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Journal ArticleDOI

New) Methods for Detection of Aspergillus fumigatus Resistance in Clinical Samples.

20 Jun 2019-Current Fungal Infection Reports (Curr Fungal Infect Rep)-Vol. 13, Iss: 3, pp 129-136
TL;DR: New molecular-based approaches for detecting triazole resistance to Aspergillus, real-time polymerase chain reaction (PCR) to detect mutations to the Cyp51A protein, have been developed which are able to detect mostTriazole-resistant A. fumigatus strains in patients with invasive aspergillosis.
Abstract: The incidence of invasive aspergillosis has increased substantially over the past few decades, accompanied by a change in susceptibility patterns of Aspergillus fumigatus with increasing resistance observed against triazole antifungals, including voriconazole and isavuconazole, the most commonly used antifungal agents for the disease. Culture-based methods for determining triazole resistance are still the gold standard but are time consuming and lack sensitivity. We sought to provide an update on non-culture-based methods for detecting resistance patterns to Aspergillus. New molecular-based approaches for detecting triazole resistance to Aspergillus, real-time polymerase chain reaction (PCR) to detect mutations to the Cyp51A protein, have been developed which are able to detect most triazole-resistant A. fumigatus strains in patients with invasive aspergillosis. Over the last few years, a number of non-culture-based methods for molecular detection of Aspergillus triazole resistance have been developed that may overcome some of the limitations of culture. These molecular methods are therefore of high epidemiological and clinical relevance, mainly in immunocompromised patients with hematological malignancies, where culture has particularly limited sensitivity. These assays are now able to detect most triazole-resistant Aspergillus fumigatus strains. Given that resistance rates vary, clinical utility for these assays still depends on regional resistance patterns.
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Journal ArticleDOI
24 Mar 2021-Mycoses
TL;DR: Invasive aspergillosis (IA) is an increasingly recognised phenomenon in critically ill patients in the intensive care unit, including in patients with severe influenza and severe coronavirus disease 2019 (COVID-19) infection as mentioned in this paper.
Abstract: Invasive aspergillosis (IA) is an increasingly recognised phenomenon in critically ill patients in the intensive care unit, including in patients with severe influenza and severe coronavirus disease 2019 (COVID-19) infection. To date, there are no consensus criteria on how to define IA in the ICU population, although several criteria are used, including the AspICU criteria and new consensus criteria to categorise COVID-19-associated pulmonary aspergillosis (CAPA). In this review, we describe the epidemiology of IA in critically ill patients, most common definitions used to define IA in this population, and most common clinical specimens obtained for establishing a mycological diagnosis of IA in the critically ill. We also review the most common diagnostic tests used to diagnose IA in this population, and lastly discuss the most common clinical presentation and imaging findings of IA in the critically ill and discuss areas of further needed investigation.

28 citations

Journal ArticleDOI
TL;DR: Current diagnostic methods are reviewed and the potential of pyrosequencing to aid in a diagnosis complete with a resistance profile to improve clinical outcomes is highlighted.
Abstract: Guidelines on the diagnosis and management of Aspergillus disease recommend a multi-test approach including CT scans, culture, fungal biomarker tests, microscopy and fungal PCR. The first-line treatment of confirmed invasive aspergillosis (IA) consists of drugs in the azole family; however, the emergence of azole-resistant isolates has negatively impacted the management of IA. Failure to detect azole-resistance dramatically increases the mortality rates of azole-treated patients. Despite drug susceptibility tests not being routinely performed currently, we suggest including resistance testing whilst diagnosing Aspergillus disease. Multiple tools, including DNA sequencing, are available to screen for drug-resistant Aspergillus in clinical samples. This is particularly beneficial as a large proportion of IA samples are culture negative, consequently impeding susceptibility testing through conventional methods. Pyrosequencing is a promising in-house DNA sequencing method that can rapidly screen for genetic hotspots associated with antifungal resistance. Pyrosequencing outperforms other susceptibility testing methods due to its fast turnaround time, accurate detection of polymorphisms within critical genes, including simultaneous detection of wild type and mutated sequences, and-most importantly-it is not limited to specific genes nor fungal species. Here we review current diagnostic methods and highlight the potential of pyrosequencing to aid in a diagnosis complete with a resistance profile to improve clinical outcomes.

21 citations

Journal ArticleDOI
TL;DR: A need exists for diagnostic tests that are effective, simple, cheap, and rapid to enable the diagnosis of bIFI in patients taking antifungals and support the design of future clinical trials in the field of clinical mycology.
Abstract: Breakthrough invasive fungal infections (bIFI) cause significant morbidity and mortality. Their diagnosis can be challenging due to reduced sensitivity to conventional culture techniques, serologic tests, and PCR-based assays in patients undergoing antifungal therapy, and their diagnosis can be delayed contributing to poor patient outcomes. In this review, we provide consensus recommendations on behalf of the European Confederation for Medical Mycology (ECMM) for the diagnosis of bIFI caused by invasive yeasts, molds, and endemic mycoses, to guide diagnostic efforts in patients receiving antifungals and support the design of future clinical trials in the field of clinical mycology. The cornerstone of lab-based diagnosis of breakthrough infections for yeast and endemic mycoses remain conventional culture, to accurately identify the causative pathogen and allow for antifungal susceptibility testing. The impact of non-culture-based methods are not well-studied for the definite diagnosis of breakthrough invasive yeast infections. Non-culture-based methods have an important role for the diagnosis of breakthrough invasive mold infections, in particular invasive aspergillosis, and a combination of testing involving conventional culture, antigen-based assays, and PCR-based assays should be considered. Multiple diagnostic modalities, including histopathology, culture, antibody, and/or antigen tests and occasionally PCR-based assays may be required to diagnose breakthrough endemic mycoses. A need exists for diagnostic tests that are effective, simple, cheap, and rapid to enable the diagnosis of bIFI in patients taking antifungals.

20 citations


Cites background from "New) Methods for Detection of Asper..."

  • ...Overall, while they remain the cornerstone of IFI diagnostics, culture-based approaches are limited by low sensitivity in patients exposed to antifungals, and delays in diagnosis are common [4]....

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Journal ArticleDOI
TL;DR: A review of the main steps of the diagnosis for systemic fungal infection, from diagnostic classifications, through methodologies considered as the gold standard, to the molecular methods currently used, and finally mentioning some of the more futuristic approaches is presented in this paper.

17 citations

References
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Book
01 Jan 2008
TL;DR: The Quality System Approach is intended to provide a guide to the development and use of such systems in the rapidly changing environment as to ensure their effectiveness and efficiency.
Abstract: ...................................................................................................................................................i Committee Membership..........................................................................................................................v Active Membership.............................................................................................................................. vii Foreword...............................................................................................................................................xv The Quality System Approach.............................................................................................................xvi

1,674 citations

Journal ArticleDOI
TL;DR: Azole resistance in Aspergillus fumigatus has now been reported from 6 continents and is emerging as a global health problem.
Abstract: Azole resistance in Aspergillus fumigatus has emerged as a global health problem. Although the number of cases of azole-resistant aspergillosis is still limited, resistance mechanisms continue to emerge, thereby threatening the role of the azole class in the management of diseases caused by Aspergillus. The majority of cases of azole-resistant disease are due to resistant A. fumigatus originating from the environment. Patient management is difficult due to the absence of patient risk factors, delayed diagnosis, and limited treatment options, resulting in poor treatment outcome. International and collaborative efforts are required to understand how resistance develops in the environment to allow effective measures to be implemented aimed at retaining the use of azoles both for food production and human medicine.

436 citations

Journal ArticleDOI
TL;DR: The use of voriconazole has become common for the management of invasive aspergillosis, however, therapy with vorIconazole still sometimes fails, more often because of unresponsive underlying disease than because of resistance of the fungus.
Abstract: To the Editor: The use of voriconazole has become common for the management of invasive aspergillosis. However, therapy with voriconazole still sometimes fails, more often because of unresponsive underlying disease than because of resistance of the fungus. Since the first description of itraconazole resistance in Aspergillus fumigatus, 1 three amino acid substitutions in the 14α-sterol demethylase cyp51A gene, which is the target site for azole drugs, have been described.2 Our laboratory receives fungal isolates for identification and susceptibility testing from throughout the Netherlands. Since 2002, using Clinical and Laboratory Standards Institute methodology, we have observed an increase in the number of . . .

349 citations

Journal ArticleDOI
TL;DR: The very low organism burdens of fungi causing infection have previously prevented direct culture and detection of antifungal resistance in clinical samples, and these findings have major implications for the sustainability of triazoles for human antIFungal therapy.
Abstract: BACKGROUND: Oral triazole therapy is well established for the treatment of invasive (IPA), allergic (ABPA), and chronic pulmonary (CPA) aspergillosis, and is often long-term. Triazole resistance rates are rising internationally. Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequency of triazole resistance. METHODS: Using an ultrasensitive real-time polymerase chain reaction (PCR) assay for Aspergillus spp., we assessed respiratory fungal load in bronchoalveolar lavage (BAL) and sputum specimens. In a subset of PCR-positive, culture negative samples, we further amplified the CYP51A gene to detect key single-nucleotide polymorphisms (SNPs) associated with triazole resistance. RESULTS: Aspergillus DNA was detected in BAL from normal volunteers (4/11, 36.4%) and patients with culture or microscopy confirmed IPA (21/22, 95%). Aspergillus DNA was detected in sputum in 15 of 19 (78.9%) and 30 of 42 (71.4%) patients with ABPA and CPA, compared with 0% and 16.7% by culture, respectively. In culture-negative, PCR-positive samples, we detected triazole-resistance mutations (L98H with tandem repeat [TR] and M220) within the drug target CYP51A in 55.1% of samples. Six of 8 (75%) of those with ABPA and 12 of 24 (50%) with CPA had resistance markers present, some without prior triazole treatment, and in most despite adequate plasma drug concentrations around the time of sampling. CONCLUSIONS: The very low organism burdens of fungi causing infection have previously prevented direct culture and detection of antifungal resistance in clinical samples. These findings have major implications for the sustainability of triazoles for human antifungal therapy.

288 citations

Journal ArticleDOI
TL;DR: A mathematical algorithm to compare and distinguish matrix-assisted laser desorption/ionization mass spectra of whole bacteria cells is developed and it has distinguished twenty five different strains of a single bacteria species, E. coli.
Abstract: We have developed a mathematical algorithm to compare and distinguish matrix-assisted laser desorption/ionization (MALDI) mass spectra of whole bacteria cells. This fingerprint matching technique eliminates the subjectivity involved in visually comparing two spectra to determine whether they match and it provides a quantitative measure of spectral similarity. Using it, we have distinguished twenty five different strains of a single bacteria species, E. coli. Cells are grown in culture, samples are prepared, and MALDI-TOF mass spectra are recorded for each strain. Pairs of spectra are compared by a modified cross-correlation procedure. This modified approach increases the sensitivity of correlation analysis to small spectral differences. The technique can be fine-tuned by varying the number of intervals into which spectra are divided.

253 citations

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