Utility of Real-time PCR for the detection ofParacoccidioides brasiliensisDNA in the diagnosis of imported paracoccidioidomycosis

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 microl 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.

Performance of Panfungal- and Specific-PCR-Based Procedures for Etiological Diagnosis of Invasive Fungal Diseases on Tissue Biopsy Specimens with Proven Infection: a 7-Year Retrospective Analysis from a Reference Laboratory

“…In recent years, the Spanish Mycology Reference Laboratory has developed a number of real-time PCR (RT-PCR) assays in order to improve the diagnosis of IFD (7)(8)(9)(10)(11). These techniques serve to confirm the diagnosis of IFD when culture results are negative and to determine the species involved in infections.…”

“…1). For the most part, the PCR assays used have been described previously (5,(7)(8)(9)(10)(11). In addition, a new multiplex PCR assay able to detect the three main species of Aspergillus was developed.…”

Chronic Paracoccidioidomycosis with adrenal involvement mimicking tuberculosis – A case report from Austria

“…Diagnosis is based on the direct identification of the fungus with microscopy, culture or histology from clinical specimen [2]. In addition, molecular biological techniques such as polymerase chain reaction (PCR) demonstrate high sensitivity suitable for diagnosis and monitoring of treatment [4], [5]. Serological tests are used for evaluation of treatment response and disease recurrence [6].…”

Paracoccidioidomycosis

“…Additionally, more sensitive serological or molecular techniques may also disclose healthy infected people [37,41,46,47,55]. However, P. brasiliensis may remain latent in the infected host giving rise to symptomatic PCM years after the initial contact, as demonstrated by the cases diagnosed outside the mycosis endemic areas, all of whom had the opportunity to become exposed to the fungus in their native countries where they had acquired the primary subclinical infection [14,34,35].…”

“…Of note, every patient had a record of previous residence in recognized endemic countries [36,37]. These cases demonstrate that P. brasiliensis primary infections can remain dormant for long periods (mean of 13 years) from the time of infection to the moment of disease manifestations [17].…”