Utility of Real-time PCR for the detection of Paracoccidioides brasiliensis DNA 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 μl of sample. This procedure proved to be very reproducible and specifi c. 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.

Important aspects of oral paracoccidioidomycosis–a literature review

“…More recently, Buitrago et al. [72] developed a molecular diagnostic technique based on real‐time PCR for the detection of P. brasiliensis DNA in both culture and patients’ clinical samples. The method employed a Molecular Beacon probe, labelled with FAM and directed at the ITS1 region of ribosomal DNA.…”

Histopathologic Diagnosis of Fungal Infections in the 21st Century

“…Alternative testing for rhinosporidiosis does not exist. Molecular diagnosis with fresh tissues has been used for several of these infections, using either specific primers for these fungi or panfungal primers (23,45,83).…”

Paracoccidioidomicosis con afectación mucocutánea. Reporte de un caso

“…Donde se llegó al consenso, basado en evidencia, que la droga de elección es el itraconazol. La anfotericina B sólo se prescribe para casos graves como tratamiento endovenoso inicial (1 mg/kg/día) 4,5,10 . El pronóstico va depender del estado de la enfermedad cuando se em- (círculo amarillo) "orejas de mickey mouse", D) Paracoccidioides brasiliensis dentro de macrófagos.…”

“…Esta enfermedad es endémica en ciertos países del sur y de centroamérica con la más alta prevalencia observada en Brasil (aproximadamente 80%) 2 . En el Perú el reporte de esta enfermedad no es obligatorio, por lo cual, no se tiene datos exactos de su prevalencia, solo el reporte de casos ha permitido conocer la distribución geográfica en zonas tropicales de la ceja de selva y selva baja amazónica 3,4 . Esto constituye una escasa cobertura en el diagnóstico y por ende un retraso en el tratamiento, lo que impide mejorar el pronóstico de esta enfermedad.…”

“…Además, las técnicas de biología molecular como la reacción en cadena de la polimerasa (PCR) demuestran una alta sensibilidad adecuada para el diagnóstico y el seguimiento del tratamiento 4,5 . Las pruebas serológicas se utilizan para la evaluación de la respuesta al tratamiento y recurrencia de la enfermedad 6 .…”