Anaplasma – PCR and Serology

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

Updates:
As of July 02, 2025, PCR testing will replace serology if a single serum sample is received as described in the Algorithm section.

*Uncontrolled print copy. Valid only on day of print: 03 July 2025.

Testing Indications

Individuals with potential exposure (prolonged hard tick bite in endemic area) and compatible clinical symptoms (acute onset of fever accompanied by chills, headache, myalgia, arthralgia, leukopenia, thrombocytopenia, and/or elevated liver enzymes) may have testing requested for Anaplasma¹.

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

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 (e.g. symptom onset date, exposure history).

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Anaplasma PCR or molecular test	General Test Requisition	Whole blood	5 ml	Collect whole blood in EDTA tubes and do not centrifuge. Avoid heparin.
Anaplasma PCR or molecular test	General Test Requisition	Serum	1.0 ml	Blood, clotted – Serum Separator Tubes (SST). Centrifuge serum samples prior to transport if using SST.
Anaplasma PCR or molecular test	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 PCR or molecular test	General Test Requisition	CSF	0.5ml	Collect CSF into sterile, leak-proof containers made of freeze-thaw and shatter-resistant plastic, without additives.
Anaplasma serology⁴	General Test Requisition	Serum	1.0ml	Blood, clotted – Serum Separator Tubes (SST). Centrifuge serum samples prior to transport if using SST.

Submission and Collection Notes

Complete all fields of PHO’s 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

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.

Whole blood PCR is preferred over serum PCR for increased sensitivity, although either specimen type is acceptable. CSF PCR is only useful if clinical conditions suggest acute meningitis.

Single serology test samples will not be tested for serology until a convalescent sample is received, and the serum will be tested by PCR instead.

Timing of Specimen Collection

Within 2 weeks of symptom onset (i.e. acute illness) the following may be collected:

Whole blood or serum sample for PCR
First serum sample (will be stored for future paired serology)

Between 2 to 12 weeks from symptom onset (i.e. convalescent phase) if the initial PCR during acute illness was negative:

Second serum sample for paired serology (not advised if PCR was positive)

For PCR, samples should ideally be collected in 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 serology, an acute serum (within 2 weeks of onset of symptoms) and a convalescent serum (between 2-12 weeks from symptom onset) is required for testing to be performed.

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. 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)

Anaplasma PCR and serology are forwarded to the National Microbiology Laboratory (NML) in Winnipeg for testing. Turnaround time is up to 30 business days from receipt at PHO.

Test Methods

Methods:

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

Serology testing is via a commercial indirect immunofluorescence assay (IFA) kit. This test provides semi-quantitation of IgG antibodies to A. phagocytophilum.

Performance:

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

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

Limitations:

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 if available.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. PCR test performed, in whole or in part, using a lab-developed test at NML which has not been fully validated.

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 EBV infections¹¹. Therefore, single serum samples are not tested by serology at PHO; only paired serum samples are eligible for testing.

Algorithm

If a whole blood, buffy coat, serum, or CSF sample is received with a request for Anaplasma PCR (or for Anaplasma without a specified test method), PCR will be performed.

If a serum sample is received with a request specifically for Anaplasma serology, 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 PCR will be performed on the serum sample instead.
If an Anaplasma serum sample was received 2-12 weeks prior and PCR was negative at the time, the prior serum sample will be reactivated, and serology will be performed on both serum samples.

Interpretation

For serology:

IFA Titre	Interpretation
≥ 1:64	Single serum titres ≥ 1:64 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 increase in titres over 2-12 weeks is required for serological interpretation.
< 1:64	No serological evidence of Anaplasma infection. A serum titre < 1:64 does not exclude the diagnosis of anaplasmosis. If acute anaplasmosis is suspected, comparison with paired serum demonstrating seroconversion or a four-fold increase in titres over 2-12 weeks is required for serological interpretation.

For PCR:

Anaplasma phagocytophilum PCR	Interpretation
Positive	A. phagocytophilum DNA detected. Clinical correlation required.
Negative	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.
Invalid	Failed detection of assay control(s). Repeat testing if clinically indicated.

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

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.