Anaplasma (Anaplasma phagocytophilum) – Serology

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Background:
This page provides testing information for Human Granulocytic Anaplasmosis (HGA/Anaplasmosis) at Public Health Ontario’s (PHO) Laboratory. The main causative agent of Anaplasmosis is Anaplasma phagocytophilum which is transmitted to humans primarily through the bite of the blacklegged tick. Transmission through blood transfusion or organ transplant is also possible although less common.

The following testing options are available for Anaplasmosis: serology (antibody) testing and molecular detection and sequencing. Anaplasma tests are referred-out; samples are sent to the PHAC-National Microbiology Laboratory (NML) in Winnipeg for testing.

Updates:
Effective July 1, 2023, Anaplasma is added under the list of diseases of public health significance (DoPHS). Positive results are reported to the Medical Officer of Health as per Health Protection and Promotion Act.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 21 nov. 2024.

Testing Indications

Appropriate clinical indications include potential exposure to blacklegged ticks within the previous 5-21 days prior to symptom onset. Clinical evidence of Anaplasmosis is characterized by an acute onset of fever with one or more of the following non-specific symptoms: chills, malaise, headache, myalgia, arthralgia, leukopenia, thrombocytopenia or elevated hepatic transaminases.¹

Acceptance/Rejection Criteria

Specimens may be subject to rejection if they are not the appropriate sample type, have insufficient volume, or are not accompanied by relevant patient information.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Anaplasma Serology	General Test Requisition	Blood or serum	5.0 ml blood or 1.0 ml serum	Vacutainer tubes (SST)
Anaplasma PCR	General Test Requisition	EDTA whole blood	5 ml blood	Collect in EDTA Tubes. Avoid Heparin. Do not centrifuge
Anaplasma PCR	General Test Requisition	CSF Buffy coat (CSF samples are accepted for PCR testing however, they are not ideal specimen types as they are typically low-yield.)	0.5 ml CSF 0.5 mL buffy coat	Collect into a sterile container without additives

Submission and Collection Notes

Complete all fields of the PHO’s General Test Requisition Form. Include the patient’s full name, date of birth, and Health Card Number (must match the specimen label), physician name and address, and mandatory clinical information:

Test(s) requests and indications for testing
Clinical information including symptom onset date
Exposure history

For clinical specimens, 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.

Timing of Specimen Collection

An acute (collected early after the onset of symptoms) and a convalescent (collected 2-3 weeks later) may be required for laboratory diagnosis.

Limitations

Grossly haemolysed, icteric, lipemic or microbially contaminated sera or plasma are not recommended for testing.

Initiation of antibiotic treatment prior to serology testing may result in decreased antibody production which will affect the outcome of serological testing. Antibiotic treatment prior to molecular testing may result in decreased bacterial genome which will affect the outcome of PCR testing. However, if infection with A. phagocytophilum is suspected based on clinical symptoms, treatment should be initiated.¹

Storage and Transport

Serology:
For serum samples, centrifuge if using SST prior to transport.

Place specimen in biohazard bag and seal. Specimens should be stored at 2-8°C following collection and shipped to PHOL on ice packs as soon as possible.

PCR:
Collect blood in EDTA tubes. AVOID HEPARIN. Do not centrifuge. Aseptically collect buffy coat or CSF into sterile, leak-proof containers made of freeze-thaw and shatter-resistant plastic, without additives. Store specimens refrigerated up to 5 days and ship with freezer packs. If > 5 days, store at -20°C and ship on dry ice.

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)

Requests for Anaplasma serology and PCR testing are forwarded to the NML in Winnipeg for testing.

Turnaround time is up to 30 business days from receipt at the PHO’s laboratory.

Test Methods

Serology:
Serology testing is via a commercial Indirect Immunofluorescence Assay (IFA) kit; This is a semi-quantitative test for the detection of IgG antibodies to A. phagocytophilum.

PCR:
Extracted DNA is screened by in-house real-time PCR assay specific for A. phagocytophilum. If positive by real-time PCR, samples are tested by conventional PCR and sequencing for confirmation and strain determination.²

PCR test is performed for investigational or research purposes only.

Algorithm

If Anaplasma testing is requested, samples will be forwarded to NML for testing and results will be returned to the PHO’s laboratory to be reported to the physician.

Interpretation

Serology¹:

IFA Titre	Interpretation
≥ 1:64	Single IgG serum endpoint titres ≥ 1:64 are suggestive of infection at an undetermined time and may be indicative of either past infection or early response to a recent infection.
< 1:64	IgG endpoint titres less than 1:64 suggests that the patient does not have a current infection.

Note: A four-fold or greater increase in IgG titre between two serum samples drawn at least 2 weeks apart and tested in parallel is considered presumptive evidence of a recent or current infection with A. phagocytophilum.

An IgG endpoint greater than 1:64 alone should not be relied on for a diagnosis of Anaplasmosis.

Results from all serological tests should be interpreted in conjunction with clinical input and epidemiological data

PCR:
PCR results are reported as either Positive or Negative.

Reporting

NML results are received back at PHO’s laboratory, and then reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

Specimens that are positive for Anaplasma are to be reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

References

NML Guide to Services – Anaplasma serology. Available at: https://cnphi.canada.ca/gts/reference-diagnostic-test/4168?labId=1019
NML Guide to Services – Anaplasma PCR. Available at: https://cnphi.canada.ca/gts/reference-diagnostic-test/4165?labId=1019