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Yeasts, Cryptococcus and dimorphic fungi

17 min

Skills you'll build4 skills
  • Read a KOH or calcofluor white preparation and state what a negative can miss
  • Confirm a presumptive yeast identification, such as possible C. auris, before release
  • Check a negative cryptococcal antigen against India ink, culture, and prozone
  • Recognize a mold that may be a dimorphic pathogen and move it into a safety cabinet

At the bench: Read an organism stain and communicate a significant preliminary finding · Reconcile organism identification with specimen source and observed growth

Try first

Try first

A KOH-calcofluor preparation of a skin scraping shows no fungal elements. How is this interpreted?

Keep going to see the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

The direct fungal preparation

Ten percent potassium hydroxide (KOH) clears keratin and debris so fungal elements stand out. Calcofluor white binds chitin and fluoresces, so adding it to KOH detects sparse organisms. The yield depends on the organism burden and the specimen. Swabs collect few fungal elements, and their fibers can look like hyphae. A negative preparation does not rule out fungal infection.2

Yeast identification

A germ tube read at 2.5 to 3 hours presumptively identifies Candida albicans or C. dubliniensis. MALDI-TOF mass spectrometry performs most species-level identification, and the library version affects the result.2 Legacy biochemical panels misidentify Candidozyma auris (Candida auris), commonly as C. haemulonii, C. duobushaemulonii or C. famata. CDC lists MALDI-TOF with a library validated for C. auris, D1-D2 or ITS sequencing, or a validated molecular assay as accurate methods. A C. haemulonii-complex identification from a sterile site is confirmed by one of them before release. Possible C. auris is reported to infection prevention and public health at once.1

Cryptococcal antigen and the prozone

Cryptococcal antigen testing on CSF or serum is the standard rapid test and runs alongside culture. In one multisite validation, the lateral flow antigen assay was 99.3% sensitive. India ink was 86% sensitive overall and 42% sensitive when CSF cultures grew fewer than 1,000 CFU/mL.3 Very high antigen loads can give a false-negative antigen result through the prozone effect. When a negative antigen result conflicts with India ink, culture or the specimen findings, dilute the specimen and retest.2,6

Handling a suspected dimorphic mold

The mold phase of a dimorphic pathogen makes infectious conidia. A white, fluffy, slow-growing mold from a respiratory specimen, a sterile site or a routine bacteriology plate, or from a patient linked to an endemic area, starts the stricter protocol. Coccidioides also grows on bacteriology media at 35 to 37 °C.2

  1. Keep the plate closed and seal the lid with laboratory film.
  2. Open it only in a certified class II biological safety cabinet.
  3. Make no slide culture, and subculture only to a screw-capped slant.
  4. Notify the supervisor and infection prevention, and document any open-bench handling.
  5. Identify it with a nucleic-acid probe in the cabinet, or refer the sealed culture.

Propagating sporulating Coccidioides, Histoplasma or Blastomyces mold cultures needs biosafety level 3 practices.4 None of these fungi is a select agent.5

Follow the bench demonstration

Sources for this step
References
  1. Centers for Disease Control and Prevention. Identification of C. auris. Accessed September 27, 2026. https://www.cdc.gov/candida-auris/hcp/laboratories/identification-of-c-auris.html
  2. Tille PM. Bailey & Scott's Diagnostic Microbiology. 15th ed. Elsevier; 2021.
  3. Boulware DR, Rolfes MA, Rajasingham R, et al. Multisite validation of cryptococcal antigen lateral flow assay and quantification by laser thermal contrast. Emerg Infect Dis. 2014;20(1):45-53. doi:10.3201/eid2001.130906
  4. Centers for Disease Control and Prevention, National Institutes of Health. Biosafety in Microbiological and Biomedical Laboratories. 6th ed. CDC/NIH; revised June 2020. Accessed September 27, 2026. https://www.cdc.gov/labs/bmbl/index.html
  5. Federal Select Agent Program. HHS and USDA select agents and toxins. Accessed September 27, 2026. https://www.selectagents.gov/sat/list.htm
  6. Centers for Disease Control and Prevention. Clinical overview of cryptococcosis. Accessed September 27, 2026. https://www.cdc.gov/cryptococcosis/hcp/clinical-overview/index.html

Watch one

Read each decision and its explanation.

CSF arrives from a man with advanced HIV infection and 3 weeks of headache. The India ink preparation of the sediment shows many round, budding yeast cells with wide clear halos. The cryptococcal antigen lateral flow assay on undiluted CSF is negative. What do you do before reporting?

  1. Read the India ink first: many budding yeasts with capsule halos.

    India ink loses sensitivity when few organisms are present, so many encapsulated yeasts indicate a high organism load.

  2. Compare the two results.

    The antigen test is far more sensitive than India ink. A negative antigen with a stain showing many organisms is a discrepancy to resolve.

  3. Identify the likely cause: the prozone effect.

    Very high antigen loads can give a false-negative result through the prozone effect.

  4. Dilute the CSF as the procedure directs and retest.

    Dilution brings the antigen into the assay's measuring range.

  5. The diluted CSF is positive. Confirm that culture is set up and continuing.

    Culture remains the gold standard and recovers the isolate.

Report the cryptococcal antigen as positive from the diluted CSF, following the procedure for titer and comments. The undiluted negative is not reported, and culture continues.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A swab is submitted for fungal culture of a skin lesion, and the KOH preparation shows scattered filaments. What does the reader conclude?

Correct. Cotton and synthetic fibers survive KOH and can look like hyphae, and a swab collects few fungal elements. A scraping from the advancing edge of the lesion gives a reliable direct examination and culture.

Incorrect. KOH dissolves keratin and debris, but swab fibers survive clearing and can imitate hyphae on direct examination.

Incorrect. Swabs collect fungal elements poorly and can still carry some. The filaments could be hyphae or fibers, so a scraping from the active edge is requested.

Hint
  1. Consider what swab fibers add to the preparation.
  2. Consider which part of a skin lesion contains the most viable organisms.

Review Specimens and direct examination

Problem 2 of 3 · Hints available

A legacy biochemical yeast panel identifies a blood-culture isolate as Candida haemulonii. What does the laboratory do before releasing the result?

Correct. Legacy systems misidentify Candidozyma auris (Candida auris), commonly as C. haemulonii, C. duobushaemulonii, or C. famata depending on the system. Accurate methods are MALDI-TOF with a library validated for C. auris, D1-D2 or ITS sequencing, or a validated molecular assay. When the laboratory cannot confirm the identification, the isolate goes through the state health department. Possible C. auris is reported immediately to infection prevention and public health.

Incorrect. A sterile-site C. haemulonii-complex result is a classic legacy-system misidentification of C. auris, so it is escalated and confirmed before release.

Incorrect. Repeating a method that cannot recognize C. auris reproduces the same identification.

Hint
  1. Consider which important yeast legacy biochemical panels are known to misidentify.
  2. Consider whether repeating the same method can give a different identification.

Review Identifying yeasts

Problem 3 of 3 · On your own

CSF from a woman with 2 weeks of headache gives a positive cryptococcal antigen lateral flow result. The India ink preparation is negative, and the culture is pending. How does the negative India ink affect the report?

India ink is the less sensitive test. A negative stain does not overrule a positive antigen result.

India ink sensitivity falls to about 42% at low burden. The positive antigen result is reported, and culture continues.

Repeating an insensitive stain delays a result the antigen test has already given.

The prozone effect causes false-negative antigen results. This result is positive.

Review Cryptococcus

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Rosalind Reagan, 66, MRN 6619043, is a retired geologist who winters in Tucson and still collects rocks.
  • A vertebral bone biopsy goes to bacteriology and mycology.
  • On day 4, a white, fluffy mold grows on the sheep blood agar plate in the 35 °C bacteriology incubator.
  • A new colleague has already lifted the lid on the open bench to look at it.
Decision 1 of 3

What do you do with the plate now?

Opening the plate on the bench releases conidia. This mold could be Coccidioides, whose arthroconidia infect by inhalation.

Kept working a suspicious mold on the open bench

A white, fluffy, slow-growing mold from a respiratory or sterile-site specimen can be the mold phase of a dimorphic pathogen, whose conidia are infectious. Opening it on the bench exposes staff. The plate stays sealed, work moves into a class II biological safety cabinet, and any open-bench handling is reported to the supervisor and infection prevention.

A white, fluffy mold from a sterile site in a patient linked to Tucson is handled as a possible dimorphic pathogen.

More growth on an open plate means more infectious conidia. Any subculture goes only to a screw-capped slant.

A mold from a sterile-site biopsy in a patient from an endemic area may be the pathogen.

Review Handling fungal cultures safely

Decision 2 of 3

What about the lid already lifted on the open bench?

Lifting the lid can release conidia without anyone smelling the plate.

Cleaning the bench is part of the response. The exposure is also reported.

The protocol requires notification and a record of any open-bench handling, so exposed staff can be followed.

Review Handling fungal cultures safely

Decision 3 of 3

How is the mold identified?

Slide cultures increase the sporulating growth that makes dimorphic pathogens hazardous. They are reserved for confirmed low-risk molds.

Morphologic conversion is obsolete and grows more of the hazardous culture.

Coccidioides is not on the select-agent list. The response is containment, notification and referral.

A probe identifies the organism without growing more of it. A laboratory without the probe sends the sealed culture to a reference or public health laboratory.

Review Handling fungal cultures safely

The handling follows from the source of the mold. A white, fluffy mold on a bacteriology plate from a sterile-site biopsy, in a patient who spends part of the year in a Coccidioides area, starts the containment protocol before any further examination.

For reference

Sources checked