AIDS patient death caused by novel Cryptococcus neoformans x C. gattii hybrid.Interspecies hybrids of Cryptococcus neoformans and C. gattii have only recently been reported. We describe a novel C. neoformans x C. gattii hybrid strain (serotype serotype /se·ro·type/ (ser´o-tip) the type of a microorganism determined by its constituent antigens; a taxonomic subdivision based thereon. se·ro·type n. See serovar. v. AB) that was previously described as C. gattii and that caused a lethal infection in an AIDS patient from Canada. ********** Cryptococcus neoformans and C. gattii are pathogenic yeasts that may cause meningoencephalitis meningoencephalitis /me·nin·go·en·ceph·a·li·tis/ (me-ning?go-en-sef?ah-li´tis) inflammation of the brain and meninges. toxoplasmic meningoencephalitis . C. neoformans primarily infects immunocompromised immunocompromised /im·mu·no·com·pro·mised/ (-kom´pro-mizd) having the immune response attenuated by administration of immunosuppressive drugs, by irradiation, by malnutrition, or by certain disease processes (e.g., cancer). patients and occurs worldwide, whereas C. gattii primarily mainly infects otherwise healthy persons and has been thought to occur in subtropical sub·trop·i·cal adj. Of, relating to, or being the geographic areas adjacent to the Tropics. subtropical Adjective of the region lying between the tropics and temperate lands regions (l-3). However, the recent outbreak of infection with C. gattii on Vancouver Island, British Columbia, Canada (4), expansion of this outbreak to mainland Canada and the Pacific Northwest region of the United States (5), and identification of C. gattii isolates in Europe (6) show that C. gattii can also occur in temperate climates. Molecular techniques can distinguish 7 haploid haploid /hap·loid/ (hap´loid) 1. having half the number of chromosomes characteristically found in the somatic (diploid) cells of an organism; typical of the gametes of a species whose union restores the diploid number. genotypic groups within C. neoformans and C. gattii (7-9; F. Hagen and T. Boekhout, unpub, data). Recently, 3 serotype BD C. neoformans var. neoformans x C. gattii hybrids were isolated from 2 HIV-negative patients in the Netherlands (10). We describe a novel C. neoformans var. grubii serotype A x C. gattii serotype B hybrid that was isolated from an HIV-positive person. The Study Strain CBS (Cell Broadcast Service) See cell broadcast. 10496 was isolated from a 31-year-old AIDS patient from Montreal, Quebec, Canada, who had traveled to Mexico [approximately equals] 15 months before cryptococcosis cryptococcosis: see fungal infection. was diagnosed. The patient died despite extensive antifungal treatment with ketoconazole ketoconazole /ke·to·co·na·zole/ (ke?to-kon´ah-zol) a derivative of imidazole used as an antifungal agent. ke·to·co·na·zole n. and amphotericin B amphotericin B (ăm'fətĕr`ĭsĭn), antibiotic that halts the growth of several disease-causing fungi. Discovered in 1956, it is produced by bacteria of the genus Streptomyces. (11). CBS10496 has been identified as C. gattii serotype B (cited as C. neoformans var. gattii) (11). Reference isolates used in this study are listed in the Table. The ploidy ploidy Number of sets of chromosomes in the nucleus of a cell. In normal human body cells, chromosomes exist in pairs, a condition called diploidy. During meiosis the cell produces sex cells (gametes), each containing half the normal number of chromosomes, a condition called of CBS10496 was determined by using flow cytometry flow cytometry (flōˑ sī·t  ˑ·m (10)
with the sequenced haploid strains CBS8710 and CBS10510 as references.
Nuclei were visualized after staining with
4',6-diamidino-2-phenylindole (10). Coloration of colonies grown on
canavanine-glycine-bromthymol blue (CGB CGB Certified Graduate Builder (professional builder designation)CGB Consumer and Governmental Affairs Bureau CGB Commonwealth Geographical Bureau (UK) CGB Game Boy Color ) medium (12) was determined after incubation at 24[degrees]C for 6 and 15 days. The serotype of CBS10496 was determined by using the CryptoCheck serotyping kit (Iatron Laboratories, Tokyo, Japan). Ten colonies of CBS10496 were used for DNA extraction (10). DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. of these colonies was used for amplified fragment length polymorphism Amplified fragment length polymorphism PCR, or "AFLP-PCR" (often AFLP), is a tool used in the study of genetics and in the practice of genetic engineering. Amplified Fragment Length Polymorphism (AFLP (AFLP) analysis (7). The partial sequence of 6 nuclear regions was determined for reference isolates CBS10488--CBS10490, CBS1622, CBS6992, and the putative hybrid isolate CBS10496. Selected nuclear regions were those for internal transcribed spacer ITS (for internal transcribed spacer) refers to a piece of non-functional RNA situated between structural ribosomal RNAs (rRNA) on a common precursor transcript. Read from 5' to 3', this polycistronic rRNA precursor transcript contains the 5' external transcribed sequence (5' ETS), (ITS) region, intergenic spacer region, laccase Laccases (EC 1.10.3.2) are copper containing oxidase enzymes that are found in many plants, fungi and microorganisms. The copper can be bound in several sites; Type 1, Type 2, and/or Type 3. When types 2 and 3 are bound together, it is called a trinuclear cluster. (CNLAC1), 2 RNA polymerase II RNA polymerase II (also called RNAP II and Pol II) is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. subunits (RPBI and RPB RPB Ruimtelijk Planbureau (Dutch: The Netherlands Institute for Spatial Research) RPB Regional Planning Body (UK) RPB Reverse-Path Broadcasting RPB Radiation Protection Bureau (Canada) 2), and translation elongation factor 1 [alpha] (TEFI [alpha]) (9,10). Mating types and serotype were determined as described (10,13). DNA content of CBS10496 was compared with that of CBS8710 and CBS10510. The G1 peak of reference strains was located at positions 31.6 (CBS8710) and 31.1 (CBS10510), and the G2 peak was located at positions 65.8 (CBS8710) and 56.4 (CBS10510). The G1 peak of CBS10496 was located at position 57.5, and the G2 peak was located at position 115.7. Thus, the G1 peak of CBS10496 coincided with the G2 peak of the haploid strains (Figure 1, panel A), which indicates that CBS 10496 has [approximately equals] 2x more DNA than haploid strains. We concluded that CBS10496 is diploid diploid /dip·loid/ (dip´loid) 1. having two sets of chromosomes, as normally found in the somatic cells; in humans, the diploid number is 46. 2. an individual or cell having two full sets of homologous chromosomes. or aneuploid an·eu·ploid n. A cell or an organism characterized by aneuploidy. Aneuploid An abnormal number of chromosomes in a cell. . Staining with 4',6-diamidino-2-phenylindole showed that cells of CBS 10496 were monokaryotic (Figure 1, panel B). Reaction of CBS10496 on CGB medium was negative, which corresponds to C. neoformans (12). The Crypto Check serotyping kit serum factors 5 (corresponding to serotype B) and 7 (corresponding to serotype A) agglutinated, which indicated that CBS10496 is a serotype AB strain. The AFLP fingerprint obtained by analysis of colonies of CBS10496 did not match any of the previously defined AFLP genotypes. The fingerprint of CBS10496 was compared with AFLP fingerprints of reference strains CBS8710 and CBS9172, which are AFLP1/VNI, and E566 and CBS 10510, which are AFLP4/VGI. The AFLP fingerprint of CBS10496 contained fragments characteristic of AFLP1/VNI and AFLP4/VGI (Figure 2), which indicated that genetic material from these 2 genotypes was present in this isolate. Two alleles representing AFLP1/VNI and AFLP4/VGI were found when fragments of RPB1, RPB2, CNLAC1, and intergenic spacer region of CBS10496 were cloned and sequenced. However, after 30 clones were sequenced, only 1 allele allele (əlēl`): see genetics. allele Any one of two or more alternative forms of a gene that may occur alternatively at a given site on a chromosome. was obtained for TEF TEF Tracheoesophageal fistula, see there 1[alpha], i.e., AFLP4/VGI, and ITS, i.e., AFLP1/VNI. Our results indicate that genetic material from AFLP1/VNI and AFLP4/VGI was present in CBS10496, although only 1 allele was obtained for TEF1 [alpha] and ITS. All sequences were submitted to GenBank (accession nos. DQ286656-DQ286676 and EF102027EF102072). [FIGURE 1 OMITTED] Amplification of CBS10496 in a PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) with the MAT[alpha] and the MAT[alpha] serotype A-specific primer pair resulted in an amplicon. When MATa and the MATa serotype A-specific PCRs were conducted, no amplicon was obtained. These findings indicate that CBS10496 has a MAT[alpha] serotype A background. All reference strains yielded amplicons with the expected primer pairs. In addition, CBS 10510, a MAT[alpha] serotype B strain, was amplified with the MAT[alpha] specific primer pair, and E566, a MATa serotype B strain, yielded an amplicon with the MATa-specific PCR. These results indicate that a C. gattii and a MAT[alpha] serotype A background are present in CBS10496. Because the mating type of the C. gattii background within CBS10496 was unknown, 30 MAT[alpha] clones of CBS10496 were sequenced to determine whether a MAT [alpha] serotype B allele could be identified. However, all clones were MAT [alpha] serotype A; no MAT [alpha] serotype B clones were found. [FIGURE 1 OMITTED] Conclusions Our results indicated that CBS10496 is a monokaryotic, diploid, or aneuploid strain with the novel AB serotype. AFLP and sequence analysis showed that the isolate contained fragments of C. neoformans var. grubii (AFLP1/ VNI VNI Visual Numerics Inc VNI Valeur Nette d'Inventaire (French) VNI Village Networks Inc. VNI Virtual Network Interface VNI Viet Nam international VNI Visual Numerics Inc. ) and C. gattii (AFLP4/VGI). We conclude that this isolate is a novel aneuploid hybrid of C. neoformans var. grubii (serotype A, AFLP1/VNI) and C. gattii (serotype B, AFLP4/VGI). CBS10496 had been identified as C. gattii on the basis of a weak positive reaction on CGB medium (11). Our results indicated that CBS 10496 was negative on CGB medium. Although a negative response on CGB medium has been shown for other C. neoformans x C. gattii hybrids (10,14), weak and delayed positive reactions on CGB medium may occur in C. neoformans x C. gattii hybrid isolates (10,14). CBS 10496 was previously identified as a serotype B strain (11). Inconsistent serotyping results have been reported for other hybrids (10,15) and may result from differences in specificity and potency among different batches of factor serum. All C. neoformans x C. gattii hybrids discovered have originated from clinical sources (13; F. Hagen and T. Boekhout, unpub, data). We expected that CBS10496 would have 2 matingtype loci loci [L.] plural of locus. loci Plural of locus, see there . However, only a serotype A MAT[alpha] background was observed. Although an amplicon was obtained with C. gattii--specific mating-type primers, the C. gattii background could not be linked to a mating type. We hypothesize hy·poth·e·size v. hy·poth·e·sized, hy·poth·e·siz·ing, hy·poth·e·siz·es v.tr. To assert as a hypothesis. v.intr. To form a hypothesis. that the serotype AB C. neoformans x C. gattii hybrid CBS10496 was formed by mating of a MATa serotype B strain with a MAT[alpha] serotype A strain and subsequent loss of the MATa serotype B allele. Detection of single ITS and TEF1[alpha] alleles in CBS10496 further supports our findings because it indicates that other alleles were also lost. Loss of genetic material has been observed in other hybrids, such as serotype AD and BD hybrids (14), and seems to be a normal process in cryptococcal hybrids. Our results show that the C. gattii parent of the serotype AB hybrid belongs to the AFLP4/VGI genotype, as was the case for serotype BD hybrids (10). The C. gattii parental sequence of all known serotype BD C. neoformans x C. gattii hybrid isolates was identical to sequences of AFLP4/VGI strains CBS 1622 and CBS6992 in all regions studied (9). Detection of 1 specific C. gattii--AFLP4/VGI subgroup in all isolated C. neoformans x C. gattii hybrids may indicate that this subgroup preferentially forms interspecies hybrids. DOI (Digital Object Identifier) A method of applying a persistent name to documents, publications and other resources on the Internet rather than using a URL, which can change over time. : 10.3201/eid1407.080122 The research of M.B. and F.H. is supported by the Odo van Vloten Foundation. E.E.K. is supported by the Renewal Fund of the Royal Netherlands Academy of Arts and Sciences The Koninklijke Nederlandse Akademie van Wetenschappen (KNAW, Royal Netherlands Academy of Arts and Sciences) is an organisation dedicated to the advancement of science and literature in the Netherlands. . Dr Bovers participated in this study while conducting doctoral research at the Centraalbureau voor Schimmelcultures--Fungal Biodiversity Centre in Utrecht. She is currently working at the Netherlands Commission on Genetic Modification in Bilthoven. Her research interests include the epidemiology and phylogeny of pathogenic yeasts. References (1.) Speed B, Dunt n. 1. A blow. D. Clinical and host differences between infections with the two varieties of Cryptococcus neoformans. Clin Infect Dis. 1995;21:28-34. (2.) Sonell TC. Cryptococcus neoformans variety gattii. Med Mycol. 2001;39:155-68. DOI: 10.1080/714031012 (3.) Kwon-Chung KJ, Bennett JE. Epidemiological differences between the two varieties of Cryptococcus neoformans. Am J Epidemiol. 1984;120:123-30. (4.) Kidd SE, Hagen F, Tscharke RL, Huynh M, Bartlett KH, Fyfe M, et al. A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). Proc Natl Acad Sci U S A. 2004;101:17258-63. DOI: 10.1073/ pnas.0402981101 (5.) MacDougall L, Kidd SE, Galanis E, Mak S, Leslie MJ, Cieslak PR, et al. Spread of Cryptococcus gattii in British Columbia, Canada, and detection in the Pacific Northwest, USA. Emerg Infect Dis. 2007;13:42-50. (6.) Viviani MA, Cogliati M, Esposto MC, Lemmer K, Tintelnot K, Colom Valiente MF, et al. European Confederation of Medical Mycology (ECMM ECMM European Community Monitor Mission (now European Union Monitoring Mission; diplomatic mission to Bosnia and Herzegovina starting 1991) ) Cryptococcosis Working Group. Molecular analysis of 311 Cryptococcus neoformans isolates from a 30-month ECMM survey of cryptococcosis in Europe. FEMS FEMS Federation of European Microbiological Societies FEMS Federation of European Materials Societies FEMS Fabrication Engineering Management System FEMS Facility Equipment Maintenance System (PMEL/TMDE) Yeast Res. 2006;6:614-9. DOI: 10.1111/j. 1567-1364.2006.00081 .x (7.) Boekhout T, Theelen B, Diaz M, Fell JW, Hop WC, Abeln EC, et al. Hybrid genotypes in the pathogenic yeast Cryptococcus Cryptococcus /Cryp·to·coc·cus/ (-kok´us) a genus of yeastlike fungi, including C. neofor´mans, the cause of cryptococcosis in humans.cryptococ´cal Cryp·to·coc·cus n. neojormans. Microbiology. 2001;147:891-907. (8.) Meyer W, Castaneda A, Jackson S, Huynh M, Castaneda E. the IberoAmerican Cryptococcal Study Group. Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerg Infect Dis. 2003;9:189-95. (9.) Bovers M, Hagen F, Kuramae EE, Boekhout T. Six monophyletic monophyletic /mono·phy·let·ic/ (mon?o-fi-let´ik) descended from a common ancestor or stem cell. mon·o·phy·let·ic adj. 1. Descended or derived from one original stock or source. lineages identified within Cryptococcus neoformans and Cryptococcus gattii by multi-locus sequence typing. Fungal Genet genet: see civet. Biol. 2007;45:400-21. DOI: 10.1016/j.fgb.2007.12.004 (10.) Bovers M, Hagen F, Kuramae EE, Diaz MR, Spanjaard L, Dromer F, et al. Unique hybrids between fungal pathogens Cryptococcus neoJbrmans and Cryptococcus gattii. FEMS Yeast Res. 2006;6:599607. DOI: l 0.1111/j. 1567-1364.2006.00082.x (11.) St-Germain G, No61 G, Kwon-Chung KJ. Disseminated cryptococcosis due to Cryptococcus neoformans variety gattii in a Canadian patient with AIDS. Eur J Clin Microbiol Infect Dis. 1988;7:587-8. DOI: 10.1007/BF01962626 (12.) Kwon-Chung K J, Polacheck I, Bennett JE. Improved diagnostic medium for separation of Cryptococcus neoformans vat. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C). J Clin Microbiol. 1982;15:535-7. (13.) Barreto de Oliveira MT, Boekhout T, Theelen B, Hagen F, Baroni FA, Lazera MS, et al. Cryptococcus neoformans shows a remarkable genotypic diversity in Brazil. J Clin Microbiol. 2004;42:1356-9. DOI: 10.1128/JCM.42.3.1356-1359.2004 (14.) Kwon-Chung KJ, Varma A. Do major species concepts support one, two or more species within Cryptococcus neoformans? FEMS Yeast Res. 2006;6:574-87. DOI: 10.111l/j.1567-1364.2006.00088.x (l5.) Viviani MA, Wen H, Roverselli A, Caldarelli-Stefano R, Cogliati M, Ferrante P, et al. Identification by polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is fingerprinting of Cryptococcus neoformans serotype AD. J Med Vet Mycol. 1997;35:355-60. DOI: 10.1080/02681219780001411 Address for correspondence: Ferry Hagen, Centraalbureau voor Schimmelcultures--ungal Biodiversity Center, Uppsalalaan 8, NL-3584CT Utrecht, the Netherlands; email: f.hagen@cbs.knaw.nl Marjan Bovers, * (1,2) Ferry Hagen, * ([dagger]) (1) Eiko E. Kuramae, * Hans L. Hoogveld, ([double dagger]) Francoise Dromer, ([section]) Guy St-Germain, (paragraph]) and Teun Boekhout * ([dagger]) * Centraalbureau voor Schimmelcultures--Fungal Biodiversity Centre, Utrecht, the Netherlands; ([dagger]) University Medical Centre Utrecht, Utrecht; ([double dagger]) Netherlands Institute of Ecology, Nieuwersluis, the Netherlands; ([section]) Institut Pasteur, Paris, France; and [paragraph] Laboratoire Sante Publique Quebec, Sainte-Anne-de-Bellevue, Quebec, Canada (1) These authors contributed equally to this article. (2) Current affiliation: Netherlands Commission on Genetic Modification, Bilthoven, the Netherlands.
Table. Cryptococcus spp. strains used in this study *
Mating type AFLP/M13
([double dagger]) genotype
Strain ([dagger]) /serotype ([section])
CBS9172 aA AFLP1/VNI
CBS8710 (H99) [alpha] A AFLP1/VNI
CBS1622 [alpha] B AFLP4/VGI
CBS6992 [alpha] B AFLP4/VGI
E566 aB AFLP4/VGI
CBS10510 (WM276) [alpha] B AFLP4/VGI
CBS10488 (AMC770616) [alpha] BaD AFLP8/NA
CBS10489 (AMC2010404) [alpha] BaD AFLP8/NA
CBS10490 (AMC2011225) [alpha] BaD AFLP8/NA
CBS10496 (LSPQ#308) [alpha] A-B AFLP9/NA
Strain ([dagger]) Source
CBS9172 Soil sample from garden of
patient with neighboring
bird colonies
CBS8710 (H99) Patient with Hodgkin disease
CBS1622 Tumor
CBS6992 Human
E566 Eucalyptus camaldulensis
CBS10510 (WM276) Debris of E. tereticornis
CBS10488 (AMC770616) Human CSF
CBS10489 (AMC2010404) Human CSF
CBS10490 (AMC2011225) Human CSF
CBS10496 (LSPQ#308) Blood of an HIV-positive patient
Strain ([dagger]) Country
CBS9172 Italy
CBS8710 (H99) United States
CBS1622 France
CBS6992 Unknown
E566 Australia
CBS10510 (WM276) Australia
CBS10488 (AMC770616) The Netherlands
CBS10489 (AMC2010404) The Netherlands
CBS10490 (AMC2011225) The Netherlands
CBS10496 (LSPQ#308) Canada
* AFLP, amplified fragment length polymorphism; CBS, Fungal
Biodiversity Centre; AMC, Netherlands Reference Laboratory for
Bacterial Meningitis, Academic Medical Center, Amsterdam, the
Netherlands; NA, not applicable; CSF, cerebrospinal fluid.
([dagger]) Origin and genetic composition of the strains are indicated.
([double dagger]) --, mating type unknown.
([section]) AFLP fingerprint genotype (7), followed by corresponding
M13 PCR fingerprint genotype (8).
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