Anaplasma – 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 molecular and antibody (serology) testing information for anaplasmosis (also known as human granulocytic anaplasmosis or HGA) at Public Health Ontario (PHO). The causative agent of anaplasmosis is the intracellular bacterium Anaplasma phagocytophilum, which is transmitted to humans primarily through the bite of hard ticks.

Updates:

Effective July 22, 2026, Anaplasma PCR testing will be routinely performed at PHO rather than being referred to the National Microbiology Laboratory (NML) in Winnipeg. Turnaround time for PCR testing will be decreased from 30 days to 7 calendar days . For PCR testing, EDTA whole blood is the main eligible specimen type; serum samples are no longer eligible for PCR testing but can continue to be collected for serology purposes where applicable.

*Uncontrolled print copy. Valid only on day of print: 22 June 2026.

Testing Indications

Individuals with potential exposure to blacklegged tick bites in risk areas and compatible clinical signs/symptoms (acute onset of fever accompanied by chills, headache, myalgia, arthralgia, leukopenia, thrombocytopenia, and/or elevated liver enzymes) may have testing requested for Anaplasma.¹

As a disease of public health significance in Ontario, additional testing and case management information is available under the Infectious Diseases Protocol Appendix 1 for Disease: Anaplasmosis (access under “A” of the Infectious Diseases Protocol section).

Acceptance/Rejection Criteria

Anaplasma PCR will only be accepted if both of the following clinical information are documented on the requisition:

Presence of sign(s)/symptom(s) compatible with anaplasmosis (please specify)
Date of sign(s)/symptom(s) onset is within 30 days of the specimen collection date

Failure to provide this information will result in test cancellation.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Anaplasma - PCR	General Test Requisition	EDTA Whole blood	5 ml	Collect whole blood in EDTA tubes and do not centrifuge. Avoid heparin.
Anaplasma - PCR	General Test Requisition	Buffy coat	0.5 ml	Collect buffy coat into sterile, leak-proof containers made of freeze-thaw and shatter-resistant plastic, without additives.
Anaplasma - Antibody	General Test Requisition	Serum	1.0 ml	Blood, clotted – Serum Separator Tubes (SST). Centrifuge serum specimens prior to transport if using SST.

Submission and Collection Notes

Complete all fields of PHO’s requisition form.

Important: include the required clinical information (signs/symptoms and date of onset) as stated above.

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

CSF is not a validated specimen type for PCR testing but may be considered if clinical conditions suggest rare acute Anaplasma meningoencephalitis. Approval from a PHO microbiologist is required prior to testing. Results should be interpreted with caution.

For Anaplasma antibody requests, single serum specimens will not be tested until a convalescent serum specimen is received within 3 months.

Timing of Specimen Collection

For Anaplasma PCR, specimens should be collected during acute illness prior to the initiation of antibiotic therapy, but treatment should never be withheld, if required based on the patient’s clinical status.

For paired Anaplasma serology, an acute serum (within 2 weeks of sign(s)/symptom(s) onset) and a convalescent serum (between 2-12 weeks from sign(s)/symptom(s) onset) is required for testing to be performed.

During the acute febrile illness phase (≤ 2 weeks):

Collect whole blood (EDTA tube) for Anaplasma PCR
Collect the first serum for Anaplasma serology (which will not be tested for Anaplasma but will be stored for future use)

During the convalescence phase (i.e., 2 to 4 weeks later):

If PCR was previously positive, do not order additional testing
If PCR was previously negative, collect the second serum for Anaplasma serology (which will be tested alongside the first serum sample)

Limitations

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

Storage and Transport

Place specimen in biohazard bag and seal. Insert the corresponding requisition in the pocket on the outside of each sealed biohazard bag. Store specimens refrigerated at 2-8°C 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)

Anaplasma PCR testing is performedonce per week at PHO. Turnaround time for PCR is up to 7 calendar days from receipt at PHO.

PairedAnaplasma serology is forwarded to the NML in Winnipeg if eligibility criteria are met. Turnaround time for paired serology is up to 30 calendar days from receipt at PHO.

Test Methods

Methods:
Anaplasma PCR is performed by PHO using a laboratory-developed real-time PCR assay specific for the msp2 gene of Anaplasma phagocytophilum.²

Paired Anaplasma serology testing is performed at NML using a commercial indirect immunofluorescence assay (IFA) kit. This test provides semi-quantitation of IgG antibodies to A. phagocytophilum.

Performance and Limitations:
For PCR, sensitivity has been estimated at 70-100% during the first week of symptom onset, 50-100% during the second week of symptom onset, and below 50% thereafter.^3,4,5 A negative PCR result does not rule out infection, especially if performed after the first week of symptom onset. Whole blood PCR has a 5 to 15% higher sensitivity than serum PCR and is preferred.⁶ PCR sensitivity may be decreased if the patient received antibiotic treatment targeting Anaplasma prior to specimen collection.⁸ However, if infection with Anaplasma is strongly suspected, treatment based on clinical assessment shouldn’t be withheld for the purpose of improving test sensitivity.

For serology, sensitivity has been estimated at 15-40% during the first week of symptom onset, 40-90% during the second week of symptom onset, and above 90% thereafter. Antibody titres tend to peak after 4 weeks from symptom onset and decrease to lower titres in the months thereafter. Reported specificity ranges from 65-100%, with improved specificity when using paired serum comparisons.^3,4,5,7 For serology, a single serum sample has limited diagnostic value since it may be negative in early infection, may remain positive for years, and may be positive in healthy individuals living in areas of endemicity.^9,10 Serological cross-reactivity has also been observed with Ehrlichia, Rickettsia, Coxiella, Orientia and EBVinfections.¹¹ Therefore, single serum samples are not tested by serology at PHO; only paired serum samples are eligible for testing.

Algorithm

If a EDTA whole blood , buffy coat, or CSF sample is received, Anaplasma PCR will be performed if meeting acceptance criteria above.

If a serum sample is received, PHO will verify if another Anaplasma serum sample was received 2 to 12 weeks prior:

If no Anaplasma serum sample was received 2-12 weeks prior, serology will be cancelled and held for up to 12 weeks until a convalescent serum is received.
If an Anaplasma serum sample was received 2-12 weeks prior, the prior serum sample will be reactivated, and serology will be performed on both serum samples.

Interpretation

For serology:

IFA Titre	Interpretation
≥1:128	Single serum titres ≥ 1:128 may suggest either an early response to a recent infection, remote/resolved infection, asymptomatic exposure in an endemic region, or cross-reactivity. If acute anaplasmosis is suspected, comparison with paired serum demonstrating seroconversion or a four-fold change in titres over 2-12 weeks is required for serological interpretation.
<1:128	No serological evidence of Anaplasma infection. A serum titre < 1:128 does not exclude the diagnosis of anaplasmosis. If acute anaplasmosis is suspected, comparison with paired serum demonstrating seroconversion or a four-fold change in titres over 2-12 weeks is required for serological interpretation.

For PCR:

Anaplasma phagocytophilum PCR	Interpretation
Detected	A. phagocytophilum DNA detected. Clinical correlation required.
Not Detected	No evidence of A. phagocytophilum DNA. A negative result does not exclude the diagnosis of anaplasmosis, especially if collected after the first week of symptom onset.
Indeterminate	May be due to a low level of target genetic material in the specimen, inadequate specimen content, or a non-specific signal. Resubmit another specimen for testing if clinically indicated.
Invalid	Failed detection of assay control(s). Repeat testing if clinically indicated.

Reporting

Results are reported to the ordering 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

Walls JJ, Aguero-Rosenfeld M, Bakken JS, Goodman JL, Hossain D, Johnson RC, Dumler JS. Inter- and intralaboratory comparison of Ehrlichia equi and human granulocytic ehrlichiosis (HGE) agent strains for serodiagnosis of HGE by the immunofluorescent-antibody test. J Clin Microbiol. 1999 Sep;37(9):2968-73. doi: 10.1128/JCM.37.9.2968-2973.1999. PMID: 10449483; PMCID: PMC85424.
Courtney JW, Kostelnik LM, Zeidner NS, Massung RF. Multiplex real-time PCR for detection of anaplasma phagocytophilum and Borrelia burgdorferi. J Clin Microbiol. 2004 Jul;42(7):3164-8. doi: 10.1128/JCM.42.7.3164-3168.2004. PMID: 15243077; PMCID: PMC446246.
Kim DY, Seo JW, Yun NR, Kim CM, Kim DM. Human granulocytic anaplasmosis in a Single University Hospital in the Republic of Korea. Sci Rep. 2021 May 25;11(1):10860. doi: 10.1038/s41598-021-90327-y. PMID: 34035378; PMCID: PMC8149831.
Dumler JS, Madigan JE, Pusterla N, Bakken JS. Ehrlichioses in humans: epidemiology, clinical presentation, diagnosis, and treatment. Clin Infect Dis. 2007 Jul 15;45 Suppl 1:S45-51. doi: 10.1086/518146. PMID: 17582569.
Schotthoefer AM, Meece JK, Ivacic LC, Bertz PD, Zhang K, Weiler T, Uphoff TS, Fritsche TR. Comparison of a real-time PCR method with serology and blood smear analysis for diagnosis of human anaplasmosis: importance of infection time course for optimal test utilization. J Clin Microbiol. 2013 Jul;51(7):2147-53. doi: 10.1128/JCM.00347-13. Epub 2013 May 1. PMID: 23637292; PMCID: PMC3697711.
Boodman C, Loomer C, Dibernardo A, Hatchette T, LeBlanc JJ, Waitt B, Lindsay LR. Using Serum Specimens for Real-Time PCR-Based Diagnosis of Human Granulocytic Anaplasmosis, Canada. Emerg Infect Dis. 2023 Jan;29(1):175-178. doi: 10.3201/eid2901.220988. PMID: 36573611; PMCID: PMC9796190.
Walls JJ, Aguero-Rosenfeld M, Bakken JS, Goodman JL, Hossain D, Johnson RC, Dumler JS. Inter- and intralaboratory comparison of Ehrlichia equi and human granulocytic ehrlichiosis (HGE) agent strains for serodiagnosis of HGE by the immunofluorescent-antibody test. J Clin Microbiol. 1999 Sep;37(9):2968-73. doi: 10.1128/JCM.37.9.2968-2973.1999. PMID: 10449483; PMCID: PMC85424.
Bakken JS, Haller I, Riddell D, Walls JJ, Dumler JS. The serological response of patients infected with the agent of human granulocytic ehrlichiosis. Clin Infect Dis. 2002 Jan 1;34(1):22-7. doi: 10.1086/323811. Epub 2001 Nov 21. PMID: 11731941.
Bakken JS, Goellner P, Van Etten M, Boyle DZ, Swonger OL, Mattson S, Krueth J, Tilden RL, Asanovich K, Walls J, Dumler JS. Seroprevalence of human granulocytic ehrlichiosis among permanent residents of northwestern Wisconsin. Clin Infect Dis. 1998 Dec;27(6):1491-6. doi: 10.1086/515048. PMID: 9868666.
Aguero-Rosenfeld ME, Donnarumma L, Zentmaier L, Jacob J, Frey M, Noto R, Carbonaro CA, Wormser GP. Seroprevalence of antibodies that react with Anaplasma phagocytophila, the agent of human granulocytic ehrlichiosis, in different populations in Westchester County, New York. J Clin Microbiol. 2002 Jul;40(7):2612-5. doi: 10.1128/JCM.40.7.2612-2615.2002. PMID: 12089287; PMCID: PMC120546.
Park JH, Heo EJ, Choi KS, Dumler JS, Chae JS. Detection of antibodies to Anaplasma phagocytophilum and Ehrlichia chaffeensis antigens in sera of Korean patients by western immunoblotting and indirect immunofluorescence assays. Clin Diagn Lab Immunol. 2003 Nov;10(6):1059-64. doi: 10.1128/cdli.10.6.1059-1064.2003. PMID: 14607867; PMCID: PMC262439.