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Identification of Culture-Negative Fungi in Blood and Respiratory Samples

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Identification of Culture-Negative Fungi in Blood and Respiratory Samples IDENTIFICATION OF CULTURE-NEGATIVE FUNGI IN BLOOD AND RESPIRATORY SAMPLES Farida P. Sidiq A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2014 Committee: Scott O. Rogers, Advisor W. Robert Midden Graduate Faculty Representative George Bullerjahn Raymond Larsen Vipaporn Phuntumart © 2014 Farida P. Sidiq All Rights Reserved iii ABSTRACT Scott O. Rogers, Advisor Fungi were identified as early as the 1800’s as potential human pathogens, and have since been shown as being capable of causing disease in both immunocompetent and immunocompromised people. Clinical diagnosis of fungal infections has largely relied upon traditional microbiological culture techniques and examination of positive cultures and histopathological specimens utilizing microscopy. The first has been shown to be highly insensitive and prone to result in frequent false negatives. This is complicated by atypical phenotypes and organisms that are morphologically indistinguishable in tissues. Delays in diagnosis of fungal infections and inaccurate identification of infectious organisms contribute to increased morbidity and mortality in immunocompromised patients who exhibit increased vulnerability to opportunistic infection by normally nonpathogenic fungi. In this study we have retrospectively examined one-hundred culture negative whole blood samples and one-hundred culture negative respiratory samples obtained from the clinical microbiology lab at the University of Michigan Hospital in Ann Arbor, MI. Samples were obtained from randomized, heterogeneous patient populations collected between 2005 and 2006. Specimens were tested utilizing cetyltrimethylammonium bromide (CTAB) DNA extraction and polymerase chain reaction amplification of internal transcribed spacer (ITS) regions of ribosomal DNA utilizing panfungal ITS primers. Positive amplicons were sequenced and compared to reference sequences in the NCBI Genbank database utilizing the BLASTn program. After testing was complete, patient characteristics including age, gender, hospital location, and length of stay were revealed and examined for potential correlations between fungal species identified and specific patient variables. Phylogenetic analysis was also performed to infer potential evolutionary relationships between the fungal species found. Our results demonstrate that basic molecular biological techniques can be utilized in clinical laboratories for rapid and sensitive identification of fungal pathogens in samples reported as culture negative for fungal pathogens, which can facilitate rapid diagnosis and appropriate treatments. iv This work is dedicated to my students, who helped me realize that to love teaching, is to love learning, and that laughter is necessary to do both well. v ACKNOWLEDGMENTS I would first like to thank my advisor, Dr. Scott O. Rogers, who didn’t know what he was getting into when he welcomed me into his lab. I don’t think you can separate the quality of one’s research from the character of the people you work with, and for this I am grateful to have worked with and for him. I would also like to thank the rest of my committee, namely Dr. W. Robert Midden, Dr. Vipaporn Phuntumart, Dr. Raymond Larsen and Dr. George Bullerjahn, for their extreme patience and understanding regarding the pace of my progress towards completion of my degree. I would also like to acknowledge the University of Michigan for partial funding of this project; particularly Mary A. M. Rogers for the organization of, access to patient data, and original idea for this study, and Duane W. Newton, for clinical samples from the University of Michigan Hospital and for answers to countless e- mails about clinical diagnostics. There are too many people to name that have helped me through my long career at BGSU so I will only name a few: special thanks to the original members of the Rogers’ lab that helped me with everything from calculations to sterile technique to PCR, including Tom D’Elia, Ram Veerapaneni, Gang Zhang, Armeria Vicol, Shin, and Chia-Jui Tsai; support staff such as Linda Treeger, Chris Hess, Steve Queen and Lorraine; micro guru Sheila Kratzner and Dr. Don Deters for help and guidance with teaching Bio 313, which influenced my belief in the importance of this research. There are three women I must thank in particular without whose support I would never have finished: Lorena Harris-Palacios, Zeynep Kocer and Irina Shilova. All are good friends, BGSU PhD graduates, and role models for women in science everywhere. Lastly, I must thank my family for believing in me, even when I didn’t. vi TABLE OF CONTENTS Page CHAPTER 1. INTRODUCTION ...................................................................................................... 1 1.1 What are fungi? Classification in the tree of life. .......................................................... 1 1.1.2 Fungal phylogeny ........................................................................................... 3 1.1.3 Cryptomycota .................................................................................................. 12 1.2 History of fungi as pathogens ......................................................................................... 13 1.2.1 Pathogenicity: Pre-adaptation, co-evolution and potential virulence factors . 13 1.2.2 Thermally dimorphic fungi ............................................................................. 16 1.2.3 Increases in the number of immunocompromised patients and opportunistic fungi ................................................................................................... 16 1.3 Rationale for this dissertation ......................................................................................... 19 1.4 Objectives ....................................................................................................................... 19 1.5 Literature cited ................................................................................................................ 20 CHAPTER 2. MATERIALS AND METHODS ............................................................................... 34 2.1 Clinical diagnosis of fungal infections............................................................................ 34 2.1.1 Phenotypic identification ................................................................................ 34 2.1.2 Molecular identification of fungi .................................................................... 39 2.1.3 Antifungal resistance ...................................................................................... 39 2.2 Sample collection, transportation and information ......................................................... 42 2.2.1 Sample collection and transportation .............................................................. 42 2.2.2 Sample and patient information ...................................................................... 42 2.3 CTAB DNA extraction ................................................................................................... 45 2.4 PCR targeting internal transcribed spacers (ITS) 1and 2 in ribosomal DNA (rDNA) .. 46 2.5 PCR purification, gel extracts and cloning .................................................................... 48 2.6 BLASTn searches using NCBI database ........................................................................ 50 vii 2.7 Phylogenetic analysis ...................................................................................................... 50 2.8 Literature cited ................................................................................................................ 51 CHAPTER 3. RESULTS AND DISCUSSION ................................................................................ 61 3.1 Detection of fungal DNA in whole blood and respiratory samples and phylogenetic analysis of organisms found .................................................................................................. 61 3.1.1 BLASTn identification and patient information ............................................. 61 3.1.2 Phylogenetic analysis ...................................................................................... 64 3.1.3 Clinical review of organisms found ................................................................ 68 3.1.3.1 Dematiaceous fungi ........................................................................ 68 3.1.3.2 Hyaline molds and Candida species ............................................... 70 3.2 Discussion ....................................................................................................................... 74 3.2.1 Patient variables .............................................................................................. 74 3.2.2 Environmental variables ................................................................................ 77 3.2.3 Clinical applications ....................................................................................... 78 3.2.4 The era of genomics ........................................................................................ 79 3.3 Conclusions .................................................................................................................... 80 3.4 Literature cited ................................................................................................................ 82 APPENDIX A. USEFUL FUNGAL DATABASES ........................................................................
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