key: cord-0696639-i13g3be9 authors: Wu, Junfang; Zhao, Mingming; Wei, Haoran; Li, Chenze; Hu, Dong; Zheng, Lemin; Wang, Dao Wen title: Neuraminidase inhibitor treatment is associated with decreased mortality in COVID-19 patients: a retrospective analysis date: 2022-03-16 journal: Eur Heart J Cardiovasc Pharmacother DOI: 10.1093/ehjcvp/pvac018 sha: 9ac778a11d7fbe5fd693aba7e6a59c3703bd7c1f doc_id: 696639 cord_uid: i13g3be9 AIMS: The aim of this study was to investigate the effects of Neuraminidase inhibitors (NI) on COVID-19 in a retrospective study. METHODS AND RESULTS: The study included an overall COVID-19 patients (n = 3267) and a 1:1 propensity score-matched patients (n = 972). The levels of plasma N-acetylneuraminic acid and neuraminidase expression were further evaluated in a panel of hospitalized and 1-month post-infection recovered COVID-19 subjects. The mortality rate in the overall patients was 9.6% (313/3267) and 9.2% (89/972) in the propensity-score matched patients. The NI treatment lowered the mortality rate (5.7% versus 10.3%) and the critically ill conversion rate (14.1% versus 19.7%) compare to those in the non-NI group in the overall patients and evaluated in the propensity score-matched patients when applying the multivariate Cox model for adjusting imbalanced confounding factors. Furthermore, NI treatment was associated with attenuated cytokine storm levels and acute heart injury but not liver or kidney injuries. Further analysis in a small panel of patients found the levels of N-acetylneuraminic acid and neuraminidase (dominantly the NEU3 isoform) were elevated in the hospitalized COVID-19 subjects and recovered at the 1-month post-infection stage, suggesting increasing desialylation in COVID-19 patients. CONCLUSION: These results suggest that NI treatment is associated with decreased mortality in COVID-19 subjects, especially for those subjects with acute heart injury. N-acetylneuraminic acid (Neu5Ac) is the major form of sialic acid in mammals. Previous studies have found that serum sialic acid is associated with cardiovascular mortality 12, 13 . It triggers myocardial injury in vitro and in vivo by activating the Rho-ROCK signaling pathway by binding to RhoA and Cdc42, which can be alleviated by NI treatment 14, 15 . Recently, Chu et al. found that coronavirus could utilize sialic acid for virus attachment and entry in both human lung epithelial cells and ex vivo human lung tissue explants 16 . Additionally, sialic acid-mediated cross reactivity with host immune lectins is known to exert an immune response in different pathological stages in coronaviruses 17 Here, we investigated the effects of NI on COVID-19 subjects from Wuhan, China in a retrospective study. We also measured the levels of plasma Neu5Ac and neuraminidase (NEU) expression in a panel of hospitalized and 1-month post-infection recovered subjects to explore the underlying mechanism. This study aimed to evaluate the efficacy of NI based on the clinical outcomes of COVID-19 patients. panel of plasma samples from COVID-19 patients, which also get approved (IRBID:TJ-IRB20200336) . The clinical data and laboratory results were exported from the electronic medical records in Tongji Hospital and checked by two study investigators independently to obtain the admission symptoms, clinical diagnosis, and treatment process for each patient. The primary outcome was defined as all-cause death during hospitalization. The secondary outcomes included acute heart injury, acute liver injury, acute kidney injury, intensive care unit, invasive mechanical ventilation, ventilator, intubation, and oxygen therapy. Of these, acute cardiac injury was defined as levels of serum cardiac Troponin I was higher than its upper limit 20, 21 . Acute liver injury was defined as a serum ALT 3-fold higher than the normal upper limit 22 . The increased in serum creatinine by 26.5 M in 48 hours was regarded as the kidney injury 23 . of NT-proBNP, CRP, and IL-6 were adjusted for the total patients since these variables are reported to be associated with the severity of COVID-19 in previous studies 20, 25 . Table 1 . The median ages were 62 (IQR, 49-70) and 61 (IQR, 48-70) years old in the NI and non-NI groups, respectively. These two groups showed no significant difference in any of the baseline characteristics on admission after 1:1 propensity-score matching analysis (N=486 for each group, Table 1 ). The baseline characteristics for all patients in both groups are presented in Table S1 . The baseline laboratory test results, including the routine blood test, biochemistry, infection-related indices, and coagulation function of all patients and the propensity scorematched patients between the NI group and non-NI group are shown in Table S2 . Most of the infection-related cytokine indices (including pro-inflammatory IL-6, IL-8, TNF- and antiinflammatory IL-10) were lower in the NI-group compared to the non-NI group in the total patients (p<0.01) after multiple comparison. After propensity scores-matching, the IL-8 levels remained lower in the NI group (p=0.009), even though the levels of IL-6 were well matched. All subjects received standard treatment following the National Guidelines for diagnosis and treatment program against pneumonia from new coronavirus infections (3rd version). For the overall patients, the percentage of some treatments, such as corticosteroids, immuneenhancement, antibiotics, antifungal drugs, and anti-HIV drugs showed a clear difference with p value less than 0.05 between the comparable groups (Table S3 ). In order to investigate the effects of NI treatment on COVID-19 subjects, propensity score matched analysis was carried out for the corticosteroids, total immune-modulators, antibiotics, antifungal drugs, and other antivirus drugs to balance other treatments that are associated with clinical outcomes. All other treatments except for NI were well matched in the sub-group (N=486 for NI and non-NI group, respectively). Among the overall COVID-19 patients, 313 out of the total 3267 patients died during the hospitalization ( Table 2) . After the propensity score-matched analysis, the mortality rate in the NI group (28 out of 486 subjects, 5.8%) was clearly lower than that in the non-NI group Figure 2A , 2C, Table 2 ). Among them, the mortality rate in the critically ill patients was also lower in the NI treatment group ( Figure 2B, 2D) Figure S1 ). It was more effective in elderly subjects that older than 60 years ( Figure S2 ), which indicated that the NI treatment is associated with decreased mortality rate in To further explore the secondary outcomes associated with NI treatment in COVID-19 subjects, the incidence of multi-organ failure and transfer to the intensive care unit was investigated in both unmatched and matched patients. In the group of NI treatment, the incidence of acute cardiac injury was lower and the utilization of ventilator was decrease in the propensity score-matched patients (Table 2, Figure 3A, 3D) . The Cox regression model consistently confirmed that the risk of developing acute heart injury in COVID-19 subjects was ameliorated in the propensity score-matched patients ( Figure 3D , adjusted HR 1.47, 95% CI 1.05-2.07, p=0.027), but not for the acute liver injury and acute kidney injury (Table 2 , Figure S5 ). The Kaplan-Meier survival curve and Cox regression analysis for different hs-cTnI (indicator of myocardial injury) and NT-proBNP (indicator of heart failure) levels further proved that NI treatment was more effective for patients with higher levels of hs-cTnI and NT-proBNP, regardless of the total and propensity score matched patients ( Figure 3B -3C, 3E-3F, Figure S6 ). These implied that NI treatment is associated with reduced incidence of acute heart injury in COVID-19 patients. The dynamic alteration of vital signs (such as pulse, diastolic blood pressure) and representative clinical indices (such as lymphocyte count, ALT, Creatinine, hs-cTnI, NT-proBNP, D-dimer, CRP, and a panel of cytokine factors) over time since admission were analyzed to evaluate the clinical manifestation that following the NI treatment in the total patients ( Figure 4) . A similar temporal pattern of vital signs and laboratory parameters were found in the propensity score-matched patients ( Figure S7 ). These parameters were tracked from day 2 after admission to day 20 at an interval of 4 days and plotted by using the median value of each period. We found a lower heart rate and higher diastolic blood pressure in the NI group compared to the non-NI group during the observational period ( Figure 4A and 4B, Figure S7A ). From trajectory plots, the majority of clinical indices in the non-NI group presented a turning point on the 4th day as the dynamic curves in NI group were more flat even there was no difference at the onset of observation (day 2). For example, the indicator of immune system reaction (levels of lymphocyte count, Figure 4D , Figure S7D and indicators of myocardial injury (hs-cTnI, Figure 4I , Figure S7I ), heart failure (NT-proBNP, Figure 4J , Figure S7J ), coagulation (D-dimer, Figure 4K , Figure S7K ), inflammation (CRP, Figure 4L , Figure S7L ), and cytokine storm (IL-6, IL-8, IL-2and TNFFigure 4M-4P, Figure S7O -7P) were increased markedly on the 4th day in the non-NI group. However, the variation trends of these indices in the NI group were more moderate, which implied the NI treatment is related to the decelerated progression of COVID-19. Indeed, the indicators of myocardial injury, heart failure, inflammation, and cytokine storm were consistently and markedly higher in the non-NI group during the observational period, suggesting the association of NI treatment and reduced inflammatory status and incidences of myocardial injury in COVID-19 subjects. Different from the hs-cTnI and NT-proBNP, the indicators for acute liver injury (ALT, Figure 4F , Figure S7F ) and kidney injury (creatinine, Figure 4H , Figure S7H ) did not show obvious difference in comparable two groups, which is in good agreement with previous incidence of liver and kidney injuries following NI treatment (Table 2, Figure S5 ). To further investigate the effects of NI on COVID-19 disease progression, the main form of sialic acid, N-acetylneuraminic acid (Neu5Ac) was evaluated in a small panel of COVID-19 subjects. Of them, the patients in COVID-H group presented higher levels of NT-proBNP, hs-cTnI and Neu5Ac, which recovered in 1-month post-infection ( Figure 5A , Then the neuraminidase (NEU), a rate-limiting enzyme for break-downing glycosides containing Neu5Ac, was measured by ELISA ( Figure 5B ) and its 3 isoforms expressed (NEU 1/3/4) in plasma were identified by western blotting (Figure 5C, 5D) . In the hospitalized patients, the NEU3 is main isoform that contributes to the increased levels of total NEU. The levels of total NEU and its isoforms were elevated in the hospitalized COVID-19 subjects and recovered at the 1-month post-infection stage, which indicated increasing desialylation during the disease development of COVID-19. The COVID-19 pandemic is a global public health crisis, with considerable mortality and morbidity exerting pressure on healthcare resources. In this retrospective study, we evaluated (Table S5) . Recently, an omics-driven study found monosialodihexosyl ganglioside enriched exosomes, which also share a Neu5Ac molecule in the tail, were also highly positively correlated with COVID-19 pathogenesis 29 . Thus, NI could serve as a competitive inhibitor of neuraminidase activity upon Neu5Ac to improve the levels of sialylation, which supported its important role in COVID-19. Moreover, NI reduced steatosis, decreased liver inflammation, and alleviated pulmonary fibrosis symptoms in experimental mouse studies 30, 31 . In line with this finding, our results suggest that the NI treatment is associated with decreased incidence of acute heart injury but not liver and kidney injuries in the matched patients. Higher levels of plasma Neu5Ac were previously reported to be associated with the risk and prognosis of cardiovascular diseases 14, 32 . In addition, cardiovascular system injury has received increasing attention as it is involved in the pathophysiology of COVID-19 33 . Chen et al. suggested that the fulminant myocarditis should be considered for those patients who deteriorate rapidly 34 . Here, from the occurrence time of acute heart injury in all COVID-19 subjects (Figure S8 ), the majority of acute heart injury occurred within 3 days (489/632, 77.4%) or in 2 weeks (591/632, 93.5%) since admission, which in accordance with the time window of fulminant myocarditis 35 or acute myocarditis 36 . It has been reported that NI treatment has a beneficial effect on fulminant myocarditis to prevent cardiac damage and cytokine storm by the released neuraminidase 35 . In our study, NI treatment was associated with a flattened trajectory curve of the disease progression by attenuating the myocardial injury/heart failure and immune system reaction ( Figure 4 ). In addition, the lower levels of plasma cytokine factors and CRP following NI treatment suggested that NI may be helpful for ameliorating the inflammatory status in patients with COVID-19. It is worth mentioning that the utilization of cardiovascular drugs (Table S6 ), in particular, did not show obvious difference in the propensity score-matched patients between NI and non-NI group, implied the beneficial effects of NI on COVID-19 with acute heart injury. In summary, our results suggest that NI treatment is associated with the decreased mortality in patients with COVID-19 and NI treatment is helpful for decelerating the disease progression and mainly alleviating the incidence of acute heart injury. Levels of Neu5Ac and higher expression of plasma neuraminidase, which is mainly driven by NEU-3, indicated the role of desialylation in the disease progression of COVID-19. This study has several limitations in this work. 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Pharmacotherapy online.Competing interests: none declared.