Abstract
An increase in immigration from endemic regions has resulted in a number of cases of paracoccidioidomycosis (PCM) being imported into Spain. A molecular diagnostic technique based on real-time PCR was developed for the detection of Paracoccidioides brasiliensis DNA in both culture and patients’ clinical samples. A Molecular Beacon probe was used, labelled with FAM and directed at the ITS1 region of ribosomic DNA. The detection limit of the technique developed was 1 fg of fungal DNA per µl of sample. This procedure proved to be very reproducible and specific. The technique was tested with cultures of 12 clinical strains and on samples from two patients with proven PCM. Real-time PCR was positive for all the culture strains, as well as those from both patients. By samples, the technique was positive in sputum and tissue biopsies but less useful on blood samples. Samples were analyzed several months after patient treatment, detecting a small amount of fungal DNA in one respiratory sample. This technique of real-time PCR is a sensitive method for rapid diagnosis of paracoccidioidomycosis and could serve to monitor patients after treatment has begun.
Acknowledgements
This work was supported by Research Projects from Instituto de Salud Carlos III (MPY 1279/05), and from RED Española de Investigación en Patología Infecciosa (REIPI, MPY 1022/07).
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Note
This paper was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007.