Detection and Species Determination of Malaria Parasites by PCR: Comparison with Microscopy and with ParaSight-F and ICT Malaria Pf Tests in a Clinical Environment

Abstract

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients. Plasmodium infections were identified with a genus-specific primer set, and species differentiation betweenPlasmodium falciparum and Plasmodium vivax was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed P. falciparum-P. vivax infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays forP. falciparum, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive forP. vivax at the time of discharge were later readmitted to the hospital with a recurrence of P. vivax infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria.

PCR‐based ELISA technique for malaria diagnosis of specimens from Thailand

“…The two GTTS-negative but MPH-positive cases were con®rmed positive by nested PCR (one mixed Pf±Pv, one Pv only). This supports other PCR-based studies, which also showed that additional malaria cases are detected by PCR among those determined as negative by microscopy or rapid antigen tests (Postigo et al 1998;Rubio et al 1999;Singh et al 1999;Tham et al 1999;Zhong & Kain 1999 1 ). The moderate correlation between parasite densities and absorbance reading, although limited to the parasite density range of 7000/ll and below, suggests that MPH may be a useful semiquantitative tool to monitor treatment outcomes and to predict recrudescence such as suggested by other PCR-based techniques (Kain et al 1994;Ciceron et al 1999).…”

“…This study is unique for its relatively high percentage of low-density (£100/ll) parasitaemias (33.0%, 34/103). This explains why sensitivity of MPH was not 100% or approaching 100% as recently reported for other PCR-based techniques (Singh et al 1999;Tham et al 1999;Zhong & Kain 1999). These levels of parasitaemia are the most likely to be missed by routine GTTS or immunochromatographic technique.…”

“…Several new malaria diagnostic techniques have recently been developed. Immunochromatographic antigen and enzyme detection of techniques are the most well-characterized (Makler et al 1998;Pieroni et al 1998;Tham et al 1999;Wongsrichanalai et al 1999). The accuracy of these techniques is generally good but sensitivity for lowdensity parasitaemias still needs to be improved (Humar et al 1997;Pieroni et al 1998;Wongsrichanalai et al 1999).…”

“…Although we used GTTS as a reference for practicality, it must be taken into consideration that no real gold standard exists for this type of study. Limitations of microscopy in distinguishing malarial species at low parasitaemia levels and in correctly identifying mixed species infection when one species tends to be dominant are well recognized (Kimura et al 1995;Tham et al 1999). If not subject to limitations of microscopy as a reference standard, speci-®city of the MPH assay would have been higher.…”

Performance of OptiMAL-IT®compared to microscopy, for malaria detection in Burkina Faso

“…2005) or to parasite density below the microscopy threshold of detection (50–100 parasites/μl) (WHO 1998). Indeed, malaria infection can be detected by PCR in samples negative for microscopy but positive by RDT (Tham et al. 1999; Moody & Chiodini 2002).…”