Clinical Evaluation of BioFire COVID-19 Test, BioFire Respiratory Panel 2.1, and Cepheid Xpert Xpress SARS-CoV-2 Assays for Sample-to-Answer Detection of SARS-CoV-2.

Background: Due to the extreme infectivity of SARS-CoV-2, sample-to-answer SARS-CoV-2 reverse transcription (RT) polymerase chain reaction (PCR) assays are urgently needed in order to facilitate infectious disease surveillance and control. The purpose of this study was to evaluate three sample-to-answer SARS-CoV-2 RT-PCR assays­BioFire COVID-19 Test, BioFire RP 2.1, and Cepheid Xpert Xpress SARS-CoV-2­using clinical samples. Methods: A total of 77 leftover nasopharyngeal swab (NP) swabs (36 positives and 41 negatives) confirmed by reference SARS-CoV-2 RT real-time (q) PCR assay were collected. The clinical sample concordance, as specified by their respective emergency use authorizations (EUAs), in comparison to the reference SARS-CoV-2 RT-qPCR assay, was assessed. Results: The results showed that all three sample-to-answer SARS-CoV-2 RT-PCR assays provided perfectly concordant results consistent with the reference SARS-CoV-2 RT-qPCR assay. The BioFire COVID-19 Test exhibited the best turnaround time (TAT) compared to the other assays, regardless of the test results, using one-way analysis of variance followed by Scheffe's post hoc test (p < 0.001). The Xpert Xpress SARS-CoV-2 showed a shorter average TAT (mean ± standard deviation, 49.9 ± 3.1 min) in the positive samples compared to that (55.7 ± 2.5 min) of the negative samples. Conclusions: Our evaluation demonstrates that the BioFire COVID-19 Test, BioFire RP 2.1, and Cepheid Xpert Xpress SARS-CoV-2 assays compare favorably to the reference SARS-CoV-2 RT-qPCR assay, along with a 100% concordance in assay results for clinical samples and an acceptable analytical performance at their guaranteed limits of detection. The addition of a widely used simultaneous sample-to-answer SARS-CoV-2 RT-PCR assay will contribute to the number of medical laboratories able to test for COVID-19.

Investigation of SARS-CoV-2 lineages and mutations circulating in a university-affiliated hospital in South Korea analyzed using Oxford Nanopore MinION sequencing.

OBJECTIVES: Despite the introduction of vaccines, treatments, and massive diagnostic testing, the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to overcome barriers that had slowed its previous spread. As the virus evolves towards increasing fitness, it is critical to continue monitoring the occurrence of new mutations that could evade human efforts to control them. METHODS: We performed whole-genome sequencing using Oxford Nanopore MinION sequencing on 58 SARS-CoV-2 isolates collected during the ongoing coronavirus disease 2019 pandemic at a tertiary hospital in South Korea and tracked the emergence of mutations responsible for massive spikes in South Korea. RESULTS: The differences among lineages were more pronounced in the spike gene, especially in the receptor-binding domain (RBD), than in other genes. Those RBD mutations could compromise neutralization by antibodies elicited by vaccination or previous infections. We also reported multiple incidences of Omicron variants carrying mutations that could impair the diagnostic sensitivity of reverse transcription-polymerase chain reaction-based testing. CONCLUSION: These results provide an understanding of the temporal changes of variants and mutations that have been circulating in South Korea and their potential impacts on antigenicity, therapeutics, and diagnostic escape of the virus. We also showed that the utilization of the nanopore sequencing platform and the ARTIC workf low can provide convenient and accurate SARS-CoV-2 genomic surveillance even at a single hospital.

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.

High Circulating Levels of Neutrophil Extracellular Traps Parameters Predicting Poor Outcome in COVID-19.

OBJECTIVE: Exploration of biomarkers to predict the severity of COVID-19 is important to reduce mortality. Upon COVID-19 infection, neutrophil extracellular traps (NET) are formed, which leads to a cytokine storm and host damage. Hence, the extent of NET formation may reflect disease progression and predict mortality in COVID-19. METHODS: We measured 4 NET parameters - cell-free double stranded DNA (cell-free dsDNA), neutrophil elastase, citrullinated histone H3 (Cit-H3), and histone - DNA complex - in 188 COVID-19 patients and 20 healthy controls. Survivors (n=166) were hospitalized with or without oxygen supplementation, while non-survivors (n=22) expired during in-hospital treatment. RESULTS: Cell-free dsDNA was significantly elevated in non-survivors in comparison with survivors and controls. The survival rate of patients with high levels of cell-free dsDNA, neutrophil elastase, and Cit-H3 was significantly lower than that of patients with low levels. These three markers significantly correlated with inflammatory markers (absolute neutrophil count and C-reactive protein). CONCLUSION: Since the increase in NET parameters indicates the unfavourable course of COVID-19 infection, patients predisposed to poor outcome can be rapidly managed through risk stratification by using these NET parameters.

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.

Rapid and accurate clinical testing for COVID-19 by nicking and extension chain reaction system-based amplification (NESBA).

We herein describe rapid and accurate clinical testing for COVID-19 by nicking and extension chain reaction system-based amplification (NESBA), an ultrasensitive version of NASBA. The primers to identify SARS-CoV-2 viral RNA were designed to additionally contain the nicking recognition sequence at the 5'-end of conventional NASBA primers, which would enable nicking enzyme-aided exponential amplification of T7 RNA promoter-containing double-stranded DNA (T7DNA). As a consequence of this substantially enhanced amplification power, the NESBA technique was able to ultrasensitively detect SARS-CoV-2 genomic RNA (gRNA) down to 0.5 copies/µL (= 10 copies/reaction) for both envelope (E) and nucleocapsid (N) genes within 30 min under isothermal temperature (41 °C). When the NESBA was applied to test a large cohort of clinical samples (n = 98), the results fully agreed with those from qRT-PCR and showed the excellent accuracy by yielding 100% clinical sensitivity and specificity. By employing multiple molecular beacons with different fluorophore labels, the NESBA was further modulated to achieve multiplex molecular diagnostics, so that the E and N genes of SARS-CoV-2 gRNA were simultaneously assayed in one-pot. By offering the superior analytical performances over the current qRT-PCR, the isothermal NESBA technique could serve as very powerful platform technology to realize the point-of-care (POC) diagnosis for COVID-19.

The Dangers of Using Cq to Quantify Nucleic Acid in Biological Samples: A Lesson From COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA quantities, measured by reverse transcription quantitative PCR (RT-qPCR), have been proposed to stratify clinical risk or determine analytical performance targets. We investigated reproducibility and how setting diagnostic cutoffs altered the clinical sensitivity of coronavirus disease 2019 (COVID-19) testing. METHODS: Quantitative SARS-CoV-2 RNA distributions [quantification cycle (Cq) and copies/mL] from more than 6000 patients from 3 clinical laboratories in United Kingdom, Belgium, and the Republic of Korea were analyzed. Impact of Cq cutoffs on clinical sensitivity was assessed. The June/July 2020 INSTAND external quality assessment scheme SARS-CoV-2 materials were used to estimate laboratory reported copies/mL and to estimate the variation in copies/mL for a given Cq. RESULTS: When the WHO-suggested Cq cutoff of 25 was applied, the clinical sensitivity dropped to about 16%. Clinical sensitivity also dropped to about 27% when a simulated limit of detection of 106 copies/mL was applied. The interlaboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI). CONCLUSION: While RT-qPCR has been instrumental in the response to COVID-19, we recommend Cq (cycle threshold or crossing point) values not be used to set clinical cutoffs or diagnostic performance targets due to poor interlaboratory reproducibility; calibrated copy-based units (used elsewhere in virology) offer more reproducible alternatives. We also report a phenomenon where diagnostic performance may change relative to the effective reproduction number. Our findings indicate that the disparities between patient populations across time are an important consideration when evaluating or deploying diagnostic tests. This is especially relevant to the emergency situation of an evolving pandemic.

Prevalence of a Single-Nucleotide Variant of SARS-CoV-2 in Korea and Its Impact on the Diagnostic Sensitivity of the Xpert Xpress SARS-CoV-2 Assay.

The sensitivity of molecular diagnostics could be affected by nucleotide variants in pathogen genes, and the sites affected by such variants should be monitored. We report a single-nucleotide variant (SNV) in the nucleocapsid (N) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), i.e., G29179T, which impairs the diagnostic sensitivity of the Xpert Xpress SARS-CoV-2 assay (Cepheid, Sunnyvale, CA, USA). We observed significant differences between the threshold cycle (Ct) values for envelope (E) and N genes and confirmed the SNV as the cause of the differences using Sanger sequencing. This SNV, G29179T, is the most prevalent in Korea and is associated with the B.1.497 virus lineage, which is dominant in Korea. Clinical laboratories should be aware of the various SNVs in the SARS-CoV-2 genome and consider their potential effects on the diagnosis of coronavirus disease 2019.

Performance Comparison of Five SARS-CoV-2 Antibody Assays for Seroprevalence Studies.

BACKGROUND: Seroprevalence studies of coronavirus disease 2019 (COVID-19) cases, including asymptomatic and past infections, are important to estimate the scale of the disease outbreak and to establish quarantine measures. We evaluated the clinical performance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody assays available in Korea for use in seroprevalence studies. METHODS: The sensitivity, specificity, cross-reactivity, and interference of five SARS-CoV-2 antibody assays were evaluated using the following: 398 serum samples from confirmed COVID-19 patients, 510 negative control samples from before 2018 (pre-pandemic), 163 serum samples from patients with SARS, Middle East respiratory syndrome (MERS), and other viral infections, and five samples for the interference study. RESULTS: The sensitivities of the five assays ranged from 92.2% to 98%, and their specificities, including cross-reactivity and interference, ranged from 97.5% to 100%. The agreement rates were excellent (kappa >0.9). Adjustment of the cutoff values could be considered through ROC curve analysis. The positive predictive values of the individual assays varied from 3.5% to 100% at a 0.1% prevalence but were as high as ≥95% when two assays were combined. CONCLUSIONS: The prevalence of COVID-19 in Korea is considered to be exceptionally low at present; thus, we recommend using a combination of two or more SARS-CoV-2 antibody assays rather than a single assay. These results could help select SARS-CoV-2 antibody assays for COVID-19 seroprevalence studies in Korea.

Clinical Performance of the Standard Q COVID-19 Rapid Antigen Test and Simulation of its Real-World Application in Korea.

The rapid antigen test (RAT) for coronavirus disease (COVID-19) represents a potent diagnostic method in situations of limited molecular testing resources. However, considerable performance variance has been reported with the RAT. We evaluated the clinical performance of Standard Q COVID-19 RAT (SQ-RAT; SD Biosensor, Suwon, Korea), the first RAT approved by the Korean Ministry of Food and Drug Safety. In total, 680 nasopharyngeal swabs previously tested using real-time reverse-transcription PCR (rRT-PCR) were retested using SQ-RAT. The clinical sensitivity of SQ-RAT relative to that of rRT-PCR was 28.7% for all specimens and was 81.4% for specimens with RNA-dependent RNA polymerase gene (RdRp) threshold cycle (Ct) values ≤23.37, which is the limit of detection of SQ-RAT. The specificity was 100%. The clinical sensitivity of SQ-RAT for COVID-19 diagnosis was assessed based on the Ct distribution at diagnosis of 33,294 COVID-19 cases in Korea extracted from the laboratory surveillance system of Korean Society for Laboratory Medicine. The clinical sensitivity of SQ-RAT for COVID-19 diagnosis in the Korean population was 41.8%. Considering the molecular testing capacity in Korea, use of the RAT for COVID-19 diagnosis appears to be limited.

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.

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.

Surveillance of Coronavirus Disease 2019 (COVID-19) Testing in Clinical Laboratories in Korea.

In response to the ongoing coronavirus disease 2019 (COVID-19) pandemic, an online laboratory surveillance system was established to monitor severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time reverse transcription-PCR (rRT-PCR) testing capacities and results. SARS-CoV-2 rRT-PCR testing data were collected from 97 clinical laboratories, including 84 medical institutions and 13 independent clinical laboratories in Korea. We assessed the testing capacities to utilize SARS-CoV-2 rRT-PCR based on surveillance data obtained from February 7th to June 4th, 2020 and evaluated positive result characteristics according to the reagents used and sample types. A total of 1,890,319 SARS-CoV-2 rRT-PCR testing were performed, 2.3% of which were positive. Strong correlations were observed between the envelope (E) gene and RNA-dependent RNA polymerase (RdRp)/nucleocapsid (N) genes threshold cycle (Ct) values for each reagent. No statistically significant differences in gene Ct values were observed between the paired upper and lower respiratory tract samples, except in the N gene for nasopharyngeal swab and sputum samples. Our study showed that clinical laboratories in Korea have rapidly expanded their testing capacities in response to the COVID-19 outbreak, with a peak daily capacity of 34,193 tests. Rapid expansion in testing capacity is a critical component of the national response to the ongoing pandemic.