Fungus in Formalin. Case 1 3/28/2017. Disclosure of Relevant Financial Relationships. Disclosure of Relevant Financial Relationships.

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1 National Center for Emerging and Zoonotic Infectious Diseases Fungus in Formalin Dr. Shawn Lockhart, PhD D(ABMM) Director, Fungal Reference Laboratory USCAP Annual Meeting 2017 March 8, 2017 Disclosure of Relevant Financial Relationships USCAP requires that all planners (Education Committee) in a position to influence or control the content of CME disclose any relevant financial relationship WITH COMMERCIAL INTERESTS which they or their spouse/partner have, or have had, within the past 12 months, which relates to the content of this educational activity and creates a conflict of interest. Dr. Atis Muehlenbachs declares he/she has no conflict(s) of interest to disclose. Disclosure of Relevant Financial Relationships Case 1 USCAP requires that all faculty in a position to influence or control the content of CME disclose any relevant financial relationship WITH COMMERCIAL INTERESTS which they or their spouse/partner have, or have had, within the past 12 months, which relates to the content of this educational activity and creates a conflict of interest. Dr. Shawn Lockhart declares he/she has no conflict(s) of interest to disclose. Brain mass The case was a 74 year old woman with a several week history of right arm weakness. developed speech difficulty over last few days prior to admission. She lived in Indiana, with recent travel to Florida. Her past medical history was unremarkable H&E brain tissue Imaging showed lobulated, peripherally enhancing mass of left parieto-occipital periventricular white matter. A brain biopsy was performed. 1

2 H&E brain tissue Differential Exophiala species Rhinocladiella species Cladophialophora bantiana Curvularia species Excerohilum species Cladophialophora of the brain Histopathology showed septate hyphae which tended to be dark rather than hyaline. DNA sequencing revealed Cladophialophora bantiana. C. bantiana is a ubiquitous mold that tends to cause fatal neurological infections in otherwise healthy individuals. No risk factors for C. bantiana have been determined. It should be strongly suspected when a mold is detected in the brain of an otherwise healthy individual. Prompt diagnosis and treatment is essential for survival. Many dematiaceous molds grow just fine at 42 C Take home point Case 2 For brain infections, might be efficacious to perform a Fontana Masson Mold endocarditis? This is a case of a 66 year old man with acute myeloid leukemia, previously healthy, status post chemotherapy. hospitalized with fever and neutropenia, started on Micafungin Beta-D glucan positive and a chest CT suspicious for fungal pneumonia, started on amphotericin B Multiple organ failure and patient died An autopsy was performed. GMS cardiac tissue 2

3 PAS cardiac tissue Differential Aspergillus species Fusarium species Scedosporium species Candida species Candida dubliniensis endocarditis! Although C. dubliniensis is a yeast, the histopathology revealed primarily hyphal forms. Many species of Candida are dimorphic and can grow as either yeast cells or hyphae in tissue. Hyphae, on cross section, can often resemble yeast. The key to differentiation is to look for any budding cells that are present. Hyphae from molds will not have any budding cells. Endocarditis is a complication of Candida sepsis in about 2-5% of cases and should be considered following sepsis. IHC cardiac tissue Take home point Keep a wide differential Case 3 Post-mastectomy wound infection F patient with breast cancer Post pre-op chemotherapy and radiation Full mastectomy, non-healing wound 3

4 Left chest wall debridement Left chest wall debridement Aspergillus fumigatus! Differential Candida species Histoplasma Aspergillus species Fusarium species Scedosporium species Candida? Take home point Looks can be deceiving What questions do I get most frequently? I could see fungal cells, why couldn t you amplify any fungal DNA? I could see fungal cells, why couldn t you amplify any fungal DNA? Tissue sat for too long in formalin Depurination blocks amplification leading to short fragments Short fragments cannot be amplified further Generally base pairs needed for good sequence 4

5 I could see fungal cells, why couldn t you amplify any fungal DNA? Too few cells in the slice that was used Distribution is not always even The next slice down may move right out of the granuloma I thought PCR only required a single cell? Technically yes, practically no I could see fungal cells, why couldn t you amplify any fungal DNA? The cells are dead! Dead cells have no or degraded DNA! Cryptococcus is notorious for this! The capsule also seems to muck up the column that captures the DNA Mucormycetes are especially troublesome Pauciseptate (sometimes erroneously called aseptate) Mucormycetes are especially troublesome Pauciseptate (sometimes erroneously called aseptate) I don t see fungal cells, but could you perform PCR just to make sure? I don t see fungal cells, but could you perform PCR just to make sure? Ritter et al (2013) Am J Path 183:

6 Sources of spurious fungal DNA in micro lab Lyticase with Saccharomyces cerevisiae CDC, unpublished Proteinase K with Fusarium CDC, unpublished Blood tubes with Aspergillus Harrison et al (2010) DMID 67: Primers, PCR reagents, master mix solutions Czurda et al (2016) J Clin Micro 54: Sources of spurious fungal DNA in pathology Dirty microtome Paraffin that has sat open on the bench for days, weeks Block that sat in a drawer and was not cleaned off before slices were taken Most frequent contaminants Alternaria and Cladosporium Do we need a genus or species, isn t just reporting Aspergillus-like mold, Mucormycete or yeast enough? Susceptibility to antifungal drugs varies widely! With new azole available such as voriconazole, posaconazole and now isavuconazole, treatment can be targeted to the genus or even the species Can our hospital start doing PCR from tissue? EORTC/MSG definitions for using FFPE tissue PCR results for genus/species determination PCR only appropriate from tissue where histopathology shows fungi Unidirectional workflow is absolutely necessary Reagents must be shown to be fungal DNA free Primers should be to rdna Positive and negative controls must be included Length of the sequence must be >150 bp Genus/species name must be consistent with pathology 6

7 Summary points Looks can be deceiving PCR should only be performed when histology suggests fungi are present Amplification of fungal DNA does not always mean that fungi are causing infection Important Information Regarding CME/SAMs The Online CME/Evaluations/SAMs claim process will only be available on the USCAP website until September 30, No claims can be processed after that date! After September 30, 2017 you will NOT be able to obtain any CME or SAMs credits for attending this meeting. Amplification of fungal DNA from FFPE should not be performed everywhere! Thank You! For more information, contact CDC CDC INFO ( ) TTY: The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. 7