Plasmodium (Malaria) – Microscopy, Antigen, PCR, Typing 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 microscopy, antigen testing (rapid diagnostic testing or RDT), polymerase chain reaction testing (PCR), antimalarial resistance typing and antibody (serology) information for malaria at Public Health Ontario (PHO). The causative agent of malaria is the parasitic apicomplexan protozoon Plasmodium. Five species are commonly known to cause malaria in humans including P. falciparum (worldwide), P. vivax (Asia, America, East Africa), P. ovale (Africa, Asia), P. malariae (worldwide) and P. knowlesi (Southeast Asia).

Updates

As of October 19, 2026, additional clarifications have been added around testing indications, acceptance criteria, after-hours testing and resistance typing processes. The antibody testing information has also been integrated into this webpage (previously listed under a separate page).

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

Testing Indications

Microscopy and antigen testing (sometimes called “malaria screen”)¹:

All patients with suspected malaria should have both routine microscopy and antigen testing ordered at the time of presentation.
Local laboratories are expected to provide STAT preliminary malaria detection and parasitemia within 2 hours of patient presentation.
Local laboratories must also ensure that they can provide preliminary identification of Plasmodium falciparum within 24 hours of patient presentation (e.g., via microscopy or antigen testing) to facilitate treatment and hospitalization decision-making.
PHO does not provide STAT preliminary detection or preliminary identification of falciparum malaria but may provide additional identification of malaria species and assist in diagnostic support following local preliminary findings.

PCR testing: PCR testing is not routinely performed at PHO and should not be routinely ordered. It is only performed in infrequent scenarios such as:

Discrepancies between antigen (or other molecular, e.g., LAMP) test results and microscopy results reported for the patient locally or at PHO.
Insufficient organism stages present by microscopy to provide definitive species identification at PHO.
Person under investigation for suspected risk group 4 (RG4) viral hemorrhagic fever (VHF) infection such as Ebola, Marburg, Lassa, or Crimean-Congo hemorrhagic fever.

Antimalarial resistance typing: Resistance typing is not routinely performed at PHO. It is only performed if there are concerns for treatment failure within 28 days of treatment, such as:2

Asexual stage parasitemia after 48 hours higher than parasitemia at presentation.
Asexual stage parasitemia after 72 hours with fever and/or with ≥ 25% of parasitemia at presentation.
Asexual stage parasitemia after 72 hours (for artemisinin-based antimalarial therapy) or after 7 days (other therapies).

Antibody testing (serology): Serology is not the recommended testing method for acute malaria and should not be routinely ordered. Its only indicated uses are in rare scenarios such as:

Linked cases of transfusion-transmitted, transplant-associated, or congenital malaria with documented negative microscopy and PCR results.
Suspected hyperreactive malarial splenomegaly (HMS) syndrome with documented negative microscopy and PCR results.

PCR, resistance typing and antibody testing require pre-approval prior to submission. To obtain pre-approval from PHO microbiologist, contact the PHO Laboratory Customer Service Center at 416-235-6556 or 1-877-604-4567.

The Committee to Advise on Tropical Medicine and Travel (CATMAT) provides further testing indications as well as background on diagnostic testing methods in the Canadian recommendations for the prevention and treatment of malaria guidance document.

Acceptance/Rejection Criteria

The following information must be provided on the test requisition, otherwise testing may be delayed or cancelled:

Travel history information
Preliminary malaria test result and method performed locally (microscopy, antigen and/or molecular method)

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Malaria or Plasmodium	General Test Requisition	Whole blood AND Unstained, unfixed thick smear⁴ AND Unstained, methanol-fixed thin smear⁴	2.0 ml 2 slides 2 slides	EDTA blood tube Slide mailer (each slide individually labelled) Slide mailer (each slide individually labelled)
Malaria or Plasmodium PCR (must be pre-approved by PHO Microbiologist)	General Test Requisition	Whole blood	2.0 ml	EDTA blood tube
Malaria or Plasmodium PCR (must be pre-approved by PHO microbiologist if needed – see indications above)	General Test Requisition	Blood or Serum	5.0 ml blood or 1.0 ml serum	Blood, clotted-Serum separator tube
Antimalarial resistance typing (must be pre-approved by PHO microbiologist – see indications above)	General Test Requisition	Whole blood	2.0 ml	EDTA blood tube

Submission and Collection Notes

Complete all fields of the requisition form.

Important: Specify all the following testing indications on the requisition. Failure to provide this information may result in delays or cancellations:

Travel history – the country/region of exposure must be stated.
Specify under ‘Clinical Information, Other’ of any recent malaria test results including smear, antigen and molecular (e.g., LAMP or CPA) results.

Label the specimen container(s), including the slide mailer itself and each individual slide, 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.

If the patient is currently under investigation for a suspected RG4 VHF infection such as Ebola, Marburg, Lassa, or Crimean-Congo hemorrhagic fever, do NOT send smear slides due to the biosafety risks; only submit an EDTA whole blood tube. Refer to Viral Hemorrhagic Fever (VHF) Viruses (e.g., Ebola, Marburg, Lassa, Crimean-Congo) – PCR.

Timing of Specimen Collection

Collect samples for testing as soon as possible and do not delay sample collection to align with cyclical fever periodicity windows. If initial testing is negative, one or two repeat collections over 24 to 48 hours should be considered or until an alternative diagnosis has been made.

Storage and Transport

Unless the patient is suspected of a VHF infection, two sets of unstained thick and thin smears must be prepared locally with finger prick capillary blood or within one hour of EDTA whole blood collection before submission to PHO. Delays greater than one hour between EDTA blood specimen collection and unstained smears preparation may lead to distortion and loss of parasite morphology, reducing microscopy accuracy. Place labelled slides and slide mailer in a biohazard bag and seal.

Place labelled EDTA blood specimen in biohazard bag and seal. EDTA blood specimens should be stored at room temperature, as refrigeration can lead to distortion and loss of parasite morphology. Both unstained smears and EDTA blood specimen must be sent to PHO as soon as possible. All clinical specimens must be shipped in accordance with the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Malaria microscopy and antigen testing are performed daily, Monday to Saturday at PHO’s laboratory-Toronto on a routine basis. Turnaround time for microscopy and antigen testing is up to 24 hours from receipt at PHO’s laboratory- Toronto site.

Malaria PCR testing is performed during weekdays upon request at PHO’s laboratory-Toronto. Turnaround time for PCR is up to 72 hours from receipt at PHO’s laboratory-Toronto.

If approved, malaria resistance typing and antibody testing are forwarded to the National Reference Centre for Parasitology (NRCP) in Montreal. Turnaround time for typing and antibody testing are up to 42 days from receipt at PHO.

STAT and Critical Specimens Testing

Urgent testing is available upon request for cases of severe malaria or treatment failure, such as:

Intensive or cardiac care unit (ICU/CCU)
Impaired consciousness/coma
Convulsions
Respiratory failure
Pulmonary edema
Shock
Abnormal bleeding
Jaundice
Hemoglobin ≤ 70 g/L
Blood glucose < 2.2 mmol/L
Arterial pH < 7.25
Bicarbonates < 15 mmol/L
Creatinine > 265 umol/L
Lactate > 2.5 mmol/L
Parasitemia ≥ 2% (children < 5 years old) or ≥ 5%
Treatment failure (see failure scenarios in testing indications above)

If urgent testing is needed, submit specimens for urgent testing as per below:

Notify PHO before sending an urgent specimen by contacting the Laboratory Customer Service Centre (business hours) at 416-235-6556 or 1-877-604-4567 or the Duty Officer (after hours) at 416-605-3113.
Indicate “Urgent” and malaria clearly on the requisition.
Indicate the travel history and preliminary malaria test results.
Indicate the rationale for urgent testing (severe malaria and/or treatment failure indications).
Package priority specimens separately from routine specimens using TDG compliant packaging and labelling.
Mark “Urgent” clearly on the outside of the package to assist with intake.
Ship urgent specimens directly to PHO’s Toronto laboratory site: Shipping and Receiving Dock, 661 University Ave, Toronto, Ontario. For delivery instructions, follow Directions to 661 University Shipping Dock for Clinical Samples.

Appropriately labelled specimens must be received by 10:30 PM at PHO’s laboratory -Toronto for urgent testing, otherwise testing will be performed the following morning.

Test Methods

Microscopy: Microscopy is performed at PHO on thick and thin blood smears stained with Giemsa’s stain. Parasitemia is reported as a percentage of red blood cells (RBCs) infected on the thin smear examined. Around 1,000 to 3,000 total RBCs are randomly examined to calculate parasitemia. Fields devoid of parasites are included if encountered. Multiply infected RBCs counted as one parasitized cell and gametocytes are excluded if present.

Antigen: Antigen testing is performed at PHO using the Abbott BinaxNOW Malaria, which detects the Plasmodium falciparum-specific histidine-rich protein 2 and 3 (HRP2/3) antigens and the pan-Plasmodium aldolase antigen.

PCR: PCR testing is performed at PHO using a laboratory-developed multiplex real-time PCR assay originally developed by Rougemont M et al (2004) and optimized by Shokoples SE et al (2009) and Phuong M et al (2014)^3-5. The assay is based on the detection of five 18s rRNA gene targets, including one generic for the Plasmodium genus and one specific for each of P. falciparum, P. vivax, P. ovale, or P. malariae.

Antimalarial resistance typing: If approved, resistance typing is performed at NRCP using a combination of laboratory-developed PCR and sequencing assays targeting the kelch13 (K13) gene for artemisinin resistance, the chloroquine resistance transporter (crt) gene for chloroquine resistance, the cytochrome b (cytb) gene for atovaquone resistance, the dihydrofolate reductase (dhfr) gene for pyrimethamine resistance, the dihydropteroate synthase (dhps) gene for sulfadoxine resistance and the multidrug resistance 1 (mdr1) gene for mefloquine/lumefantrine resistance.

Serology: If approved, serology is performed by enzyme-linked immunosorbent assay (ELISA). It uses a mixture of recombinant antigens (including the circumsporozoite protein or CSP) of all human pathogenic Plasmodium species.

Performance and Limitations:
Microscopy can provide definitive species identification of Plasmodium species, discrimination of Plasmodium co-infections and parasitemia levels. Sensitivity varies from 85% to 93% depending on microscopy expertise and stage of illness^6,7. Parasitemia levels, especially for P. falciparum infections, may vary due to the cyclical periodicity of the parasite’s replication, microvascular sequestration and microscopy technique^8,9. False positives may be due to microscopy artifacts and false negatives may be due to very low parasitemia levels.

Antigen test results cannot provide definitive species identification, cannot rule out Plasmodium coinfections and cannot provide parasitemia levels. Sensitivity is reported at 96% for P. falciparum, 69% to 87% for P. vivax and 62% or lower for P. ovale or P. malariae^10,11. Antigen tests may remain positive up to 28 days following treatment and/or due to isolated non-pathogenic gametocytemia, limiting their use for treatment monitoring. False positives may occur with autoantibodies (e.g., rheumatoid factor, anti-nuclear antibodies, heterophile antibodies). False negatives may occur with very low parasitemia levels, very high parasitemia levels (postzone or hook effect), or with P. falciparum species harboring histidine-rich protein 2/3 (HRP-2/3) mutations. Although data is limited, P. knowlesi may also cross-react with either antigen bands.

PHO-developed Plasmodium real-time multiplex PCR has a reported clinical accuracy of 100% on EDTA samples for single infections, with minor reduced sensitivity to detect coinfections⁴. Limitations of PCR include persistence of DNA detection from non-pathogenic gametocytemia and/or residual DNA after treatment, limiting its use for treatment monitoring. Although likely to be detectable, this assay has not been validated specifically for Plasmodium knowlesi.

For serology, the NRCP states a sensitivity of 96% and specificity of 99% in immunocompetent individuals. It detects antibodies against P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. Other commercial EIAs have a reported sensitivity of 53 to 91% and specificity of 80 to 100%.¹²

Algorithm

Microscopy and antigen testing is routinely performed for all incoming malaria requests without requiring preapproval.

If PCR, antimicrobial resistance typing, or serology is needed and has not already been performed for the patient, contact PHO Laboratory Customer Service Centre at 416-235-6556 or 1-877-604-4567 for pre-approval.

For patients suspected of VHF, refer to the following webpage: Viral Hemorrhagic Fever (VHF) Viruses (e.g., Ebola, Marburg, Lassa, Crimean-Congo) – PCR. Malaria testing will first be performed using the BioFire Global Fever panel. If the BioFire assay is positive for malaria, follow-up species confirmation and parasitemia calculation will be automatically performed by PCR and thin smear microscopy (without thick smear microscopy and antigen testing).

Interpretation

For routine malaria antigen and microscopy testing (“Parasitology – Malaria”):

Parasitology – Malaria	Interpretation
Malarial parasite(s) found Asexual stages such as ring forms, trophozoites, or schizonts	Active malaria infection. Requires urgent clinical assessment. The malaria species, stage(s) and parasitemia level will be reported.
Malarial parasite(s) found Sexual stage (gametocyte) only	Prior malaria infection. No clinically relevant asexual stages present therefore, no evidence of active malaria infection. The malaria species and stage will be reported, but not the parasitemia level.
Indeterminate	Inconclusive results due to positive antigen test result but negative microscopy and negative confirmatory PCR results. This may represent a recently treated malaria infection or a false positive RDT result due to natural (e.g., rheumatoid factor) or infection-induced (e.g., viral or bacterial) autoantibodies and is rarely due to other parasitic infections (e.g., schistosomiasis, filarial nematodes, toxoplasmosis, leishmaniasis, or trypanosomiasis).
Malarial parasite(s) NOT found	Negative microscopy and antigen test results. Infection cannot be ruled out if only a single specimen was submitted. If the initial testing result is negative and no alternate diagnosis is established, one or two repeat collections over 24 to 48 hours should be considered.

For malaria PCR testing (“Malaria by Real Time PCR”):

Parasitology – Malaria	Interpretation
Detected	Malaria DNA present. Does not distinguish active from prior infection. Requires urgent clinical assessment. The malaria species will be reported.
Indeterminate	Inconclusive result. An indeterminate PCR result may be due to a low level of target genetic material in the specimen, inadequate specimen content, or a non-specific signal. Please resubmit another specimen for testing if clinically indicated.
Not detected	Malaria DNA not present.
Invalid	No result available. Test results are invalid due to the failed amplification of the extraction control. Amplification failure may be due to inadequate specimen content, extraction failure, or PCR inhibition. Please resubmit another specimen for testing if clinically indicated.

For malaria serology (“Malaria Antibody – Referred Out”):

Malaria EIA Sample Value	Interpretation
≥ 1.1	Detectable level of antibodies against Plasmodium spp. Cannot distinguish between acute and remote infection. Cross-reactivity may occur with Leishmania spp.
0.8 – 1.0	Plasmodium spp. antibody status inconclusive. Repeat testing advised if clinically indicated.
< 0.8	No detectable level of antibodies against Plasmodium spp.

Reporting

Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

References

Public Health Agency of Canada. Chapter 6: Malaria diagnosis. In: Canadian recommendations for the prevention and treatment of malaria. Government of Canada; [cited 2026 Jun 24]. Available from: https://www.canada.ca/en/public-health/services/catmat/canadian-recommendations-prevention-treatment-malaria/chapter-6-malaria-diagnosis.html
World Health Organization. Methods for surveillance of antimalarial drug efficacy. Global Malaria Programme (GMP). 2009 Oc 31:90. ISBN: 9789241597531
Rougemont M, Van Saanen M, Sahli R, Hinrikson HP, Bille J, Jaton K. Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays. J Clin Microbiol. 2004 Dec;42(12):5636-43. doi: 10.1128/JCM.42.12.5636-5643.2004.
Shokoples SE, Ndao M, Kowalewska-Grochowska K, Yanow SK. Multiplexed real-time PCR assay for discrimination of Plasmodium species with improved sensitivity for mixed infections. J Clin Microbiol. 2009 Apr;47(4):975-80. doi: 10.1128/JCM.01858-08.
Phuong M, Lau R, Ralevski F, Boggild AK. Sequence-based optimization of a quantitative real-time PCR assay for detection of Plasmodium ovale and Plasmodium malariae. J Clin Microbiol. 2014 Apr;52(4):1068-73. doi: 10.1128/JCM.03477-13.
Stauffer WM, Cartwright CP, Olson DA, Juni BA, Taylor CM, Bowers SH, Hanson KL, Rosenblatt JE, Boulware DR. Diagnostic performance of rapid diagnostic tests versus blood smears for malaria in US clinical practice. Clin Infect Dis. 2009 Sep 15;49(6):908-13. doi: 10.1086/605436.
Johnston SP, Pieniazek NJ, Xayavong MV, Slemenda SB, Wilkins PP, da Silva AJ. PCR as a confirmatory technique for laboratory diagnosis of malaria. J Clin Microbiol. 2006 Mar;44(3):1087-9. doi: 10.1128/JCM.44.3.1087-1089.2006.
Beaudry JT, Fairhurst RM. Microvascular sequestration of Plasmodium falciparum. Blood. 2011 Jun 16;117(24):6410. doi: 10.1182/blood-2010-09-305102.
Manser M, Olufsen C, Andrews N, Chiodini PL. Estimating the parasitaemia of Plasmodium falciparum: experience from a national EQA scheme. Malar J. 2013 Nov 22;12:428. doi: 10.1186/1475-2875-12-428.
Farcas GA, Zhong KJ, Lovegrove FE, Graham CM, Kain KC. Evaluation of the Binax NOW ICT test versus polymerase chain reaction and microscopy for the detection of malaria in returned travelers. Am J Trop Med Hyg. 2003 Dec;69(6):589-92.
Maltha J, Gillet P, Jacobs J. Malaria rapid diagnostic tests in endemic settings. Clin Microbiol Infect. 2013 May;19(5):399-407. doi: 10.1111/1469-0691.12151.
van den Hoogen LL, Bareng P, Alves J, Reyes R, Macalinao M, Rodrigues JM, Fernandes JM, Goméz LF, Hall T, Singh SK, Fornace K, Luchavez J, Kitchen A, Chiodini P, Espino F, Tetteh KKA, Stresman G, Sepúlveda N, Drakeley C. Comparison of Commercial ELISA Kits to Confirm the Absence of Transmission in Malaria Elimination Settings. Front Public Health. 2020 Sep 9;8:480. doi: 10.3389/fpubh.2020.00480.