Molecular Detection of Aspergillus: Application of a Real-Time PCR Multiplex Assay in Tissue Samples

Sabino, Raquel; Simões, Helena; Veríssimo, Cristina

doi:10.3390/jof6010011

Cited by 6 publications

(3 citation statements)

References 21 publications

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“…An early diagnostic of invasive fungal infections is of high importance since it can lead to a more effective treatment [ 8 , 9 , 35 ]. At the moment, detection of A. fumigatus is based on PCR techniques, culturing methods, or serologic tests.…”

Section: Discussionmentioning

confidence: 99%

Establishment of a New PNA-FISH Method for Aspergillus fumigatus Identification: First Insights for Future Use in Pulmonary Samples

et al. 2020

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Aspergillus fumigatus is the main causative agent of Invasive Aspergillosis. This mold produces conidia that when inhaled by immunocompromized hosts can be deposited in the lungs and germinate, triggering disease. In this paper, the development of a method using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) is described. The PNA-FISH probe was tested in several strains and a specificity and sensitivity of 100% was obtained. Detection of A. fumigatussensu stricto was then achieved in artificial sputum medium (ASM) pre-inoculated with 1 × 102 conidia·mL−1–1 × 103 conidia·mL−1, after a germination step of 24 h. The PNA-FISH method was evaluated in 24 clinical samples (10 sputum, 8 bronchoalveolar lavage (BAL), and 6 bronchial lavage (BL)) that were inoculated with 1 × 104 conidia·mL−1 in sputum; 1 × 103 conidia·mL−1 in BL and BAL, for 24 h. Despite a specificity of 100%, the sensitivity was 79%. This relatively low sensitivity can be explained by the fact that hyphae can yield “fungal ball“ clusters, hindering pipetting procedures and subsequent detection, leading to false negative results. Nonetheless, this study showed the potential of the PNA-FISH method for A. fumigatussensu stricto detection since it takes only 1 h 30 m to perform the procedure with a pre-enrichment step of 6 h (pure cultures) and 24 h (clinical samples), and might provide a suitable alternative to the existing methods for studies in pure cultures and in clinical settings.

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Section: Discussionmentioning

confidence: 99%

Establishment of a New PNA-FISH Method for Aspergillus fumigatus Identification: First Insights for Future Use in Pulmonary Samples

et al. 2020

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“…The molecular detection of Aspergillus species from clinical samples has been achieved by amplification of parts of the rRNA region of fungi genome (White et al, 1990;Henry et al, 2000;Sabino et al, 2020). ITS1 and ITS2 have been employed in various phylogenetic studies of a variety of fungi.…”

Section: Pcr and Sequence Analysismentioning

confidence: 99%

Morphological and molecular diagnosis of invasive aspergillosis in chickens

Fagbohun¹,

Oladosu²,

Adesina³

2021

J. Vet. Med. Anim. Health

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Confirmatory diagnosis of invasive aspergillosis is paramount to ensure proper treatment and effective management of the disease in food and companion animals. Suspected invasive aspergilllosis in chickens was encountered at post-mortem. Morphological and molecular methods were employed to identify Aspergillus from samples collected from dead chickens at post-mortem. Morphologically, two species of Aspergillus were identified, namely Aspergillus fumigatus and Aspergillus flavus. Molecular identification based on polymerase chain reaction (PCR) and sequence analysis of the partial 5.8 S rRNA, complete internal transcribed spacer-2 and partial 28S rRNA sequences bolstered morphological identification to arrive at the confirmatory diagnosis of the disease. Various hotspots that differentiate A. flavus from A. fumigatus and from other Aspergillus species were identified based on multiple sequence analysis. Maximum likelihood phylogenetic tree showed that isolates from the same species were grouped in the same clade. It is important to correctly identify the Aspergillus species in order to efficiently manage the disease.

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“…Detecting azole-resistant strains is another challenging issue owing to the increasing incidence of resistant Aspergillus species [21]. Detection of resistance from culture delays diagnosis and increases the mortality rate of patients with IPA [22].…”

Section: Introductionmentioning

confidence: 99%

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

Lee,

Kim,

Cho

et al. 2023

JoF

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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.

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Molecular Detection of Aspergillus: Application of a Real-Time PCR Multiplex Assay in Tissue Samples

Cited by 6 publications

References 21 publications

Establishment of a New PNA-FISH Method for Aspergillus fumigatus Identification: First Insights for Future Use in Pulmonary Samples

Establishment of a New PNA-FISH Method for Aspergillus fumigatus Identification: First Insights for Future Use in Pulmonary Samples

Morphological and molecular diagnosis of invasive aspergillosis in chickens

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

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