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REVISIÓN
Towards a standard for Aspergillus PCR - requirements, process
and results
P. Lewis Whitea,*, Juergen Loeferb, Rosemary A. Barnesc and J. Peter Donnellyd
aPublic Health Wales, Microbiology Cardiff, UHW, Cardiff, United Kingdom
bWuerzburg University, Wuerzburg, Germany
cInfection, Immunity and Biochemistry, School of Medicine, Cardiff University, UHW, Cardiff, United Kingdom
dRadboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
* Corresponding author.
Public Health Wales,
Microbiology Cardiff, UHW, Heath Park, Cardiff CF14 4XW. UK
Tel Number: +44 (0)29 2074 6581; Fax Number: +44 (0)29 2074 2161
E-mail: lewis.white@wales.nhs.uk (P. Lewis White)
0123-9392/$ - see front matter © 2012 ACIN. Publicado por Elsevier España, S.L. Todos los derechos reservados.
Infectio
Asociación Colombiana de Infectología
www.elsevier.es/infectio
Infectio. 2012;16(Supl 3): 64-72
Abstract
The diagnosis of invasive fungal disease remains challenging. The development of the European
Organization for Research and Treatment of Cancer/Mycoses Study Group criteria has helped
standardise diagnosis, yet PCR is excluded, a result of limited standardisation and concurrent
clinical validation. In 2006, the European Aspergillus PCR Initiative working party of Interna-
tional Society of Human and Animal Mycoses was formed with the aim of establishing a standard
for PCR methodology for the diagnosis of invasive aspergillosis, to attaining accurate clinical
utility of PCR, resulting in acceptance of PCR in disease defining criteria. This manuscript will
provide an overview of the standardisation process, before describing the potential samples
available and the reasoning behind the choices made. It will summarise the key findings, provide
the European Aspergillus PCR Initiative recommendations when testing whole blood and serum,
and conclude with a synopsis of future European Aspergillus PCR Initiative perspectives and
other key processes that will improve PCR based diagnosis.
© 2012 ACIN. Published by Elsevier España, S.L. All rights reserved.
Infectio • Revista de la Asociación Colombiana de Infectología Volumen 16, Suplemento 3, 2012
ISSN: 0123-9392
Microscopía estereoscópica: Se observan cabezuelas de Aspergillus spp cultivado en caja de Petri.
Colección de fotografías de la Unidad de Micología Médica y Experimental - CIB
Volumen 16, Suplemento 3 - Infecciones Fúngicas, Diciembre de 2012
KEYWORDS
Aspergillus;
PCR;
Diagnosis;
Standardisation
PALABRAS CLAVE
Aspergillus;
PCR;
Diagnóstico;
Estandarización
Hacia un estándar para Aspergillus PCR: requisitos, proceso y resultados
Resumen
El diagnóstico de la enfermedad fúngica invasiva sigue siendo un reto. El desarrollo de los cri-
terios del grupo de trabajo de la European Organization for Research and Treatment of Cancer/
Mycoses han ayudado a estandarizar el diagnóstico, sin embargo, la prueba de PCR (reacción en
cadena de la polimerasa) se excluye por su limitada estandarización y su concurrente validación
clínica. En 2006, se formó la Iniciativa Europea Aspergillus-PCR, un grupo de trabajo de la Inter-
national Society of Human and Animal Mycoses que tenía como objetivo establecer una meto-
Towards a standard for Aspergillus PCR - requirements, process and results 65
Introduction
Molecular diagnostics brings many challenges to labora-
tories and clinicians. Orderly and systematic evaluation
of analytical, laboratory and clinical validity and utility is
required. Nowhere is this more evident than in the field
of invasive fungal disease (IFD) where traditional methods
lack sensitivity to detect disease
The European Organization for Research and Treatment
of Cancer/Mycoses Study Group (EORTC/MSG) consensus
definitions use a combination of host, clinical and myco-
logical criteria to define IFD for the purpose of clinical tri-
als1. Whilst not designed for diagnosis of IFD in other clini-
cal settings, they are used increasingly. A proven diagnosis
requires culture or histological demonstration of infection
from a sterile site and, although yeasts, dimorphic fungi,
and the occasional mould can be diagnosed by blood cul-
ture, invasive sampling procedures are required for inva-
sive aspergillosis (IA), usually in critically-ill patients and,
as consequence, are seldom performed. The “less certain”
diagnosis of probable IFD, relies on seeing “specific” radio-
logical evidence of a disease, i.e. those that are consistent
with an IFD, in combination with mycological evidence
in the form of demonstrating the presence of circulating
biomarkers (Galactomannan [GM] or β-D-glucan [BDG]) or
culture, in a patient with host factors.
PCR was not included as mycological evidence in the
EORTC/MSG criteria because of the lack of standardisation
and validity, despite significant research being done, par-
ticularly for IA2. Consequently, the value of PCR in aiding
diagnosis is often overlooked. Exclusion from the EORTC/
MSG criteria is less related to unsatisfactory perform-
ance but due to the fact that little standardisation had
been attempted and, until recently, commercial alterna-
tives were not available. Meta-analyses of GM, BDG and
Aspergillus PCR in a clinical setting show similar sensitivi-
ties and specificities (BDG: 77% (95% CI: 67-84)/ 85% (95%
CI: 79-89); GM: 78% (95% CI: 61-89)/81% (95% CI:72-88);
PCR: 75% (95% CI: 54-88)/87% (95% CI: 78-93)3-5. The
numerous publications relating to Aspergillus PCR reflect
the continued scientific interest. There is methodological
diversity with respect to PCR, though independent meta-
analysis shows its performance to be comparable to other
biomarkers that use a standard commercial approach.
Nevertheless, PCR provides similar pooled performance
to commercial standardised antigen detection, suggesting
that standardisation of an optimal PCR protocol could pro-
vide superior performance.
With this in mind, two national studies were conducted
focussing on comparing PCR methodology for the detection
of IFD, in particular Candida and Aspergillus PCR, as these
are the most frequent causative agents for IFD6,7. The UK and
Ireland fungal PCR consensus group found that Candida PCR
methods provided comparable and satisfactory performance,
achieving a reproducible detection limit of 10 organisms8.
Performance of Aspergillus PCR methods was more variable,
and even when two optimal protocols were assessed by multi-
centre evaluation performance of one assay varied with both
PCR platform and sample type, and interactions between oli-
gonucleotides and human (deoxyribonucleic acid (DNA) were
noted. Performance variability was also noted by the Ger-
man/Austrian inter-laboratory comparison of PCR methods,
and this occurred on both an intra and inter-laboratory scale9.
These studies highlighted the need for standardisation, par-
ticularly if Fungal PCR was to gain widespread use outside of
specialised molecular/mycology centres.
In 2006, the European Aspergillus PCR Initiative (EAPCRI)
working party of International Society of Human and Ani-
mal Mycoses was formed with the aim of establishing a
standard for PCR methodology for the diagnosis of IA. With
over 62 participating centres from 25 countries in four dif-
ferent continents (Europe, North and South America and
Australia), the EAPCRI is now a global initiative with sev-
eral publications to its name10,11. This article will review
the process and findings of the standardisation process for
Aspergillus PCR and attempt to describe the differences
when using different sample types.
PCR standardisation – procedural overview
Before implementing any test, it is essential to understand
both the incidence and pathology of disease, and the impact
on sample selection. These factors affect the amount and
specific target within this specimen. On agreeing a sample
type it is important to consider how best to evaluate current
methods and understand the individual stages of the testing
process that may impact on assay efficiency. This requires the
evaluation of relevant individual stages as well as of the entire
process. In the initial stages of the evaluation, logistical and
ethical issues mean it is unlikely that any samples will come
directly from infected patients. The use of animal models for
initial large scale multi-centre investigations is not possible
due the number and volume of samples required. Conse-
quently, simulated samples are usually used, and the Quality
Control (QC) panel should contain burdens and targets rep-
resentative of the clinical scenario, as well as both negative
and positive samples. The sample matrix should be screened
for contamination before use and, if clean, the panel should
be developed using good aseptic and molecular techniques, if
necessary using clean room facilities (e.g. laminar air flow) to
prevent preparation-borne contamination12.
dología estándar de la PCR para el diagnóstico de la aspergilosis invasiva y conseguir la utilidad
clínica precisa de esta prueba para definir los criterios de la enfermedad. Este estudio ofrece
una visión general del proceso de estandarización, describe las posibles muestras disponibles y el
razonamiento en el que se basaron las decisiones tomadas. Resume las principales conclusiones,
ofrece las recomendaciones de la Iniciativa Europea de Aspergillus PCR en el análisis de de la
sangre total y el suero, y concluye con una sinopsis de las futuras perspectivas europeas de la
iniciativa Aspergillus y los otros procesos clave que mejorarán el diagnóstico basado en PCR.
© 2012 ACIN. Publicado por Elsevier España, S.L. Todos los derechos reservados.
66 P.L.White et al
Once a QC panel is prepared, it should be evaluated to
determine expediency prior to storage to minimise degra-
dation. The panel should be distributed in a way that main-
tains the storage status and sample anonymity. Problems
with distribution should be recorded, particularly if they
affect the panel status and the individual recipient should
relay this back to the distributor. The panel should be
stored by the recipient as agreed until testing, using stand-
ard protocols for that centre, and meeting any pre-test
conditions set by the distributor (e.g. testing time, internal
control or replicate testing). In addition to the results,
technical details concerning the entire molecular process
should be returned. Overall multi-centre results should be
compared to the original expediency test to establish if the
panel has withstood the storage and distribution process.
Blinded meta-regression analysis of results to determine
assay performance and, both positive and negative, associ-
ations with individual technical steps should be performed.
Any findings will form the basis of technical recommenda-
tions to be conveyed to participants and assessed by the
distribution of further anonymous panels.
On return of the results and prerequisite technical
information, blinded analysis will be performed for centres
following recommendations designed to improve perform-
ance and compared to previous panels or to centres not
following the recommendations.
If performance associated with the recommendations is
shown to be superior then these are further assessed using
specimens derived from animal models of the specific dis-
ease, ahead of any multi-centre clinical evaluation using
patient samples.
Standardisation of Aspergillus PCR – the need
With the first manuscripts describing the use of PCR to aid in
the diagnosis of IA being published almost two decades ago, it
is surely time to finally decide whether PCR is clinically use-
ful or not. GM ELISA has been in use for a similar period, is
widely used for screening, diagnosis and monitoring of thera-
peutic response, and methodological standardisation has been
achieved by a single manufacturer adopting a commercial QC
of the entire manufacturing process. By contrast PCR tests are
mostly “in house” tests. Clearly methodological standardisation
is needed13. The only way to accurately determine the clinical
utility of PCR is through a prospective multi-centre study of
substantial size, and the potential role of PCR can be deter-
mined through standardisation which does not exclude com-
mercial participation, providing a much needed additional test
for inclusion into a diagnostic strategy. Once this occurs, PCR
can take its place among the tests accepted for defining IFD.
The European Aspergillus PCR Initiative
structure
Initially, the EAPCRI (http://www.eapcri.eu) created two
working groups with extensive experience in laboratory (labo-
ratory working group) and clinical (clinical working group)
diagnosis of IA. Membership to these groups initially relied
on pairing clinicians and scientists from the same centre.
Both working groups had a lead member who represented the
working parties on the steering committee, along with the
chair of the organisation, whose role was to direct the EAPCRI
process. However, it quickly became apparent that the labo-
ratory working group programme required expansion and so
additional test centres were recruited to help develop and
test the protocols required for standardisation. The amount
of information generated was such that a statistical working
party was formed to help analyse the results.
Potential specimens
A literature review of the pathology of IA, the range of
specimens tested, and how these could affect the manage-
ment of patients was undertaken before any laboratory
evaluation could take place.
Respiratory Samples
Since IA most commonly affects the lungs, a respiratory speci-
men seems the obvious choice to permit early detection of
the organism. However, sputum is of limited use, associated
with higher levels of airway contamination and can be dif-
ficult to process, whereas bronchoalveolar lavage (BAL) has
great clinical relevance but is a relatively invasive procedure.
The performance of PCR in BAL specimens is comparable to
those for GM, BDG and PCR performed on serum or blood. A
meta-analysis of Aspergillus PCR when testing BAL specimens
provided pooled sensitivity and specificity of 79% and 94%,
similar to the sensitivities and specificities generated by
respective meta-analyses of GM, BDG and Aspergillus PCR
testing blood specimens3-5,14. However, these observations do
not take into account the temporal relationships of positivity,
nor which is the earliest marker of disease, which is important
as the prognosis is dramatically improved by early treatment
led by an earlier diagnosis15. Aspergillus was detected in
blood samples of cases of prior IPA at a later stage than was
found for BAL16. However, obtaining BAL specimens may not
be possible for a variety of reasons, including thrombocyto-
penia. Moreover, Aspergillus conidia may be inhaled continu-
ally, providing multiple time points for initiation of disease,
thereby necessitating the need for frequent sampling but
also increasing the possibility of false positivity by detecting
Aspergillus airway contamination or colonisation. Although
25% of BAL specimens from healthy volunteers were PCR posi-
tive17, the meta-analysis showed that specificity was not sig-
nificantly affected in a clinical setting14. It is likely that PCR
positivity in BAL specimens taken from areas of radiologically
confirmed infection will have greater clinical significance
because of higher fungal burdens16. So, to confirm a diag-
nosis of IA at a specific time point, the testing of computed
tomography directed BAL specimens may provide the earliest
opportunity for genus or species level detection.
Blood samples
As the incidence of IA is relative low, (<10%) even among
those cohorts considered to be at high risk, it can be argued
that infrequent testing of BAL samples is not an optimal
strategy. With a low pre-test probability of disease, assays
Towards a standard for Aspergillus PCR - requirements, process and results 67
are better suited to excluding disease, relying on a high
sensitivity and negative predictive value, in other words
“screening”. This approach also reduces the need for unnec-
essary empirical therapy, thus reducing both cost and anti-
fungal associated patient toxicity18. Taking isolated samples
can result in low positivity rates, and this is compounded by
the low fungal burdens in certain sample types. Even when a
sample is positive, a second sample is needed for confirma-
tion to improve the positive predictive value of PCR5. Blood
is readily obtained but it is important to consider the poten-
tial targets and their route of entry in to the circulatory
system (Fig. 1). Normally, alveolar macrophages would clear
inhaled conidia, but failure to do so would result in germina-
tion and hyphal production that would have been countered
by the actions of the neutrophil, but these cells are absent
during neutropenia19. Infection will ensue through tissue
invasion and angioinvasion, and may coincide with the
release of fungal elements such as GM and β-D-glucan and
possibly even DNA. If unchecked, infection can then proceed
to disease with tissue destruction, haemorrhage and vascu-
lar infarction providing the typical early radiological nodule
or halo signs. Dissemination may also ensue. This simplistic
description of disease would indicate that any fungal target
in blood would be dependent on some angioinvasion, with
potential positivity being dependent on burden, itself being
reliant on the degree of angioinvasion. It is also possible that
conidia phagocytosed within macrophages could be detected
in the circulation shortly after exposure to the organism,
which is suggested by the detection of inhaled labelled
micro-particles in the bloodstream20. Detecting this poten-
tial source could help pre-empt disease, although false posi-
tivity could occur, as not every high-risk patient exposed to
Aspergillus conidia develops IA. Nevertheless, knowing that
a patient at high-risk of developing IA has become infected,
to a degree, provides clinically useful information, particu-
larly if combined with host factors (prolonged steroid use
or neutropenia, allogeneic stem cell transplantation, graft
versus host disease, genetic susceptibility to disease).
The actual source of DNA within the circulation is yet
to be determined. Blood cultures seldom yield Aspergillus
in cases of IA, yet if seeded, Aspergillus will grow in these
cultures, indicating a non-viable source in blood21. Opposing
this is the fact that the disease disseminates via the circula-
tion, and for this to occur a viable organism must be avail-
able. Morton et al. recently showed that Aspergillus would
not grow or replicate in whole blood, and it is possible that,
unlike candidaemia, where the yeast is actively growing and
budding, Aspergillus enters the circulation but remains rela-
tively inactive until it reaches its tissue destination22. Con-
sequently, the circulatory burden of the mould is likely to
be well below the detection limits of current blood culture
systems. Non-viable targets, including hyphae damaged by
platelet attachment23 and fungal cells phagocytosed within
leukocytes probably provide a cell associated DNA source,
and free circulating DNA (DNAaemia) will be present when
released through the actions of the immune system. DNAae-
mia could also be influenced by antifungal therapy through
its actions on the fungal cell wall/membrane, artificially
increasing the DNA burden despite an efficient therapeutic
response. In effectively treated cases, this effect would be
temporary, as decreasing burden and concomitant rapid
circulatory clearance of DNA should result in decreasing
burdens. These different targets (free and cell associated
DNA) would be found in different components of blood, cell
associated DNA would be associated with the leukocytes, if
present, whereas free DNA would be found in the serum or
plasma, requiring different DNA extraction techniques.
Other samples
Haematogenous transfer can lead to disseminated disease
infecting organs and other anatomical structures. This is
why histological diagnosis remains the gold standard for
diagnosis, but the invasive procedures necessary to obtain
samples can be difficult during life. Moreover, PCR testing
of these samples is limited and these specimens are usually
obtained once, only permitting a diagnostic approach. It
is of paramount importance to digest the surrounding tis-
sue efficiently when testing tissue specimens in order that
invading hyphae can be targeted. Providing QC for these
processes is difficult as developing simulated control sam-
ples is limited to specimens obtained from animal infection
models or previously tested clinical material.
With cerebral disease, PCR testing of cerebrospinal fluid
may be productive, and several studies have shown suc-
cessful applications24,25. Again testing is limited to diagno-
sis, as studies and specimens are limited. It is unclear what
the actual target within the cerebrospinal fluid sample is,
organism or free DNA.
In using PCR to diagnose disseminated disease, it can
be argued that the clinical utility of PCR testing will be
minimal, the disease is advanced and patient prognosis will
be worse compared to testing samples capable of detecting
the organism at exposure or infection. However, diagnosis
still has value when the disease continues to progress
despite antifungal therapy, as it might be due to other
moulds such as those belonging to the Mucorales. In this
scenario Aspergillus PCR testing should remain negative
and pan-fungal PCR or PCR specific to the other aetiology
would be required to confirm IFD.
The European Aspergillus PCR Initiative
strategy
At the start of the EAPCRI it was decided that using PCR
for screening provided the optimal strategy which obvi-
ously excluded the use of BAL, cerebrospinal fluid or tissue
samples, and focussed on testing blood. With the potential
targets located either in the cells or in the plasma/serum it
was decided to initially focus on whole blood to pursue the
goal of EAPCRI as there was a need for greater standardisa-
tion of DNA extraction methods. On completing this process,
it was repeated for serum, with plasma being reserved for
future investigations.
Whole blood standardisation10
The next stage was to evaluate methods currently in use.
For any molecular procedure there are two basic steps; first,
nucleic acid extraction and then amplification. While it is
important to evaluate the combined performance of these
68 P.L.White et al
processes it is also important to determine the performance
of the individual processes; this overcomes the potential
effect of combining a poor extraction technique with an
optimal PCR test, leading to poor PCR performance.
The first EAPCRI panel was developed to do both, and
comprised a panel of quantified A. fumigatus genomic DNA
to evaluate PCR alone, and a whole blood panel seeded with
different concentrations of A. fumigatus conidia to evaluate
DNA extraction and PCR10. Although conidia are unlikely to
be involved in the invasive disease process, a panel needed
to be consistently quantifiable between centres and it was
not possible to accurately quantify multinucleate hyphae. To
maintain the goal of a screening assay with a high negative
predictive value, the panel was designed to determine limits
of detection and overall sensitivity, rather than specificity,
and a designated threshold for detection was set. Participants
were asked to return results and also supply technical infor-
mation for both nucleic acid extraction and PCR amplification
to allow subsequent analysis. All data and statistical analyses
were performed in a blinded fashion to anonymise the iden-
tity of the participant and allow unbiased evaluation.
The DNA panel showed that 90% of participating centres
attained the designated threshold. However, when DNA
extraction was combined with PCR only nine (41%) of 22 cen-
tres reached the same level of performance, indicating that
DNA extraction was the rate-limiting step. Enhanced per-
Figure 1 Potential Aspergillus DNA sources and targets within the circulation.
Epithelial Uptake
Saprophytic disease Tissue Invasion
Amgioinvasion
Germination
Germination with
respiratory tract
Limited Alveolar
Macrophage phagocytosis
Macrophage translocation
to the circulation
Potential
Non-viable target via
platelet attachment or limited
neutrophilresponse
Potential PCR target:
Phagocytosed Fungall cell
Elimination via
the lymphatic system
Potential Viable target:
Hyphal fragment
Potential PCR targetPotential PCR target
Circulating hyphae
Entry into the circulation
and dissemination
Sufcient host defences
Alveolar Macrophage
phagocytosis
Macrophage translocation
to the circulation
Elimination via
the lymphatic system
Inhalation of conidia
Insufcient host defences
Colonisation
Phagocytosed Fungall CellDNAemia
Towards a standard for Aspergillus PCR - requirements, process and results 69
formance was noted with centres processing the entire 3mL
sample and the use of “bead-beating” to lyse fungal cells;
these recommendations were provided to every recipient of
the second panel. This time Aspergillus PCR performance
was significantly improved compared to the first round, and
performance for centres compliant with the recommenda-
tions provided showed further improvements, with a sensi-
tivity and specificity of 88.7% and 91.6%, respectively. Meta-
regression analysis showed positive associations between
sensitivity and compliant methods, the use of white cell
lysis, “bead-beating”, and the incorporation of an internal
control PCR. A negative association between sensitivity and
the use of elution volumes >100 µL was also noted. These
formed the basis of the recommendations for Aspergillus
PCR testing of whole blood samples (Table 1.)
Serum standardisation11
On completing whole blood PCR standardisation it was
decided to evaluate serum samples. Serum PCR, theoreti-
cally, requires less standardisation as there are no human
blood cells, fungal cell lysis is no longer required, thus
minimising any additional processing steps, and the poten-
tial target was likely to be free circulating DNA (DNAae-
mia), yet the sample still allows a screening strategy to be
employed11. It was hypothesised that when targeting such
Table 1 European Aspergillus PCR Initiative recommendations for PCR testing of whole blood10
Recommendation Evidence base
Only use EDTA-anticoagulated whole Heparin has been associated with PCR inhibition31 and sodium citrate has been
blood specimens shown to be contamination with Aspergillus32
Please note: batches of EDTA blood collection tubes have been contaminated with
Aspergillus DNA and screening of all reagents, including vacutainers, should be
performed prior to clinical use33
Use a minimum of 3mL sample Two thirds of centres attaining the designated threshold in the EAPCRI studies
used 3 mL of sample In an analytical study using lower volumes was associated
with impaired reproducibility of detection34
Red cell lysis improves performance >Two thirds of centres attaining the designated threshold in the EAPCRI studies
used 3 mL of sample In an analytical study not using red cell lysis resulted in a significant delay in Cq
values34
The use of white cell lysis is critical A significant positive association between sensitivity and white cell lysis was
determined (P:0.018)
Fungal cells should be lysed using A significant positive association between sensitivity and bead-beating was
bead-beating determined (P:0.033). It is also cheaper and quicker than an enzymatic approach
and as has been shown to effective on hyphae and conidia35
Screen all reagents before using for Contamination of various reagents used in molecular biology with fungal DNA has
routine clinical diagnosis been noted33,36,37
Use positive and negative extraction Good molecular practice to monitor for inter-assay variation
controls
Elute DNA in <100µL A significant negative association between sensitivity and elution volumes >100
µL was determined (P:0.035)
PCR testing should be performed Although most PCR assays perform to a similar standard, the fungal burden in
in duplicate clinical samples is likely to be at levels close to or beyond the reproducible limit
of detection, consequently replicate testing may be necessary to avoid the
reporting of false negative results
An internal control PCR should Good molecular practice to monitor for PCR inhibition and avoid reporting false
be performed negative results
The internal control should generate If the burden of internal control target is much higher than the target the affects
a Cq value typical to values seen of inhibition may be less evident, potentially resulting in only slight Cq delays of
when testing Aspergillus positive the internal control. As the fungal burden in blood is at the limit of QPCR
clinical samples detection any delays may result in false negative results
Human DNA should not be used for The amount of human DNA will vary per specimen, consequently PCR amplification
an internal control target is purely qualitative and it is not possible to have a reference Cq value from which
inhibition can be determined. The burden of human DNA is likely to be far greater
than the Aspergillus DNA and may not be as affected by inhibition (See above)
DNA: deoxyribonucleic acid; EAPCRI: European Aspergillus PCR Initiative; EDTA: ethylenediaminetetraacetic acid;
QPCR: quantitative polymerase chain reaction.
70 P.L.White et al
a source, simple nucleic acid extraction protocols could be
used, with commercial kits providing greater standardisation
and QC; this would be a necessary prerequisite if PCR was
to enjoy widespread use outside specialist molecular mycol-
ogy laboratories. Following the algorithm described above,
a panel of serum samples loaded with varying amounts of A.
fumigatus genomic DNA was distributed to laboratory work-
ing group members to evaluate protocols currently being
used. Again the panel was designed to principally determine
analytical sensitivity, both technical information and results
were returned, and data analysis was performed blinded to
the identity of the participant. As the previous study had
shown DNA extraction to be the rate limiting state, a panel
to evaluate PCR alone was not included.
Initially, when evaluating whole blood, less than half of
methods were able to attain the designated threshold. For
serum testing, this figure rose to 82.8% (24/29), confirming
the hypothesis that methods testing serum required less
standardisation. Overall sensitivity (86.1%) and specificity
(93.6%) values were comparable to those for whole blood
PCR compliant with EAPCRI recommendations. ROC analysis
showed excellent performance, with area under the curve
of 0.915, and optimal performance using a Cq threshold
of 43 cycles, in keeping with a previous report26. There-
fore, it was deemed unnecessary to evaluate procedural
recommendations. Instead, meta-regression analysis was
performed using the existing data. Significant positive
associations were noted between sensitivity and the use of
larger sample volumes (≥0.5 mL) (P=.023), an internal con-
trol PCR (P=.029) and PCR assays targeting the ITS region
(P=.013). Significant negative associations were noted
between sensitivity and eluting nucleic acid in volumes
>100 µL (P=.003) and using PCR assays targeting mitochon-
drial regions (P=.010). However, associations between sen-
Table 2 European Aspergillus PCR Initiative recommendations for PCR testing of serum11
Recommendation Evidence base
Use a minimum of 0.5 mL sample A significant positive association between sensitivity and sample volume was
determined (P:0.023)
Most commercially available Twenty-eight of 29 protocols evaluated were able to reach the designated
nucleic acid extraction systems threshold of detection on at least one occasion, generating a threshold
can be used to extract Aspergillus positivity rate of 91.5%
DNA using the protocols as
described by the manufacturer
Screen all reagents, including Contamination of various reagents used in molecular biology with fungal DNA has
commercially sourced kits and been noted33,36,37
reagents, before using for routine
clinical diagnosis
Use methods specific for testing Methods specifically designed to test whole blood were not able to efficiently
serum process serum samples
Use positive and negative Good molecular practice to monitor for inter-assay variation
extraction controls
Elute DNA in <100 µL A significant negative association between sensitivity and elution volumes >100 µL
was determined (P:0.003)
PCR testing should be performed Although most PCR assays perform to a similar standard, the fungal burden in
in duplicate clinical samples is likely to be at levels close to or beyond the reproducible limit
of detection, consequently replicate testing may be necessary to avoid the reporting
of false negative results
An internal control PCR should be Good molecular practice to monitor for PCR inhibition and avoid reporting false
performed, and for serum testing can negative results. By incorporating the internal control at the start of the extraction
be included at the start of the process process the efficiency of extraction for individual samples can also be monitored
The internal control should generate If the burden of internal control target is much higher than the target the affects of
a Cq value typical to values seen inhibition may be less evident, potentially resulting in only slight Cq delays of the
when testing Aspergillus positive internal control. As the fungal burden in blood is at the limit of QPCR detection any
clinical samples delays may result in false negative results
Human DNA should not be used The amount of human DNA will vary per specimen, consequently PCR amplification is
for an internal control target purely qualitative and it is not possible to have a reference Cq value from which
inhibition can be determined
Use a PCR positivity threshold This provides the greatest degree of diagnostic accuracy (DOR of 105)
of 43 cycles
DNA: deoxyribonucleic acid; QPCR: quantitative polymerase chain reaction.
Towards a standard for Aspergillus PCR - requirements, process and results 71
sitivity and specific genomic regions, whether positive or
negative, should be interpreted with caution as the number
of assays targeting the regions stated were limited and
reaction kinetics will vary according to the assay design
and optimisation, and performance may not be directly
associated with the target gene but exclusive to the indi-
vidual assay11. These results were the basis for the EAPCRI
recommendations for PCR testing of serum (Table 2).
Future considerations
The obvious next step for the EAPCRI would be to test
plasma, as this is the complete liquid component of blood
without the cell, and includes the clotting factors. Theoreti-
cally, both serum and plasma types contain the same target
(DNAaemia), but the blood clot formed prior to obtaining
serum could, in addition to trapping human erythrocytes,
leukocytes, and any fungal cells potentially reduce the
amount of free DNA in the sample; this would not happen
in plasma. Evaluating the effect of these different sample
types on the mechanics of any nucleic acid extraction tech-
nique would be relatively straightforward through testing
plasma and serum samples loaded with the same fungal DNA
burdens after the separation of the required blood fractions.
However, this would not assess the affect of clot formation
on the DNA burden. This could be assessed using simulated
samples, spiking blood collection tubes with Aspergillus DNA,
possibly prior to drawing blood, but certainly prior to clot
formation, and for plasma testing by spiking the ethylenedi-
aminetetraacetic acid blood tube prior to fractionation.
For confirming the diagnosis, specimens require more
invasive procedures, and obtaining these samples must be
weighed up against the potential benefit to the patient,
and whether a PCR diagnosis will alter patient management
or outcome. For instance, if a patient is already receiving
antifungal therapy and is responding, then obtaining a deep
tissue sample for PCR testing may not lead to a change in
therapy or alter the outcome. Post-mortem testing could
confirm a diagnosis, but there is no need for rapid diagnosis
as a histopathological/mycological approach will suffice.
The enhanced sensitivity and specificity provided by real-
time PCR provides the opportunity to pre-empt infection
from becoming manifest disease, thereby helping improve
the patient prognosis. Currently, since most cases of IA
begin in the lung, obtaining a BAL specimen is the only way
of attaining an appropriate specimen for diagnosis. Estab-
lishing testing panels with this specimen presents several
logistic hurdles, including sourcing “willing” volunteers to
provide sufficient BAL samples, and overcoming potential
airway contamination leading to Aspergillus positive BAL
material. The potential of evaluating a simulated BAL
specimen is currently being pursued.
The EAPCRI also recognises the contributions of other
organisations trying to improve the quality of molecular
diagnosis and lead to greater standardisation. The Invasive
Aspergillosis Animal model group are developing standard-
ised in vivo models of IA to provide representative pathologi-
cal samples, while the Aspergillus Technology Consortium
is developing a bank of samples from patients with IA to
provide source material for evaluating diagnostic tests.
Commercial assays are now becoming available, and pre-
liminary clinical validation looks promising, although patient
numbers are limited when compared to many evaluations
of “in-house” protocols27,28. From the beginning the EAPCRI
has sought commercial collaboration and by participation
in testing EAPCRI QC panels, commercial partners ensure
their analytical performance is comparable to well validated
methods. The commercial production and the independent
analytical performance validation of commercial assays
provide the opportunity for Aspergillus PCR to be performed
outside specialist molecular mycology laboratories.
The minimum information for publication of quantitative
real-time PCR experiments (MIQE) guidelines were published as
a guide to researchers as to the minimum information required
in manuscripts describing real-time PCR experiments29, and
this has recently been applied specifically to an Aspergillus PCR
protocol30. While we fully support this guidance, it is important
to add that the MIQE guidelines were published to encourage
researchers to provide the technical information necessary
for readers to compare and replicate assays. It is unwise to
assume that these recommendations have been followed in
papers published before these guidelines appeared, limitations
in space often precludes the inclusion of extensive technical
information, even if assay optimisation and analytical evalu-
ation has been performed as described by the current MIQE
guidelines. There needs to be direct comparisons of the recent
MIQE designed Aspergillus PCR assay with other previously
well validated assays before concluding that it will provide
improved clinical performance, particularly when our research
shows DNA extraction to be rate-limiting10.
In conclusion, the EAPCRI has made significant steps in
developing a standard for Aspergillus PCR, but recognises that
the process will not be finished until the clinical utility has
been established in formal clinical trials. Currently, samples
for PCR testing are being taken in two prospective trials,
one comparing pre-emptive and empirical strategies (EORTC
65091-06093: ClinicalTrials.gov Identifier: NCT01288378), and
the other on the prevention of invasive fungal infections in sub-
jects receiving chemotherapy for acute lymphoblastic leukae-
mia (Ambiguard: ClinicalTrials.gov Identifier: NCT01259713).
Methodological recommendations are being evaluated using
non-simulated animal model material, and collaborations
with Invasive Aspergillosis Animal model group continue. On
completion, it is hoped that PCR will have attained sufficient
standardisation and validation to be included in the EORTC/
MSG definitions of IFD.
Conflicts of interest
All authors declared no conflicts of interest.
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