Real-Time Nucleic Acid Sequence-Based Amplification Is More Convenient than Real-Time PCR for Quantification of Plasmodium falciparum

Abstract

Determination of the number of malaria parasites by routine or even expert microscopy is not always sufficiently sensitive for detailed quantitative studies on the population dynamics of Plasmodium falciparum, such as intervention or vaccine trials. To circumvent this problem, two more sensitive assays, real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) and real-time quantitative PCR (QT-PCR) were compared for quantification of P. falciparum parasites. QT-NASBA was adapted to molecular beacon real-time detection technology, which enables a reduction of the time of analysis and of contamination risk while retaining the specificity and sensitivity of the original assay. Both QT-NASBA and QT-PCR have a sensitivity of 20 parasites/ml of blood, but QT-PCR requires a complicated DNA extraction procedure and the use of 500 l of venous blood to achieve this sensitivity, compared to 50 l of finger prick blood for real-time QT-NASBA. Both techniques show a significant correlation to microscopic parasite counts, and the quantification results of the two real-time assays are significantly correlated for in vitro as well as in vivo samples. However, in comparison to real-time QT-PCR, the results of real-time QT-NASBA can be obtained 12 h earlier, with relatively easy RNA extraction and use of finger prick blood samples. The prospective development of multiplex QT-NASBA for detection of various P. falciparum developmental stages increases the value of QT-NASBA for malaria studies. Therefore, for studies requiring sensitive and accurate detection of P. falciparum parasites in large numbers of samples, the use of real-time QT-NASBA is preferred over that of real-time QT-PCR.Routine clinical diagnosis of malaria is usually based on microscopic detection of Plasmodium parasites in blood smears. However, this technique is relatively laborious when large numbers of samples need to be quantified simultaneously. Furthermore, the detection limit of microscopy, 1 to 20 parasites per l of blood, may not always be sufficiently sensitive. Parasite densities below the detection level of microscopy may play an important role in Plasmodium population dynamics and the epidemiology of the disease; therefore, the availability and use of more-sensitive detection techniques is a prerequisite for many research projects. With the rapid developments in the field of molecular biology, several nucleic acid-based amplification methods, including PCR, reverse transcriptase PCR, and nucleic acid sequence-based amplification (NASBA) (2, 3, 7-9, 11), are now available for detection of Plasmodium parasites. Because many studies require accurate and sensitive quantification of parasites, most of these techniques have been adapted for quantitative analysis (1,3,6,7,11). In preparation for vaccine trials and epidemiological studies directed at evaluation of interventions, the present study was designed to evaluate the newly developed real-time quantitative NASBA (QT-NASBA) and to compare the assay to another quantitative nucleic acidba...

Potentials and limitations of molecular diagnostic methods in food safety

“…The NASBA-based diagnostic assay for the detection of Plasmodium falciparum proved to provide results in less time than the RT-PCR equivalent [48]. RNA based techniques, such as NASBA or RT-PCR, have been mainly applied to the detection of retroviruses, leaving the diagnosis of bacteria to genomic DNA based systems.…”

Malaria Diagnostics in Clinical Trials

“…With highly sensitive assays, parasites may be detected in blood 1-4 days earlier than by microscopy. 29,83 However, it should be noted that not all molecular assays are highly sensitive and some report sensitivities more comparable to thick blood smears. In addition, quantification provided by some molecular methods makes it possible to determine the kinetics of asexual parasite growth in drug or vaccine trials.…”

“…111,112 In these studies, the usual trigger for drug treatment is blood smear-confirmed parasitemia. Because highly sensitive and quantitative molecular assays detect pre-patent parasitemia up to four days before blood smears, 29,83,113,114 it is currently possible to estimate parasite replication kinetics based on molecular assay results obtained in the days before drug treatment. 29,114,115 The CHMI trials often enroll healthy volunteers in Phase I/IIa studies and are often conducted on malaria-naive persons in non-endemic regions.…”

“…Over the past 20 years, many molecular assays for malaria have been developed. [72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91] Among published methods, 65 primer sets have been reported with at least five molecular targets used to test for as many as five human malaria species. Methods include single-step PCR with electrophoresis gel-based detection or DNA probe-based real-time detection, nested PCR with gel-based or real-time SYBR Green dye-based detection, nucleic acid-based sequence amplification (NASBA) and real-time RT-PCR with probe-based detection.…”

In vivo and in vitro gametocyte production of Plasmodium falciparum isolates from Northern Thailand

“…The recent application of more sensitive molecular detection methods such as real-time nucleic acid sequence based amplification (QT-NASBA) (Schneider et al, 2005), reverse transcription-PCR (RT-PCR) (Maeno et al, 2008) and reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) (Buates et al, 2010) have revealed that the proportion of parasite-infected individuals who harbour gametocytes is much higher than previously thought, and has led to a much clearer picture of the patterns of gametocyte carriage amongst infected individuals (Ouedraogo et al, 2009). The limit of detection of gametocytes by thicksmear microscopy is in the range of five gametocytes per µl, while molecular techniques such QT-NASBA can detect as few as 0.02 gametocytes per µl (Schneider et al, 2007).…”