2003Real-time automated polymerase chain reaction to detect Candida albicans

MycologyReal-time automated polymerase chain reaction (PCR)to detect Candida albicans and Aspergillus fumigatus DNA in whole blood fromhigh-risk patientsTodd M.Pryce*,Ian D.Kay,Silvano Palladino,Christopher H.Heath 1Department of Microbiology and Infectious Diseases,Royal Perth Hospital,Perth,Western AustraliaReceived 27January 2003;received in revised form 20May 2003AbstractWe report the development and evaluation of a real-time PCR assay using the LightCycler instrument for the detection of C.albicans and A.fumigatus DNA in whole blood.Recently published consensus criteria for the diagnosis of invasive fungal infection (IFI)were used for all patient samples.Unique and published primer pairs were developed and assessed for sensitivity,specificity,and reproducibility to detect C.albicans and A.fumigatus DNA in samples spiked with purified DNA,and whole blood samples from 8high-risk patients and 45negative controls.The real-time assay demonstrated an analytical sensitivity of 10fg of purified C.albicans and A.fumigatus DNA and was found to be specific for each species.The standardized approach was highly reproducible and detected C.albicans and A.fumigatus DNA in two patients with proven IFI and in one patient with a possible IFI.In addition,we report for the first time the use of recently published international consensus criteria for the diagnosis of IFI in the evaluation of a mildy invasive fungal diagnostic assay.Standardized clinical criteria and a more standardized approach to detect fungal DNA in less invasive patient samples,may permit a more reliable comparison of future studies.A rapid real-time detection of fungal DNA in whole blood,combined with standard clinical markers of response,may be more useful for monitoring patients at risk of developing IFI than other diagnostic methods currently available.©2003Elsevier Inc.All rights reserved.1.IntroductionInvasive fungal infections (IFIs)with yeasts and molds have emerged as the leading cause of morbidity and mor-tality in immunosuppresed patients,particularly those un-dergoing allogeneic bone marrow transplants and patients with hematologic malignancies receiving intensive cyto-toxic chemotherapy (Denning et al.,1998).The survival of these patients depends on early diagnosis and prompt initi-ation of antifungal therapy,but conventional laboratory-based tests are rarely conclusive,because blood cultures lack sensitivity and invasive procedures are often required to obtain pathologic or microbiologic confirmation (Den-ning et al.,1998;Latge,1999).The diagnosis of IFIs istherefore often only established at autopsy (Latge,1999).Hence the development of reliable less invasive techniques to facilitate the early and reliable diagnosis of IFIs is cur-rently under active investigation.Detection of circulating fungal antigens is useful for the diagnosis of IFIs,particularly invasive aspergillosis (IA).Currently,there are a number of commercially available kits for the diagnosis of IA and candidemia:the latex aggluti-nation test to detect galactomannan (Pastorex Aspergillus and Pastorex Candida ,SanofiDiagnostics Pasteur,Marnes-La-Coquette,France),a sandwich ELISA for the detection of galactomannan (Platelia Aspergillus and Platelia Can-dida ,SanofiDiagnostics Pasteur,Marnes-La-Coquette,France),and a plasma (133)-␤-D-glucan assay (Fungitec G test,Seikagaku Kogyo Corp.,Tokyo,Japan).These tech-niques show good sensitivity and varying degrees of spec-ificity in high-risk patients (Ascioglu et al.,2002;Maertens et al.,1999),but they may only yield positive test results at advanced stages of infection.In addition,the use of high-resolution CT of the chest has an emerging role in the noninvasive diagnosis of invasive pulmonary aspergillosisPresented in part:Annual Scientific Meeting of the Australian Society for Microbiology,Perth,Australia,September 2001.1Potential conflict of interest:Dr CH Heath,is a member of the Voriconazole Advisory Board,Pfizer (Australia)Pty.Ltd.*Corresponding author.Tel.:ϩ61-8-9224-2444;fax:ϩ61-8-9224-1989.E-mail address:Todd.Pryce@.au (T.M.Pryce).Diagnostic Microbiology and Infectious Disease/locate/diagmicrobio47(2003)487–4960732-8893/03/$–see front matter ©2003Elsevier Inc.All rights reserved.doi:10.1016/S0732-8893(03)00139-1(IPA)(Caillot et al.,1997;Yeghen et al.,2000).The most characteristicfindings on CT in IPA are a halo of ground-glass attenuation around focal nodules,the“halo sign”,and the air-crescent sign.Both signs are relatively sensitive for the diagnosis of IPA in high-risk patients,but these signs can be relatively non-specific and have been reported in association with a wide range of other infective and non-infective diagnoses.In addition to these non-invasive techniques,molecular techniques such as conventional PCR show great diagnostic potential with a high degree of sensitivity and specificity (Einsele et al.,1997;Hendolin et al.,2000;Loeffler et al., 1998;Van Burik et al.,1998;Williamson et al.,2000; Yamakami et al.,1996).Many published methods for con-ventional PCR include time consuming in-house DNA ex-traction protocols,requiring the use of gel-electrophoresis or other slow amplicon detection steps.Traditional PCR-gel electrophoresis also requires post-PCR manipulation which may lead to amplicon cross contamination(Loeffler et al., 1999),leading to false positive results.The use of standard-ized DNA extraction protocols and real-time PCR may address many of the limitations of conventional PCR.Re-cently,a quantitative real-time PCR assay using the Light-Cycler(LC)instrument(Roche Molecular Diagnostics, Mannheim,Germany)has been reported to show great po-tential for the rapid in vitro amplification and detection of C. albicans and A.fumigatus DNA in spiked blood samples and patient blood samples(Loeffler et al.,2000b).The use of real-time PCR technology with the LC can easily be incorporated into the routine diagnostic microbiology labo-ratory and turnaround times for results can be dramatically reduced(Palladino et al.,2001a;Palladino2001b).Real-time PCR can also provide accurate and reproducible quan-titation of fungal DNA in blood(Kami et al.,2001;Loeffler et al.,2000b),which may be useful in addition to standard clinical markers of response,in monitoring the outcome of the patient to antifungal therapy.In this study we report the development of a new real-time PCR assay using the LC instrument with FRET and melting curve analysis to detect C.albicans and A.fumiga-tus DNA in whole blood.We also report the results of a preliminary evaluation of this assay in a number of high-risk patients.In addition we have used the recently published consensus criteria for the diagnosis of IFIs,and have there-fore standardized the diagnosis of IFIs against agreed def-initions(Ascioglu et al.,2002).The use of standardized definitions of IFIs has not been previously reported in the study of new less invasive fungal diagnostic techniques.2.Materials and methods2.1.Fungal cultures and growth conditionsThe following strains of yeasts were obtained from the American Type Culture Collection(ATCC):Candida albi-cans ATCC14053,C.parapsilosis ATCC22019,C.kruseiATCC6528,C.tropicalis ATCC750,C.glabrata ATCC2238.The molds used in this study were obtained from theRoyal Perth Hospital Mycology Culture Collection:As-pergillus fumigatus RMCC75829,A.niger RMCC75830,A.flavus RMCC75831,A.terreus RMCC75832,A.nidu-lans RMCC75833.In addition,ten clinical isolates of A.fumigatus and C.albicans were included in this study.Allfungi were grown on Sabouraud2%dextrose agar for72hat30°C.Suspensions of conidia or yeast were prepared withsterile water and were adjusted to0.5McFarland standard.The suspensions werefiltered using a10␮m syringefilter to remove clumps of cells and the number of colony formingunits(CFUs)were calculated using a counting chamber.Toavoid further growth,suspensions were adjusted withoutdelay,to1ϫ106CFU/mL in sterile water for the prepara-tion of seeded samples and standard DNA templates.2.2.DNA extraction from cultured cells(fungal DNAstandards)DNA was isolated and purified from Candida spp.orAspergillus ing the High Pure PCR Template Prep-aration Kit(Roche Molecular Biochemicals,Mannheim,Germany).Briefly,suspensions of either Aspergillusconidia or Candida cells were pelleted in a1.5mL mi-crofuge tube at3,000ϫg for5min;then resuspended in200␮L of100mM Tris buffer containing10U of lyticase (Sigma,Castle Hill,NSW,Australia).Samples were incu-bated for30min at37°C.Nucleic acids were isolated fromthe treated samples following the manufacturers instruc-tions.Purified DNA was then eluted from thefilter tube with200␮L of prewarmed(70°C)elution buffer containing10 mM Tris(pH8.5).The DNA concentration was determinedusing GeneQuant spectrophotometer(Amersham PharmaciaBiotech,Sydney,Australia).All samples were stored at Ϫ70°C until use.In addition,the lysing enzyme lyticase was assessed to ensure the enzyme preparation was free ofendogenous fungal DNA that could cause a false-positivePCR result.A10-fold concentrate of lyticase(1000U)wasextracted and stored as described above.2.3.Extraction of fungal DNA from blood samplesExtraction of nucleic acids from whole blood was per-formed with equipment dedicated to DNA extraction fromclinical samples.Whole blood samples(9-mL EDTA)wereseparated into1.5mL microfuge tubes in200␮L and1mL lots,and frozen atϪ70°C until use.Once thawed10U of lyticase was added to the200␮L whole blood samples and incubated at37°C for30min.Nucleic acids from all200␮L whole blood samples were then isolated using the High Pure PCR Template Preparation Kit(Roche Molecular Bio-chemicals)according to the manufacturers instructions.All samples were stored atϪ70°C until use.488T.M.Pryce et al./Diagnostic Microbiology and Infectious Disease47(2003)487–4962.4.Amplification and detection of fungal DNA using the LC instrument and FRETSequence data from the18S small-subunit(SSU)ribo-somal RNA genes of the following fungi was obtained from GenBank database:C.albicans,accession number M60302;C.glabrata,accession number M60311;C.parapsilosis, accession number M60307;C.krusei,accession number M60305;C.tropicalis,accession number M60308;A.fu-migatus,accession number M60300;A.niger,accession number X78538;A.flavus,accession number X78537;A. terreus,accession number X78540;and A.nidulans,acces-sion number X78539.Sequences were aligned and com-pared using GeneDoc Multiple Sequence Alignment Editor and Shading Utility Version2.6.001(Pittsburgh Supercom-puting Center(PSC),Carnegie Mellon University,Univer-sity of Pittsburgh,Pittsburgh,PA,USA)(Nicholas and Nicholas,1997).A consensus sequence was generated and was used as a basis for primer selection and development using Primer3software(Whitehead Institute/MIT Center for Genome Research,Cambridge,MA,USA)(Rozen and Skaletsky,1998).The internal probe binding site was ex-cluded from the target sequence by marking the source sequence.Different primer pairs were generated and sorted according to quality and selected pairs were chosen for use by comparison of the statistical output.A number of primers were generated including primers that have been previously published(Einsele et al.,1997;Loeffler et al.,2000b). Primers(Gibco BRL,Life Technologies,Melbourne,Aus-tralia)used in this study are shown in Table1.Oligonucle-otide F1-R was also selected by Primer3software to be an alternative to PK-R using PK-F as the forward oligonucle-otide,which gave a439bp product.2.5.Sequence dependent PCR using the LC instrumentThe LC DNA Master Hybridization Probes Kit(Roche Molecular Biochemicals)was used to detect target ampli-cons and the detection of Candida albicans and A.fumiga-tus DNA was performed in separate glass capillaries.Am-plification mixtures were transferred to the glass capillary tubes containing2␮L of10ϫLC FastStart enzyme(Roche Molecular Biochemicals)concentrate(containing Taq DNA polymerase,reaction buffer,dNTP mix with dUTP instead of dTTP,and10mM MgCl2),0.8␮L of25mM MgCl2 stock solution,and sterile PCR grade water was added to a volume of15␮L.Oligonucleotides were used at a concen-tration of0.8␮M for A.fumigatus and1.0␮M for C. albicans assay.Probes were used at concentrations of0.5␮M for A.fumigatus and0.8␮M for C.albicans.5␮L of sample DNA was added to the amplification mixture to give afinal volume of20␮L.Samples consisted of fungal DNA standards,spiked fungal DNA standards,and seeded whole blood samples,high-risk patient samples and healthy neg-ative controls.The loaded glass capillaries werefilled using the LC Carousel Centrifuge(Roche Molecular Biochemi-cals).The carousel was transferred to the LC instrument and the following program was performed:an initial denatur-ation at95°C for10min to activate the FastStart Taq polymerase,followed by a45cycle program consisting of heating to95°C at20°C/s with a5s hold,cooling to62°C at20°C/s with a15s hold and heating to72°C at20°C/s with a25s hold.Signal detection was performed at the end of the annealing step with a singlefluorescence acquisition for each capillary.Following this melting curve analysis was performed to confirm positive results.The melting point of the C.albicans and A.fumigatus probe pairs wasTable1Primers and probes used for18S SSU rRNA gene amplification for detection of Aspergillus fumigatus and Candida albicans using the LC protocolPrimers and probes Sequence and label(s)(5Ј33Ј)a MeltingTemperature(°C)dPosition f GenBankaccession no.Product size(bp)PrimersF1-Forward g ATT GGA GGG CAA GTC TGG TG59.6543-562M60300503 F1-Reverse g CCG ATC CCT AGT CGG CAT AG59.71046-1027M60300PK-Forward TGT TGC AGT TAA AAA GCT CGT AGT TG62.7607-632M60300336 PK-Reverse ATG CTT TCG CAG TAG TTA GTC TTC A60.1943-919M60300PK-Forward TGT TGC AGT TAA AAA GCT CGT AGT TG62.7607-632M60300439 F1-Reverse g CCG ATC CCT AGT CGG CAT AG59.71046-1027M60300Probes gA.fumigatus a R-TGA GGT TCC CCA GAA GGA AAG GTC CAG C-P b71.1e708-681M60300F-GTT CCC CCC ACA GCC AGT GAA GGC71.2e734-711M60300C.albicans R-TGG CGA ACC AGG ACT TTT ACT TTG A-P62.9e713-737M60302F-AGC CTT TCC TTC TGG GTA GCC ATT63.7e687-710M60302a R,LC-Red640,LightCycler Red640(Roche Molecular Biochemicals,Mannheim,Germany)b P,phosphatec F,LCfluorescein dyed Based on nearest neighbour thermodynamic theory as described by Freier et al.(1986).e Based on thermodynamic approach(TIB MOLBIOL,Berlin,Germany)f Position refers to specific nucleotide locations of the primers and probes in the A.fumigatus and C.albicans18S rRNA gene sequences.g Sequences as described by Loeffler et al.(2000b).489T.M.Pryce et al./Diagnostic Microbiology and Infectious Disease47(2003)487–496determined by cooling to55°C at20°C/s with a10s holdand raising the temperature0.2°C/s to95°C.Fluorescenceacquisition was performed continuously during the meltingcurve analysis.Finally,the capillary tubes were cooled to40°C at20°C/s with a60s hold.The LC system software(Roche Molecular Biochemicals)at the end of the run thencalculates the crossing point(C p).The C p is the number ofcycles at which signal detection crosses the threshold of thecrossing line.2.6.In vitro examination of sensitivity,specificity andreproducibility of real-time PCRTo determine the sensitivity of the real-time PCR withFRET for the detection of amplified Candida or AspergillusDNA,100pg(1ϫ104genome copies)of standard DNAwas serially diluted in water(assuming a single genomecopy or1CFU is equivalent to approximately10fg offungal DNA)(Sambrook et al.,1989).The diluted DNAstandards were measured for each primer pair listed in Table1.To determine reproducibility,1pg(1ϫ102genomecopies),and100fg(1ϫ101genome copies)of standardDNA were measured eight times for each primer pair listedin Table1.To determine specificity DNA from all fungiused in this study was also tested.To determine the effi-ciency of the DNA extraction method blood samples fromhealthy volunteers were seeded with serial dilutions(1ϫ100Ϫ1ϫ104CFU)of either conidia or yeast cells.Cells were added to fresh whole blood samples and stored at Ϫ70°C;then thawed prior to DNA extraction to duplicate the method used for clinical samples.An evaluation of theanalytical sensitivity of the PCR-FRET technique to detectfungal DNA in the presence of human DNA was alsoperformed.Samples were spiked with serial dilutions ofDNA within the same range as for seeded cells(100pg-10fg).The positive control used for all experiments consistedof1pg of purified fungal DNA(1ϫ102genome copies).To detect PCR inhibition1pg of purified fungal DNA wasadded as an internal control to DNA extracts of whole bloodsamples from healthy negative controls.2.7.Clinical samples and negative controlsAll blood samples were collected from patients and vol-unteers’using9-mL Vacuette tubes(Greiner Bio-One,Longwood,FL,USA).Samples were separated into sterile1.5-mL microfuge tubes and kept atϪ70°C for retrospec-tive analysis.To reduce the risk of contamination of sam-ples with exogenous environmental fungi allfluid transferwas performed with aerosol-resistant pipette tips(MolecularBio-Products,San Diego,CA,USA).This work was carriedout in a separate research laboratory well isolated from thediagnostic mycology laboratory.Samples from8patients athigh-risk for IFIs were collected and analyzed for the pres-ence of Candida albicans or Aspergillus fumigatus DNA.Negative controls consisted of blood samples from forty-five healthy volunteers without clinical evidence of invasive fungal disease.All samples were obtained over a six-month period and information regarding the timing of specimen handling was recorded to monitor for potential exogenous environmental fungal contamination.2.8.Statistical analysisStatistical analysis of the C p values was performed using the Pearson’s correlation coefficient.3.Results3.1.In vitro examination of sensitivity of the real-time PCR assayThe C p values of the serially diluted standard DNA were plotted against the logarithmic DNA concentration for each of the primers tested(data not shown).Good linearity was achieved for each assay tested and the correlation coeffi-cient was greater than0.98for all tests.The analytical sensitivity of each primer pair was assessed using purified DNA from C.albicans and A.fumigatus in sterile water, purified DNA from A.fumigatus or C.albicans added to whole blood extracts from healthy volunteers,and blood from healthy volunteers spiked with A.fumigatus conidia or C.albicans blastoconidia.The F1-F/F1-R and the PK-F/ F1-R oligonucleotide pairs tested demonstrated a sensitivity of10fg(1genome copy)in purified A.fumigatus and C. albicans DNA,100fg(1ϫ101genome copies)from purified DNA added to DNA extracts from healthy volun-teers and10CFU from seeded blood samples.The PK-F/ PK-R primer pair showed reduced sensitivity of100fg for purified A.fumigatus DNA.Concentrations less than10fg were unable to be detected(data not shown).3.2.In vitro examination of the specificity of the real-time PCR assayThe specificity of the probes was assessed using DNA isolated from all fungi included in the study and human DNA from whole blood extracts.The probes hybridized to their specific templates only,irrespective of which primers were used.That is,the AF1and AF2probe pair,hybridized only to DNA amplified from A.fumigatus.In addition,the probes CA1and CA2hybridized only to DNA amplified from C.albicans.The probes did not hybridize with other Candida species,Aspergillus species,or human DNA from unseeded whole blood extracts.The results are in concor-dance with other investigators using the same oligonucleo-tide probes(Loeffler et al.,2000b).In addition,nofluores-cent signals were observed when a1000U(10ϫconcentrate)of lyticase was tested,indicating the absence of contaminating A.fumigatus or C.albicans DNA in this commercial enzyme preparation.490T.M.Pryce et al./Diagnostic Microbiology and Infectious Disease47(2003)487–4963.3.In vitro examination of the reproducibility of thereal-time PCR assayReproducibility was assessed for each primer pair using 1pg and100fg amounts of purified A.fumigatus and C. albicans DNA.The coefficient of variation(CV)of the C p values for1pg and100fg of A.fumigatus DNA using the F1-F/F1-R and PK-F/F1-R primers were33.16Ϯ0.70, 33.18Ϯ0.50,and33.92Ϯ5.98,34.78Ϯ4.89(meanϮstandard deviation),respectively.The CV of the C p values for1pg and100fg of C.albicans DNA for the primers F1-F/F1-R and PK-F/F1-R were32.27Ϯ0.34,32.21Ϯ0.36,and33.92Ϯ5.98,35.05Ϯ0.56,respectively.No fluorescence signals were detected in the reagent controls.3.4.Evaluation of the PCR assay from whole blood samples from patients at high-risk of IFIsThe clinical features of the eight high-risk patients stud-ied are summarized in Table2.We used the recently pub-lished consensus criteria(Ascioglu et al.,2002)to standard-ize the diagnosis of IFIs.Three patients had proven IFIs and one patient had a possible IFI.Two patients had proven IA, one patient had candidemia with proven disseminated can-didiasis and the fourth patient had possible IA.All four patients subsequently died;three deaths were directly attrib-utable to IFIs.A patient with proven disseminated A.fu-migatus infection(Patient1,Table2)had a positive real-time PCR result,but only one day before and on the day of death from IA,respectively.The PCR assay was negative on two occasions,23and9days before death,respectively. The real-timefluorescence output from the LC instrument of this patients’samples and controls using the PK-F and F1-R oligonucleotide primer pair and the A.fumigatus probes is shown in Fig.1A.A patient with proven dissem-inated A.terreus infection(Patient2;Table2)did not have a positive real-time PCR result.The third patient with a positive A.fumigatus real-time PCR result was at high-risk of an IFI because of aggressive cytotoxic chemotherapy and underlying acute myeloid leukemia,and she had possible IA (Patient3,Table2).The patient with proven disseminated C.albicans infection and candidemia had a positive real-time PCR result for C.albicans DNA,but only one day before death.In this case,no blood samples were collected earlier than one day prior to death.Analysis from the F2 channel of the LC instrument showingfluorescence output from this patients’samples and controls using the PK-F and F1-R oligonucleotide primer pair and for the C.albicans probes is shown in Fig.1B.The remaining four patients at high-risk of IFI did not develop clinical or laboratory evidence of invasive fungal disease;two died of complications relating to their primary disease.They had negative PCR assays for A.fumigatus and C.albicans DNA and their deaths were not attributable to IFIs.However neither patient had a postmortem examina-tion to unequivocally prove that they were free of IFIs prior to death.The two remaining patients survived,but had whole blood samples that were PCR positive for A.fumiga-tus DNA.In these cases there was no clinical or laboratory evidence of IA,although these patients were at high-risk of an IFI.Thefirst patient had profound immunosuppression following cardiac transplantation and had active invasive CMV disease at the time the sample was collected,but did not receive antifungal prophylaxis or therapy.The second patient had prolonged and profound immunosuppression while receiving treatment for acute lymphocytic leukemia and had received antifungal prophylaxis withfluconazole (200mg/day).At the time the sample was collected the patient was on empiric therapy with i.v.amphotericin B for febrile neutropenia non-responsive to broad-spectrum anti-biotics.Neither of these patients went on to subsequently develop clinical or laboratory evidence of IFIs and both patients currently remain well.In both these cases it is not possible to distinguish between a false positive result or fungal DNAemia that was subclinical.The second patient may also have responded to antifungal treatment without a specific focus of infection having been identified.3.5.Evaluation of the PCR assay from whole blood samples from negative controlsAll whole blood samples taken from forty-five healthy healthy controls had negative PCR results following the protocol described in the methods.4.DiscussionIFIs are life-threatening infections and most patients who succumb to invasive aspergillosis and disseminated candi-diasis have an underlying immunosuppressive disease.In most cases the diagnosis of these infections is often just before death or at autopsy because current laboratory-based tests lack sensitivity and specificity(Hopfer,1997).Blood cultures have poor sensitivity for the diagnosis of invasive fungal infections because fungemia is thought to occur intermittently with low numbers of viable fungal cells(Ͻ10 CFU/mL)circulating in the peripheral blood(Hopfer, 1997).Failure of blood cultures to detect circulating yeasts and molds may either be due to the inability of the fungi to grow or an inability for growth to be detected by automated blood culture systems.We therefore sought to evaluate a new PCR assay utilizing FRET and melting point analysis using the LC instrument for the early detection C.albicans and/or A.fumigatus DNA from whole blood samples of patients at high-risk of IFIs.The real-time PCR assay targets C.albicans and A. fumigatus DNA in whole blood,as these two fungi are responsible for the majority of IFIs,particularly in those patients undergoing bone-marrow transplantation or aggres-sive cytotoxic chemotherapy with prolonged neutropenia. Fungal PCR on blood samples appears to be very sensitive,491T.M.Pryce et al./Diagnostic Microbiology and Infectious Disease47(2003)487–496Fig.1.(A)Real-time fluorescence output of patient samples and controls with A.fumigatus probes using the PK-F and F1-R oligonucleotide pair.The ampli fication plot was generated using the LC instrument and the LC software version 3.5.The x axis is the number of cycles and the y axis is the fluorescence output at 640nm (F2channel).The samples and C p values are as follows:Positive control (1pg A.fumigatus DNA)(open circles),35.50;Positive control duplicate (1pg A.fumigatus DNA)(solid circles),35.48;Patient 1sample C (open squares),38.79;Patient 1sample C duplicate (solid squares),36.38;Patient 1sample D (large cross),42.10;Patient 1sample D duplicate (small cross),41.92;Negative control (sterile double-distilled water)(solid line).(B)Real-time fluorescence output of patient samples and controls with C.albicans probes using the PK-F and F1-R oligonucleotide pair.The ampli fication plot was generated using the LC instrument and the LC software version 3.5.The x axis is the number of cycles and the y axis is the fluorescence output at 640nm (F2channel).The samples and C p values are as follows:Positive control (1pg C.albicans DNA)(small cross),33.10;Patient 3sample A (solid circles),36.19;Patient sample A with internal positive control (1pg C.albicans DNA)(solid squares),32.89;Patient 3sample B (open squares),38.00;Patient 3sample B with internal positive control (1pg C.albicans DNA)(open circles),33.12;Negative control (sterile double-distilled water)(large cross).493T.M.Pryce et al./Diagnostic Microbiology and Infectious Disease 47(2003)487–496possibly because it depends on the degree of angioinvasion, and therefore its negative predictive value is high.Fungal PCR has been performed on whole blood(Einsele et al., 1997;Lass-Florl et al.,2001;Loeffler et al.,2000a;Loeffler et al.,2000b),serum(Williamson et al.,2000;Yamakami et al.,1996),and plasma(Loeffler et al.,2000a).Comparisons between plasma and whole blood have been reported for the detection of Aspergillus spp.DNA(Loeffler et al.,2000b) which suggest that whole blood may be preferable to both serum or plasma.However,a study using the rabbit model of systemic candidiasis has suggested that serum may be preferable to whole blood(Bougnoux et al.,1999).There were however a number of limitations in this animal model study that may not be applicable to the dynamics of fungal DNA for fungal cells in the immunosuppresed human host. Whether serum would be more useful than whole blood in immunosuppressed patients remains unanswered.There-fore,although the optimal specimen in the immunosup-pressed human host remains controversial,we chose to use EDTA-anti-coagulated whole blood for analysis,as an ali-quot of this specimen contains viable and non-viable fungal cells and free fungal DNA if present.Although the use of whole blood samples may increase PCR inhibition com-pared with serum samples,we demonstrated no evidence of PCR inhibition in our study,suggesting successful removal of PCR inhibitory substances from whole blood.In previously published studies on fungal PCR,a diver-sity of extraction methods,amplification protocols,sample volume,and primer selection,together with a range of source material studied,makes direct comparison of pub-lished studies difficult.To address this we chose a DNA extraction method based on elution of nucleic acids from fiberfleece of commercial spin columns.The use of com-mercial buffers and reagents ensures a certain degree of standardization that may make future comparisons between different PCR protocols more reliable.In our study we used a volume of200␮L of whole blood,although a larger volume could further improve the assays sensitivity.How-ever,there is a lack of suitable commercial methods of DNA extraction available for larger volumes of blood that meet the stringent degree of sterility or purity of buffers and reagents that is required to perform fungal PCR.In addition, improved reproducibility of conventional fungal PCR meth-ods is needed.In our study,the crossing points and the signal intensity results from seeded and spiked whole blood samples highly reproducible following the approach de-scribed.Other workers using real-time PCR techniques (Kami et al.,2001;Loeffler et al.,2000b)have also shown improved reproducibility,when compared with conven-tional fungal PCR assays.Finally we standardized the di-agnosis of IFIs by using the recently published agreed criteria,as evaluation of clinical studies have shown a poor correlation(Kappa statisticϭ0.253)between studies before 1997,even when only proven cases of IFIs are considered (Ascioglu et al.,2001).The use of standardized criteria for the evaluation of new less invasive fungal diagnostic tech-niques has not been previously been reported.The SSU ribosomal RNA genes of A.fumigatus and C.albicans are considered conserved multicopy genes thatshould enable increased analytical sensitivity.We found ananalytical sensitivity of10fg(1genome copy)for both theA.fumigatus and C.albicans assays,when purified DNA ofthese fungi were analyzed.The lower level of detection ofmost published fungal molecular reports varies from10-100genome copies,with few reports obtaining a sensitivity of1genome copy.In our study we used a200␮L of volume of whole blood,though the spin columns can utilize up to300␮L of whole blood.Aliquots greater than this amount can-not be processed unless multiple fractions are passed thoughthe same column and may increases the risk of exogenousDNA contamination.Thefiberfleece may also becomesaturated with human cellular DNA.Based on these limita-tions,if a whole blood sample contains5CFU/ml,then thefinal50␮L eluate from the spin column may only contain a single genome copy.If only5␮L of eluate is then used in the PCR master mix,the likelihood of the master mix containing a single genome copy,based on a100%effi-ciency of DNA extraction,is only10%.In addition,extrac-tion of fungal DNA may not be100%efficient,as not all human cellular DNA can be removed.Target DNA purified from clinical samples often co-precipitates with human DNA,which leads to nonspecific hybridization of oligonu-cleotides and mis-priming that may interfere with optimal PCR kinetics.Therefore the likelihood of fungal PCR prod-ucts being formed may be significantly less than10%. Hopefully further advances in fungal DNA extraction and purification will help resolve these problems.Probe hybridization with two probes each specific for A.fumigatus and C.albicans DNA were used in this study asthis enables a higher degree of specificity than genus-spe-cific or panfungal probes(Van Burik et al.,1998).Anotheradvantage of our approach is that additional probes can bedeveloped to improve the range of detectable fungal patho-gens.Panfungal probes are more likely to give false-positiveresults due to exogenous environmental fungal DNA orcarry over amplicon contamination.In addition,broad-range primers may compromise the PCR with amplificationof exogenous fungal DNA in addition to target fungal DNA.Contamination of fungal PCR assays has been documentedwith fungi including Aspergillus fumigatus,Saccharomycescerevisiae and Acremonium spp.(Loeffler et al.,1999).Theputative sources of exogenous fungal DNA include envi-ronmental air-borne fungal spores,and commercial prod-ucts commonly used for fungal DNA extraction includingPCR buffers or enzymes,such as zymolase and lyticase(Loeffler et al.,1999).Lyticase(Sigma)is a partially puri-fied␥-irradiated powder from a culture of Arthrobacter luteus(Scott and Schekman,1980).Loeffler et al.(1999) demonstrated contaminating fungal DNA with a band seen on gel electrophoresis from5U of lyticase,however they could not demonstrate contaminating fungal DNA in the494T.M.Pryce et al./Diagnostic Microbiology and Infectious Disease47(2003)487–496。