Comparison of three molecular methods for the detection and speciation of Plasmodium vivax and Plasmodium falciparum

Abstract

Background: Accurate diagnosis of Plasmodium spp. is essential for the rational treatment of malaria. Despite its many disadvantages, microscopic examination of blood smears remains the current "gold standard" for malaria detection and speciation. PCR assays offer an alternative to microscopy which has been shown to have superior sensitivity and specificity. Unfortunately few comparative studies have been done on the various molecular based speciation methods.

Methods: The sensitivity, specificity and cost effectiveness of three molecular techniques were compared for the detection and speciation of Plasmodium falciparum and Plasmodium vivax from dried blood spots collected from 136 patients in western Thailand. The results from the three molecular speciation techniques (nested PCR, multiplex PCR, and real-time PCR) were used to develop a molecular consensus (two or more identical PCR results) as an alternative gold standard.

Results: According to the molecular consensus, 9.6% (13/136) of microscopic diagnoses yielded false negative results. Multiplex PCR failed to detect P. vivax in three mixed isolates, and the nested PCR gave a false positive P. falciparum result in one case. Although the real-time PCR melting curve analysis was the most expensive method, it was 100% sensitive and specific and least time consuming of the three molecular techniques investigated.

Conclusion: Although microscopy remains the most appropriate method for clinical diagnosis in a field setting, its use as a gold standard may result in apparent false positive results by superior techniques. Future studies should consider using more than one established molecular methods as a new gold standard to assess novel malaria diagnostic kits and PCR assays.

Figure 1

An example of results from each of the PCR assays examined in this study. The example uses a paired single infection of Plasmodium falciparum and Plasmodium vivax. A. Image of P. falciparum (300-bp) and P. vivax (276-bp) positive samples after multiplex PCR, run on 2% agarose at 100 v for 1 hour. B. P. falciparum (205-bp) and P. vivax (120-bp) positive samples after 2^nd round nested PCR, run on 2% agarose at 100 v for 1 hour. C. Real-time PCR melt curves of P. falciparum (T_m 74.5) and P. vivax (T_m 78). Image provided by Optical Monitor Software v.3.1.