key: cord-0957034-lq1oiphf
authors: Mishra, Chandan; Meena, Suneeta; Meena, Jitendra Kumar; Tiwari, Suman; Mathur, Purva
title: Detection of three pandemic causing coronaviruses from non-respiratory samples: systematic review and meta-analysis
date: 2021-08-09
journal: Sci Rep
DOI: 10.1038/s41598-021-95329-4
sha: 52c150e9ef1e7c1bc527dde10751f70f0bdfd9f2
doc_id: 957034
cord_uid: lq1oiphf

SARS-CoV-2 has posed an unprecedented challenge to the world. Pandemics have been caused previously by viruses of this family like Middle East Respiratory Corona Virus (MERS CoV), Severe Acute Respiratory Syndrome Corona Virus (SARS CoV). Although these viruses are primarily respiratory viruses, but they have been isolated from non-respiratory samples as well. Presently, the detection rate of SARS‐CoV‐2 RNA from different clinical specimens using Real Time Reverse Transcriptase Polymerized Chain Reaction (qRT‐PCR) after onset of symptoms is not yet well established. Therefore, the aim of this systematic review was to establish the profile of detecting SARS‐CoV‐2, MERS CoV, SARS CoV from different types of clinical specimens other than the respiratory using a standard diagnostic test (qRT‐PCR). A total of 3429 non-respiratory specimens were recorded: SARS CoV (total sample—802), MERS CoV (total sample—155), SARS CoV-2 (total sample—2347). Out of all the samples studied high positive rate was seen for saliva with 96.7% (14/14; 95% CI 87.6–100.0%) for SARS CoV and 57.5% (58/250; 95% CI − 1.2 to 116.2%) for SARS CoV-2, while low detection rate in urine samples for SARS CoV-2 with 2.2% (8/318; 95% CI 0.6–3.7%) and 9.6% (12/61; 95% CI − 0.9 to 20.1%) for SARS CoV but there was relatively higher positivity in urine samples for MERS CoV with detection rate of 32.4% (2/38; 95% CI − 37.3 to 102.1%). In Stool sample positivity was 54.9% (396/779; 95% CI 41.0–68.8%), 45.2% (180/430; 95% CI 28.1–62.3%) and 34.7% (4/38; 95% CI − 29.5 to 98.9%) for SARS CoV-2, MERS CoV, and SARS CoV, respectively. In blood sample the positivity was 33.3% (7/21; 95% CI 13.2–53.5%), 23.7% (42/277; 95% CI 10.5–36.9%) and 2.5% (2/81; 95% CI 0.00–5.8%) for MERS CoV, SARS CoV-2 and SARS CoV respectively. SARS‐CoV‐2 along with previous two pandemic causing viruses from this family, were highly detected stool and saliva. A low positive rate was recorded in blood samples. Viruses were also detected in fluids along with unusual samples like semen and vaginal secretions thus highlighting unique pathogenic potential of SARS‐CoV‐2.

The search terms were combined using Boolean logic "or" and "and". Filters were set to exclude non-human studies. Finally, the article that was directed to relevant categories as indicated in the PRISMA flow diagram (Fig. 1 ).

Clinical trial and observational studies including cross-sectional studies, retrospective studies and prospective studies were selected for review. Case series and case study including even one patient per study was considered. This review is limited and focused to isolation of the Human Corona virus specifically SARS CoV, MERS CoV, SARS CoV-2 by real time Reverse Transcriptase Polymerase Chain Reaction i.e., qRT-PCR. The studies that had targeted non-respiratory sample positivity for the qRT-PCR was taken in consideration. The studies which were conducted to determine diagnostic accuracy, reviews and non-human articles were excluded.

Data extraction. The selection of the articles was done by 4 independent reviewers (SM, CM, JKM and ST) who evaluated the articles for the potential inclusion by screening the titles and abstract followed by full-text screening to determine eligibility of inclusion of the article for final study. Any discrepancies in inclusion of the articles were resolved by SM. The single specimen positive by qRT-PCR was taken into study. The number of patients tested were counted for each type of the specimen rather than the number of samples.

Result analysis. Among 80 articles included the all the positive cases were added for the study. Information was recorded if there at least one specimen tested positive (confirmed cases). Different types of non-respiratory clinical samples were recorded for SARS CoV, MERS CoV, SARS CoV-2. Various type of clinical samples like stool/faecal, anal/rectal swab, urine, semen, testes tissue, vaginal secretions/mucus, vaginal swabs, placental specimen, amniotic fluid, whole blood, serum, plasma, cord blood, ascitic fluid, peritoneal fluid, gastric fluid, pericardial fluid, pleural fluid, breast milk, saliva, ocular surface samples, ocular swab sample, conjunctival swab, tears, CSF were taken in consideration. The rectal/anal swab specimen in the studies that also included stool/ faecal specimen, the sample with maximum positivity was taken into account i.e. the stool/faecal specimen, in following studies by Huang et al. 5 www.nature.com/scientificreports/ estimate (test positivity rate/proportion) by a random-effects model using Open Meta Analyst software 85 . The results were expressed using by pooled effect estimates and their 95% confidence intervals (CIs) and a correction factor of 0.5. Heterogeneity is commonly observed within diagnostic test accuracy reviews due to the differences in study quality, sample size, method, and different outcomes for which random effects models were kept default. Heterogeneity in the analysed studies was determined using Cochrane Q-statistic test and I 2 statistic with subgrouping performed based on the types of clinical specimens. (Fig. 2 ). Between-study heterogeneity assessed using and I 2 statistic. I 2 ranges from 0 and 100% and larger values represent increasing heterogeneity (Table 1) .

Sensitivity analysis. Sensitivity analysis was done to determine the effect of individual studies on the pooled estimates. Univariate meta-regression was performed from primary studies using the random-effects model. A leave-one-out sensitivity analysis was done to assess heterogeneity between the study results. It was performed by iteratively removing one study at a time to confirm that our findings were not driven by any single study (Fig. 3a,b) . Quality assessment. The assessment was performed by four independent reviewers and further checked by two additional reviewers. As recommended by Cochrane Handbook for Systematic Reviews of Diagnostic Accuracy 86 , we adopted QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies -2) 87 to evaluate the bias and quality of selected studies. The following four domains were considered for risks of bias and application concerns as depicted in the assessment tool: (1) participant selection; (2) index test; (3) reference text; and (4) flowing and timing. Studies with more than one "high risk of bias" were excluded (Fig. 4 ).

Publication bias. Output created by Open Meta Analyst software through a cumulative forest plot was indirectly used to assess the publication bias 88 . Funnel Plot and and Egger's and Begg tests were conducted to assess the publication bias (P > 0.05) of the enclosed literature ( Table 2 ). As seen in (Fig. 5) , the funnel plot appearing symmetric, indicates absence of substantial publication bias.

Positive detection rate from non-respiratory samples. A total of 3429 non-respiratory specimens were recorded having SARS CoV (total sample-802), MERS CoV (total sample-155), SARS CoV-2 (total sample -2347). Here number of samples corresponds to number of patients. As we observed high rates of data heterogeneity further sensitivity analysis was undertaken to identify its potential source (study 

This systematic review and meta-analysis provide comprehensive data on detection of pandemic causing coronaviruses from non-respiratory samples. Our findings suggest that all the viruses are mainly respiratory, but they can cause multisystem involvement. They are detected from stool, urine, semen, testes tissue, vaginal secretions, placental specimen, amniotic fluid, whole blood, serum, plasma, cord blood, ascitic fluid, peritoneal fluid, gastric fluid, pericardial fluid, pleural fluid, breast milk, saliva, ocular specimen/tears and CSF. This finding is supported by several studies demonstrating detection of these viruses from various non-respiratory samples enlisted above. These findings show that in clinical practice non-respiratory samples are also important. SARSCoV-2 seems to have a strong predilection for the angiotensin two converting enzyme (ACE-2): the wide expression in different human tissues (as well as in the lung also in the intestine, testicle, kidney, etc.,) would also justify different theoretical modes of transmission of the virus in addition to respiratory route.

Out of all the samples studied maximum positivity was seen for saliva. This study revealed that saliva specimen had positivity of 57.5% for SARS CoV-2 detection and 96.7%for SARS CoV 40, 48, 68, 71 . Saliva has been accepted as reliable non-invasive specimen with high sensitivity in comparison to NPS and throat swab 48, 71 . Now that FDA approved kits are also available for diagnosis of covid-19 from saliva, this can be a safe alternative to respiratory samples 89 . Saliva has the advantage of being easiest to collect amongst all samples. Although positivity rate of saliva for SARS CoV-2 seems lower in our analysis as compared to previous study 3 . It could be explained by the sample size of the study by Pasomsub et al. 2020 was 200 out of which only 19 patients were positive for COVID-19 by NPS of which 18 tested positive for the saliva specimen causing rest to be outliers leading to decreased percentage of positivity. SARS CoV and SARS CoV-2 both has equally detected from saliva but there were no studies on detection of MERS.

The stool sample is well established for high sensitivity for the corona viruses 29 . This study also had same interpretation though the positivity rate for MERS CoV was lower than that of the other two viruses. Nevertheless, stool sample can be an alternate sample for diagnosis, but its clinical relevance and infectiousness needs to All the three viruses have been detected from both serum and blood during early period of viremia 28,44,70 It is highest for SARS-CoV-2 followed by MERS-CoV and SARS CoV. But the detection rate is highly variable depending on the timing of sample collection and significantly less as compared to respiratory sample. Nevertheless, it can complement diagnosis in absence of positivity from respiratory samples 90 .

Another interesting finding was detection of these viruses from genito-urinary specimen. There was 2.2% positivity in urine samples for SARS CoV-2,32.4%for MERS-CoV and 9.6%for SARS CoV. Detection of SARS CoV-2 from semen 9,33,82 sample was 9.8% and vaginal 26 swabs were 21.2%. Detection from these samples highlight unique pathogenic potential of this virus which needs to be evaluated further. www.nature.com/scientificreports/ There were studies that arises question about vertical transmission of corona viruses. Positivity in placental samples23.4% and in amniotic fluid 8.1%for SARS CoV-2 was seen in post-delivery women, but none were found for the SARS CoV. No study for the MERS CoV was found. The post-delivery cord blood sample was not positive in SARS CoV-2 and SARS CoV. Similarly, none of the three corona viruses were not detected in the breast milk samples from pregnant woman.

To our knowledge, this is the first systematic review to comprehensively examine and compare SARS-CoV-2, SARS-CoV, and MERS-CoV detection in non-respiratory samples. Our study has limitations. First, almost all patients in the included had detection from non-respiratory samples at various time points which might have affected the results. Second, our meta-analysis identified substantial study heterogeneity, probably due to differences in study population, different extraction methods and kits used in RT-PCR. Thirdly, most of the studies published are case reports or case series where patient selection may have been biased. As a result, our analyses were based on varying methods, sample timing, sample frequencies and study endpoints differing widely between the included studies impairing contrasts and strong conclusions.

We recognized a systematic review and meta-analysis published on detection of SARS-CoV-2 from different clinical specimen using RT-PCR that included studies published up until may2020. The study include 7 studies and also analysed all samples including respiratory samples 3 . Our study on the other hand has analysed 80 studies wherein a total of 3429 samples were included. Besides, we have also compared the positivity in non-respiratory specimen of the three pandemic causing viruses which has not been attempted elsewhere.

This review provides detailed understanding about the evidence available so far on detection of SARS-CoV-2, SARS-CoV, and MERS-CoV from non-respiratory samples. It has implications in understanding viral dynamics and possible transmission routes to determine preventive steps that need to be taken. Various mitigation and prevention strategies for the current ongoing pandemic should be designed and implemented keeping in mind that this virus could have non-respiratory route of transmission.

In this study, SARS-CoV-2 along with previous two pandemic causing viruses from this family, were highly detected stool and saliva. A low positive rate was recorded in blood samples. Also, viruses were also detected in fluids along with unusual samples like semen and vaginal secretions thus highlighting unique pathogenic potential of SARS-CoV-2. Thus, mitigation and prevention strategies should also focus on non-respiratory routes of transmission for the current pandemic. Table 3 . Detection of SARS, MERS, SARS CoV-2 in different non-respiratory samples.

Type of specimen SARSCoV2 + (n/N) MERS + (n/N) SARS + (n/N) 

Cultivation of viruses from a high proportion of patients with colds

Spectrum of clinical illness in hospitalized patients with "common cold" virus infections

Detection profile of SARS-CoV-2 using RT-PCR in different types of clinical specimens: A systematic review and meta-analysis

Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement

SARS-CoV-2 viral load in clinical samples of critically ill patients

Detection and analysis of nucleic acid in various biological samples of COVID-19 patients

SARS and pregnancy: A case report

Coronavirus Disease 2019 (COVID-19) During Pregnancy: A Case Series

Study of SARS-CoV-2 in semen and urine samples of a volunteer with positive naso-pharyngeal swab

Asymptomatic SARS-CoV-2 infected case with viral detection positive in stool but negative in nasopharyngeal samples lasts for 42 days

Case report: Walking pneumonia in novel coronavirus disease (COVID-19): Mild symptoms with marked abnormalities on chest imaging

A Case Report of Neonatal 2019 Coronavirus Disease in China

A case of a readmitted patient who recovered from COVID-19 in Chengdu

A first case of meningitis/encephalitis associated with SARS-Coronavirus-2

First description of SARS-CoV-2 in ascites

Frequent Convulsive Seizures in an Adult Patient with COVID-19: A Case Report

Infectious SARS-CoV-2 in feces of patient with severe COVID-19

SARS-CoV-2 detected in amniotic fluid in mid-pregnancy

Ocular manifestations of a hospitalised patient with confirmed 2019 novel coronavirus disease

SARS-CoV-2 detected in cerebrospinal fluid by PCR in a case of COVID-19 encephalitis

SARS-CoV-2 detection in the pericardial fluid of a patient with cardiac tamponade

SARS-CoV-2 Gastrointestinal infection causing hemorrhagic colitis: Implications for detection and transmission of COVID-19 disease

SARS-CoV-2 isolation from ocular secretions of a patient with COVID-19 in Italy with prolonged viral RNA detection

SARS-CoV-2-positive sputum and feces after conversion of pharyngeal samples in patients with COVID-19

The evidence of SARS-CoV-2 infection on ocular surface

Transplacental transmission of SARS-CoV-2 infection

Unlikely SARS-CoV-2 vertical transmission from mother to child: A case report

Viral RNA in blood as indicator of severe outcome in middle east respiratory syndrome coronavirus infection

Detection of novel coronavirus by RT-PCR in stool specimen from asymptomatic child

Detection of SARS coronavirus RNA in the cerebrospinal fluid of a patient with severe acute respiratory syndrome

Encephalitis as a clinical manifestation of COVID-19

Pregnancy with new coronavirus infection: Clinical characteristics and placental pathological analysis of three cases

Absence of 2019 novel coronavirus in semen and testes of COVID-19 patients

Acute polyradiculoneuritis with locked-in syndrome in a patient with Covid-19

Acute encephalopathy with elevated CSF inflammatory markers as the initial presentation of COVID-19

Assessing Viral Shedding and Infectivity of Tears in Coronavirus Disease 2019 (COVID-19) Patients. Ophthalmology [Internet

Clinical features and virological analysis of a case of Middle East respiratory syndrome coronavirus infection

COVID-19 (SARS-Cov-2) and the heart -An ominous association

COVID-19 not detected in peritoneal fluid: A case of laparoscopic appendicectomy for acute appendicitis in a COVID-19-infected patient

Detection of 2019-nCoV in saliva and characterization of oral symptoms in COVID-19 Patients

Social Science Research Network

First case of 2019 novel coronavirus in the United States

Characteristics of pediatric SARS-CoV-2 infection and potential evidence for persistent fecal viral shedding

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

Comparison of different samples for 2019 novel coronavirus detection by nucleic acid amplification tests

Evaluation of coronavirus in tears and conjunctival secretions of patients with SARS-CoV-2 infection

Evaluation of SARS-CoV-2 RNA shedding in clinical specimens and clinical characteristics of 10 patients with COVID-19 in Macau

Prolonged viral shedding in feces of pediatric patients with coronavirus disease 2019

Saliva is a reliable tool to detect SARS-CoV-2

Clinical and virological data of the first cases of COVID-19 in Europe: a case series

Comparisons of viral shedding time of SARS-CoV-2 of different samples in ICU and non-ICU patients

Consistent detection of 2019 novel coronavirus in Saliva

Detectable SARS-CoV-2 viral RNA in feces of three children during recovery period of COVID-19 pneumonia

Clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 (COVID-19) in the United States

Detection of SARS-associated coronavirus in throat wash and saliva in early diagnosis

Viral load of SARS-CoV-2 in clinical samples

Persistent shedding of viable SARS-CoV in urine and stool of SARS patients during the convalescent phase

Positive result of Sars-Cov-2 in faeces and sputum from discharged patient with COVID-19 in Yiwu

Prolonged presence of SARS-CoV-2 viral RNA in faecal samples

SARS-CoV-2 is present in peritoneal fluid in COVID-19 patients

Viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome

Persistence and clearance of viral RNA in 2019 novel coronavirus disease rehabilitation patients

Detection of severe acute respiratory syndrome coronavirus 2 in placental and fetal membrane samples

Evidence for Gastrointestinal Infection of SARS-CoV-2

Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity

Virological assessment of hospitalized patients with COVID-2019

Detection of SARS-CoV-2 by RT-PCR in anal from patients who have recovered from coronavirus disease 2019

Ocular findings and proportion with conjunctival SARS-COV-2 in COVID-19 patients

Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

Detection and Isolation of SARS-CoV-2 in serum, urine, and stool specimens of COVID-19 patients from the Republic of Korea

Interpretation of diagnostic laboratory tests for severe acute respiratory syndrome: The Toronto experience

Saliva sample as a noninvasive specimen for the diagnosis of coronavirus disease 2019: a cross-sectional study

The presence of SARS-CoV-2 RNA in the feces of COVID-19 patients

The infection evidence of SARS-COV-2 in ocular surface: A single-center cross-sectional study

Neonatal early-onset infection with SARS-CoV-2 in 33 neonates born to mothers with COVID-19 in Wuhan, China

Detection of SARS coronavirus in patients with suspected SARS

Viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection

Fecal specimen diagnosis 2019 novel coronavirus-infected pneumonia

SARS-CoV-2 in the ocular surface of COVID-19 patients

Clinical characteristics and intrauterine vertical transmission potential of COVID-19 infection in nine pregnant women: A retrospective review of medical records

Ophthalmologic evidence against the interpersonal transmission of 2019 novel coronavirus through conjunctiva

Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province

Clinical characteristics and results of semen tests among men with coronavirus disease 2019

Clinical characteristics of 19 neonates born to mothers with COVID-19

Presence of viral RNA of SARS-CoV-2 in conjunctival swab specimens of COVID-19 patients

Meta-analyst: Software for meta-analysis of binary, continuous and diagnostic data

Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy

QUADAS-2: A revised tool for the quality assessment of diagnostic accuracy studies

Persistence of chloroquine resistance alleles in malaria endemic countries: A systematic review of burden and risk factors

COVID-19) Update: FDA Issues Emergency Use Authorization to Yale School of Public Health for SalivaDirect, Which Uses a New Method of Saliva Sample Processing

Coronavirus disease 2019: Coronaviruses and blood safety

The authors declare no competing interests.

Correspondence and requests for materials should be addressed to S.M.Reprints and permissions information is available at www.nature.com/reprints.Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.