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Approaches for molecular identification and typing of the Cryptococcus species complex: an update

Feng, Xiaoboa; Yao, Zhironga; Liao, Wanqingb

Reviews in Medical Microbiology: January 2013 - Volume 24 - Issue 1 - p 1–6
doi: 10.1097/MRM.0b013e32835736c1
Original Articles

The Cryptococcus species complex is composed of two common pathogenic species, namely Cryptococcus neoformans and Cryptococcus gattii, which cause systemic infections in both immunocompromised and immunocompetent individuals. Both species have a bipolar mating system, with mating type (MATα) being predominant in clinical and environmental isolates. On the basis of genetic variation, this species complex is divided into eight major genotypes, which show differences in epidemiology, biology, virulence and antifungal susceptibility. Within each major genotype, more diverse subgroups have been discovered and could be related to specific pathogenic attributes. This review will discuss the methods that have been developed in recent years to characterize the MATs, genotypes, varieties, species and serotypes established previously in this microorganism.

aMedical Mycology Laboratory, Xinhua Hospital, Shanghai Jiaotong University School of Medicine

bShanghai Key Laboratory of Molecular Medical Mycology, Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, People's Republic of China.

Correspondence to Zhirong Yao, Medical Mycology Laboratory, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, People's Republic of China. Tel: +86 02125078999 ext. 6428; e-mail:

Received 7 May, 2012

Accepted 20 June, 2012

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The Cryptococcus species complex contains two species, namely Cryptococcus neoformans and Cryptococcus gattii, which are designated based on their differences in phenotype, biology and cladistics. A bipolar mating system with two opposite mating types, MATα and MATa, exists in both species. Several studies have demonstrated that MATα strains of C. neoformans display a hypervirulent trait in animal models, and have an enhanced predilection to penetrate the central nervous system during coinfection with MATa strains [1]. Additionally, MATα often outnumbered MATa in clinical or environmental isolates of either C. neoformans or C. gattii[2]. Phylogenetic analyses and molecular typing of a large collection of global isolates showed that the Cryptococcus species complex could be divided into eight major genotypes [3,4]. The relationship of the major genotypes with the previously established varieties and serotypes are as follows: VNI and VNII (C. neoformans var. grubii, serotype A); VNIII (AD hybrid, serotype AD); VNIV (C. neoformans var. neoformans, serotype D); and VGI, VGII, VGIII and VGIV (C. gattii, serotypes B or C) [3]. Apart from the established eight major genotypes, a restricted genotype VNB belonging to C. neoformans var. grubii from Botswana and a few novel hybrids such as AαDα and C. neoformans × C. gattii have been reported in recent studies [2,5–8]. Within each major genotype, diverse subgroups had been discovered and could be related to specific pathogenic attributes [9–11]. Differences in epidemiology, pathogenicity, biology, clinical features and drug susceptibility have been reported to be associated with species, variety, serotype, MAT and genotype in the Cryptococcus species complex [3,5,9]. This article will focus on the molecular tools that have been developed for the differentiation of these subtypes and methods used to study the molecular epidemiology or population structure of this pathogenic yeast.

To date, several methods have been developed for molecular typing of Cryptococcus species complex. For any method not described in this review, please refer to reference [12]. In these techniques, PCR fingerprinting, PCR with specific primers, PCR–restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR were popular in many studies due to their easy-to-use and low-cost qualities [3,13–18]. Amplified fragment length polymorphism (AFLP), multilocus sequence typing (MLST) or multilocus microsatellite typing (MLMT) is a more discriminative tool and is widely used in research on the molecular epidemiology or population structure of the pathogen [5,9,19,20]. New methods have been established in recent years including capillary electrophoresis, Luminex xMAP technology, loop-mediated isothermal amplification (LAMP), hyperbranched rolling circle amplification (HRCA), high-resolution melting (HRM) and matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS); all show promise and may have the potential in future laboratory diagnosis [21–26]. We shall describe a variety of identification and genotyping techniques and classify them according to the purpose for which they are performed.

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Molecular identification of species, variety or serotype

The two species C. neoformans and C. gattii, and varieties of grubii and neoformans, comprise the Cryptococcus species complex. Although serotype does not reflect the genetic structure of C. gattii, it is still used for delineation of C. neoformans[13]. PCR with specific PCR primers was developed earlier for identification of the species or varieties within this species complex. In a study, the gattii-specific and neoformans-specific primers were designed to amplify the PLB1 and SOD1 genes for differentiation of the two species. In addition, PCR with serotype-specific primers targeting the GPA1 or PAK1 allele form A or D serotype has been undertaken [16]. For performing identification in one PCR reaction, Leal et al.[18] reported a multiplex PCR assay in which 695 bp and 448 bp products were amplified for C. neoformans and C. gattii, respectively. The method successfully identified 125 C. neoformans isolates and 30 C. gattii isolates. Another study used a set of four primers designed for the LAC1 gene to differentiate serotypes A, D, B and C, and a primer pair designed for the CAP64 gene to allow serotypes D and AD to be differentiated [27]. A real-time PCR method for detection and identification of the two species was developed and evaluated in another study in which two TaqMan minor groove binder probes that distinguished between the two species were designed based on the CAP59 gene. The real-time PCR assay showed 100% specificity as evaluated against 13 reference and 300 environmental strains [28]. PCR-RFLP analysis represents a simple method and has been established in several studies for characterization of the species, variety and serotypes in the Cryptococcus species complex. Several genes such as CAP59, CAP10 and CAP64 that are required for capsule biosynthesis were chosen for RFLP analyses to differentiate between the distinct serotypes. Although reliable results were obtained in differentiation of serotype A, D, AD and BC, serotype B or C could not always be successfully discriminated [29,30].

Several new techniques were established and applied for differentiation between species or varieties. Kaocharoen et al.[24] developed four species-specific padlock probes targeting the single nucleotide polymorphisms at the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene locus for isothermal HRCA analysis. The probes were tested against 99 samples, including 94 clinical cryptococcal cultures, three closely related Cryptococcus species and two clinical specimens. The use of the padlock probes and the combination of probe signal amplification by HRCA provided a quick and sensitive assay for the accurate identification of C. neoformans var. grubii, C. neoformans var. neoformans and C. gattii. Moreover, approaches using DNA LAMP targeting the CAP59 allele and a two-step method based on HRM targeting the ITS1 region were reported for differentiation of C. neoformans var. grubii, C. neoformans var. neoformans and C. gattii[23,25]. Apart from PCR-based or DNA-based techniques, MALDI-TOF MS was reported to correctly identify 100% (113 of 113) of the two species, and distinguish 98.8% (85 of 86) isolates at the variety level for C. neoformans[26].

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Determination of mating type

The sexual development and virulence of the Cryptococcus species complex is controlled by a bipolar mating system determined by a MAT locus. The MAT locus is unusually large and contains more than 20 genes. The dramatic divergence between α and a alleles favors the identification of MATs by PCR analysis with specific primers. Among the genes, STE12, STE20 and MF were mostly used as targets in PCR analysis. For determination of MAT in C. gattii, it was established by PCR with primers specific to the STE20a, STE12α, MFa and MFα genes within MAT locus reported by Fraser et al.[31]. In another study, the MFα primers were designed from the MFα gene and were expected to amplify a 109-bp fragment from C. gattii MATα strains. The STE12α primers were designed from the STE12α gene and were expected to amplify a 150-bp fragment from C. gattii MATα strains [32]. On the basis of the previous work [31], Halliday et al.[33] developed a multiplex PCR method in which primers MFα and STE20aSF targeted at MFα and STE20a genes were conducted in one reaction to coamplify the opposite alleles in C. gattii. Furthermore, primers specific to MFα and MFa genes were developed to identify the two MATs either in C. neoformans or C. gattii[34]. For identification of MATs in C. neoformans, including various varieties and AD hybrids, primers specifically amplifying alleles of STE3a, STE3α, STE11a, STE11α, STE12α and STE12a were designed in several studies [2,35]. In addition, Cogliati et al.[36] reported a method of PCR and dot blotting using the genes MFα and MFa as probes and identified 36 of the 49 diploid isolates belonging to C. neoformans as MATa/α.

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Differentiation of major genotypes/cryptic species

Eight major genotypes in which VNI–VNIV to C. neoformans and VGI–VGIV to C. gattii have been established based on the genetic divergence; genotypes VNI, VNII, VNIV, VGI, VGII, VGIII and VGIV are now considered as genetically distinct cryptic species within the species complex. A number of molecular tools were developed to characterize the major genotypes. So far, PCR fingerprinting using a single primer M13 specific to minisatellite DNA is used as the standard for identification of the major genotypes [3]. AFLP genotyping, a more discriminatory tool also targeting the whole genome, could also identify the major genotypes [19]. Later, two PCR-RFLP approaches were established for identification. A RFLP analysis of PLB1 gene fragment digested with enzyme AvaI allowed identification of the eight major genotypes as the corresponding RFLP profiles A1–A8 [13]. Another URA5 gene RFLP analysis with enzymes HhaI and Sau96I in a double digestion grouped all 340 isolates into eight major genotypes [3]. Moreover, a PCR-based method with specific primers targeted at the intergenic spacer (IGS) region was developed to rapidly identify the VGII major genotype, the emerging pathogen related to the Vancouver Island outbreak [37].

Sequencing analysis has also been used as a more accurate tool for the differentiation of the major genotypes. The ITS region often used to identify fungal species was chosen for comparison of the distinct major genotypes by Katsu et al.[38]. In that study, the ITS types were found to correlate with PCR fingerprint genotypes; with ITS type 1, C. neoformans var. grubii, genotypes VNI and VNII, the serotype A allele of the AD hybrid and VNIII; ITS type 2, the serotype D allele of the AD hybrid, VNIII, C. neoformans var. neoformans and VNIV; ITS type 3 and ITS type 7, VGI; ITS type 4, VGII; ITS type 5, VGIII; and ITS type 6, VGIV; all corresponding to C. gattii. Diaz et al.[39] performed a phylogenetic analysis of the IGS region containing IGSI, IGSII and 5S rRNA, which revealed the presence of six major phylogenetic lineages that are also consistent with the major genotypes. MLST also proved the presence of haploid major genotypic groups in the species complex, corresponding to other typing methods described above [40].

Molecular hybridization provides a high-throughput technique for detection of pathogens. In one study, the assay used a liquid suspension hybridization format with specific oligonucleotide probes, which derived from the IGS region of the rRNA gene, are bound to fluorescent color-coded microspheres. The assay proved to be specific and sensitive, allowing discrimination of a 1-bp mismatch with no apparent cross-reactivity [41]. In a subsequent study, a Luminex suspension array was developed and tested by genotyping a set of 58 clinical isolates in which all major genotypes were included; all haploid strains were correctly assigned to the major genotypes [22].

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Characterization of variety and mating type or genotype and mating type

Due to the fact that genes within the MAT locus diverged not only according to the bipolar mating system but also according to the monophyletic lineages within the species complex, analysis of sequence traits of the genes has the potential to simultaneously delineate the MAT and genotypes. Lengeler et al.[15] developed serotype-specific and mating-type specific primers based on MF, STE11, STE12 and STE20 genes that were located at the MAT locus. This method has been widely used in later studies [2,8]. A multiplex PCR method was designed to distinguish the six MAT patterns (Aα, Dα, Aa, Da, αADa and aADα) of C. neoformans based on the NAD4, STE20 and NCP1 genes. PCR amplification identified one fragment for Aa (860 bp), Dα (413 bp) and Da (645 bp) strains; two fragments for Aα (320 and 400 bp) and aADα (860 and 413 bp) strains; and three fragments (645, 400, 320 bp) for an αADa strain [17]. In another study, sequence analysis of the MF gene revealed unique restriction enzyme sites in C. neoformans and C. gattii, after which 60 strains yielded three variety-specific or species-specific restriction patterns by RFLP analysis [34]. In our research, two PCR-RFLP analyses based on the CAP1 and GEF1 genes, which are both located at the MAT locus, were developed for simultaneous identification of the major genotypes and MATs of isolates belonging to the Cryptococcus species complex. The genotypes and MATs of all 144 cryptococcal isolates were successfully determined by both PCR-RFLP approaches. Additionally, pattern analysis of the AD hybrids revealed that the serotype A MATa allele in strains of aADα derived from genotype VNB, whereas the serotype A MATα allele among strains of αADα and αADa derived from genotype VNI [14].

In addition, Chaturvedi et al.[21] reported that MFα and MFa gene fragments were amplified with fluorescently labeled primers and analyzed by capillary electrophoresis on a DNA analyzer. Capillary electrophoresis–fragment length analysis (CE-FLAs) and capillary electrophoresis–single-strand conformation polymorphisms (CE-SSCPs) were both used to determine C. gattii, C. neoformans var. neoformans and grubii, MATs and hybrids; all 276 clinical strains tested were haploid MATα by CE-FLA. CE-SSCP analysis of MFα gene showed that 219 (79.3%) was C. neoformans var. grubii, 23 (8.3%) C. neoformans var. neoformans and 34 (12.3%) C. gattii isolates.

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Methods used for highly discriminatory typing

In many studies, AFLP genotyping, MLST and MLMT were confirmed as having potential values in highly discriminatory typing, molecular epidemiology and population genetic analysis. Previously, an AFLP analysis of the Cryptococcus species complex was firstly established by Boekhout et al.[19], and this tool has been extensively used in many studies [2,6,7,20]. In the following study, Litvintseva et al.[5] analyzed a global collection of isolates of serotype A using AFLP and MLST methods, and the results identified at least three genetically distinct subpopulations, designated groups: VNI, VNII and VNB. The MLST typing scheme using 12 polymorphic loci consisting of MPD1, TOP1, MP88, CAP59, URE1, PLB1, CAP10, GPD1, TEF1, SOD1, LAC1 and IGS1 for C. neoformans var. grubii was described for the first time and applied to type the sequence types for 102 isolates. The discriminatory power between these two methods was compared, and comparable results were obtained. In another study, MLST was conducted using five gene fragments including CAP1, FTR1, LAC, ITS and MtLrRNA for each of 78 isolates of C. neoformans var. grubii[42]. A panel of eight polymorphic loci of MLST for 202 C. gattii isolates was firstly described by Fraser et al.[9]. Subsequently, many studies used this method to genotype C. gattii isolates either for molecular epidemiology research or population structure analysis, especially for following the C. gattii outbreak on Vancouver Island [10,11,43]. In another two studies, MLST using four unlinked nuclear loci consisting of ACT1, IDE, PLB1 and URA5, and five loci of ITS, IGS, RPB1, RPB2, LAC1 and TEF1, was performed to analyze the population structure of C. neoformans and C. gattii[4,40]. Selection of distinct loci for typing in different studies hampered data exchange between laboratories and further analysis, the International Society for Human and Animal Mycology working group on ‘Genotyping of C. neoformans and C. gattii’ proposed a set of genetic loci including the housekeeping genes CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region as an international standard for MLST for C. neoformans and C. gattii, so that the sequence data are accessible and exchangeable by all researchers [44].

MLMT has been widely used for typing fungi due to its high discriminatory power, and it could be an alternative and reproducible method suitable for large-scale studies of pathogen epidemiology. For typing of C. neoformans, two microsatellite panels were developed. Hanafy et al.[45] selected three (designated CNG1, CNG2 and CNG3) microsatellite loci that were polymorphic from the H99 C. neoformans var. grubii genome. This method was applied to sequence 87 clinical and environmental C. neoformans var. grubii isolates from 12 different countries based on MLMT and the discriminatory power value was 0.992. The PCR primers of the polymorphic microsatellites amplified those loci only from strains of C. neoformans (C. neoformans var. grubii, C. neoformans var. neoformans and the AD hybrid) but not from C. gattii, suggesting a species-specific association [45]. This method was further applied to investigate the genetic relationship between clinical and environmental isolates of C. neoformans var. grubii in a study in Brazil. However, no close genetic association was found between clinical and environmental isolates [46]. In another typing scheme, nine microsatellite markers were selected for high-resolution fingerprinting in C. neoformans var. grubii. This panel of markers was applied to a collection of clinical (n = 122) and environmental (n = 68) C. neoformans var. grubii isolates from Cuba. In that study, the AFLP analysis was less discriminatory than the MLMT by which a total of 104 genotypes were distinguished. The discriminatory power value of that panel was 0.993. In phylogenetic analysis, the majority of environmental isolates (>70%) fell into one microsatellite complex containing only few clinical isolates. Clinical isolates were divided into various microsatellite complexes [20]. In their subsequent study, 19 isolates containing sequential isolates were genotyped using the panel of nine microsatellite markers, and the pattern analysis showed 14 distinctive profiles. In three patients, the recurrent infection was associated with genotypically identical isolates, whereas four other patients had relapse isolates, which were genotypically different from the initial strain [47]. In addition, 11 microsatellite loci were developed from the published genomes of C. neoformans var. neoformans to genotype the varieties and hybrids within C. neoformans[48].

For genotyping of C. gattii, Byrnes et al.[43] developed three variable number of tandem repeat (VNTR) markers (designated VNTR3, VNTR7 and VNTR15) with additional loci in which VNTR34 was for VGII and MS1 for VGI to genotype VGII (n = 38) and VGI (n = 18) major genotypes of isolates. The two studies combined MLST and VNTR markers for typing. Byrnes et al.[10] found that the VNTR markers were more polymorphic than the MLST markers. However, unfortunately, discriminatory power value was not described in those two studies.

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Identification of unusual hybrids

In previous reports, C. neoformans and C. gattii are usually haploid strains except for AD hybrids that are diploid or aneuploid [15]. Although the role of hybrids in nature is not very clear, AD hybrids exhibit hybrid vigor and are more resistant to ultraviolet light and high temperature [49]. In AD hybrids, mating patterns of αADa and aADα were reported as being the most representative [15]. However, a population of AD hybrids named H strains, which have double DNA content but only single mating-type allele, and novel αADα strains, arising from same-sex mating, were characterized [35,49]. Furthermore, several other studies discovered multiple hybrid types in which interspecies (αABa and αBDa) or intravarietal (αAAα) hybrid were included [7,8,50].

To characterize unusual hybrid strains, several molecular techniques combined with ploidy determination are involved. Among the tools, PCR fingerprinting, RFLP and AFLP analyses could characterize these hybrids due to their mixed profiles from corresponding haplotypes. In one report, novel AB hybrids could be detected by PCR-RFLP analysis of URA5 and CAP59 genes [8]. Our assay detected the αADα hybrids by RFLP analysis of either the CAP1 or GEF1 gene in which the restriction profile of αADα consisted of profiles from Aα and Dα. AFLP and PCR fingerprinting were also powerful in determination of hybrids between distinct major genotypes, and performed in identification of AB, BD and AA hybrids [6–8,50]. Additionally, serotype-specific, mating-type-specific or serotype-specific and mating-type-specific primer was conducted in separate PCRs to find these novel hybrids in several studies [6,8,49]. As well as the molecular tools mentioned earlier, Luminex suspension array, comparative genome hybridization and multigene sequencing using TA cloning were also successfully performed to verify these hybrids [6–8,49,50].

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The two species, varieties, MATs and major genotypes/cryptic species within the Cryptococcus species complex are well established and verified by many molecular approaches. Techniques with the characteristics of high-throughput, rapid, accurate identification and low cost will be increasingly used for diagnosis in microbiology laboratories. To study the molecular epidemiology of the species complex, highly discriminatory and standard typing techniques such as MLST and MLMT will be more applied in the future owing to their reproducible and interchangeable molecular data. Unlike other major genotypes, research on subtyping of VGIII and VGIV in C. gattii is less common. Elucidation of the derivation of novel hybrid or diploid strain is ongoing; its impact on evolution of this pathogenic yeast and clinical relevance remains to be determined.

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Conflicts of interest

This work was supported by grants from the National Natural Science Foundation of China (31000549) and Outstanding Young Teacher Foundation of Shanghai (jdy09131).

There are no conflicts of interest.

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Cryptococcus gattii; Cryptococcus neoformans; genotype; mating type; molecular typing; PCR

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