Evaluation of a Real-Time Polymerase Chain Reaction Assay for the Diagnosis of Malaria in Patients From Thailand

Abstract

We compared the diagnosis of malaria in 297 patients from Thailand by a real-time polymerase chain reaction (PCR) assay using the LightCycler with conventional microscopy using Giemsa-stained thick and thin blood films. The PCR assay can be completed in one hour and has the potential to detect and identify four species of Plasmodium in a single reaction by use of melting temperature curve analysis (however, we did not detect Plasmodium ovale in this study). Blood was collected, stored, and transported on IsoCode STIX, which provide a stable matrix for the archiving and rapid simple extraction of DNA. A genus-specific primer set corresponding to the 18S ribosomal RNA was used to amplify the target sequence. Fluorescence resonance energy technology hybridization probes were designed for P. falciparum over a region containing basepair mismatches, which allowed differentiation of the other Plasmodium species. The PCR results correlated with the microscopic results in 282 (95%) of 297 patient specimens. Most of these were single-species infections caused by P. vivax (150) and P. falciparum (120), along with 5 P. malariae, 2 mixed infections (P. falciparum and P. vivax), and 5 negative specimens. No negative microscopy specimens were positive by PCR (100% specificity for detection of any Plasmodium). The 15 discrepant results could not be resolved, but given the subjective nature of microscopy and the analytical objectivity of the PCR, the PCR results may be correct. The ability of the PCR method to detect mixed infections or to detect P. ovale could not be determined in this study. Within the limitations of initial equipment costs, this real-time PCR assay is a rapid, accurate, and efficient method for the specific diagnosis of malaria. It may have application in clinical laboratories, as well as in epidemiologic studies and antimalarial efficacy trials.

Malaria Diagnosis: A Brief Review

“…PCR can detect as few as 1-5 parasites/μ l of blood (≤ 0.0001% of infected red blood cells) compared with around 50-100 parasites/μ l of blood by microscopy or RDT. Moreover, PCR can help detect drug-resistant parasites, mixed infections, and may be automated to process large numbers of samples [58,59]. Some modified PCR methods are proving reliable, e.g., nested PCR, real-time PCR, and reverse transcription PCR, and appear to be useful second-line techniques when the results of traditional diagnostic methods are unclear for patients presenting with signs and symptoms of malaria; they also allow accurate species determination [58,[60][61][62].…”

Real-Time Quantitative Reverse Transcription PCR for Monitoring of Blood-Stage Plasmodium falciparum Infections in Malaria Human Challenge Trials

“…42 There are many factors to consider when choosing from the diverse molecular methods reported for malaria. [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] First, pre-analytical handling steps (such as leukocyte filtering reported in some assays) should be minimized or highly controlled to reduce the risk of contamination. Second, as the sample volume decreases, the limit of detection increases.…”

“…4 A variety of molecular methods of malaria parasite detection were developed in the past 20 years and include singlestep and nested polymerase chain reaction (PCR) with gel detection, RNA-specific nucleic acid-based sequence amplification (NASBA), and probe-based real-time PCR and reverse transcription (RT)-PCR (using various combinations of genus-or species-specific primers and TaqMan probes or molecular beacons). [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] Species identification can be achieved by SYBR Green melting curve analysis, 20 by real-time PCR with species-specific probes, 5,6 or most sensitively using laborintensive species-specific nested PCR. 24,25 In addition to diagnosing clinical cases with low parasite densities, some species-specific methods can detect pre-patent parasitemia in samples from field studies and from malaria human challenge trials.…”

Molecular Epidemiology of Malaria

“…Use of real-time quantitative PCR methods is being evaluated in clinical diagnostic laboratories in countries of endemicity with good resources (184) and reference laboratories in countries with substantial numbers of imported malaria cases (73). However, there is a limitation to the information provided by any method that samples peripheral blood for estimation of P. falciparum parasitemia, as sequestered mature asexual parasites may sometimes outnumber those in the peripheral blood (172).…”