Real-time PCR for detection and identification of Plasmodium spp

Abstract

Rapid and accurate detection of malaria parasites in blood is needed to institute proper therapy. We developed and used a real-time PCR assay to detect and distinguish four Plasmodium spp. that cause human disease by using a single amplification reaction and melting curve analysis. Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and SYBR Green was used for detection in a LightCycler instrument. Patient specimens infected at 0.01 to 0.02% parasitemia densities were detected, and analytical sensitivity was estimated to be 0.2 genome equivalent per reaction. Melting curve analysis based on nucleotide variations within the amplicons provided a basis for accurate differentiation of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. For assay validation, 358 patient blood samples from the National University Hospital in Singapore and Evanston Northwestern Healthcare in Illinois were analyzed. Of 76 blinded patient samples with a microscopic diagnosis of P. falciparum, P. vivax, or P. ovale infection, 74 (97.4%) were detected by real-time PCR, including three specimens containing mixed P. falciparum-P. vivax infections. No Plasmodium DNA was amplified in any of the 82 specimens sent for malaria testing but that were microscopically negative for Plasmodium infection. In addition, 200 blood samples from patients whose blood was collected for reasons other than malaria testing were also determined to be negative by real-time PCR. Real-time PCR with melting curve analysis could be a rapid and objective supplement to the examination of Giemsa-stained blood smears and may replace microscopy following further validation.

Figures

FIG. 1.

Real-time amplification with SYBR Green fluorescence detection. The plasmid controls for four species, water blank, and negative human control DNA are indicated. The remaining curves are patient specimens with various parasitemia levels. The graph was generated by using LightCycler Software v. 3.

FIG. 2.

Melting curve analysis, with the control plasmids, water blank, and negative human control DNA labeled. Vertical lines indicating the Tm values for each of the plasmid controls are continued in Fig. 3 through 5. The graph was generated by using LightCycler Software v. 3.

FIG. 3.

Melting curve analysis: DNA isolated from blood by using the Puregene procedure from monkeys infected with either P. malariae or P. vivax (ATCC) and purified P. falciparum genomic DNA (ATCC). The graph was generated by using LightCycler Software v. 3.

FIG. 4.

Melting curve analysis: patient samples infected with P. falciparum, P. ovale, or P. vivax prepared by using the ISOCODE card procedure for DNA extraction. Patient sample infected with both P. falciparum and P. vivax is highlighted with a dashed line, and the patient specimen with P. ovale is highlighted with solid circle points. The graph was generated by using LightCycler Software v. 3.

FIG. 5.

Melting curve analysis: discordant patient samples. Dual P. falciparum and P. vivax infections not microscopically identified are highlighted with dashed lines. The blue dashed line represents a specimen prepared by using the Puregene method, and the red dashed line is one prepared by using the standard ISOCODE method, exemplifying the slightly lower Tm peaks resulting from the former method. The black diamonds indicate microscopically diagnosed P. falciparum specimens that amplified beyond the 35 cycle cutoff limit used in the present study. The graph was generated by using LightCycler Software v. 3.