Babesia – Microscopy and PCR

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)

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.

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.

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:

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

1. 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.
2. 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.
3. 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.
4. 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.