Oro-faecal transmission of SARS-CoV-2: A systematic review of studies employing viral culture from gastrointestinal and other potential oro-faecal sources and evidence for transmission to humans

Abstract

The extent to which the oro-faecal route contributes to the transmission of SARS-CoV-2 is not established.

We systematically reviewed the evidence on the presence of infectious SARS-CoV-2 in faeces and other gastrointestinal sources by examining studies that used viral culture to investigate the presence of replication-competent virus in these samples. We conducted searches in the WHO COVID-19 Database, LitCovid, medRxiv, and Google Scholar for SARS-CoV-2 using keywords and associated synonyms, with a search date up to 28 November 2023.

We included 13 studies involving 229 COVID-19 subjects – providing 308 faecal or rectal swab SARS-CoV2 reverse transcription-polymerase chain reaction (RT-PCR)-positive samples tested with viral culture. The methods used for viral culture across the studies were heterogeneous. Three studies (two cohorts and one case series) reported observing replication-competent SARS-CoV-2 confirmed by quantitative RT-PCR (qPCR) and whole-genome sequencing, and qPCR including appropriate cycle threshold changes. Overall, six (1.9%) of 308 faecal samples subjected to cell culture showed replication-competent virus. One study found replication-competent samples from one immunocompromised patient. No studies were identified demonstrating direct evidence of oro-faecal transmission to humans.

Our review found a relatively low frequency of replication-competent SARS-CoV-2 in faecal and other gastrointestinal sources. Although it is biologically plausible, more research is needed using standardized cell culture methods, control groups, adequate follow-up, and robust epidemiologic methods, including whether secondary infections occurred, to determine the role of the oro-faecal route in the transmission of SARS-CoV-2.

Introduction

Human coronaviruses have been shown to affect the human gastrointestinal (GI) tract: Middle East respiratory syndrome coronavirus infection (MERS-CoV) was demonstrated to infect the GI tract, with intestinal epithelial cells supporting replication-competent virus [1], and SARS-CoV-1 was associated with GI symptoms and prolonged RNA shedding in faeces as demonstrated by the presence of replication-competent virus in cell cultures of faecal samples from affected patients [2].

It was shown early in 2020 that the SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) receptor as a cell entry receptor to enter ACE2-expressing cells [3]. Since the ACE2 receptor is abundant not only in lung epithelial cells but is also highly expressed on the luminal surface of intestinal epithelial cells [4], a potential route for infection via the GI system is considered biologically plausible. Studies have demonstrated that human GI tract cell lines with a brush border and colon-derived cell lines plus colonic organoids have robust viral growth after inoculation of SARS-CoV-2 and allow for persistent infection within these cell lines [5–7]. There have also been animal models, including non-human primates, which have been inoculated by intragastric intubation, bypassing the respiratory tract. These data demonstrate unequivocal evidence for direct invasion of the GI tract by SARS-CoV-2 with concomitant GI and lung pathology [8–10]. Given the findings that SARS-CoV-2 is able to replicate within human GI tract cell lines and remain infectious on excretion and the establishment of invasive infection in multiple animal models [8–10], it was reasonable to consider whether infectious SARS-CoV-2 could be found in faecal specimens or other sources representing potential oro-faecal sources, creating an oro-faecal transmission risk (also commonly referred to as faecal-oral) [11], which would be important for the application of appropriate infection control measures.

Involvement of the GI tract was one of the reasons for considering that the real rate of paediatric cases may have been higher than that officially reported, as children may present with only GI symptoms and signs. SARS-CoV-2 RNA has been identified in anal/rectal swabs and in stool specimens of COVID-19 patients even after upper respiratory tract virus clearance [12]. In contrast to what has been observed in adult patients, higher proportions of fever, vomiting, and diarrhoea have been recorded on admission in paediatric cases [13]. GI localization in children may represent an alternative site of viral shedding and transmission. For these reasons, sanitation measures in schools included interventions directed against oro-faecal transmission routes [14].

Previously, we systematically reviewed studies reporting on the possibility of transmission via the oro-faecal route and found several studies reporting that SARS-CoV-2 RNA can be shed from the GI tract [15]. However, the shedding of viral RNA does not necessarily equate to viral replication in the GI tract. Since that report, it has become more apparent that understanding the transmission of SARS-CoV-2 depends on studies using high-quality, replicable methods to assess the potential infectivity of samples, along with rigorous epidemiological data examining exposures and outcomes [16]. We therefore set out to identify, appraise, and summarize the evidence on the presence of replication-competent SARS-CoV-2 in human faecal and other source specimens that could represent faecal-oral transmission pathways, and if cases of human oro-faecal transmission of SARS-CoV-2 have been convincingly demonstrated.

Materials and methods

We performed this systematic review following our published protocol [17]. In brief, we searched for studies reporting data from participants with COVID-19 (with or without control groups) from whom biological samples were obtained. We verified whether there was a laboratory confirmation of SARS-CoV-2, and corresponding clinical data, including symptomatology, disease course, treatments, and comorbidities. We focused on studies reporting the results of viral culture from human faecal and other GI samples, coupled with reverse transcription polymerase chain reaction (RT-PCR) testing, to investigate what is known about the presence of replication-competent virus within faecal samples from individuals with COVID-19 and the demonstration of any onward oro-faecal transmission of SARS-CoV-2. To be included, studies must have complied with all the following criteria: included viral culture of faecal/GI samples from SARS-CoV-2-infected persons, and assessed the cytopathic effect and verification techniques of the isolated virus to ensure that the cultured virus was SARS-CoV-2. Studies with data only from RT-PCR testing of faecal samples from SARS-CoV-2-infected persons and/or reporting solely predictive modelling were excluded. In order to help reduce bias due to selective reporting, case studies of one or two cases were excluded as well.

We assessed the risk of bias within five domains, modified and extended from the Quality Assessment of Diagnostic Accuracy Studies criteria [18]. Further details regarding the methods are reported in the Supplementary Materials (Appendix 1).

Results

Figure 1 shows the flow of studies through the screening and inclusion/exclusion processes. The literature search identified 1,496 studies, and 14 were found from other sources, including duplications. After screening the abstracts, 53 independent studies were found eligible. We excluded 40 studies for various reasons (Appendix 2. Studies excluded on full-text screening). Finally, we included 13 studies with a total of 1,625 overall participants, regardless of Ct values [19–31].

Figure 1. Flow diagram showing the process for inclusion/exclusion of studies.

Table 1 shows the main characteristics of the included studies and viral culture information on SARS-CoV-2-positive subjects. More detailed information is presented in Table S1. Eight of the studies were cohort studies, while the remaining five were case series. The 13 studies involved mainly hospitalized patients but also some persons from ambulatory or home settings. Seven studies were conducted in Europe, four in Asia, and two in South America. The study duration ranged from 2 weeks to 15 months.

Table 1. Descriptive features of included studies and results from samples for cell culture

^† See Supplementary Materials for verification methods used.

* Faecal samples, rectal swabs, wastewater or other GI-related samples. GI = gastrointestinal. ¥ = rectal swabs. PY = publication year. NR = not reported. €: A previous protocol stated only done if <30; it was assumed the number of specimens equated to the number of patients.

^§ For Albert et al., the number of samples used for viral culture was estimated from the manuscript, since the exact number was not clear and only undiluted specimens were counted.

A Japanese case series of 13 COVID-19 patients diagnosed between January and March 2020 reported data of viral load from faeces by days and symptoms [19]. One study reported on a series of eight patients with five faecal samples investigating patients in Spanish hospitals and local wastewater samples [20]. One was a prospective multicentre Spanish cohort, which analysed 31 samples obtained from 27 patients within 2 weeks of admission with COVID-19 symptoms between March 2020 and February 2021 [24]. One was a case series from a hospital in France reporting on 46 patients admitted with COVID-19 between March and April 2020, from whom 106 stool samples were taken as part of standard care; viral culture was reported for just one patient with kidney transplantation under immunosuppressive therapy who was admitted for severe diarrhoea [25]. One study from a hospital in Brazil presented a retrospective case series using stored samples collected from four patients between January and July 2020 for the ongoing surveillance of acute gastroenteric disease [26]. A small Korean study presented data collected from five COVID-19 patients and performed a quantitative PCR (qPCR) to assess viral load [27]. Specimens positive with qPCR were subjected to virus isolation in Vero cells and they reported log copy numbers for faecal samples from three patients. Joshi presented a multicentre case series from hospitals in India using stored samples collected between May 2020 and August 2021 [28]. A further study from India described a multicentre cohort study involving an estimated 55 patients (55 samples) attending hospitals who tested positive for SARS-CoV-2 between May 2020 and August 2021 [29]. A study from three clinical centres in Austria reported data on 206 hospitalized children diagnosed between December 2020 and June 2021 [30]. A study from Denmark reported data on 28 patients diagnosed between 23 October 2020, and 17 March 2021 [31]. Another cohort study [21] described data from a slum in Rio de Janeiro, Brazil, collected from May to October 2020, regarding the SARS-CoV-2 test results of 333 rectal swabs from 33 persons, some of whom were symptomatic patients and others were their household contacts. A cluster of 4 epidemiologically linked cases (corresponding to 13 samples) managed in hospitals in Germany early in the pandemic was reported [22]. Finally, one Chinese study analysed data from stool samples of 3 of 3 patients from a cohort of 11 patients diagnosed between 22 January and 4 February 2020 [23].

Across these 13 studies, there were 308 total samples from an estimated 229 hospitalized patients, households, and other settings (exact numbers were not always clearly reported) analysed with viral culture. Among these, three studies reported finding evidence of an isolated virus (Table 1), which were confirmed positive by qPCR and/or genome sequencing of SARS-CoV-2 in the culture supernatant. One of these three studies was a cohort study, in which the positive samples identified via viral culture were obtained from an immunocompromised patient. The samples were obtained at variable time points during the course of illness, and only nine studies reported Ct values from the stool specimens at the time of initial attempts at culture. Of the studies that reported Ct values from the faecal and other samples, all the six samples found to have evidence of culturable virus had Ct values reported as ≤30, which is in the range where a high likelihood of culturable virus may be found [22, 32, 33]. Considering only the cohort studies, there was a 1.2% (3/ 258 samples) positive frequency for those specimens.

Overall, we identified a low-moderate risk of bias across the included studies in four domains but a high risk of bias in one domain, with evident deficits in reporting viral cell culture methods.

Table S2 shows the risk of bias assessment results. The risk of bias was generally considered low for the criteria for the domains of diagnosing a case clearly and appropriately, reporting of patient/population characteristics adequacy, and reporting of methods used to obtain RT-PCR results being replicable and appropriate. Reporting of patient characteristics was absent or unclear in only one (8%) study, and methods for RT-PCR were unclear in four (31%) studies. However, the risk of bias was considered high for clear reporting of viral culture methods, where it was only well reported in five studies. Analysis and appropriate reporting of the results were considered to have low-moderate risk of bias with unclear reporting in two studies.

Discussion

In this systematic review, we included 13 cohort and case series studies that subjected faecal, rectal swabs, or other GI samples (from hospitalized patients and persons in the community with confirmed COVID-19) to viral culture. Among a total of 308 samples subjected to cell culture which were found to be RT-PCR+ for SARS-CoV-2, six (1.9%) were reported as positive from cell culture. The sampling methodology was poorly described in some studies and appeared to be more of a convenience sampling as opposed to a rigorous hypothesis driven type of research protocol, with some exceptions. Sampling stool at late stages of illness with no attention paid to cycle threshold values or quantitative viral load would lead to a large number of samples that would have a high likelihood of being negative for culture given our knowledge about the natural history of COVID-19 [22, 32, 33].

Schedules of sample collection and information on symptoms across the disease course were incompletely reported. Coupled with the limited number of data points available, it was not possible to assess the relationship between the time of symptom onset, faecal sample collection, and results of viral culture. The timing of sample collection is likely to be important [34], and most of the studies reported herein are too few, varied, and unclear in their descriptions to be able to determine whether the included participants were indeed within a potential infectious window. However, the high Cts of many of the faecal and respiratory samples reported suggest that most of the included subjects were unlikely to still be infectious at the time of faecal sample collection. It is also possible that SARS-CoV-2 typically has only a brief survival time with transit through the GI tract or after exiting it; this is as yet unknown.

Nonetheless, in those samples where attention was paid to the timing of faecal sample collection with respect to the course of the illness and/or with low Ct values, higher frequencies of virus isolation were reported. It is possible that these current studies underrepresent the presence of culturable virus in human stool specimens due to poor sampling strategies.

The likelihood of transmission via the oro-faecal route is definitely biologically plausible from evidence on other human and animal coronaviruses, including MERS-CoV and SARS-CoV-1 [1, 2, 35] along with studies early in the pandemic reporting findings that suggested entry of SARS-CoV-2 into GI cells and possible replication with the GI tract, along with many experimental animal model studies [8–10].

In addition, Qian et al. [36] reported a study using electron microscopy to examine tissue obtained from the rectal mucosa of a patient undergoing rectal surgery who subsequently developed COVID-19 symptoms. The authors reported observing virions matching SARS-CoV-2 morphology in sections of the obtained tissue sample. As described above, however, viral morphology alone is not a sufficient indicator of identity [37], and in itself does not show that replication-competent virus is present; this observation could not be regarded as sufficient to indicate a transmission risk from faeces.

In February 2020, Zhang et al. [38] reported testing stool samples by cell culture in Vero cells. The authors reported on the finding of successful cell culture in one sample taken from a laboratory-confirmed COVID-19 case with severe pneumonia 15 days after the onset of symptoms. They reported that viral particles with morphology typical of SARS-CoV-2 were observed under electron microscopy; no further details were reported and again the results of a verified replication-competent virus are uncertain.

To assess a chain of transmission for SARS-CoV-2, it is necessary to have reliable epidemiology, adequate reporting of symptoms and signs, sufficient follow-up, and evidence from human samples of the presence of a replication-competent virus [39]. We previously defined viral culture as encompassing several methods that can uniquely identify the replicating agent as SARS-CoV-2 [40]. Most commonly, this would be a plaque assay combined with an RT-PCR diagnosis or immunological staining, or gene sequencing of viral RNA.

Numerous studies have identified SARS-CoV-2 RNA in faecal samples and wastewater [5], suggesting the possibility of infectious virus remaining in faeces. Although useful for surveillance purposes, RT-PCR positivity in wastewater is of little significance in understanding transmission without viral load data and cultivability [16]. To the best of our knowledge, no review of the results of viral culture on faecal samples exists.

Strengths and limitations

We attempted to collate and synthesize all the relevant data from studies using viral culture of faecal samples to investigate the presence of replication-competent virus, which would suggest the potential for oro-faecal transmission of SARS-CoV-2. We used robust methods to search for the relevant studies, and we accounted for the reporting quality of included studies. However, studies investigating the presence of replication-competent virus within faecal samples have been few and have varied in design and methods. Methodologies have generally been lacking in scientific rigour. Comparison between studies is hindered by the lack of standard approaches for sampling schedules, RT-PCR cut-offs, and viral culture methods. In addition, there are acknowledged methodological difficulties in culturing virus from faecal samples due to the presence of heavy bacterial contamination and the presence of inhibitors in stool or direct cytotoxicity of stool specimens [41].

Viral culture has been reported infrequently in respiratory samples with cycle thresholds above 25 [32, 34], and it is likely to assume similar results within faecal samples with high Ct values. Selecting samples with an RT-PCR cycle count of higher than 25 greatly reduces the likelihood of being able to culture SARS-CoV-2 [32, 38, 42]. In the studies in this review, in general, samples with relatively high RT-PCR cycle counts were subjected to viral culture testing.

Standardized, appropriate methods for viral culture studies are essential to generate reliable evidence that can be compared between studies and allow the combining of results. A weakness found in our review was the reporting of rigorous virologic methods and use of controls. Only two studies [25, 29] reported using negative/uninfected controls for the cell culture experiments; in no study were methods to reduce contamination described; and no study demonstrated an increasing viral load within cell culture supernatants related to a clear timeline and course of the disease.

Implications for research and policy

It is difficult to draw firm conclusions about the likelihood of infectiousness of faecal samples and the associated potential transmission via the oro-faecal route, given the limitations of the available data. Although our review suggests the presence of infectious virus appears to be of low frequency (1.9%) from these samples, it is important to recognize that not accounting for settings whereby Ct values are above a threshold allowing for a high likelihood of negative cultures and with testing in a non-standardized manner, the results are likely an underestimate. In addition, the study by Ribeiro et al. [21] found the presence of replicating SARS-CoV-2 in 19.4% (6/31) of the samples tested, measured by the detection of sgN mRNA, suggesting the degree of infectious virus may be higher than that found by cell culture and this approach deserves further exploration.

Our findings are consistent with the findings in a recent systematic review whereby 8.3% of fomite samples which were RT-PCR+ were found to have infectious SARS-CoV-2 [43]. They also reported that the highest frequency of detection was within 7 days of symptom onset and significantly associated with a Ct < 30. Placing our findings in context, when extrapolated to millions of COVID-19 cases globally, the real potential for an oro-faecal route of transmission in high-risk settings deserves more attention. However, it remains biologically plausible and this route of potential transmission needs further exploration. Given the stability and survivability of SARS-CoV-2 from environmental sources, taken together with our findings demonstrating the presence of infectious virus in 1.9% of faecal and/or rectal samples from humans, a network meta-analysis finding that meal-gathering by individuals had one of the highest risks associated with transmission of SARS-CoV-2 and the recent findings of infectious virus on several types of frozen foods, deli foods, meat, seafood fresh produce, and ice cream for prolonged periods of time from days to weeks, all add impetus to further explore this route of transmission [44, 45].

It is important to address the possibility of oro-faecal transmission to develop effective infection control measures, particularly in high-risk settings, such as hospitals, residential care settings, and nursing homes, where facilities may be shared and where healthcare workers have contact with a number of at-risk individuals. Evidence from studies using rigorously performed viral culture is needed to investigate the presence of replication-competent virus and relate this to cycle thresholds or other surrogates of infectivity to assess the likelihood of transmission of SARS-CoV-2 via the oro-faecal route.

Conclusions

This review concludes that there is evidence of infectious SARS-CoV-2 in faeces, rectal swabs, and other GI specimens in the settings described.

Our review highlights the need for further high-quality research, ideally from prospective cohort studies, using appropriate timing of specimen capture and using standardized high-quality methods for viral culture with appropriate negative controls, coupled with robust evidence on possible transmission events to ascertain the likelihood of transmission of SARS-CoV-2 via the oro-faecal route. Animal models and human challenge studies are both viable options to help address this research gap.