Blood Specimens - 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 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 identification of Plasmodium is done with a real-time PCR assay as described by Rougemont et al 2004. This is a dual duplex assay that detects P. falciparum and P. vivax in one reaction, and P. malariae and P. ovale in a parallel reaction, using species-specific TaqMan probes. In cases where infection by more than one Plasmodium species is suspected, there is an option to use a conventional nested PCR assay (Snounou el al, 1993) that has an improved resolution of mixed infection compared to the real-time PCR assay.

Species-specific PCR for Diagnosis of Babesia

Babesia genomic DNA is extracted in the same way as Plasmodium sp. DNA (see above). Detection of Babesia microti is done with a real-time PCR assay using B. microti–specific primers and TaqMan probe as described by Hojgaard et al 2014. For other species, we amply a fragment of the 18S rRNA gene using PCR primers specific at the genus level (Bonnet et al. 2007) and perform DNA sequencing of the amplicon to determine species.

References:

1. 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, 42(12):5636.
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.
3. Hojgaard A, Lukacik G, Piesman J. Detection of Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti, with two different multiplex PCR assays. Ticks and Tick-borne Diseases 2014 (5):349–351.
4. Bonnet S, Jouglin M, Malandrin L, Becker C, A. Agoulon A, L’Hostis M, Chauvin A. Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique. Parasitol 2007;134:197–207

For more information on molecular diagnosis for blood specimens, contact the Division of Parasitic Diseases at (404) 718-4120. For information about the technical aspects of PCR for molecular diagnosis of malaria and babesiosis, send an e-mail to dpdx@cdc.gov.