1. INTRODUCTION

Coronavirus disease 2019 (COVID-19) is a global pandemic caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). The disease was first discovered in late 2019, then rapidly spread to many countries around the world in early 2020 and gradually became a global pandemic.1 To date, more than 528 million confirmed cases have been reported worldwide, of which more than 6.287 million people have died, and the fatality rate is approximately 1.19%.2 It is one of the large-scale epidemics in human history. The common clinical manifestations of patients include fever, sore throat, dry cough, and dyspnea and, in severe cases, acute respiratory distress syndrome (ARDS) and multiple organ failure. As the spread of SARS-CoV-2 is extremely contagious, the global epidemic of COVID-19 has brought tremendous challenges to public health and medical systems around the world.

The current vaccination and promotion of vaccines have alleviated the epidemic of COVID-19 to a certain extent.3,4 However, effective treatment is still essential to treat infected people in order to prevent critical illness or even mortality. Due to the persistent effort of professionals around the world, medications including several antiviral drugs and mono-antibodies are currently available for patients infected by SARS-CoV-2.5,6 Unfortunately, the availability and accessibility of these antiviral agents and antibodies are inadequate, given their expensive cost, prescription requirement, and insufficient production capacity to catch up the speed of spread of the virus.7,8 As a result, the search for effective alternative medication for treating COVID-19 became another lesson, which in fact has been proceeding for a while since the COVID-19 pandemic. At the same time, another problem of seeking medication for COVID-19 is that the development cycle of new drugs is often time-consuming, as the development process involves the selection of targets, in vitro pharmacodynamic screening, in vivo pharmacological and toxicological verification, and confirmation of clinical safety and therapeutic effects; afterward, therapeutic drugs can be produced with clinical application value.9 Consequently, professionals may also shift the strategy on drug repurposing, which tries to use drugs that have been used clinically for a period of time with proven safety in patients with COVID-19.10 Potential drugs, such as steroids,11 quinine12 or interferon-gamma,13 for COVID-19 share some common features, including the ability to act as an antioxidant and an immune modulator to scavenge free radicals caused by hypoxic environments and reduce the systemic inflammation caused by excessive immune responses.14–16 N-acetylcysteine (NAC) is one of the potential candidates for the treatment of patients with COVID-19.17,18 In addition to its traditional pharmacological effect on promoting respiratory hygiene, studies have also revealed its potential antioxidative and anti-inflammatory effects, and it is regarded as a potential effective medication for treating COVID-19. However, current evidence for its efficacy in treating patients with COVID-19 remains controversial. Under these circumstances, we sought to perform a meta-analysis of studies evaluating the efficacy of NAC in patients with COVID-19 and to validate the results with trial sequential analysis (TSA).

2. METHODS 2.1. Study design

This systematic review with meta-analysis complies the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.19 The review was also registered prospectively on PROSPERO (CRD42022332791).

2.2. Search strategy

The Cochrane Library, PubMed, Embase, Web of Science, and Scopus databases were searched for studies published from inception to May 20, 2022. Two citation subsets were used under the framework of medical subject headings (MeSH) and text words: one including studies on concepts with COVID (“COVID 19”, “SARS-CoV-2 Infection”) and one including concepts with acetylcysteine (“N-acetyl-L-cysteine” OR “N-acetylcysteine”). We did not exclude publications by language or country. The full search strategy is recorded in Supplementary Table 1 (https://links.lww.com/JCMA/A176).

2.3. Eligibility criteria

The primary data were analyzed to evaluate if they met all of the conditions of the following criteria: the study included patients with a diagnosis of COVID-19 and allocated patients into group with intent to treat with NAC other than a standard medication and merely with standard medication. The study yielded adequate information and outcomes regarding respiratory outcomes (eg, intubation rate, oxygenation index [peripheral oxygen saturation divided by peripheral oxygen saturation ratio (SF ratio)]) and medical care outcomes (eg, mortality, duration of care unit stay) to calculate the effect estimates for meta-analysis). Studies that included randomized, controlled trials; nonrandomized, controlled trials, or observational studies were eligible for further review.

2.4. Risk of bias assessment

The methodological quality of the included studies was evaluated by the revised tool for Risk of Bias in Randomized trials tool (ROB-2)20 by two independent authors (C.-H. Chen and Y.-F. Cheng). The grading of risk was categorized into “low,” “some concern,” and “high” according to the reviewed item. Any disagreement was resolved by a project meeting and the judgment of a third author (C.-Y. Huang).

2.5. Statistical analysis

The meta-analysis was performed with a random-effects model for pooled effect size calculation. Statistical heterogeneity was evaluated by the Cochran Q test and the I2 statistic. Heterogeneity was considered low, moderate, and high at I2 values of <50%, 50%-74%, and ≥75%, respectively.21 The influence analysis of comparison between the NAC group and control group was performed with the pooled point estimates by ignoring the included studies one by one if the outcome in interest had more than two included studies. In addition, TSA was performed to evaluate whether there were type I or type II errors due to a lack of data or power. The traditional significance boundary in TSA was set from -1.96 to 1.96 under an alpha value of 0.05 and a power of 80%, and the sequential monitoring boundary varied in accordance with the analysis.22,23 Verification of a significant result would be proven if the cumulative Z score surpassed the sequential monitory boundary, while the null result would be consolidated if the Z score fell into the adjusted null area between the conventional significance boundary and would also be inferred if the Z score fell within the inner wedge of the null result.22,23 The Metaphor package of R language under the operation of R studio was used in all of the calculations for the meta-analysis, and the TSA was calculated by using TSA software version 0.9.5.10 Beta (The Copenhagen Trial Unit, Centre for Clinical Intervention Research, Copenhagen University Hospital – Rigshospitalet, Copenhagen, Denmark).22,23

3. RESULTS 3.1. Study identification and selection

The present study recognized 778 records in the initial search. After eliminating duplicates and after screening the titles and abstracts, 10 studies consequently underwent full-text review. Six studies were excluded due to irrelevant outcomes and inadequate study designs. As a result, four eligible studies were included (Fig. 1).24–27

Fig. 1:

The PRISMA flow diagram. The present study recognized 778 records in the initial search. After eliminating duplicates and after screening the titles and abstracts, 10 studies consequently underwent full-text review. Six studies were excluded due to irrelevant outcomes and inadequate study designs. As a result, four eligible studies were included. PRISMA = Preferred Reporting Items for Systematic Reviews and Meta-Analyses.

3.2. Study characteristics and risk of bias assessment

A total of 355 patients were allocated into the groups of adjunctive NAC with standard care and the control group with standard care alone. Three studies were randomized controlled trials,24,26,27 while the other study was an observational study.25 Three studies delivered NAC intravenously,24,26,27 while the other administered the medication orally.25

Regarding the evaluation of respiratory outcomes, the intubation rate was evaluated in three studies,24,25,27 and the improvement of oxygenation index was assessed in two studies.26,27 Regarding clinical endpoints, three studies evaluated the mortality between both groups.24,25,27 The duration of intensive care unit (ICU) stay was assessed in two studies,24,27 while the duration of hospital stay was checked by three included studies.24,26,27 Detailed information is presented in the Table 1.

Table 1 - Study characteristics Study Country Study type Patients Age (y, SD) Intervention number NAC delivery Outcome evaluation Main result NAC group: 58.8 (4.9) NAC group: 67 No significant difference between the NAC group and control group in the intubation rate and mortality. NAC did not significantly reduce the ICU stay length and hospital stay length de Alencar et al24 2021 Brazil Randomized controlled trial 135 Control group: 58.5 (4.7) Control group: 68 Intravenous NAC (21 g/d) Intubation rate, mortality, ICU stay length and hospital stay length NAC group: 59.4 (21.1) NAC group: 47 No significant difference between the NAC group and control group in the intubation rate, oxygenation index, mortality. NAC did not significantly reduce the ICU stay Taher et al27 2021 Iran Randomized controlled trial 92 Control group: 55.5 (16.5) Control group: 45 Intravenous NAC (40 mg/kg/d) Intubation rate, oxygenation index, mortality, ICU stay length and hospital stay length NAC group: 61.0 (12.8) NAC group: 24 NAC could improve the oxygenation index and reduce the hospital stay length Gaynitdinova et al26 2021 Russia Randomized controlled trial 46 Control group: 54.5 (12.1) Control group: 22 Intravenous NAC (1.2-1.5 g/d) Oxygenation index and hospital stay length NAC group: 61.0 (16.0) NAC group: 42 NAC could reduce the intubation rate and mortality Assimakopoulos et al25 2021 Greece Observational study 82 Control group: 64.0 (17.0) Control group: 40 Oral NAC (1.2 g/d) Intubation rate and mortality

ICU = intensive unit; NAC = N-acetylcysteine.

The risk of bias was assessed for each of the included studies. Overall, two studies were categorized as having moderate bias,26,27 while another study was scored as potentially serious.25 One study had a high risk of bias from the randomization process,25 while there was some concern about the issue in two of the included studies.26,27 One study had some concern regarding bias due to deviations from the intended intervention.26 There was some concern resulting from bias in the measurement of the outcome in two of the included studies.25,27The detailed assessment is presented in Supplementary Figs 1 and 2 (https://links.lww.com/JCMA/A176).

3.3. Outcomes 3.3.1. Comparison of the improvement of the intubation rate and oxygenation index between the NAC and control groups

Three studies compared the intubation rates between the NAC and control groups.24,25,27 Consequently, the pooled effect estimate demonstrated a nonsignificant intubation rate (OR, 0.55; 95% CI, 0.16-1.89; p = 0.34; I2 = 75%) (Fig. 2A). Meanwhile, the improvement of the oxygenation index was compared in three included studies,26,27 and the pooled result showed a nonsignificant difference in the oxygenation index between the NAC group and the control group (difference in means [MD], 80.84; 95% CI, -38.16 to 199.84; p = 0.18; I2 = 98%) (Fig. 2B).

Fig. 2:

Comparison of respiratory outcome between NAC and control group. A, The pooled result of intubation rate showed nonsignificant difference between both group (OR, 0.55; 95% CI, 0.16-1.89; p = 0.34; I 2 = 75%). B, The pooled result of SpO2/FiO2 ratio showed nonsignificant difference between both groups (MD, 80.84; 95% CI, -38.16 to 199.84; p = 0.18; I 2 = 98%). IV = inverse variance method; MD = difference in means; NAC = N-acetylcysteine; OR = odds ratio; SpO2/FiO2 = peripheral oxygen saturation divided by peripheral oxygen saturation.

3.3.2. Comparison of the duration of ICU stay and hospital stay between the NAC and control groups

In two included studies evaluating the duration of ICU stay,24,27 the pooled result showed a nonsignificant difference in the length of ICU stay between both groups (MD, -0.74; 95% CI, -3.19 to 1.71; p = 0.55; I2 = 95%) (Fig. 3A). As the total duration of hospital stay was assessed in three included studies,24,26,27 the combined effect estimate also revealed a nonsignificant difference between both groups (MD, -1.05; 95% CI, -3.02 to 0.92; p = 0.30; I2 = 90%) (Fig. 3B).

Fig. 3:

Comparison of outcome of medical care between NAC and control group. A, The pooled result of ICU duration showed nonsignificant difference between both groups (MD, -0.74; 95% CI, -3.19 to 1.71; p = 0.55; I 2 = 95%). B, The pooled result of hospitalization duration showed nonsignificant difference between both groups (MD, -1.05; 95% CI, -3.02 to 0.92; p = 0.30; I 2 = 90%). ICU = intensive care unit; IV = inverse variance method; MD = difference in means; NAC = N-acetylcysteine.

3.3.3. Comparison of mortality between the NAC and control groups

Mortality was recorded and assessed in three of the included studies,24,25,27 and the pooled result revealed a nonsignificant difference between the NAC group and the control group (OR, 0.58; 95% CI, 0.23-1.45; p = 0.24; I2 = 54%) (Fig. 4).

Fig. 4:

Comparison of outcome of mortality between NAC and control group. The pooled result of mortality rate showed nonsignificant difference between both groups (OR, 0.58; 95% CI, 0.23 to 1.45; p = 0.24; I 2 = 54%). IV = inverse variance method; NAC = N-acetylcysteine; OR = odds ratio.

3.3.4. Trial sequential analysis and influence analysis

We conducted an influence analysis by removing the included studies one by one when the outcome in interest had more than two included studies. Consequently, all the results remained within the CI of the primary result, and no outliers were identified (Supplementary Figs. 3–6, https://links.lww.com/JCMA/A176). Additionally, while all of our primary analyses were nonsignificant, the TSA for comparing intubation rate, improvement of oxygenation, duration of ICU stay and duration of hospital stay did not reach the required information size (RIS) or inner wedge of the null result, suggesting a potential insufficiency of the sample size to consolidate these pooled results. On the other hand, the TSA for the comparison of mortality indicated a null result since the cumulative z-curve lies within the inner wedge of the null result, suggesting the conclusive result of the original analysis (Supplementary Figs. 7–11, https://links.lww.com/JCMA/A176).

4. DISCUSSION

The primary result of the present study revealed that the use of NAC did not reduce the intubation rate, improve the oxygenation index, shorten the ICU and hospital stays or reduce mortality for patients infected by SARS-CoV-2. Subsequent TSA showed conclusive null results for the comparison of mortality, authentically suggesting that NAC does not reduce the mortality of patients with COVID-19. Meanwhile, the TSA for the comparisons between intubation rate, improvement of oxygenation index, duration of ICU and hospital stay did not reach the RIS, suggesting the pooled effect estimate may potentially suffer from a type II error, and further studies should be considered to consolidate the null result.

Subsequent to the outbreak of the COVID-19 pandemic, the microbiological characteristics and pathogenic mechanism of SARS-CoV-2 have been effectively understood, which can be attributed to the cooperative efforts of multidisciplinary professionals around the world and has allowed for success in the timely development of the vaccine, a key breakthrough in curbing the spread of the virus and controlling the pandemic. During the time when the notorious virus has become controllable, research on how to treat patients infected with SARS-CoV-2 has remained in full swing. For infected patients, avoiding severe illness or even mortality is the most important issue. Currently, the available medications to treat patients with COVID-19 include antiviral drugs (eg, remdesivir [Food and Drug Administration approval], ritonavir-boosted nirmatrelvir [emergency use authorization], molnupiravir [emergency use authorization]) and certain anti-SARS-CoV-2 monoclonal antibodies. However, these drugs are either expensive or are currently produced in limited capacity, and manufacturers are unable to keep up with the speed of the epidemic. Therefore, exploring drugs that can be effectively available for supportive care is also an essential goal for clinicians in the persistent pandemic. At present, treatments other than antiviral agents focus on the prevention of the overactive immune response, and these treatments include antioxidants and immunosuppressants (eg, steroids). NAC is one of the candidates that serves as an antioxidant to treat patients with COVID-19 and prevent further severe respiratory complications or even mortality. However, the current evidence on whether NAC could effectively treat patients with COVID-19 is controversial.28,29 Under these circumstances, the present study represents the first comprehensive quantitative evidence for the efficacy of NAC in treating COVID-19 by evaluating respiratory outcomes and clinical endpoints, including the ICU stay, hospital stay, and mortality.

SARS-CoV-2 is an enveloped virus with a single-stranded positive-stranded RNA base array,30 and its genome encodes four structural proteins, spike (S), envelope (E), matrix (M) and nucleocapsid (N), and nonstructural proteins, as well as a group of accessory proteins. The spike protein recognizes and binds to the angiotensin-converting enzyme 2 (ACE2) receptor present on the host cell membrane, and this binding is followed by the conformation of S2 to facilitate the integration of the viral lipid membrane into the host cell membrane, which allows the viral RNA to enter the host cell for replication.31–33 This molecular pathway illustrates why the lower respiratory tract, kidney and vessels are especially susceptible to the virus since these organs have an abundance of ACE2.34,35

The occurrence and development of severe COVID-19 involves a series of pathophysiological changes. First, as the S protein of the SARS-CoV-2 surface spike can bind to the ACE2 receptor to enter host cells, organs other than the organs of the respiratory tract have the chance to be infected by the virus, and due to the viral virulence, direct damage follows, which contributes to a part of the pathophysiology of multiple organ damage in critically ill patients with COVID-19.35 Second, SARS-CoV-2 infection and the destruction of alveoli trigger local immune responses that in turn initiate adaptive T and B-cell immune responses. As the immune response becomes uncontrolled, the dysregulated immune response may further induce a local or systemic cytokine storm and a widespread inflammatory response,36 caused by the overactivated macrophages and neutrophils that promote the release of cytokines and chemokines. The overactive inflammatory response results in acute lung injury or even ARDS, causing pulmonary edema, hypoxemia, and vascular endothelial cell injury.35,37 These would further contribute to uncontrolled disseminated intravascular coagulation and large vessel thrombosis associated with multiple organ failure in critically ill patients, and these features would generally indicate the poor prognosis of patients with COVID-19.35,37–39 Finally, after invasion by binding to the ACE2 receptor, SARS-CoV-2 eventually downregulates the receptor and reduces its activity, resulting in an increase in angiotensin II and dysregulation of the renin-angiotensin system. Excessive angiotensin II leads to pulmonary vasoconstriction, inflammation, and increased reactive oxygen species, further worsening the acute lung injury into ARDS, and this could even cause death.40

NAC is a classic expectorant drug that was first used in the 1960s. It is also well known as an antidote of acetaminophen poisoning. It is the acetylated product of L-cysteine as well as a reduced form of glutathione, which can scavenge oxygen free radicals in the body. The thiol group contained in the NAC molecule can break the disulphide bond of the peptide chain in the glycoprotein of sputum, which can thin sticky sputum and effectively improve ciliary movement, enhance ciliary clearance and act as a surfactant in alveoli. Meanwhile, NAC can scavenge oxygen free radicals in peripheral airways. Reactive oxygen intermediates (ROIs) are essential mediators of viral infection since the oxidizing environment serves as a necessary condition for virus replication. Additionally, the ROI participates in the inflammatory response of host cells induced by viruses and plays a key role as in the cytokine storm mentioned in the previous paragraph. As the ROI increases, oxidative stress further mediates the apoptosis induced by multiple physicochemical factors.17,41 Studies have illustrated that NAC serves as an antioxidant by inhibiting the nuclear translocation and phosphorylation of Mitogen-activated protein kinase p38 by nuclear factor kappa-B to exert its antioxidant effect,17,41–43 thereby inhibiting and reducing lung tissue inflammatory diseases and the pulmonary edema caused by SARS-CoV-2, decreasing the expression of toll-like receptor-4 (TLR4) protein and TLR4 messenger RNA in lung tissue, reducing myeloperoxidase (MPO) activity, increasing superoxide dismutase activity, and decreasing pro-inflammatory cytokines (eg, tumor necrosis factor alpha, interleukin [IL]-6, IL-8, and IL-1β). By improving respiratory function and counterattacking the inflammatory response, NAC was regarded as a potential cost-effective candidate for managing patients infected with COVID-19.17,41-43

Regardless of the promising evidence in basic studies and its successful application in treating other diseases, our meta-analysis suggested that there was no benefit of using NAC to improve the clinical outcomes, and the null results on improving mortality were further validated by TSA, indicating that NAC does not reverse the possible mortality of patients with COVID-19. As mentioned in the previous paragraph, the pathogenesis of COVID-19 is multifactorial, including the direct virulence of SARS-CoV-2, a subsequent hyperactive immune response and dysfunction of the renin-angiotensin system. Because NAC is a potential antioxidant, it is reasonable to consider that NAC has the ability to theoretically improve oxidative stress and respiratory conditions. However, since there are complicated and interacting pathophysiological pathways involved in the mortality caused by COVID-19, NAC is not able to block all these possible mechanisms, so NAC is not beneficial for avoiding mortality.24 Despite the result that NAC does not seem beneficial for mortality, the other outcomes, including intubation rate, improvement of oxygenation, duration of ICU stay, and hospital stay remained discussable. As a widely used medication, NAC has a positive effect on the promotion of respiratory tract hygiene and potential antioxidative and anti-inflammatory properties. In addition, the biggest advantage is the safety of this drug when compared with immune modulators, such as steroids, as NAC has fewer immunosuppressive side effects,44,45 which is a risk for patients with COVID-19 who have an unstable immune response. As the TSA suggested, a larger sample size is essential to draw a null conclusion in the results other than with mortality, but we still consider keeping NAC as a reasonable adjunctive medication until decisive evidence can be obtained.

There are several limitations noted in our study. First, evidence has shown that the difference in severity may affect the efficacy of the drug. As discussed in the previous paragraph, the pathophysiological pathway to the subsequent generation of disseminated intravascular coagulation or even ARDS and renin-angiotensin-aldosterone system dysfunction sometimes implies that the disease is already quite serious. We believe that NAC alone cannot reverse these disorders.44–46 Second, there was heterogeneity regarding disease severity, concurrent standard treatment and the favorable dose for the administration of NAC, which resulted from the varied experiences across clinicians and institutes, the formal guideline should be warranted for consistency of studies in the future. Third, with the continuous spread of SARS-CoV-2 among the population, its genome continues to mutate, from the first occurrence of S protein D614G mutation to the Alpha, Beta, Gamma, Delta, and current Omicron mutant strains,47,48 and most of the included studies did not state what kind of strain they had. We noticed that the virulence was different across the mutant strains.48 The preferred method to explore whether the potential confounder above would cause bias in the present study is to perform a subgroup analysis or a meta-regression. Unfortunately, the number of included studies in this article does not allow for a post hoc analysis, including a subgroup analysis or a meta-regression based on severity, as suggested by the Cochrane Handbook for Systematic Reviews of Interventions,49 and we regarded it as the fourth limitation of our study. Consequently, we adopted a random-effects model to account for possible confounding factors, other than a sampling error, that would influence the pooled effect size, and we performed the influence analysis to determine whether there was an outlier in the included studies, which in turn showed that there were no confounding factors. Finally, the number of included studies and sample size were relatively small. Under these circumstances, we performed TSA to test the futility of the meta-analysis. As a result, the TSA confirmed the futility in the comparison of mortality, while the futility of other outcomes was not established. For all the existing limitations, we suggest that large-scale and well-designed controlled trials are essential in the future.

ACKNOWLEDGMENTS

This work was supported by grants from the Ministry of Science (MOST-10-2622-8-075-001) and Technology and Veterans General Hospitals and University System of Taiwan Joint Research Program (VGHUST111-G6-11-2 and VGHUST111c-140).

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