Bone Marrow Transplantation (2003) 31, 1137–1142 & 2003 Nature Publishing Group All rights reserved 0268-3369/03 $25.00 www.nature.com/bmt post transplant Procalcitonin, C-reactive protein, and endotoxin after bone marrow transplantation: identification of children at high risk of morbidity and mortality from

M Sauer1, K Tiede2, D Fuchs2, B Gruhn2, D Berger3 and F Zintl2

1Department of Pediatrics, Division of Pediatric Hematology, Oncology and Bone Marrow Transplantation, University of Minnesota, MN, USA; 2Children’s Hospital, Friedrich-Schiller-University, Jena, Germany; and 3Department of Surgery, University of Ulm, Germany

Summary: (BMT).3,4 The outcome of sepsis in children undergoing BMT needs to be improved. Novel treatment strategies We prospectively evaluated the capacity of serum have been developed, targeting cytokines as mediators procalcitonin (PCT), compared with serum levels of C- of sepsis.5,6 The paucity of robust, objective criteria reactive protein (CRP) and endotoxin, to identify children by which to judge the efficacy of new approaches has been at high risk for mortality from sepsis after BMT. Of 47 a significant obstacle to conducting clinical trials. The pediatric bone marrow transplantation patients studied, 22 ideal serum biochemical parameter would be specifically had an uneventful course post-transplant (Group 1), 17 elevated during all septic events, the degree of its release survived at least one septic event (Group 2), and eight died would correlate with the severity of the sepsis, and from multiorgan failure (MOF) following septic shock its measurement would be straightforward and (Group 3). Median concentrations of PCT over the course cost-effective. of the study were 1.3, 15.2, and 102.8 ng/ml, respectively, Recently, there has been considerable interest in serum in each of the three groups (Po0.002 for each compar- procalcitonin (PCT) in the setting of infectious and ison). Median concentrations of CRP were 91, 213, and inflammatory conditions. Serum PCT measurement has 260 mg/l, respectively (Po0.001 for Group 1 vs Group 2 allowed discrimination between episodes of fever caused by and Group 3; P ¼ NS for Group 2 vs Group 3). Median , fungi, and parasites from fevers of viral and non- concentrations of endotoxin were 0.21, 0.30, and 0.93 U/l, infectious etiologies.7–9 In addition, serum PCT levels have respectively (P ¼ NS for each comparison). Median been found to correlate with severity in bacterial, fungal, concentrations of PCT, in contrast to serum CRP and and parasitic infections.10,11 endotoxin, correlated with the severity of sepsis (8.2 ng/ml In this study of pediatric BMT recipients, it was our goal in ‘sepsis’ and 22.3 ng/ml in ‘severe sepsis’, P ¼ 0.028) and to address the following questions: (1) Are serum levels of provided useful prognostic information during septic PCT specifically elevated during a septic episode? (2) Does episodes. severity of sepsis correlate with serum levels of PCT? (3) Bone Marrow Transplantation (2003) 31, 1137–1142. How predictive are serum PCT levels for the outcome of a doi:10.1038/sj.bmt.1704045 septic event? We sought to compare serum levels of PCT Keywords: procalcitonin; C-reactive-protein; endotoxin; with those of the acute-phase reactant C-reactive protein sepsis; children; survival; ; ; shock (CRP), the ‘gold standard’ inflammatory marker,12 and serum levels of endotoxin, a known trigger for severe septic events.13 To our knowledge, this is the first study to prospectively evaluate the role of PCT in sepsis risk assessment among children after BMT. The rate of death from severe sepsis ranges from 30 to 50% despite advances in critical care.1 In the United States, approximately 750 000 cases of sepsis occur each year, at Materials and methods least 225 000 of which are fatal.2 Susceptibility to infection has posed one of the most Patients and transplant characteristics formidable challenges in the clinical management of The study was approved by the Institutional Research patients undergoing bone marrow transplantation Board. We enrolled 47 consecutive patients who were admitted for BMT to the Department of Pediatrics, Friedrich-Schiller-University Jena, Germany between Correspondence: Dr M Sauer, Division of Pediatric Hematology, Oncology and Bone Marrow Transplantation, University of Minnesota, 1997 and 1999. Patient characteristics are summarized 420 Delaware Street, Minneapolis, MN, USA in Table 1. All patients older than two years of age Received 28 June 2002; accepted 9 January 2003 receiving allogeneic grafts were conditioned with total Risk assessment for sepsis after bone marrow transplantation M Sauer et al 1138 Table 1 Patient characteristics

No. Age (years) Gender Disease Transplant type Sepsis Survival 1 11 m Acute myeloid leukemia Allogeneic (6/6 urd) Yes Yes 2 20 m Acute lymphoblastic leukemia Allogeneic (6/6 SD) No No 3 13 m Acute myeloid leukemia Autologous (BM) Yes Yes 415 m Acute lymphoblastic leukemia Allogeneic (6/6 SD) No No 5 18 m Acute lymphoblastic leukemia Allogeneic (6/6 URD) Yes Yes 6 15 m Acute lymphoblastic leukemia Allogeneic (6/6 URD) Yes No 7 4m Acute lymphoblastic leukemia Allogeneic (6/6 SD) No No 8 20 m Acute lymphoblastic leukemia Autologous (6/6 URD) No No 9 17 m Acute lymphoblastic leukemia Allogeneic (6/6 URD) Yes Yes 10 24m Acute lymphoblastic leukemia Allogeneic (6/6 URD) Yes Yes 11 8 f Ewing sarcoma Autologous (BM) No No 12 6 f Clear cell carcinoma kidney Autologous (BM) No No 13 9 f Rhabdomyosarcoma Autologous (BM) No No 1413 f Ewing sarcoma Autologous (PBSC) No No 15 19 m Osteoblastic sarcoma Autologous (PBSC) No No 16 14m Ewing sarcoma Autologous (PBSC) Yes Yes 17 16 m Acute myeloid leukemia Allogeneic (6/6 URD) Yes No 18 9 f Acute lymphoblastic leukemia Allogeneic (6/6 SD) No No 19 14f Chronic myeloid leukemia Allogeneic (6/6 URD) Yes No 20 16 m Acute lymphoblastic leukemia Allogeneic (6/6 URD) No No 21 13 f Hodgkin lymphoma Autologous (PBSC) No No 22 24m Medulloblastoma Autologous (PBSC) Yes No 23 4f Acute lymphoblastic leukemia Allogeneic (6/6 SD) Yes Yes 249 f Neuroblastoma Autologous (BM) Yes Yes 25 16 m Acute myeloid leukemia Allogeneic (6/6 SD) No No 26 10 m Acute lymphoblastic leukemia Allogeneic (6/6 URD) No No 27 3 f Neuroblastoma Autologous (BM) Yes Yes 28 16 m Hodgkin Autologous (PBSC) No No 29 12 f Rhabdomyosarcoma Autologous (PBSC) No No 30 4f Wilms tumor Autologous (BM) Yes Yes 31 17 f Ewing sarcoma Autologous (PBSC) No No 32 22 m Acute lymphoblastic leukemia Allogeneic (6/6 URD) No No 33 9 m Acute lymphoblastic leukemia Allogeneic (6/6 SD) Yes Yes 3427 m CMML Allogeneic (6/6 URD) Yes Yes 35 14f Chronic myeloid leukemia Allogeneic (6/6 URD) Yes No 36 1 m Acute myeloid leukemia Allogeneic (6/6 SD) No No 37 10 f Acute lymphoblastic leukemia Allogeneic (6/6 URD) Yes Yes 38 16 m Chronic myeloid leukemia Allogeneic (6/6 URD) Yes No 39 7 m CMML Allogeneic (6/6 URD) No No 40 17 f Acute myeloid leukemia Allogeneic (6/6 URD) Yes No 41 9 f Acute lymphoblastic leukemia Allogeneic (6/6 SD) No No 42 13 m Myelodysplastic syndrome Allogeneic (6/6 URD) No No 43 11 f Severe aplastic anemia Allogeneic (6/6 SD) Yes Yes 44 15 w Chronic myeloid leukemia Allogeneic (6/6 URD) Yes No 45 5 m Neuroblastoma Autologous (BM) Yes Yes 46 14 m Acute myeloid leukemia Allogeneic (6/6 SD) Yes Yes 47 18 f Acute myeloid leukemia Allogeneic (6/6 URD) Yes Yes

6/6 SD=sibling donor matching in A, B, and DRB1; 6/6 URD=unrelated donor, matching in A, B, DRB1. BM=bone marrow PBSC=peripheral stem cells.

body irradiation (total of 1320 cGy over four consecutive (ANC) of at least 2500/ml was attained for three days) and cyclophosphamide (120 mg/kg i.v. over two consecutive days. The day of engraftment was defined as consecutive days); patients under the age of 2 years the first of two consecutive days with an ANC of at least received busulfan (1.0 mg/kg/dose i.v. every 6 h over 2 h 500/ml. All patients received infection prophylaxis consist- on four consecutive days) in place of total body ing of cefazolin, ciprofloxacin, fluconazole, and nystatin. irradiation. Autologous BMT patients received busulfan Patients were switched empirically to broad-spectrum and melphalan-based regimens. Grafts used for the coverage when the axillary temperature equaled allogeneic transplants were T-cell replete. Graft- or exceeded 38.01C. Blood cultures were obtained up to versus-host disease (GVHD) prophylaxis consisted three times a day when axillary temperature exceeded of prednisone, short-course methotrexate, and 38.01C. Antibiotic treatment was adjusted as necessary cyclosporin.14 when positive blood cultures were obtained, retaining Every patient received recombinant human granulocyte broad-spectrum coverage. On the fourth day of persisting colony-stimulating factor (G-CSF; 5 mg/kg/day) from day fevers, therapy with amphotericin B was empirically +1 post-transplant until an absolute neutrophil count initiated.

Bone Marrow Transplantation Risk assessment for sepsis after bone marrow transplantation M Sauer et al 1139 Sampling and clinical data collection levels were deemed ‘significantly elevated’, were used: 1.0 ng/ml, 50 mg/l, and 0.1 EU/ml, respectively.9,12,13 Using Enrolled patients were studied with daily serum sampling these thresholds, sensitivity and specificity were calculated. and detailed clinical evaluation from the day of graft Data were analyzed with a commercial computer soft- infusion (day 0) through day 30 post-transplant. Serum was ware package (SigmaStat version 2.0; SPSS Science). obtained each day from peripheral blood drawn from the Comparisons of group differences for continuous variables central venous catheter at 7:00 a.m. All children were were made using the Kruskal–Wallis ANOVA on ranks. assessed by means of a standardized physical examination Mann–Whitney U-tests were used for pairwise compar- conducted by one of three physicians on a daily basis, isons. A Pp0.05 was considered significant. Results are between 9:00 a.m and noon. Examination findings were given as median concentrations, with ranges between 25th documented in a standardized fashion. and 75th percentiles provided in parenthesis.

PCT, CRP, and endotoxin assays Serum concentrations of PCT were determined by an Results immunoluminometric assay (LUMItest PCT) according to the manufacturer’s specifications (BRAHMS Diagnostica, In total, 47 children were observed for 30 days after BMT. Berlin, Germany). The detection limit was 0.08 ng/ml.15 The observational time period of the first 30 days post- CRP was determined in serum samples by an immuno- transplant was subdivided into 10-day intervals. The very nephelometric method (Beckman Synchron CX-Systems) early interval included days 0–9 where the patients were (REF). Levels o5 mg/ml were considered to be normal.12 expected to develop aplasia, the following interval stretched Endotoxin concentrations were assessed using a quan- from days 10 to 19 when engraftment was most likely to be titative chromogenic limulus amebocyte lysate assay achieved, and the last interval included days 20–30 as a time (QCL-1000, Whittaker M.A. Bioproducts, Walkersville, frame where stable white count recovery was expected. No MD, USA). The detection limit for endotoxin was patients had any measurable white blood cell counts before 0.015 EU/ml.16 day 10 after transplant. All patients achieved stable engraftment (ANC4500/ml) between 15 and 30 days post-BMT. Outcomes and analysis Out of the 47 children, 22 (47%) were not diagnosed with sepsis (Group 1). None of these patients died. In all, 17 Sepsis was defined according to the ACCP/SCCM defini- children (36%) experienced a septic event (Group 2) and tions. The ACCP/SCCM defined sepsis as the systemic subsequently recovered from it. Eight children (17%) were response to infection that is manifested by two or more of diagnosed with sepsis, developed multiorgan failure the following conditions as a result of infection: tempera- (MOF), and died (Group 3). All children with a fatal ture 4381Cor 361C; heart rate 490 beats/min; res- o outcome had engrafted at the time of death. piratory rate 420 breaths/min, or PaCO 32 Torr 2 o The patients with a positive (16 of the 25 ( 4.3 kPa); white blood cell count 412 000 cells/ml, o patients with documented sepsis) are summarized in 4000 cells/ml, or with 410% ‘immature (band) forms’.17 o Table 2. In Table 3, the organisms cultured (180 Gram Adjustments for a pediatric population were made. positive and 68 Gram negative) and the origin of the Respiratory rate and heart rate were reported to be cultures are further specified. consistent with the ACCP/SCCM criteria if they differed from a normal age-matched pediatric population by more than 50%. Since leukocyte count was not an appropriate Serum PCT criterion in the BMT setting, we excluded this criterion from the definition. Over the course of the study, children in Group 1 had a Patients were divided into three groups according to their median serum PCT concentration of 1.3 ng/ml (0.45–2.1 ng/ clinical status over the course of the study. Group 1 ml). Patients in Group 2 had a median PCT concentration consisted of all the children who did not become septic of 15.2 ng/ml (10.4–27.3 ng/ml) (Po0.001). Children in during the observation period. Group 2 included all Group 3 had a median PCT concentration of 102.8 ng/ml patients with sepsis who recovered from this event. Group (16.7–173.2 ng/ml) (Po0.001 vs Group 1; P ¼ 0.002 vs 3 included all children who died during or following a septic episode. For severity of sepsis analyses, patients in Group 2 were further subdivided into a ‘sepsis group’ and a Table 2 Number of patients with positive blood cultures ‘severe sepsis group’. ‘Severe sepsis’ was defined as follows, Clinical status Number of patients Number of patients according to the AACP/SCCM Consensus Conference with a positive blood with a positive blood Criteria of 1992:17 sepsis associated with organ dysfunction, culture for Gram culture for Gram-negative hypoperfusion, or hypotension. Hypoperfusion and perfu- positive bacteria bacteria sion abnormalities may include, but were not limited to, No sepsis (n=22) 0 0 lactic acidosis, oliguria, or an acute alteration in mental Sepsis (n=13) 8 1 status. Severe sepsis (n=4) 3 0 In accordance with published norms, the following cutoff Septic shock with fatal 20 outcome (n=8) levels for serum PCT, CRP, and endotoxin, above which

Bone Marrow Transplantation Risk assessment for sepsis after bone marrow transplantation M Sauer et al 1140 Table 3 Origin of microorganisms cultured 250 Type of organism Origin of positive cultures in absolute numbers Blood Urine Throat Perineal Skin/wound 200 Gram-negative Bacteria 1 20 9 30 8 Staphylococci 9 11 17 12 18 Streptococci 41459 28 8 a Hemolytic 1 3 29 5 2 150 Nonhemolytic 1 0 18 1 0 Enterococci 2 11 12 22 6 Other 2 5 28 12 5 100

Group 2). Within Group 2, there was a significant 50 difference in serum PCT levels between those with ‘sepsis’ Serum procalcitonin levels (ng/ml) and ‘severe sepsis’ (8.2 and 22.3 ng/ml, respectively; P ¼ 0.028) (Figure 1). 0 No Sepsis Sepsis Severe Sepsis Septic Shock With the threshold set at 1.0 ng/ml, serum PCT yielded a and Death sensitivity of 56% and a specificity of 87% to differentiate patients with sepsis from those without sepsis (Table 4). Figure 1 Procalcitonin–serum procalcitonin levels are shown as 25–75th The relative risk of death during a septic episode was 3.7 percentile (box) and 5–95th percentile. Children that recovered from their sepsis (Group 2 in the text, n ¼ 17) were further subdivided into children (95% confidence interval: 2.4–4.8) among patients with with sepsis and children with severe sepsis. Serum PCT concentration PCT serum levels 41.0 ng/ml. Setting the threshold at during sepsis, severe sepsis, and septic shock differed significantly 10.0 ng/ml, the relative risk for a fatal outcome was (Po0.01). increased to 26.4(95% confidence interval: 19.6–31.4). Table 4 Diagnostic performance of the three serum parameters tested Serum CRP PCT CRP Endotoxin Over the course of the study, children in Group 1 had a median serum CRP concentration of 91 mg/l (22 mg/l– Cutoff value 1.0 ng/ml 50 mg/l 0.1 EU/ml 165 mg/l). Patients in Group 2 had a median CRP Sensitivity (%) 56 100 90 concentration of 213 mg/l (153–224mg/l) ( Po0.001). Chil- dren in Group 3 had a median CRP concentration of Specificity (%) 87 41 30 260 mg/l (179–324mg/l) ( Po0.001 vs Group 1; P ¼ NS vs Group 2). Within Group 2, there was no significant Positive predictive value (%) 69 46 20 difference in serum CRP levels between those with ‘sepsis’ Negative predictive value (%) 80 100 88 and ‘severe sepsis’ (Figure 2). With the threshold set at 50 mg/l, serum CRP yielded a sensitivity of 100% and a specificity of 41% to differentiate 450 patients with sepsis from those without sepsis (Table 4). The relative risk of death during a septic episode was 4.0 400 (95% confidence interval: 2.3–6.4) among patients with PCT serum levels 450 mg/l. Setting the threshold at 350 200 mg/l, the relative risk for a fatal outcome remained 300 unchanged. 250

Serum endotoxin 200 Over the course of the study, children in Group 1 had a median serum endotoxin concentration of 0.21 U/l (0.08– 150

0.34U/l). Patients in Group 2 had a median endotoxin Serum CRP levels (mg/l) 100 concentration of 0.3 U/l (0.12–0.82 U/l). Children in Group 3 had a median endotoxin concentration of 0.93 U/l (0.33– 50 1.47 U/l).There was no significant difference between any of the three groups (Figure 3). 0 No Sepsis Sepsis Severe Sepsis Septic Shock With the threshold set at 0.1 U/l, serum endotoxin and Death yielded a sensitivity of 90% and a specificity of 30% to differentiate patients with sepsis from those without sepsis Figure 2 C-reactive protein–serum CRP levels are shown as 25–75th (Table 4). The relative risk of death during a septic episode percentile (Box) and 5–95th percentile. Children that recovered from their sepsis (group 2 in the text, n ¼ 17) were further subdivided into children was 2.2 (95% confidence interval: 1.6–4.1) among patients with sepsis and children with severe sepsis. Serum PCT concentration with endotoxin levels 40.1 U/l. Setting the threshold at during sepsis, severe sepsis, and septic shock did not differ significantly.

Bone Marrow Transplantation Risk assessment for sepsis after bone marrow transplantation M Sauer et al 1141 1.6 and interleukin-8 (IL-8) are potent inflammatory media- tors, and their plasma concentrations have been tested as 1.4 prognostic factors in several studies.26,27 These cytokines have not discriminated reliably between patients with 1.2 infectious and noninfectious diseases. Elevated IL-8 levels were predictive of MOF and an increased likelihood of 1.0 death in one study.18 Van Dissel et al19 examined serum PCT, interleukin-10 (IL-10), and -a 0.8 (TNF-a) among an adult patient population admitted for fever and found PCT to be superior in identifying high-risk 0.6 patients with infection. New treatment strategies are being developed that 0.4 specifically target these sepsis-related cytokine cascades,5 or substitute factors, such as activated protein C, that are Serum endotoxin levels (EU/ml) 0.2 known to be modulators of the inflammation associated with severe sepsis.28 For patients undergoing BMT, such 0 No Sepsis Sepsis Severe Sepsis Septic Shock clinical trials are urgently needed. Major obstacles to the and Death implementation of these studies, however, have been the lack of reliable objective tools for the grading of sepsis, and Figure 3 Endotoxin–serum endotoxin levels are shown as 25–75th percentile (box) and 5–95th percentile. None of the four groups pictured for the determination of efficacy of novel treatments. in this figure was significantly different. Defining ‘sepsis’ and ‘severe sepsis’ in a meaningful way in this pediatric population is exceptionally challenging. In 1992, the American College of Chest Physicians and the Society of Critical Care Medicine published consensus 0.5 U/l, the relative risk for a fatal outcome was increased definitions of ‘severe inflammatory response syndrome’ to 8.1 (95% confidence interval: 6.2–10.6). (SIRS), ‘sepsis’, and ‘severe sepsis’.17 The criteria provided in these consensus definitions provide tools that will not distinguish between fever and sepsis in these cytopenic Discussion patients, let alone sepsis and severe GvHD. Despite these significant limitations, we decided to use these criteria in We have shown that serum PCT, unlike serum CRP or this study because they are the only widely accepted ones. endotoxin, correlates with the severity of sepsis in pediatric Other clinical scoring systems have been developed,6,29,30 HSCT recipients and, furthermore, that it identifies patients but they are highly examiner-dependent, limiting their use at high risk of a fatal outcome. In contrast, serum PCT did in multicenter trials. not prove to be a valuable tool for identifying sepsis at its In conclusion, we have shown that serum PCT correlates onset in these patients, with a sensitivity of only 56%. The with the severity of sepsis among profoundly immunocom- high sensitivity (100%) of CRP in this regard, which is promised pediatric BMT recipients, and that it may reliably consistent with results obtained in immunocompetent18,19 identify children at high risk for mortality. Our findings and incompetent adults,9 must be interpreted in the context suggest that monitoring serum PCT could play a valuable of its relatively low specificity (41%, compared to 87% with role in clinical trials of novel sepsis therapies in this setting, PCT). as an objective tool to grade severity. Serum endotoxin was the least discriminating of the parameters we measured. Its elevation was neither very specific for sepsis nor was it a useful tool for risk assessment. The latter finding contrasts with data from Acknowledgements other groups.13,20,21 There could be two explanations for this. First, only one septic patient had a blood culture We thank Ru¨ diger Volland, Department of Biostatistics, Friedrich-Schiller University Jena, for his expert statistical positive for Gram-negative bacteria. This distribution of advice. We thank Dr Marie Steiner, Department of Pediatrics, infectious agents reflects the shift towards infections caused University of Minnesota, and Dr Ian Thornley, Massachusetts by Gram-positive bacteria increasingly seen in immuno- General and Children’s Hospital, Boston, for a critical review of compromised patients.22 The second reason may be the the manuscript. We are especially indebted to all the nurses of short half-life of endotoxin.23 A short serum endotoxin the Pediatric Bone Marrow Transplantation Unit, Friedrich- peak has been shown to be sufficient to trigger a complex Schiller University Jena for their dedicated care of our young cascade of cytokines that can finally result in severe septic patients. complications.24 If the serum sample is not collected within a very narrow window, endotoxinemia will not be identified. It is possible that we missed this window. References During the last two decades, mounting evidence has suggested that the organ injury and mortality resulting 1 Rangel-Frausto MS, Pittet D, Costigan M et al. The natural from sepsis is to a large extent caused by the explosive history of the systemic inflammatory response syndrome release of inflammatory mediators.25 Interleukin-6 (IL-6) (SIRS). A prospective study. JAMA 1995; 273: 117–123.

Bone Marrow Transplantation Risk assessment for sepsis after bone marrow transplantation M Sauer et al 1142 2 Linde-Zwirble WT, Angus DC, Carcillo JA et al. Age specific with sepsis syndrome. AMCC Sepsis Project Working Group. incidence and outcome of sepsis in the US. Crit Care Med Clin Infect Dis 1998; 27: 582–591. 1999; 27 (Suppl.) A33. 17 Bone RC, Balk RA, Cerra FB et al. Definitions for sepsis and 3 Wald A, Leisenring W, Vanburik JA, Bowden RA. Epide- organ failure and guidelines for the use of innovative therapies miology of aspergillus infections in a large cohort of patients in sepsis. The ACCP/SCCM Consensus Conference Commit- undergoing bone marrow transplantation. J Infect Dis 1997; tee. American College of Chest Physicians/Society of Critical 175: 1459–1466. Care Medicine. Chest 1992; 101: 1644–1655. 4Williamson EC, Millar MR, Steward CG et al. Infections in 18 Harbarth S, Holeckova K, Froidevaux C et al. Diagnostic adults undergoing unrelated donor bone marrow transplanta- value of procalcitonin, interleukin-6, and interleukin-8 in tion. Br J Haematol 1999; 104: 560–568. critically ill patients admitted with suspected sepsis. Am J 5 Knaus WA, Harrell Jr, FE, LaBrecque JF et al. Use of Respir Crit Care Med 2001; 164: 396–402. predicted risk of mortality to evaluate the efficacy of anti- 19 van Dissel JT, van Langevelde P, Westendorp RG et al. Anti- cytokine therapy in sepsis. The rhIL-1ra Phase III Sepsis inflammatory cytokine profile and mortality in febrile patients. Syndrome Study Group. Crit Care Med 1996; 24: 46–56. Lancet 1998; 351: 950–953. 6 Staudinger T, Presterl E, Graninger W et al. Influence of 20 Bayston KF, Cohen J. Bacterial endotoxin and current pentoxifylline on cytokine levels and inflammatory parameters concepts in the diagnosis and treatment of endotoxaemia. J in septic shock. Inten Care Med 1996; 22: 888–893. Med Microbiol 1990; 31: 73–83. 7 Karzai W, Oberhoffer M, Meier-Hellmann A, Reinhart K. 21 Danner RL, Elin RJ, Hosseini JM et al. Endotoxemia in Procalcitonin–a new indicator of the systemic response to human septic shock. Chest 1991; 99: 169–175. severe infections. Infection 1997; 25: 329–334. 22 Anonymous. Vancomycin added to empirical combination 8 Oberhoffer M, Karzai W, Meier-Hellmann A et al. Sensitivity antibiotic therapy for fever in granulocytopenic cancer and specificity of various markers of inflammation for patients. European Organization for Research and Treatment the prediction of tumor necrosis factor-alpha and inter- of Cancer (EORTC) International Antimicrobial Therapy leukin-6 in patients with sepsis. Crit Care Med 1999; 27(9): Cooperative Group and the National Cancer Institute 1814–1818. of Canada-Clinical Trials Group. J Infectious Dis 1991; 163: 9 Chiesa C, Pacifico L, Mancuso G, Panero A. Procalcitonin in 951–958. pediatrics: overview and challenge. Infection 1998; 26: 236–241. 23 Sturk A, van Deventer SJ, Wortel CH et al. Detection and 10 Assicot M, Gendrel D, Carsin H et al. High serum clinical relevance of human endotoxemia. Z Medi Laborator- procalcitonin concentrations in patients with sepsis and iumsdiagnostik 1990; 31: 147–158. infection. Lancet 1993; 341: 515–518. 24Dandona P, Nix D, Wilson MF et al. Procalcitonin increase 11 de Werra I, Jaccard C, Corradin SB et al. Cytokines, nitrite/ after endotoxin injection in normal subjects. J Clin Endocrinol nitrate, soluble tumor necrosis factor receptors, and procalci- Metab 1994; 79: 1605–1608. tonin concentrations: comparisons in patients with septic 25 Kirsch EA, Giroir BP. Improving the outcome of septic shock shock, cardiogenic shock, and bacterial . Crit Care in children. Curr Opin Infect Dis 2000; 13: 253–258. Med 1997; 25: 607–613. 26 Gendrel D, Raymond J, Coste J et al. Comparison of 12 Jaye DL, Waites KB. Clinical applications of C-reactive procalcitonin with C-reactive protein, and protein in pediatrics. Pediatr Infect Dis J 1997; 16: 735–746; interferon-alpha for differentiation of bacterial vs viral [quiz 746-747]. infections. Pediatr Infect Dis J 1999; 18: 875–881. 13 van Deventer SJ, Buller HR, ten Cate JW et al. Experimental 27 Engel A, Mack E, Kern P, Kern WV. An analysis of endotoxemia in humans: analysis of cytokine release and interleukin-8, interleukin-6 and C-reactive protein serum coagulation, fibrinolytic, and complement pathways. Blood concentrations to predict fever, gram-negative bacteremia 1990; 76: 2520–2526. and complicated infection in neutropenic cancer patients. 14Storb R, Leisenring W, Anasetti C et al. Methotrexate and Infection 1998; 26: 213–221. cyclosporine for graft-vs-host disease prevention: what length 28 Bernard GR, Vincent JL, Laterre PF et al. Efficacy and safety of therapy with cyclosporine? Biol Blood Marrow Transplant of recombinant human activated protein C for severe sepsis. N 1997; 3: 194–201. Engl J Med 2001; 344: 699–709. 15 Meisner M, Brunkhorst FM, Reith HB et al. Clinical 29 Castellanos-Ortega A, Delgado-Rodriguez M. Comparison of experiences with a new semi-quantitative solid phase immu- the performance of two general and three specific scoring noassay for rapid measurement of procalcitonin. Clin Chem systems for meningococcal septic shock in children. Crit Care Lab Med 2000; 38: 989–995. Med 2000; 28: 2967–2973. 16 Bates DW, Parsonnet J, Ketchum PA et al. Limulus 30 Goldhill DR, Sumner A. APACHE II, data accuracy and amebocyte lysate assay for detection of endotoxin in patients outcome prediction. Anaesthesia 1998; 53: 937–943.

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