Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients

Landlinger, Christine; Preuner, Sandra; Bašková, Lenka; Grotel, Martine van; Hartwig, Nico G.; Dworzak, Michael; Mann, Georg; Attarbaschi, Andishe; Kager, Leo; Peters, Christina; Matthes‐Martin, Susanne; Lawitschka, Anita; Heuvel‐Eibrink, Marry M. van den; Lion, Thomas

doi:10.1038/leu.2010.209

Cited by 72 publications

(45 citation statements)

References 50 publications

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“…The real-time PCR assay provides a high sensitivity and specificity for the detection of fungal DNA and rapidly identifies most of clinically relevant Aspergillus species (20). Many studies have reported the correlation between the presence of proven, probable, and possible criteria of IFIs according to the EORTC-MSG criteria and PCR results of the respective patients' sera (17,21,22). The results of the present study show 5/8 patients with proven and 5/6 patients with probable criteria.…”

Section: Discussionsupporting

confidence: 52%

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Incidence of Fungal Infections in Pediatric Patients with Hematologic Neoplasms

Badiee

Zareifar

Haddadi

et al. 2017

Arch Pediatr Infect Dis

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Background: Fungal infections are one of the most important causes of morbidity and mortality in patients with hematological disorders. The frequency of these infections has increased during the past decades. Objectives: The rate of fungal infections was investigated in pediatric patients with hematological disorders, using traditional and real-time PCR methods, in order to establish proper management of these patients. Methods: Over a 13-month period, 86 patients with hematological disorders were admitted and were kept under observation for the development of fungal infections. Fungal colonization was determined and clinical samples were examined by direct microscopic examination and culture. Blood specimens were cultured by bedside inoculation into a BACTEC medium. The results of the pathology smear were collected from the patients' records. Real-time PCR was performed on all patients' sera to diagnose invasive candidiasis and aspergillosis. Results: Candida colonization was seen in 42 (48.8%) and oral candidiasis was diagnosed in 7 (8%) patients. The incidence of invasive fungal infections was 16.3% (14/86) with a mortality rate of 50% in pediatric patients. The etiologic agents were Candida albicans in 5 cases, Aspergillus flavus in 3, and Aspergillus fumigatus, Fusarium, Alterneria and Mucor, each in 1 case. In 2 cases with liver abscess, only Candida PCR was positive. Fungemia was observed in 4 patients. Conclusions:The rate of invasive fungal infections in our study was high. Early and accurate detection of these infections could result in a better outcome. In critical ill cases where only blood samples are available, molecular methods such as PCR could be more effective than culture for the detection of etiologic agents.

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Section: Discussionsupporting

confidence: 52%

“…followed by yeast infections including candidemia (20). The highest IFI-attributable mortality rates were associated with zygomycosis followed by fusariosis, aspergillosis, and candidemia (21).…”

Section: Discussionmentioning

confidence: 99%

Incidence of Fungal Infections in Pediatric Patients with Hematologic Neoplasms

Badiee

Zareifar

Haddadi

et al. 2017

Arch Pediatr Infect Dis

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“…Landlinger et al also evaluated the clinical potential of separate real-time qPCRs targeted a highly conserved region of the 28S rRNA multicopy gene, covering a wide spectrum of molds, yeasts, and the Mucormycetes (33). The sensitivity of the assay was revealed to be 96% (95% confidence interval, 82% to 99%).…”

Section: Discussionmentioning

confidence: 99%

Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays

et al. 2016

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h In a retrospective multicenter study, 102 formalin-fixed paraffin-embedded (FFPE) tissue specimens with histopathology results were tested. Two 4-to 5-m FFPE tissue sections from each specimen were digested with proteinase K, followed by automated nucleic acid extraction. Multiple real-time quantitative PCR (qPCR) assays targeting the internal transcribed spacer 2 (ITS2) region of ribosomal DNA, using fluorescently labeled primers, was performed to identify clinically important genera and species of Aspergillus, Fusarium, Scedosporium, and the Mucormycetes. The molecular identification was correlated with results from histological examination. One of the main findings of our study was the high sensitivity of the automated DNA extraction method, which was estimated to be 94%. The qPCR procedure that was evaluated identified a range of fungal genera/species, including Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Fusarium oxysporum, Fusarium solani, Scedosporium apiospermum, Rhizopus oryzae, Rhizopus microsporus, Mucor spp., and Syncephalastrum. Fusarium oxysporum and F. solani DNA was amplified from five specimens from patients initially diagnosed by histopathology as having aspergillosis. Aspergillus flavus, S. apiospermum, and Syncephalastrum were detected from histopathological mucormycosis samples. In addition, examination of four samples from patients suspected of having concomitant aspergillosis and mucormycosis infections resulted in the identification of two A. flavus isolates, one Mucor isolate, and only one sample having both R. oryzae and A. flavus. Our results indicate that histopathological features of molds may be easily confused in tissue sections. The qPCR assay used in this study is a reliable tool for the rapid and accurate identification of fungal pathogens to the genus and species levels directly from FFPE tissues.T he frequency of invasive fungal diseases (IFDs) has increased significantly over the past 3 decades, and they are associated with excessive morbidity and mortality in immunocompromised hosts, patients hospitalized with severe underlying diseases (e.g., acute myelogenous leukemia), those requiring complex surgical procedures (e.g., trauma patients), and individuals who require support in intensive care units (1-5).The most well-known cause of opportunistic filamentous mycoses is Aspergillus fumigatus (6). However, the epidemiology of IFDs due to filamentous fungi has been expanded well beyond A. fumigatus, including non-fumigatus species of Aspergillus, the mucormycetes, Fusarium and Scedosporium species, and a wide variety of melanized fungi (4, 7).Given the complexity of the population of patients at risk and the diverse and increasing arrays of fungal pathogens, which show significantly different antifungal susceptibilities, (3) early and reliable detection of a causative fungal pathogen is crucial to guide the appropriate and successful treatment of IFDs (8, 9).Formalin-fixed paraffin-embedded (FFPE) tissues obtained from patients with prov...

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“…The readout was performed by the use of a yeast-specific PCR of a panfungal real-time assay described earlier (11). Three independent experiments were performed.…”

Section: Methodsmentioning

confidence: 99%

“…Here we provide the first evidence for the presence of fungal DNA contaminants in lyophilized primers and TaqMan probes as well as in master mix solutions for real-time PCR analysis purchased at renowned biotechnology companies. Using a TaqMan-based panfungal PCR assay (11) established for sensitive screening of immunocompromised patients at risk for IFI, we detected traces of fungal DNA in several batches of the indicated reagents. To overcome the risk of false-positive PCR test results attributable to this issue, we have successfully applied a decontamination method based on the activity of a double-strand specific DNase (dsDNase).…”

mentioning

confidence: 99%

Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination

Czurda¹,

Smelik²,

Preuner-Stix³

et al. 2016

J Clin Microbiol

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Nucleic acid amplification techniques permitting sensitive and rapid screening in patients at risk for invasive fungal infections are an important addition to conventional fungal diagnostic methods. However, contamination with fungal DNA may be a serious threat to the validity of fungal amplification-based assays. Besides rigorous handling procedures to avoid false-positive test results from exogenous sources, we have implemented protocols for comprehensive assessment of fungal contamination in all materials involved in the analytical process. Traces of fungal DNA were found in different commercially available PCR reagents, including lyophilized primers, TaqMan probes, and master mix solutions. These contaminants resulted in a considerable rate of false-positive tests in panfungal real-time PCR analysis. To address this problem, we have established a decontamination protocol based on the activity of a double-strand specific DNase. Using this approach, we have significantly reduced the frequency of false-positive test results attributable to contaminated reagents. On the basis of our findings, we strongly recommend routine monitoring of all reagents used in fungal PCR assays for the presence of relevant contaminants. As long as fungal-grade reagents are not readily available, pretreatment methods facilitating elimination of fungal DNA are critical for reducing the risk of falsepositive results in highly sensitive molecular fungal detection assays. N ucleic acid amplification-based techniques can detect minute quantities of fungal DNA and can enable fast and sensitive screening in patients at risk for invasive fungal infections (IFI). They can overcome the limitations of conventional diagnostic methods that are slow or have inadequate sensitivities (1-3). However, a major drawback of highly sensitive PCR applications is that even low-level contaminations can lead to false-positive results (4). This is particularly evident when broad-range screening methods are applied because their ability to detect contaminating DNA from many different fungal species renders them more vulnerable to trace amounts of nucleic acid contamination than species-specific assays (1). This problem is already well described for universal bacterial assays, where PCR testing based on 16S rRNA gene amplification can be compromised by DNA contamination of reagents derived from bacterial sources such as Taq DNA polymerase or uracil-N-glycosylase (5, 6).Problems with fungal contamination during PCR-based assays have been previously reported. Airborne conidia or spores and trace amounts of fungal nucleic acids in commercially available enzymes used for fungal cell lysis and in components of DNA extraction kits were identified as possible sources of contamination (7-10). Here we provide the first evidence for the presence of fungal DNA contaminants in lyophilized primers and TaqMan probes as well as in master mix solutions for real-time PCR analysis purchased at renowned biotechnology companies. Using a TaqMan-based panfungal PCR assay (11) establi...

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Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients

Cited by 72 publications

References 50 publications

Incidence of Fungal Infections in Pediatric Patients with Hematologic Neoplasms

Incidence of Fungal Infections in Pediatric Patients with Hematologic Neoplasms

Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays

Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination

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