Association of SARS-COV-2 viral RNAemia, IL- 6 gene polymorphism, serum IL-6 and peripheral blood lymphocytes and monocytes with disease severity in COVID-19 patients

Coronavirus disease 2019 (COVID-19) is a fatal pandemic viral disease caused by the severe acute respiratory syndrome corona virus type-2 (SARS-CoV-2). The aim of this study is to observe the associations of IL-6, SARS-COV-2 viral load (RNAemia), IL- 6 gene polymorphism and lymphocytes and monocytes in peripheral blood with disease severity in COVID-19 patients. This study was carried out from March 2021 to January 2022. RT-PCR positive 84 COVID-19 patients and 28 healthy subjects were enrolled. Blood was collected to detect SARS-COV-2 viral RNA (RNAemia) by rRT-PCR, serum IL-6 level by chemiluminescence method, SNPs of IL-6 by SSP-PCR, immunophenotyping of lymphocytes and monocyte by flow cytometry. Serum IL-6 level (pg/ml) was considerably high among critical patients (102.02 +/- 149.7) compared to severe (67.20 +/- 129.5) and moderate patients (47.04 +/- 106.5) and healthy controls (3.5 +/- 1.8). Serum SARS-CoV-2 nucleic acid positive cases detected mostly in critical patients (39.28%) and was correlated with extremely high IL-6 level and high mortality (R =.912, P < 0.001). Correlation between IL-6 and monocyte was statistically significant with disease severity (severe group, p < 0.001, and 0.867*** and critical group p < 0.001 and 0.887***). In healthy controls, moderate, severe and critically ill COVID-19 patients, IL-6 174G/C (rs 1800795) GG genotype was 82.14%, 89.20%, 67.85% and 53.57% respectively. CC and GC genotype had strong association with severity of COVID-19 when compared with GG genotype. Significant statistical difference found in genotypes between critical and moderate groups (p < 0.001, OR-10.316, CI-3.22-23.86), where CC genotype was associated with COVID-19 severity and mortality. The absolute count of T cell, B cell, NK cell, CD4+ T cells and CD8+ T cells were significantly decreased in critical group compared to healthy, moderate and severe group (P < 0.001). Exhaustion marker CD94/NKG2A was increased on NK cells and CD8+ cytotoxic T cell among critical and severe group. Absolute count of monocyte was significantly increased in critical group (P < 0.001). Serum IL-6, IL-6 174 G/C gene and SARS-CoV-2 RNAaemia can be used in clinical practice for risk assessment;T cell subsets and monocyte as biomarkers for monitoring COVID-19 severity. Monoclonal antibody targeting IL-6 receptor and NKG2A for therapeutics may prevent disease progression and decrease morbidity and mortality.Copyright © 2023 Elsevier Inc.

Performance Evaluation of SARS-CoV-2 Viral Transport Medium Produced by Bangladesh Reference Institute for Chemical Measurements.

A viral transport medium (VTM) was developed following the Centers for Disease Control and Prevention, USA (US-CDC) standard operating procedure (SOP) DSR-052-05 with necessary improvisation and was used for storing coronavirus disease 2019 (COVID-19) swab specimens. Considering Bangladesh's supply chain and storage conditions, improvisation was essential for extending sample storage time while retaining efficiency. In-house VTM was produced using Hank's balanced salt solution (HBSS) supplemented with 1% bovine serum albumin V (BSA), 0.5 µg /mL of gentamicin sulfate, and 100 µg/mL of fluconazole. The produced VTM composition, quality, sterility, specificity, and efficiency were verified in-house and through an independent contract research organization (CRO). An accelerated stability study projected that under the recommended temperature (4 °C), it would remain stable for four months and preserve samples for over a month. The real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) test detected the targeted N gene and ORF1ab gene from the VTM stored samples. Our VTM is equally as effective as the Sansure Biotech VTM in keeping SARS-CoV-2 RNA specimens detectable in rRT-PCR (100% sensitivity and specificity in random and blinded samples). In conclusion, the BRiCM VTM will make the battle against pandemics easier by effectively collecting and storing nasopharyngeal and oropharyngeal swabs for COVID-19 detection.

COVID-19 in a Nigerian university: Modelling the spread of SARS-CoV-2 on an average university campus

Background: Currently, the world is overwhelmed with coronavirus disease 2019 (COVID-19) caused by a highly virulent virus that causes influenza-like symptoms. University administrators are confronted with challenges concerning coronavirus preparedness and response for the resumption of safe campus activities. This study aimed at assisting Nigerian Universities in COVID-19 preparation and response. Methods: We adopted the susceptible-exposed infectious-recovered (SEIR) deterministic model to appraise the transmission of SARS-CoV-2 among university staff and students and evaluated the breadth of non-pharmaceutical intervention strategies required to safely return its community to campus. The mode was parameterized to fit the resident on campus situation. The frequencies of viral screening and testing, probabilistic sensitivity analysis of model parameter was explored in this study. Results: Weekly COVID-19 screening reduced the cumulative incidence by 15% and 55.7% among university staff and students, respectively. Polymerase chain reaction (PCR) testing delay of 2-,3-,4-and 7 days reduced overall semester incidence by 65.7%, 56.9%, 50.8% and 34.4% among students;23.5%, 22.8%, 20.5% and 16.9% among university staff. Conclusions: Our simulations have revealed that extensive testing of on-campus community population may be required to curb disease explosion. While cases of hospitalization and deaths may occur, community import of COVID-19 can be curtailed with effective testing, isolation, contact tracing and quarantine. A cost-effective solution such as pool testing was proposed in this study to decrease the overall resources needed for comprehensive on-campus testing. © 2020 The author (s). Published by Zagazig University.

Evaluation of Four Rapid Antigen Tests for the Detection of SARS-CoV-2 Infection with Nasopharyngeal Swabs.

Owing to the high transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, the capacity of testing systems based on the gold standard real-time reverse transcription-polymerase chain reaction (rRT-PCR) is limited. Rapid antigen tests (RATs) can substantially contribute to the prevention of community transmission, but their further assessment is required. Here, using 1503 nasopharyngeal swabs, we compared the diagnostic performance of four RAT kits (Abbott Panbio™ COVID-19 Ag Rapid Test, SD Biosensor Standard™ Q COVID-19 Ag Test, Humasis COVID-19 Ag Test, and SG Medical Acrosis COVID-19 Ag Test) to the cycle threshold (Ct) values obtained from rRT-PCR. The precision values, area under the curve values, SARS-CoV-2 variant detection ability, and non-SARS-CoV-2 specificity of all four kits were similar. An assay using the Acrosis kit had a significantly better positive detection rate with a higher recall value and cut-off value than that using the other three RAT kits. During the current COVID-19 pandemic, the Acrosis kit is an effective tool to prevent the spread of SARS-CoV-2 in communities.

Evaluation of a high-sensitivity SARS-CoV-2 antigen test on the fully automated light-initiated chemiluminescent immunoassay platform.

OBJECTIVES: To describe a high-sensitivity SARS-CoV-2 antigen test that is based on the fully automated light-initiated chemiluminescent immunoassay (LiCA®), and to validate its analytical characteristics and clinical agreement on detecting SARS-CoV-2 infection against the reference molecular test. METHODS: Analytical performance was validated and detection limits were determined using different types of nucleocapsid protein samples. 798-pair anterior nasal swab specimens were collected from hospitalized patients and asymptomatic screening individuals. Agreement between LiCA® antigen and real-time reverse transcription polymerase chain reaction (rRT-PCR) was evaluated. RESULTS: Repeatability and within-lab precision were 1.6-2.3%. The C5∼C95 interval was -5.1-4.6% away from C50. Detection limits in average (SD) were 325 (±141) U/mL on the national reference panel, 0.07 (±0.04) TCID50/mL on active viral cultures, 0.27 (±0.09) pg/mL on recombinant nucleocapsid proteins and 1.07 (±1.01) TCID50/mL on inactivated viral suspensions, respectively. LiCA detected a median of 374-fold (IQR 137-643) lower levels of the viral antigen than comparative rapid tests. As reference to the rRT-PCR method, overall sensitivity and specificity were determined to be 97.5% (91.4-99.7%) and 99.9% (99.2-100%), respectively. Total agreement between both methods was 99.6% (98.7-99.9%) with Cohen's kappa 0.98 (0.96-1). A positive detection rate of 100% (95.4-100%) was obtained as Ct≤37.8. CONCLUSIONS: The LiCA® system provides an exceptionally high-sensitivity and fully automated platform for the detection of the SARS-CoV-2 antigen in nasal swabs. The assay may have high potential use for large-scale population screening and surveillance of COVID-19 as an alternative to the rRT-PCR test.

Performance Evaluation of Developed Bangasure™ Multiplex rRT-PCR Assay for SARS-CoV-2 Detection in Bangladesh: A Blinded Observational Study at Two Different Sites.

In this study, we evaluated the performance of the in-house developed rRT-PCR assay for SARS-CoV-2 RNA targeting the envelope (E) and nucleocapsid (N) genes with internal control as human RNase P. A total of 50 positive samples and 50 negative samples of SARS-CoV-2 were tested by a reference kit at site 1 and a subset (30 positives and 16 negatives) of these samples are tested blindly at site 2. The limit of detection (LoD) was calculated by using a replication-deficient complete SARS-CoV-2 genome and known copy numbers, where Pseudo-virus samples were used to evaluate accuracy. On site 1, among the 50 SARS-CoV-2 positive samples 24, 18, and eight samples showed high (Ct < 26), moderate (26 < Ct ≤ 32), and low (32 < Ct ≤ 38) viral load, respectively, whereas in site 2, out of 30 SARS-CoV-2 positive samples, high, moderate, and low viral loads were found in each of the 10 samples. However, SARS-CoV-2 was not detected in the negative sample. So, in-house assays at both sites showed 100% sensitivity and specificity with no difference observed between RT PCR machines. The Ct values of the in-house kit had a very good correlation with the reference kits. LoD was determined as 100 copies/mL. It also displayed 100% accuracy in mutant and wild-type SARS-CoV-2 virus. This Bangasure™ RT-PCR kit shows excellent performance in detecting SARS-CoV-2 viral RNA compared to commercially imported CE-IVD marked FDA authorized kits.

Comparison of analytical sensitivity of SARS-CoV-2 molecular detection kits.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has had a significant impact on global public health systems, making nucleic acid detection an important tool in epidemic prevention and control. Detection kits based on real-time reverse transcriptase PCR (rRT-PCR) have been used widely in clinics, but their analytical sensitivity (limit of detection, LOD) remains controversial. Moreover, there is limited research evaluating the analytical sensitivity of other molecular detection kits. METHODS: In this study, armored ribonucleic acid reference materials developed in-house were used to evaluate the analytical sensitivity of SARS-CoV-2 detection kits approved by the National Medical Products Administration. These were based on rRT-PCR and other molecular detection assays. RESULTS: The percentage retesting required with rRT-PCR kits was as follows: 0%, 7.69%, 15.38%, and 23.08% for samples with concentrations ranging from 50 000 to 781 copies/ml. In total, 93% of rRT-PCR kits had a LOD <1000 copies/ml. Only one kit had an LOD >1000 copies/ml. The LOD of other molecular detection kits ranged from 68 to 2264 copies/ml. CONCLUSIONS: The study findings can help pharmaceutical companies optimize and improve detection kits, guide laboratories in selecting kits, and assist medical workers in their daily work.

Diagnostic accuracy of saliva as a specimen for detection of SARS-CoV-2 by RT-PCR.

Context: COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging pandemic that is rapidly spreading with more than 114 million confirmed cases and 2.5 million deaths by far. Nasopharyngeal swab (NPS) in VTM has been used as the gold standard respiratory specimen for SARS-CoV-2 reverse-transcriptase real-time PCR (rRT-PCR) tests. But now the virus can also be detected in other clinical specimens like bronchoalveolar lavage, sputum, saliva, throat swab, blood, and stool specimens. Aims: The aim of this study was to determine the diagnostic potential of saliva as a sample in comparison to NPS for detection of SARS-CoV-2 by rRT-PCR. Settings and Design: A cross-sectional study was conducted among 256 paired samples (NPS and Saliva) received in the Department of Microbiology, SMS Medical College, Jaipur over a period of 2 months. Methods and Material: NPS from individuals were collected in a sterile tube containing Viral Transport Medium™. Before swab collection, whole saliva was collected by spitting from the suspected patient into a sterile container. Both were stored at room temperature and transferred to the diagnostic laboratory within four hours of collection where extraction was done using Perkin Elmer chemagic extractor and rRT- PCR was performed using NIV, Pune mastermix. Results: Sensitivity, specificity, PPV, and NPV of RT-PCR for the diagnosis of COVID-19 in saliva were 84.26%, 100%, 100%, and 54.05%, respectively. The accuracy of detection of COVID-19 by saliva samples compared to the routinely used NPS samples (considered as the standard reference) for RT PCR was 86.72%. Conclusions: Our results show that saliva as a reliable sample type for SARS-CoV-2 detection.

Comparative analysis of specificity and sensitivity between Cobas 6800 system and SARS-CoV-2 rRT-PCR to detect COVID-19 infection in clinical samples.

Fast and reliable testing for the COVID 19 infection is the need of the hour for the development of effective and reliable tools and assays. However, it is difficult to find the performance relativity among all these tests which are poorly understood. In this study, we aimed to evaluate the two different platforms where we determine the difference of sensitivity and specificity between the fully automated analyzer (Roche Diagnostics Cobas 6800 SARS-CoV-2 test) under FDA Emergency Use Authorization (EUA) and the laboratory designed test (SARS-CoV-2 rRT-PCR) based on the protocol developed by ICMR (Indian Council for Medical Research). The study was conducted for individual samples. We performed our study with two different approaches, first with validation method consisting of 188 samples (2 batches) on cobas 6800 instrument (Roche Molecular Systems, Branchburg, NJ) soon after we received US FDA EUA on 1 June 2021, all these samples were tested earlier with laboratory designed tests on 25th and 26th May 2021. Over all agreement between the two tests is of 88% and the coefficient of agreement between the two testing platform Cohen'sκ coefficient was found to be 0.76 (95% CI, 2.5897-13.4103) suggesting the substantial agreement between the two platforms. However, in some of the cases, both tests have shown a little disagreement. An overall discordance rate between two systems was found 11.1%. The difference may be due to the limit of detection, variation in the sequences of the primer design or may be due to other factors depicting the importance of comparing the two platforms used in the testing for SARS-CoV-2. Second approach includes head to head evaluation which comprises 1631 samples showed overall agreement of 99% and kappa value of 0.98. These results showed that cobas is effective and reliable assay for the detection of SARS-CoV-2 infection.

High concordance in SARSCoV-2 detection between automated (Abbott m2000) and manual (DaAn gene) RT-PCR systems: The EDCTP PERFECT-Study in Cameroon.

Molecular diagnosis of COVID-19 is critical to the control of the pandemic, which is a major threat to global health. Several molecular tests have been validated by WHO, but would require operational evaluation in the field to ensure their interoperability in diagnosis. In order to ensure field interoperability in molecular assays for detection of SARS-CoV-2 RNA, we evaluated the diagnostic concordance of SARS-CoV-2 between an automated (Abbott) and a manual (DaAn gene) realtime PCR (rRT-PCR), two commonly used assays in Africa. A comparative study was conducted on 287 nasopharyngeal specimens at the Chantal BIYA International Reference Centre (CIRCB) in Yaounde- Cameroon. Samples were tested in parallel with Abbott and DaAn gene rRT-PCR, and performance characteristics were evaluated by Cohen's coefficient and Spearman's correlation. A total of 273 participants [median age (IQR) 36 (26-46) years] and 14 EQA specimens were included in the study. Positivity was on 30.0% (86/287) Abbott and 37.6% (108/287) DaAn gene. Overall agreement was 82.6% (237/287), with k=0.82 (95%CI: 0.777-0.863), indicating an excellent diagnostic agreement. The positive and negative agreement was 66.67% (72/108) and 92.18 % (165/179) respectively. Regarding Viral Load (VL), positive agreement was 100% for samples with high VLs (CT<20). Among positive SARS-CoV- 2 cases, the mean difference in Cycle Threshold (CT) for the manual and Cycle Number (CN) for the automated was 6.75±0.3. The excellent agreement (>80%) between the Abbott and DaAn gene rRTPCR platforms supports interoperability between the two assays. Discordance occurs at low-VL, thus underscoring these tools as efficient weapons in limiting SARS-CoV-2 community transmission.

Elucidation of correlation between SARS-CoV-2 RdRp and N gene cycle threshold (Ct) by RT-PCR with age and gender.

BACKGROUND: Coronavirus disease 2019 (COVID19) caused by the new severe acute respiratory syndrome coronavirus 2 (SARSCoV2) is a global public health emergency. Age and gender are two important factors related to the risk and outcome of various diseases. Cycle threshold (Ct) value is believed to have relation with age and gender. OBJECTIVE: This study has been conducted to investigates the association between SARS-CoV-2 cycle threshold to age and gender of COVID-19 patients, to investigate whether the population-wide change of SARSCoV2 RTPCR Ct value over time is corelated to the number of new COVID19 cases and to investigate the dynamic of RdRp and N genes. METHODS: 72,811 individuals from second wave of COVID19, were observed in current study at Pure Health Lab, Mafraq Hospital, Abu Dhabi, UAE. RESULTS: 15,201/72,811 (21 %) positivity was observed. COVID-19 were more prevalent in males (59.35%) as compared to female (40.65%). The Positivity rate were significantly higher in Male than in Female cases (p-Value = 0.04). The Ct values for both targets of all the samples were ranged from 4.57 to 29.73. Longitudinal analysis showed significant increased during the study period from starting to end as were hypothesized. Interestingly, both the targets (RdRp and N) were present in age < 1 year. Which may indicate that mutated strains are not prevalent in children's < 1 year. CONCLUSION: There was no statistically significant difference in viral loads in between age-groups. Males were tending to higher viral load compared to females. The findings have implications for preventive strategies.

Monitoring SARS-CoV-2 in air and on surfaces and estimating infection risk in buildings and buses on a university campus.

BACKGROUND: Evidence is needed on the presence of SARS-CoV-2 in various types of environmental samples and on the estimated transmission risks in non-healthcare settings on campus. OBJECTIVES: The objective of this research was to collect data on SARS-CoV-2 viral load and to examine potential infection risks of people exposed to the virus in publicly accessible non-healthcare environments on a university campus. METHODS: Air and surface samples were collected using wetted wall cyclone bioaerosol samplers and swab kits, respectively, in a longitudinal environmental surveillance program from August 2020 until April 2021 on the University of Michigan Ann Arbor campus. Quantitative rRT-PCR with primers and probes targeting gene N1 were used for SARS-CoV-2 RNA quantification. The RNA concentrations were used to estimate the probability of infection by quantitative microbial risk assessment modeling and Monte-Carlo simulation. RESULTS: In total, 256 air samples and 517 surface samples were collected during the study period, among which positive rates were 1.6% and 1.4%, respectively. Point-biserial correlation showed that the total case number on campus was significantly higher in weeks with positive environmental samples than in non-positive weeks (p = 0.001). The estimated probability of infection was about 1 per 100 exposures to SARS-CoV-2-laden aerosols through inhalation and as high as 1 per 100,000 exposures from contacting contaminated surfaces in simulated scenarios. SIGNIFICANCE: Viral shedding was demonstrated by the detection of viral RNA in multiple air and surface samples on a university campus. The low overall positivity rate indicated that the risk of exposure to SARS-CoV-2 at monitored locations was low. Risk modeling results suggest that inhalation is the predominant route of exposure compared to surface contact, which emphasizes the importance of protecting individuals from airborne transmission of SARS-CoV-2 and potentially other respiratory infectious diseases. IMPACT: Given the reoccurring epidemics caused by highly infectious respiratory viruses in recent years, our manuscript reinforces the importance of monitoring environmental transmission by the simultaneous sampling and integration of multiple environmental surveillance matrices for modeling and risk assessment.

Clinical and laboratory findings and PCR results in severe and non-severe COVID19 patients based on CURB-65 and WHO severity indices.

BACKGROUND: The importance of clinicolaboratory characteristics of COVID-19 made us report our findings in the Alborz province according to the latest National Guideline for the diagnosis and treatment of COVID-19 in outpatients and inpatients (trial five versions, 25 March 2020) of Iran by emphasizing rRT-PCR results, clinical features, comorbidities, and other laboratory findings in patients according to the severity of the disease. METHODS: In this study, 202 patients were included, primarily of whom 164 had fulfilled the inclusion criteria. This cross-sectional, two-center study that involved 164 symptomatic adults hospitalized with the diagnosis of COVID-19 between March 5 and April 5, 2020, was performed to analyze the frequency of rRT-PCR results, distribution of comorbidities, and initial clinicolaboratory data in severe and non-severe cases, comparing the compatibility of two methods for categorizing the severity of the disease. RESULTS: According to our findings, 111 patients were rRT-PCR positive (67.6%), and 53 were rRT-PCR negative (32.4%), indicating no significant difference between severity groups that were not related to the date of symptoms' onset before admission. Based on the National Guideline, among vital signs and symptoms, mean oxygen saturation and frequency of nausea showed a significant difference between the two groups (P < 0.05); however, no significant difference was observed in comorbidities. In CURB-65 groups, among vital signs and comorbidities, mean oxygen saturation, diabetes, hypertension (HTN), hyperlipidemia, chronic heart disease (CHD), and asthma showed a significant difference between the two groups (P < 0.05), but no significant difference was seen in symptoms. CONCLUSION: In this study, rRT-PCR results of hospitalized patients with COVID-19 were not related to severity categories. From initial clinical characteristics, decreased oxygen saturation appears to be a more common abnormality in severe and non-severe categories. National Guideline indices seem to be more comprehensive to categorize patients in severity groups than CURB-65, and there was compatibility just in non-severe groups of National Guideline and CURB-65 categories.

SARS-CoV-2 in Egypt: epidemiology, clinical characterization and bioinformatics analysis.

COVID-19 is an infectious disease caused by SARS-CoV-2 and has spread globally, resulting in the ongoing coronavirus pandemic. The current study aimed to analyze the clinical and epidemiological features of COVID-19 in Egypt. Oropharyngeal swabs were collected from 197 suspected patients who were admitted to the Army Hospital and confirmation of the positivity was performed by rRT-PCR assay. Whole genomic sequencing was conducted using Illumina iSeq 100® System. The average age of the participants was 48 years, of which 132 (67%) were male. The main clinical symptoms were pneumonia (98%), fever (92%), and dry cough (66%). The results of the laboratory showed that lymphocytopenia (79.2%), decreased levels of haemoglobin (77.7%), increased levels of interleukin 6, C-reactive protein, serum ferritin, and D-dimer (77.2%, 55.3%, 55.3%, and 25.9%, respectively), and leukocytopenia (25.9%) were more common. The CT findings showed that scattered opacities (55.8%) and ground-glass appearance (27.9%) were frequently reported. The recovered validated sequences (n = 144) were submitted to NCBI Virus GenBank. All sequenced viruses have at least 99% identity to Wuhan-Hu-1. All variants were GH clade, B.1 PANGO lineage, and L.GP.YP.HT haplotype. The most predominant subclade was D614G/Q57H/V5F/G823S. Our findings have aided in a deep understanding of COVID-19 evolution and identifying strains with unique mutational patterns in Egypt.

Impact of SARS-CoV-2 Variants on the Analytical Sensitivity of rRT-PCR Assays.

Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with enhanced transmissibility, pathogenicity, and immune escape ability have ravaged many countries and regions, which has brought substantial challenges to pandemic prevention and control. Real-time reverse transcriptase PCR (rRT-PCR) is widely used for SARS-CoV-2 detection but may be limited by the continuous evolution of the virus. However, the sensitivity of Chinese commercial rRT-PCR kits to critical SARS-CoV-2 variants remains unknown. In this study, contrived MS2 virus-like particles were used as reference materials to evaluate the analytical sensitivity of Daan, BioGerm, EasyDiagnosis, Liferiver, and Sansure kits when detecting six important variants (Alpha, Beta, Gamma, Delta, Omicron, and Fin-796H). The Beta and Delta variants adversely affected the analytical sensitivity of the BioGerm ORF1ab gene assay (9.52% versus 42.96%, P = 0.014, and 14.29% versus 42.96%, P = 0.040, respectively), whereas the N gene assay completely failed in terms of the Fin-796H variant. The Gamma and Fin-796H variants impeded the PCR amplification efficiency for the Sansure ORF1ab gene assay (33.33% versus 66.67%, P = 0.031, and 66.67% versus 95.24%, P = 0.040, respectively), and the Delta variant compromised the E gene assay (52.38% versus 85.71%, P = 0.019). The Alpha and Omicron variants had no significant effect on the kits. This study highlights the necessity of identifying the potential effect of viral mutations on the efficacy and sensitivity of clinical detection assays. It can also provide helpful insights regarding the development and optimization of diagnostic assays and aid the strategic management of the ongoing pandemic.

Effect of heat inactivation for the detection of severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2) with reverse transcription real time polymerase chain reaction (rRT-PCR): evidence from Ethiopian study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has been a major public health importance and its specimen needs to be handled safely due to concerns of potential transmissibility to health care workers. Heat inactivation of the sample before nucleic acid isolation might permit safe testing processes. Hence, it is important to assess the effect of heat inactivation on SARS-CoV-2 RT-PCR detection in resource limited settings. METHODS: An experimental study was conducted at Ethiopian Public Health Institute (EPHI) from September 25 to October 15, 2020. A total of 188 Oro-pharyngeal swabs were collected from COVID-19 suspected cases, referred to EPHI for SARS COV-2 testing. One batch of the sample was inactivated at 56 °C heat for 30 min, and the other batch was stored at 4 °C for a similar period of time. RNA extraction and detection were done by DAAN Gene kit protocols. Abbott m2000 RT-PCR was used for amplification and detection. Data analysis was done by using SPSS version 23.0; Chi-square and Pearson correlation test for qualitative and semi-quantitative analysis were used. p-value < 0.05 was considered as statistically significant. RESULTS: Out of 188 total samples, 119 (63.3%) were positive and 69 (36.7%) were negative in the non-inactivated group. While, 115 (61.2%) of samples were positive and 73 (38.8) were negative in heat inactivated sample batch. Rate of positivity between groups did not have statistically significant difference (p > 0.05). The mean Cycle threshold (Ct) value difference between the two groups of ORF1a/b gene and N gene was 0.042 (95% CI - 0.247-0.331; t = 0.28; p = 0.774) and 0.38 (95% CI 0.097-0.682; t = 2.638; p = 0.010) respectively. CONCLUSION: Heat inactivation at 56 °C for 30 min did not affect the qualitative rRT-PCR detection of SARS-CoV-2. However, the finding showed that there was statistically significant Ct value increment after heat inactivation compared to untreated samples. Therefore, false-negative results for high Ct value (Ct > 35) samples were found to be the challenge of this protocol. Hence alternative inactivation methods should be investigated and further studies should be considered.

Association Between rRT-PCR Test Results Upon Admission and Outcome in Hospitalized Chest CT-Positive COVID-19 Patients: A Provincial Retrospective Cohort with Active Follow-up

Background: The coronavirus disease 2019 (COVID-19) pandemic imposed the most devastating challenge on healthcare systems worldwide. Iran was among the first countries that had to confront serious shortages in reverse-transcriptase-polymerase chain reaction (RT-PCR) testing for severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) and ventilators availabilities throughout the COVID-19 outbreak. Objectives: This study aimed to investigate the clinical course of hospitalized COVID-19 patients with different real-time RT-PCR test results during the first three weeks of the outbreak in Qazvin province, Iran. Methods: In this retrospective cohort study, patients with a positive chest computed tomography (CT) scan for COVID-19 who were admitted to all 12 hospitals across Qazvin province, Iran, between February 20 and March 11, 2020, were included and followed up until March 27, 2020. A multivariate logistic regression model was applied to compare the independent associates of death among COVID-19 patients. Then, patients were categorized into six groups based on admission to the intensive care unit (ICU) and rRT-PCR test status (positive, negative, or no test). Also, multilevel logistic regression was used to compare the odds of surviving in each group against the reference group (PCR negative patients not-received ICU) to show if the rational allocation of ICU occurred while its capacity is limited. Results: In this study, we included 998 patients (57% male;median age: 54 years) with positive chest CT scan changes. Among them, 558 patients were examined with rRT-PCR test and 73.8% tested positive. Case fatality rate (CFR) was 20.68 and 7.53% among hospitalized patients with positive and negative tests, respectively. While only 5.2% of patients were admitted to the ICU, CFR outside ICU was 17.70 and 4.65% in patients with positive and negative results not admitted to the ICU, respectively. Conclusions: Total CFR in all hospitalized COVID-19 patients in Qazvin province during the first three weeks of the pandemic was 11.7%. Also, according to the results, the main risk factors included a positive rRT-PCR test, age more than 70 years, and having two or more comorbidities or just immunodeficiency disorders. Hence, the ICU admission criteria or prioritized ICU beds allocation should be considered with more emphasis on rRT-PCR results when the capacity of ICU beds is low.

Evaluation of two RT-PCR techniques for SARS-CoV-2 RNA detection in serum for microbiological diagnosis.

Presence of SARS-CoV-2 RNA in serum (viremia) of COVID-19 patients has been related to poor prognosis and death. The aim of this study was to evaluate both the ability to detect viremia in COVID-19 patients of two commercial reverse real-time-PCR (rRT-PCR) tests, Cobas® and TaqPath™, comparing them with a gold standard method, and their implementation in microbiology laboratories. This retrospective cohort study included 303 adult patients (203 diagnosed with COVID-19 and 100 non-COVID-19 patients) admitted to a tertiary hospital, with at least one serum sample collected within the first 48 h from admission. A total of 365 serum samples were included: 100 from non-COVID patients (pre-pandemic and pandemic control groups) and 265 from COVID-19 patients. Serum samples were considered positive when at least one target was detected. All patients in control groups showed negative viremia. Cobas® and TaqPath™ tests showed specificity and Positive Predictive Value over 96%. Nevertheless, sensitivity (53.72 and 73.63, respectively) and Negative Predictive Value (64.78 and 75) were lower. Viremia difference between ICU and non-ICU patients was significant (p ≤ 0.001) for both techniques. Consequently, SARS-CoV-2 viremia detection by both rRT-PCR tests should be considered a good tool to stratify COVID-19 patients and could be implemented in microbiology laboratories.

Transmission of SARS-CoV-2 from humans to a 16-year-old domestic cat with comorbidities in Pennsylvania, USA.

BACKGROUND AND OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), besides causing human infection, has been shown to naturally infect several susceptible animal species including large cats (tigers, lions, pumas, spotted leopards), dogs, cats, ferrets, gorillas and minks. Cats and minks are continuing to be the most reported species with SARS-CoV-2 infections among animals but it needs to be investigated further. METHODS AND RESULTS: We report the detection of SARS-CoV-2 from a domestic cat that exhibited respiratory disease after being exposed to SARS-CoV-2 virus from humans in the same household. SARS-CoV-2 RNA was detected in two oropharyngeal swabs collected at two time points, 11 days apart; the first, when the cat was reported to be sick and the second, before euthanasia due to poor prognosis. The viral nucleic acid detected at two time points showed no genomic variation and resembled the clade GH circulating in humans in the United States. Clinical and pathological findings noted in this 16-year-old cat were consistent with respiratory and cardiac insufficiency. CONCLUSIONS: SARS-CoV-2 viral infection was likely an incidental clinical finding, as the virus was not detected in fixed lungs, heart, or kidney tissues. Only fresh lung tissue collected at necropsy showed the presence of viral nucleic acid, albeit at a very low level. Further research is needed to clarify the clinical course of SARS-CoV-2 in companion animals of advanced age and underlying cardiac disease.

Virtual system for early detection of COVID-19 infection "Etaware-CDT-2020 prototype design" (corroborated by rRT-PCR data).

The 2nd phase of COVID-19 infection outbreak experienced worldwide is an attestation to the decline in the efficiency of COVID-19 detection kits available worldwide. rRT-PCR still remains the best confirmatory test for COVID-19 infection. Sadly, most medical professionals are not conversant with the rRT-PCR protocols. Therefore, more easy-to-use alternatives are required as backup, to compensate for these lapses. "Etaware-CDT-2020" is a virtual system designed for early detection of COVID-19 infection. A comparative COVID-19 diagnosis was conducted using Etaware-CDT-2020, corroborated by rRT-PCR-confirmed COVID-19 results obtained from China (Latitude 35.8617oN and Longitude 104.1954oE), which was the epicentre for COVID-19 infection outbreak. A cross-comparison of results showed that there was a positive correlation between the output result from Etaware-CDT-2020 and rRT-PCR diagnosis from Wuhan (r = 0.92) and Hubei (r = 0.97). Furthermore, there was no significant difference between the diagnostic results of "Etaware-CDT-2020" and rRT-PCR, when compared by T-test (P(t = 0) > 0.05) and Pearson's Chi-Square test (0.04 ≥ P ≤ 0.12). Etaware-CDT-2020 is unique and can be used anywhere, anytime and by anyone. It is accessible, affordable, easy to install, simple to understand and user friendly.