key: cord-0903762-s70oxjui
authors: Giacomelli, Andrea; Pagani, Gabriele; Ridolfo, Anna Lisa; Oreni, Letizia; Conti, Federico; Pezzati, Laura; Bradanini, Lucia; Casalini, Giacomo; Bassoli, Cinzia; Morena, Valentina; Passerini, Simone; Rizzardini, Giuliano; Cogliati, Chiara; Ceriani, Elisa; Colombo, Riccardo; Rusconi, Stefano; Gervasoni, Cristina; Cattaneo, Dario; Antinori, Spinello; Galli, Massimo
title: Early administration of lopinavir/ritonavir plus hydroxychloroquine does not alter the clinical course of SARS‐CoV‐2 infection: a retrospective cohort study
date: 2020-08-10
journal: J Med Virol
DOI: 10.1002/jmv.26407
sha: 22ee9b617f0f09cc1ed1586be2cf8051257f5fa3
doc_id: 903762
cord_uid: s70oxjui

BACKGROUND: As it has been shown that lopinavir (LPV) and hydroxychloroquine (HCQ) have in vitro activity against coronaviruses, they were used to treat COVID‐19 during the first wave of the epidemic in Lombardy, Italy. METHODS: To compare the rate of clinical improvement between those who started LPV/ritonavir (LPV/r)+HCQ within five days of symptom onset (early treatment, ET) and those who started later (delayed treatment, DT). This was a retrospective intent‐to‐treat analysis of the hospitalized patients who started LPV/r+HCQ between 21 February and 20 March 2020. The association between the timing of treatment and the probability of 30‐day mortality was assessed using uni‐ and multivariable logistic models. RESULTS: The study involved 172 patients: 43 (25%) in the ET and 129 (75%) in the DT group. The rate of clinical improvement increased over time to 73.3% on day 30, without any significant difference between the two groups (Gray's test P=0.213). After adjusting for potentially relevant clinical variables, there was no significant association between the timing of the start of treatment and the probability of 30‐day mortality (adjusted odds ratio [aOR] ET vs DT=1.45, 95% confidence interval 0.50‐4.19). Eight percent of the patients discontinued the treatment because of severe gastrointestinal disorders attributable to LPV/r. CONCLUSION: The timing of the start of LPV/r+HCQ treatment does not seem to affect the clinical course of hospitalised patients with COVID‐19. Together with the severe adverse events attributable to LPV/r, this raises concerns about the benefit of using this combination to treat COVID‐19. This article is protected by copyright. All rights reserved.

The current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has seriously affected the public health systems of many countries worldwide (7, 823 ,289 cases and 431,541 deaths as of 16 June 2020) [1] .

Although most SARS-CoV-2 infections are self-limiting, about 15% of infected adults develop severe pneumonia requiring supplementary oxygen treatment, and 5% progress to critical illness requiring intensive care [2, 3] . The pathogenetic mechanisms underlying COVID-19 are still not fully understood, but increasing evidence indicates that the clinical deterioration observed during SARS-CoV-2 infection is attributable to direct viral damage followed by virus-induced immune-mediated injury [4] . The rapid spread and severity of COVID-19 has prompted clinicians to identify possible therapeutic strategies on the basis of experimental data or clinical experiences with other coronaviruses such as severe acute respiratory syndrome (SARS) and Middle Eastern respiratory syndrome (MERS).

In late February 2020, Italy was the first Western country to be hit by the COVID-19 epidemic, with the Lombardy region alone recording 91,917 cases and 16,457 deaths as of 16 June 2020 [5] . During the first weeks of the epidemic, a vademecum was provided by the Lombardy section of the Italian Society of Infectious and Tropical Diseases (SIMIT), proposed the lopinavir/ritonavir (LPV/r) and hydroxychloroquine (HCQ) combination as a therapeutic protocol for hospitalised patients with the respiratory symptoms associated with COVID-19 [6, 7] . This indication was based on experimental studies showing that HCQ (an antimalarial drug that is also widely used to treat autoimmune disorders) has in vitro antiviral activity of against SARS-CoV-1, human This article is protected by copyright. All rights reserved.

coronavirus 229E (HCoV-229E) and SARS-CoV-2 [8] [9] [10] , and it has been postulated that it may benefit patients with COVID-19 because of its modulatory effects on the production and release of tumor necrosis factor 1 (TNF-1) and interleukin-6 (IL-6), both of which are thought to be involved in the inflammatory damage associated with latestage COVID-19 [10, 11] . There were also data indicating that LPV, an HIV-1 aspartate protease, has in vitro activity against SARS-CoV-1 and MERS coronavirus (MERS-CoV) [12, 13] , and a clinical study conducted in Hong Kong in 2003 found that the addition of LPV co-formulated with ritonavir (LPV/r) to a standard treatment protocol (ribavirin plus steroid therapy) was associated with improved clinical outcomes of patients affected by SARS-CoV-1 [14] .

However, very recent studies have questioned the clinical efficacy of LPV/r and HCQ against COVID-19. In particular, one randomised controlled trial comparing the efficacy of LPV/r with that of standard of care in patients with severe COVID-19 did not find any significant differences in mortality, clinical improvement or viral shedding [15] , and an observational study carried out in New York did not find any difference in mortality between severely ill patients with COVID-19 who received HCQ and those who did not [16] . However, neither of these studies considered the possible effect of the timing of the start of treatment, although there is evidence that early treatment is crucial when assessing efficacy against acute respiratory infections [17-20].

The aim of this study was to analyse the combined effect of LPV/r and HCQ treatment on the course of COVID-19 by examining differences in the clinical outcomes of patients who started treatment within five days of the onset of symptoms and those who started later. This article is protected by copyright. All rights reserved.

This retrospective cohort study involved patients with COVID-19 pneumonia who were hospitalised at Luigi Sacco Hospital, Milan, Italy, between 21 February and 20 March 2020. COVID-19 pneumonia was diagnosed on the basis of the detection of SARS-CoV-2 RNA on nasopharyngeal swab using a real-time reverse-transcriptase polymerase chain reaction (RT-PCR) test processed using the automated ELITe InGenius system and the GeneFinder COVID-19 Plus RealAmp Kit assay (ELITechGroup, Puteaux, France) and a chest X-ray with signs of pneumonia or ≤93% oxygen saturation (SpO 2 ) while breathing room air [21] .

In accordance with the SIMIT drug protocol, all patients with COVID-19 pneumonia admitted to our hospital during the study period were offered off-label treatment with LPV/r 400/100 mg (tablet or oral solution) twice daily plus hydroxychloroquine 200 mg twice daily for a minimum of five and a maximum of 20 days depending on patients' clinical response [6, 7] . The exclusion criteria were the presence of any condition that would not allow the treatment to be safely administered (including any known allergy or hypersensitivity to the drugs used in the protocol); severe liver or kidney disease; the use of medications contraindicated with LPV/r that could not be replaced or discontinued; pregnancy or breast-feeding; known HIV infection; a history of cardiomyopathy, arrhythmias or conduction disorders; and a history of ocular macular disease or retinal damage.

The patients were included in the intention-to-treat analysis if they had received at least one dose of the scheduled treatment. Patients who died on the day of starting treatment were excluded from the analysis. This article is protected by copyright. All rights reserved.

The study was approved by hospital's ethical committee (Comitato Etico Interaziendale Area 1) , and all of the study patients gave their written informed consent to the administration of off-label treatment (informed consent was waived in the case of those undergoing mechanical ventilation).

The collected data included demographic data, the Charlson Comorbidity Index (CCI) The secondary outcomes were 30-day mortality and drug safety, including adverse events leading to premature treatment discontinuation. Adverse events were classified using the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0.

The study population was divided into two groups: an early treatment (ET) group of patients who started LPV+HCQ treatment <5 days from the onset of symptoms; and a delayed treatment (DT) group of patients who started treatment ≥5 days from the onset of symptoms.

The baseline demographic and clinical characteristics of the two groups were compared using the χ 2 (or Fisher's exact test where necessary) for categorical variables, and Wilcoxon's rank-sum test for continuous variables. The cumulative incidence of clinical improvement from day 1 (treatment start) to day 30 was estimated using death as a competing event and compared between groups using Gray's test. Uni-and multivariable logistic regression models were used to assess the influence of the timing of the start of treatment on the probability of 30-day mortality. All of the factors judged to be clinically relevant to the study outcome were considered possible confounders in the multivariable model. The data were analysed using SAS software, version 9.4, and a p-value of <0.05 was considered statistically significant. the study period, including remdesivir (n=33, 19.2%), tocilizumab (n=36, 20.9%) or both (n=10, 5.8%). The proportion of patients who received other treatments was not significantly different between the two groups: remdesivir was given to four ET patients (9.1%) and 29 DT patients (22.5%) (p=0.057), and tocilizumab was given to respectively six (13.6%) and 30 patients (23.5%) (p=0.193).

As shown in Figure 1 , the cumulative incidence of clinical improvement increased over time from 36.6% on day 10 to 66.3% on day 20 and 73.3% on day 30, with no significant difference between the two groups (p=0.213) (Fig. 2) .

At the end of the study period, 23.2% of the patients in the ET group and 17% of those in the DT group had died. The univariable analysis did not reveal any significant association between the timing of the start of LPV/r+HCQ treatment and the probability of 30-day mortality (odds ratio [ (Fig. 3) . Conversely, age per ten years more (aOR 2.21, 95%CI 1.38-3.57), obesity (aOR 3.90, 95%CI 1.19-12.82), and undergoing invasive or non-invasive mechanical ventilation (aOR 4.75, 95%CI 1.38-16.34) were all independently associated with an increased probability of death (Fig. 3 ).

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The most frequent adverse events were an increase in hepatic enzymes to at least five times above the normal values (13 patients, 7.6 %), and grade 2-3 nausea and/or diarrhoea (14 patients, 8.1% ). The treatment was discontinued in all of the 14 patients who developed grade 2-3 gastrointestinal disorders.

The spread of the COVID-19 pandemic and the exponential increase in deaths worldwide has made the demand for clinical evidence concerning new and pre-existing drugs increasingly pressing. Various molecules, including antivirals and immune modifiers, were rapidly evaluated in initial uncontrolled studies and are now being investigated in randomised controlled trials.

The search for an effective treatment of COVID-19 also needs to consider the optimal time to start the use of effective drugs, taking advantage of the emerging data concerning the pathogenetic mechanisms underlying different stages of the disease. As it has been shown that the pathogenesis of COVID-19 includes a viremic phase that peaks 5-6 days after infection, followed by an immune-mediated phase characterised by an aggressive inflammatory response that is largely responsible for airway damage [4] , it is possible to hypothesise that the early use of effective antiviral drugs would reduce the progression and mortality of COVID-19, as has been observed in the case of other acute viral respiratory illnesses [17-20]. However, our study assessing possible differences in the clinical outcomes of patients who received LPV/r+HCQ <5 or >5 days after symptom onset did not reveal any difference in the time to clinical improvement or in the probability of 30-day mortality between the two groups. This raises some doubts about the in vivo effect of LPV/r+HCQ This article is protected by copyright. All rights reserved.

treatment on SARS-CoV-2, which are also supported by emerging pharmacological questions. It has been recently estimated that the protein-adjusted 90% inhibitory concentrations (PA-IC 90 ) of LPV required to inhibit SARS-CoV-2 replication in plasma, epithelial lining fluid (ELF) and cerebrospinal fluid (CSF) are respectively 200-fold, 20fold and 2000-fold higher than those measured in vivo [24] . Moreover, a recently published mechanistic model has shown that, instead of the conventional lower dose of ≤400 mg/day, HCQ doses of >400 mg twice daily for ≥5 days would be required to obtain a rapid decrease in viral load, a reduction in the proportion of patients with detectable SARS-CoV-2 infection, and shorter treatment courses [25] , but it has been predicted that doses of >600 mg twice daily would prolong the QT interval and lead to a risk of arrhythmias, including torsade de pointes [25] .

A total of 14 (8.1%) patients in our study were unable to complete the minimum 5-day course of LPV/r+HCQ because of adverse events. The most frequent severe adverse events were gastro-intestinal disorders (nausea and/or diarrhoea) mainly attributed to LPV/r. Interestingly, a recent study has found that the trough concentrations of LPV measured in COVID-19 patients are three times higher than those measured in HIV patients, which may explain why COVID-19 patients poorly tolerate LPV [26] .

Furthermore, Cao et al. found that nearly 14% of the patients who received LPV/r in their randomised trial could not complete the full course of 14 days mainly because of gastrointestinal intolerance [15] and, as they did not find that LPV/r had a beneficial effect on the clinical course of COVID-19, they suggest that its use may expose COVID-19 patients to unnecessary toxicities.

Our study has a number of limitations. Firstly, given the emergency context in which it was carried out, it was impossible to include a control group, and so we cannot exclude the possibility that the patients whose status improved after LPV/r+HCQ treatment 

Coronavirus disease (COVID-19) Situation Report-111. Data as received by WHO from national authorities by 10:00 CEST, 15

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

30-day mortality in patients hospitalized with COVID-19 during the first wave of the Italian epidemic: A prospective cohort study

The trinity of COVID-19: immunity, inflammation and intervention

Covid-19 Regione Lombardia

Linee guida sulla gestione terapeutica e di supporto per pazienti con infezione da coronavirus COVID-19

Lombardy Section Italian Society Infectious And Tropical Diseases. Vademecum for the treatment of people with COVID-19

In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine

Human Coronavirus HCoV-229E Enters Susceptible Cells via the Endocytic Pathway

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

Induction of pro-inflammatory cytokines (IL-1 and IL-6) and lung inflammation by Coronavirus-19 (COVI-19 or SARS-CoV-2): anti-inflammatory strategies

In vitro susceptibility of 10 clinical isolates of SARS coronavirus to selected antiviral compounds

Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus

Treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study

A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19

Observational Study of Hydroxychloroquine in Hospitalized Patients with Covid-19

Clinical management of severe acute respiratory infection when COVID-19 is suspected

Clinical Characteristics of Imported Cases of Coronavirus Disease

Does lopinavir really inhibit SARS-CoV-2?

Optimizing hydroxychloroquine dosing for patients with COVID-19: An integrative modeling approach for effective drug repurposing

Lopinavir/ritonavir in Covid-19 patients: maybe yes, but at what dose?

Accepted Article Disease severity †, n (%)

µg/L 926

PaO 2 , mmHg (n=136)