10 common mistakes in laboratory mycology

Professor Arunaloke Chakrabarti
Head, Department of Medical Microbiology
Postgraduate Institute of Medical Education and Research
Chandigarh, India, and
President, International Society for Human and Animal Mycology

Pre-analytical errors

Mistake 1: Improper collection and processing

Proper sample collection is crucial to efficient laboratory diagnosis. For instance, samples from dermatophyte infections must be collected from the margins, instead of the center of a lesion. For mycetomas, a simple swab collection will not yield a good reading. Granules must be collected from the serosanguinous sinus discharge or from just under the surface of a scab covering a sinus. Granules can also adhere to the dressing, so it can be sent to the laboratory as well.

Mistake 2: Incorrect transport conditions

The table shows some recommendations for proper transport of specimens.

Specimen	Transport conditions
Sputum	Sterile screw-capped container
Bronchoscopy fluid	Sterile screw-capped container
Cerebrospinal fluid	If a delay is anticipated, specimen should be left at room temperature
Urine	If a delay beyond 2 hours is anticipated, refrigerate at 4°C
Blood	Biphasic agar broth bottles designed especially for fungal cultures or automated culture bottle
Tissue biopsy	Specimen should not be frozen or allowed to dehydrate prior to culture

Analytical errors

Mistake 3: Lack of awareness or a low index of suspicion

Clinicians may not always suspect fungal infections and may ignore sending samples to mycology laboratory. However, a vigilant laboratory person may pick up fungal infections while processing sample for the desired test of the clinician. For example, a clinician suspecting a patient of tuberculosis (TB) sends samples for mycobacterial smear and culture. An expert laboratory person may detect Cryptococcus or Histoplasma spp., which produce similar clinical presentations. Similarly, laboratory personnel should be knowledgeable enough to accept or disregard any laboratory observations. For example, blood culture that grows all mycelial fungi may not be contaminant. They could be Fusarium or Scedosporium spp. isolated from the blood. Timely communication between laboratory staff and clinicians is important in the optimal management of fungal infections.

Mistake 4: Lack of expertise in the laboratory

Training of mycologists and the technical staff is essential to prevent errors in identifying fungi microscopically (e.g. knowing the difference between real fungi and artifacts, contamination, etc.).

Mistake 5: Errors involving blood cultures

It is important for laboratory staff to be familiar with handling blood cultures, particularly in centers with no commercial blood culture system. After sub-culture on solid medium, Professor Chakrabarti recommends incubating the medium for a least 48 hours and examining it macroscopically for pin-point yeast growth, which may otherwise be missed.

Mistake 6: Errors in identification

An overdependence on commercial systems may lead to errors in identifying fungi, as in the case of Candida auris. It has been shown that C. auris cannot be reliably identified by standard biochemical identification systems because the organism is not in their databases.¹ Updating the database of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) systems may help in proper identification. Histopathologic identification of fungi in tissue sections and cytologic preparations can also be prone to error.² Fungal identification is challenging even for the best laboratories, according to Professor Chakrabarti.

Mistake 7: Errors in antifungal susceptibility testing

Most laboratories now use the commercial Vitek 2 system for antifungal susceptibility testing of yeasts. However, its results may not perfectly correlate with results using the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution reference methods. Several factors can affect CLSI/EUCAST results, such as errors in testing media, incubation times, etc. Any resistance detected by commercial system may be confirmed by standard micro-broth dilution technique.

Mistake 8: Errors involving galactomannan and beta-D-glucan assays

Mistakes can occur during:

Blood collection (contamination, collecting from an intravenous line, collecting <5 days post-dialysis);
Serum separation (hemolysis should be avoided, use of calibrated centrifuge at optimum speed, serum transfer in biosafety cabinet); and
The test itself (should be done only in biosafety cabinet, plasticware should be sterile and pyrogen-free).

Mistake 9: Errors in therapeutic drug monitoring (TDM)

Inappropriate medical indication, sample collection, method and interpretation can affect TDM results. For instance, in collecting samples, the clinician or laboratory staff must know the correct timing of collecting a peak sample and a trough sample.

Post-analytical errors

Mistake 10: Errors in interpretation

Not all positive findings require treatment, so mycologists should be familiar with guideline recommendations to know when to treat or report fungal findings. For instance, the Infectious Diseases Society of America (IDSA) guidelines state that if Candida is found in respiratory secretions or urine, antifungal treatment is not recommended unless proven true infection.³

References

Mizusawa M, et al. J Clin Microbiol 2017;55:638-640.
Sangoi AR, et al. Am J Clin Pathol 2009;131:364-375.
Pappas PG, et al. Clin Infect Dis 2016;62:e1-e50.