Babesia – Microscopy, PCR, and Antibody

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 microscopy, polymerase chain reaction (PCR), and antibody testing information for babesiosis at Public Health Ontario (PHO). The causative agent of babesiosis is the apicomplexan protozoon Babesia. The main species known to infect humans include B. microti (Northeastern and Midwestern US, East Asia, Europe, Australia), B. duncani (Western US), B. divergens (Europe), B. venatorum (Europe, East Asia), B. crassa (East Asia), B. motasi (East Asia), and are rarely reported in other species.

Updates

Consolidate microscopy, PCR, and antibody (serology) testing information into a single test information webpage.

*Uncontrolled print copy. Valid only on day of print: 19 Oct 2026.

Testing Indications

Microscopy: As per the most recent Infectious Diseases Society of America (IDSA) guidelines, microscopy is the gold standard diagnostic test for babesiosis and should be requested whenever babesiosis is suspected.¹ PCR testing is an additional test available to diagnose cases with high pre-test probability and either submicroscopic parasitemia or atypical morphology identified by microscopy.

Antibody testing (serology): Serology is not the recommended testing method for acute babesiosis and should not be routinely ordered. Its only indicated uses are in linked cases of transfusion-transmitted, transplant-associated, or congenital babesiosis with documented negative microscopy and PCR results.

Babesiosis is a Disease of Public Health Significance (DOPHS) under the Health Protection and Promotion Act (HPPA). Additional testing and management recommendations are stated in the Ontario Ministry of Health Infectious Disease Protocol Appendix 1 for Disease: Babesiosis.

Specimen Collection and Handling

Specimen Requirements

Test Requested

Required Requisition(s)

Specimen Type

Minimum Volume

Collection Kit

Babesia

General Test Requisition

Whole blood

AND

Unstained, unfixed thick smear

AND

Unstained, methanol-fixed thin smear

2.0 ml

2 slides

EDTA blood tube (labelled)

Slide mailer (each slide individually labelled)

Submission and Collection Notes

Complete all fields of the requisition form.

Two sets of unstained thick and thin smears must be prepared locally using either finger prick capillary blood or EDTA whole blood within one hour of collection prior to submission to PHO. Delays of more than one hour between EDTA blood collection and unstained smear fixation may result in distortion and loss of parasite morphology, reducing microscopy accuracy.

Label the specimen container(s), including the slide mailer itself and each individual slide, 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.

Timing of Specimen Collection

Collect samples for testing as soon as possible, and do not delay sample collection based on the cyclical fever periodicity windows. If initial testing is negative, one or two repeat collections over 24 to 48 hours should be considered or until an alternative diagnosis has been made.

Storage and Transport

Place EDTA blood specimen (and slide mailer, if collected) in biohazard bag and seal. EDTA blood specimens should be stored at room temperature, as refrigeration can lead to distortion and loss of parasite morphology. Both unstained smears AND EDTA blood specimen must be sent to PHO as soon as possible. All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Babesia microscopy testing is performed daily from Monday to Saturday at PHO’s Toronto site. Turnaround time for microscopy is up to 3 calendar days from receipt at the PHO.

Babesia PCR testing is performed at the National Microbiology Laboratory (NML) in Winnipeg. Turnaround time for PCR is up to 21 calendar days from receipt at the NML.

If approved, Babesia microti serology is forwarded to the National Reference Centre for Parasitology (NRCP) in Montreal. Turnaround time is up to 30 calendar days from receipt at PHO.

Test Methods

Microscopy: Microscopy is performed at PHO on Giemsa-stained thick and thin blood smears. Parasitemia is reported as the percentage of red blood cells (RBCs) infected, based on the examination of the thin smear. Approximately 1,000 to 3,000 RBCs are randomly assessed to determine parasitemia. Fields without parasites are included in the count if encountered. Multiply infected RBCs are counted as a single parasitized cell, and gametocytes are excluded if present.

PCR: PCR testing is performed at NML using a laboratory-developed real-time PCR assay originally developed by Persing et al. (1993)². If positive, further species-specific PCR assays and sequencing is performed at NML to identify the organism at the species level.

Serology: Babesia microti serology is performed at the NRCP by immunofluorescence assay (IFA). Serologic testing for other Babesia species (e.g. B. duncani, B. divergens) is not available in Ontario.

Performance and Limitations:
Microscopy can provide genus-level identification and parasitemia levels. Sensitivity likely varies from 80-100% depending on microscopy expertise, although evidence is limited^3,4. Microscopy may be negative very early in infection due to low parasitemia, therefore a single negative microscopic examination is not sufficient to rule out infection and repeat testing may be needed if no alternate diagnosis is established. Microscopic morphology may occasionally be difficult to differentiate from other blood pathogens such as Plasmodium (malaria), therefore results may be preliminary reported as “organisms resembling Babesia spp.” pending PCR confirmation.

PCR testing has increased sensitivity over microscopy, but nucleic acids may persist for months following treatment, therefore molecular methods are less useful for treatment monitoring.

For serology, the NRCP states a sensitivity of 100% and specificity of 99% in immunocompetent individuals for Babesia microti exposure. Other laboratories offering IFA have reported a sensitivity of 88 to 96% and specificity of 90 to 100%.¹ Serological antibody titres may be negative early in infection, in patients with impaired immunity, or in patients with impaired splenic function. False positive reactions may occur in patients with autoimmune disorders (e.g., rheumatoid arthritis). Antibody titres remain elevated for years following clearance of infection. Antibody titres ≥ 1:1024, or a four-fold increase in titres between acute and convalescent sera, may assist in distinguishing acute from chronic or past infection. Cross-reactivity may occur with Plasmodium spp. at lower antibody titre levels for IFA. Cross-reactivity is not usually reported between Babesia species-specific IFAs, therefore a negative Babesia microti IFA does not rule out infection with other Babesia species.⁵

Algorithm

At PHO, Babesia testing is routinely performed by microscopic examination. PCR is not routinely performed on all incoming requests, but is automatically performed as an additional test in the following instances:

Microscopy is positive, for species-level molecular identification;
Atypical or insufficient organisms/stages present by microscopy to provide definitive results;

Interpretation

For Babesia microscopy (“Parasite microscopy”):

Parasite microscopy	Interpretation
Parasite(s) found Babesia species	Active Babesia infection. The parasitemia level will be reported. The sample will be automatically forwarded to NML for species-level identification.
Parasite(s) not detected	Infection cannot be ruled out by a single specimen tested. If the initial testing result is negative and no alternate diagnosis is established, additional specimen(s) can be collected. Additionally, if the likelihood of babesiosis is high, you may contact our customer service to request for additional PCR testing and follow further instructions.

For Babesia PCR (“Babesia species by PCR – Referred Out”):

Babesia spp. PCR	Interpretation
Positive	Babesia spp. DNA present. Further species identification will be reported where possible. Does not distinguish active from prior infection.
Indeterminate	Inconclusive result. An indeterminate PCR result may be due to a low level of target genetic material in the specimen, inadequate specimen content, or a non-specific signal. Please resubmit another specimen for testing if clinically indicated.
Negative	Babesia spp. DNA not present.
Invalid	No result available. Test results are invalid due to the failed amplification of the extraction control. Amplification failure may be due to inadequate specimen content, extraction failure, or PCR inhibition. Please resubmit another specimen for testing if clinically indicated.

For Babesia microti serology (“Babesia antibody”):

Babesia microti IgG IFA titre	Result	Interpretation
≥ 1/64	Positive	Detectable level of IgG antibodies against Babesia microti. Cannot distinguish between acute and remote infection. Cross-reactivity may occur with Plasmodium spp.
< 1/64	Negative	No detectable level of IgG antibody against Babesia microti. This assay does not test for other Babesia species.

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.

Positive specimens (microscopy or PCR) are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

References

Krause PJ, Auwaerter PG, Bannuru RR, Branda JA, Falck-Ytter YT, Lantos PM, Lavergne V, Meissner HC, Osani MC, Rips JG, Sood SK, Vannier E, Vaysbrot EE, Wormser GP. Clinical Practice Guidelines by the Infectious Diseases Society of America (IDSA): 2020 Guideline on Diagnosis and Management of Babesiosis. Clin Infect Dis. 2021 Jan 27;72(2): e49-e64. doi: 10.1093/cid/ciaa1216.
Persing DH, Mathiesen D, Marshall WF, Telford SR, Spielman A, Thomford JW, Conrad PA. Detection of Babesia microti by polymerase chain reaction. J Clin Microbiol. 1992 Aug;30(8):2097-103. doi: 10.1128/jcm.30.8.2097-2103.1992.
Wang G, Wormser GP, Zhuge J, Villafuerte P, Ip D, Zeren C, Fallon JT. Utilization of a real-time PCR assay for diagnosis of Babesia microti infection in clinical practice. Ticks Tick Borne Dis. 2015 Apr;6(3):376-82. doi: 10.1016/j.ttbdis.2015.03.001.
Teal AE, Habura A, Ennis J, Keithly JS, Madison-Antenucci S. A new real-time PCR assay for improved detection of the parasite Babesia microti. J Clin Microbiol. 2012 Mar;50(3):903-8. doi: 10.1128/JCM.05848-11.
Sanchez E, Vannier E, Wormser GP, Hu LT. Diagnosis, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis: A Review. JAMA. 2016 Apr 26;315(16):1767-77. doi: 10.1001/jama.2016.2884.