Malaria (Plasmodium) – Microscopy, RDT, PCR and Typing

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 routine microscopy, rapid diagnostic testing (RDT), polymerase chain reaction testing (PCR), and resistance genotyping information for malaria detection and species identification at Public Health Ontario (PHO). The causative agent of malaria is the apicomplexan protist 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).

The test information described below is limited to microscopy, RDT, PCR, and resistance testing. For serology, please refer to the following link:

Malaria – Serology

*Uncontrolled print copy. Valid only on day of print: 01 May 2024.

Testing Indications

The Committee to Advise on Tropical Medicine and Travel (CATMAT) provides 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

Testing will be rejected in the following scenarios:

Specimens received in non-EDTA tubes (e.g. SST, citrate, heparin, SPS, uncoagulated/clotted)

Resistance genotyping will only be performed on positive specimens upon approval by PHO’s microbiologist.

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

PLUS

Unstained, unfixed thick smear

PLUS

Unstained, methanol-fixed thin smear

2.0 ml

2 slides

EDTA blood tube (labelled)

Slide mailer (each slide individually labelled)

Submission and Collection Notes

Complete all fields of the requisition form, including:

Test(s) requests and indications for testing
Patient setting/population
Clinical information including symptom onset date or if asymptomatic
Travel history – the country/region of exposure must be stated otherwise testing may be delayed or cancelled.

To assist in testing prioritization, state in the requisition if the patient is pregnant, has HIV, has impaired immunity, is part of an outbreak investigation, or is in the intensive care unit (ICU).

Specify under ‘Clinical Information, Other’ of any recent malaria test 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. Failure to provide this information may result in rejection or testing delay.

Storage and Transport

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’s laboratory. Delays greater than one hour between EDTA blood specimen collection and unstained smears creation 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’s laboratory as soon as possible. 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)

Malaria testing is performed daily at PHO’s Toronto laboratory site. Turnaround time is up to 24 hours from receipt at PHO’s laboratory.

STAT and Critical Specimens Testing

All malaria specimens received during regular PHO work hours will be expedited for testing. After hours, please contact PHO’s duty officer at 416-605-3113. STAT samples must be shipped separately from routine specimens in a clearly marked package indicating ‘STAT’ and handled in accordance with the Canadian Biosafety Standards and shipped in accordance with the Transportation of Dangerous Goods Regulations. Ship priority specimens directly to PHO’s Toronto laboratory site to the shipping and receiving dock at 661 University Ave., Toronto, Ontario. For delivery instructions please see Directions to 661 University Shipping Dock for Clinical Samples. Failure to ship separately will delay testing.

Test Methods

Microscopic examination is performed at PHO’s laboratory on thick and thin blood smears stained with Giemsa’s stain. Parasitemia levels are reported as a percentage of red blood cells (RBCs) infected on the thin smear examined. On average, 10 randomly selected 100X oil-immersion fields (about 1000 to 3000 total RBCs) are examined to calculate parasitemia levels, with fields devoid of parasites included if encountered, gametocytes excluded if present, and multiply-infected RBCs counted as one parasitized cell.

The RDT assay used at PHO is the Abbott BinaxNOW Malaria, which detects the Plasmodium falciparum-specific HRP-2 antigen and the pan-Plasmodium aldolase antigen.

PCR testing is performed using a laboratory-developed multiplex real-time PCR assay originally developed by Rougemont et al (2004) and optimized by Shokoples et al (2009) and Phuong et al (2014)^1,2,3. 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 genotyping is only available upon special request. For any questions related to antimalarial resistance genotyping, you may contact the Parasitology Microbiologist at PHO through our customer service centre.

Algorithm

At PHO, malaria testing is routinely performed by microscopic examination and RDT, with both assays performed in parallel.

PCR testing is not routinely performed on all malaria testing requests at this time. It is automatically performed as an additional test in the following instances:

RDT is positive but microscopy is negative for malaria;
Discordant microscopy findings between two laboratory technologists evaluating the same specimen;
Atypical or insufficient organisms/stages present by microscopy to provide definitive species identification;

PCR testing can also be requested following consultation with the Parasitology Operational Lead or Parasitology Microbiologist at PHO (e.g. if both RDT and microscopy are negative but clinical presentation is strongly suggestive of malaria).

Interpretation

RDT and microscopy results will be reported as a composite as follows:

Malaria results	Comments
Detected	Species identification by microscopy will be reported along with stage(s) seen and parasitemia level.
Not detected	Negative RDT and microscopy results. Infection cannot be ruled out by a single specimen tested. If the initial testing result is negative and no alternate diagnosis is established, two additional specimens should be collected at 12 to 24-hour intervals for testing.
Inconclusive	Positive RDT result but negative microscopy and 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 rarely due to other parasitic infections (e.g. schistosomiasis, filarial nematodes, toxoplasmosis, leishmaniasis, or trypanosomiasis).

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^4,5. Parasitemia levels, especially for P. falciparum infections, may vary due to microvascular sequestration or microscopy technique^6,7. False positives may be due to microscopy artifacts, and false negatives may be due to very low parasitemia levels.

RDT results are supportive of infection with either Plasmodium falciparum and/or a non-falciparum species. RDTs cannot provide definitive speciation, discrimination of Plasmodium co-infections, or 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^8,9. RDTs may remain positive up to 28 days following treatment and/or with isolated 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 (prozone or hook effect), or with P. falciparum species harboring histidine-rich protein 2 (HRP-2) mutations. P. knowlesi may also cross-react with the P. falciparum HRP-2 band.

PHO’s laboratory-developed Plasmodium real-time multiplex PCR has with a reported clinical accuracy of 100% on EDTA samples for single infections, with minor reduced sensitivity to detect coinfections³. Limitations of PCR include persistence of DNA detection from gametocytemia and/or residual DNA after treatment, limiting its use for treatment monitoring. The assay has not been validated for Plasmodium knowlesi.

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

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.