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Journal of Clinical Microbiology, April 2007, p. 1261-1265, Vol. 45, No. 4
0095-1137/07/$08.00+0     doi:10.1128/JCM.01839-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Molecular Identification of Cryptococcus neoformans Serotypes

A. Enache-Angoulvant,^1,2 J. Chandenier,³ F. Symoens,⁴ P. Lacube,¹ J. Bolognini,¹ C. Douchet,³ J. L. Poirot,^1,5 and C. Hennequin^1,2*

Laboratoire de Parasitologie-Mycologie, Université Pierre et Marie Curie, Faculté de Médecine Pierre et Marie Curie, site St. Antoine, F-75012 Paris, France,¹ Service de Parasitologie-Mycologie, APHP, Hôpital Tenon, F-75020 Paris, France,² Laboratoire de Parasitologie-Mycologie, Hôpital Bretonneau, F-37044 Tours, France,³ Scientific Institute of Public Health, Mycology Section, B-1050 Brussels, Belgium,⁴ Laboratoire de Parasitologie-Mycologie, APHP, Hôpital St. Antoine, F-75012 Paris, France⁵

Received 5 September 2006/ Returned for modification 16 October 2006/ Accepted 29 January 2007

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cryptococcus 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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cryptococcus neoformans is a basidiomycetous encapsulated yeast that causes life-threatening infections, mainly in immunocompromised hosts and particularly in human immunodeficiency virus-infected patients (1, 13). Based on the genetic characteristics and serologic properties of capsular polysaccharides (CPS) (9), three varieties and four nonhybrid serotypes have 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 correspond to different sexual teleomorphs, namely, Filobasidiella neoformans and Filobasidiella bacillispora for serotypes A and D and serotypes B and C, respectively. Moreover, hybrid strains that most likely correspond to either diploid or aneuploid organisms have been characterized as serotype AD strains (11, 14, 17, 20). Some epidemiologic properties, such as geographic distribution, have been associated with serotypes, as serotypes A, D, and AD are found worldwide while serotypes B and C are restricted mainly to tropical and subtropical regions (1). While serotype A and D strains are usually isolated from pigeon droppings and cause disease mainly in immunocompromised hosts, serotype B and C strains are more commonly isolated from eucalyptus trees and more often infect hosts with normal immune status, causing cryptococcoma (1). The need for prolonged treatment and surgery and sequelae including impaired visual acuity and a higher mortality rate have also been found to be more frequently associated with serotype B strains than with serotype A strains (2, 18).

Serotyping is presently performed by using agglutination with commercial (Crypto Check kit; Iatron Labs, Tokyo, Japan) or "homemade" antisera components (9) or a combination of canavanine-glycine-bromothymol (CGB) blue agar diagnostic medium and a direct immunofluorescence assay using E1 monoclonal antibody directed against CPS (3). These methods are widely used but may be either inapplicable or unreliable in the case of noncapsulated or nontypeable isolates, respectively (8, 16). Moreover, cases of misidentification have already been reported (3, 19). In the present paper, we report two molecular methods, based on the sequence characteristics of a fragment of the CAP59 gene required for capsule biosynthesis (5). Both methods allow for the rapid and reliable identification of all C. neoformans serotypes.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Strains. Seventy-two C. neoformans strains, including 33 reference strains, representing the five serotypes and one Cryptococcus uniguttulatus strain were tested (Table 1 ). The identification of all strains was confirmed by using the ID32C auxanogram panel (BioMérieux, Marcy l'Etoile, France). Strains were serotyped by agglutination with homemade antisera based on the procedure described by Ikeda et al. (9) or with a commercial kit (Crypto Check kit; Iatron Labs, Tokyo, Japan). Some strains have been serotyped by the Centre National de Référence Mycologie et Antifongiques at the Pasteur Institute of Paris by combining CGB diagnostic medium with a direct immunofluorescence assay using 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 agar plates for 2 days at 30°C before testing.

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TABLE 1. C. neoformans strains tested in this study and results of immunoserotyping and molecular identification of the serotype

CAP59 gene amplification. Sequences of CAP59 genes from the four nonhybrid serotypes of C. neoformans were retrieved from the GenBank database and the Cryptococcus neoformans Serotype B Sequencing Project of the Broad Institute of Harvard and the Massachusetts Institute of Technology (http://www.broad.mit.edu). Sequences were then aligned using ClustalX (version 1.83), and two primers (CH-Cap 59F, 5'-CCTTGCCGAAGTTCGAAACG, and CH-Cap 59R, 5'-AATCGGTGGTTGGATTCAGTGT) were designed from the conserved regions of the CAP59 genes. DNA was extracted using a rapid method based on thermal shock and the chelation of components other than nucleic acids by using a resin suspension, as previously described (7). The PCR was carried out with a 50-µl reaction volume containing 1x PCR buffer, 1.6 µM (each) primers (Eurogentec, Liege, Belgium), 0.2 mM (each) deoxynucleoside triphosphates (equimolar concentrations of dATP, dCTP, dGTP, and dTTP), and 0.5 U of Taq polymerase (New England BioLabs Inc.). A touchdown amplification program was 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; three cycles 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 and 30 s at 72°C; and a final 15-min extension at 72°C.

Restriction enzyme analysis (REA). Restriction maps of unique sites obtained using DNA Strider version 1.4x-4e (15) were used to select restriction enzymes allowing for the identification of the various serotypes (Table 2). In a first-line identification, PCR products were subjected to separate restriction procedures with BsmFI and HpaII according to the instructions of the manufacturer (New England BioLabs Inc.). Digested fragments were separated by electrophoresis in agarose gel (3% in Tris-borate-EDTA buffer) stained with ethidium bromide (0.5 µg/ml) at 90 V for 3 h. PCR products with patterns compatible with the B or C serotype were further digested with the AgeI enzyme (New England BioLabs Inc.).

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TABLE 2. Expected sizes (bp) of CAP59 gene fragments from different serotypes before and after restriction with BsmFI, HpaII, and AgeI enzymes

Fragment length polymorphism analysis. For fragment length analysis, a similar amplification protocol was performed but with a fluorolabeled (6-carboxyfluorescein dye; Applied Biosystems, Courtaboeuf, France) forward primer. A 7-base tail used to promote the addition of an extra A to the amplified fragment was added to the reverse primer in order to facilitate sizing. Each PCR product was run with an internal molecular marker (GenScan HD400Rox, ABI) for size determination on a DNA sequencer (ABI 3100; Applied Biosystems).

In the case of conflicting results, a second serotyping determination was performed with the Crypto Check commercial kit (Iatron Labs, Tokyo, Japan).

Based on fragment length results, we selected certain PCR products to be sequenced (Table 3). Fragments of a serotype AD strain (IHEM 13877) not digested by either BsmFI or HpaII and corresponding to the D or A allele, respectively, were gel purified (Gel Band purification kit; Amersham Biosciences, Freiburg, Germany) before sequencing. Direct sequencing was performed using a Big Dye terminator protocol as recommended by the manufacturer (Applied Biosystems). Double-strand sequencing was performed using the same primers as those used for PCR amplification. After manual correction, sequences were compared using the BLASTN software on the GenBank database limited to the organism C. neoformans.

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TABLE 3. Comparison of the partial sequence of the CAP59 gene with sequences in the GenBank database by using BLASTN software

Nucleotide sequence accession number. The sequence of IHEM 4159 obtained in the present study has been deposited in GenBank with accession number EF392815.

Top Abstract Introduction Materials and Methods Results Discussion References

 
All tested C. neoformans strains but not the C. uniguttulatus strain gave positive amplification.

PCR-REA. As expected from in silico analysis, enzymatic restriction with BsmFI and HpaII gave three different patterns for serotypes A, D, and B and C (Fig. 1). Serotype AD strains exhibited a mixed A and D restriction profile. All strains were tested, and no profile variation among strains of a given serotype was observed. Serotype D and serotypes B and C, while having similar profiles (unique cutting site for HpaII and no cutting site for BsmFI), can be easily differentiated based on the size difference of the digested fragments. Serotypes B and C can be further differentiated by AgeI digestion and gel electrophoresis. Two fragments corresponding to serotype B strains (one cutting site) were detected, while serotype C amplicons remained unchanged (no cutting site) (Fig. 1).

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FIG. 1. Agarose gel (3%) electrophoresis of native amplicons of the CAP59 gene fragments and restriction profiles of C. neoformans serotypes.

Length polymorphism analysis. Because in silico analysis predicted length polymorphism corresponding to serotypes in the amplified fragments (Table 2), we designed a fluorescent fragment length analysis method using a DNA sequencer. Seven different alleles accounting for eight different genotypes were detected (Table 1). Fragments amplified from the strains of serotypes A, B, C, and D used as a reference for in silico analysis were sized with the predicted length plus or minus 1 base, which is 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 380 bp (n = 2) or 382 bp (n = 14); and serotype B strains had an allele 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, of 382 and 394 bp. Fragments with sizes different from the predominant size for a given serotype were sequenced. Table 3 summarizes the results of the BLASTN comparison of these sequences with sequences in the GenBank database. In all cases, the results of the sequence comparison agreed with the molecular identifications. Sequence alignment showed polymorphism among and within serotypes, mainly in the intronic part the fragments, located between nucleotide positions 200 and 280 (data not shown).

Analysis of discrepancies between conventional and molecular serotype identifications. In a first analysis, we found seven cases of discrepancies in identifications (strains 20, 33, 47, 55, 57, 62, and 64). These discrepancies were seen only for strains for which the serotype had been determined by a homemade agglutination method. For these strains, a new serotype determination was performed using the Crypto Check kit, which agreed with the PCR-REA and length polymorphism identifications in every case.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Serotyping has been useful for the description of the epidemiologic and clinical characteristics associated with C. neoformans. More recently, it has played a major role in the investigation of the endemic infection on Vancouver Island (Canada) (10). Serotyping has no diagnostic value, but differences in treatments and outcomes corresponding to serotypes have been reported (2, 18). Thus, considering that the Crypto Check kit (Iatron, Japan) is no longer available and in view of the 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 reliable identification of the five serotypes of C. neoformans by testing a unique genomic region. Our assays based on molecular analysis of a fragment of the CAP59 gene appeared to be sensitive and specific, providing interpretable results for all C. neoformans strains tested but no amplification for a C. uniguttulatus strain.

PCR-REA is an easy-to-use approach and requires only common molecular materials. Its value for distinguishing among fungal species such as Malassezia spp. has already been emphasized (6). As restriction sites were chosen within conserved regions of the fragments, all tested strains exhibited the restriction pattern predicted by in silico analysis. A flow chart for serotype identification by combining separate digestions with BsmFI and HpaII, followed by subsequent digestion with AgeI when the pattern suggests a B or C serotype, can therefore be proposed (Table 2). Serotype AD strains exhibited a mixed A and D restriction profile. Our results were in accordance with those of previous studies suggesting that AD serotype strains are diploid or aneuploid and possess, at least for a number of genes including the CAP59 gene, both serotype A-like and serotype D-like alleles (14, 17, 19, 20). This indication was further confirmed by a sequence analysis performed with the purified fragments (Table 3).

Length polymorphism analysis showed different lengths for different serotypes, with the exception of two serotype D strains (no. 55 and 61) and a serotype B strain (no. 69), which all exhibited a 380-bp allele. Note that these particular cases concerned only strains with alleles of the minority size within the serotype considered. These difficult cases can be solved by identifying the serotype by using either REA with AgeI (only serotype B is restricted) or the results of culture on CGB blue agar, on which serotype B but not serotype D strains produce a color change in the medium (12). Fragment length polymorphism may be easier to perform but requires a DNA sequencer, which is now more frequently available in teaching hospitals in industrialized countries. Following PCR amplification, results can be obtained within 1 h and are entirely objective.

Overall, results from both molecular methods fit well with serotype identifications obtained with immunologic procedures, but the homemade technique is probably less reliable. Both methods presented in this study appear to be reliable alternatives to immunologic methods.

 
We are grateful to J. Heitman and C. Arndt (Duke University Medical Center, Durham, NC); M.A. Viviani (Istituto di Igiene e Medicina Preventiva, Milano, Italy); M. Kombila (Département de Parasitologie-Mycologie-Médecine Tropicale, FMSS, Libreville, Gabon); D. Swinne (Institut Scientifique de Santé Publique-Mycologie, Brussels, Belgium); K. Adou-Bryn (Laboratoire de Parasitologie-Mycologie, UFR des Sciences Médicales, Abidjan, Ivory Coast); Y. Buisson (IMTSSA-Le Pharo, Marseille, France); and A. Paugam, A. Datry, and M. Develoux (Laboratoires de Parasitologie-Mycologie, Hôpital Pitié-Salpêtrière, Hôpital Tenon, and Hôpital 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 Référence Mycologie et Antifongiques) directed by F. Dromer for serotyping some clinical isolates and to the Cryptococcus neoformans Serotype B Sequencing Project of the Broad Institute of Harvard and the Massachusetts Institute of Technology (http://www.broad.mit.edu) for making available C. neoformans serotype B sequences.

 
* Corresponding author. Mailing address: Laboratoire de Parasitologie-Mycologie, Faculté de Médecine Pierre et Marie Curie, site St. Antoine, 27 rue de Chaligny, 75012 Paris, France. Phone: 33-1-56016739. Fax: 33-1-56017954. E-mail: christophe.hennequin{at}laposte.net

Published ahead of print on 7 February 2007.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Casadevall, A., and J. R. Perfect. 1998. Cryptococcus neoformans. ASM Press, Washington, DC.
2
2. Chen, Y. C., S. C. Chang, C. C. Shih, C. C. Hung, K. T. Luhbd, Y. S. Pan, and W. C. Hsieh. 2000. Clinical features and in vitro susceptibilities of two varieties of Cryptococcus neoformans in Taiwan. Diagn. Microbiol. Infect. Dis. 36:175-183.[CrossRef][Medline]
3
3. Dromer, F., E. Gueho, O. Ronin, and B. Dupont. 1993. Serotyping of Cryptococcus neoformans by using a monoclonal antibody specific for capsular polysaccharide. J. Clin. Microbiol. 31:359-363.[Abstract/Free Full Text]
4
4. Franzot, S. P., B. C. Fries, W. Cleare, and A. Casadevall. 1998. Genetic relationship between Cryptococcus neoformans var. neoformans strains of serotypes A and D. J. Clin. Microbiol. 36:2200-2204.[Abstract/Free Full Text]
5
5. Garcia-Rivera, J., Y. C. Chang, K. J. Kwon-Chung, and A. Casadevall. 2004. Cryptococcus neoformans CAP59 (or Cap59p) is involved in the extracellular trafficking of capsular glucuronoxylomannan. Eukaryot. Cell 3:385-392.[Abstract/Free Full Text]
6
6. Guillot, J., M. Deville, M. Berthelemy, F. Provost, and E. Gueho. 2000. A single PCR-restriction endonuclease analysis for rapid identification of Malassezia species. Lett. Appl. Microbiol. 31:400-403.[CrossRef][Medline]
7
7. Hennequin, C., E. Abachin, F. Symoens, V. Lavarde, G. Reboux, N. Nolard, and P. Berche. 1999. Identification of Fusarium species involved in human infections by 28S rRNA gene sequencing. J. Clin. Microbiol. 37:3586-3589.[Abstract/Free Full Text]
8
8. Horta, J. A., C. C. Staats, A. K. Casali, A. M. Ribeiro, I. S. Schrank, A. Schrank, and M. H. Vainstein. 2002. Epidemiological aspects of clinical and environmental Cryptococcus neoformans isolates in the Brazilian state Rio Grande do Sul. Med. Mycol. 40:565-571.[Medline]
9
9. Ikeda, R., T. Shinoda, Y. Fukazawa, and L. Kaufman. 1982. Antigenic characterization of Cryptococcus neoformans serotypes and its application to serotyping of clinical isolates. J. Clin. Microbiol. 16:22-29.[Abstract/Free Full Text]
10
10. Kidd, S. E., F. Hagen, R. L. Tscharke, M. Huynh, K. H. Bartlett, M. Fyfe, L. Macdougall, T. Boekhout, K. J. Kwon-Chung, and W. Meyer. 2004. A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). Proc. Natl. Acad. Sci. USA 101:17258-17263.[Abstract/Free Full Text]
11
11. Kwon-Chung, K. J., J. E. Bennett, and J. C. Rhodes. 1982. Taxonomic studies on Filobasidiella species and their anamorphs. Antonie Leeuwenhoek 48:25-38.[CrossRef][Medline]
12
12. Kwon-Chung, K. J., I. Polacheck, and J. E. Bennett. 1982. Improved diagnostic medium for separation of Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C). J. Clin. Microbiol. 15:535-537.[Abstract/Free Full Text]
13
13. Kwon-Chung, K. J., T. C. Sorrell, F. Dromer, E. Fung, and S. M. Levitz. 2000. Cryptococcosis: clinical and biological aspects. Med. Mycol. 38(Suppl. 1):205-213.
14
14. Lengeler, K. B., G. M. Cox, and J. Heitman. 2001. Serotype AD strains of Cryptococcus neoformans are diploid or aneuploid and are heterozygous at the mating-type locus. Infect. Immun. 69:115-122.[Abstract/Free Full Text]
15
15. Marck, C. 1988. ‘DNA Strider': a 'C’ program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 16:1829-1836.[Abstract/Free Full Text]
16
16. Moyrand, F., B. Klaproth, U. Himmelreich, F. Dromer, and G. Janbon. 2002. Isolation and characterization of capsule structure mutant strains of Cryptococcus neoformans. Mol. Microbiol. 45:837-849.[CrossRef][Medline]
17
17. Okabayashi, K., R. Kano, Y. Nakamura, S. Watanabe, and A. Hasegawa. 2006. Capsule-associated genes of serotypes of Cryptococcus neoformans, especially serotype AD. Med. Mycol. 44:127-132.[CrossRef][Medline]
18
18. Speed, B., and D. Dunt. 1995. Clinical and host differences between infections with the two varieties of Cryptococcus neoformans. Clin. Infect. Dis. 21:28-36.[Medline]
19
19. Tanaka, R., Y. Imanishi, and K. Nishimura. 2003. Difference in FKS1 gene sequences between serotypes A and D of Cryptococcus neoformans. J. Clin. Microbiol. 41:4457-4459.[Abstract/Free Full Text]
20
20. Xu, J., G. Luo, R. J. Vilgalys, M. E. Brandt, and T. G. Mitchell. 2002. Multiple origins of hybrid strains of Cryptococcus neoformans with serotype AD. Microbiology 148:203-212.[Abstract/Free Full Text]

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Journal of Clinical Microbiology, April 2007, p. 1261-1265, Vol. 45, No. 4
0095-1137/07/$08.00+0     doi:10.1128/JCM.01839-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Enache-Angoulvant, A. Articles by Hennequin, C. Search for Related Content PubMed PubMed Citation Articles by Enache-Angoulvant, A. Articles by Hennequin, C.