Trypanosoma brucei (Human African Trypanosomiasis) – Microscopy, PCR and Serology

Background
This page provides testing information for human African trypanosomiasis (HAT, also known as sleeping sickness) at Public Health Ontario (PHO). The causative agent of HAT is the parasitic hemoflagellate Trypanosoma brucei.

Updates
This new test information page was published on October 10, 2025 and includes information that was previously found in Trypanosoma American or African-Serology and Trypanosoma-Microscopy and PCR.

*Uncontrolled print copy. Valid only on day of print: 31 Oct 2025.

Testing Indications

Trypanosoma brucei microscopy and PCR should be considered for the diagnosis of individuals with clinical and epidemiological evidence of HAT with blood, bone marrow, CSF, lymph node, and/or chancre specimens depending on the clinical presentation.¹

In addition, serology is available as an adjunct test for T. brucei subspecies gambiense (also known as western-central African HAT), however definitive diagnosis requires microscopy or PCR. Serology for T. brucei subspecies rhodesiense (also known as eastern-southern African HAT) is not available in Ontario.

Acceptance/Rejection Criteria

T. brucei testing will only be accepted if both of the following conditions are met:

Travel to an African region where T. brucei is found (see WHO Global Health Observatory map).
Symptoms consistent with human African trypanosomiasis (e.g. fever, trypanosomal chancre or rash, headache, arthralgia, lymphadenopathy, neuropsychiatric manifestations).

Failure to provide this information will result in test cancellation or testing delays.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Trypanosoma brucei microscopy	General Test Requisition	Whole blood in EDTA (see special instruction) Unstained, unfixed thick smear Unstained, methanol-fixed thin smear	4.0 ml 2 slides 2 slides	EDTA tube Slide mailer (each slide individually labelled) Slide mailer (each slide individually labelled)
Trypanosoma brucei microscopy	General Test Requisition	Bone marrow or lymph node aspirate	1.0 ml	EDTA tube or sterile container
Trypanosoma brucei microscopy	General Test Requisition	Bone marrow, lymph node, or brain biopsy	N/A	Sterile container
Trypanosoma brucei microscopy	General Test Requisition	Chancre skin biopsy	N/A	Sterile container
Trypanosoma brucei microscopy	General Test Requisition	Cerebrospinal fluid (CSF)	1.0 ml	Sterile container
Trypanosoma brucei PCR	General Test Requisition	Whole blood in EDTA	4.0 ml	EDTA tube
Trypanosoma brucei PCR	General Test Requisition	Bone marrow or lymph node aspirate	1.0 ml	EDTA tube or sterile container
Trypanosoma brucei PCR	General Test Requisition	Bone marrow, lymph node, or brain biopsy	N/A	Sterile container
Trypanosoma brucei PCR	General Test Requisition	Chancre skin biopsy	1.0 ml	Sterile container
Trypanosoma brucei PCR	General Test Requisition	Cerebrospinal fluid (CSF)	1.0 ml	Sterile container
West/Central African trypanosomiasis serology or T. brucei gambiense HAT serology	General Test Requisition	Blood or serum	5.0 ml blood or 1.0 ml serum	Blood, clotted – vacutainer tubes (SST)

Submission and Collection Notes

Complete all fields of the requisition form.

Specify all the following testing indications on the requisition. Failure to provide this information may result in rejection:

Travel to an endemic HAT region, and
Symptoms compatible with HAT

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 test cancellation or testing delay.

Timing of Specimen Collection

Due to low parasitemia, repeat microscopy may be needed if microscopy is initially negative and T. brucei gambiense is suspected.

Limitations

Grossly haemolysed, lipemic, contaminated specimens and specimens containing anti-coagulant are unsuitable for serological testing.

Storage and Transport

Place specimen container in a biohazard bag and properly seal the bag. Specimens should be stored at 2-8°C and shipped to PHO on ice packs within 48 hours of collection. All specimens must be shipped in accordance with the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

T. brucei microscopy is performed Monday to Friday at PHO’s laboratory, Toronto site. Turnaround time is up to 4 calendar days from receipt at PHO’s laboratory.

T. brucei PCR and serology are forwarded to the National Reference Centre for Parasitology (NRCP) in Montreal. Turnaround time is up to 42 calendar days from receipt at PHO’s laboratory.

STAT and Critical Specimens Testing

Priority testing for HAT is available upon request. If needed, contact PHO Laboratory Customer Service at 416-235-6556 or 1-877-604-4567 prior to sample submission.

Test Methods

T. brucei microscopy is performed at PHO using Giemsa staining. Whole blood specimens undergo buffy coat concentration. T. brucei PCR is performed at the NRCP using a laboratory-developed test. T. brucei serology is performed at the NRCP by direct agglutination testing, specifically the card agglutination test for trypanosomiasis (CATT). It uses purified and lyophilized surface antigens (mainly LiTat 3.0) from bloodstream T. brucei gambiense trypomastigotes.

Performance and Limitations:
For blood microscopy, sensitivity following concentration ranges from 45-90% (lower in T. brucei gambiense than T. brucei rhodesiense), and specificity approximates 100%. However, sensitivity may be as low as 5% if blood concentration (e.g., buffy coat) is not performed. Negative microscopy results do not rule out infection, and collection of multiple specimens for microscopy may be needed for diagnosis.

For PCR testing, the NRCP assay performance is under investigation, while other assays have a reported sensitivity of 87–100% and specificity of 92–98%. Subspecies identification cannot be made based on microscopy or PCR. Serology and PCR may remain positive for years following clearance of infection and cannot distinguish between recent and remote infection.

For serology, the NRCP states a sensitivity of 87–98% and a specificity of 95% for T. brucei gambiense, comparable to other reported ranges.^2,3Serology may be negative in the first 2 to 8 weeks following acute infection and may be lower in patients with low parasitemia. Lower sensitivity for serology has been reported in some regions (e.g. some West African loci) depending on the circulating T. brucei gambiense strain. Cross-reactivity has been observed with Toxoplasma, Strongyloides, EBV, CMV, Plasmodium (malaria), Borrelia, and other non-pathogenic Trypanosoma species. ^2,3

Interpretation

For microscopy performed at PHO:

Parasite Microscopy	Identified Organism(s)	Interpretation
Parasite(s) found.	Trypanosoma brucei trypomastigotes.	Detectable T. brucei trypomastigotes by microscopy.
No parasites found.	None.	No detectable T. brucei trypomastigotes by microscopy.

For T. brucei PCR performed at NRCP:

T. brucei PCR Result	Interpretation
Negative	No detectable Trypanosoma brucei DNA.
Indeterminate	Inconclusive result. May be due to a low level of target genetic material in the sample, inadequate sample content, or a non-specific signal. Please resubmit another specimen for testing if clinically indicated.
Positive	Detectable Trypanosoma brucei DNA. Cannot distinguish acute from remote infection.
Invalid	Test results are invalid due to the failed detection of the assay controls. May be due to inadequate sample content, extraction failure or test inhibition. Please resubmit another specimen for testing if clinically indicated.

For T. brucei gambiense serology performed at the NRCP:

T. brucei gambiense CATT Result	Interpretation
Negative	No detectable level of antibodies against Trypanosoma brucei gambiense. Serology may be negative in acute infection, consider collection for microscopy and PCR if clinically indicated.
Indeterminate	Trypanosoma brucei gambiense antibody status inconclusive. Repeat testing advised if clinically indicated.
Positive	Detectable antibodies against Trypanosoma brucei gambiense. Cannot distinguish acute from remote infection. Clinical correlation required.

Reporting

Results are received back at PHO and reports are forwarded to the ordering physician or authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

References

Büscher P, Cecchi G, Jamonneau V, Priotto G. Human African trypanosomiasis. Lancet. 2017 Nov 25;390(10110):2397-2409. doi: 10.1016/S0140-6736(17)31510-6.
Migchelsen SJ, Büscher P, Hoepelman AI, Schallig HD, Adams ER. Human African trypanosomiasis: a review of non-endemic cases in the past 20 years. Int J Infect Dis. 2011 Aug;15(8):e517-24. doi: 10.1016/j.ijid.2011.03.018.
Álvarez-Rodríguez A, Jin BK, Radwanska M, Magez S. Recent progress in diagnosis and treatment of Human African Trypanosomiasis has made the elimination of this disease a realistic target by 2030. Front Med (Lausanne). 2022 Nov 3;9:1037094. doi: 10.3389/fmed.2022.1037094.