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Test ID LMALP Malaria PCR with Parasitemia Reflex, Varies

Useful For

Detection of Plasmodium DNA and identification of the infecting species, with reflex percent parasitemia calculated using thin blood films for positive cases


An adjunct to conventional microscopy of Giemsa-stained films


Detection and confirmatory identification of Plasmodium species: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi


This test should not be used to screen asymptomatic patients.

Testing Algorithm

If positive, the percent parasitemia will be performed at an additional charge.


For more information see Malaria Laboratory Testing Algorithm.

Method Name

Real-Time Polymerase Chain Reaction (PCR)/DNA Probe Hybridization

Reporting Name

Malaria PCR with Parasitemia Reflex

Specimen Type


Ordering Guidance

1. When monitoring percent parasitemia for patient response to therapy, order MAL / Rapid Malaria/Babesia Smear, Varies (conventional blood film exam) instead of this test.

2. This test is not performed on a STAT basis and, therefore, should not be used as a primary screening test for malaria.

3. This test is used primarily to confirm a presumptive malaria diagnosis and to determine infecting Plasmodium species, particularly when the parasite morphology on traditional blood films is suboptimal.

4. Clients in the Rochester, MN area who are seeking a primary test for malaria and who can deliver the specimen within 4 hours of collection should order MAL / Rapid Malaria/Babesia Smear, Varies.

5. Laboratories that are unable to deliver a specimen within 4 hours of collection should perform an initial screen for malaria and other blood parasites in their laboratory prior to sending a specimen to Mayo Clinic Laboratories.

Shipping Instructions

Label all slides and place dry slides in a labeled slide box. Rubber band labeled slide box and labeled EDTA tube together and send to lab refrigerate.

Specimen Required

Both blood specimens and slides are required.


Specimen Type: Blood

Container/Tube: Lavender top (EDTA)

Specimen Volume: 3 mL

Collection Instructions:

1. Invert several times to mix blood.

2. Send whole blood specimen in original tube. Do not aliquot.


Specimen Type: Blood films

Container/Tube: Clean, grease-free slides in plastic slide container

Specimen Volume: 2 Thin blood films and 2 thick blood films

Collection Instructions:

1. Blood films should be made from fresh blood using fingerstick or drops of blood from needle following venipuncture. However, EDTA anticoagulated blood is also acceptable.

2. Prepare thin blood films as follows:

 a. Prepare a thin film with a "feathered edge" that is no more than a single cell thick.

 b. Allow the film to thoroughly air dry and then fix by briefly immersing in either absolute or 95% methyl alcohol.

 c. Allow to air dry after fixation.

3. Prepare thick blood films as follows:

 a. Place a large drop of blood (approximately the size of a dime and preferably from a fingerstick) on a slide.

 b. Using a corner of a second slide, spread the drop in a circular motion while applying firm pressure to literally scratch the blood onto the carrier slide. This technique allows the blood to dry quickly and adhere well to the slide. Use approximately 20 circular sweeps with the second slide. The drop of blood should be about the size of a quarter when finished.

 c. Do not fix. Air dry thoroughly (approximately 45 minutes) before placing in transport container.

Specimen Minimum Volume

Blood: 1 mL
Slides: See Specimen Required

Specimen Stability Information

Specimen Type Temperature Time Special Container
Varies Refrigerated (preferred) 7 days
  Ambient  7 days

Reject Due To

Gross hemolysis Reject
Gross lipemia Reject

Clinical Information

Malaria is a mosquito-transmitted disease caused by apicomplexan parasites in the genus Plasmodium. It is an important cause of morbidity and mortality worldwide, with the World Health Organization estimating 219 million cases and 435,000 malaria-related deaths in 2017. Malaria disproportionately affects individuals living in Africa (90% of cases), with individuals living in southeast Asia and the eastern Mediterranean regions next most affected. Malaria is also encountered outside of endemic regions, such as the United States, usually in returning travelers.


Malaria is caused primarily by 4 species of the protozoa Plasmodium: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale. A fifth Plasmodium species, Plasmodium knowlesi, is a simian parasite that may be an important source of human infection in some regions of Southeast Asia. Differentiating P falciparum and P knowlesi from other species is important since both can cause life-threatening infections. In addition, P falciparum is typically resistant to many commonly used antimalarial agents, such as chloroquine.


Microscopy of Giemsa-stained thick and thin blood films is the standard laboratory method for diagnosis and species identification of malaria parasites. Under optimal conditions, the sensitivity of the thick film microscopy is estimated to be 10 to 30 parasites per microliter of blood. However, microscopic diagnosis requires considerable expertise and may be insensitive or nonspecific when inadequate training and facilities are available. Furthermore, prolonged exposure to EDTA, transportation conditions, and prior use of antimalarial drugs may alter parasite morphology and negatively impact the ability to perform speciation by microscopy. Finally, Babesia parasites have a similar appearance to P falciparum ring forms (early trophozoites) on peripheral blood films, resulting in potential diagnostic confusion.


Polymerase chain reaction (PCR) analysis is an alternative method for malaria diagnosis that allows for sensitive and specific detection of Plasmodium species DNA from peripheral blood. PCR may be more sensitive than conventional microscopy in very low parasitemias and is more specific for species identification. It may be particularly useful when subjective microscopy does not permit certain identification of the species present. Malaria PCR can be used in conjunction with a traditional blood film or Babesia PCR when the clinical or morphologic differential includes both babesiosis and malaria. Examination of the thin film also allows for calculation of percent parasitemia, which can be used to predict prognosis and monitor response to treatment.

Reference Values



A positive result indicates the presence of Plasmodium nucleic acid, and the melting curve analysis indicates the infecting species.


Malaria is potentially a life-threatening disease, and it is imperative to test for parasites as rapidly as possible. Therefore, this test is for confirmation only, except for clients in the immediate Rochester, Minnesota area who can provide rapid delivery of specimens to Mayo Clinic Laboratories.


Assay may be negative in very low parasitemias.


Species of Plasmodium present in mixed infections may not be clearly delineated.


In some instances, the closely related species, Plasmodium ovale and Plasmodium vivax, cannot be differentiated from one another by this test. In this instance, results will be reported as "P vivax/P ovale." These 2 species have similar prognoses and treatments and can often be distinguished based on patient travel history.


This assay does not distinguish between residual nucleic acid (which may persist after adequate treatment) and viable intact parasites. It also does not distinguish between gametocytes (nonpathogenic forms that may be present in resolving infections) and virulent trophozoites.


Although the reference range is considered "negative" for individuals living in nonendemic areas, this assay may detect low-grade asymptomatic parasitemia from individuals exposed to malaria-endemic areas. However, this assay is designed to detect only Plasmodium species of clinical significance and is to be used for patients with a clinical history and symptoms consistent with malaria.


This polymerase chain reaction assay does not detect other parasites that may be present in the blood and have similar disease presentations, including Babesia and Trypanosoma species.

Supportive Data

The following supports the use of this assay for clinical testing.


Accuracy/Diagnostic Sensitivity and Specificity:

A total of 160 clinical whole blood specimens were evaluated for the presence of Plasmodium species DNA or the 18S ribosomal RNA gene using this real-time polymerase chain reaction (PCR) assay. The assay detects and differentiates DNA of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. The results were compared to results of microscopy and a nested PCR method and sequencing. The specimens comprised 48 negative and 108 positive specimens (32 P falciparum, 8 P malariae, 20 P ovale, 45 P vivax, 2 unable to determine species by morphology, and 1 mixed infection of P vivax/falciparum). The sensitivity and specificity of the real-time Plasmodium assay compared to microscopy and nested PCR and sequencing was 99% and 94%, respectively, with all species determinations by the PCR assay matching the original result. No P knowlesi clinical specimens were available, so spiking studies were performed. Thirty EDTA-anticoagulated blood specimens received in the laboratory for unrelated testing were spiked with P knowlesi plasmid near the limit of detection (50-100 targets per microliter) and tested in a blinded fashion with negative blood specimens; 100% concordance was achieved in the spiking studies.


Analytical Sensitivity/Limit of Detection:

The lower limit of detection of this assay is 10 to 50 DNA target copies per microliter in whole blood.


Analytical Specificity:

No PCR signal was obtained from extracts of 31 other bacterial, viral, rickettsial, and parasitic isolates that could be found in whole blood and cause similar symptoms, including Babesia, Borrelia, Anaplasma, and Ehrlichia species.



Interassay precision was 100% and the intra-assay precision was 100%.


Reportable Range:

This is a qualitative assay, and the results are reported as either negative or positive for the targeted Plasmodium species. Percent parasitemia will also be calculated using microscopic examination of blood smears.

Clinical Reference

1. Global Health, Division of Parasitic Diseases and Malaria: Malaria. CDC; Updated March 28, 2023. Accessed March 29, 2023. Available at

2. Swan H, Sloan L, Muyombwe A, et al: Evaluation of a real-time polymerase chain reaction assay for the diagnosis of malaria in patients from Thailand. Am J Trop Med Hyg. 2005 Nov;73(5):850-854

3. World Health Organization (WHO). Malaria. WHO; Updated December 8, 2022. Accessed March 29, 2023. Available at

Method Description

DNA from EDTA-anticoagulated whole blood is extracted and tested using real-time polymerase chain reaction (PCR) on the LightCycler 480 instrument (Roche Applied Science) with primers and fluorescence resonance energy transfer (FRET) probes. A genus-specific primer set corresponding to 18S ribosomal RNA is used to amplify target sequence. One pair of FRET hybridization probes was designed for Plasmodium falciparum over a region containing base pair mismatches allowing for differentiation of other Plasmodium species by using melting curve analysis, while a second probe set is specific for Plasmodium knowlesi.(Babady NE, Sloan LM, Rosenblatt JE, Pritt BS: Detection of Plasmodium knowlesi by real-time polymerase chain reaction. Am J Trop Med Hyg. 2009 Sept;81(3):516-518)


Slides are used to determine percentage of parasitemia if PCR is positive.

Day(s) Performed

Monday through Sunday

Report Available

Same day/1 to 3 days

Test Classification

This test was developed, and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information


87207 (if applicable)

LOINC Code Information

Test ID Test Order Name Order LOINC Value
LMALP Malaria PCR with Parasitemia Reflex 47260-5


Result ID Test Result Name Result LOINC Value
37116 Malaria PCR w/ Parasitemia 47260-5

Reflex Tests

Test ID Reporting Name Available Separately Always Performed
MALCT Plasmodium Percent Parasitemia Rflx No No