key: cord-0826777-alukeusr
authors: Gao, Chun; Zhu, Li; Jin, Cheng Cheng; Tong, Yi Xin; Xiao, Ai Tang; Zhang, Sheng
title: Prevalence and impact factors of recurrent positive SARS-CoV-2 detection in 599 hospitalized COVID-19 patients
date: 2021-02-09
journal: Clin Microbiol Infect
DOI: 10.1016/j.cmi.2021.01.028
sha: 3ca96adfa0f74a3736956fe83d03eb414d692f23
doc_id: 826777
cord_uid: alukeusr

OBJECTIVES: Re-positive tests for SARS-CoV-2 in Coronavirus Disease 2019 (COVID-19) patients were common. We aimed to investigate the rate and risk factors of recurrent positive detection of SARS-CoV-2 in hospitalized COVID-19 patients. METHODS: Oropharyngeal and nasopharyngeal swabs (n=3513) were collected to detect SARS-CoV-2 during the hospitalization. We analyzed the recurrent positive rate after consecutive negative results and its relationship to demographic characteristics. RESULTS: Among 599 enrolled COVID-19 patients, the median time for viral RNA shedding was 24 days (IQR, 19-33 days). The positive rate of RT-PCR was 35.9% (215/599), 17.0% (65/383) and 12.4% (23/185) after one, two and three consecutive negative RT-PCR test results respectively. Medians of CT-values of initial positive test, rebound positive after two consecutive negative results, and rebound positive after three consecutive negative results were 28.8, 32.8 and 36.1 respectively. Compare with male patients, females had a significant higher rate of recurrent positive RT-PCR after three consecutive negative results (18.2%, 18/99 vs. 5.8%, 5/86, p=0.013). Older age (≥55 yrs) had a significant higher rate of recurrent positive RT-PCR after one negative result (42.3%, 165/390, vs. 23.9%, 50/209, p<0.001). Nasopharyngeal swab tests produced a higher positive rate than oropharyngeal swab tests (37.3%, 152/408 vs. 35.8%, 1111/3105). CONCLUSIONS: Our study revealed the prevalence and dynamic characteristics of recurrent positive RT-PCR to SARS-CoV-2. We showed that around 17.0% (65/383) patients were tested positive for SARS-CoV-2 after two consecutive negative results. Patients with rebound positive RT-PCR test had a low viral load. Older age and female were risk factors for recurrent positive results.

To date, coronavirus disease 2019 (COVID-19) associated with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has caused a global pandemic. [1-3 2] As of the manuscript was drafted, over 85,500,000 cases were confirmed worldwide 4 with a rough case fatality rate around 2.2%. [3] 5 Chinese National Health Committee (7 th version) updated their guidelines 6 regarding discharge and discontinuing isolation, including resolution of fever, 7 improvement in respiratory symptoms and radiography findings, and with two 8 consecutive negative real-time reverse transcription polymerase chain reaction (RT-9 PCR) test results for SARS-COV-2 RNA. (from two consecutive respiratory specimens 10 collected ≥24 hours apart).

[4] Currently the US CDC updated its criteria for 11 discontinuation of transmission-based precautions for confirmed COVID-19 infected 12 patients. Negative RT-PCR test is no longer recommended.

[5] Nonetheless, increasing 13 reports on recurrent positive RT-PCR for SARS-COV-2 have aroused wide concern. [6-14 8] Prolonged viral RNA shedding in certain infected individuals and relatively high 15 false negative of viral test by RT-PCR may be responsible for the recurrence. A false 16 negative result of RT-PCR is defined as the negative test result followed by a recurrent 17 positive test. False-negative results were mainly caused by the errors in sampling and 18 detection methods. 19

The prevalence of recurrent positive results of RT-PCR test to SARS-CoV-2 were 20 reported in several studies. Here, we conducted a retrospective study to investigate the 21 dynamic viral RNA shedding and impact factors for recurrent positive test results of RT-PCR to SARS-CoV-2. 23 enrolled patients were confirmed diagnosed of COVID-19 according to the diagnosis 28 and treatment guideline for SARS-CoV-2 from Chinese National Health Committee (7 th 29 version SpO2 <94% on room air at sea level (or, for patients with chronic hypoxemia, a 45 decrease from baseline of >3%), ratio of arterial partial pressure of oxygen to fraction of 46 inspired oxygen (PaO2/FiO2) <300 mmHg, or lung infiltrates >50%) [4] [5] Oropharyngeal and nasopharyngeal swabs were collected both upon admission and 48 during hospitalization by qualified medical professionals through standard procedures 49 under level 3 biosafety protection. Real-time reverse transcription polymerase chain 50 J o u r n a l P r e -p r o o f 6 reaction (RT-PCR) assay were performed on swabs to detect SARS-CoV-2 using 51 COVID-19 test kits (Shanghai Huirui Biotechnology Co., Ltd). Once two consecutive 52 negative results were collected, the period between symptoms onset and the date of first 53 negative RT-PCR test result was considered as the length of viral RNA shedding. 54

Oropharyngeal swab samples or nasopharyngeal swab samples were collected to extract 56 RNA to confirm diagnosis of COVID-19 infection. Total RNA was extracted using 57 magnetic beads (Tianlong, Xi'an, China). Two target genes, including open reading 58 frame 1ab (ORF1ab) and nucleocapsid protein (N), were simultaneously amplified and 59 tested during the real-time RT-PCR assay. The real-time RT-PCR assay was performed 60 using a COVID-19 nucleic acid detection kit according to the manufacturer's protocol 61 (Shanghai Huirui Biotechnology Co., Ltd). The results of RT-PCR assay were 62 expressed as the cycle threshold (Ct) value. As recommend by the instruction, a CT 63 value <37 was defined as a positive test result, and a Ct value ≥39.2 was considered as a 64 negative test. Ct-value between 37 and 39.2 suggested confirmatory RT-PCR be 65 obtained using the same sample. 66

We performed statistical analyses using SPSS version 24.0 (IBM, NY, USA). 68

Continuous variables were present as mean ± standard error of mean (SEM) or medians 69 (interquartile range, IQR) and analyzed with Mann-Whitney U test. We reported 70 categorical variables as whole numbers and percentages. The cut-off value for age is 71 determined by receiver operating characteristic (ROC) curve according to prolonged 72 viral shedding (>24 days). A p value <0.05 was considered statistically significant.

We included a total of 599 patients with confirmed diagnosis of COVID-19 in our 77 study. (Table 1 ) In details, the median age of included patients was 61 years (IQR, 68) with 291 males (48.6%, 291/599) and 308 females (51.4%, 308/599). 569 patients 79 (95.0%, 569/599) were classified as moderate. No patient was transferred to intensive 80 care unit. The median time from onset of symptom to admission was 10 days (IQR, 7-81 14 days) and the median length of hospital stay was 30 days (23-40 days). The median 82 numbers of RT-PCR tests per patient was 5 (IQR, 4-8). The median length for viral 83 shedding was 24 days (IQR, 19-33 days). Supplemental Figure 2 demonstrated that 84 the percentage of patients who shed virus less than 3 weeks, between 3-6 weeks and 85 more than 6 weeks were 40.4%, 48.1% and 11.5% respectively. 86

Positive RT-PCR rates was 90.2% (540/599), 59.6% (357/599), 35.4% (212/599), 87 20.7% (124/599), 11.5% (68/599), 5.3% (32/599), 1.0% (6/599), 0.5% (3/599), 0% 88 (0/599) on week 2, week 3, week 4, week 5, week 6, week 7, week 8, week 9 and ≥ 89

week 10 after symptoms onset respectively. ( Figure. result. (Figure 1B ) As demonstrated in Figure 1C , patients older than 55yrs produced 96 a significantly higher recurrence rate after one negative result (42.3%, 165/390, vs. 97 23.9%, 50/209, p<0.001). While no significant difference of recurrent positive rate was 98 found after two or three consecutive negative RT-PCR results regardless of age. As demonstrated in Figure 1D , female patients had a significant higher rate of recurrent 100 positive RT-PCR after three consecutive negative results than male (18.2%, 18/99 vs. 101 5.8%, 5/86, p=0.013). Recurrent positive rate between genders showed no significant 102 difference after one or two consecutive negative RT-PCR results. 

We analyzed 599 patients' the viral RNA shedding time, which was grouped by age and 109 gender and shown in Table 3 . As demonstrated in Figure 3A , patients <55yrs had a 110 shorter period of viral RNA shedding than patients ≥55yrs, with a significant higher 111 percentage of end of viral shedding on week 2 (13.9%, 29/209 vs. 7.7%, 30/390, p=0.02) 112 and week 3 (33.5%, 70/209 vs. 29.0%, 113/390, p<0.01) after symptoms onset. Patients 113 ≥55yrs had a significant higher percentage for length of viral shedding on week 7 after 114 symptoms onset (9.2%, 36/390 vs. 0.5%, 1/209, p=0.03). As demonstrated in Figure 3B , 115 male patients had a shorter period of viral RNA shedding than female patients, with a 116 higher percentage of viral shedding on early stage (from week 2 to 5 after symptoms 117 onset). 118

Supplemental Figure 3A demonstrated that from week 1 to week 6 after symptoms 121 onset, nasopharyngeal swabs produced a higher positive rate for SARS-CoV-2 than 122 oropharyngeal swabs. On week 3, 4 and 5 after symptoms onset, positive rates from 123 nasopharyngeal swabs were significantly higher than that from oropharyngeal swabs vs 17.9%, 85/474 p<0.05). Supplemental Figure 3B showed that the medians of Ct-126 values of initial positive tests, rebound positive after two consecutive negative results 127 and rebound positive after three consecutive negative results from nasopharyngeal 128 swabs were higher than those from oropharyngeal swabs. From week 6 to ≥week 10 129 after onset of symptoms, positive rates for viral detection using nasopharyngeal and 130 oropharyngeal tests were not significantly different. (Supplemental Table 1 4.8% after one, two and three consecutive negative RT-PCR test results, respectively. 163

In our study we found that the positive rates of RT-PCR were 35.9%, 17.0% and 12.4% 164 after one, two and three consecutive negative RT-PCR. The positive rate (17.0%) after 165 two negative tests was close to previous reports. 166

The causes of recurrent positive tests for SARS-CoV-2 were unclear. Currently, 167 false negatives in RT-PCR tests were considered as the main reason for recurrent 168 positivity, and they could result from errors in sampling or non-infectious viral RNA 169 remnant. However, the false negative results did not include the negative results 170 achieved when the viral load is very load or reach the limit of detection assay. 171

Recurrent positives of COVID-19 have raised increasing public concern regarding the 172 infectivity of recurrent positive patients. In theory, viral transmission is determined by RNA may still be detectable by RT-PCR. According to different reports, the Ct values 177 retrieved from re-positive patients were generally higher than those from initial positive. with Ct values >35 (viral load <10 3 copies/ml) were hardly infectious. In our study, the 196 median Ct values of recurrent positive (32.8 and 36.1) suggested that the transmission 197 risk of these patients was low, even if viral shedding could still be detected by in late phase. Until now, no evidence indicating aggressive treatment or public isolation were benefit for these patients. Our results suggested that patients with Ct 200 value >36 may be considered for discharge. 201 Furthermore, we investigate the impact of age and gender on false negative rate of 202 RT-PCR to SARS-CoV-2. We found that old age and female gender patients tend to 203 have a higher false negative rate of RT-PCR test. Notably, female patients had a 204 significant higher rate of recurrent positive RT-PCR after three consecutive negative 205 results than male (18.2%, 18/99 vs. 5.8%, 5/86, p=0.013). In clinical practice, the above 206 findings may provide information for repeat viral test in selected patients. Studies also 207 showed that older age, comorbidity, low level of antibodies response, host immunity 208 status may be potential risk factors for recurrent positivity of RT-PCR test of SARS-209

CoV-2, while comprehensive intervention may be a protective factor. [25, 26] 210 Various studies investigate the dynamics of viral shedding in COVID-19 patients. 211

Severe patients had a longer duration of viral shedding [9-10]. Our study showed that 212 36.7% patients with age≥55yrs, compared with 47.4% younger patients (age<55yrs), 213 had the median viral shedding time less than 3 weeks. Male patients had a shorter viral 214 RNA shedding period than females (43.3%, 126/291 vs. 37.7%, 116/308 in 3 weeks). 215 (Table 3 ) In our study, samples from nasopharyngeal swabs produced a higher positive 216 rate than samples from oropharyngeal swabs. The mean RT-PCR CT value is higher 217 from oropharyngeal specimens than those from nasopharyngeal specimens. 218 (Supplemental Figure 3 ) Therefore, a false negative test result of RT-PCR may be 219 related to the sampling site. We suggested nasopharyngeal swabs should be the 220 standard procedure to obtain respiratory specimen for viral test.

such as laboratory characteristics, serological data and treatments were incomplete and 225 not included for analysis. Second, we only included symptomatic hospitalized patients 226 with moderate to severe illness. The results may not be applicable for asymptomatic 227 infected individuals or critical cases. Third, patients included in our study had not 228 received identical treatments, which might have an impact on the duration of viral 229

shedding. 230

In summary, in this study we for the first time performed a large-scale investigation of 232 Positive rate of SARS-CoV-2 RT-PCR, weeks after onset

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7 J o u r n a l P r e -p r o o f

Week 8

Week 9

≥Week 10 1.0% 0.5% 0% IQR, interquartile range; COVID-19, coronavirus disease 2019; RT-PCR, Real-time reverse transcription polymerase chain reaction.

J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f 

for Disease Control and Prevention. Coronavirus disease 2019 277 (COVID-19). Discontinuation of Transmission-Based Precautions and 278

Disposition of Patients with COVID-19 in Healthcare Settings

Updated

The issue of recurrently positive patients who 282 recovered from COVID-19 according to the current discharge criteria: 283 investigation of patients from multiple medical institutions in Wuhan

False-negative of RT-PCR and prolonged 286 nucleic acid conversion in COVID-19: Rather than recurrence

PCR Assays Turned Positive 289 in 25 Discharged COVID-19 Patients

Duration of SARS-CoV-2 viral shedding 303 during COVID-19 infection

Factors associated with prolonged viral RNA 306 shedding in patients with COVID-19

Associated With Prolonged Viral RNA Shedding From Respiratory Tract

Coronavirus Disease 2019: A Case Control Study

Proinflammatory cytokines are 313 associated with prolonged viral RNA shedding in COVID-19 patients

Recurrent pneumonia in a patient with new 316 coronavirus infection after discharge from hospital for insufficient antibody 317 production: a case report

Transmissibility of COVID-19

CoV-2 from diagnostic samples

Molecular Testing: Correlation of SARS-CoV-2 Culture with Molecular 334

Duration of 337 infectiousness and correlation with RT-PCR cycle threshold values in cases of 338 COVID-19

Clinical Findings of Short-Term Recurrence of Severe Acute Respiratory 343

Syndrome Coronavirus 2 Ribonucleic Acid Polymerase Chain Reaction