Persistence of SARS-CoV-2 RNA in Nasopharynx, Blood, Urine and Stool of Patients with COVID 19: A Hospital-based Longitudinal Study

Abstract

Background: This study was conducted to determine the persistence of SARS-CoV-2 RNA in the nasopharynx, blood, urine and stool of patients with COVID-19.

Methods: In this hospital based longitudinal study, 100 confirmed COVID-19 cases were recruited, between March and August 2020 in Guilan province (Northern Iran). Nasopharynx, blood, urine and stool samples were obtained from each patient at the time of hospital admission, discharge, followed by one week after discharge and every 2 weeks until all samples were negative for SARS-CoV-2 RNA by reverse transcription polymerase chain reaction (RT-PCR). Survival analysis was used to identify the duration of virus persistence over time.

Results: Positive blood, urine, stool RT-PCR were detected in 24%, 7% and 6% of patient respectively. The median duration of virus persistence in blood, urine and stool were 7 days (95% CI: 6.07–7.93), 6 days (95% CI: 4.16–8.41) and 13 days, 95% CI: 6.96–19.4), respectively.  The maximum duration of virus persistent in blood, urine and stool were 17, 11 and 42 days from admission, respectively.

Conclusions: According our results, until obtaining definite evidence of the duration of infective viral shedding, prolonged isolation duration at least 25 days from admission to hospital and strict hygienic measures for about one month were recommended. 

1. Introduction

The Coronavirus disease 2019 (COVID-19) was declared a global epidemic by the World Health Organization in December 2020 [1]. By November 22, 2020, it has infected almost 57 million people with more than 1,300,000 deaths [2]. Iran was classified as the 8th country in terms of COVID-19 morality [3].There were 1,189,203 confirmed cases of COVID-19 with 54,440 deaths in Iran, as of 15 February 2019 until 25 December 2020 [4].

The principle route of COVID-19 transmission was reported to be direct and indirect contact with respiratory droplets [5]. Although, other routes of transmission, such as: mother to fetus, fecal-oral, airborne are currently controversial and subject to future investigations [6, 7], however, the epidemiological studies have shown that COVID-19 patients had close contact or proximity in distance with someone who has COVID-19 [8].

Initially, SARS-Cov-2 was isolated and recognized in respiratory samples by real-time RT-PCR [9]. However, in recent studies, viral nucleic acids have subsequently been detected in urine, stool, and gastrointestinal mucosa [10, 11]. A study reported, after nasopharyngeal RT-PCR was negative, eight of ten children with COVID-19, had persistently positive RT-PCR on rectal swabs [12].

Base on The Centers for Disease Control and Prevention (CDC) guidelines, all patients with a positive respiratory RT-PCR test must be isolated until at least 24 hours with no fever, and at least 10 days from initial symptom onset [13]. However, there are several case reports on the persistence of positive RT-PCR in patients with COVID-19, indicating that individuals can remain positive after symptoms have resolved [14, 15]. Therefore, the persistence of the virus in body fluids can increase the potential risk of virus transmission in asymptomatic or recovered patients [15]. So, it remains uncertain whether the quarantine duration, recommended by CDC is sufficient to reduce transmission [16].

Considering that, the frequency with which SARS-CoV-2 RNA can be detected in body fluids and the period during which it remains detectable are not well understood; this longitudinal study was conducted to determine the persistence of SARS-CoV-2 RNA in the nasopharynx, blood, urine and stool of patients with coronavirus disease 2019 via collecting sequential samples every 2 weeks.

2. Materials And Methods

3. Results

During the study period, 106 hospitalized patients with positive nasopharyngeal RT-PCR of SARS-CoV-2 RNA were enrolled to the study, but 6 of these were died (5.66%). Mean age of the participants was 53.30 ± 13.03 years (range 29–86 years) and the majority of the patients were male (60%), were married (84%), had educated, less than diploma (63%), were urban residents (85%), were employed (68%) and had low socioeconomic status(82%). The result of SARS-CoV-2 RNA RT-PCR on different sample, over time is shown in Fig. 1. Only 24% of RT-PCR on blood sample was positive at admission and one sample remained positive at discharge and was cleared after 1 week. RT-PCR on urine sample was positive in 6 % of participants at admission and all were cleared at discharge. RT-PCR was positive in stool samples at admission in 4 participants and 2 remained positive at discharge, in addition, 2 new stool samples were positive at discharge. Three of them were cleared after one week and one was cleared 5 weeks after discharge. At discharge, 30% of nasopharyngeal RT-PCR was positive and all were cleared after one weak.

Participants’ clinical and demographic characteristics based on duration of virus persistence in the nasopharynx are shown in table2. Nearly 11% of patients had virus persistence in nasopharynx longer than 14 days from the first positive test. Demographic characteristics of patient were not significantly different among the 3 groups. Similarly, there was no significant association between COVID-19 symptoms and duration of nasopharyngeal persistence of the virus. Our data showed that the type of hospital treatment plan was not contributing to the virus persistence in the nasopharynx (Table 1).

The median duration of SARS-CoV-2 persistence in nasopharynx from the first positive RT-PCR test at admission time was 8 days (95% CI: 6.91–9.09) and maximum duration of virus persistence among them was 25 days from admission (Fig. 2). The median duration of SARS-CoV-2 persistence in blood from the first positive RT-PCR test at admission time was 7 days (95% CI: 6.07–7.93) and maximum duration of virus persistence among them was 17 days from admission (figur2). The median duration of SARS-CoV-2 persistence in stool from the first positive RT-PCR test was 13 days (95% CI: 6.96–19.4) and maximum duration of virus persistence among them was 42 days from admission (Fig. 2). The median duration of SARS-CoV-2 persistence in urine from the first positive RT-PCR test at admission time was 6 days (95% CI: 4.16–8.41) and maximum duration of virus persistence among them was 11 days from admission (Fig. 2).

In our analysis of factors that might be associated with durations of SARS-CoV-2 persistence by Cox regression, we did not find any statistically significant associations (data not presented).

Frequency of some COVID 19 gastrointestinal (GI) symptoms according to result of stool RT-PCR are presented in Fig. 3. Abdominal pain and diarrhea were significantly more common in stool RT-PCR positive patient than stool negative ones.

In analysis of clinical and demographic factors that might be associated with positive blood and urine RT-PCR, we did not find any statistically significant associations (data not presented).

4. Discussion

In this longitudinal study, we demonstrate that the maximum duration of nasopharyngeal (25 days) and fecal (42 days) SARS-CoV-2 RNA shedding is longer than the recommended (at least 24 hours with no fever, and at least 10 days from initial symptom onset) quarantine duration [13].

Nearly, one third of our hospitalized COVID-19 patients had positive nasopharyngeal RT-PCR at the time of discharge and about one tenth of them had virus persistence in nasopharynx longer than 14 days from the first positive test. Maximum duration of nasopharyngeal shedding was 25 days from admission. These findings are compatible with some studies that revealed, some patients continued to be upper respiratory tract RT-PCR positive after discharge from hospital, for the next few days [18, 19]. We did not identify any determinants of viral persistence, but a study in china demonstrated that the prolonged presence of the virus in upper respiratory tract was associated with disease severity [20].While, another study in Portugal revealed the viral RNA persistence not associated with disease severity and stronger immune response are the determinants of virus RNA clearance [21].

Positive blood SARS-CoV-2 RNA RT-PCR was detected in about one third of our patient, but most of them turned to negative at the time of discharge. The studies on detecting SARS-CoV-2 RNA in blood were limited, but a study in china revealed that the SARS-CoV-2 RNA detected in the blood of 6 out of 57 Chinese patients and all 6 positive bloods RT-PCR had a severe clinical picture [22]. The higher positive rate of blood RT-PCR in our study was probably due to the disease severity in our patients and hospital-based design.

Our findings revealed that the positive urine SARS-CoV-2 RNA RT-PCR was less common (7 out of 100 patients) in our study population and all of them turned to negative at the time of discharge. Our result was in accordance with studies in Turkey and China that demonstrated nearly 5–7% of COVID-19 patient had positive urine RT-PCR [23, 24].

Positive stool SARS-CoV-2 RNA RT-PCR was detected in only 6 out of 100 patients in our study, but the duration of time for a positive stool RT-PCR test to turn negative was longer than nasopharynx, urine and blood RT-PCR tests, with a maximum duration of fecal shedding of 42 days. Therefore, fecal positive hospitalized patients with positive stool RT-PCR, represent the need for precautions and protective equipment for interventional procedures involving the gastrointestinal tract in a hospital environment., Similarly, a study in china demonstrates that an asymptomatic case was positive in the stool RT-PCR for a period as long as 42 days and also, reported that in almost two third of patients, the clearance of fecal shedding takes a longer time than the nasopharyngeal sample [19].

Although, our findings demonstrate that the maximum duration of nasopharyngeal and fecal SARS-CoV-2 RNA shedding is longer than the recommended quarantine duration by the CDC ¹³, however, it is unclear whether individuals with persistent positive RNA PCR represent an infectious risk. Future studies are needed to determine whether PCR positivity is due to infective virus or non- infective nucleic acid fragments. Therefore, until such studies are concluded, prolonged isolation duration at least 25 days may be recommended, and aggressive contact tracing might also be considered. Also, patients are advised to take strict hygiene measures, especially those with gastrointestinal symptoms for about one month, in order to prevent potential fecal-oral transmission.

The strengths of the current study are the long term follow up and detecting virus RNA in respiratory and extra-respiratory sites. However, the hospital-based design is the limitation of our study, leading to more severe patient enrollment that limits the generalizability of findings. Another limitation of our study was that the day of symptom onset was not available, and all calculations and analyses were based on first nasopharyngeal RT-PCR test at admission time thus, the duration of viral shedding was underestimated. Another limitation of current study was that the, virus isolation and tests of specimens’ infectivity were not conducted and CT value of RT-PCR was not available.

5. Conclusion

Our findings demonstrate that the maximum duration of nasopharyngeal and fecal SARS-CoV-2 RNA shedding in hospitalized COVID-19 patients is longer than the recommended quarantine duration by CDC. Therefore, until obtaining definite evidence of the duration of infective viral shedding, prolonged isolation duration for at least 25 days from admission to hospital may be recommended and aggressive contact tracing might also be considered. Also, patients are advised to take public health measures such as strict personal hygiene, especially those with gastrointestinal symptoms for about one month, in order to prevent potential fecal-oral transmission.

Abbreviations

CDC: The Centers for Disease Control and Prevention

Declarations

Acknowledgments 

We thank Prof. Dr. Reza Nassiri from Departments of Pharmacology and Toxicology, Michigan State University, USA, for comments that greatly improved the manuscript. The authors gratefully acknowledge the all personnel’s in the Gastrointestinal and Liver Disease Research Center and vice-chancellor for research of Guilan University of medical science and the staff at Razi Hospital.

Author’s contributions

FA - conception, literature search, study design, data collection, data analysis, data interpretation, writing and final approval; T YK,S T,L M,HAB,A J,A P,E H- study design ,data collection, writing and final approval; MK and MS and IJ - data collection, laboratory activities (PCR); F M-G - conception, literature search, data collection, data analysis, data interpretation, writing and final approval; MN-literature search, data collection, data interpretation, writing and final approval; SM - data analysis, data interpretation, writing and final approval; MA- data collection, literature search, writing and final approval.

Funding

None.

Availability of data and materials

The datasets obtained during this study will be available upon request to the corresponding author.

Ethics approval and consent to participate

This study was registered in the Research Department of Guilan University of Medical Sciences with the ethics code of IR.GUMS.REC.1399.013. This manuscript has not been published in whole or in part. All authors have read the manuscript and have agreed that the work is ready for submission and accept responsibility for its contents.

Consent for publication

Not Applicable.

Competing Interest

Authors have no competing interests.

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Table

Table 2 not available with this version.