Malaria, Molecular Detection, PCR
NY State Approved Indicates the status of NY State approval and if the test is orderable for NY State clients.
Detection of Plasmodium DNA and identification of the infecting species
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
Real-Time Polymerase Chain Reaction (PCR)/DNA Probe Hybridization
(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)
Reporting Name A shorter/abbreviated version of the Published Name for a test; an abbreviated test name
Specimen Type Describes the specimen type needed for testing
Specimen Required Defines the optimal specimen. This field describes the type of specimen required to perform the test and the preferred volume to complete testing. The volume allows automated processing, fastest throughput and, when indicated, repeat or reflex testing.
Malaria is potentially a life-threatening disease and testing for this infection should be performed as rapidly as possible. Therefore, this test should not be used as a primary screening test for malaria, except for clients in the immediate Rochester, Minnesota area when the specimen can be delivered within several hours of collection. Laboratories that are unable to deliver a specimen within this time frame should provide an initial screen for malaria and other blood parasites in their laboratory prior to sending a specimen to Mayo Medical Laboratories. This test is useful for confirmation of a presumptive malaria diagnosis and determination of infecting Plasmodium species.
Forms: If not ordering electronically, submit a Microbiology Request Form (Supply T244) with the specimen.
Blood and slides are required.
Specimen Type: Blood
Preferred: Lavender top (EDTA)
Acceptable: Blue top (sodium citrate)
Specimen Volume: 4 mL
1. Invert several times to mix blood.
2. Do not transfer blood to other containers. Send specimen in original tube.
Specimen Stability Information: Refrigerated
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
1. Ideally, blood films should be made directly from uncoagulated blood acquired via fingerstick. 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 Stability Information: Refrigerated (preferred)/Ambient
Specimen Minimum Volume Defines the amount of specimen required to perform an assay once, including instrument and container dead space. Submitting the minimum specimen volume makes it impossible to repeat the test or perform confirmatory or perform reflex testing. In some situations, a minimum specimen volume may result in a QNS (quantity not sufficient) result, requiring a second specimen to be collected.
Blood: 1 mL/Slides: NA
Mild OK; Gross reject
Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the laboratory. Alternate acceptable temperature(s) are also included.
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Malaria is a major tropical disease infecting approximately 500 million people and causing 1.5 to 2.7 million deaths annually. Ninety percent of the deaths occur in sub-Saharan Africa and most of these occur in children <5 years old; it is the leading cause of mortality in this age group. This disease is also widespread in Central and South America, Hispaniola, the Indian subcontinent, the Middle East, Oceania, and Southeast Asia. In the United States, individuals at risk include travelers to and visitors from endemic areas.
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 similar parasite that may be an important source of human infection in some regions of Southeast Asia. Differentiating Plasmodium falciparum and Plasmodium knowlesi from other species is important since both can cause life-threatening infections. In addition, Plasmodium 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 speciation of malaria parasites. Under optimal conditions, sensitivity of 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 Plasmodium falciparum ring forms (early trophozoites) on peripheral blood films, resulting in potential diagnostic confusion.
PCR is an alternative method of 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.
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
A positive result indicates the presence of Plasmodium nucleic acid and melting curve analysis indicates the infecting species.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
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 Medical 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 "Plasmodium vivax/Plasmodium ovale." These 2 species have similar prognosis and treatment, 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 test should not be used to screen asymptomatic patients.
This PCR assay does not detect other parasites that may be present in the blood and have similar disease presentations including Babesia and Trypanosoma species.
The following supports the use of this assay for clinical testing.
Accuracy/Diagnostic Sensitivity and Specificity:
160 clinical whole blood specimens were evaluated for the presence of Plasmodium species DNA or the 18S rRNA gene using a LightCycler (MAL-K) assay. This assay detects and speciates DNA of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. The results were compared to results of microscopy, a nested PCR method and sequencing. The specimens comprised 48 negative and 108 positive specimens (32 Plasmodium falciparum, 8 Plasmodium malariae, 20 Plasmodium ovale, 45 Plasmodium vivax, 2 unable to speciate by morphology, and 1 mixed infection of Plasmodium vivax/falciparum). The sensitivity and specificity of the MAL-K assay compared to microscopy, nested PCR and sequencing was 99% and 94% respectively, with all species determinations by the PCR assay matching the original result. No Plasmodium knowlesi clinical specimens were available so spiking studies were performed. 30 EDTA-anticoagulated blood specimens received in the laboratory for unrelated testing were spiked with Plasmodium 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 (LoD) of this assay is 10 to 50 DNA target copies per microliter in whole blood.
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.
Inter-assay precision was 100% and the intra-assay precision was 100%.
This is a qualitative assay and the results are reported as either negative or positive for the targeted Plasmodium species.
Clinical Reference Provides recommendations for further in-depth reading of a clinical nature
1. 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
2. World Health Organization Malaria Page: http://www.who.int/topics/malaria/en/
3. Cox-Singh J, Davis T, Lee K et al: Plasmodium knowlesi malaria in humans is widely distributed and potentially life-threatening. Clin Infect Dis 2008 January 15;46(2):165-171
4. 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
Method Description Describes how the test is performed and provides a method-specific reference
DNA from EDTA-anticoagulated whole blood is extracted and tested using real-time PCR on the LightCycler 2.0 instrument (Roche Applied Science) with primers and fluorescence resonance energy transfer (FRET) probes. A genus-specific primer set corresponding to 18S rRNA 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 use of 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) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Sunday; Varies
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Same day/1 day
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
Performing Laboratory Location The location of the laboratory that performs the test
Test Classification Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer's instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR), Investigation Use Only (IUO) product, or a Research Use Only (RUO) product.
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 U.S. Food and Drug Administration.
CPT Code Information Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Medical Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.
LOINC® Code Information Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the result codes returned for this test or profile.
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