COVID-19 Molecular Testing in Korea: Practical Essentials and Answers From Experts Based on Experiences of Emergency Use Authorization Assays
2020; 40(6): 439-447
Ann Lab Med 2020; 40(5): 351-360
Published online September 1, 2020 https://doi.org/10.3343/alm.2020.40.5.351
Copyright © Korean Society for Laboratory Medicine.
Ki Ho Hong, M.D.1,* , Sang Won Lee, Ph.D.2,* , Taek Soo Kim, M.D.3 , Hee Jae Huh, M.D.4 , Jaehyeon Lee, M.D.5 , So Yeon Kim, M.D.6 , Jae-Sun Park, Ph.D.2 , Gab Jung Kim, Ph.D.2 , Heungsup Sung, M.D.7 , Kyoung Ho Roh, M.D.8 , Jae-Seok Kim, M.D.9 , Hyun Soo Kim, M.D.9 , Seung-Tae Lee, M.D.10 , Moon-Woo Seong, M.D.3 , Namhee Ryoo, M.D.11 , Hyukmin Lee, M.D.10 , Kye Chul Kwon, M.D.12 , and Cheon Kwon Yoo, Ph.D.2
On behalf of
Korean Society for Laboratory Medicine, COVID-19 Task Force and the Center for Laboratory Control of Infectious Diseases, the Korea Centers for Disease Control and Prevention
1Department of Laboratory Medicine, Seoul Medical Center, Seoul, Korea; 2Center for Laboratory Control of Infectious Diseases, Centers for Disease Control and Prevention, Osong, Korea; 3Department of Laboratory Medicine, Seoul National University Hospital, Seoul, Korea; 4Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; 5Department of Laboratory Medicine, Jeonbuk National University Medical School and Hospital, Jeonju, Korea; 6Department of Laboratory Medicine, National Medical Center, Seoul, Korea; 7Department of Laboratory Medicine, Asan Medical Center and University of Ulsan College of Medicine, Seoul, Korea; 8Department of Laboratory Medicine, National Health Insurance Service, Ilsan Hospital, Goyang, Korea; 9Department of Laboratory Medicine, Hallym University College of Medicine, Chuncheon, Korea; 10Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea; 11Department of Laboratory Medicine, Keimyung University School of Medicine, Daegu, Korea; 12Department of Laboratory Medicine, College of Medicine, Chungnam National University, Daejeon, Korea
Correspondence to: Hyukmin Lee, M.D.
Department of Laboratory Medicine, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea
Cheon Kwon Yoo, Ph.D.
Center for Laboratory Control of Infectious Diseases, Centers for Disease Control and Prevention Osong Health Technology Administration Complex, 187 Osongsaengmyeong 2-ro, Osonge-eup, Heungdeok-gu, Cheongju 28159, Korea
* These authors equally contributed to this study.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
The outbreak of coronavirus disease 2019 (COVID-19), which began in December 2019, is still ongoing in Korea, with >9,000 confirmed cases as of March 25, 2020. COVID-19 is a severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection, and real-time reverse transcription-PCR is currently the most reliable diagnostic method for COVID-19 around the world. Korean Society for Laboratory Medicine and the Korea Centers for Disease Prevention and Control propose guidelines for diagnosing COVID-19 in clinical laboratories in Korea. These guidelines are based on other related domestic and international guidelines, as well as expert opinions and include the selection of test subjects, selection of specimens, diagnostic methods, interpretation of test results, and biosafety.
Keywords: COVID-19, Outbreak, Laboratory diagnosis, Guidelines, Korea
In December 2019, a mass outbreak of Coronavirus disease 2019 (COVID-19) occurred in Wuhan, Hubei Province, People’s Republic of China. Currently, the disease has spread to other regions of China and around the world . In Korea, more than 9,000 patients have been confirmed as of March 25, 2020.
The World Health Organization (WHO) officially changed the name of the disease to coronavirus disease 2019 (COVID-19) from 2019 novel coronavirus (2019-nCoV) infection on February 11, 2020. The International Committee on Taxonomy of Viruses has proposed the name severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) for the causative agent of COVID-19 . This document uses COVID-19 and SARS-CoV-2, as well as 2019-nCoV infection as appropriate.
This document provides guidelines for diagnosing COVID-19 in human specimens. Some contents of this document, including new test methods, specimen selection, and biosafety recommendations, may be subject to revision later, with the emergence of new scientific evidence and based on expert review.
The guidelines presented here are based on the “Korea Laboratory Response and Preparedness for 2019 Novel Coronavirus Molecular Testing in Clinical Laboratories” project, which is a commissioned project aimed at establishing and managing COVID-19 diagnosis in hospital laboratories and commercial laboratory centers. All copyrights to these guidelines are owned by Korean Society for Laboratory Medicine (KSLM) and the Korea Centers for Disease Control and Prevention (KCDC).
The case definition is based on the 7th Edition (March 15, 2020) of the Guidelines on Response to Coronavirus Disease 2019 published by the KCDC . This case definition applies to conditions at a “serious stage” crisis alert level, in which COVID-19 imported into Korea is spreading to local communities or throughout the nation. It may be revised later based on the incidence of confirmed domestic cases, results of epidemiological studies, and the level of epidemics. The definition of a suspected case and patient under investigation (PUI) can be changed according to newly updated information from the KCDC.
Cases with confirmed infection according to the diagnostic criteria, irrespective of the clinical manifestation (diagnostic tests: COVID-19 real-time RT-PCR, virus isolation).
Cases with fever (37.5°C or higher) and/or respiratory symptoms (cough, sore throat, etc.) within 14 days of being in close contact with a confirmed case.
COVID-19 real-time RT-PCR may be performed for the purposes of:sec-type="cases"
The release criteria can be changed at any time according to newly updated information from the KCDC. See newly updated information on the KCDC website .
Although the importance of diagnosis in asymptomatic individuals who have been in close contact with confirmed COVID-19 patients remains unclear, diagnostic testing can be considered for the early detection of cases progressing to symptomatic infection.
The diagnostic testing may be performed for patients exhibiting pneumonia or respiratory syndromes with fever in order to identify the cause of the disease and to differentiate the condition from COVID-19.
Globally, various real-time RT-PCR protocols have been proposed for the diagnosis of COVID-19 [4–11]; these protocols differ in the genes they detect. Further studies are needed to identify the optimal real-time RT-PCR protocol for diagnosing COVID-19 (Table 1).
Of the various protocols, some examine two genes using a two-step interpretation algorithm [5, 8, 12]. In these protocols, the identification of one gene is used as a screening test, while that of the second gene is used as a confirmatory test. In contrast, the results of other protocols, which examine three or more genes, are considered positive only when all genes are detected. If one of the genes is not detected in these protocols, the results are often interpreted as indeterminate or negative.
These guidelines were prepared based on the guidelines provided by the KCDC and the WHO , which recommend PCR amplification of the viral
As of March 13, 2020, KSLM and the KCDC recommend a positive result determination only when all the genes are detected, even for tests using different genes from those mentioned in the above two guidelines. This recommendation is based on the opinions of numerous experts who observed nonspecific and weak amplification in the clinical specimens of patients who received final results of COVID-19 as negative.
Clinical laboratories are recommended not to use methods other than real-time RT-PCR (pan-coronavirus PCR, serological tests, etc.) , laboratory-developed tests, and reagents that are not approved for emergency use by the Korea Ministry of Food and Drug Safety for diagnostic purposes.
As of March 13, 2020, several commercially available real-time RT-PCR reagents have been approved for emergency use in Korea for the
The emergency use authorization (EUA) system is designed to respond to novel or emerging infectious diseases and is known for rapid evaluation and deliberation in emergency cases. Therefore, testing with approved reagents requires stringent and continuous quality control. The collection of data and specimens, as well as cross-testing of positive and negative specimens across laboratories are necessary to continuously analyze test performance.
All clinical laboratories should transfer their test results and all positive specimens to the KCDC. This is to ensure the quality control of reagents with EUA and laboratory surveillance of infectious diseases.
Repetitive testing may be necessary to confirm COVID-19 in suspected cases or PUIs. As the clinical significance of co-infection caused by the causative agent of COVID-19 and other infectious diseases remains unclear, the collection of a sufficient quantity of clinical specimens with proper methods is recommended.
As the current knowledge on COVID-19 is limited, it is difficult to rule out infection based on a single negative test result, especially when the test is performed using an upper respiratory tract specimen. Even if the upper respiratory tract specimen tests negative, the collection and testing of lower respiratory tract specimens are strongly recommended, especially in cases of severe or progressive disease.
The collection of specimens for diagnosis is recommended within seven days of symptom onset. See Table 2 for information on specimen containers, transport conditions, and storage periods.
Nasopharyngeal swab, oropharyngeal swab, nasopharyngeal aspirates, etc.
Sputum*, bronchial washing, tracheal aspirates, transtracheal aspirates, bronchoalveolar lavage, transbronchial lung biopsy, etc.
*In cases with no purulent sputum, ensure that the individual breathes deeply and coughs straight into the container when providing the sputum specimen. Sputum induction is not recommended.
The collection of both nasopharyngeal swabs and oropharyngeal swabs is recommended; these should be placed together in the same viral transport medium (VTM) to increase the sensitivity [14, 19]. However, the currently available VTM-swab systems are often designed for one swab. Therefore, specimen packaging and shipping should be conducted carefully, as there is a risk of leakage during transportation. When collecting only one specimen, a nasopharyngeal swab is recommended first. It may be necessary to collect lower respiratory tract specimens, such as sputum; however, sputum induction is not indicated.
Lower respiratory tract specimens, such as sputum, bronchoalveolar lavage, and tracheal aspirates, should be collected. If possible, the collection of nasopharyngeal and oropharyngeal swabs can be considered.
Lower respiratory tract specimens, such as sputum, bronchoalveolar lavage, and tracheal aspirates, should be collected.
If necessary, additional specimens, such as blood, urine, and feces, may be collected on consultation with the physician taking care of the patient and a laboratory physician. However, the diagnostic value and clinical utility of these specimens remain unclear. The collection of blood specimens may be considered for public health purposes, such as serological surveys. This may be amended based on new clinical evidence.
The guidelines for the prevention of infection and control for aerosol-producing procedures should be followed when collecting specimens using aerosol-producing procedures, such as sputum collection, tracheal aspiration, and bronchoscopy (Table 3).
Collection of upper respiratory tract specimens, especially nasopharyngeal swab specimens, requires knowledge of the anatomical structure of the respiratory system and the collection process is also invasive. Hence, it is recommended that the collection be performed by medical personnel.
When collecting blood and other non-respiratory specimens from suspected or confirmed COVID-19 patients, laboratory personnel should wear personal protective equipment (PPE; N95 or KF94 mask, disposable gloves, gown [clean, covering the entire body, long-sleeves, back-closure], and eye protection [goggles or face shield]).
*Intended for commercial kits with EUA as of March 25, 2020
Positive control (+), negative control (−)
*Independent of internal control amplification
All results of negative control (+) or positive control (−)
Invalid regardless of target gene and internal control amplification; retest is necessary. See details below.
All kits currently available in Korea can detect two or more genes. According to the interpretation criteria of some manufacturers, detection of only one of multiple genes is interpreted as COVID-19 positive. However, based on results from actual clinical specimens, KSLM recommends a determination of a positive result only when all genes are detected. When only one gene is detected, retesting or consulting the reference laboratory is recommended.
Among the reagents with EUA, some kits with three target genes use one target gene for the screening test and the other two target genes for the confirmatory test. For these kits, the confirmatory test result is deemed positive only if both confirmatory genes are detected. If one gene is not detected, the result cannot be interpreted as positive.
For kits using betacoronavirus primers for the screening test, there is a possibility of betacoronavirus rather than SARS-CoV-2.
If the internal control is also negative, the result is invalid, and a retest is necessary.
At present, there is very limited knowledge regarding the timing of virus detection in COVID-19 cases. Hence, it is difficult to rule out COVID-19 based solely on one negative result, especially when using an upper respiratory tract specimen from a suspected case. Other upper respiratory tract specimens should be collected and tested when a highly suspected COVID-19 patient tests negative using a single upper respiratory tract specimen. The lower respiratory tract specimens may be collected together and tested. Some considerations for possible false negative results are listed below:
Patient name, age (date of birth), sex, patient number (hospital number), specimen number, ward, test order date, specimen type, and specimen collection time.
*These guidelines do not clearly differentiate these terms. The laboratory physician can decide the term to use.
Note: Any unusual findings such as those regarding specimen quality.
The following procedures should be performed only in BL3 or higher facilities
The following procedures should be performed only in BL2 or higher facilities and in a certified Class II or higher BSC
The following procedures should be performed in BL2 or higher facilities
Routine tests (hematological tests, biochemical tests, etc.)
|Authors, manufacturers||Target gene||Reference|
|Ministry of Public Health, Thailand|||
|Centers for Disease Control and Prevention (USA)|||
|PowerCheck 2019-nCoV* (Kogene biotech, Seoul, Korea)||http://www.kogene.co.kr/|
|Allplex 2019-nCoV* (Seegene, Seoul, Korea)||http://www.seegene.com/|
|nCoV Real-Time Detection* (SD biosensors, Suwon, Korea)||http://sdbiosensor.com/|
|DiaPlexQ 2019-nCoV*† (Solgent, Daejeon, Korea)||http://www.solgent.com/|
|Real-Q 2019-nCoV* (BioSewoom, Seoul, Korea)||https://biosewoom.com|
*authorized for emergency use in Korea, as of Mar 13, 2020;
†The correct positions of the targets were not provided by the manufacturer.
|Types of specimens||Collection devices||Transport conditions||Storage conditions||Comments|
|Upper respiratory tract specimens: NP swab*, OP swab*, and NP aspirate||Dacron or flocked swabs in VTM||4°C||Within 5 days: 4°C|
Longer than 5 days: −70°C
|Lower respiratory tract specimen: sputum*||Sterile container||4°C||Within 48 hr: 4°C|
Longer than 48 hr: −70°C
|Lower respiratory tract specimen: bronchial washing*||Sterile container||4°C||Within 48 hr: 4°C|
Longer than 48 hr: −70°C
|Pathogens might be diluted; however, the specimen can be subjected to diagnostic testing|
|Lower respiratory tract specimens: tracheal aspirate and transtracheal aspirate||Sterile container||4°C||Within 48 hr: 4°C|
Longer than 48 hr: −70°C
|Lower respiratory tract specimen: lung biopsy||Sterile container with saline||4°C||Within 48 hr: 4°C|
Longer than 48 hr: −70°C
|Serum†||Serum separation test tube (SST): adults and children, 3–5 mL; infants, 1 mL||4°C||Within 5 days: 4°C|
Longer than 5 days: −70°C
|For serological tests, a pair of specimens is collected|
Acute phase: within 7 days of symptom onset
Convalescent period: 14 days after collection during acute phase
Dispensing of serum into another container should be conducted in a Class II or higher BSC