key: cord-0858235-blb3krbt
authors: Yoshioka, Nori; Deguchi, Matsuo; Hagiya, Hideharu; Kagita, Masanori; Tsukamoto, Hiroko; Takao, Miyuki; Yoshida, Hisao; Hamaguchi, Shigeto; Maeda, Ikuhiro; Hidaka, Yoh; Tomono, Kazunori
title: Comparison of extraction‐based and elution‐based polymerase chain reaction testing, and automated and rapid antigen testing for the diagnosis of severe acute respiratory syndrome coronavirus 2
date: 2022-03-22
journal: J Med Virol
DOI: 10.1002/jmv.27709
sha: c8e6e3f91acf0aeccf463570a8f5d4b91f6398fb
doc_id: 858235
cord_uid: blb3krbt

We aimed to compare the differences in testing performance of extraction‐based polymerase chain reaction (PCR) assays, elution‐based direct PCR assay, and rapid antigen detection tests for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). We used nasopharyngeal swab samples of patients with coronavirus disease 2019 (COVID‐19). We used the MagNA Pure 24 System (Roche Diagnostics K.K.) or magLEAD 12gC (Precision System Science Co., Ltd.) for RNA extraction, mixed the concentrates with either the LightMix Modular SARS‐CoV PCR mixture (Roche Diagnostics K.K.) or Takara SARS‐CoV‐2 direct PCR detection kit (Takara Bio Inc.), and amplified it using COBAS® z480 (Roche Diagnostics K.K.). For elution‐based PCR, we directly applied clinical samples to the Takara SARS‐CoV‐2 direct PCR detection kit before the same amplification step. Additionally, we performed Espline SARS‐CoV‐2 (Fuji Rebio Co., Ltd.) for rapid diagnostic test (RDT), and used Lumipulse SARS‐CoV‐2 antigen (Fuji Rebio Co., Ltd.) and Elecsys SARS‐CoV‐2 antigen (Roche Diagnostics K.K.) for automated antigen tests (ATs). Extraction‐based and elution‐based PCR tests detected the virus up to 214–216 and 210 times dilution, respectively. ATs remained positive up to 24–26 times dilution, while RDT became negative after 22 dilutions. For 153 positive samples, positivity rates of the extraction‐based PCR assay were 85.6% to 98.0%, while that of the elution‐based PCR assay was 73.2%. Based on the RNA concentration process, extraction‐based PCR assays were superior to elution‐based direct PCR assays for detecting SARS‐CoV‐2.

Although more than 2 years have passed since its emergence, the coronavirus disease 2019 (COVID-19) global pandemic continues. The upsurge of the Omicron variant worsens the situation, and many countries are facing unprecedented severe conditions. 1 According to the World Health Organization, more than 300 million confirmed cases and 5.5 million deaths have been reported globally. 2 Early and accurate diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for infection prevention and control, as well as appropriate treatment for the patients.

Polymerase chain reaction (PCR) has a high testing sensitivity for detecting the virus in clinical samples, which is widely used for the diagnosis of COVID-19. 3 PCR test is generally divided into the following two methods: extraction-based PCR and elutionbased PCR. In extraction-based PCR, the samples undergo purification and extraction processes, resulting in genome concentration. Although this method is laborious, timeconsuming, and expensive, it can achieve a high concentration of nucleic acids; thus, it can yield high testing sensitivity. In elution-based PCR, which is also known as direct PCR, the sample is lysed in a buffer solution without a purification step. This approach is simple, rapid, and economical but it has low sensitivity compared with the extraction-based methods.

Clinicians should be aware of the difference in testing performance of these methods, which is not well recognized. 4 Even a recent review of literature on the diagnosis of COVID-19 does not refer to this essential point. 5 An appropriate interpretation of PCR results is crucial for people in various situations such as patients with clinical symptoms and individuals with close contacts. In particular, a false negative result requires much attention because it may cause the spread of infection and underestimation of the disease prevalence. 6 Although elution-based PCR has less sensitivity, it is widely used in hospital laboratories in Japan owing to its convenience. In this study, we aimed to reveal the differences in testing sensitivity of these methods, along with rapid antigen detection tests.

This study was performed at Osaka University Hospital, a tertiary medical facility in Japan, between March 2020 and May 2021.

We used nasopharyngeal swab samples (FLOQSwab, COPAN Co., Ltd.) of patients diagnosed with COVID-19. The study protocol was approved by the ethics committee of Osaka University 3), (C) extraction-based PCR and elution-based PCR (PCR condition 3 vs.

We first examined a pooled sample of nasopharyngeal specimens to compare testing sensitivity among each condition, using four SARS-CoV-2-positive samples. After adding 2 ml of PBS to each specimen, the pooled sample was diluted with 4 n dilution. The tests were performed in a quintuplicate manner to determine the testing sensitivity. The mean threshold cycles for PCR testing and cutoff index (COI) for rapid antigen detection testing were also calculated when the testing sensitivity was 100%. We then tested 211 nasopharyngeal samples to compare the positivity rates of each testing condition. Cutoff value of the cycle threshold in PCR testing was set at 38.0, and COI of the AT was ≥1.34 for Lumipulse and ≥1.00 for Elecsys.

Comparison of testing sensitivity according to five experiments for each condition of PCR testing and rapid antigen detection tests using a single nasopharyngeal sample is summarized inTable 1. PCR conditions 1, 2, and 3 detected viral genes up to 2 14 , 2 16 , and 2 16 times dilution with 100% positivity, respectively. Thus, differences in (A) sample extraction methods and (B) PCR reagents did not markedly affect testing sensitivity.

In contrast, PCR condition 4 identified the viral genes only up to 2 10 times dilution, indicating that the extraction-based PCR conditions were 16-64 (2 4 -2 6 ) times more sensitive than the elution-based PCR. AT remained positive up to 2 4 -2 6 dilutions; thus, the differences in testing sensitivity compared with extraction-based PCR were 1024-4096 (2 10 -2 12 ) times. RDT results were negative after 2 2 dilutions, which suggested that the difference in testing sensitivity between the extraction-based PCR, and this method was up to 65 536 (2 16 ) times.

Subsequently, we examined the positivity rates of these PCR tests and rapid antigen detection tests using 211 nasopharyngeal samples ( Table 2 ). The positivity rates of PCR conditions 1, 2, 3, The RNA extraction process constitutes a major impediment to rapid testing; however, it is an important step to increase the testing sensitivity. Recent studies have proposed several methods to circumvent RNA extraction while maintaining testing sensitivity. 9 In summary, extraction-based PCR appears to be superior to elution-based PCR for detecting SARS-CoV-2. We should recognize the advantages and disadvantages of each assay and choose accordingly.

Omicron SARS-CoV-2 variant: a new chapter in the COVID-19 pandemic

World Health Organization. WHO Coronavirus Disease (COVID-19) Dashboard

SARS-CoV-2 and variant diagnostic testing approaches in the United States

Interpreting a covid-19 test result

Diagnostics for COVID-19: moving from pandemic response to control

False-negative results of initial RT-PCR assays for COVID-19: a systematic review

Efficacy of a novel SARS-CoV-2 detection kit without RNA extraction and purification

Clinical usefulness of extraction-free PCR assay to detect SARS-CoV-2

Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR

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Comparison of extraction-based and elution-based polymerase chain reaction testing, and automated and rapid antigen testing for the diagnosis of severe acute respiratory syndrome coronavirus 2

The authors would like to thank Editage (www.editage.jp) for English language editing.

The authors declare no conflicts of interest. 

The data that support the findings of this study are available from the corresponding author upon reasonable request.

http://orcid.org/0000-0003-3395-9712