Microscopic examination of stained blood smears is considered the gold
standard for diagnosis of malaria
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«
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.
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
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 Ál 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 Ál of extracted DNA is
amplified using genus specific primers; in the second step (PCR2), 1 Ál 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 Ál of extracted DNA was
amplified using B. microti specific primers, Bab1 and Bab4; in the
second step (PCR2), 1 Ál 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.
- 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.
- 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
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 firstname.lastname@example.org.