Utility of PCR in Diagnosis of Invasive Fungal Infections: Real-Life Data from a Multicenter Study

Abstract

Prospective studies addressing the clinical value of broad-range PCR using the internal transcribed spacer region (ITS) for diagnosis of microscopy-negative fungal infections in nonselected patient populations are lacking. We first assessed the diagnostic performance of ITS rRNA gene PCR compared with that of routine microscopic immunofluorescence examination. Second, we addressed prospectively the impact and clinical value of broad-range PCR for the diagnosis of infections using samples that tested negative by routine microscopy; the corresponding patients' data were evaluated by detailed medical record reviews. Results from 371 specimens showed a high concordance of >80% for broad-range PCR and routine conventional methods, indicating that the diagnostic performance of PCR for fungal infections is comparable to that of microscopy, which is currently considered part of the "gold standard." In this prospective study, 206 specimens with a negative result on routine microscopy were analyzed with PCR, and patients' clinical data were reviewed according to the criteria of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group. We found that broad-range PCR showed a sensitivity, specificity, positive predictive value, and negative predictive value of 57.1%, 97.0%, 80%, and 91.7%, respectively, for microscopy-negative fungal infections. This study defines a possible helpful role of broad-range PCR for diagnosis of microscopy-negative fungal infections in conjunction with other tests.

SNaPAfu: A Novel Single Nucleotide Polymorphism Multiplex Assay for Aspergillus fumigatus Direct Detection, Identification and Genotyping in Clinical Specimens

“…BAL and biopsy samples were conserved with liquid nitrogen (−80°C) and stored until use. DNA was extracted using a modified cetyltrimethylammonium bromide protocol [13], [15], [22] and finally resuspended in 50 µl of ultrapure water. Extracted DNA was detected with an in-house pan-fungal PCR as previously described [22], [23].…”

“…DNA was extracted using a modified cetyltrimethylammonium bromide protocol [13], [15], [22] and finally resuspended in 50 µl of ultrapure water. Extracted DNA was detected with an in-house pan-fungal PCR as previously described [22], [23]. Following DNA extraction, the DNA was stored at −20°C for few months (in this study the oldest samples were stored for 6 months).Sequencing was done with Big Dye terminators and a capillary sequencer (3500 Genetic Analyzer; Applied Biosystems).…”

New Panfungal Real-Time PCR Assay for Diagnosis of Invasive Fungal Infections

“…However, several limitations exist when the fungus causing the infection is unknown. In these cases, panfungal PCR assays have been used (27,28), but they involve a delay in the response time because they require sequencing the amplicon. As recently described, in 30% of the samples studied from patients with IFI there was no evidence concerning the fungus implicated in the infection (24), highlighting the need for new methods.…”

A new multiplex PCR for species-specific diagnosis of human candidiasis

“…Debido a sus indudables ventajas, como su facilidad de empleo, mayor rapidez, menor riesgo de contaminación y gran especificidad y sensibilidad, en las últimas décadas la PCR ha pasado a sustituir las técnicas microbiológicas tradicionales y hoy constituye la prueba de referencia para la detección de las especies causantes de un amplio número de enfermedades infecciosas (39)(40)(41).…”