3rd ADVAN AbstrAct book c E s AGAIN st A s PE r GILL os I s • January 16-19, 2008 3rd ADVANcEs AGAINst AsPErGILLosIs January 16-19, 2008 Miami Beach Resort & Spa Miami, Florida, USA
University of California, San Diego School of Medicine
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
ACKNOWLEDGEMENTS
We would like to offer very special thanks to the following organizations for their generous educational grants. Their financial support makes this conference possible
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3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
We would also like to thank the following companies for contributing their financial support and educational exhibits to this conference. We encourage you to take this opportunity to meet and share your ideas and experiences with the exhibit representatives.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
TABLE OF CONTENTS
Conference Chairmen, Scientific Committee...... 1
Faculty List...... 3
Faculty Disclosures...... 7
Scholarship Awards...... 11
Poster Abstract Index...... 13
Final Program Wednesday, January 16...... 21
Thursday, January 17...... 23
Friday, January 18...... 25
Saturday, January 19 ...... 27
Abstracts Invited Faculty...... 29
Oral Abstract Presenters...... 85
Poster Abstracts...... 91
Author Index...... 189
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
CONFERENCE CHAIRMAN AND SCIENTIFIC COMMITTEE
Chairmen
David W. Denning, MD / University of Manchester, UK
William J. Steinbach, MD / Duke University, USA
David A. Stevens, MD / Stanford University, USA
Scientific Committee
Scott E. Baker, PhD / Pacific Northwest National Laboratory, USA
Arunaloke Chakrabarti, MD / Postgraduate Institute of Medical Education & Research, India
Karl V. Clemons, PhD / Stanford University, USA
Reinhard Fischer, PhD / University of Karlsruhe, Germany
Donna Haiduven, PhD / University of South Florida, USA
Katsuhiko Kamei, MD, PhD / Chiba University, Japan
Dimitrios P. Kontoyiannis, MD / University of Texas MD Anderson Cancer Center, USA
Jean-Paul Latgé, PhD / Institut Pasteur, France
Johan Maertens, MD / University Hospital Gasthuisberg, Belgium
Richard B. Moss, MD / Stanford University, USA
David S. Perlin, PhD / UMDNJ - New Jersey Medical School
George A. Sarosi, MD / Indiana University, USA
John R. Wingard, MD / University of Florida, USA
Sally E. Wenzel, MD / University of Pittsburgh, USA
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
FACULTY
Jill Adler-Moore, MD Michael J. Day, PhD California State Polytechnic University, USA University of Bristol, UK
Ryoichi Amitani, MD David W. Denning, MD Kyoto University, Japan University of Manchester, UK
Elias J. Anaissie, MD Richard D. deShazo, MD University of Arkansas, USA University of Mississippi, USA
Arunmozhi Balajee, PhD Dennis M. Dixon, PhD Centers for Disease Control and Prevention, USA National Institute of Allergy and Infectious Diseases, USA Scott E. Baker, PhD Pacific Northwest National Laboratory, USA Richard H. Drew, PharmD Duke University, USA Joan W. Bennett, PhD Rutgers University, USA Natalie D. Fedorova, PhD The Institute for Genomic Research, USA John E. Bennett, MD National Institute of Allergy and Infectious Diseases, Scott G. Filler, MD USA Harbor-UCLA Medical Center, USA
Rosemary Boyton, PhD Reinhard Fischer, PhD Imperial College, UK University of Karlsruhe, Germany
Arunaloke Chakrabarti, MD Jens Frisvad, PhD Postgraduate Institute of Medical Education & Technical University of Denmark, Denmark Research, India David M. Geiser, PhD Pranatharthi H. Chandrasekar, MD Pennsylvania State University, USA Wayne State University, USA Donna Haiduven, PhD Thomas M. Chiller, MD University of South Florida, USA Centers for Disease Control and Prevention, USA Dominik Hartl, MD PhD Karl V. Clemons, PhD Ludwig-Maximilians-University, Germany Stanford University, USA Tobias Hohl, MD PhD Robert A. Cramer, Jr., PhD Memorial Sloan Kettering Cancer Center, USA Montana State University, USA
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Steven M. Holland, MD Jean-Paul Latgé, PhD National Institute of Allergy and Infectious Diseases, Institut Pasteur, France USA Thomas Lehrnbecher, MD PhD William W. Hope, MD Johann Wolfgang University of Frankfurt, Germany University of Manchester, UK James S. Lewis, PharmD Hiroyuki Horiuchi, PhD University of Texas - San Antonio, USA University of Tokyo, Japan Johan Maertens, MD Shahid Husain, MD University Hospital Gasthuisberg, Belgium University of Pittsburgh, USA Francisco M. Marty, MD James I. Ito, MD Harvard Medical School, USA City of Hope National Medical Center, USA Laurence F. Mirels, MD Katsuhiko Kamei, MD, PhD Stanford University, USA Chiba University, Japan Jose G. Montoya, MD Corne Klaassen, PhD Stanford University, USA Canisius Wilhelmina Hospital, The Netherlands Richard B. Moss, MD Michael Kleinberg, MD PhD Stanford University, USA University of Maryland, USA William C. Nierman, PhD Lena Klingspor, MD PhD The Institute for Genomic Research, USA Karolinska Institute, Sweden Nir Osherov, PhD Olaf Kniemeyer, PhD Tel-Aviv University, Israel Leibniz Institute for Natural Product Research and Infection Biology, Germany Alessandro C. Pasqualotto, MD PhD University of Manchester, UK Jay K. Kolls, MD University of Pittsburgh, USA Thomas F. Patterson, MD University of Texas - San Antonio, USA Dimitrios P. Kontoyiannis, MD University of Texas MD Anderson Cancer Center, David S. Perlin, PhD USA UMDNJ-New Jersey Medical School, USA
June Kwon-Chung, PhD Gordon Ramage, PhD National Institute of Allergy and Infectious Diseases, Glasgow Caledonian University, UK USA Nick Read, PhD University of Edinburgh, UK
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Luigina Romani, MD PhD Abba I. Terr, PhD University of Perugia, Italy Terr Medical Corporation, USA
Robert A. Samson, PhD Paul E. Verweij, MD PhD Centraalbureau voor Schimmelcultures University Medical Centre Nijmegen, The The Netherlands Netherlands
George A. Sarosi, MD Suresh P. Vyas, PhD Indiana University, USA Dr. Harisingh Gour University, India Mark S. Schubert, MD PhD University of Arizona Thomas J. Walsh, MD Allergy Asthma Clinic, USA National Cancer Institute, USA
Stefan Schwartz, MD David J. Weber, MD MPH Charité - Universitätsmedizin Berlin, Germany University of North Carolina, USA
Brahm H. Segal, MD Nathan P. Weiderhold, PharmD Roswell Park Cancer Institute, USA University of Texas - San Antonio, USA
Kent Sepkowitz, MD Sally E. Wenzel, MD Memorial Sloan-Kettering Cancer Center, USA University of Pittsburgh, USA
Nina Singh, MD John R. Wingard, MD University of Pittsburgh, USA University of Florida, USA
William J. Steinbach, MD De Yang, PhD Duke University, USA National Cancer Institute, USA
David A. Stevens, MD Teresa Zelante, PhD Stanford University, USA University of Perugia, Italy
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Course Description~ The Aspergillus field is in a state of rapid advancement, including wonderful genomic and molecular advances providing a huge impetus for basic and translational research. Additionally, the launch of several newer antifungals in the last few years and anticipated clinical trials of several more is a tremendous important therapeutic step forward. Despite the incidence of invasive aspergillosis increasing and the disease as the leading fungal cause of patient mortality, prior to the 1st Advances Against Aspergillosis meeting there had been insufficient communication among experts in the area. Here in 2008 is another rare chance to gather the world's aspergillosis experts in one venue. A continuing fundamental tenet of this research colloquium continues to be to engender collaborative relationships amongst clinicians, scientists, and industry to further advance the field. The sessions are purposefully designed to intermingle the clinical and scientific aspects on a specific topic
Objectives~ Recognize the therapeutic gaps in treating patients with invasive aspergillosis.
Debate the various methodologies surrounding further molecular advances.
Create new relationships between scientists and clinicians.
Statement of Need~ The content of this educational program was determined by rigorous assessment of educational need including surveys, program feedback, expert faculty assessment, literature review, research findings, medical practice and new medical knowledge.
Target Audience~ The program is intended for Infectious Disease Specialists, Mycologists, Hematologist/Oncologists, Transplantation Specialists, Chest Physicians, Allergists, Clinical Microbiologists, Molecular Biologists, Immunologists, Basic Scientists, Geneticists, Pharmacists, Epidemiologists, Public Health Specialists, Environmental/Facility, Specialists, and Veterinarians
Accreditation~ AMA: The University of California, San Diego School of Medicine is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians.
The University of California, San Diego School of Medicine designates this educational activity for a maximum of 22.5 AMA PRA Category 1 Credits™. Physicians should only claim credit commensurate with the extent of their participation in the activity.
BBS: This course meets the qualifications for 22.5 hours of continuing education credit for MFTs and/or LCSWs as required by the California Board of Behavioral Sciences. (UCSD Provider Number PCE 683)
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BRN: Nurses may claim credit for activities approved for AMA PRA Category 1 Credit™ in most states, for up to 50% of the nursing requirement for recertification. This course is designated for 22.5 AMA PRA Category 1 Credits™. (Provider # CME Category 1)
Mission Statement~ University of California, San Diego Continuing Medical Education (CME)
The Mission of the University of California, San Diego Continuing Medical Education (UCSD CME) is to provide needs based education for physicians and health care providers to improve knowledge, competence and performance and enable the optimum provision of health care.
UCSD CME employs numerous strategies to achieve our educational mission including: employing additional strategies to support physician learning and change (self-assessments, pre/post tests, reminders, feedback); participation in a system-wide process-based quality improvement platform; collaboration with stake-holders that support our mission, including hospital based quality assurance, medical societies, federal and state organizations, advocacy groups, medical schools and other health organizations; research and practical application of current adult education principles and CME literature; supporting members of our target audience in point of care learning and specific practice improvements; participation in national organizations committed to improving CME.
UCSD CME sponsors over 100 conferences per year, and produces many home-study programs. For more information visit our website at http://cme.ucsd.edu.
Cultural and Linguistic Competency ~ California Assembly Bill 1195 requires continuing medical education activities with patient care components to include curriculum in the subjects of cultural and linguistic competency. It is the intent of the bill, which went into effect on July 1, 2006, to encourage physicians and surgeons, CME providers in the state of California, and the Accreditation Council for Continuing Medical Education to meet the cultural and linguistic concerns of a diverse patient population through appropriate professional development. The planners, speakers and authors of this CME activity have been encouraged to address issues relevant in their topic area. In addition, a variety of resources are available that address cultural and linguistic competency, some of which are included in your syllabus or handout materials. Additional resources and information about AB1195 can be found on our website at http://cme.ucsd.edu.
Faculty Disclosure~ The University of California, San Diego Continuing Medical Education (UCSD CME) requires that the content of CME activities and related materials provide balance, independence, objectivity, and scientific rigor. Planning must be free of the influence or control of a commercial entity, and promote improvements or quality in healthcare. Faculty participating in UCSD sponsored CME programs are expected to disclose to the activity participants any conflict(s) of interest that may have a direct bearing on the subject matter in their role as planners or presenters. This pertains to relationships with pharmaceutical companies, biomedical device manufacturers, or other
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corporations whose products or services are related to the course content. UCSD CME has the following mechanisms in place to resolve conflicts of interest 1) altering the financial relationship with the commercial interest, 2) altering the individual’s control over CME content about the products or services of the commercial interest, and/or 3) validating the activity content through independent peer review. UCSD CME will resolve all conflicts of interest prior to an educational activity being delivered to learners. Participants will be asked to evaluate whether the speaker’s outside interests reflect a possible bias in the planning or presentation of the activity. This information is used to plan future activities.
Speaker Name Name of Commercial Interest Nature of Relevant Relationship Jill Adler-Moore, MD Molecular Express, Inc, Gilead Consultant, Research grants Sciences, Inc Elias J. Anaissie, MD Pfizer, Astellas, Enzon Grant/Research Support Pfizer, Astellas, Enzon, Amgen, Consultant Gilead Sciences, Inc. David W. Denning, MD Myconstica Shareholder Shahid Husain, MD Pfizer Grants, Speaker Astellas Grants Schering Plough Speaker Francisco M. Marty, MD Basilea Pharmceutica, Schering Grant/Research Recipient Plough, Astellas Jose G. Montoya, MD Pfizer, Schering Plough, Enzon Speakers Bureau Merck Speakers Bureau, Grant Jennifer Nielsen Kah, PhD Merck Employee David S. Perlin, PhD Myconostica Scientific Advisory Board William J. Steinbach, MD Astellas Speaker’s Bureau, Consultant Pfizer, Enzon Speaker’s Bureau Schering Plough Consultant Merck Grant Support Paul Verweij, MD, PhD Schering Plough, Pfizer, Gilead Grants/Research, Speaker’s Basilea Bureau Merck Grants/Research Grants/Research, Speaker’s Bureau, Advisor Thomas J. Walsh, MD Astellas, Vicuron Cooperative Research and Development agreements David Weber, MD Wyeth, Ortho-McNeil, Merck Speakers Bureau Sally E. Wenzel, MD GlaxoSmithKline Advisory Board, Consultant Merck, Genentech Advisory Board, Speaker Critical Therapeutics Speaker Centocor Consultant AstraZeneca Advisory Board John R. Wingard, MD Pfizer, Merck Consultant, Speaker’s Bureau, Schering Plough, MGI Pharma, Grant Support Astellas Speaker’s Bureau
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The following speakers have no relevant financial relationships to disclose:
Ryoichi Amitani, PhD Tobias Hohl, MD, PhD William C. Nierman, PhD S. Arun Balajee, PhD Steven M. Holland, MD Nir Osherov, PhD Scott E. Baker, PhD William W. Hope, MD Alessandro C. Pasqualotto, Joan W. Bennett, PhD Hiroyuki Horiuchi, PhD MD, PhD John E. Bennett, MD James I. Ito, MD Gordon Ramage, PhD Arunaloke Chakrabarti, MD Katsuhiko Kamei, MD, PhD Nick Read, PhD Thomas M. Chiller, MD Corne Klaassen, PhD Luigina Romani, MD, PhD Karl V. Clemons, PhD Lena Klingspor, MD, PhD Robert A. Samson, PhD Robert A. Cramer, Jr., PhD Olaf Kniemeyer, PhD George A. Sarosi, MD Michael J. Day, PhD Jay K. Kolls, MD Mark S. Schubert, MD Richard D. deShazo, MD Dimitrios P Kontoyiannis, MD Brahm H. Segal, MD Dennis M. Dixon, PhD June Kwon-Chung, PhD Kent Sepkowitz, MD Natalie D. Fedorova, PhD Jean-Paul Latgé, PhD David A. Stevens, MD Scott G. Filler, MD Thomas Lehrnbecher, MD PhD Abba I. Terr, MD Reinhard Fischer, PhD Johan Maertens, MD Suresh P. Vyas, PhD Jens Frisvad, PhD Richard B. Moss, MD De Yang, PhD David M. Geiser, PhD Stanford University, USA Teresa Zelante, PhD Donna Haiduven, PhD Dominik Hartl, MD
This educational activity may contain discussion of unlabeled and/or investigational uses of agents that are not approved by the FDA. Please consult the prescribing information for each product. The views and opinions expressed in this activity are those of the faculty and do not necessarily reflect the views of the University of California, San Diego.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
Scholarship Awards
International Society for Human and Animal Mycology (ISHAM) Scholar Chintala Sadavisa Reddy, India Paramee Noonim, Thailand Santosh Upadhyay, India Adriana Cuenca, Colombia
Fungal Research Trust Scholar Melissa Orzechowski Xavier, Brazil Sunita D. Deshpande, India Nansalmaa Amarsaikhan, Mongolia/Turkey
MarVista Laboratories Scholar Mark Warwas, Canada
British Society for Medical Mycology Scholar Susan Howard, UK
Foundation for Research in Infectious Diseases Scholar Marina Shevchenko, Russia
David A. and Julie A. Stevens Scholar Laïd Boukraa, Algeria
Advances Against Aspergillosis Scholar Michael Rudenko, Russia
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
POSTER # ABSTRACT & AUTHOR PAGE
1 SUBCLINICAL INVESTIGATIONS BY MEDICAL MYCOLOGISTS IN THE STUDY HUMAN 93 HABITATIONS AS USEFUL FOR CLINICAL MEDICINE IN THE CASE OF FUNGAL ASTHMA WITH UNCLEAR FUNGAL AGENT Rudenko M*, Rudenko I, Markin N, Zabelev A, Dumanov J, Shemshura A
2 ASPERGILLUS LOAD OF CIRCULATING NIGERIAN CURRENCY NOTES IN BENIN 94 CITY, NIGERIA Okungbowa F*, Egbagbe E, Oluwole S
3 ROLE OF SIALIC ACIDS ON THE SURFACE OF ASPERGILLUS FUMIGATUS 95 CONIDIOSPORES AND THE CREATION OF A NUCLEOTIDE-SUGAR TRANSPORTER- DEFICIENT MUTANT STRAIN Warwas M*, Watson J, Bennet A, Moore M
4 MOLECULAR ANALYSIS OF ADAPTATION IN RESPONSE TO ANTIFUNGAL 96 COMPOUNDS IN ASPERGILLUS SPECIES Amarsaikhan N*, Ögel Z
5 IDENTIFYING ANCHORS OF MENACING MOLD: EXTRACELLULAR THAUMATIN 97 DOMAIN PROTEIN CHARACTERIZED AS A POTENTIAL ADHESIN OF ASPERGILLUS FUMIGATUS Upadhyay SK*, Basir SF, Madan T
6 AN IN VITRO STUDY OF AYURVEDIC FORMULATIONS ON RESPIRATORY 98 ASPERGILLUS ISOLATES Deshpande SD*, Ranjan L, Bhatt M
7 SUSCEPTIBILITY PROFILES AND IN VITRO PHARMACOKINETICS OF DEVELOPING 99 ASPERGILLUS FUMIGATUS MULTICELLULAR STRUCTURES Mowat E*, Williams C, Ramage G
8 CORRELATION BETWEEN A. FUMIGATUS INDUCED ASTHMA CHARACTERS IN 100 MOUSE MODEL OF SENSITIZATION TO A. FUMIGATUS ANTIGEN AND IN NON FUNGAL ANTIGEN SENSITIZED MICE Shevchenko M*, Shekhovtsova E, Alexeeva L
9 DEVELOPMENT OF A LATERAL FLOW DEVICE FOR THE RAPID AND SPECIFIC 101 DETECTION OF INVASIVE ASPERGILLOSIS Thornton C*
10 ASPERGILLUS NIGER SUSCEPTIBILITY TO ADDITIVE ACTION OF STARCH 102 AND HONEY IN RELATION WITH DIASTASE NUMBER Boukraâ L*
11 ECOLOGY AND TAXONOMY OF OCHRATOXIGENIC ASPERGILLUS SPP. FROM 103 COFFEE BEAN IN THAILAND Noonim P*, Mahakarnchanakul W, Frisvad JC, Samson RA
12 NAVIGATING MICROBIAL SPACE USING THE STRAININFO.NET BIOPORTAL 104 Van Brabant B, De Baets B, De Vos P, Coenye T*, Dawyndt P
13 SEROLOGICAL DIAGNOSIS OF ALLERGIC BRONCHOPULMONARY ASPERGILLOSIS IN 105 PATIENTS WITH CYSTIC FIBROSIS THOUGH THE DETECTION OF IGG TO ASPERGILLUS Barton R*, Hobson R, Denton M, Peckham D, Brownlee K, Conway S, Kerr M
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POSTER # ABSTRACT & AUTHOR PAGE
14 ASPERGILLUS- AS SEEN IN A TERTIARY CARE HOSPITAL 106 Savio J*, Srinivasa H
15 STUDY OF PHYSIOLOGY AND BIOCHEMISTRY OF ASPERGILLUS SPECIES 107 ISOLATED FROM THE ATMOSPHERE OF KATHMANDU Shrestha G*, Mridha MAU
16 ASPERGILLUS GALACTOMANNAN TEST FOR PATIENTS WITH CRYPTOCOCCOSIS 108 Zhu LP*, Wu JQ, Zhang QQ, Wang YH, Xu B, Weng XH
17 ROLE OF ITRACONAZOLE IN THE TREATMENT OF INVASIVE ASPERGILLOSIS IN NON- 109 NEUTROPENIC PATIENTS: A SERIES OF CASES IN A TERTIARY HOSPITAL IN CHINA Zhu LP*, Wu JQ, Zhang QQ, Xu B, Huang YX, Weng XH
18 AFLATOXIGENIC ASPERGILLI AND AFLATOXIN CONTAMINATION OF RICE, 110 AND ITS MANAGEMENT IN INDIA Reddy CS*, Reddy KRN, Muralidharan K, Mangala UN
19 EUGENOL, AN ANTIFUNGAL COMPONENT IN CLOVE THAT CHECKS THE 111 CONTAMINATION OF ASPERGILLI IN RICE Reddy CS, Reddy KRN, Mangala UN, Muralidharan K
20 TITLE: EFFICACY OF THE DETECTION OF GALACTOMANNAN ANTIGEN IN SERUM 112 FOR THE DIAGNOSIS OF INVASIVE ASPERGILLOSIS WITH PULMONARY COMPROMISE IN PEDIATRIC ONCOHEMATOLOGIC PATIENTS Cuenca A*, Palau JM, Beltran E
21 COMPARISON OF MULTIPLE METHODS FOR THE STUDY OF THE MOLECULAR 113 EPIDEMIOLOGY OF ASPERGILLUS FUMIGATUS Vanhee L*, Symoens F, Bouchara JP, Nelis H, Coenye T
22 SURFACE-EXPOSED BETA-GLUCANS OF ASPERGILLUS FUMIGATUS TRIGGER THE 114 ACTIVATION OF ACTIVATOR PROTEIN 1 Toyotome T*, Adachi Y, Watanabe A, Ochiai E, Ohno N, Kamei K
23 THE USE OF GALACTOMANNAN AND POLYMERASE CHAIN REACTION IN BLOOD FOR 115 EARLY DIAGNOSIS OF INVASIVE ASPERGILLUS IN A HIGH-RISK PEDIATRIC POPULATION Armenian S, Nash K, Ross L, Gaynon P, Hoffman J*
24 CHEMOSENSITIZATION OF ASPERGILLUS FUMIGATUS TO ANTIMICROBIAL AGENTS 116 USING BENZO ANALOGS: TARGETING OXIDATIVE STRESS RESPONSE AND CELL WALL INTEGRITY SYSTEMS Kim J, Faria N, Chan K, May G, Campbell B*
25 THE ROLE OF BRONCHOALVEOLAR LAVAGE GALACTOMANNAN ASSAY IN THE 117 DIAGNOSIS OF INVASIVE ASPERGILLOSIS IN PEDIATRIC POPULATIONS Desai R*, Ross L, Hoffman J
26 SERUM (1-3)-BETA-D-GLUCAN AS A MEASURE OF TREATMENT OUTCOME IN 118 INVASIVEPULMONARY ASPERGILLOSIS Wiederhold N*, Vallor A, Najvar L, Kirkpatrick W, Bocanegra R, Molina D, Olivo M, Graybill J, Patterson T
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POSTER # ABSTRACT & AUTHOR PAGE
27 INCIDENCE AND SIGNIFICANCE OF ASPERGILLUS SPP. ISOLATION IN 119 BRONCHIECTASIS PATIENTS Germaud P*, Danner I, Germaud N, Cellerin L, Chailleux E
28 THE TRANSCRIPTION FACTOR SWI5 GOVERNS CONIDIA FORMATION, 120 PIGMENTATION, AND VIRULENCE IN ASPERGILLUS FUMIGATUS Ejzykowicz DE*, Cunha MM, Rozental S, Solis NV, Gravelat FN, Sheppard DC, Filler SG
29 MOLECULAR IDENTIFICATION OF CLINICAL BLACK ASPERGILLUS ISOLATES 121 AND AZOLE RESISTANCE Howard SJ*, Harrison E, Bowyer P, Denning DW
30 DIFFERENTIAL EXPRESSION AND PARTIAL CHARACTERISATION OF MYCELIAL 122 EXTRACT GLYCOPROTEIN ANTIGENS OF ASPERGILLUS ACULEATUS IN ZN2+- AND FE2+- SUPPLEMENTED LIQUID CULTURES Ajayi AO*, Adewale IO, Bakare MK, Shonukan OO
31 A CASE OF BILATERAL PULMONARY ASPERGILLOMA 123 Laroza MM*, Hernandez JFG, Cuyco EQ
32 INVASIVE INFECTIONS ATTRIBUTED TO ASPERGILLUS USTUS ARE CAUSED BY A. 124 CALIDOUSTUS SP. NOV. Varga J*, Houbraken J, van der Lee H, Verweij P, Samson R
33 TRIAZOLE RESISTANCE AMONG CLINICAL ASPERGILLUS FUMIGATUS ISOLATES 125 Lagrou K*, De Vleeschouwer J, Meersseman W., Dupont L, Verleden G, Melchers WJG., Verweij PE, Maertens J
34 SELDI-TOF TECHNOLOGY APPLIED TO THE ANALYSIS OF PROTEIN EXTRACTS OF 126 ASPERGILLUS FUMIGATUS. Pinel C, Arlotto M, Berger F, Pelloux H, Grillot R, Symoens F
35 ASPERGILLUS IN NON-NEUTROPENIC PATIENTS 127 Garbino J*, Zimmerli S, Imhof A, Flückiger U, Bille J
36 SUCCESSFUL TREATMENT OF ASPERGILLUS SINUSITIS IN A BOY WITH T ACUTE 128 LYMPHOBLASTIC LEUKEMIA (T-ALL) BY CONSERVATIVE APPROACH ONLY DESPITE DIFFICULTIES CAUSED BY DRUG TOXICITY Jabali Y, Mallatova N*, Smrcka V, Timr P, Smrckova A
37 IN VITRO ACTIVITY OF CASPOFUNGIN COMPARED WITH SIX OTHER ANTIFUNGAL 129 AGENTS AGAINST 239 PROSPECTIVELY COLLECTED CLINICAL ASPERGILLUS ISOLATES FROM THE NETHERLANDS Breuker I*, de Valk H, Debets-Ossenkopp YL, Endtz HP, Verweij PE, Meis JF, Dutch Caspo Study Group
38 ETIOLOGY OF SUPPORATIVE KERATITIS IN DELHI- ASPERGILLUS A MAJOR CAUSE? 130 Sherwal BL*, Dadeya S, Dutta R, Mehta G
39 PROTEOMICS AS A POTENTIAL TOOL FOR DIAGNOSIS OF ASPERGILLOSIS 131 Fothergill A*, Furmaga W, Mikhailov V, Sutton D, Kirkpatrick W, Najvar L, Rinaldi M, Patterson T
40 ASPERGILLOSIS IN PENGUINS: GROSS LESIONS IN 15 CASES 132 Xavier MO*, Pasqualotto AC, Soares MP, da Silva Filho RP, Araújo Meireles MC, Severo LC
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POSTER # ABSTRACT & AUTHOR PAGE
41 INFLUENCES OF ANTIMYCOTICS ON THE FUNCTION OF ANTI-FUNGAL T-CELLS 133 Tramsen L*, Koehl U, Baumgaertner N, Huenecke S, Roeger F, Latgé J-P, Klingebiel T, Lehrnbecher T
42 THE MOLECULAR BIOLOGY OF ALLERGEN EXPRESSION IN ASPERGILLUS 134 FUMIGATUS Fraczek M*; Rashid R, Denning DW, Bowyer P
43 RARE CASE OF ASPERGILLUS FUMIGATUS PERICARDITIS IN A 40 YEAR OLD 135 FEMALE PATIENT WITH CHRONIC GRANULOMATOUS DISEASE Leth Mortensen K, Petersen E, Cavling Arendrup M
44 EVOLUTION OF ASPERGILLUS FUMIGATUS IN ASPERGILLOMAS 136 Harrison E, Howard S, Anderson M, Denning D, Bowyer P*
45 EXAMINATION OF VORICONAZOLE (VRC) – POSACONAZOLE (POSA) CROSS 137 TOLERANCE IN A DROSOPHILA MODEL OF INVASIVE ASPERGILLOSIS (IA) Lamaris GA, Ben-Ami R, Lewis RE, Kontoyiannis DP*
46 1,3-BETA-D-GLUCAN ASSAY TO OVERCOME FALSE POSITIVE RESULTS OF THE 138 ASPERGILLUS GALACTOMANNAN ASSAY RELATED TO SODIUM GLUCONATE. Sulahian A, Touratier S, Peffault de Latour R, Robin M, Brethon B, Menotti J, Derouin F, Ribaud P*
47 RUTHENIUM COMPLEXES AGAINST ASPERGILLUS SPP. 139 Donnici CL, Nogueira LJ, Araújo MH, Martins CVB*, Magalhães TFF, de Resende MA
48 ACTIVITY OF AQUATIC MACROPHYTES EXTRACTS AGAINST ASPERGILLUS SPP. 140 Martins CVB*, Magalhães TFF, Nogueira LJ, Moraes R, Watanabe GA, Silva DL, Johann S, Donnici CL, de Resende MA
49 GONIOTHALAMIN ENANTIOMERS SAY NO WAY TO ASPERGILLUS SPP 141 Martins CVB, Magalhães TFF, Moraes R, Ruiz ALTG, de Carvalho JE, de Resende MA, de Fátima Â
50 MYD88-MEDIATED SIGNALING CONTRIBUTES TO EARLY PULMONARY 142 INFLAMMATORY RESPONSES TO ASPERGILLUS FUMIGATUS Chaudhary N*, Bretz C, Gersuk G, Knoblaugh S, Randolph-Habecker J, Hackman R, Staab J, Marr K
51 THE TIMING OF MYELOID CELL DEPLETION RELATIVE TO INFECTION DETERMINES 143 THE OUTCOME OF INVASIVE ASPERGILLOSIS Mircescu M*, Pamer E, Hohl T
52 INVASIVE ASPERGILLOSIS IN TRANSPLANT PATIENTS: DESCRIPTION OF TREATMENT 144 PRACTICES AND PREDICTORS OF USE OF PRIMARY COMBINATION THERAPY Baddley J*, Marr K, Wingard J, Patterson T, Waterbor J, Kempf M, Willliams O, Andes D, Kontoyiannis D, Pappas P, TRANSNET Investigators
53 A ROLE FOR TREHALOSE BIOSYNTHESIS IN A. FUMIGATUS STRESS RESPONSE AND 145 DEVELOPMENT Al-Bader N*, Sheppard DC
54 STRAIN-TO-STRAIN VARIATION IN THE VIRULENCE OF ASPERGILLUS ISOLATES 146 ASSESSED IN TOLL-DEFICIENT DROSOPHILA FLIES Ben-Ami R*, Lamaris G, Lewis R, Kontoyiannis D
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POSTER # ABSTRACT & AUTHOR PAGE
55 EVOLUTION OF CT SCAN CHANGES IN INVASIVE PULMONARY ASPERGILLOSIS 147 IN PATIENTS WITH NEUTROPENIA: KINETICS OF LESION SIZE Caillot D, Latrabe V, Thiébaut A, Herbrecht R, De Botton S, Pigneux A, Duvivier A, Mahi L*, Alfandari S, Couaillier JF
56 MELANIN, A STRUCTURAL COMPONENT OF THE CELL WALL OF ASPERGILLUS 148 FUMIGATUS CONIDIA Pihet M, Vandeputte P, Symoens F*, Renier G, Saulnier P, Georgeault S, Chabasse D, Bouchara JP
57 ASPERGILLUS FUMIGATUS – INDUCER OF IGE-INDEPENDENT MAST CELL 149 DEGRANULATION? Urb M*, Gravelat FN, Pouliot P, Olivier M, Sheppard DC
58 FUNGAL RHINOSINUSITIS DUE TO ASPERGILLUS HOLLANDICUS: A CASE REPORT 150 Shivaprakash MR*, Jain N, Gupta AK, Chakrabarti A
59 THE FUNGAL FLORA OF THE HEALTHY HUMAN LUNG 151 Harrison E*, Descalmes S, Smith J, Gore R, Denning D, Bowyer P
60 HUMAN PLATELETS INFLUENCE CELL WALL ASSEMBLY IN ASPERGILLUS 152 FUMIGATUS Perkhofer S*, Schrettl M, Haas H, Dierich MP, Lass-Flörl C
61 ASPERGILLOMA´S FREQUENCY IN PATIENTS WITH HISTORY OF CURED 153 PULMONARY TUBERCULOSIS, HEMOPTYSIS, ABNORMAL CHEST X-RAY FILMS, AND NEGATIVE SPUTUM SMEARS FOR ACID-FAST BACILLI. Casquero J*, Guevara M, Urcia F, Navarro A, Linares N, AcurioV, Huamaní L, Espinoza N, Sotomayor A, Somocurcio J, Fernández E, Asmat P, Hurtado E, Cornejo M
62 NATIVE ANTIGENS OF ASPERGILLUS FUMIGATUS FOR ASPERGILLOMA´S 154 IMMUNODIAGNOSIS USE. PRELIMINARY REPORT Casquero J*, Urcia F, Sánchez E
63 MOLECULAR DETECTION OF FOUR ASPERGILLUS SPECIES IN BLOOD AND 155 BRONCHOALVEOLAR LAVAGE (BAL) SAMPLES FROM IMUNOCOMPROMISED PATIENTS USING REAL-TIME PCR WITH TAQMAN MGB PROBES Lengerova M, Hrncirova K, Racil Z, Kocmanova I, Volfova P, Lochmanova J, Dvorakova D, Mayer J
64 UTILITY OF STANDARDIZED ANIMAL MODELS FOR THE DIAGNOSIS OF 156 INVASIVE ASPERGILLOSIS Patterson TF, Filler SG, Denning DW, Sheppard DC, Wiederhold NP, Wickes BL, Warn PA, Najvar LK, Vallor AC, Kirkpatrick WR*, Graybill JR
65 DEVELOPMENT AND VALIDATION OF A HIGH-PERFORMANCE LIQUID 157 CHROMATOGRAPHY ASSAY FOR AMINOCANDIN IN MOUSE PLASMA AND KIDNEY Parmar A*, Sharp A, Majithiya JB, Denning DW, Warn PA
66 INVOLVEMENT OF FATTY ACID CATABOLISM IN ASPERGILLUS FUMIGATUS 158 CONIDIA IN RESPONSE TO HUMAN NEUTROPHILS Sugui J*, Stanley Kim H, Zarember K, Chang Y, Gallin J, Nierman W, Kwon-Chung K
67 DETECTION OF ASPERGILLUS GALACTOMANNAN IN BAL IN THE PLATELIA® 159 ASPERGILLUS EIA, AS PERFORMED AT MIRAVISTA DIAGNOSTICS Wheat L*, LeMonte AM., Durkin M, Swartzentruber S, Knox K, Hage C, Bentsen C, Husain S, Singh N, Clancy C, Hong M, Leather H, Wingard J
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida POSTER # ABSTRACT & AUTHOR PAGE
68 QUANTIFYING ASPERGILLUS FUMIGATUS BURDEN IN INVERTEBRATE MODEL 160 Slater JL*, Denning DW, Warn PA
69 A NOVEL MODEL OF RAT INVASIVE PULMONARY ASPERGILLOSIS SUITABLE FOR 161 MONITORING DISEASE PROGRESS USING SURROGATE MARKERS Shrief RNK*, Sharp A, Parmar A, Majithiya JB, Denning DW, Warn PA
70 DEVELOPMENT AND VALIDATION OF A MICROSPHERE BASED LUMINEX ASSAY 162 FOR RAPID IDENTIFICATION OF CLINICALLY RELEVANT ASPERGILLI Etienne KA*, Kano R, Balajee SA
71 COMPARATIVE SEQUENCE ANALYSES OF MULTIPLE LOCI REVEAL RARE 163 ASPERGILLUS SPECIES IN TRANSPLANT RECIPIENTS (TRANSNET STUDY) Kano R*, Perrone G, Peterson S, Baddley JW, Marr KA, Alexander B, Andes D, Kontoyiannis DP, Pappas PG, Balajee SA
72 TLR9 IMMUNE, STRUCTURAL CELL, AND AIRWAY RESPONSES TO ASPERGILLUS 164 FUMIGATUS IN IMMUNODEFICIENT AND ALLERGIC MICE Ramaprakash H*, Kulasekaran P, O’Connor E, Kunkel SL, Hogaboam CM
73 INTERLABORATORY COMPATIBILITY OF A MICROSATELLITE BASED TYPING 165 ASSAY FOR ASPERGILLUS FUMIGATUS THROUGH THE USE OF ALLELIC LADDERS de Valk HA*, Meis JFGM, Klaassen CHW
74 EXACT AND HIGH RESOLUTION FINGERPRINTING OF ASPERGILLUS TERREUS 166 ISOLATES USING MICROSATELLITES AND AFLP de Valk HA*, Balajee SA, Meis JFGM, Klaassen CHW
75 MULTI LOCUS PHYLOGENY OF SECTION FUMIGATI 167 Hurst S, Varga J, Samson R, Peterson S, Balajee S
76 THE ROLE OF CHEMOKINE RECEPTOR 7 IN PULMONARY ANTIFUNGAL RESPONSES 168 DURING INVASIVE ASPERGILLOSIS Hartigan A*, Hogaboam C
77 A NEW MODEL SYSTEM TO SCREEN FOR VIRULENCE OF ASPERGILLUS MUTANT 169 AND WILD-TYPE STRAINS Brock M*, Jacobsen ID
78 UP REGULATION OF CYP51A GENE AS A MECHANISM OF RESISTANCE IN CLINICAL 170 ISOLATES OF ASPERGILLUS FUMIGATUS. Albarrag A*, Anderson M, Sanglard D, Denning D
79 NADPH OXIDASE ACTIVITY IN PULMONARY PHAGOCYTES RESPONDING TO 171 ASPERGILLUS FUMIGATUS CONIDIA Burritt NL, Taylor RM, Hurtgen BJ, Wang SY, Jarvis JN, Burritt JB*
80 REVIEW OF 12 PATIENTS WITH ASPERGILLUS OSTEOMYELITIS IDENTIFIED BY THE 172 PATH ALLIANCE REGISTRY Horn D*, Neofytos D, Steinbach W, Anaissie E, Fishman J, Olyaei A, Pfaller M, Webster K, Marr KA
81 EXPLORING THE CONCORDANCE OF ASPERGILLUS FUMIGATUS PATHOGENICITY 173 BETWEEN TOLL DEFICIENT FLIES AND MICE Hamilos G*, Bignell EM, Schrettl M, Lewis RE, May GS, Haas H, Kontoyiannis DP
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82 THE DEVELOPMENTAL MODULATOR MEDAP IS REQUIRED FOR ASPERGILLUS 174 FUMIGATUS VIRULENCE AND GOVERNS INTERACTIONS WITH A TYPE II PNEUMOCYTE CELL LINE. Gravelat FN*, Ejzykowicz DE, Chabot JC, Urb M, Macdonald KD, Filler SG, Sheppard DC
83 EXPOSURE LEVELS OF ASPERGILLUS FUMIGATUS FROM VARIOUS INDOOR 175 RESERVOIRS IN HEALTH CARE FACILITIES. Khan M, Gonsoulin T, Simpson S, Horner WE*
84 GENE EXPRESSION PROFILING IN HUMAN ALVEOLAR MACROPHAGES IN RESPONSE 176 TO ASPERGILLUS FUMIGATUS Dufour N, Prina E, Dubourdeau M, Philippe B, Loeffler J, Latge J-P, Ibrahim-Granet O*
85 DENDRITIC CELL BIOLOGY IN ASPERGILLOSIS 177 Bonifazi P, Silvia B, Zelante T, De Luca A, D’Angelo C, Moretti S, Fallarino F, Puccetti P, Romani L*
86 IL-10 INCREASES THE MAGNITUDE OF ASPERGILLUS FUMIGATUS-SPECIFIC 178 CD4+ T CELL RESPONSES Rivera A.*, Leiner I., Menet E., Pamer E.G.
87 THE EPIDEMIOLOGY OF INVASIVE ASPERGILLUS (IA) INFECTIONS AMONG ORGAN 179 TRANSPLANT RECIPIENTS IN THE U.S. 2001-2006: REVIEW OF TRANSNET Chiller TM*, Marr K, Kontoyiannis DP, Ito JI, Anaissie EJ, Schuster M, Alexander B, Walsh T, Wannemuehler K, Pappas PG
88 CALCINEURIN-RESPONSE ZINC FINGER (CRZA) ALSO CONTROLS HYPHAL GROWTH 180 AND PATHOGENICITY IN ASPERGILLUS FUMIGATUS Cramer Jr. RA, Perfect BZ, Zaas AK, Asfaw YG, Pinchai N, Steinbach WJ
89 FUNGAL TIP GROWTH - INFORMATION IN THE MEMBRANE? 181 Takeshita N, Fischer R*
90 PARAMETERS IN DEVELOPMENT OF A MODEL OF PULMONARY ASPERGILLOSIS IN 182 NEUTROPENIC MICE Tong AJ*, Clemons KV, Stevens DA
91 INTRAPULMONARY PHARMACOKINETICS-PHARMACODYNAMIC OF POS 183 Conte Jr. JE*, Golden JA, Krishna G, Little E, Zurlinden E
92 CADRE AND ITS ROLE IN MAINTAINING THE ASPERGILLUS NIDULANS GENOME 184 Mabey Gilsenan JE*, Anderson MJ, Bowyer P, Attwood TK, Denning DW
93 CLINICO-EPIDEMIOLOGICAL INVESTIGATION OF MOLDY CORN POISONING IN 185 MULES IN UDAYPUR DISTRICT Karki K*, Manandhar P
94 ASSESSMENT OF THE PATHOGENICITY PROPERTY OF FUSARIUM GRAMINEARUM 1 186 IN BALB/C MICE Karki KB*, Garcia GG
95 COMBINATION THERAPY WITH LIPOSOMAL AMPHOTERICIN B AND ECHINOCANDINS 187 FORTHE TREATMENT OF MURINE ASPERGILLUS FLAVUS SYSTEMIC INFECTION Constable DS, Olson JA*, Smith PJ, Adler-Moore JP
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida PROGRAM All General Sessions to be held in Mediterranean East and Center Ballroom Tuesday, January 15
5:00-7:00pm Early Registration Mediterranean Center Foyer. Main Lobby Level.
Wednesday, January 16
7:30-9:00 Registration/Continental Breakfast Mediterranean Center & Grande Promenade Foyers. Main Lobby Level.
9:00-9:10 Welcome & Explanation of sessions William J. Steinbach, MD
9:10-9:50 Keynote Interdisciplinary Lecture, Clinical aspergillosis for basic scientists David A. Stevens, MD
9:50-10:30 How can molecular biology help us understand Aspergillus and aspergillosis Aspergillus molecular biology for clinicians Joan W. Bennett, PhD
10:30-10:55 BREAK Grande Promenade
Molecular Diagnostics
10:55-11:00 Session Introduction Moderators: Dennis M. Dixon, PhD and Paul E. Verweij, MD PhD
11:00-11:25 DNA v. RNA amplification-detection platforms David S. Perlin, PhD
11:25-11:50 Proteome analysis for pathogenicity and new diagnostic markers Olaf Kniemeyer, PhD
11:50-12:15 Aspergillus PCR: Formidable challenges and progress Lena Klingspor, MD PhD
12:15-12:40 Conventional or molecular measurement of Aspergillus load Karl V. Clemons, PhD
12:40-2:15 LUNCH Grande Promenade
Pathogenesis and Clinical Manifestations in Different Hosts
2:15-2:20 Session Introduction Moderators: Scott G. Filler, MD and George A. Sarosi, MD
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
2:20-2:45 Invasive aspergillosis in chronic granulomatous disease Brahm H. Segal, MD
2:45-3:10 Difference in invasive aspergillosis in solid organ transplant recipients Shahid Husain, MD
3:10-3:35 Invasive disease in patients with leukemia Johan Maertens, MD
3:35-4:00 Invasive aspergillosis in steroid-treated patients Dimitrios P. Kontoyannis, MD
4:00-4:25 BREAK Grande Promenade
Neutrophils and Aspergillus
4:25-4:30 Session Introduction Moderators: Brahm H. Segal, MD and Michael Kleinberg, MD PhD
4:30-4:55 Neutrophils and damage during invasive aspergillosis Steve M. Holland, MD
4:55-5:20 Granulocyte products as alarmins to enhance innate and adaptive immunity De Yang, PhD
5:20-5:45 Neutrophil contribution to defense against invasive aspergillosis Tobias Hohl, MD, PhD
5:45-7:15 WELCOME RECEPTION Ocean Room - Pool Area. Lower Lobby Level.
Satellite Symposia Evaluation of Echinocandins for the Treatment of Invasive Aspergillosis Starlight Ballroom – Top of Resort
7:30-9:30 Moderator: William J. Steinbach, MD
Echinocandin activity against Aspergillus and importance of pharmacodynamic optimization James S. Lewis, PharmD
Azole resistance Paul E. Verweij, MD PhD
Paradoxical echinocandin activity: a limited in vitro phenomenon? Nathan P. Weiderhold, PharmD
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
Thursday, January 17
7:30-8:45 BREAKFAST Grande Promenade
8:00-8:45 Meet the Professors (parallel sessions)
Illuminating cases for the thinking physician George A. Sarosi, MD, John E. Bennett, MD and Laurence F. Mirels, MD Mediterranean East and Center
Sexual structures in Aspergillus - morphology, importance, and genomics David M. Geiser, PhD Regency Ballroom
Utilizing the Aspergillus Cell Wall Mediterranean East and Center
8:55-9:00 Session Introduction Moderators: David S. Perlin, PhD and Jennifer Nielsen Kahn, PhD
9:00-9:25 Keynote Interdisciplinary Lecture, Galactomannan: more than one molecule Jean-Paul Latgé, PhD
9:25-9:50 Chitin synthases of aspergilli: Functions in tip growth and morphogenesis in Aspergillus nidulans Hiroyuki Horiuchi, PhD
9:50-10:15 Glucan diagnosis for invasive aspergillosis Francisco M. Marty, MD
10:15-10:40 BREAK Grande Promenade
Aspergillus Sinusitis
10:40-10:45 Session Introduction Moderators: John E. Bennett, MD and Kent Sepkowitz, MD
10:45-11:10 Controversies surrounding categorization of fungal sinusitis Arunaloke Chakrabarti, MD
11:10-11:35 Management of invasive Aspergillus sinusitis Richard D. deShazo, MD
11:35-12:05 Aspergillus sinusitis in animals and parallels in human disease Michael J. Day, PhD
12:05-12:30 Allergic Fungal Sinusitis Mark S. Schubert, MD
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
12:30-1:50 LUNCH Grande Promenade
Selected Talks from Junior Investigators
1:50-1:55 Session Introduction Moderators: William J. Steinbach, MD and Karl V. Clemons, PhD
1:55-2:05 The role of sialic acids on the surface of aspergillus fumigatus and the characterization of an a. fumigatus sialidase Mark Warwas
2:05-2:15 The timing of myeloid cell depletion relative to infection determines the outcome of invasive aspergillosis Monica Mircescu
2:15-2:25 Human platelets influence cell wall assembly in aspergillus fumigatus Susanne Perkhofer
Aspergillus Species / Strain Differences
2:25-2:30 Session Introduction Moderators: Scott E. Baker, PhD and Katsu Kamei, MD, PhD
2:30-2:55 Criteria for Aspergillus speciation: Utrecht meeting summary Robert A. Samson, PhD
2:55-3:20 MLST vs. Microsatellites Corne Klaasen, PhD
3:20-3:45 Comparison of the two sequenced A. fumigatus strains Natalie D. Federova, PhD
3:45-4:10 A. terreus complex S. Arun Balajee, PhD
4:10-4:35 BREAK Grande Promenade
Vaccine and T Cell Immunity
4:35-4:40 Session Introduction Moderators: Luigina Romani, MD, PhD and Jill Adler-Moore, MD
4:40-5:05 Cultivated TH1 cells Thomas Lehrnbecher, MD, PhD
5:05-5:30 Host response signatures to invasive aspergillosis Teresa Zelante, PhD
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
5:30-5:55 Vaccine progress James I. Ito, MD
6:00-7:20 POSTER SESSION #1 with drinks/snacks
Session 1 - Posters 1-36 Spanish Suites (Castillian, Valencia Madrid Room). Mezzanine Level.
Session 2 - Posters 37-97 Grande Promenade. Main Lobby Level.
Satellite Symposia Risk Stratification for Invasive Aspergillosis and Prophylaxis Starlight Ballroom (Top of Resort)
7:30-9:30 Risk stratification for invasive aspergillosis: Early assessment of host susceptibility Thomas F. Patterson, MD, FACP
Prophylaxis limitations: Challenges in the management of invasive Aspergillosis Pranatharthi H. Chandrasekar, MD
Interactive case studies: Improving clinical outcomes in invasive Aspergillosis Nina Singh, MD
Friday, January 18
7:30-8:45 BREAKFAST Grande Promenade
8:00-8:45 Meet the Professors (parallel sessions)
Recognizing therapeutic failure - case illustrations and challenges Kent Sepkowitz, MD Mediterranean East and Center
Inflammation in Aspergillus immunology Luigina Romani, MD, PhD Regency Ballroom
Alternative Imaging in Invasive Aspergillosis Mediterranean East and Center
8:55-9:00 Session Introduction Moderators: Dimitrios P. Kontoyiannis, MD and Jose G. Montoya, MD
9:00-9:25 Galactomannan and beyond: MRI and PET for diagnosis Elias Anaissie, MD
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
9:25-9:50 Functionalized nanocarriers to image and target fungi infected immune cells Suresh P. Vyas, PhD
9:50-10:15 Live-cell imaging of Aspergillus in vitro Nick Read, PhD
10:15-10:40 Imaging in animals with invasive aspergillosis Thomas J. Walsh, MD
10:40-11:05 BREAK Grande Promenade
Controlling Aspergillus in buildings
11:05-11:10 Session Introduction Moderators: Thomas M. Chiller, MD and Elias J. Anaissie, MD
11:10-11:35 Infection control and HICPAC guidelines David J. Weber, MD, MPH
11:35-12:00 Nosocomial asperigllosis: review and building construction cases Donna Haiduven, PhD
12:00-12:30 Sick building debate: Is mould the cause? Abba I. Terr, MD
1:00-6:00 LUNCH and CHOICE OF THREE TOURS Board Busses in Front of Hotel
6:00-7:30 POSTER SESSION #2 with drinks/snacks
Session 1 - Posters 1-36 Spanish Suites (Castillian, Valencia Madrid Room).Mezzanine Level.
Session 2 - Posters 37-97 Grande Promenade. Main Lobby Level.
Satellite Symposia Invasive Fungal Infections in Patients With Hematologic Malignancies: Will New Strategies For Treatment Lead to New Opportunities for Improving Patient Outcome? Starlight Ballroom – Top of Resort
7:30-9:30 Amphotericin B: Past, present and future Michael Kleinberg, MD PhD
Newer antifungal delivery systems Richard H. Drew, PharmD
Cerebral aspergillosis: Tissue penetrance is the key Stefan Schwartz, MD
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
Saturday, January 19
7:30-8:45 BREAKFAST Grande Promenade
8:45-8:55 Awards and Scholarships Presentation David A. Stevens, MD Mediterranean East and Center
Aspergillus and Asthma / ABPA Cystic Fibrosis Foundation Symposia
8:55-9:00 Session Introduction Moderators: Richard B. Moss, MD and David W. Denning, MD
9:00-9:25 Phenotypes of severe asthma Sally E. Wenzel, MD
9:25-9:50 Differences in the immunobiology of Aspergillus colonization and ABPA? Jay K. Kolls, MD
9:50-10:15 Regulation of immunity in bronchiectasis and ABPA Rosemary Boyton, MD
10:15-10:40 Mechanisms behind the cystic fibrosis - ABPA link Dominik Hartl, MD
10:40-11:05 BREAK Grande Promenade
Secondary Metabolites from Aspergillus
11:05-11:10 Session Introduction Moderators: Joan Bennett, PhD and June Kwon-Chung, PhD
11:10-11:35 Genomic insights about secondary metabolite gene clusters/pathways William C. Nierman, PhD
11:35-12:00 Aspergillus metabolites in vivo Robert A. Cramer, Jr., PhD
12:00-12:25 Aspergillus and biotechnology Scott Baker, PhD
12:25-12:50 Metabolomics of A. fumigatus Jens Frisvad, PhD
12:50-2:15 LUNCH Grande Promenade
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
Aspergillus growth towards pathogenicity
2:15-2:20 Session Introduction Moderators: Reinhard Fischer, PhD and Nir Osherov, PhD
2:20-2:45 Interaction of Aspergillus with human bronchial mucosa Ryoichi Amitani, PhD
2:45-3:10 Lung epithelial cell invasion William W. Hope, MD, PhD
3:10-3:35 Biofilm formation by Aspergillus Gordon Ramage, PhD
3:35-4:00 Differences in pathogenicity and clinical syndromes due to A. fumigatus and A. flavus Alessandro C. Pasquallotto, MD PhD
4:00-4:05 Conference Farewell David W. Denning, MD
5:00- 5:45 Planning for AAA 2010 Conference Open planning session (everyone invited) to discuss ideas and plans for next meeting in 2010
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
ABSTRACTS OF INVITED FACULTY
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida CLINICAL ASPERGILLOSIS “101” FOR BASIC SCIENTISTS David A. Stevens Sta. Clara Valley Med. Ctr., Stanford Univ., and Calif. Inst. for Med. Research San Jose and Stanford, Calif.
Wednesday, January 16, 2008 9:10 – 9:50 AM Aspergillosis refers to infection with any of the species. They are important pathogens of marine organisms, insects, birds, cattle and dogs. The frequency of human invasive aspergillosis is rising worldwide. The threat to hospitalized patients has been revealed in outbreaks of infection, particularly pulmonary infection in immunocompromised hosts. The most common species infecting humans are A. fumigatus, flavus, niger, and terreus. Aspergillosis usually results from airborne conidia. It is not contagious. Invasive disease is generally a problem of compromised hosts, and more aggressive immunosuppression and anticancer therapy are important factors contributing to the rise. Often these factors are present: leukopenia, steroid therapy, cytotoxic chemotherapy, or broad-spectrum antibacterials. It is also a problem in patients with neutrophil defects. The classic picture is fever and pulmonary infiltrates or nodules, progressing to a cavity, or wedge-shaped densities resembling infarcts. The pulmonary pathology is hemorrhagic infarction and pneumonia. Pulmonary emboli are common because of the tendency to invade blood vessel walls. Targets of disseminated disease include, most commonly, the central nervous system. Typical of an aspergilloma is a fungus ball (matted hyphae and debris) in a lung cavity . The patients present with cough, hemoptysis, dyspnea, weight loss, and fatigue. Allergic bronchopulmonary aspergillosis is usually superimposed on a background of chronic asthma or cystic fibrosis, triggered in genetically susceptible persons by specific Aspergillus antigens, characterized by episodic airway obstruction, fever, eosinophilia, mucous plugs, positive sputum cultures, transient infiltrates on chest radiographs, proximal bronchiectasis, upper lobe contraction, and elevated IgE. A scratch test with Aspergillus antigens produces an immediate wheal and flare. Extrinsic allergic alveolitis is an unusual form of Aspergillus lung disease, a hypersensitivity pneumonitis with dyspnea and fever hours after inhalation of many spores, often on farms. Superficial bronchial disease and extrinsic asthma due to airborne conidia are other important pulmonary diseases. The aspergilloma, allergic, alveolitis, and superficial forms rarely progress to invasive disease. However, more invasive airway disease with tracheobronchitis occurs, particularly in compromised hosts, and may presage parenchymal invasion. Chronic necrotizing pulmonary aspergillosis occurs in patients with underlying lung disease and/or a mildly immunosuppressive condition. Locally invasive disease includes invasion of burn wounds, keratitis, external otitis, rhinitis, sinusitis, osteomyelitis or endophthalmitis. Cutaneous ulcers have been associated with use of adhesive tape. Bloodborne disease in addicts can produce foci of dissemination similar to those associated with the invasive pulmonary form. A noninvasive form of sinus disease has a predominantly allergic component and eosinophilia, responsive to drainage and steroids. Diagnosis. Antibody is detected readily in allergic disease or aspergillomas but not invasive disease. Sputum culture, good positive predictive value in the appropriate setting, is insensitive. Repeated isolations of the same species in culture correlates with invasive disease, negative results don’t rule it out. Tissue must be obtained for diagnosis, where aspergilli are seen as septate hyphae. Detection of galactomannan in serum is useful in identifying invasive disease and following response to therapy. Detection of Aspergillus DNA in blood by PCR has shown high specificity and sensitivity, and detection of glucan in serum is promising. An aggressive, invasive approach, making a diagnosis early in illness, appears to be key to survival. Computed
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
tomographic scanning of the chest may reveal a lesion with peripheral haziness (i.e., halo sign) or later an air crescent; both are highly predictive. These are radiographic correlates of edema or hemorrhage and infarction that are related to the organism’s vasculotropism. Treatment. In invasive disease, prompt aggressive antifungal therapy has improved survival, though recovery from neutropenia is a necessary accompaniment of recovery in most. The role of granulocyte transfusions, colony-stimulating factors, or cytokine therapy is unclear. When systemic chemotherapy is indicated, voriconazole proved superior in a randomized trial. Alternatives include other azoles, lipid-complexed amphotericin B and echinocandins. Clinical data to support combination therapy are limited, but given the poor record of monotherapy, combinations appears logical to explore, particularly if synergy in vitro can be demonstrated. Therapy should be continued after lesions are resolving, cultures negative, and reversible underlying predispositions have abated. Locally invasive disease may require local antifungal therapy. Surgical excision has an important role in invasion of tissue, and removal of foreign bodies at infection sites helps. Surgery may have a function in invasive pulmonary disease when chemotherapy has failed or disease impinges on major vascular structures. In aspergilloma, symptoms may relate to allergy, and improve on glucocorticoids; the role of surgery is controversial, resection has a role if recurrent, significant hemoptysis occurs. In allergic disease, systemic glucocorticoids can prevent some symptoms, bronchodilators ameliorate acute exacerbations, itraconazole lessens disease and spares steroids. The approach to extrinsic alveolitis is to avoid the stimulus. Prevention. Prophylaxis of susceptible patients using inhaled or systemic antifungals can avoid disease. Reducing airborne spores, such as by filtering hospital air and restricting contaminated materials (e.g., plants), is believed a worthwhile effort for patients who will be transiently immunosuppressed or neutropenic.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
HOW CAN MOLECULAR BIOLOGY HELP US UNDERSTAND ASPERGILLUS AND ASPERGILLOSIS?
J. W. Bennett Rutgers Univiversity, New Brunswick, NJ
Wednesday, January 16, 2008 9:50 – 10:30 AM Aspergillus species constitute a large genus of common molds that are involved in a surprisingly diverse number of biological phenomenon including biodegradation, pathogenesis (both plant and animal) and toxin formation. Aspergillosis is a term used to encompass a spectrum of human diseases ranging from hypersensitivity reactions to progressive and invasive infections. Most cases of aspergillosis are caused by just one species: Aspergillus fumigatus. Although single gene disruption studies were not able to identify specific virulence factors for A. fumigatus, comparative Aspergillus genomics and proteomics offer new tools for the molecular dissection of this fungal pathogen. Further, models that recognize the importance of host factors may inform future research to improve diagnostics and therapy.
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DNA V. RNA AMPLIFICATION-DETECTION PLATFORMS
David S. Perlin, PhD Public Health Research Institute, UMDNJ-New Jersey Medical School, Newark, New Jersey, USA
Wednesday, January 16, 2008 11:00 – 11:25 AM
The mounting prevalence of invasive fungal disease in immunocompromised patients is exacerbated by inadequate methods for pathogen detection. Conventional methodologies lack sensitivity, specificity and speed, and some infecting organisms are hard to culture. Invasive aspergillosis remains difficult to diagnose at an early stage despite advances in imaging technology and antigen-based serological testing. To overcome these limitations, nucleic acid-based amplification has been used for more than a decade to identify infecting pathogens from a variety of specimens. Unfortunately, the reliability of PCR-based home-brew assays to detect Aspergillus spp. has met with mixed success. It is now apparent that many of the problems in the past centre on non-standardized techniques for sample preparation, and nucleic acid isolation, amplification and detection. In recent years, molecular diagnostic techniques have moved into the mainstream for detection of a wide range of viral and bacterial pathogens in clinical diagnostic laboratories. This is largely due to improvements in high fidelity detection technology and the development of standardized assays. In this context, a new generation of detection modalities are in development, which will greatly improve our ability to detect infections due to Aspergillus spp.
A wide range of amplification and probe detection platforms is now available to detect Aspergillus specific DNA and RNA from respiratory samples. These platforms generally utilize ribosomal genes and their internal transcribed spacer regions to distinguish among fungal species, and Aspergillus spp. can be detected at 1 CFU or less with high fidelity. Furthermore, a new generation of probes for pan-fungal and pan-Aspergillus detection enhances existing diagnostic possibilities. PCR amplification of DNA remains the most important platform, especially when coupled with real-time probes, such as TaqMan, Scorpion or Molecular Beacons. PCR takes advantage of DNA stability, and it allows a range of annealing temperatures that can help distinguish between closely related target sequences. Alternatively, nucleic acid sequence-based amplification (NASBA) is a sensitive transcription-based amplification platform. NASBA is an isothermal reaction (41oC) that uses RNA polymerase amplification of RNA targets. It generates single-stranded RNA amplicons, which are detected in real-time by DNA probes. NASBA is more robust than PCR resulting in a 1014-fold amplification in 30-40 min., which is the equivalent of a nested PCR reaction. The target for NASBA is usually structural RNA but, in principle, high turnover transcripts can be used to distinguish between live and dead cells.
Standardized kits are beginning to emerge that combine fungal nucleic acid isolation with amplification/detection platforms. Both DNA and RNA amplification can be used to take advantage of the allele-specific properties of probes like molecular beacons to detect drug resistance markers. Finally, once new molecular diagnostic platforms are standardized and fully validated, their extended role in monitoring therapy and possibly enrolling patients at an earlier stage for clinical trials can be assessed.
D. Perlin is supported by NIH grants AI066561 and AI069397.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
PROTEOME ANALYSIS FOR PATHOGENICITY AND NEW DIAGNOSTIC MARKERS
Kniemeyer O1,3, Leßing F1,3, Schmidt A1,3, Teutschbein J1,3, Winkler R2, Haas H4, Brakhage AA1,3 1Department of Molecular and Applied Microbiology and 2Department of Biostructure Chemistry, Leibniz-Institute for Natural Product Research and Infection Biology (HKI), 07745 Jena, Germany 3Department of Microbiology and Molecular Biology, Friedrich-Schiller-University Jena, 07745 Jena, Germany 4Division of Molecular Biology, Innsbruck Medical University, 6020 Innsbruck, Austria
Wednesday, January 16, 2008, 11:25 – 11:50AM
With the completion of the Aspergillus fumigatus genome it is now possible to study protein regulation on a global scale. One of the most successful protein separation techniques is based on 2D-gel electrophoresis, in which proteins are separated by charge and size in a gel matrix. With the application of proteomic tools it is possible to gain a comprehensive overview about the proteins of A. fumigatus present or induced during environmental changes and stress conditions. During the infection process A. fumigatus has to cope with dramatic changes of the environmental conditions. Since A. fumigatus is the most prevalent airborne fungal pathogen the question arises: Does A. fumigatus possess superior adaptation strategies in comparison to other filamentous fungi? Therefore, we analysed the proteomes of A. fumigatus grown under heat stress, oxidative stress and iron depletion. During a temperature shift from 30 °C to 48 °C many stress and heat shock proteins were up-regulated. Oxidative stress induced the up-regulation of catalases, thioredoxin peroxidase and other proteins. Under iron depletion, proteins involved in siderophore biosynthesis were up-regulated, whereas iron cluster-containing proteins were down- regulated. Some of the gene products expressed under stress responses are also known fungal antigens, such as the heat shock proteins HSP 90 or the thioredoxin peroxidase AspF3. So besides pathogenicity studies, proteomics also provides tools to screen for new antigens, which could improve the diagnosis of diseases caused by A. fumigatus infections. Culture-independent clinical tests are of great interest, since there is still no sensitive, quick and unambiguous diagnostic tool available for the detection of invasive aspergillosis.. In combination with subcellular fractionation strategies immunoproteomics is an excellent tool for the screening of new Aspergillus antigens. A tool often applied for this purpose is the combination of 2D-PAGE with immunoblotting. Besides diagnosis, fungal antigens have also a great potential for the development of vaccinations against invasive mycoses.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
ASPERGILLUS PCR - FORMIDABLE CHALLENGES AND PROGRESS
Lena Klingspor, MD PhD Karolinska Institute, Department of Laboratory Medicine , Division of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden. Juergen Loeffler, University of Wuerzburg, Medical Hospital II, Wuerzburg, Germany
Wednesday, January 16, 2008, 11:50 AM – 12:15 PM
Invasive aspergillosis (IA,) are important causes of morbidity and mortality in immuno-compromised patients, with an incidence of 4-10%, and with a mortality rate of 80-90%, in allogeneic stem cell transplant recipients. Conventional diagnostic tests, such as blood culture, show poor sensitivity for the detection of Aspergillus spp. Non-culture based techniques that has been used in the past, has lacked sensitivity and specificity in immuno-compromised patients. New rapid methods which can detect IA early in the course of disease, with high sensitivity and specificity are needed since treating these infections at an early stage is often essential for a favourable outcome. Especially, the polymerase chain reaction (PCR) offers great promise for the rapid diagnosis of fungal infections, including detection of fungi that does not grow in blood cultures such as Aspergillus spp. At Karolinska University Hospital we have established an assay, using a combination of a manual extraction and a robot for automated extraction of Candida and Aspergillus DNA, in combination with real-time PCR. To asses its clinical applicability, a large number of patient samples, from patients with suspected invasive fungal infection have been analysed with real time PCR. Data will be presented with focus on Aspergillus R-T PCR results in immuno-compromised patients However, a range of different PCR assays (conventional-, nested-, real-time- based) have been developed, targeting different gene regions (cytochrome p450, heat shock proteins, 18S, 5.8S, 28S, ITS) and including a variety of amplicon detection methods, such as gel electrophoresis, hybridization with specific probes, ELISA and restriction fragment length polymorphism (RFLP). These molecular assays provide high potential in terms of sensitivity and specificity, but vary widely in their feasibility and are up until now not standardized. Despite of this progress, there are certain questions to be addresses using those assays, such as the risk of contamination with spores and amplicons (which can be minimized by the use of cabinets and real-time PCR assays), the frequency of prospective sampling as well as the number of positive results of a PCR assay required to initiate antifungal therapy. Furthermore, only few commercially available, standardized assays are available. This particular challenge will be addressed by the Working Group “EAPCRI“(European Aspergillus PCR Initiative) under the auspices of ISHAM. Twenty-four centres have started to establish an European standard for Aspergillus -PCR. The principal goal of this initiative is to achieve a standard for PCR that can be incorporated into the next revision of the EORTC/MSG definitions for IA. Besides the use of PCR assays for the diagnosis of IFI in symptomatic patients, this highly sensitive technology can be also performed to preemptively monitor patients at risk to develop IFI Thus, they might help to reduce empirical antifungal therapy and might be valuable tools for early initiation and monitoring of preemptive antifungal therapy. Future, prospective studies evaluating the potential benefits of early therapy based on R-T PCR in patients at high risk for IA infections are needed.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
CONVENTIONAL OR MOLECULAR MEASUREMENT OF ASPERGILLUS LOAD
Karl V. Clemons and David A. Stevens California Institute for Medical Research, San Jose, CA; Division of Infectious Diseases, Santa Clara Valley Medical Center, San Jose, CA; Division of Infectious Diseases and Geographic Medicine, Stanford University, Stanford, CA.
Wednesday, January 16, 2008, 12:15 – 12:40 PM
The quantification of organisms is a standard tool. Measurement of a hyphal organism, like Aspergillus, presents difficulties in that it is difficult to define what constitutes a cell. Hyphal growth occurs by hyphal tip extension, in which the hypha elongates and a septum is formed distally forming a separate compartment. However, communication between compartments and streaming of nuclei makes defining a cell of a hyphal organism difficult. When dealing with well dispersed conidial (single cells) suspensions quantitative plating of serial dilutions or hemacytometer counting are accurate, simple methods. However, the best method for quantification of the hyphal organism remains controversial. For culture-based assay, mechanical dispersion is thought to cause breakage of the hyphae into smaller viable units, which would skew estimation of fungal load. Although likely true when in vitro grown Aspergillus is dispersed, it is less obvious whether this is problematic when recovering Aspergillus from the tissues. We assessed the method of mechanical homogenization of tissues, comparing a high-speed tissue homogenizer with homogenization done by hand using a Whirlpak plastic bag. We found equivalence in the CFU recovered. However, CFU recovered temporally tend to show minimal increase, and may indicate that mechanical homogenization does not cause significant fragmentation of the hyphae in the tissues. Does the lack of increase in CFU as infection worsens inadequately reflect fungal load in the tissue? Nonculture-based methods overcome this difficulty, in part. The determination of chitin in the tissues, reported by Lehmann and White (Infect Immun 12:987-92), is done using KOH extraction and colorimetric assay of the chitosan, with fungal load based on amount of chitin. Although useful, it suffers from the number of steps and does not indicate viability. More recently, assays quantifying galactomannan by EIA or rDNA by real-time PCR have been used. Bowman, et al (Antimicrob Agents Chemother 45:3474-81) developed a real-time PCR (qPCR) methodology quantifying 18s rDNA, correlating the rDNA recovered to a conidial equivalent and demonstrated an increase in fungal load during progressive infection. Similarly, Sheppard, et al (Clin Microbiol Infect 12:376-80) recommend the use of a qPCR assay, showing it to be less variable than the EIA-based galactomannan assay and also showed progressive increase in fungal load in comparison with CFU in experimental infection. Others reported that qPCR and EIA assays can result in false negative results in comparison with CFU or histologically proven infections (Clin Diagn Lab Immunol 12:1322-7). Again, qPCR and EIA do not indicate viability, and the qPCR assay may represent a significant over-estimation, because it correlates with number of nuclei present; it also requires specialized equipment and reagents. We compared CFU and qPCR techniques (Antimicrob Agents Chemother 49:1369-76). For progressive infection, our results corroborated those of Bowman, et al., and Sheppard, et al., subsequent to our studies. However, for assessment of antifungal efficacy we found that CFU and qPCR were equivalent (Antimicrob Agents Chemother 49:1369-76) suggesting CFU determination remains a useful simple method for assessing antifungal drug efficacy. Overall, there remains no optimal, single method for determination of fungal load of Aspergillus and it may be that a combination of methods (e.g., CFU and qPCR) should be used.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
INVASIVE ASPERGILLOSIS IN CHRONIC GRANULOMATOUS DISEASE
Brahm H. Segal, MD Roswell Park Cancer Institute, USA
Wednesday, January 16, 2008, 2:20 – 2:45 PM
Invasive aspergillosis is a major cause of morbidity and mortality in highly immunocompromised persons. Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase complex in which phagocytes are defective in generating superoxide anion. Activation of preformed granular proteases is likely to be principal mechanism for NADPH oxidase-mediated destruction of pathogens. As a result of the defect in this key host defense pathway, CGD patients suffer from recurrent life-threatening bacterial and fungal infections. Invasive aspergillosis is the most important cause of mortality in CGD. Genetically engineered CGD mice recapitulate several of the features of the human disease and are highly susceptible to Aspergillus infection.
There are important differences between invasive aspergillosis in CGD compared to other immunocompromised conditions. In neutropenic animals and patients, invasive pulmonary aspergillosis is characterized by hyphal angioinvasion, coagulative necrosis and paucity of inflammatory cells. A similar pathologic phenotype may occur in non-neutropenic allogeneic hematopoietic stem cell transplant recipients (HSCT) with graft-versus-host disease (GVHD). In contrast, Aspergillus infection causes robust pyogranulomatous responses in genetically engineered p47phox -/- CGD mice. Angioinvasion was not observed in CGD mice nor is it a feature of invasive aspergillosis in CGD patients. Serum galactomannan was not elevated above baseline in CGD mice with invasive pulmonary aspergillosis, possibly a reflection of lack of vascular invasion. Lethality from pulmonary aspergillosis in CGD mice was related to excessive inflammation rather than necrosis. The CGD mouse model may be exploited to understand NADPH oxidase-independent mechanisms that defend against vascular invasive aspergillosis.
CGD is also characterized by excessive inflammatory responses that are independent of the host defense deficit. Intratracheal administration of sterile zymosan, a ligand of toll-like receptor 2 and dectin-1 led to pyogranulomatous pulmonary lesions in CGD mice that histologically resembled inflammation associated with experimental aspergillosis. Inflammation in similarly treated wildtype mice was close to nil. These findings demonstrate a key role of NADPH oxidase in downregulating inflammation induced by specific ligands of pathogen recognition receptors. Recent studies point to defective tryptophan catabolism underlying impaired host defense and pathogenic inflammation in CGD.
Prophylaxis with a mould-active agent should be offered to CGD patients. Itraconazole was safe and effective as prophylaxis in a randomized study. Bone marrow transplantation is curative in CGD, but is associated with expected frequencies of transplant-related morbidity and mortality. Gene therapy is a promising tool, but has so far been limited by lack of ability to maintain stable population gene-corrected stem cells.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
DIFFERENCE IN INVASIVE ASPERGILLOSIS IN SOLID ORGAN TRANSPLANT RECIPIENTS
Shahid Husain, MD University of Pittsburgh, USA
Wednesday, January 16, 2008, 2:45 – 3:10 PM
Invasive aspergillosis continues to be an important pathogen in the setting of solid organ transplant recipients. The incidence of invasive aspergillosis in solid organ transplant recipients varies with the type of transplant. Lung transplant recipients have the highest incidence, while kidney transplant recipients have the lowest. Recent data suggest a decline in the overall rates of IA among solid organ transplant recipients without any change in the relative prevalence rates among various types of solid organ transplants. The clinical risk factor for the development of invasive aspergillosis common to every organ is the net state of immunosuppression, while other clinical risk factors are organ specific. The TLR receptors play an important role for the susceptibility and extent of disease in humans. Median time to the onset of IA has been gradually increasing. Approximately half of the cases of invasive aspergillosis occur after initial 90 day of transplantation. The delay in time to onset of disease is primarily related to the administration of antifungal prophylaxis in solid organ transplant recipients. In lung transplant recipients the median time to onset of IA has increased to >600 days. The predominant clinical syndrome among solid organ transplant recipients is invasive pulmonary aspergillosis. Liver transplant recipients have higher rate of disseminated disease as compared to other type of solid organ transplant recipients. Most common IA presentation in lung transplant recipients had been tracheobronchitis. Pulmonary aspergillosis has now become the most common type of Aspergillus infection in lung transplant recipients. The factors responsible for the differences in the clinical presentations in solid organ transplant recipients are not clearly elucidated. The mortality associated with IA still remains high. The effect of secretion of certain cytokines such as IL 10 on mortality remains to be clearly established in human studies.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
INVASIVE DISEASE IN PATIENTS WITH LEUKEMIA
Johan Maertens, MD University Hospital Gasthuisberg, Belgium
Wednesday, January 16, 2008 3:10 – 3:35 PM
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
GLUCOCORTICOIDS AND INVASIVE ASPERGILLOSIS
Dimitrios P. Kontoyiannis, M.D., Sc.D., F.A.C.P., FIDSA Department of Infectious Diseases, Infection Control and Employee Health The University of Texas M.D. Anderson Cancer Center
Wednesday, January 16, 2008, 3:45 – 4:00 PM
Glucocorticosetroids (GCs) have pleiotropic effects on the immune system that account for the propensity of patients to potentially life-threatening invasive aspergillosis (IA). In addition, GC might enhance the "fitness" of the fungus to cause disease. Although the exact prevalence and attributed mortality of IA in GC-treated patients is difficult to assess, Aspergillus species are significant pathogens in patients with multiple myeloma, collagen vascular diseases, solid organ and especially allogeneic stem cell transplant recipients. In the latter setting, high cumulative doses of GCs administered for graft-versus-host disease (GVHD) prophylaxis and/or treatment have been shown to be associated both with the risk of acquisition and the poor outcome of IA. Finally, the use of high-potency inhaled GCs for the treatment of asthma and chronic obstructive lung disease has been implicated in isolated cases of laryngeal and pulmonary aspergillosis.
There are distinct differences in the histopathologic features of invasive pulmonary aspergillosis in GC-induced immunosuppression compared to IA caused by neutropenia. The lesions in GC- associated IA consist mainly of neutrophilic and monocytic infiltrates, inflammatory necrosis, scant intra-alveolar hemorrhage and a paucity of hyphae and angioinvasion; in contrast, coagulative necrosis, intraalveolar hemorrhage, scant mononuclear inflammatory infiltrate and higher "burden" of invading hyphal elements is observed in granulocytopenic animals. Not suprisingly, the performance of non-culture based antigen detection diagnostic methods is suboptimal in GC-associated IA, because the fungal burden is low. Furthermore, we are starting to appreciate that different classes of antifungal (such as the polyenes) might work less efficiently in GC-associated IA as it compares to neutropenia-associated IA. Since the severity of IA appears to be associated with the intensity of GC treatment, every effort should be made toward the use of the lowest GC dose for the shortest possible time, in accordance with the characteristics of the individual host.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
NEUTROPHILS AND DAMAGE DURING INVASIVE ASPERGILLOSIS
Steve Holland, MD National Institute of Allergy and Infectious Diseases, USA
Wednesday, January 16 4:30 – 4:55 PM
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
GRANULOCYTE PRODUCTS ACT AS ALARMINS TO ENHANCE INNATE AND ADAPTIVE IMMUNITY
De Yang1,2 and Joost J. Oppenheim2
1Cellular Immunology Section, Basic Research Program, SAIC-Frederick, Inc.; 2Laboratory of Molecular Immunoregulation, Center for Cancer Research; National Cancer Institute, National Institute of Health, Frederick, Maryland 21702, USA
Wednesday, January 16, 2008 4:55 – 5:20 PM
Granulocytes contribute to host defense against microbial attack through direct phagocytosis and release of soluble mediators such as antimicrobial peptides or proteins (AMPs). Aside from acting as direct antimicrobial effectors, certain AMPs, such as defensins, cathelicidins, high-mobility group box-1 protein (HMGB1), and eosinophil-derived neurotoxin (EDN), also have dual capability of mediating the recruitment and activation of antigen-presenting cells (APCs) including dendritic cells (DCs) and monocytes/macrophages. By inducing APC recruitment and activation, these AMPs demonstrate the capacity to enhance antigen-specific immune responses. Since their release through either granulocyte degranulation or epithelial cell secretion occurs early in the course of host innate immune response, these AMPs may in essence serve as endogenous signals to alarm the adaptive immune systems for the promotion of adaptive immune responses to microbial antigen(s) and we have thus proposed to classify them as ‘alarmins’.
An effort searching for additional endogenous molecules that demonstrate dual APC-recruiting and activating activities has resulted in the identification of lactoferrin and HMGN1 as potential alarmins. Lactoferrin is a major AMP in milk and can be produced by neutrophils and epithelial cells, while HMGN1 is secreted by activated peripheral blood mononuclear leukocytes. Both lactoferrin and HMGN1 are capable of inducing APC recruitment and activation and promoting antigen-specific immune responses in mouse models.
APC activation is not only critical for the induction, but also important for the determination of the type (Th1, Th2, or Th17) of adaptive immune response. Recent studies in our laboratory have demonstrated that different alarmins can preferentially enhance a particular type of antigen-specific immune responses. For example, EDN, a granule product of eosinophils, promotes an alternative antigen-specific Th2 response by signaling through DC Toll-like receptor 2. In contrast, lactoferrin, HMGB1, and HMGN1 preferentially enhance Th1 immune responses. Oveall, alarmins are not only effectors of innate immunity, but also initiators/enhancers and regulators of adaptive immunity. [Funded by NCI contract N01-CO- 12400].
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
STAGE-SPECIFIC INNATE IMMUNE RECOGNITION OF ASPERGILLUS FUMIGATUS AND MODULATION BY ECHINOCANDIN DRUGS.
Tobias M. Hohl Infectious Disease Service Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Wednesday, January 16, 2008 – 5:20 – 5:45 PM Aspergillus fumigatus conidia (spores) are ubiquitous in the environment and inhaled daily by humans. The development of invasive aspergillosis occurs in hosts with deficient or defective pulmonary immune defenses. In this setting, conidia can germinate into hyphae that invade tissues and disseminate. The innate immune system plays a critical role in clearing conidia from the airways through the action of resident alveolar macrophages and recruited neutrophils. Recent advances by several groups have identified Toll-like receptor (TLR) and lectin signaling pathways that underlie the recognition of and inflammatory responses to conidia and hyphae.
Inhaled (resting) conidia do not elicit the release of inflammatory mediators by alveolar macrophages. In contrast, swollen conidia (and germ tubes), the first two products of germination, trigger alveolar macrophage TNF/CXCL2 release and neutrophil airway recruitment. Fungal -1,3 glucan acts as a trigger for the induction of these inflammatory responses through time-dependent exposure on the surface of germinating conidia. Dectin-1, a signaling receptor that binds -1,3 glucan, mediates TNF/CXCL2 induction by swollen conidia. TLR2- and MyD88-mediated signals provide an additive contribution to macrophage activation by germinating conidia. These results suggest that the innate immune system can focus inflammatory responses on conidia with invasive potential.
Echinocandins target fungal -1,3 glucan synthesis and are used clinically to treat invasive aspergillosis. Although echinocandins do not completely inhibit in vitro growth of A. fumigatus, they induce morphologic changes in hyphae. Since -1,3 glucan activates host antifungal pathways via the dectin-1 receptor, the effect of echinocandin exposure on inflammatory responses to A. fumigatus was examined. Caspofungin or micafungin-treated conidia and germlings induced less TNF/CXCL2 secretion by macrophages than untreated counterparts. Diminished TNF/CXCL2 secretion correlates with reduced - glucan exposure on echinocandin-treated germ tubes. In contrast to drug-treated conidia and germlings, echinocandin-treated hyphae stimulated increased TNF/CXCL2 release and stained intensely with a - glucan-specific antibody, particularly at hyphal tips. Our experiments demonstrate that echinocandin- induced morphologic changes in hyphae are accompanied by increased -glucan exposure, with consequent greater dectin-1 mediated inflammatory responses by macrophages. Thus, echinocandin activity against A. fumigatus may include enhancement of innate immune responses to hyphae.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
BIOLOGICAL AND EVOLUTIONARY DIVERSITY IN THE GENUSVASPERGILLUS: WHAT IT MEANS IN MEDICAL MYCOLOGY
David Geiser Pennsylvania State University, USA
Thursday, January 17, 2008, 8:00 – 8:45 AM
The Genus Aspergillus represents an extremely diverse group of fungi, encompassing at least nine sexual (teleomorphic) genera. In this talk, I will summarize the known biological diversity of this genus, including ecological, morphological and physiological characters. Key to understanding this diversity is knowledge of the evolutionary patterns underlying it, and I will summarize recent and current attempts to characterize evolutionary relationships in this genus using genomics and multilocus phylogenetic approaches. This will include a summary of our efforts to use complete Aspergillus genome sequences to develop markers useful for making a robust inference of Aspergillus phylogeny. I will then attempt to assess the impact of a molecular evolutionary understanding of Aspergillus diversity in a practical context. Key issues include clinical diagnostics and an understanding of the evolution of traits relevant to pathogenesis.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
GALACTOMANNAN: MORE THAN ONE MOLECULE
JP Latgé Aspergillus Unit Institut Pasteur, Paris, France
Thursday, January 17 2008, 9:00 – 9:25 AM
Galactofuranose (Gal-f), the 5-membered ring form of galactose is found in many pathogenic bacteria, fungi and protozoa. In bacteria, Galf constitutes a key part of the mycobacterial cell wall. In protozoa, Galf is a major component of the lipophosphoglycan and glycophosphoinositol lipids of Leishmania and GPI-anchored proteins, mucins and lipids of Trypanosoma. Galf- containing conjugates are often necessary for viability or virulence. In Aspergillus fumigatus, Gal-f is covalently bound to the alkali insoluble fraction of the cell wall but is also found in a lipophosphoglycan, various sphingolipids and glycoproteins. In my talk, I will review the different molecules that bear a Galf moiety and discuss the pathway leading to the polymerization or/and insertion of Galf in Aspergillus molecules
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
CHITIN SYNTHASES OF ASPERGILLI: FUNCTIONS IN TIP GROWTH AND MORPHOGENESIS IN ASPERGILLUS NIDULANS
Hiroyuki Horiuchi Department of Biotechnology, The University of Tokyo Bunkyo-ku, Tokyo Japan
Thursday, January 17, 2008, 9:25 – 9:50 AM
Some filamentous fungi are known as human pathogens and their filamentous morphology is crucial to their pathogenicity. The cells of these fungi are covered with rigid cell walls that play important roles in the fungal morphogenesis.
Chitin is a homopolymer of -1,4-linked N-acetylglucosamine (GlcNAc) and one of the major cell wall components of many filamentous fungi. Chitin synthases are membrane proteins that catalyze the polymerization of GlcNAc using UDP-GlcNAc as a substrate. Recent sequence analyses of Aspergillus genomes have uncovered that Aspergillus fungi have nearly ten chitin synthase genes whose products are classified into seven classes according to their amino acid sequences similarities. To elucidate the functional differences among these gene products in hyphal tip growth and morphogenesis, we have selected Aspergillus nidulans as a model organism and analyzed their functions. There are eight chitin synthase-encoding (chs) genes in A. nidulans genome. We have cloned six chs genes, chsA, chsB, chsC, chsD, csmA, and csmB, whose gene products belong to classes II, III, I, IV, V, and VI, respectively (1 and references therein). ChsA and ChsC have overlapping functions in the growth and morphogenesis and they are suggested to be directly involved in septum formation (2, 3). ChsB plays a crucial role in the tip growth, since severe growth defects were observed in the chsB deletion mutants. ChsD seems to play non- essential roles in growth and morphogenesis. CsmA and CsmB consist of a chitin synthase domain, CSD, at their C-termini and a myosin motor-like domain, MMD, at their N-termini. No orthologs of csmA and csmB were found in the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, but they are widely distributed in the genomes of filamentous fungi and dimorphic yeasts of which cell wall chitin contents are high. csmA deletion mutants and csmB deletion mutants showed growth delay under low osmotic conditions and formed swollen hyphae called balloons. csmA csmB double deletion mutants were not viable. We showed that epitope-tagged CsmA and CsmB localized primarily at hyphal tips and forming septa (1,4). We also showed that a direct interaction of the MMD with actin was essential for the proper localization and function of CsmA (4).
Taken together, the chitin synthesis in the growth and morphogenesis of A. nidulans is a complex process and several chitin synthases are involved in it.
References 1. Takeshita, N., S. Yamashita, A. Ohta, and H. Horiuchi. 2006. Mol. Microbiol. 59:1380-1394. 2. Ichinomiya, M., E. Yamada, S. Yamashita, A. Ohta, and H. Horiuchi. 2005. Eukaryot. Cell 4:1125-1136. 3. Ichinomiya, M., A. Ohta, and H. Horiuchi. 2005. Curr. Genet. 48:171-183. 4. Takeshita, N., A. Ohta, and H. Horiuchi. 2005. Mol. Biol. Cell 16:1961-1970.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
ROLE OF (1→3)-β-D-GLUCAN IN THE DIAGNOSIS OF INVASIVE ASPERGILLOSIS
Francisco M. Marty, MD Harvard Medical School, USA
Thursday, January 17, 2008 9:50 – 10:15 AM
Measurement of serum (1→3)-β-D-Glucan (BG) is an aid in the diagnosis of fungemia and deep-seated mycoses including invasive aspergillosis (IA). BG is present in the cell wall of most pathogenic fungi (including Pneumocystis jiroveci) in significant amounts with some relevant exceptions including Cryptococcus neoformans and Zygomycetes. The two commercially available assays (manufactured by Associates of Cape Cod, Inc., Falmouth, MA, USA; and Wako Pure Chemical Industries, Ltd, Osaka, Japan) have limits of detection around 1pg/mL BG. Published validation studies have included patients with IA and other invasive fungal diseases (IFD).
BG detection appears to be more sensitive than galactomannan detection in patients with IA, but BG’s intrinsic lack of mycological specificity requires the integration of clinical, radiological, and microbiological data for proper interpretation. Serum BG kinetics are poorly understood, but detection of BG tends to be more prolonged than galactomannan in patients that receive adequate antifungal treatment and improve clinically. These test characteristics can be used, for example, to exclude IA in some clinical scenarios; to increase the certainty of IA in the presence of single positive galactomannan results or when testing follows initiation of antifungal treatment.
Serum BG detection in the absence of IFD may occur in patients undergoing hemodialysis with cellulose membranes; in patients treated with immunoglobulin, albumin, or other blood products filtered through cellulose filters containing BG; in patients with serosal exposure to glucan-containing gauze or to some IV antimicrobials. These sources of false-positivity should be considered when interpreting BG results.
BG may become a more sensitive tool for surveillance of IA and other IFD in populations at risk. Stratified IFD screening and diagnostic strategies using both galactomannan and BG should be explored. Factors affecting the production and clearance of BG during IA and other IFD need additional study to further refine its diagnostic utility.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
CONTROVERSIES SURROUNDING CATEGORIZATION OF FUNGAL SINUSITIS
Arunaloke Chakrabarti, M.D. Professor & In-Charge, Division of Medical Mycology, Department of Medical Microbiology, Postgraduate Institute of Medical Education & Research, Chandigarh, India.
Thursday, January 17, 2008 – 10:45 – 11:10 AM
Sinusitis, more accurately rhinosinusitis, is a common disorder affecting approximately 20% world population at some time of their lives. Acute rhinosinusitis (ARS) is well categorized. However, controversies surround chronic rhinosinusitis (CRS) and the role of fungus in CRS. The prevalence of fungal rhinosinusitis (FRS) and the dominant fungal pathogen appear to vary in different geographical location. Based on histopathological findings, FRS can be broadly divided into two categories: the invasive and non-invasive depending on invasion of mucosal layer. Three types of FRS are tissue- invasive infectious diseases: acute fulminant, chronic invasive, & granulomatous invasive. The two non- invasive FRS disorders are fungal ball, and fungus related eosinophilic rhinosinusitis [of which allergic fungal rhinosinusitis (AFRS) appears to be a distinct disorder]. Still, categorization of FRS remains controversial and open to discussion especially when it was suggested that fungi might be an important cause of most cases of CRS (Ponikau et al., 1999). Diversity of opinion exists on whether FRS should be characterized as an infection or an inflammatory condition.
Earlier in 1994, a chronic destructive but non-invasive form of FRS was proposed. This entity may not be a separate type but a variant of non-invasive type, in which fungal mass destroys the sinus wall by pressure. The distinction of granulomatous invasive type from chronic invasive type is also not beyond controversy as both types have a chronic course and predominant orbital involvement. Some workers even tried to classify granulomatous invasive FRS into two subtypes – one that is well defined in formation of usual granuloma, and other the hypertrophic sinus disease with chronic eosinophilic- lymphocytic granuloma and concomitant AFRS.
Maximum confusion surrounds the entity of fungus related eosinophilic rhinosinusitis, and definition of AFRS. In the diagnosis of AFRS, the detection of fungi in allergic mucin is considered important, although hyphae are sparse in sinus content. This leads to confusion in definition of this entity, especially with the description of two more closely related entities – eosinophilic fungal rhinosinusitis (EFRS) and eosinophilic mucin rhinosinusitis (EMRS). The confusion is further heightened by the alternative hypothesis of Ponikau et al., 1999, who proposed a different mechanism of AFRS and might be applied universally to encompass CRS as well. They demonstrated the presence of fungi in specimens from 93% of patients with CRS and did not find type I hypersensitivity to be prevalent in their study group. The controversy regarding the definition of AFRS is further intensified with well-documented reports of histologic invasion in possible cases of AFRS.
Currently, there are more questions than answers concerning the categorization of FRS. Recognizing the problem 5 American Societies (AAAAI, AAOA, AAO-HNS, ACAAI, ARS) tried to reach a consensus among 30 physicians on definitions and clinical research strategies of ARS, CRS with or without polyp, and AFRS (J Allergy Clin Immunol, 2004). ISHAM has also formed a working group on ‘Fungal sinusitis’ to exchange ideas in the direction of resolving the problems of definition of different categories of FRS.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
THE SYNDROMES OF INVASIVE FUNGAL SINUITIS
Richard D. deShazo, MD Billy S. Guyton Distinguished Professor University of Mississippi Medical Center Jackson, Mississippi
Thursday, January 17, 2008 – 11:10-11:35 AM
Invasive fungal sinusitis should be suspected in immunocompromised or diabetic patients with acute sinusitis, inflammation of nasal septal mucosa, unexplained fever or cough, or the orbital apex syndrome. Histopathological studies are often required to differentiate among these conditions and among syndromes of invasive fungal sinusitis.
Acute (Fulminant) Invasive Fungal Sinusitis Rhinocerebral mucormycosis is a syndrome characterized by sinusitis and a painless, necrotic black palatal or nasal septal ulcer or eschar. Without early treatment, the fungus may rapidly disseminate by the vascular route, causing death within days. It occasionally occurs in previously healthy persons. Saprophytic fungi of the order Mucorales, including rhizopus, rhizomucor, absidia, mucor, cunninghamella, mortierella, saksenaea, and apophysomyces, have caused this syndfrome, also called zygomycosis. An identical syndrome has been described with aspergillus, fusarium, and Pseudallescheria boydii infections and has been called fulminant invasive sinusitis. Fever, cough, crusting of nasal mucosa, epistaxis, and headache are the most common presenting symptoms.
Histopathological studies show hyphal invasion of blood vessels, including the carotid arteries and cavernous sinuses; vasculitis with thrombosis; hemorrhage; and tissue infarction.
When symptoms and sign are present, emergency surgery should be performed for histopathological evaluation and to debride the devitalized tissue supporting fungal growth. When histopathological studies confirm tissue invasion, treatment with amphotericin B should be initiated immediately, without waiting for the results of fungal cultures, and continued for a minimum of 14 days. Close collaboration between medical and surgical specialists is essential in the care of these patients.
Granulomatous Invasive Fungal Sinusitis Primary paranasal granuloma, also called indolent fungal sinusitis, is a curious syndrome of chronic granulomatous sinusitis associated with proptosis. Reports have come primarily from Sudan, but also from exclusively with A. flavus. There is profuse fungal growth with regional tissue invasion, noncaseating granulomas with giant cells, and plasma cells. Unless removed surgically, the resulting fibrous fungal mass may spread into the orbit, dura, and brain. Treatment with itraconazole at a dose of 8 to 10 mg per kilogram per day appears to decrease the high postoperative relaps rate.
Chronic Invasive Fungal Sinusitis Chronic invasive fungal sinusitis can be distinguished from the two other forms of invasive fungal sinusitis by its chronic course, dense accumulation of hyphae resembling a mycetoma, and association with the orbital apex syndrome, diabetes mellitus, and corticosteroid treatment. The orbital apex syndrome is characterized by decreasing vision and ocular immobility resulting from a mass in the superior portion of an orbit. Biopsy and orbital exploration show vascular invasion by fungal elements and become invasive, perhaps as a result of the immunosuppression associated with diabetes mellitus or corticosteroid treatment, and should be treated as aggressively.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
CANINE SINO-NASAL ASPERGILLOSIS: PARALLELS WITH HUMAN DISEASE
Michael J. Day Division of Veterinary Pathology, Infection and Immunity, School of Clinical Veterinary Science, University of Bristol, Langford, BS40 5DU, United Kingdom
Thursday, January 17, 2008, 11:35 AM – 12:05 PM
Canine sino-nasal aspergillosis (SNA) is characterized by the formation of a superficial mucosal fungal plaque within the nasal cavity and/or frontal sinus of systemically healthy dogs. The most common causative agent is Aspergillus fumigatus. The fungus does not invade beneath the level of mucosal epithelium but incites a severe chronic inflammatory response that leads to local destruction of nasal bone. These clinicopathological features are equivalent to those of human chronic erosive non-invasive fungal sinusitis. The clinical diagnosis of canine SNA relies on multiple modalities but local instillation of anti-fungal agents is an effective therapy with high cure-rate. Recent studies have investigated the immunopathogenesis of canine SNA. The mucosal inflammatory infiltrate involves a mixture of CD4+ and CD8+ T lymphocytes, IgG+ plasma cells and activated macrophages and dendritic cells expressing class II molecules of the major histocompatibility complex. There is active recruitment of blood monocytes and neutrophils. Real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis of mucosal tissue samples has revealed up-regulation of Th1 (IL-12, IL-18 and IFN- ), Th17 (IL-23) and pro-inflammatory (IL-6, TNF- ) cytokine mRNA with evidence of expression of genes encoding monocyte chemoattractant proteins 1 – 4. Additionally, there is significant transcription of the IL-10 gene consistent with local immunosuppression that prevents secondary immune-mediated sequelae whilst permitting chronicity of the infection. The source of this IL- 10 may be a T regulatory population or a Th1 population that switches phenotype during the course of disease. This understanding of the immunopathogenesis of canine SNA establishes this disorder as a valuable model for the equivalent human pathology.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
ALLERGIC FUNGAL SINUSITIS
Mark S. Schubert, M.D., Ph.D. Clinical Associate Professor of Medicine University of AZ College of Medicine Phoenix and Tucson, A And Allergy Asthma Clinic, Ltd. Phoenix, AZ
Thursday, January 17, 2008, 12:05 – 1:50 PM
Allergic fungal sinusitis (AFS) is a noninvasive form of chronic recurring fungal rhinosinusitis with an incidence of between 6%-9% of all rhinosinusitis requiring surgery. Regional variation in the reported incidence of AFS likely reflects geographic and climactic conditions conducive to the exposure to AFS-etiologic airborne fungi. Patients with AFS commonly present with chronic rhinosinusitis with nasal polyps, inhalant atopy, elevated total serum immunoglobulin E (IgE), and sinus-obstructing inspissates of a characteristic extramucosal “peanut buttery” eosinophil-rich material called allergic mucin that contains sparse numbers of fungal hyphae. Sinus CT shows findings of chronic rhinosinusitis that often include central areas of increased contrast (“hyperattenuation”) within abnormal paranasal sinuses that represent the presence of allergic mucin. AFS has been found to be analogous in several ways to allergic bronchopulmonary aspergillosis (ABPA). Both AFS and ABPA are chronic inflammatory respiratory tract disorders that are driven by hypersensitivity responses to the presence of small numbers of extramucosal fungi found growing within airway-impacting allergic mucin. AFS allergic mucin typically cultures positive for either dematiaceous fungi such as Bipolaris spicifera or Curvularia lunata, or Aspergillus species such as A. fumigatus, flavus or niger. However, up to 13% of AFS surgical sinus fungal cultures return negative despite histopathologic confirmation of AFS. As with ABPA, type I immediate hypersensitivity to the etiologic mold in AFS is always present. Further, both AFS and ABPA have been found to have association with specific class II major histocompatibility alleles (DQB1*0301 and *0302 for B. spicifera AFS; DRB1*1501 and *1503 for ABPA). Proper diagnosis of AFS and differentiation from the other forms of both noninvasive and invasive fungal rhinosinusitis requires adherence to strict published diagnostic criteria that include the following: 1) surgically obtained characteristic inspissated allergic mucin must be seen histopathologically and/or grossly at surgery; 2) the allergic mucin must be positive for fungal hyphae on fungal staining, and/or properly obtained surgical sinus fungal cultures must be positive in an otherwise characteristic patient; 3) there should be no histopathologic evidence for mucosal fungal invasion, mucosal necrosis, granulomas, or giant cells; 4) other fungal rhinosinusitis disorders must be excluded. Treatment of AFS has been modeled to an extent after treatment approaches for ABPA that includes the use of postoperative oral corticosteroids and aggressive antiallergic inflammation therapy. Systemic antifungal treatment has not been shown to modify the course or severity of AFS. The use of follow-up measurements of total serum IgE during treatment of both AFS and ABPA patients can help to monitor disease activity. Future AFS research will lead to further insights into chronic inflammatory diseases, improved treatments, and ultimately decreases in surgical recurrence rates for this highly recurrent rhinosinusitis disorder.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
CRITERIA FOR ASPERGILLUS SPECIES DELIMITATION: UTRECHT
Robert A. Samson, PhD Centraalbureau voor Schimmelcultures
Thursday, January 17, 2008, 2:30 – 2:00 PM
During an International Workshop held from 12-14 April 2007 in Utrecht, The Netherlands, participants discussed what an Aspergillus species is and how we delimit a species. In several sessions the species concept was presented by researchers in Aspergillus covering traditional and modern taxonomy, genetics, clinical, industrial and applied microbiology, biochemistry and genomics. Many topics were discussed including the following (1). What and how many genes should be used to delimit an Aspergillus taxon? (2) How does the phylogenetic species concept translate to practical and routine diagnoses? (3) What is the impact of Aspergillus taxonomy in terms of epidemiology, case definitions and biological understanding of disease? (4) What are the roles of Aspergillus databases for species identification? (5) What is the value and impact of polyphasic approaches for species identification? (6) What genes/methods can be used to design kits for rapid identification? (7) How should new species be proposed?
The most important recommendations include the following: (1) A polyphasic approach is recommended for describing new Aspergillus species, including molecular, morphological, physiological and ecological data. (2) Any proposed new species should show evidence for evolutionary divergence from other taxa, particularly unique DNA characters at multiple loci, in addition to any distinctive extrolites and morphological characters. (3) Media used for the description should be based on the use of media recommended by the International Commission of Penicillium and Aspergillus: Malt Extract Agar and Czapek’s Agar, with referenced formulas. (4) Type cultures of new Aspergillus species should be deposited in at least two international recognized culture collections. (5) If type cultures are not made available for the scientific community, the species will be considered invalid. (6) For the description of new taxa, multiple, independent loci are recommended for use, particularly loci for which large datasets already exist, such as ITS, ?-tubulin, calmodulin, actin, RNA polymerase. (7) Sequences must be deposited in recognized genetic databases. (8) Use dual names where necessary, single name in normal use, depending on the state (teleomorph or anamorph) that is observed. (9) A simple database for identification was proposed which would include basic sequences, photos, links to media/growth protocols, or the possibility to make a phylogenetic tree.
The recommendations will be discussed in more detail. The proceedings of this meeting has appeared in Studies in Mycology 59 (2007) (see also www.studiesinmycology.org).
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
MLST VERSUS MICROSATELLITES FOR TYPING OF ASPERGILLUS FUMIGATUS ISOLATES.
Corné HW Klaassen Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands
Thursday, January 17, 2008, 2:55 – 3:20 PM
Aspergillus fumigatus is a ubiquitous cosmopolitan fungus that may cause life-threatening complications in various immunocompromized patient populations. In order to discriminate between various isolates of different origin or to recognize isolates from a common origin, molecular typing methods offer the best available options. From the wide variety of molecular methods available, those that are highly reproducible and yield unambiguous, user independent, typing data should be preferred over other methods. Two such methods are Multi Locus Sequence Typing (MLST) and microsatellite analysis. Each of the two methods offer several advantages over the other.
The main advantage of MLST is the DNA sequence format that is accessible to a growing number of clinical laboratories. DNA sequence data are easily compared and exchanged between labs. In addition, the same targets can potentially be used for isolates belonging to different species. On the downside are the relative high costs, turn-around time and the relatively low discriminatory power. This latter is a direct consequence of the relatively low mutation rate of a given DNA sequence due to the presence of mismatch repair mechanisms.
Microsatellites are unique in their extremely high discriminatory power which is a direct result of the inherent instability, during DNA replication, of tandemly repeated DNA sequences. Fortunately, microsatellites are still sufficiently stable to allow longitudinal studies within an appropriate time window. However, interpretation of genotypically different isolates should be done with care and should take this instability into account. Microsatellites are amendable to rapid and high-throughput analyses in a modular fashion allowing large numbers of isolates to be analyzed in a timely fashion. The downside of microsatellite markers is that they usually are species specific.
Basically, the choice for either of the two methods could be appropriate and should primarily be based on the exact reason for performing strain typing. MLST seems to be most informative at the genus and/or population level whereas microsatellites are best used when high-resolution strain typing is required.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
COMPARISON OF THE TWO SEQUENCED A. FUMIGATUS ISOLATES, Af293 AND A1163
Natalie D. Fedorova, David Denning, William C. Nierman
Thursday, January 17, 2008, 3:20 – 3:45 PM
Aspergillus fumigatus isolates are thought to have low levels of genetic diversity as evidence by relative lack of sequence variation at their intergenic loci [1]. This is in contrast with the high levels of diversity observed in one of its closest sexual relatives, Neosartorya fischeri. This characteristic may limit the utility of sequence-based typing methods employed in many clinical laboratories to differentiate A. fumigatus strains. Nonetheless, whole genome sequencing of A. fumigatus isolate Af293 and array-based Comparative Genomic Hybridization (aCGH) of clinical isolates Af294 and Af71 with our Af293-based arrays has revealed that hundreds of genes were either absent or divergent in their genomes [2]. In addition, recent studies identified 14 repeat-containing polymorphic alleles (e.g. a putative cell surface protein, Afu3g08990 [3]). Finally our preliminary phenotypic characterization showed that A. fumigatus isolates can vary significantly in their pathogenicity and resistance to antifungals, indicating that their genomes contain differential genetic traits responsible for these differences.
The recent availability of the completely sequenced genome of the second A. fumigatus strain (Fedorova et al., submitted for publication), A1163, has created a unique opportunity to identify new polymorphic markers and to gain further insights into genome-scale intraspecific differences. A1163 was derived via the ectopic insertion of the A. niger pyrG gene from CEA17, which is a uridine/uracil auxotroph pyrG mutant of another clinical isolate, CEA10. Consistent with the aCGH study [2], comparative analysis of A293 and A1163 genomes revealed the presence of core, divergent and unique (isolate-specific) regions. The core portions share high sequence identity (99.8%) and comprise 98% of both genomes. In contrast, divergent loci are very small and contain orthologous genes that share very little sequence identity (37-90%). Several of them encode STAND, HET and Pfs domains predicted to function in heterokaryon incompatibility during hyphal fusion between genetically incompatible individuals. The list of divergent loci also includes the mating (MAT) locus encoding HMG- and alpha-domain proteins in A293 and A1163, respectively, consistent with a heterothallic breeding system. Unique genes, which comprise the remaining 2% of the Af293 and A1163 genomes, tend to cluster in large (up to 400 Kb) subtelomeric blocks, which seem to be the most variable segment of the genome. Remarkably 2 of 23 putative secondary metabolite biosynthesis clusters differ between Af293 and A1163.
1. Rydholm C, Szakacs G, Lutzoni F (2006) Low genetic variation and no detectable population structure in Aspergillus fumigatus compared to closely related Neosartorya species. Eukaryot Cell 5: 650-657. 2. Nierman WC, Pain A, Anderson MJ, Wortman JR, Kim HS, et al. (2005) Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438: 1151-1156. 3. Balajee SA, Tay ST, Lasker BA, Hurst SF, Rooney AP (2007) Characterization of a novel gene for strain typing reveals substructuring of Aspergillus fumigatus across North America. Eukaryot Cell 6: 1392-1399.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
ASPERGILLUS TERREUS COMPLEX
S. Arun Balajee, PhD Centers for Disease Control and Prevention, USA
Thursday, January 17, 2008, 3:45 – 4:10 PM
Based on morphological methods of identification, A. terreus is recognized as a single homogenous species within the section Terrei along with 2 other atypical variants, A. terreus var. africanus and A. terreus var. aureus. However, several recent studies have clearly shown that morphological characteristics may not be reliable markers for Aspergillus species identification and molecular methods could be powerful tools for delineating cryptic species within this genus. We recently developed a multi gene sequence approach employing three protein coding regions, Enolase [enoA], β tubulin [benA] and calmodulin [calM] [A. terreus sequencing project, Broad Institute of Harvard and MIT http://www.broad.mit.edu] to study species diversity in the section Terrei. After analyzing a large number of A. terreus isolates from clinical and environmental origins recovered from various parts of the world, we demonstrate the presence of a new, clinically relevant species Aspergillus alabamensis. Most members of this new species were recovered as colonizing isolates from immunocompetent populations and were morphologically similar to A. terreus with decreased in vitro susceptibilities to the antifungal drug amphotericin B. Further, based on phylogenetic and phenotype analyses, we also demonstrate that A. terreus var. africanus is a variant of A. terreus, whereas A. terreus var. aureus (considered as a morphological variant of A. terreus) should be raised to species within the section Terrei as Aspergillus aureus comb. nov. Results of this study also reveals that A. terreus is a truly cosmopolitan fungus with a global distribution since no population structure was revealed within the A. terreus clade.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
CULTIVATED TH1 CELLS
Thomas Lehrnbecher Pediatric Hematology and Oncology, Johann Wolfgang Goethe-University Frankfurt, Germany
Thursday, January 17, 2008, 4:40 – 5:05 PM
Invasive aspergillosis remains a serious complication in patients undergoing allogeneic stem cell transplantation. Although prolonged neutropenia is a well described risk factor for invasive aspergillosis, there is a growing body of evidence that CD4+ T-cells provide a critical secondary defense against Aspergillus fumigatus, the most frequent cause of invasive aspergillosis. Whereas the strategy of reconstituting adaptive immunity in hematopoietic transplant patients with ex vivo generated antigen-specific T-cells against cytomegalovirus or Epstein-Barr-virus is well described, there are few data on human anti-Aspergillus T-cells. In preliminary experiments, we were able to generate anti-Aspergillus T-cells in 7/7 healthy individuals. T-cells were first activated with a cellular extract of Aspergillus fumigatus, then isolated using the interferon (IFN)-γ secretion assay and expanded for up to 28 days. Phenotypic characterization revealed a memory, activated T-helper cell population (positive for CD3, CD4, CD45RO and HLA-DR). In addition, the cells secreted IFN-γ and TNF-α, but no IL-4 or IL-10, indicating that the generated cells were activated TH-1 cells. The isolated and expanded cells proliferated upon restimulation and showed a reduced alloreactivity compared to unselected CD4+ cells. Testing various fungal antigen extracts, similar proportions of IFN-γ producing CD4+ cells were obtained upon activation with antigen extracts of A.fumigatus, A.flavus, A.niger and P. chrysogenum, whereas no significant IFN-γ production was observed upon activation with antigen extracts of A. alternata and C. albicans. In addition, generated T-cells alone induced damage to A.fumigatus hyphae and significantly increased hyphal damage induced by human neutrophils. These results prompted us to develop a method for the clinical-scale generation of functionally active anti- Aspergillus T-cells according to good manufacturing practice (GMP) conditions. After stimulation of a total of 1.1x109 white blood cells derived from a leukapheresis product with Aspergillus antigens, cells were selected by the IFN-γ secretion assay and then expanded over a period of 12 days. In three independent experiments, a median number of 2x107 CD3+CD4+cells (range, 0.9-3.2x107) was obtained. The cultured cells exhibited almost exclusively a memory activated T-helper cell phenotype. In line with the preliminary results, the generated T-cells produced IFN-γ, but no interleukin IL-4 or IL-10, proliferated upon restimulation, and showed reduced alloreactivity compared to unselected CD4+ cells. This method may be suitable for future prospective trials evaluating the effect of adoptive immunotherapy in hematopoietic transplant recipients with invasive aspergillosis. Since it is unclear which patient population ultimately benefits from adoptively transferred anti-Aspergillus T-cells, we currently enumerate anti-Aspergillus T-cells in peripheral blood of hematopoietic transplant patients to test the hypothesis whether the number of anti-Aspergillus T-cells might define the risk for invasive aspergillosis in the post-transplant period and helps to stratify patients suitable for the adoptive transfer of anti-Aspergillus T cells.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
HOST RESPONSE SIGNATURES TO INVASIVE ASPERGILLOSIS
*T. Zelante, A. De Luca, C. D’Angelo, P. Bonifazi, S. Bozza, S. Moretti, F. Bistoni, P. Puccetti, L. Romani Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy
Thursday, January 17, 2008, 5:05 – 5:55 PM
The inflammatory response to fungi may serve to limit infection but an overzealous or heightened inflammatory response may contribute to pathogenicity, as documented by the occurrence of severe fungal infections in patients with immunoreconstitution disease or primary immunodeficiencies associated with hightened immune reactivity. Recent studies have suggested a greater diversification of the CD4+ T-cell effector repertoire than that encompassed by the Th1/Th2 paradigm. Th17 cells are now thought to be a separate lineage of effector Th cells contributing to immune pathogenesis previously attributed to the Th1 lineage. Th17 cells, which produce IL-17 preferentially, promote neutrophil-mediated inflammation and, although linked to the resistance to several bacterial and parasitic infections, correlate with disease severity and immunopathology in diverse infections. Recent evidence have shown that is the Th17 pathway and not the uncontrolled Th1 response that is associated with defective fungal clearance, failure to resolve inflammation and to initiate protective immune responses. Both IL-17 and IL-23 inhibited the fungicidal activity and subverted the inflammatory program of neutrophils even in the presence of IFN-gamma, a finding suggesting that the Th17 effector pathway prevails over the Th1 pathway. Protective Th1 and non protective Th17 were crossregulated in experimental models of pulmonary aspergillosis. Blockade of IL-17/IL-23 prevented pathogenic inflammatory responses, ameliorated infections and restored protective Th1 antifungal resistance, thus causally linking pathogenic inflammation to Th17 development. These new findings on the contribution of the IL-23/Th17 in promoting inflammation while subverting protective antifungal immunity provide a molecular connection between the failure to resolve inflammation and lack of antifungal immune resistance and point to strategies for immune therapy of Aspergillus infections and aspergillosis that attempt to limit inflammation to stimulate an effective immune response.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
VACCINE PROGRESS
James I. Ito, MD
Thursday, January 17, 2008, 5:30 – 5:55 PM
Despite the recent development of new anti-mould agents, there remains a significant incidence of invasive aspergillosis in the most immunocompromised hosts, the hematopoietic cell transplant (HCT) recipients on prophylaxis, and the response to these agents is still dismal (31%). There is a need for a different approach: prevention by vaccination. But this approach has been neglected because of the “Paradox of vaccination in HCT” (or in immunocompromised hosts in general). That is, since an HCT recipient cannot be adequately immunized (with standard vaccines) until at least a year after transplantation (when immune reconstituted), how would vaccination administered either before or after transplantation be expected to protect while the recipient is still immunosuppressed, i.e., under immunosuppressive therapy and not yet immune reconstituted. There are also other questions regarding the feasibility of such a vaccine in an immunocompromised host tat must be answered: (1) Is there acquired immunity to Aspergillus species?; (2) Can this immunity be boosted through infection or vaccination?; (3) Can Aspergillus antigens confer immunity as well as live organisms (pre-infection)?; (4) Can these selected Aspergillus antigens confer cross-protection against other Aspergillus species (A flavus, A niger, A terreus) or other moulds?; (5) Can protective antigens be selected and allergens avoided?; (6) Can systemic vaccination confer local (sino-pulmonary) protection?; (7) Will immunity attained while immunocompetent provide protection during subsequent immunosuppression (neutropenia, corticosteroid therapy, post-transplantation)?; (8) Can this immunity be transferred from donor to recipient under current transplantation conditions? Many of these questions were answered by Professor Romani and colleagues seven years ago when they demonstrated that by vaccinating mice prior to chemotherapy (immunosuppression) they could protect them from subsequent lethal challenge from inhaled Aspergillus conidia during neutropenia. Furthermore, they demonstrated local CD4+ cell proliferation and increased IFN-γ, IL-2 production in vaccinated animals. Also, adoptively-transferred Ag-specific CD4+ T cells producing IFN-γ and IL-2 conferred protection in naïve recipients. And, finally, there was recruitment of lymphocytes and macrophages to the site of infection in vaccinated animals despite a profound systemic neutropenia. Our laboratory demonstrated similar protection in a corticosteroid-immunosuppressed mouse model. That is, both a hyphal sonicate and a filtrate preparation used as vaccines prior to corticosteroid treatment protected mice against subsequent Aspergillus inhalational challenge. Systemic (subcutaneous) vaccination was more effective than local (intranasal) vaccination. It was noted that (non- immunocompromised) mice exposed intranasally to viable conidia responded to a protein in the 19kD range. This protein was identified by mass spectrometry as Asp f3, a known allergen. Vaccination with recombinant Asp f3 resulted in protection equivalent to the crude hyphal sonicate vaccine. Furthermore, truncated versions of rAsp f3 were created that eliminated one or both of the N-terminal or C-terminal known IgE binding sites. These “non-allergenic” truncated versions of rAsp f3 demonstrated protection against invasive aspergillosis when used as vaccines. Thus, allergenic epitopes can be separated from protective epitopes and result in a protective, non-allergenic vaccine. Furthermore, at least two polypeptides (11-mer and 13-mer) have been identified as T-cell epitopes. Current investigations involve identifying other protective T-cell epitopes on other Aspergillus proteins such di-peptidyl peptidase V (DPPV). Also, current work focuses on the transfer of protective immunity induced in a donor animal (by vaccination) to a recipient animal during HCT.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
Finally, there are other laboratories that are involved in answering the other questions (e.g., #4) posed such as will a multivalent vaccine have to be developed or is there a common cross-protective antigen(s) that will provide protection against all Aspergillus species and other moulds. Dr. David Stevens laboratory has recently shown that vaccination with Saccharomyces, live or killed, can protect against invasive aspergillosis.
In summary, there has been progress made in identifying protective, non-allergenic antigens of Aspergillus, and, hopefully, these can be tested clinically in the near future.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
RECOGNIZING THERAPEUTIC FAILURE
Kent Sepkowitz Memorial Sloan-Kettering Cancer Center, USA
Friday, January 18, 2008, 8:00 – 8:45 AM
Background: During this session, cases will be presented that raise the issues of how to (a) recognize treatment failure, including radiologic, serologic, and/or clinical definitions with special attention to assessment when available information is conflicting or ambiguous; and (b) approach management of patients with treatment failure.
Results: Just as the diagnosis of invasive aspergillus (IA) remains difficult to secure for many patients, so too is the assessment of a patient for possible treatment failure. Specifically the absence of a sensitive surrogate marker to monitor response leaves clinicians with several insensitive, non-specific and often conflicting pieces of information. For example, CT evidence of response is well-described to lag at least a week behind patient improvement and so this modality cannot be relied upon to assess daily or even weekly patient response. The clinical assessment of the patient is complicated by the presence or absence of neutropenia since patients may appear to worsen as their neutropenia recovers; conversely, patients with advanced infection may exhibit only subtle signs of IA if profoundly immunosuppressed. Finally, IA does not respond to any antifungal quickly; thus the clinician must patiently wait longer than is typical for a bacterial infection to determine whether the response is slow or simply not present.
Conclusion: Assessment of a patient with IA for treatment failure is a complicated determination that requires the clinician to synthesize incomplete and often conflicting information. Further adding to the difficulty are the morbidity and mortality of the disease and the relative lack of effectiveness of the available antifungal agents.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
INFLAMMATION IN ASPERGILLUS IMMUNOLGY
Luigina Romani Microbiology Section Department of Experimental Medicine and Biochemical Science, University of Perugia, Perugia, Italy
Friday, January 18, 2008, 8:00 – 8:45 AM
The balance between pro-inflammatory and anti-inflammatory signaling is a prerequisite for successful host/fungal interaction in aspergillosis. Although inflammation is an essential component of the protective response to fungi, its dysregulation may significantly worsen fungal diseases and limit protective antifungal immune responses. The newly described Th17 developmental pathway may play an inflammatory role previously attributed to uncontrolled Th1 cell responses. The capacity of regulatory T cells (Tregs) to inhibit aspects of innate and adaptive antifungal immunity, including functional Th17 antagonism, is required for protective tolerance to Aspergillus. The indoleamine 2,3-dioxygenase (IDO) and thyptophan catabolites contribute to such a homeostatic condition by providing the with host immune defense mechanisms adequate for protection, without necessarily eliminating fungal pathogens—which would impair immune memory—or causing an unacceptable level of tissue damage. The IDO mechanism has revealed an unexpected potential in the control of inflammation, allergy and allergic airway inflammation, all conditions in which plasmacytoid dendritic cells could have a protective function. IDO expression is paradoxically up-regulated in patients with allergy or autoimmune inflammation, a finding suggesting the occurrence of a homeostatic mechanism to halt ongoing inflammation. A unifying mechanism linking anti-inflammatory Tregs to tolerogenic Tregs via IDO appears to be at work in response to the fungus and is consistent with the revisited “hygiene hypothesis” of allergy in infections—that is, an early reduction in microbial burden may predispose to allergy. IDO has a unique and central role in this process acting as both an executor of the effector phase of anti-inflammatory Tregs and an inducer of tolerogenic Tregs. Recent data have confirmed the protective role of the IDO/Tregs axis in fungal allergy. In this model, modulation of tryptophan catabolism via the glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand, GITRL, inhibited Th2-cell responses and allergy and induced the expression of Foxp3+ Tregs through mechanisms dependent on IDO induction by components of the noncanonical NF-κB signaling pathway. Thus induction of IDO could be an important mechanism underlying the anti- inflammatory action of corticosteroids.
Together, these new findings provide a molecular connection between the failure to resolve inflammation and lack of antifungal immune resistance and point to strategies for immune therapy of fungal infections that attempt to limit inflammation in order to stimulate an effective immune response.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
CLINICAL TRIAL DESIGN FOR MOULD-ACTIVE AGENTS: TIME TO BREAK THE MOLD
Elias Anaissie, MD
Friday, January 18, 2008, 9:00 – 9:25 AM
Despite the availability of active antifungal agents, invasive aspergillosis (IA) continues to be a significant cause of morbidity, mortality, and resource utilization among the various patient populations at risk (1). New and more effective therapies are clearly needed.
Like with other drugs, the regulatory approval of new mould-active agents is based on the results of randomized clinical trials (RCT) which are particularly difficult to conduct and usually take several years to complete at very high costs. In this presentation, I will discuss the limitations of conventional RCTs for aspergillus-active agents and propose novel trial methodologies that are likely to expedite approval of therapies active against aspergillosis. My focus will be on invasive pulmonary aspergillosis in patients with hematological cancer, because it is the most common and better studied form of invasive aspergillosis.
Conventional RCTs of IA: difficult and expensive. Several factors account for these difficulties (2) including: 1) Low prevalence of IA 2) Inaccurate and non-specific disease definitions which do not mandate a microbiologic diagnosis (culture or antigen) and may lead to inclusion of patients without IA or with IA already resolving 3) Non-validated, expert-derived, outcome criteria 4) Coexistence of significant confounders such as other infections, underlying malignancy, graft versus host disease (GvHD) and others. We have recently shown (3) that the transient pulmonary deterioration of neutropenic patients with IA that is observed during neutrophil recovery may be caused by an immune reconstitution syndrome (with the infection responding) and should not be classified as “refractory” as per current outcome definitions
In addition, outcome evaluation of aspergillosis has been arbitrarily assigned at 12 weeks after starting therapy. Unfortunately, almost half of these patients die before this time endpoint, frequently from causes unrelated to aspergillosis such as progressive cancer or its complications. Because of low autopsy rates and assignment of death as treatment failure (in absence of autopsy) any death occurring during the 12 – week window may be incorrectly reported as having progressive aspergillosis making it all but impossible to accurately predict therapeutic efficacy.
Time to break the mold: a proposal for changing how RCTs of IA therapy are conducted in the hematology patient Our proposal relies on three approaches (2): 1) Selection of a validated surrogate marker as both a diagnostic tool and surrogate endpoint for clinical outcome endpoint; 2) Better patient selection for enrollment in clinical trials; and 3) Novel trial strategies.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
VALIDATED SURROGATE MARKER The Aspergillus galactomannan index (GMI) is a validated surrogate marker for diagnosis of IA in the hematology patient Frequent serum GMI testing (OD ≥0.5 cut-off) during neutropenia following chemotherapy has been shown to facilitate the early diagnosis of IA with very high sensitivity, specificity, and positive and negative predictive value even when compared to autopsy-proven aspergillosis (4,5). Because IA most commonly affects the lungs, GMI testing in BAL fluid has been studied by several investigators and shown to have a very high positive predictive value, and which reached 100% when BAL GMI was obtained in neutropenic patients with characteristic CT scan findings (6).
The serum Aspergillus GMI is a validated surrogate endpoint for outcome evaluation of IA in the hematology patient
Stringent requirements need to be met before a surrogate marker can serve as a surrogate endpoint for a clinical event (2). These include 1) biological plausibility (surrogate endpoint is in pathophysiologic causal chain, and in proximity to clinical endpoint); 2) predictive of outcome (captures net effect of intervention on outcome, consistently sensitive to and persists under the effect of the intervention, effects quantitatively similar across drugs of same and different classes, particularly across classes treating a given clinical condition, predictive of clinical outcome i.e., changes in mechanistically compatible direction, rate, temporal sequence with outcome; 3) validated in clinical trials for a specific disease and population and 4) good test attributes (standardized, reproducible, simple to measure, non-invasive, inexpensive and representative of disease burden, valid for all species/ infection sites, dichotomous and quantitative with short latency to observation of effects).
On the basis of the cumulative data, serum GMI meets all these requirements and can hence serve as a “validated” surrogate endpoint for outcome evaluation of IA in patients with hematological malignancies (2). By contrast, none of the conventional outcome endpoints for IA met these stringent criteria. In addition to the above-mentioned criteria for surrogacy, a strong correlation between surrogate end- point and objective clinical outcome such as survival or autopsy-proven aspergillosis must be demonstrated using accepted statistical methods (2). We have shown that using the kappa correlation coefficient test, strong correlations exist between serum GMI and IA outcome in the cumulative literature experience (7) and in a series of 56 consecutive cancer patients with IA cared for at our institution (8).
BETTER PATIENT SELECTION The following patient selection criteria are recommended (2): Favor adults with hematological cancer and reasonable chance of survival and marrow recovery (e.g. first remission induction chemotherapy for acute leukemia; autologous stem cell transplant) and likelihood of aspergillosis response (pulmonary, not disseminated aspergillosis), and not receiving mould-active prophylaxis.
Preferably avoid post-engraftment allogeneic stem cell transplant recipients for several reasons: a) High risk of data loss and protocol deviation because of logistics of care when patients are at home, away from treating center; b) Longer study period and hence, longer delays and higher costs; c) Outcome assessment complicated because of graft-versus-host disease and rapidly changing immunosuppressive therapies and higher rates of death and d) Serum galactomannan index (GMI) not well validated in non-neutropenic settings.
3 RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
Limit patient heterogeneity (as above) plus enroll patients with single organ aspergillosis (e. g pulmonary) and who have positive serum GMI. May expand to other populations/infection sites in subsequent phase of drug development.
Stratify according to immune and disease status
NOVEL TRIAL STRATEGIES Randomized controlled trials based on adaptive trial design could play a critical role in improving the efficiency of clinical trials. Adaptive trials represent fragmented versions of the classic RCT design, giving the investigators more opportunities for decision points rather than waiting to the end of the study to evaluate the results (8). Many variations on adaptive trials exist with different numbers of stages and endpoints, but regardless of which clinical phase is being addressed, the key decision investigators have to make is whether to continue or stop the study. Thus, critical selection of endpoints and number of patients at each stage must be made to ensure sufficient statistical power to answer the questions the trial is intended to answer at each stage. This design allows investigators to minimize sample size ensuring statistical power, reduce trial duration and costs, and reduce the number of patients exposed to potential drug toxicities, while benefiting a larger group of patient in a shorter period of time, should the drug be approved.
Conclusion Now is the time to break the mold of conventional trial design for mould-active agents. Novel strategies to evaluate risks and benefits of effective therapies for IA quickly and efficiently are urgently needed. Incorporating surrogate markers such as the Aspergillus GMI in these trials, both as a diagnostic marker and a surrogate endpoint for outcome, better patient selection and other novel trial strategies such as adaptive design will allow us to expedite approval of life-saving therapies.
References 1.Segal BH, Walsh TJ. Current approaches to diagnosis and treatment of invasive aspergillosis. Am J Respir Crit Care Med 2006;173:707-17
2.Anaissie EA. Trial design for mold-active agents: time to break the mold--aspergillosis in neutropenic adults. Clin Infect Dis. 2007 May 15;44(10):1298-306.
3.Miceli MH, Maertens J, Buvé K, Grazziutti M, Woods G, Rahman M, Barlogie B, Anaissie EJ. Immune reconstitution inflammatory syndrome in cancer patients with pulmonary aspergillosis recovering from neutropenia: Proof of principle, description, and clinical and research implications. Cancer. 2007 Jul 1;110(1):112-20.
4. Maertens JA, Klont R, Masson C, Theunissen K, Meersseman W, Lagrou K, Heinen C, Crépin B, Van Eldere J, Tabouret M, Donnelly JP, Verweij PE. Optimization of the cutoff value for the Aspergillus double-sandwich enzyme immunoassay Clin Infect Dis. 2007 May 15;44(10):1329- 36.
5. Maertens J, Verhaegen J, Demuynck H, et al. Autopsy-controlled prospective evaluation of serial screening for circulating galactomannan by a sandwich enzyme-linked immunosorbent assay for hematological patients at risk for invasive Aspergillosis. J Clin Microbiol 1999;37:3223-8
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
1.Becker MJ, Lugtenburg EJ, Cornelissen JJ, Van Der Schee C, Hoogsteden HC and De Marie S. Galactomannan detection in computerized tomography-based broncho-alveolar lavage fluid and serum in haematological patients at risk for invasive pulmonary aspergillosis. Br J Haematol 2003;121:448-57
2.Miceli MH, Grazziutti ML, Woods G, Zhao W, Kocoglu MH, Barlogie B, Anaissie E. Strong Correlation between Serum Aspergillus Galactomannan and Aspergillosis Outcome in Patients with Hematological Cancer: Clinical and Research Implications. Clin Infect Dis. (In press)
3.Woods G, Miceli MH, Grazziutti ML, Zhao W, Barlogie B, Anaissie E. Serum Aspergillus galactomannan antigen values strongly correlate with outcome of invasive aspergillosis: a study of 56 patients with hematologic cancer. Cancer. 2007 Aug 15;110(4):830-4
4.Schäfer H, Timmesfeld N, Müller HH.An overview of statistical approaches for adaptive designs and design modifications. Biom J. 2006 Aug;48(4):507-20
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
FUNCTIONALIZED NANOCARRIERS TO IMAGE AND TARGET FUNGI INFECTED IMMUNE CELLS
Suresh P. Vyas Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar (MP), INDIA
Friday, January 18, 2008, 9:25 – 9:50 AM
The ability to incorporate drugs into liganded novel carrier system(s) represents for a new paradigm in pharmacotherapy that could be used for cell-targeted drug delivery and imaging. In addition, the means to deliver a drug dose to immune cells in situ with the same specificity used to image those cells would provide for a powerful therapeutic alternative for many diseased states of immune cells including aspergillosis. The use of liposomes and emulsomes as drug delivery vehicles particularly for chronic intracellular infections like aspergillosis, tuberculosis and leishmaniasis has great potential that could revolutionise the future of chemotherapy. These nanocarriers, which are biocompatible, biodegradable, and non-immunogenic can control the delivery of drugs by targeting the drug to the site of action or by site avoidance drug delivery or by prolonged circulation of drugs. Fungal infections are very likely to be progressive in nature, and are often disseminated, ultimately becoming life threatening. The usefulness of liposomes as vehicles for amphotericin B (Amp B) has been well demonstrated in fungal diseases, such as candidiasis, aspergillosis and cryptococcosis, by reducing its toxicity and increasing its therapeutic index. Recently, targeted delivery to lung tissues, the organs infected by many fungi, via inhaled liposomal Amp B aerosol, has been shown to be a more effective approach. Furthermore, to increase the scope of liposomised Amp B, various macrophage-specific ligands, such as O-palmitoyl mannan and O-palmitoyl pollulan, were tested to dock the liposomal constituents specifically to the target cells actually in need of treatment. Pressurised packed systems based on preformed liposomal formulations in chlorofluorocarbon, aerosol propellants were evaluated for their targeting potential to alveolar macrophages. The drug localisation index, calculated after 6 h, was approximately 1.42-, 4.47- and 4.16-fold higher, respectively, for plain O-palmitoyl mannan- and O-palmitoyl pollulan-coated liposomal aerosols, when compared with plain drug solution-based aerosols. Hence, emerging nanosystems can be used for simultaneous tracking and drug delivery to those cells.
3 RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
IMAGING LIVING CELLS OF ASPERGILLUS
N.D. Read, P.C. Hickey
Friday, January 18, 2008, 9:50 – 10:15 AM
A revolutionary new perspective of the cell biology of fungal cells is arising as a result of using live-cell imaging techniques to analyse organelle and molecular dynamics at high spatial resolution. This has become possible because of the development of a wide range of fluorescent probes (vital dyes and fluorescent proteins) for living cells, as well as new microscope technologies (e.g. confocal and deconvolution microscopy), and powerful computer software and hardware for digital image processing and analysis. These innovations are having a profound impact on the experimental analysis of living fungal hyphae at the single cell level. In our presentation I will show examples of confocal live-cell imaging techniques applied to studies on Aspergillus fumigatus and A. nidulans.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
DIAGNOSTIC IMAGING OF EXPERIMENTAL INVASIVE PULMONARY ASPERGILLOSIS
Thomas J. Walsh National Cancer Institute, Bethesda, MD
Friday, January 18, 2008 10:15 – 10:40 AM
Pulmonary infiltrates in neutropenic hosts with invasive aspergillosis (IA) are caused by organism-mediated tissue injury, including vascular invasion and hemorrhagic infarction. Monitoring the dynamics of pulmonary infiltrates of invasive aspergillosis is an important tool for assessing response to antifungal therapy. Resolution of pulmonary infiltrates is one of the essential criteria for monitoring therapeutic response to antifungal agents used for treatment of invasive aspergillosis. In order to measure the effect of antifungal compounds on this organism- mediated tissue injury, we developed a procedures for monitoring the course of pulmonary infiltrates by serial ultrafast computerized tomography (UFCT) in persistently neutropenic rabbits with experimental invasive pulmonary aspergillosis. The course of pulmonary lesions treated with amphotericin B, lipid formulations of amphotericin B, antifungal triazoles, echinocandins, and combination therapy measured by serial UFCT scans correlate with those measured by histopathological resolution of lesions, microbiological clearance of Aspergillus fumigatus, and resolution of galactomannan index (GMI). Following the development of this system, we then introduced a multidimensional volumetric imaging (MDVI) method for analysis of the response of the volume of pulmonary infiltrates over time to antifungal therapy in experimental invasive pulmonary aspergillosis. We developed a semiautomatic method to measure the volume of lung lesions, which was implemented as an extension of the MEDx visualization and analysis software using ultrafast computerized tomography (UFCT). Volumetric infiltrate measures correlate with UFCT readings, histopathological resolution of lesions, microbiological clearance of A. fumigatus, and GMI. Volumetric data by MDVI correlate with conventional CT pulmonary scores. These results correlate with validated biological endpoints: pulmonary infarct scores, lung weights, residual fungal burden, and GMI. Thus, MDVI correlates with key biological markers, improves the objectivity of radiological assessment of therapeutic response to antifungal therapy, and warrants evaluation for monitoring therapeutic response in immunocompromised patients with invasive aspergillosis.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
Infection Control and HICPAC Guidelines
David J. Weber, William A. Rutala, Amada Peppercorn, University of North Carolina at Chapel Hill, Chapel Hill, NC
Friday, January 18, 2008 11:10 – 11:35 AM
Invasive aspergillosis (IA) has been increasingly recognized as a cause of severe illness and mortality in immunocompromised patients, especially patients undergoing hematopoietic stem cell transplantation. Other at risk groups include patients with advanced HIV infection (especially those with CD4 counts of <50/mm3), solid organ transplant, or undergoing chemotherapy.
Most cases of healthcare-associated IA are sporadic. Vonberg and Gastmeier have reviewed more 50 outbreaks reported in the English literature (J Hosp Infect 2006;63:246-54). Patients (N=458) involved in these outbreaks had the following underlying diseases: hematologic malignancy 65%, solid organ transplant 10%, other immunocompromising condition 17%, or normal host 8%. The overall mortality was 55%. Sources of the outbreaks included construction or renovation work (probably) 23, construction or renovation work (possible) 3, air supply problem 9, other source 6, and unknown source 12. Most nosocomial cases are felt to be acquired via inhalation due to contaminated air as a result of construction or renovation. However, airborne spread from contaminated water was been suggested as another source of infection (Clin Infect Dis 2001;33:1546-8). Surgical site infections site infections are also well described (Clin Microbiol Infect 2006;12:1060-76).
Based on the patient populations involved in outbreaks and the source of the outbreaks, prevention of healthcare-associated IA has focused on providing a protected environment for the most highly immunocompromised patients and containing Aspergillus spores associated with renovation and construction activities. Key guidelines have been published by the Healthcare Infection Control Practices Advisory Committee (HICPAC) of the CDC, Canadian Centre for Infectious Disease Prevention and Control, IDSA, ASBMT and APIC, and include Guidelines for Environmental Infection Control in Health-Care Facilities (2003), Guidelines for Preventing Health-Care-Associated Pneumonia (2003), Construction-Related Nosocomial Infections in Patients in Health Care Facilities (2001), and Guidelines for Preventing Opportunistic Infections Among Hematopoietic Stem Cell Transplant Recipients (2000).
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
NOSOCOMIAL ASPERGILLOSIS: REVIEW AND BUILDING CONSTRUCTION CASES
Donna Haiduven, PhD
Friday, January 18, 2008, 11:35-12:00
Introduction/Background: Healthcare-associated infections with aspergillus pose a serious threat for those most severely immune suppressed patients. Outbreaks of nosocomial aspergillosis have occurred mainly among neutropenic patients. These have occurred in association with environmental disturbances: hospital construction; contaminated fire-proofing materials, or air filters in the hospital ventilation system; and via contaminated carpeting. It behooves those in the practice of patient care to prevent these situations before they occur, as opposed to dealing with them once they happen.
Prevention: A process for preventing healthcare-associated infections during construction and renovation has been developed. This process is known as an Infection Control Risk Assessment or ICRA. The ICRA begins with identification of the type of construction project activity, then proceeds with specific recommendations for each class of activity rated between A-D. Type A activities are those that are non-invasive. Type B Activities include small scale, short duration activities which create minimal dust. Type C Activities include work that generates a moderate to high level of dust or requires demolition or removal of any fixed building components or assemblies. Activities that fall into Category D Major demolition and construction projects, including but not limited to: activities requiring heavy demolition or removal of a complete cabling system or new construction. The second step in the process is to identify the patient risk group that will be affected. The third step is to match the patient risk group with the construction project type (A, B, C, D) to find the Class of Precautions (I – V) required to prevent acquisition of a healthcare-associated infection with an agent such as aspergillus.
Session Outline: In this session, a review of the literature in relation to healthcare-acquired aspergillosis cases will be reviewed. This will be followed by an overview of the ICRA concepts. The session will conclude with a case presentation for application the ICRA concepts.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
SICK BUILDING SYNDROME: IS MOULD THE CAUSE?
Abba I Terr, MD
Friday, January 18, 2008, 12:00 – 12:30 PM
Concerns about indoor moulds have been increasing and have given rise to much litigation. Controversy regarding the health effects of indoor airborne moulds in humans center around the mechanisms of 1) non-specific toxicity and 2) non-specific irritation.
Mould toxicity results from the action of mycotoxins. These have been extensively studied by toxicologists, largely for the purpose of drug development, resulting in important therapeutic agents including antibiotics, immunosuppressants, and cholesterol-lowering drugs.
Human mould toxicity is almost exclusively from accidental ingestion, such as mushroom poisoning and ergotism. The one rare exception is pulmonary mycotoxicosis, an occupational disease of farmers who sustain a toxic pneumonitis from inhaling enormous quantities of fungal spores. Such spore concentrations are highly unlikely within domestic and public buildings from mould incursion through excessive moisture or water intrusion.
Stachybotrys chartarum is not uncommonly encountered on moist building materials (e.g. wallboard), which can give rise to airborne spores. Nonspecific symptoms (fatigue, headache, etc.) without objective signs of disease are sometimes attributed to this "toxic mold". No human disease has yet been proven to be caused by toxicity, infection, or allergy to Stachbotrys.
Nonspecific irritation has been postulated from indoor fungal spores. The clinical manifestions are not defined, but nasal mucous membrane, ocular, and cutaneous hyperactivity can reasonably be assumed. Ironically, no such irritation is ascribed to outdoor air, even though spore concentrations are often higher than indoors.
Fungal "aeroirritation" is based on epidemiologic questionnaires without objective evidence of the suspected disease and/or identification of the suspected cause: i.e. airborne mould spores. The suspected agent(s) are mould-generated glucans, mVOCs, and/or mycotoxins.
There is no controversy about moulds causing human disease. Three mechanisms - infection, allergy, and toxicity - account for such illnesses, each specific for the organism. The controversial aspects of this debate are largely generated by factors other than those of scientific or clinical interest.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
ASTHMA PHENOTYPES
Sally E. Wenzel, M.D.
Saturday, January 19, 2008, 9:00 – 9:25 AM
The traditional definition of asthma is based on clinical symptoms in the presende of reversible airflow limitation. This definition is very broad and likely can encompass many entities. Therefore, it is not surprising that asthma is increasing recognized as a syndrome of syndromes, as opposed to a single disease. Over the years, numerous attempts at categorizing these phenotypes have been proposed, however, likely due to the overlap among the syndromes and the lack of specific biomarkers for each syndrome, the definition of asthma phenotypes continues to evolve. General categories for asthma phenotypes include those based on clinical characteristics, those based on types of inflammation identified and those based on triggers for the symptoms. These phenotypes are perhaps easier to define in patients with more symptomatic disease, such that much of the recent work identifying phenotypes has focused on patients with more severe disease. Whether severity of asthma also can be considered a phenotype of asthma is controversial, however, among moderate to severe asthmatics, the broadest general categorization is based on disease that is highly labile, with frequent exacerbations and a range of airflow limitation, as compared to a phenotype which may have fewer or even absent exacerbations, but continuous lower level symptoms and more severe and perhaps fixed airflow limitation. A further clinical distinction can be made on the basis of age at onset with early onset asthmatics more likely to represent a more homogeneous group with a strong allergic component, while later onset asthma can be extremely heterogeneous. Currently, 3 different inflammatory phenotypes have been defined, including eosinophilic, non-eosinophilic (neutrophilic) and paucigranulocytic. Of these, the eosinophilic phenotype is the most well defined and has been associated with a more symptomatic disease with frequent exacerbations, greater reversibility and airway hyperresponsiveness. Interestingly, there may be dfifferences between eosinophilic early onset asthma and eosinophilic late onset asthma, with early onset eosinophilic asthma associated with the highest risk for extremely severe asthma exacerbations. Non-eosinophilic/neutrophilic asthma has been associated with a lower FEV1, but less wheezing/chest tightness. Finally, the association of phenotypes with triggers, such as allergic, aspirin and hormonal also has been described. Allergic asthma is more likely to be seen in childhood onset asthma, while aspirin sensitive asthma is more commonly seen in later onset disease. However, allergic bronchopulmonary aspergillosis is more likely to be seen in association with late onset asthma. In our present understanding of this area, there remains considerable overlap among the groups, with an eosinophilic asthmatic, in the face of corticosteroid treatment, more likely to be severe and exacerbating and at least in adults, likely to have gotten the disease in adult hood. Further work is necessary to more specifically identify asthma phenotypes, likely incorporating definitions from each of these areas. This broader approach should allow better biomarker identification, more precise genetic evaluation and, eventually better and more effective treatment in a targeted population.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
DIFFERENCES IN THE IMMUNOBIOLOGY OF ASPERGILLUS COLONIZATION AND ABPA?
Jay K. Kolls J.L. Kreindler, MD2, A. MaGill, B.S.1, A. Ray, Ph.D.4, P.. Ray, Ph.D.4, M. Kurs-Laskey, Ph.D.4 and J.K. Kolls, M.D.1 1Children's Hospital of Pittsburgh, Pittsburgh, PA, United States; 2Children's Hospital of Pennsylvania, Philadelphia, PA, United States; 3University of Alabama, Birmingham, AL, United States and 4University of Pittsburgh, Pittsburgh, PA, United States.
Saturday, January 19, 2008, 9:25 – 9:50 AM
ABPA is characterized by wheezing, pulmonary infiltrates, bronchiectasis, and fibrosis that affects cystic fibrosis (CF) and asthma patients. The mechanisms of inflammation in response to Aspergillus fumigatus (Af) remain incompletely characterized but evidence supports a role for glucan receptors on macrophages and dendritic cells (DC) in recognition of Af. Therefore, we hypothesized that human DCs would respond more robustly to Af heat-killed swollen conidia (HKSC). We have examined the ability of CD14+ DCs (MDCs) which lack the TLSP receptor or CD11c+ DCs (TSLP DCs) to prime for Aspergillus specific T-cell responses in patients with ABPA and controls. MDCs or TSLP DCs showed a beta-glucan dependent increase in CD86 and HLADR. MDCs pulsed with HKSC elicited low levels of IL-4 (range 0-55 pg/ml) and IL-13 (0- 78pg/ml) in CD4+ T cells in patients with ABPA. In contrast TSLP DCs showed significantly greater increases in Af specific IL-4, IL-5, IL-13 and IL-17. These responses to HKSC were dependent on both TSLP and glucan signaling. These data show that TSLP DCs are more potent at detecting Af specific T-cell responses in the peripheral blood of ABPA patients compared to MDCs. TSLP DCs detected a significant Aspergillus specific Th17 response suggesting that ABPA may be both a Th2 and Th17 disease. These data also support TSLP and glucan signaling as novel targets for therapy in ABPA. Funded By: P50HL084932
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
REGULATION OF IMMUNITY IN BRONCHIECTASIS AND ABPA
Dr Rosemary Boyton Imperial College London
Saturday, January 19, 2008, 9:50 -10:15AM
Bronchiectasis is a chronic, progressive lung disease in which longstanding airway inflammation and recurrent bacterial infection (Haemophilus influenzae, Streptococcus pneumoniae and Pseudomonas) leads to irreversible bronchial dilatation, destruction and chronic sputum production. There are several known causes and associations including congenital conditions (primary cilary dyskinesia and cystic fibrosis), immunodeficiency (common variable immunodeficiency), sequelae to toxic inhalation or aspiration (foreign body), autoimmunity (rheumatoid arthritis and inflammatory bowel disease) and postinfectious. Chronic colonisation of the lung by pathogens is believed to cause chronic inflammation in the airways and secretion of oxidants and enzymes such as neutrophil elastase and myeloperoxidase.
There is mounting evidence that both innate and adaptive immunity are implicated in the pathogenesis of bronchiectasis. Bronchiectasis is a clinical feature of transporter associated with antigen processing (TAP) deficiency syndrome where mutations in TAP1 and/or TAP2 cause reduced cell surface expression HLA class I expression, and expansion of γδ and NK cells demonstrating that bronchiectasis can occur in the context of dysregulated NK cell function. In UK patients with idiopathic bronchiectasis HLA-Cw*03 alleles and, in particular, HLA-C group 1 homozygosity are associated with the presence of bronchiectasis. Analysis of the relationship between HLA-C and KIR genes suggests a shift to activatory NK cell function. A role for adaptive immunity is implicated by the presence of CD4 and CD8 T cells in diseased lung tissue and recent evidence of an HLA-class II disease association with HLA-DR1, DQ5. This may operate through influencing susceptibility to specific pathogens or self-reactivity.
ABPA is seen in approximately 10% of cases of bronchiectasis. It is important clinically to distinguish between sensitized individuals and those where the allergic response to Asp f is contributing to progressive lung damage. The challenge scientifically is to understand the underlying immune mechanisms that drive the chronic dysregulated inflammatory process allowing invasive disease.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
MECHANISMS BEHIND THE CYSTIC FIBROSIS - ABPA LINK
Dominik Hartl Cystic fibrosis Center, Children´s Hospital of the Ludwig-Maximilians-University of Munich, Germany
Saturday, January 19, 2008, 10:15 – 10:40 AM
Allergic bronchopulmonary aspergillosis (ABPA), a pulmonary hypersensitivity disease mediated by an allergic response to Aspergillus fumigatus (A. fumigatus), occurs preferentially in disease conditions with an impaired pulmonary immunity, especially in cystic fibrosis (CF) and allergic asthma (Stevens 2000). In CF patients ABPA is diagnosed in about 10-15% of patients and frequently leads to acute worsening of the respiratory status. The pathophysiological mechanisms underlying the link between CF and ABPA are poorly understood (Knutsen 2002, 2003). ABPA is characterized by a strong Th2 immune response. Increasing evidence suggests that in CF patients and CFTR-/- mice the immune response is shifted towards a Th2 phenotype (Moss 2000, Allard 2006, Knutsen 2003). In addition, chronic infection with the CF-prototypic pathogen Pseudomonas aeruginosa (P. aeruginosa) modulates the pulmonary immune response in CF patients towards Th2 immunity (Hartl 2006). Favored by this underlying immune deviation, A. fumigatus is believed to further drive the pulmonary and systemic immune response in CF patients towards an overhelming and robust Th2 phenotype, resulting in clinically active ABPA disease. Accordingly, the majority of T cell clones in ABPA patients are Th2 cells (Chauhan 1996; Knutsen 1994) and Th2 cytokines and chemokines are highly increased in the lung fluids and the circulation of ABPA patients compared to non-ABPA CF patients and healthy controls (Hartl 2006, Walker 1994). Besides T cells, B cells from ABPA patients are more sensitive towards IL-4 stimulation (Knutsen 2004). The manifestation of clinically active ABPA is further supposed to be influenced by the underlying genotype. Accordingly, six different HLA-DR alleles (Chauhan 1996, 1997) and polymorphisms in the IL-4α receptor chain (Knutsen 2006) and in the collagen region of surfactant protein A2 (Saxena 2003) have been associated with the occurrence of ABPA. Recently, increased serum levels of the Th2 chemokines thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) were found in two different ABPA CF patient cohorts (Hartl 2006; Latzin 2007). Serum levels of TARC were highly increased in ABPA patients compared to several CF and non-CF control groups and were useful both cross-sectionally and longitudinally to discriminate ABPA from A. fumigatus colonization and sensitization and to indicate ABPA exacerbations in the clinical course of CF. Based on these studies, TARC serum levels may be useful as future biomarkers for ABPA in CF patients. Despite these intriguing insights into the immunological links between ABPA and CF, more experimental and clinical studies are needed to understand the relevance of these mechanisms for monitoring and treating ABPA in CF patients.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
SUBTELOMERIC DIVERSITY AS A MAJOR FORCE IN EVOLUTION OF ASPERGILLUS SECONDARY METABOLISM AND VIRULENCE PATHWAYS
William C. Nierman and Natalie Fedorova J. Craig Venter Institute, Rockville, MD
Saturday, January 19, 2008, 11:10 – 11:35 AM
Aspergillus is an extremely diverse and widely distributed genus of filamentous ascomycete fungi found ubiquitously in soil. The vast majority of aspergilli are saprophytic microorganisms, although many species are able to infect wounded plants and animals. Recent high-throughput genomic and post-genomic studies has created an exceptional opportunity to shed light onto genetic and evolutionary mechanisms responsible for their competitiveness in heterogeneous environments including their impressive mycotoxin repertoire and virulence attributes.
In filamentous fungi, genes responsible for secondary metabolite (SM) biosynthesis, export, and transcriptional regulation are often found in co-regulated clusters. To facilitate the analysis of SM pathways in sequenced fungal genomes, we have developed SMURF, a software tool that predicts SM clusters using high-throughput analysis data. The analysis has demonstrated that, among filamentous fungi, aspergilli have the largest (30-50 per species) mycotoxin repertoire, characterized by striking variability at both species and strain levels. This suggests that these species are in a state of a perpetual “chemical arms race” with other soil inhabitants and may thus represent an untapped source of novel pharmaceuticals.
The analysis also revealed that most SM clusters are located in subtelomeric regions, which are often associated with frequent genome rearrangements and deletions. Not surprisingly, evolutionary history of clusters involves a rapid succession of lineage-specific expansions and losses, which combined with the limited number of SM protein families, sometimes results in apparent similarities in different species. A more detailed comparison has showed that most pathways are novel and unique to each organism. The creation of new pathways appears to involve de-novo assembly, segmental duplication and accelerated differentiation. Clusters appear to go through long evolutionary intervals of relative stability, interrupted by short intervals of rapid change, when new products may emerge.
Subtelomeric diversity also appears to be one of the major forces in the evolution of pathogenicity in the genus Aspergillus. Genome-wide gene expression profiles of A. fumigatus germlings during initiation of murine infection revealed the highly coordinated program that controls metabolic and physiological adaptation to the mammalian niche. Around 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoire is induced in vivo relative to laboratory culture. Notably many up-expressed genes are physically clustered including four unknown SM clusters as well as loci directing pseurotin, gliotoxin and siderophore biosyntheses. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies against fungal infection.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
ASPERGILLUS METABOLITES
Robert A. Cramer Jr., Ph.D. Montana State University
Saturday, January 19, 2008, 11:35 AM – 12:00 PM
Like most filamentous fungi, Aspergillus fumigatus produces a wide-array of primary and secondary metabolites that may be secreted into the host environment during opportunistic fungal infections. Thus, these metabolites have the potential to affect host immune responses and alter the outcome of the fungal-host interaction. Research on metabolites produced by A. fumigatus has largely focused on the role of gliotoxin, a potent immunosuppressant secondary metabolite that has been detected in vivo in invasive pulmonary aspergillosis (IPA) patients. Several recent studies have begun to directly elucidate the role of gliotoxin in IPA, utilizing specific gene-replacement mutants in the gliotoxin biosynthesis pathway and experimental murine models of IPA.
Results from these studies suggest that gliotoxin is not required for IPA in murine models utilizing cyclophosphamide for immunosuppression, but does play a substantial role when hydrocortisone is utilized as the immunosuppressive agent. Thus, the condition of the patient’s immune system seems to be the primary determining factor for whether gliotoxin plays a critical role in IPA. Yet, an understanding of the effects of gliotoxin on the host immune response is still not precisely known.
In addition to secondary metabolites, byproducts of primary metabolism can also be secreted into the host milieu and potentially affect host defense responses. We have utilized 1H-NMR metabolite profiling of broncheoalveolar lavage fluid to identify byproducts of fungal primary metabolism in vivo in a murine model of IPA. Identification of fungal-specific metabolites gives clues to the important biochemical pathways utilized by the fungus during invasive growth in a mammalian host.
In this presentation, we will discuss the potential role of gliotoxin in invasive pulmonary aspergillosis, and present preliminary data on the potential role of specific aspects of primary metabolism in allowing A. fumigatus to survive in immunocompromised mammalian lungs.
3 RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
COMPUTATIONAL PREDICTION OF ESSENTIAL GENES IN ASPERGILLUS SPP.
Scott Baler Jette Thykaer1, Mikael R. Andersen1 and Scott E. Baker2 1 BioCentrum, Technical University of Denmark, Lyngby, Denmark 2 Fungal Biotechnology Team, Pacific Northwest National Laboratory, Richland, WA
Saturday, January 19, 2008, 12:00 – 12:25 PM
Computational metabolic flux modeling has been a great aid for both understanding and manipulating microbial metabolism. The ability to model metabolism and predict carbon flow through metabolic pathways gives researchers the ability to predict how genetic modifications, such as gene deletions, may affect the production of useful metabolites such as organic acids. The continuously expanding catalog of genomic sequence databases for individual microbes further enhances the utility of metabolic flux models. In addition to identifying potential metabolic pathways for genetic manipulation in support of production of useful metabolites, the predictive power of metabolic flux modeling can be used to identify essential biochemical pathways. A metabolic flux model for Aspergillus niger, an economically important biotechnology fungus known for protein and organic acid production was previously developed. The A. niger metabolic flux model is comprised of 1190 biochemically unique reactions that are associated with 871 open reading frames. Through a systematic in silico deletion of single metabolic reactions using this model, several essential metabolic pathways were identified for A. niger. A total of 145 genes were identified in silico as being associated with these essential biochemical pathways during growth on a minimal glucose medium. The majority of the genes involved in essential biochemical pathways grouped into cell wall biosynthesis, amino acid biosynthesis, energy metabolism and purine and pyrimidine metabolism. Based on bioinformatic analysis of genomic sequence we subsequently identified orthologous candidate essential genes and pathways in Aspergillus fumigatus. Our predictions are validated in part by the modes of action that have been identified for current antifungal drugs and by molecular genetic studies of essential genes in A. fumigatus and other fungi. The use of metabolic models to predict essential genes and pathways in Aspergillus spp. has promise to inform reverse genetic studies of gene essentiality and identify potential targets for antifungal development.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
METABOLOMICS OF ASPERGILLUS FUMIGATUS
*Jens C. Frisvad, Thomas O. Larsen and Kristian F. Nielsen Centre for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark,
Saturday, January 19, 2008, 12:25 – 12:50 PM
Aspergillus fumigatus is an impressive producer of a wide array of secondary metabolite classes1. Most of these metabolites are produced consistently by all isolates of the species. Such families of secondary metabolites include:
1.Metabolite families consistently produced by A. fumigatus: Helvolic acids, fumagillins, fumiquinazolins, trypacidins, gliotoxins, fumagaclavins, fumitremorgins, and pseurotins
2.Less cnsistently produced metabolite families: fumigatins, chloroanthraquinones, and pyripyropens being produced by 50-80% of the isolates in A. fumigatus.
3. Metabolites not being confirmed as A. fumigatus sensu stricto: asperfumin, asperfumoid and sphingofungins
4. Metabolites produced by A. fumigatus, frequency unknown: mitogillin, restrictocin, FK-463, epoxysuccinic acid, ergosterolperoxide, phthioic acid, β-transbergamoten, hexahydroxypolyprenols, ferrichrome C, and ferricrocin.
5. Metabolites not produced by A. fumigatus: antafumicins, cytochalasin E, expansolide, fumigatonin, ruakuric acid, fumifungin, cyclopiazonic acid or tryptoquivalins.
The genetics of regulation of the profile of all these secondary metabolites is complex and depends heavily on the growth medium and environmental factors. Gliotoxin, for example, is only produced on media with low or no carbohydrate source, such as yeast extract agar, and Pig-Lung agar whereas the chloroanthraquinones are produced most effectively on malt extract based media. Other secondary metabolites, such as the fumiquinazolins, are produced on most of the media tested. More secondary metabolites are produced at 25°C than at 37°C. In most strains of Aspergillus fumigatus, each biosynthetic family of secondary metabolites is represented of one or two main metabolites, often followed by a series of minor metabolites. Precursor metabolites are rarely seen after one week of growth and differentiation, except in hyperproducers, but can be detected in young cultures. Closely related species of A. fumigatus produce quite different profiles of secondary metabolites, including the species A. lentulus, A. fumigatiaffinis, A. novofumigatus and A. turcosus. However, many metabolites are common between the species in Neosartorya and Aspergillus section Fumigati , making these fungi a polythetic class2-4.
1 Larsen, T.O., Smedsgaard, J., Nielsen, K.F., Hansen, M.E., Samson, R.A. and Frisvad, J.C. 2007. Production of mycotoxins by Aspergillus lentulus and other medically important and closely related species in section Fumigati. Medical Mycology 45: 225-232. 2 Hong, S.B., Go, S.J., Shin, H.D., Frisvad, J.C. and Samson, R.A. 2005. Polyphasic taxonomy of Aspergillus fumigatus and related species. Mycologia 97: 1316-1329. 3 Hong, S.B., Cho, H.S., Shin, H.D., Frisvad, J.C. and Samson, R.A. 2006. New Neosartorya species isolated from soil in Korea. Int. J. Syst. Evol. Microbiol. 56: 439-442. 4 Hong, S.B., Shin, H.D., Hong, J., Frisvad, J.C., Nielsen, P.V., Varga, J. and Samson, R.A. 2007. New taxa of Neosartorya and Aspergillus in Aspergillus section Fumigati. Antonie van Leeuwenhoek 92, in press.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
INTERACTION OF ASPERGILLUS WITH HUMAN RESPIRATORY MUCOSA
Ryoichi Amitani, M.D. Department of Respiratory Medicine, Osaka Red Cross Hospital, Osaka, Japan
Saturday, January 19, 2008, 2:20 – 2:45 PM
Aspergillus species, most commonly A.fumigatus, are exogenous fungi and can colonize airway mucosa, particularly in patients with localized underlying bronchopulmonary disorders such as healed tuberculous cavities, cystic fibrosis and bronchiectasis, and occasionally invade the airway mucosa regardless of systemic immunocompromised conditions. However, the mechanisms of colonization and invasion of Aspergillus in the respiratory mucosa, especially the initial stage of interaction of Aspergillus with respiratory mucosa after inhalation of conidia through airways are still poorly understood.
Interaction of a clinical isolate of A.fumigatus with human bronchial mucosa in an organ culture model with an air-mucosal interface was studied. A.fumigatus conidia were inoculated onto the organ culture tissues, and incubated for up to 24h in a humidified atmosphere containing 5% CO2 at 37Ԩ. At each timepoint, after measuring the ciliary beat frequency (CBF) of bronchial mucosal epithelium by a photometric technique, adherence and invasion of the bronchial epithelium by A.fumigatus conidia (and hyphae) as well as structural changes of the epithelium were investigated by scanning and transmission electron microscopy.
These studies demonstrated that A.fumigatus caused damage to bronchial epithelium including separation of intercellular junction, extrusion and detachment of ciliated cells from neighboring cells associated with CBF slowing, and also demonstrated that part of A.fumigatus conidia were internalized within ciliated and non-ciliated epithelial cells, and hyphae penetrated through both intercellular and intracellular spaces of the epithelium. These findings suggest that there might be at least three different pathways by which Aspergillus invades the bronchial mucosa： internalization of conidia within epithelial cells (although the fate of the internalized conidia is still unclear), penetration of germinated conidia (or hyphae) through the intercellular space of ciliated epithelium, and direct penetration of hyphae through the intracellular space of the epithelial cells in association with destruction of the cytoplasmic membrane of the epithelial cells.
Although we demonstrated serial morphological changes of both human bronchial epithelium and A.fumigatus conidia (and hyphae) using an organ culture model of human bronchial tissue with an air-mucosal interface, the precise mechanisms of colonization and invasion of A.fumigatus in human respiratory tracts still remain to be elucidated and further studies will be required.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
INVASION OF THE LUNG EPITHELIUM
William W Hope The University of Manchester
Saturday, January 19, 2008, 2:45 - 3:10 PM
The lung can be directly implicated in approximately 90% of cases of invasive pulmonary aspergillosis. The alveolar-capillary barrier separates the airspace (alveolus) from the pulmonary capillary, and consists of the alveolar epithelial cells (type I and II), the basement membrane and the pulmonary capillary endothelium. Resident pulmonary alveolar macrophages are a critical innate immunological defense mechanism. The initiation of invasive disease requires the evasion of innate host defenses and transgression of the alveolar-capillary barrier. Cell culture models demonstrate that alveolar epithelial cells freely endocytose conidia, where the majority are killed in lysosomes. A fraction may escape and undergo intracellular germination to form hyphae; thus, invasion of the alveolar capillary barrier may be initiated from an intracellular location. A variety of imaging techniques demonstrate that invading hyphae pursue a direct intracellular route. An in vitro model of the alveolar-capillary barrier and a well validated neutropenic rabbit model of invasive pulmonary aspergillosis demonstrate that hyphae penetrate into the endothelial compartment (the pulmonary capillary) approximately 14-16 hours post inoculation. These models demonstrate the intimate relationship between invasion and the kinetics of the clinically relevant biomarker, galactomannan. Models of early invasive pulmonary aspergillosis suggest: (1) many of the important pathological events occur within the initial 24 hours, well before any clinical manifestations of disease are likely to be apparent; (2) small delays in the administration of appropriate antifungal therapy may have profound effects on the antifungal effect; (3) pulmonary alveolar macrophages exhibit saturable killing; (4) both innate immunological effectors and antifungal agents are required for maximal antifungal effect; and (5) the intracellular location of hyphae may be important for immunological and pharmacological evasion. An improved understanding of the pathogenesis of early fungal invasion will lead to a range of diagnostic and therapeutic strategies which will serve as an important means of optimizing the outcome of immunocompromised patients with invasive pulmonary aspergillosis. References: 1. Hope WW, Kruhlak MJ, Lyman CA, Petraitiene R, Petraitis V, Francesconi A, Kasai M, Mickiene D, Sein T, Peter J, Kelaher AM, Hughes JE, Cotton MP, Cotten CJ, Bacher J, Tripathi S, Bermudez L, Maugel TK, Zerfas PM, Wingard JR, Drusano GL and Walsh TJ. Pathogenesis of Aspergillus fumigatus and the kinetics of galactomannan in an in vitro model of early invasive pulmonary aspergillosis: implications for antifungal therapy. J Infect Dis 2007;195:455-466 2. Filler SG, Sheppard DC. Fungal invasion of normally non-phagocytic host cells. PLoS Pathog 2006;2:e129 3. Wasylnka JA, Moore MM. Uptake of Aspergillus fumigatus Conidia by phagocytic and nonphagocytic cells in vitro: quantitation using strains expressing green fluorescent protein. Infect Immun 2002;70:3156- 3163 4. Wasylnka JA, Moore MM. Aspergillus fumigatus conidia survive and germinate in acidic organelles of A549 epithelial cells. J Cell Sci 2003;116:1579-1587
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
BIOFILM FORMATION BY ASPERGILLUS FUMIGATES
Gordon Ramage University of Glasgow. Glasgow, UK
Saturday, January 19, 2008, 3:10 – 3: 35 PM
Aspergillus fumigatus is a filamentous mold that exhibits growth and development characteristics as an intertwined hyphal structure. Biofilms are defined as ‘matrix enclosed microbial populations, adherent to each other and/or one another’. This mode of growth exhibited by A. fumigatus is consistent with a biofilm description, which is a lifestyle characterised by altered phenotypic and genotypic factors. It was the aim of this study to examine A. fumigatus development into these poly-cellular structures (biofilms) using in vitro models. Conidial density was first examined to determine the optimal density to produce confluent filamentous structures on microtitre plates. It was shown that 105 condia/ml established the most coherent and stable biofilm structure. Confocal laser scanning microscopy was then used to assess to development over 24 h. We demonstrated that following germination and filamentation, biofilm architecture exponentially increased, evolving from a scant monolayer of filamenting cells to a highly differentiated structure of 150 µm (12 h), which increased to 250 µm after 24 h. Transcriptional analysis of AF293 biofilm development was then performed using a microarray approach. Total RNA was harvested during three stages of biofilm development (8, 12 and 24 h), which represented the three key phases of biofilm development. The greatest level of up-regulated gene expression was between 8 -12 h (85% ≥ 2 fold), followed by 8-24 h (55% ≥ 2 fold) and 12-24 h (6% ≥ 2 fold). SirT, which has a role in the ageing process, was identified as being maximally upregulated at 8 (×5.1),. Within the greatest phase of growth and differentiation (8 h), genes involved in cell growth (GpaA), immune resistance (RodA) and pathogenesis (Fos- 1) were all shown to be upregulated. These preliminary studies collectively illustrate that multi- cellular biofilm growth is a complex synchronised process involving key pathogenic determinants.
3RD ADVANCES AGAINST ASPERGILLOSIS January 16-19, 2008 Miami Beach, Florida
DIFFERENCES IN PATHOGENICITY AND CLINICAL SYNDROMES DUE TO A. FUMIGATUS AND A. FLAVUS
Alessandro C. Pasqualotto Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil
Saturday, January 19, 2008, 3: 35 – 4:00 PM
Aspergillus flavus commonly causes diseases to humans. It is the second leading cause of both invasive and non-invasive aspergillosis. Despite the growing importance of aspergillosis in clinical practice, most of the information available about Aspergillus infections has been originated from the study of A. fumigatus, the most frequent species in the genus. A. flavus is however particularly prevalent in regions of the world with dry and hot climate such as the Middle East and Sudan. Interestingly, A. flavus seems more virulent and more resistant to antifungal drugs than most of the other Aspergillus species, which has been demonstrated both in vitro and in animal models. Aflatoxin does not seem to be a major factor involved in the pathogenesis of A. flavus infections (A. flavus isolates produce aflatoxin B1, the most toxic and potent hepatocarcinogenic natural compound ever characterized). Some differences in the clinical syndromes associated with A. flavus and A. fumigatus are also observed. For instance, A. flavus is a common etiology of fungal sinusitis and cutaneous infections, but not fungal pneumonia. Chronic cavitary pulmonary aspergillosis has rarely been associated with A. flavus. Although A. fumigatus is responsible for the vast majority of cases of allergic bronchopulmonary aspergillosis (ABPA), A. flavus has also been implicated in some series, mostly in India. The bigger size of A. flavus spores, in comparison to A. fumigatus spores, may favor their deposit in the upper respiratory tract. A. flavus also accounts for circa 80% of Aspergillus keratitis cases. Other clinical syndromes often linked with A. flavus include post-operative wound infections, Aspergillus osteomyelitis following trauma or inoculation, and chronic granulomatous sinusitis. Outbreaks of aspergillosis involving the skin, oral mucosa, or subcutaneous tissues are usually associated with A. flavus. Most of these outbreaks have been associated with a single or a few different strains, which contrasts with what has been documented for infections caused by A. fumigatus. In summary, differences between these species justify the need for a better understanding of A. flavus infections.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
ORAL ABSTRACT PRESENTATIONS
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
THE ROLE OF SIALIC ACIDS ON THE SURFACE OF ASPERGILLUS FUMIGATUS AND THE CHARACTERIZATION OF AN A. FUMIGATUS SIALIDASE
Warwas M*, Watson J, Bennet A, Moore M
Thursday, January 17, 2008 1:55 – 2:05 PM
Purpose: Aspergillus fumigatus is an opportunistic fungal pathogen that causes life-threatening invasive aspergillosis in immunocompromised individuals. Previous research in our laboratory identified sialic acids on A. fumigatus. Sialic acids are charged monosaccharides known to play important roles in microbial pathogenesis. Although sialic acids are present on the fungal surface, the mechanism of their acquisition is unknown. Recently, we have identified a gene in A. fumigatus encoding a sialidase, an enzyme that cleaves terminal sialic acids from glycoconjugates. In bacteria and viruses, sialidase plays a role in the acquisition of sialic acids for nutrition and in pathogenesis. The purpose of this study was: 1) to determine whether removal of sialic acids from conidia affected conidial binding to fibronectin and phagocytosis by cultured murine macrophages (J774) or type 2 pneumocytes (A549), and 2) to clone and characterize a putative A. fumigatus sialidase. Methods: Surface sialic acids were enzymatically removed from conidia and the extent of binding or phagocytosis of sialidase-treated or untreated conidia was assessed using light and fluorescence microscopy, respectively. The sialidase gene was amplified from A. fumigatus genomic DNA by PCR, ligated into the pET28A+ protein expression vector in frame with a polyhistidine tag, expressed in an E. coli expression host and purified by nickel affinity chromatography. Sialidase activity of the purified enzyme was determined by measuring the release of the fluorescent product, 4-methylumbelliferone (4-MU), from a 4-MU-sialic acid substrate. Results: Enzymatic sialic acid removal significantly decreased the binding of conidia to fibronectin by 65% over controls. Removal of sialic acids also significantly decreased conidial uptake by J774 and A549 cells by 33% and 53%, respectively. Interestingly, conidial binding did not correlate with conidial uptake. The cloned A. fumigatus sialidase showed activity toward 4-MU-sialic acid. Conclusions: Sialylated molecules on A. fumigatus conidia are important in binding fibronectin and in uptake by phagocytes in vitro. We have successfully cloned and characterized an A. fumigatus sialidase, which represents the first characterization of an enzyme involved in sialic acid metabolism in fungi. It may represent a suitable target for deletion leading to the creation of a sialic acid-deficient mutant strain of A. fumigatus and allowing us assess the importance of sialic acids on A. fumigatus virulence in vivo.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
HUMAN PLATELETS INFLUENCE CELL WALL ASSEMBLY IN ASPERGILLUS FUMIGATUS
Perkhofer S.1*, Schrettl M.2, Haas H.2, Dierich M.P.1, Lass-Flörl C.1 1Department of Hygiene, Microbiology and Social Medicine, Medical University Innsbruck, Austria 2Department of Molecular Biology, Medical University Innsbruck, Austria
Thursday, January 17, 2:15 – 2:25 PM
Purpose: Aspergillus fumigatus is the most prominent causative agent of invasive aspergillosis. The ability of Aspergillus spp. to undergo morphologic changes is an important virulence factor as the onset of infection is associated with the appearance of hyphae. The fungal cell wall is essential for growth and morphogenesis, and its assembly is closely coordinated with exogenous signals. Recently, we found that human platelets exert antifungal effects as they are able to damage hyphae and significantly decrease hyphal germination and elongation (P<0.05) of Aspergillus spp. Therefore, we performed northern analysis to assess the influence of platelets on fungal ß- (1-3)- glucan synthases (fks1p) and chitinsynthases (ChsB, ChsD, ChsE) gene expressions associated to cell wall assembly. Methods: 1x105cfu/ml conidial suspension of Aspergillus fumigatus and 1x108/ml human platelets were coincubated for 16h. Untreated fungi served as controls. RNA isolation was followed by use of TRI® (Sigma-Aldrich) reagent. For northern analysis, 10µg of total RNA was electrophoresed on 1.2% (wt/vol) agarose–2.2 M formaldehyde gels and blotted onto hybond N membranes (Amersham). The hybridization probes used in this study were generated by PCR using oligonucleotides 5’-CAA CTG TCT GTG ACT CCG and 5’-TGA CGG TGG TAG TAA GCC for chsB, 5’-TTA CTA CCT CCA AGC GGG and 5’-TAC ACG CTG CTG CTT CTC for chsD, 5’-GAG TTT CTT GGT CTG GCG and 5’-CGG AGT TGG TAT CTG TGC for chsE, 5’- GGA AAG CAC GGA AAG CAG and 5’-AAA CAC ACC AGG AGC CAG for fks p which are essential for fungal growth and 5'-ATATGTTCCTCGTGCCGTTC and 5'- CCTTACCACGGAAAATGGCA for ß-tubulin encoding tubA. Results: Our findings exerted that platelets influence gene expression of A. fumigatus as the chsB, chs D, chsE and fks1p genes were up-regulated, whereas the housekeeping gene tub was not affected. Conclusions: In summary, our findings exert that platelets influence genes of A. fumigatus which are involved in key aspects of cell wall assembly. The decreased germination and hyphal growth rates due to platelet treatment suggest that significant changes in the cell wall have occurred. Taken together, our findings suggest that platelets offer antifungal properties against Aspergillus spp.
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
POSTER ABSTRACTS
3RD ADVANCES AGAINST ASPERGILLOSIS
January 16-19, 2008 Miami Beach, Florida
THE TIMING OF MYELOID CELL DEPLETION RELATIVE TO INFECTION DETERMINES THE OUTCOME OF INVASIVE ASPERGILLOSIS
*Monica M. Mircescu, Eric G. Pamer, Tobias M. Hohl Infectious Diseases Service, Memorial Sloan-Kettering Cancer Center, and Immunology Program Sloan-Kettering Institute, New York, NY
Thursday, January 17, 2008 2:05 – 2:15 PM
Purpose: Invasive aspergillosis (IA) represents an increasing problem in immunocompromised patients and is associated with mortality that exceeds 50% in high-risk groups. Since airborne Aspergillus spores (conidia) are ubiquitous in the environment, the temporal relationship between conidial inhalation, myeloid cell depletion, and the development of invasive disease remains unclear, but highly relevant from an infection control standpoint. We examined whether lethal invasive disease could develop in mice infected with Aspergillus fumigatus conidia either prior to or following myeloid cell depletion to test the hypothesis that de novo conidial inhalation is required for the development of invasive disease in the immunosuppressed state. Methods: We depleted specific myeloid subsets, either predominately neutrophils or alveolar macrophages, in C57BL6 mice through parenteral administration of the anti-Gr-1 (Ly6C/G) antibody (RB6-8C5) or intratracheal delivery of dicloromethylene disphosphonate-encapsulated liposomes (CL2-MDP). Mice were infected by intratracheal inoculation with a suspension of A. fumigatus conidia. Results: Depletion of Gr-1-expressing cells prior to infection decreased survival compared to depletion of the same subset at one day post infection. Administration of the Gr-1 depleting antibody within the first 3 hours post infection, but not later time points, rendered mice susceptible to invasive fungal disease. Contrary to our expectations, liposome-mediated depletion of alveolar macrophages prior to infection had no effect on mortality. Experiments that deplete both cell subsets are ongoing. Conclusions: These studies suggest that recruitment of Gr-1-expressing cells to murine airways plays an essential role early in anti-Aspergillus defense, i.e. within the first ~3 hours post infection. The association of invasive disease with acute infection in the setting of existing or incipient myeloid cell depletion suggests that, in clinical settings, rigorous control of ongoing conidial exposure may benefit high-risk hosts. In the experimental system examined, alveolar macrophages do not appear essential for host defense, a feature that may reflect redundancy at the cellular level.
3rd Advances Against Aspergillosis January 16-19, 2008 Miami Beach, Florida, USA
AUTHOR INDEX Poster Number Page Poster Number Page A Desai, Rishi 25 117 Ajayi, Anthony 30 122 deShazo, Richard D. Jan. 17, 50 Al-Bader, Nadia 53 145 11:10-11:35 am Albarrag, Ahmed 78 170 Deshpande, Sunita 6 98 Amarsaikhan, Nansalmaa 4 96 E Amitani, Ryoichi Jan. 19, 81 Ejzykowicz, Daniele 28 120 2:20-2:45 pm Etienne, Kizee 70 162 Anaissie, Elias J. Jan. 18, 63-66 F 9:00-9:25 am Fedorova, Natalie D. Jan. 17, 55 B 3:20-3:45 pm Baddley, John 52 144 Fischer, Reinhard 89 181 Baker, Scott E. Jan. 19, 79 Fothergill, Annette 39 131 12:00-12:45 pm Fraczek, Marlin 42 134 Balajee, Arunmohzhi Jan. 17, 56 Frisvad, Jens Jan. 19, 80 3:45-4:10 pm 12:25-12:50 pm Barton, Richard 13 105 G Ben-Ami, Ronen 54 146 Garbino, Jorge 35 127 Bennett, Joan W. Jan. 16, Geiser, David M. Jan. 17, 45 9:50-10:30 am 8:00-8:45 am Bennett, John E. Jan. 17, 33 Germaud, Patrick 27 119 8:00-8:45 am Gravelat, Fabrice 82 174 Boukraa, Laïd 10 102 H Bowyer, Paul 44 136 Haiduven, Donna Jan. 18, 71 Boyton, Rosemary Jan. 19, 75 11:35-12:00 pm 9:50-10:15 am Hamilos, Georgios 81 173 Brock, Matthias 77 169 Harrison, Elizabeth 59 151 Burritt, James 79 171 Hartigan, Adam 76 168 C Hartl, Dominik Jan. 19, 76 Campbell, Bruce 24 116 10:15-10:40 am Casquero, José 61, 62 153, 154 Hoffman, Jill 23 115 Chakrabarti, Arunaloke Jan. 17, 49 Hohl, Tobias Jan. 16, 44 10:45-11:10 am 5:20-5:45 pm Chaudhary, Neelkamal 50 142 Holland, Steven M. Jan. 16, 42 Chiller, Tom 87 179 4:30-4:55 pm Clemons, Karl V. Jan. 16, 37 Hope, William W. Jan. 19, 82 12:15-12:40 pm 2:45-3:10 pm Coenye, Tom 12 104 Horiuchi, Hiroyuki Jan. 17, 47 Conte Jr., J.E. 91 183 9:25-9:50 am Cramer Jr., Robert A. Jan. 19, 78 Horn, David 80 172 11:35-12:00 pm, Horner, Elliott 83 175 88 180 Howard, Susan 29 121 Cuenca, Adriana 20 112 Husain, Shahid Jan. 16, 39 Curfs-Breuker, Ilse 37 129 2:45-3:10 pm D Hurst, Steven 75 167 Day, Michael J. Jan. 17, 51 11:35-12:05 pm de Valk, Hanneke 73, 74 165, 166 3rd Advances Against Aspergillosis January 16-19, 2008 Miami Beach, Florida, USA
I P Ibrahim-Granet, Oumaima 84 176 Parmar, Arvindkumar 65 157 Ito, James I. Jan. 17, 59-60 Pasqualotto, Alessandro C. Jan. 19, 84 5:30-5:55 pm 3:35-4:00 pm K Perkhofer, Susanne Jan. 17, 89 Kano, Rui 71 163 2:15-2:25 pm Karki, Kedar B. 93, 94 185, 186 60 152 Kirkpatrick, William 64 156 Perlin, David S. Jan. 16, 34 Klaassen, Corne Jan. 17, 54 11:00-11:25 am 2:55-3:20 pm Pinel, Claudine 34 126 Klingspor, Lena Jan. 16, 36 R 11:50-12:15 pm Ramage, Gordon Jan. 19, 83 Kniemeyer, Olaf Jan. 16, 35 3:10-3:35 pm 11:25-11:50 am Ramaprakash, Hemanth 72 164 Kolls, Jay K. Jan. 19, 74 Read, Nick Jan. 18, 68 9:25-9:50 am 9:50-10:15 am Kontoyiannis, Dimitrios Jan. 16, 41 Ribaud, Patricia 46 138 3:35-4:00 pm, Rivera, Amariliz 86 178 45 137 Romani, Luigina Jan. 18, 62 L 8:00-8:45 am Lagrou, Katrien 33 125 85 177 Laroza, Maricel 31 123 Rudenko, Michael 1 93 Latge, Jean-Paul Jan. 17, 46 S 9:00-9:25 am Sadasiva Reddy, Chintala 18, 19 110, 111 Lehrnbecher, Thomas Jan. 17, 57 Samson, Robert A. Jan. 17, 53 4:40-5:05 pm 2:30-2:55 pm Lengerova, Martina 63 155 Sarosi, George A. Jan. 17, Leth Mortensen, Klaus 43 135 8:00-8:45 am M Savio, Jayanthi 14 106 Maertens, Johan Jan. 16, 40 Schubert, Mark S. Jan. 17, 52 3:10-3:35 pm 12:05-12:30 pm Mabey Gilsenan, Jane E. 92 184 Segal, Brahm H. Jan. 16, 38 Mahi, Lamine 55 147 2:20-2:45 pm Mallatova, Nada 36 128 Sepkowitz, Kent Jan. 18, 61 Martins, Cleide V. 47, 48, 49 139,140, 8:00-8:45 am Buzanello 141 Sherwal, Banke Lal 38 130 Marty, Francisco M. Jan. 17, 48 Shevchenko, Marina 8 100 9:50-10:15 am Shivaprakash, M.R. 58 150 Mircescu, Monica Jan. 17, 88 Shrestha, Gita 15 107 2:05-2:25pm Shrief, Raghdaa 69 161 51 143 Slater, Joanne 68 160 Mowat, Eilidh 7 99 Stevens, David A. Jan. 16, 31-32 N 9:10-9:50 am Nierman, William C. Jan. 19, 77 Sugui, Janyce 66 158 11:10-11:35 am Symoens, Françoise 56 148 Noonim, Paramee 11 103 T O Terr, Abba I. Jan. 18, 72 Okungbowa, Francisca 2 94 12:00-12:30 pm Olson, Jon A. 95 187 Thornton, Christopher 9 101 3rd Advances Against Aspergillosis January 16-19, 2008 Miami Beach, Florida, USA
Tong, Ann-Jay 90 182 Toyotome, Takahito 22 114 Tramsen, Lars 41 133 U Upadhyay, Santosh 5 97 Urb, Mirjam 57 149 V Vanhee, Lies 21 113 Varga, Janos 32 124 Vyas, Suresh P. Jan. 18, 67 9:25-9:50 am W Walsh, Thomas J. Jan. 18, 69 10:15-10:40 am Warwas, Mark Jan. 17, 87 1:55-2:05 pm Weber, David J. 3 70, 95 Jan. 18, Wenzel, Sally E. 11:10-11:35 am 73 Jan. 19, 9:00-9:25 am
Wheat, Lawrence 67 159 Wiederhold, Nathan 26 118
X
Xavier, Melissa 40 132 Y Yang, De Jan. 16, 43 4:55-5:20 pm Z Zelante, Teresa Jan. 17, 58 5:05-5:30 pm Zhu, Li-Ping 16, 17 108, 109
3rd Advances Against Aspergillosis January 16-19, 2008 Miami Beach, Florida, USA