Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii

Abstract

This communication describes the consensus multi-locus typing scheme established by the Cryptococcal Working Group I (Genotyping of Cryptococcus neoformans and C. gattii) of the International Society for Human and Animal Mycology (ISHAM) using seven unlinked genetic loci for global strain genotyping. These genetic loci include the housekeeping genes CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region. Allele and sequence type information are accessible at http://www.mlst.net/.

Introduction

Cryptococcus neoformans, the agent of cryptococcosis, had been considered a homogeneous species until 1949 when the existence of four serotypes was revealed based on the antigenic properties of its polysaccharide capsule [1]. Such heterogeneity of the species, however, remained obscure until the two morphologically distinct teleomorphs of C. neoformans were discovered during the mid 1970s [2,3]. The teleomorph Filobasidiella neoformans was found to be produced by strains of serotype A and D [2] while F. bacillispora was found to be produced by strains of serotype B and C [3]. Ensuing studies revealed numerous differences between the anamorphs of the two Filobasidiella species with regards to their ecology, epidemiology, pathobiology, biochemistry and genetics.

Presently, the etiologic agent of cryptococcosis is classified into two species [4], C. neoformans, with two varieties: C. neoformans var. grubii (serotype A) [5] and C. neoformans var. neoformans (serotype D) [6], as well as an AD hybrid, and C. gattii (serotypes B and C) [7]. Intra-species genetic diversity has also been revealed as more genotyping methods have been applied for each serotype. In addition inter-species hybrid strains of AB and BD serotypes have been described [8,9]. As a result, the number of scientifically valid species within C. neoformans has become a controversial issue because of the differing opinions among taxonomists as to the appropriate definition of a species. There are several research groups focusing on the molecular determination of the number of genetically diverse sub-groups within each serotype. The molecular methods employed by each group to define these sub-groups vary from DNA fingerprinting [10,11] and PCR fingerprinting based on microsatellite- (M13) or minisatellite-specific primers (e.g., (GACA)₄ or (GTG)₅) [12–16], over random amplification of polymorphic DNA (RAPD) analysis [17–20], amplified fragment length polymorphism (AFLP) analysis [21–23], restriction fragment length polymorphism (RFLP) analysis of the URA5 [16,24] and PLB1 genes [25], the use of IGS sequences [26], multigene sequence analysis [27, Meyer et al. unpublished data], to multi-locus sequence typing (MLST) [23,28] and multi-locus microsatellite typing (MLMT) [29,30]. This research has revealed associations between geographic origin and particular genotypes, implying an epidemiologic significance of certain genotypes. Different methods have resulted in various numbers of sub-groups or different nomenclature of those sub-groups. However, due to the lack of a cross-reference consensus between the results obtained by different genotyping method, there is currently no concordance on a universally acceptable genotyping method for this important human pathogen.

Recognizing the urgent need for a standardized globally acceptable typing method, a Cryptococcus working group I, ‘Genotyping of Cryptococcus neoformans and C. gattii’, was formed under the umbrella of the International Society of Human and Animal Mycoses (ISHAM) in the beginning of 2007 which united all the major research groups that were involved in molecular strain typing of C. neoformans complex. The members of this ISHAM working group met at the 3rd Trends in Medical Mycology (TIMM3) Meeting in Torino, Italy in October 2007, and reviewed all the typing techniques in use. The group selected multi-locus sequence typing (MLST) as the method of choice for future strain typing in light of its high discriminatory power as well as reproducibility between different laboratories. The working group also chose standard reference strains representing the eight known major molecular types of the agent of cryptococcosis as well as the nomenclature of each genotype.

Consensus genotype nomenclature

As a result of the Torino meeting, the working group recognized that the different genotyping methods used by the different research groups lead to corresponding major genotypes for the agents of cryptococcosis (Table 1). Principally, the two main typing systems being used are: PCR fingerprinting using primers specific for microsatellite (M13) [14,16] or minisatellite (GACA)₄ DNA [13,15] and AFLP analysis [21]. In both typing schemes, over 2000 isolates were grouped into eight major molecular types. With some exceptions [26,31], the molecular types of C. neoformans are correlated with the serotypes: C. neoformans var. grubii, serotype A, consists of molecular types VNI=AFLP1 and VNII=AFLP1A; the hybrid serotype AD comprises VNIII=AFLP3; and C. neoformans var. neoformans, serotype D, corresponds to VNIV=AFLP2. C. gattii consists of VGI=AFLP4, VGII = AFLP6, VGIII=AFLP5, and VGIV=AFLP7, which all correspond to both serotypes B or C [16,21, unpublished data]. Based on these findings, it was agreed by all cryptococcal working group members present in Torino to use the VNI–VNIV and VGI–VGIV nomenclature [16] since it correlated with the current concept of two species and represents the global population structure based on more than 2000 C. neoformans and C. gattii isolates among which C. neoformans var. grubii (serotype A=VNI) being the most prevalent molecular type world-wide.

Table 1.

Concordance of different molecular typing methods used for Cryptococcus neoformans and Cryptococcus gattii

Species/Variety/ Hybrid	Serotype	PCR- fingerprinting molecular type Meyer et al. [14,16,39]	PCR- fingerprinting molecular type Viviani et al. [13]	AFLP genotype Boekhout et al. [21]	AFLP genotype Litvintseva et al. [23]	URA5 RFLP type Meyer et al. [16]	PLB1 RFLP type Latouche et al. [25]	IGS genotype Diaz et al. [26,31]	ITS genotype Katsu et al. [40]
C. neoformans var. grubii	A	VNI	VN6 (VN5)	AFLP1	VNI	VNI	A1	1A/1B	ITS1
	A	VNII		AFLP1A/AFLP1B	VNB	VNII		1A	ITS1
	A	VNII	VN7	AFLP1A/AFLP1B	VNII	VNII	A2	1C	ITS1
AD Hybrid	AD	VNIII	VN3/VN4	AFLP3		VNIII	A3	2C	ITS1/ITS2
C. neoformans  var. neoformans	D	VNIV	VN1 (VN2)	AFLP2		VNIV	A4	2A/2B /2C	ITS2
C. gattii	B/C	VGI		AFLP4A/AFLP4B		VGI	A5	4	ITS3/ITS7
	B/C	VGII		AFLP6		VGII	A6	3	ITS4
	B/C	VGIII		AFLP5A/AFLP5B/ AFLP5C		VGIII	A7	5	ITS5
	B/C	VGIV		AFLP7		VGIV	A8	6	ITS6

Consensus standard strains

To enable global standardization, the working group also agreed to use a set of standard strains representing each of the eight major molecular types. This included the molecular type strains used in PCR fingerprinting or URA5-RFLP analysis [16] plus additional strains representing type cultures or strains, which are used in major cryptococcal genome projects (Table 2). All standard strains are publicly available from the CBS-Fungal Biodiversity Centre (CBS) (http://www.cbs.knaw.nl), the American Type Culture Collection (ATCC) (http://www.atcc.org) or the Fungal Genetic Stock Center (FGS) (http://www.fgsc.net). The corresponding collection numbers are listed in Table 2.

Table 2.

Standard/reference strains for Cryptococcus neoformans and Cryptococcus gattii strain typing

CBS #	ATCC#	FGS#	Other numbers	MAT & Serotype	Comments	References
Cryptococcus neoformans
Cryptococcus neoformans var. grubii
VNI (Meyer et al. [14,16])=AFLP1 (Boekhout et al. [21])=VN6 (VN5) (Viviani et al. [13])
CBS 10085	ATCC MYA-4564	10415	WM 148; W10; Brown	αA	1989, Australia, NSW, Sydney, clinical, CSF, HIV–, isolated by Sharon Chen	[14,18]
CBS 8710	ATCC 48922	9487	DUMC 135.97; H99; NYSD 1649; CBS 10515; WM 04.15	αA	1978, USA, NC, Durham, clinical, CSF, patient with Hodgkin’s lymphoma, isolated by John Perfect/Wiley Schell, type culture of C. neoformans var. grubii , genome sequence strain	[5]
VNII (Meyer et al. [14/16])=AFLP1A (Boekhout et al. [21])=VN7 (Viviani et al. [13])
CBS 10084	ATCC MYA-4565	10416	WM 626, W20; Cetin	αA	1993, Australia, NSW, Sydney, clinical, CSF, HIV–, isolated by Sharon Chen	[14,18]
AD hybrid
VNIII (Meyer et al. [14,16])=AFLP3 (Boekhout et al. [21])=VN33VN4 (Viviani et al. [13])
CBS 10080	ATCC MYA-4566	10417	WM 628; 88B5400; Zapf	αA/aD	1988, Australia, VIC, Melbourne, clinical, CSF, HIV+, isolated by Bryan Speed	[14,18]
CBS 132	ATCC 32045	-	CCRC 20528; DBVPG 6010; IFO 0608; IGC 3957; NRRL Y-2534	αA/aD	1894, Italy, environmental, fermenting fruit juice, isolated by F. Sanfelice, type culture for C. neoformans	[41]
Cryptococcus neoformans var. neoformans
VNIV (Meyer et al. [14,16])=AFLP2 (Boekhout et al. [21])=VN1 (VN2) (Viviani et al. [13])
CBS 10079	ATCC MYA-4567	10418	WM 629; B 87455, Borg, F 14	αD	1987, Australia, VIC, Melbourne, clinical, blood, HIV+, isolated by Bryan Speed	[14]
CBS 6900	ATCC 34873	10423	B-3501; DBVPG 6228; CBS 7697	αD	1975, USA, MD, Bethesda, NIH, crossing of NIH 12 x NIH 433, isolated by June Kwon-Chung	[42]
Cryptococcus gattii
VGI (Meyer et al. [16])=AFLP4 (Boekhout et al. [21])
CBS 10078	ATCC MYA-4560	10419	WM 179; Bryon; H33.1; MH56	αB	1993, Australia, NSW, Sydney, clinical, CSF, HIV −, isolated by Sharon Chen	[16,18]
CBS 6289	ATCC 32269	-	MUCL 30449, RV 20186; CBS 8273	αB	1966, Congo, Kinshasa, clinical, CSF, isolated by E. Gatti/R. Eeckels, type strain of C. neoformans var. gattii,	[43]
CBS 10510	-	-	WM 276; TCS -SC1	αB	1993, Australia, NSW, Mt Annan National Park, environmental, Eycalyptus tereticornis woody debris, isolated by Tania Sorrell/Sharon Chen, genome sequence strain	[16]
VGII (Meyer et al. [16])=AFLP6 (Boekhout et al. [21])
CBS 10082	ATCC MYA-4561	10420	WM 178; 49435; Colter; IFM 50894	αB	1991, Australia, NSW, Sydney, clinical, CSF, HIV −, isolated by Sharon Chen	[16]
CBS 10514	-	-	CDC R265; WM 02.32	αB	2001, Canada, BC, Duncan, Vancouver Island, clinical, bronchial wash, isolated by British Columbia CDC, high virulent Vancouver Island outbreak strain, VGIIa, genome sequence strain	[44]
VGIII (Meyer et al. [16])=AFLP5 (Boekhout et al. [21])
CBS 10081	ATCC MYA- 4562	10421	WM 175; WM 161; E698; 689; TP 0689; D1.13H	αB	1992, USA, California, San Diego, Blind Recreation Center/Park Boulevard UPAS street, environmental, Eucalyptus spp. woody debris, isolated by Tania Pfeifer/David Ellis	[16,19]
CBS 6955	ATCC 32608	10424	DBVPG 6225; MUCL 30454; NIH 191; CBS 6916	αC	Before 1970, USA, San Fernando, California, clinical, CSF.	[45]
VGIV (Meyer et al. [16])=AFLP7 (Boekhout et al. [21])
CBS 10101	ATCC MYA- 4563	10422	WM 779; King Cheetah; IFM 50896	αC	1994, South Africa, Johannesburg, veterinary, Cheetah, isolated by Valarie Davis	[16,46]

Consensus multi-locus sequence typing loci

To overcome problems arising from inter-laboratory reproducibility associated with the two commonly used typing techniques, such as PCR fingerprinting or AFLP analysis, the working group decided to use multi-locus sequence typing (MLST) as the method of choice for future cryptococcal strain typing. MLST has become the number one typing approach for epidemiological investigations of microorganisms [32]. MLST, originally developed for bacteria [32], indexes the sequence variation in approximately 400–500 bp of five to ten genes composed primarily of housekeeping genes. This technique has proven to be highly discriminatory for a number of human pathogenic fungi: C. albicans [33], C. glabrata [34], C. tropicalis [35], Coccidioides spp. [36] and Histoplasma capsulatum [37]. Most of the published MLST schemes are developed as tools for the wider scientific community, by being made publicly available as online databases at http://www.mlst.net/ and http://pubmlst.org/. In the case of the Cryptococcus species complex, two different MLST typing schemes have been introduced to type isolates of C. neoformans [23], and C. gattii [28], using twelve and eight unlinked loci respectively.

In the first study, 12 unlinked polymorphic loci: MPD1, TOP1, MP88, CAP59, URE1, PLB1, CAP10, GPD1, TEF1, SOD1, LAC1 and the IGS1 ribosomal RNA intergenic spacer region, which are dispersed on nine different chromosomes, were used to type 102 globally obtained serotype A strains [23]. MLST differentiated three major groups among the studied isolates, corresponding to VNI, VNII and VNB, a Botswana specific genotype closely related to VNI. In connection with this study a central web based database was created at www.mlst.net (http://cneoformans.mlst.net/) allowing for an online determination of the alleles and sequence types of C. neoformans serotype A strains.

The second study used eight unlinked polymorphic loci: SXIa or SXIα, IGS1, TEF1, GPD1, LAC1, CAP10, PLB1, and MPD1, of which two are mating type locus specific and can not be amplified for all strains, to type 202 C. gattii strains. These loci were supplemented for a more detailed analysis of 9 closely related strains by 22 additional gene loci: HOG1, BWC1, CNB1, TOR1, CAC1, CRG1, URE1, FHB1, BWC2, CNA1, CBP1, TSA1, STE7, FTR1, PAK1, CAP59, ICL1, GPA1, GPB1, RAS1, CCP1, and TRR1 to investigate the origin of the Vancouver Island outbreak isolates [28]. MLST differentiated all four major molecular types of C. gattii (VGI, VGII, VGIII and VGIV) and highlighted two possible origins (Australia or South America) for the outbreak strains.

Statistical analysis using the Simpsons’s index of diversity [38] revealed that for both previously studied MLST data sets, a minimum of seven loci are required to differentiate between the sequence types of all strains (Fig. 1). For the Litvintseva et al. [23] MLST data set, the following loci resulted in the highest discrimination of the investigated strains: CAP59, IGS1, GPD1, LAC1, PLB1, MP88 and SOD1, with a Simpson’s index of diversity of 0.9632. For the Fraser et al. [28] MLST data set, the most discriminatory loci were: GPD1, IGS1, TEF1, LAC1, MPD1, CAP10 and PLB1, which resulted in a Simpson’s index of diversity of 0.9319.

Fig. 1.

Number of genes necessary to differentiate all sequence types obtained in the two previously published cryptococcal MLST schemes based on Simpson’s index of diversity [38]. For the Fraser et al. [28] data set the mating type locus specific genes, SXIa or SXIα, have been excluded for this analysis since they can’t be amplified from all isolates.

Both MLST schemes utilized highly polymorphic loci, which resulted in stable and reproducible typing systems that were able to distinguish between closely related strains. While using as many genetic loci as possible would enhance the discriminatory power of the MLST scheme, it would be pragmatic to achieve the maximal level of differentiation with a minimal set of genetic loci. The ideal MLST scheme for the Cryptococcus species complex should fulfill two criteria: (i) it should amplify and type the same genes from all five serotypes/eight molecular types using the same set of primers, and (ii) the selected genes should contain sufficient sequence diversity to produce a discriminatory typing scheme. Taking these facts into account, the working group has selected a set of seven gene loci for a cryptococcal consensus MLST scheme based on the results obtained in the previously published studies by Litvintseva et al. [23], Fraser et al. [28], and additional unpublished data obtained by Meyer et al. and Fisher et al. Special emphasis was placed on using loci that exhibited the largest number of different allele types, as well as the potential to use the same primers with all eight major molecular types identified previously for C. neoformans and C. gattii. These gene loci included six housekeeping genes CAP59, GPD1, LAC1, PLB1, SOD1, URA5, from which three genes code for cryptococcal virulence factors: the polysaccharide capsule (CAP59), melanin synthesis (LAC1) and cell invasion (PLB1), and the intergenic spacer, IGS1, which was selected based on its high allelic diversity.

All the herein proposed MLST loci, except for the CAP59 locus, are similar to the ones used previously enabling the incorporation of, and comparisons with all previously obtained data. The region of the CAP59 locus proposed for the consensus MLST scheme represents a different fragment of the CAP59 gene used by Litvintseva et al. [23] (Fig. 2). This new locus was chosen based on the fact that it can be amplified from all eight molecular types using the same primers.

Fig. 2.

CAP59 genomic sequence of strain H99 (Cryptococcus neoformans var. grubii, VNI, http://www.broad.mit.edu) used as master sequence, indicating the location of the Cryptococcal Working Group (ISHAM) proposed consensus primers used in Fraser et al. [28] and the location of the primers used in Litvintseva et al. [23].

An additional locus, TEF1, which also showed high discriminatory power when used for C. neoformans var. grubii and for C. gattii molecular type VGII, was excluded from the consensus typing scheme. This was based on the fact that sequence data are only available for C. neoformans var. grubii and technical problems had been encountered when amplifying this locus. However, this locus may offer additional discrimination in some of the eight major molecular types.

To enable amplification of all seven loci from the eight major molecular types of C. neoformans and C. gattii, the previously published primers were tested on all eight major molecular types in three of the six laboratories (Teun Boekhout’s laboratory at the CBS, June Kwon Chung’s laboratory at the NIH, Matthew Fisher’s laboratory at the Imperial College, Wieland Meyer’s laboratory at the University of Sydney, Tom Mitchell’s laboratory at Duke University, and Maria Anna Viviani’s laboratory at the Università degli Studi di Milano) that collaborated in the development of the herein presented consensus MLST scheme. Satisfactory amplifications were obtained for all loci except for the SOD1 locus, where two different sets of primers were finally used to amplify either VNI–VNIV for C. neoformans or VGI–VGIV for C. gattii (Table 3). The specific primers and the suggested amplification conditions to amplify the seven gene loci are given in Table 3. Primer directions are listed according to the orientation in the genome sequence of the strain H99 at the Broad Institute (http://www.broad.mit.edu). Variations in the quality of the amplification products, resulting from either the Taq DNA polymerase enzyme or the PCR machine and PCR conditions used, were observed between participating laboratories. For that reason, the amplification conditions given in Table 3 should only serve as a guideline that may be optimized by individual laboratories.

Table 3.

MLST loci information

Gene locus	Gene product	Chromo- some locationa	Primer name and sequence (If not specified differently primers listed will work for C.n. and C.g.)	Amplification conditions	No. of bases analysed (bp)a	Analysed sequence fragment, start (5′) and end (3′) pointsa	Ref.
CAP59	Capsular associated protein	1	CAP59F 5′ CTCTACGTCGA GCAAGTCAAG 3′ CAP59R 5′ TCCGCTGCA CAAGTGATACCC 3′	94°C 3min; 35 cycles: 94°C 30s, 568C 30s, 72°C 1min Alternative conditions: 30 cycles: 94°C 30s, 64°C 30s, 72°C 1min or: 30 cycles: 95°C 3min, 95°C 30s, 54°C 30s, 72°C 1min	559	5′- ACGGTACGCGCCG GAGACAGAATG-3′	[28]
			Alternative primers: CAP59LF 5′ GTGAACAA GCTGCGGC 3′ CAP59LR 5′ GGATTCAG TGTGGTGGAAGA 3′	35 cycles: 94°C 30s, 60°C 30s, 72°C 1min			[current study]
GPD1	Glyceraldehyde- 3-phosphate dehydrogenase	7	GPD1F 5′ CCACCGAACCC TTCTAGGATA 3′ GPD1R 5′ CTTCTTGGCA CCTCCCTTGAG 3′	94°C 3min; 35 cycles: 94°C 45s, 63°C 1min, 72°C 2min Alternative conditions: 12 cycles; 62 − 56°C step-down 2°C every 2 cycles 95°C 3 min; 95°C 30 sec, 62 −56°C 30 s, 72°C 1 min; followed by 25 cycles: 95°C 30 s, 56°C 30 s, 72°C 1 min	543	5′- GGTTTCGGTACGG GACCCTGCCAA-3′	[28]
LAC1	Laccase	8	LAC1F 5′ AACATGTTCCCT GGGCCTGTG 3′ LAC1R 5′ ATGAGAATTG AATCGCCTTGT 3′	94°C 3min; 30 cycles: 94°C 30s, 58°C 30s, 72°C 1min Alternative conditions: 30 cycles: 95°C 30s, 50°C 30s, 72°C 1min	469	5′- GTAAGTATCAGCT CAAGCTAAACA-3′	[28]
PLB1	Phospholipase	12	PLB1F 5′ CTTCAGGCGGA GAGAGGTTT 3′ PLB1R 5′ GATTTGGCGT TGGTTTCAGT 3′	94°C 3min; 30 cycles: 94°C 45s, 61°C 45s, 72°C 1min Alternative conditions: 12 cycles; 62 −56°C step-down 2°C every 2 cycles 95°C 3 min; 95°C 30 s, 62 −56°C 30 s, 72°C 1 min; followed by 25 cycles: 95°C 30 s, 56°C 30 s, 72°C 1 min	532	5′- TGTTACTTGGATT CTGGAACATCG-3′	[23]
SOD1	Cu, Zn superoxide dismutase	5	Primers for C.n. SOD1CNF 5′AAGCCTCT CATCCATATCTT 3′ SOD1CNR 5′TTCAACCAC GAATATGTA 3′ Primers for C.g. SOD1CGF 5′ GATCCTCAC GCCATTACG 3′ SOD1CGR 5′ GAATGATG CGCTTAGTTGGA 3′	94°C 3min; 35 cycles: 94°C 30s, 52°C 30s, 72°C 1.5min	700	5′ - CCACGTGCTCGCA CCTGTCAATGC-3′	[46]
			Alternative primers for C.n.: SOD1-f 5′ TCTAATCGAAA TGGTCAAGG 3′ SOD1-r 5′ CGCAGCTGTT CGTCTGGATA 3′	12 cycles; 62 −56°C step-down 2°C every 2 cycles 95°C 3 min; 95°C 30 sec, 62 −56°C 30 sec, 72°C 1 min; followed by 25 cycles: 95°C 30 sec, 56°C 30 sec, 72°C 1 min	535	5′ -ATCGCTCACCGCT GCCCATTGTCA-3′	[23]
URA5	Orotidine monophosphate pyrophosphorylase	8	URA5F 5′ ATGTCCTCCCA AGCCCTCGAC 3′ URA5R 5′ TTAAGACCTCT GAACACCGTACTC 3′	94°C 3min; 35 cycles: 94°C 45s, 63°C 1min, 72°C 2min Alternative conditions: 30 cycles: 94°C 45s, 63°C 1min, 72°C 2min (C.n.) 26 cycles: 94°C 30 s, 68°C 30s, 72°C 30s (C.g.) or: 30 cycles: 95°C 3 min; 95°C 30 sec, 63°C 30 sec, 72°C 1min	601	5′ - TTTTCGGCAACTCT TGGAAAGCTC-3′	[16]
IGS1	Ribosomal RNA intergenic spacer	2	IGSF 5′ ATCCTTTGCAGA CGACTTGA 3′ IGSR 5′ GTGATCAGTGC ATTGCATGA 3′	94°C 3min; 35 cycles: 94°C 30s, 60°C 30s, 72°C 1min Alternative conditions: 30 cycles: 94°C 30s, 56°C 30s, 72°C 1min	723	5′ - TAAGCCCTTGTTAA AGATTTATTG-3′	[23]

Note:

^aThe sequences of the genome of strain H99 (C. neoformans var. grubii, VNI) at the Broad Institute (http://www.broad.mit.edu) were used as the master sequences. Nucleotide bases shown in bold typeface denote nucleotide bases that could vary between the different molecular types.

Automatic allele type and sequence type retrieval

Allele types for C. neoformans were assigned according to Litvintseva et al. [23] and for C. gattii according to by Fraser et al. [28], if applicable. The exact start- and endpoints for the sequence of each analyzed locus are given in Table 3 based on the H99 genome sequence at the Broad Institute (http://www.broad.mit.edu/), these may change over time if more strains are studied. The latest sequence cut points are listed at the webpage for each locus. To standardize the assignment of allele types (AT) and sequence types (ST), a centralized globally accessible MLST database will be established at www.mlst.net/. The online software NRDB (http://linux.mlst.net/nrdb/nrdb.htm) allows for an automatic retrieval of allele and sequence types and will assign a new allele and sequence type for any submitted unknown sequence. These are then uploaded to the database via a database curator. The designated curators are contactable via the website.

Conclusion

In conclusion the ISHAM working group on ‘Genotyping of Cryptococcus neoformans and C. gattii’ proposes the following set of genetic loci as an international standard for multi-locus sequence typing for C. neoformans and C. gattii: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1.