Electronic searches were carried out in Embase, PubMed, the Cochrane Library, the Web of Science, Wanfang Data, and China National Knowledge Infrastructure. The search terms we used were “SARS-CoV-2,” “Corona Virus Disease 2019,” “COVID-19,” “nitric oxide,” “NO,” “almitrine,” “iNO,” “NO and almitrine,” “nitric oxide and almitrine,” and relevant keywords for publications until 23.10.2023. The search strategies are available in Supplementary Appendix S1, appendix p9–11. Unpublished and ongoing studies were identified by searching pre-print servers including medRxiv. Searches were carried out by two reviewers (Y.W and K.Z) independently in a standardized manner, followed by screening through titles, abstracts, and full text. Disagreements were resolved by consensus with unresolved conflicts decided by a third reviewer (D.L).

Inclusion criteria were as follows: 1) Patients were confirmed COVID-19 and the SOFA score (absolute, median, and mean value) ≥2, or in accordance with the SOFA scoring tool, a certain system index (absolute, median, and mean value) should be within the scope of corresponding to the system score ≥2, such as the PaO₂/FiO₂ ratio (P/F) (absolute, median, and mean value) was less than 300 mmHg (Singer et al., 2016). According to the SpO₂/FiO₂ ratio (S/F) = 64 + 0.84*(P/F), the S/F of 315 was approximately equal to a P/F ratio of 300 mmHg (Rice et al., 2007). In this study, we defined that such COVID-19 patients were at the edge of sepsis. 2) The intervention of interest was inhaled NO, intravenous almitrine, or inhaled NO combined with intravenous almitrine with or without standard treatment. Comparator treatments included placebo, standard treatment, and no intervention. No control group studies were included. 3) Randomized clinical trials (RCTs), case-control studies, cohort studies, cross-sectional studies, case reports, case series, and grey literature were included. The language was limited to Chinese and English. Exclusion criteria were as follows: 1) Patients were not confirmed COVID-19. 2) The SOFA score (absolute, median, and mean value) ≤2 or any of the system indicators did not reach 2. 3) Data on SOFA score or certain indicators were not available in the text, Supplementary Materials, or relevant resources. 4) Studies without an available full text or incomplete or unavailable data, conference abstracts, posters, opinion articles, commentaries, animal experiments, and in vitro studies. The efficacy outcomes were 28–30 days mortality, in-hospital mortality, P/F, and intubation needs. The safety outcomes were serious adverse events (SAEs) such as acute kidney injury (AKI) (Al Sulaiman et al., 2022).

Two independent reviewers (Y.W and K.Z) extracted the eligible studies, and a third reviewer (D.L) validated them. The extracted information includes the published year, authors, country, study type, sample size, participant demographics, SOFA score, patients’ position, drug dosage, route of administration, control group, mortality outcome, safety outcome, and conclusion of authors.

Included studies were assessed for quality by three reviewers (RL.L, LP.L, and CJ.W) in a standardized process. The Risk of Bias 2.0 tool was used to assess the RCTs (Steudel et al., 1999; Sterne et al., 2019). The methodological quality of case-control and cohort studies was assessed based on the Newcastle–Ottawa Scale (NOS) (NOS, 2020). The methodological quality of the included case reports, case series, and cross-sectional studies was assessed based on JBI critical appraisal tools (JBI, 2020). The reviewers shared the quality assessment results and gained consensus through discussion. The quality of evidence was assessed by using the “Grading of Recommendations Assessment Development and Evaluation (GRADE)” tool (Granholm et al., 2019).

The Review Manager v.5.4.1 software was used for statistical analysis. For dichotomous outcomes, the total number of participants and the number of events in each group were recorded. For continuous outcomes, the total number of participants, mean, and standard deviation were recorded. If the authors reported the median and interquartile range, we estimated the mean and standard deviation (Wan et al., 2014; Luo et al., 2018). We report odds ratio (OR) for dichotomous outcomes and standard mean differences (Std MDs) for continuous outcomes. The fixed-effect model was applied when the result of the Q test was not significant (p > 0.1) and I²<50%. The I² statistic was used to measure heterogeneity (I²: 30%–60% was defined as moderate heterogeneity, and 80%–100% was defined as significant heterogeneity). Subgroup analyses would be conducted, if data are appropriate. If we could not implement a meta-analysis, we planned to comment based on the results of included studies.

A search of the electronic databases on 23 October 2023 yielded 83,090 studies. After excluding duplicate articles and screening titles and abstracts, 84 articles were evaluated for full-text review. Among these, we found 35 relevant articles (2 RCTs, 19 cohort studies, 1 case-control study, 1 cross-sectional study, and 12 case reports) (Figure 1) (Abou-Arab et al., 2020; Bagate et al., 2020; Barthélémy et al., 2020; Cardinale et al., 2020; Ferrari et al., 2020; Huette et al., 2020; Losser et al., 2020; Safaee Fakhr et al., 2020; Tavazzi et al., 2020; Caplan et al., 2021; Chandel et al., 2021; Feng et al., 2021; Garfield et al., 2021; Giri et al., 2021; Herranz et al., 2021; Heuts et al., 2021; Huette et al., 2021; Laghlam et al., 2021; Longobardo et al., 2021; Lotz et al., 2021; Moni et al., 2021; Paramanathan et al., 2021; Ziehr et al., 2021; Al Sulaiman et al., 2022; Brown et al., 2022; Kalfon et al., 2022; Lubinsky et al., 2022; Poonam et al., 2022; Vives et al., 2022; Bicakcioglu et al., 2023; Blot et al., 2023; Di Fenza et al., 2023; Mekontso Dessap et al., 2023; Saccheri et al., 2023; van Zyl et al., 2023).

In the 35 studies included, there were a total of 1,701 COVID-19 patients combined with sepsis, of whom 453 received mechanical ventilation. Ten studies reported SOFA scores of enrolled patients, of which three studies reported scores between 2 and 3 (2 for NO and 1 for almitrine), three studies reported scores between 4 and 5 (2 for NO and 1 for almitrine), and seven studies reported a score ≥ 6 (4 for NO and 3 for almitrine). The remaining 21 articles showed patients’ respiratory status, of which 19 studies included patients’ P/F ≤ 150 mmHg (15 for NO, 2 for almitrine, and 2 for NO combined with almitrine) and 2 studies reported patients’ P/F between 150 Hg and 300 mmHg(NO).

Patients usually received standard treatment (or standard of care) based on local guidelines. However, the majority patients were diagnosed with ARDS, and some of them used prone position to improve oxygenation. In addition, there is still no consensus on the dosage of NO and almitrine for such patients. The common dosage of NO is 10–80 parts per million (PPM) up to a maximum of 160–200 PPM, administered by inhalation. In some studies, patients’ dosage was adjusted according to PaO₂ in arterial blood gas analysis. The usual dosage of almitrine is 2–16 μg/kg/min up to a maximum of 0.5 mg/kg/min, administered by injection. Characteristics of included studies and patients are presented in Table 1.

The risk of bias of two RCTs was low to moderate (Supplementary Appendix S2, appendix p1–2). The methodological quality of 18 cohorts were moderate to high, and one case-control study was moderate (NOS assessment results are shown in Supplementary Appendix S2, appendix p3–22). The methodological quality of four case series was moderate, eight case reports was moderate to high, and one cross-sectional study was moderate (JBI assessment results are shown in Supplementary Appendix S2, appendix p23–35). Supplementary Appendix S2 (appendix p38–41) summarized the result of GRADE assessment for the certainty of evidence.

NO is a potent pulmonary vasodilator, while almitrine constricts pulmonary blood vessels. The combination of NO and almitrine appears to be contradictory, but some experts have utilized this combination as a rescue measure for critically ill patients (Laghlam et al., 2021). The rationale behind this combination lies in its potential to enhance the ventilation/perfusion ratio (V/Q) through selective vasoconstriction of pulmonary vessels in non-ventilated areas and selective vasodilation of pulmonary vessels in ventilated areas. Our findings indicate that this combination did not improve patient survival but did enhance oxygenation. It is crucial to exercise caution when employing this combination until further clarification regarding the mechanism, timing, and dosage is obtained. Additionally, almitrine is frequently used in conjunction with another drug called raubasine, which acts as a vasodilator, for the treatment of age-related cerebral disorders (Allain and Bentué-Ferrer, 1998) and certain pulmonary diseases. Notably, raubasine has demonstrated anti-SARS-CoV-2 effects in in vitro and animal studies, suggesting its therapeutic potential (Kumar et al., 2021; Mohseni et al., 2022).

Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome coronavirus (SARS) have caused a large number of infectious deaths in pregnant women over the past 20 years. Because pregnant women have elevated levels of progesterone and estrogen, restricted lung expansion is more susceptible to pathogens (Selim et al., 2020). At present, there is still a lack of targeted respiratory interventions for pregnant women with hypoxic respiratory failure due to COVID-19 pneumonia, other than supplemental oxygen and mechanical ventilation. In another study of six pregnant women with severe or critical forms of COVID-19, 160–200 ppm of NO was found to be applied frequently, which appears to be well tolerated and may be beneficial to the people with hypoxic respiratory failure (Safaee Fakhr et al., 2020). Studies have shown that in patients with COVID-19, this innovative breathing intervention is feasible in pregnant women. At present, there have been many studies on the application of NO in the treatment of sepsis (non-COVID-19), but the experience of applying NO in the treatment of pregnant patients with COVID-19 remains very valuable.

Most studies defined the responders as P/F increase >20% or 10 mmHg after administration (Ichinose et al., 2004; Charron et al., 2011). Whether patients with COVID-19 or without COVID-19 were “responders” to NO and almitrine and elements that predict potential responsiveness remain unclear. Manktelow C. et al. reported about 30%–40% of non-COVID-19 ARDS patients were non-respondence to inhaled NO to 67% of the patients with septic shock. They observed that septic shock was a significant predictor of NO inhalation responsiveness (Manktelow et al., 1997). Trachsel S. et al. reported endotoxin-exposed pigs that received inhaled NO responded by producing more endothelin-1, however, with higher levels in the responder group compared to the non-responder group (Trachsel et al., 2008).

As for COVID-19, among the included literature, only three studies compared the baseline characteristics of responders and non-responders, and the oxygenation of responders was lower than that of non-responders (Abou-Arab et al., 2020; Caplan et al., 2021; Garfield et al., 2021). Garfield B reported responders to inhaled NO also had higher baseline brain natriuretic peptides (Garfield et al., 2021). In addition, the prognosis of responders and non-responders is uncertain. Abou-Arab O. et al. stated that responders had a lower 28-day mortality rate (Caplan et al., 2021), and one study reported the ICU mortality of responders was similar to that of non-responders (p = 1.0) (Abou-Arab et al., 2020). During the pandemic, more in-depth research is needed on NO and almitrine responsiveness.

Infection by SARS-CoV-2 elicits a spectrum of complications, encompassing ARDS, AKI, and myocardial injury (Oxley et al., 2020; Ye et al., 2020). NO exerts selective dilation of pulmonary vessels within ventilated lung units, thereby improving ventilation/perfusion matching while averting systemic hypotension. Consequently, NO has been investigated as a potential treatment for COVID-19-associated ARDS. Moreover, NO may possess cardioprotective properties, as it can attenuate subclinical myocardial injuries normally observed during cardiopulmonary bypass procedures (Redaelli et al., 2022). Hence, this offers novel therapeutic avenues for managing COVID-19-related myocardial damage. Furthermore, inhaled NO could potentially yield favorable hemodynamic effects during cardiopulmonary bypass, thereby enhancing cardiac output and subsequently improving renal perfusion (Rezoagli et al., 2017). Utilizing NO may, thus, confer beneficial effects on cardiac output and provide renal function protection in patients with COVID-19 complicated by cardiovascular disorders.

A non-COVID-19 study showed that the plasma concentration and efficacy of almitrine increased in a dose-dependent manner, and perhaps its adverse events seemed to be also dose-dependent (Gallart et al., 1998). In this study, we found that adverse effects of 2 μg/kg/min almitrine were mild and infrequent, and the incidence was similar to that of the placebo groups (Kalfon et al., 2022). Meanwhile, no significant adverse events were observed by the investigators after the use of almitrine at a dose of 4-12 μg/kg/min (Losser et al., 2020; Caplan et al., 2021; Laghlam et al., 2021). However, considering the increase in the incidence rate of pulmonary thromboembolism in COVID-19 patients (Michard et al., 2001), some researchers recommended that almitrine should be used with caution, and the right ventricular loading conditions should be paid attention to after administration (Poissy et al., 2020).

A systematic review reported that inhaled NO might increase the risk of renal dysfunction, especially in patients with prolonged use and ARDS (non-COVID-19 (Ruan et al., 2015)). A multi-center cohort study included in this study showed that moderate-to-severe ARDS in critically ill patients of COVID-19 who received inhaled NO illustrated significantly higher odds of AKI (Al Sulaiman et al., 2022). A latest study on SAEs of NO showed that inhaled NO was associated with severe AKI and renal replacement therapy in critically ill patients of COVID-19 (Bobot et al., 2022). During the pandemic, some researchers tried to use NO for pregnant women with severe to critical COVID-19, and no acute adverse events related to NO were observed (Safaee Fakhr et al., 2020; Valsecchi et al., 2022). Based on the limited evidence, we suggest that doctors balance the benefit-to-risk ratio before prescribing NO for patients with COVID-19 (at the edge of sepsis) and pay attention to the renal function during administration.

First, some results of this study have significant heterogeneity, but due to the lack of research, subgroup analysis and meta-regression cannot be carried out. However, despite the high heterogeneity of the research results, they still reflect the trend of the efficacy of NO and almitrine. Meanwhile, the SOFA score or related indicators of included patients were mean or median, so we speculated that not all patients confirmed sepsis, but the results of the patients still reflected a trend because some of these patients might or would develop sepsis. Second, a lack of high-quality clinical research studies limited our analyses. The majority of included studies were retrospective studies; these aspects could have introduced various confounders given the lack of risk adjustment or propensity score weighting. We included studies written only in English and Chinese, which also limits the scope of the review. Third, we found that the SOFA scores of included patients varied, and factors such as ethnic differences, the use of vasoactive drugs in many patients, and prone position had uncertain effects. Few studies analyzed the impact of these factors further and drew conclusions. Both ARDS and sepsis showed individual differences, which may also increase heterogeneity. In addition, the doses of NO and almitrine in the included studies were not uniform, and differences in management schemes for patients with sepsis in different countries and additional variability during the COVID-19 pandemic would increase the heterogeneity of the findings.