Introduction of a bead beating step improves fungal DNA extraction from selected patient specimens

Scharf, Sebastian; Bartels, Anna L.; Kondakci, Mustafa; Pfeffer, Klaus; Henrich, Birgit; Haas, Rainer

doi:10.1016/j.ijmm.2020.151443

Cited by 26 publications

(30 citation statements)

References 68 publications

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“…Optimization of the protocol by adding a chemical and mechanical pre-treatment have be relevant with a significant improvement in the Candida detection rate, especially for concentrations lower than 10 4 CFU/mL (40.6% without pre-treatment vs. 66.7% with pre-treatment). This is in line with a previous study that clearly demonstrated that introducing a bead beating step to the EZ1 procedure improved fungal DNA extraction from human specimens [28]. Candida species have particularities concerning their yeast cell wall, which must have an impact on their lysis susceptibility.…”

Section: Discussionsupporting

confidence: 91%

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Evaluation of 11 DNA Automated Extraction Protocols for the Detection of the 5 Mains Candida Species from Artificially Spiked Blood

Landier

Prudent

et al. 2021

JoF

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The molecular detection of Candida plays an important role in the diagnosis of candidaemia, a major cause of morbidity and mortality. The sensitivity of this diagnosis is partly related to the efficiency of yeast DNA extraction. In this monocentric study, we investigated the suitability of 11 recent automated procedures for the extraction of low and high amounts of Candida DNA from spiked blood. The efficacy of the DNA extraction procedures to detect Candida spp. in blood samples ranged from 31.4% to 80.6%. The NucliSENSTM easyMAGTM procedure was the most efficient, for each species and each inoculum. It significantly outperformed the other procedures at the lower Candida inocula mimicking the clinical setting. This study highlighted a heterogeneity in DNA extraction efficacy between the five main Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei). Up to five automated procedures were appropriate for C. krusei DNA extraction, whereas only one method yielded an appropriate detection of low amount of C. tropicalis. In the era of the syndromic approach to bloodstream infection diagnosis, this evaluation of 11 automated DNA extraction methods for the PCR diagnosis of candidaemia, puts the choice of an appropriate method in routine diagnosis within the reach of laboratories.

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Section: Discussionsupporting

confidence: 91%

“…Few studies have compared current automated nucleic acid extraction methods for the isolation of DNA of the five main Candida species from whole blood [24,[26][27][28]. Here, all 11 extraction methods were equivalent at concentrations greater than 10 6 CFU/mL.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of 11 DNA Automated Extraction Protocols for the Detection of the 5 Mains Candida Species from Artificially Spiked Blood

Landier

Prudent

et al. 2021

JoF

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“…While culture is necessary to obtain susceptibility testing results, we conclude that in patients with an increased risk of life-threatening infections with rare fungal pathogens, a rapid detection method allowing pathogen identification like the one presented here can be an important addition. By using a method developed for efficient DNA extraction from (filamentous) fungi, an increased specificity of the branch-specific reactions of the fuPCR could shorten time to definitive diagnosis even further and help achieving the best possible outcome for the patients [ 28 ].…”

Section: Discussionmentioning

confidence: 99%

Detection of invasive Trichosporon asahii in patient blood by a fungal PCR array

Weber

Scharf

Walther

et al. 2021

Access Microbiology

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Rare invasive fungal infections are increasingly emerging in hosts with predisposing factors such as immunodeficiency. Their timely diagnosis remains difficult, as their clinical picture may initially mimic infections with more common fungal species and species identification may be difficult with routine methods or may require time-consuming subcultures. This often results in ineffective drug administration and fatal outcomes. We report on a patient in their early twenties with mixed cellularity classical Hodgkin lymphoma with a disseminated Trichosporon asahii (T. asahii) infection. Even though pathogen detection and identification was possible via the standard procedure consisting of culture followed by matrix-assisted laser desorption ionisation–time of flight (MALDI-TOF) mass spectrometry, the patient passed away in the course of multi organ failure. Herein, we report on a retrospectively applied experimental diagnostic fungal PCR-analysis used on an EDTA blood sample and consisting of two pan-fungal reactions and seven branch-specific reactions. Regarding invasive T. asahii infection, this PCR array could considerably shorten time to diagnosis and switch to a targeted therapy with triazoles.

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“…[ 17 ]. Further, Scharf et al (2020) observed that for Aspergillus , bead beating could significantly increase the amount of DNA extracted from serum and respiratory samples [ 18 ]. However, an inter-laboratory study displayed no significant effect of the method of extraction of Mucorales DNA from the serum on the diagnostic sensitivity [ 19 ].…”

Section: Dna-based Diagnosismentioning

confidence: 99%

Microbiological and Molecular Diagnosis of Mucormycosis: From Old to New

2021

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Members of the order Mucorales may cause severe invasive fungal infections (mucormycosis) in immune-compromised and otherwise ill patients. Diagnosis of Mucorales infections and discrimination from other filamentous fungi are crucial for correct management. Here, we present an overview of current state-of-the-art mucormycosis diagnoses, with a focus on recent developments in the molecular field. Classical diagnostic methods comprise histology/microscopy as well as culture and are still the gold standard. Newer molecular methods are evolving quickly and display great potential in early diagnosis, although standardization is still missing. Among them, quantitative PCR assays with or without melt curve analysis are most widely used to detect fungal DNA in clinical samples. Depending on the respective assay, sequencing of the resulting PCR product can be necessary for genus or even species identification. Further, DNA-based methods include microarrays and PCR-ESI-MS. However, general laboratory standards are still in development, meaning that molecular methods are currently limited to add-on analytics to culture and microscopy.

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Introduction of a bead beating step improves fungal DNA extraction from selected patient specimens

Cited by 26 publications

References 68 publications

Evaluation of 11 DNA Automated Extraction Protocols for the Detection of the 5 Mains Candida Species from Artificially Spiked Blood

Evaluation of 11 DNA Automated Extraction Protocols for the Detection of the 5 Mains Candida Species from Artificially Spiked Blood

Detection of invasive Trichosporon asahii in patient blood by a fungal PCR array

Microbiological and Molecular Diagnosis of Mucormycosis: From Old to New

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