Tocilizumab Association with Emerging Multi-drug Resistance Organisms and Mortality in critically ill patients with Coronavirus disease 2019 (COVID-19): A Multicenter, Retrospective Cohort Study

Background: Tocilizumab is an IgG1 class recombinant humanized monoclonal antibody that directly inhibits IL-6 receptor. Several randomized clinical trials (RCTs) have evaluated tocilizumab’s safety and ecacy in patients with COVID-19. These studies demonstrated conicting results regarding tocilizumab’s ecacy and safety. Our study aim is to determine the association between treatment with tocilizumab and emergence of multidrug-resistant bacteria and its effect on mortality in critically ill patients with Coronavirus disease 2019 (COVID-19). within 1 year, history of antibiotics exposure in the last 3 months, systemic corticosteroids use during ICU and ICU LOS. For the secondary outcomes, a multivariable logistic and generalized linear regression were used to assess the relationship between tocilizumab use and the different outcomes considered in this study after adjusting for the patients' age, SOFA score, PaO2/FiO2 ratio baseline and systemic corticosteroids during ICU. The odds ratios (OR) and estimates with the 95% condence intervals (CI) were reported for the associations.


Background
With the emergence of the novel coronavirus (SARS-CoV-2) in December 2019 in Wuhan, China (1), the world has been dealing with a new highly contagious disease. As of this 04/27/2021, the novel coronavirus disease 2019 (COVID-19) has infected over 148 million people worldwide, with mortality exceeding 3.1 million people (1). Often times, patients hospitalized with COVID-19 present with pneumonia due to excessive host immune response causing an acute respiratory distress syndrome (2). This respiratory distress has been associated with increased intensive care unit (ICU) admission and mortality (3).
The occurrence of severe respiratory distress in critically ill patients is attributed to a state known as the cytokine release syndrome (CRS), where the body produces pro-in ammatory cytokines (TNF-α, IL-1β, IL-2, and IL-6) and chemokines (IL-8) (4). Recent studies have shown that there are increased levels of cytokines, speci cally interleukin 6 (IL-6) in critically ill patients with . This suggests that elevated IL-6 may be a marker for poor prognosis in patients with COVID-19 (4). Thus, the use of therapeutic agents targeting IL-6, such as tocilizumab, in critically ill patients has been investigated (5,6,7,12) Tocilizumab is an IgG1 class recombinant humanized monoclonal antibody that directly inhibits IL-6 receptor (9). Several randomized clinical trials (RCTs) have evaluated tocilizumab's safety and e cacy in patients with COVID-19 (5,6,7,10,11). These studies demonstrated con icting results regarding tocilizumab's e cacy. A randomized controlled trial that included 389 COVID-19 pneumonia patients who did not receive mechanical ventilation (MV) reported a decrease in the likelihood of the progression to MV or death when compared to standard therapy by day-28(6). The bene t of tocilizumab was not demonstrated in randomized controlled trials that included mild to moderate patients with COVID-19 pneumonia. Additionally, tocilizumab was well tolerated compared to standard therapy in most studies (6,7,10).
Although tocilizumab was associated with a lower incidence of serious infections (11), new evidence emerged portraying an increased risk of new Delhi Metallo-Beta-Lactamase-Producing Carbapenem-Resistant Enterobacteria (NDM-CRE) acquisition in patients with COVID-19 (13). The incidence and risk factors for the development of resistant co-infections among patients who receive tocilizumab for COVID-19 was not adequately assessed in previous studies. Thus, we sought to evaluate the incidence of multidrug-resistant bacteria, and its effect on mortality among critically ill patients with COVID-19 who received tocilizumab.

Study design
This was a multicenter retrospective, cohort study evaluating the risk of developing multi-drug resistance organisms (MDRO) in adult COVID19 critically ill patients (aged ≥ 18 years) who received tocilizumab between March 01, 2020 and January 31, 2021. COVID-19 was diagnosed using reverse transcriptasepolymerase chain reaction (RT-PCR) obtained from nasopharyngeal or throat swabs. Patients were excluded if the ICU length of stay (LOS) was less than a day or if they had a "Do-Not-Resuscitate" code status within 24 hours of ICU admission.
Eligible patients were classi ed into two groups based on tocilizumab use during ICU stay. Bacteria and fungus were identi ed in the blood, urine, wound, drainage, cerebrospinal uid (CSF), and respiratory specimens. Microbial isolates de ned as sputum or endotracheal aspiration shows a growth of ≥ 100,000 CFU/mL; bronchoalveolar lavage (BAL) shows a growth of ≥ 10,000 CFU of single organism/mL for protected specimen brushes (PSBs), and ≥ 100,000 CFU of single organism/mL for BAL uid.
Additionally, urine cultures were considered signi cant if showing a growth of ≥ 100,000 CFU/mL of no more than two species of microorganisms 13

Data collection
Demographic data, comorbidities, vital signs and laboratory tests, severity scores (i.e., Acute Physiology and Chronic Health Evaluation II (APACHE II), Sequential Organ Failure Assessment (SOFA) scores), Glasgow Coma Score (GCS), acute kidney injury (AKI), the needs for MV and MV parameters (e.g., PaO 2 /FiO 2 ratio, FiO 2 requirement) within 24 hours of ICU admission were collected from the electronic health record (See additional le 1). Additionally, renal pro le, liver function tests (LFTs), coagulation pro le (i.e., INR, aPTT, brinogen), and in ammatory markers (CRP, procalcitonin) within 24 hours of ICU admission were collected. Moreover culture information, including the presence of resistant organisms were collected during the ICU stay. Tocilizumab and systemic corticosteroids use were recorded for the eligible patients. All patients were followed until they were discharged from the hospital or died during the in-hospital stay, whichever occurred rst.

Outcomes
The primary outcomes were to evaluate the incidence of microbial isolation, resistant organisms, and carbapenem-resistant Enterobacteriaceae (CRE) in COVID-19 critically ill patients who received tocilizumab. Secondary outcomes included 30-day ICU mortality, in-hospital mortality, hospital LOS, ICU LOS, MV duration, and ICU-related complications during ICU stay (i.e. acute kidney injury, acute liver injury, respiratory failure requires MV, thrombosis/infraction).

De nition (s)
Multidrug-resistant (MDR) is non-susceptibility to at least three or more agents, extensively drugresistant (XDR) is non-susceptibility to at least one agent in all but two or fewer antimicrobial categories remain susceptible, pandrug-resistant (PDR) is non-susceptibility to all agents in all antimicrobial categories. Susceptibility of gram-positive/negative bacteria created using documents and breakpoints based on Clinical Laboratory Standards Institute (CLSI) (15,18).
Respiratory failure was de ned as one of following types; hypoxemic respiratory failure with PaO2 < 60 mm Hg and normal or low arterial carbon dioxide tension or hypercapnic respiratory failure (PaCO2 > 50 mm Hg) that requires mechanical ventilation (18).
Acute liver injury was de ned as alanine aminotransferase (ALT) exceeding three times the upper limit of normal or double in patients with elevated baseline ALT(18).

Data management and Statistical analysis
Categorical data were expressed as number and percentage, continuous variables were expressed as mean and standard deviation (SD), or median and lower quartile (Q1) and upper quartile (Q3), as appropriate.
Categorical variables were analyzed using Chi-square or Fisher's exact test, and continuous variables were analyzed using student t-test or Mann-Whitney U test, as appropriate. A multivariable logistic regression was used to evaluate the primary outcomes after adjusting for possible co-founders including patient's comorbidities (i.e. diabetes mellitus (DM), chronic kidney disease (CKD) on dialysis), history of hospitalization or invasive procedure (surgery) within 1 year, history of antibiotics exposure in the last 3 months, systemic corticosteroids use during ICU and ICU LOS. For the secondary outcomes, a multivariable logistic and generalized linear regression were used to assess the relationship between tocilizumab use and the different outcomes considered in this study after adjusting for the patients' age, SOFA score, PaO2/FiO2 ratio baseline and systemic corticosteroids during ICU. The odds ratios (OR) and estimates with the 95% con dence intervals (CI) were reported for the associations.
We assessed model t using the Hosmer-Lemeshow goodness-of-t test. No imputation was made for missing data as the cohort of patients in our study was not derived from random selection. We considered a p-value of < 0.05 to be statistically signi cant. All statistical analyses were carried using SAS version 9.4 for all statistical analyses.

Results
A total of 738 patients met our inclusion criteria and were included in the analysis. Of these 262 (35.5%) received tocilizumab and 476 (64.5%) did not receive tocilizumab and were included in the control group.

Demographic and Clinical Characteristics
For all patients, the mean (SD) age was 61 years (14.7), with the majority of patients being male (72.1%). There were some notable differences between the two groups. Patients who received tocilizumab were younger in age (59.2 vs. 61.9 years, p = 0.014), had higher C-reactive protein (CRP) (167 vs. 143, p < 0.001), had lower median PaO 2 /FiO 2 ratio within 24 hours of admission (75.5 vs. 87.8, p < 0.001) and were more likely to have received systemic corticosteroid use in the ICU (93.4% vs. 84.9%, p < 0.001).
Patients who received tocilizumab were less likely to have a history of hospitalization or surgery within a year (7.4% vs. 20.5%, p < 0.001) compared with the control group, respectively (Additional le 1 - Table 1). Additionally, prevalence of diabetes, chronic kidney disease, and ischemic heart disease were signi cantly lower in the tocilizumab group compared with the control group (Additional le 2 - Table 2).
Among non-mechanically ventilated patients within 24 hours of ICU admission, there was a signi cant difference in the incidence of respiratory failure requiring MV between the two groups (OR 2.27; 95% CI, 1.05-4.89 p = 0.03). On the ip side, there were no signi cant differences between the control and tocilizumab groups in terms of incidence of acute kidney injury, thrombosis, and duration of mechanical ventilation during ICU stay after adjusting for possible confounders ( Table 2). The follow-up in ammatory markers were statistically signi cant among the groups, D-dimer and iron were higher in tocilizumab group but brinogen was lower in tocilizumab group. CRP and procalcitonin were similar among the groups, as described in Table 3.

Discussion
In our retrospective cohort study of critically ill COVID-19 patients, we found that patients who received tocilizumab did not have a signi cantly higher risk of acquiring multidrug-resistant bacteria such as MDR, XDR, PDR nor had higher risk of CRE. Moreover, the tocilizumab group had similar in-hospital mortality, 30 day ICU mortality, days on MV during ICU stay, ICU length of stay, and hospital length of stay to those who did not receive tocilizumab. However, patients who received tocilizumab did have a signi cantly higher risk of respiratory failure that required MV.
Our primary outcomes ndings were observed after adjusting for the patient's comorbidities, history of hospitalization or invasive procedure within one year, history of antibiotics exposure in the last three months, systemic corticosteroids during ICU, and ICU LOS. The nding of no difference in secondary infections rate is similar to previous studies that conducted adjusted matched analyses for tocilizumab and controls and showed no difference between the arms only (26 in the treatment arm, 25 in the control arm; 95% CI for difference, − 0.15 to 0.13; P = 1.00) (20,21). This was also demonstrated in another multicenter cohort study of 4485 adults with COVID-19 admitted to (ICUs) has found no differences in secondary infection rate (140 [32.3%] vs 1085 [31.1%]) (22,23 ). This is also concurrent with a recent meta-analysis that evaluated tocilizumab use in critically ill patients and found no difference in secondary infection between tocilizumab and the control group (24). However, other published trials found lower rates of secondary infections with tocilizumab (7,10,11), while another study found higher rates of secondary infections with tocilizumab (25). The differences in outcomes in these studies could be attributed to the unadjusted analysis in some studies and if studied on critically ill patients or other hospitalized patients as critical care settings are more prone to infections. Further randomized studies are needed to con rm these ndings.
We found no signi cant difference in 30-day ICU and in-hospital mortality rates between the two groups. Previous studies depicted mixed results in the mortality outcome. In one randomized control study comparing tocilizumab with usual care in non ICU COVID-19 patients with moderate to severe pneumonia, there was also no difference in mortality at 28-days (10). Similarly, a recent meta-analysis in ICU patients found no signi cant difference in mortality rates between the tocilizumab group and the comparator groups (24). Alternatively, a previous retrospective cohort study in ICU COVID-19 patients found signi cantly lower ICU mortality in the tocilizumab group but no difference in the 28-day mortality (23). Another retrospective cohort study for non-ICU patients found lower mortality with tocilizumab (25). In the recent RECOVERY trial that included non-ICU COVID-19 patients, there was a signi cant lower 28-days mortality in the tocilizumab group compared with placebo. However, it is important to note that this bene t may be attributed to the combination with corticosteroids, as more than 80% on included patients were recieveing corticosteroids at the same time, a confounder that we took in consideration and adjusted for it (26).
Furthermore, a randomized clinical trial evaluated tocilizumab versus usual care in ICU patients with COVID-19 and found no signi cant difference in 15-day, in-hospital and 28-days mortality between the two groups. However, this trial was prematurely interrupted after the rst interim analysis due to an excess number of deaths at day 15 in the tocilizumab group (27). Overall, the mortality bene t with tocilizumab in an ICU setting is still debatable, but other potential clinical bene ts could justify its use and improve COVID-19 patients course of illness.
Intersteningly, our study demonstrated that the progression of care towards the use of mechanical ventilator was higher among patients who received tocilizumab. This could be due to the increase in IL-6 levels and the cytokine storm shortly after the infusion, which was associated with worsening PaO 2 /FIO 2 ratio and decline in the lung function as reported by Rossotti R,et al (28). These nding are contradicted by the result of two randomized controlled trials conducted by Hermine O, et al (10) and Salama C, et al (6), which showed that the use of tocilizumab was associated with reduced risk of intubation and need for mechanical ventilation.
In this study, tocilizumab use was not associated with any statistically signi cant differences in the ICU and hospital length of stay. In contrast to our ndings is the reports from COVACTA trial (5) were they found that the median time until patients were discharged from the hospital or was 20 days in the tocilizumab group and 28 days in the placebo group. Also, they report shorter ICU Los, the median duration of ICU stay was 9.8 days in the tocilizumab group and 15.5 days in the placebo group, for a difference of 5.8 days.
Among patient who received tocilizumab, we found differences in in ammatory markers levels compared with the control group. The D-dimer levels were higher in the tocilizumab group compared with placebo. In line with this results, Rossotti et al. found that d -dimer rise by day 5, then it decreased but without returning to baseline values (28). This nding is consistent with previous observational studies (30,31) were C-reactive protein, IL-6 improved; however, D-dimer levels increased signi cantly and is concerning as it might increase the risk of thromboembolic events, which is increased among patient with covid-19 (32). Fibrinogen levels during ICU stay were lower in the tocilizumab group, which was expected as tocilizumab tends to decrease in ammatory markers.
Our study has some limitations that need to be addressed. The retrospective and observational nature of the study puts the study at risk for residual confounding, despite adjustment for possible confounders.
Furthermore, the decision to prescribe tocilizumab to COVID-19 patients was guided by the institutional and the Ministry of Health treatment protocols which continued to change with the emergence of new data. Moreover, the subsequent doses of tocilizumab and the timing between them could affect some of the clinical outcomes, which warrant further studies.

Conclusion
Overall, our study demonstrated that tocilizumab use in critically ill COVID-19 patients was not associated with different incidence of microbial isolation , emergence of resistant organisms, detection of CRE organisms, or mortality bene ts. Tocilizumab was associated with an increased risk of respiratory failure requiring mechanical ventilation. Further randomized clinical and interventional studies are required to con rm our ndings.

Funding
None.

Availability of data and material
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Participants' con dentiality was strictly observed throughout the study by using the anonymous unique serial number for each subject and restricting data only to the investigators. Informed consent was waived because of the study's retrospective nature, and the analysis used anonymous clinical data as per the policy of the local research center.