Impact of ITS-Based Sequencing on Antifungal Treatment of Patients with Suspected Invasive Fungal Infections

Guenter, Sara; Gorkiewicz, Gregor; Halwachs, Bettina; Kashofer, Karl; Thueringer, Andrea; Wurm, Philipp; Zollner‐Schwetz, Ines; Valentin, Thomas; Prattes, Juergen; Wunsch, Stefanie; Ullrich, Elisabeth; Zurl, Christoph; Hoenigl, Martin; Krause, Robert

doi:10.3390/jof6020043

Cited by 6 publications

(6 citation statements)

References 35 publications

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“…Diagnostics such as sequencing have a positive impact and can inform antifungal treatment strategies and therefore play a crucial role in dealing with rare species. Larger datasets are needed for correlation with clinical disease and to establish trends [ 19 ].…”

Section: Discussionmentioning

confidence: 99%

Cryptic aspergillosis: a rare entity and a diagnostic challenge

Lavanya

Ninan

Kurien

et al. 2022

Access Microbiology

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Introduction. Cryptic aspergillosis, caused by cryptic species of Aspergillus, is increasingly reported in humans and causes significant morbidity and mortality in immunocompromised individuals. The main aim of this study was to describe the occurrence of this entity at a large tertiary care centre and analyse the challenges in identifying them in a routine diagnostic laboratory. Methods. This was a retrospective case review of all patients diagnosed with cryptic Aspergillus species from April 2019 to February 2020. The isolates were identified using conventional microbiological techniques, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF MS), 28S rRNA and internal transcribed spacer (ITS) sequencing. Results. The species identified were Aspergillus tamarii, Aspergillus lentulus and Aspergillus sydowii. Identification by MALDI- TOF MS and sequencing was concordant for all except A. sydowii, with MALDI- TOF MS misidentifying it as Aspergillus thermomutans. All isolates showed low minimum inhibitory concentrations (MICs) for the panel of antifungal drugs. Conclusion. Aspergillosis caused by cryptic Aspergillus species presents a diagnostic challenge. This study confirms the importance of molecular methods for accurate identification.

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Section: Discussionmentioning

confidence: 99%

Cryptic aspergillosis: a rare entity and a diagnostic challenge

Lavanya

Ninan

Kurien

et al. 2022

Access Microbiology

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“…ITS sequencing was performed as previously described. [9][10][11] Briefly, DNA extraction from formalinfixed paraffin-embedded (FFPE) tissues was performed via mechanical tissue disruption (MagNA Lyser; Roche, Indianapolis, IN, USA) and via an semiautomated extraction system (Maxwell Tissue DNA Purification; Promega, Madison, WI, USA). DNA concentration was measured by Picogreen fluorescence (Promega) and fungal ITS sequences were amplified with the primers ITS1 5 0 -TCCGTAGGTGAACCTGCGG-3 0 and ITS2 5 0 -GCTGCGTTCTTCATCGATGC-3 0 .…”

Section: Methodsmentioning

confidence: 99%

“…All sequences were bioinformatically processed as previously described. [9][10][11] The phylogenetic relatedness of detected Mucorales ITS sequences was inferred by using the maximum likelihood method and the Tamura-Nei model, 12 conducted with MEGA11. 13,14…”

Section: Methodsmentioning

confidence: 99%

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The mutual value of histopathology and ITS sequencing in the diagnosis of mucormycosis

Zacharias,

Thüringer,

Krause

et al. 2024

Histopathology

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AimsMucormycosis is a fast‐progressing disease with a high mortality rate. The most important factor determining survival of patients is early and accurate diagnosis. Although histopathology often recognises invasive mould infections at first, histomorphology alone is insufficient in providing an accurate diagnosis. Unbiased molecular methods to detect and identify fungi are promising, yet their role in complementing routine histopathological workflows has not been studied sufficiently.Methods and resultsWe performed a retrospective single‐centre study examining the clinical value of complementing histopathology with internal transcribed spacer (ITS) sequencing of fungal DNA in the routine diagnosis of mucormycosis. At our academic centre, we identified 14 consecutive mucormycosis cases diagnosed by histopathology and subsequent ITS sequencing. Using histomorphological examination, fungal hyphae could be detected in all cases; however, morphological features were unreliable regarding specifying the taxa. Subsequent ITS sequencing identified a remarkable phylogenetic diversity among Mucorales: the most common species was Rhizopus microsporus (six of 14; 42.9%), followed by Lichtheimia corymbifera (three of 14, 21.4%) and single detections of Rhizopus oryzae, Actinomucor elegans, Mucor circinelloides, Rhizomucor pusillus and Rhizomucor miehei (one of 14; 7.1%, respectively). In one case, we additionally detected Pneumocystis jirovecii in the same lung tissue specimen, suggesting a clinically relevant co‐infection. Fungal culture was performed in 10 cases but yielded positive results in only two of 10 (20%), revealing its limited value in the diagnosis of mucormycosis.ConclusionsOur study demonstrates that a combination of histopathology and ITS sequencing is a practically feasible approach that outperforms fungal culture in detecting Mucorales in tissue‐associated infections. Therefore, pathologists might adapt diagnostic workflows accordingly when mucormycosis is suspected.

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“…ELISA GM testing suffers from many limitations, including cost and turnaround time, particularly in settings where bulk testing is not commonly done or samples are sent to a central lab [14,[19][20][21][22]. Molecular tests such as polymerase chain reaction (PCR) are widely used to diagnose IA [19,23,24], although there is a lack of standardization among assays [25] and a large variation in diagnostic performance across studies and settings [21,26]. Aspergillus PCR from blood shows particularly poor performance for the diagnosis of breakthrough infections [27] in settings that use mold-active prophylaxis [28,29].…”

Section: Introductionmentioning

confidence: 99%

The Aspergillus Lateral Flow Assay for the Diagnosis of Invasive Aspergillosis: an Update

Jenks

Miceli

Prattes

et al. 2020

Curr Fungal Infect Rep

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Purpose of Review To review the data on the Aspergillus lateral flow assay for the diagnosis of invasive Aspergillosis. Recent Findings Aspergillus spp. cause a wide spectrum of disease with invasive aspergillosis (IA) as its most severe manifestation. Early and reliable diagnosis of disease is crucial to decrease associated morbidity and mortality, and enable prompt initiation of treatment for IA. Most recently, non-culture-based tests, such as Aspergillus galactomannan (GM), have been useful in early identification and treatment of patients with IA. However, cost, turnaround time, and variable performance indifferent populations at risk for IA remain significant drawbacks to the use of this test. Several diagnostic tests for IA have been developed, including the sōna Aspergillus GM Lateral flow assay (GM-LFA) rapid test. Summary The GM-LFA has shown excellent performance for the diagnosis of IA in patients with hematologic malignancy and may be a viable option for settings where ELISA GM testing is not feasible. Further evaluation of the GM-LFA in the non-hematology setting is ongoing, including in solid organ transplant recipients and patients in the intensive care unit.

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Impact of ITS-Based Sequencing on Antifungal Treatment of Patients with Suspected Invasive Fungal Infections

Cited by 6 publications

References 35 publications

Cryptic aspergillosis: a rare entity and a diagnostic challenge

Cryptic aspergillosis: a rare entity and a diagnostic challenge

The mutual value of histopathology and ITS sequencing in the diagnosis of mucormycosis

The Aspergillus Lateral Flow Assay for the Diagnosis of Invasive Aspergillosis: an Update

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