Comparison of Nucleocapsid Antigen with Strand-specific Reverse-Transcription PCR for Monitoring SARS-CoV-2 Infection

Background . Tests that sensitively detect the presence of actively replicating SARS-CoV-2 may improve patient care by allowing the safe and timely discontinuation of isolation. Correlates of active replication include nucleocapsid antigen and virus minus-strand RNA. Methods . Qualitative agreement of the DiaSorin LIAISON SARS-CoV-2 nucleocapsid antigen chemiluminescent immunoassay (CLIA) with minus-strand RNA was determined using 402 upper respiratory specimens from 323 patients previously tested using a laboratory-developed SARS-CoV-2 strand-specific RT-qPCR. Nucleocapsid antigen levels, minus-strand and plus-strand cycle threshold values, as well as virus culture, were used to evaluate discordant specimens. Receiver operating characteristic curves were also used to identify virus RNA thresholds for active replication, including values harmonized to the World Health Organization International Standard. Results . Overall agreement was 92.0% [95% confidence interval (CI): 89.0 – 94.5], positive percent agreement was 90.6% (95% CI: 84.4 – 95.0), and negative percent agreement was 92.8% (95% CI: 89.0 – 95.6). The kappa coefficient was 0.83 (95% CI: 0.77 – 0.88). Discordant specimens contained low levels of nucleocapsid antigen and minus-strand RNA. 84.8% (28/33) were negative by culture. Sensitivity-optimized plus-strand RNA thresholds for active replication were 31.6 cycles or 3.64 log10 IU/mL;resulting in 100.0% sensitivity (95% CI: 97.6 to 100.0) and 55.9 specificity (95% CI: 49.7 to 62.0). Conclusions . Detection of nucleocapsid antigen by CLIA performs equivalently to minus-strand detection via strand-specific RT-qPCR, though these methods may overestimate replication-competent virus compared to culture. Careful implementation of biomarkers for actively replicating SARS-CoV-2 has the potential to inform infection control decision-making and patient management.

Standardization of SARS-CoV-2 Cycle Threshold Values: Multisite Investigation Evaluating Viral Quantitation across Multiple Commercial COVID-19 Detection Platforms.

The demand for testing during the coronavirus disease 2019 (COVID-19) pandemic has resulted in the production of several different commercial platforms and laboratory-developed assays for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This has created several challenges, including, but not limited to, the standardization of diagnostic testing, utilization of cycle threshold (CT) values for quantitation and clinical interpretation, and data harmonization. Using reference standards consisting of a linear range of SARS-CoV-2 concentrations quantitated by viral culture-based methods and droplet digital PCR, we investigated the commutability and standardization of SARS-CoV-2 quantitation across different laboratories in the United States. We assessed SARS-CoV-2 CT values generated on multiple reverse transcription-PCR (RT-PCR) platforms and analyzed PCR efficiencies, linearity, gene targets, and CT value agreement. Our results demonstrate the inappropriateness of using SARS-CoV-2 CT values without established standards for viral quantitation. Further, we emphasize the importance of using reference standards and controls validated to independent assays, to compare results across different testing platforms and move toward better harmonization of COVID-19 quantitative test results. IMPORTANCE From the onset of the COVID-19 pandemic, the demand for SARS-CoV-2 testing has resulted in an explosion of analytical tests with very different approaches and designs. The variability in testing modalities, compounded by the lack of available commercial reference materials for standardization early in the pandemic, has led to several challenges regarding data harmonization for viral quantitation. In this study, we assessed multiple commercially available RT-PCR platforms across different laboratories within the United States using standardized reference materials characterized by viral culture methods and droplet digital PCR. We observed variability in the results generated by different instruments and laboratories, further emphasizing the importance of utilizing validated reference standards for quantitation, to better harmonize SARS-CoV-2 test results.

Cepheid Xpert Xpress Flu/RSV evaluation performed by minimally trained non-laboratory operators in a CLIA-waived environment.

The COVID-19 pandemic highlighted the significance of readily available and easily performed viral testing for surveillance during future infectious pandemics. The objectives of this study were: to assess the performance of the Xpert Xpress Flu and/or RSV test, a multiplex PCR assay for detecting influenza A and B virus and respiratory syncytial virus nucleic acids in respiratory tract specimens, relative to the Quidel Lyra Influenza A+B assay and the Prodesse ProFlu+ assay, and the system's ease of use by minimally trained operators. Overall, the Xpert Xpress Flu/RSV test demonstrated a high positive and negative percent agreement with the comparator assays, and was easy to use and interpret results, based on the operators' feedback. We concluded that the Xpert Xpress Flu/RSV test is sensitive, specific, and easy to use for the diagnosis of influenza and RSV by minimally trained operators and can be a valuable tool in future infectious clusters or pandemics.

Soluble Immune Checkpoints Are Dysregulated in COVID-19 and Heavy Alcohol Users With HIV Infection.

Immune checkpoints (ICPs) consist of paired receptor-ligand molecules that exert inhibitory or stimulatory effects on immune defense, surveillance, regulation, and self-tolerance. ICPs exist in both membrane and soluble forms in vivo and in vitro. Imbalances between inhibitory and stimulatory membrane-bound ICPs (mICPs) in malignant cells and immune cells in the tumor immune microenvironment (TIME) have been well documented. Blockades of inhibitory mICPs have emerged as an immense breakthrough in cancer therapeutics. However, the origin, structure, production regulation, and biological significance of soluble ICPs (sICPs) in health and disease largely remains elusive. Soluble ICPs can be generated through either alternative mRNA splicing and secretion or protease-mediated shedding from mICPs. Since sICPs are found in the bloodstream, they likely form a circulating immune regulatory system. In fact, there is increasing evidence that sICPs exhibit biological functions including (1) regulation of antibacterial immunity, (2) interaction with their mICP compartments to positively or negatively regulate immune responses, and (3) competition with their mICP compartments for binding to the ICP blocking antibodies, thereby reducing the efficacy of ICP blockade therapies. Here, we summarize current data of sICPs in cancer and infectious diseases. We particularly focus on sICPs in COVID-19 and HIV infection as they are the two ongoing global pandemics and have created the world's most serious public health challenges. A "storm" of sICPs occurs in the peripheral circulation of COVID-19 patients and is associated with the severity of COVID-19. Similarly, sICPs are highly dysregulated in people living with HIV (PLHIV) and some sICPs remain dysregulated in PLHIV on antiretroviral therapy (ART), indicating these sICPs may serve as biomarkers of incomplete immune reconstitution in PLHIV on ART. We reveal that HIV infection in the setting of alcohol misuse exacerbates sICP dysregulation as PLHIV with heavy alcohol consumption have significantly elevated plasma levels of many sICPs. Thus, both stimulatory and inhibitory sICPs are present in the bloodstream of healthy people and their balance can be disrupted under pathophysiological conditions such as cancer, COVID-19, HIV infection, and alcohol misuse. There is an urgent need to study the role of sICPs in immune regulation in health and disease.

Serum from COVID-19 patients early in the pandemic shows limited evidence of cross-neutralization against variants of concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results in a variety of clinical symptoms ranging from no or mild to severe disease. Currently, there are multiple postulated mechanisms that may push a moderate to severe disease into a critical state. Human serum contains abundant evidence of the immune status following infection. Cytokines, chemokines, and antibodies can be assayed to determine the extent to which a patient responded to a pathogen. We examined serum and plasma from a cohort of patients infected with SARS-CoV-2 early in the pandemic and compared them to negative-control sera. Cytokine and chemokine concentrations varied depending on the severity of infection, and antibody responses were significantly increased in severe cases compared to mild to moderate infections. Neutralization data revealed that patients with high titers against an early 2020 SARS-CoV-2 isolate had detectable but limited neutralizing antibodies against the emerging SARS-CoV-2 Alpha, Beta and Delta variants. This study highlights the potential of re-infection for recovered COVID-19 patients.

Excessive Matrix Metalloproteinase-1 and Hyperactivation of Endothelial Cells Occurred in COVID-19 Patients and Were Associated With the Severity of COVID-19.

BACKGROUND: Systemic vascular injury occurs in coronavirus disease 2019 (COVID-19) patients; however, the underlying mechanisms remain unknown. METHODS: To clarify the role of inflammatory factors in COVID-19 vascular injury, we used a multiplex immunoassay to profile 65 inflammatory cytokines/chemokines/growth factors in plasma samples from 24 hospitalized (severe/critical) COVID-19 patients, 14 mild/moderate cases, and 13 healthy controls (HCs). RESULTS: COVID-19 patients had significantly higher plasma levels of 20 analytes than HCs. Surprisingly, only 1 cytokine, macrophage migration inhibitory factor (MIF), was among these altered analytes, while the rest were chemokines/growth factors. Additionally, only matrix metalloproteinase-1 (MMP-1) and vascular endothelial growth factor A (VEGF-A) were significantly elevated in hospitalized COVID-19 patients when compared to mild/moderate cases. We further studied MMP-1 enzymatic activity and multiple endothelial cell (EC) activation markers (soluble forms of CD146, intercellular adhesion molecule 1 [ICAM-1], and vascular cell adhesion molecule 1 [VCAM-1]) and found that they were highly dysregulated in COVID-19 patients. CONCLUSIONS: COVID-19 patients have a unique inflammatory profile, and excessive MMP-1 and hyperactivation of ECs are associated with the severity of COVID-19.

Identifying Risk Factors That Distinguish Symptomatic Severe Acute Respiratory Syndrome Coronavirus 2 Infection From Common Upper Respiratory Infections in Children.

Background Demographic and clinical risk factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children presenting with respiratory viral symptoms are not well defined. An understanding of risk factors for SARS-CoV-2 infection can help prioritize testing. Methodology We evaluated potential demographic and clinical factors in children who had respiratory viral symptoms and were tested by polymerase chain reaction (PCR) for SARS-CoV-2 and other respiratory viral infections. Results Among the 263 symptomatic children tested for routine seasonal respiratory viruses by PCR, 18 (6.8%) tested positive for SARS-CoV-2. Overall, 22.2% of SARS-CoV-2-infected children and 37.1% of SARS-CoV-2-uninfected children had infection with one or more non-SARS-CoV-2 pathogens (p = 0.31). Higher proportions of children with compared to without SARS-CoV-2 infection were male (77.8 vs. 51.8%, p = 0.05), Hispanic (44.4% vs. 9.8%, p < 0.001), or had the symptoms of fatigue (22.2% vs. 2.5%, p = 0.003) or anosmia/ageusia (11.1% vs. 0%, p = 0.004). History of hypoxic-ischemic encephalopathy (HIE) and obesity were more common in children with versus without SARS-CoV-2 infection (11.1% vs. 1.2%, p = 0.04, and 11.1% vs. 0%, p = 0.004, respectively). In a multivariate analysis, Hispanic ethnicity, symptoms of fatigue or anosmia/ageusia, and presence of obesity (as noted on physical examination) or HIE were independently associated with SARS-CoV-2 infection. Numbers in each category were small, and these preliminary associations require confirmation in future studies. Conclusions In this area of the United States, infection with other viruses did not rule out infection with SARS-CoV-2. Additionally, children with respiratory viral symptoms who were of Hispanic ethnicity, had symptoms of weakness/fatigue, or had obesity or HIE were at an increased risk for SARS-CoV-2 infection. Future studies should assess if these factors are associated with risk in populations in other areas of the United States.

Persistent SARS-CoV-2 infectivity greater than 50 days in a case series of allogeneic peripheral blood stem cell transplant recipients.

The coronavirus disease 19 (COVID-19) pandemic has infected tens of millions across the world, but there is a significant gap in our understanding about COVID-19 in the hematopoietic stem transplant (HSCT) recipient population. Prolonged viral shedding is frequently observed with severe acute respiratory syndrome coronavirus-2 (SARSCoV-2), but studies suggest viral loads decline 10 days after symptom onset. Current CDC guidance suggests that severely ill and immunocompromised hosts are no longer infectious after 20 days from symptom onset. Cycle threshold (Ct) values are inversely proportional to the viral load and are used to detect SARS-CoV-2 RNA concentration. Specimens with reverse transcriptase PCR (RT-PCR) Ct values > 33-34 have been associated with inability to culture virus, and have been used as a surrogate for diminished infectivity. We report two cases of  allogeneic peripheral blood stem cell transplant (PBSCT) recipients who had prolonged durations of infectivity with SARSCov-2, based on culture positivity and persistently low Ct values for greater than 50 days.

Multicenter Evaluation of the QIAstat-Dx Respiratory Panel for Detection of Viruses and Bacteria in Nasopharyngeal Swab Specimens.

The QIAstat-Dx Respiratory Panel (QIAstat-Dx RP) is a multiplex in vitro diagnostic test for the qualitative detection of 20 pathogens directly from nasopharyngeal swab (NPS) specimens. The assay is performed using a simple sample-to-answer platform with results available in approximately 69 min. The pathogens identified are adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, human metapneumovirus A and B, influenza A, influenza A H1, influenza A H3, influenza A H1N1/2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, rhinovirus/enterovirus, respiratory syncytial virus A and B, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae This multicenter evaluation provides data obtained from 1,994 prospectively collected and 310 retrospectively collected (archived) NPS specimens with performance compared to that of the BioFire FilmArray Respiratory Panel, version 1.7. The overall percent agreement between QIAstat-Dx RP and the comparator testing was 99.5%. In the prospective cohort, the QIAstat-Dx RP demonstrated a positive percent agreement of 94.0% or greater for the detection of all but four analytes: coronaviruses 229E, NL63, and OC43 and rhinovirus/enterovirus. The test also demonstrated a negative percent agreement of ≥97.9% for all analytes. The QIAstat-Dx RP is a robust and accurate assay for rapid, comprehensive testing for respiratory pathogens.