Clinical application of the Panbio™ COVID-19 Ag rapid test device and SSf-COVID19 kit for the detection of SARS-CoV-2 infection.

OBJECTIVE: We evaluated the sensitivity and specificity of the Panbio™ COVID-19 Ag rapid test device using nasal swabs and those of the SSf-COVID19 kit, one of RT-PCR tests, using saliva specimens. These tests were compared with RT-PCR tests using nasopharyngeal swabs for the diagnosis of SARS-CoV-2 infection. The three diagnostic tests were simultaneously conducted for patients aged ≥ 18 years, who were about to be hospitalized or had been admitted for COVID-19 confirmed by RT-PCR in two research hospitals from August 20 to October 29, 2021. Nasal swabs were tested using the Panbio™ COVID-19 Ag rapid test device. More than 1 mL of saliva was self-collected and tested using the SSf-COVID19 kit. RESULTS: In total, 157 patients were investigated; 124 patients who were about to be hospitalized and 33 patients already admitted for COVID-19. The overall sensitivity and specificity of the Panbio™ COVID-19 Ag rapid test device with nasal swabs were 64.7% (95% confidence interval [CI] 47.9-78.5%) and 100.0% (95% CI 97.0-100.0%), respectively. The median time to confirm a positive result was 180 s (interquartile range 60-255 s). The overall sensitivity and specificity of the SSf-COVID19 kit with saliva specimens were 94.1% (95% CI 80.9-98.4%) and 100.0% (95% CI 97.0-100.0%), respectively.

Guidelines for the Laboratory Diagnosis of Monkeypox in Korea.

While the coronavirus disease 2019 pandemic is ongoing, monkeypox has been rapidly spreading in non-endemic countries since May 2022. Accurate and rapid laboratory tests are essential for identifying and controlling monkeypox. Korean Society for Laboratory Medicine and the Korea Disease Prevention and Control Agency have proposed guidelines for diagnosing monkeypox in clinical laboratories in Korea. These guidelines cover the type of tests, selection of specimens, collection of specimens, diagnostic methods, interpretation of test results, and biosafety. Molecular tests are recommended as confirmatory tests. Skin lesion specimens are recommended for testing in the symptomatic stage, and the collection of both blood and oropharyngeal swabs is recommended in the presymptomatic or prodromal stage.

SARS-CoV-2 shedding dynamics and transmission in immunosuppressed patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern have been emerging. However, knowledge of temporal and spatial dynamics of SARS-CoV-2 is limited. This study characterized SARS-CoV-2 evolution in immunosuppressed patients with long-term SARS-CoV-2 shedding for 73-250 days, without specific treatment. We conducted whole-genome sequencing of 27 serial samples, including 26 serial samples collected from various anatomic sites of two patients and the first positive sample from patient 2's mother. We analysed the intrahost temporal dynamics and genomic diversity of the viral population within different sample types. Intrahost variants emerging during infection showed diversity between individual hosts. Remarkably, N501Y, P681R, and E484K, key substitutions within spike protein, emerged in vivo during infection and became the dominant population. P681R, which had not yet been detected in the publicly available genome in Korea, appeared within patient 1 during infection. Mutually exclusive substitutions at residues R346 (R346S and R346I) and E484 (E484K and E484A) of spike protein and continuous turnover of these substitutions occurred. Unique genetic changes were observed in urine samples. A household transmission from patient 2 to his mother, at least 38 days after the diagnosis, was characterized. Viruses may differently mutate and adjust to the host selective pressure, which could enable the virus to replicate efficiently for fitness in each host. Intrahost variants could be candidate variants likely to spread to the population eventually. Our findings may provide new insights into the dynamics of SARS-CoV-2 in response to interactions between the virus and host.

Update of Guidelines for Laboratory Diagnosis of COVID-19 in Korea.

Korean Society for Laboratory Medicine and the Korea Disease Prevention and Control Agency have announced guidelines for diagnosing coronavirus disease (COVID-19) in clinical laboratories in Korea. With the ongoing pandemic, we propose an update of the previous guidelines based on new scientific data. This update includes recommendations for tests that were not included in the previous guidelines, including the rapid molecular test, antigen test, antibody test, and self-collected specimens, and a revision of the previous recommendations. This update will aid clinical laboratories in performing laboratory tests for diagnosing COVID-19.

Guidelines for Mobile Laboratories for Molecular Diagnostic Testing of COVID-19.

With the rapid spread of the coronavirus disease (COVID-19), the need for rapid testing and diagnosis and consequently, the demand for mobile laboratories have increased. Despite this need, there are no clear guidelines for the operation, maintenance, or quality control of mobile laboratories. We provide guidelines for the operation, management, and quality control of mobile laboratories, and specifically for the implementation and execution of COVID-19 molecular diagnostic testing. These practical guidelines are primarily based on expert opinions and a laboratory accreditation inspection checklist. The scope of these guidelines includes the facility, preoperative evaluation, PCR testing, internal and external quality control, sample handling, reporting, laboratory personnel, biosafety level, and laboratory safety management. These guidelines are useful for the maintenance and operation of mobile laboratories not only in normal circumstances but also during public health crises and emergencies.

Longitudinal proteomic profiling provides insights into host response and proteome dynamics in COVID-19 progression.

In managing patients with coronavirus disease 2019 (COVID-19), early identification of those at high risk and real-time monitoring of disease progression to severe COVID-19 is a major challenge. We aimed to identify potential early prognostic protein markers and to expand understanding of proteome dynamics during clinical progression of the disease. We performed in-depth proteome profiling on 137 sera, longitudinally collected from 25 patients with COVID-19 (non-severe patients, n = 13; patients who progressed to severe COVID-19, n = 12). We identified 11 potential biomarkers, including the novel markers IGLV3-19 and BNC2, as early potential prognostic indicators of severe COVID-19. These potential biomarkers are mainly involved in biological processes associated with humoral immune response, interferon signalling, acute phase response, lipid metabolism, and platelet degranulation. We further revealed that the longitudinal changes of 40 proteins persistently increased or decreased as the disease progressed to severe COVID-19. These 40 potential biomarkers could effectively reflect the clinical progression of the disease. Our findings provide some new insights into host response to SARS-CoV-2 infection, which are valuable for understanding of COVID-19 disease progression. This study also identified potential biomarkers that could be further validated, which may support better predicting and monitoring progression to severe COVID-19.

Sequential Analysis of Viral Load in a Neonate and Her Mother Infected With Severe Acute Respiratory Syndrome Coronavirus 2.

We report changes in viral load over time in a 27-day-old neonate with coronavirus disease 2019 who presented with fever, cough, and vomiting. Severe acute respiratory syndrome coronavirus 2 RNA was detected in the nasopharynx, oropharynx, stool, saliva, plasma, and urine. The highest viral RNA copies in nasopharynx decreased over time while viral load in stool remained high.

Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals.

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were composed of immunoglobulin heavy variable 3-53 (IGHV3-53) or IGHV3-66 and immunoglobulin heavy joining 6 (IGHJ6) genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different IGHV chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in 6 of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

In-depth blood proteome profiling analysis revealed distinct functional characteristics of plasma proteins between severe and non-severe COVID-19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over forty million patients worldwide. Although most coronavirus disease 2019 (COVID-19) patients have a good prognosis, some develop severe illness. Markers that define disease severity or predict clinical outcome need to be urgently developed as the mortality rate in critical cases is approximately 61.5%. In the present study, we performed in-depth proteome profiling of undepleted plasma from eight COVID-19 patients. Quantitative proteomic analysis using the BoxCar method revealed that 91 out of 1222 quantified proteins were differentially expressed depending on the severity of COVID-19. Importantly, we found 76 proteins, previously not reported, which could be novel prognostic biomarker candidates. Our plasma proteome signatures captured the host response to SARS-CoV-2 infection, thereby highlighting the role of neutrophil activation, complement activation, platelet function, and T cell suppression as well as proinflammatory factors upstream and downstream of interleukin-6, interleukin-1B, and tumor necrosis factor. Consequently, this study supports the development of blood biomarkers and potential therapeutic targets to aid clinical decision-making and subsequently improve prognosis of COVID-19.

Erratum: COVID-19 Molecular Testing in Korea: Practical Essentials and Answers From Experts Based on Experiences of Emergency Use Authorization Assays.

[This corrects the article on p. 439 in vol. 40.].

Evidence of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection After Recovery from Mild Coronavirus Disease 2019.

BACKGROUND: Positive results from real-time reverse-transcription polymerase chain reaction (rRT-PCR) in recovered patients raise concern that patients who recover from coronavirus disease 2019 (COVID-19) may be at risk of reinfection. Currently, however, evidence that supports reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been reported. METHODS: We conducted whole-genome sequencing of the viral RNA from clinical specimens at the initial infection and at the positive retest from 6 patients who recovered from COVID-19 and retested positive for SARS-CoV-2 via rRT-PCR after recovery. A total of 13 viral RNAs from the patients' respiratory specimens were consecutively obtained, which enabled us to characterize the difference in viral genomes between initial infection and positive retest. RESULTS: At the time of the positive retest, we were able to acquire a complete genome sequence from patient 1, a 21-year-old previously healthy woman. In this patient, through the phylogenetic analysis, we confirmed that the viral RNA of positive retest was clustered into a subgroup distinct from that of the initial infection, suggesting that there was a reinfection of SARS-CoV-2 with a subtype that was different from that of the primary strain. The spike protein D614G substitution that defines the clade "G" emerged in reinfection, while mutations that characterize the clade "V" (ie, nsp6 L37F and ORF3a G251V) were present at initial infection. CONCLUSIONS: Reinfection with a genetically distinct SARS-CoV-2 strain may occur in an immunocompetent patient shortly after recovery from mild COVID-19. SARS-CoV-2 infection may not confer immunity against a different SARS-CoV-2 strain.

Viral RNA Load in Mildly Symptomatic and Asymptomatic Children with COVID-19, Seoul, South Korea.

Along with positive SARS-CoV-2 RNA in nasopharyngeal swabs, viral RNA was detectable at high concentration for >3 weeks in fecal samples from 12 mildly symptomatic and asymptomatic children with COVID-19 in Seoul, South Korea. Saliva also tested positive during the early phase of infection. If proven infectious, feces and saliva could serve as transmission sources.

Nationwide External Quality Assessment of SARS-CoV-2 Molecular Testing, South Korea.

External quality assessment (EQA) is essential for ensuring reliable test results, especially when laboratories are using assays authorized for emergency use for newly emerging pathogens. We developed an EQA panel to assess the quality of real-time reverse transcription PCR assays being used in South Korea to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the participation of 23 public health organization laboratories and 95 nongovernmental laboratories involved in SARS-CoV-2 testing, we conducted qualitative and semiquantitative performance assessments by using pooled respiratory samples containing different viral loads of SARS-CoV-2 or human coronavirus OC43. A total of 110 (93.2%) laboratories reported correct results for all qualitative tests; 29 (24.6%) laboratories had >1 outliers according to cycle threshold values. Our EQA panel identified the potential weaknesses of currently available commercial reagent kits. The methodology we used can provide practical experience for those planning to conduct evaluations for testing of SARS-CoV-2 and other emerging pathogens in the future.

COVID-19 Molecular Testing in Korea: Practical Essentials and Answers From Experts Based on Experiences of Emergency Use Authorization Assays.

Coronavirus disease 2019 (COVID-19) is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Early detection of COVID-19 and immediate isolation of infected patients from the naive population are important to prevent further pandemic spread of the infection. Real-time reverse transcription (RT)-PCR to detect SARS-CoV-2 RNA is currently the most reliable diagnostic method for confirming COVID-19 worldwide. Guidelines for clinical laboratories on the COVID-19 diagnosis have been recently published by Korean Society for Laboratory Medicine and the Korea Centers for Disease Control and Prevention. However, these formal guidelines do not address common practical laboratory issues related to COVID-19 real-time RT-PCR testing and their solutions. Therefore, this guideline is intended as a practical and technical supplement to the "Guidelines for Laboratory Diagnosis of COVID-19 in Korea".

A cluster of tertiary transmissions of 2019 novel coronavirus (SARS-CoV-2) in the community from infectors with common cold symptoms.

BACKGROUND/AIMS: As the global impact of the novel coronavirus disease 2019 (COVID-19) has been severe, many countries have intensified containment activities to eliminate virus transmission, through early detection and isolation strategies. To establish a proper quarantine strategy, it is essential to understand how easily the virus can spread in the communities. METHODS: In this study, we collected detailed information on the circumstances in which human-to-human transmission occurred in the tertiary transmission cases of COVID-19 in the community. RESULTS: On January 26, 2020, an imported case of COVID-19 was confirmed, and by February 10, 2020, one secondary transmission and three tertiary transmissions were identified. Secondary transmission occurred on the first day of illness of the infector, and his symptoms were suggestive of a common cold. The transmission occurred during a 90-minute long meal together in a restaurant. The people were sitting within one meter of each other, and had no direct contact. The tertiary transmission also occurred on the first-day illness of the other infector, and his only symptom was slight chills. The transmission occurred at a church during 2-hour-long worship, and two rows separated them. CONCLUSION: Our findings suggest that mildly symptomatic patients with COVID-19 could transmit the virus from the first day of illness through daily activities in the community. Early detection and isolation of patients with COVID-19 may be challenging.

In vitro activity of lopinavir/ritonavir and hydroxychloroquine against severe acute respiratory syndrome coronavirus 2 at concentrations achievable by usual doses.

BACKGROUND/AIMS: As the coronavirus disease-2019 global pandemic progresses, screening of antiviral agents effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is urgently needed. In addition, considering the viral load kinetics of SARS-CoV-2, which peaks early in the illness, and the massive burden of the disease, which may increase in the near future, identifying well-tolerated oral antivirals becomes increasingly important. We examined the in vitro activity of lopinavir/ritonavir and hydroxychloroquine on SARS-CoV-2, at concentrations which can be used to treat coronavirus-19 patients with little concern of toxicity. METHODS: Lopinavir/ritonavir (7/1.75 µg/mL), hydroxychloroquine base (1 or 2 µg/mL), or a combination thereof were administered 1 hour after the inoculation of SARS-CoV-2 to Vero cells at a multiplicity of infection of 0.05. We examined cytopathic effects of virus 48 hours after administration of the respective treatments and measured viral loads at three time points (0, 24, and 48 hours post-treatment) by quantitative real-time reverse-transcription polymerase chain reaction, and compared the results obtained from the different antiviral regimens tested. RESULTS: The severity of cytopathic effects was lower in lopinavir/ritonavir-treated cells, and viral load was significantly reduced in this group compared with the control group (p < 0.001). However, hydroxychloroquine did not show significant inhibitory effects on anti-SARS-CoV-2-mediated cytotoxicity or on viral load at either concentration. CONCLUSION: Lopinavir/ritonavir showed significant inhibitory effects on SARS-CoV-2 in vitro at its usual plasma concentration. However, the in vitro antiviral activity of hydroxychloroquine at concentrations commonly used in humans was minimal, whether used alone or in combination with lopinavir/ritonavir.

Virus Isolation from the First Patient with SARS-CoV-2 in Korea.

Novel coronavirus (SARS-CoV-2) is found to cause a large outbreak started from Wuhan since December 2019 in China and SARS-CoV-2 infections have been reported with epidemiological linkage to China in 25 countries until now. We isolated SARS-CoV-2 from the oropharyngeal sample obtained from the patient with the first laboratory-confirmed SARS-CoV-2 infection in Korea. Cytopathic effects of SARS-CoV-2 in the Vero cell cultures were confluent 3 days after the first blind passage of the sample. Coronavirus was confirmed with spherical particle having a fringe reminiscent of crown on transmission electron microscopy. Phylogenetic analyses of whole genome sequences showed that it clustered with other SARS-CoV-2 reported from Wuhan.