Impact of Prolonged SARS-CoV-2 Viral Shedding on COVID-19 Disease Outcome and Viral Dynamics

Abstract

This article aimed to review the current literature on the impact of continuous shedding of the COVID-19 virus in infected patients in relation to disease outcome variables and viral dynamics. Electronic databases PubMed, Google Scholar, and MedlinePlus were searched using relevant keywords, restricting the selection to thirty-two peer-reviewed articles and four gray literatures from the WHO websites. Findings from this study showed that several variables such as sex, age, immune status, treatments, and vaccines were found to affect the outcomes associated with the COVID-19 virus shedding. These findings highlight the need for further research using longitudinal whole-genome sequencing of the virus and its variants to increase the understanding.

Introduction

The World Health Organization (WHO) suggests that close contact interactions are the main factor in viral transmission. Close contact situations may result in any or all the following routes, such as short and long-range airborne transmission, droplet transmission, and the transfer to mucosal surfaces after contacting infected surfaces [1]. The evolution of a virus is due to the process of changing or mutations and selection of successive variants that can result in a variant that adapts better to its environment compared with the original virus, whereby it can spread more easily due to altered transmission or cause more severe disease [2]. The reproductive number denoted by the letter R subscripted by a point in time, thus making the start of the pandemic R₀ is a value that estimates the likelihood of a viral spread by an individual. A number lower than 1 indicates the viral spread is in a state of stagnation, while a number greater than 1 indicates an active viral spread [3]. The evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted to a pattern of genetic diversity in the genome across geographical regions in the human population which can determine the transmissibility of the virus and the severity of coronavirus disease 2019 (COVID-19) disease (Fig. 1); the driving forces in the viral evolution could be due to the genetic variations, adaptation to new animal hosts, and immune differences in the human populations [4, 5]

Fig. 1.

Genomic epidemiology of SARS-CoV-2 variants with subsampling focused globally, generated from data and diagrams created by Nextstrain.org

There are many studies with a focus on the shedding of SARS-CoV-2 with the Delta variant as the most concerning as its duration of shedding is much longer than other variants. The Delta variant can shed easily for longer than 1-month. This is even more important for asymptomatic individuals as it would be possible for them to unwittingly spread the infection to more people for a much longer duration as compared to Omicron [6–8]. For the immunocompromised, it is not uncommon for their infection to actively shed for a prolonged period ranging from 73 to 250 days [6, 9]. Several factors may also categorize individuals as immunocompromised, such as older age, HIV/AIDS, chemotherapy, radiotherapy, cancer, or long-term administration of corticosteroids. It is possible to see prolonged shedding lasting for months, confirmed by reverse transcription polymerase chain reaction (RT-PCR) [10, 11]. Omicron, which is currently causing outbreaks around the world, is the most altered version to have emerged to date [12]. It has mutations similar to those noted in earlier variants of concern associated with partial resistance to vaccine-induced immunity and increased transmissibility [12]. The Omicron XBB.1.5 variant a sub lineage of XBB, which is a recombinant of two BA.2 sub lineages have been reported from 38 countries between 22 October 2022 and 11 January 2023 with 5 288 sequences. Majority of these sequences are reported from the United States of America (82.2%), the United Kingdom (8.1%), and Denmark (2.2%) [13].

The sudden and rapid global spread of COVID-19 and its variants has resulted in a multitargeted and multifaceted approach to its virology, giving rise to not only answers but more questions [4, 9, 14]. A host's immune system or the pathogen's capacity to adapt to its surroundings play a role in the spread of infection; in addition, it may not be a spread but a reinfection [6, 7, 9, 15]. This paper offers a focused perspective on the impact of prolonged, persistent shedding of the SARS-CoV-2 virus on the COVID-19 disease. It discusses how contributing factors may influence overall patient outcomes, the viral dynamics of various strains in relation to prolonged shedding, effect on the immune responses, and the impact antiviral treatment and vaccines may have during viral replication.

Methodology

The electronic databases PubMed, Google Scholar, and MedLinePlus were searched for literature reviews. The publications were chosen if they contained key phrases like ‘viral evolution’, ‘viral adaptation’, ‘viral dynamics’ ‘sex, immune response and viral infection’, and COVID-19-causing extended ribonucleic acid (RNA) viral shedding of SARS-CoV-2. The articles were then examined for relevance to the subject and included. For the study, the selection was restricted to thirty-two peer-reviewed articles and four gray literatures from the WHO websites.

SARS-CoV-2 Dynamics: Clinical Outcomes and Persistent Viral Shedding

According to the WHO, as of November 2022, there have been over 631,935,687 confirmed cases of COVID-19 which have included 6,588,850 deaths [16]. Many of the infected patients have presented with mild to moderate symptoms [17]. The commonly reported symptoms include fever, cough, weakness, shortness of breath, malaise, myalgia, and taste dysfunction [18, 19]. Yet, in some incidents, patients have developed severe outcomes leading to the use of mechanical ventilation, hospitalization, and even death [20]. Since the start of the SARS-CoV-2 outbreak in 2019, nucleic acid amplification tests (NAATs), particularly real-time RT-PCR assays have been the operational standard by which detection, diagnosis, and confirmation of the virus have been made [21, 22]. Studying persistent viral RNA shedding has helped in the understanding of long-term effects and resolution of many viral infections such as avian influenza, Zika, and Ebola [23]. Viral shedding is induced by the immune status of the host, the source of the initial infection, the severity of the disease, and the viral load [23].

Regarding SARS-CoV-2, the continuous viral RNA shedding in the upper respiratory tract is defined as a positive RT-PCR test after 14 days from the onset of symptoms or the first positive test [21, 24]. Unremitting, prolonged shedding of SARS-CoV-2 is correlated with the number of critical cases leading to hospitalized patients with the infection [8, 10, 25, 26]. Overwhelmingly, it has been found that an association exists between persistent viral shedding and increased 6-month mortality in these individuals [23]. Many studies have been conducted to understand factors impacting the delayed clearance of viral RNA. One study investigated 109 participants, between the ages of 3–90 years, with 52% being male [24]. Within this sample population, 92% had neurologic, 90% had upper respiratory, 87% had lower respiratory, 84% had constitutional, and 64% had gastrointestinal symptoms from the infection. After rRT-PCR-positive specimens were collected and analyzed, it was found that 51/109 (47%) participants had indeterminate viral shedding, 51/58 (88%) had persistent viral shedding, and 7/58 (12%) did not show persistent RNA shedding. From the start of their symptoms when they tested positive until their negative rRT-PCR test result, the probability of viral resolution at 25 days after symptom onset was approximately 63% in the sampled group. In addition, those participants < 18 years old had fewer persistent cases and an earlier resolution of viral RNA shedding in comparison to those in older age groups (≥ 50) which took longer to shed and were more persistent (Fig. 2) [24]. In another study with 703 individuals, intergroup comparisons revealed a long viral shedding (> 28 days) with higher frequency among females, older age group greater than 60 years, longer interval from symptom onset to hospital admission and longer length of hospitalization (Figs. 3, 4) [10].

Fig. 2.

Depicts the SARS-CoV-2 RNA shedding resolution over time stratified by age groups among participants in the transmission investigation study (March to May 2020) conducted by Owusu et al. (2021) [24]. Note. Persistent: Positive test result on rRT-PCR after 14 days of start of symptoms or first rRT-PCR positive test result. Indeterminant: Participants who withdrew from the study but were still positive at the end of research and observed less than 14 days from the start of symptoms, are those who could not be categorized due to the testing interval

Fig. 3.

Long viral shedding (> 28 days) by age and sex Long et al. (2021) [10]

Fig. 4.

Long viral shedding (> 28 days) by interval from symptom onset to hospital admission and length of hospitalization Long et al. (2021) [10]

Discussion

Sex, Age, and Immunity: Impact on COVID-19 Outcomes

It is proposed that numerous variables such as sex, age, immunity, treatments, and vaccines may alter the outcome of the COVID-19 infection. For this discussion, "sex" refers to biological differences in development between males and females because of the sex chromosomes of an individual [27]. In addition, age and sex significantly influence immune responses to infection, as studies have shown that severe COVID-19 transpires more in males than in females [27]. Furthermore, the differentiation of gonadal tissue into testes and ovaries in XY and XX individuals contributes to the difference in the secretions of the sex steroids [27]. The immune cells also carry cytoplasmic receptors for sex hormones allowing for gene expression and regulation of the immune response, causing biological females to have a better immune response and viral clearance than biological males [27]. Data shows that male COVID-19 patients are admitted to intensive care units (ICUs) more often than their female counterparts and are 30% more likely to result in mortality than females [27]. Clinical and epidemiological studies continue to show differences in male and female immunity, as it occurs with many types of viral infections, autoimmune diseases, and inflammatory diseases, and not just SARS-CoV-2 [27].

Sex Disparities in Immune Responses to Viral Infections: A TLR Perspective

Studies have ascertained that females are less susceptible to viral infections than males due to a higher expression of toll-like receptors (TLRs) and hypermediated T-cell immune responses [10, 28]. Various microbes and pathogens bind to TLRs; these receptors aid the host cell in recognizing itself and mounting an appropriate immune response against microbes and pathogens [28]. Females display an increased expression of TLR3, TLR7, and TLR9, which recognize viral deoxyribonucleic acid (DNA) and RNA. In contrast, males show an increase in TLR2, and TLR4, which bind to the bacterial cell wall [29]. The results of a study conducted by Torcia et al. [28], which evaluated mononuclear cells after being stimulated by various TLRs and pathogens, indicated that males produced lower levels of interferon-alpha (IFN-α) and increased amounts of interleukin 10 (IL10). IL10 is a cytokine that promotes immunosuppression and could be related to plasma levels of sex hormones in both groups [28], as data showed that following a viral infection, the mononuclear cells from males yielded more IL10 than females [28]. Estrogen impairs the negative selection of auto-reactive B-cells with a high affinity for self, modulates B-cell activity, and initiates a T-helper 2 (Th2) response [29]. This increased pro-inflammatory state, along with an increased number of antibodies, could be a reason for females experiencing significant side effects such as an enhanced post-infection or post-vaccination immune reaction [29]. In contrast, testosterone promotes immunosuppression by initiating a T-helper 1 (Th1) response, activating a cluster of differentiation 8 (CD8) cells, and creating an anti-inflammatory state [29], indicating that males may have a higher affinity for a Th1 response [10].

Aging and SARS-CoV-2: Prolonged RNA Shedding and Immune Responses

Older ages tend to be associated with prolonged SARS-CoV-2 RNA shedding [30, 31]. Furthermore, as other studies have indicated, older demographics have been directly linked to a higher likelihood of developing acute respiratory distress syndrome (ARDS) and a higher mortality risk [31]. These complications are mainly due to a weaker immune response, affecting the effectiveness of clearing out viral infections and prolonging viral shedding [31]. Aging can also lead to inequality in the regulation of Th1 and Th2 responses, as older patients are more likely to have a Th2-type response, as seen in a study by Long et al. in Wuhan, China [10]. The study, which had a median patient age of 63 years and ranged from 10 to 92 years, saw 2726 respiratory samples obtained from 703 COVID-19 patients with symptoms [10, 24]. Data from the study, of which 47.2% were male, indicated that viral loads are the highest during the initial stages of infection. Subsequently, they declined over time, with a significant lingering of the virus in the elderly and female populations [10].

Antibody Dynamics in COVID-19 Patients: Insights from Diagnostic Testing

To evaluate the humoral immune response, a study done by Song et al. [32] used an RT-PCR to detect the viral load and antibodies against SARS-CoV-2 in 4 patients who tested positive on RT-PCR after recovering from COVID-19 [32]. The RT-PCR is an indirect enzyme-linked immunosorbent assay based on recombinant nucleocapsid protein that detects IgG and IgM antibodies [32]. IgM and IgG appear between week 3 and week 4 after the onset of symptoms, with IgM levels decreasing by week 5 and IgG levels persisting past week 7 [32]. The study found that the IgM and IgG antibodies peaked approximately 1-month after the onset of symptoms in all 4 patients, followed by a plateauing of IgG titers and a decrease in IgM titers, regardless of RT-PCR results [32]. Interestingly, while the IgM and IgG antibodies were still detectable in patients, the IgM and IgG levels did not indicate any rise in titers after the 1-month despite post-negative positive RT-PCR results [32]. These findings imply that given the patient history, redetecting the virus at this stage is more likely caused by remnants of residual viral RNA particles as opposed to re-infection or viral reactivation [32]. Unfortunately, one significant downside to the RT-PCR test is that it cannot differentiate infectious viral pathogens from non-infectious remnants of viral RNA [32]. Hence the need for using longitudinal whole-genome sequencing of the virus and its variants to increase the understanding.

The time to negativity was the number of days between onset and the date SARS-CoV-2 RNA became negative [10]. Long et al. used the 2726 nasopharyngeal samples obtained from 703 COVID-19-positive patients to determine if there were any changes to the viral loads in patients after being admitted to the hospital throughout the three stages of an outbreak [10]. Throughout the outbreak period, the study targeted two SARS-CoV-2 genes, the nucleocapsid protein (the N gene) and open reading frame 1ab or ORF1ab (the ORF gene), used for viral load detection [10]. Samples positive for both gene were labeled "positive." In addition, the study would utilize a seroFlash SARS-CoV-2 IgG/IgM enzyme-linked immunoassay (ELISA) fast kit and a recombinant SARS-CoV-2 antigen (S protein) to measure serum samples for SARS-CoV-2-specific IgM and IgG antibodies [10]. This result indicated a correlation between low levels of specific IgM antibodies and high viral loads, highlighting the patients' inadequate humoral response. Moderate viral shedding was still detected 3 weeks after the onset of symptoms. In comparison, prolonged viral shedding has been recorded in other studies anywhere from 20 to 30 days [10]. Fecal shedding of SARS-CoV-2 RNA was commonly detected in COVID-19 patients and exceeded half among those with gastrointestinal (GI) symptoms [30]. In some cases, fecal RNA was seen for 3 weeks after onset, approximately a week longer than the respiratory tract [30]. The median time to obtain a negative nasopharyngeal sample was 26 days (day 53 being the 95th percentile), despite some studies showing it could take longer depending on the severity [10]. Based on these findings, detecting viral RNA at day 60 is a low probability (> 95th percentile) [10].

Chronic SARS-CoV-2 Infections: Origins, Variants, and Transmission Dynamics

According to viral sequencing data, the source of origin is thought to be chronically infected people [12]. Further investigation has shown that SARS-CoV-2 may develop the ability to escape antibodies through two deletions in the N-terminal domain and a mutation in the spike (S) protein when subjected to a potent immunological response [12]. While RNA shedding can last for weeks after the resolution of symptoms, current data supports that chronic SARS-CoV-2 infections can last for months [33]. In contrast to "long COVID," a condition where symptoms continue despite the body clearing out the virus, chronic SARS-CoV-2 infection is characterized by having a prolonged period of active viral replication as well as viral RNA shedding [33]. Immunocompromised patients usually have difficulty clearing viral infections from the body; this defect creates the opportunity for these patients to develop chronic SARS-CoV-2 infections [33]. It is also currently hypothesized that variants of concern may emerge from chronic SARS-CoV-2 infections. This theory is also supported as the mutations found in the S protein, the N-terminal domain, and the receptor-binding domain of chronic SARS-CoV-2 are identified as lineage-defining mutations in variants of concern, such as the Omicron variant [33]. The study revealed that evolutionary mutational patterns of viruses from chronic infections are reminiscent of that in a variant of concerns with a few exemptions and that viral rebound could be predictive of antibody evasion mutation. Partial penetration of antibodies to a particular niche such as an infected organ or within it could lead to viral rebound coupled with antibody evasion; thus, viral clearance is prevented with an accompanying selection of antibody evasion mutations suggesting that a weak immune response is the driver of adaptive evolution in the virus. However, in general, viruses emerging in chronic COVID-19, lack the potential for onward transmission [33].

Within host Sars-Cov-2 variants and transmission using robust methods was studied by Lythgoe et al. 2021; a consistent pattern of low within-host diversity, purifying selection, and narrow transmission bottlenecks were reported. Furthermore, during early infection, when viral loads are high, within-host emergence of vaccine and therapeutic escape mutations is presumed to be rare [34]. Recent data indicate that reducing the amount of viral load and inhibiting or reducing viral shedding time are ways to prevent transmission from person-to-person [35]. In a cohort study of symptomatic and asymptomatic patients with SARS-CoV-2 infection who were isolated in a community treatment center in Cheonan, Republic of Korea, the diagnostic Ct values in asymptomatic patients were similar to those in symptomatic patients with prolonged viral shedding; thus, indicating that asymptomatic patients with SARS-CoV-2 may be a key factor in community viral transmission [36].

Antiviral Efficacy in COVID-19 Treatment: Assessing Viral Shedding

Prolonged viral shedding may have a useful implication for the development of treatments and vaccines for COVID-19. The duration of viral shedding is a benchmark for assessing the efficacy of drugs that could treat Covid-19, as seen in clinical trials evaluating treatments with lopinavir, ritonavir, nirmatrelvir, and meplazumab [35, 37]. Those antivirals that indicate a low efficacy for inhibiting viral shedding may prove to be hindering, as some studies have shown that antivirals with low efficacy may result in a paradoxical longer shedding time [35]. While using antivirals prophylactically or for tracing contacts based on exposure before the onset of symptoms may decrease viral shedding time, antiviral therapy starting after the onset of symptoms may not be effective in shortening viral shedding time. There is a need to align treatment strategy for Covid-19 with prolonged viral shedding in persitent infection [38].

Comprehensive Strategies Against COVID-19: An Approach to Addressing Viral Infections and Enhancing Pandemic Mitigation

To develop a comprehensive and adaptive strategy against COVID-19, further research is imperative. This research should focus on understanding the impact of multiple viral infections on the severity of the disease. A multifaceted approach is recommended, encompassing the development and deployment of next-generation vaccines, continuous surveillance of virus variants, effective transmission control measures, global vaccination initiatives, exploration of treatment approaches, and sustained adherence to safety measures [39–41]. This holistic approach aims to address the evolving nature of the pandemic and enhance the capacity to mitigate its impact.

It is crucial to acknowledge the limitations of this review. The existing literature is limited in certain patient populations, features variable follow-up times, and lacks comprehensive data on the prevalence of pre-existing clinical conditions (comorbidities). Furthermore, additional studies are warranted, particularly focusing on the use of antivirals prophylactically before symptom onset, to assess their potential in reducing viral shedding within specific populations. Recognizing these limitations underscores the necessity for more extensive and targeted research in this field, fostering a deeper understanding of the dynamics surrounding persistent viral shedding and informing effective public health interventions to curb transmission.

Conclusion

Persistent viral RNA shedding is defined as a positive rRT-PCR test ≥ 14 days after symptom onset or the first positive test. While RNA shedding can last for weeks after the resolution of symptoms, current data supports that chronic SARS-CoV-2 infections can last for months. Prolonged shedding of SARS-CoV-2 is correlated with the number of critical cases leading to hospitalized patients with worsening outcomes. Of note, there is a dynamic profile of viral loads detected by the N gene and ORF gene among COVID-19 patients across the outbreak periods. It has been found that an association exists between persistent viral shedding and increased 6-month mortality in these individuals. Delayed antiviral use, immunocompromised patients, older age (≥ 50), and sex all had a significant impact on infection outcomes and have been linked to underlying differences in immune responses to infection. Males were noted to be at an increased risk for worse outcomes and had higher susceptibility to infections. More research is needed to predict accurately the emergence of variants causing chronic illness and identify mutations affecting transmissibility. Clinical trials evaluating treatments with antivirals have given hope, however, some have been shown to have low efficacy for inhibiting viral shedding with low efficacy and may result in a paradoxical longer shedding time. This review highlights the utmost importance of more research in this area for greater understanding. This review is limited to scarce literature in certain patient populations, variable follow-up times, and the prevalence of pre-existing clinical conditions (comorbidities). More studies are also needed on antivirals being used prophylactically before the onset of symptoms to monitor if they will decrease viral shedding in specific populations. Persistent viral shedding can contribute to increased viral evolution of SARS-CoV-2 and the emergence of new variants at the population level; hence, more studies are required due to its clinical and public health significance.

Authors contribution

Conceptualization, AS and RI. Original draft preparation, AM, SP, VB, KH, NH, AFA, ZH, KY. Revised and editing, AS, RI, SS, OB. Approved the version to be published, AS. Agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved, AS.

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The authors declare that they have no conflict of interest.

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