Detection of Candida species associated with Candida vaginitis by real-time PCR and pyrosequencing

Detection and identification of Candida species associated with Candidavaginitis by real-time PCR and pyrosequencingJason P.Trama,Eli Mordechai,Martin E.Adelson *Medical Diagnostic Laboratories,L.L.C,2439Kuser Road,Hamilton,NJ 08690,USAAccepted for publication 22October 2004AbstractReal-time polymerase chain reaction (PCR)is currently considered the most sensitive method to detect low abundance DNA of pathogens in clinical samples.Furthermore,obtaining DNA sequence is the ‘gold standard’of precise molecular detection.Here we combine species-specific real-time PCR and pyrosequencing to rapidly amplify and sequence ribosomal DNA from Candida albicans ,Candida glabrata ,Candida parapsilosis ,and Candida tropicalis ,which are commonly associated with candida vaginitis (CV).A standard curve was developed from plasmids containing the target DNA for each of the Candida species.A minimum real-time PCR and pyrosequencing detection limit of 100copies per reaction was achieved.The combined technique was applied to the identification of the four Candida species in DNA extracts from vaginal samples.The results from 231samples were compared with conventional PCR methods of identification.The results of both methods agreed on all but two samples,which were determined by both methods to contain C.albicans ,but real-time PCR and pyrosequencing identified a second species that went undetected by conventional PCR.This is the first application of real-time PCR and pyrosequencing to DNA from vaginal samples for identification of four Candida species associated with CV,without the need for time-consuming culture methods.q 2004Elsevier Ltd.All rights reserved.Keywords:Candida;Vaginitis;Gynecology;Real-time PCR;Pyrosequencing1.IntroductionVaginal candidiasis causes 20–25%of infectious vagi-nitis cases,second only to the 40–50%of cases caused by bacterial vaginosis [1].Candida vaginitis (CV)is marked by pruritis,soreness,a change in discharge,dyspareunia,vulvar erythema,edema,and fissures [1,2].The condition is rare before puberty,but by the age of 25,nearly one-half of all women will have had at least one clinician-diagnosed episode of CV.Overall,it is estimated that 75%of all women will experience an episode of CV in their lifetime [1,3].Among the Candida species causing infections,Candida albicans ,Candida glabrata ,Candida parapsilosis ,and Candida tropicalis account for 80–90%of fungal isolates encountered worldwide [4,5].Although C.albicans is implicated in 85–95%of all cases of CV [1,6],the widespread use of azole antifungal drugs is postulated to have promoted the shifting of vaginal colonization and selection of more naturally resistant species,such as C.glabrata [7–10].The 2004guidelines for the treatment of candidiasis put forth by P.G.Pappas,et al.state that knowledge of the infecting species is highly predictive of likely drug susceptibility and that this information should be used as a guide for selecting therapy [11].Therefore,rapid and specific identification of Candida species will facilitate appropriate antifungal selection and improve patient monly,Candida in vaginal samples is identified by microscopic examination of a wet mount with potassium hydroxide (KOH,amine test).This technique detects budding yeast cells in only 50–70%of women with CV [12,13]and may fail to detect non-albicans species [14].Alternatively, C.albicans and C.tropicalis can be distinguished by growth on chromogenic agar medium and other Candida spp.can be identified by enzymatic tests.However,each of these tests requires isolated organisms to0890-8508/$-see front matter q 2004Elsevier Ltd.All rights reserved.doi:10.1016/j.mcp.2004.10.004Molecular and Cellular Probes 19(2005)145–152/locate/ymcpr*Corresponding author.Tel.:C 16095701015;fax:C 16095701030.E-mail address:madelson@ (M.E.Adelson).be grown on solid medium for24–48h before they can be performed or interpreted[15,16].In addition,the‘gold standard’for definitive biochemical identification requires analysis of assimilation and fermentation,taking up to30 days to complete[16].In recent years,numerous DNA-based techniques have been developed to improve the identification of Candida species.PCR amplification of Candida target DNA[17–22] is particularly promising because of its simplicity,speci-ficity,and sensitivity.However,these strategies require post-amplification analyses and are considered to have lower sensitivity than real-time PCR techniques that directly monitor amplification viafluorescent probes[23].Real-time PCR strategies have been developed to identify Candida species[24–27],but these methods were designed and optimized for detection of Candida in blood or blood culture.Strategies for the detection of Candida species in DNA extracted from vaginal samples,especially without time-consuming culture,are lacking.In addition,current DNA-based Candida detection methods do not take into account the fact that DNA sequencing is generally accepted as the most precise method for discriminating among closely related species.The purpose of this study was to develop a rapid method for the detection of four Candida species associated with CV in DNA extracted directly from vaginal samples.The detection method combines the sensitivity and specificity of real-time PCR with the unambiguous species identification and validation capability of DNA sequencing by pyrose-quencing,an established bioluminometric technique that employs a cascade of coupled enzymatic reactions to monitor DNA synthesis[28].2.Materials and Methods2.1.Clinical samples and DNA extractionA total of231vaginal samples from female subjects were tested.The subjects’symptoms,HIV status,and clinician diagnoses were not disclosed.Patient care providers collected specimens from a vaginal sampling using a Cellmatics swab(BD,Sparks,MD),which was then placed in2ml of its accompanying transport medium.Upon receipt,swabs were immediately processed for PCR analysis.Established procedures for SDS/proteinase K lysis and phenol/chloroform DNA extraction[29]from 470m l of swab transport media were used.DNA concen-tration was calculated by absorbance260/280readings and was adjusted to0.2m g/m l prior to PCR analysis.2.2.Conventional PCR assayThe primers utilized for species-specific amplification of Candida ribosomal DNA and reaction conditions were previously described[18].All PCR reactions were carried out with1m g of extracted DNA in50m l total volume and were analyzed by electrophoresis through a2%agarose gel containing0.5m g/ml ethidium bromide.Positive controls consisted of DNA extracted from C.albicans,C.glabrata, C.parapsilosis,and C.tropicalis purchased from ATCC. Negative controls consisted of the substitution of nuclease and pyrogen-free water for DNA.100%specificity and 100%sensitivity of these PCR amplifications was pre-viously reported[18].Additionally,PCR amplifications with each primer pair exhibited no cross-reactivity among the four Candida species or a panel of genomic DNA extracted from34different bacterial,viral,and fungal pathogens(data not shown).2.3.Primer andfluorescent probe designAll primers,probes,and modifications were synthesized by Integrated DNA Technologies(IDT,Coralville,IA). Sequences for the internal transcribed spacer2(ITS2) regionflanked by the5.8S and28S rDNAs of C.albicans,C. glabrata,C.parapsilosis,C.tropicalis,and Saccharomyces cerevisiae(GenBank accession numbers L07796, AF218994,L11352,L47112,and AJ275936,respectively) were aligned using MegAlign version 5.51software (Lasergene suite,DNASTAR Inc.,Madison,WI).The sequence of the real-time PCR and pyrosequencing primers and probes are listed in Table1.Successful amplification of C.albicans,C.glabrata,C.parapsilosis,or C.tropicalis resulted in a261,298,251,or249bp product,respectively (Fig.1).Sequencing primers were selected within the regions amplified by the PCR primers for each of the four Candida species.2.4.Positive control plasmidsPositive controls for each Candida species were generated by subcloning amplicons derived from using the ITS3and ITS4universal fungal primer pair[30]and template DNA extracted from C.albicans,C.glabrata,C. parapsilosis,and C.tropicalis ATCC-purchased controls. Amplicons were subcloned into the pCRII-TOPO vector of the TOPO TA Cloning Dual Promoter kit(Invitrogen, Carlsbad,CA)according to the manufacturer’s instructions. DNA concentration was calculated by260/280absorbance readings.2.5.Real-time PCR assayEach25m l reaction contained0.5m g of extracted DNA, 300nM each of bio-ITS3,CA-SHIN,CG-JPT2L,CP-SHIN, and CT-SHIN,200nM of CANFAM,and12.5m l of2X concentration Platinum Quantitative PCR Supermix-UDG (Invitrogen,Carlsbad,CA).The real-time PCR reactions were performed on a Rotor-Gene3000instrument(Corbett Research,Sydney,Australia)and included an initial incubation at508C for2min followed by958C for2min.J.P.Trama et al./Molecular and Cellular Probes19(2005)145–152 146Next,45cycles of denaturation (958C,20s)and annea-ling/extension (608C,60s)were performed with fluor-escence acquisition (470nM source/510nM detection)immediately following each annealing/extension step.A final extension (728C,10min)was performed.Positive controls consisted of positive control plasmid DNA at 106,104,and 102copies per reaction.Negative controls consisted of the substitution of nuclease and pyrogen-free water for DNA.Normalized fluorescence was analyzed on the Rotor-Gene 3000Software,Version 5(Build 47)with dynamic tube normalization and slope correction.2.6.PyrosequencingFor PCR product purification prior to pyrosequencing analysis,the bio-ITS3primer (Table 1)was synthesized with a 50biotin modification which was incorporated into the amplicon during the amplification process.The biotinylated PCR product was captured with streptavidin Sephadex (Amersham Biosciences,Uppsala,Sweden),then purified and denatured with a vacuum prep workstation according to the manufacturer’s instructions (Biotage,Uppsala,Sweden).For the pyrosequencing reaction,0.5m M of each sequencing primer in the sequencing primer pool (Table 1)was utilized to prime the biotinylated amplification products.A pyrosequencing 96MA System(Biotage,Uppsala,Sweden)was programmed with 10cycles of an AGCT dispensation order.The resulting pyrosequencing data,or pyrograms,were analyzed with the PSQ 96MA version 2.0.2software.The best quality DNA sequence resolved was used in subsequent analyses.3.Results3.1.Analysis of real-time PCR conditionsTo confirm amplification quality,the real-time PCR products generated from 5!106copies of the positive control plasmid,10ng of DNA extracted from an isolate purchased from ATCC,and 0.5m g of DNA extracted from a positive vaginal sample (confirmed by conventional PCR)of each Candida species were subjected to agarose gel electrophoresis (Fig.1).The product generated from each template type was a single band of the expected size and lacked the formation of any primer dimers.This indicates the ability of the real-time PCR to efficiently amplify a specific target not only from the positive control plasmids,but also from more complex DNAs (isolated Candida genomic DNA)and mixtures of complex DNAs (DNA extracted from a vaginal sample).To further validate the specificity of the real-time PCR,DNA was extracted fromTable 1Primers and probes for Candida real-time PCR and pyrosequencing Method Function Name Sequence (50to 30)Real-time PCRPrimerBio-ITS3a Bio b /GCA TCG ATG AAG AAC GCA GC CA-SHIN c GGA CGT TAC CGC CGC AAG CAA TCG-JPT2L CCG AGT TGG TAA AAC CTA ATA CAG TAT TAA C CP-SHIN c TGG AAG AAG TTT TGG AGT TTG TAC C CT-SHIN c GGC CAC TAG CAA AAT AAG CGT TTT GProbe CANFAM c 6-FAM d /AAA YGA CGC TCA AAC AGG CAT GCC C/BHQ1e PyrosequencingPrimerCA-MOD ACG TTA CCG CCG CAA GCA AT CG-seq1CGA GTT GGT AAA ACC TAA TACP-SHIN c TGG AAG AAG TTT TGG AGT TTG TAC C CT-MODGGC CAC TAG CAA AAT AAG CGTa Redesigned from a previously described primer [30].b 50Biotin modification.c Redesigned from a previously described primer [26].d 506-Carboxy-fluorescein modification.e30Black Hole Quencher 1modification.Fig.1.Agarose gel electrophoresis of real-time PCR products.The end products from real-time PCR amplification with 5!106copies of the positive control plasmid (plas),10ng of DNA extracted from an isolate purchased from ATCC (gen),0.5m g of DNA extracted from a positive vaginal sample (confirmed by conventional PCR,clin)of each Candida species (CA,C.albicans ;CG,C.glabrata ;CP,C.parapsilosis ;CT,C.tropicalis ),0.5m g of DNA extracted from a negative vaginal sample (confirmed by conventional PCR,neg),and nuclease and pyrogen-free water (NTC)as a template were analyzed on a 2%agarose gel containing 0.5m g/ml ethidium bromide.J.P.Trama et al./Molecular and Cellular Probes 19(2005)145–15214742potentially cross-reacting human pathogens of bacterial,viral,and fungal origin,including potentially cross-reacting Candida ,Aspergillus ,and Saccharomyces species pur-chased from ATCC.Two hundred nanograms of genomic DNA from each pathogen were examined under test conditions for cross-reactivity and none was observed (data not shown).To determine the sensitivity of the real-time PCR for the target,the positive control plasmid was 10-fold serially diluted from 108to 10copies and each dilution addedasFig.2.Positive control plasmid DNA standard curve for real-time PCR.A,C,E,and G:Real-time PCR amplification curves from duplicate 10-fold dilutions of the positive control plasmid (108to 102or 10copies per reaction,from left to right)containing the target for C.albicans ,C.glabrata ,C.parapsilosis ,and C.tropicalis ,respectively.B,D,F,and H:The corresponding standard curves for C.albicans ,C.glabrata ,C.parapsilosis ,and C.tropicalis ,respectively.J.P.Trama et al./Molecular and Cellular Probes 19(2005)145–152148a template to duplicate PCR reactions(Fig.2).The linear detection range for the C.albicans, C.glabrata,and C.parapsilosis plasmids was from108to100copies per reaction with r2values of0.995,0.995,and0.998, respectively.The linear detection range for the C.tropicalis plasmid was from108to10copies per reaction with an r2 value of0.996.To verify that components of a clinical vaginal sample DNA extraction do not alter the efficiency of detection,0.5m g of a Candida-negative DNA extract (confirmed by conventional PCR)were added to PCR reactions of each positive control plasmid dilution series.In their respective linear ranges,no significant difference in C T score was apparent between the presence and absence of the vaginal DNA extract(data not shown).3.2.Analysis of pyrosequencing conditionsTo determine the ability of the primer pool to specifically discriminate among the four Candida species,0.2m g of genomic DNA extracts from C.albicans, C.glabrata, C.parapsilosis,C.tropicalis,C.krusei,and S.cerevisiae isolates purchased from ATCC were amplified in separate real-time PCR reactions using the PCR primer and probe pool(Table1).The products of each PCR reaction were then sequenced using the pyrosequencing primer pool (Table1).Sequences obtained from C.albicans, C. glabrata,C.parapsilosis,and C.tropicalis were identical to the expected sequences shown in Table2,except for the stretch of6A’s in C.parapsilosis,which was occasionally resolved as5A’s.The resolution of homopolymeric stretches is a known limitation of the pyrosequencing technique[28].No readable sequence was obtained from C. krusei or S.cerevisiae.To determine the minimum copy number initially present in a PCR reaction necessary to generate readable sequence,PCR products of each positive control plasmid dilution series(as in Fig.2)were sequenced with the pyrosequencing primer pool.Readable sequence was obtained for all four species with as few as100copies of positive control plasmid initially present in the PCR reaction(data not shown).The lengths of the best quality sequences were significantly shorter than those from purified genomic DNA,but provided enough sequence (C.albicans,15nucleotides;C.glabrata,18nucleotides;C.parapsilosis,eight nucleotides;and C.tropicalis,12 nucleotides)to differentiate the four Candida species by identity to the expected sequences.3.3.Clinical application of the real-time PCRand pyrosequencing assay.To access the quality of the real-time PCR and pyrosequencing method for identifying Candida species from clinical samples,DNA extracts from231vaginal samples were analyzed.Typical real-time PCR(Fig.3A) and pyrosequencing results(Fig.3B–F)from clinical samples were similar to the results obtained from genomic DNA isolated from purified Candida isolates.The lengths of the best quality sequences resolved from the pyrograms of clinical samples were usually shorter than those from purified genomic DNA,but provided enough sequence to differentiate the four Candida species by identity to the expected sequences.To assist in rapid speciation,sequencing primers and the nucleotide dispensation order were chosen to provide easily identifiable pyrogram patterns within thefirst dispensation cycle.As expected from Table2,each sequence generated species-specific peaks within thefirst cycle of four nucleotide dispensations:C.albicans is identified by a G and T peak(Fig.3B),C.glabrata is identified by a C peak (Fig.3C),C.parapsilosis is identified by an A and T peak (Fig.3D),and C.tropicalis is identified by a T peak(Fig. 3E).As shown in Fig.3F,it is also possible to resolve the sequences of two Candida species present in a clinical sample by inspection of the pyrogram.The A,G,and T peaks in thefirst cycle of four nucleotide dispensations positively identify a combination of C.albicans and C.parapsilosis.As shown in Table2,a combination of C.albicans and C.tropicalis is not as easily identified in the first dispensation cycle due to similarities with C.albicans. However,a T peak in the third dispensation cycleTable2Expected DNA sequences for pyrosequencing identification of Candida speciesSpecies DNA sequence(reverse complement)GenBank accession no.C.albicans GT5/G2T2/AG/AC2T/A2GC2/AT2/GT/C/A3GC/G L07796C.glabrata C/AGT/AT2/A2C5/GC2/GCT/C/GC/GC/A2C AF218994C.parapsilosis A2T/G/AGT/G2/A6C2T/AT/C2/AT2/AGT3/AT L11352C.tropicalis T3/G2/AT/A3C2T/A2GT/C/GCT2/A4T/A2GT3/C2L47112C.albicans C C.glabrata GCT5/AG3T3/A2GT2/A3C7T/A2G2C4/AGCT3/GCT/GC2/A3G2C2/A3GCC.albicans C C.parapsilosis A2GT6/G3T2/A2G2T/AG2C2T/A8GC4T/A2T3/GC2T/ACT2/A4G2CT3/AGTC.albicans C C.tropicalis GT8/G4T2/A2GT/A4C4T2/A4G2C2T/ACT2/G2CT3/A4CT/A5G2CT3/GC2C.glabrata C C.parapsilosis A2CT/AG2T/A2GT3/A2G2C5/A6GC4T/AGCT2/C3/AGCT2/AG2CT3/A4CTC.glabrata C C.tropicalis CT3/AG3T/A2T3/A5C7T/A2G2C2T/GC2T/GC2T2/A4GCT/A2G2CT3/A3C3C.parapsilosis C C.tropicalis A2T4/G3/A2GT2/A3G2C2T/A8GC2T2/ACT/GC3T2/A5T3/A3G2T6/AC2TSubscript numbers indicate the number of repeats of the preceding nucleotide in the expected sequence.Slashes divide the expected sequence by pyrosequencing AGCT nucleotide dispensation cycles.J.P.Trama et al./Molecular and Cellular Probes19(2005)145–152149(dispensation 12),which is absent in C.albicans ,positively identifies a combination of C.albicans and C.tropicalis .To access the specificity and sensitivity of the real-time PCR and pyrosequencing method for identifying Candida species from clinical samples,the results were compared to those obtained from conventional PCR identification of C.albicans ,C.glabrata ,C.parapsilosis ,and C.tropicalis .The two PCR-based methods amplify different regions of the rDNA.As shown in Table 3,the real-time PCR method generated no false negatives or false positives with respect to the absence (96/96)or presence of DNA from any of the four Candida species (135/135).When speciated by pyrosequencing,results from 133of the 135positive samples agreed.The two discordant samples were found to contain C.albicans by both identification methods,but the sequencing data obtained from pyrosequencing unam-biguously identified a second Candida species.One sample contained both C.albicans and C.parapsilosis (Fig.3F)Fig.3.Real-time PCR and pyrosequencing identification of Candida species in DNA extracted from vaginal samples.A:Representative real-time PCR amplification curves from reactions using DNA extracted from clinical samples,with C.albicans positive control plasmid standards (dashed lines;106,104,and 102copies per reaction,from left to right).B–F:Representative pyrograms and resolved sequence (dispensations boxed and corresponding sequence shown below)for C.albicans ,C.glabrata ,C.parapsilosis ,C.tropicalis ,and a combination of C.albicans and C.parapsilosis (with sequence from C.albicans in bold and C.parapsilosis in italics),respectively.J.P.Trama et al./Molecular and Cellular Probes 19(2005)145–152150and the other contained both C.albicans and C.tropicalis.A separate species-specific real-time PCR[26]confirmed the presence of the second species in both samples and agreed with the pyrosequencing results(data not shown).4.Discussion and conclusionsThe development of a rapid,sensitive,and unambiguous method for identification of Candida species for aiding diagnosis and treatment of CV is critical mainly because of species-specific differences in susceptibility to antifungal drugs.To meet this need,the real-time PCR and pyrosequencing method described here is rapid,taking only approximately2h to complete(10min for real-time PCR preparation,75min real-time PCR run-time,15min pyrosequencing preparation,and20min minimum for pyrosequencing run-time and analysis)after DNA is extracted directly from a vaginal sample with no need for culturing or isolation of Candida.The method is also sensitive and specific,being able to detect as few as100 copies of a plasmid control,showing no cross-reactivity to closely related fungi,and generating no false positive or false negative results when compared to conventional PCR methods.Finally,precise identification of four common Candida species associated with CV is accomplished using the most accepted data for reliable differentiation of closely related species,the DNA sequence.The simultaneous detection of multiple Candida species by the described real-time PCR method also raises an inherent limitation to this particular assay. When multiple Candida species are present in a single clinical sample,all of them will be amplified,which has no great effect on qualitative analysis,but many-fold differences in the amount of the target from each species may lead to the preferential amplification of the target in excess.Poor amplification of the lesser target may not generate enough material to meet the threshold for sequence identification by pyrosequencing,therefore potentially allowing the Candida species in lesser quantity to escape detection.This phenomenon may result from the shared forward amplification primer(bio-ITS3),the shared probe(CANFAM),and perhaps other reagents.The development of distinct sets of primers and probes toward each of the four Candida species was avoided,however,to minimize potential cross-reactivity inherent to the incorporation of eight primers and four probes into a single reaction.The use of real-time PCR and the pyrosequencing platform make this Candida identification method amenable to additions and improvements.For example,C.krusei is a rare but important cause of CV[31]and its detection is becoming increasingly important because of its natural resistance tofluconazole[7,31–33].Currently,amplification and sequencing primers are being developed to include the unambiguous detection of C.krusei.In addition,sequence analysis can be automated by linking sequence output from the pyrosequencing software to a searchable database of expected sequence,but given the limitations in resolving homopolymeric nucleotide stretches,another possible alternative is pyrogram pattern recognition.The sequences used to identify the four Candida species and all possible combinations of two species in as few as13nucleotide dispensations using a cyclic AGCT dispensation order was designed to generate easily recognized pyrogram patterns. However,identification by pattern recognition must be performed manually.Given the fact that pyrosequencing utilizes a96well format permitting the simultaneous screening of a large number of specimens,the development of a pyrogram pattern recognition algorithm that can be integrated with the pyrosequencing analysis software would greatly simplify such analyses.References[1]Sobel JD.Vaginitis.N Engl J Med1997;337:1896–903.[2]Diagnosis of Candida vaginitis.J Fam Pract1985;20:19–20.[3]Sobel JD.Pathogenesis and epidemiology of vulvovaginal candidia-sis.Ann NY Acad Sci1988;544:547–57.[4]Pfaller MA.Nosocomial candidiasis:emerging species,reser-voirs,and modes of transmission.Clin Infect Dis1996;22(2): S89–S94.Table3Agreement of real-time PCR and pyrosequencing with conventional PCR for Candida species identificationConventional PCRNEG CA CG CP CT CA C CP CA C CT TOTALReal-time PCR and pyrosequen-cing NEG9696 CA6767 CG3232 CP1818 CT1313 CA C CP123 CA C CT112 Total966932181321231NEG,negative;CA,C.albicans;CG,C.glabrata;CP,C.parapsilosis;CT,C.tropicalis.J.P.Trama et al./Molecular and Cellular Probes19(2005)145–152151[5]Hazen KC,Baron EJ,Colombo AL,Girmenia C,Sanchez-Sousa A,del Palacio A,et parison of the susceptibilities of Candida spp.tofluconazole and voriconazole in a4-year global evaluation using disk diffusion.J Clin Microbiol2003;41:5623–32.[6]Sobel JD.Vulvovaginitis in healthy pr Ther1999;25:335–46.[7]Pfaller MA,Diekema DJ,Messer SA,Boyken L,Hollis RJ.Activitiesoffluconazole and voriconazole against1,586recent clinical isolates of Candida species determined by Broth microdilution,disk diffusion, and Etest methods:report from the ARTEMIS Global Antifungal Susceptibility Program,2001.J Clin Microbiol2003;41:1440–6. [8]Snydman DR.Shifting patterns in the epidemiology of nosocomialCandida infections.Chest2003;123:500S–3S.[9]Hazen KC.New and emerging yeast pathogens.Clin Microbiol Rev.1995;8:462–78.[10]Fidel Jr PL,Vazquez JA,Sobel JD.Candida glabrata:review ofepidemiology,pathogenesis,and clinical disease with comparison toC.albicans.Clin Microbiol Rev1999;12:80–96.[11]Pappas PG,Rex JH,Sobel JD,Filler SG,Dismukes WE,Walsh TJ,Edwards JE.Guidelines for treatment of candidiasis.Clin Infect Dis 2004;38:161–89.[12]Elliott KA.Managing patients with vulvovaginal candidiasis.NursePract1998;23:44–6,49–53.[13]ACOG technical bulletin.Vaginitis.Number226-July1996(replacesNo.221,March1996).Committee on Technical Bulletins of the American College of Obstetricians and Gynecologists.Int J Gynaecol Obstet54(1996)293–302.[14]ACOG technical bulletin.Vaginitis.Number221–March1996(replaces no.135,November1989).American College of Obste-tricians and Gynecologists.Int J Gynaecol Obstet53(1996)271–80.[15]Odds FC,Bernaerts R.CHROMagar Candida,a new differentialisolation medium for presumptive identification of clinically import-ant Candida species.J Clin Microbiol1994;32:1923–9.[16]Warren NG,Hazen KC.Candida,Cryptococcus,and other yeasts ofmedical importance.In:Murray PR,Barton EJ,Pfaller MA, Tenover FC,Yolken RH,editors.Manual of clinical microbiology.Washington,DC:American Society for Microbiology;1995.p.723–37.[17]Chen YC,Eisner JD,Kattar MM,Rassoulian-Barrett SL,LaFe K,Yarfitz SL,et al.Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer2region of the rRNA genes.J Clin Microbiol2000;38:2302–10.[18]Luo G,Mitchell TG.Rapid identification of pathogenic fungi directlyfrom cultures by using multiplex PCR.J Clin Microbiol2002;40: 2860–5.[19]Evertsson U,Monstein HJ,Johansson AG.Detection and identifi-cation of fungi in blood using broad-range28S rDNA PCR amplification and species-specific hybridisation.Apmis2000;108: 385–92.[20]Tamura M,Watanabe K,Imai T,Mikami Y,Nishimura K.New PCRprimer pairs specific for Candida dubliniensis and detection of the fungi from the Candida albicans clinical isolates in Japan.Clin Lab2000;46:33–40.[21]Wahyuningsih R,Freisleben HJ,Sonntag HG,Schnitzler P.Simpleand rapid detection of Candida albicans DNA in serum by PCR for diagnosis of invasive candidiasis.J Clin Microbiol2000;38: 3016–21.[22]Elie CM,Lott TJ,Reiss E,Morrison CJ.Rapid identification ofCandida species with species-specific DNA probes.J Clin Microbiol 1998;36:3260–5.[23]Holland PM,Abramson RD,Watson R,Gelfand DH.Detection ofspecific polymerase chain reaction product by utilizing the50–30 exonuclease activity of Thermus aquaticus DNA polymerase.Proc Natl Acad Sci USA1991;88:7276–80.[24]Guiver M,Levi K,Oppenheim BA.Rapid identification of Candidaspecies by TaqMan PCR.J Clin Pathol2001;54:362–6.[25]Borst A,Leverstein-Van Hall MA,Verhoef J,Fluit AC.Detection ofCandida spp.in blood cultures using nucleic acid sequence-based amplification(NASBA).Diagn Microbiol Infect Dis2001;39:155–60.[26]Shin JH,Nolte FS,Holloway BP,Morrison CJ.Rapid identification ofup to three Candida species in a single reaction tube by a50 exonuclease assay usingfluorescent DNA probes.J Clin Microbiol 1999;37:165–70.[27]Selvarangan R,Bui U,Limaye AP,Cookson BT.Rapid identificationof commonly encountered Candida species directly from blood culture bottles.J Clin Microbiol2003;41:5660–4.[28]Ronaghi M,Uhlen M,Nyren P.A sequencing method based on real-time pyrophosphate.Science1998;281:363–5.[29]Ausubel F,Brent R,Kingston RE,Moore DD,Seidman JG,Smith JA,et al.Short protocols in molecular biology.3rd ed.New York:Wiley;1997.[30]White TJ,Burns TD,Lee SB,Taylor JW.Amplification and directsequencing of fungal ribosomal RNA genes for phylogenetics.In: Innis MA,Helfand DH,Sninsky JJ,White TJ,editors.PCR protocols.San Diego:Academic Press,Inc;1990.p.315–22.[31]Singh S,Sobel JD,Bhargava P,Boikov D,Vazquez JA.Vaginitis dueto Candida krusei:epidemiology,clinical aspects,and therapy.Clin Infect Dis2002;35:1066–70.[32]Pfaller MA,Diekema DJ.Twelve years offluconazole in clinicalpractice:global trends in species distribution andfluconazole susceptibility of bloodstream isolates of Candida.Clin Microbiol Infect2004;10(1):11–23.[33]Pfaller MA,Messer SA,Boyken L,Hollis RJ,Rice C,Tendolkar S,et al.In vitro activities of voriconazole,posaconazole,andfluconazole against4,169clinical isolates of Candida spp.and Cryptococcus neoformans collected during2001and2002in the ARTEMIS global antifungal surveillance program.Diagn Microbiol Infect Dis2004;48: 201–5.J.P.Trama et al./Molecular and Cellular Probes19(2005)145–152 152。