Microscopic Observation and Culture of Aspergillus species: The Traditional Way

1. Microscopic Observation and Culture of Aspergillus species: The Traditional Way Nancy McClenny, MPA, CLS/MT Associate Program Director, CLS Internship Program, San Francisco State University San Francisco, CA mcclenny@sfsu.edu

2. What’s the Role of the Clinical Laboratory? • Who is a Clinical Laboratory Scientist (CLS)? • Are microscopy and culture still the methods of choice for fungi? • Who will do the work?

3. Diagnostic Specimen for Fungus Clinical Laboratory Pathology Gram stain Wet mount (KOH) Culture Serology Fungal stains Immunodiagnostics

4. MICROSCOPY

5. Hyphae septate, 45° branching Septum cmazzanti, CMVL,Stanford What fungus is shown in this Gram stain?

6. Microscopy in the Clinical Lab: The Critical Tools • Fluorescent dye with KOH • Blankophor • Calcofluor • Uvitex 2B • Calibrated micrometer • Expert readers!

7. How do we know it’s Aspergillus? Stipe Accessory conidia Stanford Medical Center Wiley Schell, Duke Univ. A. niger A. terreus

8. How do we know it’s NOT Aspergillus? Annelloconidia Phialoconidium Phialide Wiley Schell, Duke Univ. Stanford Medical Center Scedosporium apiospermum Fusarium species

9. How do we know it’s NOT Aspergillus? Dematiaceous hyphae Methenamine Silver KOH Stanford Medical Center Stanford Medical Center Dactylaria gallopava

10. Why do we need culture? Stipes of A. niger, not hyphae of Mucor. Conidia of A. niger, not Candida Wiley Schell, Duke Univ.

11. CULTURE

12. How can culture for Aspergilli be improved? • Sporulation media for primary isolation • Incubation at both 35-37°C and 30°C. • Daily inspection of cultures • Microaerophilic environment (Tarrand, Kontoyiannis, and Han. Abstract M-909, 42nd ICAAC, 2002.) nmcclenny nmcclenny A. versicolor on Potato Dextrose Agar nmcclenny A. versicolor on Sabouraud Agar

13. Rapid Identification ofA. fumigatus 24-48 hours at 35-37 C ±1 hr 3-7 days

14. Unusual or Atypical Isolates nmcclenny nmcclenny Poorly sporulating A. fumigatus Neosartorya fischeri with masses of cleistothecia nmcclenny

15. Phenotypic Identification of Other Aspergillus Species nmcclenny nmcclenny Aspergillus terreus Jharris, Texas Dept. of Health

16. “You just get used to how they look.”

17. Who Will Do the Work? • A 53-56% reduction in CLS training programs in the past 12 years • 34% of professionals in microbiology laboratories 50 years old Available at: http://www.asm.org/Policy/index.asp?bid=28422

18. Case StudyCLS Internship Program S. F. State University • April, 2004: $10.3 million is cut. • CLS Internship Program must become self-supporting. • August 2004: Director accepts a job offer outside of SFSU. • September, 2004: CLS program is suspended.

19. Where Have All the Students Gone? Class 54 CLS Interns, SFSU Class??

20. Summary • Microscopy and culture remain common, necessary diagnostic tools. • Procedural enhancements to microscopy and culture are available. • Recruitment and training of laboratory scientists must be concurrent with the integration of phenotypic and genotypic methods.