Clinical Implementation of β-Tubulin Gene-Based Aspergillus Polymerase Chain Reaction for Enhanced Aspergillus Diagnosis in Patients with Hematologic Diseases: A Prospective Observational Study

Abstract

The β-tubulin (benA) gene is a promising target for the identification of Aspergillus species. Assessment of the clinical implementation and performance of benA gene-based Aspergillus polymerase chain reaction (PCR) remains warranted. In this study, we assessed the analytical performance of the BenA probe PCR in comparison with the Aspergenius kit. We prospectively collected bronchoalveolar lavage (BAL) fluid via diagnostic bronchoscopy from adult patients with hematologic diseases. BenA gene-based multiplex real-time PCR and sequential melting temperature analysis were performed to detect the azole resistance of Aspergillus fumigatus. In total, 76 BAL fluids in 75 patients suspicious of invasive pulmonary aspergillosis (IPA) were collected. Before the application of PCR, the prevalence of proven and probable IPA was 32.9%. However, after implementing the benA gene-based PCR, 15.8% (12 out of 76) of potential IPA cases were reclassified as probable IPA. The analytical performance of the BenA probe PCR in BAL samples was comparable to that of the Aspergenius kit. The diagnostic performance was as follows: sensitivity, 52.0%; specificity, 64.7%; positive predictive value, 41.9%; negative predictive value, 73.3%; positive likelihood ratio, 1.473; and negative likelihood ratio, 0.741. Moreover, benA gene-based Aspergillus PCR discriminated all major sections of Aspergillus, including cryptic species such as Aspergillus tubingensis. Sequential melting temperature analysis successfully detected 2 isolates (15.4%) of A. fumigatus carrying resistant mutations. BenA gene-based Aspergillus PCR with melting temperature analysis enhances diagnostic accuracy and detects not only cryptic species but also resistant mutations of A. fumigatus. It shows promise for clinical applications in the diagnosis of IPA.