Excretion of SARS-CoV-2 RNA in feces has no prognostic benefit in the outcome of COVID-19: A clinical and immunological study

This study explores the correlation between immunological and clinical characteristics in coronavirus disease 2019 (COVID-19) patients with detectable severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in feces, analyzing data from 251 patients admitted to Mostar University Clinical Hospital (UCH) from December 2021 to January 2022. Methods involved reverse transcription quantitative polymerase chain reaction (RT-qPCR) from nasopharyngeal (NP) swabs and feces, alongside serological tests for anti-SARS-CoV-2 spike IgGs. Demographic and clinical data were collected through questionnaires and medical records. The data analyses were performed using SPSS statistical software. Death occurred in 53 patients (21.1%, P < 0.001), mostly in the elderly (47/53, 88.7%, P ═ 0.001) and immunocompromised (19/53, 35.8%, P ═ 0.05), particularly those developing acute respiratory insufficiency (ARI) (46/53, 86.8%, P ═ 0.004), and severe/critical disease (46/53, 86.8%, P ═ 0.002). Among the patients with positive anti-SARS-CoV-2 IgG antibodies (86/251, 34.3%, P < 0.001), 41 (47.7%) were vaccinated and 45 (52.3%) unvaccinated (P ═ 0.666), showing no significant differences in clinical outcomes or mortality. Unvaccinated patients with a negative antibody titer had a higher incidence of ARI (96/123, 78%, P ═ 0.029) and intensive care unit (ICU) admission (22/123, 17.9%, P ═ 0.026), than those with a positive antibody titer. Forty-seven (62.7%) patients, out of the 75 hospitalized who provided a feces sample, were positive for SARS-CoV-2 RNA (P ═ 0.028), without statistical differences between fecal SARS-CoV-2 positive and negative groups regarding vaccination status (15/47, 31.9%, P ═ 0.493), antibody status (18/47, 38.3%, P ═ 0.628), or death outcome (5/47, 10.6%, P ═ 0.706). In conclusion, unvaccinated hospitalized patients with a severe COVID-19 presentation and a negative anti-spike SARS-CoV-2 IgG titer had adverse outcomes more frequently. This suggests cautious consideration for the diagnostic use of fecal samples compared to NP swabs.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID- 19), remains a threat to global health, with various genetic lineages emerging and circulating globally [1].The gold standard for the diagnosis of COVID-19 is reverse transcription quantitative polymerase chain reaction (RT-qPCR) which determines the presence of genomic material of SARS-CoV-2 in samples from different origins.The viral RNA is commonly detected in nasopharyngeal (NP) swabs, but it can also be detected in sputum, urine, lung, serum, plasma, and feces samples [2,3].The value of the cycle threshold (Ct value) determined by RT-qPCR is inversely proportional to viral load [4].A prior study showed that the viral load from NP swabs was the highest at the beginning of the symptoms or a few days after, followed by a significant decline two weeks after the onset of symptoms [5], whereas viral loads in feces samples usually peak later on, indicating later viral clearance in feces samples [6].Higher viral load in NP swabs positively correlates to virus infectivity and COVID-19 severity, while SARS-CoV-2 RNA shedding duration and feces viral load dynamics require further research, including the virus transmissibility and clinical significance [5,6].
In addition to RT-qPCR testing, serological tests are used to identify currently or previously SARS-CoV-2-infected individuals, based on the detection of specific antibodies (IgM, IgG, IgA immunoglobulins) to SARS-CoV-2 antigens [7].The IgG antibodies have affinity to its antigen and a high efficiency for pathogen neutralization resulting in mostly systemic protection against COVID-19 [8].They appear later in the immune response and are associated with long-term immunity following infection or vaccination [9].Consequently, the specific antibody titer is an accurate method to detect neutralizing antibodies from recently resolved or past infections.Antibody titers are decreasing following infection resolution [10].The neutralizing antibodies are stalling infection if antibody titers are at an optimal level.Accordingly, one of the main goals of vaccination is the induction of antibody titers at a similar or higher level as in convalescent individuals.In addition, vaccination can expand and improve antibody clonal lineages induced by previous infection, thus preventing COVID-19 severity against novel virus strains [11].
Patients in our hospital were tested by NP swab, although there is evidence that SARS-CoV-2 RNA can be detected in samples from different origins [12].Members of the coronavirus family, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), are known to be excreted in the feces of infected patients, suggesting a potential for fecal-oral transmission [13].Similar to these viruses, SARS-CoV-2 was also found in the feces of a significant number of patients with COVID-19 [14].Recent studies have shown no correlation between the excretion of SARS-CoV-2 RNA in the feces and the outcome of COVID-19 [15,16].
This study aimed to determine the association of fecal virus excretion with immunological and clinical characteristics, as well as clinical outcomes of COVID-19 patients.

Study design
A total of 251 SARS-CoV-2-positive participants admitted to the COVID department of the University Clinical Hospital (UCH) Mostar between December 2021 and January 2022 were enrolled in the study.The sociodemographic and clinical data were collected from questionnaires and medical records.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
NP swabs were collected from patients at hospitalization between the fifth and seventh day of symptom onset and were routinely analyzed using RT-qPCR.The criteria for SARS-CoV-2/COVID-19 positive tests were Ct values ≤ 38 for both the RNA-dependent RNA polymerase (RdRp) and nucleocapsid protein (N) genes, according to the manufacturer's instructions.Based on PCR test results and clinical status, patients were hospitalized and consequently included in our study.
Four SARS-CoV-2 genes were analyzed after the swabs underwent extra processing: RdRp, N, non-structural protein 14 (nsp14), and envelope protein (E).Using an RNA extraction kit and reverse transcription, RNA was transcribed into complementary DNA (cDNA) using a one-step RT-qPCR assay (qScript XLT One-Step RT-qPCR ToughMix, Quanta Bio), following the manufacturer's protocol.Eurofins Genomics (Vienna, Austria) produced and supplied the primers and probes.The reaction was carried out by a magnetic induction cycler (MIC PCR, Bio Molecular Systems) and the results were interpreted as previously reported [17].
Feces samples were collected within the first three days of hospitalization, and analyzed by RT-qPCR after short-term storage at +4 °C.30 mg of feces was suspended in 600 μL of lysis buffer (RLys buffer) for every sample to obtain 5% w/v suspension.Feces suspensions were then homogenized by vortexing for 60 s, followed by centrifugation for 2 min at 15,000 rotations per minute.Supernatants were then used as starting points for RNA extraction, which was performed by using an RNA extraction kit for tissues (EXTRACTME total RNA kit, BLIRT, S.A.), according to the manufacturer's protocols.Obtained RNA eluates were then analyzed for four genes (RdRP, E, N, and nsp14) by the same protocol that was previously described for NP swabs.Confirmed-positive samples were used as positive controls for all RT-qPCR reactions, while no-template controls served as negative controls.

Serological testing
Serum samples, after routine biochemical processing, were stored at -20 °C until analysis.The serum concentration of SARS-CoV-2 IgGs was quantitatively determined using the SARS-CoV-2 IgG reagent kit (REF-11207376, Siemens Healthcare Diagnostic Inc., USA) on the ADVIA Centaur XPT analyzer (Siemens Healthcare Diagnostics Inc., USA), as described previously [18].

Ethical statement
The study was conducted in accordance with the ethical standards stated in the 1964 Declaration of Helsinki and its subsequent amendments.Ethical approval was acquired from the Ethical Committee at UCH Mostar, number 1035/21.

Statistical analysis
Descriptive and analytical statistical methods were used in data processing.Data were presented as mean ± SD or median and number (percentage) for categorical variables.A chi-squared or Fisher's exact test was used for the analysis of categorical data.Pearson (r) and Spearman (Rho) tests were used for the correlation of continuous and discrete data, respectively.We first converted the continuous variable into categorical variables using the median and quartile analysis, for the correlation test, and then the Spearman test with a two-tailed test of significance was performed for the correlation.All statistical analyses were performed using SPSS version 23.0 software (SPSS Inc., Chicago, IL, USA).

The influence of the viral load on the clinical characteristics and outcomes of COVID-19
To analyze the influence of the viral load on the clinical manifestation of the disease, we analyzed the epidemiological and clinical parameters of patients stratified by the Ct values.Our results showed that patients with lower viral load presented by Ct value over 30 (51/100, 51%, P < 0.001) had higher levels of virus-specific antibodies (Table 3).Immunocompromised patients had a higher viral load with 30.4% of patients with Ct ≤ 30 (42/138, P = 0.034).Among patients with Ct ≤ 30, 38 (27.5%) patients died, compared to the group of patients with Ct > 30 (14/100, 14.0%, P = 0.017).Other clinical and epidemiological parameters did not statistically differ between patients with lower and higher viral load (Table 3).

The influence of previous infection on the clinical characteristics and outcomes of COVID-19
To examine the influence of previous infection, we stratified unvaccinated patients according to the SARS-CoV-2 seropositivity.Unvaccinated seronegative patients had a higher incidence of ARI (96/123, 78%; P = 0.029) and ICU admission (22/123, 17.9%, P = 0.026).No statistically significant difference  was observed when other clinical and epidemiological parameters were compared between seropositive and seronegative patients (Table 4).

The role of vaccination in acquiring antiviral protection
Our next aim was to determine the impact of positive antibody status on the clinical characteristics and outcomes of COVID-19 in vaccinated, as well as unvaccinated patients.Among the patients with positive IgGs, 41 (47.7%) were vaccinated and 45 (52.3%) were unvaccinated (P = 0.666), with no statistical difference regarding clinical manifestations or mortality.An observed trend indicated that unvaccinated patients exhibited an increased propensity for developing pneumonia and more severe disease manifestations, although this difference did not reach statistical significance (P = 0.107) (Table 5).
Seropositive patients were stratified according to the antibody titers into high/low groups.A moderate positive correlation between a high antibody titer and higher Ct values was observed (P = 0.008), while other variables did not show a correlation with the antibody titer (Table S3).

Discussion
We included 251 patients with manifested COVID-19 requiring hospital treatment in this study.The results indicated that SARS-CoV-2 IgGs were more frequently present in patients with lower viral load and in immunocompetent patients.Unvaccinated patients without previous SARS-CoV-2 infection had a higher incidence of ARI as well as ICU admission, while other clinical and epidemiological parameters, as well as mortality rate, did not show a statistically significant difference between patients with positive or negative antibody titers in this group.Patients with positive antibody titers were similarly distributed between those who had been vaccinated and those who had resolved COVID-19, with no statistical difference regarding clinical manifestations or death outcomes.Although the difference was not statistically significant, unvaccinated patients with positive antibody titers were more likely to develop pneumonia and more severe disease compared to unvaccinated patients with negative antibody titers.Finally, there were no differences in age, gender, vaccination status, comorbidities, and death outcome depending on SARS-CoV-2 RNA presence in feces samples.
In line with previous findings, our study further confirms that older age is the primary risk factor for mortality, followed by ICU admission, immunocompromised status, and disease severity [19].
We used Ct values as an indicator of a viral load in each tested sample in this study.The Ct value could indicate potential infectivity [20].We found higher levels of SARS-CoV-2-specific antibodies, indicating the presence of previously acquired neutralizing anti-spike SARS-CoV-2 antibodies as a result of vaccination or resolved infections in patients with lower viral loads [10].Furthermore, immunocompromised patients exhibited a higher viral load compared to their immunocompetent counterparts, likely due to a diminished immune response to the infection [21].
Taking into account the time of our research conduction, we assume that the dominant variant in the study was the Omicron variant of SARS-CoV-2 [22,23].It is well established that the Omicron variant has developed several defense mechanisms against the immunity acquired by a previous infection [24,25] or vaccination, compared to the previous SARS-CoV-2 variants [26].Unvaccinated patients with a negative antibody status upon hospital admission demonstrated an increased likelihood of developing ARI and requiring ICU admission.These findings underscore the critical role of vaccination, particularly for individuals with no prior exposure to SARS-CoV-2.Additionally, the results revealed no significant difference in patients with a positive titer of specific IgGs, regardless of vaccination status.
Within the initial weeks following vaccination, patients who receive the vaccine exhibit milder clinical manifestations compared to those unvaccinated when contracting COVID-19 [27,28].Similarly, prior vaccination provides benefits for hospitalized patients [28][29][30].Notably, the patients included in this study were vaccinated with only two doses of the COVID vaccine, although three doses of the vaccine were recommended by the WHO during the study period [31].Furthermore, most of our patients received a second dose of the vaccine five or more months before infection occurred.In addition, the lack of the third dose administration among patients in this study could also affect the effectiveness of vaccination against the dominant variants of SARS-CoV-2 during the study period.Also, a possible explanation for the lack of greater advantage of the COVID-19 vaccine observed in this study could be that mostly the elderly with comorbidities and immunocompromised people were enrolled, rather than immunocompetent younger people [17,32,33].
Our recent study demonstrated that previous contact with SARS-CoV-2, irrespective of vaccination, has a protective effect during subsequent infection, indicating that immunity acquired by previous infection might play an essential role in the prevention of symptomatic disease, especially the appearance of high fever, and loss of taste and smell [17].This could be explained by the stronger mucosal immunity that arises as a result of a natural infection.Available COVID-19 vaccines are most often administered intramuscularly and primarily elicit IgG antibody response, with a weak response of mucosal IgA antibodies, which have a superior antiviral effect at the entry points of infection [34][35][36].Therefore, hybrid immunity gained by vaccination and infection creates a better immune defense, with circulating IgG antibodies and mucosal IgA antibodies [37].
Previous research has shown that 40%-60% of COVID-19 patients have a positive feces sample for SARS-CoV-2.Furthermore, SARS-CoV-2 RNA could be detected in feces even after the negativization of NP swabs [16,38].Also, the presence of viral particles in the tissue of the gastrointestinal tract suggests acute viral replication [39] and potential for fecal-oral transmission of the virus [40].
One of the questions of this research was the correlation between SARS-CoV-2 RNA-positive feces samples and clinical manifestations of COVID-19.There is conflicting evidence on the association between a positive feces PCR test for SARS-CoV-2 RNA and the severity of the patient's clinical manifestation [41,42], but several previous studies have shown that there is no significant correlation between the detection of the virus in the feces and the severity of clinical manifestations [38,43].However, the presence of viral particles in the feces does not necessarily mean that the virus can be transmitted via the feces.Our results are in line with previous research, showing no difference in the clinical manifestations, course of the disease, or outcome in patients who tested positive for SARS-CoV-2 RNA in feces, compared to patients with negative SARS-CoV-2 RNA results in feces samples.
This study has several limitations.The study only included hospitalized patients, and as such, the results cannot be extrapolated to the overall population infected by SARS-CoV-2.All vaccinated patients received only two doses of vaccine in the study period which might affect the vaccine effectiveness against the Omicron variant.Furthermore, the Omicron variant was presumed rather than identified in each study participant.However, the study period overlaps with the peak of the COVID-19 prevalence in Bosnia and Herzegovina, as well as the Omicron predominance during the pandemic.Some of the observed trends in our results were not statistically significant due to the small number of participants.We were not able to detect previously recovered asymptomatic COVID-19 among investigated patients based on the anamnestic records and SARS-CoV-2 IgG detection method used in our study.Since the detection of SARS-CoV-2 IgGs in our study did not present neutralizing capacity, further study to clarify this issue should be conducted.

Conclusion
In this study, we showed that hospitalized unvaccinated patients without previous infection had a higher incidence of ARI and ICU admission.Despite the significant presence of comorbidities among vaccinated patients, ARI and pneumonia occurred to a lower extent compared to unvaccinated patients.Given that these were hospitalized patients, the impact of vaccination and the presence of antibodies did not affect the clinical outcome of COVID-19.Fecal excretion of SARS-CoV-2 RNA had no impact on the clinical outcome of COVID-19.Moreover, in one-third of patients with SARS-CoV-2-positive NP swabs, SARS-CoV-2 RNA was not detected in the feces.Thus, caution should be taken when using feces as a diagnostic sample for detecting SARS-CoV-2 infection.