Conditions: Lymphoma Infection Leukemia
Keywords: Myelodysplastic and Myeloproliferative Diseases
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 600
Healthy volunteers: no

Brief summary:
The study is designed as a Phase III, randomized, double-blind, multicenter, prospective, comparative study of fluconazole versus voriconazole for the prevention of fungal infections in allogeneic transplant recipients. Recipients will be stratified by center and donor type (sibling vs. unrelated) and will be randomized to either the fluconazole or voriconazole arm in a 1:1 ratio.

Detailed description:
BACKGROUND:

Allogeneic blood and marrow transplant patients are highly susceptible to invasive fungal infection prior to engraftment, due to neutropenia and mucosal injury. After engraftment, an impairment of cell mediated immunity from graft-versus-host disease (GVHD) and the use of aggressive immunosuppressive therapies, such as corticosteroids, leave patients vulnerable to invasive fungal infections. Recipients of alternate donor transplants are especially susceptible due to slow reconstitution of cell mediated immunity.

Fluconazole prophylaxis in prospective randomized trials of both autologous and allogeneic transplant recipients has been demonstrated to reduce invasive fungal infections due to yeasts prior to engraftment. A prolonged course of fluconazole given during the first 75 days (to cover the early post-engraftment period of risk) is highly effective in the prevention of early and later yeast infections. This has translated into a survival benefit. A recent analysis of long-term outcomes of these individuals demonstrated a continuing benefit beyond the course of prophylaxis with a further benefit in survival. In another study of various factors associated with survival after matched unrelated donor transplants, fluconazole prophylaxis was an independent predictor for overall survival in a multivariate analysis. Fluconazole prophylaxis has been found to be effective and safe with few substantive drug interactions and has been widely adopted by transplant clinicians.

DESIGN NARRATIVE:

This is a randomized, double-blind, multicenter, prospective, comparative study of fluconazole versus voriconazole for the prevention of fungal infections in allogeneic hematopoietic transplant recipients and cord blood recipients in children under the age of 12. Prior to the start of the pre-transplant conditioning regimen, participants will give written informed consent and be screened for eligibility. Participants who meet all entry criteria will be assigned randomly to voriconazole or fluconazole within 72 hours of Day 0. Participants will begin the study drug on Day 0 (after completion of the conditioning regimen). Day 0 is defined as the day infusion of the stem cell product is completed. The study drug will be continued until Day 100 following transplant or until one or more criteria for early withdrawal are met. Continuation of the study drug beyond Day 100 is permitted for participants who meet specific criteria. The development of any fungal infection during prophylaxis will be classified according to the definitions listed in the protocol.

Arms:
arm 1: The dose of fluconazole is 400 mg by mouth or intravenous drip. arm 2: The dose of oral voriconazole is 200 mg twice daily. When voriconazole must be given intravenously, it will be given at a dose of 200 mg every 12 hours for the duration of intravenous therapy.

Interventions: intervention 1: Fluconazole intervention 2: Voriconazole
Intervention descriptions: intervention 1: Fluconazole will be administered orally once daily. Fluconazole capsules should be taken at least one hour before or one hour after a meal. If oral drug is not possible, it will be given intravenously once daily in a total volume of 200 mL in patients \> 12 years. For adults, each 200 mL infusion will be administered over 2 hours. In patients \< 12 years, intravenous doses will be prepared. intervention 2: Voriconazole will be administered orally twice daily. Voriconazole capsules should be taken at least one hour before or one hour after a meal. Taken concomitantly with food, bioavailability of voriconazole is reduced. If oral drug is not possible, it will be given intravenously at a dosage of 200 mg every 12 hours over two hours in patients \> 12 years. Each voriconazole dose will be diluted to a total volume of 200 mL in patients \> 12 years. Volumes of the formulation required to provide 4 mg/kg doses for children age \< 12 years.

Protocol document:
Prot_SAP_ICF_000.pdf: 
BMT CTN 0101 
A Randomized Double-blind Trial of Fluconazole vs. 
Voriconazole for the Prevention of Invasive Fungal 
Infection in Allogeneic Blood and Marrow 
Transplant Patients 
NCT00075803 


 
 
A Randomized Double-blind Trial of Fluconazole vs. Voriconazole 
for the Prevention of Invasive Fungal Infections in Allogeneic 
Blood and Marrow Transplant Patients 
 
 
BMT CTN PROTOCOL 0101 
VERSION 7.0 
 
 
Study Chairpersons 
John Wingard, M.D.1 
 
Thomas Walsh, M.D.2 
 
 
Protocol Team 
Shelly Carter, Sc.D.3 
 
Brent Logan, Ph.D.6 
Dennis Confer, M.D.4 
 
Kieren Marr, M.D.7  
Iris Gersten, M.S.3  
 
Trudy Small, M.D.8 
Joanne Kurtzberg, M.D.5  
 
 
Sponsored by the National Institutes of Health 
National Heart, Lung and Blood Institute 
National Cancer Institute 
 
 
 
 
 
 
 
1 University of Florida, Shands Hospital 
2 National Cancer Institute 
3 The EMMES Corporation 
4 National Marrow Donor Program 
5 Duke University Medical Center 
6 Center for International Blood and 
Marrow Transplant Research, Medical 
College of Wisconsin 
7 Fred Hutchinson Cancer Research Center 
8 Memorial Sloan-Kettering Cancer Center 
 


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CORE STUDY PARTICIPANTS 
Case Western Reserve University Consortium  
Oregon Health & Science University 
St. Louis Children’s Hospital 
University Hospitals of Cleveland 
Washington University 
City of Hope National Medical Center 
Dana Farber/Partners Cancer Center 
Duke University Medical Center 
Fred Hutchinson Cancer Research Center 
Johns Hopkins University 
Memorial Sloan-Kettering Cancer Center 
Pediatric Blood and Marrow Transplant Consortium 
Cardinal Glennon Children’s Hospital-St. Louis University 
Children’s Mercy Hospital, University of Missouri 
Children’s Memorial Hospital, Chicago 
Children’s National Medical Center, Washington, DC 
Hackensack University Medical Center 
Texas Transplant Institute 
 
 
Stanford Hospital and Clinics 
UCSD/SCRIPPS School of Medicine 
University of Florida College of Medicine 
University of Michigan Medical Center 
University of Minnesota 
University of Nebraska Medical Center 
University of Pennsylvania Hospital 
University of Texas, MD Anderson Cancer Research Center 
 
NON-CORE STUDY PARTICIPANTS 
H. Lee Moffitt Cancer Center 
Indiana University Medical Center 
Kansas City Cancer Centers, Central BMT 
Karmanos Cancer Institute 
Hackensack University Medical Center 
Roswell Park Cancer Institute 
Texas Transplant Institute 
University of Alabama at Birmingham 
Virginia Commonwealth University/MCV Hospitals 
Wake Forest University Baptist Medical Center 
 
 


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PROTOCOL SYNOPSIS – BMT CTN PROTOCOL 0101 
 
A Randomized Double-blind Trial of Fluconazole versus Voriconazole for the Prevention 
of Invasive Fungal Infections in Allogeneic Blood and Marrow Transplant Recipients 
 
Co-Principal Investigators: 
John R. Wingard, M.D., Thomas Walsh, M.D. 
Accrual Objective:  
Allogeneic blood or marrow transplant recipients and cord blood 
recipients in children under the age of 12 will be targeted for accrual.  
Per study arm, approximately 300 recipients (a total of 600 recipients) 
will be accrued. 
Accrual Period:  
The estimated accrual period is three years. 
Study Design: 
The study is designed as a Phase III, randomized, double-blind, 
multicenter, prospective, comparative study of fluconazole versus 
voriconazole in the prevention of fungal infections in allogeneic 
transplant recipients.  Recipients will be stratified by center and donor 
type (sibling vs. unrelated) and will be randomized to either the 
fluconazole or voriconazole arm in a 1:1 ratio. 
Primary Objective:  
The primary objective is to compare the fungal-free survival rates 
between the two study arms through Day 180. 
Secondary Objectives:  
The secondary objectives will be to compare the frequency of invasive 
fungal infection, time to invasive fungal infection, survival rate, 
duration of amphotericin B or caspofungin therapy for possible 
invasive fungal infection, time to neutrophil and platelet engraftment, 
time to and severity of acute and chronic GVHD, and to perform 
exploratory analyses of quantitative aspects of the galactomannan 
assay.  The relative safety of the two antifungals will also be assessed 
through the collection of adverse events and routine laboratory 
monitoring. 
Eligibility Criteria: 
Recipients must be diagnosed with leukemia or myelodysplastic 
syndrome (MDS).  Lymphoma patients with chemosensitive disease 
and a related donor are eligible.  Recipients must receive a 
myeloablative, 5/6 or 6/6 HLA-matched allogeneic blood or marrow 
transplant, be two years of age or older, have adequate physical 
function and give signed informed consent prior to enrollment.  
Treatment Description:  
Recipients will begin the study drug on Day 0 (day infusion of stem 
cell product is initiated).  The development of any fungal infection 
during 
prophylaxis 
will 
be 
classified 
according 
to 
revised 
EORTC/MSG definitions (see Tables 3.1.1a and 3.1.1b).  Study drug 
will be continued until Day 100 post-transplant or until invasive 
infection occurs, or the recipient develops a Grade III or IV toxicity 
attributable to the study drug.  For recipients of any type of graft 
receiving at least 1.0 mg/kg/day of prednisone (or equivalent steroid 
dose) on Day 90-100 or for recipients of T cell depleted grafts, 
receiving immunoprophylaxis post-transplant or having CD4+ counts 
< 200/L on Day 90-100, study drug will be continued until Day 180. 


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Study Duration:  
Recipients will be followed for a minimum of one year post-transplant.  
Fluconazole 
or 
voriconazole 
will 
be 
taken 
(depending 
on 
randomization arm) beginning at Day 0 of transplant for at least 100 
days (or longer as specified above).   


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TABLE OF CONTENTS 
 
 
1. 
BACKGROUND AND RATIONALE .............................................................................. 1-1 
1.1. 
Study Background............................................................................................................... 1-1 
1.2. 
Human Clinical Pharmacokinetics of Voriconazole ............................................................. 1-3 
1.3. 
Clinical Efficacy of Voriconazole ....................................................................................... 1-5 
1.4. 
Rationale for Study ............................................................................................................. 1-7 
2. 
STUDY DESIGN............................................................................................................... 2-1 
2.1. 
Study Overview .................................................................................................................. 2-1 
2.2. 
Hypotheses and Specific Objectives .................................................................................... 2-1 
2.2.1. 
Primary Hypothesis............................................................................................................. 2-1 
2.2.2. 
Secondary Hypothesis ......................................................................................................... 2-1 
2.2.3. 
Study Objectives ................................................................................................................. 2-1 
2.3. 
Patient Eligibility ................................................................................................................ 2-2 
2.3.1. 
Eligibility Criteria ............................................................................................................... 2-2 
2.3.2. 
Exclusion Criteria ............................................................................................................... 2-3 
2.4. 
Treatment Plan .................................................................................................................... 2-4 
2.4.1. 
Drug Dosages ..................................................................................................................... 2-4 
2.4.2. 
Administration Procedures .................................................................................................. 2-5 
2.4.3. 
Dosage and Formulation Adjustments ................................................................................. 2-7 
2.4.4. 
Study Drug Supply.............................................................................................................. 2-8 
2.4.5. 
Pharmaceutical Information ................................................................................................ 2-8 
2.4.6. 
Early (Premature) Withdrawal of Study Drug .................................................................... 2-11 
2.4.6.1. 
Patient early withdrawal from study treatment ................................................................... 2-11 
2.4.6.2. 
Breaking the blind ............................................................................................................. 2-13 
2.4.7. 
Contraindications, Drug Interaction and Monitoring .......................................................... 2-14 
2.4.7.1 
Voriconazole and fluconazole ........................................................................................... 2-14 
2.4.7.2 
Voriconazole drug interactions .......................................................................................... 2-15 
2.4.8. 
Duration of Study Drug ..................................................................................................... 2-16 
2.4.9. 
Recommendations for Treatment of Documented Fungal Infections .................................. 2-16 
2.4.10. 
Guidelines for Empirical Antifungal Therapy with Amphotericin B or Caspofungin Usage for 
Patients not “Off Study”.................................................................................................... 2-17 
2.4.10.1. 
Required tests prior to use of empirical antifungal therapy ............................................. 2-17 
2.4.10.2. 
Pre-engraftment requirements and amphotericin B/caspofungin dose ............................. 2-17 
2.4.10.3. 
Post-engraftment requirements and amphotericin B/caspofungin dose ............................ 2-17 
2.4.10.4. 
Continuation of study drug during empirical antifungal therapy ..................................... 2-18 
2.4.11. 
Other Antifungal Therapy ................................................................................................. 2-18 
2.4.12. 
Use of the Galactomannan Assay ...................................................................................... 2-18 
2.4.13. 
Supportive Care ................................................................................................................ 2-19 
2.4.13.1. 
Prophylaxis against infections ........................................................................................ 2-19 
2.4.13.2. 
Identification of opportunistic infections ........................................................................ 2-20 
2.4.14. 
Diagnosis of GVHD .......................................................................................................... 2-21 
2.4.14.1. 
Acute GVHD................................................................................................................. 2-21 
2.4.14.2. 
Chronic GVHD ............................................................................................................. 2-21 
2.4.15. 
Risks and Toxicities .......................................................................................................... 2-21 
2.4.16. 
Genetic Variation Study Sampling: Single Nucleotide Polymorphisms from Donor ........... 2-22 


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3. 
STUDY ENDPOINTS ....................................................................................................... 3-1 
3.1. 
Primary Endpoint ................................................................................................................ 3-1 
3.2. 
Secondary Endpoints........................................................................................................... 3-6 
3.3. 
Safety Monitoring Endpoints............................................................................................... 3-7 
4. 
PATIENT ENROLLMENT AND EVALUATION ......................................................... 4-1 
4.1. 
Enrollment and Randomization ........................................................................................... 4-1 
4.1.1 
Screening and Enrollment Procedures ................................................................................. 4-1 
4.1.2 
Randomization .................................................................................................................... 4-1 
4.2. 
Study Monitoring ................................................................................................................ 4-1 
4.2.1. 
Follow-up Schedule ............................................................................................................ 4-1 
4.2.2. 
Assessments........................................................................................................................ 4-4 
4.2.3. 
Fungal Infection (possible, presumptive, probable or proven) .............................................. 4-7 
4.2.4. 
Discontinuation of Study Drug ............................................................................................ 4-7 
4.2.5. 
GVHD Monitoring .............................................................................................................. 4-8 
4.2.6. 
Serious Adverse Event Reporting ........................................................................................ 4-8 
5. 
STATISTICAL CONSIDERATIONS.............................................................................. 5-1 
5.1. 
Study Design ...................................................................................................................... 5-1 
5.2. 
Sample Size and Power Considerations ............................................................................... 5-1 
5.3. 
Interim Analysis and Stopping Guidelines ........................................................................... 5-2 
5.3.1. 
Interim Analysis for Efficacy .............................................................................................. 5-2 
5.3.2. 
Guidelines for Safety Monitoring ........................................................................................ 5-3 
5.4. 
Demographic and Baseline Characteristics .......................................................................... 5-5 
5.5. 
Analysis of Secondary Endpoints ........................................................................................ 5-6 
5.6. 
Safety Analysis ................................................................................................................... 5-8 
 
 
 
LIST OF APPENDICIES 
 
APPENDIX A 
REFERENCES 
APPENDIX B 
CONSENT FORMS 
APPENDIX C 
LABORATORY PROCEDURES 
APPENDIX D 
GALACTOMANNAN ASSAY 
APPENDIX E 
HUMAN SUBJECTS 
APPENDIX F 
VORICONAZOLE AND FLUCONAZOLE DILUTION AND INFUSION 
INSTRUCTIONS 
APPENDIX G 
DOSING TABLES 
APPENDIX H 
ORDER FORM FOR DOSE ADJUSTMENT OF STUDY DRUG 
APPENDIX I 
DERIVATION OF THE SPRT FOR UNCENSORED EXPONENTIAL SURVIVAL 
TIMES 
 


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CHAPTER 1 
 
 
1. 
BACKGROUND AND RATIONALE 
 
1.1. Study Background 
 
Allogeneic blood and marrow transplant patients are highly susceptible to invasive fungal 
infection prior to engraftment due to neutropenia and mucosal injury.  After engraftment, an 
impairment of cell mediated immunity from GVHD and the use of aggressive 
immunosuppressive therapies such as corticosteroids [1] leave patients vulnerable to invasive 
fungal infections.  Recipients of alternate donor transplants are especially susceptible due to slow 
reconstitution of cell mediated immunity.   
 
Fluconazole prophylaxis in prospective randomized trials [2, 3] of both autologous and 
allogeneic transplant recipients has been demonstrated to reduce invasive fungal infections due 
to yeasts prior to engraftment.  A prolonged course of fluconazole given during the first 75 days 
(to cover the early post-engraftment period of risk) is highly effective in prevention of early and 
later yeast infections [4].  This has translated into a survival benefit.  A recent analysis of long-
term outcomes of these individuals demonstrated a continuing benefit beyond the course of 
prophylaxis with a further benefit in survival [5].  In another study of various factors associated 
with survival after matched unrelated donor transplants, fluconazole prophylaxis was an 
independent predictor for overall survival in a multivariate analyses [6].  Fluconazole 
prophylaxis has been found to not only be effective but safe with few substantive drug 
interactions and has been widely adopted by transplant clinicians. 
 
The recently published guidelines for preventing opportunistic infections among hematopoietic 
stem cell transplant recipients, a joint effort of the Centers for Disease Control, the Infectious 
Disease Society of America, and the American Society for Blood and Marrow Transplantation 
[7], noted that “No regimen has been reported to be clearly effective or superior in preventing 
aspergillosis, and therefore no recommendation can be made.  Further studies are needed to 
determine the optimal strategy for aspergillosis prevention.”  At a “State of the Science” Meeting 
on Hematopoietic Stem Cell Transplantation held April 1, 2000, jointly sponsored by the 
American Society for Blood and Marrow Transplantation and the National Institutes of Health, 
the Infectious Complications Working Group deemed the highest priority study would be testing 
of new antifungals and development of strategies to control invasive mould infections after BMT 
[8]. 
 
Voriconazole, a triazole with broad-spectrum antifungal activity [9-18 and reviewed in 19], is 
available in both oral and intravenous formulations.  Although there are more drug interactions 
with voriconazole than fluconazole, it is well tolerated in clinical use [13-15, 18, 20, 21].  As 
empirical therapy in febrile neutropenic patients, voriconazole proved to be associated with 
fewer breakthrough infections than liposomal amphotericin B (AmBisome) with less toxicity in a 
prospective randomized comparison [20].  The availability of an oral formulation of 
voriconazole makes it appealing for use long-term and its availability as an intravenous 
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gastrointestinal toxicities frequently seen in BMT recipients which might impair compliance 
with an oral agent.  Accordingly, prolonged use through 100 days, like fluconazole, may be 
associated not only with reduced frequency of invasive fungal infection but also with a survival 
benefit and prolonged protection even beyond cessation of drug. 
 
Two studies have evaluated another azole, itraconazole, as long-term antifungal prophylaxis after 
allogeneic BMT [22, 23].  Itraconazole has similar broad-spectrum antifungal activity including 
activity against aspergillus as voriconazole does.  However, itraconazole is less reliably and well 
absorbed by mouth (20%) and has substantial interpatient varability in contrast to excellent, 
reliable absorbance (96%) of voriconazole.  Itraconazole can be difficult to tolerate orally, and a 
pilot trial including a pharmacokinetic analysis of itraconazole at Stanford was unable to be 
completed.  Itraconazole also has substantial negative inotropic effects that have proven to be 
deleterious in patients who have received anthracyclines or in those receiving concomitant high 
dose cyclophosphamide.  This cardiotoxicity of itraconazole has resulted in a warning from the 
FDA; it is not shared by voriconazole.  In one of the two itraconazole trials by Winston et al. 
[22], a reduction in invasive fungal infections was noted (in comparison to fluconazole), but 
there were inadequate numbers of aspergillus cases to determine how effective it is as 
prophylaxis against aspergillosis.  Of concern was an excess of deaths in the itraconazole group 
(21 of 71 versus 12 of 67) and an increase in adverse events leading to discontinuation of drug in 
the itraconazole group (6 versus 1).  The second randomized trial comparing itraconazole and 
fluconazole by Marr et al. [23] used a higher dose of itraconazole to ensure therapeutic blood 
concentrations.  The trial was stopped prematurely after nearly 300 patients were enrolled 
(substantially more enrollees than in the Winston study) due to excessive toxicity in the 
itraconazole arm.  A significant excess of renal and hepatic toxicity was noted in the patients in 
the itraconazole arm.  The smaller sample size of the Winston study may have missed important 
toxicities of itraconazole due to too few patients.  Thirty-six percent of patients on itraconazole 
stopped the drug due to toxicity.  There was no reduction in the frequency of fungal infections in 
the itraconazole arm in the Marr trial, although among those who were able to tolerate it in a 
posthoc subset analysis, there were fewer infections in patients who could take itraconazole.  
Taking both itraconazole HCT trials together, there currently is insufficient data to state 
definitively that itraconazole is safe and effective in the allogeneic BMT setting for longterm 
prophylaxis. 
 
The risk of invasive aspergillosis after allogeneic BMT ranges between 7% and 24% in various 
series [4, 24-32].  In the center with the lowest rate, 7% (seen in patients transplanted between 
1990-1992) [4, 5], a larger analysis over time indicated an increase from 5.7 to 11.2% spanning 
the earlier report and more recently [31].  A still more recent survey of aspergillosis at that center 
found an infection rate of 12% [56].  Most centers find rates of invasive aspergillosis in the range 
of 11-15%.  Several of the series included both low-risk patients (autologous transplant 
recipients) along with high-risk patients (allogeneic transplant recipients).  By excluding low-risk 
transplant recipients (autologous BMT recipients), including other fungal pathogens in addition 
to aspergillus (those organisms not controlled by fluconazole), adding cases of probable 
aspergillosis and incorporating the galactomannan assay into the diagnostic criteria, we estimate 
the rate of invasive fungal infection in patients receiving fluconazole to be at least 15% and are 
using that as the estimate in this trial. 
 


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Because of poor sensitivity and specificity of clinical criteria and difficulty in performing 
invasive diagnostic procedures in this patient population, more accurate and less invasive 
diagnostic assays have been sought.  Recently, the double-sandwich ELISA assay for 
galactomannan has been found to be highly sensitive and specific [33-35]; it performs well in the 
BMT recipient patient population [29, 33].  Maertens et al identified 12 episodes of proven 
invasive aspergillosis in 99 consecutive allogeneic stem cell transplant recipients plus 3 episodes 
of probable invasive aspergillosis.  Using a sandwich ELISA technique, serum samples were 
assayed for the presence of galactomannan twice weekly.  Two or more consecutive sera tested 
positive in all episodes documented by clinical and microbiologic criteria.  Galactomannan was 
detected prior to any clinical signs of invasive aspergillosis in 5 of the 12 patients and preceded 
fever or other symptoms in 9 of the 12.  In 93% of culture positive episodes, galactomannan was 
detected before isolation of aspergillosis.  Galactomannan was detected prior to the presence of 
radiographic abnormalities in 91%.  Using classical criteria for initiation of empirical antifungal 
therapy, the assay was positive a median of 6 days prior to standard criteria for empirical therapy 
in 87% of cases.  Autopsies of patients suspected of having invasive fungal infection but with 
negative galactomannan assays demonstrated no histologic evidence of invasive aspergillus.  In a 
retrospective survey using banked serum from patients found to have proven invasive 
aspergillosis when compared with controls, Marr showed that the galactomannan assay has a 
high degree of sensitivity and specificity [29].  As with the Maertens study, Marr found the 
presence of galactomannan in the serum prior to the diagnosis in two-thirds of patients using 
classical diagnostic criteria.   
 
Incorporation of the galactomannan assay along with the classical criteria will improve accuracy 
and may increase the number of cases documented (by upgrading possible cases not proven by 
classical criteria to proven or probable cases with the galactomannan assay) in the context of a 
clinical trial.  The assays will be performed at a certified contract laboratory or a certified 
hospital laboratory associated with the transplant center (see Appendix C).  The data will be 
analyzed in grading aspergillosis cases as either possible, presumptive, probable or proven in 
accordance with revised EORTC/MSG consensus case definitions (see Section 3.1).   
 
1.2. Human Clinical Pharmacokinetics of Voriconazole 
 
Voriconazole has been administered to over 3000 healthy volunteers or patients with fungal 
infections in single or multiple dosing regimens, by oral and intravenous routes.  
 
In order to improve its solubility, voriconazole is formulated with sulphobutylether-cyclodextrin 
(SBECD) for intravenous administration.  Volunteer studies show that the cyclodextrin is 
completely renally cleared at a rate consistent with creatinine clearance.  Multiple dosing 
indicates that there is no accumulation of SBECD and the mean elimination half-life after 10 
days dosing is 1.6 hours.  
 
Voriconazole is widely distributed throughout body fluids and the volume of distribution is about 
2 L/kg.  Following oral administration of voriconazole, absorption is rapid with a mean time to 
maximum plasma concentration of 1 to 2 hours post dose.  Single and multiple dose 
administration of voriconazole in the presence of a high-fat meal delays absorption.  Multiple 
dosing in the presence of food reduces systemic exposure by 32% compared to the fasted state.  


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As a precaution, it is recommended that voriconazole should not be taken within one hour of 
eating.  
 
Voriconazole in adults has non-linear pharmacokinetics with regard to dose [57, 58].  For 
example, a two-fold increase in single oral dose resulted in a three to four fold increase in the 
AUC0-last (the area under the concentration time curve from time 0 to the last observed 
concentration above the limit of detection).  For the intravenous formulation, the single dose 
pharmacokinetics are linear up to 4 mg/kg, thereafter they become non-linear with similar 
increase in the AUC seen with a doubling of dose.  For oral multiple dosing, comparison across 
cohorts suggests that for a 1.5 fold increase in dose there is a three to four fold increase in the 
AUC (the area under the concentration time curve from time 0 to the end of the dosing interval).   
 
There is wide inter-patient variability in the pharmacokinetics of voriconazole with the 
coefficient of variation of 126% for AUC for a 3 mg/kg Q12H oral dose and 76% for 
intravenous 3 mg/kg Q12H dosing.  Multiple dosing at intravenous doses of 3 mg/kg Q12H and 
oral doses of 200 mg Q12H results in a mean accumulation of approximately three fold and a 
maximum accumulation of five fold which is not predictable from the single dose data.  For both 
intravenous and oral administration at single and multiple doses, voriconazole has a mean 
terminal phase half-life of about six hours.  Steady state, for both intravenous and oral dosing, is 
achieved within six days.  Use of a loading dose (6 mg/kg) results in steady state being achieved 
by 24 hours of dosing.  
 
The pharmacokinetic data from phase II studies showed that voriconazole exhibits a high degree 
of inter-patient variability.  In the 175 patients analyzed, the average AUC, maximum plasma 
concentration and time to maximum plasma concentration were consistent with healthy volunteer 
data at equivalent doses.  The average bioavailability was high, 96% of subjects had observed 
plasma voriconazole concentrations within the range 1-10 mcg/mL. 
 
Salivary levels of voriconazole are approximately 65% of plasma levels (range 51 to 79%).  This 
is consistent with a plasma protein binding of about 58%.  Comparison of the oral and 
intravenous systemic exposure following single doses and at steady state after multiple dosing 
indicates that the bioavailability of the oral dose lies within the range of 58 to 90%.   
 
Plasma pharmacokinetics of intravenous voriconazole in immunocompromised children aged 2–
11 were assessed and compared with those of healthy adults.  In contrast to adults where 
nonlinear kinetics were observed, in children, the elimination of voriconazole was linear after 
doses of 3 and 4 mg/kg [60].  Comparison of observed and simulated pediatric and adult 
pharmacokinetic data indicated that plasma concentrations and area under the concentration-time 
curve  of 3 and 4 mg/kg in children were lower than the corresponding dosages in adults.  
Nevertheless, the peak plasma concentrations in pediatrics still substantially exceed the reported 
mean inhibitory concentrations (MICs) for most species of Candida, aspergillus and other 
filamentous fungi.  Doses of 4 mg/kg in children appear to achieve AUCs that approximate the 
AUCs in adults dosed at 3 mg/kg. 
 


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1.3. Clinical Efficacy of Voriconazole 
 
The clinical efficacy, safety and tolerance of voriconazole have been tested in three Phase II 
studies in Europe; oropharyngeal candidiasis in HIV positive patients [36], acute aspergillosis in 
immunocompromised patients [37] and chronic fungal infections in non-neutropenic patients.  In 
addition, preliminary efficacy data are available in pediatric patients who received voriconazole 
compassionately.  
 
In a seven day double-blind, dose ranging study in 165 HIV positive patients with oropharyngeal 
candidiasis (dose levels 50 mg QD, 200 mg QD or 200 mg Q12H), clinical efficacy (cured + 
improved) in the intention to treat (ITT) analysis was 80% at 50 mg QD, 100% at 200 mg QD 
and 89% at 200 mg Q12H.  Mycological efficacy (reduction in colony counts < 11 
colonies/culture) in the ITT analysis increased with dose from 33% at 50 mg QD to 74% at 200 
mg Q12H.  The drug was generally well tolerated and the only drug-related adverse event of any 
significance was transient visual disturbance.  The visual events had resolved fully in all patients 
at follow-up.   
 
Based upon the plasma pharmacokinetic profile of voriconazole, there may be a dose-response 
relationship between higher dosages and improved antifungal efficacy.  The geometric-mean 
inhibitory concentrations (GMIC) of voriconazole vs. aspergillus spp. range from 0.19 to 0.58 
ug/mL.  Although the GMIC cannot be as closely correlated with clinical efficacy as MIC can 
for an antibiotic, it is generally considered desirable for plasma concentrations of an antifungal 
agent to cover the GMIC.  Plots of Cmin data showed that a dosage of 4 mg/kg IV BID resulted 
in trough plasma levels of voriconazole > 0.5 g/mL in 6 of 7 subjects.  However, a dosage of  
3 mg/kg IV BID resulted in trough levels of voriconazole > 0.5 g/mL in only 4 of 14 subjects. 
 
A second open, non-comparative study was conducted using 200 mg PO Q12H of voriconazole 
for up to 24 weeks in chronic aspergillosis or candidiasis of non-neutropenic patients.  At the 
interim analysis of clinical efficacy in 25 patients, favorable clinical response (complete or 
partial resolution of clinical signs and symptoms, as determined by the investigator) was 
achieved in 70% (12/17).  Mycological response was available in three patients at the end of 
therapy (EOT) and all three had negative cultures.  The compound was generally well tolerated 
in those patients.  Visual disturbance, skin rash and elevated liver function tests were observed 
and reported as adverse events possibly related to therapy.  In two patients, elevation of liver 
function tests (LFTs) above the protocol-specified maximum resulted in their withdrawal from 
the study.   
 
The third study was an open, non-comparative study of voriconazole (6 mg/kg IV Q12H for one 
day then 3 mg/kg IV Q12H for 6-27 days followed by 200 mg PO Q12H for up to a total of 24 
weeks) in acute invasive aspergillosis of neutropenic and other immunocompromised patients.  
Interim analysis of clinical efficacy in 71 patients indicated a favorable clinical response 
(complete/partial resolution or stable clinical signs and symptoms, as determined by the 
investigator) was achieved in 83% (54/65).  Mycological response at EOT was available in 31 
patients; three patients had their infection eradicated, four had persistent infection and 24 were 
not evaluable.  The drug was generally well tolerated by these severely ill patients.  LFT 


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elevations above the protocol-specified maximum resulted in withdrawal of three patients from 
the study.   
 
The safety and efficacy of voriconazole in children treated within the compassionate release 
program who were intolerant of, or refractory to, conventional antifungal therapy was recently 
reported [38].  Seventy-two patients (ages 9 months to 15 years, median 7 years) received 
voriconazole; 63 had a proven or probable fungal infection.  Among these 63 patients, 27 had 
hematological malignancies and 14 had chronic granulomatous disease as the most frequent 
underlying conditions.  Forty-two patients had aspergillosis, 8 scedosporiosis, 4 invasive 
candidiasis, and 4 had other invasive fungal infections.  The median duration of voriconazole 
therapy was 93 days.  At EOT 27 patients (44%) had a complete or partial response.  Four 
patients (7%) had a stable response, 25 (43%) failed therapy, and 4 (7%) were discontinued from 
voriconazole because of intolerance.  Success rates were highest in patients with CGD (57%) and 
lowest in patients with hematological malignancies (25%).  Two patients experienced treatment-
related serious adverse events (ulcerated lips with rash, elevated hepatic transaminases, or 
bilirubin).  Voriconazole-related adverse events included rash (8), increased hepatic 
transaminases or bilirubin (5), blurred vision (2), and vomiting (1).  These data support the use of 
voriconazole for treatment of invasive fungal infections in pediatric patients who are intolerant 
of, or refractory to, conventional antifungal therapy. 
 
The results of an open, randomized comparison of voriconazole versus amphotericin B followed 
by other licensed antifungal therapy for primary treatment of invasive aspergillosis were recently 
described [39].  Patients with invasive aspergillosis were randomized to receive amphotericin B 
1 mg/kg or voriconazole intravenously at 6 mg/kg Q12 hours (2 doses) then 4 mg/kg Q12H.  
Oral formulation at a dosage of 200 mg Q12H could be given following 4 mg/kg IV dosing.  
Other licensed antifungal therapy was allowed after initial randomization therapy.  Blinded 
teams assessed eligibility and response, and followed modified EORTC/MSG definition criteria 
for probable or proven infection.  Favorable outcomes were complete response of clinical and 
radiological findings or partial response, which was significant clinically and > 50% radiological 
improvement.  Unfavorable outcomes were unchanged or worsened condition.  The primary 
outcome was assessed at week 12.  Secondary endpoints included outcome of study therapy and 
survival through week 12.  Three hundred ninety-two patients were enrolled over three years in 
92 centers in 19 countries.  One hundred forty-four voriconazole and 133 amphotericin B 
patients had confirmed invasive aspergillosis and received > 1 dose of therapy.  Underlying 
diseases represented were allogeneic bone marrow transplant in 26 and 23%, autologous bone 
marrow transplant or hematologic disease in 56 and 63%, and other in 18 and 14% of 
voriconazole and amphotericin B patients, respectively.  Pulmonary aspergillosis was present in 
83% of the voriconazole and 84% of the amphotericin B treated patients.  Disseminated disease 
occurred in 8.3% voriconazole versus 7.5% amphotericin B-treated and CNS disease in 1.4% 
versus 0.8%, respectively.  At week 12, a complete/partial response of 52.8% with voriconazole 
versus 31.6% with amphotericin B was observed (95% CI for the difference was 32.9% to 
10.4%).  Survival on the voriconazole arm was 70.8% for voriconazole versus 57.9% on the 
amphotericin B arm (Hazard ratio 0.59; 95 CI 0.40 to 0.88).  Study therapy was continued for a 
median of 77 days on the voriconazole arm versus 11 days on the amphotericin B arm.  End of 
therapy response was 53.5% on the voriconazole arm versus 21.8% on the amphotericin B arm 
(95% CI for the difference was 42% to 21%).  These data suggest voriconazole was more 


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effective than amphotericin B followed by other licensed antifungal therapy at week 12. 
Moreover, voriconazole improved survival in patients with invasive aspergillosis. 
 
Recently reported data on the efficacy of voriconazole against rare or resistant fungal pathogens 
(C. neoformans, F. solani, P. lilacinus, Trichosporon spp, S.imitis and H. capsulatum) appear 
promising [40-43].  Patients with documented invasive infections caused by rare or resistant 
fungal pathogens and enrolled into voriconazole clinical studies or for compassionate use were 
included in the analysis.  Fifty-nine subjects were analyzed; those receiving primary therapy 
(n=8 success=3, failure=5) and those receiving salvage therapy (n=51 success=21 failure=30). 
Outcomes were grouped according to underlying condition: AIDS/HIV (n=13 success=3, 
failure=10), hematologic malignancy (n=15 success=5, failure=8), drug or disease induced 
immune suppression (n=11 success=4, failure=7), injury/trauma or post surgical (n=5 success=1, 
failure=4), non-malignant hematological disorders (n=6 success=3, failure=3).  Fifteen of the 59 
patients had hematologic diseases (n=15 success=7, failure=8).  Successful outcomes were seen 
in 17 of 44 patients without these risk factors.  Outcomes based on pathogens were also 
analyzed: Cryptococcus spp was successfully treated in 4 of 13 patients, Fusarium spp in 6 of 16 
infections, and infections with other rare fungi, 14 of 30.  Therefore, voriconazole appears to be a 
promising antifungal agent in severely immunocompromised patients who have failed or are 
intolerant to other treatment options, with potent activity against a wide variety of resistant and 
rare fungal pathogens. 
 
The recently completed MSG-42 study of voriconazole versus liposomal amphotericin B (L-
AMB) for empirical antifungal therapy in persistently febrile neutropenic patients suggests 
voriconazole is a safe and effective alternative therapy in this population [44].  Voriconazole was 
compared to L-AMB in a randomized, multicenter, international trial.  Eight hundred forty-nine 
randomized patients (voriconazole n=421, L-AMB n=428) receiving at least one dose of study 
drug were evaluable for success.  Composite success rates were 26% with voriconazole and 31% 
with L-AMB (95% CI for difference; -10.6% to 1.6%).  These were independent of 
administration of colony stimulating factors or antifungal prophylaxis.  Documented 
breakthrough fungal infections were fewer among patients treated with voriconazole 8 (1.9%) 
versus 21 (5.0%).  Severe infusion-related reactions (p<0.01) as well as nephrotoxicity (p<0.001) 
were less in patients receiving voriconazole versus L-AMB.  Hepatoxicity was similar for both 
drugs.  More cases of transient visual changes (22% versus 1%) (p<0.001) and hallucination 
(4.3% versus 0.5%) occurred on the voriconazole arm than on the L-AMB arm.  Ninety-two 
patients (22%) changed to oral formulation of voriconazole with a reduction of mean duration of 
hospitalization by one day in all patients and by two days in high-risk patients (p<0.05).  The 
authors concluded that voriconazole is an effective alternative for empirical antifungal therapy in 
patients at risk.  
 
1.4. Rationale for Study 
 
Given the high mortality rate associated with documented fungal infections in BMT patients 
despite aggressive therapy with amphotericin B, strategies to prevent infections are warranted [7, 
8] and may be more effective than trying to treat a documented established infection.  Allogeneic 
BMT patients are at special risk for infection due to aggressive or prolonged immunosuppressive 
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voriconazole can be administered orally, have good activity against several fungal pathogens and 
are less toxic than amphotericin B.  The availability of intravenous formulations of both agents 
permit continued compliance with the regimen in the event that the patient cannot tolerate oral 
medications.   
 
Although voriconazole has a wider antifungal spectrum than fluconazole, there are more drug 
interactions requiring greater vigilance over the immunoprophylaxis regimen, and there is the 
potential for greater toxicity.  Whether or not, on balance, the potential benefit outweighs these 
potential risks can only be determined in a prospective, randomized trial. 
 


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CHAPTER 2 
 
 
2. 
STUDY DESIGN 
 
2.1. Study Overview 
 
This is a randomized, double-blind, multicenter, prospective, comparative study of fluconazole 
versus voriconazole in the prevention of fungal infections in allogeneic hematopoietic transplant 
recipients and cord blood recipients in children under the age of 12.  Prior to the start of the pre-
transplant conditioning regimen, patients will give written informed consent and be screened for 
eligibility.  Patients who meet all entry criteria will be assigned randomly to voriconazole or 
fluconazole within 72 hours of Day 0.  Patients will begin study drug on Day 0 (after completion 
of the conditioning regimen).  Day 0 is defined as the day infusion of the stem cell product is 
initiated.  Study drug will be continued until Day 100 following transplant or until one or more 
criteria for study drug early withdrawal are met as defined in Section 2.4.6.  Continuation of 
study drug beyond Day 100 is permitted for patients who meet criteria specified in Section 2.4.8.  
The development of any fungal infection during prophylaxis will be classified according to the 
definitions listed in Section 3.1.1.  
 
2.2. Hypotheses and Specific Objectives 
 
2.2.1. 
Primary Hypothesis 
 
Voriconazole prophylaxis will be associated with fewer invasive fungal infections and reduce 
morbidity and mortality due to fungal infection.  This will result in an improvement of fungal-
free survival (survival without invasive fungal infection). 
 
2.2.2. 
Secondary Hypothesis 
 
Reduction in fungal infectious rates will be associated with improvement in post-transplant 
survival. 
 
2.2.3. 
Study Objectives  
 
This trial is designed to compare the efficacy of fluconazole 400 mg/day PO (or IV at the same 
dose if the patient is unable to tolerate it PO) with voriconazole 200 mg PO BID (or IV at the 
same dose if the patient is unable to tolerate it PO) in prevention of fungal infections in 
allogeneic hematopoietic transplant recipients during the first 180 days after transplant.  Children 
aged < 12 years will receive age-appropriate dosing, as indicated in Section 2.4.1.  The primary 
objective is to compare the fungal-free survival rates between the two study arms during the first 
180 days.  Secondary objectives will be to compare invasive fungal infection rates, overall 
mortality due to fungal infection, engraftment rates, acute and chronic GVHD rates, reasons for 
failure (e.g., plasma concentrations, tolerance, fungal sensitivity, etc.), and to perform 
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the two antifungals also will be assessed through the collection of post-transplant toxicities, 
serious adverse event reports and routine laboratory monitoring.   
 
2.3. Patient Eligibility 
 
2.3.1. 
Eligibility Criteria 
 
Patients fulfilling all of the following criteria will be eligible for this study: 
 
1. Patients who receive an allogeneic peripheral blood or marrow transplant from a family 
or unrelated donor, or for children under the age of 12, a cord blood transplant from 
either a sibling or other donor. 
2. Patients must have a 5 or 6 of 6 HLA-matched donor.  The match may be determined at 
serologic level for HLA-A and HLA-B loci.  For sibling donors, matching may be 
determined at serologic level for HLA-DR; for unrelated donors, matching for HLA-
DRB1 must be at the high-resolution molecular level.   
3. Patients two years of age or older. 
4. Patients and/or legal guardian able to provide informed consent  
5. Patients with one of the following underlying diseases: 
a) AML, with or without a history of myelodysplastic syndrome, in first or second 
complete remission (see Section 3.4) or in early relapse (< 30% blasts in bone 
marrow with no circulating blasts in peripheral blood and no extramedulary 
leukemia); or 
b) ALL, in first or second complete remission (see Section 3.4); or 
c) AUL (acute undifferentiated leukemia) in first or second complete remission (see 
Section 3.4); or 
d) Acute biphenotypic leukemia in first or second complete remission (see Section 3.4); 
or 
e) CML in either chronic or accelerated phase; or 
f) One of the following myelodysplastic syndrome(s) defined by the following: 
 
1) Refractory anemia 
2) Refractory anemia with ringed sideroblasts 
3) Refractory cytopenia with multilineage dysplasia 
4) Refractory cytopenia with multilineage dysplasia and ringed sideroblasts 
5) Refractory anemia with excess blasts-1 (5-10% blasts) 
6) Refractory anemia with excess blasts-2 (10-20% blasts) 
7) Myelodysplastic syndrome, unclassified 
8) MDS associated with isolated del (5q) 


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9) CMML; or 
g) Lymphoma (including Hodgkin’s) with chemosensitive disease (≥ 50% response to 
chemotherapy) and receiving a related donor transplant 
6. Patients receiving myeloablative conditioning regimens. 
7. Patients with adequate physical function, within six weeks of initiation of conditioning 
(preferably within four weeks) unless otherwise specified, as measured by: 
a) Cardiac: 
 
Asymptomatic or, if symptomatic, then left ventricular ejection 
fraction at rest must be > 40% and must improve with exercise, or 
shortening fraction > 26% 
b) Hepatic: 
 
< 5 x ULN ALT (within 72 hours of Day 0) 
< 2.5 mg/dL total serum bilirubin 
c) Renal: 
 
Serum creatinine within normal range for age or if serum 
creatinine above upper limit of normal range for age then renal 
function (creatinine clearance) > 50% LLN for age 
d) Pulmonary: 
DLCO, FEV1, FVC (capacity) > 45% of predicted value (corrected 
for hemoglobin) or O2 saturation > 85% of room air 
8. Patients must have baseline galatomannan blood samples drawn within 30 days prior to 
randomization with the results available prior to randomization (72 hours prior to 
transplant) 
9. Patients must have chest CT scans within six weeks prior to randomization if the results 
of the baseline galactomannan blood sample are not available prior to randomization (72 
hours prior to transplant) 
 
2.3.2. 
Exclusion Criteria 
 
Patients with the following will be ineligible for randomization on this study: 
 
1. Patients with an invasive yeast infection within the eight weeks prior to conditioning 
regimen initiation.  Patients are eligible if colonized or have had superficial infection. 
Patients with a history of candidemia > 8 weeks prior to conditioning must have a 
negative blood culture within 14 days of conditioning (within 7 days is recommended), 
no clinical signs of candidemia, and may not still be requiring antifungal therapy. 
2. Patients with a presumptive, proven or probable aspergillus or other mould infection or 
deep mycoses (including hepatosplenic candidiasis) within 4 months prior to conditioning 
regimen initiation.  
3. Patients with an uncontrolled viral or bacterial infection at the time of study registration. 
4. Women who are pregnant (positive -HCG) or breastfeeding.  Women of child-bearing 
age must avoid becoming pregnant while receiving antifungal agents. 
5. Patients with a Karnofsky performance status < 70% or Lansky < 50% for patients < 16 
years old unless approved by the Medical Monitor or protocol Chair. 


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6. Patients with a history of allergy or intolerance to azoles (fluconazole, itraconazole, 
voriconazole, posaconazole, ketoconazole, miconazole, clotrimazole). 
7. Patients requiring therapy 
with rifampin, rifabutin, 
carbamazepine, 
cisapride 
(Propulsid®), terfenadine; (Seldane®), or astemizole (Hismanal®), ergot alkaloids, long-
acting barbiturates, or who have received > 3 days treatment with rifampin or 
carbamazepine within 7 days prior to conditioning regimen initiation.  Patients on 
therapeutic anticoagulation with coumadin (1 mg/day for port prophylaxis is permitted). 
8. Patients receiving sirolimus. 
9. Patients with prolonged QTc syndrome at study entry.  
10. Patients who are HIV positive.  
11. Patients receiving another investigational drug unless cleared by the medical monitors. 
12. Patients who have received a prior allogeneic or autologous transplant. 
13. Patients with active CNS disease. 
14. Patients on fungal prophylaxis during conditioning regimen (it is recommended that 
fungal prophylaxis be suspended once patient is enrolled). 
15. Patients with prior malignancies except resected basal cell carcinoma or treated 
carcinoma in-situ.  Cancer treated with curative intent < 5 years previously will not be 
allowed unless approved by the Medical Monitor or Protocol Chair.  Cancer treated with 
curative intent > 5 years previously will be allowed. 
 
2.4. Treatment Plan 
 
The immediate pre-transplant evaluation will be carried out according to the operating 
procedures of the participating institutions and should be in keeping with the data reporting 
requirements of this study.  Similarly, special orders and procedures will be those defined by the 
operations manuals of the Clinical Centers.  All patients enrolled on this protocol will be 
hospitalized in accordance with isolation procedures for recipients of allogeneic blood and 
marrow transplants as defined by the given institution. 
 
2.4.1. 
Drug Dosages  
 
The study drugs will be masked by overencapsulation to obscure their identity.  Patients 
randomized to voriconazole will receive one dose in the morning and one dose in the evening.  
Fluconazole is appropriately efficacious if administered once daily.  Those patients randomized 
to fluconazole will receive their full daily dose in the morning followed by placebo in the 
evening.  
 
Study drug will begin on Day 0, day of transplant.  Patient should be given an AM and PM dose. 
If necessary, the AM dose may be administered prior to stem cell infusion. 
 


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If a patient misses a dose, it should be taken within four hours of the scheduled time.  If more 
than four hours have passed, the patient should not take an additional dose until his next 
scheduled dose. 
 
In efforts to maintain the blind, both intravenous voriconazole and fluconazole will be 
administered in equivalent volumes as specified in Appendix F. 
 
Adults:  The dose of oral voriconazole is 200 mg twice daily.  When voriconazole must be given 
intravenously, it will be given at a dose of 200 mg every 12 hours for the duration of intravenous 
therapy.  Doses will not be reduced for renal dysfunction. 
 
The dose of fluconazole is 400 mg by mouth or intravenous drip.  Doses will be reduced for 
renal dysfunction in accordance with Section 2.4.3 and Appendix G. 
 
Children (age < 12 years old):  Oral doses will be given as follows:  In patients weighing > 20 
kg, voriconazole will be administered at a dose of 100 mg twice daily.  Patients weighing < 20 
kg will receive oral voriconazole at a dosage of 50 mg twice daily.  If intravenous administration 
is needed, voriconazole will be at a dose of 4 mg/kg (total body weight) every 12 hours (not to 
exceed the equivalent oral dose) for the duration of intravenous therapy (see Appendix F).   
 
For oral administration, patients weighing > 20 kg will receive fluconazole at a dose of 200 mg 
once daily.  For those weighing < 20 kg, a dose of 100 mg once daily will be used.  Doses will be 
reduced for renal dysfunction in accordance with Section 2.4.3 and Appendix G.  When 
intravenous administration is received, fluconazole will be given at 6 mg/kg/day total body 
weight (with maximal IV dose not to exceed the equivalent oral dose per weight) once daily (see 
Appendix F).   
 
Both study drugs will be given orally whenever possible.  Both voriconazole and fluconazole are 
well absorbed after oral administration with bioavailability exceeding 90%.  Study drugs should 
be taken at least one hour after or one hour before a meal.  The intravenous formulation will be 
given only when the oral medication cannot be tolerated.  Oral study drug should be resumed as 
quickly as possible when oral medication can be given. 
 
Total duration of the prophylaxis study drug will be 100 days, or until study drug early 
withdrawal criteria have been met (Section 2.4.6) including criteria for a presumptive, probable 
or proven fungal infection (Section 3.1.1).  Study drug will be continued beyond Day 100 in 
patients who meet criteria in Section 2.4.8.   
 
Fungal prophylaxis must not be given after protocol-specified treatment plan is completed on 
Day 100 (or 180). 
 
2.4.2. 
Administration Procedures 
 
1. Voriconazole 
Voriconazole will be administered orally twice daily.  Voriconazole capsules should be 
taken at least one hour before or one hour after a meal.  Taken concomitantly with food, 


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bioavailability of voriconazole is reduced.  If oral drug is not possible, it will be given 
intravenously at a dosage of 200 mg Q12H over two hours in patients > 12 years.  Each 
voriconazole dose will be diluted to a total volume of 200 mL in patients > 12 years.  
Volumes of the formulation required to provide 4 mg/kg doses for children (age < 12 
years) of various body weights, and the dilution and infusion instructions are in Appendix 
F.  To maintain the blind in children, a standardized volume defined in Appendix F will 
be used for all doses.  Accordingly, dilutions to the specified volume, if necessary, should 
be made. 
 
2. Fluconazole  
Fluconazole will be administered orally once daily.  Fluconazole capsules should be 
taken at least one hour before or one hour after a meal.  If oral drug is not possible, it will 
be given intravenously once daily in a total volume of 200 mL in patients > 12 years.  For 
adults, each 200 mL infusion will be administered over 2 hours.  In patients < 12 years, 
intravenous doses will be prepared in accordance with Appendix F in standardized 
volumes and administered over two hours. 
 
3. Incompatibilities 
Concomitant medications should not be infused in the same line as voriconazole, 
fluconazole or amphotericin B.  
 
4. Accountability 
The investigator (and/or hospital pharmacist) will ensure that all study drug is stored in a 
secured area under recommended storage conditions and is dispensed by qualified staff 
members.  All study drug will be accounted for on medication inventory sheets. 
 
The BMT CTN clinical monitor will be allowed at intervals, and upon request during the 
study, to check unused supplies.  Accounting for the use of supplies will be by reference 
to each center's record of supplies received, the dispensing records for the total number of 
patients enrolled at each center and the unused and returned supplies.   
 
Patients who are discharged from the hospital during the time they are receiving oral 
voriconazole or fluconazole therapy will be provided with ample supply of study 
medication between visits.  The patient will be requested to return all containers of study 
drug, including empty containers and containers with unused capsules, at the next visit.  
Unused drug may be returned to patient if therapy continued. 
 
The investigator is responsible for maintaining drug accountability records.  Drug 
accountability records will be reviewed during monitoring visits.  Study drug must be 
administered only to patients enrolled in this study as per the protocol. 
 
5. Labeling 
The investigator or pharmacist will record the patient number on the label of all 
containers containing voriconazole or fluconazole capsules before dispensing to the 
patient.  The lot number of the bottle should be recorded on the patient's dispensing 
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6. Cardiac Monitoring 
In subjects assessed by the treating physician to be at risk for cardiac arrhythmia, the 
following procedure must be performed: Continuous cardiac monitoring during the first 
three days of study medication.  If the cardiac monitoring reveals a significant arrhythmia, 
the administration of study medication must be held, and the subject must undergo further 
assessment by a cardiologist to evaluate the significance of the findings.  The 
cardiologist’s assessment and opinion on the significance of any abnormal finding will be 
summarized in a report and submitted on an Adverse Event Form.  If the PI and 
cardiologist conclude that it is not related to the study drug, study drug can be resumed.  
Otherwise, the patient will be withdrawn from study drug.   
 
2.4.3. 
Dosage and Formulation Adjustments  
 
Oral intolerance: Oral study medication will be temporarily discontinued in patients who 
become unable to take or to tolerate oral medications; the parenteral antifungal formulation 
should be substituted in the dose according to Section 2.4.1.  Patients will be changed back to 
oral medications as soon as tolerable.  If the patient is an outpatient and requires IV study drug 
for more than 14 days and cannot tolerate oral study drug, the patient must be taken off study 
drug. 
 
Empirical therapy with Amphotericin B or Caspofungin: Patients who develop a possible 
invasive fungal infection as defined in Section 3.1.1, but do not meet the criteria for failure of 
prophylaxis, may be treated empirically with an amphotericin B formulation or caspofungin 
according to the provisions of Section 2.4.10.  These patients will remain on study and continue 
to be assessed for more definitive evidence of fungal infection.  During empirical therapy, the 
study drug should be continued.  Prior to initiation of any empirical trial of amphotericin B or 
caspofungin, at least two blood samples for the diagnostic galactomannan assay should have 
been collected during the last seven days (with at least one within 48 hours).  No systemic 
antifungal agent, other than one of the amphotericin B formulations or caspofungin, is permitted 
while the patient is on study. 
 
Renal dysfunction: In the setting of renal dysfunction, fluconazole doses will be adjusted as 
follows: estimated CrCl < 50ml/min = 50% dose reduction.  For pediatric patients with CrCl  
< 20ml/min/1.73m2, fluconazole dose reduction = 75%.  Calculations of creatinine clearance 
should be made based on the method of Cockgroft and Gault [45] for adult males [CrCl = (140-
age in years) X kg weight (ideal) /serum creatinine x72].  For adult females, the equation is 
multiplied by 0.85.  Ideal body weight (IBW) is to be calculated as follows:  Male IBW = 50kg + 
2.3 kg/inch over 5 feet, Female IBW = 45.5 kg + 2.3 kg/inch over 5 feet.  If less than 5 feet, 
subtract 2.3 kg/inch.  For children, the Schwartz calculation will be used (CrCl = k value X 
height (cm)/plasma creatinine).  K values for children and adolescent girls = 0.55, adolescent 
boys = 0.70.  Guidelines for oral dose adjustment are given in Appendix G.  No dose adjustments 
for renal dysfunction will be made in the voriconazole arm.  For patients with moderate renal 
insufficiency (estimated CrCl < 50 mL/min), accumulation of the voriconazole IV vehicle can 
occur.  Oral voriconazole should be administered to these patients unless a benefit/risk 
assessment justifies the use of IV voriconazole.  However, if CrCl declines to 25 mL/min and 


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persists for more than 14 days and the patient must receive IV drug and is unable to switch to 
oral drug, then the study drug should be discontinued.  Study drug should be discontinued in the 
setting of hemodialysis (see Section 2.4.6.1.f). 
 
To maintain the blind in patients receiving intravenous study drug, for patients randomized to 
receive voriconazole who meet the renal impairment criteria, the volume of voriconazole will be 
adjusted to mimic the volume the patient would receive if randomized to fluconazole.  To 
maintain the blind in patients receiving oral study drug, the number of voriconazole capsules 
taken in the morning will be adjusted to mimic the number the patient would receive if 
randomized to fluconazole (see Appendix G). 
 
The physician must order dose adjustments for all patients randomized into the study using an 
order conforming to type listed in Appendix H.  The pharmacist will make the appropriate 
adjustments based on randomization and the type of renal impairment. 
 
2.4.4. 
Study Drug Supply 
 
An initial supply of study drug (oral and IV, adult and pediatric doses, if appropriate) will be 
automatically sent to the investigational pharmacy upon enrollment of the transplant center’s first 
patient onto Segment A (see Section 4.1.1).  The transplant center’s investigational pharmacist 
will be responsible for subsequent ordering, receiving, storing, distributing and accounting of the 
study drug.  He/she will be responsible for recording the receipt and administration of all trial 
drug supplies, for ensuring the supervision of the storage and allocation of these supplies, and for 
maintaining the drug distribution and accountability logs.  These logs must be available for 
inspection during routine site monitoring visits.  The investigator will ensure that all protocol 
drugs are stored in a secured area under recommended storage conditions and are dispensed by 
qualified staff members.   
 
2.4.5. 
Pharmaceutical Information 
 
1. Source 
Voriconazole and fluconazole are manufactured by Pfizer, Inc., Groton, CT.  
 
2. Toxicity 
Voriconazole:  Voriconazole had been administered to over 3000 healthy volunteers or 
patients with fungal infections in single or multiple dosing regimens, by oral and 
intravenous routes at the time of FDA review for licensure.  The following safety issues 
have been identified.  There were 286 serious adverse events recorded.  Of these, a total 
of eight were reported by the investigators to be potentially causally related to 
voriconazole.  Other events were assessed by the investigators to be related to the disease 
under study or to other factors.  The eight events that were causally related to study drug 
were: three cases of elevated liver function test (LFT) values, three cases of skin 
reactions, one case of exacerbated hypoglycemia and one case of atypical pneumonia 
with hypoglycemia. 
 


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Overall, 30% of volunteers and patients have reported adverse events related to vision, 
regardless of route of administration.  The most consistent description offered was of a 
transient altered/increased perception of light or blurred vision.  The events generally 
lasted less than one hour, with onset occurring between 30 and 60 minutes after an oral 
dose.  Of these, three of four were mild.  The remainder were moderate, with 1-2% being 
severe.  All of these events resolved.  Abnormal vision is therefore reversible, even 
during continued therapy, and is not considered to represent a serious risk.  
 
Ophthalmologic tests (visual evoked response), performed in volunteers, did not reveal 
any abnormalities and provide no basis for a potential mechanism.  Also there have been 
no ophthalmologic or brain/optic nerve histological changes in animals treated for up to 
six months with high doses of voriconazole.  These data suggest that pathological 
changes to the visual pathways are unlikely.   
 
Analysis of the data from patient studies has shown cases of elevated alkaline 
phosphatase (ALP) and bilirubin, potentially indicative of cholestasis, which appear to be 
temporally related to onset of voriconazole therapy.  Occasionally, when these levels rose 
to greater than three times the upper limit of normal (ULN) therapy was discontinued; a 
total of 16 patients fulfilled this criterion.  One of these cases resulted in prolonged 
hospitalization and therefore was considered to be a serious adverse event. 
 
Also, a number of cases of elevated transaminases (alanine aminotransferase and 
aspartate aminotransferase, ALT and AST, respectively) have occurred.  When levels 
reached five times the ULN, therapy was discontinued in nine patients from two studies.  
Two of these cases were hospitalized for observation and therefore were considered 
serious events.  Elevations in liver function tests (LFTs) have only rarely occurred in 
studies of voriconazole in healthy volunteers. 
 
There have been a small number of skin reactions recorded with the total incidence of 
rash being 7%.  These reactions were largely localized to the sun exposed areas of the 
skin and may, therefore, indicate a photosensitizing potential with voriconazole.  Other 
azole antifungal agents have been reported to cause skin reactions.   
 
In addition to the visual disturbances, elevated liver function tests, and skin reactions 
discussed above, the following treatment-related adverse events have occurred at a rate of 
< 1% in human volunteers and patients that have received voriconazole to date: 
abdominal pain, headache, nausea, dizziness, asthenia, hypokalemia and injection site 
reactions.  
 
The following serious adverse events have been reported in patients receiving other azole 
antifungal agents, and could potentially occur in patients receiving voriconazole: cardiac 
dysrhythmias (reported in patients receiving itraconazole in combination with terfenadine 
or astemizole), and anaphylaxis (reported with fluconazole and ketoconazole). 
 


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Pediatric patients have received voriconazole in Phase II studies.  Patients received 
between 200-1000 mg per day and tolerated voriconazole without any attributable serious 
adverse events.   
 
Fluconazole:  Sixteen percent of over 4,000 patients treated with fluconazole in clinical 
trials of seven days or more experienced adverse events.  Discontinuation of therapy was 
required in 1.5% of patients due to adverse clinical events and in 1.3% of patients due to 
laboratory test abnormalities.  Treatment-related clinical adverse events occurring at an 
incidence of 1% or greater included nausea in 7.7%, headache in 1.9%, skin rash in 1.8%, 
vomiting in 1.7%, abdominal pain in 1.7%, and diarrhea in 1.5%.   
 
In both combined clinical trials and marketing experience there have been rare cases of 
serious hepatic reactions thought probably causally associated.  The spectrum of these 
reactions has ranged from mild transient elevations in transaminases to clinical hepatitis, 
colestasis, and fulminant hepatic failure including fatalities.  Generally, liver function 
returned to baseline on discontinuation of fluconazole.   
 
Other side effects noted with uncertain causal association include seizures, exfoliative 
skin disorders including Stevens Johnson syndrome and toxic epidermal necroliasis, 
alopecia, leukopenia, including neutropenia, thrombocytopenia, hypercholesteremia, 
hypertriglyceridemia, and hypercalcemia.   
 
In children, 13% of 577 patients experienced treatment-related adverse events.  These 
included vomiting in 5%, abdominal pain in 3%, nausea in 2% and diarrhea in 2%.  
Treatment was stopped in 2.3% of patients due to adverse reactions and in 1.4% of 
patients due to laboratory test abnormalities.  The majority of the laboratory 
abnormalities were elevations of transaminases or alkaline phosphatase.   
 
3. Formulation and Preparation 
a) Voriconazole  
Voriconazole for oral use is presented as 50 mg and 200 mg tablets.  Tablets will be 
overencapsulated by the BMT CTN Central Pharmacy to maintain the blind.  
Capsules must not be opened or altered.  Voriconazole for intravenous injection is 
presented in a 30 mL vial as a lyophilized powder for reconstitution equivalent to 200 
mg Voriconazole per vial.  The lyophile must be stored at room temperature (15o-
30oC).  After reconstitution with 19 mL of water for injection USP, each mL contains 
10 mg of voriconazole and 160 mg of SBECD.  The pH of the reconstituted solution 
ranges from 5.5 to 7.5.  Refer to Appendix F for reconstitution guidelines for IV 
administration.    
 
b) Fluconazole  
Fluconazole for oral use is presented as 100 mg and 200 mg tablets.  Tablets will be 
overencapsulated by the BMT CTN Central Pharmacy to maintain the blind.  
Capsules must not be opened or altered.  Oral fluconazole suspension is not available 
for this trial.  Fluconazole for injection is packaged in Viaflex Plus plastic containers 
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concentration of 2 mg/mL).  Refer to Appendix F for reconstitution guidelines for IV 
administration.  Intravenous fluconazole will be stored and prepared according to the 
manufacturer’s instructions provided in the package insert. 
 
c) Placebo 
The oral formulations of voriconazole and fluconazole differ in appearance.  
Accordingly, all oral medications will be masked by overencapsulation to prevent 
identification.  Patients receiving oral fluconazole will receive active masked drug in 
the morning and an identical appearing placebo in the evening.  Similarly, patients 
receiving IV fluconazole therapy will receive an equivalent volume of normal saline 
(weight-adjusted as per Appendix F) for the evening infusion. 
 
4. Stability and Storage 
The safe storage of study drug supplies will be arranged by the investigator until they are 
dispensed to the patient(s).  They will be stored in the hospital’s investigational pharmacy 
under the supervision of the investigational pharmacist. 
 
 
a) Voriconazole  
Voriconazole capsules should be stored at controlled room temperature between 15º-
30ºC (59º-86ºF).   
 
Vials containing unreconstituted lyophilized voriconazole should be stored at 
controlled room temperature between 15º-30oC (59º-86ºF).  Vials of reconstituted 
voriconazole solution must be stored between 2º-8ºC (37º-46ºF) for no more than 24 
hours.  They should not be frozen.  The reconstituted solution does not contain a 
preservative, and each vial is intended for single use only.  
 
 
b) Fluconazole 
Fluconazole capsules should be kept below 30ºC (86ºF).   
 
Fluconazole for injection is packaged in Viaflex® Plus plastic containers and should 
be stored between 5º-25ºC (41º-77ºF). 
 
2.4.6. 
Early (Premature) Withdrawal of Study Drug 
 
2.4.6.1. 
Patient early withdrawal from study treatment 
 
Patients will be prematurely withdrawn from study treatment prior to day 100 (or 180) if: 
1. There is evidence at any time during the study of a presumptive, probable or proven 
invasive fungal infection as defined by criteria (Section 3.1.1).  
2. Any of the following toxicities occur.  However, if another etiology is judged by the local 
PI to be the likely cause of the toxicity and an interval of no more than 14 days has 
lapsed, then the study drug can be resumed at original dose.  If the same toxicity recurs, 
the patient will be permanently withdrawn from the study treatment. 


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a) For hepatic toxicity:  If ALT exceeds ten times the upper limit of normal and is felt to 
be at least possibly related to study drug, the study drug should be held until the 
causality clarifies and until the toxicity resolves to a Grade II or less.  If ALT exceeds 
five times the upper limit of normal and is felt to be at least possibly related to study 
drug, the study drug may be held at the discretion of the treating physician until the 
causality clarifies and until the toxicity resolves to less than two and a half times the 
upper limit of normal. 
b) For visual toxicity:  Photopsia (visual disturbances) is an occasional side effect of 
voriconazole.  It typically is transient and is not associated with organic or enduring 
sequelae.  It does not represent grounds for withdrawal.  If a patient experiences loss 
of vision (blindness), it is unlikely that it is due to the study drug; however, the study 
drug should be held until etiology is established.  Cyclosporine and tacrolimus can 
cause loss of vision and should be held.  Retinal hemorrhage is also a potential cause 
of visual loss and platelet transfusions and optimization of coagulation parameters 
should be considered where appropriate.  Ophthalmic evaluation should be promptly 
carried out.  
c) For cutaneous toxicity:  A skin rash can occasionally occur.  This may be provoked or 
exacerbated by sun exposure.  Other potential causes of rash should be investigated, 
including graft-versus-host disease and drug sensitivity.  A skin biopsy may be useful 
in the evaluation of other causes.  This ordinarily will not constitute grounds for 
withdrawal unless there is skin necrosis or ulceration or generalized exfoliative 
dermatitis. 
d) For neurologic toxicity:  An infrequent observation in patients receiving voriconazole 
is hallucinations.  A preliminary analysis suggests this is due to potentiation of opiate 
or benzodiazepine effect.  If such should occur, attempts should be made to reduce 
opiate and/or benzodiazepine dosages.  If necessary after at least 24 hours after 
reduction of the dose(s) of concomitant opiates or benzodiazepines, study drug can be 
held (without withdrawal) up to 14 days as specified above to allow reduction of 
doses of these concomitant medications.   
e) For cardiac arrhythmia:  If a significant arrhythmia occurs, study medication must be 
held, and the subject must undergo further assessment by a cardiologist to evaluate 
the significance of the findings.  The cardiologist’s assessment and opinion on the 
significance of any abnormal finding will be summarized in a report and submitted 
with an Adverse Event Form.  If the PI and cardiologist conclude that it is not related 
to the study drug, study drug can be resumed as specified above.  Otherwise, the 
patient will be withdrawn from study treatment.   
f) For renal insufficiency:  The patient experiences serious renal impairment requiring 
hemodialysis and IV study drug. 
g) For any other Grade III or IV toxicity according to the NCI Common Terminology 
Criteria for Adverse Events (CTCAE) Version 3.0 which is not typically expected in 
the course of BMT and may be possibly related to study drug. 
3. Systemic amphotericin B (or one of the lipid formulations) or caspofungin is given for 
more than 14 consecutive days.   


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If a patient is prematurely withdrawn from study drug prior to Day 100 for reasons 
other than a presumptive, probable or proven fungal infection, fluconazole will be 
allowed for fungal prophylaxis until Day 100 with permission of the Medical 
Monitor or Protocol Chair.  No other antifungal agents are allowed. 
4. The patient requires terfenadine, astemizole, cisapride or sirolimus, maintenance 
phenytoin/anticonvulsant therapy, or any of the drugs prohibited at study entry (Section 
2.3.2).  
5. The patient is an outpatient and requires IV study drug for more than 14 consecutive days 
and cannot tolerate oral study drug.  IV study drug must be prepared by the 
investigational pharmacist at the transplant center (and not by home health care) in order 
to maintain the blind. 
6. The patient’s creatinine clearance declines to < 25 mL/min and persists for more than 14 
days and the patient requires IV study drug and cannot tolerate oral study drug. 
7. The patient has failed to engraft and requires chemotherapy. 
8. The patient has relapsed and requires chemotherapy. 
9. The patient becomes pregnant. 
10. The patient withdraws consent. 
 
The medical monitor or one of the protocol chairs should be contacted for advice about any of 
the above considerations for holding or withdrawal of the study drug due to adverse events. 
 
All early study drug withdrawals should be reported to Data Coordinating Center within three 
business days.  Even if withdrawal from study drug occurs for any of the above reasons, all study 
assessments should continue through the remainder of the one-year period. 
 
If a patient is prematurely withdrawn from study drug prior to Day 100 (or 180) for reasons other 
than a probable or proven invasive fungal infection, only fluconazole will be allowed as 
prophylaxis until Day 100 (or 180) with permission of the Medical Monitor or Protocol Chair.  
No other antifungals are allowed for prophylaxis. 
 
Refer to NCI’s Common Terminology Criteria for Adverse Events (CTCAE) as a guideline for 
toxicity assessment.  The CTCAE Document Version 3.0 can be accessed at the following web 
site: http://ctep.cancer.gov/reporting/ctc.html. 
 
2.4.6.2. 
Breaking the blind 
 
Study drug assignment may be revealed only for reasons relating to the patient’s safety or when 
critical therapeutic decisions are contingent upon knowing the assigned study drug.  A decision 
to break the blind must be discussed with the medical monitor or one of the protocol chairs in 
advance.  Withdrawal of a patient from the study treatment is not a sufficient reason to break the 
study blind.  Suggestions for antifungal therapy for patients withdrawn due to presumptive, 
probable or proven infection are provided in Section 2.4.9. 
 


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2.4.7. 
Contraindications, Drug Interaction and Monitoring 
 
2.4.7.1 
Voriconazole and fluconazole 
 
Voriconazole is metabolized by three separate CYP 450 isoenzymes, including CYP34A, 
CYP2C9 and CYP2C19.  Fluconazole is also metabolized by the cytochrome P450 34A enzyme 
system and may interact with several medications.  Clinically and potentially significant drug 
interactions that have been observed with fluconazole, as well as voricoazole (i.e., both azoles), 
include the following: 
 
Co-administration of terfenadine, cisapride, astemizole, quinidine, pimozide and dofetilide 
with voriconazole and fluconazole is contraindicated.  Serious cardiovascular adverse events, 
including death, ventricular tachycardia, torsades de pointes and prolonged QTc have been 
observed in patients taking other azoles concomitantly with these drugs.  Voriconazole causes 
increased concentrations of terfenadine, cisapride and astemizole.  
 
Other medications with documented drug interactions with fluconazole and voriconazole whose 
serum concentrations should be closely monitored, or the end effect closely monitored (e.g., 
blood sugar levels with sulfonylureas), include phenytoin, phenobarbital, digoxin, omeprazole, 
warfarin and sulfonylureas (can increase plasma levels which may lead to hypoglycemia) [46-
51]. 
 
1. Oral Hypoglycemics: Clinically significant hypoglycemia may be precipitated by the use 
of fluconazole or voriconazole with oral hypoglycemic agents.  One fatality has been 
reported from hypoglycemia in association with combined fluconazole and glyburide use.  
Fluconazole reduces the metabolism of tolbutamide, glyburide and glipizide, and 
increases the plasma concentration of these agents.  Blood glucose concentrations should 
be carefully monitored and the dose of the sulfonylurea should be adjusted as necessary.  
2. Coumadin-Type Anticoagulants: Prothrombin time may be increased with concomitant 
fluconazole or voriconazole and coumadin-type anticoagulants.  Careful monitoring of 
prothrombin time is recommended. 
3. Phenytoin: Fluconazole or voriconazole increase the plasma concentrations of phenytoin.  
Careful monitoring of phenytoin concentrations is recommended.  
4. Cyclosporine: Fluconazole or voriconazole may significantly increase cyclosporine 
levels in renal transplant patients with or without renal impairment.  Careful monitoring 
of cyclosporine concentrations and serum creatinine is recommended [52].  
5. Rifampin: Rifampin enhances the metabolism of fluconazole.  Depending on clinical 
circumstances, consideration should be given to increasing the dose of fluconazole if 
administered with rifampin.  
6. Theophylline: Fluconazole (and probably voriconazole) increases the 
serum 
concentrations of theophylline.  Careful monitoring of serum theophylline concentrations 
is recommended.  


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7. Rifabutin: There have been reports of uveitis in patients to whom fluconazole and 
rifabutin were co-administered.  Patients should be carefully monitored.  The 
concomitant administration of voriconazole and rifabutin is contraindicated (see 
inclusion criteria). 
8. Tacrolimus: There have been reports of nephrotoxicity in patients in whom fluconazole 
or voriconazole and tacrolimus were co-administered.  Patients should be carefully 
monitored. 
 
Grapefruit juice should be avoided since it is known to interfere with the cytochrome P450 
system.  Certain herbal medicines (e.g., St. John’s Wort) should be avoided for similar reasons. 
 
2.4.7.2 
Voriconazole drug interactions 
 
Co-administration of voriconazole and immunosuppressive agents metabolized by cytochrome 
P450 3A4 may result in alterations in drug metabolism as follows: 
 
1. Cyclosporine: In stable transplant recipients, voriconazole increased cyclosporine Cmax 
and AUC (area under the blood concentration time curve to the last quantifiable 
measurement) by at least 13% and 170%, respectively.  Trough cyclosporine 
concentrations increased by an average of 2.5 fold [52].  These observations have led to 
the recommendation that when initiating voriconazole in patients already receiving 
cyclosporine, the cyclosporine dose be halved and blood cyclosporine levels carefully 
monitored [59].  When voriconazole is discontinued, blood cyclosporine levels must be 
carefully monitored and the dose increased as necessary. 
2. Tacrolimus: In healthy volunteers, voriconazole increased tacrolimus (0.1 mg/kg single 
dose) Cmax and AUC by 117% and 221%, respectively.  These observations have led to 
the recommendation that when initiating voriconazole in patients already receiving 
tacrolimus, the tacrolimus dose should be reduced to at least one third of the original dose 
and blood tacrolimus levels carefully monitored.  When voriconazole is discontinued, 
blood tacrolimus levels must be carefully monitored and the dose increased as necessary. 
3. Sirolimus: In healthy volunteers, voriconazole increased sirolimus Cmax and AUC by 
6.6 fold and 11.1 fold, respectively.  Voriconazole should not be co-administered with 
sirolimus.  
4. Benzodiazepines:  In vitro studies demonstrate the potential for voriconazole to inhibit 
the metabolism of benzodiazepines (and thereby increasing plasma exposure).  Frequent 
monitoring for adverse events and toxicity (especially prolonged sedation) related to 
benzodiazepines metabolized by cytochrome P450 3A4 (e.g., midazolam, triazolam, 
alprazolam) is recommended.  Adjustment of benzodiazepine dosage may be needed. 
5. Statins:  In vitro studies demonstrate the potential for voriconazole to inhibit metabolism 
(thereby increasing plasma exposure).  Frequent monitoring for adverse events and 
toxicity related to statins is advised.  Increased statin concentrations in plasma have been 
associated with rhabdomyolysis.  Adjustment of the statin dosage may be needed.   
6. Calcium channel blockers:  Voriconazole may increase the serum concentrations of this 
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events and toxicity related to calcium channel blockers is advised.  Adjustment of 
calcium channel blocker dosage may be required. 
 
2.4.8. 
Duration of Study Drug 
 
Study drug will be continued without interruption through Day 100 unless the patient has met the 
early drug withdrawal criteria or has met the criterion for a presumptive, probable or proven 
fungal infection.  In three circumstances, study drug may be continued beyond Day 100 and 
should be given in those instances until Day 180.  An assessment between Day 90 and 100 
should be made to determine if either of the following three conditions are met: 
 Any patient who is receiving at least 1.0 mg/kg/d of prednisone or other steroid 
equivalent (based on actual or ideal body weight) during that assessment should continue 
study drug until Day 180. 
 Recipients of T cell depleted transplants receiving post-transplant immunosuppression 
between Day 90 and 100 should continue to receive study drug until Day 180.   
 Recipients of T cell depleted transplants with CD4+ counts < 200/uL should continue 
study drug until Day 180. 
 
2.4.9. 
Recommendations for Treatment of Documented Fungal Infections 
 
In the event of the patient demonstrating a presumptive, probable or proven invasive fungal 
infection as per the criteria of Section 3.1, the patient has reached the primary study endpoint, 
should stop study drug and the clinician is free to treat the infections according to local 
institutional guidelines.  The following are offered as suggestions. 
 
One option is to use amphotericin B and voriconazole together.  This would provide adequate 
therapy for most fungal pathogens irrespective of which study drug the patient was assigned to 
and this strategy would ordinarily not require breaking the blind.  Voriconazole is to be given at 
a dose of 6 mg/kg/dose IV twice daily for two days, then at a dose of 4 mg/kg/d thereafter.  One 
can switch to the equivalent dose orally once the patient is stable and taking oral drugs 
satisfactorily.  The dose of amphotericin B is 1.0 mg/kg/d if the conventional formulation is used 
or is 4.0-5.0 mg/kg/d if a lipid formulation is used.  If the pathogen is mucormycoses, an 
amphotericin B formulation is the therapy of choice; voriconazole is not active against this 
pathogen and is not needed. 
 
For patients with proven mucormycoses or probable fungal infection with filamentous fungal 
forms on cytologic or histologic section, which cannot exclude mucormycetes, an amphotericin 
B formulation (or other investigational therapy) is recommended.  Voriconazole is not active 
against mucormycosis. 
 


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2.4.10. Guidelines for Empirical Antifungal Therapy with Amphotericin B or Caspofungin 
Usage for Patients not “Off Study” 
 
Limited empirical antifungal therapy may be given using amphotericin B (or one of the lipid 
formulations of amphotericin B) or caspofungin.  No other systemic antifungal is permitted 
without approval of the medical monitor.  Empirical antifungal therapy is intended for patients 
strongly suspected to have invasive fungal infections where exhaustive diagnostic evaluation has 
excluded other etiologies but has also failed to demonstrate a presumptive, probable or proven 
invasive fungal infection as defined in Table 3.1.1a and b.  It is generally intended for patients in 
whom the criteria for possible invasive fungal infection in Tables 3.1.1a and b are met.  The 
guidelines for empirical therapy for the most common situations are below.  In no case should it 
be continued beyond 14 consecutive days.  During an empirical trial of antifungal therapy, 
efforts should continue to be undertaken to ascertain the etiology of the fever through cultures 
and biopsies of pertinent tissue specimens. 
 
2.4.10.1. 
Required tests prior to use of empirical antifungal therapy 
 
All focal lesions suspicious for fungi must be biopsied if clinically possible.  Cultures of urine 
and blood must be obtained.  Isolator culture methods are preferred if available in your 
institution.  At least two blood samples for the diagnostic galactomannan assay should have been 
obtained during the seven days (with at least one within 48 hours) before institution of empirical 
antifungal therapy and should continue to be drawn along with blood samples for investigational 
fungal diagnostic assays during the empirical therapy.  In addition, one Pharmokinetics (PK) 
blood sample should be obtained prior to the start of empirical antifungal therapy. 
 
2.4.10.2. 
Pre-engraftment requirements and amphotericin B/caspofungin dose 
 
If the patient is neutropenic, has persistent fever (≥  38°C) of unknown origin refractory to at 
least 96 hours of broad-spectrum antibiotics or fever that recurs after 96 hours of antibiotics, and 
a specific etiology cannot be established, an empirical trial of either amphotericin B at a dose of  
1.0 mg/kg/d (or one of the lipid formulations at a dose of 4-5 mg/kg/d) or caspofungin (in 
accordance with manufacturer prescribing guidelines) is permitted.  Generally, the loading dose 
for caspofungin is 70 mg on day one and 50 mg/day on subsequent days for patients over 12 
years of age.  This may be continued until resolution of neutropenia (ANC > 500) but should not 
be continued beyond 72 hours after resolution of neutropenia and not longer than 14 consecutive 
days. 
 
2.4.10.3. 
Post-engraftment requirements and amphotericin B/caspofungin dose 
 
If the patient is post-engraftment and meets the criteria for possible infection in Table 3.1.1b and 
attempts to document etiology have failed, an empirical trial of an amphotericin B formulation or 
caspofungin is permitted.  Continued radiographic procedures, cultures, biopsies, and other 
pertinent diagnostic evaluations should be performed in an effort to elucidate the suspected 
infection.  If these measures fail, then the empirical trial should be terminated after no more than 
14 days.  If the physician feels that additional therapy is warranted this should be discussed with 
the medical monitor or one of the protocol chairs.  The dose of amphotericin B or one of the lipid 


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formulations of amphotericin B should be 1.0 mg/kg/d (for conventional amphotericin B) or 4-5 
mg/kg/d for the lipid formulations.  The dose of caspofungin should be in accordance with 
manufacturing guidelines, generally, 70 mg on the first day followed by 50 mg/day on 
subsequent days for patients over 12 years of age. 
 
2.4.10.4. 
Continuation of study drug during empirical antifungal therapy  
 
There are no data to suggest antagonism of voriconazole or fluconazole with amphotericin B or 
caspofungin.  So as to avoid fluctuations in the drug levels of concomitant immunosuppressive 
agents, it is recommended that the study drug be continued during empirical therapy.   
 
If the clinician does not discontinue empirical amphotericin B or caspofungin within the 14 days, 
then the study drug will be withdrawn.  The patient will continue to be monitored for all study 
assessments for the remainder of the study duration (one year) and continue to have 
galactomannan assays performed according to the study schedule. 
 
2.4.11. Other Antifungal Therapy 
 
Topical antifungal therapy is permitted and should be documented on the appropriate case report 
form.  Nebulized or inhaled amphotericin B is not permitted.  Systemic amphotericin B (or one 
of its lipid preparations) and caspofungin are the only systemic antifungal agents permitted on 
study but are only permitted as specified as empirical therapy for possible infection (or for 
presumptive, probable or proven invasive infection) as specified above in Section 2.4.10.   
 
If a patient is prematurely withdrawn from study drug prior to Day 100 (or 180) for reasons other 
than a probable or proven invasive fungal infection, only fluconazole will be allowed as 
prophylaxis until Day 100 (or 180) with permission of the Medical Monitor or Protocol Chair.  
No other antifungals are allowed for prophylaxis. 
 
Fungal prophylaxis must not be given after protocol-specified treatment plan is completed on 
Day 100 (or 180). 
 
If a patient develops a presumptive, proven or probable fungal infection as specified in Table 
3.1.1a and b, the clinician can employ other antifungal therapy as appropriate. 
 
2.4.12. Use of the Galactomannan Assay 
 
The galactomannan assay is used with host and clinical factors to determine the likelihood of 
invasive aspergillus infection.  The galactomannan assay detects only aspergillus and 
penicillium.  The sensitivity and specificity of the galactomannan assay are reported as 90% and 
98% respectively [32].  The positive and negative predictive values are 88% and 98% 
respectively [32].  Many invasive fungi other than aspergillus can cause invasive infection 
without galactomannan positivity.  Thus, a negative test does not exclude the possibility of 
infection by fungi. 
 


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A positive galactomannan assay satisfies only the microbiologic criterion for invasive fungal 
infection as defined in Tables 3.1.1a and b.  In the event that a positive result is obtained, 
additional radiographic, cultural, and other diagnostic tests should be performed, and a repeat 
specimen for the galactomannan assay should be sent.  The study drug should be continued while 
these investigations are underway. 
 
There have been reports of false positive results using the galactomannan assay in patients 
receiving concomitant piperacillin-tazobactem (Zosyn).  In addition, certain lots of Zosyn have 
tested positive.  However, piperacillin alone does not test positive.  As a result, positive 
galactomannan results in patients receiving piperacillin/tazobactam (Zosyn) should NOT be 
considered a microbiological criterion to support documentation of an invasive fungal infection. 
Other diagnostic methods should be pursued.  Subsequent positive galactomannan results should 
not be considered as microbiological criteria until the patient has been off of Zosyn for 5 days. 
 
One other secondary endpoint to be evaluated is the utility of the galactomannan assay to 
determine response to therapy.  Accordingly, if the patient develops a presumptive, probable or 
proven invasive fungal infection, twice weekly monitoring of treatment galactomannan samples 
should be collected at onset and after initiation of treatment for the infection for four weeks, then 
once every two weeks for eight weeks for a total of 12 samples.  These samples will be frozen 
and batch shipped to the Repository.  The assays will be performed after completion of study 
enrollment at Dr. Kieren Marr’s laboratory at FHCRC.  The investigators will be blinded to the 
results. 
 
For the galactomannan assays, all specimens are to be collected, processed, and transported in 
accordance with the procedures in Appendices C and D. 
 
2.4.13. Supportive Care 
 
Blood products must be irradiated. 
 
Antibiotics and intravenous immunoglobulin should be given according to institutional practice. 
 
Growth factors should be given according to institutional practice. 
 
Granulocyte transfusions are not permitted as part of routine care. 
 
2.4.13.1. 
Prophylaxis against infections 
 
Patient should be given prophylaxis for: 
1. Pneumocystis carinii: According to institutional practice. 
2. Herpes viruses (HSV, VZV): According to institutional practice until neutrophil 
engraftment for HSV+ recipients. 
3. Encapsulated bacteria: According to institutional practice. 
 


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The Manual of Procedures for Infection should be followed for patients with cytomegalovirus 
(CMV) infections.  In general, the guidelines for CMV prevention jointly developed by the 
CDC/IDSA/ASBMT should be used.  These can be accessed from the CDC website: 
http://www.cdc.gov/mmwr/PDF/RR/RR4910.pdf. 
 
2.4.13.2. 
Identification of opportunistic infections 
 
In the event that a patient develops fever, sinusitis, interstitial pneumonia, diarrhea, or hepatitis, 
all efforts will be made to identify the responsible organism.  Cultures will include routine 
bacterial, mycobacterial, fungal, and viral cultures.  Bronchial lavages and open lung biopsies 
will also be evaluated for pneumocystis carinii.  If possible, these samples will also be evaluated 
for RSV, other respiratory viral pathogens, and legionella.  Stool samples will also be evaluated 
for C. difficile toxin, cryptosporidium, and rotavirus.  Samples will not be routinely sent for EM 
studies.  If a GI biopsy is performed, evaluation for CMV with immunofluorescence and PCR 
should be considered.  
 
When a fungal infection is suspected, radiographic studies and invasive diagnostic procedures 
will be obtained as deemed necessary by the investigator.  CT scans of the chest are 
recommended in persistently febrile patients.  CT scans of the sinuses are recommended in 
patients with symptoms of sinusitis.  Biopsies should be obtained whenever possible of tissue 
from sites suspected to be infected.  If biopsy is not feasible, BAL or a percutaneous needle 
biopsy should be performed for evaluation of pulmonary infiltrates.  Assays for bacteria, CMV, 
fungi, PCP and respiratory viruses should be performed.  If visual symptoms occur, an 
ophthalmologic consult should be obtained if signs or symptoms are persistent.  Blood and urine 
fungal cultures should be obtained in all febrile patients and fungal cultures and stains obtained 
of all specimens of suspected infection. 
 
For non-fungal infections, a Post-Transplant Infection Report Form should be completed for 
each infectious episode with a severity grade assigned for each known agent contributing to the 
episode.  A Fungal Infection Report Form incorporating the fungal infection-specific definitions 
will be used for fungal infections. 
 


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2.4.14. Diagnosis of GVHD 
 
2.4.14.1. 
Acute GVHD 
 
Acute GVHD generally develops within the first three months after transplantation and appears 
as a characteristic dermatitis often accompanied by hepatic cholestasis and enteritis.  The clinical 
appearance of skin GVHD can be mimicked by toxicity of the transplant conditioning regimen 
and by drug reactions.  Therefore, documentation of the diagnosis by skin biopsy is 
recommended.  Severity of liver GVHD is usually described according to the serum bilirubin 
level.  Hepatic GVHD cannot be assessed solely on clinical grounds in patients who have 
concurrent drug toxicity, viral hepatitis, or toxicity caused by the pre-transplant chemotherapy 
and irradiation.  Liver biopsy can be helpful but often cannot be done because of clinical 
contraindications such as thrombocytopenia.  Gastrointestinal GVHD is characterized by watery 
diarrhea with anorexia, nausea and vomiting accompanied in more severe cases by abdominal 
cramps, gastrointestinal hemorrhage, and ileus.  Eating often exacerbates symptoms.  The 
volume of diarrhea has been used as an indicator of the severity of gut GVHD, but this can be 
inaccurate and highly variable from day to day.  In many cases, it can be difficult to distinguish 
GVHD from infectious enteritis, and endoscopic biopsy is often helpful and should be done 
wherever possible.  Prospective data related to symptoms of acute GVHD will be collected.  
 
2.4.14.2. 
Chronic GVHD 
 
Manifestations of chronic GVHD typically do not occur until three to twelve months after 
transplantation.  Initial symptoms frequently include nausea, anorexia and weight loss, ocular 
and oral sicca, and skin changes.  Rash characteristically appears with pigmentary changes, 
vitiligo, mottling, erythema, plaques, papules, nodules, poikiloderma, or exfoliation progressing 
to sclerosis and contractures.  Hair loss and onychodystrophy may also indicate chronic GVHD.  
Cough and dyspnea with wheezing, rales, and abnormal PFTS may indicate pulmonary 
involvement.  Diarrhea and abdominal pain may occur but are relatively infrequent.  Liver 
involvement may be indicated by increased bilirubin and alkaline phosphatase and less 
frequently by increased transaminase levels. 
 
2.4.15. Risks and Toxicities 
 
Recipients of allogeneic blood or marrow transplants incur risks from pre-transplant conditioning 
and post-transplant therapy, which must be weighed against the risk of the disease for which the 
transplant is prescribed.  Major risks following transplantation include: 1) Infection which can be 
of a fungal, bacterial, viral or parasitic nature.  Often, these infections are life-threatening, 
particularly when caused by viral or fungal agents, and are associated with a high mortality rate 
in the transplant population; 2) GVHD, either acute or chronic in nature, may occur following 
allogeneic blood or marrow transplants.  The degree of GVHD varies from mild cutaneous 
reactions to extensive widespread and systemic involvement of skin, liver, and gastrointestinal 
tract.  Probably due to a direct association, the incidence of fatal infection is greater in patients 
developing GVHD; 3) Graft Failure can occur and is associated with a high risk of mortality; 4) 
End Organ Damage of all or any of the major organs may occur as a result of reactions to drugs 
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infection, GVHD, etc.) or radiotherapy, and may have a fatal outcome; 5) for patients 
transplanted for malignant disorders, Relapse of the underlying disease may occur, especially in 
patients with advanced disease status at time of transplant; 6) Unknown Toxicities may occur in 
any individual patient due to multiple events and cumulative effects which may involve any and 
all organs, including the brain.  Brain damage can result in severe loss of cognitive or neurologic 
function; and 7) Death. 
 
Damage to major body organs may include the brain, eyes, heart, lung, liver, and kidneys.  
Possible late effects may include growth retardation deformities, cataracts, changes in endocrine 
function, sterility, learning disabilities or brain damage, bone and joint abnormalities, and 
secondary malignancy. 
 
For specific study drug risks/interactions refer to Sections 2.4.5 and 2.4.7.   
 
For toxicity assessment, refer to NCI’s Common Terminology Criteria for Adverse Events 
(CTCAE) as a guideline for toxicity assessment.  The CTCAE Document Version 3.0 can be 
accessed at the web site:  http://ctep.cancer.gov/reporting/ctc.html. 
 
2.4.16. Genetic Variation Study Sampling: Single Nucleotide Polymorphisms from Donor 
 
Subtle differences in different molecules of innate immunity may contribute to either the 
predisposition or clinical course of infection with filamentous fungi.  The allelic frequencies of 
24 different genes [MBL2 (mannose-binding lectin 2), CCR5 (chemokine receptor 5), IL1RN 
(interleukin-1 receptor antagonist), IL1A (interleukin-1 alpha), IL1B (interleukin-1 beta), IL6 
(interleukin-6), IL8 (interleukin-8), IL8RA (interleukin-8 receptor type A), IL8RB (interleukin-8 
receptor type B), IL10 (interleukin-10), TNFA (tumor necrosis factor alpha), TNFB (tumor 
necrosis factor beta), MPO (myeloperoxidase), NRAMP1 (natural resistance-associated 
macrophage protein 1), CHIT1 (chitotriosidase 1), FCGR2A (Fc-gamma receptor 2a), FCGR3A 
(Fc-gamma receptor 3a), FCGR3B (Fc-gamma receptor 3b), MICA (MHC, major 
histocompatibility complex, class I chain-related gene A), MICB (MHC class I chain-related 
gene B), TLR4 (toll-like receptor 4), CD14, HBD-1 (beta defensin), IL-18 (interleukin 18) and 
their intragenic polymorphic forms will be analyzed and this data will be compared to the 
incidence and severity of fungal infection.  The purpose of this study is to identify a group of 
molecules of innate immunity, which may influence the risk and severity of invasive fungal 
infection.  Single Nucleotide Polymorphisms (SNPs) of the genes encoding the following two 
cytochrome P450 proteins (3A4 and 2C19) will also be studied for a total of 26 genes.  


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CHAPTER 3 
 
 
3. 
STUDY ENDPOINTS 
 
3.1. Primary Endpoint 
 
The primary endpoint for this study of prevention of invasive fungal infections in allogeneic 
blood and marrow transplants is fungal-free survival (proportion of patients alive and free from 
proven, probable or presumptive invasive fungal infection) at 180 days post-transplant. 
 
Fungal infections graded as colonization, superficial infection or possible invasive infection 
(defined below) will not be counted as events for this endpoint.  A fungal infection will be 
defined in accordance with the following consensus EORTC/MSG1 criteria [53]: 
 
Colonization: Defined as fungus or mould present in surveillance cultures in the absence of 
clinical findings of fungal infection.  
 
Superficial Infection: Includes cutaneous fungal infections, oropharyngeal candidiasis and 
vulvovaginal candidiasis defined as: 
 
1. Typical signs of mucocutaneous fungal infection  
2. Positive fungal culture of scrapings, swab or exudate   
3. Hyphae or pseudohyphae noted on Gram or other stain, or biopsy demonstrating invasive 
fungal elements  
4. Patients who develop superficial infections will be treated with topical antifungals and 
continued on study medication 
 
Invasive Infection:  Defined as per Table 3.1.1a and 3.1.1b. 
                                                   
1 European Organization for Research and Treatment of Cancer-Invasive Fungal Infection Cooperative 
Group/National Institutes of Allergy and Infectious Dieases-Mycoses Study Group. 


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Table 3.1.1a 
Definitions of Invasive Fungal Infections in Patients with Cancer and Recipients of 
Hematopoietic Stem Cell Transplants 
 
Category, Type of Infection 
Description 
Proven invasive fungal infections 
 
Deep tissue infections 
 
Mouldsa 
Histopathologic or cytopathologic examination showing hyphae from needle 
aspiration or biopsy specimen with evidence of associated tissue damage 
(either microscopically or unequivocally by imaging); or positive culture 
result for a sample obtained by sterile procedure from normally sterile and 
clinically or radiologically abnormal site consistent with infection, 
excluding urine and mucous membranes 
Yeastsa 
Histopathologic or cytopathologic examination showing yeast cells (Candida 
species may also show pseudohyphae or true hyphae) from specimens of 
needle aspiration or biopsy excluding mucous membranes; or positive 
culture result on sample obtained by sterile procedure from normally sterile 
and clinically or radiologically abnormal site consistent with infection, 
excluding urine, sinuses, and mucous membranes; or microscopy (India ink, 
mucicarmine stain) or antigen positivityb for Cryptococcus species in CSF 
Fungemia 
 
Mouldsa 
Blood culture that yields fungi, excluding aspergillus species and Penicillium 
species other than Penicillium marneffei, accompanied by temporally 
related clinical signs and symptoms compatible with relevant organism 
Yeastsa 
Blood culture that yields Candida species and other yeasts in patients with 
temporally related clinical signs and symptoms compatible with relevant 
organism 
Endemic fungal infectionsc 
 
Systemic or confined to lungs 
Must be proven by culture from site affected, in host with symptoms 
attributed to fungal infection; if culture results are negative or unattainable, 
histopathologic or direct microscopic demonstration of appropriate 
morphological forms is considered adequate for dimorphic fungi 
(Blastomyces, Coccidioides and Paracoccidiodes species) having truly 
distinctive appearance; Histoplasma capsulatum variant capsulatum may 
resemble Candida glabrata 
Disseminated 
May be established by positive blood culture result or positive for urine or 
serum antigen by means of RIA [17] 
Probable invasive fungal infections 
At least 1 host factor criterion (see Table 3.1.1b); and 1 microbiological 
criterion; and either a) 1 major (or 2 minor) clinical criteria from abnormal 
site consistent with infection for sites other than lower respiratory tract, or 
b) one of the clinical criteria for lower respiratory tract site 


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Category, Type of Infection 
Description 
Presumptive invasive fungal infection 
 
At least 1 host factor criterion (see Table 3.1.1b) and 1 clinical criterion 
for lower respiratory tract infection as listed below.  A microbiological 
criterion is NOT required.  The clinical criterion for lower respiratory 
tract infection must be consistent with the microbiological findings, if 
any, temporally related to current episode and other potential causes 
must have been eliminated, along with: 
­ The presence of one of the following “specific” imaging signs on 
CT: (1) Halo sign, (2) wedge-shaped infiltrate, (3) air crescent 
sign, OR 
­ The presence of a new non-specific focal infiltrate, PLUS at least 
one of the following: pleural rub, pleural pain, or haemoptysis, 
AND no evidence of other etiology demonstrated by 
bronchoscopic examination 
 
Or if lacking a host criterion, but otherwise meets the microbiological 
and clinical criteria for a presumptive, probable or proven invasive 
fungal infection.  The clinical criterion for lower respiratory tract 
infection must be consistent with the microbiological findings, if any, 
temporally related to current episode and other potential causes must 
have been eliminated, along with: 
­ The presence of one of the following “specific” imaging signs on 
CT: (1) well-defined nodule with or without a halo sign, (2) halo 
sign, (3) wedge-shaped infiltrate, (4) air crescent sign, (5) cavity 
within area of consolidation OR 
­ The presence of a new non-specific focal infiltrate, PLUS at least 
one of the following: pleural rub, pleural pain, or haemoptysis 
AND no evidence of other etiology demonstrated by 
bronchoscopic examination 
 
 


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Table 3.1.1a cont’d 
 
Category, Type of Infection 
Description 
Possibled invasive fungal infections 
At least 1 host factor criterion; and 1 microbiological criterion; or either a) 1 
major (or 2 minor) clinical criteria from abnormal site consistent with 
infection for sites other than lower respiratory tract, or b) one of the clinical 
criteria for lower respiratory tract site  
 
Notes for Table 3.1.1a 
a  Append identification at genus or species level from culture, if available. 
b  False-positive cryptococcal antigen reactions due to infection with Trichosporan beigelli (1), infection with Stomatococcus 
mucilaginosis (2), circulating rheumatoid factor (3), and concomitant malignancy (4) may occur and should be eliminated if 
positive antigen test is only positive result in this category. 
c  Histoplasmosis, blastomycosis, coccidioidomycosis, and paracoccidioidomycosis. 
d  This category is not recommended for use in clinical trials of antifungal agents but might be considered for studies of 
empirical treatment, epidemiological studies, and studies of health economics 
 
 
 
Table 3.1.1b 
Host Factor, Microbiological, and Clinical Criteria for Invasive Fungal Infections in 
Patients with Cancer and Recipients of Hematopoietic Stem Cell Transplants 
 
Type of Criteria 
Criteria 
Host factors 
Neutropenia (< 500 neutrophils/mm3 for > 10 days) 
Persistent fever (≥ 38°C) for > 96 h refractory to appropriate broad-
spectrum antibacterial treatment in high-risk patients 
Body temperature either > 38oC or < 36oC and any of the following 
predisposing conditions: prolonged neutropenia (> 10 days) in previous 
60 days, recent or current use of significant immunosuppressive agents 
in previous 30 days, proven or probable invasive fungal infection during 
previous episode of neutropenia, or coexistence of symptomatic AIDS 
Signs and symptoms indicating graft-versus-host disease, particularly 
severe (Grade > 2) or extensive chronic disease 
Prolonged (> 3 weeks) use of corticosteroids in previous 60 days 
Microbiological 
Positive result of culture for mould (including aspergillus, Fusaruim, or 
Scedosporium species or Zygomycetes) or Cryptococcus neoformans or 
an endemic fungal pathogena from sputum or bronchoalveolar lavage 
fluid samples 
Positive result of culture or findings of cytologic/direct microscopic 
evaluation for mould from sinus aspirate specimen 
Positive findings of cytologic/direct microscopic evaluation for mould or 
Cryptococcus species from sputum or bronchoalveolar lavage fluid 
samples 
Positive result for aspergillus antigen in blood samples as defined in 
Section 3.2 
Positive result for aspergillus antigen in specimens of bronchoalveolar 
lavage fluid or CSF 
Positive result for cryptococcal antigen in blood sampleb 
Positive findings of cytologic or direct microscopic examination for fungal 
elements in sterile body fluid samples (e.g., Cryptococcus species in 
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Table 3.1.1b cont’d 
 
Type of Criteria 
Criteria 
Microbiological (cont’d) 
Positive result for Histoplasma capsulatum antigen in blood, urine, or CSF 
specimens [17] 
Two positive results of culture of urine samples for yeasts in absence of 
urinary catheter 
Candida casts in urine in absence of urinary catheter 
Positive result of blood culture for Candida species 
Clinical 
Must be related to site of microbiological criteria and temporally related to 
current episode 
Lower respiratory tract infection 
 
The presence of one of the following “specific” imaging signs on 
CT: (1) well defined nodule(s) of at least 1 cm in diameter with 
or without a halo sign, (2) wedge-shaped infiltrate, (3) air 
crescent sign, or (4) cavity within area of consolidationc, OR 
 
The presence of a new non-specific focal infiltrate, PLUS at least 
one of the following: pleural rub, pleural pain or hemoptysis 
Sinonasal infection 
 
Major 
Suggestive radiological evidence of invasive infection in sinuses (i.e., 
erosion of sinus walls or extension of infection to neighboring structures, 
extensive skull base destruction) 
Minor 
Upper respiratory symptoms (e.g., nasal discharge, stuffiness); nose 
ulceration or eschar of nasal mucosa or epistaxis; periorbital swelling; 
maxillary tenderness; black necrotic lesions or perforation of hard palate 
CNS infection 
 
Major 
Radiological evidence suggesting CNS infection (e.g., mastoiditis or other 
parameningeal foci, extradural empyema, intraparenchymal brain or 
spinal cord mass lesion) 
Minor 
Focal neurological symptoms and signs (including focal seizures, 
hemiparesis, and cranial nerve palsies); mental changes; meningeal 
irritation findings; abnormalities in CSF biochemistry and cell count 
(provided that CSF is negative for other pathogens by culture or 
microscopy and negative for malignant cells) 
Disseminated fungal infection 
Papular or nodular skin lesions without any other explanation; intraocular 
findings suggestive of hematogenous fungal chorioretinitis or 
endophthalmitis 
Chronic disseminated candidiasis 
Small, peripheral, target-like abscesses (bull’s-eye lesions) in liver and/or 
spleen demonstrated by CT, MRI, or ultrasound, as well as elevated 
serum alkaline phosphatase level; supporting microbiological criteria are 
not required for probable category 
Candidemia 
Clinical criteria are not required for probable candidemia; there is no 
definition for possible candidemia 
Notes for Table 3.1.1.b 
a  H. capsulatum variant capsulatum, Blastomyces dermatitidis, Coccidioides immitis, or Paracoccidioides brasiliensis. 
b  See Table 3.1.1a footnote b for causes of false-positive reactions that must be considered and eliminated from consideration. 
c In absence of infection by organisms that may lead to similar radiological findings including cavitation, such as 
Mycobacterium, Legionella, and Nocardia species. 
 
 


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3.2. Secondary Endpoints 
 
The secondary endpoints are: 
1. Frequency of invasive fungal infections 
2. Time to invasive fungal infection 
3. Six-month and one-year patient survival 
4. Empirical use of amphotericin B or caspofungin, frequency and duration of use 
5. Time to and severity of acute and chronic graft-versus-host disease (GVHD) 
6. Utility of galactomannan assay in diagnosis of aspergillus and response to therapy 
7. Time to neutrophil engraftment 
8. Time to platelet engraftment 
9. Freedom from possible, presumptive, probable, or proven invasive fungal infection, 
death, or withdrawal of study drug due to toxicity, intolerance, or an empirical trial of 
amphotericin B or caspofungin > 14 consecutive days. 
 
Other complications that are ordinarily seen during the course of BMT will be captured in BMT 
CTN and IBMTR follow-up forms.  Planned data to be collected, tabulated and analyses to be 
performed include: association of CD4 counts with infection risk, rates of hepatotoxicity and 
hepatic GVHD in the two cohorts, drug levels on Day 14 and 28 as they relate to subsequent 
infection and toxicities, in vitro fungal susceptibility and occurrence of breakthrough infection 
and response to therapy, and rates of possible fungal infections, fever of uncertain etiology, and 
pulmonary infiltrates of uncertain etiology.  Galactomannan levels will be collected, tabulated 
and analyses of these data with various endpoints will be performed. 
 
Neutrophil Engraftment: Neutrophil engraftment is defined as achieving ANC  500/mm3 for 
three consecutive measurements over three or more days by Day 42.  The first of the three 
measurements may occur on Day 42.  A patient receiving a second stem cell infusion prior to 
Day 42 will be considered a graft failure. 
 
Platelet Engraftment: Platelet engraftment will be defined as the first day of a minimum of 
three consecutive measurements over three or more days such that the patient: 
1. Has achieved a platelet count > 50,000/mm3, and 
2. Is platelet transfusion independent for a minimum of seven days. 
 
Acute and Chronic GVHD: Acute GVHD usually develops within the first three months after 
transplantation and appears as a characteristic dermatitis often accompanied by cholestasis and 
enteritis.  Initial symptoms of chronic GVHD frequently include nausea and anorexia with ocular 
and oral sicca.  Rash characteristically appears with pigmentary changes progressing to sclerosis 
and contractures.  Other organs may be involved.  Symptoms may mimic those seen in patients 
with scleroderma and other autoimmune disorders.   
 


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The staging of acute GVHD will follow NMDP guidelines but will include weekly capture of 
symptoms and characterization of alternative causes.  The highest level of organ abnormalities, 
the etiologies contributing to the abnormalities and biopsy results pertaining to GVHD will be 
identified.  Staging and grading of acute GVHD are listed in the BMT CTN Manual of 
Procedures (MOP). 
 
Chronic GVHD typically does not occur until three or more months after transplantation.  Details 
regarding the definition and diagnosis are listed in the BMT CTN MOP. 
 
Infection Grading: Definitions for fungal infections are provided in Section 3.1.1.  Non-fungal 
infections will be graded according to the severity scale defined in the BMT CTN Data 
Management Handbook. 
 
Galactomannan Positivity: A positive galactomannan assay is defined as two positives on the 
same specimen or two consecutive positives on different specimens, i.e., a repeat test on the first 
specimen was not performed.  Sera with an index less than 0.5 are considered to be negative.  
Sera with an index greater than or equal to 0.5 are considered to be initially positive. 
 
Whenever a sample has an index  0.5, positivity must be confirmed by re-testing the sample, 
including repeating the heat treatment on a new aliquot, and by testing another sample obtained 
from the patient.  This confirmation is necessary in order to eliminate any false-positive results 
due to contamination of the sample after collection.   
 
3.3. Safety Monitoring Endpoints 
 
The rates of neural, cardiac, hepatic, and renal toxicities will be monitored up to 100 days post-
transplant, separately in each treatment arm.  The definitions of these toxicities are described 
below: 
1. Neural toxicity is defined as convulsions. 
2. Cardiac toxicity, defined as grade III toxicity on the Bearman scale, is severe EKG 
abnormalities with no or partial response to medical intervention; or, heart failure with no 
or minor response to medical intervention; or, decrease in voltage by more than 50%. 
3. Hepatic toxicity, defined as grade III toxicity on the Bearman scale, is severe hepatic 
dysfunction with bilirubin > 20 mg/dL; or, hepatic encephalopathy; or, ascites 
compromising respiratory function. 
4. Renal toxicity is defined as use of dialysis. 
 
Monitoring will be performed monthly until enrollment to that treatment arm is closed.  Each 
month, the null hypothesis that the rate of convulsions is less than or equal to 10% will be tested 
against the alternative that it is greater than 10%.  Similarly, the rate of hepatic and renal 
toxicities will be compared to thresholds of 8% and 10% respectively.  The expected threshold 
for cardiac toxicity is 2% and a formal monitoring plan was not developed.  If the calculated 
rates are significantly greater than the threshold rates (as defined in section 5.3.2), the NHLBI 
will be consulted to determine if the Data Safety and Monitoring Board should convene to 
review the data. 


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3.4 
Definition of Morphologic Complete Remission 
 
Complete remission (CR) will be defined as all of the following according to the revised 
recommendations of the international working group [61]: 
1. A bone marrow aspirate containing spicules with < 5% blasts with a count of at least 200 
nucleated cells and no Auer rods seen.  If spicules are absent in the aspirate, a bone 
marrow biopsy should confirm that < 5% blasts are present. 
2. No evidence of a persistently abnormal leukemic population by flow cytometry. 
3. ANC > 1000/L and platelet count > 100,000/L. 
4. No extramedullary leukemia. 
5. No blasts in peripheral blood. 
 
 


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CHAPTER 4 
 
 
4. 
PATIENT ENROLLMENT AND EVALUATION 
 
4.1. Enrollment and Randomization 
 
4.1.1 Screening and Enrollment Procedures 
 
Patients will be registered using the BMT CTN Electronic Data Capture System 
(AdvantageEDCSM).  The following procedures should be followed: 
 
1. An authorized user at the clinical center completes the initial screening by entering 
patient demographics, Segment O information (date informed consent signed) and 
Segment A information (inclusion/exclusion criteria and HLA typing information) of the 
Eligibility form preferably within 10 days prior to the initiation of the conditioning 
regimen and no later than 72 hours prior to transplant (Day 0).  
2. If patient is eligible, an ALT test should be obtained within 72 hours of Day 0. 
3. An authorized user at the clinical center completes the enrollment process by entering 
Segment B (dates of conditioning and proposed transplant, ALT value, baseline GM 
sample and CT scan) of the Eligibility Form within 72 hours of Day 0. 
4. If the patient is eligible, a study number and random treatment assignment is generated. 
 
If a connection is interrupted during a randomization session, the process is completely canceled 
and logged.  A backup manual registration and randomization system will also be available to 
provide for short-term system failure or unavailability. 
 
4.1.2 Randomization  
 
Patients will be randomized within 72 hours prior to Day 0.  Patients will be stratified by center 
and donor type (related vs. unrelated).  Patients will be assigned to either voriconazole or 
fluconazole in a 1:1 ratio. 
 
4.2. Study Monitoring 
 
4.2.1. 
Follow-up Schedule 
 
The Follow-up Schedule for scheduled study visits is outlined in Table 4.2.1.  A detailed 
description of each of the forms and the procedures required for forms completion and 
submission can be found in the Data Management Handbook and User’s Guide. 
 
Follow-up Assessments: The timing of follow-up visits is based on the date of allogeneic blood 
or marrow transplant.  Following randomization, the Transplant Center can print a Patient Visit 
Schedule listing target dates for assessments.  Weeks 1-14, Day 120, and Day 150 visits are 


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primarily for fungal assessments.  The subsequent visits are for follow-up reports.  
Galactomannan (GM) and investigational fungal diagnostic assays will be done up to twice 
weekly during the first 100 days.  In addition, blood for GM and fungal diagnostic assays will be 
collected from patients with possible, presumptive, probable or proven infection as stated in 
Section 4.2.2.  All efforts should be made to obtain the samples.  Failure to obtain all of the 
samples does not constitute a protocol deviation. 
 
Criteria for Forms Submission: Criteria for timeliness of submission for all study forms are 
detailed in the Data Management Handbook and User’s Guide.  Forms that are not submitted to 
the DCC within the specified time will be considered delinquent.  Transplant Centers can view 
past due forms via the web-based electronic data capture system.  A missing form will continue 
to appear until the form is entered into AdvantageEDC, or until an exception is granted and 
entered into the Missing Form Exception File, as detailed in the Data Management Handbook 
and User’s Guide. 
 
Reporting Patient Deaths: The Recipient Death Information must be entered into the web-
based electronic data capture system within one business day of knowledge of a patient’s death.  
If the cause of death is unknown at that time, it need not be recorded at that time.  However, once 
the cause of death is determined, the form should be updated. 
 
CIBMTR Data Reporting: All transplant centers will be required to pre-register all of their 
transplant patients, not just those participating in BMT CTN protocols, with the Center for 
International Blood and Marrow Transplant Research (CIBMTR).  The IBMTR Day 100 Report 
Form (including the Core, Graft and Disease Inserts) and IBMTR Follow-up Form (including the 
Core and Disease Inserts) for patients participating in the BMT CTN protocols will be submitted 
directly to the CIBMTR at the times specified in the Data Management Handbook and User’s 
Guide. 
 


BMT CLINICAL TRIALS NETWORK 
 
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Table 4.2.1 
FOLLOW-UP SCHEDULE 
 
Assessment Time 
Target Day1 
(Days Post-BMT) 
1 week2 
7 days 
2 week 
14 days 
3 week 
21 days 
4 week 
28 days 
5 week 
35 days 
6 week 
42 days 
7 week 
49 days 
8 week 
56 days 
9 week 
63 days 
10 week 
70 days 
11 week 
77 days 
12 week 
84 days 
13 week 
91 days 
100 day 
100 days 
120 day 
120 days3 
150 day 
150 days3 
6 month 
180 days3 
9 month 
270 days3 
12 month 
365 days3 
 
1 Target day range =  2 days up to Day 100,  14 days for Day 120 and 150, and  28 days for 
Day 180, 270 and 365 post-transplant. 
2 Samples for galactomannan (GM) assays will be drawn twice weekly on two non-consecutive 
days, preferably three days apart (e.g., Mondays and Thursdays), during the first 60 days, once 
weekly during Days 61-100 unless ONE of the following is met: (1) patient received a T cell 
depleted transplant and received post-transplant GVHD prophylaxis, or (2) patient is on 
steroids, or (3) patient has or has had acute GVHD requiring systemic therapy.  These select 
patients will have GM samples collected twice weekly during Days 61-100.  Any patient that 
has possible, presumptive, probable or proven infection will have additional samples collected 
for GM assays as described in Section 4.2.2.  Investigational fungal diagnostic blood samples 
will be collected at each of these time points. 
3 Data will be collected by telephone contact with the patient and/or treating physician if patient 
is unable to return to the transplant center. 


BMT CLINICAL TRIALS NETWORK 
 
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4.2.2. 
Assessments 
 
Pre-transplant required for study 
1. History, physical examination, weight, height, and BSA according to institutional 
practice (preferably within one week of initiation of the conditioning regimen).  Vital 
signs within 48 hours of initiation of the conditioning regimen. 
2. Serum creatinine, aspartate aminotransferase, alanine aminotransferase, alkaline 
phosphatase, and total bilirubin within 30 days of initiation of the conditioning regimen 
(preferably within 7 days) 
3. A CT scan of the chest (with contrast) and/or sinuses (without contrast) must be obtained 
within three weeks (one week preferable) prior to initiation of conditioning regimen if the 
patient has respiratory or sinus symptoms, or a history of pulmonary infection within the 
preceding three months 
4. Patients with signs or symptoms of respiratory infection or hepatosplenic candidiasis 
(e.g., elevated alkaline phosphatase) should be evaluated with appropriate CT scans 
within three weeks (one week preferable) prior to initiation of conditioning regimen 
5. A pregnancy test must be obtained in women of childbearing age within 30 days prior to 
start of the conditioning regimen (test must be negative to be eligible for study) 
6. Karnofsky or Lansky performance status within one week of initiation of the conditioning 
regimen 
7. Whole blood (5 mL) to be cryopreserved at the transplant center for future testing (e.g., 
infectious disease testing) if consent obtained.  This should be collected in a red top tube 
and processed per Appendix C. 
8. Whole blood or stem cell product from donor (5 mL) for SNPs assay if consent obtained.  
This should be collected in a green top tube and processed per Appendix C.  
9. EKG to exclude prolonged QTc syndrome 
 
Pre-transplant generally performed for transplant eligibility 
1. CBC (including hemoglobin and hematocrit), differential, platelet count within 48 hours 
of initiation of the conditioning regimen 
2. Serum creatinine, aspartate aminotransferase, alanine aminotransferase, alkaline 
phosphatase, and total bilirubin within one week of initiation of conditioning regimen 
3. Urinalysis < 48 hours of initiation of the conditioning regimen 
4. ABO and Rh typing 
5. Serum chemistries including sodium, potassium, chloride, bicarbonate, blood urea 
nitrogen, phosphate, calcium, albumin, and total protein < one week of initiation of 
conditioning regimen and ≤ 72 hours of Day 0 
6. Cardiac evaluation: echocardiogram or MUGA with ejection fraction (or shortening 
fraction if appropriate) in accordance with institutional policies 


BMT CLINICAL TRIALS NETWORK 
 
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7. Pulmonary function evaluation:  pulmonary function tests in accordance with institutional 
policies 
8. Serology for CMV, HSV, HIV, toxoplasmosis, varicella, hepatitis B-surface antigen, 
hepatitis B-core antibody and hepatitis C in accordance with institutional policies 
9. Sample of peripheral blood (3 mL) from donor and recipient to be stored for chimerism 
studies at the home institution.  This should be collected in a purple top tube and 
processed according to local institutional practices. 
10. A lumbar puncture is recommended if the patient has a history of CNS disease. 
 
Post-transplant required for study 
1. Pre-engraftment: CBC at least 3 times weekly until neutrophil engraftment (once ANC  
≥  500/mm3 for three consecutive days).  Differential if WBC ≥ 500/mm3. 
2. CBC and Absolute CD4 counts measured at Day 100 post-transplant. 
3. Peripheral blood samples (5 mL) for analysis of fluconazole or voriconazole 
concentrations collected at Day 14 and Day 28, and at onset of possible, presumptive, 
probable or proven infection (prior to start of amphotericin B or caspofungin) and at time 
of any suspected study drug toxicities delineated in Section 2.4.6.  Blood is to be 
collected during one of the windows relative to study drug administration as delineated in 
Appendix C.  Pediatric patients less than 12 years old will provide 2.5 mL of blood. 
4. Serum creatinine, aspartate aminotransferase, alanine aminotransferase, alkaline 
phosphatase, and total bilirubin, on a weekly basis during the first 100 days or more 
frequently if dictated by the condition of the patient, and if the patient is still on study 
drug at Day 120, 150 and 180. 
5. Blood samples (5 mL for adult, 2.5 mL for pediatric patients less than 12 years old) will 
be obtained twice weekly on two non-consecutive days, preferably three days apart (e.g., 
Mondays and Thursdays), from Day 0 to Day 60 for diagnostic galactomannan testing 
and processed in accordance with Appendices C and D.  In addition, blood samples will 
be obtained once weekly from Days 61 to 100 unless ONE of the following is met: (1) 
patient received a T cell depleted transplant and received post-transplant GVHD 
prophylaxis, or (2) patient is on steroids, or (3) patient has or has had acute GVHD 
requiring systemic therapy.  Blood samples from these patients will be collected twice 
weekly from Days 61 to 100.  In addition, at least two samples within the week before 
(and one within the last 48 hours) should be collected at initiation of empirical antifungal 
therapy for possible infection.  Collection of blood for diagnostic galactomannan (GM) 
assays should be continued twice weekly in patients receiving empirical antifungal 
therapy (per Section 2.4.10) for possible invasive fungal infection.   
6. Collection of blood for diagnostic GM samples should be continued once to twice weekly 
until Day 100 in patients who have been prematurely withdrawn from study drug for 
reasons other than presumptive, probable or proven fungal infection (see Section 2.4.6.1). 
7. For patients prematurely withdrawn from study drug due to presumptive, probable or 
proven aspergillus infection, monitoring of treatment blood GM samples (5 mL for adult, 
2.5 mL for pediatric patients < 12 years old) should be collected at onset and twice 


BMT CLINICAL TRIALS NETWORK 
 
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weekly for four weeks and then once every two weeks for eight weeks (for a total of 12 
samples) to monitor response.  Collection of blood for diagnostic GM samples after 
treatment of presumptive, probable or proven infection is at the discretion of the 
investigator. 
8. Blood samples (10 mL for adult; 5.0 mL for pediatric patients less than 12 years old) for 
investigational fungal diagnostic (IFD) assays will be collected at the same frequency as 
the diagnostic GM blood samples.  Collection of IFD blood samples for patients with 
presumptive, probable or proven fungal infection will be at the same frequency as the 
monitoring of treatment GM samples and then 1-2x weekly until Day 100. 
9. The center may make adjustments in blood volumes collected from patients less than 12 
years old to meet the institution’s Human Investigation Committee Guidelines. 
10. For any patient receiving a bronchoalveolar lavage, a 1 mL sample should be taken and 
stored in a cryovial at –70C. 
 
Post-transplant generally performed for transplant recipient 
1. CMV surveillance should be performed according to institutional policy. 
2. Post-engraftment: CBC and platelet count 3 times weekly until discharge from initial 
hospitalization; Post-discharge: CBC and platelet count weekly until PRBC and platelet 
transfusion independent, and at Day 100, 180, and 365.  Differential should be done if 
WBC is between 500-1500/mm3 at any time post-engraftment and at Day 100, 120, 150, 
180 and 365. 
3. Vital signs taken at least weekly up to Day 100 post-transplant and obtained from local 
health provider at Day 120, 150, 180 and 365. 
4. Clinical assessment weekly until Day 100, then by telephone contact at Day 120, 150, 
180 and 365 to ascertain whether any signs, symptoms or clinical events have occurred 
that suggest infection. 
5. Serum chemistries including sodium, potassium, chloride, bicarbonate, blood urea 
nitrogen, phosphate, calcium, and albumin, total protein on a weekly basis during the first 
100 days or more frequently if dictated by the condition of the patient, and at Day 120, 
150 and 180. 
6. Serum creatinine, aspartate aminotransferase, alanine aminotransferase, alkaline 
phosphatase and total bilirubin at Day 120, 150 and 180. 
7. Karnofsky/Lansky assessments by history and physical examination at Day 180 and 365. 
8. Cyclosporine (or tacrolimus) blood levels should be assessed closely during the first one 
to two weeks of study and doses adjusted in accordance with institutional policy.  This is 
important for patient safety because of drug interactions (see Section 2.4.7).  
Measurement of cyclosporine or tacrolimus levels three times or more during the first 
week may be necessary initially until drug levels are optimized.  Clinical and laboratory 
assessments of cyclosporine or tacrolimus toxicities should also be monitored closely. 
9. Sample of peripheral blood (3 mL) or bone marrow from patient for chimerism studies to 
document engraftment. 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
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4.2.3. 
Fungal Infection (possible, presumptive, probable or proven) 
1. Specimens from sites of suspected infection and blood will be obtained and analyzed for 
fungi as clinical symptoms warrant.  All isolates of yeast or mould organisms from blood 
or tissue, if available, should be obtained by the PI, stored at the local institution and 
shipped to the Study Repository in accordance with Appendix C.  Antifungal 
susceptibility testing will be conducted by the University of Florida.   
2. CT scans of chest and sinuses should be obtained at diagnosis and preferably at least after 
6 and 12 weeks of therapy and otherwise as clinically appropriate. 
3. For patients with possible aspergillus infection, at least two blood samples within the 
week before (and one within the last 48 hours) should be collected for diagnostic 
galactomannan assays upon initiation of empirical antifungal therapy with amphotericin 
B or caspofungin for possible infection.  Collection of blood for diagnostic 
galactomannan assays should be continued twice weekly in patients receiving empirical 
antifungal therapy (per Section 2.4.10) for possible invasive fungal infection.  These 
samples should be shipped immediately to a certified reference lab.  Investigational 
fungal diagnostic samples should be collected at the same frequency as the diagnostic 
galactomannan samples. 
4. For patients with presumptive, probable or proven aspergillus infection, monitoring of 
treatment galactomannan samples should be collected at onset and twice weekly for four 
weeks and then once every two weeks for eight weeks (for a total of 12 samples) to 
monitor response.  These samples should be frozen and batched shipped quarterly to the 
repository.  Investigational fungal diagnostic samples should be collected at the same 
frequency as the monitoring of treatment galactomannan samples and then 1-2x weekly 
until Day 100. 
5. Peripheral blood sample (5 mL for adult patients, 2.5 mL for pediatric patients less than 
12 years old) for analysis of fluconazole or voriconazole concentrations to be collected 
and processed as per Appendix C. 
6. The center may make adjustments in blood volumes collected from patients less than 12 
years old to meet the institution’s Human Investigation Committee Guidelines. 
7. Paraffin blocks of tissue specimens demonstrating fungal infection should be saved and 
shipped to the Study Repository in accordance with Appendix C.  
 
4.2.4. 
Discontinuation of Study Drug 
1. Upon discontinuation of study drug, all patients will be assessed with regards to drug 
efficacy in terms of the fungal definition criteria described in Section 3.1.1.   
2. A physical examination including vital signs will be performed within 72 hours of 
discontinuing study drug.   
3.  If previously positive, CT scans of chest or sinus should be performed within a 72-hour 
window of study drug discontinuation and if abnormality suspected or proven to be 
fungal in nature is still present then pertinent studies should be repeated approximately 


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two weeks following study drug completion and subsequently repeated until resolution of 
clinical or radiographic signs of infection, generally at no more than 6 week intervals.   
4. Other follow-up diagnostic tests should be obtained as deemed appropriate by each 
center's principal investigator.  
5. Peripheral blood sample (5 mL for adult patients, 2.5 mL for pediatric patients less than 
12 years old) for analysis of fluconazole or voriconazole concentrations (as per Appendix 
C). 
6. Any patient removed from study drug still should continue to have all study assessments 
performed up through Day 365. 
 
4.2.5. 
GVHD Monitoring 
 
GVHD should be monitored in accordance with BMT Clinical Trials Network guidelines as 
specified in the Manual of Procedures, i.e., weekly until Day 70. 
 
Follow-up GVHD assessments should be obtained at Day 90, 120, 180 and 365. 
 
4.2.6. 
Serious Adverse Event Reporting 
 
Unexpected grade 3-5 Adverse Events (AEs) will be reported through an expedited AE reporting 
system via the web-based electronic data capture system, AdvantageEDC.  Unexpected grade 4-5 
AEs must be reported within 24 hours of knowledge of the event.  Unexpected grade 3 AEs must 
be reported within 3 business days of knowledge of the event.  Toxicities will be reported using 
NCI’s Common Terminology Criteria for Adverse Events (CTCAE) Version 3.0 at regular 
intervals as defined on the Form Submission Schedule.  Decision to discontinue or re-administer 
the study drug will be made on a case-by-case basis after discussion with the Medical Monitor or 
one of the Protocol Chairs. 


BMT CLINICAL TRIALS NETWORK 
 
 
 
 
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Table 4.2.2  REQUIRED ASSESSMENTS 
 
X = Required 
 
Days Post Transplant 
 
O = Generally Performed 
Baseline 7 
7 
14 
28 
30 
42 
60 
90 
100 
120 
150 
180 
270 
365 
Pregnancy test (females only) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
CT scan of the chest1  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
CT scans of sinus and abdomen2 
X 
X-At time of possible, presumptive, probable or proven fungal infection, Refer to Section 4.2.3 
History, physical exam, weight, height, BSA, and 
Karnofsky/Lansky performance status 
X 
 
 
 
 
 
 
 
 
 
 
O3 
 
O3 
 
CBC4, differential5 and platelet count4 
X 
X-Refer to Section 4.2.2 for frequency 
O 
O 
O 
O 
 
O 
CD4 Counts 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
EKG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signs and clinical assessment 
X 
Generally performed weekly until Day 100 
O 
O 
O 
 
O 
Serum chemistries 
X 
X-Refer to Section 4.2.2 for frequency but at least one per week until 
Day 100 
X9 
X9 
X9 
 
 
Diagnostic galactomannan assay8 and investigational fungal 
diagnostic (IFD) assay10 
X 
X-Refer to Section 4.2.4 for frequency; twice weekly until Day 60 and at least once weekly Day 61-100.  Also prior to 
and during empirical trials of amphotericin B or caspofungin for possible fungal infection. 
Monitoring of treatment galactomannan assay8 and IFD 
assay 
 
X-At onset and twice weekly for 4 weeks and then once every 2 weeks for 8 weeks for a total of 12 samples for 
patients with presumptive, probable or proven aspergillus infection.  IFD continue 1-2x weekly until Day 100. 
Whole blood sample: 5 mL (Pharmacokinetics) 
 
 
X 
X 
X-Day 14 and 28 and at onset of possible, presumptive, probable or proven fungal infection 
or serious suspected drug toxicity as described in Section 2.4.6. 
Donor whole blood or stem cell product 5 mL for SNPs 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Whole blood 5 mL6 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Bronchoalveolar Lavage 
 
X-At any time procedure is performed 
Fungal Isolates 
 
X-At time of possible, presumptive, probable or proven infection and every seven days if available following the 
initiation of antifungal therapy or if infection relapses after therapy discontinued 
Paraffin Blocks of Infected Tissues 
 
X-At time of infection 
 
1  Obtain within 6 weeks (one week preferable) prior to randomizaton if the results of a baseline galactomannan 
assay are not available prior to randomization. 
2  Refer to Section 4.2.2.4 for conditions. 
3  Physical exam only. 
4  Post-discharge CBC & platelet count must be done weekly until PRBC and platelet transfusion independent and at 
Day 100, 180, 365. 
5  Differential must be done if WBC = 500-1500/mm3 at any time post-engraftment and at Day 100, 180, 270, 365. 
6  To be cryopreserved at the transplant center for future testing. 
7  Baseline refers to prior to conditioning.  Hepatic function tests 
should be repeated within 72 hours of start of study drug.  See 
Section 4.2.2. 
8  Refer to Section 4.2.2 for frequency in patients with possible, 
presumptive, probable or proven infections. 
9  If patient is on study drug until Day 180. 
10 Investigational fungal diagnostic assays are not required at baseline. 
. 
 


BMT CLINICAL TRIALS NETWORK 
 
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Version 7.0 dated March 29, 2006 
 
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CHAPTER 5 
 
 
5. STATISTICAL CONSIDERATIONS 
 
5.1. Study Design 
 
The study is designed as a Phase III, randomized, blinded, multicenter, prospective comparative 
study of fluconazole versus voriconazole in the prevention of fungal infections in allogeneic 
transplant recipients.  The target enrollment is 600 patients. 
 
Accrual 
 
It is estimated that three years of accrual will be necessary to enroll the targeted sample size.  
Both Core and non-Core Centers will enroll patients on this study.  Accrual will be reported by 
race, ethnicity, gender, and age (pediatric patients will be defined as < 18 years). 
 
Randomization 
 
Patients will be registered using the electronic data capture system, AdvantageEDC.  Only 
patients who have completed the screening process as described in Chapter 4 will be eligible for 
randomization.  All patients will be randomized within 3 days prior to the day of transplant.  
Randomization will be performed in a 1:1 ratio using random block sizes for the voriconazole 
and fluconazole arms.  Randomization will be stratified by transplant center and donor type 
(sibling versus unrelated donor). 
 
Primary Endpoint 
 
The primary endpoint is the fungal-free survival proportion at 180 days post-transplant.  The 
primary analysis will be performed using the intent-to-treat principle so that all randomized 
patients will be included in the analysis.  Death or the occurrence of a presumptive, probable or 
proven invasive fungal infection by Day 180 will be considered events for this endpoint. 
 
Primary Hypothesis 
 
The primary hypothesis of the study is that the voriconazole treatment will improve Day 180 
fungal-free survival as compared to the fluconazole treatment. 
 
Ho: 
pv = pf 
Ha: 
pv  pf 
 
5.2. Sample Size and Power Considerations 
 
Fungal-free survival at 180 days will be compared between the standard and experimental 
therapy arms using the standardized difference in the Kaplan Meier estimates of survival.  The 
final analysis will be performed after all patients have been followed for a minimum of 180 days 


BMT CLINICAL TRIALS NETWORK 
 
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post-transplant.  At this time point, all individuals will have been completely observed for the 
primary outcome.  However, the Kaplan Meier test will be used rather than a binomial test of 
proportions in order to ensure consistency with the results of interim analysis conducted with 
incomplete follow-up.  The sample size of 300 patients per group is sufficient to maintain type I 
error of 5% across all planned interim analyses (see below) while providing > 80% statistical 
power for a two-sided test to detect an increase in the proportion surviving fungal-free at 180 
days from 0.50 in the standard therapy arm to 0.62 in the experimental arm. 
 
5.3. Interim Analysis and Stopping Guidelines 
 
Interim analyses for efficacy will be conducted at times coincident with regularly scheduled 
meetings of the NHLBI-appointed Data and Safety Monitoring Board (DSMB) at approximately 
six month intervals.  Monitoring of key safety endpoints will be conducted monthly, and if rates 
significantly exceed pre-set thresholds, the NHLBI will be consulted to determine if the DSMB 
should be advised and if an ad-hoc meeting will be convened.  Policies and composition of the 
DSMB are described in the BMT CTN's Manual of Procedures. 
 
5.3.1. 
Interim Analysis for Efficacy 
 
Analyses will be performed as described below for the primary endpoint.  Toxicity, adverse 
events, and other safety endpoints will be monitored regularly and reported to the DSMB at each 
interim analysis.  
 
At the time of each interim analysis, the test statistic based on the Kaplan-Meier proportion will 
be compared to the critical value shown below.  All patients randomized prior to the time of the 
interim analyses will be used to compute the Kaplan-Meier estimate.  If the test statistic exceeds 
the critical value, the DSMB will discuss the continuation of the trial. 
 
At each interim analysis time point, a two-sided test to detect either an increase or decrease in the 
proportion of patients with fungal-free survival at 180 days will be performed, as described 
above for the final analysis.  In order to preserve the over-all type I error rate at 5%, the critical 
value for the test statistic will be inflated above 1.96, the value that would be used if no repeated 
testing were used.  Equivalently, the nominal p-value at which an observed difference is declared 
significant will be reduced below 0.05.  The actual critical values and nominal p-values will be 
computed using statistical methods for group sequential testing with O’Brien Fleming 
boundaries.   
 
As an example, Table 5.3.1a shows the critical values and nominal p-values for tests conducted 
at 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 years after the study opens to enrollment.  The column labeled 
“followed to 180 days” shows the average number of individuals who have reached the 180-day 
post-transplant follow-up time point, assuming uniform accrual over a three-year period.  The 
fraction of patients followed to 180 days, as compared to a denominator comprised of the total 
sample of 600, quantifies the “statistical information” from which the critical values, nominal p-
values and cumulative type I error are computed.   
 


BMT CLINICAL TRIALS NETWORK 
 
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Table 5.3.1a Critical Values and Nominal P-Values 
 
Calendar Time 
Followed to 180 Days 
Critical Value 
Nominal P-value 
Cumulative Type I Error 
1.0 
100 
5.3660 
.00000 
.00000 
1.5 
200 
3.7107 
.00021 
.00021 
2.0 
300 
2.9697 
.00284 
.00305 
2.5 
400 
2.5386 
.00905 
.01210 
3.0 
500 
2.2522 
.01605 
.02815 
3.5 
600 
2.0447 
.02185 
.05000 
 
In practice, the rate of accrual or timing of DSMB meetings may not be as anticipated.  To 
permit necessary flexibility in scheduling interim analyses, the critical values will be recomputed 
to correspond to the actual available statistical information using the “use-function” approach of 
Lan and DeMets. 
 
Operating Characteristics of the Design 
 
Under the assumption that time to infection or death is exponentially distributed, the statistical 
power to reject the null hypothesis of equal 180 day survival is shown below under a variety of 
scenarios. 
 
Table 5.3.1b – Power to Reject the Null Hypothesis under Various Scenarios 
 
N 
Proportion Surviving  
Power at Interim and Final Analyses 
Overall 
Power 
per 
Fungal-Free at Day 180 
By Year of Scheduled Analysis 
Arm 
Standard 
Experiment 
1.0 
1.5 
2.0 
2.5 
3.0 
3.5 
 
275 
.50 
.50 
.0000 
.0003 
.0030 
.0114 
.0167 
.0239 
.0553 
275 
.50 
.62 
.0000 
.0337 
.1847 
.2609 
.2053 
.1198 
.8044 
300 
.50 
.50 
.0000 
.0005 
.0039 
.0099 
.0170 
.0218 
.0531 
300 
.50 
.62 
.0006 
.0400 
.2061 
.2736 
.2085 
.1068 
.8355 
325 
.50 
.50 
.0000 
.0002 
.0036 
.0102 
.0155 
.0224 
.0519 
325 
.50 
.62 
.0001 
.0475 
.2251 
.2829 
.2088 
.1021 
.8665 
* from simulation with 10,000 replications, assuming exponential time to failure 
 
 
5.3.2. 
Guidelines for Safety Monitoring 
 
The rates of neural, hepatic and renal toxicities will be monitored up to 100 days post-transplant, 
separately in each treatment arm for all transplanted patients.  Patients will be analyzed 
according to their randomized treatment assigment.  Refer to Section 3.3 for definitions of these 
safety monitoring endpoints.  Monitoring will be performed monthly until enrollment to that 
treatment arm is closed.  We expect approximately 20 patients to be on study at the time of the 
first look.  Each month, the null hypothesis that the rate of convulsions is less than or equal to 
10% will be tested against the alternative that it is greater than 10%.  Similarly, the rate of 
hepatic and renal toxicities will be compared to thresholds of 8% and 10% respectively.   
 


BMT CLINICAL TRIALS NETWORK 
 
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An extension to the Sequential Probability Ratio Test (SPRT) will be used for each endpoint, as 
described in greater detail below and in Appendix I.  This sequential testing procedure conserves 
type I error across all of the monthly examinations for a single endpoint, but not across the 
multiple safety endpoints.  Thus for a single endpoint, the type I error is approximately 5%, and 
across all three safety endpoints, the study-wise type I error is < 15% per arm for a total of 30% 
study-wide. 
 
The rationale for not conserving type I error across multiple safety endpoints is twofold.  First, 
adjusting the size of the test for multiple comparisons would reduce statistical power to detect 
adverse outcomes, which seems imprudent.  Secondly, the procedure is a guideline for requiring 
additional review by the Data and Safety Monitoring Board, and is not a formal “stopping rule” 
that would mandate automatic closure of study enrollment.   
 
The SPRT can be represented graphically.  At each monthly interim analysis, the total time on 
study is plotted against the total number of endpoints, (e.g., patients with convulsions).  The 
continuation region of the SPRT is defined by two parallel lines.  Only the lower boundary will 
be used for monitoring each treatment arm to protect against poor 100-day rates of convulsions.  
If the graph falls below the lower boundary, the SPRT rejects the null hypothesis, and concludes 
that there are more convulsions than predicted by the observed time on study.  Otherwise, the 
SPRT continues until enrollment to the treatment arm reaches the target goal. 
 
This procedure assumes an exponential distribution for the time until failure during the first 100 
days, but censors follow-up time after 100 days.  Only endpoints that occur on or before the 
patient has been followed for 100 days post-randomization are counted.  Total time on study is 
computed as time from randomization to event, or to 100 days, whichever comes first, summed 
for all individuals on study.  Death is a competing risk for the event, and time on study will be 
censored at the time of death.  
 
The usual measures of performance of an SPRT are the error probabilities  and  of rejecting 
H0 when  = 0 and of accepting H1 when  = 1, respectively, and the expected sample size 
E(N|i).  The tests to be used in this protocol were developed from the following SPRTs:   
 
 A SPRT contrasting 10% versus 15% 100-day rate of convulsions, with nominal type I 
and II errors of 9% and 15%, respectively.  The common slope of the parallel lines is 
2.076 and the intercepts are –10.755 and 8.635. 
 A SPRT contrasting 8% versus 13% 100-day rate of hepatic toxicity, with nominal type I 
and II errors of 7% and 15%, respectively.  The common slope of the parallel lines is 
2.513 and the intercepts are –12.233 and 8.939. 
 A SPRT contrasting 10% versus 15% 100-day rate of use of dialysis, with nominal type I 
and II errors of 9% and 15%, respectively.  The common slope of the parallel lines is 
2.076 and the intercepts are –10.755 and 8.635. 
 
Note that since the test uses only the lower boundary, and is truncated by a finite sample size, 
both the size and power of the test will be lower than nominal levels.  
 


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The actual operating characteristics of the truncated test, shown in Table 5.3.2, were determined 
in a simulation study that assumed uniform accrual of 300 individuals over a three-year time 
period, and exponential time to failure after randomization.  Since 100,000 replications were 
used, the estimates have two digits of precision. 
 
Table 5.3.2 
Operating Characteristics of Sequential Testing Procedure 
from a Simulation Study with 100,000 Replications 
 
 
Neural Toxicity (Convulsions) 
True 100-Day Rate 
10% 
15% 
20% 
Probability Reject Null 
0.06 
0.79 
1.00 
Mean Month Stopped 
35.8 
21.8 
10.7 
Mean # Endpoints in 100 days 
28.4 
25.2 
15.3 
Mean # Patients Enrolled 
290.9 
179.8 
89.1 
 
 
Hepatic Toxicity 
True 100-Day Rate 
8% 
13% 
18% 
Probability Reject Null 
0.05 
0.81 
1.00 
Mean Month Stopped 
36.1 
21.2 
10.2 
Mean # Endpoints in 100 days 
22.9 
21.3 
13.0 
Mean # Patients Enrolled 
292.7 
175.3 
85.3 
 
Renal Toxicity (Use of Dialysis) 
True 100-Day Rate 
10% 
15% 
20% 
Probability Reject Null 
0.06 
0.79 
1.00 
Mean Month Stopped 
35.8 
21.8 
10.7 
Mean # Endpoints in 100 days 
28.4 
25.2 
15.3 
Mean # Patients Enrolled 
290.9 
179.8 
89.1 
 
For example, the testing procedure for convulsions rejects the null hypothesis in favor of the 
alternative 6% of the time when the true 100 day convulsion rate is 10%, and 79% of the time 
when the rate is 15%.  This corresponds to a type I error rate of  = .06 and a type II error rate of 
 = .21.  When the true 100 day convulsion rate is 15%, on average, the Data and Safety 
Monitoring Board will be consulted 21.8 months after opening, when 25.2 convulsions have 
been observed in 179.8 patients.   
 
Since the expected rate of serious cardiac events is 2%, a formal monitoring rule will not be 
used. 
 
5.4. Demographic and Baseline Characteristics 
 
Demographics and baseline characteristics will be summarized for all patients.  Characteristics to 
be examined are: age, gender, race/ethnicity, performance status, HLA match, primary disease, 
risk status, prior history of fungal infections, source of stem cells, donor age, donor gender, and 


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donor ethnicity.  Between group comparisons will be performed for continuous variables via a t-
test and for categorical variables, via the chi-square test. 
 
5.5. Analysis of Secondary Endpoints 
 
1. Frequency of invasive fungal infections by Day 180:  The Cochran-Mantel Haenszel 
(CMH) test will be used to compare the proportion of patients with invasive fungal 
infection between the treatment groups. 
2. Time to invasive fungal infection: Cumulative incidence curves will be computed by 
treatment.  A stratified log-rank test will be used to compare the two arms.  Also, a Cox 
proportional hazards model will be fit to control for important prognostic variables. 
3. Patient survival: Survival distribution will be estimated by the Kaplan- Meier curve. The 
two treatment arms will be compared using the stratified log-rank test.  A Cox 
proportional hazard model will be fit to control for important prognostic variables.  All 
patients will be followed for a minimum of one year post transplant for mortality. 
4. Empirical use of amphotericin B or caspofungin: This will be assessed by 
measurement of proportions of patients with empirical use of amphotericin B or 
caspofungin and total number of days on amphotericin B or caspofungin and compared 
between the two treatment groups via the CMH and 2-way ANOVA respectively.  Rank 
(or other) data transformation may be used for the number of days on amphotericin B or 
caspofungin for the 2-way ANOVA.  The impact of use of amphotericin B or 
caspofungin on the incidence of systemic fungal infections will be examined using 
logistic regression.  Duration of amphotericin B or caspofungin and the proportion of 
patients who received > 14 days of consecutive amphotericin B or caspofungin use will 
be assessed. 
5. Time to acute or chronic graft-versus-host disease (GVHD): Cumulative incidence 
curves will be computed by treatment.  A stratified log-rank test will be used to compare 
the two arms.  Also, a Cox proportional hazards model will be fit to control for important 
prognostic variables. 
6. Exploratory analyses of quantitative aspects of galactomannan assay in diagnosis 
and treatment of aspergillus in the presence of different patient conditions: The 
objectives of the analyses are (1) to explore quantitative aspects of the assay to optimize 
sensitivity and specificity in the setting of different host factors and manifestations of 
aspergillus, (2) to determine if cases first detected by the galactommanan assay have 
improved treatment outcomes as compared to cases first documented by other diagnostic 
tests, and if galactomannan kinetics are predictive of clinical response to antifungal 
therapy. 
 
Objective 1 
 
It is hypothesized that the performance of the assay will be dependent on circulating 
fungal burden, which is potentially impacted by multiple host factors, and various 
manifestations of aspergillus.  Specifically, it is anticipated that pulmonary infections 


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may be less likely detected by GM than disseminated infection, and sinus infections 
will be the least likely to be detected by GM early. 
 
Host factors that will be considered include: age, neutropenia or ANC > 500, receipt 
of mould-active antifungal therapy, primary graft failure versus engrafters, and 
receipt of voriconizole vs. fluconozole.  Manifestations of aspergillus that will be 
considered include: (1) site of infection (lung, sinus, extra pulmonary); (2) stage of 
infection (localized versus disseminated); and, (3) causative aspergillus species.    
 
Patients who develop aspergillus infection will be classified according to whether the 
infection was first detected by GM positivity or by conventional means.  The effects 
of the host and infection factors listed above on the probability of first detection by 
GM will be explored by conditional logistic regression modeling.  If infection status 
is not ascertained in all patients, more complex models that incorporate non-response 
will be considered. 
 
The proportion of patients diagnosed with aspergillus infection, and the median time 
to first detection of the first episode of aspergillus, will be compared according to 
whether the infection was first detected by GM positivity or conventional means.  
However, the results are not expected to differ because the GM result will be reported 
in real time to clinicians and will trigger a clinical work-up.   
 
It is anticipated that one-third of cases of “possible” aspergillosis will be upgraded to  
“probable” by incorporation of the GM assay.  To test this hypothesis, the proportion 
of cases reclassified from “possible” to “probable,” and its associated confidence 
interval, will be estimated.  More generally, the rates of reclassification will be 
investigated using methods appropriate for ordinal responses on paired samples.   
 
The performance (e.g., sensitivity and specificity) of diagnostic criteria that 
incorporate the GM assay will be compared to a “gold standard” of the conventional 
diagnostic criteria, using Receiver Operator Curve (ROC) methodology.  The 
semiquantitative GM index will be compared with baseline to determine what relative 
increase in GM index occurs at the time of diagnosis by other diagnostic tests.  Other 
thresholds for determining a positive GM response will be investigated.  
 
Objective 2 
 
It is hypothesized that cases first detected by GM positivity will be less advanced 
infections and will be more likely associated with survival than those detected by 
other conventional diagnostic means.  For cases detected by conventional means, 
67% die.  It is believed that the case fatality rate for GM-detected cases will be lower.   
 
A survival model with two time-dependent indicator variables, representing the 
absorbing states of first diagnosis by GM positivity, and first diagnosis by 
conventional means will be employed.  The hypothesis that GM-detected cases will 


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have improved survival will be tested using a likelihood ratio test to compare the 
coefficients for these terms. 
 
In patients treated for aspergillosis, the temporal change in galactomannan level will be 
tested as a guide to response to treatment and compared with clinical, cultural, and 
radiographic parameters of response. 
7. Engraftment: Rate of primary graft failure will be estimated using cumulative incidence 
estimates.  The treatment arms will be compared using the stratified log-rank test. 
Neutrophil and platelet engraftment will be computed with cumulative incidence 
estimates.  The number of autologous recoveries will be reported. 
8. Freedom from possible, presumptive, probable or proven infection, death or 
withdrawal of study drug.  Cumulative incidence curves will be computed by treatment.  
A stratified log-rank test will be used to compare the two arms.  Also, a Cox proportional 
hazards model will be fit to control for important prognostic variables. 
 
Two-sided tests will be used throughout.  A p-value of 0.05 or less will be considered 
statistically significant. 
 
5.6. Safety Analysis 
 
All entered patients will be included in the safety analysis: 
1. Serious Adverse Events: All reported serious adverse events potentially associated with 
study drug will be carefully examined with respect to the severity and relationship to 
study drug.  Adverse events will be graded according to the NCI Common Terminology 
Criteria for Adverse Events Version 3.0.  The incidence for each reported study drug 
associated adverse event delineated in Section 2.4.5 will be presented for each group.  
2. Clinical Laboratory Tests: Laboratory tests will be performed during the study. 
Descriptive statistics will be calculated for each laboratory test.  Abnormal laboratory test 
results will be summarized in the report.  Laboratory data will be cross-classified into 
below, within, and above the reference range according to their values at screening and 
during the study.  
 
 


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APPENDIX A 
 
REFERENCES 


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APPENDIX A 
 
REFERENCES 
 
 
1. Wingard JR.  Fungal infections after bone marrow transplant.  Biol Blood Marrow 
Transplant 5:55-68, 1999. 
2. Goodman JL, Winston DJ, Greenfield RA:  A controlled trial of Fluconazole to prevent 
fungal infections in patients undergoing bone marrow transplantation.  N Eng J Med 
326:845, 1992. 
3. Rotstein C, Bow EJ, Laverdiere M, et al.  Randomized placebo-controlled trial of 
Fluconazole prophylaxis for neutropenic cancer patients: benefit based on purpose and 
intensity of cytotoxic therapy.  Clin Infect Dis 28:331, 1999. 
4. Slavin MA, Osborne B, Adams R, et al.  Efficacy and safety of Fluconazole prophylaxis for 
fungal infections after marrow transplantation – a prospective, randomized, double-blind 
study.  J Infect Dis 171:1545, 1995. 
5. Marr KA, Seidel K, Slavin M, et al.  Outcome of blood and marrow transplantation in 
patients who received prolonged Fluconazole prophylaxis: long-term follow-up of a 
randomized, placebo-controlled trial.  Blood 96:12055, 2000. 
6. Hansen JA, Gooley TA, Martin PJ, et al.  Bone marrow transplants from unrelated donors for 
patients with chronic myeloid leukemia.  N Eng J Med 338:962, 1998.  
7. Recommendations of CDC, the Infectious Diseases Society of America, and the American 
Society of Blood and Marrow Transplantation.  Guidelines for preventing opportunistic 
infections among hematopoietic stem cell transplant recipients.  Biol Blood Marrow 
Transplant 6:659-734, 2000.  
8. Wingard JR, Chairman of the IBMTR/ASBMT/NIH State of Science BMT Meeting,  
Infectious Complications Working Group, April 1, 2000, personal communication.   
9. Marco F, Pfaller MA, Messer S, et al.  In vitro activities of Voriconazole (UK-109-496) and 
four other antifungal agents against 394 clinical isolates of Candida sp.  Antimicrob Agents 
Chemother 42:161, 1998. 
10. Johnston D, Zhou X, Fukuoka T, et al.  Voriconazole is highly active against the 14-
demethylase of Candida krusei.  40th Interscience Conference on Antimicrobial Agents and 
Chemotherapy, September 26-29, 1999, San Francisco, CA, abstract 2073. 
11. Clancy CJ, Fothergill, Rinaldi MG, et al.  SCH56592 and Voriconazole against emerging 
yeasts: in vitro efficacy compared with Fluconazole and Itraconazole.  40th Interscience 
Conference on Antimicrobial Agents and Chemotherapy, September 26-29, 1999, San 
Francisco, CA, abstract 1518. 


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12. Pai MP, Prause JL, Danziger LH, et al.  In vitro activity of Voriconazole and other azoles 
against Candida isolates.  40th Interscience Conference on Antimicrobial Agents and 
Chemotherapy, September 17029, 2000, Toronto, Canada, poster presentation. 
13. Schlamm HT, Corey L, Brown J, et al.  Voriconazole for salvage treatment of invasive 
aspergillosis.  40th Interscience Conference on Antimicrobial Agents and Chemotherapy, 
September 17029, 2000, Toronto, Canada, abstract 304. 
14. Perfect J, Lutsar I, Gonzalea-Ruiz A.  Voriconazole (VORI) for the treatment of resistant 
and rare fungal pathogens.  40th Interscience Conference on Antimicrobial Agents and 
Chemotherapy, September 17029, 2000, Toronto, Canada, abstract 303. 
15. Green BA, Breen A, Corey L.  Voriconazole for salvage therapy of fungal infections in 
patients with hematological malignancies and stem cell transplantation.  40th Interscience 
Conference on Antimicrobial Agents and Chemotherapy, September 17029, 2000, Toronto, 
Canada, Abstract 41. 
16. Johnston D, Cannom RRM, Filler SG.  Amino acid substitutions in the aspergillus fumigatus 
ERG 11 gene product render it resistant to itraconazole yet susceptibility to Voriconazole is 
maintained.  40th Interscience Conference on Antimicrobial Agents and Chemotherapy, 
September 17029, 2000, Toronto, Canada. Abstract.  
17. Torre-Cisneros J, Gonzalez-Ruiz A, Hodges MR.  Voriconazole for the treatment of 
S.apiospermum and S.prolificans infection.  40th Interscience Conference on Antimicrobial 
Agents and Chemotherapy, September 17029, 2000, Toronto, Canada, Abstract 305.  
18. Sabo JA, Abdel-Rahman SM.  Voriconazole:  a new triazole antifungal.  Ann Pharmacother 
34:1032, 2000. 
19. Dupont B, Ally R, Burke J, et al.  A double-blind, randomized, multicenter trial of 
Voriconazole (VORI) vs Fluconazole (FLU) for the treatment of esophageal candidiasis (EC) 
in immunocoompromised adults.  40th Interscience Conference on Antimicrobial Agents and 
Chemotherapy, September 17-20, 2000, Toronto, Ontario, Canada, Abstract 706. 
20. Walsh TJ, Pappas P, Winston D, et al.  Voriconazole versus liposomal amphotericin B for 
empirical antifungal therapy of persistently febrile neutropenic patients: a randomized, 
international, multicenter trial.  40th Interscience Conference on Antimicrobial Agents and 
Chemotherapy, September 17-20, 2000, Toronto, Ontario, Canada, abstract L-1. 
21. Denning DW, Patterson TF, Herbrecht R, et al.  Utility of criteria to assess outcome of 
invasive aspergillosis: consistency of response assessments in a clinical trial.  40th 
Interscience Conference on Antimicrobial Agents and Chemotherapy, September 17029, 
2000, Toronto, Canada, abstract 1325.  
22. Winston DJ, Maziarz RT, Chandrasekar PH, et al.  Intravenous and oral intraconazole versus 
intravenous and oral fluconazole for long-term antifungal prophylaxis in allogeneic 
hematopoietic stem-cell transplant recipients.  A multicenter, randomized trial.  Ann Itern 
Med. 2003; 138:705-713. 
23. Marr, K, Crippa F, Leisenring W, et al.  Intraconazole vs Fluconazole for antifungal 
prophylaxis in allogeneic HSCT recipients: results of a randomized trial.  Blood 2002; 11: 
(311), 215a (Abstrat 806). 


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24. Jantunen E, Ruutu P, Hiskanen L, et al.  Incidence and risk factors for invasive fungal 
infections in Allogeneic BMT patients. Bone Marrow Transplant 19:801, 1997. 
25. Wald A, Leisenring W, van Burik J-A, et al.  Epidemiology of aspergillus infections in a 
large cohort of patients undergoing bone marrow transplantation.  J Infect Dis 175:1459, 
1997. 
26. Grow WB, Moreb JS, Roque D, et al.  Late onset of invasive aspergillosis infection in bone 
marrow transplant patients at a university hospital.  Blood 94:341a, Abstract #1524,1999. 
27. Ribaud P, Chastang C, Latge JP, et al.  Survival and prognostic factors of invasive 
aspergillosis after allogeneic bone marrow transplantation.  Clin Infect Dis 28:322-330, 1999.  
28. Morrison VA, Haake RJ, Weisdorf DJ. The spectrum of non-candida fungal infections 
following bone marrow transplantation.  Medicine 72:78, 1993.  
29. Marr KA, Balajee A, Leisenring W, et al.  Utility of galactomannan detection for the 
diagnosis of invasive aspergillosis in HSCT recipients.  Blood 96:786a, Abstract 3400, 2000.  
30. Iwen PC, Reed EC, Armitage JO, et al.  Nosocomial invasive aspergillosis in lymphoma 
patients treated with bone marrow or peripheral stem cell transplants.  Infect Control Hosp 
Epidemiol 14:131, 1993. 
31. Wald A, Leisenring W, van Burik J-A, et al.  Epidemiology of aspergillus infection in a 
large cohort of patients undergoing bone marrow transplantation.  J Infect Dis 175:1459, 
1997.  
32. Maertens JA, Verhaegen J, Lagrou K, et al.  Screening for circulating galactomannan as a 
non-invasive diagnostic tool for invasive aspergillosis in prolonged neutropenic patients and 
stem cell transplantation recipients:  a prospective validation.  Blood 97:1604-1610, 2001. 
33. Salonen J, Lehtonen O-P, Terasjarvi M-R, et al.  Aspergillus antigen in serum, urine and 
bronchoalveolar lavage specimens of neutropenic patients in relation to clinical outcome.  
Scand J Infect Dis 32:485-490, 2000. 
34. Denning DW.  Early diagnosis of invasive aspergillosis.  Lancet 355:423, 2000. 
35. Becker MJ, de Marie S, Willemse D, et al.  Quantitative galactomannan detection is superior 
to PCR in diagnosing and monitoring invasive pulmonary aspergillosis in an experimental rat 
model.  J Clin Microbiol 38:1434-1438, 2000.  
36. Ally R, Schurman D, Kreisel W, et al.  A randomized, double-blind, double-dummy, 
multicenter trial of voriconazole and fluconazole in the treatment of esophageal candidiasis 
in immunocompromised patients.  Clin Infect Dis 33:1447-1454, 2001. 
37. Denning DW, Ribaud P, Milpied N, et al.  Efficacy and safety of voriconazole in the 
treatment of acute invasive aspergillosis.  Clin Infect Dis 34:563-571, 2002. 
38. Walsh TJ, Lutsar I, Driscoll T, DuPont B, Roden M, Gharamani P, Hodges M, Groll AH, 
and Perfect JR: Voriconazole in the treatment of aspergillosis, scedosporiosis, and other 
invasive fungal infections in children.  Ped Infect Dis J 21: 240-248, 2002. 
39. Herbrecht R, Denning DW, Patterson TF, et al.  Open, randomized comparison of 
voriconazole (VRC) and amphotericin B (AmB) followed by other licensed antifungal 


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therapy of invasive aspergillosis (IA). Abstract J-680. p. 378 41st Annual Intersci Conf 
Antimicrob Agents Chemother.  2001. 
40. McGinnis MR, Nordoff N, Li RK, et al.  Sporothrix schenkii sensitivity to voriconazole, 
itraconazole and amphotericin.  Med Mycol 39:369-371, 2001. 
41. Fernandez-Torres B, Carrillo AJ, Martin E, et al.  In vitro activities of 10 antifungal drugs 
against 508 dermatophyte strains.  Antimicrob Agents Chemother 45:2524-2528, 2001. 
42. Carrillo AJ, Guarro J.  In vitro activities of four novel triazoles against scedosporium spp.  
Antimicrob Agents Chemother 45:2151-2153, 2001. 
43. Meletiadis J, Meis JF, Mouton JW, et al.  In vitro activities of new and conventional 
antifungal agents against clinical scedosporium isolates.  Antimicrob Agents Chemother 
46:62-68, 2001. 
44. Walsh TJ, Pappas P, Winston DJ, et al.  Voriconazole compared with liposomal amphotericin 
B for empirical antifungal therapy in patients with neutropenia and persistent fever.  N Engl J 
Med 346:225-234, 2002. 
45. Cockgroft DW, Gault MH.  Prediction of creatinine clearance from serum creatinine.  
Nephron 16:31-41, 1976.  
46. Ghahramani P, Purkins L, Kleinermans, et al.  Voriconazole potentiates warfarin-induced 
prolongation of prothrombin time.  40th Interscience Conference on Antimicrobial Agents 
and Chemotherapy, September 17029, 2000, Toronto, Canada, abstract 846.  
47. Ghahramani P, Purkins L, Wood ND, et al.  Drug interactions between Voriconazole and 
phenytoin.  40th Interscience Conference on Antimicrobial Agents and Chemotherapy, 
September 17029, 2000, Toronto, Canada, abstract 847.  
48. Ghahramani P, Wood ND, Purkins L, et al.  Effect of omeprazole on the pharmacokinetics of 
Voriconazole.  40th Interscience Conference on Antimicrobial Agents and Chemotherapy, 
September 17029, 2000, Toronto, Canada, abstract 843.  
49. Ghahramani P, Purkins L, Wood ND, et al.  The pharmacokinetics of Voriconazole and its 
effect on prednisolone disposition.  40th Interscience Conference on Antimicrobial Agents 
and Chemotherapy, September 17029, 2000, Toronto, Canada, abstract 842.  
50. Ghahramani P, Purkins L, Wood ND, et al.  Effects of rifampicin and rifabutin on the 
pharmacokinetics of Voriconazole.  40th Interscience Conference on Antimicrobial Agents 
and Chemotherapy, September 17029, 2000, Toronto, Canada, abstract 844. 
51. Ghahramani P, Purkins L, Klleinermans D, et al.  Voriconazole does not affect the 
pharmacokinetics of digoxin.  40th Interscience Conference on Antimicrobial Agents and 
Chemotherapy, September 17029, 2000, Toronto, Canada, abstract 849. 
52. Ghahramani P, Romero AJ, Wood ND, et al.  The effect of Voriconazole on the 
pharmacokinetics of cyclosporin.  40th Interscience Conference on Antimicrobial Agents and 
Chemotherapy, September 17029, 2000, Toronto, Canada, abstract 845. 
53. Ascioglu S, Res JH, de Pauw B, et al.  Defining opportunistic invasive fungal infections in 
immunocompromised patients with cancer and hematopoietic stem cell transplants:  an 
international consensus.  Clin Infect Dis 34:7-14, 2002. 


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54. National Committee for Clinical Laboratory Standards. Reference method for broth dilution 
antifungal susceptibility testing of yeasts.  Approved standard M27-A. National Committee 
for Clinical Laboratory Standards, Wayne, Pa, 1997. 
55. National Committee for Clinical Laboratory Standards.  Reference method for broth dilution 
antifungal susceptibility testing of conidium-forming filamentous fungi. Proposed standard 
M38-P. National Committee for Clinical Laboratory Standards, Wayne, Pa, 1998. 
56. Marr KA, Carter RA, Cripa F, et al.  Eidemiology and outcome of mould infections in 
hematopoietic stem cell transplant recipients.  Clin Infect Dis 34:909-917, 2002.   
57. Purkins L, Wood N, Chahramani P, et al.  Pharmacokinetics and safety of voriconazole 
following intravenous to oral-dose escalation regimens.  Antimiccrob Agents Chemother 
46:2546-2553, 2002. 
58. Lazarus HM, Blumer JL, Yanovich S, et al.  Safety and pharmacokinetics of oral 
voriconazole in patients at risk of fungal infection: a dose escalation study.  J Clin Pharmacol 
42:395-402, 2002. 
59. Romero AJ, Pogamp PL, Nilsson L-G, et al.  Effect of voriconazole pharmacokinetics on 
cyclosporine in renal transplant patients.  Clin Pharmacol Ther 71:226-234, 2002. 
60. Walsh TJ, Wood N, Milligan P, et al.  Pharmacokinetics of intravenous voriconazole in 
children after single and multiple dose administration.  Abstract A17.  In:  41st Interscience 
Conference on Antimicrobial Agents and Chemotherapy, Chicago, September 22 to 25, 
2001.  Washington, DC:  American Society for Microbiology, 2001:2. 
61. Cheson BD, Bennett JM, Kopecky KJ, Buchner T, Willman CL, Estey EH, et al.  Revised 
recommendations of the international working group for diagnosis, standardization of 
response criteria, treatment outcomes, and reporting standards for therapeutic trials in acute 
myeloid leukemia.  J Clin Oncol 21(24): 4642-9, 2003. 
 


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APPENDIX B-1 
 
CONSENT FORMS 
 
 
 
PARTICIPANT INFORMED CONSENT 
 
 
 
 


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Informed Consent to Participate in Research 
 
 
 
 
 
 
 
 
 
 
 
If you are a parent, as you read the information in this Consent Form, you should put yourself 
in your child’s place to decide whether or not to allow your child to take part.  Therefore, for the 
rest of the form, the word “you” refers to your child. 
 
If you are a child, adolescent or adult who is reading this form, the word “you” refers to you. 
 
 
You are being asked to take part in a research study.  This form tells you about 
the study.  The Principal Investigator (the person in charge of this research) or a co-
worker of the Principal Investigator will also describe this study to you and answer all 
of your questions.  Before you decide whether or not to take part, read the information 
below and ask questions about anything you do not understand.  Your taking part is 
entirely your choice. 
 
1. 
Name of the Subject (“Study Subject”) 
 
 
2. 
Title of Research Study 
 
 
A Randomized Double-blind Trial of Fluconazole Versus Voriconazole for the 
Prevention of Invasive Fungal Infections in Allogeneic Blood and Marrow 
Transplant Patients 
 
3. 
Principal Investigator Contact Information 
 
 
 
 
 


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Contact information for emergencies after hours or on weekends or holidays: 
 
Call (xxx) xxx-xxxx, the in-patient Bone Marrow Transplant Unit.  Ask to speak 
to the Charge Nurse.  
 
4. 
Sponsor and Source of Funding or Other Material Support 
 
 
The sponsor of this study, The National Institutes of Health (NIH), is providing the 
study drugs and research tests free of charge to study subjects through the Blood and 
Marrow Transplantation Clinical Trials Network (BMT CTN), a group of 32 U.S. 
transplant centers. 
 
5. 
What is the purpose of this study? 
 
 
You are being asked to take part in this research study because you are having a 
blood or bone marrow transplant (BMT) using cells from another person. 
 
 
One of the most common side effects of BMT is a decrease in the number or 
strength of infection fighting cells (white blood cells) in the body.  Infections are caused by 
bacteria, viruses or fungi.  When white blood cell numbers are low or the cells are weak, 
the body can’t fight infections.  For this reason, all BMT patients take drugs to prevent 
infection by these germs. 
 
 
Fungal infections are one of the worst forms of infection.  One of the drugs used to 
both prevent and treat fungal infections is called fluconazole.  Fluconazole prevents some, 
but not all, types of fungal infections.  Voriconazole is a newer antifungal drug that can 
treat more types of fungal infections than fluconazole.  But, voriconazole has more side 
effects than fluconazole.  Although voriconazole is used to treat severe fungal infections, it 
has not yet been tested for the prevention of fungal infections. 
 
 
The purpose of this study is to compare fluconazole and voriconazole in the 
prevention of fungal infections in blood or marrow transplant patients where another 
person is the donor.  About 600 patients will take part in this study at many centers 
around the country. 
 


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6. 
What will be done if you take part in this research study? 
 
 
Before starting treatment in this study, your doctor will check your general health. 
This will include a physical exam and taking blood (about 3 teaspoons or 10-20 mL for 
adult patients).  A CT scan of the chest will be required if the results of the baseline 
galactomannan blood draw are not available.  A CT scan is a type of x-ray that takes many 
detailed pictures.  A chest CT scan results in an estimated dose of 0.8 rem, equivalent to 
2.25 years of naturally occurring background radiation in the United States.  A CT scan of 
the abdomen and/or sinuses may be required.  An abdomen CT results in 1.1 rem, 
equivalent to 3 years.  A head (sinus) CT results in 0.55 rem, equivalent to 1.7 years of 
background.  If you are a female able to have children, a pregnancy test will also be 
performed.  If you are pregnant, you will not be able to take part in this study. 
 
 
If you choose to take part in this study, you will be randomly assigned (much like 
the toss of a coin) to receive either fluconazole or voriconazole.  You and your study 
doctor will not know whether you are receiving fluconazole or voriconazole.  This is called 
a double-blind study.  In an emergency, the name of your drug can be obtained quickly.  
Treatment will begin on the day of your transplant (Day 0).   
 
 
Since voriconazole is given twice per day and fluconazole only once per day, a 
placebo (a pill with no active drug or a salt water infusion) will be used to keep us from 
knowing which drug you are receiving.  You will take the assigned drug twice a day.  
Whenever possible, the drug will be given as pills.  If you are unable to take pills, the drug 
will be given over two hours by an IV infusion through your catheter.  If you have been 
assigned to fluconazole, one of the pills (or infusions) will be a placebo since fluconazole is 
normally taken once daily. 
 
 
You will continue the study drug until 100 days after your transplant.  At around 
90 to 100 days, your doctor will test your immune function and review your 
immunosuppressive drugs.   If your risk of fungal infection is high, you will stay on study 
drug until 180 days.  The study drug will stop by 180 days.  But your doctor can give you 
an antifungal drug for longer if he/she believes you need it. 
 
 
While you are in the study, your doctor will conduct routine and study-specific 
tests (such as x-rays, CT scans, or blood tests) and check you for a fungal infection.  The 
table below shows when you will have study-required blood tests.  The tests are described 
after the table. 
 


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Time 
Amount of Blood 
per Draw (adults)* 
Type of Test / Purpose** 
Standard of CareS / InvestigationalI 
Pre transplant 
1 teaspoon (5 mL)  
1 teaspoon (5 mL)  
1 teaspoon (5 mL)  
Liver function testsS/ eligibility 
Galactomannan assayS/ eligibility 
Future testingI/ research  
3x/week until 
engraftment 
1 teaspoon (5 mL) 
CBCS/ blood chemistry 
1x/week until Day 100.  
Also on Day 120, 150 
and 180 if patient 
remains on study drug 
1 teaspoon (5mL) 
Liver function testsS/ check for bad side effects 
Day 14 and 28 
1 teaspoon (5 mL)  
Pharmacokinetics (PK) I/ test for drug levels 
2x/week, Days 1 – 60 
1 tablespoon (15 mL)   
 
Galactomannan (GM) assayS and Investigational 
Fungal DiagnosticsI/ tests for fungal infections  
1x/week***, Days 61 – 
100 
1 tablespoon (15 mL)   
 
GM assayS and Investigational Fungal 
DiagnosticsI/ tests for fungal infections 
If you have bad side 
effects 
1 teaspoon (5 mL)  
 
PKI/ test for drug levels 
 
If doctor suspects you 
have a possible fungal 
infection 
4 teaspoons (20 mL) at 
onset 
 
 
1 tablespoon (15 mL)  
2x/week for up to 14 
days while on empirical 
antifungal therapy 
PKI/ test for drug levels, GM assayS and 
Investigational Fungal DiagnosticsI/ tests for 
fungal infections 
 
GM assayS and Investigational Fungal 
DiagnosticsI/ tests for fungal infections 
If you get a fungal 
infection 
4 teaspoons (20 mL) at 
onset 
 
 
1 tablespoon (15 mL)  
2x/week for 4 weeks 
and then once every 2 
weeks for 8 weeks, for 
a total of 12 samples 
PKI/ test for drug levels, GM assayI and 
Investigational Fungal DiagnosticsI/ tests for 
fungal infections  
 
GM assayI and Investigational Fungal 
DiagnosticsI/ measure response to treatment 
 
* 
Pediatric patients less than 12 years old will provide one-half of the required amounts of blood.  
The center may make adjustments in blood volumes collected from patients less than 12 years old 
to meet the institution’s Human Investigation Committee Guidelines. 
** Tests that are typically done for your care are considered standard of care.  These tests will be 
charged to you.  Tests that are done solely for research purposes are considered investigational and 
will not be charged to you. 
*** Patients will be tested twice weekly from Days 61-100 if they (1) received a T cell depleted 
transplant (a transplant where certain white cells have been removed to reduce the risk of GVHD) 
and post transplant GVHD prophylaxis (medications designed to prevent GVHD), or (2) have or 
have had GVHD (a condition in which the donated cells recognize the recipient’s cells as non-self 
and attack them) requiring systemic therapy, or (3) are taking steroids. 
 


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Blood (1 tablespoon or 15 mL) will be taken prior to the transplant to determine 
eligibility for the study.  A small sample (1 teaspoon or 5 mL) will be saved for future 
testing. 
 
 
Blood samples (1 teaspoon or 5 mL) will also be taken 3x/week to determine 
engraftment (when the infused stem cells begin to multiply its own cells in your bone 
marrow).  Engraftment typically occurs within 1-4 weeks post-transplant. 
 
 
Weekly blood tests (about 1 teaspoon or 5 mL) will be taken for the first 100 days 
(and on Day 120, 150 and 180 if you remain on study drug) to check for bad side effects.  
Blood (1 teaspoon or 5 mL) will also be taken to measure drug levels at about Day 14 and 
28, plus if you develop any bad side effects possibly related to the study drug, and at the 
start of a fungal infection.  Usually, blood can be taken from your central venous catheter.  
In addition, we will also take blood (about 1 tablespoon or 15 mL), twice a week for the 
first 60 days of the study, and extra samples if your doctor suspects you have an infection 
to see if new tests can detect a serious fungal infection earlier and more easily than current 
tests.   
 
 
Blood samples will also be taken once per week from Days 61-100 unless: (1) you 
received a T cell depleted transplant and received post-transplant GVHD prophylaxis or, 
(2) you are on steroids, or (3) you have or have had acute GVHD requiring systemic 
therapy.  Then blood samples will be taken twice weekly from Days 61-100.  Other tests 
will also be done to see which treatment is more effective in treating infections.   
 
 
If your doctor suspects a fungal infection he/she may give you a drug 
(amphotericin B, Ambisome, Abelcet, Amphotec or Caspofungin) to treat it while he/she 
is reviewing your symptoms.  You will continue to receive the study drug.  If the tests do 
not show a fungal infection, your doctor will stop the other drug within 14 days.  If your 
doctor believes that you need the other drug after 14 days, then he/she will continue it but 
the study drug will stop.  You will continue to be tested as part of the study for the full 
year.  Your doctor will still do the research tests, including the blood tests described above 
just as if you were taking the study drug.   
 
 
If you get a fungal infection, the study drug will be stopped and another therapy 
will be discussed with you.  This could include voriconazole if your doctor believes it to be 
the best choice.  Your blood will be tested twice weekly for four weeks and then once 
every two weeks for eight weeks, for a total of 12 samples, to test your response to 
treatment.   
 
 
This study will employ a new test approved by the FDA for detecting fungal 
infections in adults using blood samples.  It is called the galactomannan test.  


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Galactomannan is a part of the fungus that gets into the blood of infected patients.  Some 
(1 teaspoon or 5 mL) of the blood samples described above will be used for this test. 
 
 
This galactomannan test will be used with other standard tests to check you for 
certain kinds of fungal infection.  The results will be used along with your medical history, 
physical check up, and other laboratory tests to judge whether you have or may have an 
infection.  If you have a procedure on your lungs known as a bronchoscopy to test for an 
infection, a small part of the fluid obtained will be saved for later testing by the 
galactomannan test and other investigational tests to see if these new tests are better than 
the tests used now.  The galactomannan test will also be used to test response to treatment 
if you develop an infection.  
 
 
It is not yet clear if the galactomannan test will be better than the old tests and it is 
possible that it may not be as good in patients receiving one of the study drugs and certain 
other antifungal drugs, including amphotericin B and caspofungin.  One good thing about 
this new test is that it requires only a sample of blood rather than a biopsy of tissue or 
other more uncomfortable tests.  It also may show infection earlier.  However, it may not 
be as exact.  This could mean that if the test is positive and you really are not infected, you 
may receive treatment you don’t need or if the test is negative and you really are infected, 
you may not receive treatment you need.  For these reasons, your doctor will be using 
several kinds of tests for your safety.  Results from this study will be used to measure the 
new test’s value. 
 
 
After your treatment is complete, we would like to keep track of your progress 
until 12 months after your transplant.  To do this, we will contact you or your local doctor. 
 
7. 
Will You Provide Samples for Research? 
 
Genetic material is any sample of blood, tissue, fluid, etc. that is obtained from 
you.  You will be asked to provide samples of blood, tissue and fluid to be used solely 
for future research and testing in laboratories where we are studying fungal infections.  
These samples will not require additional procedures.  Your name will not be on these 
samples.  You do not have to agree to provide these research samples to participate in 
the study. 
 
 
If you agree, a small blood sample (1 teaspoon or 5 mL) will be drawn pre-
transplant and saved for future testing.  Additional blood samples (2 teaspoons or 10 
mL) will be taken, if you agree, each time samples are drawn to test for galactomannan.  
Usually the blood can be drawn from your central venous catheter at the time of other 
blood collections.  If this is not possible, then it would be drawn directly from a vein. 
 


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If you agree, small samples of tissue or fluid (1 mL) will be collected post-
transplant and saved for future testing.  These samples will be taken from samples that 
are collected for diagnosis of potential fungal infections.  Additional procedures are not 
required. 
 
 
The samples collected for research purposes will be sent to laboratories that have 
contracts with the National Marrow Donor Program (NMDP) to conduct these research 
tests.  They will be labeled with unique codes that do not contain information that could 
identify you.  A link to this code does exist.  The link is stored at the Data Coordinating 
Center for the Blood and Marrow Transplant Clinical Trials Network (BMT CTN).  The 
staff at the laboratories where your samples are being tested do not have a link to this 
code.  Your samples will be stored at these laboratories until the entire sample has been 
used for the research tests or until the end of the study. 
 
 
If any of your samples are leftover after the research studies are completed, these 
samples will either be destroyed or be sent to the National Heart Lung and Blood 
Institute (NHLBI) sample repository in Maryland.  If your leftover samples are sent to 
the repository, they will be given an anonymous code.  These leftover samples stored at 
the repository can never be linked to you.  Any research performed on these left-over 
samples must first be approved by an advisory panel at the NHLBI. 
 
 
If you agree to allow your blood, tissue and fluids to be kept for research, you are 
free to change your mind at any time.  We ask that you tell [the Principal Investigator] in 
writing and let him/her know you are withdrawing your permission for your samples to 
be used for research.  The mailing address is on the first page of this form.  Any unused 
samples will be destroyed.  You are free not to take part in this future research and still 
take part in the other parts of the study.  There will be no change in your care if you 
decide not to give these extra samples.   Please mark your choice below for each sample:  
 
(1) Pre-transplant 5 mL blood sample 
 I agree to have a blood sample drawn pre-transplant for future research and testing. 
 I do not agree to have a blood sample drawn pre-transplant for future research and 
testing. 
 
(2) Post-transplant 10 mL blood samples taken each time blood samples are taken for 
the galactomannan (GM) assay  
 I agree to have blood samples drawn post-transplant for future research and testing. 
 I do not agree to have blood samples drawn post-transplant for future research and 
testing. 
 


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(3) Post-transplant tissue and fluid samples each time samples are taken post-
transplant for diagnosis of fungal infections 
 I agree to have tissue and fluid samples collected post-transplant for future research 
and testing. 
 I do not agree to have tissue and fluid samples collected post-transplant for future 
research and testing. 
 
____________________________________                             _______________________ 
             Signature                                                                                     Date 
 
8. 
What are the possible discomforts and risks? 
 
 
Cancer treatments can cause bad side effects, some of which may be life 
threatening or could kill you.  The drugs used in this study may cause all, some, or none 
of the side effects listed below.  Also, there is always the chance of unexpected new side 
effects. 
 
 
The most common side effect in patients who have received voriconazole has 
been a change in eyesight that was often described as a “brightness” in vision that 
usually lasts less than one hour.  Some patients treated with voriconazole have reported 
temporary glare or blurred vision.  The effect may begin within 30 minutes after taking 
a dose of voriconazole and lasts an average of one-half hour but may last up to one 
hour.  You should not drive or operate machinery during these periods.  The next most 
common side effect was abnormal liver tests.   
 
The following side effects have been reported in more than 1% of patients who 
have received voriconazole: a reaction at the site of injection, photosensitivity (a rash 
caused by sunlight), weakness, dizziness, headache, trouble sleeping, dry mouth, 
nausea, stomach pain, and low potassium levels.  Other side effects that have been 
reported in at least one patient that was treated with voriconazole include: skin rash, 
skin erythema (redness), psoriasis (scaling) with eosinophilia (an abnormal blood 
condition), hypoglycemia (low blood sugar), hepatitis (liver disease), nausea, vomiting 
and jaundice (yellow skin), pancreatitis (a disease of the pancreas with pain in the 
stomach), decrease in the number of cells in the bone marrow (this was reported in a 
patient who had leukemia before starting voriconazole), and irregular heartbeat.  An 
irregular heartbeat resulting in death occurred during treatment in a patient who had a 
history of an irregular heartbeat before entering the study.  The patient also had a 
history of cancer and was on chemotherapy and many other drugs (including a 
potassium infusion, which may have affected the heart beat).  In addition, an irregular 
heartbeat possibly related to taking voriconazole with the medications pimozide, 


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quinidine, dofetilide, and quinupristin-dalfopristin (an antibiotic) has been reported.  
One patient with leukemia, who was also taking this antibiotic, was hospitalized 
because of faintness and palpitations (rapid heartbeats).   
 
 
Voriconazole may increase or decrease levels of other commonly prescribed 
drugs and non-prescription drugs.  Drugs known to interact with voriconazole include 
warfarin, phenytoin, cyclosporine, tacrolimus, sirolimus, rifabutin, ompreazole and 
rifampin.  Other drugs that may interact with voriconazole include ritonavir, lovastatin 
(and other statins), benzodiazepines, sulfonylureas, clarithromycin, terfenadine, 
astemizole, cisapride, dihydropyridine calcium channel blockers (verapamil, diltiazem), 
certain chemotherapy drugs (vinca alkaloids) and cabamazepine.  We ask that you 
consult with your doctor before starting new drugs or increasing the dose of any drug 
including non-prescription drugs or herbs such as St. John’s Wort.  Grapefruit juice 
should be avoided.  Other drugs that you may be taking may need to be changed or 
stopped in order for you to receive voriconazole. 
 
 
The most frequent side effects after treatment with fluconazole affect the 
gastrointestinal tract (stomach and intestines) such as nausea and vomiting.  Other side 
effects include dizziness, headache, skin rash, and liver problems.  Serious side effects 
which have been reported with fluconazole include: liver damage which included 
deaths primarily in patients with serious medical conditions; elevated liver tests; severe 
skin disorders; hepatitis (liver disease), jaundice (yellow skin), irregular heartbeats or 
heart function changes; and acute allergic reactions.   
 
 
The risks of drawing blood from a vein include discomfort at the site of puncture; 
possible bruising and swelling around the puncture site; rarely, an infection; and, 
uncommonly, faintness from the procedure. 
 
 
Only you, the person for whom it has been prescribed, can take the study drug.  If 
the drug is not packaged in a childproof container, you should keep it out of the reach of 
children and persons who have limited ability to read or understand.  By federal law, this 
drug cannot be given or transferred to anyone for whom it is not prescribed. 
 
 
As noted earlier, a new test, the galactomannan test, will be used along with other 
tests to see if you have an infection and will be used to test your response to treatment if 
you develop an infection.  It is possible that this test may be less exact in patients taking 
one of the study drugs (voriconazole) when compared to the other (fluconazole).  It is 
hoped that information from the study will help show that the new test can quickly and 
easily detect fungal infections.   
 


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Throughout the study, the researchers will tell you of new information that might 
affect your decision to remain in the study. 
 
 
If you wish to discuss the information above or any other discomforts you may 
experience, you may ask questions now or call the Principal Investigator or contact 
person listed on the front page of this form. 
 
9a. 
What are the possible benefits to you? 
 
 
Although this study cannot be guaranteed to be of benefit to you, it is hoped that 
your taking part may prevent you from getting a fungal infection.  A possible advantage 
of this study is that one antifungal treatment may prevent fungal infections better than the 
other.  However, you may not benefit from this treatment.  The use of the galactomannan 
test along with other diagnostic tests may allow us to more quickly and more easily 
diagnose fungal infections and determine how well you respond to treatment if you 
develop an infection.  However, there is no guarantee that this test is better than the 
currently available tests.   
 
9b. 
What are the possible benefits to others? 
 
 
Future patients may benefit from the results of this study. 
 
10. 
If you choose to take part in this study, will it cost you anything? 
 
 
Your cost for care on this research study will not be higher than for standard 
treatment for this disease.  The BMT CTN will cover the cost of the study drug.  You 
will remain responsible for the costs of standard treatment for your disease.  Your 
insurance provider may not cover all or part of these costs.  You or your family will 
have to pay installments based on your verified ability to pay.  Any questions about 
these charges should be discussed with the Principal Investigator of the study. 
 
11. 
Will you receive payment for taking part in this research study? 
 
 
No. 
 


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12. 
What if you are injured because of the study? 
 
 
If you experience an injury that is directly caused by this study, only the 
professional medical care you receive at the [participating clinical facility] will be provided 
without charge.  You or your insurance provider will pay hospital expenses.  No other 
compensation is offered.  If you have any questions about study-related injuries, you may 
call [insert name of person at institution] at [insert phone number]. 
 
13. 
What other options or treatments are available if you do not want to be in this 
study? 
 
 
Taking part in this study is entirely up to you.  You are free to refuse to be in the 
study, and your refusal will not affect current or future health care you receive at this 
institution.  You and your doctor will discuss any other treatment options available to you. 
 
 
The current standard therapy for preventing fungal infections in patients receiving 
bone marrow or stem cell transplant is fluconazole.  Your doctor will review other 
treatments with you.   
 
14a. 
How can you withdraw from this research study? 
 
 
If you agree to be in this study, you are free to change your mind.  At any time you 
may withdraw your consent to be in this study and for us to use your data.  If you 
withdraw from the study, you will continue to have access to health care at [participating 
clinical facility].  If you decide to withdraw, we ask that you tell [the Principal 
Investigator] in writing; his/her mailing address is on the first page of this form.  If you do 
withdraw your consent, there will be no penalty and you will not lose any benefits to 
which you are otherwise entitled.  You will be asked to return any unused study drug.  
You will also be asked to return for a checkup before you stop your study drug.  Even if 
you withdraw, or your doctor withdraws you, from the study, you are asked to have 
research tests conducted and allow the study investigators to collect that information. 
 
 
If you have any questions about your rights as a study subject, you may call the 
Institutional Review Board (IRB) office at (xxx) xxx-xxxx. 
 
14b. 
If you withdraw, can information about you still be used and/or collected? 
 
 
If you withdraw from the study, we ask that you agree that we can continue using 
all information about you that has already been collected as part of the study prior to your 
withdrawal, and to continue to allow your doctor to tell us about your progress until 12 
months after your transplant.  You may, of course, say no. 


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14c. 
Can the Principal Investigator withdraw you from this research study? 
 
 
You can be taken off the study (with or without your consent) for any of the 
following reasons: 
 You do not qualify to be in the study because you do not meet the study 
requirements.  Ask your doctor if you would like more information about this. 
 You need a medical treatment not allowed in this study. 
 The investigator decides that continuing in the study would be harmful to you. 
 The study treatments have a bad effect on you. 
 You become pregnant and the study treatment could be harmful to the fetus. 
 You are unable to keep appointments or take study drugs as directed. 
 Other study-specific reasons; for example, if the dose of study drug you are taking 
has been found to be unsafe. 
 The study is cancelled by the Food and Drug Administration (FDA) or the National 
Institutes of Health (NIH). 
 
15. 
How will your privacy and the confidentiality of your research records be 
protected? 
 
 
Study records that have your name will be kept private as required by law.  You 
will not be identified by name in the central study records.  Your records will be given a 
unique code number.  The key to the code will be kept in a locked file in the Principal 
Investigator’s office.  Authorized persons from [the participating clinical facility], the 
hospital or clinic (if any) involved in this research, and the Institutional Review Board 
have the legal right to review your research records and will protect their confidentiality 
to the extent permitted by law.  This research study is sponsored by and conducted with 
funds from the National Institutes of Health; therefore, the sponsor, the Blood and 
Marrow Transplant Clinical Trials Network (BMT CTN), the investigators conducting this 
study and the FDA also have the legal right to review your research records.  Otherwise, 
your research records will not be shown to anyone without your consent unless required 
by law or a court order. 
 
 
If the results of this research are published or presented at scientific meetings, your 
name will not be disclosed. 
 
 
Information related to or resulting from your stem cell transplant will be reported to the 
Center for International Blood and Marrow Transplant Research (CIBMTR).  The CIBMTR is a 
voluntary organization of basic and clinical scientists working together in an effort to gather 


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information on results of stem cell and marrow transplants.  This information is used to guide 
clinical decisions and identify ways to improve transplant outcomes.   Scientific data or medical 
information (not identifiable with you) that could be useful to others may be presented at 
meetings and/or published in medical journals. 
 
16. 
Expiration date for retention of records 
 
 
The study results will stay in your research record at (insert Institution) for at least 
six years or until after the study is completed, whichever is longer.  At that time either the 
research information not already in your medical record will be destroyed or your name 
and other identifying information will be removed from such study results.  Research 
information in your medical record will be kept indefinitely. 
 
17. 
How will the researcher(s) benefit from your being in this study? 
 
 
In general, presenting research results helps the career of a scientist.  Therefore, the 
Principal Investigator may benefit if the results of this study are presented at scientific 
meetings or in the scientific press.  In addition, the sponsor is paying the Principal 
Investigator to conduct this study. 
 
18. HIPAA2 authorization to use and disclose individual health information for 
research purposes 
 
a. Purpose:  As a research participant, I authorize the Principal Investigator and the 
researcher’s staff to use and disclose my individual health information for the 
purpose of conducting the research study entitled A Randomized, Double-blind, 
Trial of Fluconazole vs. Voriconazole for Prevention of Invasive Fungal Infections in 
Allogeneic Blood and Bone Marrow Transplant Patients. 
 
b. Individual Health Information to be Used or Disclosed: My individual health 
information that may be used or disclosed to conduct this research includes: 
demographic information (e.g., age, date of birth, sex, weight), medical history 
(e.g., diagnosis, complications with prior treatment), physical examination 
findings, and laboratory test results obtained at the time of work up and after 
transplantation (e.g., CT scan, blood tests, biopsy results).  
 
                                                   
2 HIPAA is the Health Insurance Portability and Accountability Act of 1996, a federal law related to privacy of health 
information 


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c. Parties Who May Disclose My Individual Health Information: The researcher 
and the researcher’s staff may obtain my individual health information from: 
(list hospitals, clinics or providers from which health care information can be requested) 
 
______________________________________________________________________ 
 
______________________________________________________________________ 
 
______________________________________________________________________ 
 
d. Parties Who May Receive or Use My Individual Health Information: The 
individual health information disclosed by parties listed in item c and 
information disclosed by me during the course of the research may be received 
and used by the following parties: 
 Principal Investigator and the researcher’s staff 
 Dr. John Wingard, Study Chairperson and staff/laboratories at University of 
Florida College of Medicine  
 Dr. 
Thomas 
Walsh, 
Study 
Chairperson 
and 
staff/laboratories 
at 
NIH/NCI/POB 
 Staff/laboratories identified in the protocol for the evaluation of other 
laboratory samples; e.g., Dr. Patricia Fraser/Harvard 
 National Heart, Lung and Blood Institute (NHLBI) and the National Cancer 
Institute (NCI), both of the National Institutes of Health (NIH), study 
sponsors  
 Blood and Marrow Transplant Clinical Trials Network (BMT CTN), data 
coordinating center 
 U.S. government agencies that are responsible for overseeing research such as 
the Food and Drug Administration (FDA) and the Office of Human Research 
Protections (OHRP) 
 U.S. government agencies that are responsible for overseeing public health 
concerns such as the Centers for Disease Control (CDC) and federal, state and 
local health departments. 
 
e. Right to Refuse to Sign this Authorization: I do not have to sign this 
Authorization.  If I decide not to sign the Authorization, I will not be allowed to 
participate in this study or receive any research-related treatment that is 
provided through the study.  However, my decision not to sign this 


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authorization will not affect any other treatment, payment, or enrollment in 
health plans or eligibility for benefits.   
 
f. Right to Revoke:  I can change my mind and withdraw this authorization at any 
time by sending a written notice to the Principal Investigator to inform the 
researcher of my decision.  If I withdraw this authorization, the researcher may 
only use and disclose the protected health information already collected for this 
research study.  No further health information about me will be collected by or 
disclosed to the researcher for this study. 
 
g. Potential for Re-disclosure: My individual health information disclosed under 
this authorization may be subject to re-disclosure outside the research study and 
no longer protected.  Examples include potential disclosures for law enforcement 
purposes, mandated reporting or abuse or neglect, judicial proceedings, health 
oversight activities and public health measures. 
 
h. 
This authorization does not have an expiration date. 


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19. 
Consent and Assent Instructions 
 
Consent: 
Subjects 18 years and older must sign on the subject line below. 
For subjects under 18, consent is provided by the Legally Authorized 
Representative 
 
Assent: 
Is required for subjects under the age of 18, using the Assent Section on 
the following page. 
 
I have been informed about this study’s purpose, procedures, possible benefits and 
risks.  I have been given the chance to ask questions.  My questions have all been 
answered satisfactorily.  I understand that I can ask other questions at any time. 
 
I voluntarily agree to take part, or to allow my child to take part, in this study. 
 
By signing this consent form, I have not given up any of the legal rights that I (my child) 
otherwise would have as a subject in a research study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Subject‘s Signature  
 
 
 
 
Date 
 
If you are not the subject, please print your name 
 
 
 
 
 
and indicate one of the following: 
 
 
 
The subject’s parent  
 
     The subject’s guardian 
 
 
A surrogate  
 
 
     A durable power of attorney 
 
 
A proxy 
 
 
 
     Other, please explain: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Legally Authorized Representative Signature  
Date 
 
 
As a representative of this study, I have explained the purpose, the procedures, the 
benefits, and the risks that are involved in this research study: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature of person conducting informed consent  
Date 
 


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ASSENT SIGNATURES:  For subjects under the age of 18 years. 
 
 
Assent of Minor 
I have been told what I will be asked to do if I am in this study.  I have been told that I 
don’t have to be in this study.  I may quit the study at any time, and no one will be mad 
at me.  I have had a chance to discuss the study and ask questions.  My questions have 
been answered.  I agree to be in the study and do what I am asked to do so long as I 
continue in the study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature of Minor  
 
 
Date  
 
Age (years) 
 
Study Personnel 
 
I have explained the purposes, procedures, and risks involved in this research study in 
detail to: 
 
 
 
 
 
 
 
 
 
 
 
 
 
          
Print name(s) of Parents/Authorized Consenting Party, and  
 
 
 
 
, who in my opinion 
IS/ 
IS NOT capable of assenting to 
participate in this study. 
 
Print child’s name 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature of Person Conducting Assent   
 
Date 
 
 


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APPENDIX B-2 
 
CONSENT FORMS 
 
 
 
DONOR INFORMED CONSENT 
FOR SNP ASSAY 
 


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CO-PRINCIPAL INVESTIGATORS: John R. Wingard, M.D. and Thomas J. Walsh, M.D.  
STUDY TITLE: A Randomized, Double-blind, Trial of Fluconazole vs. Voriconazole for 
Prevention of Invasive Fungal Infections in Allogeneic Blood and Bone Marrow 
Transplant Patients  
 
 
INTRODUCTION 
 
We invite you to take part in a research study sponsored by the National Institutes of 
Health (NIH). 
 
First, we want you to know that: 
Taking part in NIH research is entirely voluntary. 
You may choose not to take part, or you may withdraw from the study at any time.  In 
either case, you will not lose any benefits to which you are otherwise entitled.  
You may receive no benefit from taking part.  The research may give us knowledge that 
may help people in the future. 
 
Second, some people have personal, religious or ethical beliefs that may limit the kinds of 
medical or research treatments they would want to receive (such as blood transfusions).  If 
you have such beliefs, please discuss them with your doctors or research team before you 
agree to the study. 
 
Now we will describe this research study.  Before you decide to take part, please take as 
much time as you need to ask any questions and discuss this study with your family, 
friends, or your personal physician or other health professional. 
 
 
PURPOSE 
 
During the course of this study, we will attempt to learn about genetic factors that may 
have an influence on infections due to invasive fungal infections.  This type of infection 
occurs in individuals with a breakdown in the body’s immune system, due to either 
medications, (such as corticosteroids) or in those who have had bone marrow 
transplantation.  We are interested in studying the small variations or differences in genes, 
called polymorphisms or variants that could effect the body’s ability to fight off invasive 
fungal infections.  We also will study genes that help the body eliminate fluconazole and 
voriconazole from the blood stream.  We have identified a collection of genes (twenty-six), 
each of which has one or more polymorphism.  We would like to analyze your DNA for 
these twenty-six genes to see if any of these genes are associated with fungal infections.  
We invite you to participate in this study so that we can learn more about these genetic 


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factors that may influence the susceptibility and severity of fungal infections in those who 
undergo blood or marrow transplantation. 
 
 
PROCEDURES 
 
Five (5) milliliters of blood or stem cell product will be used for the genetic analysis.  We 
ask that you submit a sample of no more than 5 mL, equivalent to one teaspoon.  It may be 
necessary to perform a buccal swab of the inside of your mouth to obtain genomic DNA if 
blood is unable to be drawn.  If you withdraw from the study, your samples will not be used 
for other research studies or tested further.   
 
Clinical information (e.g., HLA typing) about you/your child will be collected.  NIH will 
not have access to the names of the patients enrolled in this study.  The clinical information 
will be coded and compared to the genetic analysis of the twenty-six genes chosen for our 
study: MBL2, CCR5, IL1RN, IL1A, IL1B, IL6, IL8, IL8RA, IL8RB, IL10, TNFA, TNFB, 
MPO, NRAMP1, CHIT1, FCGR2A, FCGR3A, FCGR3B, MICA, MICB, TLR4, CD14, 
HBD-1, IL-18 and two cytochrome P450 genes (3A4 and 2C19).  In the laboratory, we will 
isolate DNA from your blood or stem cell product and test for each of these genes by 
standard techniques.  We shall determine normal and variant sequences in DNA and 
compare this information to the clinical information collected.  
 
 
ALTERNATIVES 
 
You may choose not to participate in this part of the study.  The decision to participate in 
this study will not affect the care given to you by your physicians.  
 
 
RISK AND DISCOMFORT 
 
It is possible that the information from this study could be important for family members 
(even though no blood and no analysis of family members are proposed).  However 
individual results will not be reported directly to you.   
 
There is a small risk of an infection or fainting from the blood draw.  If blood is not 
available, a buccal swab from the inside of your mouth needs to be performed by a health 
care provider.  This may result in minimal discomfort in the mouth during the time the 
cotton swab is rubbed against the inside of the cheek of your mouth.   
 
At no time will this information be made available to those not directly involved in the 
study without your written consent.  Patient information will not be made public. Only the 
results of the proposed analysis will be collected, presented and published.  At no time, will 


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the name or an identifier of patients be available to anyone except those conducting the 
study.  The investigators who will conduct the genetic analysis will not have access to the 
names of the patients enrolled in this study.  The clinical information will be coded and 
compared to the genetic analysis of the twenty-six genes chosen for the study. 
 
 
BENEFITS 
 
This study will increase our understanding of the factors that influence the risk for 
developing and treating fungal infections during bone marrow transplantation.  We hope 
that it will eventually contribute to improvements in medical care, treatment and prevention 
of these types of infection.  There may be no direct benefit to you or your family from this 
study.  If there are any questions, we will attempt to answer them with the most recent 
information.   
 
 
SAMPLES FOR RESEARCH AND FUTURE TESTING 
 
Genetic material is any sample of blood, tissue, fluid, etc. that is obtained from you.  You 
will be asked to provide a sample of blood, stem cell or buccal product to be used solely for 
future research and testing in laboratories where we are studying fungal infections.  This  
sample will not require additional procedures.  You do not have to agree to provide this 
research sample to participate in the study. 
 
A portion of your 5 mL blood sample, stem cell product or buccal swab product, if you 
agree, will be saved for future testing and research related to infectious diseases or the 
immune system.  These tests may include genetic analysis of your DNA beyond the 26 
genes identified in this document. 
 
The samples collected for research purposes will be sent to laboratories that have contracts 
with the National Marrow Donor Program (NMDP) to conduct these research tests.  They will 
be labeled with unique codes that do not contain information that could identify you.  A link 
to this code does exist.  The link is stored at the Data Coordinating Center for the Blood and 
Marrow Transplant Clinical Trials Network (BMT CTN).  The staff at the laboratories where 
your samples are being tested do not have a link to this code.  Your samples will be stored at 
these laboratories until the entire sample has been used for the research tests or until the end 
of the study. 
 
If any of your samples are leftover after the research studies are completed, these samples will 
either be destroyed or be sent to the National Heart Lung and Blood Institute (NHLBI) sample 
repository in Maryland.  If your leftover samples are sent to the repository, they will be given 
an anonymous code.  These leftover samples stored at the repository can never be linked to 


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you.  Any research performed on these leftover samples must first be approved by an advisory 
panel at the NHLBI. 
 
If you agree to allow your blood, stem cell or buccal product to be kept for research, you are 
free to change your mind at any time.  We ask that you tell [the Principal Investigator] in 
writing and let him/her know you are withdrawing your permission for your sample to be used 
for research.  The mailing address is on the first page of this form.  Any unused sample will be 
destroyed.  You are free not to take part in this future research and still take part in the 
other parts of the study.  There will be no change in your care if you decide not to give 
this extra sample.  Please mark your choice below:  
 
 
 
 I agree to use of a sample of my blood, stem cell or buccal swab product for additional 
research or future testing.  
 
 I do not agree to use a sample of my blood, stem cell or buccal swab product for any 
additional research or future testing.  
 
 
________________________________________________________ __________________ 
Signature  
 
 
 
 
 
 
 
Date 
 
 
HIPAA3 AUTHORIZATION TO USE AND DISCLOSE INDIVIDUAL HEALTH 
INFORMATION FOR RESEARCH PURPOSES 
 
1.  Purpose:  As a research participant, I authorize the Principal Investigator and the 
researcher’s staff to use and disclose my individual health information for the purpose of 
conducting the research study entitled A Randomized, Double-blind, Trial of Fluconazole 
vs. Voriconazole for Prevention of Invasive Fungal Infections in Allogeneic Blood and 
Bone Marrow Transplant Patients. 
 
 
2.  Individual Health Information to be Used or Disclosed: My individual health 
information that may be used or disclosed to conduct this research includes: Demographic 
information (e.g., age, date of birth, sex, weight), medical history, physical examination 
findings, and genetic test results.  
                                                   
3 HIPAA is the Health Insurance Portability and Accountability Act of 1996, a federal law related to privacy of health 
information. 


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3. 
Parties Who May Disclose My Individual Health Information: The researcher and the 
researcher’s staff may obtain my individual health information from (list hospitals, clinics 
or providers from which health care information can be requested): 
 
___________________________________________________________________ 
 
___________________________________________________________________ 
 
___________________________________________________________________ 
 
 
4.  Parties Who May Receive or Use My Individual Health Information: The individual health 
information disclosed by parties listed in item 3 and information disclosed by me during the 
course of the research may be received and used by the following parties: 
 
Principal Investigator and the researcher’s staff 
 
Dr. John Wingard, Study Chairperson and staff/laboratories at University of Florida 
College of Medicine  
 
Dr. Thomas Walsh, Study Chairperson and staff/laboratories at NIH/NCI/POB 
 
Staff/laboratories identified in the protocol for the evaluation of other laboratory 
samples; e.g., Dr. Patricia Fraser/Harvard 
 
National Heart, Lung and Blood Institute (NHLBI) and National Cancer Institute 
(NCI), both of the National Institutes of Health (NIH), study sponsors  
 
Blood and Marrow Transplant Clinical Trials Network (BMT CTN), data 
coordinating center 
 
U.S. government agencies that are responsible for overseeing research such as the 
Food and Drug Administration (FDA) and the Office of Human Research Protections 
(OHRP) 
 
U.S. government agencies that are responsible for overseeing public health concerns 
such as the Centers for Disease Control (CDC) and federal, state and local health 
departments. 
 
 
5.  Right to Refuse to Sign this Authorization: I do not have to sign this Authorization.  If I 
decide not to sign the Authorization, I will not be allowed to participate in this study or 
receive any research-related treatment that is provided through the study.  However, my 
decision not to sign this authorization will not affect any other treatment, payment, or 
enrollment in health plans or eligibility for benefits.   
 


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6.  Right to Revoke:  I can change my mind and withdraw this authorization at any time by 
sending a written notice to the Principal Investigator to inform the researcher of my decision.  
If I withdraw this authorization, the researcher may only use and disclose the protected health 
information already collected for this research study.  No further health information about me 
will be collected by or disclosed to the researcher for this study. 
 
 
7.  Potential for Re-disclosure: My individual health information disclosed under this 
authorization may be subject to re-disclosure outside the research study and no longer 
protected.  Examples include potential disclosures for law enforcement purposes, mandated 
reporting or abuse or neglect, judicial proceedings, health oversight activities and public 
health measures. 
 
 
8. This authorization does not have an expiration date. 
 
 


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OTHER PERTINENT INFORMATION 
 
1.  Confidentiality. When results of an NIH research study are reported in medical journals or at scientific meetings, the people 
who take part are not named and identified.  In most cases, the NIH will not release any information about your research 
involvement without your written permission.  However, if you sign a release of information form, for example, for an insurance 
company, the NIH will give the insurance company information from your medical record.  This information might affect (either 
favorably or unfavorably) the willingness of the insurance company to sell you insurance. 
 
The Federal Privacy Act protects the confidentiality of your NIH medical records.  However, you should know that 
the Act allows release of some information from your medical record without your permission, for example, if it is required by 
the Food and Drug Administration (FDA), members of Congress, law enforcement officials, or other authorized people. 
 
2.  Policy Regarding Research-Related Injuries.  The Clinical Center will provide short-term medical care for any injury 
resulting from your participation in research here.  In general, no long-term medical care or financial compensation for research-
related injuries will be provided by the National Institutes of Health, the Clinical Center, or the Federal Government.  However, 
you have the right to pursue legal remedy if you believe that your injury justifies such action. 
 
3.  Payments.  The amount of payment to research volunteers is guided by the National Institutes of Health policies.  In general, 
patients are not paid for taking part in research studies at the National Institutes of Health. 
 
4.  Problems or Questions.  If you have any problems or questions about this study, or about your rights as a research 
participant, or about any research-related injury, contact the Principal Investigator at (xxx) xxx-xxxx. 
 
5.  Consent Document.  Please keep a copy of this document in case you want to read it again. 
 
COMPLETE APPROPRIATE ITEM BELOW, A or B 
A. Adult Patient's Consent. 
I have read the explanation about this study and have been 
given the opportunity to discuss it and to ask questions. I 
hereby consent to take part in this study. 
 
 
Signature of Adult Patient & Date Signed 
 
 
 
THIS CONSENT DOCUMENT HAS BEEN APPROVED 
FOR USE FROM _________ THROUGH __________. 
B. Parent's Permission for Minor Patient. 
I have read the explanation about this study and have been 
given the opportunity to discuss it and to ask questions. I 
hereby give permission for my child to take part in this study. 
(Attach NIH 2514-2, Minor’s Assent, if applicable) 
 
Signature of Parent(s)/Guardian & Date Signed 
If other than parent, specify relationship:  
 
 
 
Child's Verbal Assent (if applicable). 
The information in the above consent form has been 
adequately described to my child in language that my child 
can understand, and my child willingly agrees to participate 
in the study. 
 
Signature of Parent(s)/Guardian & Date Signed 
 
Signature of Investigator & Date Signed 
 
Signature of Witness & Date Signed 


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APPENDIX C 
 
LABORATORY PROCEDURES 


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APPENDIX C 
 
LABORATORY PROCEDURES 
 
 
LABORATORY SPECIMEN COLLECTION, STORAGE AND SHIPPING 
PROCEDURES 
 
Standard procedures for collection, storage, and shipping of specimens will be followed 
according to the NMDP and the NHLBI guidelines.  Samples will be given a unique 
alphanumeric code that contains no personal identifiers.  Transplant Center Coordinators will 
hold the link to the code.  Laboratory staff will not have access to the link. 
 
 
LABORATORY CONTRACTS AND REMAINING SAMPLES 
 
All laboratory studies will be performed at laboratories under contract with the NMDP on behalf 
of the BMT CTN.  The laboratory contract specifies that any remaining sample must be stored at 
the laboratory for the duration of the study.  If the investigators choose to perform additional 
studies on these remaining samples, a formal amendment will be made to the protocol.  Any 
amendments to the protocol are subject to the DSMB and IRB approval process. 
 
At the end of the study, the BMT CTN will either instruct the laboratory to destroy any 
remaining samples or to transfer the remaining samples to the NHLBI sample repository in 
Maryland.  These samples will be paired with the respective donor or recipient sample and given 
unique bar code designations that cannot be linked back to the donor or the recipient.  An 
NHLBI Biologic Specimen Repository Utilization Committee will advise the Institute on 
requests for specimens to perform research with these anonymous samples.  If an investigator 
request for these samples is approved by the committee, the NHLBI may provide a panel of the 
specimens requested using unique code numbers.  Laboratory test results, clinical information, 
etc., associated with the coded samples are provided to the investigator only after completion of 
his/her research protocol.  Samples sent to researchers cannot be linked with any remaining 
sample at the repository. 
 
 
PHARMACOKINETICS 
 
Blood for analysis of fluconazole and voriconazole concentrations will be obtained on Day 14 
and 28 of therapy, at onset of serious suspected study drug toxicities as delineated in Section 
2.4.6 and at onset of possible, presumptive, probable or proven infection (prior to start of 
antifungal therapy).  Blood should be drawn in relationship to administration of the study drug in 
one of three windows of time after the start of the infusion or administration of the oral dose: 
Window 1 is 1-5 hours after start of the infusion; Window 2 is 5-8 hours after start of infusion; 
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time of the drug and the time of the blood draw.  The form will calculate from which of the three 
windows the blood was drawn.  Testing of pharmacokinetic samples will be conducted at Dr. 
Tom Walsh’s lab at NCI. 
 
Collection and Processing:  Collect 5 mL (2.5 mL for patients less than 12 years old) of blood in 
green top vacutainer; centrifuge for 20 minutes within 120 minutes (preferably 60 minutes) of 
collection at 450 x g or 1500 rpm; transfer plasma to a cryovial; sample may stored at 2-8ºC for 
24 hours prior to cryopreservation at -70ºC; batch ship frozen samples on dry ice quarterly to the 
repository. 
 
 
ANTIFUNGAL SUSCEPTIBILITY TESTING 
 
Collection and Processing Procedures:  All fungal isolates will be collected and identified to the 
species level at participating centers.  Isolates will then be streaked onto Sabouraud dextrose agar 
slants, grown at 35°C until confluent, and stored at –20°C until use.  Serial isolates from a given 
patient will be collected every seven days when possible following initiation of antifungal 
therapy, or if infection relapses following the discontinuation of therapy.  Frozen isolates will be 
shipped in batch quarterly to the Study Repository in compliance with the shipping procedures 
specified in the BMT CTN MOP.  The antifungal susceptibility testing will be performed by Dr. 
Nguyen at the University of Florida College of Medicine.  
 
Assay Procedures:  The antifungal susceptibility testing will be performed according to the 
NCCLS recommendations (NCCLS M27-A for yeast and NCCLS M38-P for moulds) [54, 55].  
The susceptibility testing will be performed in batch, and repeated twice on two different days to 
ensure that the results are reproducible.  The reference isolate Aspergillus flavus ATCC 204304 
and the quality control strain Candida parapsilosis ATCC 22019 will be included in each batch 
of testing as control isolates.  
 
1.  Hypothesis 
The hypothesis, for patients treated with fluconazole, is that breakthrough fungal infections 
will be caused by yeasts or moulds exhibiting elevated fluconazole minimum inhibitory 
concentrations (MICs).  A number of these organisms, however, will exhibit low MICs of 
voriconazole.  The hypothesis, for patients treated with voriconazole, is that breakthrough 
fungal infections will be caused by yeasts or moulds exhibiting elevated MICs of both 
fluconazole and voriconazole.  These organisms will also exhibit elevated MICs of 
itraconazole, consistent with broad azole cross-resistance.  In addition, we hypothesize that 
mould infections in patients receiving fluconazole will be primarily caused by aspergillus 
spp., whereas mould infections in patients receiving voriconazole will be caused by Rhizopus 
or moulds other than aspergillus spp. 
 
2.  Antifungal Agents 
All isolates will be tested in vitro against fluconazole (Pfizer Inc., New York, NY), 
itraconazole (Janssen Pharmaceutical, Titusville, NJ), and voriconazole (Pfizer Inc., New 
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3.  Assay Medium  
The medium used will be RPMI 1640 medium without sodium bicarbonate and with L-
glutamine.  It will be buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid, and 
sterilized by filtration.  
 
4.  Drug Concentration Ranges  
Drug dilutions will be prepared at 100 times the final concentration in 100% dimethyl 
sulfoxide followed by further dilutions (1:50) in RPMI-1640 medium to yield twice the final 
concentrations required for the test.  The final concentrations of the tested drugs will range 
from 0.125 to 64 µg/mL for fluconazole and 0.03 to 16 µg/mL for itraconazole and 
voriconazole. 100 µl of twofold serial dilutions of the antifungal drugs will be dispensed into 
each well of sterile flat-bottomed plastic microtitre plates.  Two drug-free-medium wells will 
be used per row as sterility and growth controls.  The plates will be prepared in batch and 
stored at –70°C until used. 
 
5.  Inoculum Preparations  
Yeasts: Two days before the susceptibility test, the yeast strains will be streaked for isolation 
from the –70oC stock onto Sabouraud Dextrose Agar (SDA).  These strains will be streaked 
again onto SDA a day before the susceptibility test and incubated at 35oC.  The yeast 
suspension will then be prepared from five colonies of each culture to match the turbidity of a 
McFarland 0.5 standard as determined by a standard spectrophotometric method.  Further 
dilutions will be made in RPMI 1640 medium to achieve a final yeast inoculum of 0.5 × 103 
to 2.5 × 103 CFU/mL. 
 
Moulds:  Stock inoculum suspensions will be prepared as described in the NCCLS M38-P 
document from 7-day-old cultures grown on potato dextrose agar slants and adjusted 
spectrophotometrically to optical density that ranges from 0.09 to 0.3 (82 to 60% 
transmittance).  This stock solution will contain mainly conidia.  The nongerminated conidial 
inoculum suspensions will be diluted 1:50 in medium.  The final concentration of stock 
inoculum will range from 0.5 × 106 to 4.5 × 106 CFU/mL. 
 
6.  Determination of MICs 
On the day of the test, the microtitre plates containing drug will be allowed to thaw at room 
temperature.  A 100 µl aliquot of the tested inoculum will be added to each well.  The plates 
will then be incubated at 350C and examined at the time points described.  MIC will be 
determined by visual inspection.  For yeasts, the MIC will be determined at both 24 and 48h 
according to a 0-to-4 scale, with 0 indicating an optically clear culture, 1 indicating a slightly 
hazy culture, 2 indicating a prominent decrease in turbidity, 3 indicating a slight decrease in 
turbidity, and 4 indicating no reduction in turbidity.  The MIC will be defined as the lowest 
concentration of a drug for which the score is 2 [54].  For moulds, the MIC will be 
determined at 48h, and will be defined as the lowest drug concentration that yields a 
prominent inhibition of growth compared to the growth control well [55].  
 


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INVESTIGATIONAL FUNGAL DIAGNOSTIC ASSAYS TO DIAGNOSE 
ASPERGILLUS AND OTHER INFECTIONS 
 
New assays, including antigen, antibody, metabolite, and PCR techniques, are being developed 
to improve diagnostic accuracy.  Whole blood will be collected for storage and assay by future 
investigational tests.  These blood samples will be collected twice weekly on two non-
consecutive days, preferably three days apart (e.g., Mondays and Thursdays), during the first 60 
days post-transplant and once weekly on Days 60-100 unless one of the following criteria is met: 
(1) patient received a T cell depleted transplant and received post-transplant GVHD prophylaxis; 
or, (2) patient is on steroids; or, (3) patient has or has had acute GVHD requiring systemic 
therapy.  Investigational fungal diagnostic samples will be collected twice weekly from these 
select patients.  Investigational fungal diagnostic samples will also be collected at onset of 
possible, presumptive, probable and proven infections and twice weekly from patients receiving 
empirical antifungal therapy for possible invasive infection.  Investigational fungal diagnostic 
samples will also be collected from patients with presumptive, probable or proven infection 
twice weekly for four weeks, then once every two weeks for eight weeks and then once to twice 
weekly until Day 100.  The investigational assays will be conducted at Dr. John Wingard’s lab at 
the University of Florida College of Medicine. 
 
Collection and Processing: 
Serum: Collect 5 mL (2.5 mL for patients less than 12 years old) of blood in red top vacutainer; 
allow 15-30 minutes for clotting; centrifuge at 900 x g or 2100 rpm for 10 minutes; (clot and 
serum should be distinct); carefully remove the serum without disturbing the clot (leaving behind 
a small volume of serum if necessary) and transfer to a cryovial; sample may stored at 2-8ºC for 
24 hours prior to cryopreservation at -70ºC; batch ship frozen samples on dry ice quarterly to the 
repository. 
 
Blood: Collect 5 mL (2.5 mL for patients less than 12 years old) of blood in green top 
vacutainer; transfer whole blood, without centrifugation, to a cryovial; sample may stored at 2-
8ºC for 24 hours prior to cryopreservation at -70ºC; batch ship frozen samples on dry ice 
quarterly to the repository. 
 
 
GENETIC VARIATION STUDY SAMPLING SINGLE NUCLEOTIDE 
POLYMORPHISMS (SNPs) 
 
A major goal of human genetics is to understand the contribution of genetic diversity to disease 
susceptibility and outcome.  With the completion of a first generation map of the human genome, 
a catalogue of known genes and their annotated sequence variations has begun to take shape1, 2.  
 
DNA sequence variations include insertions, deletions, variable numbers of repeat sequences, 
and single nucleotide polymorphisms (SNPs).  The latter, SNPs, are the most common variants 
that occur and create alleles that can be investigated for their association with a specific, defined 
disease outcome3.  Current estimates are that there could be as many as 10 million SNPs (defined 
on the basis of the frequency of the minor allele being greater than 1% in at least one 
population)4.  Until recently, the ability to perform genetic association studies with SNPs has 
been limited by the paucity of known SNPs.  Advances in high-throughput sequence analysis and 


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DNA chip arrays systems are generating an expanding set of reagents that can be used for 
investigating the relationship between genetic variations and disease.  For the past five years, we 
have exclusively utilized the candidate SNP approach5.  However, new technical developments 
and comprehensive SNP catalogues should not only improve the utility of SNP association 
studies, but also in the near future allow their use in classical linkage studies; however, for the 
immediate future, this approach is not viable nor is it economically feasible to search for such 
genetic markers.  Accordingly, we will continue to concentrate on candidate SNPs to dissect 
their genetic contribution to complex diseases6.  The focus will shift from the study of previously 
characterized SNPs of known biological importance, towards the identification of novel SNPs 
and haplotypes in groups of genes belonging to key pathways.  Haplotypes comprise a set of 
genetic variants residing on one chromosome and represent ancestral segments inherited by 
descent from shared ancestors7. 
 
In order to validate SNPs and standardize assays, the laboratory devised a program that has been 
adapted and supported by the Cancer Genome Anatomy Program (CGAP).  This web-based 
resource, the SNP500Cancer (http://snp500cancer.nci.nih.gov).  SNP500Cancer is a new 
database designed to publicly address the immediate needs for candidate SNPs and candidate 
gene approaches to mapping complex diseases. SNP500Cancer provides bi-directional 
sequencing information on a set of control DNA samples derived from anonymized subjects (102 
Coriell samples representing four self-described ethnic groups: African/African-American, 
Caucasian, Hispanic and Pacific Rim).  All SNPs are chosen from public databases and reports, 
and the choice of genes includes a bias towards nonsynonymous and promoter SNPs in genes 
that have been implicated in one or more cancers.  The website is searchable for SNPs by gene, 
chromosome and by known dbSNP ID.  For each analyzed SNP, gene location and over 200 bps 
of surrounding annotated sequence (including nearby SNPs) are provided, with frequency 
information in total and per sub-population.  A calculation of Hardy-Weinberg Equilibrium is 
included.  Moreover, genotyping assays (5’-exonuclease, hybridization-triggered fluorescence, 
and/or MALDI-TOF) were developed for SNPs with minor allele frequencies > ~5%.  These 
assays have been performed on the same 102 samples and the results compared for concordance 
with the sequencing results.  The website will soon post the conditions (including sequences for 
probes, primers, etc.) for validated genotyping assays.  Estimation of haplotypes is currently 
provided for select genes, with the goal of expanding this effort rapidly.  SNP500Cancer 
provides an invaluable resource for investigators world-wide to select validated candidate SNPs 
linked to established genotyping assays and sequence data.  In addition, this project forms the 
cornerstone for the assay validation pipeline of the Core Genotyping Facility. 
 
In this prospective study, the purpose is to study genetic variants in genes of innate immunity, 
which could contribute to fungal infections.  Already, the paradigm has been established in a 
number of infections, such as malaria, filariasis, RSV infection and most recently in candidiasis 
in leukemia patients (Choi et al in press JID, 2003).  Moreover, the initial rationale was based 
upon the search for genetic determinants of infectious complications using immune SNPs in a 
well-defined population receiving comparable therapy, but the design is suitable for investigating 
pharmacogenomic questions at a later time8.  The choice of genes was made because on the basis 
of three criteria: (1) well defined SNPs altered function of the gene (either the protein sequence 
or expression), (2) a frequency for the variants was greater than 5% (most are between 20 and 
35% for the minor alleles), which provides adequate opportunity for statistical analysis and (3) 


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prior association studies 8-9.  A total of 20 genes will be studied by Dr. Tom Walsh at 
NIH/NCI/POB.  The genes were chosen from several pathways within innate immunity, 
specifically, because these pathways are not substantially altered by changes in lymphocyte or 
phagocyte populations following myelotoxic therapy.  The 18 genes involved in intrinsic host 
defense include MBL2, CCR5, IL1RN, IL1A, IL1B, IL6, IL8, IL8RA, IL8RB, IL10, TNFA, TNFB, 
MPO, NRAMP1, CHIT1, FCGR2A, FCGR3A, and FCGR3B.  The other two genes will be 
studied for the metabolism of voriconazole (3A4 and 2C19).  The products of the 20 following 
genes mediate their respective functions (indicated in parentheses): MBL2 (involved in innate 
host recognition of fungal pathogens); CCR5, IL8, IL8RA, and IL8RB (mediate recruitment of 
PMNs); IL1RN, IL1A, IL1B, and IL6, TNFA, TNFB (pro-inflammatory cytokine mediators); IL10 
(suppression of phagocytic host defenses); MPO (mediator of phagocytic oxidative injury); 
NRAMP (mediates intracellular phagocytic injury and divalent metal efflux); CHIT1 (mediates 
chitin and fungal cell wall hydrolysis); FCGR2A, FCGR3A, and FCGR3B (antibody receptors); 
and 3A4 and 2C19 (cytochrome P450-dependent hepatic microsomal enzymes involved in 
metabolic clearance of voriconazole).  Additional genes mediating host response and metabolism 
will be studied as new technology is developed.   
 
Additional SNP analyses in six other genes involved in the innate immune response to fungal 
infection will be conducted by Dr. Patricia Fraser at the Center for Blood Research at Harvard 
Medical School.  The six genes and their respective functions (indicated in parenthesis) include 
MICA and MICB (augment cytolysis mediated by  T cells and NK cells and co-stimulate 
CD8+ T cells in pathogen-specific immune response), TLR4 and CD14 (TLR4 mediates cell 
signaling from exposure to microbial pathogens such as fungi and complexes with CD14 which 
facilitates monocyte activation by Aspergillus hyphae), HBD-1 (expresses antifungal properties) 
and IL-18 (confers protection against pulmonary and disseminated infections). 
 
Collection and Processing:  Collect 5mL of blood or stem cell product (2.5 mL for patients less 
than 12 years old) from related donor in green top vacutainer; transfer whole blood, without 
centrifugation, to two cryovials; samples may be stored at 2-8ºC for 24 hours prior to 
cryopreservation at -70ºC; batch ship frozen samples on dry ice to the repository. 
 
Sample Analysis  
 
1.  Dr. Tom Walsh at NIH/NCI/POB and Dr. Patricia Fraser at the Center for Blood 
Research at Harvard Medical School will conduct extraction of genomic DNA by 
standard technique under SOPs filed at their laboratories. 
 
2. Samples will be given a numerical number and a record linking the number to patient 
information will be kept confidential.  It will only be available for investigators 
participating in this study.  Samples will be linked to clinical data collected under the 
supervision of Dr. Walsh.  Genomic samples will not be shared with other investigators 
unless approved by an IRB amendment and informed consent is given. 
 
3.  Sample tracking will be accomplished using a numerical code assigned each consecutive 
patient enrolled on study.  Clinical data will be entered on datasheets containing the 
polymorphism/variant data on the 26 genes of the study.  


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4. Genetic analysis will be performed on fragments amplified by the polymerase chain 
reaction (PCR) and subjected to analysis designed to distinguish between known alleles 
in the following genes: MBL2, CCR5, IL1RN, IL1A, IL1B, IL6, IL8, IL8RA, IL8RB, IL10, 
TNFA, TNFB, MPO, NRAMP1, CHIT1, FCGR2A, FCGR3A, FCGR3B, 3A4, 2C19, 
MICA, MICB, TLR4, CD14, HBD-1and IL-18.  Each study will be confirmed at least 
twice before entry into the spread sheet data bank. 
 
Data Evaluation 
 
1.a 
Sample analysis will be performed as they are made available.  
 
1.b 
The genomic DNA of each patient will be genotyped for allelic variations in the 26 
genes: MBL2, CCR5, IL1RN, IL1A, IL1B, IL6, IL8, IL8RA, IL8RB, IL10, TNFA, 
TNFB, MPO, NRAMP1, CHIT1, FCGR2A, FCGR3A, FCGR3B, 3A4, 2C19, MICA, 
MICB, TLR4, CD14, HBD-1and IL-18.  The genotype determination for each gene 
will be considered to be a separate variable for each patient. 
 
1.c 
Data will be analyzed with respect to each gene in terms of allelic frequency and 
separately in terms of gene distribution (wild type, heterozygotes and homozygotes 
for the gene being analyzed).  
 
1.d 
Frequencies will be compared to the general population norms- both published and 
established in our laboratory.  Comparison for statistical significance will be 
evaluated within the cohort to determine if specific alleles are over or under-
represented. 
 
2.  Epidemiological data on the infectious complications of all enrolled will be evaluated and 
entered into the database program for comparison with genotypic evaluation of individual 
patients.  Continuous and non-continuos variable analysis will be performed.  
 
3.  The primary outcome will be a direct comparison of the allelic frequencies of a specific 
gene or combination of genes in the identified population to established controls (already 
established in our laboratory and in the literature).  Secondary outcomes that will undergo 
statistical analysis are continuous variables (e.g., complications, response to therapy and 
presenting parameters).  
 
4.  After univariate analyses are performed in an exploratory mode for the individual loci, 
multivariate analyses of the possibly correlated outcomes will be performed. This 
analysis will be performed with as few as two loci and as many as all 26 loci.  
 
5. Complex trait analysis will be performed on the database. All of the variables included in 
the infectious events will be analyzed with respect to cohorts of patients determined by 
allelic frequency. 
 


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PARAFFIN BLOCKS OF INFECTED TISSUE SPECIMENS 
 
Paraffin blocks from tissues found to be infected should be saved and batch sent quarterly at 
room temperature to the Study Repository in compliance with the shipping procedures specified 
in the BMT CTN MOP.  Dr. John Wingard at the University of Florida will analyze these 
specimens for identification of fungal pathogen where cultures were negative but organisms were 
seen microscopically. 
 
INVESTIGATIONAL FUTURE TESTING 
 
Blood for investigational future testing will be collected at baseline prior to initiation of 
conditioning.  The identification and location of test(s) to be performed have not been 
determined. 
 
Collection and Processing:  Collect 5 mL (2.5 mL for patients less than 12 years old) of blood in 
red top vacutainer; allow 15-30 minutes for clotting; centrifuge at 900 x g or 2100 rpm for 10 
minutes; (clot and serum should be distinct); carefully remove the serum without disturbing the 
clot (leaving behind a small volume of serum if necessary) and transfer equally to two cryovials; 
samples may stored at 2-8ºC for 24 hours prior to cryopreservation at -70ºC; batch ship frozen 
samples on dry ice quarterly to the repository. 
 
DIAGNOSTIC GALACTOMANNAN ASSAYS 
 
Incorporation of the “real-time” diagnostic GM assay along with classical criteria will improve 
accuracy and may increase the number of documented invasive aspergillosis cases (by upgrading 
possible cases not proven by classical criteria to proven or probable cases with the GM assay).  
Blood samples for the GM assays will be collected twice weekly on non-consecutive days, 
preferably three days apart (e.g., Mondays and Thursdays), during the first 60 days post-
transplant and once weekly on Days 60-100 unless one of the following criteria is met: (1) 
patient received a T cell depleted transplant and received post-transplant GVHD prophylaxis; or, 
(2) patient is on steroids; or, (3) patient has or has had acute GVHD requiring systemic therapy.  
GM blood samples will be collected twice weekly from these select patients.  Diagnostic GM 
samples should also be collected at onset of possible infections and twice weekly from patients 
receiving empirical trials of amphotericin B or caspofungin for possible invasive infection.  
 
GM samples may be processed at contract laboratories or hospital laboratories associated with 
the transplant center if the laboratory is certified by CLIA, CAP or JCAHO.  Samples to be 
processed at these certified laboratories must be collected twice weekly on two non-consecutive 
days, preferably three days apart.  Results are required within 72 hours of collection.  It is 
recommended that these certified laboratories complete Bio-Rad’s proficiency panel and use the 
criteria for galactomannan positivity as defined in §3.2 of the protocol, i.e., two consecutive 
positives on one sample. 
 
Collection and Processing:  Collect 5 mL of blood (2.5 mL for patients less than 12 years old) in 
gold top plastic serum separator tube; allow 15-30 minutes for clotting; centrifuge at 900 x g or 
2100 rpm for 10 minutes but do not decant or transfer unless instructed otherwise by reference 


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lab; store at 2-8°C for up to 72 hours if necessary; ship sample on cold packs immediately via 
priority overnight to the reference lab according to the lab’s instructions.  
 
 
INVESTIGATIONAL MONITORING OF TREATMENT GALACTOMANNAN ASSAYS 
  
For patients with presumptive, probable or proven aspergillus infection, monitoring of treatment 
GM samples will be collected at onset and twice weekly for four weeks and then once every two 
weeks for eight weeks for a total of 12 samples. These samples will be processed at the Fred 
Hutchinson Cancer Research Center at a later date.  Results will not be made available to the 
transplant center. 
 
Collection and Processing:  Collect 5 mL of blood (2.5 mL for patients less than 12 years old) in 
gold or red top, plastic or glass serum separator tube; allow 15-30 minutes for sample to clot; 
centrifuge at 900 x g or 2100 rpm for 10 minutes (clot and serum should be distinct); carefully 
remove the serum without disturbing the clot (leaving behind a small volume of serum) and 
transfer to cryovial; sample may stored at 2-8ºC prior to cryopreservation at -70ºC; batch ship on 
dry ice quarterly to the repository. 
 
 
INVESTIGATIONAL GALACTOMANNAN ASSAYS OF BAL 
 
Whenever a bronchoscopy is performed, 1 mL of BAL (bronchoalveolar lavage) will be 
collected in a cryovial, frozen at -70ºC, and batch shipped quarterly to the Repository.  These 
samples will be processed at the Fred Hutchinson Cancer Research Center at a later date.  
Results will not be made available to the transplant center. 
 
 
REPOSITORY INFORMATION 
 
Room temperature and frozen samples should be batched and shipped quarterly to the Repository 
in compliance with the shipping procedures specified in the BMT CTN MOP and the BMT CTN 
0101 Laboratory Sample Information Guide:  
 
Misti Dowell 
NHLBI Repository 
SeraCare BioServices 
217 Perry Parkway 
Gaithersburg, Maryland  20877 
Phone: (301) 208-8100 
Fax: 
(301) 208-8829 
Email: 
nhlbi@bbii.com 
 
 


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REFERENCES IN APPENDIX C 
 
1. 
Venter JC, Adams MD, Myers EW, et al. The sequence of the human genome. Science 
2001; 291:1304-51. 
2. 
Lander ES, Linton LM, Birren B, et al. Initial sequencing and analysis of the human 
genome. Nature 2001; 409:860-921. 
3. 
Kruglyak L, Nickerson DA. Variation is the spice of life. Nat Genet 2001; 27:234-6. 
4. 
Taylor JG, Choi EH, Foster CB, Chanock SJ. Using genetic variation to study human 
disease. Trends Mol Med 2001; 7:507-12. 
5. 
Chanock S. Candidate genes and single nucleotide polymorphisms (SNPs) in the study of 
human disease. Dis Markers 2001; 17:89-98. 
6. 
Schork NJ, Fallin D, Thiel B, et al. The future of genetic case-control studies. Adv Genet 
2001; 42:191-212. 
7. 
Fallin D, Cohen A, Essioux L, et al. Genetic analysis of case/control data using estimated 
haplotype frequencies: application to APOE locus variation and Alzheimer's disease. 
Genome Res 2001; 11:143-51. 
8. 
Chanock SJ, Foster CB. SNPing away at innate immunity. J Clin Invest 1999; 104:369-
70. 
9. 
Foster CB, Chanock SJ. Mining variations in genes of innate and phagocytic immunity: 
current status and future prospects. Curr Opin Hematol 2000; 7:9-15. 
 


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Table C-1 
LABORATORY SCHEDULE 
 
 
TEST 
TYPE OF  
SAMPLE 
(collection container) 
TYPE OF STORAGE 
DATES SAMPLES OBTAINED 
SHIPPING 
SPECIFICATIONS 
LOCATION OF 
TEST PERFORMED 
Pharmacokinetics 
5 mL peripheral blood1 
(green top tube) 
Centrifuge for 20 min 
within 120 min of 
collection; transfer 
plasma to cryovial 
Store at –70oC 
Day 14 & 28 of therapy, at onset of serious 
suspected drug toxicities as delineated in Section 
2.4.6 and at onset of possible, presumptive, 
probable or proven infection (prior to start of 
antifungal therapy). 
Frozen shipment quarterly 
to Repository as specified 
in the BMT CTN 
MOP/BMT CTN 0101 
Laboratory Sample 
Information Guide 
Dr. Walsh’s Lab at NCI 
Antifungal 
Susceptibility Test 
Fungal isolate from any 
infected site, if 
available 
Streak on agar slant 
Store at –20oC 
Serial isolates every 7 days if available following 
initiation of antifungal therapy, or if infection 
relapses following discontinuation of therapy. 
Frozen shipment quarterly 
to Repository as specified 
in the BMT CTN 
MOP/BMT CTN 0101 
Laboratory Sample 
Information Guide 
Dr. Nguyen’s Lab at 
University of Florida 
College of Medicine 
Investigational 
Fungal Diagnostic 
Assays 
5 mL peripheral blood1 
(red top tube) 
 
 
 
 
 
 
 
___________________ 
5 mL peripheral blood1 
(green top tube) 
Sit for 15-30 minutes to 
allow for clotting 
centrifuge for 10 min; 
transfer serum to 
cryovial 
May store at 2o-8° C for 
up to 24 hours 
For longer storage 
–70°C 
 
___________________ 
Transfer to cryovial 
without centrifugation 
May store at 2 o-8° C 
for up to 24 hours 
For longer storage 
 –70°C 
2x/week from Day 0 to Day 60.  After Day 60, 
samples will be collected once per week until Day 
100 unless one of the following is met: (1) patient 
received a T cell depleted transplant and received 
post-transplant GVHD prophylaxis, or (2) patient is 
on steroids, or (3) patient has or has had acute 
GVHD requiring systemic therapy.  These patients 
will provide samples 2x/week until Day 100.  
Samples will also be collected at onset of possible 
fungal infection and 2x/week in patients receiving 
an empirical trial of amphotericin B or caspofungin 
for possible invasive fungal infection.  For patients 
with presumptive, probable or proven aspergillus 
infection, 2x/week for 4 weeks and then once every 
2 weeks for 8 weeks for a total of 12 samples, and 
then 1-2x/week until Day 100. 
Frozen shipment quarterly 
to Repository as specified 
in the BMT CTN 
MOP/BMT CTN 0101 
Laboratory Sample 
Information Guide 
Dr. John Wingard’s Lab 
at University of Florida 
College of Medicine 
Donor SNPs (related 
donor) 
(Single Nucleotide 
Polymorphisms) 
5 mL peripheral blood 
or stem cell product 
from donor (green top 
tube) 
 
Transfer without 
centrifugation equally 
to two 3.6 mL cryovials 
Store at –70ºC 
At baseline prior to transplant. 
Frozen shipment quarterly 
to Repository as specified 
in the BMT CTN 
MOP/BMT CTN 0101 
Laboratory Sample 
Information Guide 
Dr. Walsh’s Lab at NCI 
and Dr. Fraser’s Lab at 
Harvard 


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TEST 
TYPE OF  
SAMPLE 
(collection container) 
TYPE OF STORAGE 
DATES SAMPLES OBTAINED 
SHIPPING 
SPECIFICATIONS 
LOCATION OF 
TEST PERFORMED 
Paraffin Blocks of 
Infected Tissues 
Paraffin blocks 
Room temperature 
At time of infection. 
Ship at room temp. 
quarterly to Repository as 
specified in the BMT CTN 
MOP/BMT CTN 0101 
Laboratory Sample 
Information Guide 
Dr. Wingard’s Lab at 
the University of 
Florida College of 
Medicine 
Diagnostic 
Galactomannan 
Assays2 
5 mL peripheral blood1 
(gold hemogard tube)  
 
Sit for 15-30 minutes to 
allow for clotting 
Centrifuge for 10 min 
(but do not decant or 
transfer), leave in gold 
hemogard collection 
tube unless instructed 
otherwise by reference 
lab 
Store at 2 o–8°C  
2x/week from Day 0 to Day 60.  After Day 60, 
samples will be collected once per week until Day 
100 unless one of the following is met: (1) patient 
received a T cell depleted transplant and received 
post-transplant GVHD prophylaxis; or, (2) patient 
is on steroids; or, (3) patient has or has had acute 
GVHD requiring systemic therapy.  These patients 
will provide samples 2x/week until Day 100.  
Samples will also be collected prior to and at onset 
of possible fungal infection and 2x/week in patients 
receiving an empirical trial of amphotericin B or 
caspofungin for possible invasive fungal infection.   
Cold pack shipment 
PRIORITY OVERNIGHT 
to reference lab in 
compliance with shipping 
procedures specified in the 
BMT CTN MOP/BMT 
CTN 0101 Laboratory 
Sample Information Guide 
Certified contract lab 
or certified hospital lab 
associated with the 
transplant center 
Investigational 
Monitoring of 
Treatment 
Galactomannan 
Assays 
5 mL peripheral blood1 
(gold hemogard tube)  
 
Sit for 15-30 minutes to 
allow for clotting 
Centrifuge for 10 min; 
transfer serum to 
cryovial  
May store at 2º-8ºC for 
up to 24 hours. Freeze 
at –70ºC  
For patients with presumptive, probable or proven 
aspergillus infection, at onset and 2x/week for 4 
weeks and then once every two weeks for a total of 
12 samples. 
Frozen shipment quarterly 
to Repository as specified 
in the BMT CTN 
MOP/BMT CTN 0101 
Laboratory Sample 
Information Guide 
Dr. Kieren Marr’s Lab 
at Fred Hutchinson 
Cancer Research Center 
Investigational 
Galactomannan 
Assays of BAL 
(bronchoalveolar 
lavage) 
1 mL BAL 
(cryovial) 
May store at 2º-8ºC for 
up to 24 hours.  
 Freeze at –70ºC 
Anytime bronchoscopy is performed. 
Frozen shipment quarterly 
to Repository as specified 
in the BMT CTN 
MOP/BMT CTN 0101 
Laboratory Sample 
Information Guide 
Dr. Kieren Marr’s Lab 
at Fred Hutchinson 
Cancer Research Center 


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TEST 
TYPE OF  
SAMPLE 
(collection container) 
TYPE OF STORAGE 
DATES SAMPLES OBTAINED 
SHIPPING 
SPECIFICATIONS 
LOCATION OF 
TEST PERFORMED 
Investigational 
Future Testing 
5 mL peripheral blood1 
(red top tube) 
Sit for 15-30 minutes to 
allow for clotting 
Centrifuge for 10 min; 
transfer serum to two 
cryovials. 
May store at 2º-8ºC for 
up to 24 hours.  For 
longer storage  
–70ºC 
Baseline. 
Frozen shipment quarterly 
to Repository as specified 
in the BMT CTN 
MOP/BMT CTN 0101 
Laboratory Sample 
Information Guide 
TBD 
 
Notes for Table C-1:   
1 Collect 2.5 mL for pediatric patients less than 12 years old  
2 Diagnostic GM samples must be collected twice weekly on two non-consecutive days preferably three days apart (e.g., Monday and Thursday). 
 


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D-1 
 
APPENDIX D 
 
GALACTOMANNAN ASSAY 
 
 
 
Platelia® Aspergillus Laboratory Protocol 
with 
Platelia® Aspergillus Kit Receipt Log 
Platelia® Aspergillus Package Insert 
Platelia® Aspergillus Worksheets 
Platelia® Aspergillus Quick Reference Guide 
 
 


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D-2 
 
APPENDIX D 
TABLE OF CONTENTS 
 
 
INTENDED USE .............................................................................................................. D-3 
SUMMARY AND EXPLANATION ..................................................................................... D-3 
PRINCIPLE OF THE PROCEDURE ................................................................................. D-4 
STUDY DESIGN .............................................................................................................. D-5 
TECHNICAL INFORMATION ........................................................................................... D-9 
MATERIALS AND METHODS .......................................................................................... D-9 
  A. Materials provided by Bio-Rad Laboratories ................................................................ D-9 
  B. Materials not Provided.............................................................................................. D-10 
PRECAUTIONS ............................................................................................................. D-11 
  A. Health and Safety Precautions ................................................................................. D-11 
  B. Test Precautions ...................................................................................................... D-11 
SPECIMEN COLLECTION / TESTING ........................................................................... D-12 
  A. Specimen Collection ................................................................................................ D-12 
  B. Specimen Testing .................................................................................................... D-12 
  C. Calculation of Results .............................................................................................. D-14 
  D. Limitations of the Procedure ..................................................................................... D-15 
TRAINING / PROFICIENCY TESTING ........................................................................... D-15 
  A. Proficiency Testing Documentation Form .................................................................. D-17 
DATA COLLECTION ...................................................................................................... D-18 
 
 
ATTACHMENT A:  PATELIA® ASPERGILLUS KIT RECEIPT LOG.................................. D-19 
ATTACHMENT B:  PATELIA® ASPERGILLUS PACKAGE INSERT ................................. D-21 
ATTACHMENT C:  PATELIA® ASPERGILLUS WORKSHEETS ...................................... D-45 
ATTACHMENT D:  PATELIA® ASPERGILLUS QUICK REFERENCE GUIDE .................. D-48 
 


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D-3 
PLATELIA® ASPERGILLUS LABORATORY PROTOCOL 
for 
“A Randomized Double-blinded Trial of Fluconazole vs. Voriconazole 
for the Prevention of Invasive Fungal Infections in Allogeneic Blood and Marrow 
Transplant Patients” 
 
 
Read the entire protocol, including the appendices, before beginning the study.  Instructions 
for collecting, handling, and testing specimens are provided in detail in this protocol. 
 
 
INTENDED USE 
The Platelia® Aspergillus EIA Galactomannan Assay is a 1-stage immunoenzymatic assay utilizing the 
sandwich microplate technique for the detection of galactomannan in human serum.  This assay is to be 
used and test results interpreted in conjunction with other conventional diagnostic procedures such as 
microbiological culture, histologic examination of biopsy samples, and other signs and symptoms for 
detection of aspergillus infection.  Positive Platelia® Aspergillus EIA test results are an aid in the early 
diagnosis of Invasive Aspergillosis, and, along with other diagnostic tests and clinical signs of disease, 
allow for effective early treatment of disease.  Negative Platelia® Aspergillus EIA test results, along with 
other diagnostic tests, can be used to prevent the unnecessary use of anti-fungal agents in the absence 
of Invasive Aspergillosis.   
 
SUMMARY AND EXPLANATION 
Aspergillus infections usually occur following inhalation of aspergillus spores, which are present in the 
environment.  Invasive forms, which have been on the increase for more than 10 years, constitute the 
most serious infections.  They mainly occur in neutropenic patients (following anticancer treatment) and in 
patients treated with immunosuppressants (organ transplantations, particularly bone marrow 
transplantation) and corticosteroids4.  AIDS-related cases of aspergillus have also been reported5.   
The efficacy of treatment depends on early introduction of treatment, but the diagnosis of Aspergillosis is 
still very difficult.  The test for anti-aspergillus antibodies is not adapted to the diagnosis of invasive 
Aspergillosis in immunocompromised patients.  Aspergillus is rarely isolated from blood culture.  The 
diagnosis is often based on nonspecific or late criteria (clinical symptoms, radiology, etc.)   
At the present time, the test for soluble aspergillus antigens in the serum appears to be the only 
serological method able to improve both the rapidity and specificity in the diagnosis of the infection6,7.  
Among the various antigens present in body fluids of patients suffering from invasive Aspergillosis,  
carbohydrates and particularly galactomannan appear to be well-suited for the diagnosis of the 
infection8,9. 
                                                   
4 Denning DW, Stevens DA. 1990. Anti-fungal and surgical treatment of invasive Aspergillosis: review of 2121 published cases.  
Rev. Infect. Dis. 12:1147 - 1201. 
5 Lortholary O, Meyohas MC, Dupont B, et al.  1993.  Invasive aspergillosis in patients with acquired immunodeficiency syndrome: 
report of 33 cases.  Am J. Med. 95:177 - 187. 
6 Andriolet VT. 1993.  Infections with Aspergillus species.  Clin Infect Dis. 17 (suppl 2) : S481-6. 
7 De Repentigny L, Kaufman L. Cole GT, Druse K, Latge JP, Matthews RC. 1994.  Immunodiagnosis of invasive fungal infections. J. 
Med Vet Mycol.  32 (S1): 239 - 252. 
8 De Repentigny L. 1989.  Serological techniques for diagnosis of fungal infection.  Eur. J. Clin Microbiol Infect Dis. 8 (4) : 362 - 375. 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-4 
 
PRINCIPLE OF THE PROCEDURE 
The Platelia® Aspergillus EIA is a one-stage immunoenzymatic sandwich microplate assay that detects 
galactomannan in human serum.  The assay uses the rat monoclonal antibody EBA-2, which is directed 
against aspergillus galactomannan, and has been characterized during previous studies10 11 12.  The 
monoclonal antibody is used to sensitize the wells of the microplate, bind the antigen and as the detector 
antibody in the conjugate reagent (peroxidase-linked monoclonal antibody).  Serum samples are heat-
treated in the presence of EDTA in order to dissociate the immune-complexes and to precipitate serum 
proteins, which could possibly interfere with the EIA.  The treated serum samples and conjugate are 
added to the wells coated with the monoclonal antibody, and incubated.  A monoclonal antibody - 
galactomannan - monoclonal antibody / peroxidase complex is formed in the presence of aspergillus 
antigen.  The strips are washed to remove any unbound material.  Next, the substrate solution is added, 
which will react with the complexes bound to the well to form a blue color reaction.  The enzyme reaction 
is stopped by the addition of acid, which changes the blue color to yellow.  The optical absorbance of 
specimens and controls is determined with a spectrophotometer set at 450 and 620/630-nm wavelength. 
                                                                                                                                                                    
9 Latge JP. 1995.  Tools and trends in the detection of Aspergillus fumigatus.  Curr Top Med Mycol, in press. 
10 Latge JP, Kobayashi H, Debeaupuis JP, Diaquin M, Sarfati J, Wieruszeski JM, Parra E, Bouchara JP, Fournet B. 1994.  
Chemical and immunological characterization of the extracellular galactomannan secreted by Aspergillus fumigatus.  Infect Immun. 
62: 5424 -5433. 
11 Stynen D, Sarfati J, Symoens F, Goris A, Nolard N, Latge JP.  1991.  Rat monoclonal antibodies against exocellular carbohydrate 
antigens of Aspergillus and dermatophytes.  In: “Fungal Cell Wall and Immune Response”, 181 -193.  Ed. By J.P. Latge and D. 
Boucias,  Springer Verlag. 
12 Stynen D, Sarfati J,  Goris A, Prevost MC, Lesour M, Kamphuis H, Darras V, Latge JP.  1992.  Rat monoclonal antibodies against 
Aspergillus galactomannan.  Infect Immun. 60: 2237 - 2245. 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-5 
STUDY DESIGN 
for 
Laboratory Protocol for 
 
 
BMT CTN Protocol 0101 – “A Randomized Double-blinded Trial of Fluconazole versus Voriconazole for 
the Prevention of Invasive Fungal Infections in Allogeneic Blood and Marrow Transplant Recipients” 
 
Objectives: 
 
1) To perform exploratory analyses of quantitative aspects of the Platelia® Aspergillus EIA in 
detecting galactomannan antigen in serum as an aid in the diagnosis of invasive Aspergillosis in 
allogeneic blood and marrow transplant patients receiving prophylactic treatment with 
Fluconazole or Voriconazole (diagnostic phase). 
 
2) To perform exploratory analyses of quantitative aspects of the Platelia® Aspergillus EIA in 
determining response to anti-fungal therapy in allogeneic blood and marrow transplant patients 
with proven or probable invasive Aspergillosis (monitoring phase). 
 
Population: 
 
Allogeneic hematopoietic transplant recipients randomized to receive post-transplant prophylaxis with 
either Fluconazole (N = 300) or Voriconazole (N = 300).  The length of these studies is expected to be 
three years. 
 
Study Design: 
 
This is a randomized, double-blinded, multicenter, prospective, comparative study of Fluconazole versus 
Voriconazole prophylaxis in the prevention of invasive fungal infections in allogeneic hematopoietic 
transplant recipients.  The Platelia® Aspergillus assay for detection of galactomannan is being used in 
conjunction with host and clinical factors (see Table 3.1.1 a and b in BMT CTN Protocol 0101) to 
determine the likelihood of invasive aspergillus infection. 
 
Sample Collection and Testing: 
 
DIAGNOSTIC PHASE 
A serum specimen for the Platelia® Aspergillus assay will be collected prior to the start of the conditioning 
regimen.  Serum blood samples will be obtained up to twice weekly from Day 0 to Day 100 for testing with 
the Platelia® Aspergillus assay.   
 
The Platelia® Aspergillus EIA will be performed “real-time” for this protocol, and positive or negative assay 
results will be reported to the clinician.  Positive results will elicit further clinical evaluation for the 
presence of invasive aspergillus. 
 
The assay testing will be performed at a certified reference laboratory.  Testing will be performed in all 
sera following the manufacturer’s package insert instructions.  Results of each assay will be 
communicated to each site’s clinical coordinator within three business days.  Index Values and positive or 
negative results will be reported. 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-6 
MONITORING PHASE 
 
During this monitoring phase, two serum samples will be collected within 7 days of initiation of anti-fungal 
therapy, with one of the samples collected 48 hours before.  After anti-fungal therapy has been initiated 
and during therapy monitoring, a serum sample will be collected twice a week for four weeks and then 
once every two weeks for eight weeks.  In all, 12 samples will be collected and tested with the Platelia® 
Aspergillus assay after the initiation of drug therapy.  Samples will be frozen at –70oC and batch shipped 
quarterly to the Repository.  These samples should be clearly marked “Monitoring of Treatment.”  Results 
of this testing will not be reported to the clinician. 
 
Testing Algorithm: 
 
DIAGNOSTIC PHASE 
 
If a sample is negative in the Platelia® Aspergillus assay, the sample is reported as negative.  If a sample 
is initially positive in the Platelia® Aspergillus assay, the result is not reported and a new treatment of the 
same sample will be retested as soon as possible.  If the repeat test result using a new treatment is also 
positive, the result will be reported as positive and another serum sample will be requested for testing 
with the Platelia® Aspergillus assay.  In addition, additional diagnostic procedures will be initiated.  These 
will be evaluated in conjunction with clinical signs to determine the likelihood of invasive aspergillus 
infection.   
 
MONITORING PHASE 
 
The monitoring phase of the study will begin for any patient who is diagnosed with invasive aspergillosis 
and begins anti-fungal therapy.  Samples will be collected as described above.  Testing will be performed 
at a later date.  Test results are to be blinded and not reported to the clinician. 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-7 
TESTING ALGORITHM 
 
Diagnostic Phase 
 
 
600 post-transplant patients 
Fluconazole (N = 300) or Voriconazole (N = 300) 
Platelia ®  Aspergillus EIA 
Negative Result 
(Index < 0.5) 
Positive Result 
(Index > 0.5) 
Report as Negative 
Re-test Same Sample 
(new treatment) 
Do not report 
Negative Result 
(Index < 0.5) 
Positive Result 
(Index > 0.5) 
Report as Positive 
Collect a second sample and 
test with Platelia®  Aspergillus. 
Sample QNS 
for retest
Request another serum sample. 
Initiate additional diagnostic 
procedures.  Evaluate in 
conjunction with clinical signs to 
determine if invasive   aspergillus 
infection. 
 
 
 
 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-8 
 
TESTING ALGORITHM 
 
Monitoring Phase  
(No reporting of results) 
 
 
Invasive Aspergillosis Patients on Anti-Fungal Therapy 
Platelia ® Aspergillus EIA 
Negative Result 
(Index < 0.5) 
Positive Result 
(Index > 0.5) 
No Report 
Re-test Same Sample 
(new treatment) 
Negative Result 
(Index < 0.5) 
Positive Result 
(Index > 0.5) 
No Report 
Collect a second sample and 
test with Platelia®  Aspergillus 
Sample QNS 
for retest 
 
 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-9 
 
TECHNICAL INFORMATION 
Store the Platelia® Aspergillus assay kits at 2-8 °C.  The kit lot number as well as the expiration date will 
be on the box containing the reagents. 
 
All printouts must be signed in blue or black ink by the person actually performing the Platelia®  
Aspergillus assay and should be sent by facsimile or electronically weekly to the study monitor:  
 
 
Lisa McLaughlin 
 
Phone: 425-498-1774 
 
 
Bio-Rad Laboratories 
 
Fax: 
425-498-1651 
 
 
6565 185th N.E.  
 
E-mail: lisa_McLaughlin@bio-rad.com 
 
 
Redmond, WA 98052 
 
The investigator, or a responsible party designated by the investigator, must maintain an inventory record 
of devices/reagents received (see Attachment A to Appendix D) to assure that the materials will not be 
used for any purpose other than what is stated in this protocol.  These records must include all dates, lot 
numbers, quantities received, and identification of the subject.  If any portion of the device is lost, i.e. 
wastage, the name of the responsible individual, the date, and the quantity must be documented.  The 
study reagents must be used only at the institution specified in this protocol. 
 
Return the completed Platelia® Aspergillus Receipt Log and all unused devices/reagents in their original 
containers to the attention of the study monitor at Bio-Rad Laboratories at the conclusion or termination of 
the study.  Return unused serum samples to the Repository. 
 
MATERIALS AND METHODS 
A. Materials Provided by Bio-Rad Laboratories 
 
Platelia® Aspergillus Kits 
 
7 Member panel for Training / Proficiency    


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-10 
 
Platelia® Aspergillus Kit Composition  [Product No. 62797 (96 Tests)] 
Component 
Contents 
Quantity 
R1 - Microwell Strip 
Plate 
 96 wells coated with antigalactomannan 
monoclonal antibody 
1 plate containing 12 strips 
with 8 wells each 
R2 - Wash Solution, 
10X Concentrate 
 Tris NaCl buffer 
 1% Tween 20 
 0.01% sodium merthiolate 
1 x 100 mL 
R3 - Negative Control 
 Freeze-dried human negative serum 
 Negative for anti-HIV-1, anti-HIV-2, anti-HCV 
antibodies and HBs antigen 
2 x 1 mL 
R4 – Cut-off Control 
 Freeze-dried 
human 
serum 
containing 
galactomannan 
 Negative for anti-HIV-1, anti-HIV-2, anti-HCV 
antibodies and HBs antigen 
2 x 1 mL 
R5 - Positive Control 
 Freeze-dried 
human 
serum 
containing 
galactomannan 
 Negative for anti-HIV-1, anti-HIV-2, anti-HCV 
antibodies and HBs antigen 
2 x 1 mL 
R6 - Conjugate 
 Peroxidase-labeled 
antigalactomannan 
monoclonal antibody 
 0.01% sodium merthiolate 
1 x 8 mL 
R7 - Serum Treatment 
Solution 
 EDTA acid solution 
1 x 10.5 mL 
R8 - Substrate buffer 
 Citric acid 
 Sodium acetate 
 0.009% Hydrogen peroxide 
 4% Dimethylsulfoxide (DMSO) 
1 x 60 mL 
R9 - Chromogen 
 Dimethylsulfoxide (DMSO) 
 Tetramethylbenzidine (TMB)* 
1 x 1 mL 
R10 - Stopping 
Solution 
 1.5 N Sulphuric acid 
1 x 12 mL 
Plate sealers 
 Adhesive sheets for microplates 
6 
*NOTE: TMB (Tetramethylbenzidine) is a non-carcinogenic and non-mutagenic chromogen for 
peroxidase. Store the kit at 2-8C.  Bring all reagents to room temperature (15-30C) before use.  Return 
all reagents, except controls, to 2-8C immediately after use.  After reconstitution, unused Negative 
Control, Cut-off Control, and Positive Control must be frozen at -20C.  Return unused strips/plates to 
pouch and reseal.  Do not remove desiccant.  Strips should be used within 5 weeks of opening and 
resealing the pouch.  After dilution, wash solution can be kept for 15 days at 2-8C.  All other reagents are 
stable until expiration after opening.  Reagents are supplied in sufficient quantity to perform 96 
determinations in a maximum of 6 batches. 
 
B. Materials Not Provided  
1. 
Distilled or deionized water, for dilution of Wash Solution. 
2. 
Sterile purified water, for reconstitution of control sera. 
3. 
Absorbent paper. 
4. 
Disposable gloves. 
5. 
Protective glasses. 
6. 
Sodium hypochlorite (bleach) and sodium bicarbonate. 
7. 
Pipettes or multi-pipettes, adjustable or fixed, to measure and dispense 50 µl, 100 µl, 300 µl, and 
1000 µl. 
8. 
1.5 mL Eppendorf (Sarstedt Cat. # 72.692.105) or similar polypropylene tubes with airtight 
stoppers, able to support heating to 100C. 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-11 
9. 
Laboratory bench centrifuge for Eppendorf tubes capable of obtaining 10,000g (Brinkman Cat.# 
22-36-280-1 or VWR Scientific Cat.# 20901-051or equivalent).  
10. 
Vortex agitator. 
11. 
Boiling water bath or heating block (VWR Scientific Cat. # 13259-005). 
12. 
Micro-plate incubator at 37  1C. 
13. 
Manual or semi-automatic or automatic plate washer. 
14. 
Microplate reader equipped with 450 nm and 620/630 nm filters. 
15. 
Printer. 
 
PRECAUTIONS 
 
A. Health and Safety Precautions  
1. 
The Positive Control, Cut-off Control, and Negative Control are heat-inactivated, human serum.  
They have been tested and found to be negative for anti-HIV-1, anti-HIV-2, and anti-HCV 
antibodies, as well as HBs antigen.  However, all the reagents should be handled as though 
capable of transmitting infection.  All tests should be conducted using the precautions 
recommended for bloodborne pathogens, as defined by OSHA regulations. 
2. 
Wear protective clothing and disposable gloves while handling the kit reagents and patient 
samples.  Wash hands thoroughly after performing the test. 
3. 
Do not pipette by mouth. 
4. 
Do not smoke, drink, or eat in areas where specimens or kit reagents are being handled. 
5. 
Avoid splashing samples or solutions containing them. 
6. 
Biological spills not containing acid should be wiped thoroughly with an effective disinfectant.  
Disinfectants that can be used include (but are not limited to) a solution of 10% bleach (0.5% 
solution of sodium hypochlorite), 70% ethanol, or 0.5% Wescodyne™.  Spills containing acid 
should be wiped dry or neutralized with sodium bicarbonate and then cleaned with one of the 
chemical disinfectants.  Materials used to wipe up spills should be disposed of as biohazardous 
waste.  CAUTION: Do not place solutions containing bleach in the autoclave. 
7. 
Dispose of all specimens and materials used to perform the test as though they contain an 
infectious agent.  Disposal should comply with all applicable waste disposal requirements. 
8. 
CAUTION: The Stopping Solution is an acid.  Wipe up spills immediately and flush the areas with 
water. 
R36/38: Irritating to eyes and skin. 
S:2-26-30: Keep out of reach of children.  In case of contact with eyes, flush immediately and 
thoroughly with water and consult a physician.  Never pour water into this product. 
9. 
Avoid contact of Substrate Buffer, Chromogen, and Stopping Solution with eyes, skin, and 
mucosa (risk of toxicity, irritation, and burns). 
 
B. Test Precautions  
 
Please follow these guidelines in order to maintain consistency and reliability of results when using the 
Bio-Rad Laboratories PlateliaAspergillus: 
1. 
Do not use kit or any kit reagents after the stated expiration date. 
2. 
Do not mix reagents from other kits that have the same or different lot numbers. 
3. 
Store Platelia® Aspergillus kit reagents at 2 - 8C.  Bring all reagents to room temperature before 
use.  Return to storage at 2 - 8C immediately after use. 
4. 
Mix thoroughly while reconstituting reagents, exercising care to avoid microbial contamination. 
5. 
Do not conduct the test in the presence of reactive vapors (acids, alkalis, aldehydes) or dust, 
which could affect the enzymatic activity of the conjugate. 
6. 
Use clean, disposable polypropylene plastic containers to prepare the chromogen solution.  If 
glassware must be used, clean thoroughly and rinse with deionized water.   
7. 
Always use a fresh disposable pipette tip for each specimen and when making reagent dilutions. 
8. 
When aliquots of reagents are removed from their stock bottles, take care to avoid chemical and 
microbial contamination.  Always use a fresh disposable pipette or pipette tip for these transfers. 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-12 
9. 
To ensure adequate washing of the wells, comply with the recommended number of wash cycles 
and ensure that all wells are completely filled and then completely emptied. 
10. 
Use plastic disposable containers to dispense the conjugate and substrate solution.  Do not use 
the same container for the conjugate and substrate solutions. 
11. 
Do not allow conjugate or substrate solutions to come into contact with metal or metallic ions. 
12. 
Use clean, dust-free materials (tubes, tips, containers, etc.) to minimize the possibility of 
contamination with aspergillus spores in the environment.  Because galactomannan is heat-
stable, sterilization of material used does not guarantee the absence of contaminating antigen.  
Pyrogen-free materials are optimal, but standard material can be used with adequate 
precautions. 
13. 
Never perform the test in the same room where aspergillus or other molds are being cultured or 
otherwise manipulated. 
14. 
Limit exposure of solutions (sera, treatment solution, conjugate) or open containers (plates, 
tubes, pipettes) to free air.  Do not leave microplate wells exposed to air.  Perform the assay as 
soon as possible after removing the strips from the packet.  Remove only the number of strips 
necessary for testing samples. 
15. 
Add Conjugate to the microplate wells before the sample is added. Do not pour any unused 
conjugate back into the original container.  
16. 
The substrate-chromogen reaction solution must be colorless.  The appearance of a blue color 
after dilution indicates the reagent is contaminated and should not be used.  Discard and prepare 
fresh reagent. 
 
SPECIMEN COLLECTION / TESTING 
 
A.  Specimen Collection:  
Collect a blood sample according to current practice. The test is performed on undiluted serum.  Extract 
the serum from the clots as soon as possible to avoid hemolysis.  Serum samples must be 
uncontaminated with fungal spores or bacteria.  Transport and store samples in sealed tubes, unexposed 
to air.  Samples can be stored at 2-8C unopened for up to 5 days prior to testing. After initial opening, 
samples may be stored at 2-8C for 48 hours prior to testing. For longer storage, store the serum at          
–70C. 
 
B.  Specimen Testing:  
Procedural Comments: 
Test patient samples according to the Platelia® Aspergillus package insert (See Attachment B of 
Appendix D).  Note: Samples should be tested in batches for best utilization of assay reagents.  Each kit 
has reagents in sufficient quantity to perform 96 determinations in a maximum of 6 batches. 
 
BRING ALL REAGENTS TO ROOM TEMPERATURE (15 - 30C) BEFORE USE AND RETURN THEM 
IMMEDIATELY AFTER USE TO 2 - 8C. 
 
If samples have been frozen, thaw at room temperature and mix thoroughly (vortex) before testing. 
Negative, Positive, and Cut-off Controls must be run on each plate to validate the test results. 
 
Reagent preparation and storage / Reconstitution of Controls (Negative, Cut-off, and Positive 
Controls 
 
Working Wash Solution 
 
Prepare Working Wash Solution as needed by adding one part Concentrated Wash Solution to 9 parts 
deionized or distilled water.  The Working Wash Solution can be stored for 15 days at 2-8C.  Prepare a 
sufficient amount of Working Wash Solution to complete the run (i.e., minimum of 160 mls for each 2 
strips of 8 wells). 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-13 
Negative Control Serum   
 
Reconstitute the contents of one bottle of control with 1000 µl (1 mL) of sterile purified water (preferably 
pyrogen-free).  The sera must be rehydrated just before performing the test.  Mix thoroughly after allowing 
2-3 minutes for re-hydration of the serum.  Aliquot 300 µl into each of 3 Eppendorf tubes.  Immediately 
freeze at -20C the two tubes not used on the day of re-hydration. Note: control sera that have been 
previously rehydrated and immediately frozen at -20C may be thawed and used without further 
rehydration. Frozen rehydrated controls may be stored at -20C for up to 5 weeks. Handle the control 
sera in the same manner as patient specimens.  
 
Cut-off, and Positive Control Serum 
 
PREPARE AS DESCRIBED ABOVE FOR THE NEGATIVE CONTROL. 
 
Substrate-Chromogen Reaction Solution 
 
Prepare Substrate-Chromogen Reaction Solution by adding one part Concentrated Chromogen Solution, 
R9, to 50 parts Substrate Buffer, R8 (e.g., 200 µl of R9 + 10 mL of R8).  Prepare 4 ml of Substrate-
Chromogen Reaction Solution per strip.  The solution is stable for 6 hours when stored in the dark at 
room temperature. 
 
Treatment of Sera / Controls  
 
1. 
Pipette 300 µl of each test serum and control into individual 1.5 mL polypropylene tubes. 
2. 
Add 100 µl of treatment solution (R7) to each tube. 
3. 
Mix tubes thoroughly by vigorous homogenization or vortex to mix thoroughly.  Heat tubes in 
boiling water bath or heat block for 3 minutes at 100C. 
4. 
Centrifuge tubes at 10,000 x g for 10 minutes. 
5. 
Test the supernatants using the following procedure. After preparation, the supernatant may be 
removed and stored at 2-8C for up to 72 hours prior to testing.  If analysis of the results indicate 
retesting is required, another aliquot of serum must be treated for testing. 
 
 
EIA Procedure  
1. 
Prepare a Platelia® Aspergillus Worksheet (see Attachment C of Appendix D) and indicate the 
identification of test sera and controls in the microplate on the platemap.  Use one well for the 
Negative Control Serum (R3), two wells for the Cut-off Control (R4), and one well for the Positive 
Control Serum (R5). 
See suggested platemap below: 
 
 
 
 
 
 
 
 
 
 
 
 
 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
A 
R3 
 5 
 
 
 
 
 
 
 
 
 
 
B 
R4 
 6 
 
 
 
 
 
 
 
 
 
 
C 
R4 
 7 
 
 
 
 
 
 
 
 
 
 
D 
R5 
 8 
 
 
 
 
 
 
 
 
 
 
E 
Sample 1 
 9 
 
 
 
 
 
 
 
 
 
 
F 
 2 
 10 
 
 
 
 
 
 
 
 
 
 
G 
 3 
 11 
 
 
 
 
 
 
 
 
 
 
H 
 4 
 etc. 
 
 
 
 
 
 
 
 
 
 
 = Null strip / empty well 
 
2. 
Remove the plate holder and microwell strips (R1) from the plate pouch.  Return any strips to the 
pouch that will not be used, with the desiccant, and reseal the pouch. 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-14 
3. 
Add 50 µl of conjugate (R6) to each well.  Next, add 50 µl of treated serum supernatant to each 
well, as designated above.  Do not add serum samples to the wells before the conjugate. 
4. 
Cover plate with plate sealer, ensuring that entire surface is covered and watertight. 
5. 
Incubate the microplate in a water bath or dry microplate incubator for 90 + 5 minutes at       
37C( +1C). 
6. 
Remove the plate sealer.  Wash the plate 5 times, using a minimum of 370 µl of Working Wash 
Solution.  After the last wash, invert the microplate and gently tap on absorbent paper to remove 
remaining liquid. 
7. 
Add 200 µl of Substrate-Chromogen Reaction Solution (R8 + R9) to each well, avoiding exposure 
to bright light. 
8. 
Incubate microplate in the dark at room temperature (18 to 25C) for 30  5 minutes.  Do not use 
adhesive film during this incubation. 
9. 
Add 100 µl of Stopping Solution (R10) to each well, utilizing the same order for addition of 
Substrate Solution. 
10. 
Thoroughly wipe the bottoms of each plate. 
11. 
Read the optical density of each well at 450 nm (reference filter of 620/630 nm).  Micro-plates 
must be read within 30 minutes of addition of Stopping Solution.  Avoid exposure of plate to light 
before reading.  
 
C.  Calculation of Results 
The presence or absence of galactomannan antigen in the test sample is determined by calculation of an 
index for each sample.  The Index (I) is the OD value of the specimen divided by the mean optical density 
of the wells containing the cut-off control specimen. 
 
Calculation of the Mean Cut-off Control OD:  
Add the OD values of the two wells containing Cut-off control (R4) and divide the total by 2. 
 
Calculation of an index (I) for R3 and R5 control:   
Calculate the following ratio for R3 and R5 controls: 
 
 
 
      OD R3 /or R5 control 
I =  --------------------------------------------------------- 
 
               Mean Cut-off Control OD 
 
 
Assay Validation 
Calculate an index (I) for the Positive and Negative controls as described above.  In order for the assay to 
be valid, the following criteria must be met: 
 
 
Cut-off Control: The optical density of each Cut-off control must be between 0.3 and 0.8 
 
Positive Control: Index (I) must be greater than 2.0 
 
Negative Control: Index (I) must be less than 0.4 
 
Interpretation of Results 
The presence or absence of galactomannan antigen in the test sample is determined by calculation of an 
index for each patient specimen. The Index (I) is the OD value of the specimen divided by the mean 
optical density of the wells containing the cut-off control specimen 
 
Calculation of an index (I) for each patient sample 
Calculate the following ratio for each patient sample: 
 
 
 
      OD patient sample 
I =  --------------------------------------------------------- 
 
               Mean Cut-off Control OD 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-15 
 
Sera with an Index less than 0.5 are considered to be negative. 
 
Sera with an Index greater than or equal to 0.5 are considered to be positive. 
 
Whenever a sample has an Index  0.5, positivity must be confirmed by re-testing the sample, including 
repeating the heat treatment on a new aliquot, and/or by testing another sample obtained from the 
patient.   
 
D. Limitations of the Procedure 
1. 
A negative test cannot rule out the diagnosis of invasive Aspergillosis. Patients at risk for invasive 
Aspergillosis should be tested twice a week. 
2. 
The Platelia® Aspergillus Procedure and the Interpretation of Results must be followed when 
testing samples for the presence of aspergillus antigen.  The user of the kit is advised to read the 
package insert carefully prior to conducting the test.  In particular, the test procedure must be 
carefully followed for sample and reagent pipetting, plate washing, and timing of the incubation 
steps. 
3 
Failure to add specimen or reagent as instructed in the procedure could result in a falsely 
negative test.  Repeat testing should be considered where there is clinical suspicion of infection 
or procedural error. 
4. 
Contamination of negative patient specimen wells by positive control/patient specimen wells is 
possible if the contents of one well spill over into another well due to rough handling of the 
microplate or poor pipetting technique while adding reagents. 
 
TRAINING / PROFICIENCY TESTING 
 
The purpose of training is to provide the technologist with familiarity in using the Bio-Rad Platelia® 
Aspergillus and instruments.  Proficiency in running the assay will be assessed and demonstrated by 
testing a panel of specimens provided by Bio-Rad Laboratories with the Bio-Rad Platelia® Aspergillus EIA 
kit.  Testing will be done according to package insert instructions (See Attachment B of Appendix D). 
 
Each technologist who will be testing study specimens with the Platelia® Aspergillus EIA must 
demonstrate proficiency in running the assay before testing study specimens. 
 
Proficiency will be assessed and demonstrated by testing a 7 member -training panel provided by Bio-
Rad Laboratories. Each of the 7 panel members will be pretreated by heating 300l of serum with 100l 
Treatment Solution to 100C as instructed in the Platelia® Aspergillus assay package insert.  After 
centrifugation, the supernatant will be assayed in triplicate.  See the suggested plate map below. 
Complete testing according to package insert instructions. 
 
The within plate accuracy and reproducibility for each panel member will be calculated by the monitor. 
 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
A 
R3 
X 
AF 
AI 
 
 
 
 
 
 
 
 
B 
R4 
X 
AF 
 
 
 
 
 
 
 
 
 
C 
R4 
Z 
AF 
 
 
 
 
 
 
 
 
 
D 
R5 
Z 
AH 
 
 
 
 
 
 
 
 
 
E 
W 
Z 
AH 
 
 
 
 
 
 
 
 
 
F 
W 
AC AH 
 
 
 
 
 
 
 
 
 
G 
W 
AC 
AI 
 
 
 
 
 
 
 
 
 
H 
X 
AC 
AI 
 
 
 
 
 
 
 
 
 
 
R3 = Negative Control Serum         R4 = Cut-off Control          R5 = Positive Control Serum 
W,X,Z,AC,AF,AH,AI = Panel Members  = Empty well  
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-16 
The study monitor will complete the Proficiency Testing Documentation Form (page D-17) for each 
technologist who will be testing specimens with the Platelia® Aspergillus antigen assay and will indicate 
on the form if proficiency testing is acceptable or unacceptable. If proficiency testing is unacceptable, the 
technologist must repeat proficiency testing as instructed by the study monitor. If proficiency testing is 
acceptable, the technologist can begin testing study specimens on the date indicated on the form. 
 
Testing of study specimens can begin when proficiency testing is acceptable and a copy of the 
signed and dated Proficiency Testing Documentation Form is received from the study monitor. 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-17 
 
 
Proficiency Testing Documentation Form 
 
 
Site: __________________________________________________________ 
Name of Technologist: _______________________________Date of Testing ________________ 
 
Platelia®Aspergillus Lot #_________________________Expiration Date____________________ 
R3 =___________ 
R4 =1.___________ 2.___________ Mean = _____________ 
R5 =___________ 
Cutoff = mean optical density of R4 (Cut-off Control) = ____________ 
 
Panel 
Member 
Mean Index (I) 
Interpretation 
SD 
CV% 
Acceptable Range 
Index (I) 
Acceptable 
CV% 
W 
 
 
 
 
0.10-0.21 
< 25% 
X 
 
 
 
 
2.60-3.45 
< 10% 
Z 
 
 
 
 
3.88-5.44 
< 10% 
AC 
 
 
 
 
2.75-3.56 
< 10% 
AF 
 
 
 
 
1.68-2.27 
< 10% 
AH 
 
 
 
 
0.85-1.33 
< 15% 
AI 
 
 
 
 
0.47-0.78 
< 25% 
 
___ Proficiency Testing Not Acceptable. Repeat proficiency testing. 
___ Proficiency Testing Acceptable. Testing of specimens can begin on:____________________ 
(Attach a copy of the printout of OD values). 
 
 
 
 
Signature of the Study Monitor:___________________________________ Date:___________________ 
 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-18 
DATA COLLECTION 
 
Platelia® Aspergillus Data 
 
Data will be collected following the NIH Clinical Protocol guidelines. 
 
Patient Clinical Data 
 
Blood samples will be collected as part of the clinical protocol (“A Randomized Double-blind Trial of 
Fluconazole vs. Voriconazole for the Prevention of Invasive Fungal Infections in Allogeneic Blood and 
Marrow Transplant Patients”).  These samples will be tested with the Platelia® Aspergillus assay and the 
results correlated with patient clinical data. 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-19 
 
 
 
 
APPENDIX D 
ATTACHMENT A:  PLATELIA® ASPERGILLUS KIT RECEIPT LOG 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-20 
PLATELIA® ASPERGILLUS KIT RECEIPT LOG 
 
DATE 
RECEIVED 
PLATELIA® ASPERGILLUS 
LOT NUMBER 
NUMBER OF KITS 
RECEIVED 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
______________________________________   ________________________ 
Investigator’s Signature  
 
Date 
(Make copies of this form if additional space is needed) 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-21 
 
 
 
APPENDIX D 
 
ATTACHMENT B:  PLATELIA ASPERGILLUS PACKAGE INSERT 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-22 
 
 
PLATELIA® ASPERGILLUS EIA 
 
96 TESTS: 
62793 
480 TESTS: 
62794 
 
 
 
THE PLATELIA® ASPERGILLUS EIA IS AN IMMUNOENZYMATIC SANDWICH 
MICROPLATE ASSAY FOR THE DETECTION OF ASPERGILLUS 
GALACTOMANNAN ANTIGEN IN SERUM  
 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-23 
1. INTENDED USE 
The Platelia Aspergillus EIA is an immunoenzymatic sandwich microplate assay for the 
detection of Aspergillus galactomannan antigen in serum. 
 
2. INDICATIONS FOR USE 
The Platelia Aspergillus EIA is a test which, when used in conjunction with other diagnostic 
procedures such as microbiological culture, histological examination of biopsy samples and 
radiographic evidence, can be used as an aid in the diagnosis of Invasive Aspergillosis. 
 
3. SUMMARY AND EXPLANATION 
Aspergillus infections usually occur following inhalation of Aspergillus spores which are present 
in the environment.  Invasive forms, which have been on the increase for the past 10 years, 
constitute the most serious infections.  They mainly occur in neutropenic patients (following anti-
cancer treatment) and in patients treated with immuno-suppressants (organ transplantations, 
particularly bone marrow transplantation) and corticosteroids2. 
 
Aspergillus is rarely isolated from blood culture.  The diagnosis is often based on nonspecific 
diagnostic or radiological evidence (clinical symptioms, CT scan, chest x-ray, etc.). 
 
At the present time, the test for soluble Aspergillus antigen in serum appears to be a serological 
method able to aid in the diagnosis of Invasive Aspergillosis1,3,6,17,21. 
 
 
4. PRINCIPLE OF THE PROCEDURE11 
The Platelia Aspergillus EIA is a one-stage immunoenzymatic sandwich microplate assay 
which detects galactomannan in human serum.  The assay uses the rat monoclonal antibody 
EBA-2, which is directed against Aspergillus galactomannan, and has been characterized 
during previous studies8,12.  The monoclonal antibody is used, (1) to coat the wells of the 
microplate and bind the antigen, and (2) as the detector antibody in the conjugate reagent 
(peroxidase-linked monoclonal antibody). 
 
Serum samples are heat-treated in the presence of EDTA in order to dissociate the immune 
complexes and to precipitate serum protieins that could possibly interfere with the test7.  The 
treated serum samples and conjugate are added to the wells coated with the monoclonal 
antibody, and incubated.  A monoclonal antibody - galactomannan - monoclonal antibody / 
peroxidase complex is formed in the presence of Aspergillus antigen. 
 
The strips are washed to remove any unbound material.  Next, the substrate solution is added, 
which will react with the complexes bound to the well to form a blue color reaction.  The enzyme 
reaction is stopped by the addition of acid, which changes the blue color to yellow.  The optical 
absorbance of specimens and controls is determined with a spectrophotometer set at 450 and 
620/630 nm wavelength. 
 
5. REAGENTS 
Platelia Aspergillus  
 
 
product No. 62793 (96 Tests) 
 
 
 
product No. 62794 (480 Tests) 
 
Store the kit at 2-8C.  Bring all reagents to room temperature (18-25C) before use.  Return all 
reagents, except controls, to 2-8C immediately after use.  After reconstitution, unused Negative 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-24 
Control, Cut-off Control, and Positive Control must be frozen at -20C.  Return unused 
strips/plates to pouch and reseal.  Do not remove desiccant.  Strips should be used within 5 
weeks of opening and resealing the pouch.  After dilution, wash solution can be kept for 15 days 
at 2-8C.  All other reagents are stable until expiration after opening.  Reagents are supplied in 
sufficient quantity to perform 96 determinations in a maximum of 6 batches or 480 tests in a 
maximum of 12 batches. 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-25 
 
 
Component 
Contents 
Quantity 
 
 
96 
Test Kit 
480 
Test Kit 
R1 
Microwell 
Strip Plate 
 96 wells (12 strips of 8 wells each) 
coated with antigalactomannan 
monoclonal antibody 
1 Plate / 
12 x 8 Wells 
5 Plates / 
12 x 8 Wells 
R2 
Wash 
Solution 10X 
Concentrate 
 Tris NaCl buffer 
 1% Tween® 20 
 0.01% thimerosal 
1 x 100 mL 
3 x 100 mL 
R3 
Negative 
Control 
Serum 
 Freeze-dried human negative serum 
 Negative for anti-HIV-1, anti-HIV-2, 
anti-HCV antibodies and HBsAg 
2 x 1 mL 
4 x 1 mL 
R4 
Cut-off 
Control 
Serum 
 Freeze-dried human serum containing 
galactomannan 
 Negative for anti-HIV-1, anti-HIV-2, 
anti-HCV antibodies and HBsAg 
2 x 1 mL 
4 x 1 mL 
R5 
Positive 
Control 
Serum 
 Freeze-dried human serum containing 
galactomannan 
 Negative for anti-HIV-1, anti-HIV-2, 
anti-HCV antibodies and HBsAg 
2 x 1 mL 
4 x 1 mL 
R6 
Conjugate 
 Anti-galactomannan monoclonal 
antibody/peroxidase labeled 
 0.01% thimerosal 
1 x 8 mL 
4 x 8 mL 
R7 
Treatment 
Solution 
 EDTA acid solution 
1 x 10.5 mL 
5 x 10.5 mL 
R8 
Substrate 
Buffer 
 Citric acid 
 Sodium acetate 
 0.009% Hydrogen peroxide 
 4% Dimethylsulfoxide (DMSO) 
1 x 60 mL 
3 x 60 mL 
R9 
Chromogen 
 Dimethylsulfoxide (DMSO) 
 Tetramethylbenzidine (TMB)* 
1 x 1 mL 
3 x 1 mL 
R10 
Stopping 
Solution 
 1.5 N Sulphuric acid (H2SO4) 
1 x 12 mL 
5 x 12 mL 
Plate sealers 
 Adhesive sheets for microplates 
1 x 6 sheets 
2 x 6 sheets 
 
*Note: TMB (Tetramethylbenzidine) is a non-carcinogenic and non-mutagenic chromogen for peroxidase. 
 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-26 
6. WARNINGS FOR USERS 
 
1. For in vitro diagnostic use. 
2. For professional use only. 
3. Use of this test kit with samples other than human serum is not recommended. 
4. The Positive Control, Cut-off Control, and Negative Control are heat-inactivated, human 
serum.  They have been tested and found to be negative for anti-HIV-1, anti-HIV-2, and anti-
HCV antibodies, as well as HBsAg with French approved tests.  However, all the reagents 
should be handled as though capable of transmitting infection.  All tests should be 
conducted using the precautions recommended for blood borne pathogens, as defined by 
OSHA regulations. 
5. Wear protective clothing and disposable gloves while handling the kit reagents and patient 
samples.  Wash hands thoroughly after performing the test. 
6. Do not pipette by mouth. 
7. Do not smoke, drink, or eat in areas where specimens or kit reagents are being handled. 
8. Avoid splashing samples or solutions containing them. 
9. Biological spills not containing acid should be wiped thoroughly with an effective 
disinfectant.  Disinfectants that can be used include (but are not limited to) a solution of 10% 
bleach (0.5% solution of sodium hypochlorite), 70% ethanol, or 0.5% Wescodyne™.  Spills 
containing acid should be wiped dry or neutralized with sodium bicarbonate and then 
cleaned with one of the chemical disinfectants.  Materials used to wipe up spills should be 
disposed of as biohazardous waste.  CAUTION: Do not place solutions containing bleach in 
the autoclave. 
10. Dispose of all specimens and materials used to perform the test as though they contain an 
infectious agent.  Disposal should comply with all applicable waste disposal requirements. 
11. CAUTION: The Stopping Solution is an acid.  Wipe up spills immediately and flush the areas 
with water. 
R36/38: Irritating to eyes and skin. 
S:2-26-30: Keep out of reach of children.  In case of contact with eyes, flush 
immediately and thoroughly with water and consult a physician.  Never pour 
water into this product. 
 
12. Avoid contact of Substrate Buffer, Chromogen, and Stopping Solution 
with eyes, skin, and mucosae (risk of toxicity, irritation, and burns). 
 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-27 
7. PRECAUTIONS FOR USERS 
1. FROZEN SERUM SAMPLES STORED IN UNKOWN CONDITIONS MAY GIVE FALSE 
POSITIVE RESULTS DUE TO CONTAMINATION WITH FUNGUS AND/OR BACTERIA. 
2. Do not use kit or any kit reagents after the stated expiration date. 
3. With the exception of the 10x Wash Solution (R2) and the Stopping Solution (R10), do not 
mix reagents from other kits that have the same or different lot numbers.  Note: Platelia 
10X Wash Solution (R2) may be used on any Platelia brand assay. 
4. Bring all reagents to room temperature for at least 15 minutes before use. 
5. Mix thoroughly while reconstituting reagents, exercising care to avoid microbial 
contamination. 
6. Do not conduct the test in the presence of reactive vapors (acids, alkalis, aldehydes) or 
dust, which could affect the enzymatic activity of the conjugate. 
7. Use clean, disposable polypropylene plastic containers to prepare the chromogen solution.  
If glassware must be used, clean thoroughly and rinse with deionized water. 
8. For manual pipetting of controls and specimens, use individual pipette tips to prevent 
carryover of samples. 
9. To ensure adequate washing of the wells, comply with the recommended number of wash 
cycles and ensure that all wells are completely filled and then completely emptied.  Washing 
should not be performed manually with a squeeze bottle. 
10. Do not use the same container for the conjugate and substrate solutions. 
11. Do not allow conjugate or substrate solutions to come into contact with metal or metallic 
ions. 
12. Avoid exposing the Chromogen or the Substrate-Chromogen Reaction Solution to strong 
light during storage or incubation.  Do not allow the chromogen solutions to come into 
contact with an oxidizing agent. 
13. Avoid contact of the Stopping Solution with any oxidizing agent.  Do not allow the Stopping 
Solution to come into contact with metal or metallic ions. 
14. Use clean, dust-free materials (tubes, tips, containers, etc.) to minimize the possibility of 
contamination with Aspergillus spores from the environment.  Because galactomannan is 
heat-stable, sterilization of material used does not guarantee the absence of contaminating 
antigen.  Pyrogen-free materials are optimal, but standard material can be used with 
adequate precautions. 
15. Limit exposure of solutions (sera, treatment solution, conjugate) or open containers (plates, 
tubes, pipettes) to the air. 
16. Do not pour any unused conjugate back into the original container. 
17. The Substrate-Chromogen Reaction Solution must be colorless.  The appearance of a blue 
color after dilution indicates the reagent is contaminated and should not be used.  Discard 
and prepare fresh reagent. 
 
 
8. REAGENT PREPARATION AND STORAGE 
Wash Solution 
Prepare Working Wash Solution as needed by adding one part Concentrated Wash Solution to 
9 parts deionized or distilled water.  The Working Wash Solution can be stored for 15 days at 2-


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-28 
8C.  Prepare a sufficient amount of Working Wash Solution to complete the run (i.e., minimum 
of 160 mls for each 2 strips of 8 wells). 
 
Negative Control Serum 
Reconstitute the contents of one bottle of control with 1000 µl (1 mL) of sterile, purified water.  
The sera must be rehydrated just before performing the test.  Mix thoroughly after allowing 2-3 
minutes for rehydration of the serum.  Aliquot 300 µl into each of 3 Eppendorf tubes.  
Immediately freeze at -20C any remaining Eppendorf tubes that will not used after rehydration. 
 
Note: Control sera that have been previously rehydrated and immediately fozen at -20C may 
be thawed and used without further rehydration.  Frozen rehydrated controls may be stored at -
20C for up to five weeks.  Handle the control sera in the same manner as patient specimens. 
 
Cut-off Control Serum and Positive Control Serum 
Prepare as described above for Negative Control Serum. 
 
Substrate-Chromogen Reaction Solution 
Prepare Substrate-Chromogen Reaction Solution by adding one part Concentrated Chromogen 
Solution, R9, to 50 parts Substrate Buffer, R8 (e.g., 200 µl of R9 + 10 mL of R8).  Prepare  
4 mL of Substrate-Chromogen Reaction Solution per strip.  The solution is stable for 6 hours 
when stored in the dark at room temperature. 
 
 
9. SPECIMEN COLLECTION 
Collect blood samples according to standard laboratory procedures.  The test is performed on 
serum.  Serum samples must be uncontaminated with fungal spores and/or bacteria.  Transport 
and store samples in sealed tubes, unexposed to air.  Unopened samples can be stored at 2-
8C for up to 5 days prior to testing.  After initial opening, samples may be stored at 2-8C for 48 
hours prior to testing.  For longer storage, store the serum at –70C. 
 
 
10. PROCEDURE 
Materials provided 
See REAGENTS section. 
 
Materials required but not provided 
1. Distilled or deionized water, for dilution of Wash Solution. 
2. Sterile, purified water for reconstitution of control sera. 
3. Absorbent paper. 
4. Disposable gloves. 
5. Protective glasses. 
6. Sodium hypochlorite (bleach) and sodium bicarbonate. 
7. Pipettes or multipipettes, adjustable or fixed, to measure and dispense 50 µl, 100 µl, 300 µl, 
and 1000 µl. 
8. 1.5 mL Eppendorf (Sarstedt Cat. #72.692.105) or similar polypropylene tubes with airtight 
stoppers, able to support heating to 100C. 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-29 
9. Laboratory bench centrifuge for polypropylene tubes capable of obtaining 10,000g 
(Brickman Cat. #22-36-280-1 or VWR Scientific Cat. #20901-051 or equivalent). 
10. Round, floating micro-centrifuge rack for 1 L beaker (VWR Scientific Cat. # 60986-100 or 
Nalgene #5974-1015 or equivalent). 
11. Vortex agitator. 
12. Boiling waterbath. 
13. Microplate incubator at 37  1C. 
14. Manual or semi-automatic plate washer. 
15. Microplate reader equipped with 450 nm and 620/630 nm filters. 
 
Procedural Comments 
1. Negative, Positive, and Cut-off Controls must be tested on each run to validate the test 
results. 
 
Treatment of the sera 
1. Pipette 300 µl of each test serum and control into individual 1.5 mL polypropylene tubes. 
2. Add 100 µl of Treatment Solution (R7) to each tube. 
3. Mix tubes thoroughly by vigorous homogenization or vortexing to mix thoroughly.  Heat 
tubes for 3 minutes in boiling waterbath at 100C. 
4. Carefully remove hot tubes from the boiling waterbath and place in a centrifuge.  Centrifuge 
tubes at 10,000 x g for 10 minutes. 
5. Test the supernatants using the following procedure.  After preparation, the supernatant 
may be removed and stored at 2-8C for up to 72 hours prior to testing.  If analysis of the 
results indicates retesting is required, another aliquot of serum must be treated for testing. 
 
EIA Procedure 
1. Bring reagents to room temperature (18 - 25C) for at least 15 minutes before use. 
2. Prepare Wash Solution, Substrate-Chromogen Reaction Solution, and Negative, Positive, 
and Cut-off Controls. 
3. Prepare a chart for identification of test sera and controls in the microplate.  Use one well for 
the Negative Control Serum (R3), two wells for the Cut-off Serum (R4), and one well for the 
Positive Control Serum (R5). 
4. Remove the plateholder and microwell strips (R1) from the plate pouch.  Return any strips 
that will not be used to the pouch, with the desiccant, and reseal the pouch. 
5. Add 50 µl of Conjugate (R6) to each well.  Next, add 50 µl of treated serum supernatant to 
each well, as designated above.  Do not add serum samples to the wells before the 
conjugate. 
6. Cover plate with plate sealer, or other means to prevent evaporation, ensuring that entire 
surface is covered and watertight. 
7. Incubate the microplate in a dry microplate incubator for 90 ± 5 minutes at 37C (± 1 C). 
8. Remove the plate sealer.  Wash the plate 5 times, using a minimum of 370 µl of Working 
Wash Solution.  After the last wash, invert the microplate and gently tap on absorbent paper 
to remove remaining liquid. 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-30 
9. Add 200 µl of Substrate-Chromogen Reaction Solution (R8 + R9) to each well, avoiding 
exposure to bright light. 
10. Incubate microplate in the dark at room temperature (18 to 25C) for 30  5 minutes.  Do 
not use adhesive film during this incubation step. 
11. Add 100 µl of Stopping Solution (R10) to each well, utilizing the same order for addition of 
Substrate Solution.  Mix well. 
12. Thoroughly wipe the bottoms of each plate. 
13. Read the optical density of each well at 450 nm (reference filter of 620/630 nm).  
Microplates must be read within 30 minutes of addition of Stopping Solution. 
 
 
11. QUALITY CONTROL (VALIDITY CRITERIA) 
 
Cut-off Control:  The O.D. of each Cut-off Control Serum must be ≥ 0.3 and ≤ 0.8. 
 
Positive Control:  The index of the Positive Control Serum must be greater than 2. 
 
I = 
OD Positive Control (R5) 
> 2 
Mean Cut-off Control OD 
 
Negative Control:  The index of the Negative Control Serum must be less than 0.4. 
 
I = 
OD Negative Control (R3) 
< 0.4 
Mean Cut-off Control OD 
 
Failure of any of the controls to meet the validity criteria described above renders the assay 
invalid, and patient specimen results should not be reported.  The operator may decide to 
repeat the assay, after reviewing the procedure, or may contact the manufacturer for 
assistance.  If a repeat assay is performed, then a new aliquot of the same sample should be 
used in the repeat assay. 
 
 
 
Example Calculation: 
 
Sample 
Absorbence (OD) 
Negative Control (R3) OD 
0.117 
Cut-off Control (R4) OD 
0.596 
0.576 
Positive Control (R5) OD 
2.602 
 
Calculations 
 
Mean Cut-off Control Value 
To calculate the mean Cut-off Control (R4) OD, add the OD values for each Cut-off Control 
replicate together and divide the result by 2: 
 
(0.596 + 0.576)  2 = 0.586 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-31 
Negative Control Index 
To calculate the index of the Negative Control, divide the OD of the Negative Control by the 
mean Cut-off Control OD: 
 
I = 
0.117 
= 0.20 
0.586 
 
Positive Control Index 
To calculate the index of the Positive Control, divide the OD of the Positive Control by the mean 
Cut-off Control OD: 
 
I = 
2.602 
= 4.44 
0.586 
 
Validity 
 
In the above example: 
 
Each Cut-off Control OD is ≥ 0.3 and ≤ 0.8, indicating that the Cut-off Control is valid. 
 
The index of the Negative Control is < 0.4, indicating that the Negative Control is valid. 
 
The index of the Positive Control is > 2, indicating that the Positive Control is valid. 
 
The test run in this example is considered to be valid since the results meet the validity criteria 
for each control. 
 
 
 
12. INTERPRETATION OF RESULTS 
 
The presence or absence of Aspergillus antigen in the test sample is determined by calculation 
of an index for each patient specimen.  The Index (I), is the OD value of the specimen divided 
by the mean optical density of the wells containing Cut-off Control Serum. 
 
Calculation of the mean Cut-off Control optical density: 
Add the optical densities of the two wells containing Cut-off Control Serum (R4) and divide the 
total by 2. 
 
Calculation of an index (I) for each test serum: 
Calculate the following ratio for each test serum: 
 
I = 
OD sample 
Mean Cut-off Control OD 
 
Sera with an index < 0.5 are considered to be negative for galactomannan antigen. 
 
Note: A negative result may indicate that the patient’s result is below the detectable level 
of the assay. 
 
Note: Negative results do not rule out the diagnosis of Invasive Aspergillosis.  Repeat testing is 
recommended if the result is negative, but the disease is suspected. 
 


BMT CLINICAL TRIALS NETWORK 
 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-32 
Sera with an index > 0.5 are considered to be positive for galactomannan antigen. 
 
Note: The Platelia Aspergillus EIA is intended to be used as an aid in the diagnosis of 
Invasive Aspergillosis.  Positive results obtained with the Platelia Aspergillus EIA 
should be considered in conjuction with other diagnostic procedures such as 
microbiological culture, histological examination of biopsy samples and radiographic 
evidence. 
 
For all positive patients, it is recommended that a new aliquot of the same sample be repeated 
as well as collection of a new sample from the patient for follow-up testing. 
 
Note: An absorbance value of less than 0.000 OD may indicate a procedural or instrument error 
which should be evaluated.  That result is invalid and the specimen must be re-run. 
 
Example Calculation: 
 
Sample 
Absorbence (OD) 
Negative Control (R3) OD 
0.117 
Cut-off Control (R4) OD 
0.596 
0.576 
Positive Control (R5) OD 
2.602 
Patient Sample #1 
0.134 
Patient Sample #2 
0.436 
Patient Sample #3 
1.196 
 
Calculations 
 
Refer to the Quality Control (Validity Criteria) section for an example of calculations to 
determine the validity of the assay controls. 
 
Mean Cut-off Control Value 
To calculate the mean Cut-off Control (R4) OD, add the OD values for each Cut-off Control 
replicate together and divide the result by 2: 
 
(0.596 + 0.576)  2 = 0.586 
 
Patient Sample #1 
To calculate the index of Patient #1, divide the OD of Patient Sample #1 by the mean Cut-off 
Control OD: 
 
I = 
0.134 
= 0.23 
0.586 
 
In this example, Patient Sample #1 is negative, since the Index of 0.23 is < 0.5. 
 


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Patient Sample #2 
To calculate the index of Patient #2, divide the OD of Patient Sample #2 by the mean Cut-off 
Control OD: 
 
I = 
0.436 
= 0.74 
0.586 
 
In this example, Patient Sample #2 is positive, since the Index of 0.74 is > 0.5. 
 
Patient Sample #3 
To calculate the index of Patient #3, divide the OD of Patient Sample #3 by the mean Cut-off 
Control OD: 
 
I = 
1.196 
= 2.04 
0.586 
 
In this example, Patient Sample #3 is positive, since the Index of 2.04 is > 0.5. 
 
 
13. LIMITATIONS OF THE PROCEDURE 
1. A negative test cannot rule out the diagnosis of Invasive Aspergillosis.  Patients at 
risk for Invasive Aspergillosis should be tested twice a week. 
2. The Platelia Aspergillus Procedure and the Interpretation of Results must be followed when 
testing samples for the presence of Aspergillus antigen.  The user of the kit is advised to 
read the package insert carefully prior to conducting the test.  In particular, the test 
procedure must be carefully followed for sample and reagent pipetting, plate washing, and 
timing of the incubation steps. 
3. Failure to add specimen or reagent as instructed in the procedure could result in a falsely 
negative test.  Repeat testing of additional samples should be considered where there is 
clinical suspicion of Invasive Aspergillosis or procedural error. 
4. Contamination of negative patient specimen wells by positive control/patient specimen wells 
is possible if the contents of one well spill over into another well due to rough handling of the 
microplate or poor pipetting technique while adding reagents. 
5. The performance of the Platelia Aspergillus EIA has not been evaluated with neonate or 
pediatric serum samples. 
6. The Platelia Aspergillus EIA may exhibit reduced detection of galactomannan in patients 
with chronic granulomatous disease (CDG) and Job’s syndrome19,20. 
7. The concomitant use of mold-active anti-fungal therapy in some patients with Invasive 
Aspergillosis may result in reduced sensitivity with the Platelia Aspergillus EIA. 
8. The Platelia Aspergillus EIA has not been evaluated for use with plasma or other sample 
types such as urine, BAL, or CSF. 
9. The performance of the Platelia Aspergillus EIA has not been established for manual 
reading and/or visual result determination. 
10. Other genera of fungi such as Penicillium and Paecilomyces have shown reactivity with the 
rat, EBA-2 monoclonal antibody used in the assay for the detection of Aspergillus 
galactomannan15.  These species are rarely implicated in invasive fungal disease. 
 


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14. EXPECTED VALUES 
The expected prevalence of Invasive Aspergillosis varies with the patient population; rates from 
5-20% have been reported2,5.  A clinical study was conducted on a total of 1890 serum samples 
from 179 bone marrow transplant (BMT) and leukemic patients diagnosed with and without 
Invasive Aspergillosis, at three testing centers in North America to determine the performance 
characteristics of the Platelia Aspergillus EIA.  The average prevalence rate for this study was 
14%.  The distribution of index values for these populations is represented in the following 
charts. 
 
Patients diagnosed with Invasive Aspergillosis (control population) 
A total of 1362 frozen serum samples obtained from 148 bone marrow transplant (BMT) and 
leukemic patients at three testing centers in North America were tested with the Platelia 
Aspergillus EIA test.  The distribution of index values is shown in the following chart. 
 
 
This scatter plot depicts 
galactomannan assay 
results for the 1362 serum 
samples from 148 control 
patients in this study 
(patients undergoing 
immunosuppressive therapy 
for HSCT, or to treat 
hematological malignancy). 
 
 


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Patients diagnosed with Invasive Aspergillosis 
 
 
This scatter plot depicts 
galactomannan assay results for 
the 528 serum samples from 31 
patients in this study diagnosed 
with proven or probable Invasive 
Aspergillosis as defined by 
EORTC/NIAID definitions.  Not 
every serum sample from each 
patient is expected to be positive.  
The expected prevalence of 
Invasive Aspergillosis varies with 
the patient population; rates from 5-
20% have been reported 2,5.  The 
prevalence rate for this study was 
14%. 
 
 
 
The following graphs represent examples of a patient without clinical signs or symptoms of 
Invasive Aspergillosis (negative for Aspergillus) and a patient with proven or probable Invasive 
Aspergillosis (positive for Aspergillus) respectively. 
 
Negative Patient 
 
 
 
 
 
 
 
 
 
 
 
 
 
Positive Patient 
 
 
 


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15. SPECIFIC PERFORMANCE CHARACTERISTICS 
 
A. Reproducibility Studies 
 
Inter-assay and Intra-assay variability for the Platelia Aspergillus EIA were determined in a 
study using a panel of 6 pooled patient serum samples (one negative, one low positive, two 
positive, and two high positive) obtained from actual clinical trial sites.  Each of the 6 panel 
members was tested in triplicate (x3) on 3 different days, on one lot, at two sites (total number 
of replicates at each site = 9).  Each of the 6 panel members was tested in duplicate (x2) on 3 
different days, on 1 lot, at a third site (total number of replicates at the third site = 6).  One (1) 
operator performed all precision testing at each site.  The data were analyzed according to the 
National Committee for Clinical Laboratory Standards (NCCLS).  The mean optical density (D) 
and mean index value, standard deviation (SD), percent coefficient of variation (%CV), within 
run precision (intra-assay) and within site (inter-assay) precision for each panel member at each 
site are illustrated below in the following tables. 


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Site 1 
Panel Member 
Neg 
Low Pos 
Pos #1 
Pos #2 
High Pos #1 
High Pos #2 
Neg Control 
CO Control 
Pos Control 
 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
N 
9 
9 
9 
9 
9 
9 
9 
9 
9 
9 
9 
9 
3 
3 
6 
6 
3 
3 
Mean 
0.052 
0.09 
0.445 
0.74 
0.702 
1.17 
0.931 
1.563 
1.227 
2.06 
2.887 
4.83 
0.046 
0.08 
0.606 
1.00 
2.216 
3.67 
Within Run 
(intra-assay)1SD 
0.002 
0.00 
0.022 
0.03 
0.059 
0.09 
0.044 
0.08 
0.051 
0.09 
0.089 
0.17 
N/A 
N/A 
0.02 
0.03 
N/A 
N/A 
% CV 
N/A 
N/A 
4.8% 
4.4% 
8.4% 
7.6% 
4.7% 
5.1% 
4.2% 
4.4% 
3.1% 
3.6% 
N/A 
N/A 
3.7% 
3.4% 
N/A 
N/A 
Total 
(inter-assay)2SD 
0.036 
0.04 
0.051 
0.08 
0.070 
0.14 
0.044 
0.25 
0.058 
0.29 
0.169 
0.58 
N/A 
N/A 
0.102 
0.03 
0.317 
0.12 
% CV 
N/A 
N/A 
11.5% 
10.4% 
10.0% 
11.6% 
4.7% 
15.7% 
4.7% 
14.3% 
5.9% 
11.9% 
N/A 
N/A 
16.9% 
2.8% 
14.3% 
3.3% 
 
Site 2 
Panel Member 
Neg 
Low Pos 
Pos #1 
Pos #2 
High Pos #1 
High Pos #2 
Neg Control 
CO Control 
Pos Control 
 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
N 
9 
9 
9 
9 
9 
9 
9 
9 
9 
9 
9 
9 
3 
3 
6 
6 
3 
3 
Mean 
0.040 
0.10 
0.280 
0.70 
0.364 
0.89 
0.602 
1.49 
0.801 
2.01 
1.361 
3.43 
0.074 
0.18 
0.415 
1.00 
1.197 
2.97 
Within Run 
(intra-assay)1SD 
0.006 
0.01 
0.041 
0.09 
0.023 
0.07 
0.045 
0.11 
0.046 
0.10 
0.047 
0.11 
N/A 
N/A 
0.00 
0.01 
N/A 
N/A 
% CV 
N/A 
N/A 
14.5% 
13.0% 
6.4% 
7.6% 
7.5% 
7.1% 
5.7% 
4.8% 
3.5% 
3.2% 
N/A 
N/A 
1.1% 
1.1% 
N/A 
N/A 
Total 
(inter-assay)2SD 
0.006 
0.03 
0.058 
0.19 
0.083 
0.18 
0.057 
0.28 
0.042 
0.53 
0.079 
1.00 
N/A 
N/A 
0.094 
0.01 
0.068 
0.54 
% CV 
N/A 
N/A 
20.8% 
27.0% 
22.7% 
19.8% 
9.5% 
18.7% 
5.3% 
26.5% 
5.8% 
29.2% 
N/A 
N/A 
22.7% 
0.9% 
5.7% 
18.2% 
 
Site 3 
Panel Member 
Neg 
Low Pos 
Pos #1 
Pos #2 
High Pos #1 
High Pos #2 
Neg Control 
CO Control 
Pos Control 
 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
OD 
Index 
N 
6 
6 
6 
6 
6 
6 
6 
6 
6 
6 
6 
6 
3 
3 
6 
6 
3 
3 
Mean 
0.049 
0.10 
0.388 
0.81 
0.652 
1.36 
0.830 
1.73 
1.158 
2.41 
2.378 
4.96 
0.059 
0.12 
0.480 
1.00 
1.652 
3.45 
Within Run 
(intra-assay)1SD 
0.003 
0.01 
0.009 
0.02 
0.082 
0.17 
0.068 
0.14 
0.094 
0.20 
0.126 
0.25 
N/A 
N/A 
0.028 
0.06 
N/A 
N/A 
% CV 
N/A 
N/A 
2.4% 
2.4% 
12.5% 
12.2% 
8.2% 
8.2% 
8.1% 
8.2% 
5.3% 
5.1% 
N/A 
N/A 
5.8% 
5.8% 
N/A 
N/A 
Total 
(inter-assay)2SD 
0.012 
0.03 
0.078 
0.13 
0.068 
0.15 
0.104 
0.25 
0.082 
0.15 
0.111 
0.34 
N/A 
N/A 
0.028 
0.04 
0.056 
0.23 
% CV 
N/A 
N/A 
20.0% 
15.8% 
10.5% 
11.1% 
12.5% 
14.3% 
7.1% 
6.2% 
4.7% 
6.8% 
N/A 
N/A 
5.8% 
4.1% 
3.4% 
6.6% 
 
N/A = not applicable 
 
1NCCLS EP5-A, Vol. 19, No. 2, Page 24, Equation (C2) 
2NCCLS EP5-A, Vol. 19, No. 2, Page 25, Equation (C3) and Equation (C4) 
 


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B. Cross Reactivity 
 
A study to evaluate the effect of potentially interfering medical conditions unrelated to Invasive 
Aspergillosis was performed with one lot of the Platelia Aspergillus EIA kit.  The following 
serum samples were tested for cross-reactivity with the Platelia Aspergillus EIA.  A total of 151 
sera were tested. 
 
Pathology 
#Samples Tested 
# Positives 
Rheumatoid Factor 
10 
0 
ANA Positive 
10 
0 
IgG Hypergammaglobulinemia 
10 
0 
IgM Hypergammaglobulinemia 
10 
0 
Cancer* 
11 
0 
Non-Viral Cirrhosis (primary biliary; 
alcohol induced; drug induced) 
10 
0 
Multiple Transfusions 
10 
0 
Multiparous Females 
10 
0 
HAV 
10 
0 
HCV 
10 
0 
Rubella 
10 
0 
CMV 
10 
0 
Syphilis (RPR+) 
10 
0 
Toxoplasmosis 
10 
0 
Mycoplasma 
10 
0 
 
*One each of bladder, breast (2), colon, endometrial, lung, prostate, renal, and squamous (3). 
 
 
C. Clinical Testing 
 
Clinical testing to evaluate the sensitivity, specificity, and predictive value of the Platelia 
Aspergillus EIA was conducted at three sites located in the U.S. and Canada.  The study was 
conducted retrospectively using a total of 1890 serum samples collected from 179 patients from 
the following populations*: 
 
 
patients without signs of Invasive Aspergillosis (control patients) 
 
patients with probable Invasive Aspergillosis 
 
patients with proven Invasive Aspergillosis 
 
* The Invasive Fungal Infection Cooperative Group (IFICG) of the European Organization for 
Research (EORTC) and the Mycosis Study Group (MSG) of the National Institute of Allergy and 
Infectious Diseases (NIAID) have defined criteria for diagnosis of Invasive Aspergillosis (IA) in 
patients with hematologic malignancy or hematopoetic stem cell transplant. 
 
 
Proven Invasive Aspergillosis is defined by positive microbiological culture obtained by sterile 
procedure from the site affected, and histopathological demonstration of the appropriate 
morphological forms in a host with symptoms attributed to the fungal infection. 
 


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Probable Invasive Aspergillosis is defined as a least one microbiological criterion, and one 
major or two minor clinical criteria from a site consistent with infection, in a host with symptoms 
attributed to the fungal infection. 
 
Possible Invasive Aspergillosis is defined as at least one microbiological criterion, or one 
major or two minor clinical criteria from a site consistent with infection, in a host with symptoms 
attributed to the fungal infection. 
 
Given the relative rarity of Probable and Proven Invasive Aspergillosis, we offer the following 
definition of clinical sensitivity and specificity for the purposes of this study. 
 
SENSITIVITY 
Results from this study have been analyzed in terms of patient sensitivity.  Sensitivity testing 
was conducted using the Platelia Aspergillus EIA at three sites on a combined total of 31 Bone 
Marrow Transplant (BMT) and Leukemia patents diagnosed with Proven or Probably Invasive 
Aspergillosis. 
 
1. Proven Aspergilliosis (as defined by IFICG / EORTC; see above) 
Combined Sites 
 
N = 11 (patients) 
11 patients: 
 
6 patients diagnosed with Proven Invasive Aspergillosis of the lung 
 
5 patients diagnosed with Proven Invasive Aspergillosis of the sinus 
Sensitivity: 81.8% (9/11). 
Note: The 95% confidence interval could not be calcuated due to insuffient sample size. 
 
2. Probable Aspergillosis (as defined by IFICB / EORTC; see above) 
Combined Sites 
 
N = 20 (patients) 
20 patients: 
 
16 patients diagnosed with Probable Invasive Aspergillosis of the lung 
 
4 patients diagnosed with Probable Invasive Aspergillosis of the sinus  
Sensitivity: 80.0% (16/20) 
Note: The 95% confidence interval could not be calculated due to insufficient sample size. 
 
3. Combined Proven and Probable Aspergillosis (as defined byIFICG / EORTC; see 
above) 
Combined Sites 
 
N = 31 (patients) 
31 patients: 
 
22 patients diagnosed with Proven or Probable Invasive Aspergillosis of the lung 
 
9 patients diagnosed with Proven or Probable Invasive Aspergillosis of the sinus 
Sensitivity: 80.7% (25/31).  The 95% confidence interval is 64.0 – 97.3% 


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SPECIFICITY 
Specificity testing was conducted using the Platelia Aspergillus EIA at three sites on a 
combined total of 1362 samples obtained from 148 Bone Marrow Transplant (BM) and 
Leukemia patients without signs of Invasive Aspergillosis (control patients). 
 
Site 1  
 
N = 33 patients 
449 sera obtained from: 
 
16 BMT patients without signs of Invasive Aspergillosis 
 
11 BMT patients colonized with Aspergillus and/or Candida sp. 
 
1 BMT patient diagnosed with Invasive Fusariosis 
 
3 BMT patients diagnosed with Candidemia 
 
1 patient with blood cultures positive for Lecythophora mutabilis 
 
1 patient diagnosed with Invasive Pseudoallescheria boydii 
 
Site 1 
Specificity 
95% Confidence Interval 
Patients (27/33) 
81.8% 
66.1 – 97.5% 
Patients after repeat testing (31/33) 
 
 
 
Site 2  
 
N = 77 patients 
560 sera obtained from: 
 
67 Leukemic patients without signs of Invasive Aspergillosis 
 
8 Leukemic patients with Fungemia (Candida, Fusarium, Trichosporon, or Aureobasidium) 
 
1 Leukemic patient diagnosed with Probable Fusarium pneumonia 
 
1 Leukemic patient diagnosed with Candida pneumonia 
 
Site 2 
Specificity 
95% Confidence Interval 
Patients (71/77) 
93.4% 
87.1 – 99.8% 
Patients after repeat testing (74/77) 
 
 
 
Site 3  
 
N = 38 patients 
353 sera obtained from: 
 
28 BMT patients without signs of Invasive Aspergillosis 
 
5 Leukemic patients receiving a second course of cytotoxic therapy 
 
5 BMT patients being treated for Graft Versus Host Disease 
 
Site 3 
Specificity 
95% Confidence Interval 
Patients (34/38) 
89.5% 
77.8 – 100% 
Patients after repeat testing (38/38) 
 
 


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Combined Sites 
N = 148 patients* 
Combined Sites 
Specificity 
95% Confidence Interval 
Patients (132/148) 
89.2% 
83.8 – 94.6% 
Patients after repeat testing (143/148) 
 
 
 
PREDICTIVE VALUE 
Positive and negative predictive values have been analyzed for the patient population in this 
study, based on the actual average 14% prevalence rate observed in this study.  Positive and 
negative predictive values have been calcualted for both the initial test result and after repeat 
testing. 
Actual Prevalence of 14% 
PPV 
NPV 
Patients 
54.8% 
96.6% 
Patients after repeat testing  
68.3% 
95.5% 
 
The expected prevalence of Invasive Aspergillosis varies with the patient population; rates from 
5-20% have been reported2,5.  For patient populations on the lower end of the published 
prevalence, the positive and negative prevalence have been re-calculated using a 5% 
prevalence rate. 
Calculated Prevalence of 5% 
PPV 
NPV 
Patients 
12.5% 
96.0% 
Patients after repeat testing  
31.3% 
96.3% 
 
 
*Note: A total of 1362 sera obtained from 148 patients were tested.  1343 of the 1362 sera were 
intitially negative, resulting in a sample agreement of 98.6% with a 95% confidence interval of 
97.9 – 99.3%.  On repeat testing, 1355 of the 1362 sera were negative. 
 


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16. APPENDIX D BIBLIOGRAPHY 
 
1. de Repentigny L., L. Kaufman, G.T. Cole, D. Kruse, J.P. Latge, R.C. Matthews.  1994.  
Immunodiagnosis of Invasive Fungal Infections.  J Med Vet Mycol.  32: 239-252. 
 
2. Denning, D.W. 1998.  Invasive Aspergillosis.  Clin. Infect. Dis. 26: p. 781-803. 
 
3. Erjavec, Z. and P.E. Verweij, 2002.  Recent progress in the diagnosis of fungal infections in 
the immunocompromised host.  Drug Resist. Updat. 5: p. 3-10. 
 
4. Herbrecht, R., V. Letscher-Bru, C. Oprea, B. Lioure, J. Waller, F. Campos, O. Villard, 
K.L. Liu, S. Natarajan-Ame, P. Lutz, P. Dufour, J.P. Bergerat, and E. Candolfi. 2002.  
Aspergillus galactomannan detection in the diagnosis of Invasive Aspergillosis in cancer 
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5. Herbrecht, R., D. Denning, T. Patterson, J. Bennett, R. Greene, J. Oestmann, W. Kern, 
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Durand, D. Caillot, E. Thiel, P. Chandrasekar, M. Hodges, H. Schlamm, P. Troke, B. 
DePauw.  2002.  Voriconazole Versus Amphotericin B for Primary Therapy of Invasive 
Aspergillosis.  N Engl J Med. 347, 6: p. 408-415. 
 
6. Latge, J.P.  1995.  Tools and trends in the detection of Aspergillus fumigatus.  Curr Top 
Med Mycol 6, p. 245-281. 
 
7. Latge, J.P.  1999.  Aspergillus fumigatus and Aspergillosis.  Clin Microbiol Rev 12[2], 310-
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8. Latge, J.P., H. Kobayashi, J.P. Debeaupuis, M. Diaquin, J. Sarfati, J.M. Wieruszeski, E. 
Parra, J.P. Bouchara, and B. Fournet.  1994.  Chemical and immunological 
characterization of the extracellular galactomannan of Aspergillus fumigatus.  Infect. Immun. 
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9. Maertens, J., J. Verhaegen, H. Demuynck, P. Brock, G. Verhoef, P. Vandenberghe, J. 
Van Eldere, L. Verbist, and M. Boogaerts.  1999.  Autopsy-controlled prospective 
evaluation of serial screening for circulating galactomannan by a sandwish enzyme-linked 
immunosorbent assay for hematological patients at risk for invasive Aspergillosis.  J. Clin. 
Microbiol. 37: p. 3223-3228. 
 
10. Maertens, J., J. Verhaegen, K. Lagrou, J. Van Eldere, and M. Boogaerts.  2001. 
Screening for circulating galactomannan as a noninvasive diagnostic tool for Invasive 
Aspergillosis in prolonged neutropenic patients and stem cell transplantation recipients: a 
prospective validation.  Blood 97: p. 1604-1610. 
 
11. Stynen D, A. Goris, J. Sarfati, and J.P. Latge.  1995.  A new sensitive sandwich enzyme-
linked immunosorbent assay to detect galactofuran in patients with Invasive Aspergillosis.  
J. Clin Microbiol.  33: 497-500. 
 
12. Stynen D., J. Sarfati, A. Goris, M.C. Prevost, M. Lesourd, H. Kamphuis, V. Darras, and 
J.P. Latge.  1992.  Rat monoclonal antibodies against Aspergillus galactomannan.  Infect. 
Immun. 60: p. 2237-2245. 


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13. Sulahian, A., F. Boutboul, P. Ribaud, T. Leblanc, C. Lacroix, and F. Derouin.  2001.  
Value of antigen detection using an enzyme immunoassay in the diagnosis and prediction of 
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prospective study.  Cancer 91: p. 311-318. 
 
14. Sulahian, A., M. Tabouret, P. Ribaud, J. Sarfati, E. Gluckman, J.P. Latge, and F. 
Derouin.  1996.  Comparison of an enzyme immunoassay and latex agglutination test for 
detection of galactomannan in the diagnosis of Invasive Aspergillosis.  Eur. J. Clin. 
Imcrobiol. Infect. Dis. 15: p. 139-145. 
 
15. Swanink, C.M., J.F. Meis, A.J. Rijs, J.P. Donnelly, and P.E. Verweij.  1997.  Specificity of 
a sandwish enzyme-linked immunosorbent assay for detecting Aspergillus galactomannan.  
J Clin. Microbiol. 35: p. 257-260. 
 
16. Verweij, P.E., E.C. Dompeling, J.P. Donnelly, A.V. Schattenberg, and J.F. Meis.  1997.  
Serial monitoring of Aspergillus antigen in the early diagnosis of Invasive Aspergillosis.  
Preliminary investigations with two examples.  Infection 25: p. 86-89. 
 
17. Verweij, P.E. and J.F. Meis.  2000.  Microbiological diagnosis of Invasive Fungal Infections 
in transplant recipients.  Transpl. Infect. Dis. 2: p. 80-87. 
 
18. Verweij, P.E., D. Stynen, A.J. Rijs, B.E. de Pauw, J.A. Hoogkamp-Korstanje, and J.F. 
Meis.  1995.  Sandwish enzyme-linked immunosorbent assay compared with Pastorex latex 
aggluination test for diagnosing Invasive Aspergillosis in immunocompromised patients.  J. 
Clin. Microbiol. 33: p. 1912-1914. 
 
19. Verweij, P.E., C.M. Weemaes, J.H. Curfs, S. Bretagne, J.F. Meis.  2000. Failure to detect 
circulating Aspergillus markers in a patient with chronic granulomatous disease and Invasive 
Aspergillosis.  J Clin. Microbiol. 38: p. 3900-3901. 
 
20. Walsh, T.J., R.L. Schaufele, T. Sein, J. Gea-Banacloche, M. Bishop, N. Young, R. 
Childs, J. Barrett, H.L. Malech, and S.M. Holland.  2002.  Reduced expression of 
galactomannan antigenemia in patients with Invasive Aspergillosis and chronic 
granulomatous disease or Job’s syndrome.  Abstracts of the 40th Annual Meeting of the 
Infectious Diseases Society of America.  Arlington, VA. P. 105; Abstr. 345. 
 
21. Yeo, S.F. and B. Wong.  2002.  Current Status of Nonculture Methods for Diagnosis of 
Invasive Fungal Infections.  Clin. Microbiol. Rev. 15: p. 465-484. 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-44 
 


BMT CLINICAL TRIALS NETWORK 
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Version 7.0 dated March 29, 2006 
 
D-45 
 
 
 
APPENDIX D 
 
ATTACHMENT C:  PLATELIA ASPERGILLUS WORKSHEETS 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-46 
R1 Microwell Plates
Date p ouch op ened:
R2 Concentrated Wash
Date d iluted:
Fresh
(c irc le
Frozen
one)
Fresh
(c irc le
Frozen
one)
Fresh
(c irc le
Frozen
one)
R6 Conjugate
R7 Serum Treatment Solution
R8 Peroxidase Substrate Buffer
R9 Chromogen
R10 Stopping Solution
Boiling water bath or heat block
Temp:
Centrifuge for Eppendorf tubes
Revolution:
xG
Washer
Incubator
Temp:
Reader
Wa velength:
Heat in water bath or heat block
3 minutes
Heat in water bath or heat block
3 minutes
Heat in water bath or heat block
3 minutes
Centrifuge at 10,000 g
10 minutes
Centrifuge at 10,000 g
10 minutes
Centrifuge at 10,000 g
10 minutes
Incubation of conjugate and treated sera
90 minutes
Substrate-chromogen incubation
30 minutes +/- 5
Stopping solution added
Plate read
within 30 min of stop
N/A
N/A
N/A
TREATMENT OF SERA                              TREATMENT OF SERA                              TREATMENT OF SERA
EIA PROCEDURE                                        EIA PROCEDURE                                        EIA PROCEDURE
START TIME
FINISH TIME
ACCEPTABLE
PROCEDURE TIMING VERIFICATION
ASSAY STEP
/
R3 Negative Control Serum
R4 Threshold Control Serum
R5 Positive Control Serum
Date rec onst:
Date rec onst:
Date rec onst:
INSTRUMENTATION USED
INSTRUMENT
MANUFACTURER
SERIAL NO.
COMMENTS
(THIS PAGE TO BE HAND-WRITTEN)
REAGENT
COMMENTS
LOT #
Run No:______________________________     Date Performed:____________________     Technologist:____________________
Platelia Aspergillus Kit Lot #:_________________________     Expiration Date:_________________________
KIT REAGENTS
Clinical Trial Site:______________________________________________________________________


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
D-47 
 
1
2
3
4
5
6
A
B
C
D
E
F
G
H
7
8
9
10
11
12
A
B
C
D
E
F
G
H
(THIS PAGE TO BE HAND-WRITTEN)
Run No:______________________________     Date Performed:____________________     Technologist:____________________
Platelia Aspergillus Kit Lot #:_________________________     Expiration Date:_________________________
Clinical Trial Site:____________________________________________________________
 


BMT CLINICAL TRIALS NETWORK 
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Version 7.0 dated March 29, 2006 
 
D-48 
 
 
 
APPENDIX D 
 
ATTACHMENT D:  QUICK REFERENCE GUIDE 
 
 


BMT CLINICAL TRIALS NETWORK 
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Version 7.0 dated March 29, 2006 
 
D-49 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
E-1 
 
 
 
APPENDIX E 
 
HUMAN SUBJECTS 
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
E-2 
 
 
APPENDIX E 
 
HUMAN SUBJECTS 
 
 
Subject consent: Candidates for the study will be identified as described in Section 2.0 of the 
protocol.  The PI or his/her designee at each transplant center will contact the candidates and 
enroll them onto the study.  The study coordinator at each center will provide the patient with 
information about the purpose of the study and obtain consent.  A template of the consent form 
will be provided by the network to each center.  Each center will customize the template 
according to their local requirements and submit it for review by the local IRB.  Each center 
must provide evidence of IRB approval. 
 
Confidentiality: Confidentiality will be maintained by individual names being masked and 
assigned a patient identifier code.  The code relaying the patient’s identity with the ID code will 
be kept separately at the center.  The ID code will be transmitted to the network.   
 
Participation of women and minorities, children and other populations: Women and ethnic 
minorities will be included in this study.  The lower age limit of participants is age 2.  
 
Accrual will be monitored within each center with the expectation that the enrolled patient 
population is representative of the transplanted patient population at each center.  Representation 
will be examined by comparing gender, race, ethnicity and age distributions.  Accrual of 
minority patients will be expected to be in proportion to the number of minority patients 
transplanted at each center.  The DCC and NHLBI will discuss enrollment anomalies with the 
centers. 
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
F-1 
 
 
APPENDIX F 
 
VORICONAZOLE AND FLUCONAZOLE 
DILUTION AND INFUSION INSTRUCTIONS 
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
F-2 
 
APPENDIX F 
 
VORICONAZOLE AND FLUCONAZOLE  
DILUTION AND INFUSION INSTRUCTIONS 
 
A.    VORICONAZOLE DILUTION AND INFUSION INSTRUCTIONS 
 
Voriconazole is manufactured and supplied by Pfizer Pharmaceuticals, Groton, Connecticut, 
USA and will be supplied by the Blood and Marrow Transplant Clinical Trials Network (BMT 
CTN).  Voriconazole is packaged as a lyophilized powder for injection, 200 mg in 30 mL vials, 
with each vial also containing 3200 mg sulfobutyl ether beta-cyclodextrin sodium (SBECD).  
Prior to reconstitution vials should be stored at room temperature 15o-30oC.  The drug does not 
contain a preservative, and is suitable for single use only.  If not used immediately, the in-use 
storage times and conditions prior to use are the responsibility of the individual user and should 
not be longer than 24 hours at 2o-8oC (37o-46oF). 
 
RECONSTITUTION FOR PEDIATRIC DOSES - Under aseptic conditions: 
1. Voriconazole should be reconstituted with 19 mL Sterile Water for Injection, USP.  Each 
mL will contain 10 mg voriconazole.   
2. Use the table below to determine the number of vials needed for each dose. 
3. The volume of 10 mg/mL concentrated voriconazole solution required based on the 
patients weight should be calculated (see below) and withdrawn into a syringe. 
 
VORICONAZOLE - Patients < 12 years 
Body Weight (kg) 
Dose 
Volume of Voriconazole Concentrated 
(10 mg/mL) Solution Required for  
~4 mg/kg Dose (Number of Vials) 
 10 
50 mg 
5 mL (1) 
10 – 14.99 
50 mg 
5 mL (1) 
15 – 19.00 
100 mg 
10 mL (1) 
20 – 24.99 
100 mg 
10 mL (1) 
25 – 29.99 
100 mg 
10 mL (1) 
30 – 34.99 
150 mg 
15 mL (1) 
35 – 39.99 
150 mg 
15 mL (1) 
 40 
200 mg 
20 mL (1) 
* NOTES: 
1. Dose capped for children weighing > 40 kg. 
2. It is expected that weight fluctuations during study periods will not be substantial enough to 
warrant dosing changes. 
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
F-3 
4. This volume should be withdrawn and injected into an empty Viaflex bag, and then 
further diluted with an approved solution (these solutions will not be provided by the 
BMT CTN).   
5. Approved diluent:  Sodium Chloride 0.9%, USP. 
6. The final volume is dependent upon the age of the patient.  For pediatric patients the 
final concentration of voriconazole will vary, with the final volume varying according to 
body weight. 
 
 
Body Weight (kg) 
VORICONAZOLE - Patients < 12 Years 
Volume concentrated 
solution (10mg/mL) 
Additional Fluid for Final 
Reconstitution 
Total Volume 
 10 
5.0 mL 
45 mL 
50 mL 
10 – 14.99 
5.0 mL 
45 mL 
50 mL 
15 – 19.99 
10.0 mL 
40 mL 
50 mL 
20 – 24.99 
10.0 mL 
65 mL 
75 mL 
25 – 29.99 
10.0 mL 
65 mL 
75 mL 
30 – 34.99 
15.0 mL 
85 mL 
100 mL 
35 – 39.99 
15.0 mL 
85 mL 
100 mL 
 40 
20.0 mL 
80 mL 
100 mL 
 
NOTE: for children < 30 kg, a single vial of voriconazole 200 mg may be used to prepare both AM and PM 
doses.  In such circumstances doses should be prepared at the same time and given an expiration of 24 hours 
under refrigerated conditions. 
 
Administration:  Voriconazole is to be administered over 2 hours. 
 
RECONSTITUTION FOR ADULT (patients aged 12 years or greater) DOSES - Under 
aseptic conditions: 
1.  Voriconazole should be reconstituted with 19 ml Sterile Water for Injection, USP.  Each 
mL will contain 10 mg voriconazole.   
2. Use the table below to determine the number of vials needed for each dose. 
3. The volume of 10 mg/mL concentrated voriconazole solution required based on the 
patients weight should be calculated (see below) and withdrawn into a syringe. 
 
Volume of Voriconazole Concentrated (10 mg/mL) Solution required: 
All adult patients (i.e., those 12 years of age or greater) will receive a standard intravenous 
dose of voriconazole of 200 mg twice daily.  The volume of concentrated solution to make 
each dose is 20mL. 
 
4. This volume should be withdrawn and injected into an empty Viaflex bag, and then 
further diluted with an approved solution (these solutions will not be provided by the 
BMT CTN).   


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
F-4 
5. Approved diluent:  Sodium Chloride 0.9%, USP. 
6. The final volume is dependent upon the age of the patient.   
 
 
Age 
VORICONAZOLE – Adult Patients ( 12 years) 
Volume 
concentrated 
solution (10 mg/mL) 
Additional Fluid for Final 
Reconstitution 
Total Volume 
> 12 years 
(Normal Renal 
Function) 
20.0 mL 
180 mL 
200 mL 
> 12 years 
(CrCl  
< 50 mL/min) 
20.0 mL 
80 mL 
100 mL 
Notes:  Voriconazole should not be infused into the same catheter as other drug infusions, 
including parenteral nutrition.  Infusion of blood products and infusion of electrolyte 
supplementation must not occur simultaneously with voriconazole infusion. 
 
 
B.    FLUCONAZOLE DILUTION AND INFUSION INSTRUCTIONS 
 
Fluconazole (Diflucan) is manufactured by Pfizer, Inc., Groton, Connecticut and will be 
supplied by the Blood and Marrow Transplant Clinical Trials Network.  Fluconazole for 
injection is packaged in Viaflex Plus plastic containers containing 200 mg of fluconazole in a 
volume of 100 mL in sodium chloride (concentration 2 mg/mL). 
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
F-5 
RECONSTITUTION FOR PEDIATRIC (< 12 years of age) PATIENTS: 
1. The dose of fluconazole required should be calculated, and the corresponding volume of 
fluid should be withdrawn from the manufacturer’s Viaflex bag (see below for 
volumes). 
 
FLUCONAZOLE – Patients < 12 years 
Body 
Weight (kg) 
Volume of Fluconazole 
Concentrated (2 mg/mL) 
Solution Required for 6 mg/kg 
Dose in Pediatrics 
(Number of Bags) 
Additional Fluid 
for Final 
Reconstitution 
Total Volume 
< 10 
30 mL (1) 
20 mL 
50 mL 
10 – 14.99 
30 mL (1) 
20 mL 
50 mL 
15 – 19.99 
45 mL (1) 
5 mL 
50 mL 
20 – 24.99 
60 mL (1) 
15 mL 
75 mL 
25 – 29.99 
75 mL (1) 
Nil 
75 mL 
30 – 34.99 
90 mL (1) 
10 mL 
100 mL 
> 35 
100 mL (2) 
Nil 
100 mL 
 
2. This solution should be injected into an empty Viaflex bag, and further diluted as 
directed in the table above with an approved diluent (see below) 
3. Approved diluent:  Sodium Chloride 0.9%, USP. 
 
RECONSTITUTION FOR PEDIATRIC (< 12 years of age) PATIENTS WITH 
CREATININE CLEARANCE <50mL/min/1.73m2: 
 
FLUCONAZOLE – Patients < 12 years (CrCl <50 mL/min/1.73m2) 
Body 
Weight (kg) 
Volume of Fluconazole 
Concentrated (2 mg/mL) 
Solution Required for 3 mg/kg 
Dose in Pediatrics 
(Number of Bags) 
Additional Fluid 
for Final 
Reconstitution 
Total Volume 
< 10 
15 mL (1) 
35 mL 
50 mL 
10 – 14.99 
15 mL (1) 
35 mL 
50 mL 
15 – 19.99 
22.5 mL (1) 
27.5 mL 
50 mL 
20 – 24.99 
30 mL (1) 
45 mL 
75 mL 
25 – 29.99 
37.5 mL (1) 
37.5 mL 
75 mL 
30 – 34.99 
45 mL (1) 
55 mL 
100 mL 
> 35 
50 mL (1) 
50 mL 
100 mL 
 
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
F-6 
RECONSTITUTION FOR PEDIATRIC (< 12 years of age) PATIENTS WITH 
CREATININE CLEARANCE < 20mL/min/1.73m2: 
 
FLUCONAZOLE – Patients < 12 years (CrCl < 20 mL/min/1.73m2) 
Body 
Weight (kg) 
Volume of Fluconazole 
Concentrated (2 mg/mL) 
Solution Required for 1.5 mg/kg 
Dose in Pediatrics 
(Number of Bags) 
Additional Fluid 
for Final 
Reconstitution* 
Total Volume 
< 10 
7.5 mL (1) 
42.5 mL 
50 mL 
10 – 14.99 
7.5 mL (1) 
42.5 mL 
50 mL 
15 – 19.99 
11.25 mL (1) 
38.75 mL 
50 mL 
20 – 24.99 
15 mL (1) 
60 mL 
75 mL 
25 – 29.99 
18.75 mL (1) 
56.25 mL 
75 mL 
30 – 34.99 
22.5 mL (1) 
77.5 mL 
100 mL 
≥ 35 
25 mL (1) 
75 mL 
100 mL 
 
4. This solution should be injected into an empty Viaflex bag, and further diluted as 
directed in the table above with an approved diluent (see below) 
5. Approved diluent:  Sodium Chloride 0.9%, USP. 
 
 
RECONSTITUTION FOR ADULT (> 12 years of age) PATIENTS: 
1. A total volume of 200 mL should be withdrawn from the manufacturer’s Viaflex bag.  
This solution is to be transferred into an empty sterile Viaflex bag (250 mL bag). 
2. No further dilution is required. 
 
Administration: Doses should be administered over 2 hours for both adults and pediatrics. 
 
C.    PLACEBO SOLUTION INSTRUCTIONS FOR FLUCONAZOLE-RANDOMIZED 
        PATIENTS 
 
PLACEBO – Patients < 12 years 
Body Weight (kg)  
Volume of Sodium Chloride 
< 10 
50 mL  
10 – 14.99 
50 mL 
15 – 19.99 
50 mL 
20 – 24.99 
75 mL 
25 – 29.99 
75 mL 
30 – 34.99 
100 mL 
 35 
100 mL 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
F-7 
 
 
PLACEBO SOLUTION FOR FLUCONAZOLE RANDOMIZED  
ADULT ( 12 years of age) PATIENTS 
 
PLACEBO – Patients  12 years 
 
Volume of Sodium Chloride 
 12 years age 
(Normal Renal 
Function) 
200 mL 
 12 years age 
(CrCl < 50 mL/min) 
100 mL 
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
G-1 
 
 
APPENDIX G 
 
DOSING TABLES 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
G-2 
 
ORAL DOSING TABLE 
 
 
Age 
Drug 
Route 
Renal 
Function 
Dosage 
Pills in AM 
Pills in PM 
Adult 
fluconazole 
PO 
normal 
400 mg/d 
 
 
 
 
voriconazole 
PO 
normal 
200 mgQ12H 
 
 
 
Adult * 
fluconazole 
PO 
< 50 mg/min 
200 mg/d 
 
 
 
voriconazole 
PO 
< 50 mg/min 
200 mgQ12H 
 
 
Age < 12 and 
> 20 kg 
fluconazole 
PO 
normal 
200 mg/d 
 
 
   
 
voriconazole 
PO 
normal 
100 mgQ12H 
  
 
  
 
Age < 12 and 
> 20 kg * 
fluconazole 
PO 
< 50 mg/min 
100 mg/d 
   
   
 
voriconazole 
PO 
< 50 mg/min 
100 mgQ12H 
  
 
  
 
Age < 12 and 
< 20 kg 
fluconazole 
PO 
normal 
100 mg/d 
 
 
 
voriconazole 
PO 
normal 
 50 mgQ12H 
 
 
* RENAL INSUFFICIENCY based on calculated creatinine clearance of < 50 mL/min 
RENAL INSUFFICIENCY in a patient age < 12 and < 20kg should be treated by IV infusion in order to achieve a 
dosage of 50 mg/d fluconazole.  To maintain blind, voriconazole patient should be treated by IV. 
 
 
LEGEND: 
Real Drug 
 
200mg flu  
 
200mg vori  
 
100mg flu  
 
50mg vori  
 
Placebo 
 
200 mg flu / 200 mg vori 
 
100 mg flu / 50 mg vori 
 
 
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
G-3 
 
IV DOSING TABLE 
 
 
Age 
Drug 
Route 
Renal 
Function 
Agents 
Number of Doses/Dose# 
Time 
Adult  
[ 12] 
Fluconazole 
IV 
Normal 
1 FLUC 
NS 
400 mg/200 mL 
200 mL NS 
AM 
PM 
Voriconazole 
IV 
Normal 
1 VORI 
1 VORI 
200 mg/200 mL 
200 mg/200 mL 
AM 
PM 
Adult 
[ 12] 
Fluconazole 
IV 
< 50mL/min 1 FLUC 
NS 
200 mg/100 mL 
100 mL NS 
AM 
PM 
Voriconazole 
IV 
< 50mL/min 1 VORI 
1 VORI 
200 mg/100 mL 
200 mg/100 mL 
AM 
PM 
Pediatric 
[< 12 years] 
Fluconazole 
IV 
Normal 
1 FLUC 
 
NS 
6 mg/kg/weight adjusted 
volume* 
Weight adjusted volume* 
AM 
 
PM 
Voriconazole 
IV 
Normal 
1 VORI 
 
1 VORI 
4 mg/kg/weight adjusted 
volume* 
4 mg/kg/weight adjusted 
volume* 
AM 
 
PM 
Pediatric 
[< 12 years] 
Fluconazole 
IV 
< 50mL/min 1 FLUC 
 
NS 
3 mg/kg/weight adjusted 
volume* 
Weight adjusted volume* 
AM 
 
PM 
Fluconazole 
IV 
< 20mL/min 1 FLUC 
 
NS 
1.5 mg/kg/weight adjusted 
volume* 
Weight adjusted volume* 
AM 
 
PM 
 
Voriconazole 
IV 
< 50mL/min 1 VORI 
 
1 VORI 
4 mg/kg/weight adjusted 
volume* 
4 mg/kg/weight adjusted 
volume* 
AM 
 
PM 
 
# Doses to be rounded up to the nearest 5kg bracket. 
* See Appendix F for volume guidelines. 
If the patient is an outpatient, and requires study IV drug for more than 2 weeks and cannot tolerate 
oral study drug, the patient must be taken off study drug. 
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
H-1 
 
 
APPENDIX H 
 
ORDER FORM FOR DOSE ADJUSTMENT OF STUDY DRUG 
 
 
 
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
H-2 
 
 
 
   
 
 
DATE 
TIME 
PHYSICIAN’S ORDERS (Provider ID # Required) 
SMS # 
 
 
Orders for IRB#******,  A Randomized , Double-blind  
 
 
 
Trial of Fluconazole vs. Voriconazole for the Prevention of Invasive Fungal  
 
 
 
Infections in Allogeneic Blood and Marrow Transplant Patients. 
 
 
 
1. Subject meets inclusion/exclusion criteria and is enrolled in IRB # ***  
 
 
 
2. Patient’s serum creatinine:________mg/dL, patient weight (kg):___________ 
 
 
 
3. Calculate Creatinine Clearance using Cockroft-Gault equation in 2.4.3 protocol 
 
 
 
    for adults and Schwartz equation for pediatrics or 24-hour urine Creatinine  
 
 
 
    Clearance. 
 
 
 
    Creatinine Clearance (mL/min):__________________mL/min 
 
 
 
4. Dosage adjustments: 
 
 
 
    If CrCl  50 mL/minute – no dose adjustment required 
 
 
 
    If CrCl <  50 mL/minute - adjust the doses of study medications as follows: 
 
 
 
    Fluconazole:  dose reduction of 50% PO and IV.   
 
 
 
     Initial dose:_________mg, new dose ___________mg.  OR 
 
 
 
    If CrCl < 20 mL/min/1.73m2 for pediatric patients (i.e. < 12 years) 
 
 
 
    Fluconazole:  dose reduction of 75% PO and IV.   
 
 
 
     Initial dose:_________mg, new dose ___________mg. 
 
 
 
    Voriconazole:  no dose adjustment PO.  Accumulation of the voriconazole 
 
 
 
    IV vehicle can occur.  Oral voriconazole should be administered to these  
 
 
 
    patients unless a benefit/risk assessment justifies the use of IV voriconazole. 
 
 
 
    Medications to be reconstituted in the same volume of fluid to maintain the  
 
 
 
     blind. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
Rev. 8/00
15-0610-1
DO NOT WRITE BELOW THIS LINE 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
I-1 
 
 
APPENDIX I 
 
DERIVATION OF THE SPRT FOR UNCENSORED EXPONENTIAL 
SURVIVAL TIMES 
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
I-2 
 
 
APPENDIX I 
 
DERIVATION OF A SEQUENTIAL TEST STATISTIC FOR CENSORED 
EXPONENTIAL DATA 
 
 
Background – The Sequential Probability Ratio Test 
 
Let 
)
(.,
f
be the density function for random variable X.  According to Neyman and Pearson, the most 
powerful test of 
o
H


:
0
 versus
1
1 :


H
 decides in favor of 
1
H  or 
0
H if 

c
Ln 
 or 

c
Ln 
, 
respectively, where 


n
i
i
i
n
x
f
x
f
L
)
;
(
/)
;
(
0
1


is the likelihood ratio, and 

c is determined to have the 
size .  When the sample size is not fixed in advance, further improvement is possible by using Wald’s 
Sequential Probability Ratio Test (SPRT).  The SPRT continues to sample as long as 
A
L
B
n 

 for some 
constant 
A
B

1
, stops sampling and decides in favor of 
1
H as soon as 
A
Ln 
, and stops sampling and 
decides in favor of 
0
H as soon as 
B
Ln 
. 
 
The usual measures of performance of such a procedure are the error probabilities 
and of rejecting 
0
H when 
0


, and of accepting 
0
H when 
1


, respectively, and the expected sample size 
)
(
)
|
(
N
E
N
E
j
j 

.  Wald and Wolfowitz showed that among all tests, sequential or not, for which 


)
reject 
(
Pr
0
0
H
 and 


)
reject 
(
Pr
0
1
H
, and for which 
)
(N
E j
 are finite, j=0,1, the SPRT with error 
probabilities 
 and minimizes 
)
(
0 N
E
 and 
)
(
1 N
E
.  If, in addition, the 
,...
2
,1 x
x
 are independent and 
identically distributed (i.i.d.) with density function 
)
,
( 
x
f
, with monotone likelihood ratio in 
)
(x

, then 
any SPRT for testing 
0
against 
)
(
0
1


 has non-decreasing power function. 
 
For the SPRT with error probabilities 
and , the SPRT boundaries are given approximately by 

/)
1( 

A
 and 
)
1
/(




B
.  The operating characteristics of the SPRT are given by 
)
/(
)1
(
)
,
,
,
,
(
)
(
)
(
)
(
1
0








h
h
h
B
A
A
O



 where 
)
(
h
is the non-trivial solution to the equation 

1
)
;
(
))
,
(
/)
;
(
(
)
(
2
1
dx
x
f
x
f
x
f
h




.   
 
The formula 
)
;
(
/]
log
)
(
log
)]
(
1
[[(
)
;
(




z
E
B
O
A
O
N
E



provides the average sample number for an 
arbitrary .  The sample size distribution is very highly skewed, 
2
)]
(
[
)
(
N
E
N
Var

.  Thus we will consider 
a truncated test with maximum sample size of 
0
N  and simulate to obtain the operating characteristics of 
the test. 
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
I-3 
Derivation of the SPRT for Uncensored Exponential Survival Times 
 
For example, we wish to construct a sequential test for the composite null hypothesis that the rate of 
convulsions at 100 days is less than or equal to 10% versus the alternative hypothesis that it is greater than 
or equal to 10%.  For the derivation of the uncensored SPRT, we will require that the type I error of the 
test be less than 5%, and that the test provide 80% power to reject the null hypothesis under a specified 
alternative that the true rate is 15%.  A maximum sample size of 300 patients will be permitted. 
 
Let us assume that the survival times, 
nT
T
T
,...,
,
2
1
, are completely observed (uncensored) and are i.i.d. with 
exponential density function 
T
e
T
f





)
,
(
.  These assumptions will be relaxed to incompletely observed 
data subsequently.  In the exponential parameterization, a 100-day survival rate of 90% translates into a 
mean survival of 2.599 years (

0

.3848), and 85% translates into a mean survival of 1.685 years 
(

1

.5936). 
 
The SPRT is derived with reference to a simple null and alternative hypothesis, in this case, 


o
H


:
0
 
.3848 versus
1
1 :


H
 = .5936.  However, since the log-likelihood ratio for the exponential, 








n
i
n
i
n
i
i
i
i
T
n
x
f
x
f
)
(
))
log(
)
(log(
)
,
(
log
)
;
(
log
0
1
0
1
0
1






, is a monotone function of 
n
i
iT , 
the power of the test is non-decreasing in .  Thus the SPRT is a one-sided level .05 test of a composite 
null (
3848
.
:
0


o
H


) versus a composite alternative (
3848
.
:
1


o
H


), with power of 
80
.
1


at the selected alternative 


1


.5936. 
 
The SPRT can be represented graphically.  The continuation region is bounded by two parallel lines with 
common slope 



)
/(
)
log
(log
1
0
1
0




2.076, and intercepts 


)
/(
log
1
0


A
-13.28 and 


)
/(
log
1
0


B
7.462, for the lower and upper bounds, respectively.  As each individual unit is put on 
trial and observed to fail, the cumulative sum of failure times,
n
i
iT , is recomputed, and plotted against 
the current sample size, n .  When this graph crosses the lower boundary, the null hypothesis is rejected. 
 
The maximum sample size of 300 patients requires that the SPRT be truncated.  We choose to truncate the 
SPRT by declaring that if the test has failed to terminate after 300 patients, that the null hypothesis will be 
accepted.  Since the probability that the untruncated SPRT would reject the null at a sample size of 300 is 
negligible, it makes little difference how the final boundary value is selected, and this rule is chosen for 
simplicity.   
 
Derivation of a Modified SPRT for Censored Exponential Data 
 
The assumption of uncensored exponential survival times is flawed.  However, we consider it reasonable 
to assume the hazard for convulsions is constant over the first 100 days post-transplant, and we will 
restrict our attention to this time interval.  Furthermore, it is not practical to conduct a clinical study by 
putting each individual on trial, and waiting until that individual is observed to fail.  We relax our 
assumptions as follows.  Firstly, each individual’s time on study will be computed as time from transplant 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
I-4 
to failure, or to the 100 day time point, whichever comes first.  Secondly, we will put individuals on trial 
as soon as they become available, without waiting for the previous individual to fail. 
 
Let us consider the impact of relaxing these assumptions one at a time.  In a fixed sample size trial with 
uncensored exponential failure times, mean survival time is estimated by the sample mean of the failure 
times, or total time on study divided by the number of individuals enrolled.  When censoring is 
introduced, the estimate becomes the total time on study divided by the number of observed (non-
censored) failures.  This suggests that in an exponential SPRT test modified to incorporate censoring, we 
replace the observed failure times, 
nT
T
T
,...,
,
2
1
, with censored failures times, 
nx
x
x
,...,
,
2
1
, and the current 
sample size, n , with the number of observed failures, d .   
 
Now we relax the second assumption, and put individuals on trial as soon as they become available, 
without waiting for the previous individual to fail.  Assume that three years are required for accrual of 300 
patients to the study, and that the final analysis takes place 100 days after the last patient is entered.  
Putting all of this together, we propose a modified truncated SPRT, where at any interim time point, s , 
ranging from 0 to 3 years 100 days, the sum of observed time on study, 
n
i
i s
X
)
(
is plotted against the 
number of observed failures, 
)
(s
d
.  In practice, monitoring will be scheduled monthly after the start of 
enrollment to the study.  A further modification to the SPRT was to only use the lower boundary for 
stopping since the primary focus of the monitoring is to protect against unacceptable 100-day convulsion 
rates. 
 
Operating Characteristics of the Modified SPRT Test for Censored Exponential Data 
 
Recall that the uncensored SPRT targeted a drop in convulsion-free survival at Day 100 from 90% to 
85%, with type I and II errors of 5% and 20%.  Since only the lower boundary is used for monitoring, the 
continuation region of the test was bounded below by a line with a slope of 2.076 and intercept of –13.28.  
The effect of truncation is to reduce the power of the test.  In order to compensate for this, we raise the 
lower boundary to make it easier to cross.  Under the further assumption of uniform accrual over a three 
year period, and monthly interim analyses over the course of the study, the operating characteristics of the 
modified SPRT were obtained from a simulation study.  These simulation show that an intercept of –
10.555, corresponding to setting parameters 
 and to 9% and 15%, result in empirical type I and II 
error rates of 5% and 20%.   
 


BMT CLINICAL TRIALS NETWORK 
Fungal Prophylaxis Protocol – 0101 
Version 7.0 dated March 29, 2006 
 
I-5 
Table I-1   Operating Characteristics of Sequential Testing Procedures from a 
Simulation Study with 100,000 Replications 
 
 
Neural Toxicity (Convulsions) 
True 100-Day Rate 
10% 
15% 
20% 
Probability Reject Null 
0.06 
0.79 
1.00 
Mean Month Stopped 
35.8 
21.8 
10.7 
Mean # Endpoints in 100 days 
28.4 
25.2 
15.3 
Mean # Patients Enrolled 
290.9 
179.8 
89.1 
 
While the motivation for this testing procedure is largely heuristic rather than theoretical, the simulation 
results validate the approach.  When the true rate of convulsions on or before Day 100 was 10%, the test 
crossed the lower boundary in 5977 of 100,000 replications, for an estimated type I error rate of 6%.  
When the true rate of convulsions on or before Day 100 was 15%, the test failed to cross the boundary in 
the in 21483 of 100,000 replications, for an estimated type II error rate of 21%.  The test is almost certain 
(100%) to reject the null hypothesis when the true rate is 20%, and on average, the boundary will be 
crossed at 10.7 months, with probability less than 1% of crossing in the first 3 months. 
 
It is interesting to note that the SPRT derived above for exponential failure times with censoring at 100 
days, has operating characteristics which are similar to those of a more traditional SPRT, derived for 
binomial variates with success probability equal to the 100 day failure rate.  Using time to failure rather 
than a simple binary indicator of failure, leads to little improvement in power when failure times are 
censored relatively soon after entry on study.  We speculate that if the constant hazard rate over the first 
100 days were high, the exponential test would reject faster than the binomial test, but have not conducted 
simulation studies to demonstrate this.