Development of a Real-Time PCR Assay for Detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for Routine Clinical Diagnosis

ABSTRACT A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites—Plasmodium falciparum, P. ovale, and P. vivax—was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/μl for P. falciparum, P. vivax, and P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic malaria tests. Future studies are warranted to investigate the clinical value of this technique.

[1]  M. Uhlén,et al.  An integrated system using immunomagnetic separation, polymerase chain reaction, and colorimetric detection for diagnosis of Plasmodium falciparum. , 1997, The American journal of tropical medicine and hygiene.

[2]  G. Snounou,et al.  A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. , 1999, The American journal of tropical medicine and hygiene.

[3]  T. McCutchan,et al.  Structurally distinct, stage-specific ribosomes occur in Plasmodium. , 1987, Science.

[4]  U. Morgan,et al.  Molecular detection of parasitic protozoa , 1999, Parasitology.

[5]  A. Moody.  Rapid Diagnostic Tests for Malaria Parasites , 2002, Clinical Microbiology Reviews.

[6]  J. Iqbal,et al.  Comparison of the OptiMAL Test with PCR for Diagnosis of Malaria in Immigrants , 1999, Journal of Clinical Microbiology.

[7]  R. S. Phillips,et al.  Current Status of Malaria and Potential for Control , 2001, Clinical Microbiology Reviews.

[8]  K. Kain,et al.  Evaluation of a Colorimetric PCR-Based Assay To Diagnose Plasmodium falciparum Malaria in Travelers , 1999, Journal of Clinical Microbiology.

[9]  K. Kain,et al.  Imported malaria: prospective analysis of problems in diagnosis and management. , 1998, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[10]  W. Jarra,et al.  Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. , 1993, Molecular and biochemical parasitology.

[11]  E. Tjitra,et al.  Immunochromatographic Test for Detection of Plasmodium falciparum and Plasmodium vivax in Patients with a Presumptive Clinical Diagnosis of Malaria in Eastern Indonesia , 1999 .

[12]  D. Warhurst,et al.  ACP Broadsheet no 148. July 1996. Laboratory diagnosis of malaria. , 1996, Journal of clinical pathology.

[13]  K. Kain,et al.  Evaluation of a rapid and inexpensive dipstick assay for the diagnosis of Plasmodium falciparum malaria. , 1999, Bulletin of the World Health Organization.

[14]  S. Hoffman,et al.  Diagnosis of malaria by detection of Plasmodium falciparum HRP-2 antigen with a rapid dipstick antigen-capture assay , 1994, The Lancet.

[15]  G. Dettori,et al.  Identification of Plasmodium falciparum, P. vivax, P. ovale and P. malariae and detection of mixed infection in patients with imported malaria in Italy. , 2003, The new microbiologica.

[16]  S. Isomura,et al.  Sequence Variation in the Small-Subunit rRNA Gene of Plasmodium malariae and Prevalence of Isolates with the Variant Sequence in Sichuan, China , 1998, Journal of Clinical Microbiology.

[17]  L. Bruce-Chwatt.  FROM LAVERAN'S DISCOVERY TO DNA PROBES: NEW TRENDS IN DIAGNOSIS OF MALARIA , 1987, The Lancet.

[18]  W. Jarra,et al.  High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. , 1993, Molecular and biochemical parasitology.

[19]  J. M. Rubio,et al.  Usefulness of Seminested Multiplex PCR in Surveillance of Imported Malaria in Spain , 1999, Journal of Clinical Microbiology.

[20]  E. Linders,et al.  Detection of Plasmodium falciparum malaria parasites in vivo by real-time quantitative PCR. , 2001, Molecular and biochemical parasitology.

[21]  W. Klaskala,et al.  Evaluation of the OptiMAL Test for Rapid Diagnosis of Plasmodium vivax and Plasmodium falciparum Malaria , 1998, Journal of Clinical Microbiology.

[22]  C. Tang,et al.  Real-Time Fluorescence-Based PCR for Detection of Malaria Parasites , 2002, Journal of Clinical Microbiology.

[23]  P. Chiodini,et al.  Accuracy of routine laboratory diagnosis of malaria in the United Kingdom. , 1994, Journal of clinical pathology.

[24]  Y. Wataya,et al.  Sequence variation in the 18S rRNA gene, a target for PCR-based malaria diagnosis, in Plasmodium ovale from southern Vietnam , 1996, Journal of clinical microbiology.