Reverse Transcriptase-polymerase Chain Reaction Assay Research Articles

Influence of diverse pH and temperatures on the gene expression of toxin-antitoxin systems in Klebsiella pneumoniae

Background: The type II toxin-antitoxin system consists of tightly linked genetic units that produce a toxin and its specific antitoxin. Under typical circumstances, the antitoxin neutralizes the toxic effects caused by the toxin. However, the toxin-antitoxin systems during stress regulate bacterial growth and persistence by affecting translation or DNA replication. This study aimed to assess how variations in temperature and pH stress influence the regulation of type II toxin-antitoxin gene expression within Klebsiella pneumoniae isolates.Methods: Sixty-five Klebsiella pneumoniae isolates were identified by the VITEK®2 compact system and confirmed identity by Polymerase Chain Reaction assay by amplified rpoB gene (Housekeeping gene). The isolates were subjected to Reverse Transcriptase-polymerase chain Reaction assay to determine the transcriptional variations of the type II toxin-antitoxin system genes at diverse temperature and pH values.Results: The results of the current study demonstrated higher expression levels of the antitoxin hipB gene at (37℃, 30℃, 44℃, pH 7, pH 9), mqsR toxin gene at (37℃, 30℃, pH 7, pH 5, pH 11) and relE toxin gene at (pH 5, pH 11).Conclusion: These findings suggest the possible influence of pH and temperature on toxin-antitoxin systems, which might enhance bacterial antibiotic tolerance and persistence.Keywords: Toxin-antitoxin systems; pH stress; Temperature stress; Klebsiella pneumoniae; Gene expression; Reverse Transcriptase PCR

From the discovery of a novel arepavirus in diseased arecanut palms (Areca catechu L.) in India to the identification of known and novel arepaviruses in bee and plant transcriptomes through data-mining

Arecanut palm is a commercially important plantation crop valued for its nut. In this investigation, we report the discovery of a putative novel arepavirus, named areca palm necrotic ringspot virus 2 (ANRSV2), in necrotic ringspot diseased areca palms in Bantwal, Dakshina Kannada, Karnataka, India through RNA-sequencing and transmission electron microscopy. Further, the presence of ANRSV2 in the diseased samples was confirmed through reverse transcriptase-polymerase chain reaction assays. In addition, by mining public domain transcriptome data for arepaviral sequences, we identified a putative novel arepavirus in Psychotria rubra, a non-palm host. We recovered the genome sequences of the areca palm necrotic ringspot virus in honey bees, tomato, Onobrychis viciifolia, and Rhamnus heterophylla. These findings broaden our comprehension of arepaviral diversity and host range, and suggest an intriguing possibility of pollen-mediated arepaviral transmission that necessitates empirical validation. Further studies are needed to understand the biology of identified putative novel arepaviruses.

A new strategy of multiplex real-time RT-qPCR assay for differentiating Omicron variants from other SARS-CoV-2 lineages

The global dissemination of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) Omicron variants has been characterized by a distinct 9-base pair (bp) deletion within the nucleocapsid (N) gene, a genomic feature absent in previously circulating variants. In response to this unique characteristic, we have developed an innovative amplicon-competition strategy to differentiate Omicron lineages from other SARS-CoV-2 variants using a real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay. This novel methodology demonstrates high specificity and sensitivity, effectively detecting viral loads ranging from 1500 to 1.5x106 copies per milliliter in nasopharyngeal swab specimens. The potential applications of this approach extend beyond current needs, providing a robust framework for the genomic characterization of future variants exhibiting unique genetic signatures.

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

BackgroundThe 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.MethodsThis 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.ResultsA 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).ConclusionThis 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.

Diagnostic Precision of Molecular Testing for Severe Acute Respiratory Syndrome Coronavirus 2 versus Chest Computer Tomography

Abstract Background: Molecular detection is considered benchmark criteria for SARS-CoV-2 diagnosis. Both computer tomography (CT) chest imaging and reverse transcriptase polymerase chain reaction (RTPCR) can give false-negative test results in the initial state of coronavirus infection. This study aimed to assess the precision of computer tomography (CT) chest imaging and RTPCR assay and to compare the role of CT chest imaging and molecular testing (RTPCR) in the early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Methods: Retrospective analysis of 588 clinically suspected coronavirus disease cases was performed over from April 2021 to June 2021. RTPCR test result was compared with chest computer tomography and diagnostic precision of computer tomography (CT) scan was determined using RTPCR as reference standard. Categorical variables were statistically analyzed as frequencies and percentages. Results: Out of 465 patients who tested positive by chest computer tomography (CT) scan findings, 132 (28.39%) tested RTPCR positive and 333 (71.61%) tested RTPCR negative. 306/465 (65.81%) cases showed moderate-to-severe infection and 159/465 (34.19%) had mild infection. Diagnostic precision of imaging chest computer tomography (CT) scan was observed to be 38.44%. Low sensitivity of RTPCR detection method in the early identification of coronavirus disease was observed. Conclusion: The use of computer tomography (CT) scan in the early detection of coronavirus disease is supplemental to RTPCR assay and is limited to the detection of laboratory-confirmed coronavirus disease cases as imaging characteristics of SARS-CoV-2 pneumonia superimposed with other types of pneumonia.

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.

AGXT2 Suppresses the Proliferation and Dissemination of Hepatocellular Carcinoma Cells by Modulating Intracellular Lipid Metabolism.

Alanine glyoxylate aminotransferase (AGXT) family members are crucial in cancer processes, but their role in hepatocellular carcinoma (HCC) metabolism is unclear. This study investigates AGXT2's function in HCC. AGTX2 expression was studied using bioinformatics, real-time reverse transcriptase-polymerase chain reaction (RT-qPCR), Western blot, and Enzyme-linked immunosorbent assay (ELISA). A lentivirus-induced AGTX2 overexpression cell model was analyzed with RNA sequencing (RNA-seq) and liquid chromatography-mass spectrometry (LC-MS). Cholesterol levels were confirmed by Oil Red O staining. AGTX2 effects were evaluated through cell cycle analysis, wound healing, and transwell migration assays.Tumorigenic effects were observed in NOD-SCID IL2Rγnull (NTG) mice in subcutaneous experiments. Protein interaction was examined through co-immunoprecipitation methods. We observed a significant reduction in AGXT2 mRNA and protein levels in both HCC tumor tissues and serum samples from patients with liver cancer, which was associated with a worse prognosis. The activation of AGXT2 has been shown to effectively decrease cholesterol levels in liver cancer cells, serving as an antagonist in the cholesterol metabolism pathway. An increase in low density lipoprotein receptor (LDLR) mRNA was noted in cells overexpressing AGXT2, accompanied by a decrease in LDLR protein and an elevation in proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and protein levels. Molecular docking and co-immunoprecipitation experiments further elucidated the interaction between AGXT2 and LDLR proteins. AGXT2 was observed to suppress the migratory and invasive capabilities of HCC cells, inducing cell cycle arrest in the G2/M phase. AGXT2 activation inhibited subcutaneous liver cancer tumor growth in NTG mice. AGXT2 was found to lower cholesterol levels in liver cancer cells, possibly through interactions with the LDLR protein and modulation of PCSK9-mediated LDLR degradation. This mechanism may impede cholesterol transport to liver cancer cells, thereby suppressing their growth and metastasis.

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.