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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
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.
A positive result indicates the presence of Plasmodium nucleic acid and 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 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.
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