key: cord-1055851-034muhvm
authors: Kitajima, Heita; Tamura, Yoshitaka; Yoshida, Hiroko; Kinoshita, Hitomi; Katsuta, Hiroki; Matsui, Chika; Matsushita, Akane; Arai, Tsuyoshi; Hashimoto, Shoji; Iuchi, Atsuhiko; Hirashima, Tomonori; Morishita, Hiroshi; Matsuoka, Hiroto; Tanaka, Toshio; Nagai, Takayuki
title: Clinical COVID-19 diagnostic methods: comparison of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and quantitative RT-PCR (qRT-PCR)
date: 2021-03-26
journal: J Clin Virol
DOI: 10.1016/j.jcv.2021.104813
sha: 2c7ad66768a44946477254b6ff534d7d33470fc8
doc_id: 1055851
cord_uid: 034muhvm

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is a major public health concern. Accurate and rapid diagnosis of COVID-19 is critical for disease control. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification assay similar to reverse transcription-polymerase chain reaction (RT-PCR), the former being a simple, low cost, and rapid method. OBJECTIVES: This study aimed to compare the RT-LAMP assay with RT-PCR using the Loopamp(TM) SARS-CoV-2 Detection Kit. STUDY DESIGN: One hundred and fifty-one nasopharyngeal swab and 88 sputum samples obtained from individuals with suspected or confirmed COVID-19 were examined. RESULTS: RT-LAMP had high specificity (98.5% (95% CI: 96.9–100%)), sensitivity (87.0% (95% CI: 82.8–91.3%)), positive predictive value (97.9% (95% CI: 96.1–99.7%)), negative predictive value (90.2% (95% CI: 86.4–94.0%)), and concordance rate (93.3% (95% CI: 90.1–96.5%)). Nasopharyngeal and sputum samples positive in RT-LAMP contained as few as 10.2 and 23.4 copies per 10 μL, respectively. RT-LAMP showed similar performance to RT-PCR for samples with cycle threshold value below 36. CONCLUSIONS: These results indicate that RT-LAMP is a highly reliable and at least equivalent to RT-PCR in utility, and potentially applicable in settings that are more diverse as a point-of-care tool.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, Hubei Province, China, and rapidly spread worldwide [1, 2] . Approximately 2 months after the first report, the World Health Organization declared a COVID-19 pandemic on March 11, 2020. By November 3, 2020, nearly 50 million confirmed cases of COVID-19, including more than one million deaths, had been reported in 219 countries [3] . A serious consequence of the pandemic is that the medical-care system has become overwhelmed. Therefore, the accurate and rapid diagnosis of COVID-19, as well as the development of effective vaccines and treatments, is critical to control this disease and its spread within populations.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of upper and lower respiratory swab samples is the current standard for the diagnosis of COVID-19 [2, 4] . However, it relies on sophisticated equipment, specific skills, and requires 1.5-2 h to complete the reaction [5, 6] . Reverse transcription loop-mediated isothermal amplification (RT-LAMP) does not require expensive equipment such as thermal cyclers, is easy to perform, and only requires 1 h to obtain results [7] [8] [9] . Hence, RT-LAMP is regarded as a point-of-care testing tool, with a reported detection limit of

The study protocol was approved by the institutional review board of Osaka Habikino Medical Center on April 30, 2020 (Approval No. 1017). Written informed consent was obtained from all enrolled patients.

We enrolled patients with suspected or confirmed COVID-19, who had been admitted to the Osaka Habikino Medical Center between April 1 and July 31, 2020.

Nasopharyngeal swab samples were collected in 1 mL of sterile saline, with sputum samples collected by the patients themselves. All samples were stored at -30 °C until analyzed. RNA was extracted from the specimens using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer's instructions.

The extracted RNA was aliquoted and stored at -80 °C until it was used for amplification by RT-LAMP and RT-PCR. A flowchart of the study protocol is provided in Fig. 1 . In addition, LAMP positive reactions can also be detected visually, through fluorescence, using calcein staining; therefore, we reassessed RT-LAMP reactions using this visual endpoint detection [9] .

RT-PCR was performed using a protocol devised by the National Institute of Infectious Diseases (NIID), which is a national recommendation for SARS-CoV-2 detection in Japan [11] . In this assay, the QuantiTect ®︎ Probe RT-PCR kit (QIAGEN, Hilden, Germany) and LightCycler ®︎ 480 (Roche, Penzberg, Germany) were used with the following primers and probe targeting the SARS-CoV-2 N gene: 500 nM of forward (Table 1) . (1 nasopharyngeal specimen), or < 10 copies/10 μL (4 sputum and 8 nasopharyngeal specimens; Table 2 ). J o u r n a l P r e -p r o o f

Because of its transmissibility and rapid spread, COVID-19 is a major public health concern. Although PCR is considered the gold standard for the diagnosis of COVID-19, early and accurate means of diagnosis, such as RT-LAMP, are urgently required. Interestingly, one sample each from the nasopharyngeal and sputum samples tested negative for SARS-CoV-2 in RT-PCR, but positive in RT-LAMP (Table 2) . Any association with Tt values is unclear. This discrepancy may depend on the characteristics of the RT-LAMP method, which uses a set of four types of primers based on six distinct regions of the target gene, as opposed to the two primers used in RT-PCR [7] .

The primary limitation of this study is the small number of samples analyzed. In addition, samples were obtained from patients hospitalized with symptoms. Hence, further studies with larger sample sizes are required. In conclusion, this study supports the applicability of the RT-LAMP assay for nasopharyngeal swabs and sputum samples for the detection of SARS-CoV-2. RT-LAMP is a simple, low-cost, rapid, and highly J o u r n a l P r e -p r o o f loads. It is also adaptable to more diverse diagnostic settings, similar to the ones during the current pandemic.

None of the authors have financial conflicts of interest to disclose. The study was not funded by any organization.

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The authors thank the patients, their families, and all members of the COVID-19 care team at Osaka Habikino Medical Center.J o u r n a l P r e -p r o o f