Molecular Diagnosis

Specimen Collection
Microscopic examination of stained blood smears is considered the gold standard for diagnosis of malaria and babesiosis.  When species determination cannot be made by microscopic examination, analysis by polymerase chain reaction (PCR) is helpful.  Collect a 1-5 ml blood sample in Vacutainer® EDTA tubes prior to anti-parasitic therapy and ship at 4°C to a reference laboratory.  Alternatively, blood can be collected on filter papers (e.g., products available through Whatman® http://www.whatman.com).  Punching the spots may increase the risk of cross-contamination among specimens.  Spot the paper directly from whole blood or finger stick.  Follow all shipment guidelines and requirements.  Blood smears should always accompany the EDTA blood sample.  The blood smears will be examined first; PCR will be performed only if species determination cannot be made from the blood smears.

The following procedure describes how a specimen will be accepted for PCR analysis at CDC.  Prior arrangements should be made to determine the appropriateness of PCR as an adjunct for the diagnosis of malaria and babesiosis.  At this time, PCR analysis takes approximately one week for completion.

DNA has to be extracted from the blood specimens for PCR detection.  Click to view the DNA extraction protocols recommended for molecular diagnosis of malaria and babesiosis.

Species-specific PCR for Diagnosis of Malaria
Plasmodium sp. genomic DNA is extracted from 200 µl whole blood using the QIAamp Blood Kit (Cat. No. 29106; Qiagen Inc., Chatsworth, CA) or a similar product that can yield the comparable concentration of genomic DNA from the same volume of blood.  Detection and speciation of Plasmodium is done with a two step nested PCR using the primers of Snounou et al 1993.  In the first step (PCR1), 1 µl of extracted DNA is amplified using genus specific primers; in the second step (PCR2), 1 µl of PCR1 amplification product is further amplified using primers specific for each Plasmodium species.  Ten microliters of each PCR2 amplified DNA product is electrophoretically resolved on a 2% agarose gel, stained for 15 min with ethidium bromide and visualized by UV illumination for analysis of results.

Species-specific PCR for Diagnosis of Babesia
Babesia sp. genomic DNA is extracted in the same way as Plasmodium sp. DNA (see above).  Detection of Babesia microti is done with a two step nested PCR using the primers of Persing et al.  In the first step (PCR1), 1 µl of extracted DNA was amplified using B. microti specific primers, Bab1 and Bab4; in the second step (PCR2), 1 µl of PCR1 amplification product was further amplified using internal primers, Bab2 and Bab3.  Ten microliters of PCR2 amplified DNA product was electrophoretically resolved on a 2% agarose gel, stained for 15 minutes with ethidium bromide and visualized by UV illumination for analysis of results.

References:

1. Persing D, Mathiesen D, Marshall WF, Telford SR, Spielman A, Thomford JW, Conrad PA. Detection of Babesia microti by Polymerase Chain Reaction. J Clin Microbiol 1992;30:2097-2103.
2. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario VE, et al. High sensitivity detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parastiol 1993;61:315-320.