A Laboratory-Developed Assay for the Simultaneous Detection of <i>Aspergillus fumigatus</i> and <i>Pneumocystis jirovecii</i> Pulmonary Pathogens
Invasive fungal diseases are a significant threat in immunocompromised patients, underscoring the need for rapid and accurate diagnostics. This study describes the development and validation of a real-time PCR-based laboratory-developed assay (LDA) on the Panther Fusion system for the simultaneous d...
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MDPI AG
2025-04-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/11/4/280 |
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| author | Margherita Cacaci Debora Talamonti Giulia Menchinelli Damiano Squitieri Riccardo Torelli Elena De Carolis Giulia De Angelis Maurizio Sanguinetti Brunella Posteraro |
| author_facet | Margherita Cacaci Debora Talamonti Giulia Menchinelli Damiano Squitieri Riccardo Torelli Elena De Carolis Giulia De Angelis Maurizio Sanguinetti Brunella Posteraro |
| author_sort | Margherita Cacaci |
| collection | DOAJ |
| description | Invasive fungal diseases are a significant threat in immunocompromised patients, underscoring the need for rapid and accurate diagnostics. This study describes the development and validation of a real-time PCR-based laboratory-developed assay (LDA) on the Panther Fusion system for the simultaneous detection of <i>Aspergillus fumigatus</i> (AF) and <i>Pneumocystis jirovecii</i> (PJ) in bronchoalveolar lavage fluid (BALF) samples. The assay was evaluated using 239 clinical BALF samples, including cases confirmed positive for AF or PJ by reference mycological methods. Rigorous optimization ensured compatibility with the automated workflow of the Panther Fusion system, which addresses challenges such as BALF viscosity and fungal DNA recovery. No cross-reactivity with non-target fungal species was observed, and the assay demonstrated high analytical sensitivity and specificity. Only two false-negative results were reported, which could plausibly be reclassified as true negatives when interpreted alongside the serum beta-<span style="font-variant: small-caps;">d</span>-glucan and galactomannan assay results. For PJ detection, the assay showed excellent concordance with the OLM PneumID assay, supporting its reliability in clinical settings. The dual-target approach facilitates the simultaneous detection of both pathogens within a single workflow, improving diagnostic efficiency. The AF/PJ LDA represents a robust and scalable alternative to existing molecular assays, with the potential to enhance routine diagnostics for pulmonary fungal infections. |
| format | Article |
| id | doaj-art-7a87a42a28284bc983fb027c9751d0b8 |
| institution | OA Journals |
| issn | 2309-608X |
| language | English |
| publishDate | 2025-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj-art-7a87a42a28284bc983fb027c9751d0b82025-08-20T02:18:14ZengMDPI AGJournal of Fungi2309-608X2025-04-0111428010.3390/jof11040280A Laboratory-Developed Assay for the Simultaneous Detection of <i>Aspergillus fumigatus</i> and <i>Pneumocystis jirovecii</i> Pulmonary PathogensMargherita Cacaci0Debora Talamonti1Giulia Menchinelli2Damiano Squitieri3Riccardo Torelli4Elena De Carolis5Giulia De Angelis6Maurizio Sanguinetti7Brunella Posteraro8Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, ItalyDipartimento di Scienze di Laboratorio ed Ematologiche, Fondazione Policlinico Universitario A. Gemelli IRCCS, Largo A. Gemelli 8, 00168 Rome, ItalyDipartimento di Scienze di Laboratorio ed Ematologiche, Fondazione Policlinico Universitario A. Gemelli IRCCS, Largo A. Gemelli 8, 00168 Rome, ItalyDipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, ItalyDipartimento di Scienze di Laboratorio ed Ematologiche, Fondazione Policlinico Universitario A. Gemelli IRCCS, Largo A. Gemelli 8, 00168 Rome, ItalyDipartimento di Scienze di Laboratorio ed Ematologiche, Fondazione Policlinico Universitario A. Gemelli IRCCS, Largo A. Gemelli 8, 00168 Rome, ItalyDipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, ItalyDipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, ItalyDipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, ItalyInvasive fungal diseases are a significant threat in immunocompromised patients, underscoring the need for rapid and accurate diagnostics. This study describes the development and validation of a real-time PCR-based laboratory-developed assay (LDA) on the Panther Fusion system for the simultaneous detection of <i>Aspergillus fumigatus</i> (AF) and <i>Pneumocystis jirovecii</i> (PJ) in bronchoalveolar lavage fluid (BALF) samples. The assay was evaluated using 239 clinical BALF samples, including cases confirmed positive for AF or PJ by reference mycological methods. Rigorous optimization ensured compatibility with the automated workflow of the Panther Fusion system, which addresses challenges such as BALF viscosity and fungal DNA recovery. No cross-reactivity with non-target fungal species was observed, and the assay demonstrated high analytical sensitivity and specificity. Only two false-negative results were reported, which could plausibly be reclassified as true negatives when interpreted alongside the serum beta-<span style="font-variant: small-caps;">d</span>-glucan and galactomannan assay results. For PJ detection, the assay showed excellent concordance with the OLM PneumID assay, supporting its reliability in clinical settings. The dual-target approach facilitates the simultaneous detection of both pathogens within a single workflow, improving diagnostic efficiency. The AF/PJ LDA represents a robust and scalable alternative to existing molecular assays, with the potential to enhance routine diagnostics for pulmonary fungal infections.https://www.mdpi.com/2309-608X/11/4/280pulmonary fungal infectionlaboratory-developed assaymolecular diagnosticsOpen AccessPanther Fusion |
| spellingShingle | Margherita Cacaci Debora Talamonti Giulia Menchinelli Damiano Squitieri Riccardo Torelli Elena De Carolis Giulia De Angelis Maurizio Sanguinetti Brunella Posteraro A Laboratory-Developed Assay for the Simultaneous Detection of <i>Aspergillus fumigatus</i> and <i>Pneumocystis jirovecii</i> Pulmonary Pathogens Journal of Fungi pulmonary fungal infection laboratory-developed assay molecular diagnostics Open Access Panther Fusion |
| title | A Laboratory-Developed Assay for the Simultaneous Detection of <i>Aspergillus fumigatus</i> and <i>Pneumocystis jirovecii</i> Pulmonary Pathogens |
| title_full | A Laboratory-Developed Assay for the Simultaneous Detection of <i>Aspergillus fumigatus</i> and <i>Pneumocystis jirovecii</i> Pulmonary Pathogens |
| title_fullStr | A Laboratory-Developed Assay for the Simultaneous Detection of <i>Aspergillus fumigatus</i> and <i>Pneumocystis jirovecii</i> Pulmonary Pathogens |
| title_full_unstemmed | A Laboratory-Developed Assay for the Simultaneous Detection of <i>Aspergillus fumigatus</i> and <i>Pneumocystis jirovecii</i> Pulmonary Pathogens |
| title_short | A Laboratory-Developed Assay for the Simultaneous Detection of <i>Aspergillus fumigatus</i> and <i>Pneumocystis jirovecii</i> Pulmonary Pathogens |
| title_sort | laboratory developed assay for the simultaneous detection of i aspergillus fumigatus i and i pneumocystis jirovecii i pulmonary pathogens |
| topic | pulmonary fungal infection laboratory-developed assay molecular diagnostics Open Access Panther Fusion |
| url | https://www.mdpi.com/2309-608X/11/4/280 |
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