Efficacy of DNA amplification in tissue biopsy samples to improve the detection of invasive fungal disease

Buitrago, María J.; Aguado, J. M.; Ballen, Aida; Bernal‐Martínez, Leticia; Prieto, Mª Isabel; García‐Reyne, Ana; García-Rodríguez, Julio; Rodríguez-Tudela, Juan Luis; Cuenca‐Estrella, Manuel

doi:10.1111/1469-0691.12110

Cited by 69 publications

(58 citation statements)

References 18 publications

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“…Similar to our finding, in a retrospective study by Buitrago et al, the ability of the real-time PCR procedures targeting ITS1-ITS2 in the rDNA gene was evaluated to detect and identify the fungal DNA at the species level (32). A total of 89 FFPE biopsy samples from 84 patients with proven IFDs were tested.…”

Section: Discussionsupporting

confidence: 66%

Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays

et al. 2016

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h In a retrospective multicenter study, 102 formalin-fixed paraffin-embedded (FFPE) tissue specimens with histopathology results were tested. Two 4-to 5-m FFPE tissue sections from each specimen were digested with proteinase K, followed by automated nucleic acid extraction. Multiple real-time quantitative PCR (qPCR) assays targeting the internal transcribed spacer 2 (ITS2) region of ribosomal DNA, using fluorescently labeled primers, was performed to identify clinically important genera and species of Aspergillus, Fusarium, Scedosporium, and the Mucormycetes. The molecular identification was correlated with results from histological examination. One of the main findings of our study was the high sensitivity of the automated DNA extraction method, which was estimated to be 94%. The qPCR procedure that was evaluated identified a range of fungal genera/species, including Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Fusarium oxysporum, Fusarium solani, Scedosporium apiospermum, Rhizopus oryzae, Rhizopus microsporus, Mucor spp., and Syncephalastrum. Fusarium oxysporum and F. solani DNA was amplified from five specimens from patients initially diagnosed by histopathology as having aspergillosis. Aspergillus flavus, S. apiospermum, and Syncephalastrum were detected from histopathological mucormycosis samples. In addition, examination of four samples from patients suspected of having concomitant aspergillosis and mucormycosis infections resulted in the identification of two A. flavus isolates, one Mucor isolate, and only one sample having both R. oryzae and A. flavus. Our results indicate that histopathological features of molds may be easily confused in tissue sections. The qPCR assay used in this study is a reliable tool for the rapid and accurate identification of fungal pathogens to the genus and species levels directly from FFPE tissues.T he frequency of invasive fungal diseases (IFDs) has increased significantly over the past 3 decades, and they are associated with excessive morbidity and mortality in immunocompromised hosts, patients hospitalized with severe underlying diseases (e.g., acute myelogenous leukemia), those requiring complex surgical procedures (e.g., trauma patients), and individuals who require support in intensive care units (1-5).The most well-known cause of opportunistic filamentous mycoses is Aspergillus fumigatus (6). However, the epidemiology of IFDs due to filamentous fungi has been expanded well beyond A. fumigatus, including non-fumigatus species of Aspergillus, the mucormycetes, Fusarium and Scedosporium species, and a wide variety of melanized fungi (4, 7).Given the complexity of the population of patients at risk and the diverse and increasing arrays of fungal pathogens, which show significantly different antifungal susceptibilities, (3) early and reliable detection of a causative fungal pathogen is crucial to guide the appropriate and successful treatment of IFDs (8, 9).Formalin-fixed paraffin-embedded (FFPE) tissues obtained from patients with prov...

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Section: Discussionsupporting

confidence: 66%

Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays

et al. 2016

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“…DNA extractions from other specimens were also performed by using the Qiamp DNA minikit, without modifications. DNAs from paraffin-embedded tissues were isolated as described previously (51).…”

Section: Methodsmentioning

confidence: 99%

A Multiplex Real-Time PCR Assay for Identification of Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii in Samples from AIDS Patients with Opportunistic Pneumonia

et al. 2014

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A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r 2 of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 l reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log 10 copies/20 l reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications.

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“…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).…”

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confidence: 99%

“…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.…”

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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

et al. 2014

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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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Efficacy of DNA amplification in tissue biopsy samples to improve the detection of invasive fungal disease

Cited by 69 publications

References 18 publications

Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays

Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays

A Multiplex Real-Time PCR Assay for Identification of Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii in Samples from AIDS Patients with Opportunistic Pneumonia

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

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