RNA loads of severe acute respiratory syndrome coronavirus 2 in patients with breakthrough coronavirus disease 2019 caused by the Delta and Omicron variants.

OBJECTIVES: To compare the RNA loads of severe acute respiratory syndrome coronavirus 2 in nasopharyngeal specimens collected from patients with breakthrough coronavirus disease 2019 (COVID-19) caused by the Delta variant with those in specimens collected from patients with breakthrough COVID-19 caused by the Omicron variant. METHODS: A retrospective, observational study was conducted, including 240 consecutive adult out-patients, of whom 121 (74 females; median age, 40 years) had COVID-19 due to the Omicron variant and 119 (65 females; median age, 48 years) had COVID-19 caused by the Delta variant. The viral RNA load was quantitated using the TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Waltham, MS, USA). The viability platinum chloride reverse transcription-PCR assay was used to discriminate between potentially infectious viral particles and free (encapsidated) viral RNA. RESULTS: Overall, the viral RNA loads were significantly higher (p 0.003) for the Omicron variant (median, 8.1 log10 copies/mL; range, 4.0-10.9 log10 copies/mL) than for the Delta variant (median, 7.5 log10 copies/mL; range, 3.0-11.6 log10 copies/mL). A trend towards higher viral loads was noticed for Omicron compared with that for Delta across the following time frames since vaccination: 16-90 days (median, 6.83 vs. 5.88 log10 copies/mL, respectively; range, 3.91-10.68 vs. 3.67-9.66 log10 copies/mL, respectively; p 0.10), 91-180 days (median, 8.09 vs. 7.46 log10 copies/mL, respectively; range, 4.30-10.92 vs. 3.03-11.56 log10 copies/mL, respectively; p 0.003) and 181-330 days (median, 8.56 vs. 8.10 log10 copies/mL, respectively; range, 6.51-10.29 vs. 3.03-10.61 log10 copies/mL, respectively; p 0.11). The platinum chloride treated or untreated reverse transcription-PCR cycle threshold ratio for the nucleocapsid gene as the target was slightly higher for Omicron than for Delta (median, 0.62 vs. 0.57, respectively; range, 0.57-0.98 vs. 0.61-0.87, respectively), although statistical significance was not reached (p 0.10). CONCLUSION: The time elapsed since vaccination has a major impact on the RNA loads of severe acute respiratory syndrome coronavirus 2 in nasopharyngeal specimens, particularly for the Omicron variant. The Omicron variant may be better adapted for replication in the upper respiratory tract than the Delta variant, in which this is unlikely given its more efficient generation of viral particles.

Understanding Host-Virus Interactions: Assessment of Innate Immune Responses in Mastomys natalensis Cells after Arenavirus Infection.

Mastomys natalensis is the natural host of various arenaviruses, including the human-pathogenic Lassa virus. Homologous arenaviruses, defined here as those having M. natalensis as a natural host, can establish long-lasting infection in M. natalensis, while these animals rapidly clear arenaviruses having another rodent species as a natural host (heterologous viruses). Little is known about the mechanisms behind the underlying arenavirus-host barriers. The innate immune system, particularly the type I interferon (IFN) response, might play a role. In this study, we developed and validated RT-PCR assays to analyse the expression of M. natalensis interferon-stimulated genes (ISGs). We then used these assays to study if homologous and heterologous viruses induce different IFN responses in M. natalensis cells. Infection experiments were performed with the homologous Lassa and Morogoro viruses and the related but heterologous Mobala virus. Compared to the direct induction with IFN or Poly(I:C), arenaviruses generally induced a weak IFN response. However, the ISG-expression profiles of homologous and heterologous viruses were similar. Our data indicate that, at least in M. natalensis cells, the IFN system is not a major factor in the virus-host barrier for arenaviruses. Our system provides a valuable tool for future in vivo investigation of arenavirus host restrictions at the level of the innate immune response.

Differential gene expression analysis of common target genes for the detection of SARS-CoV-2 using real time-PCR.

COVID-19 currently is the main cause of the severe acute respiratory disease and fatal outcomes in human beings worldwide. Several genes are used as targets for the detection of SARS-CoV-2, including the RDRP, N, and E genes. The present study aimed to determine the RDRP, N, and E genes expressions of SARS-CoV- 2 in clinical samples. For this purpose, 100 SARS-CoV-2 positive samples were collected from diagnostic laboratories of Mazandaran province, Iran. After RNA extraction, the real-time reverse transcription PCR (real-time RT-PCR) assay was performed for differential gene expressions' analysis of N, E, and RDRP. The threshold cycle (Ct) values for N, RDRP, and E targets of 100 clinical samples for identifying SARS-CoV-2 were then evaluated using quantitative real-time PCR (qRT-PCR). This result suggests N gene as a potential target for the detection of the SARS-CoV-2, since it was observed to be highly expressed in the nasopharyngeal or oropharynges of COVID-19 patients (P < 0.0001). Herein, we showed that SARS-CoV- 2 genes were differentially expressed in the host cells. Therefore, to reduce obtaining false negative results and to increase the sensitivity of the available diagnostic tests, the target genes should be carefully selected based on the most expressed genes in the cells.

Diagnostic sensitivity of RT-PCR assays on nasopharyngeal specimens for detection of SARS-CoV-2 infection: A Systematic Review and Meta-Analysis.

Background: Reverse transcription polymerase chain reaction (RT-PCR) is the current standard of reference in the diagnosis of SARS-CoV-2 infection. In outpatient clinical practice, nasopharyngeal swab RT-PCR testing is still the most common procedure. The purpose of this systematic review and meta-analysis was to evaluate the sensitivity of RT-PCR nasopharyngeal assays. Methods: We searched three databases, including PubMed/MEDLINE, EMBASE, and Cochrane Library, using a comprehensive strategy. Studies investigating the sensitivity of SARS-CoV-2 RT-PCR nasopharyngeal assays in adults were included. Two reviewers extracted data and assessed trial quality independently. Pooled sensitivity and its confidence interval were computed using the meta package in R. Results: Thirteen studies were found eligible for the inclusion in the systematic review. Out of these, 25 different sub-studies were identified and included in the meta-analysis, which reported the sensitivities of 25 different nasopharyngeal RT-PCR assays. Finally, the overall pooled sensitivity resulted 89% (95% CI, 85.4 to 91.8%). Conclusion: Our study suggests that RT-PCR assays on nasopharyngeal specimens have a substantial sensitivity for diagnosing SARS-CoV-2 infection.

Comparison of Chest CT and RT-PCR Assay for Indication of Disease Course of Coronavirus Disease 2019 (COVID-19) Pneumonia.

BACKGROUND: COVID-19 patients' courses vary in length, indicating a variable prognosis. The disease duration revealed by different examination methods may differ. OBJECTIVE: The study aims to compare the differences in the disease course of patients with COVID-19 by chest computed tomography (CT) and reverse-transcription polymerase chain reaction (RT-PCR) assay and explore the factors that affect the course of the illness. METHODS: 106 patients confirmed with COVID-19 were enrolled and divided into two groups (age <60 years and age ≥60 years). The clinical characteristics of the two groups were analyzed. The intervals from symptoms onset to initial positive time point (ISIP), symptoms onset to the initial negative time point (ISIN), and initial positive to initial negative time point (IIPN) indicated by chest CT and RTPCR assay were analyzed. Multiple regression analysis was performed to assess the correlations between independent factors and the intervals. RESULTS: Chest CT showed an earlier positive time point, a later negative time point, and a longer disease duration than the RT-PCR assay (P<.001, respectively). Older patients over 60 years old showed a later negative time point and a longer disease duration by chest CT than younger patients (P<.01 vs. P<.05, respectively). The CT score and clinical grades of older patients were greater than those of younger patients (P<.001, respectively). Age and clinical grades were significantly correlated with the disease course shown by chest CT (P<.05, respectively), and CT score was positively correlated with the illness course shown by chest CT and RT-PCR assay (P<.01, respectively). CONCLUSION: The disease course revealed by chest CT and RT-PCR assay was asynchronous. Chest CT showed a 17-day longer period compared to the RT-PCR assay. Older patients had a longer duration than younger ones. A prolonged course is predicted by increasing age, CT score, and clinical grades.

Pooling for SARS-CoV2 Surveillance: Validation and Strategy for Implementation in K-12 Schools.

Repeated testing of a population is critical for limiting the spread of the SARS-CoV-2 virus and for the safe reopening of educational institutions such as kindergarten-grade 12 (K-12) schools and colleges. Many screening efforts utilize the CDC RT-PCR based assay which targets two regions of the novel Coronavirus nucleocapsid gene. The standard approach of testing each person individually, however, poses a financial burden to these institutions and is therefore a barrier to using testing for re-opening. Pooling samples from multiple individuals into a single test is an attractive alternate approach that promises significant cost savings-however the specificity and sensitivity of such approaches needs to be assessed prior to deployment. To this end, we conducted a pilot study to evaluate the feasibility of analyzing samples in pools of eight by the established RT-PCR assay. Participants (1,576) were recruited from amongst the Tufts University community undergoing regular screening. Each volunteer provided two swabs, one analyzed separately and the other in a pool of eight. Because the positivity rate was very low, we spiked approximately half of the pools with laboratory-generated swabs produced from known positive cases outside the Tufts testing program. The results of pooled tests had 100% correspondence with those of their respective individual tests. We conclude that pooling eight samples does not negatively impact the specificity or sensitivity of the RT-PCR assay and suggest that this approach can be utilized by institutions seeking to reduce surveillance costs.

Clinical evaluation of the molecular-based BD SARS-CoV-2/Flu for the BD MAX™ system.

BACKGROUND: COVID-19 and influenza (flu) share similar clinical symptoms. Therefore, differential detection of these viruses during the respiratory virus season will be an important component for proper patient triage, management, and treatment. OBJECTIVES: Establish the diagnostic performance related to SARS-CoV-2 and Flu A/B detection for the BD SARS-CoV-2/Flu for BD MAX™ System ("MAX SARS-CoV-2/Flu") multiplex assay. MATERIALS AND METHODS: Two hundred and thirty-five (235) retrospective nasopharyngeal specimens were obtained from external vendors. The BD BioGx SARS-CoV-2 Reagents for BD MAX™ System ("BioGx SARS-CoV-2″) and the Cepheid Xpert® Xpress Flu/RSV ("Xpert Flu/RSV") were utilized as reference methods. RESULTS: By reference methods, 52 specimens were SARS-CoV-2-positive, 59 were Flu A-positive, and 60 were Flu B-positive. MAX SARS-CoV-2/Flu had positive percent agreement (PPA) and negative percent agreement (NPA) values for SARS-CoV-2 detection of 96.2% ([95%CI]:87.0-98.9) and 100% [95%CI:88.7-100], respectively; PPA values for Flu A and Flu B of 100% [95%CI:93.9-100] and 98.3% [95%CI:91.1-99.7], respectively, and NPA values for Flu A and Flu B of 98.9% [95%CI:94.0-99.8] and 100% [95%CI:95.9-100], respectively. CONCLUSIONS: The MAX SARS-CoV-2/Flu assay met FDA-EUA performance criteria for SARS-CoV-2 (≥95% for PPA and NPA) and FDA clearance criteria for Flu A/B (PPA ≥90%; lower bound of the 95%CI ≥80% and NPA ≥95%; lower bound of the 95%CI ≥90%).

Rapid detection of human respiratory syncytial virus A and B by duplex real-time RT-PCR.

Respiratory syncytial virus (RSV) is a common cause of acute respiratory disease worldwide, especially in young children. The World Health Organization (WHO) has initiated an RSV Surveillance Pilot program that aims to perform worldwide RSV surveillance, requiring the development of reliable and rapid molecular methods to detect and identify RSV. A duplex real-time RT-PCR assay developed for simultaneous detection of both A and B subtypes of RSV was included as part of this program. This duplex assay targeted a conserved region of the RSV polymerase gene and was validated for analytical sensitivity, specificity, reproducibility and clinical performance with a wide range of respiratory specimens. The assay was highly specific for RSV and did not react with non-RSV respiratory pathogens, including the SARS-CoV-2 virus.

A Rapid SARS-CoV-2 RT-PCR Assay for Low Resource Settings.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.

A case of imported COVID-19 diagnosed by PCR-positive lower respiratory specimen but with PCR-negative throat swabs.

A 35-year-old woman presented with fever and mild diarrhoea without any respiratory symptoms 9 days after travelling to Japan from Wuhan, China. Her computed tomography scan revealed pneumonia. The first polymerase chain reaction (PCR) test on throat swab for the novel corona virus upon admission was negative. Therefore, she was treated for community-acquired pneumonia, but fever persisted. On hospital day 5, PCR test on induced sputum was positive, but a second polymerase chain reaction test on throat swab remained negative. She was discharged, fully recovered, on hospital day 12. A lower respiratory tract specimen should be obtained for better diagnosis of corona virus disease 2019, even in the absence of respiratory symptoms for patients with significant travel or exposure history.