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

Abstract

A retrospective analysis of real-time PCR (RT-PCR) results for 151 biopsy samples obtained from 132 patients with proven invasive fungal diseases was performed. PCR-based techniques proved to be fast and sensitive and enabled definitive diagnosis in all cases studied, with detection of a total of 28 fungal species. Most of the methods used in microbiology laboratories for the diagnosis of invasive fungal diseases (IFDs) present limitations (1, 2). Cultures are too slow and ineffective for early diagnosis, although the gold standard methods to prove IFD continue to be isolation in culture from a sterile sample or demonstration of invasion by fungal structures in tissues (3,4).Classic histopathological studies have significant drawbacks. Microscopic examinations are quite variable, depending on experience, but the most important limitation of tissue observation is the impossibility of differentiating species, which is essential for determining therapy, given the differences in sensitivity to antifungal agents among fungal species (5).Molecular methods, such as those based on PCRs, have been used recently for fungal DNA detection in both fresh and paraffinembedded tissue samples, using different targets (4). The great advantages of these molecular techniques are the determination of the specific agent and greater sensitivity (6). A limitation is the lack of standardization of techniques, which are mostly homemade.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 (7-11). These techniques serve to confirm the diagnosis of IFD when culture results are negative and to determine the species involved in infections. In this work, a retrospective analysis of RT-PCR results for biopsy samples was performed. The survey included samples analyzed between 2006 and 2013. A total of 151 biopsy specimens from 132 patients with diagnoses of IFD, proven by histopathological examinations, and negative culture results were analyzed. The samples were sent to the Spanish Mycology Reference Laboratory by hospitals throughout Spain and had very heterogeneous origins, depending on the patients' symptoms, with lung, skin, liver, and brain samples being most common.Fourteen patients had more than one sample for diagnosis; the samples had the same origins (duplicates) for seven patients, and the origins of the samples were different for the rest of them. The biopsy specimens were fresh (n ϭ 92) or embedded in paraffin (n ϭ 59). For samples embedded in paraffin, the blocks were cut into 10-m sections. Three to 10 cuts were used to extract the DNA, in order to obtain approximately 25 mg of tissue. Biopsy specimens were deparaffinized by lavage with 1.5 ml of xylene (100%) followed by two washes with 1.2 ml of ethanol (96 to 100%). The tissue was incubated at 37°C for about 10 min to evaporate the remaining ethanol.DNA from fresh and paraffin-embedded tissues was extracted using a QIAmp Tissue DNA minikit (Qiagen, Izasa, Madrid, Spain), foll...

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