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Background  The misdiagnosis of  Plasmodium knowlesi  by microscopy has prompted a re-evaluation of the geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this report, a specific probe for  P. knowlesi , that can be used in a previously described TaqMan real-time PCR assay for detection of  Plasmodium  spp., and  Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae  and  Plasmodium ovale , was designed and validated against clinical samples.    Methods  A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the  P. knowlesi  small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing  P. knowlesi  DNA and genomic DNA of  P. falciparum, P. knowlesi, P. malariae, P. ovale  and  P. vivax  isolated from clinical samples. DNA samples of the simian malaria parasites  Plasmodium cynomolgi  and  Plasmodium inui  that can infect humans under experimental conditions were also examined together with human DNA samples.    Results  Analytical sensitivity of the  P. knowlesi -specific assay was 10 copies/μL and quantitation was linear over a range of 10-10 6  copies. The sensitivity of the assay is equivalent to nested PCR and  P. knowlesi  DNA was detected from all 40 clinical  P. knowlesi  specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67  Plasmodium  DNA samples (31  P. falciparum , 23  P. vivax , six  P. ovale , three  P. malariae , one  P. malariae/P. ovale , one  P. falciparum/P. malariae, one P. inui and one P. cynomolgi)  and four samples of human DNA.    Conclusions  This test demonstrated excellent sensitivity and specificity, and adds  P. knowlesi  to the repertoire of  Plasmodium  targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.

A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi