Aspergillus PCR: One Step Closer to Standardization

White, P. Lewis; Bretagne, Stéphane; Klingspor, Lena; Melchers, Willem J. G.; McCulloch, Elaine; Schulz, Bettina; Finnstrom, Niklas; Mengoli, Carlo; Barnes, Rosemary A.; Donnelly, J. Peter; Loeffler, Juergen

doi:10.1128/jcm.01767-09

Cited by 296Antifungal resistance and susceptibilityFungal and yeast genetics researchBacterial Identification and Susceptibility Testing

Aspergillus PCR: One Step Closer to Standardization

Open access

White, P. Lewis, Bretagne, Stéphane, Klingspor, Lena, Melchers, Willem J. G., McCulloch, Elaine, Schulz, Bettina, Finnstrom, Niklas, Mengoli, Carlo +3 more·2010·Journal of Clinical Microbiology

DOI 10.1128/jcm.01767-09PMC PMC2849576

Open this paper in CoChat — free account

Abstract

PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus PCR Initiative was formed. The aim of the initiative was to provide optimal standardized protocols for the widespread clinical evaluation of the Aspergillus PCR to determine its diagnostic role and allow inclusion in disease diagnosis criteria. Quality control panels were developed and circulated to centers for evaluation of the existing methodology before recommendations based on the initial results were proposed for further panels. The centers were anonymously classified as “compliant” or “noncompliant,” according to whether they had followed the proposed recommendations before the performance parameters were determined and meta-regression analysis was performed. Most PCR amplification systems provided similar detection thresholds, although positivity was a function of the fungal burden. When PCR amplification was combined with DNA extraction, 50% of the centers failed to achieve the same level of detection. Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the proposed recommendations and the use of bead beating, white cell lysis buffer, and an internal control PCR. The use of elution volumes above 100 μl showed a negative correlation with sensitivity. The efficiency of the Aspergillus PCR is limited by the extraction procedure and not by PCR amplification. For PCR testing of whole blood, it is essential that large blood volumes (≥3 ml) be efficiently lysed before bead beating to disrupt the fungal cell and performance of an internal control PCR to exclude false negativity. DNA should be eluted in volumes of <100 μl.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

Integrity signals

Registry

Crossref · 10.1128/jcm.01767-09

Cites

3 of 3 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Among patients with documented MRSA infections, delays of > 24-48 hours until antibiotic administration are associated with increased mortality in some studies, but not in others.
“Among patients with documented MRSA infections, delays of > 24-48 hours until antibiotic administration are associated with increased mortality in some studies (143-147), but not in others (148-154).”
From Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock 2021. (2021) · cites this paper as [146]
The Abstract discusses mortality in invasive aspergillosis and the early initiation of antifungal therapy, not documented MRSA infections. Searches for MRSA, delayed antibiotic administration, and a 24–48-hour mortality association found no passage addressing the claim. (LLM, conf=0.78)
Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Revised Definitions of Invasive Fungal Disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group
De Pauw, B., Walsh, T. J., Donnelly, J. P., Stevens, D. A., Edwards, J. E., Calandra, T., Pappas, P. G., Maertens, J., Lortholary, O., Kauffman, C. A., et al. (2008) · Clinical Infectious Diseases · doi:10.1086/588660 · Cited by 4,917 · see citing works
Self-citation
Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD initiative
Bossuyt, P. M., Reitsma, J. B., Bruns, D. E., Gatsonis, C. A., Glasziou, P. P., Irwig, L. M., Lijmer, J. G., Moher, D., Rennie, D., & de Vet, H. C. W. (2003) · BMJ · doi:10.1136/bmj.326.7379.41 · Cited by 3,215
Laboratory diagnosis of invasive aspergillosis
Hope, W., Walsh, T., & Denning, D. (2005) · The Lancet Infectious Diseases · doi:10.1016/s1473-3099(05)70238-3 · Cited by 495 · see citing works

Flagged first, then by citations

Research works printed but not linked: 30

[1]Ascioglu, S., J. H. Rex, B. de Pauw, J. E. Bennett, J. Bille, F. Crokaert, D. W. Denning, J. P. Donnelly, J. E. Edwards, Z. Erjavec, D. Fiere, O. Lortholary, J. Maertens, J. F. Meis, T. F. Patterson, J. Ritter, D. Selleslag, P. M. Shah, D. A. Stevens, T. J. Walsh, and the Invasive Fungal Infections Cooperative Group of the European Organization for Research and Treatment of Cancer, Mycoses Study Group of the National Institute of Allergy and Infectious Diseases. 2002. Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus. Clin. Infect. Dis. 34:7-14. [DOI] [PubMed] [Google Scholar]
[3]Caillot, D., J. F. Couaillier, A. Bernard, O. Casasnovas, D. W. Denning, L. Mannone, J. Lopez, G. Couillault, F. Piard, O. Vagner, and H. Guy. 2001. Increasing volume and changing characteristics of invasive pulmonary aspergillosis on sequential thoracic computed tomography scans in patients with neutropenia. J. Clin. Oncol. 19:253-259. [DOI] [PubMed] [Google Scholar]
[5]Donnelly, J. P. 2006. Polymerase chain reaction for diagnosing invasive aspergillosis: getting closer but still a ways to go. Clin. Infect. Dis. 42:487-489. [DOI] [PubMed] [Google Scholar]
[6]Einsele, H., H. Hebart, G. Roller, J. Löffler, I. Rothenhofer, C. A. Muller, R. A. Bowden, J. van Burik, D. Engelhard, L. Kanz, and U. Schumacher. 1997. Detection and identification of fungal pathogens in blood by using molecular probes. J. Clin. Microbiol. 35:1353-1360. [DOI] [PMC free article] [PubMed] [Google Scholar]
[7]Garcia, M. E., J. L. Blanco, J. Caballero, and D. Gargallo-Viola. 2002. Anticoagulants interfere with PCR used to diagnose invasive aspergillosis. J. Clin. Microbiol. 40:1567-1568. [DOI] [PMC free article] [PubMed] [Google Scholar]
[8]Greene, R. E., H. T. Schlamm, J. W. Oestmann, P. Stark, C. Durand, O. Lortholary, J. R. Wingard, R. Herbrecht, P. Ribaud, T. F. Patterson, P. F. Troke, D. W. Denning, J. E. Bennett, B. E. de Pauw, and R. H. Rubin. 2007. Imaging findings in acute invasive pulmonary aspergillosis: clinical significance of the halo sign. Clin. Infect. Dis. 44:373-379. [DOI] [PubMed] [Google Scholar]
[9]Herrera, M. L., A. C. Vallor, J. A. Gelfond, T. F. Patterson, and B. L. Wickes. 2009. Strain-dependent variation in 18S ribosomal DNA copy numbers in Aspergillus fumigatus. J. Clin. Microbiol. 47:1325-1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
[11]Irwig, L., P. Bossuyt, P. Glasziou, C. Gatsonis, and J. Lijmer. 2002. Evidence base of clinical diagnosis: designing studies to ensure that estimates of test accuracy are transferable. BMJ 321:669-671. [DOI] [PMC free article] [PubMed] [Google Scholar]
[12]Kami, M., T. Fukui, S. Ogawa, Y. Kazuyama, U. Machida, Y. Tanaka, Y. Kanda, T. Kashima, Y. Yamazaki, T. Hamaki, S. Mori, H. Akiyama, Y. Mutou, H. Sakamaki, K. Osumi, S. Kimura, and H. Hirai. 2001. Use of real-time PCR on blood samples for diagnosis of invasive aspergillosis. Clin. Infect. Dis. 33:1504-1512. [DOI] [PubMed] [Google Scholar]
[13]Klingspor, L., and S. Jalal. 2006. Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real-time PCR. Clin. Microbiol. Infect. 12:745-753. [DOI] [PubMed] [Google Scholar]
[14]Kontoyiannis, D. P., and K. A. Marr. 2009. Therapy of invasive aspergillosis: current consensus and controversies, p. 491-502. In J.-P. Latge and W. J. Steinbach (ed.), Aspergillus fumigatus and aspergillosis. ASM Press, Washington, DC.
[15]Lengerova, M., K. Hrncirova, I. Kocmanova, Z. Racil, B. Weinbergerova, J. Vorlicek, and J. Mayer. 2008. Detection of Aspergillus fumigatus in bronchoalveolar lavage samples from immunocompromised patients using real-time PCR with fungal DNA preamplification, p.630. Abstr. 48th Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, Washington, DC.
[16]Loeffler, J., N. Schultze, H. Hebart, and H. Einsele. 2005. Detection of genomic DNA from Aspergillus species using the LightCycler technology, poster M-721. Abstr. 45th Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, Washington, DC.
[17]Loeffler, J., N. Henke, H. Hebart, D. Schmidt, L. Hagmeyer, U. Schumacher, and H. Einsele. 2000. Quantification of fungal DNA by using fluorescence resonance energy transfer and the LightCycler system. J. Clin. Microbiol. 38:586-590. [DOI] [PMC free article] [PubMed] [Google Scholar]
[18]Loeffler, J., H. Hebart, R. Bialek, L. Hagmeyer, D. Schmidt, F. P. Serey, M. Hartmann, J. Eucker, and H. Einsele. 1999. Contaminations occurring in fungal PCR assays. J. Clin. Microbiol. 37:1200-1202. [DOI] [PMC free article] [PubMed] [Google Scholar]
[19]Mennink-Kersten, M. A., J. P. Donnelly, and P. E. Verweij. 2004. Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis. Lancet Infect. Dis. 4:349-357. [DOI] [PubMed] [Google Scholar]
[20]Millon, L., R. Piarroux, E. Deconinck, C. E. Bulabois, F. Grenouillet, P. Rohrlich, J. M. Costa, and S. Bretagne. 2005. Use of real-time PCR to process the first galactomannan-positive serum sample in diagnosing invasive aspergillosis. J. Clin. Microbiol. 43:5097-5101. [DOI] [PMC free article] [PubMed] [Google Scholar]
[21]Pfeiffer, C. D., J. P. Fine, and N. Safdar. 2006. Diagnosis of invasive aspergillosis using a galactomannan assay: a meta-analysis. Clin. Infect. Dis. 42:1417-1427. [DOI] [PubMed] [Google Scholar]
[22]Rimek, D., A. P. Garg, W. H. Haas, and R. Kappe. 1999. Identification of contaminating fungal DNA sequences in zymolyase. J. Clin. Microbiol. 37:830-831. [DOI] [PMC free article] [PubMed] [Google Scholar]
[23]Schulz, B., K. Weber, C. Radecke, C. Scheer, and M. Ruhnke. 2009. Effect of different sample volumes on the DNA extraction of Aspergillus fumigatus from whole blood. Clin. Microbiol. Infect. 15:686-688. [DOI] [PubMed] [Google Scholar]
[24]Schwartz, S., and M. Ruhnke. 2009. Aspergillus sinusitis and cerebral aspergillosis, p. 301-318. In J.-P. Latge and W. J. Steinbach (ed.), Aspergillus fumigatus and aspergillosis. ASM Press, Washington, DC.
[25]Sterne, J. A. C., M. J. Bradburn, and M. Egger. 2001. Meta-analysis in Stata™, p. 347-369. In M. Egger, G. Davey Smith, and D. G. Altman (ed.), Systematic reviews in health care: meta-analysis in context, 2nd ed. BMJ Publishing Group, London, United Kingdom.
[26]Tong, K. B., C. J. Lau, K. Murtagh, A. J. Layton, and R. Seifeldin. 2009. The economic impact of aspergillosis: analysis of hospital expenditures across patient subgroups. Int. J. Infect. 13:24-36. [DOI] [PubMed] [Google Scholar]
[27]Upton, A., K. A. Kirby, P. Carpenter, M. Boeckh, and K. A. Marr. 2007. Invasive aspergillosis following hematopoietic cell transplantation: outcomes and prognostic factors associated with mortality. Clin. Infect. Dis. 44:531-540. [DOI] [PubMed] [Google Scholar]
[28]Van Burik, J. A., H. R. W. Schreckhise, T. C. White, R. A. Bowden, and D. Myerson. 1998. Comparison of six extraction techniques for isolation of DNA from filamentous fungi. Med. Mycol. 36:299-303. [PubMed] [Google Scholar]
[29]White, P. L., and R. A. Barnes. 2009. Aspergillus PCR, p. 373-392. In J.-P. Latge and W. J. Steinbach (ed.), Aspergillus fumigatus and aspergillosis. ASM Press, Washington, DC.
[30]White, P. L., R. Barton, M. Guiver, C. J. Linton, S. Wilson, M. Smith, B. L. Gomez, M. J. Carr, P. T. Kimmitt, S. Seaton, K. Rajakumar, T. Holyoake, C. C. Kibbler, E. Johnson, R. P. Hobson, B. Jones, and R. A. Barnes. 2006. A consensus on fungal polymerase chain reaction diagnosis?: a United Kingdom-Ireland evaluation of polymerase chain reaction methods for detection of systemic fungal infections. J. Mol. Diagn. 8:376-384. [DOI] [PMC free article] [PubMed] [Google Scholar]
[31]White, P. L., C. J. Linton, M. D. Perry, E. M. Johnson, and R. A. Barnes. 2006. The evolution and evaluation of a whole blood polymerase chain reaction assay for the detection of invasive aspergillosis in hematology patients in a routine clinical setting. Clin. Infect. Dis. 42:479-486. [DOI] [PubMed] [Google Scholar]
[32]White, P. L., and R. A. Barnes. 2006. Aspergillus PCR—platforms, strengths and weaknesses. Med. Mycol. 44(Suppl.):191-198. [DOI] [PubMed] [Google Scholar]
[33]Williamson, E. C. M. 2001. Molecular approaches to fungal infections in immunocompromised patients. MD thesis. University of Bristol, Bristol, United Kingdom.

Sorted by citations

Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock 2021.
Evans, L., Rhodes, A., Alhazzani, W., Antonelli, M., Coopersmith, C. M., French, C., Machado, F. R., Mcintyre, L., Ostermann, M., Prescott, H. C., et al. (2021) · Critical Care Medicine · doi:10.1097/ccm.0000000000005337 · Cited by 2,935 · see citing works
ECIL recommendations for the use of biological markers for the diagnosis of invasive fungal diseases in leukemic patients and hematopoietic SCT recipients
Marchetti, O., Lamoth, F., Mikulska, M., Viscoli, C., Verweij, P., Bretagne, S., & Groups, A. T. E. C. O. I. I. L. (. L. W. (2012) · Bone Marrow Transplantation · doi:10.1038/bmt.2011.178 · Cited by 275 · see citing works
Self-citation
Performance of Galactomannan, Beta- <scp>d</scp> -Glucan, Aspergillus Lateral-Flow Device, Conventional Culture, and PCR Tests with Bronchoalveolar Lavage Fluid for Diagnosis of Invasive Pulmonary Aspergillosis
Hoenigl, M., Prattes, J., Spiess, B., Wagner, J., Prueller, F., Raggam, R. B., Posch, V., Duettmann, W., Hoenigl, K., Wölfler, A., et al. (2014) · Journal of Clinical Microbiology · doi:10.1128/jcm.00467-14 · Cited by 233 · see citing works
Clinical Performance of Aspergillus PCR for Testing Serum and Plasma: a Study by the European Aspergillus PCR Initiative
White, P. L., Barnes, R. A., Springer, J., Klingspor, L., Cuenca-Estrella, M., Morton, C. O., Lagrou, K., Bretagne, S., Melchers, W. J. G., Mengoli, C., et al. (2015) · Journal of Clinical Microbiology · doi:10.1128/jcm.00905-15 · Cited by 140 · see citing works
Self-citation
Surviving sepsis campaign: international guidelines for management of sepsis and septic shock 2021
Evans, L., Rhodes, A., Alhazzani, W., Antonelli, M., Coopersmith, C. M., French, C., Machado, F. R., Mcintyre, L., Ostermann, M., Prescott, H. C., et al. (2021) · Intensive Care Medicine · doi:10.1007/s00134-021-06506-y · Cited by 5,082 · see citing works
Diagnosis of aspergillosis by PCR: Clinical considerations and technical tips
Barnes, R. A., White, P. L., Morton, C. O., Rogers, T. R., Cruciani, M., Loeffler, J., & Donnelly, J. P. (2018) · Medical Mycology · doi:10.1093/mmy/myx091 · Cited by 68 · see citing works
Self-citation
Diagnosis of invasive aspergillosis: recent developments and ongoing challenges
Arvanitis, M., & Mylonakis, E. (2015) · European Journal of Clinical Investigation · doi:10.1111/eci.12448 · Cited by 56 · see citing works
The current management landscape: aspergillosis: Table 1.
Maertens, J. A., Blennow, O., Duarte, R. F., & Muñoz, P. (2016) · Journal of Antimicrobial Chemotherapy · doi:10.1093/jac/dkw393 · Cited by 29 · see citing works
Diagnostic Approaches for Invasive Aspergillosis—Specific Considerations in the Pediatric Population
Lehrnbecher, T., Hassler, A., Groll, A. H., & Bochennek, K. (2018) · Frontiers in Microbiology · doi:10.3389/fmicb.2018.00518 · Cited by 29 · see citing works
Diagnostics and Susceptibility Testing in <i>Aspergillus</i>
Bernal-Martínez, L., Alastruey-Izquierdo, A., & Cuenca-Estrella, M. (2016) · Future Microbiology · doi:10.2217/fmb.15.140 · Cited by 25 · see citing works
Evaluating the use of PCR for diagnosing invasive aspergillosis
Buchheidt, D., Reinwald, M., Hofmann, W.-K., Boch, T., & Spiess, B. (2017) · Expert Review of Molecular Diagnostics · doi:10.1080/14737159.2017.1325735 · Cited by 17 · see citing works
PCR Technology for Detection of Invasive Aspergillosis
Barnes, R., & White, P. (2016) · Journal of Fungi · doi:10.3390/jof2030023 · Cited by 8 · see citing works
“Initial studies using whole blood showed that sample input volume, sample processing steps (red and white cell lysis and bead beating), fungal cell lysis through bead beating and elution volume all affected PCR performance and led to the conclusion that Aspergillus PCR efficiency is limited by extraction and not by PC…”
MethodSelf-citation

Authors: White, P. L., Bretagne, S., Klingspor, L., Melchers, W. J. G., McCulloch, E., Schulz, B., Finnstrom, N., Mengoli, C., Barnes, R. A., Donnelly, J. P., & Loeffler, J.
Affiliations: University Hospital of Wales (GB)
Henri Mondor Hospital, Créteil, France
Karolinska University Hospital (SE)
Radboud University Medical Center (NL)
Royal Hospital for Children (GB)
Charité – Universitätsmedizin Berlin (DE)
Cepheid AB, Toulouse, France
University of Padua (IT)
Cardiff University (GB)
University of Würzburg (DE)
ORCID: White, P. Lewis
Klingspor, Lena
Melchers, Willem J. G.
Mengoli, Carlo
Barnes, Rosemary A.
Donnelly, J. Peter
Loeffler, Juergen
Venue: Journal of Clinical Microbiology
Publisher: American Society for Microbiology
Published: 2010-04
Volume: 48
Issue: 4
Pages: 1231-1240
ISSN: 0095-1137 (ISSN-L)
1098-660X
DOI: 10.1128/jcm.01767-09
PMC: PMC2849576
OpenAlex: W2104808759
Cited by: 296
References: 3
Citations by year: 2026: 6 · 2025: 15 · 2024: 8 · 2023: 8 · 2022: 6 · 2021: 11 · 2020: 13 · 2019: 13 · 2018: 18 · 2017: 19 · 2016: 27 · 2015: 23 · 2014: 27 · 2013: 33 · 2012: 32
FWCI: 21.16
Type: journal article
Language: en
Open access: green
www.ncbi.nlm.nih.gov
Licence: journals.asm.org/non-commercial-tdm-license (tdm)
Locations: Journal of Clinical Microbiology (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
ORCA Online Research @Cardiff (Cardiff University) (acceptedVersion)
Radboud Repository (Radboud University) (submittedVersion)
Funders: Univerzita Karlova v Praze
Cardiff University
Radboud Universiteit
Università degli Studi di Padova
Hospital for Sick Children
Università degli Studi di Verona
Topics: Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Bacterial Identification and Susceptibility Testing
Keywords: Aspergillus PCR, invasive aspergillosis, diagnostic standardization, DNA extraction, bead beating, detection sensitivity, fungal burden
MeSH terms: Animals, Aspergillosis, Aspergillus, DNA, Fungal, False Negative Reactions, Mycology, Sensitivity and Specificity, Polymerase Chain Reaction, Clinical Laboratory Techniques

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review

Aspergillus PCR: One Step Closer to Standardization

Abstract

Integrity signals

Claims the paper makes

Claims other papers make about this one

Self-check

Cites

Cited by

Record

About these checks