Real-time Reverse Transcriptase-polymerase Chain Research Articles

Evaluation of Fecal Sample Pooling for Real-Time RT-PCR Testing SARS-CoV-2 in Animals

During the COVID-19 pandemic, veterinary diagnostic laboratories tested both human and animal samples and needed to ensure that they could accurately perform large numbers of diagnostic tests in a timely manner. Sample pooling, a methodology used effectively for over 80 years as a surveillance tool for screening large numbers of potentially infected individuals, was employed. Given its sensitivity, real-time polymerase chain reaction (PCR) is more suitable for employing this strategy, as compared to other less sensitive testing methods. In this study, we evaluated the capability of detecting SARS-CoV-2 in both 5-sample and 10-sample pools of feces using real-time reverse transcriptase polymerase chain reaction (rRT-PCR) as well as determined the level of sensitivity. A blinded method test (BMT) by an independent laboratory was conducted to assess the five-sample fecal pool. To complement detection capability, the stability of the genome within a PBS fecal suspension was measured under various time and temperature conditions across a 28-day period. Our results showed that the limit of detection for 5-sample and 10-sample fecal pools is 12.8 and 6.4 genome copies in a 25 µL PCR, respectively. The 5-sample and 10-sample pooling resulted in a cycle threshold (Ct) value loss of 2.35 and 3.45, as compared to Ct values of known positive individual samples, but consistent detection was still achieved in pools containing positive samples with an original Ct below 36 and 34, respectively. The simulation of clinical five-sample pooling showed that all positive samples could be detected regardless of the number (1–3) of positive samples in each pool. The BMT results demonstrated excellent sensitivity (100 copies/reaction) in five-sample pools for the detection of SARS-CoV-2 RNA even though a fecal matrix effect was observed. Finally, our results show that the SARS-CoV-2 genome remains stable over a wide range of time and temperature variations. Overall, our findings provide solid data to scale up SARS-CoV-2 testing capacity in veterinary diagnostic laboratories.

A contemporary study: Molecular approach to histological analysis of meat products by reverse-transcriptase PCR

Meat and meat products constitute an important part of human diet. Yet, in order to meet consumers' increasing demand for meat products and manufacturers’ demand to lower the expenses, usage of offal tissues in meat products are prevalent as food adulteration. Industrial food processing techniques enable producers hide the tissue texture; therefore, histological analysis methodology often fails to detect the fraud. Here, we present a contemporary and unbiased, molecular approach to traditional histological analysis of meat products by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Our study includes the bioinformatic approach to select relevant transcripts that are expressed only in offal tissues but not in muscle and adipose tissues. The selected transcripts were particularly paid attention to have high expression levels for a higher possibility of being detected in processed meat products. The developed qualitative RT-PCR method was also validated by specificity, sensitivity, repeatability and reproducibility parameters.

Longitudinal wastewater-based surveillance of SARS-CoV-2 during 2023 in Ethiopia.

Although wastewater-based epidemiology (WBE) successfully functioned as a tool for monitoring the coronavirus disease 2019 (COVID-19) pandemic globally, relatively little is known about its utility in low-income countries. This study aimed to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater, estimate the number of infected individuals in the catchment areas, and correlate the results with the clinically reported COVID-19 cases in Addis Ababa, Ethiopia. A total of 323 influent and 33 effluent wastewater samples were collected from three Wastewater Treatment Plants (WWTPs) using a 24-h composite Moore swab sampling method from February to November 2023. The virus was captured using Ceres Nanotrap® Enhancement Reagent 2 and Nanotrap® Microbiome A Particles, and then nucleic acids were extracted using the Qiagen QIAamp Viral RNA Mini Kit. The ThermoFisher TaqPath™ COVID-19 kit was applied to perform real-time reverse transcriptase polymerase chain reaction (qRT-PCR) to quantify the SARS-CoV-2 RNA. Wastewater viral concentrations were normalized using flow rate and number of people served. In the sampling period, spearman correlation was used to compare the SARS-CoV-2 target gene concentration to the reported COVID-19 cases. The numbers of infected individuals under each treatment plant were calculated considering the target genes' concentration, the flow rate of treatment plants, a gram of feces per person-day, and RNA copies per gram of feces. SARS-CoV-2 was detected in 94% of untreated wastewater samples. All effluent wastewater samples (n = 22) from the upflow anaerobic sludge blanket (UASB) reactor and membrane bioreactor (MBR) technology were SARS-COV-2 RNA negative. In contrast, two out of 11 effluents from Waste Stabilization Pond were found positive. Positive correlations were observed between the weekly average SARS-CoV-2 concentration and the cumulative weekly reported COVID-19 cases in Addis Ababa. The estimated number of infected people in the Kality Treatment catchment area was 330 times the number of COVID-19 cases reported during the study period in Addis Ababa. This study revealed that SARS-CoV-2 was circulating in the community and confirmed previous reports of more asymptomatic COVID-19 cases in Ethiopia. Additionally, this study provides further evidence of the importance of wastewater-based surveillance in general to monitor infectious diseases in low-income settings. Wastewater-based surveillance of SARS-CoV-2 can be a useful method for tracking the increment of COVID-19 cases before it spreads widely throughout the community.

A-279 Performance Evaluation of the Respiratory Viral for BD MAX™ System in Geriatric Population

Abstract Background Respiratory Viral Infection is one of the most common infection in the geriatric population and cause major concern in the Long-Term Care Facilities. Influenza A and B, SARS-CoV-2, and respiratory syncytial virus (RSV) are responsible for most of the hospitalization and deaths among elderly patients. Rapid identification, treatment, and isolation are among the most important steps to fight the spreading of the infection. Methods The BD Respiratory Viral Panel for BD MAX™ System is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT- PCR) test intended for the simultaneous, qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) nucleic acid in nasopharyngeal swab (NPS) and anterior nasal swab (ANS) specimens from individuals with signs and symptoms of respiratory tract infection. The assay is targeting RNA from the nucleocapsid phosphoprotein gene (N1 and N2 regions) of the SARS-CoV-2, a conserved region of the matrix protein M1 gene for influenza A, conserved regions of the matrix protein M1 gene and HA gene for influenza B, conserved regions of the N and M genes for RSV, and the human RNase P gene. The assay was evaluated for: precision, reproducibility, accuracy, and correlation with BioGx for SARS-CoV-2, Solana (Quidel) for influenza A, influenza B and RSV; the percentage agreement for the evaluation steps was calculated. We ran 86 samples collected from patients residing in Long-Term Care Facilities. Statistical analyses were done using Analyse-it. Results The percentage agreement for precision, reproducibility, and accuracy were 100%. The patients’ correlation was 100% when compared to the existing molecular testing in the laboratory. Female patients accounted for 62.8% (54 female and 32 male); 2 patients were positive for both influenza A and RSV, and one patient was positive for both influenza A and SAR-CoV-2. Conclusions The Respiratory Viral panel for BD MAX™ System gave the benefit of a fully automated, high precision, accuracy and acceptable sensitivity assay; in addition, it offers easier collection (one swabs versus multiple swabs), faster turnaround time for four targets in less than three hours which allow for earlier isolation to prevent the spreading of the infection and initiating treatment when needed. As with every molecular assay avoiding contamination is very important to eliminated false positive results.

Assessment of COVID-19 patients’ outcome based on clinical profile, laboratory parameters, and clinical management: A retrospective observational study

ABSTRACT Background: The coronavirus disease 2019 (COVID-19) pandemic has presented an unprecedented challenge to the global healthcare system, prompting an urgent need to understand the factors influencing patient outcomes. Critical to improving treatment protocols and reducing mortality rates is an in-depth assessment of the clinical profile, laboratory findings, and management strategies employed in treating COVID-19 patients. This research provides valuable insights that could influence future therapeutic approaches and public health strategies, ultimately aiming to reduce the morbidity and mortality associated with COVID-19. The study aimed to assess mortality predictors in patients admitted to the intensive care unit (ICU) due to COVID-19. Methods: This study employed a retrospective approach, utilizing patient data from medical records. The collected data encompassed demographic and clinical profiles and details regarding the duration of admission and treatment. The evaluation focused on patients admitted to the ICU for COVID-19 between March 2020 and July 2021, with confirmation through real-time reverse transcriptase polymerase chain reaction (RT-PCR). Rigorous statistical analysis was conducted to compare outcomes between discharged and deceased patients. Results: The study included a total of 202 ICU patients admitted for COVID-19. Among the cases, 147 (72.8%) were males and 55 (27.2%) were females. The mean age was 58.42 years, with a standard deviation of 15.59 years. Fever (92%) emerged as the most frequently encountered symptom, followed by cough (48.5%) and dyspnea (35%). Patients with underlying comorbidities exhibited a higher susceptibility to developing a severe or critical disease. Hypertension (n = 38) was identified as the most prevalent comorbidity, followed by type 2 diabetes mellitus (n = 36). Hypertension has demonstrated a significant association with disease outcomes. Body temperature, respiratory rate, oxygen saturation, and mechanical ventilation played substantial roles in patient outcomes. Conclusion: The study revealed that underlying comorbidities and complications, such as acute respiratory distress syndrome (ARDS), were linked to significantly higher mortality rates among COVID-19 patients. Abnormal laboratory parameters also exhibited significant differences in the outcomes of ICU patients.

High expression of transcription factor EGR1 is associated with postoperative muscle atrophy in patients with knee osteoarthritis undergoing total knee arthroplasty

BackgroundMuscle atrophy is a typical affliction in patients affected by knee Osteoarthritis (KOA). This study aimed to examine the potential pathogenesis and biomarkers that coalesce to induce muscle atrophy, primarily through the utilization of bioinformatics analysis.MethodsTwo distinct public datasets of osteoarthritis and muscle atrophy (GSE82107 and GSE205431) were subjected to differential gene expression analysis and gene set enrichment analysis (GSEA) to probe for common differentially expressed genes (DEGs) and conduct transcription factor (TF) enrichment analysis from such genes. Venn diagrams were used to identify the target TF, followed by the construction of a protein-protein interaction (PPI) network of the common DEGs governed by the target TF. Hub genes were determined through the CytoHubba plug-in whilst their biological functions were assessed using GSEA analysis in the GTEx database. To validate the study, reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and Flow Cytometry techniques were employed.ResultsA total of 138 common DEGs of osteoarthritis and muscle atrophy were identified, with 16 TFs exhibiting notable expression patterns in both datasets. Venn diagram analysis identified early growth response gene-1 (EGR1) as the target TF, enriched in critical pathways such as epithelial mesenchymal transition, tumor necrosis factor-alpha signaling NF-κB, and inflammatory response. PPI analysis revealed five hub genes, including EGR1, FOS, FOSB, KLF2, and JUNB. The reliability of EGR1 was confirmed by validation testing, corroborating bioinformatics analysis trends.ConclusionsEGR1, FOS, FOSB, KLF2, and JUNB are intricately involved in muscle atrophy development. High EGR1 expression directly regulated these hub genes, significantly influencing postoperative muscle atrophy progression in KOA patients.

Low-cost high-throughput targeted sequencing for the accurate detection of respiratory tract pathogens.

The current gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis by real-time reverse transcriptase polymerase chain reaction (RT-PCR) is limited by the number of genes that can be detected. In this study, we developed a low-cost and high-throughput next-generation sequencing technology that can overcome the limitations of real time RT-PCR. A targeted sequencing panel (TSP) consisting of approximately 500 amplicons was designed. This panel could simultaneously detect a broad range of gene loci of SARS-CoV-2, and genes for the most common infectious viruses that affect the respiratory tract, in a single run and could include up to 96 samples. Four hundred and forty-eight samples and 31 control samples were analyzed independently with both TSP and RT-PCR, and the results were compared for accuracy and other indicators. TSP identified 50 SARS-CoV-2 positive samples with a 99.33% match to RT-PCR results. It is not surprising that TSP also identified multiple infections from the 96 samples, whereas RT-PCR could not. Thus, TSP was able to accurately diagnose the samples which could not be identified based on single RT-PCR test. Our data demonstrated that TSP is a fast and accurate testing method for identifying multiple pathogen infections of the respiratory tract.

Comparison of diagnostic accuracy of rapid antigen tests for COVID19 compared to the viral genetic test in adults: a systematic review and meta-analysis.

The objective of this review is to determine the diagnostic accuracy of the currently available and upcoming point-of-care rapid antigen tests (RATs) used in primary care settings relative to the viral genetic real-time reverse transcriptase polymerase chain reaction (RT-PCR) test as a reference for diagnosing COVID-19/SARS-CoV-2 in adults. Accurate COVID-19 point-of-care diagnostic tests are required for real-time identification of SARS-CoV-2 infection in individuals. Real-time RT-PCR is the accepted gold standard for diagnostic testing, requiring technical expertise and expensive equipment that are unavailable in most primary care locations. RATs are immunoassays that detect the presence of a specific viral protein, which implies a current infection with SARS-CoV-2. RATs are qualitative or semi-quantitative diagnostics that lack thresholds that provide a result within a short time frame, typically within the hour following sample collection. In this systematic review, we synthesized the current evidence regarding the accuracy of RATs for detecting SARS-CoV-2 compared with RT-PCR. Studies that included nonpregnant adults (18 years or older) with suspected SARS-CoV-2 infection, regardless of symptomology or disease severity, were included. The index test was any available SARS-CoV-2 point-of-care RAT. The reference test was any commercially distributed RT-PCR-based test that detects the RNA genome of SARS-CoV-2 and has been validated by an independent third party. Custom or in-house RT-PCR tests were also considered, with appropriate validation documentation. The diagnosis of interest was COVID-19 disease and SARS-CoV-2 infection. This review considered cross-sectional and cohort studies that examined the diagnostic accuracy of COVID-19/SARS-CoV-2 infection where the participants had both index and reference tests performed. The keywords and index terms contained in relevant articles were used to develop a full search strategy for PubMed and adapted for Embase, Scopus, Qinsight, and the WHO COVID-19 databases . Studies published from November 2019 to July 12, 2022, were included, as SARS-CoV-2 emerged in late 2019 and is the cause of a continuing pandemic. Studies that met the inclusion criteria were critically appraised using QUADAS-2. Using a customized tool, data were extracted from included studies and were verified prior to analysis. The pooled sensitivity, specificity, positive predictive, and negative predictive values were calculated and presented with 95% CIs. When heterogeneity was observed, outlier analysis was conducted, and the results were generated by removing outliers. Meta-analysis was performed on 91 studies of 581 full-text articles retrieved that provided true-positive, true-negative, false-positive, and false-negative values. RATs can identify individuals who have COVID-19 with high reliability (positive predictive value 97.7%; negative predictive value 95.2%) when considering overall performance. However, the lower level of sensitivity (67.1%) suggests that negative test results likely need to be retested through an additional method. Most reported RAT brands had only a few studies comparing their performance with RT-PCR. Overall, a positive RAT result is an excellent predictor of a positive diagnosis of COVID-19. We recommend that Roche's SARS-CoV-2 Rapid Antigen Test and Abbott's BinaxNOW tests be used in primary care settings, with the understanding that negative results need to be confirmed through RT-PCR. We recommend adherence to the STARD guidelines when reporting on diagnostic data. PROSPERO CRD42020224250.

Study of MicroRNA-124 in Patients with Lupus Nephritis.

Lupus nephritis is associated with a six-fold increase in mortality compared with the general population. MicroRNAs studies revealed that increased MicroRNA -21 and MicroRNA -155 levels represent risk factors for active LN patients. MicroRNAs can be used as biomarkers in the diagnosis of clinical stages of LN. The present study aimed to determine the level of miR-124 in patients with lupus nephritis by reverse transcriptase real-time polymerase chain reaction compared to healthy control and correlate its levels with biochemical findings in those patients. The study was a case-control study that included fifty patients with lupus nephritis in addition to fifty healthy controls. Blood samples from the participants were subjected to the determination of serological markers of SLE. Moreover, real-time PCR was used for the determination of miR-124. The comparison of Micro-RNA124 between patients and control subjects revealed a statistically significant decrease in Micro-RNA124 in patients (1.193 ± 0.56) compared to the control (3.36 ± 0.50, p <0.001); the comparison of the level of MicroRNA 124 in the patients with different clinical and serological findings of SLE revealed a significant decrease in the level of MicroRNA 124 in patients with muscular findings (1.02 ± 0.5) compared to the patients with negative manifestations (1.47 ± 0.5, p =0.005). In the present study, a comparison of MicroRNA-124 in LN patients with different stages compared to normal control showed a statistically significant decrease in Micro-RNA124 in patients with lupus nephritis p <0.001 with significant correlation to the patients' different clinical and serological findings of SLE. Therefore, it may be used as a new noninvasive therapeutic approach to monitor response to therapy, predict relapses, and identify the degree of the activity of the disease or the progression to the chronic stage.

Anti-Inflammatory Efficacy of Nanobody Specific to β-Glucan on a Fungal Cell Wall in a Murine Model of Fungal Keratitis.

Purpose: to explore the anti-inflammatory effects of a nanobody (Nb) specific to β-glucan on fungal keratitis (FK). Methods: in order to verify the therapeutic and anti-inflammatory efficacy of Nb in FK, the severity of inflammation was assessed with inflammatory scores, hematoxylin-eosin (HE) staining, and myeloperoxidase (MPO) assays. In corneas of mice of FK model and human corneal epithelial cells stimulated by fungal hyphae, real-time reverse transcriptase polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were used to detect the expression levels of inflammatory cytokines and pattern recognition receptors (PRRs). In vivo, macrophages and neutrophils infiltration in the cornea stroma was detected by immunofluorescence (IFS) staining. Results: In murine models infected with Aspergillus fumigatus (A. fumigatus), Nb treatment could reduce the inflammatory scores. HE staining and MPO results showed Nb significantly alleviated corneal edema and reduced inflammatory cell infiltration 3 days post-infection. In addition, the expression levels of LOX-1 and Dectin-1 were significantly decreased in the Nb group in vivo. The expression of chemokines CCL2 and CXCL2 also decreased in the Nb group. Compared with the PBS group, the number of macrophages and neutrophils in the Nb group was significantly decreased, which was shown in IFS results. Moreover, Nb attenuated the expression of Dectin-1, LOX-1, and inflammatory mediators, including IL-6 and IL-8 in vitro. Conclusion: our study showed that Nb could alleviate FK by downregulating the expression of PRRs and inflammatory factors as well as reducing the infiltration of macrophages and neutrophils.

Effect of Quorum Sensing Systems on Biofilm Formation and Virulence in Salmonella

In recent years, as the paradigm of communication between cells has been clarified, the ability of bacteria to change their gene expression patterns in response to various extracellular signals has attracted great interest. In particular, intracellular and intercellular communication between bacterial populations, called quorum sensing (QS), is essential for coordinating physiological and genetic activities. QS studies are critical, particularly in elucidating the regulatory mechanisms of infectious processes in food-borne pathogens. Elucidating the QS mechanisms in Salmonella is effective in silencing the virulence factors in the fight against this bacterium. The aims of this study were; to create luxS gene mutants that play a vital role in the QS activity of Salmonella and to determine the effect of this mutation on the expression of virulence genes in the bacteria and to determine the impact of synthetic N-hexanoyl-homoserine lactone (C6HSL) on biofilm formation and AI-2 signaling pathway of Salmonella wild strain and luxS gene mutants. luxS gene mutants were constructed by recombining the gene region with the chloramphenicol gene cassette based on homologous region recombination. In the luxS mutants obtained in this way, the expression of eight different virulence genes (hilA, invA, inv, glgC, fimF, fliF, lpfA, gyrA), which have essential roles in Salmonella pathogenicity, was determined by quantitative real-time reverse transcriptase polymerase chain reaction (rRT-qPCR) method and compared with natural strains. As a result of these studies, it was determined that the expression of each gene examined was significantly reduced in luxS mutant strains. The relative AI-2 activities of Salmonella strains were analyzed depending on time. It was determined that the highest activity occurred at the fourth hour and the AI-2 activities of luxS mutants were reduced compared to the wild strain. Finally, it was determined that C6HSL increased the biofilm activity of Salmonella Typhimurium DMC4, SL1344 wild strains, and mutants, mainly at the 72nd hour. In conclusion, our results proved that C6HSL stimulated QS communication in all strains and increased biofilm of Salmonella formation and autoinducer activity. This situation determines that Salmonella responds to external signals by using QS systems. In addition, this research contributed to provide additional information on interspecies communication mechanisms to develop strategies to prevent biofilm formation of this pathogen.

Expression profile of long noncoding RNAs and comprehensive analysis of lncRNA-cisTF-DGE regulation in condyloma acuminatum

ObjectiveTo identify differentially expressed long noncoding RNAs (lncRNAs) in condyloma acuminatum (CA) and to explore their probable regulatory mechanisms by establishing coexpression networks.MethodsHigh-throughput RNA sequencing was performed to assess genome-wide lncRNA expression in CA and paired adjacent mucosal tissue. The expression of candidate lncRNAs and their target genes in larger CA specimens was validated using real-time quantitative reverse transcriptase polymerase chain reaction (RT‒qPCR). Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used for the functional enrichment analysis of these candidate lncRNAs and differential mRNAs. The coexpressed mRNAs of the candidate lncRNAs, calculated by Pearson’s correlation coefficient, were also analysed using GO and KEGG analysis. In addition, the interactions among differentially expressed lncRNAs (DElncRNAs)-cis-regulatory transcription factors (cisTFs)-differentially expressed genes (DEGs) were analysed and their network was constructed.ResultsA total of 546 lncRNAs and 2553 mRNAs were found to be differentially expressed in CA compared to the paired control. Functional enrichment analysis revealed that the DEGs coexpressed with DElncRNAs were enriched in the terms of cell adhesion and keratinocyte differentiation, and the pathways of ECM-receptor interaction, local adhesion, PI3K/AKT and TGF-ß signaling. We further constructed the network among DElncRNAs-cisTFs-DEGs and found that these 95 DEGs were mainly enriched in GO terms of epithelial development, regulation of transcription or gene expression. Furthermore, the expression of 3 pairs of DElncRNAs and cisTFs, EVX1-AS and HOXA13, HOXA11-AS and EVX1, and DLX6-AS and DLX5, was validated with a larger number of specimens using RT‒qPCR.ConclusionCA has a specific lncRNA profile, and the differentially expressed lncRNAs play regulatory roles in mRNA expression through cis-acting TFs, which provides insight into their regulatory networks. It will be useful to understand the pathogenesis of CA to provide new directions for the prevention, clinical treatment and efficacy evaluation of CA.

Construction and Bioinformatics Analysis of ceRNA Regulatory Networks in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive form of pulmonary fibrosis of unknown etiology. Despite ongoing research, there is currently no cure for this disease. Recent studies have highlighted the significance of competitive endogenous RNA (ceRNA) regulatory networks in IPF development. Therefore, this study investigated the ceRNA network associated with IPF pathogenesis. We obtained gene expression datasets (GSE32538, GSE32537, GSE47460, and GSE24206) from the Gene Expression Omnibus (GEO) database and analyzed them using bioinformatics tools to identify differentially expressed messenger RNAs (DEmRNAs), microRNAs (DEmiRNAs), and long non-coding RNAs (DElncRNA). For DEmRNAs, we conducted an enrichment analysis, constructed protein-protein interaction networks, and identified hub genes. Additionally, we predicted the target genes of differentially expressed mRNAs and their interacting long non-coding RNAs using various databases. Subsequently, we screened RNA molecules with ceRNA regulatory relations in the lncACTdb database based on the screening results. Furthermore, we performed disease and functional enrichment analyses and pathway prediction for miRNAs in the ceRNA network. We also validated the expression levels of candidate DEmRNAs through quantitative real-time reverse transcriptase polymerase chain reaction and analyzed the correlation between the expression of these candidate DEmRNAs and the percent predicted pre-bronchodilator forced vital capacity [%predicted FVC (pre-bd)]. We found that three ceRNA regulatory axes, specifically KCNQ1OT1/XIST/NEAT1-miR-20a-5p-ITGB8, XIST-miR-146b-5p/miR-31-5p- MMP16, and NEAT1-miR-31-5p-MMP16, have the potential to significantly affect IPF progression. Further examination of the underlying regulatory mechanisms within this network enhances our understanding of IPF pathogenesis and may aid in the identification of diagnostic biomarkers and therapeutic targets.

The Impact of Cycle Threshold Value in Influencing the Performance of COVID-19 Antigen

COVID-19 antigen is an alternative test for detecting SARS-CoV-2 infection. Viral load represented by the Cycle Threshold (CT) in the Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) affects the diagnostic performance of the test. Higher CT values result in reduced sensitivity of the SARS-CoV-2 antigen. The main objective of this study was to determine the highest CT value in rRT-PCR that still yielded reactive results in the COVID-19 antigen test. This cross-sectional study was conducted at the Fever Outpatient Clinic in Dr. Cipto Mangunkusumo Hospital from July 2020 to June 2021. Two hundred and thirty-five naso-oropharyngeal swabs were taken from patients with confirmed and suspected COVID-19 diagnoses. About 24.7% of subjects were tested positive. The median highest CT value giving reactive COVID-19 antigen results was 28.22 (13.33-39.16), while the median CT value for non-reactive antigen results was 34.45 (26.08-39.65). At a CT value &lt; 40, the COVID-19 antigen test demonstrated 63.8% sensitivity, 99.4% specificity, 89.3% Negative Predictive Value (NPV), and 97.4% Positive Predictive Value (PPV). At the CT value &lt; 25, the test showed 92.3% sensitivity, 99.4% specificity, 99.4% NPV, 92.3% PPV, 163.4 LR+, and 0.1 LR-. The identified cut-off point for the CT value was 29.82, with a sensitivity of 64.9% and specificity of 81%. In conclusion, COVID-19 antigen is a valuable test for screening patients with symptoms of SARS-CoV-2 infection. Understanding the influence of cycle threshold can enhance the interpretation and reliability of the antigen test.

Gastric juice piR-015551: A promising prognostic biomarker for gastric cancer.

e16074 Background: Emerging reports demonstrated that PIWI-interacting RNAs (piRNAs) played an indispensable role in tumorigenesis. However, it still remains elusive whether piR-015551 in gastric juice specific in stomach could be employed as a biomarker for gastric cancer (GC). The present work is aiming at exploring the possibility of piR-015551 in gastric juice as a potential marker to judge for diagnosis and prognosis of gastric cancer. Methods: Gastric juice was collected from 66 GC patients and 66 healthy individuals. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was employed to measure the levels of piR-015551 expression. Then, the pattern of piR-015551 expression in gastric juice was determined between GC patients and healthy individuals. A receiver operating characteristic (ROC) curve was constructed for distinguishing GC from healthy individuals. Results: Gastric juice piR-015551 levels in GC were higher than those of controls (P &lt; 0.0001). The value of area under ROC (AUC) was 0.885 (sensitivity, 90.9%; specificity, 74.2%; 95% confidence interval, 0.8286 to 0.9414). High gastric juice piR-015551 expression was signally correlated with tumor size (P = 0.013) and TNM stage (P = 0.001). GC patients with high piR-015551 expression in gastric juice exerted a poorer overall survival (OS) (P = 0.0152) and progression-free survival (PFS) (P = 0.013). COX regression analysis verified that gastric juice piR-015551 expression was an independent prognostic risk variable for OS (P &lt; 0.05). Conclusions: The current study suggested that piR-015551 in gastric juice had the potential to be a useful biomarker for GC detection and prognosis prediction.

Characteristics, risk factors, and outcomes related to Zika virus infection during pregnancy in Northeastern Thailand: A prospective pregnancy cohort study, 2018-2020.

In response to the 2015-2016 Zika virus (ZIKV) outbreak and the causal relationship established between maternal ZIKV infection and adverse infant outcomes, we conducted a cohort study to estimate the incidence of ZIKV infection in pregnancy and assess its impacts in women and infants. From May 2018-January 2020, we prospectively followed pregnant women recruited from 134 participating hospitals in two non-adjacent provinces in northeastern Thailand. We collected demographic, clinical, and epidemiologic data and blood and urine at routine antenatal care visits until delivery. ZIKV infections were confirmed by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Specimens with confirmed ZIKV underwent whole genome sequencing. Among 3,312 women enrolled, 12 (0.36%) had ZIKV infections, of which two (17%) were detected at enrollment. Ten (83%, 3 in 2nd and 7 in 3rd trimester) ZIKV infections were detected during study follow-up, resulting in an infection rate of 0.15 per 1,000 person-weeks (95% CI: 0.07-0.28). The majority (11/12, 91.7%) of infections occurred in one province. Persistent ZIKV viremia (42 days) was found in only one woman. Six women with confirmed ZIKV infections were asymptomatic until delivery. Sequencing of 8 ZIKV isolates revealed all were of Asian lineage. All 12 ZIKV infected women gave birth to live, full-term infants; the only observed adverse birth outcome was low birth weight in one (8%) infant. Pregnancies in 3,300 ZIKV-rRT-PCR-negative women were complicated by 101 (3%) fetal deaths, of which 67 (66%) had miscarriages and 34 (34%) had stillbirths. There were no differences between adverse fetal or birth outcomes of live infants born to ZIKV-rRT-PCR-positive mothers compared to live infants born to ZIKV-rRT-PCR-negative mothers. Confirmed ZIKV infections occurred infrequently in this large pregnancy cohort and observed adverse maternal and birth outcomes did not differ between mothers with and without confirmed infections.

The Emergence of Linezolid-Resistant Staphylococcus Epidermidis in the COVID-19 Hospitalized Intubated Patients in North Khorasan, Iran.

The present study aimed to investigate secondary bacterial infections among patients infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Coagulase-negative Staphylococci can infect immunocompromised patients. Linezolid resistance among Staphylococcus epidermidis is one of the most critical issues. In 2019, 185 SARS-CoV-2-positive patients who were admitted to North Khorasan Province Hospital (Bojnurd, Iran), were investigated. Patients having positive SARS-CoV-2 reverse transcriptase real-time polymerase chain reaction (RT-PCR) test results, who had a history of intubation, mechanical ventilation, and were hospitalized for more than 48 hours were included. After microbiological evaluation of pulmonary samples, taken from intubated patients with clinical manifestation of pneumonia, co-infections were found in 11/185 patients (5.94%) with S. epidermidis, Staphylococcus aureus, and Acinetobacter baumani, respectively. Remarkably, seven out of nine S. epidermidis isolates were linezolid resistant. Selected isolates were characterized using antimicrobial resistance patterns and molecular methods, such as Staphylococcal cassette chromosome mec (SCCmec) typing, and gene detection for ica, methicillin resistance (mecA), vancomycin resistance (vanA), and chloramphenicol-florfenicol resistance (cfr) genes. All of the isolates were resistant to methicillin, and seven isolates were resistant to linezolid. Nine out of 11 isolated belonged to the SCCmec I, while two belonged to the SCCmec IV. It should be noted that all patients had the underlying disease, and six patients had already passed away. The increasing linezolid resistance in bacterial strains becomes a real threat to patients, and monitoring such infections, in conjunction with surveillance and infection prevention programs, is very critical for reducing the number of linezolid-resistant Staphylococcal strains. A preprint of this study was published at https://europepmc.org/article/ppr/ppr417742.

Genetic Sequence and Pathogenicity of Infectious Bursal Disease Virus in Chickens in Egypt During 2017-2021.

The continued circulation of infectious bursal disease virus (IBDV) in Egypt, despite the use of various vaccines, is a serious problem that requires continuous detection of IBDV. In the current study, real-time reverse transcriptase polymerase chain reaction testing of 100 diseased chicken flocks during 2017-2021 revealed the presence of very virulent IBDV (vvIBDV) in 67% of the flocks, non-vvIBDV in 11%, and a mixture of both vvIBDV and non-vvIBDV in 4%. Twenty-nine IBDV isolates were submitted for partial sequencing of the viral protein 2 hypervariable region (VP2-HVR), and 27 isolates were confirmed to be genogroup A3 (vvIBDV) with 96.3%-98.5% similarity to the global A3 (vvIBDV) and 88.9%-97% similarity to genogroup A1 vaccine strains. The remaining two isolates were non-vvIBDV and showed 91.1% and 100% identity with classical genogroup A1 strains, respectively. Furthermore, the sequence and phylogenetic analysis of VP1 (amino acids 33-254) of two selected isolates of A3, 5/2017 and 98/2021, clustered them as B2, vvIBDV-like, strains with high similarity (99.5%) to four Egyptian, 99% to Chinese and European, and 97.7% to Chinese and Polish vvIBDV isolates. Experimental infection of commercial broiler chickens with two vvIBDV-A3B2 isolates (5/2017 and 98/2021) showed no mortality despite typical tissue lesions, clear histopathological changes, and strong ELISA antibody response. Isolate 98/2021 was more pathogenic, as confirmed by histopathology, whereas isolate 5/2017 induced a stronger serological response. In conclusion, vvIBDV (A3B2) strains with two amino acid (aa) substitutions in VP1 as V141I and V234I as well as VP2 as Y220F and G254S are still circulating in Egypt.

Detection of SARS-CoV-2 variants distributed from March 2020 to May 2022 and their effect on the infection severity in Iraqi population

New SARS-CoV-2 variants appeared in late December 2020 as Mutations accumulated in the original virus. This study aimed to provide a local database about variants of COVID-19 circulating in the Iraqi population from 2020 to 2022 and the time of emergence of new strains each month since very few local studies have documented its existence in the country. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays were employed to 319 collected and analyzed nasal swabs to determine whether an infection had occurred. A sophisticated diagnostic kit that sorted the distinctive mutations was implemented to evaluate the variants. Results showed Younger patients were more likely to be infected with the Alpha variant (66 patients) than older people (43 patients). Additionally, patients with wild-type infestations had more robust viral load and lower Ct threshold values, culminating in an increase in severity during infection with wild-type virus 26/32 (81.250). Meanwhile, 65/109(59.63%) of patients infected with the Alpha variant developed severe and critical illness and 51/84(60.71%)were infected with Delta or Delta plus variants. In conclusion, the Alpha variant had the highest infection percentage of 109(46.6%), followed by Delta or Delta plus variant 84(26.33%), Beta or Gamma variants 47(20.1%), Omicron variant 46(19.6%), and finally wild-type virus of 32(13.7%). February 2020 witnessed a preliminary finding of the wild-type, while the Alpha variant emerged in December 2020, Beta/Gamma variances were recognized in December 2020, Delta/Delta plus variances began in April 2021, and the Omicron variant debuted in March 2022. Keywords: SARS-CoV-2, Mutation, rRT-PCR, Coronavirus disease 2019, TaqPath, cycle threshold (Ct) value

Detection of SARS-CoV-2 variants distributed from March 2020 to May 2022 and their effect on the infection severity in Iraqi population

New SARS-CoV-2 variants appeared in late December 2020 as Mutations accumulated in the original virus. This study aimed to provide a local database about variants of COVID-19 circulating in the Iraqi population from 2020 to 2022 and the time of emergence of new strains each month since very few local studies have documented its existence in the country. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays were employed to 319 collected and analyzed nasal swabs to determine whether an infection had occurred. A sophisticated diagnostic kit that sorted the distinctive mutations was implemented to evaluate the variants. Results showed Younger patients were more likely to be infected with the Alpha variant (66 patients) than older people (43 patients). Additionally, patients with wild-type infestations had more robust viral load and lower Ct threshold values, culminating in an increase in severity during infection with wild-type virus 26/32 (81.250). Meanwhile, 65/109(59.63%) of patients infected with the Alpha variant developed severe and critical illness and 51/84(60.71%)were infected with Delta or Delta plus variants. In conclusion, the Alpha variant had the highest infection percentage of 109(46.6%), followed by Delta or Delta plus variant 84(26.33%), Beta or Gamma variants 47(20.1%), Omicron variant 46(19.6%), and finally wild-type virus of 32(13.7%). February 2020 witnessed a preliminary finding of the wild-type, while the Alpha variant emerged in December 2020, Beta/Gamma variances were recognized in December 2020, Delta/Delta plus variances began in April 2021, and the Omicron variant debuted in March 2022. Keywords: SARS-CoV-2, Mutation, rRT-PCR, Coronavirus disease 2019, TaqPath, cycle threshold (Ct) value