key: cord-0995592-ycsguxnw
authors: Anjos, Déborah; Fiaccadori, Fabiola Souza; Servian, Carolina do Prado; da Fonseca, Simone Gonçalves; Guilarde, Adriana Oliveira; Borges, Moara Alves Santa Bárbara; Franco, Fernanda Craveiro; Ribeiro, Bergmann Morais; Souza, Menira
title: SARS-CoV-2 loads in urine, sera and stool specimens in association with clinical features of COVID-19 patients
date: 2021-12-16
journal: Journal of clinical virology plus
DOI: 10.1016/j.jcvp.2021.100059
sha: dc308175c50ff7bb51742cbb2eeda70f3e5f0bba
doc_id: 995592
cord_uid: ycsguxnw

Background COVID-19 pandemic continues to be a priority in public health worldwide, and factors inherent to SARS-CoV-2 pathogenesis and genomic characteristics are under study. Investigations that evaluate possible risk factors for infection, clinical manifestations, and viral shedding in different specimens also need to clarify possible associations with COVID-19 prognosis and disease outcomes. Study design In this study, we evaluated SARS-CoV-2 positivity and estimated viral loads by real-time RT-PCR in stool, sera, and urine samples from 35 patients, with a positive SARS-CoV-2 RNA molecular test in respiratory sample, attended at a University COVID-19 referral hospital in Goiania, Goias, Brazil. Whole-genome sequencing was also performed in samples with higher viral load. Results The positivity index was 51.43%, 14.28%, and 5.71% in stool, sera, and urine specimens, respectively. The median viral load was 8.01 × 106 GC/g, 2.03 × 106 GC/mL, and 1.36 × 105 GC/mL in stool, sera, and urine, respectivelly. Of all patients, 88.57% had previous comorbidities, and 48.39% of them had detectable SARS-CoV-2 RNA in at least one type of clinical specimen evaluated by this study (stool, sera or urine). A higher viral load was observed in patients with more than two previous comorbidities and that were classified as severe or critical conditions. Samples with the highest viral loads were sequenced and characterized as B.1.1.33 variant. Conclusion We conclude that SARS-CoV-2 RNA is present in more than one type of clinical specimen during the infection, and that the most critical patients had detectable viral RNA in more than one clinical specimen at the same time point.

.28%, and 5.71% in stool, sera, and urine specimens, respectively. The median viral load was 8.01x10 6 GC/g, 2.03x10 6 GC/mL, and 1.36x10 5 GC/mL in stool, sera, and urine, respectivelly. Of all patients, 88.57% had previous comorbidities, and 48.39% of them had detectable SARS-CoV-2 RNA in at least one type of clinical specimen evaluated by this study (stool, sera or urine). A higher viral load was observed in patients with more than two previous comorbidities and that were classified as severe or critical conditions. Samples with the highest viral loads were sequenced and characterized as B.1.1.33 variant. Conclusion: We conclude that SARS-CoV-2 RNA is present in more than one type of clinical specimen during the infection, and that the most critical patients had detectable viral RNA in more than one clinical specimen at the same time point.

Keywords: SARS-CoV-2 RNA; viral shedding, Comorbidity, B.1.1.33 variant; wholegenome sequencing

The COVID-19 pandemic caused by SARS-CoV-2 has, to date, affected over 216,000,000 people worldwide, causing more than 4,490,000 deaths [1] . The SARS-CoV-2 was classified in the family Coronaviridae together with MERS-CoV and SARS-CoV, and it was immediately associated with severe respiratory syndrome [2, 3] .

Clinical manifestations of SARS-CoV-2 infection ranges from asymptomatic presentation to severe, usually associated with pneumonia, coagulopathy, and sepsis, which can culminate with death [4] . Main symptoms or signs of this illness include fever, dyspnea, dry cough, and fatigue. Other reported symptoms are headache, coryza, diarrhea, vomiting, and abdominal pain [4, 5] .

Even though the virus can spread to various sites in the infected body [6] , not many studies have assessed viral loads in clinical specimens, other than respiratory tract samples [6] [7] [8] [9] . Therefore, in the present study, we evaluated the positivity and estimated loads of SARS-CoV-2 in different clinical specimens obtained from patients that were hospitalized in a COVID-19 referral University Hospital in Goiás, Brazil, in association with clinical aspects of the infection and comorbidities presented by the patients.

This is a single-center study conducted at a University Hospital (Hospital das Clínicas-HC/Universidade Federal de Goiás-UFG) located in Goiânia, Goiás, Brazil.

The hospital is a regional reference for COVID-19 patients with underlying chronic conditions and pregnancy. The study was approved by the Ethics Committee of the Hospital das Clínicas/Brazil (CAEE 30804220.2.0000.5078). Positive SARS-CoV-2 RNA molecular test in respiratory samples was considered as inclusion criteria. Therefore, patients that met these criteria treated at the HC/UFG from May to October 2020 were enrolled in the study.

Clinical specimens (stool, urine, and serum) were collected after the participant signed the Informed Consent Form or by their responsible relative. After samples were collected, they were immediately transported to the laboratory (LABCOVID/UFG), under proper packaging, where they were processed and stored at -80°C until further testing.

Stool, urine, and serum samples were processed according to the previously described protocol [8] . After processing, all samples were subjected to RNA extraction using the commercial Qiagen viral RNA mini kit (Qiagen, Hilden, Germany). 

Stool samples with the highest viral loads were sequenced following Midnight protocol as previously described [10] using MinION (Oxford, UK). After sequencing, a quality threshold was set and only sequences with high coverage of at least 200x per base pair, coverage length of >98% of the genome, were accepted. The FASTA file with consensus sequences was uploaded in the Nextclade Beta (https://clades.nextstrain.org/) [11] to identify mutations profile, considering PANGO linages. The alignment was performed by MAFFT v.7.477 [12] and checked manually.

The data set of sequences was constituted of sequences available at the GISAID EpiCoV database [13] , collected between August 1 st and October 31 st 2020, in Goias,

Brazil. The Maximum-likelihood phylogenetic tree was built by IQ-tree web [14] server and visualized in FigTree v1.4.4 [15] .

The patients' clinical profiles were classified according to the National Institute of Health categories [5] : mild, moderate, severe, or critical COVID-19, according to clinical observations and laboratory results, obtained from medical records, and were validated by two infectious diseases physicians of the collaborative group. The statistical analysis of the data was performed by the software R version 4.0.5 [16] , using descriptive analysis.

Following the inclusion criteria, 35 patients were enrolled in the study, and one stool, serum, and urine sample each were obtained from each patient. The SARS-CoV-2 RNA positivity index in stool samples was 51.43% (18/35), with viral loads ranging from 2.0x10 4 GC/g to 6.53x10 10 GC/g, with a median of 8.01x10 6 GC/g. In urine samples, the positivity index was 5.71% (2/35), with viral loads ranging from 2.22x10 4 G.C./mL to 2.49x10 5 G.C./mL, and a median of 1.36x10 5 G.C./mL. In sera, the positivity was 14.28% (5/35) with viral loads ranging from 7.86x10 4 GC/mL to 3.98x10 6 GC/mL (median of 2.03x10 6 GC/mL). As shown in figure   1 , considering the patient's clinical profile, the highest positivity index was detected in The three stool samples with the highest viral loads were submitted to wholegenome sequencing, one collected in August 2020 and two collected in October 2020.

The genomes were deposited in GISAID EPICoV database: EPI_ISL_3769334, EPI_ISL_3769414, EPI_ISL_3769304. According to the Pango Linagens platform, all samples were classified as B.1.1.33 variant. By submitting the nucleotide sequences on the NextClade platform, it was possible to observe the profile of mutations found in these genomes. All mutations found were described in Table 2 . Of notice, the three genomes presented the D614G mutation in the Spike protein (Gene S). The Maximum-likelihood phylogenetic tree was built on data set that contained the three sequences of this study plus 23 sequences deposited in GISAID between August 1 st and October 31 st 2020, collected in Goias, Brazil (Figure 2 ). Following the phylogenetic analyses, the sample collected in August 2020 was similar to others

samples collected in the same period in Goias. Furthermore, the same pattern was observed in the two samples collected in October 2020. 

This study detected the RNA and estimated SARS-CoV-2 loads in different specimens of COVID-19 patients. Although it was not possible to obtain periodically serially collected samples from each patient during infection, the data found here regarding positivity is in accordance with data from other studies that also reported SARS-CoV-2 RNA positivity in stool in more than 50% of the patients [7, 17, 18] , and lower positivity rates in urine samples [9, 17, 19, 20] .

The SARS-CoV-2 RNA detection in other specimens than respiratory tract samples (urine, sera and stool) has been reported by other studies [8, 9, [20] [21] [22] [23] [24] . However a defined period of shedding in those clinical specimens remains unclear. In this study, detection of SARS-CoV-2 RNA was more frequent up to 15 days after the first symptom, considering stool and urine samples. This data, coupled with the concomitant positivity observed in more than one clinical specimen, suggest the importance of exploring clinical specimens other than nasopharyngeal swabs for the diagnosis of COVID-19, since they may remain positive after SARS-CoV-2 RNA is no longer detected in the respiratory tract [25] . Futhermore, the viral sheeding in stool and urine bring on the possibility of new routes of transmission that need to be better elucidated.

The two patients who had detectable SARS-CoV-2 RNA in urine did not present renal dysfunctions during the hospitalization. However, they were classified into severe or critical COVID-19. Few other studies have been able to report viral shedding in urine, and the possible correlation between viral shedding in the urine and disease severity is reinforced by our data [22, 23, 26] . The presence of SARS-CoV-2 RNAemia has been already linked to disease severity [17, 24, 27, 28] , as we could also observed in this study. Also, RNAmia may suggest systemic infection and more studies should be conducted to clarify this matter.

The cohort followed by this study presented, in its majority, previous comorbidities, and more than half of the patients developed severe disease. We could also notice a pattern that the presence of previous comorbidities constitutes a risk factor [27] [28] [29] for the development of severe COVID-19, which has also been shown by other studies. Furthermore, we could observe that the patients included in this study who had more than one comorbidity had the highest viral loads, when compared to those patients that had only one type of comorbidity.

The main symptoms recorded at admission were dyspnea, cough, and fever, similar to what has been already reported [17, 26, 30, 31] . A small number of patients who presented positivity in stool, had also diarrhea, nausea or vomiting. However, shedding in the stool is not directly related to gastrointestinal symptoms, as it has been reported [8, 17, 23, 25] . As for comorbidities as predictors of disease severity, some studies [17, [30] [31] [32] [33] have reported obesity, hypertension, and diabetes mellitus as common comorbidities among COVID-19 hospitalized patients, as also indicated by our results.

The three samples sequenced in this study were characterized asthe same variant, B.1.1.33, predominant in the state of Goiás, according to the sequences in GISAID collected during the same period. Furthermore, this variant was the predominant one circulating in Brazil at the beginning of the pandemic [34, 35] . The D614G mutation was present in all three of our samples. This mutation is characteristic of the B.1.1.33 lineage and has previously been associated with a greater affinity for the ACE-2 receptor and, therefore, higher transmissibility and infectivity [36] .

This study has some limitations, starting with the fact that we could only obtain one sample of each clinical specimen per patient during their hospitalization period.

Also, the number of enrolled patients was low, and therefore it was not possible to acess statistical significance of the data, but still we could observe a trend of higher viral loads in stool and positivity in other specimens such as urine and sera as an indicator of illness severity. Also, that SARS-CoV-2 positivity is found in concomitance in stool and sera. We believe that our findings may contribute to the knowledge of SARS-CoV-2 RNA-positivity and viral loads in distinct clinical specimens and the clinical characteristics of COVID-19. The samples obtained during the study will be included in a biobank for future analysis on the molecular and antigenic characteristics of SARS-CoV-2.

Ethical approval: Ethical approval was obtained from the Research Ethics Committees of Clinical Hospital -Federal University of Goiás/protocol: 30804220.2.0000.5078

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