Pathogen-Specific DNA Enrichment Does Not Increase Sensitivity of PCR for Diagnosis of Invasive Aspergillosis in Neutropenic Patients

Springer, Jan; Loeffler, Juergen; Heinz, Werner; Schlossnagel, Hannes; Lehmann, Marc; Morton, Oliver; Rogers, Thomas R.; Schmitt, Corinna; Frosch, Matthias; Einsele, Hermann; Kurzai, Oliver

doi:10.1128/jcm.01679-10

Cited by 38 publications

(31 citation statements)

References 12 publications

(18 reference statements)

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“…No crossreactivity with other fungi or human genomic DNA was observed (18). This assay detected all clinically relevant Aspergillus species (16).…”

Section: Methodsmentioning

confidence: 91%

“…All DNA extraction steps were performed in a class II laminar-flow cabinet and were compliant with EAPCRI recommendations (9). WB was extracted as described previously (15,16). Briefly, red and white cells from 3 ml of EDTA blood were lysed and centrifuged.…”

Section: Methodsmentioning

confidence: 99%

“…In Wuerzburg, an Aspergillus-specific real-time PCR assay targeting the internal transcribed spacer 1 (ITS1)-5.8S rRNA gene region (16,18) was used to detect fungal DNA. Briefly, 21-l reaction mixtures contained 0.3 M primer Asp fum_F degen, 0.6 M primer Fungi 5.8_R, 0.15 M hydrolysis probe ITS-PF, 10 l TaqMan gene expression master mix (Applied Biosystems), and 10 l template DNA.…”

Section: Methodsmentioning

confidence: 99%

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Multicenter Comparison of Serum and Whole-Blood Specimens for Detection of Aspergillus DNA in High-Risk Hematological Patients

et al. 2013

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. Forty-seven cases (proven and probable IA) and 31 controls (no evidence of IA) were selected retrospectively for this case-control study, comprising 803 samples, in order to determine the performance of whole-blood PCR, serum PCR, and serum galactomannan testing. Although no single assay was able to detect every case of IA, a combination of different assays provided the best performance. There was no significant difference between the use of whole-blood and serum specimens for PCR-based diagnosis of IA, but there was a trend for whole blood to be more sensitive (85% versus 79%) and to yield an earlier positive result (36 days versus 15 days) than for serum. However, DNA extraction from serum specimens is easier and faster than that from whole-blood specimens, and it allows the same specimen to be used for both galactomannan and PCR assays. In conclusion, the appropriate sample type for DNA extraction should be determined by the local requirements and the technical platforms available at each individual center. A combination of biomarker tests offered the best diagnostic utility for detecting IA.

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“…No crossreactivity with other fungi or human genomic DNA was observed (18). This assay detected all clinically relevant Aspergillus species (16).…”

Section: Methodsmentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Multicenter Comparison of Serum and Whole-Blood Specimens for Detection of Aspergillus DNA in High-Risk Hematological Patients

et al. 2013

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“…Although the array of DNA-based molecular diagnostic tests that outperform the conventional methods has expanded over the past two decades, these platforms were not included in the European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSG) consensus definitions because of the lack of standardization. It is clear that there is an urgent need to develop effective and reliable diagnostics for IA due to the continuing growth of publications working at incorporating DNA based applications into routine clinical diagnostics to reduce costs and to improve sensitivity and specificity respectively [25][26][27][28][29][30][31] and supporting diagnostic driven therapy.…”

Section: Discussionmentioning

confidence: 99%

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

Paholcsek

Leiter

Markovics

et al. 2014

Acta Microbiologica Et Immunologica Hungarica

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Despite concerted efforts, diagnosis of aspergillosis is still a great challenge to clinical microbiology laboratories. Along with the requirement for high sensitivity and specificity, species-specific identification is important. We developed rapid, sensitive and species-specific qPCR assays using the TaqMan by Aspergillus fumigatus and Aspergillus terreus, which merits future diagnostic studies. The assays were sensitive enough to detect a few genomic equivalents in blood samples.

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“…At UKW, DNA was extracted from 1.0 ml of 306 undiluted serum samples and 0.5 to Ͻ1.0 ml of 114 undiluted serum samples using the commercially available Qiagen UltraSens virus kit, following the manufacturer's instructions with the following modifications: (i) no carrier RNA was used, (ii) lysate centrifugation was adjusted to 3,000 ϫ g, and (iii) the elution buffer volume was increased to 70 l and incubated on the column at room temperature for 2 min before tubes were centrifuged for 2 min (3). At UKW, the WBP was extracted using standardized methods as described previously (1,12). Briefly, red and white cells from 3 ml of EDTA blood were lysed and centrifuged.…”

Section: Methodsmentioning

confidence: 99%

Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations

et al. 2016

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cStandardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P ‫؍‬ 0.0019; for serum, P ‫؍‬ 0.0049; and for WBP, P ‫؍‬ 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P ‫؍‬ 0.0238) and plasma (53%, P ‫؍‬ 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time to diagnosis may be enhanced by testing multiple contemporaneously obtained sample types. Standardized methodologies for the molecular detection of invasive aspergillosis (IA) when testing whole blood (WB), serum, and plasma have been established by the European Aspergillus PCR Initiative (EAPCRI), but direct comparisons of performance for methods compliant with the recommendations are limited (1-8). The published evidence from the data generated is contradictory. Serum was shown to be superior to the cell pellet in one study, only for another to recommend the use of WB over plasma (4,5). A recent study showed no significant difference in PCR performance when testing serum or WB (6). Differences between the methodologies used in the studies, particularly sample volumes and DNA extraction protocols, make comparisons difficult, and if suboptimal methods were used for one particular sample type, the assay performance and conclusions could be biased (1, 2).It is important to compare the different specimen types using optimal methodology and to co...

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Pathogen-Specific DNA Enrichment Does Not Increase Sensitivity of PCR for Diagnosis of Invasive Aspergillosis in Neutropenic Patients

Cited by 38 publications

References 12 publications

Multicenter Comparison of Serum and Whole-Blood Specimens for Detection of Aspergillus DNA in High-Risk Hematological Patients

Multicenter Comparison of Serum and Whole-Blood Specimens for Detection of Aspergillus DNA in High-Risk Hematological Patients

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

Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations

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