Molecular Identification of Cryptococcus neoformans Serotypes (PDF Download Available)
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31.39Université Paris-Sud 11+ 433.25Belgian Scientific Institute for Public Health41.58Pierre and Marie Curie University - Paris 6Show more authorsAbstractCryptococcus neoformans is a fungal pathogen that causes life-threatening infections primarily in immunocompromised hosts. Based on the genetic characteristics
and serologic properties of capsular polysaccharides, three varieties and five serotypes have been defined: C. neoformans var. neoformans (serotype D), C. neoformans var. grubii (serotype A), hybrid serotype AD, and C. neoformans var. gattii (serotypes B and C). Epidemiologic features, such as geographic distribution and ecologic niche, and clinical characteristics
have been shown to be associated with serotypes. At the present time, serotyping is based on agglutination tests with either
commercial or “homemade” antisera or on immunofluorescence assays using a monoclonal antibody directed against the capsule
polysaccharide. In this paper, we describe two molecular methods (PCR-restriction enzyme analysis and length polymorphism
analysis) for C. neoformans serotype identification. Both are based on the sequence characteristics of a fragment of the CAP59 gene required for capsule biosynthesis. Testing of 72 C. neoformans strains including representatives of the five serotypes demonstrated the reliability of these methods.Discover the world's research14+ million members100+ million publications700k+ research projectsFigures
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2007, p.
Vol. 45, No. 4/$08.00?0 doi:10.1128/JCM.01839-06Copyright (C) 2007, American Society for Microbiology. All Rights Reserved.Molecular Identification of Cryptococcus neoformans Serotypes?A. Enache-Angoulvant,1,2J. Chandenier,3F. Symoens,4P. Lacube,1J. Bolognini,1C. Douchet,3J. L. Poirot,1,5and C. Hennequin1,2*Laboratoire de Parasitologie-Mycologie, Universite? Pierre et Marie Curie, Faculte?deMe?decine Pierre et Marie Curie, site St. Antoine,F-75012 Paris, France1; Service de Parasitologie-Mycologie, APHP, Ho^pital Tenon, F-75020 Paris, France2; Laboratoire deParasitologie-Mycologie, Ho^pital Bretonneau, F-37044 Tours, France3; Scientific Institute of Public Health,Mycology Section, B-1050 Brussels, Belgium4; and Laboratoire de Parasitologie-Mycologie, APHP,Ho^pital St. Antoine, F-75012 Paris, France5Received 5 September 2006/Returned for modification 16 October 2006/Accepted 29 January 2007Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections primarily in immunocom-promised hosts. Based on the genetic characteristics and serologic properties of capsular polysaccharides, threevarieties and five serotypes have been defined: C. neoformans var. neoformans (serotype D), C. neoformans var. grubii(serotype A), hybrid serotype AD, and C. neoformans var. gattii (serotypes B and C). Epidemiologic features, such asgeographic distribution and ecologic niche, and clinical characteristics have been shown to be associated withserotypes. At the present time, serotyping is based on agglutination tests with either commercial or “homemade”antisera or on immunofluorescence assays using a monoclonal antibody directed against the capsule polysaccha-ride. In this paper, we describe two molecular methods (PCR-restriction enzyme analysis and length polymorphismanalysis) for C. neoformans serotype identification. Both are based on the sequence characteristics of a fragment ofthe CAP59 gene required for capsule biosynthesis. Testing of 72 C. neoformans strains including representatives ofthe five serotypes demonstrated the reliability of these methods.Cryptococcus neoformans is a basidiomycetous encapsulatedyeast that causes life-threatening infections, mainly in immu-nocompromised hosts and particularly in human immunodefi-ciency virus-infected patients (1, 13). Based on the geneticcharacteristics and serologic properties of capsular polysaccha-rides (CPS) (9), three varieties and four nonhybrid serotypeshave been defined, namely, C. neoformans var. neoformans(serotype D), C. neoformans var. grubii (serotype A), and C.neoformans var. gattii (serotypes B and C) (4). Serotypes cor-respond to different sexual teleomorphs, namely, Filobasidiellaneoformans and Filobasidiella bacillispora for serotypes A andD and serotypes B and C, respectively. Moreover, hybridstrains that most likely correspond to either diploid or aneu-ploid organisms have been characterized as serotype ADstrains (11, 14, 17, 20). Some epidemiologic properties, such asgeographic distribution, have been associated with serotypes,as serotypes A, D, and AD are found worldwide while sero-types B and C are restricted mainly to tropical and subtropicalregions (1). While serotype A and D strains are usually isolatedfrom pigeon droppings and cause disease mainly in immuno-compromised hosts, serotype B and C strains are more com-monly isolated from eucalyptus trees and more often infecthosts with normal immune status, causing cryptococcoma (1).The need for prolonged treatment and surgery and sequelaeincluding impaired visual acuity and a higher mortality ratehave also been found to be more frequently associated withserotype B strains than with serotype A strains (2, 18).Serotyping is presently performed by using agglutinationwith commercial (Crypto Check
Iatron Labs, Tokyo, Ja-pan) or “homemade” antisera components (9) or a combina-tion of canavanine-glycine-bromothymol (CGB) blue agar di-agnostic medium and a direct immunofluorescence assay usingE1 monoclonal antibody directed against CPS (3). These meth-ods are widely used but may be either inapplicable or unreli-able in the case of noncapsulated or nontypeable isolates,respectively (8, 16). Moreover, cases of misidentification havealready been reported (3, 19). In the present paper, we reporttwo molecular methods, based on the sequence characteristicsof a fragment of the CAP59 gene required for capsule biosyn-thesis (5). Both methods allow for the rapid and reliable iden-tification of all C. neoformans serotypes.MATERIALS AND METHODSStrains. Seventy-two C. neoformans strains, including 33 reference strains,representing the five serotypes and one Cryptococcus uniguttulatus strain weretested (Table 1). The identification of all strains was confirmed by using theID32C auxanogram panel (BioMe?rieux, Marcy l’Etoile, France). Strains wereserotyped by agglutination with homemade antisera based on the proceduredescribed by Ikeda et al. (9) or with a commercial kit (Crypto Check
IatronLabs, Tokyo, Japan). Some strains have been serotyped by the Centre Nationalde Re?fe?rence Mycologie et Antifongiques at the Pasteur Institute of Paris bycombining CGB diagnostic medium with a direct immunofluorescence assayusing E1 monoclonal antibody specific for CPS, as described by Dromer et al. (3).Isolates were stored at ?80°C and were grown on yeast-peptone-glucose agarplates for 2 days at 30°C before testing.CAP59 gene amplification. Sequences of CAP59 genes from the four nonhy-brid serotypes of C. neoformans were retrieved from the GenBank database andthe Cryptococcus neoformans Serotype B Sequencing Project of the Broad Insti-tute of Harvard and the Massachusetts Institute of Technology (http://www.broad.mit.edu). Sequences were then aligned using ClustalX (version 1.83), andtwo primers (CH-Cap 59F, 5?-CCTTGCCGAAGTTCGAAACG, and CH-Cap59R, 5?-AATCGGTGGTTGGATTCAGTGT) were designed from the con-served regions of the CAP59 genes. DNA was extracted using a rapid methodbased on thermal shock and the chelation of components other than nucleic acidsby using a resin suspension, as previously described (7). The PCR was carried outwith a 50-?l reaction volume containing 1? PCR buffer, 1.6 ?M (each) primers* Corresponding author. Mailing address: Laboratoire de Parasitolo-gie-Mycologie, Faculte?deMe?decine Pierre et Marie Curie, site St. An-toine, 27 rue de Chaligny, 75012 Paris, France. Phone: 33-1-.Fax: 33-1-. E-mail: christophe..?Published ahead of print on 7 February 2007.1261
TABLE 1. C. neoformans strains tested in this study and results of immunoserotyping and molecular identification of the serotypeStrainno.Strain nameIsolationaImmunologic serotyping Molecular identificationOrigin Site MethodbResultREApatterncFragment size(s)(bp)d1 H99 United States CSF Commercial kit A A 3942 IHEM 11752 Congo CSF Commercial kit A A 4003 IHEM 11753 Congo CSF Commercial kit A A 3944 IHEM 11756 Congo CSF Commercial kit A A 3945 IHEM 11757 Congo CSF Commercial kit A A 3946 IHEM 11876 Congo CSF Commercial kit A A 3947 IHEM 11877 Congo CSF Commercial kit A A 3948 IHEM 11880 Congo CSF Commercial kit A A 3949 IHEM 11882 Congo CSF Commercial kit A A 39410 Tou Mali CSF Homemade Ab A A 39411 Fat Congo CSF Homemade Ab A A 40012 coc 119 Cochin, India CSF Homemade Ab A A 39413 333 Gabon CSF Homemade Ab A A 39414 2598 P Ivory Coast CSF Homemade Ab A A 39415 g 57497 Gabon CSF Homemade Ab A A 39416 Oug 1 Uganda CSF Homemade Ab A A 39417 PP38 Cambodia CSF Homemade Ab A A 39418 RV66254 Rwanda CSF Homemade Ab A A 39419 3/768 Congo CSF Homemade Ab A A 39420 Lim France CSF Homemade Ab D A 39421 C3 Cambodia CSF Homemade Ab A A 39422 C6 Cambodia CSF Homemade Ab A A 39423 C7 Cambodia CSF Homemade Ab A A 39424 Oug 7 Uganda CSF Homemade Ab A A 39425 PP29 Cambodia CSF Homemade Ab A A 39426 RV68535 Rwanda CSF Homemade Ab A A 39427 RV68528 Rwanda CSF Homemade Ab A A 39428 Tnn Ci France CSF Homemade Ab A A 39429 g 59546 Gabon CSF Homemade Ab A A 39430 RV66254 Rwanda CSF Homemade Ab A A 39431 C57 Cambodia CSF Homemade Ab A A 39432 C68 Cambodia CSF Homemade Ab A A 39433 Oug 3 Uganda CSF Homemade Ab AD A 39434 Oug 9 Uganda CSF Homemade Ab A A 39435 PP43 Cambodia CSF Homemade Ab A A 39436 241 Gabon CSF Homemade Ab A A 39437 Tnn Mb France Blood mAb IF A A 39438 Tnn Da France Blood mAb IF A A 39439 Tnn Mo France CSF mAb IF A A 39440 Bel Belgium CSF Commercial kit A A 39441 IHEM 4157 Belgium NS Commercial kit AD AD 382, 39442 IHEM 13877 Belgium CSF Commercial kit AD AD 382, 39443 IHEM 14440 Belgium CSF Commercial kit AD AD 382, 39444 IHEM 16079 Belgium Voice prosthesis Commercial kit AD AD 382, 39445 IHEM 16513 Peru CSF Commercial kit AD AD 382, 39446 IHEM 17861 Peru CSF Commercial kit AD AD 382, 39447 RV 66423 Rwanda CSF Homemade Ab A D 38248 JEC21 NS NS Commercial kit D D 38249 B 3501 A NS NS Commercial kit D D 38250 NIH D52 United States CSF Commercial kit D D 38251 IHEM 15038 Peru NS Commercial kit D D 38252 IHEM 18620 Belgium Skin Commercial kit D D 38253 IHEM 16711 Belgium Skin Commercial kit D D 38254 IHEM 4158 Italy NS Commercial kit D D 38255 AKD 30 Ivory Coast CSF Homemade Ab A D 38056 T 6073 France CSF Homemade Ab D D 38257 2815 Cambodia CSF Homemade Ab A D 38258 Tnn Re France BAL fluid mAb IF D D 38259 JLP34 France CSF mAb IF D D 38260 JLP23 France Urine mAb IF D D 38261 IHEM 11758 Congo CSF Commercial kit D D 38062 C9 Cambodia CSF Homemade Ab A D 38263 IHEM 14934 Brazil NS Commercial kit B B 37364 IHEM 4161eUnited States Sputum Commercial kit B B 37365 IHEM 11796eZaire CSF Commercial kit B B 373Continued on following page1262 ENACHE-ANGOULVANT ET AL. J. CLIN.MICROBIOL.
(Eurogentec, Liege, Belgium), 0.2 mM (each) deoxynucleoside triphosphates(equimolar concentrations of dATP, dCTP, dGTP, and dTTP), and 0.5 U of Taqpolymerase (New England BioLabs Inc.). A touchdown amplification programwas performed as follows: 7 min at 94°C; three cycles of 30 s at 94°C, 30 s at 60°C,and 30 s at 72°C; three cycles of 30 s at 94°C, 30 s at 58°C, and 30 s at 72°C; threecycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C; 28 cycles of 30 s at 52°C and30 s at 72°C; and a final 15-min extension at 72°C.Restriction enzyme analysis (REA). Restriction maps of unique sites obtainedusing DNA Strider version 1.4x-4e (15) were used to select restriction enzymesallowing for the identification of the various serotypes (Table 2). In a first-lineidentification, PCR products were subjected to separate restriction procedures withBsmFI and HpaII according to the instructions of the manufacturer (New EnglandBioLabs Inc.). Digested fragments were separated by electrophoresis in agarose gel(3% in Tris-borate-EDTA buffer) stained with ethidium bromide (0.5 ?g/ml) at 90V for 3 h. PCR products with patterns compatible with the B or C serotype werefurther digested with the AgeI enzyme (New England BioLabs Inc.).Fragment length polymorphism analysis. For fragment length analysis, a sim-ilar amplification protocol was performed but with a fluorolabeled (6-carboxy-fluorescein
Applied Biosystems, Courtaboeuf, France) forward primer. A7-base tail used to promote the addition of an extra A to the amplified fragmentwas added to the reverse primer in order to facilitate sizing. Each PCR productwas run with an internal molecular marker (GenScan HD400Rox, ABI) for sizedetermination on a DNA sequencer (ABI 3100; Applied Biosystems).In the case of conflicting results, a second serotyping determination was per-formed with the Crypto Check commercial kit (Iatron Labs, Tokyo, Japan).Based on fragment length results, we selected certain PCR products to besequenced (Table 3). Fragments of a serotype AD strain (IHEM 13877) notdigested by either BsmFI or HpaII and corresponding to the D or A allele,respectively, were gel purified (Gel Band purification
Amersham Bio-sciences, Freiburg, Germany) before sequencing. Direct sequencing was per-formed using a Big Dye terminator protocol as recommended by the manufac-turer (Applied Biosystems). Double-strand sequencing was performed using thesame primers as those used for PCR amplification. After manual correction,sequences were compared using the BLASTN software on the GenBank data-base limited to the organism C. neoformans.Nucleotide sequence accession number. The sequence of IHEM 4159 obtainedin the present study has been deposited in GenBank with accession numberEF392815.RESULTSAll tested C. neoformans strains but not the C. uniguttulatusstrain gave positive amplification.PCR-REA. As expected from in silico analysis, enzymaticrestriction with BsmFI and HpaII gave three different patternsfor serotypes A, D, and B and C (Fig. 1). Serotype AD strainsexhibited a mixed A and D restriction profile. All strains weretested, and no profile variation among strains of a given sero-type was observed. Serotype D and serotypes B and C, whilehaving similar profiles (unique cutting site for HpaII and nocutting site for BsmFI), can be easily differentiated based onthe size difference of the digested fragments. Serotypes B andC can be further differentiated by AgeI digestion and gel elec-trophoresis. Two fragments corresponding to serotype Bstrains (one cutting site) were detected, while serotype C am-plicons remained unchanged (no cutting site) (Fig. 1).Length polymorphism analysis. Because in silico analysis pre-dicted length polymorphism corresponding to serotypes in theamplified fragments (Table 2), we designed a fluorescent frag-ment length analysis method using a DNA sequencer. Sevendifferent alleles accounting for eight different genotypes weredetected (Table 1). Fragments amplified from the strains of se-rotypes A, B, C, and D used as a reference for in silico analysiswere sized with the predicted length plus or minus 1 base, whichis the instrument resolution provided by the manufacturer.A small degree of intraserotype polymorphism was seen:serotype A strains exhibited an allele of either 394 bp (n ? 38)or 400 bp (n ? 2); serotype D strains had an allele of either 380bp (n ? 2) or 382 bp (n ? 14); and serotype B strains had anallele of 371 bp (n ? 3), 373 bp (n ? 4), or 380 bp (n ? 1),while the two serotype C strains tested had an allele of 366 bp(Table 1). The six serotype AD strains exhibited two alleles, of382 and 394 bp. Fragments with sizes different from the pre-TABLE 1—ContinuedStrainno.Strain nameIsolationaImmunologic serotyping Molecular identificationOrigin Site MethodbResultREApatterncFragment size(s)(bp)d66 IHEM 4170 Malaysia NS Commercial kit B B 37367 IHEM 4164 China CSF Commercial kit B B 37168 WM 276 Australia Environment Commercial kit B B 37169 VanR265 Canada BWF Commercial kit B B 38070 RV68522 Rwanda CSF Homemade Ab D B 37171 IHEM 10079 NS CSF Commercial kit C C 36672 IHEM 4159eUnited States CSF Commercial kit C C 366aNS, not
CSF, cerebrospinal
BAL, bronchoalveolar
BWF, bronchial wash
Congo, Democratic Republic of the Congo.bThe commercial kit used was the Crypto Check kit (Iatron Labs, Tokyo, Japan). Homemade Ab, agglutination with homemade antisera (9); mAb IF, immuno-fluorescence with monoclonal antibody specific for CPS (3).cREA pattern deduced from in silico analysis (Table 2).dPCR fragments are supposed to be 8 bases longer than the sequences due to a 7-base tail added to the reverse primer used to promote the addition of an extraA to the amplified fragment.eType strain.TABLE 2. Expected sizes (bp) of CAP59 gene fragments fromdifferent serotypes before and after restriction with BsmFI,HpaII, and AgeI enzymesAssay or fragmentExpected size(s) (bp) for serotypea:ABCDNative amplicon 387 373 359 375REA withBsmFI206, 191 NRS NRS NRSREA withHpaIINRS 270, 101 256, 101 211, 162REA with AgeI NRS 269, 100 NRS NRSaNRS, serotype A, strain H99 (GenBank accession numberAF337639); serotype B, strain R265 (http://www.broad.mit.edu/annotation/genome/cryptococcus_neoformans_b/Assembly.html); serotype C, strain IHEM4159 (sequence obtained in the present study); serotype D, JEC21 strain(GenBank accession number AE017341).VOL. 45, 2007 MOLECULAR IDENTIFICATION OF C. NEOFORMANS SEROTYPES 1263
dominant size for a given serotype were sequenced. Table 3summarizes the results of the BLASTN comparison of thesesequences with sequences in the GenBank database. In allcases, the results of the sequence comparison agreed with themolecular identifications. Sequence alignment showed poly-morphism among and within serotypes, mainly in the intronicpart the fragments, located between nucleotide positions 200and 280 (data not shown).Analysis of discrepancies between conventional and molec-ular serotype identifications. In a first analysis, we found sevencases of discrepancies in identifications (strains 20, 33, 47, 55,57, 62, and 64). These discrepancies were seen only for strainsfor which the serotype had been determined by a homemadeagglutination method. For these strains, a new serotype deter-mination was performed using the Crypto Check kit, whichagreed with the PCR-REA and length polymorphism identifi-cations in every case.DISCUSSIONSerotyping has been useful for the description of the epide-miologic and clinical characteristics associated with C. neofor-TABLE 3. Comparison of the partial sequence of the CAP59 gene with sequences in the GenBank database by using BLASTN softwareStrain SerotypeFragment length(bases)Results (best hit) Accession no.IHEM 11752 A 392 Best score obtained when aligned with 2 fragments fromserotype A strain M9253 (accession no. AB066120)EF392812Partial 5? end: 99% identity over a 203-nucleotidealignmentPartial 3? end: 97% identity over a 131-nucleotidealignmentBetter score for partial 3? end obtained with sequencefrom serotype A strain H99: 98% identity over a 173-nucleotide alignmentIHEM 13877 AD 387 (A allelea)99% identity over a 382-nucleotide alignment withsequence from serotype A strain H99 (accession no.AF337639)EF392816375 (D alleleb)98% identity over a 375-nucleotide alignment withsequence from serotype D strain JEC21 (accessionno. AF337639)EF392813IHEM 11796 B 373 99% identity over a 324-nucleotide alignment withsequence from serotype B strain M9244 (accessionno. AB066134)EF392816IHEM 4159 C 359 99% identity over a 312-nucleotide alignment withsequence from serotype C strain M9247 (accessionno. AB066126)cEF392815aNo restricted fragment after digestion with HpaII.bNo restricted fragment after digestion with BsmFI.cComplete sequence of the fragment not available in the GenBank database.FIG. 1. Agarose gel (3%) electrophoresis of native amplicons of the CAP59 gene fragments and restriction profiles of C. neoformans serotypes.1264 ENACHE-ANGOULVANT ET AL. J. CLIN.MICROBIOL.
mans. More recently, it has played a major role in the inves-tigation of the endemic infection on Vancouver Island(Canada) (10). Serotyping has no diagnostic value, but differ-ences in treatments and outcomes corresponding to serotypeshave been reported (2, 18). Thus, considering that the CryptoCheck kit (Iatron, Japan) is no longer available and in view ofthe relative complexity of obtaining monoclonal antibodies,new techniques need to be developed for the typing of C.neoformans strains.Both methods developed in this work allow the reliableidentification of the five serotypes of C. neoformans by testinga unique genomic region. Our assays based on molecular anal-ysis of a fragment of the CAP59 gene appeared to be sensitiveand specific, providing interpretable results for all C. neofor-mans strains tested but no amplification for a C. uniguttulatusstrain.PCR-REA is an easy-to-use approach and requires onlycommon molecular materials. Its value for distinguishingamong fungal species such as Malassezia spp. has already beenemphasized (6). As restriction sites were chosen within con-served regions of the fragments, all tested strains exhibited therestriction pattern predicted by in silico analysis. A flow chartfor serotype identification by combining separate digestionswith BsmFI and HpaII, followed by subsequent digestion withAgeI when the pattern suggestsaBorCserotype, can there-fore be proposed (Table 2). Serotype AD strains exhibited amixed A and D restriction profile. Our results were in accor-dance with those of previous studies suggesting that AD sero-type strains are diploid or aneuploid and possess, at least for anumber of genes including the CAP59 gene, both serotypeA-like and serotype D-like alleles (14, 17, 19, 20). This indica-tion was further confirmed by a sequence analysis performedwith the purified fragments (Table 3).Length polymorphism analysis showed different lengths fordifferent serotypes, with the exception of two serotype Dstrains (no. 55 and 61) and a serotype B strain (no. 69), whichall exhibited a 380-bp allele. Note that these particular casesconcerned only strains with alleles of the minority size withinthe serotype considered. These difficult cases can be solved byidentifying the serotype by using either REA with AgeI (onlyserotype B is restricted) or the results of culture on CGB blueagar, on which serotype B but not serotype D strains producea color change in the medium (12). Fragment length polymor-phism may be easier to perform but requires a DNA se-quencer, which is now more frequently available in teachinghospitals in industrialized countries. Following PCR amplifi-cation, results can be obtained within 1 h and are entirelyobjective.Overall, results from both molecular methods fit well withserotype identifications obtained with immunologic proce-dures, but the homemade technique is probably less reliable.Both methods presented in this study appear to be reliablealternatives to immunologic methods.ACKNOWLEDGMENTSWe are grateful to J. Heitman and C. Arndt (Duke UniversityMedical Center, Durham, NC); M.A. Viviani (Istituto di Igiene eMedicina Preventiva, Milano, Italy); M. Kombila (De?partement deParasitologie-Mycologie-Me?decine Tropicale, FMSS, Libreville, Ga-bon); D. Swinne (Institut Scientifique de Sante? Publique-Mycologie,Brussels, Belgium); K. Adou-Bryn (Laboratoire de Parasitologie-My-cologie, UFR des Sciences Me?dicales, Abidjan, Ivory Coast); Y. Buis-son (IMTSSA-Le Pharo, Marseille, France); and A. Paugam, A. Datry,and M. Develoux (Laboratoires de Parasitologie-Mycologie, Ho^pitalPitie?-Salpe^trie`re, Ho^pital Tenon, and Ho^pital Cochin, APHP, Paris,France) for the generous gift of some of the strains tested in this study.We are also indebted to the CNRMA (Centre National de Re?fe?renceMycologie et Antifongiques) directed by F. Dromer for serotypingsome clinical isolates and to the Cryptococcus neoformans Serotype BSequencing Project of the Broad Institute of Harvard and the Massa-chusetts Institute of Technology (http://www.broad.mit.edu) for mak-ing available C. neoformans serotype B sequences.REFERENCES1. Casadevall, A., and J. R. Perfect. 1998. Cryptococcus neoformans. ASMPress, Washington, DC.2. Chen, Y. C., S. C. Chang, C. C. Shih, C. C. Hung, K. T. Luhbd, Y. S. Pan, andW. C. Hsieh. 2000. Clinical features and in vitro susceptibilities of twovarieties of Cryptococcus neoformans in Taiwan. Diagn. Microbiol. Infect.Dis. 36:175–183.3. Dromer, F., E. Gueho, O. Ronin, and B. Dupont. 1993. 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