Microscopic Observation and Culture of Aspergillus species:

The Traditional Way

Nancy McClenny, MPA, CLS/MTAssociate Program Director, CLS Internship Program,

San Francisco State UniversitySan Francisco, CA

mcclenny@sfsu.edu

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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?

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Diagnostic Specimen for Fungus

Pathology

Fungal stainsImmunodiagnostics

Clinical Laboratory

Gram stainWet mount (KOH) CultureSerology

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MICROSCOPY

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What fungus is shown in this Gram stain?

Hyphaeseptate, 45°

branching

Septum

cmazzanti, CMVL,Stanford

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Microscopy in the Clinical Lab: The Critical Tools

• Fluorescent dye with KOH– Blankophor– Calcofluor– Uvitex 2B

• Calibrated micrometer

• Expert readers!

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How do we know it’s Aspergillus?

Accessory conidia

A. niger A. terreus

Stipe

Stanford Medical Center Wiley Schell, Duke Univ.

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How do we know it’s NOT Aspergillus?

Phialide

Phialoconidium

Fusarium species

Annelloconidia

Scedosporium apiospermum

Wiley Schell, Duke Univ. Stanford Medical Center

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How do we know it’s NOT Aspergillus?

Dactylaria gallopava

Methenamine Silver KOH

Stanford Medical Center

Dematiaceous hyphae

Stanford Medical Center

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Stipes of A. niger, not hyphae of Mucor.

Conidia of A. niger,

not Candida

Wiley Schell, Duke Univ.

Why do we need culture?

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CULTURE

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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.)

A. versicolor onSabouraud Agar

nmcclenny

nmcclenny

A. versicolor on Potato Dextrose Agar

nmcclenny

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Rapid Identification of A. fumigatus

±1 hr

3-7 days24-48 hours at 35-37 C

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Poorly sporulatingA. fumigatus

Unusual or Atypical Isolates

nmcclennyNeosartorya fischeri with

masses of cleistothecia

nmcclennynmcclenny

15Aspergillus terreus Jharris, Texas Dept. of Health

nmcclenny

Phenotypic Identification of Other Aspergillus Species

nmcclenny

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

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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

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Case Study CLS 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.

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Where Have All the Students Gone?

Class 54 CLS Interns, SFSU

Class??

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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.