Trypanosoma cruzi (Chagas Disease) – Antibody, Microscopy, and PCR

Background
This page provides antibody (serology), microscopy, and PCR testing information for Chagas disease at Public Health Ontario (PHO). The causative agent of Chagas disease is the parasitic hemoflagellate Trypanosoma cruzi.

Updates

Updated to consolidate microscopy, PCR, and antibody (serology) testing information into a single test information webpage.

*Uncontrolled print copy. Valid only on day of print: 19 Oct 2026.

Testing Indications

Trypanosoma cruzi serology should be considered for the diagnosis of individuals with clinical and epidemiological evidence of chronic or congenital Chagas disease.¹

Trypanosoma cruzi microscopy and PCR should be considered for the diagnosis of individuals with clinical and epidemiological evidence of acute, congenital, or reactivated Chagas disease. For the diagnosis of chronic Chagas disease or for the primary screening of asymptomatic individuals, microscopy and PCR are not routinely recommended.²

Acceptance/Rejection Criteria

T. cruzi microscopy and PCR will only be accepted in cases with any of the following documented:

Travel to a Latin American region (including Mexico, Central America, and South America)
Family history of known Chagas disease
Transplantation, transfusion, or congenital exposure

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 cruzi Antibody	General Test Requisition	Serum, or Blood, clotted	1.0 ml 5.0 ml	Serum separator tube (SST), Or Plain or pro-coagulation tube
Trypanosoma cruzi 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 cruzi microscopy	General Test Requisition	Other body fluid or tissue specimens (e.g. tissue biopsy, lymph node aspirate, cerebrospinal fluid)	1.0 ml (fluid) or 0.2 mg (tissue)	Sterile container
Trypanosoma cruzi PCR	General Test Requisition	Whole blood in EDTA (see special instruction)	4.0 ml	EDTA tube
Trypanosoma cruzi PCR	General Test Requisition	Other body fluid or tissue specimens (e.g. tissue biopsy, lymph node aspirate, cerebrospinal fluid)	1.0 ml (fluid) or 0.2 mg (tissue)	Sterile container

Submission and Collection Notes

Complete all fields of the requisition form.

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

Documented travel history to a Latin American region
Family history of Chagas disease
Transplantation, transfusion, or congenital exposure

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 test cancellation or testing delay.

Timing of Specimen Collection

Serology: Serology may be negative in the first 2 to 8 weeks following acute infection and is not recommended for the diagnosis of acute Chagas disease. In cases of suspected congenital Chagas disease, serology may need to be repeated when the infant is 9 to 12 months of age to allow for interpretation without transferred maternal IgG antibodies.

Microscopy and PCR: In cases of congenital Chagas disease, parasitemia levels may be low at birth and peak around 30 days of life, therefore, newborns with negative microscopy and PCR results may require repeat testing one month later.

Limitations

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

Storage and Transport

Centrifuge tube if using SST.

Place specimen(s) into the appropriate collection container and securely seal the container. Then, place the sealed container into a biohazard bag and ensure the bag is properly sealed. 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 to the Transportation of Dangerous Good Act.

Special Instructions

For congenital Chagas disease specimen collection, umbilical cord blood or neonate venous blood is preferable to collect. See timing of specimen collection above.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

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

T. cruzi PCR and antibody testing is forwarded to the National Reference Centre for Parasitology (NRCP) in Montreal. Turnaround time is up to 42 calendar days from receipt at PHO.

Test Methods

T. cruzi antibody testing is performed at the NRCP by enzyme-linked immunoassay (ELISA) based on antibody capture using antigens from cultured epimastigotes of the Tulahuan and Brazil strains.

T. cruzi microscopy is performed at PHO using Giemsa staining. Whole blood specimens undergo buffy coat concentration.

T. cruzi PCR is performed at the NRCP using a laboratory-developed test that qualitatively amplifies a family of DNA repeats constituting approximately 9% of the nuclear DNA of T. cruzi.

Performance and Limitations:
For serology, the NRCP reports a sensitivity of 100% in individuals with the indeterminate or chronic forms of Chagas, along with a specificity of 96% overall. Other ELISA tests have reported a sensitivity averaging 90% and specificity averaging 98%.³ Serology may be negative in the first 2 to 8 weeks following acute infection. Serology may remain positive for years following treatment in chronic disease and cannot distinguish current from resolved or past infection, but seroreversion may occur within months in acute or congenital cases.⁴ Cross-reactivity has been observed with Leishmania, Plasmodium (malaria), and syphilis infections.⁵

For blood microscopy, sensitivity following concentration ranges from 40–90% in acute infections, and specificity approximates 100%.⁶Microscopic parasitemia may be low in the initial days following acute infection and usually peaks 20 to 30 days later followed by a decrease to submicroscopic levels within 90 days. Microscopy sensitivity is low in chronic or early congenital Chagas infections.

For PCR testing, the NRCP assay performance has a stated sensitivity of 85% and specificity of 100%, while other assays have a reported sensitivity of 82-88% and specificity of 98–99% in acute infections. PCR sensitivity in chronic infections is reportedly reduced to 65–89%.⁷PCR may be positive prior to microscopy during acute infection and may remain intermittently positive (but is often negative) in chronic infection, therefore a single positive PCR result does not confirm reactivation, and a single negative PCR result does not rule out chronic infection. Quantitative PCR testing is not available in Ontario currently.

Interpretation

For antibody testing performed at the NRCP:

Optical Density (OD) Sample Value	Result	Interpretation
≥ 0.40	Positive	Detectable antibodies against Trypanosoma cruzi. Does not distinguish current from resolved or past infection. Clinical correlation required.
0.30 to 0.39	Indeterminate	Trypanosoma cruzi antibody status inconclusive. Repeat testing advised if clinically indicated.
< 0.30	Negative	No detectable level of antibodies against Trypanosoma cruzi. False negative results may occur in the first 2 months of symptom onset.

For microscopy performed at PHO:

Parasite Microscopy	Interpretation
Parasite(s) found: Trypanosoma cruzi	Detectable T. cruzi organisms by microscopy.
No parasites found.	No detectable T. cruzi organisms by microscopy. Does not rule out infection. Refer to PCR for additional test results.

For T. cruzi PCR performed at NRCP:

T. cruzi PCR Result	Interpretation
Positive	*Detectable Trypanosoma cruzi* DNA. Cannot distinguish acute from chronic infection.**
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.
Negative	No detectable Trypanosoma cruzi DNA.
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.

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

Forsyth CJ, Manne-Goehler J, Bern C, Whitman J, Hochberg NS, Edwards M, Marcus R, Beatty NL, Castro-Sesquen YE, Coyle C, Stigler Granados P, Hamer D, Maguire JH, Gilman RH, Meymandi S. Recommendations for Screening and Diagnosis of Chagas Disease in the United States. J Infect Dis. 2022 May 4;225(9):1601-1610. doi: 10.1093/infdis/jiab513.
Bern C. Chagas' Disease. N Engl J Med. 2015 Jul 30;373(5):456-66. doi: 10.1056/NEJMra1410150.
Afonso AM, Ebell MH, Tarleton RL. A systematic review of high quality diagnostic tests for Chagas disease. PLoS Negl Trop Dis. 2012;6(11):e1881. doi: 10.1371/journal.pntd.0001881. Epub 2012 Nov 8.
Bern C, Montgomery SP, Herwaldt BL, Rassi A Jr, Marin-Neto JA, Dantas RO, Maguire JH, Acquatella H, Morillo C, Kirchhoff LV, Gilman RH, Reyes PA, Salvatella R, Moore AC. Evaluation and treatment of chagas disease in the United States: a systematic review. JAMA. 2007 Nov 14;298(18):2171-81. doi: 10.1001/jama.298.18.2171.
Caballero ZC, Sousa OE, Marques WP, Saez-Alquezar A, Umezawa ES. Evaluation of serological tests to identify Trypanosoma cruzi infection in humans and determine cross-reactivity with Trypanosoma rangeli and Leishmania spp. Clin Vaccine Immunol. 2007 Aug;14(8):1045-9. doi: 10.1128/CVI.00127-07. Epub 2007 May 23.
Bisio MMC, Rivero R, Gonzalez N, Ballering G, D'Amico I, Kessler C, Moroni S, Moscatelli G, Ruiz AM, Altcheh J. Diagnostic Accuracy of Two Molecular Tools for Diagnosis of Congenital Chagas Disease. Mol Diagn Ther. 2021 Nov;25(6):791-801. doi: 10.1007/s40291-021-00553-3.
Pascual-Vázquez G, Alonso-Sardón M, Rodríguez-Alonso B, Pardo-Lledías J, Romero Alegría A, Fernández-Soto P, Muñoz Bellido JL, Muro A, Belhassen-García M. Molecular diagnosis of Chagas disease: a systematic review and meta-analysis. Infect Dis Poverty. 2023 Oct 16;12(1):95. doi: 10.1186/s40249-023-01143-7.