SARI HÄMÄLÄINEN

Severe Sepsis in Neutropenic

Haematological Patients

ISBN 978-951-27-1372-1ISBN 978-951-27-1389-9 (PDF)ISSN 1235-0303

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KUOPION YLIOPISTON JULKAISUJA D. LÄÄKETIEDE 472 KUOPIO UNIVERSITY PUBLICATIONS D. MEDICAL SCIENCES 472

KUOPION YLIOPISTON JULKAISUJA D. LÄÄKETIEDE 472KUOPIO UNIVERSITY PUBLICATIONS D. MEDICAL SCIENCES 472

SARI HÄMÄLÄINEN

Severe Sepsis in NeutropenicHaematological Patients

Doctoral dissertation

To be presented by permission of the Faculty of Medicine of the University of Kuopio for public examination in Conference Hall, Olavinlinna Castle, Savonlinna

on Monday 21st December 2009, at 12 noon

Department of MedicineKuopio University Hospital and

University of Kuopio

KUOPIO 2009

Distributor: Kuopio University Library P.O. Box 1627 FI-70211 KUOPIO Tel. +358 40 355 3430 Fax +358 17 163 410 http://www.uku.fi /kirjasto/julkaisutoiminta/julkmyyn.shtml

Series Editors: Professor Raimo Sulkava, M.D., Ph.D. School of Public Health and Clinical Nutrition

Professor Markku Tammi, M.D., Ph.D. Institute of Biomedicine, Department of Anatomy

Author´s address: Department of Medicine Kuopio University Hospital P.O.Box 1777 FI-70211 KUOPIO Tel. +358 17 173 311 Fax +358 17 173 931

Supervisors: Docent Esa Jantunen, M.D., Ph.D. Department of Medicine University of Kuopio and Kuopio University Hospital

Auni Juutilainen, M.D., Ph.D. Department of Medicine University of Kuopio and Kuopio University Hospital

Irma Koivula, M.D., Ph.D. Department of Medicine Kuopio University Hospital

Taru Kuittinen, M.D., Ph.D. Department of Medicine Helsinki University Central Hospital

Reviewers: Docent Esa Rintala, M.D., Ph.D. Department of Medicine Satakunta Central Hospital

Sari Karlsson, M.D., Ph.D. Department of Medicine Tampere University Hospital

Opponent: Docent Veli-Jukka Anttila, M.D., Ph.D. Department of Medicine Helsinki University Central Hospital

ISBN 978-951-27-1372-1 ISBN 978-951-27-1389-9 (PDF)ISSN 1235-0303

Suomen Graafi set Palvelut Oy LtdKuopio 2009Finland

Hämäläinen, Sari. Severe Sepsis in Neutropenic Haematological Patients.Kuopio University Publications D. Medical Sciences 472. 2009. 99 p.ISBN 978-951-27-1372-1ISBN 978-951-27-1389-9 (PDF)ISSN 1235-0303

ABSTRACT

Neutropenic fever and septic infections are common in patients with acute myeloidleukaemia (AML) and in autologous stem cell transplant (ASCT) recipients. Severe sepsis isimportant cause of treatment-related morbidity and mortality in both patient groups. Readilyavailable circulatory markers for severe sepsis could be useful.

This study evaluated epidemiology, microbiology and outcome of neutropenic fever andsevere sepsis in haematological patients with special reference to the kinetics of C-reactiveprotein (CRP), vascular endothelial growth factor (VEGF) and amino-terminal pro-brainnatriuretic peptide (NT-proBNP).

Retrospective series included 84 AML patients and 319 ASCT recipients. In theprospective series of 70 patients with the same diagnoses CRP, VEGF and NT-proBNP weredetermined at the beginning of the neutropenic fever and then daily.

In retrospective series, severe sepsis was found in 13% of AML patients and in 5% ofASCT recipients. In the prospective study, severe sepsis was found in 14% of febrile periods.Severe sepsis was associated with 27% mortality in AML patients and 53% in ASCTrecipients. In the prospective study including careful supportive care, the mortality rate forsevere sepsis was 15%. Gram-positive microbes were more common in blood cultures than gram-negative ones.During 1996-2006 Pseudomonas spp. bacteraemia was found in 30% of neutropenic periodswith severe sepsis in ASCT recipients. VEGF concentrations were generally low. Less than 24 hours after the start of neutropenicfever the VEGF concentrations were higher in patients with severe sepsis than those without.Neither serial NT-proBNP nor CRP showed early predictive value for development of severesepsis.

Despite of intensive interventions, mortality remained high in haematological patients whodeveloped severe sepsis. Higher CRP values coincided with severe sepsis but could notpredict it. VEGF was a more rapid indicator for severe sepsis than CRP. Neither serial NT-proBNP nor CRP showed early predictive value for development of severe sepsis. Severe sepsis is frequent in neutropenic haematological patients and is associated withsignificant mortality. Careful patient monitoring and supportive care are needed to improvethe outcome of severe sepsis in haematological patients.

National Library of Medicine Classification: WC 240, WH 120, WH 250, QU 325, QZ 350Medical Subjects Headings: Sepsis; Bacteremia; Neutropenia; Fever; Epidemiology;Microbiology; Hematologic Diseases; Leukemia, Myeloid, Acute; Stem Cell Transplantation;Transplantation, Autologous; C-Reactive Protein; Vascular Endothelial Growth Factor A;Natriuretic Peptide, Brain; Mortality; Gram-Positive Bacteria; Pseudomonas; Monitoring,Physiologic.

Pieni ja hento ote ihmisestä kiinni Aivan sama tunne kuin koskettava tuuli Pieni ja hento ote - siinä kaikki

Dave Lindholm

ACKNOWLEDGEMENTS

This thesis was carried out in the Department of Medicine at Kuopio University Hospitalbetween 2006 and 2009. I owe my sincere thanks to Professor Leo Niskanen and DocentSeppo Lehto, Chief Physician of the Department of Medicine, Kuopio University Hospital forarranging me facilities to perform this study. I also wish to express my warmest thanks toMatti Huttunen, Chief Physician of the Department of Medicine, Savonlinna Central Hospitalfor providing me with the opportunity to carry out this study.

I am most grateful to the supervisors of this thesis.

Docent Esa Jantunen who considered his task in coordinating these studies with unfailingexpertise and enthusiasm. He has taught me the fundamentals of scientific writing, and,subsequently, has spent numerous hours revising my manuscript. I admire him for hispositive attitude and availability in all phases of writing this thesis. These qualities and hisunfaltering support have been invaluable throughout this process.

Taru Kuittinen, M.D., PhD., who guided me through the very first steps in statistical andscientific analysing. As a mentor and a supervisor, Taru has been demanding yet alwayskind and friendly. Without her bright attitude, endless support and encouragement when it ismost needed, this work would never have been completed.

Auni Juutilainen, M.D., PhD., who took the responsibility for statistical analyses during theprospective phase of this study. Her intelligent and analytical guidance has been preciousand simply indispensable in writing of this thesis. I admire her unfailing logic, and, at thesame time, her sophisticated and academic approach. In many ways Auni has been the soulof this thesis, and a loyal mentor regardless of time of day.

Irma Koivula, M.D., PhD., who has been my principal teacher in infectious diseases. I greatlyadmire her unbeatable logic and sharp scientific thinking. Irma has provided me with thefacilities to continue this study in the middle of clinical whirl, and without her continuoussupport, this study would have never been made.

Yet more than challenging moments, we have enjoyed laughter, long lasting dinners andsome sparkling wine together. Dear friends, this has been a privilege

I wish to express my warmest thanks to co-authors Professor Esko Ruokonen and DocentTapio Nousiainen, M.D. My thanks go especially to Professor Kari Pulkki and Docent IrmaMatinlauri for their persistency and kindly offered guidance in matters of clinical chemistryand laboratory techniques.

I wish to express my gratitude to the official reviewers of this dissertation, Docent EsaRintala, M.D., and Sari Karlsson, M.D., PhD., for their strict criticism, constructive commentsand valuable advice during the final phase of this thesis. This thesis has substantiallyimproved thanks to their contribution. My warmest thanks to Docent David Laaksonen, M.D.,for proof-reading this thesis.

I am most grateful to the nursing staff at the haematology ward of Kuopio University Hospitalfor their accurate patient monitoring and tireless updating of the patient records. I admiretheir strenuous work in nursing of critically ill patients, especially when there is very little hopeleft. I owe much to Ms Tuulikki Pelkonen and Ms Tuija Nenonen for their administrativesupport always offered with patience. I warmly thank Ms Seija Laitinen, RN, chief medical

laboratory technologist, for performing the VEGF analyses, and our unofficial study nurse MsRaija Isomäki for her unfailing help and support.

I want to express my deepest gratitude to all our study patients. Your willingness toparticipate in this study during a very difficult period in your lives has been simply admirable.

I am privileged to have had this opportunity to work and study medicine in several Finnishhospitals. I want to thank all friends and colleagues at Jyväskylä Central Hospital, at KuopioUniversity Hospital, at Helsinki University Hospital and at Oulu University Hospital for sharingthis journey with me. Especially I want to thank colleagues at Savonlinna Central Hospital formany unforgettable moments in clinical practise and, in particular, around the lunch table.Jaana and Mika, this is for you.

I want to thank my friends for being there for me when I was writing this thesis, and I hopeyou will be there in future, too. To Eija for being a truthful friend and a teacher in practise ofinfectious disease. To Ulla-Maija and her family for their ex tempore dinner parties and theendless conversations we shared. To Merja for early mornings in the swimming pool andsupportive coffee breaks. To Marjut and her family for many exhilarating moments atTaiteiden yö happening in Helsinki, and for being a friend in truly difficult times. To Tarja andall her boys for their tales and laughter as well as for speedy and dangerous situations! ToHelena for being a colleague and a teacher during the very first steps in my career as adoctor as well as for all the heavy metal concerts and horseback riding in Ireland! To all myfriends at the stables and yards, especially to Saila and her family for raising a foal togetherbetween spinning lessons. To Salla and her family for having being both a riding instructorand a loyal friend for over a decade now. To Selina and her family for being there both for thepony, Juulia and myself.

Above all, my warmest thoughts belong to my family. To my mother Ritva and my step-fatherPaavo for helping out in everyday crisis, always having the coffee ready and keeping thepugs fat. For my brother Janne and his family for practical guiding in everyday life. Andspecial thanks to my nephew Juuso for helping out with the typing, and being the bestgodson ever. To my niece Juulia for ceaselessly bringing sunshine and laughter into my life.And last but not least, to my sarcastic sister Sanna for splendid holidays and priceless cross-border caring of my pugs.

The study was financially supported by grants from the Finnish Society of Haematology,Blood Research Foundation, Finnish Cultural Foundation and EVO funding of KuopioUniversity Hospital.

Kuopio, December 2009.

ABBREVIATIONS

AML acute myeloid leukaemiaASCT autologous stem cell transplantationAUC area under curveBEAC carmustine, etoposide, cytarabine and cyclophosphamideBEAM carmustine, etoposide, cytarabine and melphalanBNP brain natriuretic peptideCD4 helper T-cellCD8 cytotoxic T-lymphocyteCI confidence intervalCLL chronic lymphocytic leukaemiaCRP C-reactive proteinEBMT the European Group of Blood and Marrow TransplantationESBL extended-spectrum betalactamaseELISA enzyme-linked immunosorbent assayHDT high-dose chemotherapyHL Hodgkin lymphomaIL interleukinICU intensive care unitIDSA Infectious Diseases Society of AmericaMM multiple myelomaMOF multiorgan failureMRSA methicillin-resistant Staphylococcus aureusNHL non-Hodgkin lymphomaNT-proBNP amino-terminal pro-brain natriuretic peptidePaC02 arterial partial pressure of C02

PAMPs pathogen-associated molecular patternsPCR polymerase chain reactionPCT procalcitoninROC receiver operating characteristic curveSIRS systemic inflammatory response syndromeSOFA sepsis-related organ failure assessmentTh1 type-1 helper T-cellTh2 type-2 helper T-cellTLRs toll-like receptorsSPSS statistical package for the social sciencesS-TnT serum cardiac troponin TTNF- tumor necrosis factor-alphaVEGF vascular endothelial growth factorVRE vancomycin-resistant enterococci

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following papers, which will be referred to by their Roman

numerals.

I. Hämäläinen S, Kuittinen T, Matinlauri I, Nousiainen T, Koivula I, Jantunen E.

Neutropenic fever and severe sepsis in adult AML patients receiving intensive

chemotherapy: causes and consequences. Leuk Lymphoma. 49:495-501, 2008

II. Hämäläinen S, Kuittinen T, Matinlauri I, Nousiainen T, Koivula I, Jantunen E.

Severe sepsis in autologous stem cell transplant recipients: microbiological

aetiology, risk factors and outcome. Scand J Infect Dis. 41:14-20, 2009

III. Hämäläinen S, Juutilainen A, Matinlauri I, Kuittinen T, Ruokonen E, Koivula I,

Jantunen E. Serum vascular endothelial growth factor in adult haematological

patients with neutropenic fever: a comparison with C-reactive protein. Eur J

Haematol. 83;251-257, 2009

IV. Hämäläinen S, Juutilainen A, Kuittinen T, Nousiainen T, Matinlauri I, Pulkki K,

Koivula I, Jantunen E. Serum amino-terminal pro-brain natriuretic peptide in

haematological patients with neutropenic fever: a prospective comparison with

C-reactive protein [Submitted]

CONTENTS

1. INTRODUCTION ....................................................................................................................15

2. REVIEW OF THE LITERATURE .............................................................................................16

2.1. Infections in neutropenic haematological patients ..........................................................152.1.1. Bloodstream infections in patients with neutropenic sepsis .............................172.1.2. Infectious complications in patients with acute myeloid leukaemia ..................192.1.3. Infectious complications in autologous stem cell transplant recipients .............20

2.2. Sepsis and severe sepsis..............................................................................................212.2.1. Pathophysiology of sepsis ..............................................................................23

2.2.1.1. Innate immunity response................................................................232.2.1.2. Adaptive immunity response............................................................242.2.1.3. Complement system, coagulation and inflammation.........................252.2.1.4. Endothelium and inflammation.........................................................262.2.1.5. Multiorgan failure.............................................................................26

2.2.2. Epidemiology of sepsis...................................................................................272.2.3. Microbiological aetiology of sepsis..................................................................28

2.3. Markers for severe sepsis .............................................................................................292.3.1. C-reactive protein...........................................................................................302.3.2. Vascular endothelial growth factor..................................................................332.3.3. Amino-terminal pro-brain natriuretic peptide....................................................342.3.4. Other markers ................................................................................................35

2.3.4.1. Lactate ............................................................................................35 2.3.4.2. Procalcitonin....................................................................................36 2.3.4.3. Interleukins......................................................................................37

3. AIMS OF THE STUDY .......................................................................................................38

4. PATIENTS AND METHODS.…………………………………………………………………………. 39

4.1. Patients.........................................................................................................................394.1.1 Patients in retrospective studies (I–II) .............................................................39

4.1.1.1. Characteristics of patients with acute myeloid leukaemia (I).............394.1.1.2. Characteristics of autologous stem cell transplant recipients (II).......39

4.1.2. Patients in prospective studies (III–IV) ............................................................39

4.2. Methods........................................................................................................................434.2.1. Definitions ......................................................................................................434.2.2. Clinical management and supportive care measures (I-IV)…………………….. 43

4.2.2.1. Chemotherapy..................................................................................454.2.2.2. Antimicrobial therapy.......................................................................48

4.2.3. Laboratory methods .......................................................................................484.2.3.1. Blood cultures .................................................................................504.2.3.2. Serum C-reactive protein.................................................................504.2.3.3. Serum vascular endothelial growth factor.........................................524.2.3.4. Plasma amino-terminal pro-brain natriuretic peptide.........................52

4.2.4. Statistical methods .........................................................................................53

4.3. Approval of the Ethics Committee..................................................................................53

5. RESULTS..................................................................................................................................54.

5.1. Retrospective studies (I-II).............................................................................................545.1.1. Epidemiological features of severe sepsis.........................................................54

5.1.2. Microbiological findings and site of infection....................................................555.1.3. Factors associated with severe sepsis............................................................565.1.4. Serum C-reactive protein in relation to severe sepsis......................................60

5.2. Serum vascular endothelial growth factor in patients with neutropenic fever: acomparison with C-reactive protein (III)..........................................................................605.2.1. Epidemiological features of severe sepsis ......................................................605.2.2. Microbiological findings and site of infection....................................................605.2.3. C-reactive protein...........................................................................................615.2.4. Serum vascular endothelial growth factor .......................................................63

5.3. Serum amino-terminal pro-brain natriuretic peptide in patients with neutropenicfever: a comparison with C-reactive protein (IV).............................................................645.3.1. Epidemiological features of severe sepsis ......................................................645.3.2. Microbiological findings and site of infection....................................................685.3.3. Factors associated with severe sepsis............................................................685.3.4. C-reactive protein...........................................................................................685.3.5. Amino-terminal pro-brain natriuretic peptide....................................................695.3.6. Association of amino-terminal pro-brain natriuretic peptide with fluid

intake and previous cardiovascular disease....................................................69

6. DISCUSSION .........................................................................................................................75

6.1. Patients and methods....................................................................................................75

6.2. Epidemiological features and outcome of severe sepsis ................................................766.2.1. Patients with acute myeloid leukaemia ...........................................................766.2.2. Autologous stem cell transplant recipients ......................................................776.2.3. Prospective study...........................................................................................78

6.3. Microbiological findings .................................................................................................78

6.4. Kinetics of C-reactive protein in patients with neutropenic fever (Studies I-II) .................80

6.5. Comparison of serum vascular endothelial growth factor and C-reactive proteinin patients with neutropenic fever (Study III) ..................................................................80

6.6. Comparison of serum amino-terminal pro-brain natriuretic peptide and C-reactiveprotein in patients with neutropenic fever (Study IV) ......................................................82

6.7. Study limitations.... .......................................................................................................83

6.8. Concluding remarks ......................................................................................................846.8.1. Implications for further research .....................................................................85

7. CONCLUSIONS......................................................................................................................87

8. REFERENCES .......................................................................................................................88

ORIGINAL PUBLICATIONS I – IV

15

1. INTRODUCTION

Treatment of haematological malignancies has improved during recent decades. This is due

to modern effective drugs and regimens but also due to improvements in supportive care.

About 40% of younger patients with acute myeloid leukaemia (AML) are cured with intensive

chemotherapy (1, 2). Relatively high percentage of patients with relapsed non-Hodgkin

lymphoma (NHL) (3) or Hodgkin lymphoma (HL) (4) are cured by high-dose therapy supported

by autologous stem cell transplantation (ASCT).

Intensive chemotherapy has certain disadvantages. Chemotherapy induces breakdown of

mucosal barriers, usually leading to high risk of neutropenic fever, which is associated with

development of severe sepsis and high mortality. Thus, early initiation of antimicrobial therapy

(5-7) and other supportive measures are vital in the management of haematological patients

receiving intensive chemotherapy. In spite of some obvious advances, the need for

improvement remains.

Several studies have evaluated the epidemiology and prognostic factors of septic patients

admitted to intensive care units (ICU) (8-10). However, only a minority of these patients has

had haematological malignancies. Therefore, the risk factors for severe sepsis and outcome of

sepsis in these ICU-based studies are not necessarily applicable for patients with neutropenic

sepsis. In fact, there are very limited data available on the incidence, risk factors and outcome

of severe sepsis in neutropenic haematological patients receiving intensive chemotherapy.

C-reactive protein (CRP) has traditionally been used to evaluate the severity of infection and

also the response to antimicrobial therapy in patients with febrile neutropenia, even though its

predictive value has been controversial (11-14). Other markers of septic infections that are

also used to predict outcome in the ICU, like serum vascular endothelial growth factor (VEGF)

(15) or amino-terminal pro-brain natriuretic peptide (NT-proBNP) (16) have not been

prospectively evaluated in a haematological ward setting. Even though marked improvements

have been observed in the treatment of severe sepsis in ICUs (8), there are only limited data

available on careful patient monitoring and vigilant supportive care in haematological wards of

patients with severe sepsis.

The aim of this study was to evaluate the epidemiological features, microbiological aetiology

and outcome of severe sepsis in neutropenic haematological patients. Early prognostic

markers for the development of severe sepsis were also evaluated. These markers included

VEGF and NT-proBNP, with special reference to the kinetics of CRP. In addition, the purpose

of the prospective treatment protocol was to improve the clinical management of septic

neutropenic patients in haematological ward.

16

2. REVIEW OF THE LITERATURE

2.1. Infections in neutropenic haematological patients

Unfortunately, as long as patients are neutropenic it is likely that some of them will develop

infectious complications

GP Bodey (17)

The mechanisms and pathophysiology of sepsis in neutropenic patients is similar to that in

patients without neutropenia but with the exception of pancytopenia. Neutropenia enables the

infection to progress in a more insidious and aggressive way. The role of neutrophils in septic

infections is controversial. They are thought to be essential for the eradication of pathogens

but at the same time, excessive release of oxidants and proteases by neutrophils might be

responsible for tissue injury (18). In studies carried out with granulocyte colony-stimulating

factor in patients with pneumonia the markedly increased neutrophil count was not deleterious

but did not have obvious clinical benefits, either (19). Neutropenic patients with septic

infections have higher mortality rates emphasizing the protective role of an appropriate acute

inflammatory response (20).

Infections have been recognised as complications of leukaemia for the first time as early as

in 1845 (21). Until 1948, there was no specific treatment for acute leukaemia (22). When very

little was to do for the natural course of the disease, there were no major interest to study or

understand the possibility of infectious complications. Although neutropenia is a common

consequence of acute leukaemia, its role in infection was not fully recognised until the early

1960´s (17).

Patients with haematological malignancies are immunocompromised because of both the

underlying malignancy and the therapy employed to manage it. Therapeutic interventions such

as corticosteroids, stem cell transplant and radiotherapy also produce deficiencies in host

defenses. Normal human skin and mucosal colonisation change during chemotherapy and

hospitalisation. Within all this, neutropenia acts as a common risk factor for severe bacterial

infections. It is estimated that at least 30-60% of neutropenic patients develop an infection, of

whom 13-37% develop bacteraemia (23-26). During the last three decades the overall

mortality rate due to septic infections in neutropenic patients has decreased from 21% to 7%

(27), but febrile neutropenia still remains a major reason for significant morbidity and mortality

in this patient group (28). The depth and duration of neutropenia is directly related to incidence

17

of serious bacterial infections. The risk of severe infection is significantly greater at low

neutrophil counts (absolute neutrophil count <0.1x109/l) (29).

Neutropenia alters the host’s inflammatory response and makes the infection difficult to

detect because the classic signs and symptoms of infection are often missing (30, 31). The

endogenous pyrogen (IL-1) is produced by mononuclear cells, not by granulocytes, and these

mononuclear cells include also fixed-tissue macrophages that persist after chemotherapy. This

explains the presence of fever despite the otherwise poor inflammatory response in

neutropenic patients (32). Fever is the principal sign of infection, and it is often the only

evidence of infection in neutropenic patients. In clinical practice, careful history of possible

symptoms and physical examination to seek for subtle signs of infection remain essential. The

prompt initiation of empirical antibiotics has been the most important advance in the treatment

of febrile neutropenic patients (33) – prior to this policy the mortality from gram-negative

infections was as high as 80% (29, 34). The importance of early antimicrobial treatment for

febrile neutropenia was for the first time demonstrated in the study of Schimpff et al (35).

Today the overall survival rate for febrile neutropenic patients is over 90%.

In Finland empirical antimicrobial treatment in febrile neutropenic patients includes usually a

combination of a third generation cephalosporin and an aminoglycoside. If Pseudomonas

aeruginosa bacteraemia is suspected, antimicrobial treatment should be initiated with

antipseudomonal penicillin together with an aminoglycoside. Vancomycin is only rarely used

empirically (36).

2.1.1. Bloodstream infections in patients with neutropenic sepsis

Over the last three decades, several studies have demonstrated a shift in the aetiology of

bacteraemic infections from predominance of gram-negative rods to gram-positive cocci (37,

38). Prior to the availability of methicillin, penicillin-resistant Staphylococcus aureus was the

main threat to neutropenic patients and mortality rate from Staphylococcus aureus infections

exceeded 50%. Methicillin became available during the 1960s, curing most neutropenic

infections caused by Staphylococcus aureus (17). During the subsequent two decades gram-

negative bacteria emerged as the main causative agents (39). Infections with Pseudomonas

aeruginosa were common and were associated with a high mortality rate (40).

By the end of the 1980s and lately during 1990s, gram-positive microbes re-emerged (41,

42). The most common infective agent was coagulase-negative staphylococci, mainly

Staphylococcus epidermidis. The cause for this change has not been clearly identified.

Possible factors responsible for this process include widespread use of indwelling central

18

venous catheters, a higher incidence of severe oropharyngeal mucositis and bowel damage

with the use of more intensive chemotherapy regimens and more profound and prolonged

neutropenia. In addition, use of quinolone-based prophylactic antibiotics suppressing aerobic

gram-negative microbes colonizing the gastrointestinal tract but failing to suppress sufficiently

gram-positive flora and use of other antibiotics with selective pressure may be potential

factors. Furthermore, administration of histamine H2 receptor blockers or proton-pump

inhibitors may promote infections by reducing gastric pH and promoting overgrowth of

oropharyngeal gram-positive flora (43-45).

Mucositis, which is a well-known consequence of cytotoxic chemotherapy (46), predisposes

patients to infections arising from patient’s own flora – primarily of oropharyngeal and

gastrointestinal origin. Mainly because of this, clinically important gram-positive microbes

include also viridans group streptococci (e.g. Streptococcus milleri or Streptococcus mitis),

which belong to the normal flora of the oropharynx and gastrointestinal tract. Among

neutropenic patients both the morbidity and mortality to streptococcal bacteraemia remains

high (6, 47). Other new gram-positive microorganism include species belonging to Enterococci,

Stomatococci, Leuconostoc, Lactobacillus and Corynebacterium species – microbes that were

traditionally considered contaminants in blood cultures.

There is also an interesting difference in the incidence of gram-negative bacteraemias

between developed and developing countries (48-51). In developing countries, the amount of

gram-negative bacteraemias remains high, probably due to the less frequent use of central

lines and prophylactic antibiotics (52, 53). This distribution may nevertheless be reversing

since in some western centers the gram-negative microbes are making a comeback (48, 52,

54). Even though the general incidence of gram-negative microbes as a cause of bacteraemic

infections in developed countries has declined, they are nonetheless still a problem. This is

primarily because of their traditional association with high mortality rates and also the alarming

rise of multidrug-resistant strains (49, 55). In recent study the distribution of bacteraemias in

neutropenic cancer patients was gram-positive in 57%, gram-negative in 34% and

polymicrobial in 10% (56). The mortality rates were 5%, 18% and 13%, respectively. The most

common causative agents of gram-negative rods are Klebsiella and Enterobacter species as

well as Stenotrophomonas maltophilia and Burkholderia cepacia, not forgetting Pseudomonas

species (44, 57).

19

2.1.2. Infectious complications in patients with acute myeloid leukaemia

In patients with acute myeloid leukaemia (AML) intensive chemotherapy regimens enable

complete remission in 80-85% of patients below 65 years of age. About 40% of patients are

cured with intensive consolidation courses (2, 58).

Infections are the major cause of morbidity and mortality in patients with AML. The treatment

protocol of AML induces long lasting periods of neutropenia, which predisposes patients to

recurring infections. These arise mainly during the first course of induction chemotherapy. This

may cause delays in consolidation therapy and increase the risk of leukaemia relapse. Also

consolidation courses are associated with long periods of neutropenia and the risk of fatal

infections both in adults and children (59, 60).

The infectious mortality during AML treatment ranges between 5.5 -13% (60-64). The initial

source of infection often remains unknown and therefore the first antimicrobial therapy choice

is empirical. Therapy is directed primarily against gram-negative microbes. Like in other septic

neutropenic infections, the spectrum of causative pathogens has changed over the past

decades. Coverage of coagulase-negative staphylococci is not necessary for the initial

antimicrobial therapy. Coverage of oropharyngeal streptococci is sufficient when combination

therapy or monotherapy with highly active third-generation cephalosporins or carbapenems are

used (43).

In recent studies the amount of bloodstream infections among neutropenic AML patients has

varied between 34% and 38% (24, 59, 65). In the study of Madani (24) the frequency of

isolation of gram-positive cocci was 74.2% and gram-negative bacilli 12.1%. The most

common pathogens in the gram-positive group were coagulase-negative staphylococci

(34.8%) followed by Streptococcus viridans (22.7%) and in the gram-negative group Klebsiella

pneumoniae (3%) followed by Enterobacter cloacae (1.5%). Ciprofloxacin prophylaxis was

used during the neutropenic period. In spite of this there were still gram-negative

bacteraemias. The amount of resistant microbes was not discussed. In this study, the site of

infection was identified in 81% of all febrile episodes. Mucositis (21.7%), pneumonia (13.2%)

and central venous catheter infection (12.4%) were the most common sites of infection.

Bloodstream infections (37.9%) were commonly associated with cellulitis, mucositis and

central venous catheter infection.

In a study analysing the infections occurring in newly diagnosed AML patients under 65

years of age the amount of bloodstream infections was 34% (59). During induction phase 13%

of neutropenic patients had bloodstream infection caused by gram-negative rods and 21% by

gram-positive cocci. The case fatality rates for gram-negative and gram-positive bacteraemias

20

were 10% and 8%, respectively. When the potential age-related differences in nosocomial

infections between younger (<60 years old) and older leukaemia patients with neutropenic

fever were evaluated, no significant age-related differences were found in the overall incidence

of infections, nosocomial pattern, or overall outcome(66) .

2.1.3. Infectious complications in autologous stem cell transplant recipients

High-dose chemotherapy (HDT) supported by ASCT is a commonly applied treatment for

haematological malignancies, mainly lymphomas and multiple myeloma (MM) (67, 68).

Annually more than 15 000 procedures are performed in Europe (69). The procedure is

considered to be relatively safe with a low incidence of severe complications and transplant-

related mortality. However, approximately 1-5% of neutropenic periods after HDT are

complicated by fatal infections (70-74).

After ASCT, there is an unavoidable period of neutropenia, where the risk for infections is

high. The conditioning regimen causes neutropenia and defects in mucosal and cutaneous

barriers. Neutropenic periods are shorter than in AML treatment, but the intensity of treatment

in ASCT recipients is greater and the mucosal damage may thus become more serious.

Hence, mucositis and indwelling central venous catheters commonly lead to bacterial and

fungal bloodstream infections. During the first 4 weeks after haematopoietic stem cell

transplant, bacterial infections and invasive fungal infections predominate. Between 30 and

100 days after transplant, impaired cellular immunity increases the risk of viral infections (75).

The percentage of bloodstream infections varies between 12% and 20% in ASCT recipients

(72-74, 76). As in all other neutropenic bacteraemias, gram-positive microbes cause at least

half of the bloodstream infections occurring after ASCT. In a recent study 60.3% of the positive

blood cultures were gram-positive cocci and 28.9% gram-negative rods (70). The most

common isolates in the gram-positive group were coagulase-negative staphylococci (43.4%)

followed by Streptococcus viridans (5.7%) and in the gram-negative group Stenotrophomonas

maltophilia (10.1%), followed by Acinetobacter baumannii (6.9%). Of the 314 study patients 12

(3.8%) died because of infectious complications – 6/12 patients because of gram-negative

bacteraemia (Stenotrophomonas maltophilia, Acinetobacter baumannii and Pseudomonas

aeruginosa). In that study norfloxacin was used as a prophylactic antibiotic. During the study

period (1994-2005) Pseudomonas aeruginosa was resistant to ceftazidime and

aminoglycosides in half of the cases and to fluoroquinolones in 25-50% of the cases.

Fluoroquinolone resistance seemed to diminish during the study period. The patients who died

due to infection had markedly longer median time of severe neutropenia (70).

21

2.2. Sepsis and severe sepsis

Inflammation is a reactive state of the organism against disturbances of homeostasis with the

goal of healing and repair of the injured tissue

JB Cone (77)

In 1991 the American College of Chest Physicians/Society of Critical Care Medicine

Consensus Conference (ACCP/SCCM) compiled the current definition of sepsis as a systemic

inflammatory syndrome in response to infection. When this was associated with hypotension,

hypoperfusion or acute organ dysfunction sepsis was considered to be severe (78, 79). Before

these definitions the terms sepsis, septicemia, sepsis syndrome and bacteraemia were used

without precise characterisation.

When the ACCP/SCCM consensus conference released these definitions they also

proposed a new term to describe inflammatory processes that occur in parallel with systemic

infection (78). Systemic inflammatory response syndrome (SIRS) has several clinical

manifestations, including abnormalities in body temperature, respiratory and heart rate.

The definition of SIRS has been noted to be controversial and challenging for clinical use.

American and European critical care societies reconsidered the definitions of ACCP/SCCM in

2001. The decision was that although the concepts based on SIRS were too sensitive and

indefinite, these were useful as a baseline construction for the diagnosis of sepsis (79).

Subsequently, these definitions have been used widely both in clinical practice and in research

worldwide (Table 1).

22

Table 1. Criteria for the systemic inflammatory response syndrome, sepsis and severe sepsisaccording to the Consensus Conference of the ACCP/SCCM (79)_________________________________________________________________________Term Definition_________________________________________________________________________

SIRS Systemic inflammatory response syndrome.

The systemic inflammatory response is manifested bytwo or more of the following criteria:

Fever (body temperature > 38°C) or hypothermia(<36°C)

Tachycardia (heart rate > 90 beats/min)

Tachypnea (>20 breaths/min) or PaC02 < 4.3 kPa

Leucocytosis or leucopenia (white blood cell count >12,000 or < 4000/mm3 or > 10% immature forms

SEPSIS Presence of SIRS in response to infection. SIRS ismanifested by two or more of the criteria mentionedabove

SEVERE SEPSIS Sepsis associated with organ dysfunction,hypoperfusion or hypotension. Organ dysfunction andhypoperfusion abnormalities may include, but are notlimited to lactatic acidosis, oliguria or alteration inmental status

Septic hypotension is defined as a systolic bloodpressure < 90 mmHg or a decrease in systolic bloodpressure by 40 mmHg or more from the baseline

_________________________________________________________________________Abbreviations: PaC02, arterial partial pressure of C02; kPa, kiloPascal.

23

2.2.1. Pathophysiology of sepsis

The microorganisms that seem to have it in for us…turn out…to be rather more like

bystanders…It is our response to their presence that makes the disease. Our arsenal for

fighting off bacteria are so powerful…that we are more in danger from them than the invaders

Lewis Thomas (80)

The English word sepsis is derived from the Greek term ptik s for rotten or “to make

putrid”. Sepsis, defined as the systemic host response to microorganisms in previously sterile

tissue, is a syndrome related to severe infections. In its severe form, sepsis is characterised

by end-organ dysfunction often far away from the primary site of infection.

The pathophysiology of sepsis is understood as a continuum. The normal response of host

to infection is both to identify pathogen invasion and to start tissue repair. The immune

response consists of innate immunity and adaptive immunity responses. The innate immunity

response embodies front-line reaction towards invading pathogens. It includes recognition of

microbial components, activation of local phagocytosis (e.g. neutrophils, eosinophils,

monocytes and macrophages), complement system and coagulation as well as production of

acute phase proteins. Adaptive immunity comprises responses of cell-mediated and humoral

immunity. These phenomena progress in parallel and are contingent on each other. These

both give rise to anti-inflammatory and proinflammatory responses (81). If this continuum is

disturbed, a chain of events occurs in which the promotion and liberation of mediators leads

inevitably to sepsis (82).

2.2.1.1. Innate immunity response

The natural physical barriers to host invasion are formed externally by the skin and internally

by mucous membranes lining the gastrointestinal, genitourinary and respiratory tracts. These

form a mechanical barrier towards invading pathogens in co-operation with local normal flora.

In the hospital environment patients’ indwelling catheters, intravenous cannulas and urinary

catheters must be noted as possible sources of infection (83). Sepsis may be caused by

numerous invasive pathogens, including bacteria, yeasts, viruses and parasites.

The structural components of the microbe responsible for triggering the septic process are

important not only for understanding the mechanisms of infection and inflammation, but also

for identifying potential therapeutic targets. Endotoxin is a lipopolysaccharide present in the

outer portion of gram-negative bacteria. Exposure to endotoxins, exotoxins produced by gram-

24

positive bacteria, or other types of microbial components triggers intracellular events in

immune cells and the epithelium, endothelium and neuroendocrine system through microbial-

associated molecular patterns (84).

The initiation of the response involves pattern of recognition receptors which recognise

specific structures of microbes (85). Part of this family are toll-like receptors (TLRs), which are

transmembrane proteins on the surface of immune cells. These are capable of sensing

invading microbes. Microbes have unique cell-wall molecules known as pathogen-associated

molecular patterns (PAMPs). PAMPs bind to TLRs on the surface of immune cells. This

binding activates, in turn, intracellular signaling pathways. At the end of this pathway,

proinflammatory cytokines are released. Also macrophages and monocytes participate in the

secretion of proinflammatory cytokines. Neutrophils and endothelial cells are activated to

produce adhesion molecules, which help to kill pathogens, but also cause damage to the

endothelium (85). Macrophages release VEGF-like mediators, which increase vascular

permeability and vascular proliferation, contributing to coagulation and inflammatory processes

(86).

2.2.1.2. Adaptive immunity response

Following the initial host-microbe interaction there is a widespread activation of adaptive

immune response, which coordinates defence response involving both humoral and cellular

immune systems.

The humoral immune response is mediated by secreted antibodies (immunoglobulins)

produced in the cells of B lymphocyte lineage (plasma cells). Secreted antibodies bind to

antigens on the surfaces of invading microbes, marking them for destruction by components of

innate immune system (87, 88). Pathogens are destroyed by phagocytic cells (e.g. neutrophils,

macrophages or natural killer cells) with the help of complement activation or recognition by

antibodies (89, 90).

Cellular immunity is an immune response mediated by T-cells. It involves activation of

macrophages, natural killer cells, antigen-specific cytotoxic T-lymphocytes and the release of

various cytokines in response to an antigen. Cytotoxic T-lymphocytes (CD8) are able to

destroy cells infected by viruses or cells with intracellular bacteria. Helper T cells (CD4)

secrete cytokines by differentiating into type 1 helper T-cells (Th1) and type 2 helper T-cells

(Th2) (or other subsets). Th1 secretes pro-inflammatory cytokines and Th2 anti-inflammatory

cytokines. Proinflammatory mediators (e.g. interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor

necrosis factor-alpha (TNF- )) contribute to eradication of invading pathogens and anti-

25

inflammatory mediators (e.g. interleukin-4 (IL-4) and interleukin-10 (IL-10)) to control this

response (91, 92). This proinflammatory response leads inevitably to damage of the host

tissues, whereas the anti-inflammatory response causes leukocyte reprogramming and

changes immune status (93). During this time sequence, circulatory abnormalities (e.g. intra-

vascular volume depletion, peripheral vasodilatation and myocardial depression) lead to an

imbalance between systemic oxygen delivery and demand.

2.2.1.3. Complement system, coagulation and inflammation

The complement system and coagulation are the major components of plasma cascades.

They are closely related and are activated in a contiguous manner by common stimuli (e.g.

infection, trauma). They both contribute to inflammation and mutually interact at several stages

(94, 95).

Complement is not only a part of the innate immune system, but also an effector of antibody-

mediated immunity. The major functions are the defence against pyogenic bacterial infections

bridging innate and adaptive immunity, and clearance of immune complexes and products of

inflammatory injury. Circulating components of the complement are activated via three

pathways: 1) the classical pathway initiated by the binding of component C1q to antigen-

antibody complex, 2) the lectin pathway initiated by binding of mannose-binding lectin to

sugars present in the bacterial wall, and 3) an alternative pathway initiated after exposure to

surface molecules of invading pathogens. At the end of these pathways several convertases

(e.g. C3, C5) are released and they in turn facilitate the phagocytosis of opsonised pathogens

by macrophages and neutrophils, act as inflammatory mediators and, further, participate in the

lysis of bacterial cell membranes. The regulatory mechanisms of the complement system are

sensitively balanced. The aim is to focus the activation of complement on the surface of

invading pathogens, while on limiting deposition on normal cells and tissues (94, 96).

A delicate balance of pro- and anticoagulant factors maintains haemostasis. In normal

conditions three anticoagulant pathways prevent systemic activation of coagulation:

antithrombin, activated protein C and tissue factor pathway inhibitor. During inflammation-

induced activation of coagulation all three functions are impaired; IL-6 release triggers tissue

factor upregulation, initiating the coagulation cascade, and TNF- mediates the suppression of

natural anticoagulants. The latter can lead to disseminated intravascular coagulation, which

has been thought to be central in the pathogenesis of multiorgan failure (MOF) in sepsis

patients. Thus, sepsis is a chaotic environment associated with exacerbated coagulation,

decreased anticoagulation and impaired fibrin removal (95, 97, 98).

26

Complement and coagulation cascades are intended to act locally. If they are activated

systemically as a result of a failure of the relevant control mechanisms, the effect of this broad

reaction can be irreversible.

2.2.1.4. Endothelium and inflammation

Vascular endothelial cells play an active role in the regulation of blood vessel tone,

permeability, coagulation, angiogenesis and both leukocyte and platelet activation. Endothelial

cells synthesise several pro- and anti-inflammatory molecules. They also synthesise proteins

which increase vascular permeability to fluid and large molecules (e.g. antibodies and

components of complement) (95, 99).

Several microbes can adhere to the endothelium, causing localised inflammation and

recruitment of neutrophils and monocytes. Increased vascular permeability allows leukocyte

migration into surrounding tissue as a response to chemotactic factors generated at the site of

injury. Bacterial endotoxins or other foreign particles induce neutrophil activation and release

of pro-inflammatory cytokines. Strong adhesion initiates the coagulation cascade (97, 99). The

endothelium is one of the key organs involved in the pathogenesis of sepsis. Endothelial

damage may result in global tissue hypoxia or shock (99). Worsening tissue hypoxia may

ultimately lead to MOF and death.

2.2.1.5. Multiorgan failure

The ultimate cause of death in patients with severe sepsis is multiorgan failure. The exact

mechanisms for MOF are unknown. Earlier observations indicate that the number of failing

organs or organs with dysfunction correlates with mortality, with an increase of 20% for each

additional organ failure (100, 101). In-hospital mortality with the failure of at least two organ is

four-hold compared with patients who have only a single organ failure (102).

Mechanisms of multiorgan failure include widespread microvascular occlusion, development

of tissue exudates that further compromise tissue oxygenation and disorders in microvascular

homeostasis, which all result from elaboration of vasoactive substances (e.g. histamine).

Cellular infiltrates (mainly neutrophils) damage tissue directly by releasing lysosomal

enzymes. Proinflammatory cytokines (mainly TNF- ) mediate increased production of nitric

oxide, causing further vascular instability. This may also contribute to the direct myocardial

depression that occurs commonly in sepsis (82) .

27

Ongoing tissue hypoxia acts as an indicator for progressive MOF. The extent of oxygen

debt, i.e. the amount by which oxygen requirements exceed oxygen delivery, is related to the

outcome of sepsis. Also impaired endocrine responses to sepsis may result due to cytokines

and metabolic and ischaemic derangements in hypothalamic-pituitary axis and adrenal

glands. Deficiencies in adrenal gland function and vasopressin production contribute to

hypotension and eventual death (103, 104).

2.2.2. Epidemiology of sepsis

Despite extensive study and new therapeutic modalities, sepsis is still a challenge in clinical

medicine. Severe sepsis and septic shock are both relatively common and represent an

important cause of morbidity and mortality.

Both the incidence and mortality in severe sepsis varies in different studies. In Scandinavia,

there are only a few nationwide studies, the most important of which are the Norwegian study

in 1999 and the FINNSEPSIS study in 2004-2005. The national health database in Norway

was retrospectively screened for cases of severe sepsis. The incidence of severe sepsis was

1.5/1000 of the population. Severe sepsis was diagnosed in 31.8% of sepsis patients, and the

hospital mortality for severe sepsis was 27% (9). The FINNSEPSIS study was a prospective

study in the ICUs of 21 hospitals in Finland. Severe sepsis was diagnosed in 470 of 4500

patients (10.5%) admitted to ICUs. The incidence of ICU-treated severe sepsis in Finland was

0.38/1000 of the adult population. The ICU mortality rate was 15.5% and the number of organ

failures affected the mortality rate. In patients with a single organ failure the mortality was

11.5% but in patients with three organ failures mortality increased up to 34% (10).

In the EPISEPSIS study sepsis was prospectively evaluated among all admissions of 206

adult ICUs of public hospitals in France (8). The incidence of severe sepsis was 0.95/1000.

Altogether 14.6% of patients had severe sepsis or septic shock. The in-hospital mortality rate

was 35% at 30 days. This study was a reappraisal for an earlier study (105) in which 8.4% of

patients were found to have severe sepsis or septic shock and 56% of them died at hospital.

Although the admission rate of severe sepsis in French ICUs appears to have increased,

hospital mortality has decreased suggesting improved management (105). In a large

retrospective British study, the incidence of severe sepsis was 0.46-0.66/1000 of population.

Of adult patients admitted to ICU, 27% met criteria for severe sepsis during the first 24 hours

(in-hospital mortality 47%). In the multicentre prospective European SOAP-study the

corresponding percentage was 25% (total in-hospital mortality 36%) (106). Authors found a

considerable variation among the European countries, with a strong correlation between the

28

frequency of sepsis and the intensive care unit mortality rates. In two large retrospective

epidemiological studies, the incidence of severe sepsis was 0.76/1000 of the population and

in-hospital mortality 31% in Australia. In the United States, the incidence of severe sepsis was

3/1000 in population and overall hospital mortality rate 29% (100, 107).

With the exception of the FINNSEPSIS-study, the occurrence and mortality rates of severe

sepsis seem to be rather similar. Most studies were made in ICUs and in adult population,

considering all patient groups admitted to intensive care (surgical and non-surgical). The

different classifications of intensive care units between countries should also be noted – ICU

and lighter step-up/ step-down units might have been classified together in these studies. The

amount of malignancies among study patients varied between 4.6% and 11.6%, but the

number of haematological patients with neutropenia was not mentioned (10, 108, 109).

An important finding in FINNSEPSIS study was that even though there were national

guidelines for the treatment of severe sepsis in adults (110) the compliance to the guidelines

was noted to be poor. This was also the case in the international setting (111, 112), even

though relative simple therapeutic interventions might save a significant number of lives (113).

It is well known that the time window for interventions in the evolution of sepsis is short. The

transition from a mild to serious situation occurs during the critical “golden hours” when

recognition and aggressive treatment provide maximal benefit in terms of outcome (114).

2.2.3. Microbiological aetiology of sepsis

Blood cultures have been positive in 30-60% of the patients (9, 10, 115) in ICU- based

sepsis studies. In most studies the percentage of positive blood cultures was less than 60% -

underlining the fact that severe sepsis must primarily be a clinical diagnosis.

Among the ICU studies including both community- and hospital-acquired infections the

respiratory tract has been the most common site of infection, followed by the abdomen, urinary

tract and skin or soft tissue infections (5, 8, 100, 109, 115, 116). In the recent FINNSEPSIS

study (10) blood cultures were positive in 40% of patients with severe sepsis. Of these, the

proportion of gram-positive bacteria was 59% and gram-negative bacteria 33%, respectively.

The most common site of infection was the respiratory system (43%) followed by intra-

abdominal (32%) and skin or soft tissue (10%) infections. In patients older than 65 years the

urinary tract has been observed to be a common site of infection (117).

Since the late 1980s, gram-positive organisms have slowly replaced gram-negative bacteria

as a cause of sepsis (116, 118). This was demonstrable in most studies during this decade (8,

107, 115) with some exceptions. In a large Chinese study (108) gram-negative microbes were

29

the most common isolates in blood cultures. This was also the situation in the study by Valles

et al (119). In that study 339 patients were admitted to ICU with community-acquired

bacteraemia (25% with sepsis, 20% with severe sepsis and 55% with septic shock). The most

common pathogens were Escherichia coli (25%), Streptococcus pneumoniae (16%) and

Staphylococcus aureus (14%).

It has been showed that initiation of effective antimicrobial therapy within the first hour

following the onset of septic shock-related hypotension is associated with an 80% survival.

Every additional hour of delay in the initiation of antimicrobial therapy decreased survival by an

average of 7.6% (5). The importance of broad coverage in the initial treatment was illustrated

by the poor prognosis of patients in whom the first line antibiotics were found to be ineffective

in sensitivity assays (120).

At presentation both the source of infection and the microbial nature of the disease are often

unknown. Antibiotic treatment should be therefore guided by symptoms and signs, bedside

clinical findings and knowledge of local bacterial resistance. The epidemiological change of

nosocomial bloodstream infections can be insidious, which was observed recently in Taiwan

and the Republic of Korea (121-123). This was characterised by the notable predominance of

gram-negative microbes with increasing antimicrobial resistance.

2.3. Markers for severe sepsis

In severe sepsis and other serious infections, the circulating levels of biomarkers depend on

the origin and extent of the infection. In addition, microbes may induce a distinct response in

various organs, resulting in a variable diversity of circulating biomarkers and mediators. It is

obvious that any infection is far too complex to be reduced to a single cutoff of any biomarker.

Nevertheless, the dynamics of biomarker levels have prognostic implications, and increasing

levels are associated with an unfavourable outcome. On the contrary, decreasing levels

suggest favourable outcome (124). Biomarkers may be valuable and helpful tools in the

diagnostic dilemma of severe sepsis, especially in distinguishing severe sepsis from less

severe forms of infections in early phase of the disease.

Various treatment strategies are effective in septic infections, but the disease should be

diagnosed early to be effectively treated. Early diagnosis with an unknown microbiological

aetiology, but early initiation of sepsis therapy is more effective than specific, but lately initiated

sepsis therapy (125). This is especially true in neutropenic sepsis, where symptoms and

clinical findings of infection may be obscure or even absent. Thus, early prognostic markers of

bacterial infections are warranted in the treatment of neutropenic patients.

30

These markers should be released and regulated independently of neutrophil cell counts and

activity of the underlying disease. An ideal prognostic marker should be also able to distinguish

septic infections from several other causes of the systemic inflammatory response syndrome.

It should also reflect the severity of the infection and distinguish periods with high risk of

complications from those with low risk (126).

Leukocyte count and leukocyte differentiation are among the oldest markers of infection, but

are useless in patients with severe neutropenia. CRP, procalcitonin (PCT) and

proinflammatory cytokines (IL-6 and IL-8) are all useful markers of inflammation. In addition,

VEGF, natriuretic peptides, lactate and procalcitonin have been a persistent interest of study.

Among other potential future candidates are lactoferrin, neopterin and prostaglandins (126).

2.3.1. C-reactive protein

CRP is a member of the acute-phase reactants, because its level rises rapidly in response

to inflammatory processes. CRP is produced by hepatocytes, predominantly under

transcriptional control by IL-6 (Table 2). About 90% of healthy population has CRP

concentrations < 3 mg/l (127). Synthesis after the inciting stimulus starts rapidly, and serum

concentrations of CRP rise above 5 mg/l by about 6 hours. The peak values are achieved in

around 48 hours. The sole determinant of circulating CRP concentration is the synthesis rate,

and it reflects directly the intensity of the pathological process stimulating CRP production

(128). When the stimulus terminates, the concentration of circulating CRP falls rapidly.

CRP production is part of the nonspecific acute-phase response to most forms of

inflammation, infection and tissue damage. Persistent increases in CRP can also occur in

chronic inflammatory disorders, including autoimmune diseases and malignancy. It has been

shown that CRP has an important role in host defence by complement action, opsonization

and by inducing phagocytosis (129, 130). CRP has been used clinically for monitoring

infections and autoimmune disorders. CRP concentrations are most useful clinically when

combined with full knowledge of all other clinical and pathological results (126, 131). Of note,

in severe liver diseases CRP is generated at a markedly lower pace than in patients without

liver pathology (132).

The use of CRP is common in many European countries, mainly because of its low costs

and easy availability in everyday clinical practise. In many studies, CRP has usually been used

as a comparator for other markers of inflammation, mainly because its well-known kinetics and

broad use in various infectious diseases (133).

31

The time from suspicion to diagnosis in septic infections is critical because uncontrolled

infections can rapidly lead to severe sepsis, with a mortality rate of 20-52% (81, 82, 141). In

many recent sepsis studies, the initial slow kinetics of CRP has diminished its value as an

early marker of septic infection. In the study by Rintala et al (142) CRP peaked 24h later than

procalcitonin (PCT) in bacteraemic patients. The finding was similar in another study (143),

where the PCT concentrations reacted more quickly than CRP to the severity of sepsis in ICU

patients. The diagnostic utility of CRP and other inflammation markers was studied in cancer

patients with suspected infection (144). Only PCT seemed to be a good marker to discriminate

bacteraemic patients from other patients.

In studies with neutropenic patients, the results are in line with the studies in sepsis patients

without known neutropenia. CRP identifies patients with infection, but when the time interval is

short (<12 hours), predictive capacity of CRP declines significantly. When CRP kinetics during

infections in leukaemia patients was evaluated, it was concluded that CRP-level over 100 mg/l

was predictive for infection (145). Manian (12) found that an increase in CRP level of > 40 mg/l

over 2 or 3 days may suggest infection. A CRP level > 200 mg/l for over 5 days during

neutropenia was associated with a mortality rate of 50%. In another study CRP levels were

significantly higher in bacteraemic haematological patients and in patients whose infection

focus was identified than in those with fever of unknown origin (11). On the other hand,

Yonemori and colleagues (13) found no predictive value for serial measurement of CRP in

their study of 47 neutropenic patients. In these studies, CRP kinetics was not compared

between periods with severe sepsis and those without.

Persson et al (14) studied prospectively the predictive value of systemic inflammation

markers to determine the clinical course of febrile neutropenic patients. CRP did not differ at

any time point between patients with and those without complications. Another study from the

same group evaluated the ability of CRP and other inflammatory markers to predict

bacteraemia during the first 48 hours of fever in neutropenic patients (146). During the first 10

hours, CRP had sensitivity of 42% and specificity of 76% for bacteraemia. The positive

predictive value was 33%. CRP reached the highest levels after 20 to 30 hours of neutropenic

fever in patients with bacteraemia. In another study there were no differences in early CRP

concentrations between bacteraemic and non-bacteraemic neutropenic patients (147).The

result was similar in the study of Sandri and colleagues (148), where PCT concentrations

increased early only in bacteraemic patients with the highest levels at day +1 after the onset of

fever. CRP reached its peak level also at day +1 after the onset of fever but could not

distinguish bacteraemic patients from non-bacteraemic patients. Based on these studies, CRP

is useless from a predictive point of view.

32

Table 2. Markers for severe sepsis discussed in this thesis (90, 95, 128, 133-140).Marker Main source T½ Nature Major activities

CRP Liver (hepatocytes) 19 hours Acute phasereactantBoth anti- andproinflammatoryproperties

Ability to bindphosphocholineand recogniseforeign pathogensActivation ofcomplementsystemInduction ofinflammatorycytokines andtissue factor inmonocytes

Procalcitonin MonocytesParenchymal cells

25-30 hours Precursor ofcalcitonin

Mediator insystemicinfections, exactfunction unknown

IL-4 Helper T cells (Th2)B cellsMast cellsBasophilsEosinophilsStromal cells

Few minutes(NA)

Anti-inflammatory Activation oflymphocytes andmonocytes

IL-6 Helper T cells (Th2)MonocytesMacrophagesEndothelial cells

< 60 minutes ProinflammatoryAnti-inflammatory

Activation oflymphocytes,differentiation of B-cells, stimulation ofthe production ofacute-phaseproteins

IL-8 T cellsMonocytesMacrophagesEndothelial cells

< 10 hours ProinflammatoryAnti-inflammatory

Chemotaxis ofneutrophils,basophils and T-cells

IL-10 MonocytesLymphocytesMacrophages

Approximately2 minutes

Anti-inflammatory Inhibits productionof proinflammatorycytokinesRegulates T- andB cell proliferationStimulates Th2-mediated immunity

VEGFs(VEGF-A,-B,-C,-D,-E,-Fand placentalgrowth factorPIGF)

LymphocytesMacrophagesPlateletsVascular smoothmuscle cellsLung epithelium

Approximately3 minutes

Hypoxia inducedmediators

AngiogenesisMediator ofvascularpermeability

NT-proBNP Inactive metaboliteof BNP, which inturn is secreted bycardiomyocytes

120 minutes Inactive metaboliteof BNP

Maintaincardiovascularhomeostasis

Abbreviations: CRP, C-reactive protein; IL, interleukine; VEGF, vascular endothelial growth factor; NT-pro-BNP, amino terminal pro-brain natriuretic peptide; BNP, brain natriuretic peptide; T½, half-life.

33

There are no prospective studies evaluating predictive capacity of CRP kinetics in relation to

development of severe sepsis in haematological patients with neutropenic fever.

2.3.2. Vascular endothelial growth factor

Vascular endothelial growth factors (VEGFs) are important signaling proteins involved in

both vasculogenesis (de novo formation of the embryonic circulatory system) and

angiogenesis (growth of blood vessels from pre-existing vasculature). VEGF is one of the most

potent factors regulating angiogenesis and microvascular permeability (149-151). VEGF

production is induced in hypoxaemic cells. When a cell is deficient in oxygen, it produces

hypoxia-inducible factor (HIF). HIF in turn stimulates the release of VEGF (152). The role of

VEGF as a mediator of vascular permeability is important in severe infections. VEGF has been

found to cause vasodilatation influenced by endothelial nitric oxidase synthase in patients with

sepsis (153). In the study of Yano and colleagues (154), experimental septic infections were

associated with time-dependent increase in circulating levels of VEGF. They also showed that

peak VEGF concentration occurred during the first 24 hours after the onset of experimental

sepsis, and the concentration of VEGF remained elevated for several days.

Two groups have reported that VEGF levels increase in patients with severe sepsis. Pickkers

and colleagues (155) studied meningococcal septic shock in children as a prototype of gram-

negative septic shock. VEGF plasma concentrations were measured during the first 48 hours

and were highest in the presence of septic shock. VEGF concentrations at admission also

correlated with the severity of infection. Van der Flier and colleagues (156) measured plasma

VEGF levels in patients with severe sepsis in the adult ICU. They found that VEGF levels were

significantly elevated in patients with severe sepsis compared with healthy controls. Moreover,

VEGF levels in non-survivors were higher than in survivors. Increased VEGF levels at study

entry also correlated with the severity of MOF during the course of disease. Karlsson and

colleagues (15) evaluated serum VEGF values in ICU patients with severe sepsis in order to

predict organ dysfunction and mortality. They found that VEGF values were elevated in

patients with severe sepsis compared with healthy controls. Low circulating VEGF levels were

associated with haematological and renal dysfunction, suggesting possible disturbed

production of VEGF in severe sepsis. Furthermore, very low concentrations of VEGF were

associated with more severe forms of organ dysfunction and mortality, possibly because of

endothelial injury.

There are no previous prospective studies available on VEGF in patients with neutropenic

fever. Kraft and colleagues (157) analysed the VEGF values of 212 patients with various

34

malignant tumours without known neutropenia and compared the results with the VEGF values

of both healthy controls and patients with non-malignant disease. Elevated levels of VEGF

were detectable in 0-20% of patients with localised cancer but in 11-69% of patients with

metastatic cancer. VEGF levels in acute infection (non-severe sepsis) were elevated

compared with levels in healthy individuals.

The pathogenesis of sepsis, including the production of VEGF, is congruent in patients with

and without neutropenia. It has been shown that in cancer patients VEGF production

correlated with platelet count after chemotherapy-induced thrombocytopenia (158).

Furthermore, a peak in VEGF production followed platelet recovery. Several other studies

have shown that circulating VEGF resides not only in platelets but also in granulocytes, mainly

in neutrophils (159-162). This might influence VEGF production in patients with neutropenic

fever.

2.3.3. Amino-terminal pro-brain natriuretic peptide

Brain natriuretic peptide (BNP) is a prohormone secreted by cardiomyocytes. Secretion is

primarily a response to the increased myocardial wall stress and is aimed to maintain

cardiovascular homeostasis through its natriuretic, diuretic and vasodilatory properties (163).

NT-proBNP is an inactive metabolite of BNP. NT-proBNP has markedly longer plasma half-life

and better stability than BNP, which makes it better applicable for clinical use (164) (Table 2).

Increased levels of both BNP and NT-proBNP have been identified as early markers of

myocardial dysfunction and increased mortality in the ICU setting (165-167). Elevated levels of

natriuretic peptides have been found to be markers of unfavourable prognosis in patients with

severe sepsis and septic shock (16, 168, 169). Varpula and colleagues (138) found that the

NT-proBNP values were frequently increased in severe sepsis and septic shock in patients

admitted to the ICU. Of note, NT-proBNP values were higher in non-survivors than survivors.

Also patients with less severe infections seem to have elevated levels of natriuretic peptides

(170).

A correlation between increased plasma levels of BNP and IL-6 in patients with septic shock

has been shown earlier and in recent studies both BNP and NT-pro-BNP secretion have been

linked to general inflammation (171). Rudiger and colleagues (172) showed a correlation

between NT-pro-BNP and CRP levels in a small group of haemodynamically unstable patients.

NT-pro-BNP or BNP levels did not differ significantly between patients with acute cardiac

failure and those with septic shock. The result was similar in a study by Shor and colleagues

(173), in which BNP levels correlated positively with CRP in septic patients without systolic

35

myocardial dysfunction. In another study the focus was in cancer patients with multiple co-

morbidities (174). The BNP-levels of these patients were elevated but without association with

volume overload or left ventricular dysfunction. Nonetheless, there was a significant

association with both sepsis and 30-day mortality in patients with markedly elevated BNP-

values. Nikolaou and colleagues (170) showed that BNP levels were elevated also in the acute

phase of community-acquired microbial infections without severe sepsis or septic shock.

There are no earlier studies of the kinetics or predictive use of NT-pro-BNP in patients with

neutropenic fever.

2.3.4. Other markers

2.3.4.1. Lactate

Plasma lactate is a result of the balance between lactate production and consumption.

Hyperlactataemia is typically present in patients with severe sepsis or septic shock. In these

patients not only high lactate but also poor lactate clearance has been recognized as an early

marker of mortality (175). Hyperlactatemia may be secondary to anaerobic metabolism due to

tissue hypoperfusion. The prognostic value of raised serum lactate levels has been well

established in patients with septic shock, especially if the high levels persist (176, 177). Serial

lactate measurements have been considered to be better markers for mortality and organ

failure than a single lactate determination (178). Evaluation of serum lactate levels is essential

to identify tissue hypoperfusion in patients who are not yet hypotensive, but who are at high

risk for septic shock.

The estimation of lactate levels in septic patients is not always straightforward. For example,

elevated lactate levels may result from decreased clearance by the liver or lactate acidosis

rather than from global hypoperfusion. In the study by Ramzi and colleagues (179) elevated

lactate (>3 mmol/l) and low serum bicarbonate (<17 mmol/l) at the onset of bacteraemia were

useful biomarkers in predicting septic shock and mortality in neutropenic patients. In a

multivariate analysis, especially two variables, pulmonary infection and serum lactate >3

mmol/l, were associated strongly with septic shock.

There are only limited data available on the kinetics or prognostic usefulness of lactate in

haematological patients with neutropenic fever. In a recent study elevated plasma lactate level

at start of neutropenic fever was not common, and did not distinguish severe sepsis. In

contrast, a high lactate level and impaired lactate decrease signified a fatal course of

neutropenic fever (180).

36

2.3.4.2. Procalcitonin

Calcitonin and its precursor procalcitonin (PCT) were initially used as a serum marker for

detection and follow-up of therapy for neuroendocrine tumours. Later on PCT levels were

found to be increased in patients with severe systemic inflammation (e.g. trauma, systemic

bacterial infection and sepsis). Levels of procalcitonin are undetectable in healthy individuals

(181).

The production and biological function of PCT involves a complex and time-dependent

mechanism. Significant production of PCT has been observed in adherent monocytes, but not

in circulating leukocytes. Monocytes produce PTC only for a limited time. Parenchymal cells

start to produce PTC after interaction with adherent monocytes. Local or systemic

inflammations affecting the parenchyma and monocyte adhesion are the preconditions for PCT

production. This explains also why PCT is induced by not only local or systemic inflammation,

but also after tissue trauma (182). In neutropenic patients, monocytes and other leukocytes are

absent, leaving tissue-based mechanisms of host defence (124). PCT elevates rapidly (within

2-4 hours) in severe forms of systemic inflammation or bacterial infections. It has been shown

in many settings that PTC serum concentrations react more rapidly than CRP concentrations

in sepsis patients (142, 147).

In Switzerland PCT has been used to guide antibiotic therapy in non-neutropenic community-

acquired pneumonia with success (183). The results have been similar when decrease in

serial PCT concentrations was used to predict favourable outcome of febrile episodes in

neutropenic haematological patients (184). In recent review by Sakr and colleagues (185) PCT

was shown to discriminate fever due to systemic forms of infection from fever of non-infectious

origin. It had only a minimal role in discriminating gram-negative from gram-positive infections.

Of note, PCT was not superior to interleukin-6 or CRP concentrations in outcome prediction in

patients with febrile neutropenia. In a large meta-analysis the results were in line with the

findings of Sakr and colleagues: although high PCT occured commonly in infection, it was also

elevated in many non-infectious conditions (186). There were also observations of febrile

septic patients with documented bacteraemia having PCT values within normal range.

Accordingly, PCT is not a specific indicator of either infection or sepsis and publications

concerning its prognostic utility have been contradictory (187-189). The authors point out that

the most commonly applied assay is not sensitive enough to detect potentially important mild

elevations or trends and that clinical studies with a more sensitive PCT assay are needed

(125, 183).

37

2.3.4.3. Interleukins

Interleukins 6 (IL-6) and 8 (IL-8) are proinflammatory cytokines, mainly produced by

monocytes. They are an important part of the cytokine cascade, together with inhibitory

cytokines (126). The kinetics of these cytokines are very fast (releasing within less than 1-2

hours), but concentrations may also decline within a short time (Table 2). IL-6 is a

multifunctional cytokine that regulates B- and T-cell function and acute-phase response such

as secretion of CRP. IL-8 is an inflammatory cytokine that mainly functions as a neutrophil

chemo-attractant and activating factor (190).

Both IL-6 and IL-8 have been demonstrated to be reliable predictors of sepsis in patients with

neutropenic fever (190). Engel and colleagues (191) showed that both IL-6 and IL-8 had

predictive value in neutropenic patients with bacteraemia. Especially IL-8 might have predictive

capacity in this respect.

Anti-inflammatory cytokines, mainly interleukin 4 (IL-4) and 10 (IL-10), are produced to down-

regulate the systemic inflammatory response in sepsis. The value of anti-inflammatory

cytokines in different studies is rather obscure. Loisa and colleagues (192) studied the anti-

infammatory response in the development of MOF in the ICU setting, but overproduction of IL-

10 was not observed. In another study the simultaneus detection of 17 different cytokines

demonstrated that both pro- and anti-inflammatory cytokines were significantly higher in

patients with septic shock than in patients with severe sepsis (193). Thus, higher cytokine

concentrations were associated with severity and evolution of organ dysfunction, but anti-

inflammatory cytokines had no specific role in this setting. In adult patients the measurement of

IL-10 was of limited value in predicting the outcome of febrile neutropenia (194).

38

3. AIMS OF THE STUDY

The aim of this study was to examine the nature of severe sepsis and possible predictive

factors of severe sepsis in neutropenic haematological patients. The specific study questions

were:

1. What are the epidemiological features, microbiological aetiology and outcome of severe

sepsis patients with AML and ASCT? (I, II)

2. Are the kinetics of CRP associated with severe sepsis in neutropenic patients with AML

and ASCT? (I, II)

3. Does serum VEGF have predictive value in severe neutropenic sepsis compared with

CRP? (III)

4. Does serum NT-proBNP have predictive value in severe neutropenic sepsis when

compared with CRP? (IV)

39

4. PATIENTS AND METHODS

4.1. Patients

4.1.1. Patients in retrospective studies (I-II)

4.1.1.1. Characteristics of patients with acute myeloid leukaemia (I)

The study included altogether 84 patients aged 70 years with AML treated with intensive

chemotherapy in the Department of Medicine, Kuopio University Hospital in 1996-2005. There

were 44 males and 40 females with a median age of 50 years at diagnosis (range 18-69

years).

4.1.1.2. Characteristics of autologous stem cell recipients (II)

The study population consisted of 319 adult patients who received ASCT at the Department

of Medicine, Kuopio University Hospital in 1996-2006. There were 189 males and 130 females,

with a median age of 55 years (range 16-73). The most common diagnosis was NHL (n=160,

50%) followed by MM (n=113, 35%). The patient and transplant characteristics are presented

in Table 3.

4.1.2. Patients in prospective studies (III-IV)

From first of December 2006 to 30th of November 2008 adult patients ( 70 years of age)

treated in the haematology ward of Kuopio University Hospital were enrolled in the prospective

part of the study. Patients were eligible if they had either AML or received high-dose

chemotherapy supported by ASCT and if they had neutropenic fever after chemotherapy.

Study III included all eligible patients treated during the first 12 months (n=42) and study IV all

eligible patients during a 24-month period (n=70). Patient characteristics and chemotherapy

given for study III are presented in Table 4 and for study IV patients in Table 5.

In study III, only the first episode of neutropenic fever was included in the study. In study IV,

fourteen patients with AML had more than one period of neutropenic fever after induction and

consolidation chemotherapy. Thus, the number of periods with neutropenic fever was 94 in 70

patients.

40

Table 3. Characteristics of ASCT patients transplanted in 1996-2006___________________________________________________________________________Total number of patients 319___________________________________________________________________________

Male 189 (59%)Female 130 (41%)

Age (years)median (range) 55 (16-73)

< 60 years 223 (70%) 60 years 96 (30%)Diagnosis NHL 160 (50%) MM 113 (35%) HL 22 (7%) CLL 8 (3%) Others 16 (5%)High-dose regimen BEAC 132 (41%) BEAM 54 (17%) HD-MEL 118 (37%) Other 15 (5%)Stem cell source PB 317 (99%) BM 2 (1%)___________________________________________________________________________Abbreviations: ASCT, autologous stem cell transplant recipients; NHL, non-Hodgkin lymphoma; MM,multiple myeloma; HL, Hodgkin lymphoma; CLL, chronic lymphocytic leukaemia; PB, peripheral blood;BM, bone marrow; BEAC, carmustine, etoposide, cytarabine and cyclophosphamide; BEAM,carmustine, etoposide, cytarabine and melphalan; HD-MEL, high-dose melphalan.

41

Table 4. Characteristics of patients in study III.

Number of patients 42

MaleFemale

26 (62%)16 (38%)

Age: median, range 57, 18-70

> 60 years, number (%) 16 (38%)

Diagnosis, number (%)

AML 15 (36%)

NHL 13 (31%)

MM 7 (17%)

HL 6 (14%)

CLL 1 (2%)

High-dose regimen,number (%)

BEAC 15 (36%)

BEAM 5 (12%)

HD-MEL 9 (21%)

IAT 6 (14%)

HD-AraC-Ida 5 (12%)

Mito-HDAraC 2 (5%)

Abbreviations:AML, acute myeloid leukaemia;NHL, non-Hodgkin lymphoma;MM, multiple myeloma; HL,Hodgkin lymphoma;CLL, chronic lymphocytic leukaemia;BEAC, carmustine, etoposide, cytarabine andcyclophosphamide; BEAM: carmustine, etoposide, cytarabine and melphalan; HD-MEL, high-dosemelphalan; IAT, idarubicin, cytarabine, thioguanine; HD-AraC-Ida, high-dose cytarabine, idarubicin;Mito-HDAraC, mitoxantrone, high-dose cytarabine.

42

Table 5. Characteristics of patients in study IV._________________________________________________________________________Number of patients 70Male 44 (63%)Female 26 (37%)Age: median, range 56, 18-70

60 years, number (%) 23 (33%)

Diagnosis, number (%) AML 19 (27%) NHL 25 (36%) MM 14 (20%) HL 9 (13%) Other 3 (4%)

Previous cardiovascular disease, number (%) 10 (14%)Hypertension and coronary heart disease 2Hypertension and chronic atrial fibrillation 2Previous coronary by-pass 2Cardiomyopathy (anthracycline) 2Mixed type cardiomyopathy 1Hypertension and aortic valve sclerosis 1

High-dose regimen during first period of neutropenic fever, number (%) BEAM 20 (29%) BEAC 15 (21%) HD-MEL 16 (23%) IAT 8 (11%) HDAraC-Ida 6 (9%) IdAraC-Ida 3 (4%) Mito-HDAraC 2 (3%)

Prior anthracycline therapy, number (%) 60 (86%) Doxorubicin 41 (59%) Idarubicin 19 (27%)_________________________________________________________________________

Abbreviations: NHL, non-Hodgkin lymphoma; AML, acute myeloid leukaemia; MM, multiple myeloma;HL, Hodgkin lymphoma; BEAM, carmustine, etoposide, cytarabine and melphalan; BEAC, carmustine,etoposide, cytarabine and cyclophosphamide; HD-MEL, high-dose melphalan; IAT, idarubicin,cytarabine, thioguanine; HD-AraC-Ida, high-dose cytarabine, idarubicin; IdAraC-Ida, intermediate-dosecytarabine, idarubicin; Mito-HDAraC, mitoxantrone, high-dose cytarabine.

43

4.2. Methods

4.2.1. Definitions

Neutropenic fever was defined using the criteria from the Infectious Diseases Society of

America (IDSA) for the definition of neutropenia and neutropenic fever (6). Neutropenia was

defined as a neutrophil count of <0.5 x 109/l, or a count of <1 x 109/l with a predicted decrease

to <0.5 x 109/l. Fever was defined as a single oral temperature of 38.3°C or a temperature of

38.0°C for 1 h.

Sepsis was defined as a clinical syndrome in which a systemic inflammatory response was

present with infection. Severe sepsis was present if sepsis was complicated by organ

dysfunction, hypoperfusion or hypotension. The definiton for septic hypotension included

systolic arterial pressure <90 mmHg, a mean arterial pressure <60 mmHg or a reduction in

systolic blood pressure of >40 mmHg from baseline despite adequate volume resuscitation, in

the absence of other causes of hypotension (78, 79).

4.2.2. Clinical management and supportive care measures (I-IV)

In retrospective studies (I-II) the information of chemotherapy regimen used, number of days

with neutropenia, number of febrile days (>38°C) and blood culture findings were collected

from individual patient charts for each febrile neutropenic period (Figure 1). Haematological

wards are accustomed to taking care of critically ill patients and individual monitoring charts

are familiar. These are also commonly used, which makes the collection of data much easier.

In retrospective part of the study each patient’s temperature, blood pressure and heart rate

were observed twice a day after chemotherapy by the nursing staff of the haematological ward

as a part of the normal treatment routine. In case of neutropenic fever bed-side monitoring

continued at a two or three hour interval. In case of severe sepsis, the monitoring was

constinuous. A potential localisation of infection was searched for during each neutropenic

period. In addition, re-induction chemotherapy cycles and intensive consolidation cycles were

taken into account in patients with AML.

In the prospective part of the study (studies III-IV) the clinical aim was to improve

management of haematological patients with neutropenic fever with close monitoring and

vigilant supportive care in the haematology ward setting. The start of prospective study was

preceeded by careful education of the whole nursing staff.

44

Information from each neutropenic fever period

- Chemotherapy regimen used- Number of days with febrile neutropenia

- Blood culture findings

- CRP-valueso Baseline CRP <48 h prior to the rise of

neutropenic fevero CRP 2-3 days after the rise of neutropenic

fever

- Signs or findings of local infectiono Radiological findingso Urine culture findingso Stool culture findings

- Blood pressure- Heart rate- Breath rate

CRP-

slope

velocity

Patient chart

Fulfilment of criteria for severe sepsis?

Neutropenic fever

period with severe

sepsis

Neutropenic fever

period without

severe sepsis

YES NO

Figure 1. Collection of information from neutropenic patients and fever periods in studiesI (AML) and II (ASCT).

45

Each patient was examined daily for signs and possible sources of infections. Special

attention was paid to features indicating the development of severe sepsis. When a patient

became neutropenic, monitoring (body temperature, blood pressure, heart rate, breath rate

and peripheral blood oxygen saturation) began bedside twice a day. From the beginning of

neutropenic fever monitoring was either hourly or continual – depending on the individual

patient situation (Table 6). Daily fluid intake (intravenous and per os) and urine output were

registered every 12 hours during the first three days of neutropenic fever by haematological

ward nursing staff. The patient’s weight was monitored once a day. The chemotherapy used,

number of days with neutropenia (<0.5 x 109/l), number of febrile days (>38°C) and blood

culture findings were also noted for each febrile neutropenic period (Figure 2). In case of fever

during neutropenia, fluid resuscitation using saline infusion was used.

Indwelling central venous catheters were used during the study period in all AML patients.

Central venous catheters were used in about 80% of ASCT recipients. Granulocyte colony-

stimulating factor (filgrastim or peg-filgrastim) was routinely used in ASCT recipients, whereas

only a minority of neutropenic AML patients received growth factors as a part of supportive

care, on an individual basis. Red blood cell concentrates were used to keep haemoglobin >80

g/l, and platelet concentrates were given routinely if the morning platelet count was less than

20 x109/l.

4.2.2.1. Chemotherapy

During the retrospective study period, patients with AML were treated with intensive

induction and consolidation chemotherapy courses according to the Finnish Leukaemia Group

prospective protocols. The chemotherapy for AML is given in the haematological wards of

university hospitals. The intensive induction course takes weeks, with long lasting neutropenia

until the bone marrow recovers. After this AML patients may leave hospital for a few days

before consolidation courses begin. Peripheral blood samples are monitored on a regular

basis. When cytopenia or neutropenic fever developes, patients return to the hospital. The

AML-92 protocol (2) (Table 7) was used for 65 patients and the AML-2003 protocol for 19

patients. In the AML-2003 trial, the induction course is according to randomisation idarubicin,

low-dose cytarabine and thioguanine (IAT) or intermediate-dose cytosine arabinoside plus

idarubicin.

46

Table 6. Example of a follow-up chart used in haematological ward in patients with neutropenicfever.

47

Figure 2. Collection of information from neutropenic patients and fever periods in studies III(VEGF) and IV (NT-proBNP).

Information from the first

neutropenic fever period

(Study III)

Patient chart

Information from each

neutropenic fever period

(Study IV)

Neutropenic fever

with

severe sepsis

Neutropenic fever

without

severe sepsis

Neutropenic fever

with

severe sepsis

Neutropenic fever

without

severe sepsis

Information from each neutropenic fever period

Chemotherapy regimen used

Number of days with febrile neutropenia

Blood culture findings

Signs or findings of local infection

- Radiological findings

- Urine culture findings

- Stool culture findings

- Blood pressure

- Heart rate

- Breath rate

- Peripheral oxygen saturation

- Daily fluid intake and diuresis (every 12 hours)

NT-proBNP values

CRP values

VEGF values

CRP values

in case of neutropenic

fever either every one

hour or constantly

48

The second course is high-dose cytosine arabinoside plus idarubicin. Responding patients

receive risk-adapted consolidation courses all including high-dose cytosine arabinoside with an

anthracycline or mitoxantrone.

HDT supported by ASCT is given in university hospitals. Chemoterapy characteristics for

studies II-IV are presented in Tables 3-5. Additionally, in study IV, sixty patients (86%) had a

history of anthracycline treatment. Nineteen of these patients (27%) received idarubicin

(median dose 36, range 24-60 mg/m2) and 41 (59%) received doxorubicin or epirubicin

(median dose 300, range 100-500 mg/m2).

4.2.2.2. Antimicrobial therapy

In case of neutropenic fever, 2-3 blood culture sets were drawn from cubital veins at a one-

hour interval by laboratory technicians. Empirical antibiotic treatment was started immediately

after blood cultures were obtained. A combination of a broad-spectrum betalactam and an

aminoglycoside was used. Antimicrobial treatment was changed when needed, according to

the microbiological findings, radiological or clinical findings. If the fever persisted for 3-5 days,

new blood cultures were drawn, and the initial antimicrobial therapy was re-considered. In case

of mucosal symptoms during neutropenia, oral fluconazole (200 mg/d) was started. In case of

persistent fever, amphotericin B deoxycholate was given as empirical antifungal therapy. More

recently caspofungin has been used for empirical antifungal therapy. Vancomycin was

considered only if there were signs of infection in central venous catheter or if simultaneus

blood cultures revealed coagulase-negative staphylococci. No systematic antibacterial

prophylaxis was used until January 2008, when oral ciprofloxacin (500mg b.i.d) was given to

neutropenic NHL patients receiving ASCT as a part of clinical protocol.

General infection control measures (e.g. isolation policies, hospital hygiene including

cleaning and hand-washing policies) remained constant during the whole study period.

4.2.3. Laboratory methods

In the retrospective part of the study (I-II) serum concentrations of C-reactive protein (CRP)

were collected from the individual patient charts. CRP was routinely determined three times

per week during neutropenia. During clinically significant infection, CRP measurements were

performed at 1-2 day intervals.

49

Table 7. Chemotherapy protocol in the AML-92 study of the Finnish Leukaemia Group (2)

_________________________________________________________________________

Therapy Dosage Administration Days administered_________________________________________________________________________

First course (IAT)

Idarubicin 12 mg/m2 i.v., 10–15 min 1, 3, 5

Cytarabine 50 mg/m2 i.v., bolus 1

100 mg/m2 i.v., continuously 1(–7)–9

Thioguanine 75 mg/m2 p.o., twice daily 1(–7)–9

Second course (HDAraC-Ida)

Cytarabine 1500 mg (1.0 g)/m2 i.v., 3 h, twice daily 1–5

Idarubicin 8 mg/m2 i.v., 30 min 6–8

Third course (MEA)

Etoposide 100 mg/m2 i.v., 1 h 1–4

Cytarabine 1000 mg (0.5 g)/m2 i.v., 2 h, twice daily 1–4

Mitoxantrone 12 mg (8 mg)/m2 i.v., 30 min 2–5

Fourth course (Amsa-HDAraC)

Amsacrine 115 mg/m2 i.v., 2 h 1–5

Cytarabine 3000 mg/m2 i.v., 3 h, twice daily 1–2

Fifth course (HDAraC-Ida)

Cytarabine 1500 mg (1.0 g)/m2 i.v., 3 h, twice daily 1–5

Idarubicin 8 mg/m2 i.v., 10–15 min 6–8

_________________________________________________________________________

Dosage and days administered in parenthesis indicate patients 56 years old.Abbreviations: i.v, intravenously; p.o, orally.

For the prospective part of the study (III-IV), the first blood samples for the measurement of

CRP, vascular endothelial growth factor (VEGF) and amino-terminal pro-brain natriuretic

peptide (NT-proBNP) serum concentrations were drawn at the beginning of neutropenic fever

concomitantly with samples taken for blood cultures. Further samples were collected next

morning and then every 24 hours up to three or five days (Figure 3). Platelet and leukocyte

counts were routinely examined daily throughout the neutropenic period.

50

Blood samples for VEGF and NT-proBNP were centrifuged (2200 G 10 minutes) by

laboratory technicians. VEGF samples were then stored at -20°C for later analysis and NT-

proBNP samples were analysed within 12 hours.

4.2.3.1. Blood cultures

In case of neutropenic fever (neutropenia and a single oral measurement of 38.3°C or

>38°C for one hour) 2-3 blood cultures were drawn from cubital veins at 1 hour interval by

laboratory technicians. Blood cultures were processed using the automated

blood culture system Bactec 9240 (Becton Dickinson, Sparks, USA). Incubation period for both

aerobic and anaerobic bottles were 7 days and for MYCO F / Lytic bottles 42 days.

A single positive blood culture was considered significant if the microbe was a clinically

relevant cause of infection. Common skin contaminants (e.g. coagulase-negative

staphylococci) were considered significant only if they were found in two consecutive blood

cultures or if there was a concurrent skin or catheter infection.

4.2.3.2. Serum C-reactive protein

The concentration of serum CRP was measured with an immunoturbidometric method

(Otsuji, 1982, Clin Chem) using a multichannel Hitachi 717 Automatic Analyzer (Hitachi Ltd,

Tokyo, Japan) until the year 2000 and then with a Konelab60i Clinical Chemistry Analyzer (Lab

systems CLD, Konelab, Helsinki, Finland). The reference value for serum CRP in healthy

persons was <10 mg/l.

In the retrospective studies (I-II) also CRP slope velocity was analysed. For all febrile

neutropenic periods following intensive chemotherapy, baseline CRP (< 48 h prior to rise of

fever, CRP0) and the CRP level 2-3 days after the onset of fever (CRP2-3) was registered. Peak

CRP and the time from the rise of fever to the highest CRP value of a given neutropenic period

was also registered. CRP slope velocity was defined as the difference between CRP2-3 and

CRP0, divided by the number of days and expressed as mg/l/d.

CRP2-3 – CRP0CRP slope velocity = -------------------------------------- Number of days (2 or 3)

In the prospective studies (III-IV), CRP was measured at the start of neutropenic fever and

then every morning during the study period (Figure 3).

51

Figure 3. Prospective study protocol.

Written informed consent from all patients•with AML < 70 years•with HDT supported by ASCT

On the first morning after the beginningof neutropenic feverstudy blood samples (d1)

Neutropenic fever:neurophil count < 0.5 x 109/l andfever (single oral temperature 38.3°or a temperature 38.3° for 1 h)

No neutropenic fever

On the second morning from the beginningof neutropenic feverstudy blood samples (d2)

On the third morning from the beginningof neutropenic feverstudy blood samples (d3)

On the fourth morning from the beginningof neutropenic feverstudy blood samples (d4)

Study bloodsamples werenot collected

At the beginning of neutropenic feverat the same time when blood culturesand other clinically important samples were obtainedstudy blood samples (d0)

52

4.2.3.3. Serum vascular endothelial growth factor

Serum VEGF concentrations were measured using a commercial enzyme-linked

immunosorbent assay (ELISA) kit (Quantikine ®, R&D Systems; Minneapolis, MN), which

recognises the soluble isoforms (VEGF121 and VEGF165). The same VEGF ELISA method was

used and described in detail in a recent Finnish study called FINNSEPSIS (15). In brief, the

optical density at 450 nm was measured with wavelength correction at 540 nm (Multiscan RC

plate reader; Thermolabsystems, Helsinki, Finland) and serum VEGF concentration was

determined with Genesis (Life Sciences, UK) computer software capable of generating a four

parameter logistic curve fit.

The intra-assay coefficient variation (CV) for a control sample (mean concentration 345

pg/ml) was 5.7% (n=10) and for pooled serum (mean concentration 96 pg/ml) 6.5% (n=10).

The inter-assay CV for two control samples (mean concentration 167 pg/ml and 1002 pg/ml)

was 8.9% and 4.3% (n=12). For pooled serum the inter-assay CV was 8.5% (n=12). In the

FINNSEPSIS study, the samples of 30 healthy adult controls were collected. The mean age of

these controls was 36±7 years. The number of males and females was equal (M/F 15/15). The

median concentration of VEGF in the control group was 260 (IQR 126-459, range 63-809)

pg/ml, which was used as the control value in the FINNSEPSIS study (15).

4.2.3.4. Plasma amino-terminal pro-brain natriuretic peptide

Samples for the measurement of serum NT-proBNP were drawn by venipuncture into Li

heparin-containing tubes from patients lying in the supine position. Plasma was separated and

NT-proBNP was determined within 24 hours using an electrochemiluminescence

immunoassay (ECLIA, Roche Diagnostics) on a Cobas e 601 analyzer (Hitachi High

Technology Co, Tokyo, Japan). The measuring range, defined by the lower detection limit and

the maximum of the master curve provided by the manufacturer was 0.6-35 000 ng/l. The

reference values were given according to age and gender of the patients: for males under 50

years of age 0-84 ng/l, 50-65 years of age 0-194 ng/l and over 65 years of age 0-229 ng/l. For

females under 50 years of age 0-155 ng/l, 50-65 years of age 0-222 ng/l and over 65 years of

age 0-352 ng/l.

53

4.2.4. Statistical methods

Statistical analysis was performed with SPSS version 14.0 for Windows (SPSS, Inc.,

Chicago, IL, USA). For the analysis of nominal data, the Chi-square test was employed.

Continuous variables were expressed as medians with ranges (interquartile ranges or ranges

from minimum to maximum). Categorical variables were expressed as absolute numbers

(percentages).

The correlation between continuous variables was evaluated by the non-parametric

Spearman’s correlation. For single measurements, differences between subgroups were

evaluated by the Mann-Whitney U-test, and by Student’s t-test for log-transformed variables. In

study III the time period from the beginning of neutropenic fever to achieve maximal VEGF

concentration was compared to the respective time to achieve maximal CRP concentration

using Wilcoxon signed ranks test. The Youden index (sensitivity + specificity –1) (195), a

summary measure of receiver-operating-characteristics curve analysis, was used to evaluate

the goodness of CRP vs. VEGF on the first days of neutropenic fever to predict the

development of severe sepsis (a high Youden index indicates good test performance).

Repeated measures ANOVA was used to assess the impact of severe vs. non-severe sepsis

(defined as between-subjects factor) on the VEGF and CRP concentrations. The within-

subjects factor was defined as the VEGF concentration on day 0, 1, 2, and 3. The receiver-

operating characteristic (ROC) curve analysis was used to evaluate the predictive

discrimination of VEGF and CRP for severe sepsis on day 0 and day 1 of neutropenic fever.

In the study IV a logarithmic transformation was used to correct the skewed distribution of

NT-proBNP and CRP. After logarithmic transformation, a near-normal distribution was

achieved. When patient-related variables were evaluated, only the first episode of neutropenic

fever for each patient was included in the analyses. When fever period-related variables were

evaluated, all fever periods were included. ANOVA for repeated measurements was used to

evaluate possible differences between subgroups in NT-proBNP and CRP levels during days

0-3. A p-value < 0.05 was considered significant.

4.3. Approval of the Ethics Committee

The study was approved by the Ethic Committee of the Kuopio University Hospital. All study

patients gave their written informed consent.

54

5. RESULTS

5.1. Retrospective studies (I-II)

5.1.1 Epidemiological features of severe sepsis

In study I, there were altogether 290 neutropenic periods in 84 AML patients (median 3,

range 1-7). In 280 periods (97%) neutropenic fever was present. Severe sepsis was observed

in 35 periods (13%) and in 13 periods (5% of all neutropenic fever periods) severe sepsis led

to ICU admission (Table 8). In 17 periods (49%) the criteria for severe sepsis was hypotension

despite of fluid resuscitation. In 11 periods (31%) the criteria for severe sepsis was septic

shock and in 3 periods (8%) respiratory failure. In 3 periods (8%) septic shock developed into

MOF. One patient (3%) died due to septic shock on the way to the hospital (Table 9).

The median time from the onset of the fever to the point where criteria for severe sepsis

were met was 2 days (range 0-39). Thirteen patients were treated in the ICU due to severe

sepsis, with a median stay of 3 days (1-42). Five patients (38%) recovered and eight patients

died due to irreversible septic shock a median of 2 days (1-7) after admission to the ICU.

After the first induction course in study I, neutropenic fever was found in 80 out of 84 patients

(95%). Severe sepsis was observed in 10 patients (13%) and 7 patients (8%) were admitted to

the ICU. Four patients (5%) died after the first induction course due to infectious complications.

In study II there were 319 ASCT recipients with 265 (83%) febrile neutropenic periods.

Because of the treatment protocol, only one period of neutropenic fever was included for each

patient. Severe sepsis was observed in 17 patients (5%). In 5 patients (29%) the criteria for

severe sepsis was hypotension despite fluid resuscitation. In 12 patients (71%) the criteria for

severe sepsis was septic shock (Table 10). In nine occasions (3%) severe sepsis led to ICU

admission.

The median time from the onset of fever to the point, where criteria for severe sepsis were

met was 3 days (range 1-10). The median time to ICU admission was 7 days after the start of

fever (range 2-25), and the median treatment time at ICU was 3 days (1-17). Seven patients

(78%) died due to irreversible septic shock a median of 2.5 days (1-12) after the admission to

ICU. Only two patients treated in the ICU (22%) recovered.

Severe sepsis was the most common severe early (<30 d) complication in ASCT recipients

(17/20 patients, 85%). Other severe early complications included pneumonia (n=1), severe

cardiotoxicity (n=1) and a combination of liver and cardiac failure (n=1).

55

Table 8. Studies I-IV

Study I Study II Study III Study IV

_________________________________________________________________________Number of patients 84 319 42 70

Number of patients with severe sepsis 30 (36%) 17 (5%) 5 (12%) 13 (19%)

Number of neutropenic feverperiods 280 265 42 94

Number of periods with severe sepsis 35 (13%) 17 (5%) 5 (12%) 13 (14%)

Length of neutropenia, daysmedian (range)

Periods with severe sepsis 23 (7-85) 11 (6-25) 9 (3-25) 12 (3-25) Periods without severe sepsis 24 (5-227) 9 (3-63) 10 (4-23) 10 (4-30)

Mortality associatedwith severe sepsis 9/84 (11%) 9/319 (2%) 1/42 (2%) 2/70 (3%)

Case fatality rate for periodswith severe sepsis 9/35 (27%) 9/17 (53%) 1/5 (20%) 2/13 (15%)________________________________________________________________________

In study I neutropenic septic infection was the cause of death in 9 patients. Thus mortality

due to neutropenic sepsis was 11% (9/84). Neutropenic sepsis was suspected cause of death

also in 2 other patients: one patient (number 27 in table 9) died due to a septic shock outside

hospital (paramedic care) and one patient (number 26 in table 9) with refractory leukaemia

died without ICU admission in the hematology ward. The case fatality rate for periods with

severe sepsis was 27% (9/35). In study II neutropenic sepsis was the cause of death in 9

patients and mortality due to neutropenic sepsis was 2% (9/319). The case fatality rate for

periods with severe sepsis was 53% (9/17). The mortality due to severe sepsis was higher in

NHL patients when compared to other patient groups (6% vs. 0%, p=0.003).

5.1.2 Microbiological findings and site of infection

In study I clinically significant microbiological findings were found by blood cultures in 165 out

of 280 episodes with neutropenic fever (59%) (Table 11). Gram-positive cocci were the most

commonly identified causes of neutropenic fever (94 episodes, 57%), followed by gram-

56

negative rods (70 episodes, 43%). Only one fungal sepsis was observed during this time. In

cases of severe sepsis, gram-negative rods were found in 15/35 of periods (43%).

In study II clinically significant microbiological growth in blood cultures was seen in 68 out of

265 periods with neutropenic fever (26%) (Table 11). Gram-positive cocci (44 episodes, 65%)

were the most common identified cause of neutropenic fever, followed by gram-negative rods

(23 episodes, 34%). Only one fungal bloodstream infection was found, concomitantly with

gram-positive bacteraemia. In cases of severe sepsis, gram-positive cocci were found in 7 out

of 17 periods (41%). Gram- negative rods were found in six periods (35%). Pseudomonas

aeruginosa was the most common (5 episodes, 30%).

The lungs and upper respiratory airways were the most common site of infection (48

episodes, 17%) in study I. The number of catheter-related infections was 45 (16%) and skin

(including perineal region) infections 17 (6%). In study II, the localisation of infection remained

in most cases unknown. The percentage of catheter-related infections was 11%.

5.1.3. Factors associated with severe sepsis

In both studies, the neutropenic febrile periods were divided into those with and those

without severe sepsis. In study I there were no differences in the median length of neutropenia,

but in study II the median length of neutropenia was longer in ASCT recipients with severe

sepsis (11 vs. 9, p=0.05).

In study I gram-negative bacteria were found more often in blood cultures (43% vs. 22%,

p=0.03) and blood culture findings were more often positive (66% vs. 58%, NS) in periods with

severe sepsis than those without. In study II severe sepsis was more common in patients with

NHL than in other patients (9% vs. 3%, NS). Patients with severe sepsis were also older than

those without (59 vs. 55, p=0.007). Blood culture findings were significantly more often positive

(76% vs. 22%, p<0.001) and gram-negative bacteria were found more often (35% vs. 7%,

p=0.001) among patients with severe sepsis than those without. Of note, Pseudomonas

aeruginosa was found in 30% (n=5) of patients with severe sepsis and only in 1% (n=3) of

those without severe sepsis (p<0.001).

57

Table 9. Description of AML patients (n=30) with periods of severe sepsis (n=35) in study I.____________________________________________________________________________

Number Gender/age Treatment Conditioning Criteria for ICU Blood culture

protocolregimennumber severe sepsis

Recovered1 F/57 AML-92 IV Hypotension No Gemella morbillorum2 M/54 AML-92 II Hypotension No Klebsiella pneumoniae3 F/69 AML-92 V Hypotension No Enterobacter cloacae4 F/43 AML-92 IV Hypotension No Staphylococcus epidermidis5 M/38 AML-92 I Hypotension No Culture negative6 IV Hypotension No Escherichia coli7 F/59 AML-92 I Hypotension No Culture negative8 F/57 AML-92 IV Hypotension No Klebsiella pneumoniae9 M/62 AML-92 IV Respiratory failure No Haemophilus influenzae

10 M/44 AML-92 III Hypotension No Escherichia coli11 M/66 AML-92 III Hypotension No Entrococcus faecium12 M/25 AML-92 I Hypotension No Culture negative13 M/38 AML-92 IV Hypotension No Staphylococcus epidermidis14 M/57 AML-92 V Hypotension No Staphylococcus epidermidis15 M/55 AML-92 I Septic shock Yes Enterococcus faecalis16 F/46 AML-92 I Septic shock Yes Culture negative17 F/52 AML 2003 I Respiratory failure No Culture negative18 IV Septic shock Yes Escherichia coli19 F/55 AML-92 III Respiratory failure Yes Klebsiella pneumoniae20 F/39 AML-92 I Hypotension Yes Culture negative21 M/59 AML 2003 II Hypotension No Klebsiella pneumoniae22 V Hypotension No Enterococcus faecium

Died23 F/59 AML-92 II Hypotension No Culture negative24 III Hypotension No Staphylococcus epidermidis25 IV Septic shock Yes Klebsiella pneumoniae26 M/20* AML-92 II Septic shock No Escherichia coli

27 M/64* AML-92 III Septic shock NoDied on the way to thehospital

28 F/33 AML-92 I Septic shock Yes Haemophilus influenzae29 M/38 AML-92 I Septic shock Yes Culture negative

30 F/39 AML-92 II Septic shock YesStenotrophomonasmaltophilia

31 M/58 AML 2003 III Septic shock Yes Culture negative32 M/61 AML 2003 I Septic shock, MOF Yes Streptococcus mitis33 M/58 AML-92 I Septic shock, MOF Yes Staphylococcus epidermidis34 F/58 AML 2003 III Septic shock, MOF Yes Culture negative35 M/53 AML-92 VI Septic shock No Pseudomonas aeruginosa

Abbreviations: ICU, intensive care unit; F, female; M, male; MOF, multiorgan failure.

* Patients with suspected neutropenic sepsis

58

Table 10. Description of adult patients with severe sepsis (N=17) after ASCT.

NoGender

/Age Dg

ConditioningRegimen /

Year ofTreatment

Criteria for SevereSepsis ICU Blood Culture Finding

Recovered:

1. M/54 MM HD-MEL/2005 Hypotension No Negative

2. F/59 MM HD-MEL/2004 Hypotension No Pseudomonas aeruginosa

3. M/59 NHL BEAC/2003 Hypotension No Negative

4. M/61 MM HD-MEL/2001 Hypotension No Negative

5. M/64 NHL BEAC/2003 Hypotension No Staphylococcus epidermidis

6. F/58 MM HD-MEL/2005 Septic shock, MOF Yes Pseudomonas aeruginosa

7. M/62 NHL BEAC/2005 Septic shock Yes Streptococcus viridans

8. F/59 NHL BEAC/2006 Septic shock No Staphylococcus hominis

Died:

9. M/62 NHL BEAC/2003 Septic shock Yes Negative

10. M/67 NHL BEAC/2005 Septic shock, MOF Yes Staphylococcus epidermidis

11. M/62 NHL BEAM/2006 Septic shock Yes Pseudomonas aeruginosa

12. F/66 NHL BEAC/2006 Septic shock, MOF Yes Enterococcus faecium

13. M/50 NHL BEAC/1996 Septic shock No Enterococcus faecalis

14. M/40 NHL BEAM1998 Septic shock, MOF YesStreptococcus pneumoniae,Candida albicans

15. M/64 NHL BEAM/1999 Septic shock, MOF Yes Klebsiella pneumoniae

16. F/58 NHL BEAM/2000 Septic shock Yes Pseudomonas aeruginosa

17. M/59 NHL BEAC/2003 Septic shock No Pseudomonas aeruginosaAbbreviations: M, male; F, female; ICU, intensive care unit; NHL, non-Hodgkin lymphoma; MM,multiple myeloma; BEAC, carmustine, etoposide, cytarabine and cyclophosphamide; BEAM,carmustine, etoposide, cytarabine and melphalan; HD-MEL, high-dose melphalan; MOF,multiorgan failure.

59

Table 11. Blood culture findings and site of infection in patients with neutropenic fever periodsin retrospective studies (I and II).________________________________________________________________________

Study I Study II p-value N=84 N=319________________________________________________________________________

Number of neutropenic feverperiods 280 265

Localisation of infection

Unknown 137 (49%) 198 (75%) <0.001

Lungs and upper respiratory airways 48 (17%) 9 (3%) <0.001

Catheter-related infection 45 (16%) 29 (11%) NS

Skin (including perineal region) 17 (6%) 2 (1%) <0.001

Other 33 (12%) 27 (10%) NS

Positive blood culture 165 (59%) 68 (26%) <0.001

Gram positive cocci 94 (57%) 44 (65%) <0.001

Staphylococcus epidermidis 51 (31%) 28 (41%) 0.011

Streptococcus mitis 9 (5%) 5 (7%) NS

Enterococcus faecium 9 (5%) 2 (3%) NS

Others 25 (15%) 9 (13%) 0.008

Gram negative rods 70 (43%) 23 (34%) <0.001

Klebsiella pneumoniae 19 (12%) 9 (13%) NS

Pseudomonas aeruginosa 5 (3%) 8 (12%) NS

Escherichia coli 27 (16%) 4 (6%) <0.001

Others 19 (12%) 2 (3%) <0.001

Fungal 1 (0.6%) 1 (1%) NS

Candida albicans 1 (0.6%) 1 (1%) NS

Negative blood culture 114 (41%) 198 (74%) <0.001

60

5.1.4. Serum C-reactive protein in relation to severe sepsis

In study I the median time from the onset of the fever to peak CRP value was 4 days (range

1-39). The median peak CRP value was higher in patients with severe sepsis (261 mg/l vs.

153 mg/l, p<0.001). CRP2-3 was higher in patients with severe sepsis (190 mg/l vs. 96 mg/l,

p<0.001) than those without. In this study the CRP slope velocity was steeper in patients with

severe sepsis (48 vs. 34 mg/l/d, p= 0.02) (Table 12).

In study II the median time from the onset of fever to the peak CRP value was 3 days (range

1-7). CRP2-3 was higher in patients with severe sepsis (148 mg/l vs. 90 mg/l, p<0.001) as well

as the median peak CRP value (217 mg/l vs. 111 mg/l, p<0.001). The CRP slope velocity was

again found to be steeper in patients with severe sepsis (54 vs. 30 mg/l/d, p= 0.007) (Table

12).

5.2. Serum vascular endothelial growth factor in patients with neutropenic

fever: a comparison with C-reactive protein (III)

5.2.1. Epidemiological features of severe sepsis

All patients with severe sepsis had received ASCT for NHL. The median time after the onset

of fever to the fulfillment of the criteria for severe sepsis was 1 day (range 1-3). The diagnostic

criteria for severe sepsis were fulfilled in five out of 42 patients (12%) (Table 8): one of them

had septic shock requiring vasopressor support in the ICU, one patient had septic respiratory

failure requiring CPAP ventilation and three patients had fluid-responsive hypotension. One

patient (2%) died due to septic shock in the ICU during the first day after admission (Table 13).

5.2.2. Microbiological findings and site of infection

Blood cultures remained negative in the majority of the patients (36/42, 85%). Positive

findings in blood cultures included Staphylococcus epidermidis (n=3) and Staphylococcus

haemolyticus, Enterobacter cloacae and Escherichia coli, one each. The lungs were the most

common site of infection (n=10, 24%), followed by skin infections (including perineal region)

(n=8, 19%). The percentage of catheter related infections was 10%. In 18 periods (43%), the

site of infection remained unknown.

61

Table 12. Comparison of CRP in neutropenic fever periods with or without severesepsis in studies I and II.

Study I Study II

With

severe sepsis

Without

severe

sepsis

p

With

severe

sepsis

Without

severe

sepsis

P

N=35 N=245 N=17 N=248

Baseline CRP (mg/l)

median (range)17 (5-194) 24 (5-227) NS 27 (5-127) 15 (5-118) NS

CRP (mg/l) 2 to 3

days median (range)190 (26-379) 96 (3-359) <0.001 148 (76-375) 90 (5-253) <0.001

Peak CRP (mg/l)

median (range)261 (57-563) 153 (22-492) <0.001 217 (76-545) 111 (5-304) <0.001

Time to peak CRP

(d) median (range)4 (1-39) 5 (1-59) NS 3 (1-7) 3 (0-13) NS

CRP slope velocity

(mg/l/day) median

(range)

48 (8-167) 34 (0-162) 0.02 54 (4-120) 30 (0-125) 0.007

Abbreviations: CRP, C-reactive protein.

5.2.3. C-reactive protein

The median CRP concentration was 35 mg/l (range 5-253 mg/l) at the beginning of the fever

(d0), 65 mg/l (range 11-234 mg/l) on day 1 (d1), 83 mg/l (range 7-257 mg/l) on day 2 (d2) and

82 mg/l (range 12-241 mg/l) on day 3 (d3) in the whole group of patients.

The peak concentration of CRP was achieved on d0 in 10%, on d1 in 19%, on d2 in 40% and

on d3 in 31% of the patients. There were no statistically significant differences in CRP values

on any day between the groups with severe and non-severe sepsis (Table 14). In the analysis

of variance of repeated measures the overall difference in CRP between patients with and

without severe sepsis was not statistically significant either (p=0.344).

62

Table 13. Patients with severe sepsis in studies III and IV.

Number Gender/age Diagnosis Conditioningregimennumber

Criteria forseveresepsis

ICU Bloodculture

Recovered:

1. F/70* AML II Hypotension No Enterobactercloacae

2. F/53 AML II Hypotension No Negative

3. M/63 AML II Respiratoryfailure

No Enterococcusfaecalis

4. M/61 AML II Hypotension No Negative

5. F/36 HL After ASCT Hypotension No Negative

6. F/51 AML II Hypotension No Enterococcusfaecium

7. M/44* NHL After ASCT Hypotension No Negative

8. F/58* NHL After ASCT Hypotension No Negative

9. M/61* NHL After ASCT Respiratoryfailure

No Negative

10. F/42 HL After ASCT Hypotension No Negative

11. F/60 NHL After ASCT Hypotension No Negative

Died:

12. M/54 AML II Septicshock

Yes Negative

13. M/55* NHL After ASCT Septicshock

Yes Negative

Abbreviations: ICU, intensive care unit; F, female; M, male; AML, acute myeloid leukaemia;HL: Hodgkin lymphoma; NHL, non-Hodgkin lymphoma; ASCT, auologous stem celltransplantation.* included in both prospective studies (III and IV).

63

Table 14. Comparison of laboratory measures on days 0 to 3 from the beginning of theneutropenic fever of the study patients with severe sepsis and without severe sepsis. The dataare expressed as medians (range).

Patients with

severe sepsis

(N=5)

Patients without

severe sepsis

(N=37)

p

VEGF (pg/ml)

d0 77 (71 – 100) 52 (14 – 210) 0.061

d1 82 (71 – 102) 56 (9 – 163) 0.048

d2 54 (44 – 60) 34 (9 – 162) NS

d3 37 (27 –54) 39 (9 – 123) NS

CRP (mg/l)

d0 23 (16 – 253) 36 (5 – 212) NS

d1 65 (28 – 215) 64 (11 – 234) NS

d2 69 (57 – 86) 92 (7 – 257) NS

d3 70 (59 – 132) 85 (12 – 241) NS

Platelet count

(109/l)

d0 27 (13 – 121) 26 (8 – 83) NS

d1 25 (20 – 144) 38 (9 – 100) NS

d2 62 (6 – 78) 35 (11 – 74) NS

d3 37 (20– 62) 34 (11 – 69) NS

Abbreviations: VEGF, vascular endothelial growth factor; CRP, C-reactive protein; d, day.

5.2.4. Serum vascular endothelial growth factor

The median VEGF concentration at the beginning of the fever (d0) was 53 pg/ml (range 14-

210 pg/ml), on d1 58 pg/ml (range 9-163 pg/ml), on d2 48 pg/ml (range 9-162 pg/ml) and on d3

39 pg/ml (range 9-123 pg/ml) in the whole group. On d1 the median VEGF concentration was

higher in patients with severe sepsis than in patients with non-severe sepsis (82 pg/ml vs. 56

pg/ml, p=0.048) (Table 14). The same was seen on day 0 but the difference did not reach

statistical significance (77 pg/ml vs. 52 pg/ml, p=0.061).

The peak concentration of VEGF was achieved on d0 in 45%, on d1 in 24%, on d2 in 14%,

and on d3 in 17% of the patients (Figure 4). Time to achieve peak VEGF concentration was

significantly shorter than time to achieve peak CRP concentration (mean 1.02 with S.E. 0.18

64

days vs. mean 1.93 days with S.E. 0.15 days, p=0.002). On d0 and d1 VEGF seems to

distinguish the groups with severe and without severe sepsis better than CRP (Table 14,

Figure 5). There was no significant correlation between VEGF and platelet levels at any time

point. There were no statistically significant correlations between CRP and VEGF levels,

either. In the analysis of variance of repeated measures the overall difference in VEGF

between patients with and without severe sepsis was not statistically significant (p=0.310).

The advantage of CRP compared to VEGF in the prediction of severe sepsis was evaluated

based on receiver operating characteristics curve analysis (ROC) and the Youden index. In the

ROC curve analysis, the AUC for CRP was 0.56 (95% CI 0.40-0.72, p=0.480) on day 0 and

0.49 (95% CI 0.33-0.64, p=0.880) on day 1. AUC for VEGF on day 0 was 0.76 (95% CI 0.62-

0.90, p=0.060) and 0.80 (0.67-0.94 p=0.048) on day 1 (Figure 6). The predictive capacity of

VEGF was better for severe sepsis during the first 24 hours after the beginning of neutropenic

fever.

5.3. Serum amino-terminal pro-brain natriuretic peptide in patients withneutropenic fever: a comparison with C-reactive protein (IV)

5.3.1. Epidemiological features of severe sepsis

There were altogether 70 patients with 94 episodes of neutropenic fever when all

neutropenic fever periods were included (fourteen patients with AML had more than one period

of neutropenic fever). The diagnostic criteria for severe sepsis were fulfilled in 13/94

neutropenic fever periods (14%) (Table 8). As it is shown in table 13 there were only one

neutropenic fever period with severe sepsis for each patient. In 9 patients (69%, 9/13) the

criteria for severe sepsis was hypotension despite of fluid resuscitation. In 2 patients (15%,

2/13) the criteria for severe sepsis was septic shock and in 2 patients (15%, 2/13) respiratory

failure. Two patients (15% of the patients with severe sepsis) died due to septic shock in

intensive care unit; one ASCT recipient one day after admission and one after second

laparotomy because of appendicitis while still neutropenic following the second induction

course for AML. The median time after the onset of fever to the point when the criteria for

severe sepsis were fulfilled was 1 day (range 1-7).

65

Figure 4. The day of the peak concentration of VEGF (the upper panel) and of CRP (the lowerpanel) in patients with neutropenic fever.

0

2

4

6

8

10

12

14

16

18

20

0

2

4

6

8

10

12

14

16

18

20

Num

ber o

f pat

ient

sN

umbe

r of p

atie

nts

d0 d1 d2 d3

d0 d1 d2 d3

Day of Maximal CRP concentration

Day of Maximal VEGF concentrationDay of maximal VEGF concentration

Day of maximal CRP concentration

66

Figure 5. VEGF (pg/ml) (in the upper panel) and CRP (mg/l) (in the lower panel) in patientswith and without severe sepsis on days 0 to 3 after the start of neutropenic fever. The crosslines indicate the mean values and the p-values significances for differences between patientswith and without severe sepsis.

VEGF

Day 0 Day 1 Day 2 Day 3 Day 0 Day 1 Day 2 Day 30

50

100

150

200

VEG

F(pg

/ml)

CRP

Day 0 Day 1 Day 2 Day 3 Day 0 Day 1 Day 2 Day 30

50

100

150

200

250

CRP

(mg/

l)

VEGF No severe sepsis Severe sepsis

CRP No severe sepsis Severe sepsis

p = 0.310

p = 0.344

p-va

lue

0.06

1

p-va

lue

0.04

8

p-va

lue

0.56

7

p-va

lue

0.83

6

p-va

lue

0.89

3

p-va

lue

0.93

7

p-va

lue

0.55

7

p-va

lue

0.76

6

No severe sepsis

Severe sepsisNo severe sepsis

Severe sepsis

CR

P (m

g/l)

VE

GF

(pg/

ml)

67

Figure 6. In the ROC curve analysis for VEGF, AUC was 0.80 (0.67-0.94), p=0.048 on day1 to discriminate between severe and non-severe sepsis among neutropenic fever periods.The diagonal line (with dashes) represents results no better than chance.

68

5.3.2. Microbiological findings and site of infection

Blood cultures were positive in 22 (23%) of the periods with neutropenic fever. Positive

findings included Staphylococcus epidermidis (n=4), Escherichia coli (n=3), Klebsiella

pneumoniae (n=2), Enterobacter cloacae (n=2), Rothia mucilaginosa (n=2) and

Staphylococcus haemolyticus, Staphylococcus aureus, Streptococcus pneumoniae,

Streptococcus mitis, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa,

Stenotrophomonas maltophilia and Morganella morganii, one each.

In about half of the neutropenic febrile periods the site of infection remained unknown. The

lungs and upper respiratory airways were the most common known sites of infection (19

episodes, 20%), followed by skin infections (including the perineal region) (16 episodes, 16%)

and catheter-related infections (8 episodes, 9%).

5.3.3. Factors associated with severe sepsis

The median age of patients in the subgroups with and without severe sepsis was similar. The

median length of neutropenia was 10 days (range 3-30) without statistically significant

difference between periods with and without severe sepsis.

The frequency of severe sepsis was similar in patients with and without previous

cardiovascular disease. The incidence of severe sepsis was 14% (6/43) in AML patients and

14% (7/51) in ASCT recipients of all episodes of neutropenic fever.

5.3.4. C-reactive protein

The peak CRP level was achieved on d2 (Figure 7). The median CRP was 35 (17-61) mg/l

on d0, 79 (38-79) mg/l on d1, 109 (56-109) mg/l on d2, 90 (54-160) mg/l on d3, and 84 (44-

141) mg/l.

There were no differences between the subgroups with and without severe sepsis in the

levels of CRP on d0 to d2 but the difference was found in CRP values between the subgroups

on d3 (147 mg/l vs. 86 mg/l, p=0.052) and on d4 (179 mg/l vs. 79 mg/l, p=0.015) (Table 15,

Figure 8). Even though in the repeated measures ANOVA from day 0 to 3 no difference was

found in the levels of CRP (p=0.454) between subgroups, the differences of CRP were

significant between d0-d1 and between d1-d2 with p-values of < 0.001 and < 0.001,

respectively.

69

5.3.5. Amino-terminal pro-brain natriuretic peptide

The peak median NT-proBNP concentration was achieved on d4. The median NT-proBNP

was 127 (57-393) ng/l on d0, 236 (78-752) ng/l on d1, 318 (126-734) ng/l on d2, 351 (134-

1023) ng/l on d3, and 542 (194-1385) ng/l on d4 (Figure 7).

There were no statistically significant differences between the subgroups with and without

severe sepsis in the level of NT-proBNP at any time point (Table 15, Figure 8). After excluding

subjects with previous cardiovascular disease, the findings remained similar. In the repeated

measures ANOVA from day 0 to 3 no difference was found in the levels of NT-proBNP

(p=0.474) between subgroups with and without severe sepsis, but the differences between d0-

d1 and between d1-d2 NT-proBNP levels were significant (p= 0.001 and 0.011, respectively).

There was no correlation between NT-proBNP and the level of the same day CRP on the

first three days of neutropenic fever. However, NT-proBNP on d3 correlated slightly with CRP

on d4 (r=0.24, p=0.050), and NT-proBNP on d4 correlated with CRP on d3 (r=0.35, p=0.010)

and on d4 (r=0.33, p=0.015).

5.3.6. Association of amino-terminal pro-brain natriuretic peptide with fluidintake and previous cardiovascular disease

There was a slight negative correlation between NT-proBNP and the level of the fluid intake

on the same day for d0 and d1 of neutropenic fever (d0: r=-0.24, p=0.038; d1: r=-0.25,

p=0.021). Day 0 fluid intake correlated negatively with d4 NT-proBNP (r=-0.35, p=0.020).

The levels of NT-proBNP were higher in the subgroup of patients with previous

cardiovascular disease than those without throughout the course of the neutropenic fever

(Table 16, Figure 9). In the repeated measures ANOVA from d0 to d3 a significant difference

was found in the levels of NT-proBNP (p<0.001) between subgroups with and without previous

cardiovascular disease.

70

Table 15. Comparison of the laboratory values and fluid intake from the beginning of theneutropenic fever (n=94). The data are expressed as medians (range).

Severe sepsis

(N=13)

Without severe sepsis

(N=81)

p-value

NT-proBNP (ng/l)

d0 53 (18-11376) 137 (19-25529) NS

d1 85 (17-2501) 273 (22-15331) NS

d2 177 (50-3020) 321 (34-9016) NS

d3 253 (50-3107) 352 (18-11162) NS

d4 768 (50-3681) 521 (15-24368) NS

CRP (mg/l)

d0 39 (16-253) 34 (5-239) NS

d1 72 (28-218) 80 (9-272) NS

d2 88 (57-251) 110 (7-333) NS

d3 147 (59- 340) 86 (7-320) 0.052

d4 179 (44-452) 79 (8-239) 0.015

Fluid intake (l)

d1 4.5 (3.0- 6.8) 3.9 (2.0-7.1) NS

d2 4.0 (2.0-8.5) 4.4 (1.2-11.4) NS

d3 4.6 (2.0-5.3) 4.5 (2.0-8.6) NSAbbreviations: NT-proBNP, amino-terminal pro-brain natriuretic peptide; CRP, C-reactive protein;d, day.

71

Figure 7. The median concentrations with interquartile ranges of NT-proBNP (solid line) andCRP levels (dotted line) on days 0-4 from the onset of neutropenic fever.

0

500

1000

1500

0

50

100

150

200

NT-proBNP

d0 d1 d2 d3 d4

d0 d1 d2 d3 d4

CRPmg/l

ng/l

d0 d1 d2 d3 d4

d0 d1 d2 d3 d4

NT-proBNP

CRP

d0 d1 d2 d3 d4

d0 d1 d2 d3 d4

ng/l

mg/l

1500

1000

500

0

200

150

100

50

0

72

Figure 8. The median concentrations with interquartile ranges of NT-proBNP and CRP ondays 0-4 from the onset of neutropenic fever in periods with (dotted lines) and without (solidlines) severe sepsis.

10

100

1000

10000

10

100

1000

NT-proBNP

CRP

d0 d1 d2 d3 d4

ng/l

mg/l

d0 d1 d2 d3 d4

73

Table 16. Comparison of the laboratory values and fluid intake on days 0 to 4 from thebeginning of the neutropenic fever for all neutropenic fever periods in patients with and withoutprevious cardiovascular disease. The data are expressed as medians (range).

Previous cardiovascular

disease

(N=16)

Without previous

cardiovascular disease

(N=78)

P

(Mann-Whitney

U-test)

NT-proBNP (ng/l)

d0 868 (22-25529) 100 (18-11376) 0.001

d1 1096 (46-15331) 214 (17-6957) <0.001

d2 1908 (37-9016) 264 (34-6929) 0.001

d3 2072 (146-7145) 243 (18-11162) <0.001

d4 1915 (204-7107) 260 (15-24368) <0.001

CRP (mg/l)

d0 24 (5-194) 37 (5-253) NS

d1 47 (16-249) 85 (9-272) NS

d2 113 (12-264) 102 (7-333) NS

d3 134 (8-241) 85 (7-340) NS

d4 103 (10-220) 79 (8-452) NS

Fluid intake (l)

d1 3.2 (2.0-6.1) 4.0 (2.0-7.1) NS

d2 3.7 (1.2-6.2) 4.5 (2.0-11.4) NS

d3 3.8 (2.0-5.3) 4.6 (2.0-8.6) NS

Abbreviations: NT-proBNP, amino-terminal pro-brain natriuretic peptide; CRP, C-reactiveprotein.

74

Figure 9. The median concentrations with interquartile ranges of NT-proBNP and CRP ondays 0-4 from the onset of fever in neutropenic fever periods in patients with (dotted lines) andwithout (solid lines) previous cardiovascular disease.

10

100

1000

10000

10

100

1000

NT-proBNP

CRP

d0 d1 d2 d3 d4

ng/l

mg/l

d0 d1 d2 d3 d4

75

6 DISCUSSION

6.1 Patients and methods

The present series of studies are based on two retrospective studies (I-II) and a prospective

still ongoing study (III-IV). The intention of the study was to evaluate the various aspects of

severe sepsis in haematological patients, especially in patients with a high risk of neutropenic

fever. Therefore, only patients with AML and those receiving ASCT were included.

Chemotherapy regimens used in the treatment of AML cause a markedly longer period of

neutropenia than HDT supported by ASCT (Table 8). The intensity of the HDT in ASCT

recipients, on the other hand, causes more severe mucosal damage in the patients. These

patient groups present different sides of high-risk neutropenic fever and were analysed

separately in the retrospective part of the present series. For prospective study, these patient

groups were combined because interest was focused on clinical management of

haematological patients with severe sepsis.

The definitions for neutropenia, neutropenic fever and severe sepsis were based on

internationally used definition criteria of both the Infectious Diseases Society of America (6)

and 2001 International Sepsis Definitions Conference (79). Even though the definition of SIRS

and severe sepsis have been available for almost two decades (78), these have not been

systematically applied in haematological patients receiving intensive therapy. The definition of

SIRS includes leukocytosis or leucopenia, fever, tachypnea and tachycardia (Table 1), and two

of these criteria must be manifested to fulfill the criteria of SIRS. All of the study patients met at

least two of these other criteria than leucopenia. Thus, leucopenia is a consequence of the

treatment, and this has also been taken into account in this study.

Despite the criteria for neutropenic fever and severe sepsis are definitive, the backgrounds

for both entities are often heterogeneous. The treatment strategies for septic infections in

haematological wards may differ, but the amount of supportive care remains crucial. Vigorous

volume resuscitation as used in the prospective study might thus prevent or shorten the

duration of severe sepsis - with obvious clinical benefits. Severe sepsis that responds quickly

to supportive therapy may be different from the situation where hypotension responds more

slowly and eventually necessitates ICU admission.

Based on retrospective studies it was evident that hard endpoints (ICU admission, death)

were rather infrequent in this patient cohort. Because of this, severe sepsis was used as an

endpoint in prospective studies and as a model to evaluate prognostic utility of various

76

laboratory markers. Therefore, the findings of the present studies do not as such apply to the

ICU setting although the kinetics of various markers are likely to be the same.

During the study period, the empirical antimicrobial treatment practice and hospital infection

control measures (e.g. isolation policies and hospital hygiene, including cleaning and hand-

washing policies) remained constant. Systematic antibiotic prophylaxis was not used, except

during the last study year in NHL patients receiving ASCT. In the prospective studies, each

patient was daily carefully monitored for clinical signs and possible sources of infections.

Special attention was paid to features indicating the development of severe sepsis. In addition,

supportive care was provided including aggressive volume resuscitation when needed.

In regard to laboratory methods, serum CRP measurements have been routine for more than

two decades, and the method is standard. Also serum NT-proBNP measurements have been

in clinical use in ISLAB (Eastern Finland Laboratory Centre, Kuopio, Finland) for several years.

NT-proBNP was preferred to BNP because of its markedly longer plasma life and better

stability (164). VEGF was determined from serum samples instead of plasma. This was

because it was the only method used by the laboratory (ISLAB) and because reference values

were available only for serum VEGF based on the FINNSEPSIS study.

6.2. Epidemiological features and outcome of severe sepsis

The available data of incidence of severe sepsis in neutropenic patients is limited. One

reason for this might be that most of the studies regarding incidence of severe sepsis or septic

shock are made in patients treated at ICUs. The policy among admission practice of patients

with malignant diseases to ICUs varies between centres (196, 197). In present studies the

admission of patients with neutropenia and severe sepsis to ICU has had a relatively low

threshold. The admission criteria to ICU may have had slight changes during the study period,

and this naturally may influence the outcome of patients.

6.2.1. Patients with acute myeloid leukaemia

In AML patients, the criteria for severe sepsis were fulfilled in 13% of periods with

neutropenic fever after intensive induction or consolidation courses. Nine out of 84 AML

patients (11%) died due to severe sepsis. This underlines the importance of severe sepsis as a

cause of death in AML patients treated with modern intensive chemotherapy. In other studies,

the infection mortality rate has ranged between 5-13% (2, 60-64). In elderly patients, the risks

may be even higher (198).

77

Even though the induction course may be the most dangerous in relation to severe or fatal

infections, also the consolidation courses are associated with high risk of neutropenic fever (2,

62). Therefore, it is obvious that all measures aiming to decrease mortality due to severe

sepsis might improve overall outcome of AML patients. Unfortunately, in the present study only

38% of patients treated in ICU recovered. At least in some patients the late referral to ICU

might be a possible reason for this. During the study period (1996-2005) the practises of

admission to ICU have changed: life-sustaining treatments are begun outside ICUs in different

step-up/ step-down units. Moreover, the threshold admission to the ICU has become lower for

patients with malignancies and severe co-morbidities. In AML patients, the goal of the

intensive treatment is curative, and early ICU admission in severe sepsis may improve

prognosis. Close clinical monitoring of these patients is therefore mandatory in the

haematology ward.

6.2.2. Autologous stem cell transplant recipients

In ASCT recipients the incidence of severe sepsis was much lower (5%) than in AML

patients, but patients receiving a transplant for NHL were at a higher risk than the rest of ASCT

recipients. In earlier studies the percentage of septic bloodstream infections in ASCT recipients

has varied between 12% and 20% (72-74, 76).

The case fatality rate in patients with severe sepsis was, however, high (9/17, 53%) and all

patients who died due to severe sepsis after ASCT had NHL. Only 18% of patients admitted to

ICU recovered. This figure is clearly inferior than in some ICU- based studies of

haematological patients (199), but the reason for admission may effect ICU outcome in an

important way (e.g. septic hypotension in young neutropenic patients vs. neutropenic septic

shock in patients with co-morbidies).

The mortality for severe sepsis was 6% in patients with NHL. This is a high figure compared

with other series where an early mortality of 2-4% has been observed (70-72, 74). One

potential reason for the high mortality rate in this study may be the relatively high number of

elderly NHL patients receiving ASCT. In an EBMT (European Group of Blood and Marrow

Transplantation) study, the risk of early treatment-related mortality in older NHL patients (>60

years of age) was significantly higher (4.4%) than in younger patients (2.8%) (200). Other

reasons for higher mortality of NHL patients due to severe sepsis might be the combination of

longer neutropenia together with the known cardiotoxicity of prior anthracycline treatment

(201). Therefore, a prospective treatment protocol for NHL patients receiving ASCT was

initiated in January 2008. This includes oral ciprofloxacin prophylaxis during neutropenia,

78

cardiac pre-evaluation with radio-cardiography, ACE-inhibitor treatment in patients with

decreased left ventricular ejection fraction and use of BEAM chemotherapy instead of earlier

BEAC. The latter is known to be associated with early cardiotoxicity (201).

6.2.3. Prospective study

In the prospective series the incidence of severe sepsis was 14% without a difference

between AML patients and ASCT recipients. Even though the incidence of severe sepsis was

not lower than in retrospective series, the case fatality rate for periods with severe sepsis

seemed to be lower (15%). There are many possible reasons for this. Improved supportive

care during the prospective treatment protocol and thus possibly earlier detection of severe

sepsis may be important factors. This includes starting effective treatment by the trained

nursing personnel earlier. However the number of periods with severe sepsis is still relatively

low in the prospective series and more experience is needed before drawing firm conclusions.

Haematological patients themselves may today be better informed and aware of the risk of

septic infections during chemotherapy. This in turn helps them to recognise symptoms of

septic infections and contact hospitals earlier than other patients with community-acquired

sepsis.

6.3. Microbiological findings

In general, the incidence of microbiologically documented bloodstream infections has been

between 7.7% and 19% in patients with neutropenic fever (23, 25, 26). In the present studies,

the percentage varied between 23 and 59%. In AML patients (study I) the amount of

bloodstream infections was 59%, which is higher than in earlier studies (24, 66).

There are some obvious reasons for the high percentage of bloodstream infections in AML

patients. The duration of neutropenia in AML patients during intensive induction and

consolidation chemotherapy is markedly longer than in any other treatment protocol for

malignancies. During the long-lasting treatment period, the use of long-term indwelling

catheters (up to six months) is common. This predisposes patients to catheter-related

infections. In addition, intensive chemotherapy itself causes mucosal damage. This in turn

increases the risk of infections caused by microbes from the normal oro-pharyngeal and

gastrointestinal flora. Long-lasting febrile neutropenia increases the number of blood cultures

taken and hence, also the number of positive culture results.

79

As expected, gram-positive microbes caused the majority of bloodstream infections. This

finding is in accord with other recent studies (24, 37, 74). The proportion of gram-negative

microbes in blood cultures was high (9-25%) and especially as a cause of severe sepsis (35-

41%). The presumable reason for this is the lack of prophylactic antibiotics in present series –

the situation is similar in other centres where antibiotic prophylaxis has not been used (202-

204).

Among gram-positive microbes causing bloodstream infections the most common finding

was Staphylococcus epidermidis, which is in line with earlier observations (59, 205, 206). The

amount of bloodstream infections arising from central venous catheters varied between 9%

and 16%, with the highest percentage in AML patients. The plausible explanation for this was

the long-term use of indwelling catheters in this patient group. The lungs and upper respiratory

airways were the most common known localisation of infection in febrile neutropenic patients,

as observed in other studies (66, 70).

Candida albicans was found in 1% of febrile neutropenic periods in both AML patients and

ASCT recipients. The apparent reason for the small amount of fungaemia was the use of

fluconazole prophylaxis during prolonged neutropenia and mucosal symptoms (207). In the

Finnish nationwide series the risk of invasive Candida-infections was also <1% among 1188

ASCT recipients (208). In addition, the lack of systematic antibacterial prophylaxis may have

had an impact.

In contrast to some other recent series (38, 209), there were not a single case of

multiresistant bacteria like MRSA, VRE, extended-spectrum -lactamase (ESBL) -producing

gram-negative rods or multi-drug resistant gram-negative microbes. The explanation for this

might be our treatment practice in Finland – antibacterial prophylaxis is not commonly used.

Furthermore, empirical use of vancomycin has been restricted during the whole study period

and the overall use of wide spectrum antibiotics has been under control.

In the beginning of 2008 the use of oral ciprofloxacin was started for NHL patients receiving

ASCT. This was mainly because of the fatal Pseudomonas aeruginosa-bloodstream infections

in NHL patients during years 1996-2006 (study II). The mortality with Pseudomonas

aeruginosa bloodstream infections was high and this finding is in line with earlier studies (210-

213). The present prophylaxis practice is experimental and is part of a prospective clinical

protocol. Although preliminary, the early impression seems to be promising, because there has

not been a single case of severe sepsis among fourteen consecutive patients treated within

this protocol (214). The amount of patients who received ciprofloxacin prophylaxis during the

present study time was low and probably does not have a major influence on the study

findings.

80

6.4. Kinetics of serum C-reactive protein in patients with neutropenic fever(Studies I-II)

In the retrospective studies (I-II) the median time from the onset of neutropenic fever to the

point where criteria for severe sepsis were met was 2 (study I) and 3 days (study II). CRP

concentrations elevated in paralle with this but the peak CRP was achieved later than the

criteria for severe sepsis. Neither CRP slope velocity nor CRP on days 2-3 reliably distinguish

periods with severe sepsis from those without in advance. In the study of Persson and

colleagues (14) the CRP concentrations on days 2 and 3 of febrile neutropenia were lower in

those patients who did not develop complications (e.g. pneumonia). This is in line with our

findings.

Although several parameters of CRP kinetics were statistically associated with severe

sepsis, in most instances CRP parameters evaluated coincided or even followed the diagnosis

of severe sepsis. This was observed also in earlier studies both in patients with febrile

neutropenia (146) and those without (142, 143). CRP identifies patients with infection but when

the time interval is short, the predictive capacity of CRP declines markedly. This is mostly

explained by the biochemistry of CRP – the peak values are reached 48 hours after the

beginning of secretion independently of the reason for secretion (128).

6.5. Comparison of serum vascular endothelial growth factor and C-reactiveprotein in patients with neutropenic fever (Study III)

The peak serum VEGF level was reached during the first 24 hours and the peak CRP values

on day 2 after the onset of neutropenic fever in patients with severe sepsis. Serum VEGF

levels were continuously low, but slightly higher in patients who developed severe sepsis than

in those who did not. The difference reached statistical significance for VEGF after the first 24

hours but there were no statistically significant differences in CRP values on any day between

the groups with severe and non-severe sepsis.

The serum VEGF values on d0 in study III were notably lower than in patients with severe

sepsis in FINNSEPSIS study (median 423 pg/ml) or in healthy controls (median 260 pg/ml)

(Figure 10) (15). However, during the first 24 hours the VEGF levels in neutropenic patients

with severe sepsis were higher than in those without. This latter finding is in line with earlier

studies (215-217), although those studies included patients without known neutropenia or

thrombocytopenia.

81

Figure 10. The median concentrations with interquartile ranges of VEGF (mg/ml) on days 0

and 3 in subjects with neutropenic sepsis (n=42) (solid line) and in septic patients participating

in the FINNSEPSIS-study (dotted line) (n=242) in 24 multidisciplinary intensive care units in

Finland(15) The comparison is shown with the kind permission of the FINNSEPSIS Study

Group.

An apparent reason for the low VEGF values observed in this study may be

thrombocytopenia and neutropenia in all patients. The close association of serum VEGF and

platelet count was first considered when changes in VEGF levels were found to mirror changes

in platelet counts during chemotherapy for breast cancer (158). Platelets tend to accumulate

VEGF, acting as a storage for circulating VEGF both in healthy subjects and in cancer patients

(218, 219). The result was similar in the study of Benoy and colleagues (220). Several studies

have also shown that circulating VEGF resides not only in platelets, but also in granulocytes,

mainly in neutrophils (159-161). The study patients all had severe neutropenia during the study

period. Low values also in patients with severe sepsis support the theory that platelets and

granulocytes are the major source of VEGF. The early increase in VEGF levels may be due to

liberation of VEGF from endothelial stores caused by endothelial injury associated with sepsis.

10

100

1000

D0 D3VE

GF

conc

entra

tion

(pg/

mL)

Neutropenic sepsis Sepsis (Finsepsis data)FINNSEPSIS data

Neutropenic sepsis

82

In patients with neutropenic fever, there are no previous studies available on the kinetics and

prognostic utility of serum VEGF. In the present study, it was demonstrated that also

thrombocytopenic and neutropenic patients are capable of producing VEGF during fever.

Although haematological patients had low VEGF concentrations during sepsis, the VEGF

concentrations in severely septic patients were higher than in other febrile patients, especially

on the first days after the start of fever. This implies that VEGF is a more rapid indicator than

CRP in haematological patients with neutropenic fever, which deserves further study.

6.6. Comparison of serum amino-terminal pro-brain natriuretic peptide and C-reactive protein in patients with neutropenic fever (Study IV)

There were no statistically significant differences in the level of NT-proBNP between patients

with and without severe sepsis at any time point analysed. CRP concentrations did not show

any predictive utility either; the median level of CRP was significantly higher in the subgroup

with severe sepsis only on day 3 and day 4. Taking into account that the median time to the

fulfilment of criteria for severe sepsis was only one day in this prospective study, it is obvious

that these markers are unsuitable for clinical use to predict severe sepsis.

Previous myocardial infarction, acute coronary syndrome, valvular disease, ventricular

hypertrophy and cardiomyopathy are known cardiological reasons for increased values of

natriuretic peptides. Non-cardiac factors include age, female gender, renal failure,

glucocorticoid use and acute pulmonary embolism (138, 221, 222). In the present study,

patients with a history of previous cardiovascular disease had significantly higher levels of NT-

proBNP than those without, even though none of the study patients had clinically severe

cardiovascular disease. Significant release of NT-proBNP continued during the entire study

period. Of note, the amount of daily fluid intake did not show any significant association with

NT-proBNP levels.

There are some previous studies showing a significant association with sepsis and ICU

mortality in patients with markedly elevated BNP- and NT-proBNP values (16, 138, 168, 169).

Nikolau and his colleagues showed that BNP levels were elevated also in the acute phase of

community-acquired microbial infections without severe sepsis or septic shock (170). It was

therefore reasonable to assume that natriuretic peptides would serve as prognostic markers

also in the haematology ward setting. This was, however, not the case in the present study

where NT-proBNP was used.

Neither serial NT-proBNP nor CRP showed any early predictive value in neutropenic periods

with severe sepsis compared to those without. NT-proBNP seemed to reflect cardiac distress

83

more than the inflammatory response in this patient population. It might thus serve as a useful

adjunct to optimise management of patients with previous cardiovascular disease during

sepsis.

6.7. Study limitations

Because of the retrospective nature, studies I-II have several unavoidable limitations. The

criteria for severe sepsis were well applicable for all study subjects, although the retrospective

classification of patients with severe sepsis was challenging. All periods regarded to represent

severe sepsis were confirmed by consultation between the clinical haematologist (EJ) and

infectious disease specialist (SH), but uncertainties remain in patients who were not treated in

the ICU.

CRP values on the first day of the fever and particularly 12-24 hours from the onset of fever

were often missing. For this reason the values 2 or 3 days after the onset of fever were used.

Further, in patients with clinical signs of severe infection CRP values were evaluated

apparently more often than febrile neutropenic patients without these signs. The observers

were not blinded to CRP values when evaluating febrile neutropenic periods.

In studies I and IV all febrile neutropenic periods in study patients were included. This is

common practice in haematological studies, but it also causes a situation where the same

patient is analysed repeteadly during new periods of neutropenic fever. It is unknown how

inflammatory markers behave in repeating periods of febrile neutropenia in the same

individual. However, in present series only four patients (study I) (Table 9) had more than one

episode of severe sepsis. As with these patients and with all other study patients, it was

impossible to predict the febrile period that will be complicated with severe sepsis. For this

reason, all periods with neutropenic fever are included.

The site of infection remained unknown in most of the febrile neutropenic periods.

Neutropenia alters the host’s inflammatory response and makes the infection difficult to detect

because the classic signs and symptoms of infection are usually missing. This is mainly

because of an almost total lack of neutrophils. In this respect traditional laboratory methods are

also useless. Fever is often the only sign of infection in accordance with criteria of severe

sepsis (e.g. hypotension). Sometimes medications like corticosteroids can obscure especially

fever. Only patients with both neutropenia and fever ( 38.3°C or 38.0°C for 1 h) were

included into the present studies. It is possible that there have been also patients with

neutropenic infection but without fever. In septic infections, however, this is unlikely. Patients

84

are closely monitored during the treatment period. Even without fever, the other signs of

severe infection (e.g. hypotension, dyspnoea) would have been noted.

Study III was the first prospective study published evaluating VEGF kinetics in cytopenic

haematological patients. The number of patients was relatively small. Consequently, the

number of patients who had severe sepsis was also small. The number of endpoints (severe

sepsis, septic shock or death) was low in both prospective studies, but in line with the

incidences observed in retrospective studies.

Sepsis-related Organ Failure Assesment (SOFA) or another comparable method would have

been useful in patient monitoring. Classification of neutropenic patients with SOFA in

haematological wards has some inevitable limitations. Patients have a low number of platelets

because of chemotherapy even in the absence of infection, and platelet count is one of the

main criteria for SOFA. A more important reason for not using SOFA is that usage of invasive

monitoring is not routine outside ICUs.

More frequent early measurements (e.g. between 2-6 hour intervals after the beginning of

fever) of both NT-proBNP and CRP might have been more informative in prospective studies.

However, such a frequent sampling may not be applicable in clinical practice outside intensive

care units. Considering the kinetics of these measures, it is also unlikely that more frequent

sampling would have led to different conclusions.

6.8. Concluding remarks

The frequency of severe sepsis was lowest among ASCT recipients (study II) (5%) and

highest in study IV (14%), where both AML patients and ASCT recipients were prospectively

included. The outcome of severe sepsis was rather poor in the retrospective studies with a

mortality of 11% in AML patients and 2% in ASCT recipients during intensive therapy. The

case fatality rate for periods with severe sepsis were 27% and 53%, respectively. In the

prospective study with close patient monitoring and supportive care, the mortality during

intensive therapy was 3% and the case fatality rate for periods with severe sepsis 15%.

Altough the incidence of severe sepsis seems to remain the same, the outcome seems to have

been better in the prospective part of the study.

The microbial aetiology of severe sepsis in neutropenic patients did not change during the

study period (1995-2008). Gram-positive microbes were the predominant findings in blood

cultures, which is well in line with other studies (37, 38, 223). Because antibacterial prophylaxis

was not used, the proportion of gram-negative microbes in bloodstream infections was

considerable compared with studies where prophylactic antibiotics were used. The incidence

85

of Pseudomonas aeruginosa was nevertheless alarming, and changed the policy concerning

prophylactic antimicrobial use in patients with greatest risk of fatal septic infections. During the

study period, not a single strain of MRSA, VRE or ESBL were found, which is an important

finding concerning the hospital hygiene policies in our hospital.

CRP was one of the infection markers chosen for this study protocol. It is easily available

and familiar as a reference marker for sepsis. Other chosen markers were compared with

CRP. As demonstrated in the earlier studies (14, 142, 146), CRP was slow to change in the

early phase (< 24 hours) of severe sepsis. The situation was similar with the NT-proBNP

(study IV). The peak values came far too late for predictive purposes in patients with

neutropenic sepsis.

The results with serum VEGF showed some promise. Study III was the first study published

evaluating VEGF kinetics in haematological patients with neutropenic fever. It was

demonstrated that also thrombocytopenic and neutropenic patients can produce VEGF during

fever. Although the VEGF concentrations were in general low, the VEGF concentrations in

severely septic patients were higher than in other febrile patients. The difference was obvious

during the first critical hours from the start of neutropenic fever. This implies that VEGF might

be a rapid indicator for severe sepsis in haematological patients with neutropenic fever. The

problem with VEGF is the current lack of an appropriate methodology in clinical laboratories

that can provide results within 12-24 hours. The definition of practical cut-off values with

evaluation of positive and negative predictive capacity in neutropenic and thrombocytopenic

patients is also needed.

6.8.1. Implications for further research

The major focus of this series of studies was on febrile neutropenic patients treated in a

haematological ward. In these circumstances, the conditions and equipment are inferior to

those in ICUs. Furthermore, the possibilities for respiratory and circulatory support are limited.

In retrospective studies only 38% of the AML patients and 22% of the ASCT recipients

admitted to the ICU recovered. This contrasts with the results of the FINNSEPSIS study (10),

where 86% of the patients admitted for sepsis to ICU recovered. In recent years, intensive care

treatment with step-down units has become more available, and admission criteria have

probably changed. In addition, the treatment strategies in haematological wards have

changed. Although the supportive care in haematological ward has been rather efficient also in

the near past, the need for improvement is obvious. The possibilities in the ward are, however,

86

limited and a flexible consultation system is essential to get these patients early into step-up-

step-down units or the ICU when needed.

The question of antibiotic prophylaxis in neutropenic haematological patients needs to be

reconsidered. Based on the findings in study II a new prospective protocol was started in the

beginning of January 2008. One of its main principles is to determine the benefits and possible

disadvantages of fluoroquinolone prophylaxis in high-risk ASCT recipients. Mucositis caused

by intensive chemotherapy is in closely associated with this – if the incidence of mucositis

could be reduced (224, 225), the amount of neutropenic infections and of course, septic

infections would diminish. There are interesting possibilities for the development of locally

administered nonpharmacological measures and pharmacotherapeutics to restore mucosal

barriers in the future (226).

Readily available early circulatory markers for severe sepsis could be helpful in clinical

practice, especially in haematological wards. Severe sepsis proceeds rapidly. Commonly used

laboratory methods recognise this phenomenon too late and too slowly. Serum VEGF might be

one option for further research. At present, the lack of an appropriate methodology applicable

in clinical laboratories to get the result within 12-24 hours and the lack of practical cut-off

values with known positive and negative prediction capacity are problems. Many other early

markers are of potential interest in relation to the pathogenesis of sepsis in neutropenic

haematological patients as well as polymerase chain reaction (PCR) based assays for fast

microbial recognition (135, 227).

While continuing the search for clinically useful laboratory markers, it is of utmost importance

that febrile neutropenic patients be carefully monitored for vital functions with optimised

supportive care. In haematological wards, severe sepsis is still primarily a clinical diagnosis,

and the experience of caregiving physicians is vitally important. Close co-operation with

colleagues in ICUs and other supportive care units is crucial in the treatment of patients with

severe neutropenic sepsis.

87

7. CONCLUSION

I Severe sepsis was observed in 13 % of periods with neutropenic fever in AML patients

and 5% of periods in ASCT recipients. Gram-negative rods were more commonly

found in blood cultures of periods with severe sepsis in both patient groups. In ASCT

recipients, 30% of blood stream infections were caused by Pseudomonas sp. The

case fatality rate of severe sepsis was 28 % in patients with AML and 53 % in ASCT

recipients.

II Peak CRP values were higher and CRP slope velocity steeper in periods with severe

sepsis in both patient groups. CRP kinetic merely coincided than preceded the

development of severe sepsis.

III VEGF concentrations were in general low but < 24 hours after the start of neutropenic

fever VEGF levels were higher in patients with severe sepsis. VEGF seems to be a

more rapid indicator of severe sepsis than CRP in haematological patients with

neutropenic fever.

IV Neither serial NT-proBNP nor CRP showed any early predictive value for the

development of severe sepsis in haematological patients with neutropenic fever. NT-

proBNP values were significantly higher in patients with previous cardiovascular

disease.

88

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SARI HÄMÄLÄINEN

Severe Sepsis in Neutropenic

Haematological Patients

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• Severe Sepsis in Neutro

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KUOPION YLIOPISTON JULKAISUJA D. LÄÄKETIEDE 472 KUOPIO UNIVERSITY PUBLICATIONS D. MEDICAL SCIENCES 472