Babesia – Microscopy and PCR

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Background
This page provides routine microscopy and polymerase chain reaction (PCR) testing information for babesiosis at Public Health Ontario (PHO). The causative agent(s) of babesiosis is the apicomplexan protist 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 rarely other species have been reported.

The test information described here is limited to microscopy and PCR testing. For serology, please refer to following link: Babesia – Serology

Updates
Effective July 1, 2023, babesiosis was added under the list of diseases of public health significance (DoPHS) in Ontario. Positive results are reported to the Medical Officer of Health as per Health Protection and Promotion Act. Additional testing and management recommendations are stated in the Infectious Disease Protocol Appendix 1 for Disease: Babesiosis.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 01 mai 2025.

Testing Indications

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 suspected¹. PCR is an additional test available to diagnose cases with high pre-test probability and either submicroscopic parasitemia or atypical morphology identified by microscopy (see algorithm below).

Acceptance/Rejection Criteria

Testing will be rejected in the following scenarios:

Specimens received in non-EDTA tubes (e.g. SST, citrate, heparin, SPS, uncoagulated/clotted)

Specimen Collection and Handling

Specimen Requirements

Test Requested

Required Requisition(s)

Specimen Type

Minimum Volume

Collection Kit

Babesia

General Test Requisition

Whole blood

PLUS

Unstained, unfixed thick smear

PLUS

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, including:

Test(s) requests and indications for testing
Patient setting/population
Clinical information including symptom onset date or if asymptomatic
Tick exposure history
Travel history

To assist in testing prioritization, state in the requisition if the patient is pregnant, has HIV, has impaired immunity, is part of an outbreak investigation, or is in the intensive care unit (ICU).

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. Failure to provide this information may result in rejection or testing delay.

Storage and Transport

Two sets of unstained thick and thin smears must be prepared locally with finger prick capillary blood or within one hour of EDTA whole blood collection before submission to PHO’s laboratory. Delays greater than one hour between EDTA blood specimen collection and unstained smear fixation could lead to distortion and loss of parasite morphology, reducing microscopy accuracy. Place labelled slide mailer in a biohazard bag and seal.

Place EDTA blood specimen 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’s laboratory 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 is performed daily at PHO’s laboratory Toronto site. Turnaround time is up to 24 hours from receipt at the PHO’s laboratory. Babesia PCR is performed at the National Microbiology Laboratory (NML). Turnaround time is up to 21 calendar days from receipt at the NML.

Test Methods

Microscopic examination is performed at PHO’s laboratory on thick and thin blood smears stained with Giemsa’s stain. Parasitemia levels are reported as a percentage of red blood cells (RBCs) infected on the thin smear examined. On average, 10 randomly selected 100X oil-immersion fields (about 1000 to 3000 total RBCs) are examined to calculate parasitemia levels, with fields devoid of parasites included if encountered, and multiply-infected RBCs counted as one parasitized cell.

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.

Performance and Limitations:
Microscopy can provide genus-level identification. Sensitivity likely varies from 80-100% (compared to PCR) 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, 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.

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;
Discordant microscopy findings between two laboratory technologists evaluating the same specimen;
Atypical or insufficient organisms/stages present by microscopy to provide definitive results;

PCR testing can also be requested following consultation with the Parasitology Operational Lead or Parasitology Microbiologist at PHO (e.g. if microscopy is negative but clinical presentation is strongly suggestive of babesiosis).

Interpretation

Babesia microscopy results will be reported as follows:

Microscopy results	Organism identified	Comments
Parasite(s) detected	*Babesia* species. Further species-level identification to follow.	Genus identification by microscopy will be reported along with parasitemia level. The sample will be automatically forwarded to NML for species-level identification.
Parasite(s) not detected	N/A	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.

Babesia PCR results will be reported as positive or negative for the species tested.

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.