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.

Plasmodium malariae and Plasmodium ovale – the ‘bashful’ malaria parasites

“…These assays enable detection of the four Plasmodium parasite species that infect humans at densities ~100 times lower than the limit of LM detection [47] and have evolved from simple species-specific amplification strategies to multiplex approaches that incorporate semiquantitative assessments [31,[49][50][51][52][53][54][55][56]. We have greatly extended the potential use of PCR diagnosis for a wide range of epidemiological studies through our recent efforts to develop a ligase detection reaction fluorescent microsphere assay (LDR-FMA) [52,57] to evaluate all four malaria parasites of humans in a single-well multiplex format.…”

Plasmodium falciparum Na+/H+ Exchanger (pfnhe-1) Genetic Polymorphism in Indian Ocean Malaria-Endemic Areas

“…26 The parasite species was confirmed by real-time polymerase chain reaction (PCR) as described in the work by Mangold and others. 27 Amplification and sequencing of the ms4760 locus in the P. falciparum Na + /H + exchanger gene (pfnhe-1) was performed in accordance with the protocol described earlier.…”

Prevalence of Plasmodium falciparum and P. vivax in an area of transmission located in Pará State, Brazil, determined by amplification of mtDNA using a real-time PCR assay

“…Although light microscopy performed under optimal conditions can detect parasitemia as low as 5 parasites/mL (0.0001%) on thick blood smears (Warrell, 2002), molecular assays, particularly those involving qPCR, are well documented to have various advantages over microscopic examination. Compared with the gold-standard nested-PCR assay, qPCR methods have shown high sensitivity (100%) and specificity (100%) for the simultaneous detection of Plasmodium species and have the additional advantage of yielding results in less than 3 h (Perandin et al, 2004;Mangold et al, 2005;Boonma et al, 2007). To bypass one of the main limitations of the standard PCR technique, which is the need for multiple PCR assays on each sample because the target gene is present in only a limited number of copies, Cunha et al (2009) standardized a PCR method to detect P. falciparum and P. vivax through amplification of an mtDNA sequence, which is present in a large number of copies in infected cells.…”