Title

Identification of culture-negative fungi in blood and respiratory samples

Date of Award

2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Scott Rogers, Ph.D.

Second Advisor

George Bullerjahn, Ph.D. (Committee Member)

Third Advisor

Raymond Larsen, Ph.D. (Committee Member)

Fourth Advisor

W. Robert Midden, Ph.D. (Committee Member)

Fifth Advisor

Vipaporn Phuntumart, Ph.D. (Committee Member)

Abstract

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, and the latter is complicated by atypical phenotype 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.