Reverse Transcriptase-polymerase Chain Reaction Results Research Articles

Detection of Enteropathogens in Human Immunodeficiency Virus and Non-Human Immunodeficiency Virus-Infected Children with Acute Diarrhea in an Indonesian Tertiary Hospital Using Multiplex Real-Time Polymerase Chain Reaction.

Diarrhea is one of the leading causes of mortality in children living in developing countries. The etiology of acute diarrhea in each healthcare center varies depending on place, time, and population. This study aimed to identify pathogen patterns in human immunodeficiency virus (HIV)-infected and non-HIV children suffering from acute diarrhea, using multiplex real time reverse transcriptase polymerase chain reaction (RT-PCR), in an Indonesian tertiary hospital. This cross-sectional study was conducted at Dr. Cipto Mangunkusumo National Hospital from March 2019 to April 2020. The study showed that multiplex RT-PCR results were positive in 58.9% of the specimens, with more positive results in HIV-infected children than in non-HIV-infected children (70% vs. 54.7%). Altogether 72 enteropathogens were detected from all specimens. Enteropathogens in non-HIV children with acute diarrhea consisted of bacteria (70.6%) and viruses (29.4%) with a predominance of enteroaggregative Escherichia coli (25.4%), followed by Campylobacter spp. (11.8%), enteropathogenic E. coli (9.8%), Norovirus GII (7.8%), and Clostridium difficile (7.8%). Enteropathogens in HIV-infected children consisted of viruses (57.1%), bacteria (28.6%), and parasites (14.3%) comprising Norovirus GII (24%), Cryptosporidium spp. (14.3%), Campylobacter spp. (14.3%), Norovirus GI (14.3%), and Astrovirus (14.3%). Cryptosporidium spp. was the only parasite found in this study and was found only in HIV-infected children. In non-HIV children with acute diarrhea, most pathogens were invasive bacteria, while in HIV-infected children, more viral and parasite infections occurred, primarily caused by opportunistic pathogens. The pattern of enteropathogens can help clinicians determine further examinations and appropriate empirical antimicrobial therapy for the patient.

The relationship between thorax computed tomography findings and prognosis in patients diagnosed with COVID-19.

This study aimed to demonstrate the relationship between thorax computed tomography (CT) findings at the time of admission and prognosis using a semiquantitative CT severity scoring system in patients diagnosed with coronavirus disease 2019 (COVID-19) who tested positive for reverse transcriptase polymerase chain reaction (RT-PCR). A total of 305 patients aged 18 years and older who were diagnosed with COVID-19 confirmed by RT-PCR and underwent thorax CT at the time of admission, were included in the study between March and July 2020. The demographic data of the patients, their presenting complaints at the time of admission, RT-PCR results, and thorax CT images were scanned retrospectively from electronic medical records. Lesions on thorax CT were evaluated for the presence of ground glass opacity, consolidation, and septal thickening and scoring. No significant relationship was found between mortality and CT score or other parameters. A significant relationship was found between admission to the intensive care unit and CT scoring (p=0.014), aortic diameter (p=0.032), chronic pulmonary disease (p=0.004), halo sign (p=0.031), mortality (p<0.001), fever (p=0.038), and dyspnea (p=0.031). A statistically significant difference was detected in the score parameter between discharged patients and intensive care unit patients who survived and those who died (p<0.001). In the parameter of the number of lobes, a statistically significant difference was found only between discharged patients and intensive care unit patients who survived (p=0.016). Thorax CT is an advisor for early diagnosis, treatment, and prognosis assessment of the disease. Semiquantitative CT severity scoring can provide valuable information about the prognosis of the patient.

First Report of Viral Nervous Necrosis in Asian Sea Bass, Lates Calcarifer Cultured in Sri Lanka

Viral Nervous Necrosis (VNN) caused by Betanodavirus is a devastating disease in aquatic animals. The virus infects both marine and freshwater fish worldwide. Vacuolating necrosis of neural cells of the brain, the retina of the eye, and the spinal cord of the infected fish are the primary histological lesions of the condition. It causes up to 100% mortality in larvae and juvenile fish and can cause significant death in adult fish. The present study detected viral nervous necrosis in larvae and fry of Asian sea bass (Lates calcarifer) with progressive mortality of up to 95% in one week during the Northeast monsoon when the mean water temperature was 27 to 29°C. Histopathological examination of the moribund fish revealed extensive vacuolation and gliosis in the olfactory bulb, the optic lobe of the forebrain, and the inner and outer layer of the retina. Furthermore, tissues of the brain and the retina had intracellular inclusion bodies suggesting viral etiology, further justified by the negative results of the bacterial and parasitic examinations. The Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) test additionally confirms the etiology diagnosis using specific primers designed previously. The histopathology and RT-PCR results suggest that the mortalities of Asian sea bass were due to the VNN. The present finding is the first report of the VNN associated with mass mortalities in Asian seabass cultured in Sri Lanka. These crucial findings emphasize the need for quarantine and control strategies to prevent the spread of the virus and outbreak of the disease.

Viral Detection by Reverse Transcriptase Polymerase Chain Reaction in Upper Respiratory Tract and Metagenomic RNA Sequencing in Lower Respiratory Tract in Critically Ill Children With Suspected Lower Respiratory Tract Infection.

Viral lower respiratory tract infection (vLRTI) contributes to substantial morbidity and mortality in children. Diagnosis is typically confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) of nasopharyngeal specimens in hospitalized patients; however, it is unknown whether nasopharyngeal detection accurately reflects presence of virus in the lower respiratory tract (LRT). This study evaluates agreement between viral detection from nasopharyngeal specimens by RT-PCR compared with metagenomic next-generation RNA sequencing (RNA-Seq) from tracheal aspirates (TAs). This is an analysis of of a seven-center prospective cohort study. Seven PICUs within academic children's hospitals in the United States. Critically ill children (from 1 mo to 18 yr) who required mechanical ventilation via endotracheal tube for greater than or equal to 72 hours. We evaluated agreement in viral detection between paired upper and LRT samples. Results of clinical nasopharyngeal RT-PCR were compared with TA RNA-Seq. Positive and negative predictive agreement and Cohen's Kappa were used to assess agreement. Of 295 subjects with paired testing available, 200 (68%) and 210 (71%) had positive viral testing by RT-PCR from nasopharyngeal and RNA-Seq from TA samples, respectively; 184 (62%) were positive by both nasopharyngeal RT-PCR and TA RNA-Seq for a virus, and 69 (23%) were negative by both methods. Nasopharyngeal RT-PCR detected the most abundant virus identified by RNA-Seq in 92.4% of subjects. Among the most frequent viruses detected, respiratory syncytial virus demonstrated the highest degree of concordance (κ = 0.89; 95% CI, 0.83-0.94), whereas rhinovirus/enterovirus demonstrated lower concordance (κ = 0.55; 95% CI, 0.44-0.66). Nasopharyngeal PCR was more likely to detect multiple viruses than TA RNA-Seq (54 [18.3%] vs 24 [8.1%], p ≤ 0.001). Viral nucleic acid detection in the upper versus LRT reveals good overall agreement, but concordance depends on the virus. Further studies are indicated to determine the utility of LRT sampling or the use of RNA-Seq to determine LRTI etiology.

Performance of Rapid Antigen Tests to Detect Symptomatic and Asymptomatic SARS-CoV-2 Infection : A Prospective Cohort Study.

The performance of rapid antigen tests (Ag-RDTs) for screening asymptomatic and symptomatic persons for SARS-CoV-2 is not well established. To evaluate the performance of Ag-RDTs for detection of SARS-CoV-2 among symptomatic and asymptomatic participants. This prospective cohort study enrolled participants between October 2021 and January 2022. Participants completed Ag-RDTs and reverse transcriptase polymerase chain reaction (RT-PCR) testing for SARS-CoV-2 every 48 hours for 15 days. Participants were enrolled digitally throughout the mainland United States. They self-collected anterior nasal swabs for Ag-RDTs and RT-PCR testing. Nasal swabs for RT-PCR were shipped to a central laboratory, whereas Ag-RDTs were done at home. Of 7361 participants in the study, 5353 who were asymptomatic and negative for SARS-CoV-2 on study day 1 were eligible. In total, 154 participants had at least 1 positive RT-PCR result. The sensitivity of Ag-RDTs was measured on the basis of testing once (same-day), twice (after 48 hours), and thrice (after a total of 96 hours). The analysis was repeated for different days past index PCR positivity (DPIPPs) to approximate real-world scenarios where testing initiation may not always coincide with DPIPP 0. Results were stratified by symptom status. Among 154 participants who tested positive for SARS-CoV-2, 97 were asymptomatic and 57 had symptoms at infection onset. Serial testing with Ag-RDTs twice 48 hours apart resulted in an aggregated sensitivity of 93.4% (95% CI, 90.4% to 95.9%) among symptomatic participants on DPIPPs 0 to 6. When singleton positive results were excluded, the aggregated sensitivity on DPIPPs 0 to 6 for 2-time serial testing among asymptomatic participants was lower at 62.7% (CI, 57.0% to 70.5%), but it improved to 79.0% (CI, 70.1% to 87.4%) with testing 3 times at 48-hour intervals. Participants tested every 48 hours; therefore, these data cannot support conclusions about serial testing intervals shorter than 48 hours. The performance of Ag-RDTs was optimized when asymptomatic participants tested 3 times at 48-hour intervals and when symptomatic participants tested 2 times separated by 48 hours. National Institutes of Health RADx Tech program.

Lessons from the Lab: SARS-CoV-2 Detection Rate amongst the Vaccinated Travel Lane and Non-Vaccinated Travel Lane Travellers into Malaysia.

The vaccinated travel lane (VTL) between Malaysia and Singapore was implemented to facilitate travelling between countries without the need for quarantine. i) Observe the rate of positive SARS-CoV-2 test results amongst inbound international travellers; ii) Explore possible factors associated with the outcome of test results between VTL and non-VTL travellers. This is a retrospective cross-sectional study involving air travellers arriving in Malaysia via the Kuala Lumpur International Airport (KLIA) or Kuala Lumpur International Airport 2 (KLIA2) who were tested for SARS-CoV-2 by reverse transcriptase polymerase chain reaction (RT-PCR) from 29 November 2021 to 15 March 2022. Subject demographics and RT-PCR results were retrieved from the laboratory information system and statistically analysed. Out of 118,902 travellers, mostly were Malaysian nationals (62.7%) and VTL travellers (68.2%) with a median age of 35 years old. A total of 699 (0.6%) of travellers tested positive on arrival, out of which 70.2% had cycle threshold (Ct) values > 30 (70.8% of VTL and 70.0% of non-VTL cohorts). Non-VTL travellers were 4.5 times more likely to test positive compared with VTL travellers (1.25% versus 0.28%; P < 0.001). Tighter entry requirements including vaccination status and testing frequency, the use of sensitive detection methods on arrival and similar public health policies between countries may have contributed to the VTL being a safe and cost-effective mode of travel.

AN ANALOGY OF HRCT CHEST AND ANTIGEN ASSAY WITH RT- PCR TEST IN THE DIAGNOSIS OF COVID-19

Introduction We aim to compare Rapid Antigen Test and HRCT chest with Reverse Transcriptase-Polymerase chain reaction (RT-PCR) for diagnosis of COVID-19. MethodsThis observational cross-sectional analytical study included patients tested with RT- PCR and at least one Rapid Antigen test or HRCT chest, which was conducted at ACS Medical college Chennai, from June 2021 to June 2022 after obtaining the ethical clearance. Data analysis was done using statistical packages for social sciences version 16. Results 315 of 755(41.7%) patients had positive RTPCR and 18.4% of 733 patients had positive antigen tests. Of 600 patients, 106(14.0%) were found to have CO-RADS 1,152(20.1%) had CORADS 2, 77(10.2%) had CO-RADS 3, 18(2.4%) had CO-RADS 4 and 247(32.7%) had CO-RADS 5 score on HRCT. Rapid Antigen Test was found to have 43.6% sensitivity, 98.6% specicity, 95.6% Positive predictive value (PPV), and 72.1% negative predictive value (NPV).CO-RADS scoring system was able to distinguish between RT-PCR positive and RT-PCR negative results with an average Area under curve 0.787. CO-RADS 5 had a sensitivity of 71.6%, specicity of 85.1%, PPVof 80.6%, and NPVof 77.6% for a positive RT-PCR result. Acombination of CO-RADS 1 in HRCT chest and a Negative Antigen test can predict PCR negative results with 23.6% sensitivity, 97.3% specicity, 90.5% PPV, and 54.0% NPV. Conclusions Rapid Antigen Test is a better tool for conrmation rather than screening for COVID-19. HRCT chest with higher CO-RADS can be useful to diagnose COVID-19 in suspected patients, even if RT-PCR is negative.

Vertical Transmission of Severe Acute Respiratory Syndrome Coronavirus 2: Current Evidence and Perspectives

Vertical Transmission of Severe Acute Respiratory Syndrome Coronavirus 2: Current Evidence and Perspectives

Diagnostic accuracy of autoverification and guidance system for COVID-19 RT-PCR results

BackgroundTo date, most countries worldwide have declared that the pandemic of COVID-19 is over, while the WHO has not officially ended the COVID-19 pandemic, and China still insists on the personalized dynamic COVID-free policy. Large-scale nucleic acid testing in Chinese communities and the manual interpretation for SARS-CoV-2 nucleic acid detection results pose a huge challenge for labour, quality and turnaround time (TAT) requirements. To solve this specific issue while increase the efficiency and accuracy of interpretation, we created an autoverification and guidance system (AGS) that can automatically interpret and report the COVID-19 reverse transcriptase-polymerase chain reaction (RT-PCR) results relaying on computer-based autoverification procedure and then validated its performance in real-world environments. This would be conductive to transmission risk prediction, COVID-19 prevention and control and timely medical treatment for positive patients in the context of the predictive, preventive and personalized medicine (PPPM).MethodsA diagnostic accuracy test was conducted with 380,693 participants from two COVID-19 test sites in China, the Hong Kong Hybribio Medical Laboratory (n = 266,035) and the mobile medical shelter at a Shanghai airport (n = 114,658). These participants underwent SARS-CoV-2 RT-PCR from March 28 to April 10, 2022. All RT-PCR results were interpreted by laboratorians and by using AGS simultaneously. Considering the manual interpretation as gold standard, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were applied to evaluate the diagnostic value of the AGS on the interpretation of RT-PCR results.ResultsAmong the 266,035 samples in Hong Kong, there were 16,356 (6.15%) positive, 231,073 (86.86%) negative, 18,606 (6.99%) indefinite, 231,073 (86.86%, negative) no retest required and 34,962 (13.14%, positive and indefinite) retest required; the 114,658 samples in Shanghai consisted of 76 (0.07%) positive, 109,956 (95.90%) negative, 4626 (4.03%) indefinite, 109,956 (95.90%, negative) no retest required and 4702 (4.10%, positive and indefinite) retest required. Compared to the fashioned manual interpretation, the AGS is a procedure of high accuracy [99.96% (95%CI, 99.95–99.97%) in Hong Kong and 100% (95%CI, 100–100%) in Shanghai] with perfect sensitivity [99.98% (95%CI, 99.97–99.98%) in Hong Kong and 100% (95%CI, 100–100%) in Shanghai], specificity [99.87% (95%CI, 99.82–99.90%) in Hong Kong and 100% (95%CI, 99.92–100%) in Shanghai], PPV [99.98% (95%CI, 99.97–99.99%) in Hong Kong and 100% (95%CI, 99.99–100%) in Shanghai] and NPV [99.85% (95%CI, 99.80–99.88%) in Hong Kong and 100% (95%CI, 99.90–100%) in Shanghai]. The need for manual interpretation of total samples was dramatically reduced from 100% to 13.1% and the interpretation time fell from 53 h to 26 min in Hong Kong; while the manual interpretation of total samples was decreased from 100% to 4.1% and the interpretation time dropped from 20 h to 16 min at Shanghai.ConclusionsThe AGS is a procedure of high accuracy and significantly relieves both labour and time from the challenge of large-scale screening of SARS-CoV-2 using RT-PCR. It should be recommended as a powerful screening, diagnostic and predictive system for SARS-CoV-2 to contribute timely the ending of the COVID-19 pandemic following the concept of PPPM.

Comparison of Rapid Antigen Tests' Performance Between Delta and Omicron Variants of SARS-CoV-2 : A Secondary Analysis From a Serial Home Self-testing Study.

It is important to document the performance of rapid antigen tests (Ag-RDTs) in detecting SARS-CoV-2 variants. To compare the performance of Ag-RDTs in detecting the Delta (B.1.617.2) and Omicron (B.1.1.529) variants of SARS-CoV-2. Secondary analysis of a prospective cohort study that enrolled participants between 18 October 2021 and 24 January 2022. Participants did Ag-RDTs and collected samples for reverse transcriptase polymerase chain reaction (RT-PCR) testing every 48 hours for 15 days. The parent study enrolled participants throughout the mainland United States through a digital platform. All participants self-collected anterior nasal swabs for rapid antigen testing and RT-PCR testing. All Ag-RDTs were completed at home, whereas nasal swabs for RT-PCR were shipped to a central laboratory. Of 7349 participants enrolled in the parent study, 5779 asymptomatic persons who tested negative for SARS-CoV-2 on day 1 of the study were eligible for this substudy. Sensitivity of Ag-RDTs on the same day as the first positive (index) RT-PCR result and 48 hours after the first positive RT-PCR result. A total of 207 participants were positive on RT-PCR (58 Delta, 149 Omicron). Differences in sensitivity between variants were not statistically significant (same day: Delta, 15.5% [95% CI, 6.2% to 24.8%] vs. Omicron, 22.1% [CI, 15.5% to 28.8%]; at 48 hours: Delta, 44.8% [CI, 32.0% to 57.6%] vs. Omicron, 49.7% [CI, 41.6% to 57.6%]). Among 109 participants who had RT-PCR-positive results for 48 hours, rapid antigen sensitivity did not differ significantly between Delta- and Omicron-infected participants (48-hour sensitivity: Delta, 81.5% [CI, 66.8% to 96.1%] vs. Omicron, 78.0% [CI, 69.1% to 87.0%]). Only 7.2% of the 69 participants with RT-PCR-positive results for shorter than 48 hours tested positive by Ag-RDT within 1 week; those with Delta infections remained consistently negative on Ag-RDTs. A testing frequency of 48 hours does not allow a finer temporal resolution of the analysis of test performance, and the results of Ag-RDTs are based on self-report. The performance of Ag-RDTs in persons infected with the SARS-CoV-2 Omicron variant is not inferior to that in persons with Delta infections. Serial testing improved the sensitivity of Ag-RDTs for both variants. The performance of rapid antigen testing varies on the basis of duration of RT-PCR positivity. National Heart, Lung, and Blood Institute of the National Institutes of Health.

Evaluation of rapid antigen test (RAT) screening test among people attending fever clinic of Shimoga Institute of Medical Sciences, Shivamogga: An analytical cross-sectional study

Background: COVID-19 pandemic continues to be a public health threat. Rapid antigen tests (RATs) for the detection of SARS-CoV-2 infection will help in formulation of clinical and public health strategies for the control of transmission. In India, 49% of COVID-19 tests done are RATs.1 The sensitivity of RAT is 50.6–84%. RAT has specificity ranging from 99.3% to 100%.2 The present study aims at evaluating the RAT screening test done on people attending fever clinic of Shimoga Institute of Medical Sciences, Shivamogga. Aims and Objectives: The objectives of the study were to evaluate the COVID-19 RAT screening test with reverse transcriptase-polymerase chain reaction (RT-PCR) as the gold standard test done at fever clinic of SIMS, Shimoga. Materials and Methods: An observational, cross-sectional analytical study was conducted for a period of 1 month, May 2021. The study participants included all the people attending fever clinic of SIMS, Shimoga. Assuming the sensitivity of RAT to be 85%, power of 80%, and precision of 5%, the calculated sample size was 204. Considering non-response rate of 10%, the final sample size was 224. Secondary data regarding contact number of the people attending fever clinic were collected from the COVID-19 test register. Oral consent was taken after explaining about study and assuring confidentiality. Telephonic interview was done to collect relevant information. Analysis was done using Epi Info software version 7.2.4.0. Descriptive statistics such as percentages and analytical statistics such as Student’s t-test and Chi-square test were used. Results: Overall positivity rate was 43.7%. About 71% of people had contact history. Sensitivity and specificity of RAT test were found to be 64.2% and 97.2%, respectively, and were comparable with the previous studies. Significant difference was found (P&lt;0.05) between RAT and RT-PCR results. Conclusion: Significant difference was found between RAT and RT-PCR results which indicate that RAT is not diagnostic for people who test positive in RT-PCR. Sensitivity of RAT is relatively less in our study (but crucial in detecting disease early) and hence we strongly recommend that RT-PCR for those who test negative for RAT test.

Concordance of SARS-CoV-2 RNA in Aerosols From a Nurses Station and in Nurses and Patients During a Hospital Ward Outbreak

Aerosol-borne SARS-CoV-2 has not been linked specifically to nosocomial outbreaks. To explore the genomic concordance of SARS-CoV-2 from aerosol particles of various sizes and infected nurses and patients during a nosocomial outbreak of COVID-19. This cohort study included patients and nursing staff in a US Department of Veterans Affairs inpatient hospital unit and long-term-care facility during a COVID-19 outbreak between December 27, 2020, and January 8, 2021. Outbreak contact tracing was conducted using exposure histories and screening with reverse transcriptase-polymerase chain reaction (RT-PCR) for SARS-CoV-2. Size-selective particle samplers were deployed in diverse clinical areas of a multicampus health care system from November 2020 to March 2021. Viral genomic sequences from infected nurses and patients were sequenced and compared with ward nurses station aerosol samples. SARS-CoV-2. The primary outcome was positive RT-PCR results and genomic similarity between SARS-CoV-2 RNA in aerosols and human samples. Air samplers were used to detect SARS-CoV-2 RNA in aerosols on hospital units where health care personnel were or were not under routine surveillance for SARS-CoV-2 infection. A total of 510 size-fractionated air particle samples were collected. Samples representing 3 size fractions (>10 μm, 2.5-10 μm, and <2.5 μm) obtained at the nurses station were positive for SARS-CoV-2 during the outbreak (3 of 30 samples [10%]) and negative during 9 other collection periods. SARS-CoV-2 partial genome sequences for the smallest particle fraction were 100% identical with all 3 human samples; the remaining size fractions shared >99.9% sequence identity with the human samples. Fragments of SARS-CoV-2 RNA were detected by RT-PCR in 24 of 300 samples (8.0%) in units where health care personnel were not under surveillance and 7 of 210 samples (3.3%; P = .03) where they were under surveillance. In this cohort study, the finding of genetically identical SARS-CoV-2 RNA fragments in aerosols obtained from a nurses station and in human samples during a nosocomial outbreak suggests that aerosols may have contributed to hospital transmission. Surveillance, along with ventilation, masking, and distancing, may reduce the introduction of community-acquired SARS-CoV-2 into aerosols on hospital wards, thereby reducing the risk of hospital transmission.

Autopsy Histopathologic Cardiac Findings in 2 Adolescents Following the Second COVID-19 Vaccine Dose

Autopsy Histopathologic Cardiac Findings in 2 Adolescents Following the Second COVID-19 Vaccine Dose

Identification of a Novel Deep Intronic Variant by Whole Genome Sequencing Combined With RNA Sequencing in a Chinese Patient With Menkes Disease

Background: Menkes disease (MD) is a rare X-linked connective tissue disorder of copper metabolism caused by pathogenic variant(s) in ATP7A gene. The aim of the present study is to determine the clinical characteristics and molecular basis of one patient with MD. Methods: One 10-month-old Chinese boy who met the clinical manifestations of MD was enrolled in this study. Whole genome sequencing (WGS) was performed in the patient in order to identify the variant(s), followed by Sanger sequencing. RNA sequencing (RNA-seq) from whole blood was subsequently applied to assess the effect of variant on transcription levels, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed for further validation. In addition, X chromosome inactivation (XCI) status of the patient’s mother at the DNA level was measured by capillary electrophoresis. Results: The patient suffered from intermittent convulsions for more than 6 months, with psychomoto retardation and neurodegenerations. The patient also had curly hair, hypopigmented skin, cutis laxa, decreased muscle strength and hypotonia. MRI showed the intracranial arteries were tortuous with some “spiral” changes. The patient’s serum ceruloplasmin level was low. WGS revealed one novel hemizygous variant, c.2627-501C > T (NM_000,052.7), located in the deep intronic sequence of ATP7A gene. Sanger sequencing confirmed that the variant was inherited from his mother. RNA-seq confirmed the variant itself, and identified a pseudo-exon inserted between exons 12 and 13 in mRNA of ATP7A. The sequencing results of RT-PCR from the patient confirmed this finding, while neither of his parents detected aberrant splicing. The Capillary electrophoresis results showed that the patient’s mother had a skewed XCI. Conclusion: Our finding of the variant enlarges the variant spectrum in the ATP7A gene. This is a novel deep intronic variant which leads to the activation of a pseudo-exons in the ATP7A gene, and it demonstrates the usefulness of WGS combined with RNA-seq, in terms of revealing disease-causing variants in non-coding regions. Furthermore, the fact that the deep intronic variants cause disease by the activation of pseudo-exon inclusion indicates that in MD this might be an important mechanism.

Comparison of Clinical and Laboratory Features in RT-PCR Positive and RT-PCR Negative Covid-19 Patients Admitted in High Dependency Unit in a Tertiary Care Hospital, SGRH, Lahore

Introduction: Corona virus disease 2019 (COVID-19) is currently diagnosed mainly using reverse transcriptase polymerase chain reaction (RT-PCR). Yet a significant proportion of patients have negative RT-PCR result. A comparative study of RT-PCR negative patients with RT-PCR positive patients will help understand clinical characteristic and differences of this diseased population. Objective: To compare the clinical and laboratory features of RT-PCR Positive and RT-PCR negative patients admitted in high dependency unit. Patients and methods: In this retrospective cohort study, the data of 128 patients (59 patients with RT-PCR positive result and 69 patients with RT-PCR negative results) was obtained. These patients had been admitted in high dependency unit of a community hospital. Demographics, clinical characteristics and laboratory abnormalities were noted and a comparison was done using statistical analysis. Results: In our study-total 128 patients were enrolled out of which 68 (53.1%) were males and 60 (46.9%) were females. 59 (46.1%) patients were RT-PCR positive and 69 (53.9%) patients were RT-PCR negative. Median age was 55.34 years (18 to 95). No significant difference was noted in most of clinical symptoms (fever, sputum production, rhinorrhea, dyspnea, myalgia, nasal congestion, vomiting, diarrhea, urinary symptoms, altered level of consciousness), comorbidities (diabetes mellitus, hypertension, prior lung disease, prior ischemic heart disease, prior kidney disease), laboratory abnormalities (elevated creatinine, elevated liver enzymes, elevated ferritin, elevated C-reactive protein, elevated d-dimers, elevated procalcitonin, abnormal electrocardiogram). Cough was significantly found to be more prevalent in RT-PCR positive patients (p=0.042) and severe disease was also more prevalent in these patients significantly (p=0.000). Conclusion: Our study shows that patients admitted and diagnosed to be suffering from COVID-19 infection had remarkable similarities in clinical features and laboratory parameters regardless of RT-PCR status, however RT-PCR positive patients suffered from more severe pneumonia as compared to RT-PCR negative patients. Keywords: COVID-19, RT-PCR positive, RT-PCR negative

Utility of Routine Clinical Laboratory Tests in COVID 19

The novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection leading to COVID-19 pandemic has affected many countries all over the world including India. Diagnostic laboratories play crucial role in containment of pandemic as they enable the rapid identification for isolation and treatment of COVID-19 positive cases. Gold standard test for COVID 19 detection is the reverse transcriptase Polymerase Chain Reaction (RT-PCR)-based assays performed on respiratory specimen. The role of other laboratory parameters in COVID-19 cases has not been definitely established. Clinical laboratory investigations need to be studied in larger populations to understand the pathogenesis and to understand whether these parameters are useful to triage COVID-19 suspect patients before the results of RT PCR are available. This study was aimed to analyze and compare routine clinical laboratory parameters in COVID-19 positive and negative patients at the time of presentation to our tertiary care referral center situated in western region of India from April, 2020 till November, 2020. Patients were divided into two groups based on the results of RT-PCR for COVID-19. The outcome of different laboratory parameters like CBC, CRP, AST, ALT, LDH, ferritin were evaluated in cases with positive RT-PCR and compared with negative RT-PCR group. We found no statistically significant differences between COVID-19 positive and negative group on routine clinical laboratory parameters of 4602 patients.

Oncological Surgery During the COVID-19 Pandemic: Effectiveness of Preoperative Screening and Factors Associated with Postoperative SARS-CoV-2 Infection.

BackgroundRoutine preoperative screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with reverse transcriptase-polymerase chain reaction (RT-PCR) may reduce in-hospital SARS-CoV-2 transmission.MethodsThis was a prospective, observational, cohort study. The endpoints were the incidence of asymptomatic patients with positive preoperative RT-PCR results and the incidence and factors associated with postoperative SARS-CoV-2 infection in patients with cancer referred for elective surgery. Patients with elective surgery between May and October 2020 were included. RT-PCR of nasopharyngeal swabs was performed preoperatively for all patients. Postoperative SARS-CoV-2 infection was assessed within 30 postoperative days.ResultsA total of 1636 preoperative screening RT-PCR tests were performed. Of these, 102 (6.2%) cases were positive, and 1,298 surgical procedures were analyzed. The postoperative SARS-CoV-2 infection rate was 0.9%. The length of stay (odds ratio [OR] 1.08; 95% confidence interval [CI] 1.04–1.11; p < 0.001), surgical time (OR 1.004; 95% CI 1.001–1.008; p = 0.023), intensive care unit admission (OR 7.7; 95% CI 2.03–29.28; p = 0.003), and hospital readmissions (OR 9.56; 95% CI 2.50–36.56; p = 0.001) were associated with postoperative coronavirus disease (COVID-19). Using unadjusted and adjusted logistic regression, length of stay (OR 1.08; 95% CI 1.04–1.11; p < 0.001), and readmission (OR 9.02; 95% CI 2.30–35.48; p = 0.002) were independent factors of postoperative COVID-19.ConclusionsScreening patients preoperatively may reduce in-hospital SARS-CoV-2 transmission. Length of stay and readmission were independently correlated with postoperative COVID-19.

Evaluation of Using Direct Viral Transport Medium Samples without Nucleic Acid Isolation for SARS-CoV-2 Diagnosis by RT-PCR.

Diagnosis with reverse transcriptase polymerase chain reaction (RT-PCR) test is a very important step for the control of the COVID-19 pandemic. The aim of this study is to compare the RT-PCR results of the samples taken directly from the viral transport medium (VTM) without extraction with the RT-PCR results of two different extraction methods, one automated and the other manual, in the diagnosis of COVID-19. Among the respiratory tract samples sent to Sakarya Training and Research Hospital Microbiology Laboratory for COVID-PCR study, 20 negative and 43 positive samples with different cycle threshold (CT) values were included in the study. Both manual nucleic acid isolation with the vNAT isolation kit (Bioeksen, Turkey) and automatic nucleic acid isolation with the EZ1 Virus Mini Kit v2.0 in the isolation device were performed simulta-neously from the patient samples included in the study and the results were compared. The mean Ct values of the samples were found to be 21.58 using manual vNAT as the extraction method, 17.63 using the automated magnetic bead method, and 21.45 in PCR from direct VTM without extraction. When the automatic magnetic beads extraction method was taken as the reference method, the sensitivity of direct PCR was 97.3%, the specificity was 95%, the positive predictive value was 97.3%, and the negative predictive value was 95%. Phi coefficients were found to be 0.927 between vNAT and direct PCR, 1 between vNAT and EZ1, and 0.922 between direct PCR and EZ1. Direct PCR has advantages such as eliminating RNA extraction and purification steps, providing a shorter detection time, and using less labor and less consumables without reducing the diagnostic accuracy. It is thought that this method can help as a useful process management for the control of the epidemic in countries with limited resources.

Clinical evaluation of single-swab sampling for rapid COVID-19 detection in outbreak settings in Dutch nursing homes

To assess whether one swab can be used to perform both the antigen-detection rapid diagnostic test (Ag-RDT) and reverse transcriptase polymerase chain reaction (RT-PCR) for COVID-19 detection during an outbreak in the nursing home (NH) setting. The single-swab method (SSM), where the Ag-RDT is performed with the transport medium used for RT-PCR, was evaluated in three Dutch NHs and compared to the laboratory setting. We collected Ag-RDT and RT-PCR results, NH resident characteristics and symptomatology. In addition, two focus groups were held with the involved care professionals to gain insight into the feasibility of the SMM in the NH setting. In the NH setting, the SSM had a sensitivity of 51% and a specificity of 89% compared to RT-PCR. These were lower than in the laboratory setting (69% and 100% respectively). Yet, when stratified for cycle threshold values, the sensitivity became comparable between the settings. Symptoms occurred more frequent in the Ag-RDT+ group than Ag-RDT- group. Resident characteristics did not differ between these groups. Based on the focus groups, the SSM was feasible to perform if certain requirements, such as availability of staff, equipment and proper training, were met. However, the rapid availability of the test results were perceived as a dilemma. The advantages and disadvantages need to be considered before implementation of the SSM can be recommended in the NH setting. For the vulnerable NH residents, it is important to find the right balance between effective testing policy and the burden this imposes.

Multireader evaluation of radiologist performance for COVID-19 detection on emergency department chest radiographs

BackgroundChest radiographs (CXR) are frequently used as a screening tool for patients with suspected COVID-19 infection pending reverse transcriptase polymerase chain reaction (RT-PCR) results, despite recommendations against this. We evaluated radiologist performance for COVID-19 diagnosis on CXR at the time of patient presentation in the Emergency Department (ED). Materials and methodsWe extracted RT-PCR results, clinical history, and CXRs of all patients from a single institution between March and June 2020. 984 RT-PCR positive and 1043 RT-PCR negative radiographs were reviewed by 10 emergency radiologists from 4 academic centers. 100 cases were read by all radiologists and 1927 cases by 2 radiologists. Each radiologist chose the single best label per case: Normal, COVID-19, Other – Infectious, Other – Noninfectious, Non-diagnostic, and Endotracheal Tube. Cases labeled with endotracheal tube (246) or non-diagnostic (54) were excluded. Remaining cases were analyzed for label distribution, clinical history, and inter-reader agreement. Results1727 radiographs (732 RT-PCR positive, 995 RT-PCR negative) were included from 1594 patients (51.2% male, 48.8% female, age 59 ± 19 years). For 89 cases read by all readers, there was poor agreement for RT-PCR positive (Fleiss Score 0.36) and negative (Fleiss Score 0.46) exams. Agreement between two readers on 1638 cases was 54.2% (373/688) for RT-PCR positive cases and 71.4% (679/950) for negative cases. Agreement was highest for RT-PCR negative cases labeled as Normal (50.4%, n = 479). Reader performance did not improve with clinical history or time between CXR and RT-PCR result. ConclusionAt the time of presentation to the emergency department, emergency radiologist performance is non-specific for diagnosing COVID-19.