Microsporidia (Microsporidiosis) – Microscopy and PCR

Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.

Background
This page provides testing information for microsporidiosis at Public Health Ontario (PHO). The causative agents of microsporidiosis are intracellular organisms of the Microsporidia phylum including Anncaliia, Encephalitozoon, Endoreticulatus, Enterocytozoon, Microsporidium, Nosema, Pleistophora, Trachipleistophora, Tubulinosema, and Vittaforma.

Updates
This webpage has been updated to include polymerase chain reaction (PCR) testing along with background, testing indications, acceptance criteria, performance and limitations, interpretations, and expected turnaround times based on calendar days instead of business days.

*Uncontrolled print copy. Valid only on day of print: 17 Dec 2025.

Testing Indications

Microscopy is the primary diagnostic method for individuals with clinical features suggestive of microsporidiosis, particularly in patients with impaired immunity presenting with symptoms such as chronic diarrhea, encephalitis, keratitis, myositis, or systemic infection.

PCR is a supplemental test that may be performed only upon approval, typically when:

the pre-test probability of microsporidial infection is high and
microscopy results are negative or inconclusive.

Note: Currently, antigen and antibody testing for Microsporidia is not available at Public Health Ontario (PHO).

Acceptance/Rejection Criteria

Accepted Submissions:
Testing will only be accepted in cases with any of the following documented on the requisition form:

Impaired immune system (e.g. HIV, transplant recipient, steroids use)
Signs or symptoms consistent with microsporidiosis (e.g. chronic diarrhea, keratitis, encephalitis)

Rejected or Cancelled Submissions:

Enteric specimens received without sodium acetate, acetic acid, and formalin (SAF) preservation are ineligible and will be cancelled.
Only one specimen per patient per collection date will be tested. Any additional specimens collected on the same date will be cancelled. If submitting multiple specimens, ensure each is collected at least 1 to 2 days apart for them to be eligible for testing.

Note: PCR testing, if needed, is only performed upon approval by a PHO microbiologist and must meet specific testing indication criteria. If PCR testing is being considered, please review the testing indications carefully and contact PHO’s Laboratory Customer Service at 416-235-6556 or 1-877-604-4567 prior to specimen submission.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Microsporidia - Microscopy	General Test Requisition	Enteric specimens (e.g., stool, intestinal biopsy / aspirate / scraping, Entero-Test)	1.0 ml	SAF vial
Microsporidia - Microscopy	General Test Requisition	Other body fluid or tissue specimens (e.g. aspirates, biopsies, urine)	1.0 ml or 2.0 g	SAF or empty sterile vial
Microsporidia - PCR	General Test Requisition	Enteric specimens or other specimens (e.g., stool, intestinal biopsy / aspirate / scraping, Entero-Test, urine)	1.0 ml or 2.0 g	Empty sterile vial

Submission and Collection Notes

Complete all fields of the requisition form.

Important: Specify any of the following testing indications on the requisition. Failure to provide this information may result in rejection:

Impaired immune system (e.g. HIV, transplant, steroids)
Signs or symptoms consistent with microsporidiosis

Important: Make sure that the enteric specimen and SAF fluid is mixed thoroughly as soon as collection occurs to preserve the specimen fully.

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.

Limitations

For enteric specimens: Avoid antacids or antimicrobials at least 2-3 weeks before collection as it can alter the intestinal microbiome. Avoid laxatives/enemas (e.g. mineral/castor oil), nonabsorbable antidiarrheal preparations (e.g. bismuth), and kaolin at least 7-10 days before collection as it can affect the staining process. Avoid contrast dyes (e.g. barium) at least 3 weeks before collection as it can affect the staining process.

Storage and Transport

Place specimen container in a biohazard bag and properly seal the bag. SAF specimens can be stored at room temperature (or alternatively 2-8°C) and shipped to PHO within 48 hours of collection. All specimens must be shipped in accordance with the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Microscopy is performed daily from Monday to Friday at PHO’s Toronto laboratory site. Turnaround time is up to 7 calendar days from receipt at PHO.

If approved upon request, PCR is forwarded to the Associated Regional and University Pathologists (ARUP) laboratory in Utah, U.S.A. Turnaround time is up to 21 business days from receipt at PHO.

Test Methods

At PHO, specimens are stained using modified trichrome staining technique.

If approved upon request, PCR is performed at ARUP using a laboratory-developed assay targeting the ribosomal operon of Encephalitozoon species and the internal transcribed spacer (ITS) region of Enterocytozoon species.

Performance and Limitations:
Microscopy sensitivity from a single stool specimen set is usually < 50% and a negative result cannot rule out infection. Collection of multiple specimen sets is required for diagnosis. Inadequate volume or delayed mixing of the enteric specimen and SAF fluid in the vial may lead to poor preservation and uninterpretable results. Species identification cannot be reliably made based on microscopic examination alone and cannot be performed on SAF-preserved specimens. ^1-4

For PCR, ARUP’s assay has no reported clinical accuracy data but has an analytical sensitivity of 16,000 copies/ml for Enterocytozoon bieneusi and 4,400 copies/ml for Encephalitozoon species. There is no analytical cross-reactivity with most other relevant organisms. The assay does not distinguish between Encephalitozoon species and does not test for organisms other than Encephalitozoon species or Enterocytozoon bieneusi. Other assays have reported a sensitivity of 90-100% and specificity of 90-100% with fecal and urine specimens.^4-7

Interpretation

Microscopy:

Microsporidia Microscopy	Interpretation
*Microsporidia* spores found	Species level identification cannot be made by microscopy. If species identification is needed, submit an unpreserved sample for PCR.
No microsporidia spores found	No evidence of Microsporidia spores. Due to the limited test sensitivity, testing of additional specimens may be considered if clinically indicated.

PCR:

Result	Interpretation
Detected	DNA detected for the specified microsporidial species.
Not detected	Enterocytozoon/Encephalitozoon DNA NOT detected. Test performance has not been evaluated for microsporidial organisms other than Enterocytozoon and Encephalitozoon..

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.

References

Han B, Pan G, Weiss LM. Microsporidiosis in Humans. Clin Microbiol Rev. 2021 Dec 15;34(4):e0001020. doi: 10.1128/CMR.00010-20. Epub 2021 Jun 30. PMID: 34190570; PMCID: PMC8404701.
Moniot M, Nourrisson C, Faure C, Delbac F, Favennec L, Dalle F, Garrouste C, Poirier P. Assessment of a Multiplex PCR for the Simultaneous Diagnosis of Intestinal Cryptosporidiosis and Microsporidiosis: Epidemiologic Report from a French Prospective Study. J Mol Diagn. 2021 Apr;23(4):417-423. doi: 10.1016/j.jmoldx.2020.12.005. Epub 2020 Dec 30. PMID: 33387699.
Clarridge JE 3rd, Karkhanis S, Rabeneck L, Marino B, Foote LW. Quantitative light microscopic detection of Enterocytozoon bieneusi in stool specimens: a longitudinal study of human immunodeficiency virus-infected microsporidiosis patients. J Clin Microbiol. 1996 Mar;34(3):520-3. doi: 10.1128/jcm.34.3.520-523.1996. PMID: 8904406; PMCID: PMC228838.
Wang Z, Orlandi PA, Stenger DA. Simultaneous detection of four human pathogenic microsporidian species from clinical samples by oligonucleotide microarray. J Clin Microbiol. 2005 Aug;43(8):4121-8. doi: 10.1128/JCM.43.8.4121-4128.2005. PMID: 16081959; PMCID: PMC1233985.
Wolk DM, Schneider SK, Wengenack NL, Sloan LM, Rosenblatt JE. Real-time PCR method for detection of Encephalitozoon intestinalis from stool specimens. J Clin Microbiol. 2002 Nov;40(11):3922-8. doi: 10.1128/JCM.40.11.3922-3928.2002. PMID: 12409353; PMCID: PMC139654.
Taniuchi M, Verweij JJ, Sethabutr O, Bodhidatta L, Garcia L, Maro A, Kumburu H, Gratz J, Kibiki G, Houpt ER. Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples. Diagn Microbiol Infect Dis. 2011 Dec;71(4):386-90. doi: 10.1016/j.diagmicrobio.2011.08.012. Epub 2011 Oct 6. PMID: 21982218; PMCID: PMC3217099.
Tanida K, Hahn A, Eberhardt KA, Tannich E, Landt O, Kann S, Feldt T, Sarfo FS, Di Cristanziano V, Frickmann H, Loderstädt U. Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples. Pathogens. 2021 May 26;10(6):656. doi: 10.3390/pathogens10060656. Erratum in: Pathogens. 2022 Feb 17;11(2):256. doi: 10.3390/pathogens11020256. PMID: 34073403; PMCID: PMC8229491.