Primary diagnostic approaches of invasive aspergillosis – molecular testing

Received 15 March 2010 ; Rece

Accepted 11 July 2010

Correspondence: St é phane B

Mycologie, H ô pital Henri Mond

81 36 41; fax: � 33 1 49 81 36 0

Original Article

Primary diagnostic approaches of invasive

aspergillosis – molecular testing

ST É PHANE BRETAGNE

Groupe hospitalier Chenevier-Mondor, APHP, Laboratoire de Parasitologie-Mycologie, Créteil; Institut Pasteur, Centre National de

Référence de Mycologie et des Antifongiques, Paris; and Université Paris Est-Créteil, Créteil, France

© 2011 ISHAM

Medical Mycology April 2011, 49(Suppl. 1), S48–S53

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The PCR methods published for the diagnosis of invasive aspergillosis (IA) are diverse in terms of amplifi cation protocols and methods, equipment, fl uorescent detection dyes, PCR chemistries, and clinical specimens used. This explains why PCR is still not included in the revised EORTC/MSG defi nitions of IA despite encouraging results. Therefore, achieving consensual PCR procedures at the international level is manda-tory. When using PCR as a diagnostic tool, emphasis must be put on limiting false positive results due to contamination either with previously amplifi ed products or with environmental commensals. Internal amplifi cation controls are compulsory to evi-dence false negative results. For most of these aspects, quantitative PCR (qPCR) should improve both the results ’ reliability and the clinicians ’ confi dence. A checklist of items (Minimum information for publication of quantitative real-time PCR experiments) has been proposed to help scientists and reviewers. Currently, the main limitation relies in the DNA extraction procedure the choice of which dramatically depends on the still unknown origin of the Aspergillus DNA to amplify. There is an urgent need for basic studies to elucidate the origin and kinetics of Aspergillus DNA in blood. Once a techni-cal consensus is achieved, clinical studies should be initiated to integrate qPCR in the diagnostic armentarium of IA.

Keywords invasive aspergillosis , quantitative PCR , diagnosis

Introduction

PCR has extremely rapidly emerged as a powerful tool in

numerous and diverse fi elds of applications. Less than ten

years elapsed between the fi rst publication and the attribu-

tion of the Nobel Prize in Chemistry to Kary B. Mullis for

his discovery of PCR. Therefore, the number of reports

dealing with detection of microorganisms by PCR either

because of their small amounts in human specimens or

their low growth yield has exploded. For the diagnosis of

invasive aspergillosis (IA), as the classical mycological

ived in fi nal revised from 23 June 2010;

retagne, Laboratoire de Parasitologie-

or-APHP, Cr é teil, France. Tel: � 33 1 49

1; E-mail: [email protected]

diagnostic tools lacks sensitivity and specifi city, indirect

biomarkers of infection have been investigated, e.g., mainly

antigens and DNA detection. However, whereas both

galactomannan (GM) and beta-glucan (BG) detection are

now included in the European Organization for Research

and Treatment of Cancer and the Mycoses Study Group

(EORTC/MSG) consensus defi nitions for diagnosing IA,

it is not the case for PCR tools [1].

A recent meta-analysis of PCR tests performed on blood

specimens from patients with haematological diseases

reported a global sensitivity of 75% (95% CI: 54 – 88) and

88% (95% CI: 75 – 95) and a specifi city of 87% (95% CI:

78 – 93) and 75% (95% CI: 63 – 84) when two or one PCR

positive results are considered, respectively [2]. This

performance is close to that reported with GM in the same

population [3]. It may therefore be surprising that PCR

results were not included in the IA criteria. The main

DOI: 10.3109/13693786.2010.508186

Molecular diagnosis of invasive aspergillosis S49

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reason is lack of standardization [1]. Indeed, GM and BG

detection are performed using a limited number of com-

mercially available kits. This insures a relative uniformity

of the reagents and therefore some reproducibility of the

results. In contrast, there is a huge diversity of the PCR

protocols reported (Table 1). As multicentric enrollment is

the rule for epidemiological studies and therapeutic trials

evaluating diagnostic or therapeutic strategies for IA, it is

mandatory to reach a technical consensus [4,5].

Technical issues

To set up a PCR assay is simple since only knowledge of

the DNA sequence to amplify is required, and many soft-

ware packages are available to design specifi c primers.

Positive and negative controls are usually included. How-

ever, the implementation of PCR for routine use requires

numerous additional precautions [4,6].

The fi rst one is the management of false-positive results

due to previously amplifi ed products disseminated through

aerosolization. In a recent collaborative study, 6 out of 20

laboratories experienced possible false-positive results [7].

Control of contamination is crucial for the diagnosis of

infections when the microorganism load is very low, as

always observed during IA. As every step of the amplifi ca-

tion process must be optimized to detect this very low load,

© 2011 ISHAM, Medical Mycology, 49(Suppl. 1), S48–S53

every minute amount of contaminating amplicons could

lead to false positive results. With contamination below ten

copies, positive and negative results can be alternatively

observed according to Poisson ’ s law and can lead to falsely

reassuring negative results in the negative controls. Two

means are currently available to prevent these false positive

results. The fi rst one is the use of enzymes such as uracyl-

N-glycosylase able to cut previously amplifi ed products

[8]. The second and major one is the use of real-time quan-

titative PCR (qPCR). As amplifi cation and detection take

place in the same tube without need to open it, this dra-

matically decreases the risk of amplifi ed DNA aerosols

contaminating the environment. This is the major break-

through that allows PCR assays to reach the routine mycol-

ogy laboratory [4].

Another possible major source of false positive results

is the presence of fungal spores or DNA in the environment

and reagents [9,10]. Manipulating under laminar airfl ow

hoods can control contamination with fungal spores but not

with fungal DNA. A lot of commercial enzymes are pro-

duced by fungi, which can be source of residual fungal

DNA in the fi nal reagents. Therefore, the DNA extraction

process should limit the amount and number of reagents

used and avoid unnecessary enzymes [7]. Commercial

tubes containing heparin and sodium citrate have been

associated with fungal DNA contamination [11,12]. These

contaminations are diffi cult to avoid. Regular tests of both

commercial and ‘ in-house ’ reagents using negative extrac-

tion controls might be a way to uncover/evidence them.

The second requirement is the control of amplifi cation ’ s

yield. Once a PCR assay is validated, the same amplifi ca-

tion effi ciency is to be obtained for every clinical sample,

otherwise there is a risk of false-negative results. Many

commercial DNA extraction kits are currently available

and proved to be effi cient to avoid residual PCR inhibitors.

However, a certain level of PCR inhibition can be evi-

denced in 10 – 20% of the tubes used for blood or serum

collection [11,12]. Thus, amplifi cation ’ s performances

must be monitored by an internal control [13]. Choosing

to amplify a human gene can be falsely reassuring. Indeed,

the amount of human DNA in a clinical specimen is huge

compared with that of the targeted microorganisms. There-

fore, even with low amplifi cation ’ s yield, a positive PCR

signal can be observed leading to a falsely validated result.

One effi cient means is to use a specifi c control for every

primer set [8]. However, this can be expensive when diag-

noses are done for multiple infectious diseases. A good

compromise is the use of heterogeneous DNA such as plas-

mids [14], virus [15] or mouse DNA [16]. This internal

control is intended to be included in the PCR mix. As the

added quantity is known, identical results using qPCR

should be obtained in the absence of any PCR inhibitors

whatever the clinical specimen.

Table 1 Technical details of the 16 studies included in a metaanalysis

[2] showing the huge differences in the PCR protocols used.

Item n

Sample type

Whole blood

Serum

10

6Sample volume

10 ml

2 – 5 ml

500 – 700 μ l

200 μ l

Not available

3

7

3

1

2Enzymatic cell wall disruption

Zymolyase

Lyticase

None

6

6

4DNA extraction methods

Commercial kits

Automated extraction

Phenol-chloroform

11

1

4PCR format

PCR-ELISA

Nested PCR

qPCR

Conventional PCR

5

6

3

2Gene target

18S rRNA

28S rRNA

Mitochondrial DNA

11

2

2

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Another major interest of controlling the PCR yield is

quantifi cation of the results to follow the treatment effi -

cacy. This is widely used in virology for HIV infection or

to follow some parasitic diseases [17]. Unfortunately, for

Aspergillus DNA detection in blood or serum, the fungal

load is always low. With these low loads, reliable quanti-

fi cation is diffi cult due to the random dispersion of the few

DNA copies.

Minimum Information for publication of

Quantitative real-time PCR Experiments

(MIQE)

Even if qPCR has become the format of choice for the

detection and quantifi cation of nucleic acids in a research,

diagnostic, forensic and biotechnology setting, variable

pre-analytical conditions and assay design have resulted in

the recurrent publication of disputable data [6,18,19]. To

improve these points, Bustin has recently published a set

of guidelines that propose a minimum standard for the pro-

vision of information for qPCR experiments ( ‘ MIQE ’ )

[20]. This set presents 60 items, mainly for RNA experi-

ments, but also for qPCR validation (Table 2). These items

are classifi ed as ‘ D ’ for desirable and ‘ E ’ for essential.

Among these items, two can be underlined. The fi rst

one is the need to calculate the effi ciency of the amplifi ca-

tion. This is achieved with plotting the quantifi cation cycle

(Cq), previously known as the threshold cycle (Ct) or

crossing point (Cp), of serial dilutions of the targeted

DNA on a semi-log scale. This gives access to the effi -

ciency of the amplifi cation. Therefore, by adding the same

quantity of internal control in the PCR mix (see above),

the same Cq must be obtained to validate the result with

a given clinical specimen. The second one is the limit of

detection (LOD). The most sensitive LOD theoretically

possible is three copies per PCR, assuming a Poisson dis-

tribution, a 95% chance of including at least one copy in

the PCR, and single-copy detection. Therefore, to have a

consistently positive control for a PCR assay, this limit

cannot be inferior to 10 copies. Results below this theo-

retical threshold should not be reported.

Nested PCR assays have been reported with the goal to

enhance analytical sensitivity. In nested PCR, a fi rst run of

amplifi cation is followed by a second one using primers

that amplify a smaller fragment within the fi rst amplicon.

Therefore, nested PCR requires opening of the PCR tubes

between the two runs of amplifi cations and prevents enzy-

matic prevention of contamination. Both dramatically

increase the risk of false positive results. Moreover, nested

PCR cannot give access to the yield of amplifi cation and

prevents any control of false negative results with the use

of an internal control. Additionally, nested-PCR is not

more sensitive than a single run PCR which is optimized

with a LOD of few copies per reaction. The theoretical

LOD cannot be improved precisely because of the PCR

format used. As a consequence, the two PCR reactions in

a nested format are only designed to compensate for the

low yield of a single run PCR test. To obtain reliable and

exchangeable results, it is therefore preferable to opt for a

single run optimized qPCR [4,21].

Basic studies

One of the major limits to adopt a PCR consensual

technique is our ignorance of the origin of the Aspergillus

DNA amplifi ed from blood or serum. This knowledge

will impact the DNA extraction step. Indeed, a high ana-

lytical sensitivity is essential as the fungal burden circulat-

ing in the bloodstream is low [22,23]. A solution could be

to increase the volumes tested. However, if the overall

amount of fungal DNA is expected to increase, paradoxi-

cally, too much human DNA may decrease the PCR effi -

ciency, hence the necessity of the internal control to avoid

false negative results.

It is unlikely that spores circulate in blood since blood

cultures are massively negative, and a positive culture is

often deemed to be an environmental contamination [1].

However, non-viable hyphae fragments can be engulfed in

white cells and dissemination through bloodstream can

occur. Another hypothesis is the released of cell-free DNA

by the fungus, as observed with cancer or foetal cells [24].

Knowledge of the DNA source will dramatically infl uence

the clinical specimens and the DNA extraction to use. To

disrupt the fungal wall to free fungal DNA will require

very stringent methods [7], a control of this DNA extrac-

tion step [25], and blood would then be the preferred spec-

imen. For cell-free DNA, serum should be the best

Table 2 Minimum information for publication of quantitative real-time

PCR experiments (MIQE) checklist for qPCR validation from reference

[19]. Essential information must be submitted with the manuscript and

desirable information should be submitted if available.

Item Importance

Evidence of optimization (from gradients) DesirableSpecifi city EssentialCalibration curves EssentialPCR effi ciency calculated from slope EssentialCIs for PCR effi ciency or SEs of the means of

estimated PCR effi ciencies

Desirable

r 2 of calibration curve EssentialLinear dynamic range EssentialCq variation at limit of detection EssentialCIs throughout range DesirableEvidence of limit of detection EssentialIf multiplex, effi ciency and limit of detection

of each assay

Essential

Cq: quantifi cation cycle.



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specimen, and it is more amenable to automatic DNA

extraction [22,26].

Another basic question is the timing between GM pro-

duction and the availability of amplifi able DNA, as several

biomarkers are expected to be used in a screening strategy

[22,27 – 29]. If serial detection of Aspergillus DNA were on

average 68 days earlier than GM detection and if false

positive results are excluded, an explanation should be

found for the physiopathology of IA [28].

European Aspergillus PCR Initiative

With the fi nal goal of assessing the usefulness of PCR for

the diagnosis and the management of IA, a European

Aspergillus PCR Initiative (EAPCRI) was launched at the

ISHAM meeting in 2006 with the aim of providing optimal

standardized protocols for evaluation of Aspergillus PCR.

Based on a previous UK initiative [30], panels of quality

control (QC) circulated to different centres to get recom-

mendations on the technical aspects of the PCR. Two kinds

of QC were evaluated.

One QC panel consisted of blood spiked with serial

dilutions of Aspergillus fumigatus DNA previously

extracted from conidia. The LOD was calculated to be

around 27 rRNA gene copies per μ l eluate [7]. Most qPCR

amplifi cation systems provided similar detection thresh-

olds. There was no statistical difference between the mean

PCR effi ciency for the different platforms used. There was

no signifi cant correlation between sensitivity and PCR tar-

get neither. The most frequent target was multicopy rRNA

and the primers used were specifi c for A. fumigatus [2,7].

Another possible strategy is to use ‘ pan fungal primers ’

that share signifi cant similarity between fungal species.

Unfortunately, these primers share also similarity with

homologous human genes and this may result in both false

positivity and false negativity. Additionally, once the diag-

nosis of fungal infection is done, an additional specifi c test

is mandatory to choose the right antifungal treatment since

the antifungal drugs have different activity spectra. There-

fore, the panfungal strategy does not seem justifi ed for a

routine diagnosis. To ascertain the nature of the PCR prod-

ucts, most of the centres used probes instead of dissocia-

tion curves [7]. Probes give results more easily transferable

between laboratories.

Another QC consisted in blood spiked with A. fumiga-tus conidia. Two blood panels were distributed to 24

centres and analyzed for 20 of them. Six centres were

subsequently excluded because of the possibility of posi-

tive results arising from contamination [7]. Half of the

centres failed to reach the level of detection achieved

with blood spiked with DNA. This underlines the neces-

sity of fi nding a relevant positive control of the extraction

step, i.e., a sample correctly mimicking IA setting to


routinely monitor extraction performance. Meta-regression

analysis showed positive correlations between sensitiv-

ity and extraction protocols incorporating the use of

bead-beating, white cell lysis buffer and internal control.

An elution volume below 100 μ l for the fi nal step of

DNA extraction is also associated with an increased

sensitivity.

Broncho-alveolar lavage fl uids

The performances of PCR have been investigated to over-

come the low sensitivity of culture from broncho-alveolar

lavage (BAL) fl uids. A meta-analysis calculated a mean

sensitivity of 79% (95% CI: 72.8 – 83.1) and a mean speci-

fi city of 94% (95% CI: 92.1 – 95.0) for PCR assays [31].

Therefore, BAL fl uids provide an additional useful sample

for the diagnosis of IA. The technical requirement for the

PCR assays as the same as those for PCR on blood speci-

mens [21]. A very stringent DNA extraction step is required

since whole fungal cells and not cell-free DNA are thought

to be present in the respiratory tract. However, PCR results,

as culture results, cannot differentiate between colonisation

and infection. A quantitative cut-off could be defi ned to

distinguish the two situations. However, quantifi cation

with BAL fl uid is highly dependant on the quality and the

recovered volume. This can make the defi nition of a quan-

titative threshold questionable. Commercial kits are emerg-

ing for this specifi c application [32].

Other specimens

A further application of molecular tools is the confi rmation

of histological fi ndings, as identifi cation of the invading

fungus may be possible by molecular methods, when cul-

ture has not been done or is inhibited by empirical antifun-

gal treatment. However, the diagnostic goal is then

different than for blood/serum or BAL samples. For blood

and serum, the aim is to screen patients. For BAL, the aim

is to improve the sensitivity of direct examination and cul-

ture. For tissue specimens, the issue is to identify the fun-

gus seen in biopsy. The starting material is fresh and

formalin-fi xed, paraffi n embedded (PE) sections. Given

that more than 200 fungal species have been reported to

cause disease in humans, a species-specifi c or even a

genus-specifi c assay is limited. Panfungal PCR assays, on

the other hand, have the potential to detect all fungal spe-

cies. The majority of PCR assays target the ribosomal

DNA genes (18S, 28S, and 5.8S) and the internal tran-

scribed spacer (ITS) regions, in order to obtain enough

amplifi ed material for sequencing. Sequence-based identi-

fi cation of PCR products is a reliable method provided that

accurate sequences have been submitted to public data-

bases [33]. The main technical limit is then to obtain DNA

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fragments long enough to be sequenced. Formalin-fi xed

tissues often yield short DNA fragments, which preclude

correct identifi cation [34,35]. To overcome this issue,

probe and primers designed for known pathogens can con-

fi rm the identifi cation, but only for the predefi ned species

[36]. Indeed, this strategy does not allow the identifi cation

of new and emerging agents.

Clinical use

Once a consensual procedure for the detection of fungal

DNA is defi ned, the goal is to use it for the diagnosis and

the management of patients with IA. The mortality rate of

IA remain high, between 40 and 70%, despite the avail-

ability of new effective drugs [37]. Frequent PCR testing

in patients with a high risk of IFI may enable a diagnosis

of IFI before the onset of symptoms and an earlier start of

treatment [5]. Several designs are possible depending on

whether the reliance is on the positive predictive value or

the negative predictive value, keeping in mind that the sen-

sitivity of Aspergillus PCR testing can be limited during

antifungal therapy [38].

In a fi rst study investigating the impact of PCR as a

diagnostic tool for guiding pre-emptive antifungal therapy,

patients who underwent allogeneic stem cell transplanta-

tion were randomized to PCR based pre-emptive (group

A, n � 198) as opposed to empirical treatment (group B,

n � 211) with liposomal amphotericin B [39]. Twelve

patients in group A and 16 patients in group B developed

proven invasive fungal infection. Survival curves showed

signifi cant better survival until day 30 when close PCR

monitoring was performed but there was no difference at

day 100. In another recent study, PCR on peripheral blood

was a poor indicator of invasive fungal infection early

after reduced-intensity conditioning allogeneic haemato-

poietic stem cell transplantation [40]. In these studies, GM

was not used as a diagnostic criterion that could have

modifi ed the treatment strategy [29].

A second way of viewing the interest of PCR is to rely on

the predictive negative value [27]. A cohort of 130 high-risk

haematology and stem cell transplant patients was enrolled

in a neutropenic care pathway in which targeted diagnostic

testing replaced empiric antifungal treatment. Patients were

screened twice a week by PCR and GM testings. No excess

morbidity or mortality was seen in patients in whom empiric

antifungal treatment was withheld, and there were substantial

savings in antifungal drug expenditure.

Conclusion

There are more and more evidence of the role of qPCR for

IA diagnosis, and more generally of biomarkers, in a

screening strategy to improve the prognosis of IA and to

restrain the use of unjustifi ed antifungal drugs [5]. Reli-

ance for clinical use depends on the procedure used. Some

technical issues regarding PCR assays are already solved

such as the use of qPCR as long as MIQE requirements

are fulfi lled. It is not necessary to defi ne a unique PCR

assay but to follow consensual procedures. Currently, the

variability in current performance mainly, but not exclu-

sively, appears to be a direct consequence of variable

performances in the DNA extraction techniques. Besides,

there is an urgent need for basic studies to elucidate the

origin of the fungal DNA present in blood in order to

rationally design DNA extraction procedures. Even if

these additional issues are not yet solved, clinical studies

should be initiated in using the consensus already

achieved.

Declaration of interest: The author does consultancy

work for Myconostica.

References

De Pauw B, Walsh TJ, Donnelly JP, 1 et al . Revised defi nitions 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. Clin Infect Dis 2008; 46 : 1813 – 1821.

Mengoli C, Cruciani M, Barnes RA, Loeffl er J, Donnelly JP. Use of 2

PCR for diagnosis of invasive aspergillosis: systematic review and

meta-analysis. Lancet Infect Dis 2009; 9 : 89 – 96.

Pfeiffer CD, Fine JP, Safdar N. Diagnosis of invasive aspergillosis 3

using a galactomannan assay: a meta-analysis. Clin Infect Dis 2006;

42 : 1417 – 1427.

Bretagne S, Costa JM. Towards a molecular diagnosis of invasive 4

aspergillosis and disseminated candidosis. FEMS Immunol Med Microbiol 2005; 45 : 361 – 368.

de Pauw BE, Donnelly JP. Timely intervention for invasive fungal 5

disease: should the road now lead to the laboratory instead of the

pharmacy? Clin Infect Dis 2009; 48 : 1052 – 1054.

Bustin SA. Why the need for qPCR publication guidelines? The 6

case for MIQE. Methods 2010; 50 : 217–226.

White PL, Bretagne S, Klingspor L, 7 et al . Aspergillus PCR: one

step closer towards standardisation. J Clin Microbiol 2010; 48 :

1231 – 1240.

Bretagne S, Costa JM, Marmorat-Khuong A, 8 et al . Detection of

Aspergillus species DNA in bronchoalveolar lavage samples by

competitive PCR. J Clin Microbiol 1995; 33 : 1164 – 1168.

Loeffl er J, Hebart H, Bialek R, 9 et al . Contaminations occurring in

fungal PCR assays. J Clin Microbiol 1999; 37 : 1200 – 1202.

Rimek D, Garg AP, Haas WH, Kappe R. Identifi cation of contami-10

nating fungal DNA sequences in Zymolyase. J Clin Microbiol 1999;

37 : 830 – 831.

Garcia ME, Blanco JL, Caballero J, Gargallo-Viola D. Anticoagulants 11

interfere with PCR used to diagnose invasive aspergillosis. J Clin Microbiol 2002; 40 : 1567 – 1568.

Harrison E, Bowyer P, Sugrue MW, 12 et al . Fungal DNA contamina-tion of blood collection tubes . In: 48th Interscience Conference on

Antimicrobial Agents and Chemotherapy. Washington, DC: American

Society for Microbiology; 2008.



Med

Myc

ol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cgill

Uni

vers

ity o

n 11

/17/

14Fo

r pe

rson

al u

se o

nly.

Paterson RR. Internal amplifi cation controls have not been employed 13

in fungal PCR hence potential false negative results. J Appl Microbiol 2007; 102 : 1 – 10.

White PL, Linton CJ, Perry MD, Johnson EM, Barnes RA. The 14

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 2006; 42 :

479 – 486.

van Doornum GJ, Guldemeester J, Osterhaus AD, Niesters HG. 15

Diagnosing herpes virus infections by real-time amplifi cation and

rapid culture. J Clin Microbiol 2003; 41 : 576 – 580.

Costa JM, Ernault P, Gautier E, Bretagne S. Prenatal diagnosis of 16

congenital toxoplasmosis by duplex real-time PCR using fl uorescence

resonance energy transfer hybridization probes. Prenat Diagn 2001;

21 : 85 – 88.

Costa JM, Pautas C, Ernault P, 17 et al . Real-time PCR for diagnosis

and follow-up of Toxoplasma reactivation after allogeneic stem cell

transplantation using fl uorescence resonance energy transfer hybrid-

ization probes. J Clin Microbiol 2000; 38 : 2929 – 2932.

Bohlenius H, Eriksson S, Parcy F, Nilsson O. Retraction. 18 Science

2007; 316 : 367.

Madej RM, Davis J, Holden MJ, 19 et al . International standards and

reference materials for quantitative molecular infectious disease

testing. J Mol Diagn 2010; 12 : 133 – 143.

Bustin SA, Benes V, Garson JA, 20 et al . The MIQE guidelines: mini-

mum information for publication of quantitative real-time PCR

experiments. Clin Chem 2009; 55 : 611 – 622.

Khot PD, Ko DL, Hackman RC, Fredricks DN. Development and 21

optimization of quantitative PCR for the diagnosis of invasive

aspergillosis with bronchoalveolar lavage fl uid. BMC Infect Dis

2008; 8 : 73.

Costa C, Costa JM, Desterke C, 22 et al . Real-time PCR coupled

with automated DNA extraction and detection of galactomannan

antigen in serum by enzyme-linked immunosorbent assay for

diagnosis of invasive aspergillosis. J Clin Microbiol 2002; 40 :

2224 – 2227.

Loeffl er J, Henke N, Hebart H, 23 et al . Quantifi cation of fungal DNA

by using fl uorescence resonance energy transfer and the light cycler

system. J Clin Microbiol 2000; 38 : 586 – 590.

Swarup V, Rajeswari MR. Circulating (cell-free) nucleic acids – a 24

promising, non-invasive tool for early detection of several human

diseases. FEBS Lett 2007; 581 : 795 – 799.

Raddadi N, Belaouis A, Tamagnini I, 25 et al . Characterization of poly-

valent and safe Bacillus thuringiensis strains with potential use for

biocontrol. J Basic Microbiol 2009; 49 : 293 – 303.

Suarez F, Lortholary O, Buland S, 26 et al . Detection of circulating

Aspergillus fumigatus DNA by real-time PCR assay of large serum

volumes improves early diagnosis of invasive aspergillosis in high-

risk adult patients under hematologic surveillance. J Clin Microbiol 2008; 46 : 3772 – 3777.


Barnes RA, White PL, Bygrave C, 27 et al . Clinical impact of enhanced

diagnosis of invasive fungal disease in high-risk haematology and

stem cell transplant patients. J Clin Pathol 2009; 62 : 64 – 69.

Cuenca-Estrella M, Meije Y, Diaz-Pedroche C, 28 et al . Value of serial

quantifi cation of fungal DNA by a real-time PCR-based technique

for early diagnosis of invasive Aspergillosis in patients with febrile

neutropenia. J Clin Microbiol 2009; 47 : 379 – 384.

Millon L, Piarroux R, Deconinck E, 29 et al . Use of real-time PCR to

process the fi rst galactomannan-positive serum sample in diagnosing

invasive aspergillosis. J Clin Microbiol 2005; 43 :5097 – 5101.

White PL, Barton R, Guiver M, 30 et al . A consensus on fungal poly-

merase chain reaction diagnosis? A United Kingdom-Ireland evalua-

tion of polymerase chain reaction methods for detection of systemic

fungal infections. J Mol Diagn 2006; 8 : 376 – 384.

Tuon FF. A systematic literature review on the diagnosis of 31

invasive aspergillosis using polymerase chain reaction (PCR) from

bronchoalveolar lavage clinical samples. Rev Iberoam Micol 2007;

24 : 89 – 94.

Lass-Fl ö rl C, Cui X, Hughes M, 32 et al . Clinical Performance of FXG: RESP (Asp � ) Assay for Aspergillus on lung and other tissue samples .

In: Interscience Conference on Antimicrobial Agents and Chemother-

apy. Washington, DC: American Society for Microbiology; 2008.

Balajee SA, Nickle D, Varga J, Marr KA. Molecular studies reveal 33

frequent misidentifi cation of Aspergillus fumigatus by morphotyping.

Eukaryot Cell 2006; 5 : 1705 – 1712.

Hewitt SM, Lewis FA, Cao Y, 34 et al . Tissue handling and specimen

preparation in surgical pathology: issues concerning the recovery of

nucleic acids from formalin-fi xed, paraffi n-embedded tissue. Arch Pathol Lab Med 2008; 132 : 1929 – 1935.

Lau A, Chen S, Sorrell T, 35 et al . Development and clinical application

of a panfungal PCR assay to detect and identify fungal DNA in tissue

specimens. J Clin Microbiol 2007; 45 : 380 – 385.

Rantakokko-Jalava K, Laaksonen S, Issakainen J, 36 et al . Semiquantita-

tive detection by real-time PCR of Aspergillus fumigatus in broncho-

alveolar lavage fl uids and tissue biopsy specimens from patients with

invasive aspergillosis. J Clin Microbiol 2003; 41 : 4304 – 4311.

Walsh TJ, Anaissie EJ, Denning DW, 37 et al . Treatment of aspergillo-

sis: clinical practice guidelines of the Infectious Diseases Society of

America. Clin Infect Dis 2008; 46 : 327 – 360.

Hummel M, Spiess B, Cornely OA, 38 et al . Aspergillus PCR testing:

results from a prospective PCR study within the AmBiLoad trial.

Eur J Haematol 2010: Apr 1. Epub ahead of print.

Hebart H, Klingspor L, Klingebiel T, 39 et al . A prospective randomized

controlled trial comparing PCR-based and empirical treatment with

liposomal amphotericin B in patients after allo-SCT. Bone Marrow Transplant 2009; 43 : 553 – 561.

Blennow O, Remberger M, Klingspor L, 40 et al . Randomized PCR-

based therapy and risk factors for invasive fungal infection following

reduced-intensity conditioning and hematopoietic SCT. Bone Marrow Transplant 2010: Mar 1. Epub ahead of print.

This paper was fi rst published online on Early Online on 20 August

2010.

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Primary diagnostic approaches of invasive aspergillosis – molecular testing