Assessing the Clinical Improvement in Patients with COVID-19 using Lopinavir-Ritonavir: A Systematic Review

Aims: Globally the focus is towards finding an effective treatment for COVID-19 patients in order to suppress the spread of this pandemic disease. An antiviral combination of lopinavir-ritonavir is considered to be effective in treating COVID-19 patients. Therefore, the present study aims to assess the clinical improvements of lopinavir-ritonavir in COVID-19 patients. 
Study Design: a systematic review study was conducted and articles published since December 2019 were included. The statistical analysis of quantitative data was performed using Review Manager (RevMan) to generate forest plots. 
Results: The study showed that there was no significant difference in COVID-19 patients treated with lopinavir-ritonavir or in combination with anti-viral therapy or other conventional methods. Conclusion: the use of lopinavir-ritonavir resulted in greater adverse consequences among COVID-19 patients. It further recommends conducting meta-analysis studies with a greater number of studies to highlight the clinical improvement associated with the use of Lopinavir-ritonavir.


INTRODUCTION
For many years, strains of coronavirus have been circulating in the animal and human populations. The viruses of this family cause respiratory tract infections in humans [1]. Recently, an outbreak of severe acute respiratory syndrome occurred in Wuhan, China in December 2019 which is now commonly known as coronavirus disease   [2]. In March 2020, this outbreak was declared a global pandemic by the World Health Organization [3]. The main symptoms of COVID-19 include cough, fever and shortness of breath [2]. Older individuals and those with underlying health conditions are more susceptible to this disease; therefore, the disease mortality rate is higher in these individuals.
In this age of pandemic, there is a dire need for a safe and effective treatment for COVID-19. A combination of protease inhibitor with nucleoside analogue is known as lopinavir-ritonavir (LPVr) and it is used to treat human immunodeficiency virus (HIV) type 1 [4]. Previously, LPVr has been administered to patients suffering from severe acute respiratory syndrome and it produced promising results. The drug considerably reduced the viral load after 48 hours of administration and the incidence of adverse clinical outcomes also decreased after 21 days [5,6]. Therefore, worldwide clinical trials are being conducted to determine the effectiveness of LPVr as a treatment for COVID-19 and the most prominent of them is the SOLIDARITY and RECOVERY trial being conducted by World Health Organization [7]. Monitoring of treatments is important along with the examination of the benefit-risk profile of all medications. However, some countries are using lopinavir-ritonavir as a standard treatment for COVID-19.
The plasma half-life of this drug is increased by inhibiting cytochrome P450. A previous study suggested adding lopinavir-ritonavir (400 mg and 100 mg respectively) to ribavirin for reducing adverse clinical outcomes such as acute respiratory distress syndrome or SARS [6]. It is difficult to assess the effect of lopinavir-ritonavir because of the concomitant use of glucocorticoids and lack of randomization/contemporary control group. The activity of lopinavir has been observed in an animal model [2] and in vitro [8] for Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Previous studies have also shown virologic clearance and survival of patients after administrating a combination of lopinavirritonavir with ribavirin and interferon Alfa [9][10][11]. Clinical trials have shown promising results for MERS [12,13]; however, there is a lack of studies about the efficacy of this approach in humans [11].
The effectiveness of lopinavir-ritonavir has been observed in several international clinical trials; however, it failed to gain the approval of the Food and Drug Administration as a treatment option in the current COVID-19 pandemic. Consequently, only three pharmacologically different therapies, at the time of writing this work, have been approved to treat COVID-19: immunotherapy (convalescent plasma therapy), antibiotic-hydroxychloroquine and antiviralremdesivir [14,15]. One of the clinical trials conducted for lopinavir-ritonavir showed negative outcomes as severe COVID-19 patients who were treated with lopinavir-ritonavir showed no clinical improvement beyond standard care and reduced mortality rate after 28 days [16]. At present, this medicine is considered as tenable evidence of efficacy because this combination is available in the therapeutic guidelines of countries including the USA [17], Ireland (Health Protection Surveillance Centre Treatment guidelines for COVID-19 in Ireland HPSC 2020) and Saudi Arabia [18]. However, there is a steady emergence of negative and conflicting results about lopinavir/ritonavir combination which highlights the need of assessing its safety and efficacy in treating COVID-19. The current study aims to assess the extent of clinical improvement in COVID-19 patients treated with lopinavir-ritonavir combination by gathering data from published researches.

Search Strategy and Selection Criteria
A systematic review has been conducted considering the basics of Cochrane Handbook for Systematic Reviews of Interventions. Higgins et al [19] as stated by Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) statement [20,21]. Electronic databases including PubMed, Wiley online library, Medline and Embase were searched for selecting articles published between December 2019 and June 2020. The treatment of COVID-19 patients with lopinavir/ritonavir was the focus of this review. The primary outcome was related to the efficacy of lopinavir/ritonavir in treating COVID-19 and the secondary outcome focused on the adverse impact of its administration.
The study selected only readily accessible peerreviewed complete articles, clinical trials and observational cohort studies. There was no age limit for COVID-19 patients to be included in the sample; they just had to be lab-confirmed COVID-19 patients. The keywords used for searching included: COVID-19, novel coronavirus, combination, lopinavir, ritonavir, efficacy, treatment, clinical trial, retrospective, cohort and prospective. The articles including editorials, case reports, duplicate articles, letters to editors and reviews were excluded from the study.

Data Extraction and Analysis
The authors screened the title and abstracts of all the shortlisted articles separately. Full texts of the relevant articles were reviewed for further evaluation. PRISMA diagram was followed to record the inclusion and exclusion of articles ( Fig. 1). The categorization of articles was done on the basis of cohort studies and clinical trials. The data extracted from the selected studies were as follows: author, year of publication, study design and methods, intervention details, control therapies, treatment outcome and adverse events.

Risk of Bias
To undertake the quality assessment of the included studies, the revised Cochrane Risk of Bias Tool was used for randomized controlled studies [22]. Newcastle Ottawa Scale was used for observational cohort studies [23] and ROBINS-I Tool was used for non-randomized interventional studies [21]. Checking was done for appropriate critical appraisal checklists for each study design. The possibility of the bias of these tools was evaluated by the investigators.

Assessment of Heterogeneity
The study used either mean difference or odds ratio to conduct estimations at 95% confidence interval as all the data were continuous. Metaanalysis was performed by using Mantel Haxel Method for dichotomous data and Inverse Variance Method for continuous data in the absence of significant clinical heterogeneity. A random effect model was utilized and conservative approach was employed to produce wider confidence intervals (See Supplemental Data), as compared to the fixed effect model [19]. The statistical analysis was conducted and forest plots were generated by using the Review Manager (Version 5.3, Oxford, UK; The Cochrane Collaboration 2014).

RESULTS AND DISCUSSION
Total 4 literature databases were screened and 65 non-duplicate articles were identified. These articles were further evaluated by screening their titles and abstracts. Among these articles, 25 articles were selected for full-text screening and at the final stage, 11 articles (1192 patients) were chosen for qualitative analysis and six articles (594 patients) were chosen for quantitative analysis ( Table 1).
The comparison of lopinavir-ritonavir with no antiviral therapy on the basis of its safety and efficacy has been shown in six studies [6,[24][25][26][27][28]. Virologic cure was reported by three studies, with n=117 for no antiviral conventional therapy and n=171 for lopinavir-ritonavir [24,25,28]. A significant mean difference was observed in both the treatment modalities considering the virological cure (mean difference=0.71 day; 95% CI, −4.34 to 2.71; P = .006, I2 =70%) (Fig. 2a). The results revealed that administration of lopinavir-ritonavir in comparison with no anti-viral therapy reduced the number of days of the patients' care.
Three of the included studies carried out comparison of lopinavir-ritonavir and umifenovir on day 7 post initiation of the therapy [24,27,28]. Virologic cure was reported by these studies with n=87 for umifenovir and n=127 for lopinavir/ritonavir. A significant mean difference was observed in both the treatment modalities considering the virological cure (mean difference = 0.85 day; 95% CI, −1.01 to 3.00; P = .008, I2 = 48%) (Fig. 2b).
Two of the studies on virological cure conducted comparisons between lopinavir/ritonavir and umifenovir along with lopinavir/ritonavir with respect to their efficacies [25,28]. Virologic cure was reported by these studies with n=75 for umifenovir plus lopinavir/ritonavir and n=93 for lopinavir/ritonavir. A significant mean difference was observed in both the treatment modalities considering the virological cure (mean difference = -0.73 day; 95% CI, −2.35 to 0.68; P = .56, I2 = 0%) (Fig. 2c).
The current study has also focused on the clinical factors that lead to the improvement of symptoms in the COVID-19 patients including the normalization of body temperature, reduction in cough, and improvement in chest CT. The association between time duration and normalization of body temperature was reported by two studies that compared the efficacies of umifenovir (n=71) and lopinavir/ritonavir (n=93) [24,28]. A significant mean difference was observed in both the treatment modalities considering the virological cure (OR = 0.77 day; 95% CI, 0.32 to 1.68; P = .51, I2 = 0%) (Fig. 2d). Similarly, the association between time duration and normalization of body temperature was also reported by two studies that made a comparison between the effects of no antiviral therapy (n=75) and lopinavir/ritonavir (n=93) [24,28]. A significant mean difference was observed in both the treatment modalities considering the virological cure (OR = 0.89 day; 95% CI, 0.39 to 1.89, P = .25, I2 = 0%) (Fig. 2e).
Alleviation in cough was reported by two studies that compared the efficacies of umifenovir (n=71) and lopinavir/ritonavir (n=93) [24,28]. The results revealed a significant decrease in the coughing period after using lopinavir/ritonavir by 0.52 (95% CI 0.05 to 5.43, P = .01; I2 = 71%) (Fig. 3a). Similarly, alleviation in cough was reported by two studies that compared the effect of no antiviral therapy (n=75) and lopinavir/ritonavir (n=93) [24,28]. No significant difference was observed in both the treatment modalities (OR = 0.7 7 days; 95% CI, 0.00 to 27.06; P = .07, I2 = 57%) (Fig. 3b). Decrease in the duration of coughing was, however, observed in comparison with no anti-viral therapy or with umifenovir after the treatment for 7 days. Median time was shortened in the control group from the start of study treatment to obtain negative nasopharyngeal swab.
Mortality rate was zero.
Ye et al. [33] Retrospective cohort study Confirmed cases of COVID-19 treated with lopinavir/ritonavir or not during hospitalization Lopinavir/ritonavir was administered to 42 patients, along with umifenovir and IFN-α1b Umifenovir with FN -α1b was administered to 5 patients only.
Normal body temperature was restored in the patients given the combination of lopinavir/ritonavir, Umifenovir, and IFN-α1b Standard care was provided to the patients, who were in dire need of medical assistance.
Yuan et al. [34] Retrospective cohort study Confirmed cases of COVID-19 presented with fever, diarrhea, and fatigue IFN -α + Lopinavir/ritonavir was given to 46 patients. IFN -α + Lopinavir/ritonavir along with ribavirin was administered to 94 patients. No significant differences observed between different treatment groups.
Majority of the patients were <40 years of age.

. Rate of alleviation in cough; (a) Lopinavir-ritonavir vs umifenovir; (b) Lopinavir-ritonavir vs no anti-viral therapy
No significant difference was observed between the treatment with lopinavir-ritonavir alone and the treatment with umifenovir plus lopinavirritonavir considering the cure from the viral infection after 7 days. However, a study conducted on a small cohort sample showed promising results when a combination of lopinavir-ritonavir and umifenovir was administered [29]. Another study conducted by Lian et al. [30] showed that the duration of the hospital stays increased in patients treated with umifenovir in comparison with other patients. It is known that umifenovir, which is currently used in the treatment of COVID-19 patients, was initially used to treat MERS-CoV and SARS-CoV infections [31].
Radiological progression after the treatment with lopinavir-ritonavir was evident; however, a few patients also showed radiological progression after being treated with umifenovir or anti-viral therapy for 7 days. These results showed no significant difference in all the treatments that include lopinavir-ritonavir, umifenovir, and antiviral therapy. Further, the current study showed that administration of lopinavir-ritonavir in COVID-19 patients caused some adverse effects in them, which were not reported in patients receiving umifenovir or anti-viral treatment. The adverse events associated with the use of lopinavir-ritonavir included vomiting, nausea, acute gastritis, diarrhea, acute kidney injury, and bleeding in gastrointestinal tract [28].
The efficacy of a combination of lopinavirritonavir and IFN-α1b was assessed to test the clinical improvements in COVID-19 patient and the result revealed that inclusion of ribavirin was much safer as compared to the administration of lopinavir-ritonavir alone [32]. Rapid body temperature normalization was observed in patients after the administration of a combination of lopinavir-ritonavir umifenovir and IFN-α1b [33]. However, a decrease in the therapeutic responses was reported in COVID-19 patients in terms of viral clearance after the administration of lopinavir-ritonavir in combination with IFN-α1b. The study conducted by Yuan et al [34] showed that there was no significant difference in IFN-α1b combined with lopinavir-ritonavir or IFN-α1b combined with lopinavir-ritonavir and ribavirin with respect to the average negative conversion time of polymerase chain reaction.
The findings of this study are limited since it included and reviewed only a few studies that investigated clinical improvement in COVID-19 patients. Moreover, on account of large methodological differences, the study failed to assess clinical improvement with respect to the use of lopinavir/ritonavir in combination with other agents or no antiviral therapy or control.

CONCLUSION
The current study revealed no significant clinical improvement in COVID-19 patients after their treatment with lopinavir-ritonavir or other antiviral or conventional treatments. However, this systematic review revealed much greater adverse effects associated with the administration of lopinavir-ritonavir in COVID-19 patients. Considering the study limitation, it is suggested that future studies need to include a greater number of studies with large randomized clinical trials to evaluate clinical improvements in COVID-19 patients after their treatment with lopinavir-ritonavir.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable. and Refad Magadmi for their help in figures and table formatting.