Comparison of three real-time PCR methods with blood smears and rapid diagnostic test in Plasmodium sp. infection

Abstract

In cases of malaria, rapid and accurate diagnosis of Plasmodium sp. is essential. In this study three different quantitative, real-time PCR methods were compared with routine methods used for malaria diagnosis. A comparative study was conducted prospectively in the laboratories of Montpellier and Nîmes University Hospitals. The methods used for routine diagnostic malaria testing consisted of microscopic examination of Giemsa-stained blood smears and rapid diagnostic tests. Three quantitative real-time PCR methods (qRT-PCR) were tested: qRT-PCR1 amplified a specific sequence on the P. falciparum Cox1 gene, qRT-PCR2 amplified a species-specific region of the multicopy 18S rDNA, and qRT-PCR3 amplified a mitochondrial DNA sequence. Among the 196 blood samples collected, 73 samples were positive in at least one of the five tests. Compared with the routine method, there were no false negatives for P. falciparum diagnosis in either qRT-PCR1 or qRT-PCR3. In all P. ovale, P. vivax and P. malariae infections diagnosed from blood smears, qRT-PCR1 was negative, as expected, whereas qRT-PCR2 and qRT-PCR3 were positive and concordant (simple kappa coefficient = 1). One negative sample from microscopy was positive with both qRT-PCR2 and qRT-PCR3. Together, qRT-PCR3 and the combined qRT-PCR1 and qRT-PCR2 were concordant with routine methods for malaria diagnosis (99% and 99.5%, respectively). These three rapid, molecular qRT-PCR methods, used alone or in association, showed excellent results, with high concordance, accuracy and reliability in malaria diagnosis.

Cytochrome b Gene Quantitative PCR for Diagnosing Plasmodium falciparum Infection in Travelers

“…The sensitivity of microscopy, about 5 to 100 parasites/l of blood (12,19), is surpassed by that of the cytb PCR assay, with a limit of detection of around 20 parasites/400 l. Many qPCR assays have been described; most of them target the 18S rRNA-encoding gene of Plasmodium spp. (3,9,13,17,22,25,26,29). The mitochondrial genes have been less frequently studied (3,11,26).…”

“…We developed a qPCR assay targeting the mitochondrial cytochrome b gene and compared our results to those of an already published qPCR method targeting the 18S rRNA-encoding gene (17). The 18S rRNA gene is one of the most often reported targets in qPCR (1,3). However, there are some reports suggesting that mitochondrial targets could be more sensitive than ribosomal ones (11,28).…”

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 the qPCR method requires specific material and is more expensive than mi-Prevalence of P. falciparum and P. vivax in Pará State croscopy and conventional PCR (Berry et al, 2005), it presents advantages of rapidity, lower contamination, and better standardization and can be used for routine testing, particularly for the study of populations in endemic areas in which patients may be asymptomatic (Mens et al, 2007;Boonma et al, 2007;Gama et al, 2007;Veron et al, 2009) and in patients who have negative results on routine methods but are strongly suspected of having malaria (Bourgeois et al, 2010). Thus, qPCR assay based on the detection of mitochondrial cytochrome c oxidase genes may be the method of choice for specific situations, including detection in low-parasitized individuals.…”

“…Nevertheless, the need for a more sensitive and time-efficient assay has led to the development of molecular assays using real-time PCR (qPCR), a procedure that can detect low levels of parasitemia, identify mixed infections, and allow for the precise differentiation of species via melting curve analysis or TaqMan fluorescence-labeled probes. Since the first report on this technique in 2001 (Hermsen et al, 2001), at least 17 assays have been developed, most of which use small subunit ribosomal RNA as the target (Review in Bourgeois et al, 2010).…”

Molecular Testing for Plasmodium falciparum by Use of Serum or Plasma and Comparison with Microscopy and Rapid Diagnostic Testing in Febrile Nigerian Patients

“…Although Plasmodium nucleic acids have been detected in serum and plasma, there is little published data describing the performance of malaria NAATs using these specimen types (4)(5)(6)(7). Rather, most studies have used whole blood or dried blood spots (DBS) (8)(9)(10)(11)(12). While whole blood and DBS provide ample amounts of Plasmodium nucleic acids, they present significant challenges for (i) long-term storage and/or (ii) nucleic acid extraction with removal of inhibitors for optimal NAAT performance (13,14).…”