Reference Identification – Yeast, Filamentous Fungi and Nocardia/Aerobic Actinomycetes

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Background
This page provides testing information for identification of isolates such as yeast, filamentous fungi and Nocardia/aerobic actinomycetes submitted to Public Health Ontario (PHO). This page is for information specific to reference identification of cultured isolated. For information regarding other testing options, refer to the following PHO webpages:

Updates
The following labstract(s) have been removed and information is included in this test information sheet:

LAB-SD-S-131 Candida auris, reference identification and susceptibility.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 21 nov. 2024.

Testing Indications

Fungi and Nocardia/Aerobic Actinomycetes being investigated as causative agents of disease should be submitted as pure cultures for identification if submitting laboratory is unable to identify.

Many fungi are ubiquitous in the environment and can be common laboratory contaminants or represent transient flora. Fungi are often encountered in routine bacteriology specimens but may not be involved in disease.

Consider submitting fungal isolates for identification when mycology testing is specifically requested by the treating physician (exceptions include if isolation of suspect dimorphic fungi occurs as an incidental finding on bacterial cultures; in this case specify the request to rule in/out a dimorphic fungus).
Review the patient history to determine if fungal identification is warranted (i.e. Is this an incidental finding? Were fungal elements observed in the specimen upon microscopic exam? is there a single colony at the edge of the plate that may represent contamination?)

Acceptance/Rejection Criteria

Isolates must be received as pure culture only.

Samples received in a contaminated state (contaminated with other fungi or bacteria) will be cancelled upon receipt.

Special Instruction for Submission of Potential Risk Group 3 Fungal Isolates

For sending potential Risk Group 3 Fungi, it is required to call PHO’s laboratory Customer Service at 1-877-604-4567 or 416-235-6556 and advice that you are sending an isolate for RG3 rule-out but pre-approval to send is not required.

Please clearly indicate on the requisition the request to rule-in/out RG3 fungi and ship following Transportation of Dangerous Good Act.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Yeast Identification	Reference Bacteriology Requisition	Pure culture of yeast isolate	N/A	Appropriate culture media for growth (slant or plate) or Swab from pure culture. (see note # 3 below)
Filamentous fungi Identification	Reference Bacteriology Requisition	Pure culture of filamentous fungus isolate	N/A	Appropriate culture media for growth (slant or plate) or Swab from pure culture. (see note # 3 below)
RG3 Fungal Isolate Rule Out	Reference Bacteriology Requisition	Pure culture of suspected RG3 fungal isolate	N/A	Appropriate culture media for growth (slant or plate) or Swab from pure culture. (see note # 3 below)
Nocardia and aerobic Actinomycetes Identification	Reference Bacteriology Requisition	Pure culture of suspected Nocardia spp. or other Aerobic Actinomycetes isolates (eg. Gordonia spp., Tsukamurella spp.)	N/A	Appropriate culture media for growth (slant or plate) or Swab from pure culture. (see note # 3 below)

Submission and Collection Notes

Complete all fields of the requisition form, fields a-d are mandatory

Test(s) requests and indications for testing
Patient setting/population/source
Immune status – VERY important to note on the requisition if patient is ICU, has had a transplant, has a haematological malignancy, or is otherwise immunocompromised etc as this will impact level of identification performed and reporting practices
Requests for specific organisms (dimorphics, Aspergillus spp., Fusarium spp., etc)
Any available direct microscopic exam results from submitting lab (including if fungal elements were observed on Gram stain)
Any preliminary culture findings or presumptive identification
Any information indicating if the isolate has been cultured repeatedly.

For clinical specimens, label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. For additional information see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay.

Place culture of the isolate in a biohazard bag and seal. Store cultures at room temperature or 28°C until submitted. Specimens should be shipped to PHO’s laboratory as soon as possible.

Limitations

Culture must be submitted in pure state.

Isolates received with bacterial contamination or mixed growth will be rejected. If there are extenuating circumstances where this is not possible, consult with PHO’s Laboratory- Mycology section.

PHO’s laboratory does not offer any identification services for yeast/filamentous fungi that have been isolated from environmental sources. Exceptions may be made for Candida auris if there is a link to a confirmed patient case. See below in ‘Special Instructions’ for more details.

Storage and Transport

Specimens should be placed in a biohazard bag, sealed and stored at room temperature or 28°C following isolation and shipped to PHO’s laboratory with ASAP. All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Special Instructions

Submission of Candida auris isolates:

Patient Isolates
As C. auris is an emerging pathogen and little is known about its prevalence and public health impact in Canada, all sterile and non-sterile clinical and patient screening isolates suspected of being C. auris should be sent to PHO’s laboratory for confirmation of identification and as a part of provincial and national surveillance programs. While this is not a mandatory program, it is strongly recommended that all laboratories participate in order for the detection and epidemiology of this emerging threat be understood. The site of isolation and whether the yeast was isolated from a clinical sample or from a screening/surveillance program must be indicated on the requisition.

Please note, PHO’s laboratory will not accept primary surveillance/screening swabs directly. Only isolated colonies of yeast suspected of being C. auris will be accepted for reference identification and susceptibility testing from patient screens.

Direct clinical specimens will continue to be processed as outlined in our Test Information Sheets (see Fungal Culture - Systemic).

Environmental Isolates
C. auris is known to colonize or remain viable in healthcare environments and be a source of nosocomial outbreaks. Testing isolates grown from environmental sources requires approval by a PHO microbiologist. Contact PHO’s laboratory Customer Service Centre prior to submission. Testing to confirm the identification of yeast suspected to be C. auris from an environmental source will be considered if there is a link to a confirmed patient case.

Only isolated colonies of yeast suspected of being C. auris will be accepted for identification and no susceptibility testing will be performed on these isolates.

Information required on the requisition form for C. auris identification includes:

information on type of specimen (clinical or surveillance or environmental), source
presumptive identification and method used for identification
whether this is part of an outbreak investigation (ensure the outbreak or investigation number is included)
any known risk factors for C. auris (eg. direct contact with a C. auris case, admission to a health care facility with known C. auris cases, hospitalization outside of Canada within the last 12 months, etc.)

Yeast presumed to be Candida auris will be identified using a MALDI-ToF MS system which has been validated for the identification of C. auris, and/or through PCR and sequencing. Testing will be performed at PHO Laboratory in Toronto.

Typing and National Surveillance
All confirmed C. auris isolates are forwarded to the National Microbiology Laboratory for typing by next generation sequencing as part of a national effort to better understand the epidemiology of this emerging pathogen in Canada. In order to expedite this process, information on any risk factors for acquisition of C. auris (eg. hospitalization outside of Canada, or at an institution known to have cases of C. auris) should be included on the original requisition.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Fungal culture reference identification testing is done daily Monday to Friday.

Yeasts, filamentous fungi and Nocardia/Aerobic Actinomyctes are identified at PHO’s Toronto laboratory.

Turnaround Time: dependent on the organism(s)

Yeast: typically, 3 business days (up to 5 business days) from date of receipt at PHO’s laboratory
Filamentous fungi: up to 14 business days from date of receipt at PHO’s laboratory
Nocardia/Aerobic Actinomycetes: up to business 14 days from date of receipt at PHO’s laboratory

Results are reported to the ordering physician or health care provider as indicated on the requisition.

STAT and Critical Specimens Testing

Not available.

Reach out to the PHO Microbiologist responsible for Mycology for time sensitive requests by calling PHO’s laboratory Customer Service at 1-877-604-4567 or 416-235-6552.

Test Methods

Reference identification uses various conventional methods, including macroscopic and microscopic morphology examination, biochemical reactions, growth temperatures and culture characteristics. Molecular methods such as polymerase chain reaction (PCR), sequencing and matrix-assisted laser desorption/ ionization –time of flight- mass spectrometry (MALDI-TOF MS) will be used when needed.

All isolates will be evaluated for dimorphic fungi (.i.e. Blastomyces dermatitidis/gilchristii, Histoplasma capsulatum, Coccidioides immitis/posadasii).

Filamentous fungi isolates are screened for pathogenic potential by checking the growth at 37⁰C. Isolates that fail to grow at 37⁰C may not be identified to genus/species level. If there is clinical information leading to the belief that the organism is indeed involved in disease process, please clearly state on the requisition as this may impact isolate work up.

Interpretation

The following table provides possible test results with associated interpretations:

Organism	Expected Result	Notes on ID
Yeast identification	Identification to species level in most cases.	Testing methods include chromogenic agar, microscopic features, MALDI-TOF MS and PCR and sequence analysis when required.
Filamentous fungi identification	Identification to genus/ species level for sterile sites immunocompromised patients, underlying malignancies.	Testing methods include macroscopic and microscopic morphology, biochemical reactions, temperature tests, MALDI-TOF MS and PCR and sequence analysis when required.
Filamentous fungi identification – isolate not growing at 37⁰C NON-HIGH RISK PATIENT	Filamentous fungi isolated. Please note that the organism submitted did not grow at 37⁰C and is indicative of probable contaminant.	Please consult with PHOL Mycology Microbiologist if further identification is warranted, as per note above.
Filamentous fungi identification – isolate not growing at 37⁰C HIGH RISK PATIENT	Identification to species level in most cases.	Testing methods include chromogenic agar, microscopic features, MALDI-TOF MS and PCR and sequence analysis when required.
Risk Group 3 Rule-In/Out	Isolate will be screened for dimorphic potential. Any isolates suggestive of RG3 dimorphic fungi will be subject to full identification. Isolates that are ruled-out as dimorphic fungi may not be identified to genus/species. The report will indicate that RG3 has been ruled-out.	Please consult with PHOL Mycology microbiologist if further identification is warranted, as per note above. Testing methods include macroscopic and microscopic morphology, biochemical reactions, RT-PCR and/or PCR and sequence analysis when required.
Aerobic actinomycetes (Nocardia spp., Gordonia spp., Tsukarmurella spp. etc)	Identification to genus/species level.	Testing methods include macroscopic and microscopic morphology, MALDI-TOF MS and molecular sequence analysis when required.

Reporting

Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

For isolates known to cause reportable diseases: specimens are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

References

Clinical and Laboratory Standards Institute (CLSI). Principles and Procedures for Detection and Culture of Fungi in Clinical Specimens. 2nd ed. CLSI guideline M54 (ISBN 978-1-68440-098-0 {Print}; ISBN 978-1-68440-099-7 {Electronic}). Clinical and Laboratory Standards Institute; 2021
Hazen, K., Howell, S.A.. Mycology and antifungal susceptibility testing. In: Leber, A.M. Editor. Clinical Microbiology Procedures Handbook. 4th ed. Washington, D.C.; ASM Press; 2016.
de Hoog, G.S., Guarro, J., Gene, J., Ahmed, S.A., Al-Hatmi, A.M.S., Figueras, M.J., Vitale, R.G.. Atlas of Clinical Fungi. 4th ed. Hilversum; Foundation Atlas of Clinical Fungi; 2020.
Ontario Agency for Health Protection and Promotion (Public Health Ontario). Focus On: Candida auris. Toronto, ON: King's Printer for Ontario; 2023.
Ontario Agency for Health Protection and Promotion (Public Health Ontario), Provincial Infectious Diseases Advisory Committee. Interim guide for infection prevention and control of Candida auris. Toronto, ON: Queen's Printer for Ontario; 2019.