Clinical Infectious Diseases Advance Access published June 25, 2015
1 Aspergillus PCR: a systematic review of evidence for clinical use in comparison with antigen testing
cr ipt
P. Lewis White1, John R. Wingard2, Stéphane Bretagne3, Jürgen Löffler4, Thomas F. Patterson5, Monica A. Slavin6, Rosemary A. Barnes7, Peter G. Pappas8 and J. Peter Donnelly9 Health Wales, Microbiology Cardiff, UK
Diderot University, Saint Louis Hospital-APHP, France
4Wuerzburg 5The
University, Wuerzburg, Germany
M an
3Paris
of Florida Health, Gainsville, Florida, USA
University of Texas Health Science Center and South Texas Veterans Health
Care System, San Antonio, Texas, USA
Peter MacCallum Cancer Centre, University of Melbourne, Australia
7Infection,
Immunity and Biochemistry, Cardiff University, UK
8University
of Alabama, Birmingham, Alabama, USA
University Medical Centre, Nijmegen, The Netherlands
pt
9Radboud
ed
6The
Corresponding Author: Dr P. Lewis White, Public Health Wales, Microbiology
ce
Cardiff, University Hospital of Wales, Heath Park, Cardiff, UK. CF14 4XN. Telephone number: +44 (0)29 2074 6581, Fax number: +44 (0)29 2074 2161,
Ac
Email: [email protected]
© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons AttributionNonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact [email protected].
Downloaded from http://cid.oxfordjournals.org/ at University of Pennsylvania Library on July 14, 2015
2University
us
1Public
2 Key Point: There is sufficient evidence, that is, at least, equivalent to that used to include galactomannan-EIA and β-D-Glucan testing, for PCR to be included in the
cr ipt
EORTC/MSG definitions.
Abstract
Aspergillus PCR was excluded from the EORTC/MSG definitions of invasive fungal
us
being revised. A systematic literature review was performed to identify
analytical and clinical information available on inclusion of galactomannan-EIA
M an
(2002) and β-D-Glucan (2008), providing a minimal threshold when considering PCR.
Categorical parameters and statistical performance were compared. When incorporated, galactomannan-EIA and β-D-Glucan sensitivities and
ed
specificities, for diagnosing invasive aspergillosis, were 81.6% and 91.6%, and 76.9% and 89.4%, respectively. Aspergillus PCR has similar sensitivity and specificity (76.8-88.0% and 75.0-94.5%, respectively) and comparable utility.
pt
Methodological recommendations and commercial PCR assays assist
ce
standardisation. While all tests have limitations, currently, PCR is the only test with independent quality control.
Ac
We propose there is sufficient evidence, that is at least equivalent to that used to
include galactomannan-EIA and β-D-Glucan testing, and PCR is now mature enough for inclusion in the EORTC/MSG definitions.
Downloaded from http://cid.oxfordjournals.org/ at University of Pennsylvania Library on July 14, 2015
disease because of limited standardisation and validation. The definitions are
3 INTRODUCTION PCR to aid diagnosis of invasive aspergillosis (IA) has been used for over two decades, but is not included in the European Organisation for the Research and
cr ipt
Treatment of Cancer/Mycoses Study Group (EORTC/MSG) definitions of invasive fungal disease (IFD). In the 2002 definitions, PCR was excluded on the basis of potential false positive results and lack of standardised commercial testing
platforms (1). It was reconsidered when the definitions were revised in 2008,
us
because there was no standard, and clinically validation was limited (2).
M an
The EORTC/MSG definitions are currently under review. To provide impartial review of Aspergillus PCR technology we reassessed information that was available at the time of inclusion of previous biomarkers, galactomannan enzyme linked immnuno-sorbent assay (GM-EIA) in 2002, and -D-Glucan (BDG) in2008.
ed
This baseline information provides a systematic dataset for categorical and standardised comparison of PCR, and any other emerging or future diagnostic assays (e.g. Aspergillus Lateral flow device; (3,4). The evidence forms the basis
pt
for full review of manuscripts describing Aspergillus PCR allowing the
ce
EORTC/MSG consensus committee to consider PCR in future definitions. This manuscript describes the findings of this initial review and is a joint effort of
Ac
the European Aspergillus PCR initiative (EAPCRI), a working group of the
International Society for Human and Animal Mycology (ISHAM), the EORTC Infectious Disease Group and the Mycoses Study Group (MSG).
Downloaded from http://cid.oxfordjournals.org/ at University of Pennsylvania Library on July 14, 2015
but despite continued development molecular methods were not to be included
4
REVISION PROCESS Evaluating the GM-EIA and BDG evidence available prior to incorporation
cr ipt
into disease defining definitions. Manuscripts describing the use of GM-EIA and BDG were identified by literature searches using Medline/PubMed using the following key words (Aspergillus
diagnosis, galactomannan EIA or ELISA, Aspergillus antigen, aspergillosis for GM-
us
for BDG). To confirm the quality of the literature searches and identify missed
M an
articles, bibliographies of representative meta-analyses were screened (5-12). Only manuscripts published before incorporation of both assays into the EORTC/MSG definitions were included.
The focus of studies was determined by reviewing the title and abstract. If the
ed
study was a technical description or an animal model evaluation it was assessed for any potential useful information (e.g. cross reactivity with other fungal species, inter-assay reproducibility or potential kinetics of antigen release) but
pt
excluded from performance analysis. For clinical evaluation, factors deemed
ce
important with respect to diagnostic use were identified (Table 1). As the BDG assay detects a broader range of fungi, performance data specific to the detection
Ac
of IA were retrieved from the original manuscript, and manuscripts were excluded if it was not possible.
Evaluating the evidence base for Aspergillus PCR testing. Manuscripts describing the use of PCR were identified by literature searches using PubMed. More than200 manuscripts were available for review (13, 14).
Downloaded from http://cid.oxfordjournals.org/ at University of Pennsylvania Library on July 14, 2015
EIA, and glucan, beta-glucan, fungal infection (diagnosis), mycoses, Aspergillus
5 Many were available before the last EORTC/MSG revision so the focus was on important developments in the field of Aspergillus PCR, particularly, after 2008. Relevant meta-analyses were used, representing an independent and statistically
cr ipt
sound representation of clinical performance (15-19).
Statistical analysis
Sensitivity, specificity, positive and negative predictive values, likelihood ratios
us
retrospective studies were analysed both separately, and combined. The 95%
M an
confidence values (95% CI) were calculated for proportions and when comparing parameters between different assays, Fisher’s exact test was used to generate a two-sided P value, with value of ≤0.05 being deemed significant
ed
EVIDENCE FOR ANTIGEN TESTING
GM-EIA– Manuscript characteristics
Sixty-one articles were retained for further review. Sixteen were subsequently
pt
excluded as they did not describe the use of the BioRad GM-EIA or a pre-cursor
ce
of this test (e.g. Latex agglutination or “in-house” ELISA tests). A breakdown of
Ac
the remaining manuscripts is shown in Supplementary Figure 1.
GM-EIA – Analytical evidence Six manuscripts evaluated GM-EIA detection and provided information pertinent to clinical performance. Cross reactivity with non-Aspergillus fungal species was determined (Supplementary Table 1, (20)). Of greater concern was the
Downloaded from http://cid.oxfordjournals.org/ at University of Pennsylvania Library on July 14, 2015
(LR) and diagnostic odds ratio (DOR) were calculated. Prospective and
6 generation of false positive results (8/40) associated with bacteraemia and candidaemia in febrile neutropenic patients (20). Intra and inter-assay reproducibility of the GM-EIA was investigated. While the
cr ipt
qualitative agreement (positive/negative) between centres was excellent, the quantitative inter-assay precision was only 25.6% (21).
GM-EIA – Pre-inclusion clinical performance
us
validations. Fifteen of the prospective studies described the performance of
M an
testing serum from 2,067 adults with an IA incidence of 13.9%. (22-36). Six of the retrospective studies described performance testing serum from 214 adults with an IA incidence of 25.7% (37-42). Neither sensitivity nor specificity differed significantly between prospective or retrospective studies (P: 0.57 and P: 0.45,
ed
respectively). The performance of serum GM-EIA pre-incorporation into the EORTC/MSG definitions is summarised in table 2. Three studies described the use of GM-EIA in 458 paediatrics patients, with an IA
pt
incidence of 5.5% (25, 43-44). The pooled sensitivity and specificity were 100%
ce
(95% CI: 86.7-100) and 90.1% (95% CI: 86.8-93.2), respectively. The testing of broncho-alveolar lavage (BAL) samples was limited with pooled sensitivities and
Ac
specificities of 89.5% (95% CI: 68.6-97.1) and 93.3%, 95% CI: 81.4-97.6), respectively (31, 42). Evidence for GM-EIA testing of cerebrospinal fluid was limited to case reports or small series (35, 45-48).
Downloaded from http://cid.oxfordjournals.org/ at University of Pennsylvania Library on July 14, 2015
Of the 26 articles describing clinical performance, 17 were prospective
7 Characteristics and clinical utility of GM-EIA before 2002 A summary of GM-EIA characteristics is described in Table 1. Methodological standardisation was evident, since only one commercial assay available.
cr ipt
However, since the original EORTC-MSG definitions the analytical threshold for positivity has been reduced from an optical density index (ODI) of 1.5 to 0.5 (21, 23-24, 49-50). The choice of optimal threshold will be defined by the strategy
chosen. For highly sensitive screening a lower ODI ≤0.5 is preferable, whereas to
us
1.5. The specificity of GM-EIA as calculated by both meta-analyses (including
M an
studies using the lower threshold) is significantly lower than those calculated for studies performed before 2002 using an ODI ≥1.5 (P:
1 Aspergillus PCR: a systematic review of evidence for clinical use in comparison with antigen testing
cr ipt
P. Lewis White1, John R. Wingard2, Stéphane Bretagne3, Jürgen Löffler4, Thomas F. Patterson5, Monica A. Slavin6, Rosemary A. Barnes7, Peter G. Pappas8 and J. Peter Donnelly9 Health Wales, Microbiology Cardiff, UK
Diderot University, Saint Louis Hospital-APHP, France
4Wuerzburg 5The
University, Wuerzburg, Germany
M an
3Paris
of Florida Health, Gainsville, Florida, USA
University of Texas Health Science Center and South Texas Veterans Health
Care System, San Antonio, Texas, USA
Peter MacCallum Cancer Centre, University of Melbourne, Australia
7Infection,
Immunity and Biochemistry, Cardiff University, UK
8University
of Alabama, Birmingham, Alabama, USA
University Medical Centre, Nijmegen, The Netherlands
pt
9Radboud
ed
6The
Corresponding Author: Dr P. Lewis White, Public Health Wales, Microbiology
ce
Cardiff, University Hospital of Wales, Heath Park, Cardiff, UK. CF14 4XN. Telephone number: +44 (0)29 2074 6581, Fax number: +44 (0)29 2074 2161,
Ac
Email: [email protected]
© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons AttributionNonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact [email protected].
Downloaded from http://cid.oxfordjournals.org/ at University of Pennsylvania Library on July 14, 2015
2University
us
1Public
2 Key Point: There is sufficient evidence, that is, at least, equivalent to that used to include galactomannan-EIA and β-D-Glucan testing, for PCR to be included in the
cr ipt
EORTC/MSG definitions.
Abstract
Aspergillus PCR was excluded from the EORTC/MSG definitions of invasive fungal
us
being revised. A systematic literature review was performed to identify
analytical and clinical information available on inclusion of galactomannan-EIA
M an
(2002) and β-D-Glucan (2008), providing a minimal threshold when considering PCR.
Categorical parameters and statistical performance were compared. When incorporated, galactomannan-EIA and β-D-Glucan sensitivities and
ed
specificities, for diagnosing invasive aspergillosis, were 81.6% and 91.6%, and 76.9% and 89.4%, respectively. Aspergillus PCR has similar sensitivity and specificity (76.8-88.0% and 75.0-94.5%, respectively) and comparable utility.
pt
Methodological recommendations and commercial PCR assays assist
ce
standardisation. While all tests have limitations, currently, PCR is the only test with independent quality control.
Ac
We propose there is sufficient evidence, that is at least equivalent to that used to
include galactomannan-EIA and β-D-Glucan testing, and PCR is now mature enough for inclusion in the EORTC/MSG definitions.
Downloaded from http://cid.oxfordjournals.org/ at University of Pennsylvania Library on July 14, 2015
disease because of limited standardisation and validation. The definitions are
3 INTRODUCTION PCR to aid diagnosis of invasive aspergillosis (IA) has been used for over two decades, but is not included in the European Organisation for the Research and
cr ipt
Treatment of Cancer/Mycoses Study Group (EORTC/MSG) definitions of invasive fungal disease (IFD). In the 2002 definitions, PCR was excluded on the basis of potential false positive results and lack of standardised commercial testing
platforms (1). It was reconsidered when the definitions were revised in 2008,
us
because there was no standard, and clinically validation was limited (2).
M an
The EORTC/MSG definitions are currently under review. To provide impartial review of Aspergillus PCR technology we reassessed information that was available at the time of inclusion of previous biomarkers, galactomannan enzyme linked immnuno-sorbent assay (GM-EIA) in 2002, and -D-Glucan (BDG) in2008.
ed
This baseline information provides a systematic dataset for categorical and standardised comparison of PCR, and any other emerging or future diagnostic assays (e.g. Aspergillus Lateral flow device; (3,4). The evidence forms the basis
pt
for full review of manuscripts describing Aspergillus PCR allowing the
ce
EORTC/MSG consensus committee to consider PCR in future definitions. This manuscript describes the findings of this initial review and is a joint effort of
Ac
the European Aspergillus PCR initiative (EAPCRI), a working group of the
International Society for Human and Animal Mycology (ISHAM), the EORTC Infectious Disease Group and the Mycoses Study Group (MSG).
Downloaded from http://cid.oxfordjournals.org/ at University of Pennsylvania Library on July 14, 2015
but despite continued development molecular methods were not to be included
4
REVISION PROCESS Evaluating the GM-EIA and BDG evidence available prior to incorporation
cr ipt
into disease defining definitions. Manuscripts describing the use of GM-EIA and BDG were identified by literature searches using Medline/PubMed using the following key words (Aspergillus
diagnosis, galactomannan EIA or ELISA, Aspergillus antigen, aspergillosis for GM-
us
for BDG). To confirm the quality of the literature searches and identify missed
M an
articles, bibliographies of representative meta-analyses were screened (5-12). Only manuscripts published before incorporation of both assays into the EORTC/MSG definitions were included.
The focus of studies was determined by reviewing the title and abstract. If the
ed
study was a technical description or an animal model evaluation it was assessed for any potential useful information (e.g. cross reactivity with other fungal species, inter-assay reproducibility or potential kinetics of antigen release) but
pt
excluded from performance analysis. For clinical evaluation, factors deemed
ce
important with respect to diagnostic use were identified (Table 1). As the BDG assay detects a broader range of fungi, performance data specific to the detection
Ac
of IA were retrieved from the original manuscript, and manuscripts were excluded if it was not possible.
Evaluating the evidence base for Aspergillus PCR testing. Manuscripts describing the use of PCR were identified by literature searches using PubMed. More than200 manuscripts were available for review (13, 14).
Downloaded from http://cid.oxfordjournals.org/ at University of Pennsylvania Library on July 14, 2015
EIA, and glucan, beta-glucan, fungal infection (diagnosis), mycoses, Aspergillus
5 Many were available before the last EORTC/MSG revision so the focus was on important developments in the field of Aspergillus PCR, particularly, after 2008. Relevant meta-analyses were used, representing an independent and statistically
cr ipt
sound representation of clinical performance (15-19).
Statistical analysis
Sensitivity, specificity, positive and negative predictive values, likelihood ratios
us
retrospective studies were analysed both separately, and combined. The 95%
M an
confidence values (95% CI) were calculated for proportions and when comparing parameters between different assays, Fisher’s exact test was used to generate a two-sided P value, with value of ≤0.05 being deemed significant
ed
EVIDENCE FOR ANTIGEN TESTING
GM-EIA– Manuscript characteristics
Sixty-one articles were retained for further review. Sixteen were subsequently
pt
excluded as they did not describe the use of the BioRad GM-EIA or a pre-cursor
ce
of this test (e.g. Latex agglutination or “in-house” ELISA tests). A breakdown of
Ac
the remaining manuscripts is shown in Supplementary Figure 1.
GM-EIA – Analytical evidence Six manuscripts evaluated GM-EIA detection and provided information pertinent to clinical performance. Cross reactivity with non-Aspergillus fungal species was determined (Supplementary Table 1, (20)). Of greater concern was the
Downloaded from http://cid.oxfordjournals.org/ at University of Pennsylvania Library on July 14, 2015
(LR) and diagnostic odds ratio (DOR) were calculated. Prospective and
6 generation of false positive results (8/40) associated with bacteraemia and candidaemia in febrile neutropenic patients (20). Intra and inter-assay reproducibility of the GM-EIA was investigated. While the
cr ipt
qualitative agreement (positive/negative) between centres was excellent, the quantitative inter-assay precision was only 25.6% (21).
GM-EIA – Pre-inclusion clinical performance
us
validations. Fifteen of the prospective studies described the performance of
M an
testing serum from 2,067 adults with an IA incidence of 13.9%. (22-36). Six of the retrospective studies described performance testing serum from 214 adults with an IA incidence of 25.7% (37-42). Neither sensitivity nor specificity differed significantly between prospective or retrospective studies (P: 0.57 and P: 0.45,
ed
respectively). The performance of serum GM-EIA pre-incorporation into the EORTC/MSG definitions is summarised in table 2. Three studies described the use of GM-EIA in 458 paediatrics patients, with an IA
pt
incidence of 5.5% (25, 43-44). The pooled sensitivity and specificity were 100%
ce
(95% CI: 86.7-100) and 90.1% (95% CI: 86.8-93.2), respectively. The testing of broncho-alveolar lavage (BAL) samples was limited with pooled sensitivities and
Ac
specificities of 89.5% (95% CI: 68.6-97.1) and 93.3%, 95% CI: 81.4-97.6), respectively (31, 42). Evidence for GM-EIA testing of cerebrospinal fluid was limited to case reports or small series (35, 45-48).
Downloaded from http://cid.oxfordjournals.org/ at University of Pennsylvania Library on July 14, 2015
Of the 26 articles describing clinical performance, 17 were prospective
7 Characteristics and clinical utility of GM-EIA before 2002 A summary of GM-EIA characteristics is described in Table 1. Methodological standardisation was evident, since only one commercial assay available.
cr ipt
However, since the original EORTC-MSG definitions the analytical threshold for positivity has been reduced from an optical density index (ODI) of 1.5 to 0.5 (21, 23-24, 49-50). The choice of optimal threshold will be defined by the strategy
chosen. For highly sensitive screening a lower ODI ≤0.5 is preferable, whereas to
us
1.5. The specificity of GM-EIA as calculated by both meta-analyses (including
M an
studies using the lower threshold) is significantly lower than those calculated for studies performed before 2002 using an ODI ≥1.5 (P:
