Microscopic Observation and Culture of Aspergillus species: The Traditional Way

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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_at_sfsu.edu

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Whats 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
Clinical Laboratory
Pathology
Gram stain Wet mount (KOH) Culture Serology
Fungal stains Immunodiagnostics
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MICROSCOPY
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Hyphae septate, 45 branching
Septum
cmazzanti, CMVL,Stanford
What fungus is shown in this Gram stain?
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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 its Aspergillus?
Stipe
Accessory conidia
Stanford Medical Center
Wiley Schell, Duke Univ.
A. niger
A. terreus
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How do we know its NOT Aspergillus?
Annelloconidia
Phialoconidium
Phialide
Wiley Schell, Duke Univ.
Stanford Medical Center
Scedosporium apiospermum
Fusarium species
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How do we know its NOT Aspergillus?

Dematiaceous hyphae
Methenamine Silver
KOH
Stanford Medical Center
Stanford Medical Center
Dactylaria gallopava
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Why do we need culture?
Stipes of A. niger, not hyphae of Mucor.
Conidia of A. niger, not Candida
Wiley Schell, Duke Univ.
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CULTURE
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How can culture for Aspergilli be improved?

• Sporulation media for primary isolation
• Incubation at both 35-37C and 30C.
• 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
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Rapid Identification of A. fumigatus
24-48 hours at 35-37? C
1 hr
3-7 days

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Unusual or Atypical Isolates
nmcclenny
nmcclenny
Poorly sporulating A. fumigatus
Neosartorya fischeri with masses of
cleistothecia
nmcclenny
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Phenotypic Identification of Other Aspergillus
Species
nmcclenny
nmcclenny
Aspergillus terreus
Jharris, Texas Dept. of Health
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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.as
p?bid28422
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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.