Rapid Identification of Sporothrix Species by T3B Fingerprinting

Abstract

This article describes PCR fingerprinting using the universal primer T3B to distinguish among species of the Sporothrix complex, S. brasiliensis, S. globosa, S. mexicana, and S. schenckii. This methodology generated distinct banding patterns, allowing the correct identification of all 35 clinical isolates at the species level, confirmed by partial calmodulin (CAL) gene sequence analyses. This methodology is simple, reliable, rapid, and cheap, making it an ideal routine identification system for clinical mycology laboratories.

TEXT

Sporotrichosis is a globally distributed subcutaneous mycosis with areas of high endemicity (11, 21) that is caused by the dimorphic fungus Sporothrix schenckii (22). Sporotrichosis has been regarded as a job-related disease occurring as isolated cases or small outbreaks affecting people exposed to plants or soil (1, 6, 7). Rio de Janeiro State, Brazil, is a region of sporotrichosis hyperendemicity where several human and animal cases have been described since 1998 (5, 23).

The diagnosis of sporotrichosis is attained by clinical, epidemiological, and laboratorial data, including culture and analysis of phenotypic characteristics. The first description of PCR for sporotrichosis' diagnosis was reported in 2001 (9). A diagnostic nested PCR assay targeting the S. schenckii 18S rRNA gene was further evaluated and showed high sensitivity and specificity, indicating that PCR may be clinically useful for diagnosis (8).

S. schenckii was long considered a single taxon, although great genetic variation within this species has been described (10). By associating phenotypic and genotypic features, Marimon et al. (13) recognized three new species, Sporothrix brasiliensis, Sporothrix globosa, and Sporothrix mexicana, and proposed an identification key for these Sporothrix species. S. globosa has worldwide distribution (12, 18), whereas S. brasiliensis is apparently restricted to Brazil (13) and S. mexicana to Mexican environmental samples (13), although the latter was recently identified in Portugal (3). Additionally, these authors have proposed the promotion of S. schenckii var. luriei to the status of a new species, Sporothrix luriei (14). However, identification based only on phenotypic characteristics is often inconclusive due to phenotypic variability within these species. Therefore, new rapid and reliable identification strategies are necessary (19).

Analysis of tRNA intergenic spacers was first used to distinguish Streptococcus species (15). It has also been applied successfully for Candida identification (2, 16, 24). Here, we evaluate T3B PCR fingerprinting to differentiate clinical Sporothrix strains at the species level in comparison to analysis of partial calmodulin (CAL) gene sequences (19).

Thirty-five Sporothrix spp. isolates from the Fungal Culture Collection of IPEC/Fiocruz were included in this study approved by the Ethics Commission of the same institution. Among them, S. brasiliensis type strain CBS 120339 (IPEC16490), S. globosa IPEC27135 (18), S. schenckii IPEC29334 (IOC1226) (19), and S. mexicana (MUM11.02) (3) were used as controls. All 35 strains were previously phenotypically characterized (3, 19), and 15 isolates could not be identified (Table 1).

Table 1.

Comparison of tools applied in the characterization of strains of the Sporothrix complex

Strain	Phenotypic identificationa	Genotypic characterization
		Final identificationb	GenBank accession no.c	Referenced
IPEC16490	S. brasiliensis	S. brasiliensis	AM116899	19
IPEC27445-3	S. brasiliensis	S. brasiliensis	HQ426950	19
IPEC27052	Sporothrix sp.*	S. brasiliensis	HQ426941	19
IPEC27135	Sporothrix sp.*	S. globosa	GU456632	3
IPEC27387	Sporothrix sp.*	S. brasiliensis	HQ426948	19
IPEC34067	Sporothrix sp.*	S. brasiliensis	HQ426952	19
IPEC27372	Sporothrix sp.*	S. brasiliensis	HQ426947	19
IPEC25011	S. brasiliensis	S. brasiliensis	HQ426935	19
IPEC33605	Sporothrix sp.*	S. brasiliensis	HQ426957	19
IPEC27930	Sporothrix sp.*	S. brasiliensis	HQ426951	19
IPEC28772	Sporothrix sp.*	S. brasiliensis	HQ426955	19
IPEC28457	S. brasiliensis	S. brasiliensis	JN995607	This study
IPEC34007	Sporothrix sp.*	S. brasiliensis	HQ426959	This study
IPEC27177-2	Sporothrix sp.*	S. brasiliensis	HQ426944	19
IPEC27087	S. brasiliensis	S. brasiliensis	HQ426942	19
IPEC27288	Sporothrix sp.*	S. brasiliensis	HQ426945	19
IPEC27209	Sporothrix sp.*	S. brasiliensis	HQ426946	19
IPEC28604	S. brasiliensis	S. brasiliensis	HQ426953	19
IPEC26945	Sporothrix sp.*	S. brasiliensis	HQ426939	19
IPEC27130	Sporothrix sp.*	S. brasiliensis	HQ426943	19
IPEC25521	Sporothrix sp.*	S. brasiliensis	HQ426936	19
IPEC16919	S. brasiliensis	S. brasiliensis	HQ426930	19
IPEC18782A	S. brasiliensis	S. brasiliensis	HQ426933	This study
IPEC28329	S. schenckii*	S. brasiliensis	JN995610	This study
IPEC27022	S. brasiliensis	S. brasiliensis	HQ426940	19
IPEC28487	S. brasiliensis	S. brasiliensis	HQ426928	19
IPEC28665	S. brasiliensis	S. brasiliensis	JN995606	This study
IPEC28790	S. brasiliensis	S. brasiliensis	HQ426956	19
IPEC29334	S. schenckii	S. schenckii	HQ426962	19
IPEC26961	S. schenckii	S. schenckii	JN995605	This study
IPEC27157-1	S. schenckii	S. schenckii	JN995604	This study
IPEC27100	S. schenckii*	S. brasiliensis	JN995609	This study
IPEC27133	S. schenckii*	S. brasiliensis	JN995608	This study
MUM 11.02	S. mexicana or S. schenckii*	S. mexicana	JF970258	4
IPEC27722	S. mexicana*	S. schenckii	HQ426961	19

^aPerformed according to reference 19. Asterisks indicate incorrect phenotypic identifications.

^bCalmodulin sequencing and T3B concordant identification.

^cOf strain used for characterization.

^dReference from which the partial gene calmodulin sequencing result was obtained.

Genomic DNA was extracted from the mycelial phase (25), and PCR was performed with the primer T3B (5′-AGGTCGCGGGTTCGAATCC-3′) (24). T3B PCR fingerprinting reproducibility was confirmed by repeating the assays at least 3 times under the same conditions at two different laboratories. The T3B profiles were analyzed using the software Bionumerics 5.1 (Applied Maths BVBA, Saint-Martens-Latem, Belgium). Similarity coefficients were calculated using the Dice algorithm, and cluster analysis was performed by means of the unweighted-pair group method using average linkages (UPGMA). Sequencing of the CAL gene was performed as previously described (17) using the sequencing platform at Fiocruz, Brazil (20). Sequences were edited with Sequencer 4.6 (Genes Codes Corporation), aligned with MEGA 4.0.2 software, and compared with sequences available from NCBI GenBank by BLAST. Phylogenetic analyses were performed using MEGA with 1,000 bootstrap replicates (4) (http://www.megasoftware.net/).

T3B PCR fingerprinting of control strains showed profiles with DNA fragments ranging from 300 to 1,500 bp that allowed a clear distinction of S. brasiliensis, S. globosa, S. mexicana, and S. schenckii (Fig. 1). A dendrogram derived from analysis of the T3B profiles of all isolates splits Sporothrix strains into three groups, not the expected four, with IPEC27722, the only strain phenotypically characterized as S. mexicana, grouping into the S. schenckii cluster. Regardless of this discrepancy, the T3B profiling showed a high correspondence between clusters and Sporothrix species, with all isolates clustering with their respective control strain (Fig. 2). The T3B fingerprinting identification was confirmed by comparison with the CAL gene partial sequences obtained along with sequences from the NCBI database, AM398393.1 (S. mexicana), AM398382.1 (Sporothrix albicans), AM116908 (S. globosa), and AM117444.1 (S. schenckii). The phylogenetic tree of the CAL locus analyzed by neighbor joining revealed five distinct clades represented by the five species (Fig. 3). Overall, there was 100% agreement between T3B PCR fingerprinting and CAL locus sequencing on species identification. Comparing these results with phenotypic analysis, 14 of the 15 isolates with inconclusive phenotypes grouped within the S. brasiliensis cluster and IPEC27135 grouped within the S. globosa cluster. The genotypic analyses also allowed the correction of phenotypic misidentifications, such as for the isolate IPEC27722, which was phenotypically characterized as S. mexicana but grouped within the S. schenckii cluster, as well as for the isolates IPEC27133, IPEC27100, and IPEC28329, which identified as S. schenckii but clustered with S. brasiliensis.

Fig 1.

Representative T3B PCR fingerprinting profiles of the Sporothrix complex. (1 and 7) Molecular marker DNA ladder, 100 bp (Invitrogen). (2) S. brasiliensis (IPEC 16490). (3) S. globosa (IPEC 27135). (4) S. mexicana (MUM 11.02). (5) S. schenckii (IPEC27722). (6) Negative control.

Fig 2.

Dendrogram showing the degrees of similarity of T3B fingerprinting profiles among the Sporothrix isolates by using the Dice coefficient and the UPGMA cluster method. The cophenetic correlation coefficient (0.97) indicates a very good fit for this analysis.

Fig 3.

Consensus tree of Sporothrix based on partial calmodulin (CAL) gene sequences of 35 strains and the NCBI public GenBank sequences AM398393.1 (S. mexicana), AM398382.1 (S. albicans), and AM117444.1 (S. schenckii) that was constructed with MEGA version 4.0.2 and 1,000 bootstrap replicates.

Identification of the Sporothrix species complex has been based on a polyphasic approach using a combination of phenotypic methodologies and sequencing (13, 14, 18, 19). The proposed identification key based on phenotypic tests (13) was reported as easy and reliable for species differentiation without the need of molecular techniques. However, the results are often inconclusive or ambiguous, and some species are too closely related to show phenotypic differences. This work describes the assessment of DNA polymorphism within the Sporothrix complex by genomic DNA amplification with the single nonspecific primer T3B. The T3B PCR profiles were highly informative and generated clearly distinct banding patterns for each species, allowing their differentiation. S. brasiliensis strains showed bands sharing similarity higher than 80%, but intraspecies variation was observed. This variation was previously demonstrated for Candida (2), and band-sharing values observed are within the variation range (70 to 85%) expected for strains within the same species (16).

Comparison of the inconclusive phenotypic identification of isolates with the results obtained by T3B PCR fingerprinting demonstrated the sensitivity of the latter, since species of those strains could all be determined. This proposed identification technique is simple, reliable, rapid, and cheaper and requires less technical expertise than sequencing. The generated, computer-scanned PCR profiles can form the basis of a database which can be used for future identifications of atypical or unidentifiable Sporothrix isolates. Furthermore, these advantages are especially valuable in a laboratory with limited facilities, making it an ideal identification methodology for clinical mycology laboratories.

Nucleotide sequence accession numbers.

All sequences were deposited in the GenBank database under accession numbers GU456632, HQ426928 to HQ426962, and JN995604 to JN995610.