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 Note to Nancy: Culture, n. The growing of microbes in a nutrient medium. V, to cultivate. To develop microbes in a cultue medium. Such a growth or colony Why was I invited to the party? Bench technologist and mycology specialist for lots of years…roughly the same vintage of Mike Rinaldi. In other words, I was one the professionals who made sure that the lab reports you receive were accurate and timely. Basically I’d like to give a brief overview of what happens now in the lab and hopefully address misconceptions you might have about the limitations of phenotypic methods as they relate to Aspergillus. Assuming that “experience leads to expertise” (get source from Steinbach, et al), I am an expert in recognizing and identifying moulds in a clinical environment.

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? Discuss data from the ASM Benchmarking Survey re 89% of reporting labs use culture, 16% use serology and 5% use molecular (probably genprobes for dimorphic fungi….note that Genprobe is the only FDA (CLIA) approved molecular test that the CDC offers per Mary Brandt. The phenotypic methods for detecting and identifying Aspergillus and similar moulds have changed little since the days of Sabourauds. Now, given the value of rapid diagnosis to patient survival, the emphasis is on: how to do it more quickly and how to get the message to the physician efficiently. Despite the exciting and indeed wonderful advances in molecular diagnostics, the CLS in the microbiology laboratory of most, if not all, hospitals detects Aspergillus phenotypically, not with automated amplication systems, and identifies them phenotypically, not by gene sequencing. However obvious this fact might be, it is important to realize that the clinical laboratory has a much different agenda than does the research laboratory. For physicians, therefore, it is important to understand the basic lab techniques used, how the techniques might be enhanced to speed diagnosis, and how physicians are to interpret results. When the organizers encouraged NEW information, I was a bit flummoxed. What’s new with phenotypic methods? Well, a LOT…not all of which is positive…but we’ll get to that later.

Diagnostic Specimen for Fungus Pathology Clinical Laboratory Fungal stains Immunodiagnostics Gram stain Wet mount (KOH) Culture Serology The Traditional route

M I C R O S C O P Y

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

Microscopy in the Clinical Lab: The Critical Tools Fluorescent dye with KOH Blankophor Calcofluor Uvitex 2B Calibrated micrometer Expert readers! Dissecting microscope for detection of conidial heads, cleistothecia in slow-growing, unusual strains. If hyphae are seen on gram stain, oil can be removed, and the slide stained with Blankophor. Ruchel suggests maceration of tissue with the KOH/Blankophor and heating and 1-30 mins will usually do it. Specimens can be centrifuged to concentrate the hyphal elements. Ruchel discussed a case in which immunofluorescence was performed using asperillus antibodies, then Blankophor was added and ANOther mold fluoresced. Further testing showed that the other mold was a zygomycete. Uvitex 2B is being used to diagnosis microsporidium and Isospora. It appears to yield the most intense fluorescence and stains red cells, etc. less well than does Calcofluor. There are other stains, too, in the lit….

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

How do we know it’s NOT Aspergillus? Annelloconidia Phialoconidium Phialide Others include acremonium, paecilomyces, and many others. Emphasize the importance of culture to confirm and rule out a mixed infection. Wiley Schell, Duke Univ. Stanford Medical Center Scedosporium apiospermum Fusarium species

How do we know it’s NOT Aspergillus? Dematiaceous hyphae This is brain tissue…female, very complicated course. Mention the pathologists and their input first…. Aspergillus spp Hyphae 2.5-8µ wide. Acute angle branching Hyphal tips branch into two parts of equal lengths. Hyphae arranged in a tree- or fan-like pattern. Chandler, Color Atlas…. 1980 Larone. Med. Imp. Fungi, 4th ed., 2002. Schell. Oto. Clin. No. Amer., 33, April, 2000. Methenamine Silver KOH Stanford Medical Center Stanford Medical Center Dactylaria gallopava

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

C U L T U R E Discuss data from the ASM Benchmarking Survey re 89% of reporting labs use culture, 16% use serology and 5% use molecular (probably genprobes for dimorphic fungi….note that Genprobe is the only FDA (CLIA) approved molecular test that the CDC offers per Mary Brandt. The phenotypic methods for detecting and identifying Aspergillus and similar moulds have changed little since the days of Sabourauds. Now, given the value of rapid diagnosis to

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 “The media makes the mould!” Mention Tarrand’s ICAAC abstract from 2002 that compares Aspergillus inocula all adapted to a tissue-like environment in vitro were plated on sabs Emmons and held in ambient air at 25 degrees C and on sabsE and incubated at 35 degrees in 6% O2 and 10% CO2 to simulate the tissue environment. 12 of 12 inocula grew at modified conditions…NONE grew at standard conditions. Maren prefers malt extract for macroscopic and Czapek for microscopic. Microaerophilic atmosphere by Terrand 12 of 12 isolates grew in 10% oxygen and 10% CO2 with nitrogen. Media make the mould….give examples of media used for plant pathogens. A. versicolor on Sabouraud Agar

Rapid Identification of A. fumigatus 24-48 hours at 35-37 C ±1 hr 3-7 days American Heritage Dictionary of the English Language, 1969. Under definition of dogma.. “The dogmas of the quiet past are inadequate to the stormy present.” Lincoln

Unusual or Atypical Isolates Balajee SA, Weaver M, Imhof A, et al. Aspergillus fumigatus variant with decreased susceptibility to multiple antifungals. Antimicrob Agents Chemother 2004; 48: 1197-1203. White AFUM. Typical sporulation after 10-12 days. Higher MICS to itra, vori, caspo and a few to AMB. The original study was of itra resitstance retrospectively on 128 isolates. 6 with itra MICs 1 and 1 with low MICs had the atypical, white colony type. All 7 had high vori MICs and high caspo MECs. 2 had high AMB MICs This is where molecular identification will be most helpful for poorly growing, atypical, and unusual isolates. Mary Brandt….we would never separate the two…phenotypic and genotypic…methods of identification. nmcclenny nmcclenny Poorly sporulating A. fumigatus Neosartorya fischeri with masses of cleistothecia nmcclenny

Phenotypic Identification of Other Aspergillus Species nmcclenny Note that the LPB prep is available at the Kaminski Digital Library on the Univ of Adelaide mycology website from Dr. David Ellis. nmcclenny Aspergillus terreus Jharris, Texas Dept. of Health

“You just get used to how they look.”

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 Use the Pseudallecheria case from maryland as an example. Available at: http://www.asm.org/Policy/index.asp?bid=28422

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. Because funding has not been secured

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

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. We need people who are familiar with BOTH phenotypic and genotypic methods for identification of fungi. Mary Brandt, chief, fungus reference and Molecular Subtyping Unit, CDC and Prevention says that specialty labs usually know those who are experts in the field and can contact them for assistance. We frequently call upon Steve Peterson (USDA in Peoria) for help with non-sporulating white Aspergilli that might also be Neosartoryas. It can be a field of landmines for people who are not familiar with BOTH phenotypic and molecular data