Effect of Ribavirin on Recovery Time in COVID-19 Patients: A Real-world Retrospective Cohort Study

Yuanlong Hu Shandong University of Traditional Chinese Medicine Xue Zhu A liated Hospital of Shandong University of Traditional Chinese Medicine Ning Shen A liated Hospital of Shandong University of Traditional Chinese Medicine Xinhua Jia A liated Hospital of Shandong University of Traditional Chinese Medicine Xingcai Zhang A liated Hospital of Shandong University of Traditional Chinese Medicine Jian Han A liated Hospital of Shandong University of Traditional Chinese Medicine Miao Yue Shandong Provincial Chest Hospital Chengmin Yuan Jinan Infectious Disease Hospital Zhanjun Qiu A liated Hospital of Shandong University of Traditional Chinese Medicine Huijie Ma Anqiu Traditional Chinese Medicine Hospital Hui Li A liated Hospital of Shandong University of Traditional Chinese Medicine Yingying Liu A liated Hospital of Shandong University of Traditional Chinese Medicine Wei Zhang (  huxizhijia@126.com ) A liated Hospital of Shandong University of Traditional Chinese Medicine


Introduction
Up to now, there is still no speci c drug against COVID-19. Ribavirin is a broad-spectrum nucleoside antiviral drug, which can interfere with the early transcriptional events of the virus to inhibit the replication and spread of the virus. There is only in silico [1][2] and in vitro experimental evidence [3] for treatment of COVID-19 with Ribavirin, and there is no clear clinical evidence for Ribavirin's bene ts to  patients. In addition, Guideline for COVID-19 Management in China (Trial) (Fifth Edition) [4] also comes from the clinical experience during the outbreak of SARS and MERS [5] . We conducted a real-world retrospective study to evaluate the e cacy of Ribavirin for SARS-CoV-2 infection.

Study design and population
This study was designed as a real-world retrospective cohort study based on EMRs. A total of 418 con rmed COVID-19 cases discharged from hospital from Shandong Provincial Chest Hospital, Jinan Infectious Diseases Hospital, Liaocheng Infectious Diseases Hospital, Qingdao Thoracic Hospital, Qingdao Municipal Hospital, Linyi Central Hospital, Binzhou Central Hospital, Weihai Thoracic Hospital, Linyi People's Hospital, Dezhou People's Hospital, Yantai Qishan Hospital and Huanggang Central Hospital between February 26, 2020 and March 9, 2020 were analyzed in this retrospective study. And, the baseline data of patient's EMRs must be complete.
The data for this study were obtained retrospectively from hospital EMRs. All con rmed cases in this study received basic antiviral therapy with interferon or Lopinavir-Ritonavir. The exclusion criteria were: (I) Patients received antiviral treatment with interferon or Lopinavir-Ritonavir combined with Ribavirin for less than 3 days; (II) Patients received no interferon or Lopinavir-Ritonavir combined with Ribavirin within 5 days of admission.
In Shandong Province, China, the government organized experts to consult COVID-19 patients. As the head of the Expert Committee on Prevention and Control of Epidemic Infectious Diseases and Emergency Treatment of Traditional Chinese Medicine in Shandong Province, doctors from the a liated Hospital of Shandong University of traditional Chinese Medicine were sent to designated hospitals for consultation. This study was approved by the Research Ethics Commission of A liated Hospital of Shandong University of Traditional Chinese Medicine, which waived the requirement for informed consent, as previously mentioned (KY-2020-001).

Data collection
Case Report Form was used to extract demographic information, comorbidities and clinical records from hospital EMRs. Before data extraction, personal identity information was replaced with codes for privacy concerns. All data were reviewed by two clinicians and a third-party researcher adjudicated any difference in interpretation between the two primary reviewers.

De nitions
The diagnosis and disease severity of COVID-19 were de ned according to the Guideline for COVID-19 Management in China (version 7.0) [6] . Recovery time was regarded as the clinical outcome, de ned as the time from the onset of the disease to the discharge of the patient. Pre-hospital delay was de ned as the time from the onset of the disease to the admission of the patient.

Statistical analysis
All data analysis was run using R software (version 3.6.3) and Rstudio (version 1.2). Measurement data were described as mean±standard deviation or median with interquartile range, whereas count data were presented as numbers and percentages. Shapiro-Wilk test was performed to test the normality. Parametric or nonparametric tests were used to compare the baseline characteristics of COVID-19 patients receiving no treatment combined with Ribavirin and receiving treatment combined with Ribavirin. The linear regression model was used to evaluate the effect of Ribavirin on recovery time. Missing data was processed by Multivariate Imputation (MI) method using mice package (version 3.8.0), in which data is imputed multiple times to create 20 versions of the entire data set.

Baseline characteristics of participants
Of the 418 COVID-19 patients recruited by searching medical records, 342 were enrolled in the study. Table 1 shows no statistical difference between baseline characteristics of the two groups.   Table 3 shows the relationship between Ribavirin and recovery time after adjusting for age, disease severity status and pre-hospital delay. Compared with treatment uncombined with Ribavirin, treatment combined with Ribavirin can shorten the recovery time after adjusting confounding variables.

The results of subgroup analysis
The strati ed analysis of the relationship between Ribavirin and recovery time are presented in Table 4. The test for interactions were not statistically signi cant in age, gender, underlying disease and disease severity status (P for interaction=0.904, 0.240, 0.347 and 0.059, respectively). This negative effect was evident in all subgroups considered except the severe subgroup and after ne adjustments. In the severe subgroup, patients receiving treatment combined with ribavirin had a shorter recovery time than those receiving no treatment combined with Ribavirin, but the difference was not signi cant (β = 2.99, 95%CI =-6.17 to 12.16, p = 0.508).

Discussion
On February 11, 2020, the new coronavirus-infected pneumonia was named COVID-19 by WHO [7] . China's diagnosis and treatment plan "New Coronavirus-infected Pneumonia Diagnosis and Treatment Plan (Trial)" pointed out that the antiviral plan can adopt Alpha interferon Nebulized inhalation and oral administration of Lopinavir/Ritonavir. In the fth edition, Ribavirin was added [4] , and in the sixth edition, Chloroquine Bis and Abidor were added [8] .
The purpose of this study was to explore whether Ribavirin can bene t COVID-19 patients. What is encouraging is that in both unadjusted and adjusted models, the treatment combined with Ribavirin can indeed shorten the recovery time of COVID-19 patients. However, the treatment combined with Ribavirin in severe and critical patients was positively correlated with the recovery time. In addition, age, disease severity status and pre-hospital delay By searching PubMed, we found no clinical studies evaluating the e cacy of Ribavirin on COVID-19. However, the clinical experience of SARS and MERS outbreaks shows that Ribavirin can provide clinical bene ts to patients without septicemia and organ failure [9] .
Ribavirin is a nucleoside analog with broad-spectrum antiviral effect, which was discovered in the 1970s.
During the SARS epidemic in 2003, doctors tried to treat SARS patients with Ribavirin. Poutanen [10] reported on the experience of using Oseltamivir, Ribavirin combined with broad-spectrum antibiotics in the treatment of 7 SARS patients, of which 5 patients improved signi cantly, 1 improved, and 1 died.
Booth [11] conducted a retrospective analysis of the clinical characteristics, treatment, and short-term prognosis of 144 SARS patients. Among them, 126 (88%) treatment regimens included intravenous infusion of Ribavirin. The results showed that patients receiving Ribavirin treatment had obvious improvement, but more adverse reactions occurred.
Ribavirin is also used in the treatment of MERS, though the effect is controversial. In a retrospective cohort study by Omrani [12] , 20 patients with severe MERS were divided into 2 groups. One group received oral Ribavirin combined with subcutaneous injection of polyethylene glycol IFNα2a, and the other group did not use these two drugs. The survival rates of the two groups were 70% (14/20) and 29% (7/14) respectively, with statistically signi cant difference between the two groups(P = 0.004); after 28 days, the survival rates of the two groups were 30% (6/20) and 17% (4/24) respectively, without statistically signi cant difference between the groups (P = 0.54). Morra [13] collected 8 non-randomly controlled MERS treatment studies for meta-analysis. Among 116 patients, 68 were subcutaneously injected with IFN (IFNα2a for 34 cases, IFNα2b for 22 cases, IFNα1a for 12 cases) combined with oral or intravenous infusion of Ribavirin (observation group), and 48 cases did not use IFN and Ribavirin (control group). The mortality rates of the two groups were similar [71% (48/67) to 71% (34/48), P = 1.000], and there was no statistically signi cant difference between the mortality of patients receiving different types of IFN (P = 0.650).
Our analysis shows that patients with non-severe COVID-19 can bene t from the treatment combined with Ribavirin, while severe patients do not.
Lopinavir is an anti-HIV protease inhibitor. Chu [14] reported that Lopinavir and/or Ritonavir have anti-SARS-CoV activity in an in vitro virus sensitivity test; SARS patients were treated with Lopinavir/Ritonavir and Ribavirin, and the incidence and mortality of acute respiratory distress syndrome [2.4% (1/41) and 0 (0/41)] after 21 days of medication were signi cantly lower than that in historically controlled patients treated with Ribavirin alone [22.5 % (25/111) and 6.3% (7/111)], with statistically signi cant difference (both P <0.001).
Bin Cao's [16] study showed that, in the hospitalized severe COVID -19 adult patients, the treatment with Lopinavir/Ritonavir brought no bene ts beyond the standard treatment. 199 patients with laboratorycon rmed SARS- Our study has several limitations. Firstly, this study is a real-world retrospective study, which provides only weak evidence. Secondly, all the data in this study are obtained from EMRs, which limits the collection of confounding variables. Thirdly, due to the limitation of sample size, the evaluation of the e cacy of Ribavirin in severe COVID-19 patients is not accurate. Finally, although MI was used to deal with the missing values, and the results obtained by complete case analysis and multiple imputation analysis were compared, the accuracy of statistical inference in this study was still affected.
In summary, this study shows that interferon or Lopinavir-Ritonavir combined with Ribavirin can shorten the recovery time of patients with non-severe COVID-19. These preliminary data can provide a research basis for Ribavirin in curing COVID-19.
[Authors' contributions] WZ was responsible for the conception and design, analytical plan, critical revision of the manuscript for important intellectual content, approval of the nal version to be published and agreement to be accountable for all aspects of the work. YL and XZ were responsible for the conception and design, drafting of the manuscript, critical revision of the manuscript for important intellectual content, approval of the nal version to be published and agreement to be accountable for all aspects of the work. NS, XJ, XCZ, JH, ZQ, HL, YL and HM collected the data, critically revised the manuscript for important intellectual content and gave nal approval for the version to be published. MY and CY were responsible for the analytical plan, critical revision of the manuscript for important intellectual content, approval of the nal version to be published and agreement to be accountable for all aspects of the work. All authors read and approved the nal manuscript. [Acknowledgements] We thank all the colleagues who have helped with the research data collection. [Funding] Funding was provided by Shandong Province Major Science and Technology Innovation Project (2020SFXGY04-1) and Taishan Scholars (201712096).
[Ethical approval and consent to participate] This article belongs to the nal editorial and is exempt from ethical review.
[Availability of data and materials] The raw/processed data required to reproduce these ndings cannot be shared at this time as the data also forms part of an ongoing study.

[Competing interests]
All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declared that Xinhua Jia, Yingying Liu, Huijie Ma and Zhanjun Qiu are members of Shandong Province's medical team assisting Wuhan; there are no nancial relationships with any organizations that might have an interest in the submitted work in the previous three years; there are no other relationships or activities that could appear to in uence the submitted work.