Novel and sensitive qPCR assays for the detection and identification of aspergillosis causing species

Paholcsek, Melinda; Leiter, Éva; Markovics, Arnold; Bíró, Sándor

doi:10.1556/amicr.61.2014.3.3

Cited by 2 publications

(3 citation statements)

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“…The detection and identification of Mucorales and Aspergillus from FFPE samples played an important role in the diagnosis and management of aspergillosis and mucormycosis, whereas microscopy, serology, and culture were restricted by several disadvantages (Jensen et al, 1997;Frater et al, 2001;Rickerts et al, 2007;Hofman et al, 2010;Hamilos et al, 2011). Numerous RQ-PCR assays have been described for detection of Mucorales (Bernal-Martinez et al, 2013;Millon et al, 2013;Lengerova et al, 2014) and Aspergillus (Lass-Florl et al, 2011;Fricke et al, 2012;Paholcsek et al, 2014;Pini et al, 2015). In this study, we evaluated two Mucorales RQ-PCR assays, two Aspergillus RQ-PCR assays, and four combined tests to rapidly detect and identify Mucorales and Aspergillus.…”

Section: Discussionmentioning

confidence: 99%

Detection and identification of Mucorales and Aspergillus in paraffin-embedded samples by real-time quantitative PCR

Jiang

Jiang²,

Ye³

2023

Front. Cell. Infect. Microbiol.

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BackgroundIn this study, we used real-time quantitative PCR (RQ-PCR) to rapidly detect Mucorales and Aspergillus in formalin-fixed, paraffin-embedded (FFPE) samples, targeting 18SrRNA gene and 28SrRNA gene. Identification of Mucorales and Aspergillus was analysed by combining Mucorales RQ-PCR (Mucorales18SrRNA and Mucorales28SrRNA) with Aspergillus RQ-PCR (Aspergillus18SrRNA and Aspergillus28SrRNA).ObjectivesThe aims of this study were to compare the diagnostic performances of four RQ-PCR assays as single and combined diagnostic and identification tools.MethodsWe collected 12 control group samples and 81 experimental group samples diagnosed by histopathology, including mucormycosis (19 patients, 21 FFPE samples), aspergillosis (54 patients, 57 FFPE samples) and mucormycosis with aspergillosis (3 patients, 3 FFPE samples). All samples were detected by four RQ-PCR tests to compare and analyze diagnostic performance.ResultsThe sensitivities of Mucorales18SrRNA and Mucorales28SrRNA were both 75%, with the tests having specificities of 97.10% and 94.20%. The sensitivities of Aspergillus18SrRNA and Aspergillus28SrRNA were 73.33% and 65%, with the tests having specificities of 87.88% and 81.82%. The values of the evaluation indexes of the combined detection of Mucorales28SrRNA and Aspergillus18SrRNA (M28A18) were the highest with a kappa coefficient value of 0.353, followed by M18A18. M28A18 had a sensitivity of 67.90% and a specificity of 100%.ConclusionsWe recommend using the combination of Mucorales RQ-PCR and Aspergillus RQ-PCR as a screening tool to detect samples suspected of mucormycosis and/or aspergillosis.

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Section: Discussionmentioning

confidence: 99%

Detection and identification of Mucorales and Aspergillus in paraffin-embedded samples by real-time quantitative PCR

Jiang

Jiang²,

Ye³

2023

Front. Cell. Infect. Microbiol.

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“…The real time PCR detection of A. fumigatus specific DNA was based on the Streptomyces factor C orthologous gene ( facC -PCR): this was most likely horizontally transferred to a few filamentous fungi including A. fumigatus as previously described [ 11 ]. Since the facC gene that was acquired by horizontal gene transfer [ 12 ] is only present in a few fungal species [ 13 ] and they diverged millions of years ago, we were able to design species-specific assays that do not cross-react even with closely related species that carry this gene therefore the analytical specificity is increased as compared to other assays.…”

Section: Methodsmentioning

confidence: 99%

“…Species specific hydrolysis assays were used that targeted the facC orthologous genes present in A. fumigatus [ 11 ]. A. fumigatus specific duplex TaqMan-LNA hydrolysis assay was used targeting facC orthologous genes (AFUA_3G14910, and AFUA_5G00540) present in two copies (on Chr#3 and on Chr#5 respectively) in A. fumigatus genome.…”

Section: Methodsmentioning

confidence: 99%

Combining standard clinical methods with PCR showed improved diagnosis of invasive pulmonary aspergillosis in patients with hematological malignancies and prolonged neutropenia

et al. 2015

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BackgroundWe assessed the diagnostic value of standard clinical methods and combined biomarker testing (galactomannan assay and polymerase chain reaction screening) in a prospective case–control study to detect invasive pulmonary aspergillosis in patients with hematological malignancies and prolonged neutropenia.MethodsIn this observational study 162 biomarker analyses were performed on samples from 27 febrile neutropenic episodes. Sera were successively screened for galactomannan antigen and for Aspergillus fumigatus specific nucleic acid targets. Furthermore thoracic computed tomography scanning was performed along with bronchoscopy with lavage when clinically indicated. Patients were retrospectively stratified to define a case-group with “proven” or “probable” invasive pulmonary aspergillosis (25.93 %) and a control-group of patients with no evidence for of invasive pulmonary aspergillosis (74.07 %). In 44.44 % of episodes fever ceased in response to antibiotic treatment (group II). Empirical antifungal therapy was administered for episodes with persistent or relapsing fever (group I). 48.15 % of patients died during the study period. Postmortem histology was pursued in 53.85 % of fatalities.ResultsConcordant negative galactomannan and computed tomography supported by a polymerase chain reaction assay were shown to have the highest discriminatory power to exclude invasive pulmonary aspergillosis. Bronchoalveolar lavage was performed in 6 cases of invasive pulmonary aspergillosis and in 15 controls. Although bronchoalveolar lavage proved negative in 93 % of controls it did not detect IPA in 86 % of the cases. Remarkably post mortem histology convincingly supported the presence of Aspergillus hyphae in lung tissue from a single case which had consecutive positive polymerase chain reaction assay results but was misdiagnosed by both computed tomography and consistently negative galactomannan assay results. For the galactomannan enzyme-immunoassay the diagnostic odds ratio was 15.33 and for the polymerase chain reaction assay it was 28.67. According to Cohen’s kappa our in-house polymerase chain reaction method showed a fair agreement with the galactomannan immunoassay. Combined analysis of the results from the Aspergillus galactomannan enzyme immunoassay together with those generated by our polymerase chain reaction assay led to no misdiagnoses in the control group.ConclusionThe data from this pilot-study demonstrate that the consideration of standard clinical methods combined with biomarker testing improves the capacity to make early and more accurate diagnostic decisions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-015-0995-8) contains supplementary material, which is available to authorized users.

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Novel and sensitive qPCR assays for the detection and identification of aspergillosis causing species

Cited by 2 publications

References 32 publications

Detection and identification of Mucorales and Aspergillus in paraffin-embedded samples by real-time quantitative PCR

Detection and identification of Mucorales and Aspergillus in paraffin-embedded samples by real-time quantitative PCR

Combining standard clinical methods with PCR showed improved diagnosis of invasive pulmonary aspergillosis in patients with hematological malignancies and prolonged neutropenia

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