Improved performance with saliva and urine as alternative DNA sources for malaria diagnosis by mitochondrial DNA-based PCR assays

Saliva and urine from malaria-infected individuals contain trace amounts of Plasmodium DNA, and therefore, could be used as alternative specimens for diagnosis. A nested PCR targeting the mitochondrial cytochrome b gene (Cytb-PCR) of four human malaria species and Plasmodium knowlesi was developed and tested with 693 blood samples from febrile patients living in diverse malaria-endemic areas of Thailand, and compared with microscopy and nested PCR targeting small-subunit rRNA (18S-PCR). Cytb-PCR was 16% and 39.8% more sensitive than 18S-PCR and microscopy, respectively, in detecting all of these malarial species in blood samples. Importantly, 34% and 17% of Plasmodium falciparum and Plasmodium vivax mono-infections, respectively, detected by microscopy were, in fact, mixed P. falciparum and P. non-falciparum infections. Analysis of matched blood, saliva and urine from 157 individuals showed that microscopy and Cytb-PCR of saliva yielded no significant difference in detecting P. falciparum and P. vivax. However, Cytb-PCR of saliva was more sensitive than microscopy for diagnosis of mixed-species infections. A combination of Cytb-PCR of saliva and of urine significantly outperformed microscopy (p = 0.0098 for P. falciparum, p = 0.006 for P. vivax, and p = 0.0002 for mixed infections). Furthermore, Plasmodium malariae and P. knowlesi could also be identified in saliva and urine with this method. Therefore, the Cytb-PCR developed herein offers a high potential for the use of both saliva and urine for malaria diagnosis, with a sensitivity comparable with or superior to that of microscopy.