one missense variant. RT-PCR showed that the missense variant c.9542G>A (p.R3181Q) altered pre-mRNA splicing to generate a truncated protein p. (Ser3082Asnfs*6). Retinal dystrophy (RD) was noted in all the patients, followed by metabolism disturbance (obesity or acanthosis nigricans) in 66.7% and hearing impairment in 61.1% of the patients. Patient systemic symptom numbers and their age at evaluation showed a significant positive correlation, and BCVA and age at the last examination showed a moderate correlation. All patients exhibited early-onset RD and severe visual impairment. The exception was one patient carrying homozygous p. R3181Q, who showed a mild visual defect and atypical retinal phenotype. Conclusion: Our findings expand the pathogenic variant spectrum of ALMS1 and provide the first verification of a novel missense variant caused AS by aberrant pre-mRNA splicing. Patients with AS might demonstrate varied clinical spectra; therefore, genetic analysis is vital for the early and accurate diagnosis of patients with atypical AS.

Urinary Mitochondrial Deoxyribonucleic Acid: A Novel Biomarker of Coronavirus Disease 2019-associated Acute Kidney Injury in Renal Transplant Recipients

Abstract Background: Acute kidney injury (AKI) is one of the common manifestations of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. An early identification of AKI is of paramount importance to modulate the management of AKI, limiting the severity, avoiding nephrotoxic medicines, and modifying the drug dose depending on renal function. Aim: To determine the utility of urinary mitochondrial deoxyribonucleic acid (umt-DNA) and neutrophil gelatinase-associated lipocalin (NGAL) in predicting coronavirus disease 2019 (COVID-19)-associated AKI and mitochondrial stress. Materials and Methods: Live-related renal transplant recipients (RTRs) (n = 66), who acquired SARS-CoV-2 infection and were admitted to a COVID hospital were included and subclassified into AKI (n = 19) with &gt; 1.5-fold rise in serum creatinine level from the pre-COVID-19 serum creatinine level, and non-AKI (n = 47) whose serum creatinine value remained stable, or a rise of &lt;1.5-fold of the baseline values of pre-COVID-19. A 50 mL urine sample was collected and urinary mt-DNA and NGAL was determined by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays methods. Results: Both the urinary mitochondrial gene NADH dehydrogenase (ND-1) and NGAL level were significantly higher in the AKI group compared to non-AKI. The mean ND-1 gene Ct in the AKI group was (19.44 ± 2.58 a.u) compared to non-AKI (21.77 ± 3.60; P = 0.013). The normalized ND-1 gene cycle of threshold (Ct) in AKI was 0.79 ± 0.11 a.u compared to non-AKI (0.89 ± 0.14; P = 0.007). The median urinary NGAL level in AKI group was (453.53; range, 320.22–725.02, 95% confidence interval [CI]) ng/mL compared to non-AKI (212.78; range, 219.80–383.06, 95% CI; P = 0.015). The median urine creatinine normalized uNGAL was 4.78 (0.58–70.39) ng/mg in the AKI group compared to 11.26 ng/mg (0.41–329.71) in the non-AKI group. The area under curve of ND-1 gene Ct was 0.725, normalized ND-1 Ct was 0.713, uNGAL was 0.663, and normalized uNGAL was 0.667 for detecting the AKI and mitochondrial stress. Conclusions: Urinary mt-DNA quantification can detect the post-COVID mitochondrial distress with higher sensitivity than uNGAL in RTRs.

In Silico Analysis of Neutralizing Antibody Epitopes on The Hepatitis C Virus Surface Glycoproteins.

Despite of antiviral drugs and successful treatment, an effective vaccine against hepatitis C virus (HCV) infection is still required. Recently, bioinformatic methods same as prediction algorithms, have greatly contributed to the use of peptides in the design of immunogenic vaccines. Therefore, finding more conserved sites on the surface glycoproteins (E1 and E2) of HCV, as major targets to design an effective vaccine against genetically different viruses in each genotype was the goal of the study. In this experimental study, 100 entire sequences of E1 and E2 were retrieved from the NCBI website and analyzed in terms of mutations and critical sites by Bioedit 7.7.9, MEGA X software. Furthermore, HCV-1a samples were obtained from some infected people in Iran, and reverse transcriptase-polymerase chain reaction (RTPCR) assay was optimized to amplify their E1 and E2 genes. Moreover, all three-dimensional structures of E1 and E2 downloaded from the PDB database were analyzed by YASARA. In the next step, three interest areas of humoral immunity in the E2 glycoprotein were evaluated. OSPREY3.0 protein design software was performed to increase the affinity to neutralizing antibodies in these areas. We found the effective in silico binding affinity of residues in three broadly neutralizing epitopes of E2 glycoprotein. First, positions that have substitution capacity were detected in these epitopes. Furthermore, residues that have high stability for substitution in these situations were indicated. Then, the mutants with the strongest affinity to neutralize antibodies were predicted. I414M, T416S, I422V, I414M-T416S, and Q412N-I414M-T416S substitutions theoretically were exhibited as mutants with the best affinity binding. Using an innovative filtration strategy, the residues of E2 epitopes which have the best in silico binding affinity to neutralizing antibodies were exhibited and a distinct peptide library platform was designed.

Clinical features and viral etiology of acute respiratory infection in an outpatient fever clinic during COVID-19 pandemic in a tertiary hospital in Nanjing, China.

Clinical feature and viral etiology for acute respiratory infection (ARI) in the community was unknown during coronavirus disease 2019 (COVID-19) pandemic. In a retrospective study, we aimed to characterize the clinical feature and etiology for the ARI patients admitted to the outpatient fever clinic in Nanjing Drum Tower Hospital between November 2020 and March 2021. Fifteen common respiratory pathogens were tested using pharyngeal swabs by multiplex reverse transcriptase-polymerase chain reaction assays. Of the 242 patients, 56 (23%) were tested positive for at least one viral agent. The predominant viruses included human rhinovirus (HRV) (5.4%), parainfluenza virus type III (PIV-III) (5.0%), and human coronavirus-NL63 (HCoV-NL63) (3.7%). Cough, sputum, nasal obstruction, and rhinorrhea were the most prevalent symptoms in patients with viral infection. Elderly and the patients with underlying diseases were susceptible to pneumonia accompanied with sputum and chest oppression. Three (5.4%) patients in virus infection group, whereas 31 (16.7%) in non-viral infection group (p=0.033), were empirically prescribed with antiviral agents. Among 149 patients who received antibiotic therapy, 30 (20.1%) patients were later identified with viral infection. Our study indicated the importance of accurate diagnosis of ARI, especially during the COVID-19 pandemic, which might facilitate appropriate clinical treatment.