Lab Test Aspergillosis

Review

IntroductionAspergillus spp are ubiquitous opportunistic moulds thatcause both allergic and invasive syndromes. The genuscomprises approximately 180 species, of which 33 havebeen associated with human disease. Most infections arecaused by Aspergillus fumigatus, Aspergillus flavus,Aspergillus terreus, and Aspergillus niger;1 less commonly,Aspergillus nidulans can be implicated as the causativepathogen, especially in the setting of chronicgranulomatous disease.2

An accurate diagnosis of invasive aspergillosis isimportant for clinical reasons; an earlier diagnosis isassociated with improved patient survival3 and tests witha high negative predictive value may allow expensive andpotentially toxic antifungal drugs to be withheld. Newdrugseg, voriconazoleexhibit differential mouldactivity; the ability to specifically exploit their anti-aspergillus properties requires a rapid and accuratelaboratory diagnosis. The epidemiology of invasiveaspergillosis is changing; invasive disease is increasinglyobserved in the non-neutropenic phase ofhaematopoietic stem cell transplantation46 and in non-classic settings such as critically ill patients in intensivecare units.7 Aspergillus spp other than A fumigatussomeof which demonstrate inherent resistance to antifungaldrugsare increasingly recognised.810 An internationalcollaborative effort recently produced standardiseddefinitions for invasive fungal infections.11 Thus, a reviewof the diagnostic modalities and their use in establishinga diagnosis of invasive aspergillosis is timely.

Diagnostic toolsDirect techniquesThe advantages of direct techniques over culture includesuperior sensitivity and a relatively rapid turn aroundtime. The principal disadvantage is the inability todefinitively distinguish other filamentous fungi (eg,

Penicillium spp and Scedosporium spp) or implicateAspergillus spp as the causative pathogen in circumstancesin which there are atypical or non-specific morphologicalfeatures. This disadvantage may compromise diagnosticaccuracy and hence estimates of therapeutic efficacy ifpatients are recruited to clinical trials solely on the basis ofhyphae that resemble Aspergillus spp. Within tissuesections, Aspergillus spp typically appear as slender septatehyphae that exhibit angular dichotomous branching(figure 1).

Lancet Infect Dis 2005; 5: 60922

DWD and WWH are at the Schoolof Medicine, University ofManchester and WythenshaweHospital, Manchester, UK; TJW isat the Pediatric Oncology Branch,National Cancer Institute,National Institutes of Health,Bethesda, MD, USA. WWH is alsoat the Pediatric Oncology Branch,National Cancer Institute.

Correspondence to: Professor David W Denning,Education and Research Centre,Wythenshawe Hospital,Southmoor Road, ManchesterM23 9LT, UK. Tel +44 (0)161 291 5811; fax +44 (0)161 291 5806;[email protected]

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Invasive aspergillosis occurs in a wide range of clinical scenarios, is protean in its manifestations, and is still associatedwith an unacceptably high mortality rate. Early diagnosis is critical to a favourable outcome, but is difficult to achievewith current methods. Deep tissue diagnostic specimens are often difficult to obtain from critically ill patients. Newerantifungal agents exhibit differential mould activity, thus increasing the importance of establishing a specific diagnosisof invasive aspergillosis. For these reasons, a range of alternate diagnostic strategies have been investigated. Mostinvestigative efforts have focused on molecular and serological diagnostic techniques. The detection of metabolitesproduced by Aspergillus spp and a range of aspergillus-specific antibodies represent additional, but relatively underused,diagnostic avenues. The detection of galactomannan has been incorporated into diagnostic criteria for invasiveaspergillosis, reflecting an increased understanding of the performance, utility, and limitations of this technique.Measurement of (1,3)--D glucan in blood may be useful as a preliminary screening tool for invasive aspergillosis,despite the fact that this antigen can be detected in a number of other fungi. There have been extensive efforts directedtoward the detection of Aspergillus spp DNA, but a lack of technical standardisation and relatively poor understanding ofDNA release and kinetics continues to hamper the broad applicability of this technique. This review considers theapplication, utility, and limitations of the currently available and investigational diagnostic modalities for invasiveaspergillosis.

Laboratory diagnosis of invasive aspergillosisW W Hope, T J Walsh, D W Denning

Figure 1: The appearance of Aspergillus spp in histological sections(A) Gomori methanamine silver (GMS) stain of rabbit lung in experimental invasive pulmonary aspergillosis(magnification x400). (B) A similar section stained with periodic acid-Schiff (PAS) (magnification x400). (C) and (D)show acute angle dichotomous branching, which is typical of Aspergillus spp (magnification x630).The GMS sectionsdemonstrate the prominent staining and stark appearance of hyphae. By contrast, with PAS there is preservation ofbackground histological detail and hyphal morphology, but hyphae are less conspicuous against the background.

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Wet mounts, potassium hydroxide preparations, and use ofroutine stainsAll specimens obtained in scenarios in which fungi arepossible aetiological agents should be subject to a series ofroutine direct procedures; these procedures may varyaccording to the specimen, degree of clinical urgency, andthe individual laboratory. Specimens may be examined asa wet mount preparation with or without the addition of10% potassium hydroxide, which aids in the visualisationof hyphal elements through the partial digestion andclearing of proteinaceous material while leaving thefungal cell wall intact.12 Subsequently, a smear is made ona slide, fixed and subjected to a variety of stainingprocedures. A Gram stain should be done as a matter ofroutine, but cytological stains (eg, Papanicolaou stain),fungal stains, and fluorescent stains may improvesensitivity.

Fungal stainsFungal-specific stains should be applied in all cases inwhich invasive aspergillosis is considered a diagnosticpossibility.13 Hyphal elements are stained withhaematoxylin and eosin, although they may be difficultto visualise if sparse, fragmented, or present in thesetting of substantial tissue necrosis. Fungal-specificstainseg, Gomoris methenamine silver stain (GMS)and periodic acid-Schiff (PAS)can be applied tohistological sections and smears (figure 1). On occasion,GMS is referred to as Grocotts stain or the Grocott-Gomori silver stainRobert Grocott demonstrated thatGMS, which was initially designed as a stain forglycogen and mucin, also readily stained fungalelements.14 PAS has the advantage of providing acounter stain that reveals the background host cellulardetail, tissue architecture, and inflammatory response.By contrast, the GMS counter stain removes the finedetails of background host cells and tissues, butprovides a more sensitive stain for detecting smallfragments of cell wall that may be otherwise obscuredby surrounding tissue elements. Thus, for detection ofhyphal elements, the use of the GMS stain may be moresensitive; whereas PAS provides more of the cellulardetail and architecture that may be of help inestablishing relations between the fungus and otherelements of tissue. This may be important in definingthe individual aspergillus-related syndromes that varyaccording to the immunological status of the host. Inthis regard, GMS and PAS are complementary.

Fluorescent techniquesFluorescent dyeseg, Calcofluor white, Uvitex 2B, andBlankophorare water-soluble colourless dyes thatselectively bind to beta-glycosidically linked poly-saccharides within fungal cell walls. They are not specificfor Aspergillus spp, but have the advantages of relativelyhigh sensitivity, rapid turnaround time, and broadapplicability. They may be applied to frozen sections,

paraffin-embedded tissue, and other fresh clinicalspecimenseg, bronchoalveolar lavage fluid (BAL) orcorneal scrapings.15,16

Immunohistochemistry, immunofluorescence, and in-situhybridisationImmunohistochemistry (using the monoclonal antibodyWF-AF-117 or EB-A118,19), immunofluorescence,20 and in-situ hybridisation21,22 have been studied as diagnosticmodalities. Collectively, these techniques have thepotential to provide genus and species specific data, whichmay be important to improve diagnostic certainty whenhyphae are seen invading tissue, but cultures or otheradjunctive diagnostic data are negative. The availability ofthese modalities in routine clinical microbiologylaboratories is variable.

CultureA culture yielding Aspergillus spp, in addition to enabling adiagnosis of invasive aspergillosis, may further definetherapeutic options via susceptibility testing or theisolation of a species possessing inherent antifungalresistance; examples of the latter include A terreus andA nidulans, which are both resistant to amphotericin B.10,23

The main disadvantage of culture is that it is relativelyslow (the process takes days), is relatively insensitive,24 andrequires specialised expertise for species determination.

In common with other pathogenic fungi, the ability togrow at 37C distinguishes Aspergillus spp from other non-pathogenic environmental moulds. Aspergillus spp can berecovered on most routine solid and liquidmicrobiological media (eg, blood agar, chocolate agar,brain heart infusion broth). A fungal-specific mediumeg, Sabouraud dextrose agarshould be included at thetime of initial specimen set-up in clinical scenarios inwhich Aspergillus spp (or other moulds) are consideredpossible pathogens, because of superior yield.25 Theaddition of antibioticseg, chloramphenicol andgentamicinto the medium is required for the recoveryof Aspergillus spp from specimens obtained from non-sterile sites, since they prevent bacterial overgrowth.Cycloheximide, a eukaryotic protein synthesis inhibitor, isfrequently added to fungal media to inhibit theovergrowth of cultures by non-pathogenic environmentalmoulds; however, on occasion, cycloheximide may inhibitthe growth of Aspergillus spp.26

The identity of a laboratory isolate can often be inferredon the basis of colonial morphology and colour. Definitiveidentification, however, is dependent on a detailedinspection of conidial morphology and ontogeny andrequires a microscopic examination of a simple teasedpreparation or a slide culture (a procedure in whichsporulation is induced and the relevant diagnostic featuresare visualised on the under-surface of a cover-slip). Theappearance and diagnostic features of individual species isbeyond the scope of this review and readers are referred todefinitive texts,27 useful guides,28 and excellent websites.

Seehttp://www.aspergillus.man.ac.uk

http://www.mycology.adelaide.edu.au and

http://www.doctorfungus.org

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Several additional issues pertaining to culture requireemphasis. First, the growth characteristics andmorphological appearances of Aspergillus spp areprotean and in some circumstances quite atypical; inthis regard, Aspergillus spp are great mimics and shouldalways be included in the list of diagnostic possibilitiesfor an unidentified mould. Second, at least on occasion,sporulation may be difficult or impossible to induce,29

and other modalities must be used for the purposes ofidentification. In this circumstance, moleculartechniques are perhaps best placed to enable rapid andaccurate identification.

Serological techniquesGalactomannan Galactomannan is a heat-stable heteropolysaccharidepresent in the cell wall of most Aspergillus and Penicilliumspecies.30 The molecule is comprised of a non-immunogenic mannan core with immunoreactive side-chains of varying lengths containing galactofuranosylunits.30 The composition of galactomannan varies betweengenera and strains, as well as the strain and conditionsused for its production, extraction, and purification.30

There are two commercial assays for the detection ofgalactomannanthe Pastorex kit (Sanofi DiagnosticsPasteur,Marnes-La-Coquette, France) and Platelia ELISA(BioRad, Marnes-La-Coquette, France). Pastorex is nowrarely used, while Platelia has been available in Europe forapproximately 10 years and has recently been licensed inthe USA. There has been a progressive increase in theunderstanding of the diagnostic utility of galactomannanto a point that has enabled its incorporation intodiagnostic criteria.11 However, galactomannan testing isnot universally available to clinicians; the decision to offergalactomannan testing within a hospital microbiologylaboratory depends on resources, the institutionalincidence of invasive aspergillosis, and the hospital case-mix.

Details surrounding the release and kinetics ofcirculating galactomannan remain largely undefined. Thegrowth phase, microenvironment, host immune status,and pathology may all influence galactomannan release.31

An abundance of data supports the notion thatgalactomannan production is proportional to fungal loadin tissue;3234 furthermore, galactomannan levels appear tohave prognostic significance, with high unremitting levelsin the face of antifungal therapy associated with anunfavourable outcome.10,3337

Assays to detect galactomannan have mostly usedserum and BAL fluid. Galactomannan can also bedetected in tissue and a number of bodily fluids includingCSF, peritoneal fluid, urine, and pericardial fluid,although data to support its use at these sites is relativelyscant, and is likely to remain that way.38

Galactomannan assays use EB-A2, a monoclonalantibody derived from rats, which is directed towards the (1,5)-linked galactofuranoside side-chain residues of the

galactomannan molecule.39 Four or more epitopes arerequired for antibody binding.31,39 Detection is achievedusing a sandwich ELISA format, which is made possibleby multiple immunoreactive epitopes on a singlegalactomannan molecule.39

There are a number of important determinants ofanalytical sensitivity of galactomannan assays. First, thebinding of EB-A2 requires four or moregalactofuranoside epitopessensitivity may becompromised by the inability to detect secreted antigensthat bear fewer residues.31 Second, the Platelia assay isdependent on a pretreatment step, the goal of which is toremove complexing antibody that may block EB-A2binding. However, the acid-sensitive galactofuranosideresidues may be degraded by the edetic acid used in thisstep.31 Finally, the limit of detection using the sandwichELISA format is lower (1 ng/L) than that achievableusing latex agglutination (15 ng/L).40 In terms of theanalytical specificity, cross reactivity with otherfilamentous fungi, bacteria, drugs, and cotton swabshave been documented,4145 but whether this is due to(exogenous) galactomannan or unrelated cross-reactivemolecules is unclear.

There have been considerable efforts in establishing theappropriate galactomannan ELISA cut-off to maximiseclinical sensitivity and specificity. The ELISA endpoint is acontinuous variable and the optimal cut-off should bedetermined after defining the receiveroperator curverelation (ie, the relation between sensitivity and1specificity).46 The cut-off level of 15 ng/L initiallyrecommended by BioRad and used in many early studieshas been progressively revised downwards; a cut-off of05 ng/mL is now currently accepted by the US Food andDrug Administration (FDA), while a level of 07 ng/L iscommonly used in Europe.47

The clinical sensitivity of galactomannan ELISA issomewhat variable, with a range of 29100%.31 There are anumber of potential reasons for these disparate results.First, the performance of the assay may differ according tothe host group and therefore the underlying pathologicalprocess. In studies of profoundly immunocompromisedpatients, sensitivity has been generally reported to be inexcess of 90%,48,49 while in other settingseg, chronicgranulomatous disease50 and solid organ transplan-tationsensitivity appears to be somewhat lower.5153

Second, accumulating evidence suggests that concomitantantifungal therapy leads to a decrease in the sensitivity ofgalactomannan.32,36,54 Finally, inadequate samplingstrategies could conceivably compromise clinicalsensitivity; the optimal sampling strategy for screeninghas not been rigorously defined, but the twice weeklydetermination of antigen levels has been generally used inpatients deemed to be at risk of invasive aspergillosis. Bycontrast, galactomannan levels should be determinedimmediately in a host with a constellation of clinicalfeatures indicative of invasive aspergillosis to facilitate adefinitive diagnosis.


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The clinical specificity of galactomannan is generallyestimated to be greater than 90%.32,36,4749,55 The specificity ofgalactomannan in neonates and children appears to belower, which is possibly due to the ingestion of extraneousgalactomannan (in food and water) and translocationacross a damaged or immature gut wall.45,47,56 Antibioticsrepresent an additional source of extraneousgalactomannan that may compromise clinical specificity.The in-vitro reactivity of a range of antibiotics ingalactomannan assays was originally reported in 1997.57

More recently, positive galactomannan results in patientsreceiving piperacillin-tazobactam have beendocumented.58,59 This phenomenon has been furtherexplored in vitro and in vivo and probably relates to thepresence of galactomannan within the drug itself.60,61 Thisfinding has forced some institutions to change theirantibacterial protocols and the FDA to issue a warning.62

(1,3)--D glucanThere has been an emergence of clinical data pertaining tothe diagnostic utility of the cell wall component, (1,3)--Dglucan.6367 (1,3)--D glucan assays have been developed byWako Pure Chemical Industries (Tokyo, Japan),Seikagaku Kogyo Corporation (Tokyo, Japan), MaruhaCorporation (Tokyo, Japan) and Associates of Cape Code(Falmouth, USA); the assay developed by Associates ofCape CodeFungitellhas been approved by the FDA inthe USA for the diagnosis of invasive fungal infections. -D glucan is present in the cell wall of most fungi; thenotable exceptions are Cryptococcus spp and thezygomycetes.67 The molecule is ubiquitous in theenvironment and has been used as a marker of fungalbiomass.68 The presence of (1,3)--D glucan in fungalspecies other than Aspergillus spp (eg, Candida spp,Fusarium spp, Acremonium spp, and Pneumocystis jiroveci)means that its role in establishing a specific diagnosis ofinvasive aspergillosis is not straightforward.

Assays to detect (1,3)--D glucan typically use serum.The common feature of all of the glucan assays is theability of (1,3)--D glucan to activate a coagulation cascadewithin amoebocytes derived from the haemolymph ofhorseshoe crabs. Horseshoe crab lysate preparations werefirst used to detect endotoxin using the limulus test orlimulus reaction (named after one type of horseshoe crab,Limulus polyphemus). Endotoxin induces clot formation viaa serine protease zymogen named factor C (figure 2).Subsequently, evidence emerged that (1,3)--D glucan-induced clot formation independently of factor C, via asecond serine protease zymogen, factor G, thus providingthe impetus for the development of the current assays.

The analytical sensitivity of the Fungitell assay is in theorder of 1 pg/mL, which is less than the cut-off of60 pg/mL used in a recent clinical study.67 A technicalconsideration pertinent to the analytical sensitivity of (1,3)--D glucan assays is that human plasma contains anumber of inhibitors of serine proteases that need to beremoved in a pretreatment step; this removal can be

achieved by an alkali reagent method (Fungitell), or by theaddition of Triton X-100 and heating to 70C for10 minutes (Wako assay). The alkali pretreatment step inthe Fungitell assay also converts triple-helix glucans intosingle-helix structures, which appear to be more reactive.Since both endotoxin and (1,3)--D glucan activate thehorseshoe crab coagulation pathway, an assay thatspecifically detects (1,3)--D glucan requires removal ofendotoxin from the specimen or the endotoxin-specificpathway from the lysate; correspondingly, endotoxin isinactivated by the addition of polymyxin in thepretreatment step in the Wako assay, while the Fungitellassay uses factor C to deplete limulus lysate. Thepretreatment step also enhances analytical specificity viathe removal of non-specific activators of serine proteasespresent in human serum.

There are no data that address the clinical sensitivity ofthe (1,3)--D glucan assays specifically for Aspergillus spp.The positive cut-off of 60 pg/mL was defined in a non-neutropenic group of patients with candidaemia.69 Theperformance of (1,3)--D glucan in the context ofantifungal therapy has not been rigorously studied. False-positive (1,3)--D glucan results have been documented inhaemodialysis, cardiopulmonary bypass, treatment withimmunoglobulin products, and exposure to glucan-containing gauze (eg, following major surgery).69

Environmental (1,3)--D glucan contamination may alsocompromise specificity.

Antibodies directed toward Aspergillus sppThe demonstration of specific antibody is required toestablish the diagnosis of chronic pulmonaryaspergillosis.69 Traditionally, antibody detection has notbeen considered useful for the diagnosis of acute invasiveaspergillosis, following an early study that failed todocument antibody formation in 15 patients with invasiveaspergillosis.70 Subsequently, antibody has beendocumented in approximately one-third of patients withinvasive aspergillosis.47,71 The detection of antibody may


Endotoxin (1,3)--D glucan

Factor C Activated factor C Activated factor G Factor G

Factor B Activated factor B

Clotting enzymeProclotting enzyme

Clotting enzyme activity detected via cleaving of synthetic chromagenicsubstrate or turbidimetric assay

Figure 2: The pathways for the activation of the amoebocyte lysate byendotoxin and (1,3)--D glucan and the use of this pathway for thedetection of (1,3)--D glucan

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prove to be the best non-invasive means of establishingthe diagnosis of subacute invasive aspergillosis in non-neutropenic patients with invasive aspergillosis, asillustrated by a recent case report describing invasivepulmonary aspergillosis in an individual with chronicgranulomatous disease.72 Furthermore, antibody detectioncould be useful as a means of establishing a retrospectivediagnosis of invasive aspergillosis in profoundlyimmunocompromised hosts who have undergoneimmunological reconstitution, although more work isrequired in this regard.

The detection of antibodyMany assay formats have been used to detect antibodies toAspergillus spp, including immunodiffusion, counterimmunoelectrophoresis, complement fixation, particle-haemagglutination, indirect-immunofluoresence, radio-immunoassay, and ELISA.73,74 The large number ofepitopes in crude extracts may compromise specificity.The use of recombinant antigenseg, dipeptidyl-peptidases,75 superoxide dismutase,75,76 catalase,75 metallo-protease,75 mitogillin,77 and galactomannoprotein71,78mayrectify this situation. One potential advantage of usingassays with a single antigen is the prospect of studyingprotective epitopes and thereby facilitating the generationof assays that may also confer prognostic information.

MetabolitesAspergillus spp produce a range of extracellular enzymes(eg, metalloproteases, phospholipases) as well asprimary (eg, mannitol)33 and secondary metabolites (eg,gliotoxin),79 all of which at least have the potential toserve as diagnostic markers for invasive aspergillosis.The ability of Aspergillus spp to produce D-mannitol hasbeen known for many years80 and its diagnostic potentialexamined in several experimental models of invasiveaspergillosis,33,81 although it is limited in terms of itsbroad applicability as a diagnostic tool because of thecomplexity of measurements, which are done by gasliquid chromatography and mass spectroscopy. Recentwork suggests that gliotoxin is produced by most Afumigatus strains and the possibility of using it as adiagnostic marker has been entertained.82 Acomprehensive summary of the various secondarymetabolites (mycotoxins) produced by Aspergillus sppcan be found at http://www.aspergillus.man.ac.uk. Thedetection of metabolites represents an under-researchedarea in terms of their possible application as diagnosticmodalities for invasive aspergillosis.

Nucleic acid testsAs far as the amplification of nucleic acid and diagnosis ofinvasive aspergillosis is concerned, PCR technology hasdominated. A limited number of publications have usedthe isothermal technique nucleic acid sequence-basedamplification.83,84 Only PCR will be discussed here. Thelack of standardisation of technical issues has and

continues to represent a considerable barrier for thewidespread application of PCR as a diagnostic modalityfor invasive aspergillosis and this is the focus of thefollowing discussion.85

Clinical specimensMany studies have addressed the detection of nucleicacid from various fractions of blood (serum, plasma,whole blood) to establish a diagnosis of invasiveaspergillosis, but PCR may also be applied to BALspecimens86,87 and tissue,88 including paraffin-embeddedsections.89,90 The optimal blood fraction for the detectionof aspergillus DNA remains unknown. One study, usingquantitative PCR (qPCR), suggested that the yield ofDNA from serum, plasma, and white cell pellet wassimilar,91 while another demonstrated that the(qualitative) PCR signal from whole blood was superiorto plasma.92 Serum has the advantage that it enablesconcomitant antigen testing91 and does not require theaddition of anticoagulants (eg, sodium citrate, edetic acid,or heparin) that may inhibit PCR.93

DNA extractionThere are a multitude of extraction techniques; theprincipal technical issues are summarised in table 1.The chosen extraction method represents acompromise between efficiency, freedom fromexogenous contamination, and applicability to routinehigh-throughput laboratories. The fungal cell wallclearly represents the major hurdle to high-efficiencyextraction of fungal DNA. DNA may be extracted usingin-house methods, commercial kits (eg, Qiagen QIAmpTissue Kit [Hilden, Germany]), and automatedcommercial techniques (eg, MagNA Pure LC [RocheDiagnostics, Basel, Switzerland). Automatedcommercial techniques are probably required to makefungal DNA detection a viable option for routine clinicallaboratories. The efficiency of extraction of fungal DNAmay vary considerably between commercial kits.94 Highspeed cell disruption incorporating chaotropic reagentsand lysing matrices provide efficient and high yields ofDNA from Aspergillus spp and other filamentous fungi.95

Fungal contamination of extraction systems andreagents has been documented.96 Considerabledifferences in DNA extraction protocols andperformance is one aspect of molecular assays thathinders the comparison of studies.

Primer targetFor clinical diagnostic purposes, the detection of a broadrange of fungi is important, as is the ability to ultimatelyidentify the specific pathogen(s). The optimal approach, inthis regard, involves the application of broad-rangingpanfungal primers with post-amplification analysis forspecies determination. Panfungal primers are directedtoward conserved regions, usually within multicopygenes, which flank sequences containing species specific


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polymorphisms that can be exploited in post-amplificationanalysis.

The ribosomal DNA (rDNA) complex is the mostcommon target. This complex contains both conservedand variable sequences and there is a large volume of datadeposited in public databases for a wide range of generaand species. The recent genome sequencing ofA fumigatus, using strain Af293, revealed 35 repeatingunits;97 the structure of the gene complex is illustrated infigure 3.98 The mitochondrial genes encoding some of thetRNA genes91 and (apo)cytochrome b99 have also been usedas primer targets. Mitochondrial targets can be consideredmulticopy because of a multiple number ofmitochondria per cell nucleus; in Af293, there were12 copies of the mitochondrial genome present for everycopy of the nuclear genome.97

Amplification formatNested PCR formats have been widely used for Aspergillusspp in an attempt to optimise analytical sensitivity, but the

requirement to open reaction tubes means that there isconsiderable risk of contamination and the subsequentgeneration of false-positive results. Real-time formats havebeen increasingly used and are likely to dominate in thenear future.

Post-amplification analysisPost-amplification detection techniques provide genusor species specific data but may also increasesensitivity and specificity.100,101 Real-time detectiontechniques (eg, TaqMan, LightCycler, molecularbeacons) are automated, rapid, and reproducible, thusfacilitating comparisons between studies. Southernblotting has had a valuable role in the evolution of PCRas a diagnostic modality, but is unlikely to have anysubstantial future role in routine clinical assays.Single-strand conformational polymorphism,102,103

restriction fragment length polymorphism digestpattern,104 Line Probes,105 fragment size deter-mination,106 and PCR-ELISA107 may have a limited role


Feature Definitions and goals Specific considerations with respect to invasive aspergillosis

Sample Sample type, volume, transport, and handling Serum and white cell pellet equivalent and possibly superior to plasma as sampleshould be defined Heparin and citrate inhibitory to PCR

Sample handling varies between studies (some have demonstrated stability at room temperature for 48 h, others recommend immediate freezing)

DNA extraction Target is of an adequate concentration and quality Extraction efficiency for fungal DNA is low due to the requirement to break the cell wallfor amplification. PCR inhibitors and DNA nucleases Contamination rate 33% in one series, commercial reagents may be contaminated with removed fungal DNA

Ideally negative and positive extraction controls should be usedRemoval of red and white cells Red cell lysis buffer and white cell lysis bufferDisruption of cell wall Enzymatic methods (eg, lyticase, zymolase)

Chemical (eg, boiling in dilute alkali) Physical (eg, glass bead milling, freeze-thawing, sonication, grinding in liquid nitrogen)

Disruption of cell membrane Usually achieved with lysis buffer (sodium dodecyl sulphate, beta-mercaptoethanol, EDTA)Precipitation of protein and purification of DNA Phenol-chloroform

Silica fibres (eg, Qiagen Tissue Kit) Capture of DNA Alcohol precipitation

Magnetic beads (eg, MagNA Pure)Silica fibres (eg, Qiagen Tissue Kit)

Amplification Nested PCR, real-time formats, PCR-ELISA represent Nested formats potentially allow for optimal analytical sensitivity but are associated withthe commonest formats contamination and are difficult to compare

Real-time formats will probably dominate in the futureAmplification controls Negative and positive controls are required

Analytical The smallest number of target organisms reliably and Multicopy target preferablesensitivity reproducibly detected by the assay Assessed by serial dilution of Aspergillus spp (conidia or purified DNA) using the appropriate

clinical specimen as the diluent Circulating DNA in invasive aspergillosis is typically less than 10 colony forming units per mL or less than 30 fgExtraction method, primer target, and detection method all influence analytical sensitivity

Analytical Does the test detect only what it purports to? Specific primer and probe sequences initially identified from public databasesspecificity Amplicon ideally should be sequenced and a BLAST search done

Cross reactivity studies with a range of fungal and bacterial pathogens as well as human DNA are requiredPost-amplification detection probe(s) may enhance specificity

Inhibition Inhibitors of DNA polymerase (eg, heparin) Spiking with purified aspergillus DNA and analysing in a separate reaction controls Spiking with a plasmid construct containing different size and sequence or label to the target

Amplifying a human housekeeping gene (eg, betaglobin, HLA2), which also allows some determination of specimen adequacy, although the relative dominance of human DNA in clinical samples may mask low levels of inhibitors which could interfere with target amplification

Contamination Uracil-D-glycolase, appropriate number of control negative controls

Table 1: Technical variables required for a robust and reproducible PCR assay

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in specific instances, such as the identification oflaboratory isolates.

Analytical sensitivity and specificityThe analytical sensitivity of a molecular assay is usuallydetermined by serial dilution of the infectious agent inpooled non-infectious clinical material as the diluent.108

Such a paradigm immediately presents a problem forAspergillus spp or any other mould, since accurate andindeed meaningful dilution of hyphae is not possible.Two commonly used approaches include serial dilutionof conidia or DNA (either purified genomic DNA or aplasmid construct), although neither are ideal; theformer does not mimic a biologically valid scenario,since hyphae rather than conidia are the invasive form,while the latter does not control for issues in extractionefficiency. If it is intended that more than one species isdetectable then DNA from those species should beincluded in the assessment of analytical sensitivity.109

The analytical sensitivity of published assays varies byseveral orders of magnitude; however, most studiesreport detection limits in the order of 110 fg DNA;variability in the detection limit is yet another issue thatcompromises study comparability.

Studies differ considerably in terms of the methods andextent to which analytical specificity is determined; there

are no standard techniques or criteria (table 2). Primertargets are generally identified by aligning sequencesretrieved from public databases. This practice should beviewed as a necessary but insufficient step in establishingthe analytical specificity of an assay and further validationprocedures are required. Ideally, relatively early in assay


IGS

5'

5'

IGS IGSITS1 ITS2

58S18SSSU

28SLSU

3'

3'

IGS

Figure 3: The structure of the ribosomal DNA complexShaded areas denote areas of variability that are present throughout the complex that can be exploited to designassays of varying levels of specificity. IGS=intergenic spacer; ITS=internal transcribed spacer; LSU=long subunit;SSU=short subunit. Adapted from reference 98.

Primer target Assay format Intended BLAST search of primer and probe sequences; isolates Method by which analytical specificity determined and result Referencespecificity with same probability match as intended target

18S rRNA PCR-ELISA Aspergillus spp Aspergillus spp, Penicillium italicum, Penicillium commune, Penicillium Cross-reactivity studies: Einsele chryosogenum, Penicillium brevicompactum, Penicillium phialosporum, Amplification of Aspergillus fumigatus, Aspergillus flavus, Aspergillus et al110

Penicillium tardum, Penicillium allii, Penicillium expansum, Ajellomyces terreus, Aspergillus niger, Aspergillus nidulans, Aspergillus versicolor, capsulatus (telemorph of Histoplasma capsulatum), Paracoccidiodes Histoplasma capsulatumbrasiliensis, Eupenicillium spp, Penicilliopsis spp No amplification of Malassezia furfur (3 strains), Fusarium spp (3 strains),

Trichosporon cutaneum (2 strains), Mucor spp (3 strains), Penicillium spp (2 strains), Pseudallescheria boydii (1 strain), Paecilomyces spp (2 strains), Saccharomyces cerevisiae (2 strains)

18S rRNA TaqMan Aspergillus spp Aspergillus spp, P italicum, Penicillium glabrum, P commune, Cross-reactivity studies: Kami P chryosogenum, P brevicompactum, P phialosporum, Penicillium Ampification of A fumigatus, A niger, A terreus, A flavus, Aspergillus oryzae et al111

purpurogenum, P tardum, Penicillium verruculosum, Penicillium hirsutum, No amplification of Candida albicans, Candida tropicalis, Candida krusei, Penicillium radicum, Penicillium funiculosum, Penicillium siamense, Candida parapsilosis, Candida glabrata, Candida guilliermondiiPenicillium pittii, Penicillium minioluteum, Penicillium pinophilum, Penicillium variabile, Penicillium rugulosum, Penicillium crateriforme, Penicillium variotii, Eupenicillium spp, and others

Mitochondrial Competitive PCR Aspergillus spp A fumigatus Cross-reactivity studies: BretagneDNA (tRNA) with PCR-ELISA Ampification of 30 isolates of A fumigatus, A niger, A terreus, A flavus et al112

No amplification of A nidulans, C albicans, C tropicalis, C krusei, C parapsilosis, C glabrata, Cryptococcus neoformans

Mitochondrial Competitive PCR A fumigatus, No database matches Amplicon sequenced: revealing A fumigatus and A flavus Bretagne DNA (tRNA) with PCR-ELISA A flavus Cross-reactivity studies: et al113

No ampification of A niger, A terreus, A nidulans, Aspergillus ustus, Penicillium purporogenum, Scopulariopsis brevicaulis

Mitochondrial LightCycler A fumigatus A fumigatus None, although clinical specificity assessed using 20 serum samples from Costa DNA (tRNA) healthy individuals et al91

Mitochondrial LightCycler A fumigatus Eupenicillium shearii, Neosartorya fischerii, A fumigatus Cross-reactivity studies: Spiess DNA (cyto- Amplification of A fumigatus, Aspergillus clavatus et al99

chrome b) No amplification of Candida spp, other Aspergillus spp, P chryosegenum, P expansum, P funiculosum, P variotii, Rhizopus oryzae, Fusarium proliferatum

The BLAST searches were done at http://www.ncbi.nlm.nih.gov, using search for short nearly exact matches. The primer and probe sequences were searched simultaneously and were separated by a string of at least ten nucleotides toensure only the specified sequences were matched in the search algorithm. Only matches identical to those of the intended target are displayed.

Table 2: Selected examples of issues in establishing the analytical specificity of PCR assays to detect Aspergillus spp

Review

development, the amplicon should be sequenced and aBLAST search done to verify that the intended target hasbeen amplified.109 Subsequently, the assay should bechallenged with organisms that have a high likelihood ofcross-reacting with the target; in the case of Aspergillusspp, genera that are close phylogenetic relationseg,Penicillium spp and Paecilomyces spp114 are especiallyimportant to consider (figure 4). A further considerationis that sequences are being continuously deposited inpublic databases; a unique sequence at the time of primerdesign may subsequently align with a sequence from anunrelated species or genus deposited at a later date. Somehave suggested that BLAST searches are done on anannual basis to ensure there is no cross reactivity withrecently submitted sequence.109 A final consideration isthat false-positive reactions due to carry-overcontamination of amplicon from previous reactions maybe prevented with the addition of uracil-D-glycolase.85

Clinical sensitivity and specificityThere are a number of factors that potentially have animpact upon the clinical sensitivity of PCR. Themagnitude of the quantitative PCR signal falls withantifungal therapy in both experimental models and inclinical contextsthis may account for false-negative PCRresults.110,111,115,116 Patients at risk for invasive aspergillosisare also often prescribed a multitude of drugs and fluids,all of which may act as non-specific inhibitors of PCR; as a

result, inhibition controls are mandatory and may take theform of spiking the sample with aspergillus DNA, aplasmid construct, or amplification of a human gene suchas betaglobin (table 1).

The application of diagnostic modalitiesLaboratory isolatesGiven the distinct differences in disease manifestations,prognosis, and antifungal susceptibility betweendifferent fungal genera and species, a rapid diagnosiswill assume increasing importance. The inherentproblems with identification using culture methods havebeen outlined. An increasing number of studies haveexamined the use of PCR to enable the accurate andrapid detection of laboratory isolates (table 3). The rapididentification of laboratory isolates using microarraytechnology with a panfungal chip is possible and nodoubt the relevant studies will emerge in the near future.

Clinical specimensThe application of diagnostic modalities to tissue,respiratory tract secretions, and blood in the context of thepathophysiology of invasive pulmonary aspergillosis isillustrated in figure 5.

Tissue and sterile fluidsHistological and culture techniques applied to tissue formthe reference diagnostic standard for invasive


005

100

100

100

100

100

58

100

100

Candida krusei

Candida lusitaniaeCandida albicans

Taphrina deformans

Aureobasidium pullulans

Histoplasma capsulatum

Blastomyces dermatitidis

Coccidioides immitis

Trichophyton rubrum

Eremascus albusAscosphaera apis

Penicilllum marneffei

Paecilomyces variotii

Peniclllium chrysogenum

Aspergillus flavusAspergillus fumigatus

Aspergillus terreus

Aspergillus nigerAspergillus nidulans

Eurotium fubrumMonascus purpureus

Neurospora crassa

Colletotrichum gloeosporiodesOphiostoma uimi

Ophiostoma stenoceras

Sporothrix schenckii

Candida viswanathiiCandida tropicalis

Schizosaccharomyces pombe

Endomyces geotrichum

Figure 4: The phylogenetic relations between Aspergillus spp and other fungi based on the 18S rRNA complexAdapted from reference 114.

Review

aspergillosis11 and have been, and continue to be, thestandard tools by which tissue invasion and destruction byhyphae is documented. Within this context, the followingpoints specifically deserve emphasis. First, difficulties inobtaining deep tissue specimens in patients who are leastable to tolerate invasive procedures have been exhaustivelydocumented and remain one of the principal factorsdriving the development of new diagnostic techniques.Second, the analytical sensitivity of both histology andculture is relatively low, meaning that invasive disease iswell established by the time that culture and histology arepositive. Third, the specificity of the reference standard forAspergillus spp is optimised with the combination ofhistological and culture data and this rigorous standardhas been used in some recent clinical trials.48,120 Theproblem, however, is that Aspergillus spp can only berecovered from tissue in the context of positive histologyin 3050% of cases.24 Finally, the possibility of accepting apositive PCR result in tissue as the reference standard forinvasive aspergillosis deserves increasing attention.Certainly, data from experimental models suggests thatvalidated PCR is more sensitive than culture for thedetection of Aspergillus spp in tissue, especially in thesetting of substantial tissue necrosis;115,121 the key in thisregard is assay validation.

Non-sterile sitesIn the absence of tissue specimens, samples obtainedfrom contiguous non-sterile siteseg, the upper andlower respiratory tractserve as a surrogate with whichto establish the diagnosis of invasive aspergillosis. Inthe case of invasive pulmonary aspergillosis, viablehyphal elements or related serological or molecularmarkers are shed into the respiratory tract from infectedparenchyma (figure 5). A body of data suggests thisshedding occurs relatively late in the natural history,thus compromising attempts to establish an earlydiagnosis using this approach.25,122,123 The isolation ofAspergillus spp (or related serological, molecular, orbiochemical markers) in the respiratory tract may

represent one of three scenarios: (1) evidence of currentdisease, (2) true colonisation, or (3) a marker for thefuture development of invasive disease. An example ofthe latter is provided by a study that demonstrated that apositive PCR result from BAL at the time of bonemarrow transplant conditioning was predictive of thesubsequent development of invasive pulmonaryaspergillosis.124

There are a number of points to make about usingBAL specimens to secure a diagnosis of invasivepulmonary aspergillosis. First, although BAL is a safeprocedure, even in patients with substantialimmunological impairment, it is not a trivialundertaking and requires a dedicated and competentbronchoscopist and an adequate commitment ofresources. Second, the overall sensitivity (using cultureand microscopy) is relatively low and generally estimatedto be in the order of 50%.122,123,125,126 Variations in BALtechnique,127 the location, size, and type of pulmonarylesions,128130 and the timing of bronchoscopy122 are allimportant determinants of the overall estimate. Theimpact of antifungal therapy in terms of the recovery ofaspergillus and related markers in the respiratory tractremains poorly defined. Third, the specificity of theisolation of Aspergillus spp from the respiratory tract inpatients with substantial immunological impairmenteg, those with allogeneic haematopoietic stem celltransplantation or neutropeniais very high,131 but forother patient groups, the likelihood of underlyinginvasive pulmonary aspergillosis varies enormously.25,131

Fourth, qPCR may prove to be especially useful indetermining the relation between the fungal burden inthe respiratory tract and the probability of underlyinginvasive disease;87,99,115 however, at the current time, thebenefit of PCR over conventional culture remains to befurther defined. Finally, the diagnostic yield from BALfluid is potentially optimised with the application ofmore than one test; a recent study demonstratedsensitivity was improved with the concomitantapplication of galactomannan and PCR.130


Specimen Target Demonstrated specificity PCR format Detection method Reference

Cultures ITS1-58S rRNA-ITS2 Aspergillus fumigatus, Aspergillus flavus, Aspergillus Conventional Sequencing of amplicon Henry et al117

terreus, Aspergillus niger, Aspergillus ustusCultures ITS1-58S rRNA-ITS2 A fumigatus, A flavus, Aspergillus nidulans, Aspergillus

versicolor Conventional Line probe Martin et al105

Cultures ITS1-58S rRNA-ITS2 A fumigatus, A flavus, A terreus, A niger, A nidulans Conventional SSCP Rath et al103

Cultures ITS1-58S rRNA-ITS2 A fumigatus Multiplex PCR Ethidium bromide Luo et al118

Cultures ITS1-58S rRNA-ITS2 A fumigatus Nested Ethidium bromide Zhao et al119

Cultures 58S rRNA-ITS2 region A fumigatus, A flavus, A terreus, A niger Conventional Automated fluorescent capillary electrophoresis (detection of different length of amplicon) Turenne et al106

Cultures 18S rRNA A fumigatus, A terreus Conventional SSCP Walsh et al102

Cultures and ITS2 Aspergillus spp and Penicillium spp, A fumigatus, A flavus, Conventional PCR-ELISA De Aguirre et al107

tissue A terreus, A niger, A nidulans, A ustus, A versicolor

SSCP=single-strand conformational polymorphism

Table 3: The use of PCR in the identification of Aspergillus spp

Review

BloodBlood sampling represents the optimal non-invasivediagnostic approach for invasive aspergillosis. Despitetheir propensity for vascular invasion, Aspergillus spp areonly very infrequently isolated from blood usingconventional culture techniques, hence the traditionaldependence on tissue specimens to secure a definitivediagnosis of invasive aspergillosis. There is an extensivebody of literature examining the diagnostic utility ofmolecular and serological techniques in blood.Galactomannan has been incorporated into diagnosticcriteria for invasive aspergillosis and the technical issuesrequired for PCR to be applied in the same manner have

been discussed. However, there remain some additionalpertinent issues. First, specific sampling strategies are yetto be systematically studiedyield is almost certainly afunction of the volume and frequency of sampling, as isthe case with blood cultures. Second, the appropriateinterpretation of a positive galactomannan or validatedPCR result in a patient at risk of invasive aspergillosis, butwithout subsequent evidence of invasive disease, remainsunclear and difficult to resolve; the most conservativeinterpretation in this context is that all single positiveresults are false-positive, but at least on occasion, suchresults may reflect true invasive disease that has abortedor is non-progressive. Third, a body of evidence suggests


Appearance of viable or non-viablehyphal elements or surrogate markers(galactomannan, DNA) in the contiguousrespiratory tract

Pathogenesis of IPA

Respiratory tract

Inhalation of conidia,germination, mucosalsurfaces breached

Blood

Dissemination tonon-contiguous sites

A B

Lung andsinus tissue Tissue

invasionanddamage

Haematogenousdissemination

Compartmental characteristicsand inter-compartmental relations

Appearance of Aspergillus sppand associated markers occurslate in the natural history of IA

Quantitative relation betweenfungal load in tissue and respiratorytract difficult to determine with culture;quantitative PCR may be useful

Positive predictive value dependson underlying disease

Destruction and invasion of tissue byhyphal elements represents the conceptualunderpinning for IA; the demonstration ofAspergillus spp in tissue is the referencestandard for IA

Sensitivity of tissue sampling may be low due to sampling error, prior antifungal therapy, fungal tissue load beneath the analytical sensitivity of histology and culture

Highest specificity achieved with combination of histology and culture

Blood cultures typically negative

Risk factors for haematogenousdissemination remain poorly defined

Clinical samples andsampling strategies

Bronchoalveolar lavageSensitivity ~50%

Sputum examinationPositive sputum culturesoccur relatively late in thenatural history

Fine needle aspiration May be more sensitive than bronchoscopy depending on the radiological pattern of disease

Complications include pneumothorax,haemoptysis, haemorrhagiccomplications, seeding of needle tract

Open biopsySensitivity compromised by infarctionand necrosisIncreased diagnostic certainty may not translate to improved patient outcome

Blood samplingBlood cultures generally not helpfulPCR and galactomannan from blood potentially usefulOptimal sampling strategies yet to berigorously defined

Figure 5: Compartmental characteristics, inter-compartmental relations, and sampling strategies as they relate to the pathogenesis of invasive pulmonaryaspergillosis(A) Hepatosplenic aspergillosis (courtesy of Damon Eisen). (B) Cerebral abscess due to Aspergillus fumigatus. IA=invasive aspergillosis.

Review

that both PCR and galactomannan may enable a specificdiagnosis to be established earlier than is possible using aconventional approach.32,49,132 Fourth, the combination ofdifferent diagnostic modalitieseg, concomitant meas-urement of galactomannan and (1,3)--D glucanis astrategy that may optimise diagnostic accuracy.63 Finally, itseems likely that both PCR and galactomannan engenderimportant prognostic information; a falling galacto-mannan titre or a positive-turning-negative PCR signal inthe context of antifungal therapy is usually associated witha successful outcome. However, at the current time,galactomannan and PCR have not been systematicallyused to guide antifungal therapy.

The incorporation of diagnostic data into managementstrategiesGalactomannan (and validated PCR) applied to blood canbe used as screening tools to further improve theidentification of patients at high risk of developinginvasive aspergillosis.133 A positive result may enable thestart of early targeted antifungal chemotherapy, whileexpensive and potentially toxic antifungal drugs can bewithheld with persistently negative results. Testing for(1,3)--D-glucan could be also be useful in this regard.When the assays are used in this manner, a positive resultshould also serve as a trigger for additional diagnosticevaluationeg, a high-resolution computed tomographyscan of the thoraxto investigate the possibility of asubclinical focus of infection. The success of galac-tomannan (and validated PCR) as a screening tool islargely dependent on the underlying prevalence ofinvasive aspergillosis, which varies according to thespecific host group and institution; thus, the requirementand extent of galactomannan screening may varyaccordingly.

An alternate diagnostic strategy is to reservegalactomannan and validated PCR for situations inwhich clinical and radiological data are suggestive ofinvasive aspergillosis; in this scenario, galactomannanand validated PCR applied to serum, and other tissuesand fluids, may enable a definitive diagnosis of invasiveaspergillosis to be secured. Although this approach doesnot facilitate early antifungal therapy, it may minimisethe use of invasive diagnostic modalities. Furthermore, amore definitive diagnosis enables the administration ofspecific anti-aspergillus therapy and would be of

considerable benefit for future diagnostic andtherapeutic research.

Future challengesInvasive aspergillosis continues to pose many challenges.From a diagnostic point of view, improving the testaccuracy remains a priority for patient care, therapeuticresearch, and future diagnostic research. The question, ofcourse, is the manner in which these improvements canbe achieved. The progressive refinement of existingtechniques and development of new diagnostic technolo-gies is clearly a priority. Substantial work remains in areasrelated to cost-effectiveness and whether patients whoundergo intensive diagnostic testing have improvedoutcome. Just as importantly, however, is the generationof a clinical environment and culture that is amenable tohigh quality diagnostic research, the provision of adequatefunding, multicentre participation, international collabo-ration, and rigorous study design.

Conflicts of interestWWH is supported by an unrestricted educational grant from Merck & Coand the Fungal Research Trust. TJW and DWD have no conflicts ofinterest to declare.

AcknowledgmentsWe thank Ruta Petraitiene for the photomicrographs in figure 1.

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