key: cord-1047375-yi6hd6j8 authors: Raess, Natalie; Schuetz, Philipp; Cesana‐Nigro, Nicole; Winzeler, Bettina; Urwyler, Sandrine A.; Schaedelin, Sabine; Rodondi, Nicolas; Blum, Manuel R.; Briel, Matthias; Mueller, Beat; Christ‐Crain, Mirjam; Blum, Claudine A. title: Influence of Prednisone on Inflammatory Biomarkers in Community‐Acquired Pneumonia: Secondary Analysis of a Randomized Trial date: 2021-05-25 journal: J Clin Pharmacol DOI: 10.1002/jcph.1914 sha: ea5914b09a4848c6af0ff29c076a150d7e3c9ed1 doc_id: 1047375 cord_uid: yi6hd6j8 Glucocorticoids are frequently prescribed in inflammatory diseases and have recently experienced a boom in the treatment of COVID‐19. Small studies have shown an effect of glucocorticoids on inflammatory marker levels, but definitive proof is lacking. We investigated the influence of prednisone on inflammatory biomarkers in a previous placebo‐controlled, randomized‐controlled multicenter trial which compared a 7‐day treatment course of 50 mg prednisone to placebo in patients hospitalized with community‐acquired pneumonia (CAP). We compared levels of C‐reactive protein (CRP), procalcitonin (PCT), leukocyte and neutrophil count between patients with and without glucocorticoid treatment at baseline and on days 3, 5, 7, and at discharge by Wilcoxon tests and variance analysis. 356 patient data sets in the prednisone group and 355 in the placebo group were available for analysis. Compared to placebo, use of prednisone was associated with reductions in levels of CRP on days 3, 5, and 7, (mean difference of 46%, p < 0.001 for each time point). For PCT, no such difference was observed. Leukocyte and neutrophil count were higher in the prednisone group at all time points (mean difference of 27% for leukocytes and 33% for neutrophils, p < 0.001 for all time points). We conclude that after administration of glucocorticoids in CAP, patients had lower CRP levels and increased leukocyte and neutrophil count as compared to the placebo group. PCT levels were not different between treatment groups. PCT levels thus may more appropriately mirror the resolution of infection compared to more traditional inflammatory markers. This article is protected by copyright. All rights reserved Blood biomarkers mirroring severity and resolution of inflammation are commonly used to monitor patients with systemic infections in the in-hospital setting. Traditionally, these markers include leukocyte count and C-reactive protein (CRP), which may not only be influenced by inflammation per se, but also by immunomodulating treatments such as glucocorticoids. [1] [2] [3] Glucocorticoids are frequently prescribed in inflammatory diseases 4 and have recently experienced a boom in the treatment of coronavirus disease 2019 (COVID- 19) . 5, 6 Glucocorticoid-induced leukocytosis has been described as early as 1964. 7 Some trials showed an elevation of neutrophil counts by glucocorticoids, 8, 9 while others reported that neutrophilia is rather caused by the underlying disease. 10 Also, most studies, 3, 9, 11, 12 but not all, 13 show that CRP levels are diminished by glucocorticoid treatment in comparison to placebo. The effect is thought to be mediated by inhibition of IL-6 synthesis, which is a strong CRP stimulator. [14] [15] [16] [17] [18] Another marker of inflammation, procalcitonin (PCT), has been shown to be more specific towards bacterial infections and is thus preferably used for the purpose of antibiotic stewardship. 19 Several small cohorts suggested that in contrast to leukocytes and CRP, glucocorticoid administration seems to have little effects on PCT levels. 2, 3, 13, 20, 21 Yet, these studies were very limited by observational designs, small patient cohorts, or lack of systematic measurement of different markers of inflammation during hospital stay. Therefore, large-scale validation is needed to better understand the influence of corticosteroids on biomarker levels. Herein, we conducted a preplanned secondary analysis in 711 patients of a double-blind, multicenter, randomized, placebo-controlled trial, which originally investigated adjunct prednisone therapy vs. placebo in 785 patients with community-acquired pneumonia (CAP). We aimed to determine the influence of prednisone on the inflammatory biomarkers CRP, PCT, leukocyte and neutrophil cell count, during the course of hospitalization, as compared to placebo. 22 This article is protected by copyright. All rights reserved. The conduct of the trial adhered to the declaration of Helsinki and Good Clinical Practice Guidelines, and the ethical committees of all participating hospitals approved the study before patient recruitment (EKBB, Basel, Switzerland, ethics committee Lausanne, Switzerland, ethics committee Aargau/Solothurn, Switzerland, KEK Bern, Switzerland). Furthermore, all patients, or if not possible their relatives and an independent physician, provided written informed consent before enrollment into this trial. In the cases in which the informed consent was given by their relatives and an independent physician, the written informed consent was seeked from the patients themselves as soon as it was possible. This is a pre-planned subproject of a multicenter, randomized, placebo-controlled trial which was conducted in the following centers in Switzerland: University Hospital Basel, Kantonsspital Aarau, Hôpital du Jura, Delémont, Hospital Liestal and Bruderholz, Kantonsspital Baselland, Bürgerspital Solothurn, and Inselspital Bern. The effect of adjunct prednisone on the time to clinical stability in patients with CAP was investigated. The study protocol of the initial STEP-trial has been published elsewhere. 22 In short, patients presenting with CAP were screened and enrolled at the emergency department or hospital ward in seven tertiary care hospitals in Switzerland between 1 st December 2009 and 21 st May 2014, within 24 hours (respectively 36 hours on weekends) of hospitalization. After informed consent was confirmed, hospitalized patients fulfilling inclusion criteria were randomized to prednisone 50 mg or placebo. Included patients met the following criteria: age over 18 and hospitalized with CAP specified by a new infiltrate on thorax radiograph and the presence of at least one of the following acute respiratory signs or symptoms: cough, sputum production, dyspnea, core body temperature of 38°C or more, auscultatory finding of pathologically breath sounds This article is protected by copyright. All rights reserved. or rales, or leukocyte count over 10 or less than 4 x 10 3 /µl (G/L). 23 Exclusion criteria were: persistent disability to give informed consent, pre-existing need of over 0.5 mg/kg prednisone or equivalent per day, active intravenous drug use, gastrointestinal hemorrhage within the past three months, pre-existing adrenal insufficiency, acute burn injury, pregnancy or breast feeding, or severe immunosuppression (previously known HIV-infection and CD4 cell count < 350 x 10 3 /µl (G/L), immunosuppressive therapy after solid organ transplantation, neutropenia below 500 x 10 3 /µl (G/L) or neutrophil count between 500 and 1'000 x 10 3 /µl (G/L) while ongoing chemotherapy with an expected decrease to counts < 500 x 10 3 /µl (G/L) 24 , active tuberculosis or cystic fibrosis). Allocation of patients was based on a pre-specified computer-generated randomization list and concealed through a centralized password-secured website. Assignment to prednisone or placebo group was done in 1:1 proportion, using variable blocks in size of 4 to 6. Generators and executors of assignment were separated, and patients were randomly allocated to study medication containing either 7 pills of 50 mg prednisone or placebo. Medication was prepared before onset of the study, wrapped up and numbered by the Pharmacology Department of the University Hospital Basel. Attending physicians, patients, examiners and data assessors were blinded to treatment allocation. Figure 1 shows the study flow chart. Subsequently after obtained informed consent, baseline blood samples were taken. Thereafter, study medication was given for seven days either as 50 This article is protected by copyright. All rights reserved. mg prednisone or placebo. Antibiotic treatment was started at the discretion of the attending physician, following the European Respiratory Society (ERS) / European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines adapted for Switzerland. 25 Inflammatory biomarkers were measured during hospitalization on day 1 before randomization and on days 3, 5, 7, and at discharge if it was later than day 7. If the patient was discharged earlier than day 7, no additional follow-up measurements were performed. The objective of this analysis was the preplanned analysis of the effect of prednisone on inflammatory biomarkers, i.e. CRP, PCT, leukocytes and neutrophils during the course of hospitalization. 22 Specifically, we assessed how the inflammatory biomarkers (CRP, leukocytes, neutrophils, and PCT) differed between the two treatment groups when adjusting for differences at day 1. The main primary outcome of the original trial protocol was time to clinical stability according to official guidelines as normalization of body temperature, oxygen saturation, blood pressure and heart rate, as well as resolution of ability to eat and normal mentation. 22, 26, 27 Secondary endpoints were all-cause mortality, time to effective hospital discharge, and side effects of glucocorticoid treatments, among others. The main results have been published elsewhere. 24 In brief, time to clinical stability was shortened by 1.4 days in the prednisone group. Furthermore, time to discharge and duration of intravenous antibiotic treatment were reduced by one day without an increase in CAP complications. This article is protected by copyright. All rights reserved. Calculations were done with R 3.5.1 (R Foundation for Statistical Computing, www.rproject.org). Tests were performed at the two-sided 5% significance level. The data were analyzed according to the per-protocol principle, i.e. patients not adhering to the trial protocol were excluded from the analysis. Binary data are summarized as counts and percentages and continuous data as means and standard deviations (SD). Binary data were analyzed by Fisher's exact or Chi-square test, while continuous data were compared by Wilcoxon rank-sum test. P-values were not adjusted for multiple testing. Data was transformed by natural logarithm (ln) if not following normal distribution. First, we performed a Wilcoxon rank-sum test, comparing means at day 1, 3, 5, 7, and at discharge between the two groups. Second, we performed a variance analysis (ANOVA) of a mixed-model, which accounts for repeated measures. The effects of prednisone, the Pneumonia-severity Index (PSI) 28 at hospital admission, and the number of comorbidities on the biomarkers were tested using an F-test. Significant variables were further investigated in the variance analysis. The mixedmodel consisted of the variable (inflammatory biomarkers) and the tested effects of prednisone, the PSI, and the number of comorbidities as fixed effects and study subject as random effect. This article is protected by copyright. All rights reserved. This analysis contains 711 per-protocol treated patients, including patients with discharge before day 7, as this was allowed by the protocol and mirrors clinical practice. 22 A total of 356 patients were in the prednisone group and 355 in the placebo group (see figure 1 , study flow chart, for details on the inclusion algorithm). In Table 1 , a summary of the baseline characteristics is shown. In brief, 272 (38 %) patients were female, mean age was 69.5 ± 17.3 SD years, and they presented with a median of 1 comorbidity (SD 2). Mean inflammatory biomarker levels in the overall cohort and per randomization group at baseline are also shown in table 1. There was no significant difference in measured inflammatory biomarkers at baseline (= day 1). Figure 2A , CRP levels were not different between groups at day 1 (p = 0.815). We detected lower CRP levels in the prednisone group on days 3, 5, and 7 (overall mean difference of -46%, all p < 0.001). At discharge after day 7, CRP levels did not differ any more (p = 0.548). (Table 2, Figure 2A ). The CRP in PSI classes I-III was not significantly different from PSI classes IV and V (p = 0.361), whereas the patients with 3 comorbidities showed higher CRP levels (mean difference 22.99 %, p=0.009), and those with 5 comorbidities showed lower CRP levels (mean difference -56.64%, p = 0.002). This article is protected by copyright. All rights reserved. As shown in Figure 2B , leukocyte count was not different between groups on day 1 (p = 0.814). We detected higher leukocyte counts in the prednisone group compared to placebo on days 3, 5, 7, and discharge (overall mean difference of +27%, all p < 0.001). (Table 2, Figure 2B ). Leukocyte count was slightly but statistically significantly higher in PSI classes IV and V as compared to PSI classes I-III (mean difference +4.78%, p = 0.034). The number of comorbidities (1 -4: all p > 0.1) showed no association with leukocyte counts, except the three patients with five comorbidities had lower leukocyte counts (p < 0.001), and one patient with six comorbidities had higher leukocyte counts (p=0.035). As shown in Figure 2C , day 1 was not different between groups (p = 0.491). We discovered significantly higher neutrophil counts in the prednisone group on days 3, 5, 7 and discharge (overall mean difference of +33%, all p < 0.001). (Table 2, Figure 2C ). PSI class IV and V had significantly higher neutrophil levels than PSI classes I-III (mean difference + 10.79%, p < 0.001). The number of comorbidities (1 -4: all p > 0.1) did not affect neutrophil levels, except the three patients with five comorbidities had lower neutrophil counts (mean difference -54.74%, p < 0.001), and one patient with six comorbidities had higher neutrophil counts (mean difference 131.11%, p=0.019). This article is protected by copyright. All rights reserved. As shown in Figure 2D , PCT levels were not significantly different between groups in Wilcoxon analysis on day 1 (p = 0.670), day 3 (p = 0.374), day 5 (p = 0.135), and discharge (p = 0.929). The marginal effect on day 7 (p = 0.049) was not confirmed in variance analysis (day 7: p = 0.170, see also Table 2, Figure 2D , and Table S1 in the Supplemental Online Material). In variance analysis, prednisone (all p > 0.1) and comorbidities (all p > 0.1) were not influencing factors, whereas PSI class IV and V had higher PCT levels than PSI classes I-III (mean difference + 39.54%, p < 0.001; see also Table S1 in the Supplemental Online Material for detailed results). Our main finding in this large trial of patients hospitalized with CAP is that prednisone treatment in a dose of 50 mg per day -as compared to placebo -significantly decreased CRP levels, increased leukocyte and neutrophil counts, and had no effects on procalcitonin levels. We showed a lower CRP in the prednisone group at day 3, 5, and 7, as compared to the placebo group. In patients hospitalized longer than the 7-day course of prednisone treatment, CRP levels at discharge were not different from patients in the placebo group, indicating a rebound effect of CRP after stopping prednisone. This analysis confirms the results of several other smaller studies. For example, an experimental endotoxin study showed attenuated CRP reaction after dexamethasone in healthy volunteers. 9 Mysler et al. found reduced CRP levels after prednisone administration in patients with rheumatoid arthritis, 11 and Sin et al. showed a reduction of CRP levels after both oral and inhaled intake of prednisone compared to This article is protected by copyright. All rights reserved. placebo. 12 Previous glucocorticoid administration in the COPD group, possibly also as inhaled glucocorticoids, might have influenced the results of Perren et al, who detected no difference in the decline of CRP levels between prednisone and placebo in 10 COPD and 10 non-COPD patients with CAP. 13 For new antirheumatic agents like anti-IL-6 agents, it has been shown that levels of CRP are diminished due to the lower synthesis of IL-6, which is a strong stimulator of CRP. 15, 16 As glucocorticoids interfere with cytokine synthesis and inhibit IL-6 synthesis, 15, 17, 18 it is plausible that glucocorticoid administration also attenuates CRP levels. This is of important clinical relevance, as changes in CRP level upon glucocorticoid treatment might be misleading if used as a parameter for improvement of CAP. However, there was no difference in recurrent pneumonia or relapse between prednisone and placebo groups. The question of potential relapse after stopping glucocorticoids has been discussed widely due to the results of another, smaller trial of 213 patients, which had shown an increase of CAP relapse after 72 hours in the glucocorticoid group. 29 This finding was not reproduced in an individual patient data meta-analysis including data from both trials. 30 Nevertheless, the meta-analysis indicated an increased risk for CAP-related readmission within 30 days. Therefore, we believe that clinicians should be alert of potential discrepancies of CRP levels and clinical presentation in patients receiving prednisone. We showed a rise of leukocytes in the prednisone group at day 3, 5, 7, and discharge, even after adjusting for disease severity and number of comorbidities. Glucocorticoids in general are known to be able to induce leukocytosis. This has been shown as early as 1964 in a small cohort of 11 patients, 31 and in a larger cohort by Shoenfeld et al. 32 This effect is mainly driven by an elevation of neutrophils. 9 Neutrophils were elevated at day 3, 5, 7 and discharge in the prednisone group. Peretti et al showed that glucocorticoids upregulate Annexin A1 and thereby lead to more neutrophil detachment, less neutrophil transmigration, more neutrophil apoptosis and phagocytosis of This article is protected by copyright. All rights reserved. apoptotic neutrophils. 33 This explains our finding of higher neutrophil counts after glucocorticoid administration. We also detected a small but statistically significant effect of PSI on leukocyte count at admission, which was mainly driven by neutrophil count. Severe pneumonia (PSI class IV and V) caused higher neutrophil counts. Therefore, our results support the conclusion that neutrophil counts are strongly influenced by the underlying disease. 10 We detected no significant effect of prednisone on PCT. We are therefore able to confirm previous results of two small cohorts. 3, 21 The anti-inflammatory action of glucocorticoids acts partially through the induction of Il-1 decoy receptor II, which is expressed in monocytes and neutrophils. 2 The majority of infection-related secretion of PCT is tissue-related and largely independent of leukocytes, 20 but cytokine-mediated through TNF-alpha, IL-2 and IL-6. 34 Apparently, the effect of IL-6 on PCT stimulation is much smaller than in CRP and not of clinical relevance. PCT was largely affected by disease severity, indicating its potential for prognostic use and for antibiotic stewardship. 2, 35 We also confirmed previous studies that the effect of the PSI is stronger for PCT than for CRP or leukocyte count. 36 However, other biomarkers like copeptin or MR-proANP have been shown to have a better prognostic value than PCT. 37 We believe these results have a potentially large impact on current practice. CRP is an important and widely used inflammatory marker. There are estimations that one in five adults in the United States receives a short-time prescription for glucocorticoids within three years, 38 and about 10% of outpatients with acute respiratory tract infections. 39 Therefore, a clinician may often easily be misled by falling CRP levels, believing in treatment response, but really seeing the diminishing effect of glucocorticoids on CRP levels. Procalcitonin may This article is protected by copyright. All rights reserved. be a more suitable and reliable parameter for treatment response patients receiving glucocorticoids. Not all patients had inflammatory marker measurement until day 7 or even later, as many were discharged earlier: median length of stay was 6 days (IQR 4 -9) in the prednisone group and 7 days (IQR 4 -11) in the placebo group. This could also have influenced the measured means, as patients hospitalized for seven days or longer had probably suffered from more severe pneumonia than earlier discharged patients. Due to multiple measurements, there is an increased probability of a type 1 error. However, our multivariate analysis showed that the effect of prednisone on CRP, leukocyte and neutrophil count was independent of PSI or comorbidities. Furthermore, as this was a randomized trial with balanced baseline characteristics, the expected bias would be small. We acknowledge that we did not pro forma separate viral and bacterial CAP. However, an inclusion criterion was the presence of a new infiltrate on chest x-ray or CT scan, thus predominantly including patients with classical pneumonia rather than atypical pneumonia, the latter traditionally indicating either viral pneumonia or atypical bacteria. We have analyzed the microbiology data in another secondary analysis and found a similar distribution of viral pneumonia in the prednisone (12%) and the placebo group (9.6%). 40 Furthermore, it is unknown whether the results of this analysis are also applicable to COVID- Major strengths of this analysis are the large number of patients, the observation of the biomarkers during the course of hospitalization, and the randomized placebo-controlled design. We found diminished CRP levels and increased leukocyte and neutrophil counts during treatment with prednisone compared to placebo in a large-scale cohort of hospitalized CAP patients. PCT levels were not different between treatment groups. Therefore, PCT -in comparison to other inflammatory markers -may be a more suitable and reliable parameter for treatment response in CAP patients receiving glucocorticoids. This article is protected by copyright. All rights reserved. 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