key: cord-0805871-6jabqim5 authors: Hiroi, Satoshi; Kubota‐Koketsu, Ritsuko; Sasaki, Tadahiro; Morikawa, Saeko; Motomura, Kazushi; Nakayama, Emi E.; Okuno, Yoshinobu; Shioda, Tatsuo title: Infectivity assay for detection of SARS‐CoV‐2 in samples from patients with COVID‐19 date: 2021-06-29 journal: J Med Virol DOI: 10.1002/jmv.27145 sha: 3c85070a220741d0f8e9f2523598faabd54fcb9c doc_id: 805871 cord_uid: 6jabqim5 Since the coronavirus disease 2019 (COVID‐19) outbreak, laboratory diagnosis has mainly been conducted using reverse‐transcription polymerase chain reaction (RT‐PCR). Detecting the presence of an infectious virus in the collected sample is essential to analyze if a person can transmit infectious severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). However, there have been no quantitative investigations conducted for infectious SARS‐CoV‐2 in clinical samples. Therefore, in the present study, a rapid and simple focus‐forming assay using the peroxidase‐antiperoxidase technique was developed to quantify infectious SARS‐CoV‐2 titers in 119 samples (n = 52, nasopharyngeal swabs [NPS]; n = 67, saliva) from patients with COVID‐19. Furthermore, the study findings were compared with the cycle threshold (Ct) values of real‐time RT‐PCR. The infectious virus titers in NPS samples and Ct values were inversely correlated, and no infectious virus could be detected when the Ct value exceeded 30. In contrast, a low correlation was observed between the infectious virus titers in saliva and Ct values (r = ‐0.261, p = 0.027). Furthermore, the infectious virus titers in the saliva were significantly lower than those in the NPS samples. Ten days after the onset of COVID‐19 symptoms, the infectious virus was undetectable, and Ct values were more than 30 in NSP and saliva samples. The results indicate that patients whose symptoms subsided 10 days after onset, with Ct values more than 30 in NSP and saliva samples, were less likely to infect others. 2020, the number of cases exceeded than that of initially reported during the first wave in 2020. February 1, 2020 under the Japanese Infectious Disease Control Law. Hence, it is now possible to implement hospitalization measures and employment restrictions to the patients. Individuals with confirmed COVID-19 infection would be hospitalized regardless of their symptoms. Furthermore, discharge from the hospital requires confirmatory negative polymerase chain reaction (PCR) tests from at least two consecutive samples collected more than or equal to 24 h apart. 1 However, many infected people test positive by repeated PCR tests, even if they are asymptomatic. These factors lead to hospital beds being occupied by asymptomatic patients as well as those with mild symptoms, thereby hindering the hospitalization of critically ill patients. Moreover, testing numerous negative samples during recovery phase imposes a heavy burden on laboratories that conduct PCR tests in terms of time and labor. In consideration of this situation, the Japanese government reviewed the discharge criteria in mid-June 2020 and revised that it is not always necessary to have a negative PCR test. Currently, the standard method for laboratory testing of COVID-19 is quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) that determines the cycle threshold (Ct) values of viral RNA as an index. [2] [3] [4] High Ct values may indicate low infectious virus titers; however, there is little evidence to support this possibility. Moreover, it is important to confirm the presence of infectious virus in the clinical samples to determine whether a patient infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belonging to the family Coronaviridae, genus Betacoronavirus, can infect others. [5] [6] [7] [8] [9] [10] [11] [12] However, cell culture assays that detect infectious viruses require specialized skills and experience, must be safely performed, and are rarely conducted for SARS-CoV-2. A few studies have reported the detection of cytopathic effects (CPEs) of infectious viruses, [13] [14] [15] whereas others used the focus-forming assay. 16 However, at present, there have been no quantitative investigations performed on infectiousness of SARS-CoV-2 in clinical samples. Therefore, in this study, a focus-forming assay using the peroxidase-antiperoxidase (PAP) staining technique, typically applied to numerous types of viruses, was developed. [17] [18] [19] [20] [21] Here, infectious SARS-CoV-2 titers in patients with COVID-19 and their corresponding Ct values by RT-PCR were measured. The findings of the present study will significantly contribute toward the control of COVID-19 transmission and consequently, reduce the number of patients requiring long-term hospitalization. Vero-E6/TMPRSS2 (JCRB 1819) cells were seeded in 24-well plates (2 × 10 5 cells/well) and incubated in an atmosphere containing 5% CO 2 at 37°C. On the following day, the medium in each well-containing cell monolayers was removed. The cell monolayers were then inoculated with 100 µl of serially diluted VeroE6/TMPRSS2 cells were seeded in 24-well plates (2 × 10 5 cells/well) one day before the experiment. The medium was re- Figure 1C ). (A) (B) Although the viral RNA load is slightly higher in the sputum than in NPS samples, [23] [24] [25] [26] NPSs are optimal for detecting viral shedding from the upper respiratory tract, which contributes to the transmission of infection. [24] [25] [26] [27] It has been demonstrated that saliva contains a large copy number of viral genes and the burden of saliva collection is small. Consequently, medical personnel is at low risk of exposure to the virus. 4, [28] [29] [30] [31] [32] [33] In the present study, Ct values and infectious virus titers were determined using NPS and saliva samples. Detection of infectious SARS-CoV-2 has been mainly performed using viruses propagated in cultured cell lines. 5, 10, [12] [13] [14] [15] [16] 29, 34 Although virus culture is a qualitative method, it has been applied to investigate the presence of infectious viruses over time. 5, 8, 34 Examples of techniques to measure infectious viruses include plaque and focus-forming assays to quantitatively employ a stock preparation of SARS-CoV-2. 16 A study found a significant correlation between infectious virus titers obtained using plaque and focus-forming assays. 34 The Ct values and infectious virus titers of NPS and saliva samples were measured, and the significance of the correlations was investigated ( Figure 2 ). In the same Ct value groups, we found that infectious virus titers in the saliva were significantly lower than those in NPS samples ( The present study demonstrated that when the Ct value was more than 30, the infectious virus was difficult to detect in NPS or saliva samples (Figure 2) , which is consistent with the results of previous studies. 5, [11] [12] [13] 32 One of the studies in which analysis of virus cultures prepared from 183 RT-PCR-positive specimens (n = 9, sputum; n = 174, In Japan, individuals infected with SARS-CoV-2 with a positive PCR test are eligible for admission to a designated medical institution, and this standard is strictly enforced when the number of patients is small. However, with an increase in the number of patients, hospital admission may not be possible, which significantly increases the number of patients who must wait elsewhere before they can be treated. The present study demonstrated that in patients with positive PCR test results, who were asymptomatic or mildly infected, the infectious virus produced was with very low titers when the Ct value was more than or equal to 30, which indicated that others were less likely to be infected. These findings will significantly aid efforts to mitigate the severe stress on the healthcare system in Japan, as well as in other countries, should the COVID-19 pandemic continue at the present alarming pace. 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