Real-time Reverse Transcriptase-polymerase Chain Research Articles

Telomerase Reverse Transcriptase and Telomerase RNA Component Gene Expression as a Novel Biomarkers for Alzheimer's disease.

Alzheimer's disease (AD) is a neurological, age-related condition that causes cognitive decline and memory loss; it induces dementia in the elderly. Telomerase is a reverse transcriptase ribonucleoprotein that adds nucleotides to the end of DNA. This study aimed to compare human telomerase reverse transcriptase (hTERT) and telomerase RNA component (TERC) expression in different phases of AD and healthy cohorts. Sixty participants were divided into 30 who had dementia and 30 who did not. After collecting blood samples, total RNAs were extracted from the plasma. Screening for hTERT and TERC gene expression was carried out by quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) using the relative quantification method to estimate the expression changes in hTERT and TERC. The RT-qPCR results show that hTERT and TERC gene expression was significantly down-regulated in Alzheimer's patients compared to the health subjects (P-value= <0.0001,0.005), respectively. The area under curve AUC was 0.773 for hTERT and 0.703 for TERC. The Mini-Mental State Examination scores revealed a significant difference between dementia and non-dementia subjects (P=<0.0001). We conclude down-regulations in both hTERT and TERC gene expression in AD patients, which supports our hypothesis that the telomerase expression gene in the blood of AD patients can serve as a non-invasive, early, and novel diagnostic marker of AD.

Could self-reported symptoms be predictors of RT-PCR positivity in suspected COVID-19 cases? The Libya experience.

COVID-19 has symptoms similar to several other respiratory and non-respiratory diseases, which makes differentiating them a challenging task and could lead to unnecessary use of realtime reverse transcriptase polymerase chain reaction (RT-PCR) resources. The study aimed to assess self-reported symptoms as predictors for RT-PCR positivity in suspected COVID-19 cases. This was a cross-sectional study. We retrospectively reviewed the database of COVID-19 care centres in the eastern district of Tripoli, Libya, from May to December 2020. Presenting symptoms and RT-PCR test data were extracted. Of the 4593 subjects, 923 (20.1%) had positive RT-PCR result. Sensitivity for COVID-19 disease diagnosis was very low (≤ 18.2%) for all symptoms, except for myalgia (82.1%). Specificity was high for all symptoms (90.7-99.8%), except for myalgia (11.0%). Loss of taste and smell had the highest positive likelihood ratio (LR) for RT-PCR positivity (LR+ = 3.59, 95% CI: 2.95-4.37). In the multiple logistic regression, three symptoms maintained significant contribution to RT-PCR positivity; these were loss of taste and smell (odds ratio (OR) = 3.90, 95% CI: 3.04-4.99), sore throat (OR = 1.50, 95% CI: 1.02-2.19), and myalgia (OR = 0.65, 95% CI: 0.49-0.85). Other significant predictors were history of contact with a COVID-19 case (OR = 0.50, 95% CI: 0.39-0.62), and being female (OR = 1.33, 95% CI: 1.15-1.55). The findings of this study do not support the use of self-reported symptoms for the confirmation of COVID-19 disease in suspected cases because of their poor diagnostic properties.

Intercellular mitochondrial transfer as a means of revitalizing injured glomerular endothelial cells.

BACKGROUNDRecent studies have demonstrated that mesenchymal stem cells (MSCs) can rescue injured target cells via mitochondrial transfer. However, it has not been fully understood how bone marrow-derived MSCs repair glomeruli in diabetic kidney disease (DKD).AIMTo explore the mitochondrial transfer involved in the rescue of injured glomerular endothelial cells (GECs) by MSCs, both in vitro and in vivo.METHODS In vitro experiments were performed to investigate the effect of co-culture with MSCs on high glucose-induced GECs. The transfer of mitochondria was visua lized using fluorescent microscopy. GECs were freshly sorted and ultimately tested for apoptosis, viability, mRNA expression by real-time reverse transcri ptase-polymerase chain reaction, protein expression by western blot, and mitochondrial function. Moreover, streptozotocin-induced DKD rats were infused with MSCs, and renal function and oxidative stress were detected with an automatic biochemical analyzer and related-detection kits after 2 wk. Kidney histology was analyzed by hematoxylin and eosin, periodic acid-Schiff, and immunohistochemical staining.RESULTSFluorescence imaging confirmed that MSCs transferred mitochondria to injured GECs when co-cultured in vitro. We found that the apoptosis, proliferation, and mitochondrial function of injured GECs were improved following co-culture. Additionally, MSCs decreased pro-inflammatory cytokines [interleukin (IL)-6, IL-1β, and tumor necrosis factor-α] and pro-apoptotic factors (caspase 3 and Bax). Mitochondrial transfer also enhanced the expression of superoxide dismutase 2, B cell lymphoma-2, glutathione peroxidase (GPx) 3, and mitofusin 2 and inhibited reactive oxygen species (ROS) and dynamin-related protein 1 expression. Furthermore, MSCs significantly ameliorated functional parameters (blood urea nitrogen and serum creatinine) and decreased the production of malondialdehyde, advanced glycation end products, and ROS, whereas they increased the levels of GPx and superoxide dismutase in vivo. In addition, significant reductions in the glomerular basement membrane and renal interstitial fibrosis were observed following MSC treatment.CONCLUSIONMSCs can rejuvenate damaged GECs via mitochondrial transfer. Additionally, the improvement of renal function and pathological changes in DKD by MSCs may be related to the mechanism of mitochondrial transfer.

Diagnostic Value of Natural Killer Cells, CD56+ CD16+ Natural Killer Cells, NLRP3, and Lactate Dehydrogenase in Severe/Critical COVID-19: A Prospective Longitudinal Study According to the Severe/Critical COVID-19 Definitions.

Innate immunity, as the first line of defense of our immune system, plays a crucial role in defending against SARS-CoV-2 infection and also its immunopathogenesis. We aim to investigate the immune status of natural killer (NK) cells, natural killer T (NKT) cells, and NLRP3 gene expression in COVID-19 patient blood samples. The immunophenotype of NK cell subsets and NKT cells was detected by flow cytometry and the expression of NLRP3 gene assessed by reverse transcriptase real-time polymerase chain reaction in 44 COVID-19 patients and 20 healthy individuals. The percentage of most of NK cell subpopulation and NKT cells was significantly decreased in COVID-19 patients. The percentage of CD56dim CD16- NK cell subsets, and NLRP3 gene expression increased. The percentage of total NK cells, CD56+ CD16+ NK cells, and NLRP3 gene expression had acceptable sensitivity and specificity for assisting diagnosis of severe/critical COVID-19. O2 saturation% and lactate dehydrogenase levels showed valuable diagnostic value to identify critical cases. The declined NK and NKT cells in COVID-19 patients and enhanced NLRP3 gene expression were associated with disease severity. Total NK cells, CD56+ CD16+ NK cells, and NLRP3 gene expression might be used as meaningful indicators for assisting diagnosis of severe/critical COVID-19.

Should Pneumothorax Developing During the Recovery Period After COVID-19 in Patients with Previously Healthy Lungs be Considered a Primary Spontaneous Pneumothorax or a Secondary Spontaneous Pneumothorax?

OBJECTIVE:It is still unknown how to call the pneumothorax that develops during the recovery period after coronavirus disease 2019. Patients who developed pneumothorax during the recovery period after coronavirus disease 2019 were compared with those who had a primary or secondary spontaneous pneumothorax without a coronavirus disease 2019 history.MATERIAL AND METHODS:Between 2020 and 2021, 160 patients with pneumothorax were retrospectively analyzed. Twenty-three patients had a history of coronavirus disease 2019 (coronavirus disease recovery) confirmed by real-time reverse transcriptase-polymerase chain reaction, whereas the remaining 137 patients did not have a history of coronavirus disease 2019 (18 of the patients with secondary spontaneous pneumothorax group and 119 patients with primary spontaneous pneumothorax group).Results:The median time between discharge and readmission to the hospital because of pneumothorax was 9 days in the coronavirus disease recovery group. There were statistically significant differences in regards to age (P < .001), gender (P = .02), the presence of bullae (P = .02), and dystrophic severity lung score (P = .04) between the coronavirus disease recovery and primary spontaneous pneumothorax groups, whereas no difference was found between the coronavirus disease recovery and the secondary spontaneous pneumothorax groups (P > .05). The prolonged air leak was observed in 17.6% (n = 25). Patients who had prolonged air leak were statistically higher in the coronavirus disease recovery group than the primary spontaneous pneumothorax group (56.5% vs. 10.1%), although it was almost similar between the coronavirus disease recovery and secondary spontaneous pneumothorax groups (P = .951). On logistic regression analysis, the coronavirus disease recovery group was the independent factor for prolonged air leak (odds ratio = 9.900, 95% CI = 1.557-62.500, P = .01).Conclusion:Pneumothorax may be developed during the recovery period after coronavirus disease 2019 in patients with previously healthy lungs, and it should be called as secondary spontaneous pneumothorax.

RT-PCR Ct values combined with age predicts invasive mechanical ventilation and mortality in hospitalized COVID-19 patients in a MERS-CoV-endemic country

BackgroundSeveral risk factors have been used to predict severity of coronavirus disease 2019 (COVID-19), real-time reverse transcriptase polymerase chain reaction (RT-PCR) cycle threshold (Ct) values have not been included. MethodsA retrospective analysis of laboratory-confirmed COVID-19 patients who were hospitalized between March 2 and September 1, 2020, in an academic hospital in Riyadh that serves as a Middle East respiratory syndrome coronavirus (MERS-CoV) referral center was conducted. Nasopharyngeal (NP) and endotracheal (ET) samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by RT-PCR, and viral load (VL) was determined based on the Ct values of E genes. The Ct values were distributed into four groups, with group Ct1 (≤19) indicating the highest VL and Ct4 (≥31) indicating the lowest VL. Univariate logistic regression was used to analyze age, gender, and comorbidities in relation to Ct groups for a primary endpoint of either invasive mechanical ventilation (IMV) or mortality. Significant variables were further analyzed by multivariate logistic regression. ResultsThe analysis included 728 patients hospitalized with COVID-19 (38% female; median age = 53 years; 41.3% diabetic; 39.4% hypertensive). Overall, 13.6% of these patients required IMV, and the in-hospital mortality rate was 15.5%. The IMV rate was higher in the Ct1 and Ct2 groups (15.2% and 15.5%, respectively) than in the Ct4 group (6.4%; p = 0.01). The mortality rate was also higher in the Ct1 and Ct2 groups (19.4% and 18.9%, respectively) than in the Ct4 group (8.9%; p = 0.02). The univariate analysis showed that lower Ct values and increasing age were associated with an increased risk of IMV (OR: 1.03; 95% CI: 1.01, 1.04; P < 0.0001) and mortality (OR: 1.04; 95% CI: 1.03, 1.06; P < 0.0001). The multivariate analysis showed that Ct1 was associated with the highest risk of mortality (OR: 2.29; 95% CI: 1.16, 5.52; P = 0.016), while Ct2 was associated with the highest risk of IMV (OR: 3.1; 95% CI: 1.47, 6.53; P = 0.003). ConclusionThe SARS-CoV-2 RT-PCR Ct values of hospitalized COVID-19 patients can be used as predictors of IMV and mortality, and this effect increases when combined with age. Clinicians could use these predictors to triage older patients for risk stratification and allocate IMV.

Validation of the COVID-19 IgG/IgM Rapid Test Cassette (BNCP - 402 and BNCP402) in a South African setting.

BackgroundDifferent diagnostic tools could improve early detection of coronavirus disease 2019 (COVID-19). A number of antibody-based serological point-of-care tests have been developed to supplement real-time reverse transcriptase polymerase chain reaction (RT-PCR)-based diagnosis. This study describes the validity of an antibody test, namely the immunoglobulin G (IgG)/immunoglobulin M (IgM) Rapid Test Cassette® (BNCP – 402 and BNCP402), manufactured by Spring Healthcare Services.MethodsA prospective cohort validation study was undertaken at Chris Hani Baragwanath Academic Hospital between 16 July 2020 and 12 August 2020. A total of 101 patients admitted as COVID-19 cases under investigation were included in the study. They were divided into two categories depending on time since symptom onset: testing performed within seven days (early cohort) and after seven days (late cohort). The rapid antibody test was compared to the RT-PCR.ResultsOverall, the test has a sensitivity and specificity of 85.2% and 80.0%, respectively, for a combination of IgG and IgM. Sensitivity and specificity of IgG testing alone were 81.5% and 85%. Sensitivity improved for testing with increasing time from symptom onset; however, specifity was not significantly different.ConclusionThe study data adds to the body of evidence that because of relatively low sensitivity and specificity, there is a limited role for antibody-based point-of-care testing in the acute phase of COVID-19 infection, as was the case with this IgG/IgM Rapid Test Cassette (BNCP – 402 and BNCP402). There may exist a role for such testing in patients recovered from prior COVID-19 infection or in seroprevalence studies; however, additional evaluations at later timepoints from symptom onset are required.

Assessment of Clinical and Virological Characteristics of SARS-CoV-2 Infection Among Children Aged 0 to 4 Years and Their Household Members

Few studies have prospectively assessed SARS-CoV-2 community infection in children aged 0 to 4 years. Information about SARS-CoV-2 incidence and clinical and virological features in young children could help guide prevention and mitigation strategies. To assess SARS-CoV-2 incidence, clinical and virological features, and symptoms in a prospective household cohort and to compare viral load by age group, symptoms, and SARS-CoV-2 lineage in young children, older children, and adults. This prospective cohort study enrolled 690 participants from 175 Maryland households with 1 or more children aged 0 to 4 years between November 24, 2020, and October 15, 2021. For 8 months after enrollment, participants completed weekly symptom questionnaires and submitted self-collected nasal swabs for SARS-CoV-2 qualitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) testing, quantitative RT-PCR testing, and viral lineage determination. For the analyses, SARS-CoV-2 Alpha and Delta lineages were considered variants of interest or concern. Sera collected at enrollment and at approximately 4 months and 8 months after enrollment were assayed for SARS-CoV-2 spike and nucleocapsid protein antibodies. Incidence, clinical and virological characteristics, and symptoms of SARS-CoV-2 infection by age group and correlations between (1) highest detected viral load and symptom frequency and (2) highest detected viral load and SARS-CoV-2 lineage. Among 690 participants (355 [51.4%] female and 335 [48.6%] male), 256 individuals (37.1%) were children aged 0 to 4 years, 100 (14.5%) were children aged 5 to 17 years, and 334 (48.4%) were adults aged 18 to 74 years. A total of 15 participants (2.2%) were Asian, 24 (3.5%) were Black, 603 (87.4%) were White, 43 (6.2%) were multiracial, and 5 (0.7%) were of other races; 33 participants (4.8%) were Hispanic, and 657 (95.2%) were non-Hispanic. Overall, 54 participants (7.8%) had SARS-CoV-2 infection during the surveillance period, including 22 of 256 children (8.6%) aged 0 to 4 years, 11 of 100 children (11.0%) aged 5 to 17 years, and 21 of 334 adults (6.3%). Incidence rates per 1000 person-weeks were 2.25 (95% CI, 1.28-3.65) infections among children aged 0 to 4 years, 3.48 (95% CI, 1.59-6.61) infections among children aged 5 to 17 years, and 1.08 (95% CI, 0.52-1.98) infections among adults. Children aged 0 to 17 years with SARS-CoV-2 infection were more frequently asymptomatic (11 of 30 individuals [36.7%]) compared with adults (3 of 21 individuals [14.3%]), with children aged 0 to 4 years most frequently asymptomatic (7 of 19 individuals [36.8%]). The highest detected viral load did not differ between asymptomatic vs symptomatic individuals overall (median [IQR], 2.8 [1.5-3.3] log10 copies/mL vs 2.8 [1.8-4.4] log10 copies/mL) or by age group (median [IQR] for ages 0-4 years, 2.7 [2.4-4.4] log10 copies/mL; ages 5-17 years: 2.4 [1.1-4.0] log10 copies/mL; ages 18-74 years: 2.9 [1.9-4.6] log10 copies/mL). The number of symptoms was significantly correlated with viral load among adults (R = 0.69; P < .001) but not children (ages 0-4 years: R = 0.02; P = .91; ages 5-17 years: R = 0.18; P = .58). The highest detected viral load was greater among those with Delta variant infections (median [IQR], 4.4 [3.9-5.1] log10 copies/mL) than those with infections from variants not of interest or concern (median [IQR], 1.9 [1.1-3.6] log10 copies/mL; P = .009) or those with Alpha variant infections (median [IQR], 2.6 [2.3-3.4] log10 copies/mL; P = .006). In this study, SARS-CoV-2 infections were frequently asymptomatic among children aged 0 to 4 years; the presence and number of symptoms did not correlate with viral load. These findings suggest that symptom screening may be insufficient to prevent outbreaks involving young children.

Pandemic driven innovation: A pilot evaluation of an alternative respiratory pathogen collection device

BackgroundThe nasopharyngeal swab is the gold standard collection method for COVID-19, but is invasive and painful, subsequently resulting in poor patient acceptance. This investigation explores the process of developing and validating an alternative respiratory pathogen collection device that relies on a nasopharyngeal irrigation mechanic. The primary objective was to determine if sufficient pathological sampling can be achieved by mechanism of nasopharyngeal irrigation that is proportionate to the nasopharyngeal swab method. MethodsThe study device was designed using Shapr3D modeling software and fabricated on a fused deposition modeling printer. Fifteen participants were enrolled with each receiving a saline nasopharyngeal washing using the study device. Specimen adequacy was evaluated by two real-time reverse transcriptase polymerase chain reaction (PCR) testing methods to identify the presence of the human RNase P gene. Results were evaluated quantitatively through interpretation of the PCR cycle threshold (Ct). ResultsAll 15 specimens tested positive for the presence of RNaseP, demonstrating specimen cellularity, adequate extraction of nucleic acids, and the absence of inhibitors to amplification. The mean Ct value was 29.5 (Applied Biosystems TaqPath RT-qPCR) and 30.7 (NECoV19). All participants felt the study device irrigation procedure was faster than the nasopharyngeal swab, with none experiencing any discomfort from the irrigation mechanism. ConclusionThe importance of early diagnostic testing and its role in countermeasures for communicable diseases such as COVID-19 is well established in the literature. Innovation to bolster our testing infrastructure is more important now than ever. This study was successful in developing and validating an alternative nasopharyngeal respiratory pathogen collection device that utilizes fluid debridement as its core mechanic. Data from this pilot study demonstrated the study device was successful in producing high-quality specimens for PCR testing. Feedback from the study participants was also in favor of the study device when compared to the nasopharyngeal swab.

Identification of a Five-MiRNA Expression Assay to Aid Colorectal Cancer Diagnosis

Introduction: One-third of colorectal cancer (CRC) patients present with advanced disease, and establishing control remains a challenge. Identifying novel biomarkers to facilitate earlier diagnosis is imperative in enhancing oncological outcomes. We aimed to create miRNA oncogenic signature to aid CRC diagnosis. Methods: Tumour and tumour-associated normal (TAN) were extracted from 74 patients during surgery for CRC. RNA was isolated and target miRNAs were quantified using real-time reverse transcriptase polymerase chain reaction. Regression analyses were performed in order to identify miRNA targets capable of differentiating CRC from TAN and compared with two endogenous controls (miR-16 and miR-345) in each sample. Areas under the curve (AUCs) in Receiver Operating Characteristic (ROC) analyses were determined. Results: MiR-21 (β-coefficient:3.661, SE:1.720, p = 0.033), miR-31 (β-coefficient:2.783, SE:0.918, p = 0.002), and miR-150 (β-coefficient:−4.404, SE:0.526, p = 0.004) expression profiles differentiated CRC from TAN. In multivariable analyses, increased miR-31 (β-coefficient:2.431, SE:0.715, p &lt; 0.001) and reduced miR-150 (β-coefficient:−4.620, SE:1.319, p &lt; 0.001) independently differentiated CRC from TAN. The highest AUC generated for miR-21, miR-31, and miR-150 in an oncogenic expression assay was 83.0% (95%CI: 61.7–100.0, p &lt; 0.001). In the circulation of 34 independent CRC patients and 5 controls, the mean expression of miR-21 (p = 0.001), miR-31 (p = 0.001), and miR-150 (p &lt; 0.001) differentiated CRC from controls; however, the median expression of miR-21 (p = 0.476), miR-31 (p = 0.933), and miR-150 (p = 0.148) failed to differentiate these groups. Conclusion: This study identified a five-miRNA signature capable of distinguishing CRC from normal tissues with a high diagnostic test accuracy. Further experimentation with this signature is required to elucidate its diagnostic relevance in the circulation of CRC patients.

Identification of proline-rich protein 11 as a major regulator in mouse spermatogonia maintenance via an increase in BMI1 protein stability.

Spermatogenesis accompanied by self-renewal and differentiation of spermatogonia under complicated regulation is crucial for male fertility. Our previous study demonstrated that the loss of the B-lymphoma Mo-MLV insertion region 1 (BMI1) could cause male infertility and found a potential interaction between BMI1 and proline-rich protein 11 (PRR11); however, the specific co-regulatory effects of BMI1/PRR11 on spermatogonia maintenance remain unclear. The expression of PRR11 was downregulated in a mouse spermatogonia cell line (GC-1) via transfection with PRR11-siRNAs, and PRR11 knockdown was verified by real-time reverse transcriptase polymerase chain reaction (RT-qPCR). The proliferative activity of GC-1 cells was determined using the cell counting kit (CCK-8), colony formation, and 5-ethynyl-2-deoxyuridine (EdU) incorporation assay. A Transwell assay was performed to evaluate the effects of PRR11 on GC-1 cell migration. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to measure GC-1 cell apoptosis. Furthermore, co-immunoprecipitation, RT-qPCR, and western blot analyses were used for investigating the regulatory mechanisms involved in this regulation. It was found that downregulation of PRR11 could cause a marked inhibition of proliferation and migration and induced apoptosis in GC-1 cells. Moreover, silencing of PRR11 obviously led to a reduction in the BMI1 protein level. PRR11 was found to interact with BMII at the endogenous protein level. PRR11 knockdown produced a decrease in BMI1 protein stability via an increase in BMI1 ubiquitination after which derepression in the transcription of protein tyrosine phosphatase receptor type M (Ptprm) occurred. Importantly, knockdown of Ptprm in PRR11-deficient GC-1 cells led to a reversal of proliferation and migration of GC-1 cells. This study uncovered a novel mechanism by which PRR11 cooperated with BMI1 to facilitate GC-1 maintenance through targeting Ptprm. Our findings may provide a better understanding of the regulatory network in spermatogonia maintenance.

Investigation of Polymorphisms of ACEII Gene in People With Coronavirus With Severe and Mild Symptoms or Asymptomatic

Introduction: Angiotensin-converting enzyme 2 (ACE2) is the central receptor of coronavirus disease 2019 (COVID-19) in host cells. Genetic polymorphisms in the ACE2 gene may promote cardiovascular disease and systemic inflammatory injury in a patient affected by COVID-19. Thus, the genetic background may account for the substantial inter-individual diversity in illness susceptibility or severity. Our study was conducted to find a significant relationship between ACE2 rs4646142 and rs2285666 polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: In this study, we randomly selected 230 samples, including 76 patients with severe clinical symptoms and 154 patients with mild clinical symptoms (the positive case of COVID-19 was confirmed by real-time reverse transcriptase polymerase chain reaction [RT-PCR] assay). Then, we performed DNA extraction and investigated the polymorphisms of rs2285666 and rs4646142 by RFLP-PCR method with TaqI and Alu1 restriction enzymes. Results: The study population included 107 men and 123 women, and the mean (±SD) age of the participants was 42.66±10.2. First, the levels of IgM and IgG were examined, and a significant association was observed in the level of IgM between the two groups of COVID-19 patients with mild and severe symptoms, as opposed to IgG. Meanwhile, no significant difference was observed between ACE2 rs4646142 and rs2285666 polymorphisms and the severity of COVID-19. Conclusion: To better understand the genetic variations in people’s susceptibility to COVID-19, this study was designed to evaluate the association between various ACE2 polymorphisms and the infection risk of SARS-CoV-2. However, no statistical difference was discovered.

Epidemiology of Hepatitis C Virus Infection Among High-risk Populations in Northeastern Iran

Background: Chronic hepatitis C virus (HCV) infection affects 58 million people globally. The frequency of HCV infection in the general Iranian population is less than 0.5%; however, a concentrated epidemic was reported among people who use drugs, particularly those with a history of drug injection. Objectives: This cross-sectional study was performed to assess the prevalence of HCV infection among high-risk groups outside of prison in northeastern Iran. Methods: A total of 962 participants in Razavi Khorasan province were enrolled from 2018 to 2022. They included drug users referred to drug treatment and harm reduction centers and individuals with a history of crimes such as drug use or imprisonment who worked in a private industrial unit. Serum anti-HCV antibodies were assessed using a rapid or ELISA kit, and seroreactive samples were confirmed by single-step reverse transcriptase or qualitative real-time polymerase chain reactions. A P-value &lt; 0.05 was considered statistically significant. Results: The mean age of 707 males and 255 females was 39.8 ± 10.2 years. Anti-HCV antibodies were detected from 129 samples (13.41%), of which 117 were available for polymerase chain reaction testing. HCV RNA was detected in 88 cases (75.2%); the total viremia rate was calculated as 9.26% (88/950). Logistic regression analysis revealed that HCV infection among drug users was significantly associated with older age (P = 0.002), being single (P = 0.009), and history of drug injection (P &lt; 0.001) or incarceration (P = 0.04). Conclusions: The findings showed a considerably high prevalence of HCV infection among people who use drugs in northeastern Iran. To achieve the global goal of viral hepatitis elimination by 2030, we strongly recommend stricter screening and treatment of this infection among such hard-to-access populations in Iran.

A Review on Newer Detection Technologies for the Diagnosis of COVID-19 Infection

COVID-19 is a sickness brought about by coronavirus responsible for causing simple to extreme complications in people. COVID-19 first case was seen in Wuhan, Hubei Province, China, on December, 2019. The World Health Organization (WHO) to pronounce it as a worldwide pandemic on March 11, 2020, as the pandemic has spread quickly all through the world. Regardless of extensive endeavors made to contain the infection, the infection has proceeded with its pervasiveness in numerous nations with changing levels of clinical signs. Henceforth, in this report, we discuss the various strategies, for example, serological and nucleic acid-based procedures which are accessible for the determination and successful counteraction of coronavirus. With expanding the rate of coronavirus cases, the precise and early identification of the COVID-19 is the need of great importance for viable avoidance with treatment and just as to check its spread. Reverse transcriptase-real time polymerase chain reaction (PCR) measures are viewed as the highest quality level for the early identification of infection. This diagnostic technique is being utilized worldwide with recommendations from WHO and Center for Disease Control and Prevention. Reverse transcriptase real-time quantitative PCR (RT-qPCR) is being done compulsory before any medical procedures and major surgeries for early detection, prevention, and management in due time course. Rapid antigen test is also a screening test used widely in hospitals for screening of COVID-19 and before the admission in hospitals. Other nucleic acid amplification test widely done for the detection of COVID-19 are RT-qPCR, next-generation sequencing, clustered regularly interspaced short pallindromic repeats, reverse transcription -loop-mediated isothermal amplification, droplet digital PCR. Some immunological tests are lateral flow, ELISA.

SC2Net: A Novel Segmentation-Based Classification Network for Detection of COVID-19 in Chest X-Ray Images.

The pandemic of COVID-19 has become a global crisis in public health, which has led to a massive number of deaths and severe economic degradation. To suppress the spread of COVID-19, accurate diagnosis at an early stage is crucial. As the popularly used real-time reverse transcriptase polymerase chain reaction (RT-PCR) swab test can be lengthy and inaccurate, chest screening with radiography imaging is still preferred. However, due to limited image data and the difficulty of the early-stage diagnosis, existing models suffer from ineffective feature extraction and poor network convergence and optimisation. To tackle these issues, a segmentation-based COVID-19 classification network, namely SC2Net, is proposed for effective detection of the COVID-19 from chest x-ray (CXR) images. The SC2Net consists of two subnets: a COVID-19 lung segmentation network (CLSeg), and a spatial attention network (SANet). In order to supress the interference from the background, the CLSeg is first applied to segment the lung region from the CXR. The segmented lung region is then fed to the SANet for classification and diagnosis of the COVID-19. As a shallow yet effective classifier, SANet takes the ResNet-18 as the feature extractor and enhances high-level feature via the proposed spatial attention module. For performance evaluation, the COVIDGR 1.0 dataset is used, which is a high-quality dataset with various severity levels of the COVID-19. Experimental results have shown that, our SC2Net has an average accuracy of 84.23% and an average F1 score of 81.31% in detection of COVID-19, outperforming several state-of-the-art approaches.

Pan-cancer analysis identifies CDCA3 as a novel prognostic marker associated with immune infiltration in lung adenocarcinoma through bioinformatics analysis.

BackgroundLung adenocarcinoma (LUAD) is the most common subtype of lung malignancy. However, the expression of cell division cycle-associated protein-3 (CDCA3) and its significance in LUAD remain unclear. In this study, we investigated the functional role of CDCA3 in LUAD through bioinformatics analysis and expected to provide a new direction for clinical treatment.MethodsThe expression of CDCA3 was analyzed by online database. The association between the expression of CDCA3 and clinical parameters with LUAD was explored in TCGA. Survival and independent prognostic analysis were performed by TCGA database and the GSE30219 and GSE31210 datasets. Furthermore, Enrichment analyses were conducted to analyze the functions of CDCA3. Afterward, the relationship between CDCA3 and immune infiltration was investigated. Additionally, a competing endogenous RNA (ceRNA) regulatory network related to CDCA3 was constructed. Finally, CDCA3 expression was validated in clinical tissues by immunohistochemistry (IHC), real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and western blotting (WB).ResultsCDCA3 expression was upregulated in 20 tumors and was significantly higher in LUAD compared with normal tissues in3 datasets. In addition, CDCA3 was significantly correlated with age, gender, stage, N, and smoking status. Kaplan-Meier survival curves showed that LUAD samples with higher CDCA3 expression were associated with poorer overall survival (OS) and disease-free survival (DFS). Univariate Cox regression analysis showed that the p value of CDCA3 expression was less than 0.05 (P<0.05) and it appeared in the results of multivariate Cox regression analysis (HR ≥1), indicating that CDCA3 can be used as an independent prognostic factor for LUAD. Intriguingly, Gene Set Enrichment Analysis (GSEA) suggested that CDCA3 was correlated with DNA-related terms and metabolic-related pathways in LUAD. CDCA3 expression was correlated with four immune scores and 14 immune cells in different groups. Next, a ceRNA network was constructed with CDCA3, and the experimental results of IHC, qRT-PCR, and WB were consistent with the bioinformatic analysis.ConclusionsCDCA3 could serve as a prognostic biomarker for LUAD.

Utility of Molecular Analysis of Peritoneal Fluid in Staging Laparoscopy of Advanced Esophagogastric Junction and Gastric Cancer Prior to Neoadjuvant Treatment.

Molecular analysis of peritoneal fluid in staging laparoscopy of gastric cancer is performed to improve the detection of free intraperitoneal tumor cells. Nevertheless, its significance is controversial, especially in patients with negative cytology but positive molecular analysis. The aim of this study was to analyze the sensitivity of molecular analysis and its prognostic value. A retrospective analysis from April 2011 to October 2019 was performed. Cytology (Cyt) and molecular analysis were analyzed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) of the carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) tumor makers. During the study period, 138 staging laparoscopies were performed. Macroscopic carcinomatosis was found in 12.3%. Of the remaining 87.7%, 9.9% were Cyt + and 11.6% were Cyt- RT-PCR + . Of the latter, 9 responded to chemotherapy and underwent radical surgery. The sensitivity of cytology and molecular analysis was 0.70 and 0.76, respectively (p = 0.67). The 2-year overall survival (OS) of Cyt- RT-PCR + vs. Cyt + was similar (p = 0.1). The 2-year OS of Cyt-RT-PCR + subgroup who underwent radical surgery vs. Cyt-RT-PCR- patients was similar (p = 0.69), but disease-free survival was shorter in the first group (p = 0.005). Our results show that the sensitivity of molecular analysis is similar to that of cytology. The prognostic value of positive molecular analysis was similar to positive cytology in terms of 2-year overall survival, except in the subgroup of operated patients in whom the overall survival was similar to that of those with a negative molecular analysis, albeit with a shorter disease-free survival.

Detection of circulating Influenza A and B virus by real-time reverse transcriptase polymerase chain reaction at a tertiary care center in Chennai, Tamil Nadu, India

Objective: The aim of this study was to determine the proportion of influenza-like illnesses (ILIs) caused by influenza A and influenza A H1N1 and to determine the proportion of influenza B in a smaller group of samples with ILIs and influenza A H1N1 negative by qualitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) using TaqMan-based assay at a tertiary health-care center in Chennai, Tamil Nadu. Material and Methods: Laboratory samples of participants from all age groups who had ILIs were included in this study. The study was conducted from January 2018 to January 2019 at a tertiary health-care center in Chennai, Tamil Nadu. The sample size of the study was 1755. This is a cross-sectional study. RNA extraction was performed using QIAamp Viral RNA Mini Kit (Qiagen, U.S.A) as per the manufacturer instructions. The assay is a TaqMan®-based real-time detection of circulating novel influenza A H1N1 and H3N2. Real-time PCR for influenza B virus was performed in influenza A H1N1-negative patients using artus Infl/H1 LC/RG RT-PCR kit (Cat 4523003, Qiagen, Germany). Samples that had a crossing threshold value 15–35 cycles were considered positive. Results: The majority of the participants were in the pediatric and young adult age group (&lt;30 years) (41%). The incidence of influenza was in the range of 32.34–41% up to 60 years. Beyond 60 years, the frequency of detection reduced to 25.9%, and in those above 71 years, it was 22.3%. About 45.4% (n = 798) were positive for influenza A, of which 32.7% (n = 575) were positive for influenza A H1N1. Both influenza A H1N1 and influenza A other than H1N1 incidence started to rise in September and spiked between October and December. Among patients with persistent ILI, screening for influenza B was done in 48 samples. Among 48 samples, 18% (n = 8) had influenza B. Conclusion: The need for increased vaccination is demonstrated through the high influenza A H1N1 positivity rate among pediatric patients with ILIs. Detection of influenza B among influenza A H1N1-negative individuals demonstrates the need for influenza B screening. Incidence of influenza is highest in cooler months. The implementation of vaccination against influenza before the beginning of the cooler seasons could possibly reduce the burden of influenza on the health-care system. The importance of surveillance for the continued screening of influenza could be expanded in the private sector as a majority of the disease burden is observed in that sector.

SPR Based Biosensing Chip for COVID-19 Diagnosis-A Review.

Surface Plasmon Resonance (SPR) techniques are highly accurate in detecting biomolecular like blood group measurement, food adulteration, milk adulteration and recently developing as a rapid detection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. In order to validate the clinical diagnosis, Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of nasopharyngeal swabs has been utilized, which is time consuming and expensive. For fast and accurate detection of the SARS-CoV-2 virus, SPR based biosensing chips are described in this review article. SPR sensors have the potential to be employed for fast, accurate, and portable SARS-CoV-2 virus diagnosis. To combat the SARS-CoV-2 pandemic, there is considerable interest in creating innovative biosensors that are quick, reliable, and sensitive for COVID-19 diagnosis.

Does Molecular Profiling of KRAS-Mutant Non-Squamous Non-Small Cell Lung Cancer (NSCLC) Help in Treatment Strategy Planning?

Background: Several studies suggest that patients with KRAS-mutant NSCLC fail to benefit from standard systemic therapies and do not respond to EGFR inhibitors. Most recently, KRAS 12c data suggest specific treatment for improving ORR and OS. There is a clear need for therapies specifically developed for these patients. Moreover, data that might be suggestive of a response to specific therapies, such as BRCA1, are needed, and two mutations that were studied in other malignancies show more response to PARP inhibitors. Molecular profiling has the potential to identify other potential targets that may provide better treatment and novel targeted therapy for KRAS-mutated NSCLC. Methods: We purified RNA from archived tissues of patients with stage I and II NSCLC with wild-type (wt) and mutant (mt) KRAS tumors; paired normal tissue adjacent to the tumor from 20 and 17 patients, respectively, and assessed, using real-time reverse transcriptase–polymerase chain reaction (RT-PCR), the expression of four genes involved in DNA synthesis and repair, including thymidylate synthase (TS), BRCA1, ECCR1, RAP80, and the proto-oncogene SRC. Additionally, we assessed the expression of PD-L1 in mt KRAS tumors with immunohistochemistry using an antibody against PD-L1. Results: Our results show that in mt KRAS tumors, the level of expression of ERCC1, TS, and SRC was significantly increased in comparison to paired normal lung tissue (p ≤ 0.04). The expression of BRCA1 and RAP80 was similar in both mt KRAS tumors and paired normal tissue. Furthermore, the expression of BRCA1, TS, and SRC was significantly increased in wt KRAS tumors relative to their expression in the normal lung tissue (p < 0.044). The expression of ERCC1 and RAP80 was similar in wt KRAS tumors and paired normal tissue. Interestingly, SRC expression in mt KRAS tumors was decreased in comparison to wt KRAS tumors. Notably, there was an expression of PD-L1 in the tumor and stromal cells in a few (5 out of 20) mt KRAS tumors. Our results suggest that a greater ERCC1 expression in mt KRAS tumors might increase platinum resistance in this group of patients, whereas the greater expression of BRCA1 in wt KRAS tumor might be suggestive of the sensitivity of taxanes. Our data also suggest that the combination of an SRC inhibitor with a TS inhibitor, such as pemetrexed, might improve the outcome of patients with NSCLC and in particular, patients with wt KRAS tumors. PD-L1 expression in tumors, and especially stromal cells, suggests a better outcome. Conclusion: mt KRAS NSCLC patients might benefit from a treatment strategy that targets KRAS in combination with therapeutic agents based on pharmacogenomic markers, such as SRC and BRCA1. mt KRAS tumors are likely to be platinum-, taxane-, and pemetrexed-resistant, as well as having a low level of PD-L1 expression; thus, they are less likely to receive single-agent immunotherapy, such as pembrolizumab, as the first-line therapy. wt KRAS tumors with BRCA1 positivity tend to be sensitive to taxane therapy and, potentially, platinum. Our results suggest the need to develop targeted therapies for KRAS-mutant NSCLC or combine the targeting of oncogenic KRAS in addition to other therapeutic agents specific to the molecular profile of the tumor.