ABSTRACT
Objectives: Gastrointestinal endoscopy (GIE) is useful for the early detection and treatment of many diseases;however, GIE is considered a high-risk procedure in the coronavirus disease 2019 (COVID-19) pandemic era. This study aimed to explore the rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positivity in saliva and gastrointestinal fluids to which endoscopy medical staff are exposed. Method(s): The study was a single-center cross-sectional study. From June 1 to July 31, 2020, all patients who underwent GIE at Yokohama City University Hospital were registered. All patients provided 3 mL of saliva. For upper GIE, 10 mL of gastric fluid was collected through the endoscope. For lower GIE, 10 mL of intestinal fluid was collected through the endoscope. The primary outcome was the positive rate of SARS-CoV-2 in saliva and gastrointestinal fluids. We also analyzed serum-specific antibodies for SARS-CoV-2 and patients' background information. Result(s): A total of 783 samples (560 upper GIE and 223 lower GIE samples) were analyzed. Polymerase chain reaction (PCR) on saliva samples did not show any positive results in either upper or lower GIE samples. However, 2.0% (16/783) of gastrointestinal fluid samples tested positive for SARS-CoV-2. No significant differences in age, sex, purpose of endoscopy, medication, or rate of antibody test positivity were found between PCR positive and PCR negative cases. Conclusion(s): Asymptomatic patients, even those with no detectable virus in their saliva, had SARS-CoV-2 in their gastrointestinal tract. Endoscopy medical staff should be aware of infection when performing procedures. The study was registered as UMIN000040587. Text S1 Detection of SARS-Co-V-2 genomic RNA and serological tests for SARS-CoV-2 antibodies. Detection of SARS-CoV-2 genomic RNA was performed according to the Manual for the Detection of Pathogen 2019-nCoV Ver.2.61) provided by the National Institute of Infectious Diseases in Japan. RNA extraction was performed from a 140-microl sample using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. The final elution was performed with 60 microl of elution buffer, and 5 microl of extracted RNA was subject to evaluation by real-time quantitative PCR (RT-qPCR). The RT-qPCR reaction mix was prepared using TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific, Foster City, CA, USA) and primer/probe N2 (2019-nCoV) (TakaRa, Tokyo, Japan) according to the manufacturer's protocol. Primer sequences are shown in Supplementary Table 1. RT-PCR was conducted by StepOnePlusTM Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, MA. USA) The denaturation and annealing/extension steps were repeated for 45 cycles. A well containing absolutely quantified artificial synthetic template RNA was evaluated as a positive control, and a well without template RNA was evaluated as a negative control. According to the Manual for the Detection of Pathogen 2019-nCoV Ver.2.6, the assay was considered valid when the following criteria were met: (a) 50 copies/well of template RNA successfully detected before 40 cycles and (b) nonspecific amplification not detected in the well lacking template RNA up to 45 cycles. Samples showing amplification of SARS-CoV-2 genomic RNA before 40 cycles were defined as positive for COVID-19. We previously reported a method for serological testing2),3). Briefly, an enzyme-linked immunosorbent assay was performed to detect and quantify anti-SARS-CoV-2 antibodies in plasma. We used an N-terminally-truncated nucleocapsid protein (NP) and the receptor-binding domain of the spike protein (SP) as antigens (100 ng/well). Following blocking with phosphate-buffered saline (PBS) containing 3 % skimmed milk for 2 h, 100 microl of diluted plasma (1:100) were added and incubated for 1 h. After three washes with PBS-T, wells were incubated with 100 microl of diluted horseradish peroxidase-conjugated goat anti-human immunoglobulin G antibody (1:20000) for 1 h. Afte five additional washes with PBS-T, 100 microl of Tetramethylbenzidine Substrate (Kirkegaard & Perry Laboratories, Maryland, USA) was added and incubated for 10 mins. The reaction was terminated by adding 50 microl of 2-M H2SO4, and optical density was measured at 450 nm using a plate reader. In the serological test, COVID-19 antibody positivity was defined as a value greater than 1.139 in the NP test and greater than 0.277 in the SP test. In this study, a positive result in either the NP test or the SP test was defined as a positive antibody test. Copyright © 2022 Japan Gastroenterological Endoscopy Society. All rights reserved.