Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Method for Discrimination between Molecular Types of Cryptococcus neoformans and Cryptococcus gattii

Abstract

We evaluated the usefulness of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for Cryptococcus identification at the species and subspecies levels by using an in-house database of 25 reference cryptococcal spectra. Eighty-one out of the 82 Cryptococcus isolates (72 Cryptococcus neoformans and 10 Cryptococcus gattii) tested were correctly identified with respect to their molecular type designations. We showed that MALDI-TOF MS is a practicable alternative to conventional mycology or DNA-based methods.

TEXT

Two pathogenic basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii, are known to cause meningoencephalitis in immunocompromised and apparently immunocompetent human hosts, respectively. Cryptococcus neoformans and C. gattii are considered two separate species (20), with the former including two varieties, C. neoformans var. grubii and C. neoformans var. neoformans (15), as well as the intravarietal serotype AD hybrids (4). By grouping >2,000 cryptococcal isolates collected globally, eight major molecular types of C. neoformans (VNI to VNIV) and (VGI to VGIV) have been identified by means of two main typing systems, namely, PCR fingerprinting (9, 28) and amplified fragment length polymorphism (AFLP) analysis (3). The molecular types VNI and VNII correspond to C. neoformans var. grubii, type VNIII corresponds to AD hybrids, and type VNIV corresponds to C. neoformans var. neoformans, whereas types VGI, VGII, VGIII, and VGIV correspond to C. gattii (3, 28). Also, multilocus sequence typing (MLST), which is becoming the method of choice for Cryptococcus strain genotyping (27), allowed the identification of another cluster, VNB, among a set of C. neoformans var. grubii isolates from Botswana (21). However, these techniques are generally laborious, time-consuming, and expensive.

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), through the generation of characteristic fingerprints of intact microbial cells, has been successfully applied for the rapid characterization and identification of bacteria and filamentous fungi (2, 16). With regard to pathogenic yeasts, a simple and fast protein extraction step is still required to obtain reliable results (35, 36). Lower identification scores were obtained with C. neoformans isolates (25, 30) than with Candida species isolates, leading to the claim that reduction of the scores required for species level identification may improve the diagnostic usefulness of MALDI-TOF MS.

In the present study, an in-house database of MALDI-TOF MS reference Cryptococcus spectra was established and evaluated for the capability to provide species or subspecies level identification of a number of C. neoformans and C. gattii challenge isolates. Results were compared with those obtained using DNA-based typing methods.

A total of 107 Cryptococcus isolates, including 89 of C. neoformans and 18 of C. gattii, were studied. Among the clinical isolates, 56 were collected in the mycology laboratory of the Università degli Studi di Milano (Milan, Italy) from 1991 to 2001 and 34 were collected in the mycology laboratory of the Università Cattolica del Sacro Cuore (Rome, Italy) from 1990 to 2009. Additional isolates were obtained from the National Institutes of Health (Bethesda, MD) (three isolates), the National Institute of Mental Health and Neurosciences (Bangalore, Karnataka, India) (two isolates), the Westmead Millennium Institute (Sydney, Australia) (four isolates), and the Institute Pasteur (IP, Paris, France) (one isolate). Seven type or reference strains were purchased from the American Type Culture Collection (Manassas, VA). For all C. neoformans isolates, the mating-type allelic pattern was determined by multiplex PCR (14), whereas molecular types were identified using PCR fingerprinting with the minisatellite (GACA)₄-specific primer (9). All of the C. gattii isolates and most of the C. neoformans isolates have been previously characterized at the molecular level by DNA-based typing methods (9, 10, 38).

For MALDI-TOF MS analysis, protein extracts were prepared from cryptococcal isolates grown on Sabouraud dextrose agar (Kima, Padua, Italy) for 48 h at 30°C and suspended in 10% formic acid (Sigma-Aldrich, Milan, Italy). One microliter of the mixture was spotted onto a polished steel target plate (Bruker Daltonics, Bremen, Germany), air dried, and overlaid with 1 μl of absolute ethanol (Sigma-Aldrich). After air dehydration, 1 μl of a saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile–2.5% trifluoroacetic acid (Bruker Daltonics) was added and the mixture was allowed to cocrystallize at room temperature. Measurements were performed with a microflex LT mass spectrometer (Bruker Daltonics), and spectra were recorded in the positive linear mode (laser frequency, 20 Hz; ion source 1 voltage, 20 kV; ion source 2 voltage, 16.7 kV; lens voltage, 8.5 kV) (11). Seventeen C. neoformans and eight C. gattii isolates representative of the eight known molecular types (see Table S1 in the supplemental material) were selected to generate MALDI-TOF MS reference spectra at m/z ratios of 2,000 to 20,000. These spectral data were added to the Bruker Daltonics BioTyper 3.0 library database (containing spectra of 3,740 microorganisms), which already included the spectra of four C. neoformans and two C. gattii isolates. Each database entry was generated as a composite of 10 to 12 spectra, resulting in a main spectrum (MSP) that contains the average mass, the average intensity, and the frequencies of the most significant peaks (11).

Raw spectra from a set of challenge isolates (72 of C. neoformans and 10 of C. gattii) were used for pattern matching (with default parameter settings) against the extended BioTyper 3.0 database using the BioTyper 3.0 software (Bruker Daltonics). Results of this process were expressed with log(score) values as proposed by the manufacturer; i.e., values of ≥2.0 are rated as identification at the species level, values of ≥1.7 and <2.0 are rated as identification at least at the genus level, and values of <1.7 are rated as unsuitable for identification. Samples from two biological replicates, i.e., separate cultivations of the same strain (11), or eight technical replicates of a given sample analyzed at different times (1) yielded results that were reproducible (data not shown). Also, hierarchical cluster analysis was conducted with the integrated statistical tool Matlab 7.1 of the BioTyper 3.0 software package using default settings. Briefly, a dendrogram was generated by similarity scoring of a set of MSPs to obtain graphical distance values between the cryptococcal isolates, which were calculated by a correlation function, through the use of an average statistical algorithm as implemented in the BioTyper 3.0 software. Species and subspecies with distance levels of <500 are reliably classified.

All 82 Cryptococcus isolates were correctly identified at the species level, as both C. neoformans and C. gattii isolates displayed log(score) values of spectra of >2.0 (Table 1), whereas nonidentification or misidentification with the 3,734 spectra from the other microorganisms contained in the database did not occur. In agreement with the DNA-based typing results, 81 (98.8%) of the 82 isolates were unambiguously assigned to a molecular type on the basis of the MALDI spectrum matching the expected one in the reference database (Table 1). The C. gattii isolate (IUM 93-6682) showing discordant results was identified as VGI instead as VGII (Table 1), suggesting that more C. gattii strains need to be analyzed to firmly establish the discriminatory power of the MALDI-TOF method. A cluster analysis with reference and challenge isolates based on a pairwise correlation matrix was performed for both the C. neoformans and C. gattii species, in order to assess the ability of the method to display the phylogenetic relationships of the strains. As depicted in Fig. 1, the resulting dendrogram for all cryptococcal isolates showed separate clusters corresponding to the eight molecular types of the two Cryptococcus species. However, the C. neoformans var. neoformans VNIV strains did not form a single cluster but did partially group (one isolate) within the C. neoformans var. grubii VNI cluster, showing that the cluster analysis mostly, but not fully, resolved C. neoformans var. neoformans. On the other hand, the C. gattii VGIII strains did cluster together with the other C. gattii strains of molecular types VGI and VGII, but they were completely separate according to their designated serotypes, perhaps implying a lack of discriminatory power of the molecular method.

Table 1.

Comparison of identification results obtained by MALDI-TOF MS analysis and DNA-based methods for 82 challenge and 25 reference isolates of C. neoformans and C. gattii

Isolate	Molecular characterization			MALDI-TOF MS
	Species	Mating-type allele	Molecular type	Species	Molecular type	Log(score)a
						First match	Second match
IUMb 97-4877	C. neoformans	αA	VNI	C. neoformans	VNI	2.559	2.401
IUM 98-3592	C. neoformans	αA	VNI	C. neoformans	VNI	2.355	2.137
IUM 97-4515	C. neoformans	αA	VNI	C. neoformans	VNI	2.308	2.088
IUM 98-0977	C. neoformans	αA	VNI	C. neoformans	VNI	2.437	2.345
IUM 98-2450	C. neoformans	αA	VNI	C. neoformans	VNI	2.489	2.116
IUM 98-4519	C. neoformans	αA	VNI	C. neoformans	VNI	2.237	2.175
IUM 98-4640	C. neoformans	αA	VNI	C. neoformans	VNI	2.350	2.314
IUM 99-1838	C. neoformans	αA	VNI	C. neoformans	VNI	2.271	2.260
IUM 99-5678	C. neoformans	αA	VNI	C. neoformans	VNI	2.468	2.172
IUM 98-5021	C. neoformans	αA	VNI	C. neoformans	VNI	2.255	2.209
IUM 99-5690	C. neoformans	αA	VNI	C. neoformans	VNI	2.198	2.178
IUM 99-5719	C. neoformans	αA	VNI	C. neoformans	VNI	2.237	2.204
IUM 01-4726	C. neoformans	αA	VNII	C. neoformans	VNII	2.217	2.205
IUM 98-4520	C. neoformans	αA	VNI	C. neoformans	VNI	2.146	2.020
IUM 94-5982	C. neoformans	αA	VNI	C. neoformans	VNI	2.297	2.262
IUM 94-4725	C. neoformans	αA	VNI	C. neoformans	VNI	2.232	2.214
IUM 94-3443	C. neoformans	αA	VNI	C. neoformans	VNI	2.381	2.330
CRc 15-422	C. neoformans	αA	VNI	C. neoformans	VNI	2.647	2.505
CR 16-423	C. neoformans	αA	VNI	C. neoformans	VNI	2.443	2.291
CR 28	C. neoformans	αA	VNI	C. neoformans	VNI	2.361	2.252
CR 37	C. neoformans	αA	VNI	C. neoformans	VNI	2.130	2.115
CR 38	C. neoformans	αA	VNI	C. neoformans	VNI	2.664	2.471
CR 40	C. neoformans	αA	VNI	C. neoformans	VNI	2.498	2.490
CR 42	C. neoformans	αA	VNI	C. neoformans	VNI	2.509	2.208
IUM 93-3233	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.520	2.355
IUM 94-2361	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.613	2.475
IUM 93-3922	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.444	2.278
IUM 93-1656	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.332	2.208
IUM 93-2095	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.430	2.423
IUM 93-0631/2	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.063	2.033
IUM 93-0333	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.264	2.005
IUM 93-0323	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.121	2.070
IUM 92-4211	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.234	2.202
IUM 92-0891	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.273	2.221
IUM 92-0160	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.114	2.100
IUM 92-6093	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.059	1.992
IUM 96-4739	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.046	1.996
IUM 93-4941	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.173	2.125
CR 2-415	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.358	2.314
CR 3-416	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.381	2.293
CR 10-417	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.488	2.391
CR 11-418	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.577	2.448
CR 12-419	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.552	2.371
CR 26	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.329	2.327
CR 27	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.274	2.274
CR 29	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.331	2.322
CR 31	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.503	2.494
CR 32	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.534	2.422
CR 33	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.382	2.341
CR 35	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.568	2.486
IUM 93-4941	C. neoformans	αD	VNIV	C. neoformans	VNIV	2.173	2.157
IUM 91-1871	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.155	2.117
IUM 99-3615	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.421	2.405
IUM 94-5754	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.231	2.196
IUM 93-1666	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.368	2.349
IUM 92-2562	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.271	2.033
IUM 92-4734	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.224	2.117
CR 14-421	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.551	2.446
CR 17-424	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.448	2.328
CR 18-425	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.359	2.292
CR 19-426	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.437	2.430
CR 20-427	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.458	2.419
CR 36	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.549	2.487
CR 39	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.281	2.220
CR 25	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.457	2.286
CR 41	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.503	2.483
CR 43	C. neoformans	αAaD	VNIII	C. neoformans	VNIII	2.600	2.430
IUM 92-6198	C. neoformans	aAαD	VNIII	C. neoformans	VNIII	2.432	2.360
IUM 92-4686	C. neoformans	aAαD	VNIII	C. neoformans	VNIII	2.133	2.125
CR 22	C. neoformans	aAαD	VNIII	C. neoformans	VNIII	2.453	2.322
CR 23	C. neoformans	aAαD	VNIII	C. neoformans	VNIII	2.474	2.404
CR 24	C. neoformans	aAaD	VNIII	C. neoformans	VNIII	2.482	2.423
IUM 92-6682d	C. gattii	αB	VGII	C. gattii	VGI	2.120	2.078 (VGIII)
IUM 91-6492	C. gattii	αB	VGI	C. gattii	VGI	2.298	2.067
WMe 163	C. gattii	αB	VGI	C. gattii	VGI	2.009	1.712 (VGIV)
IUM 92-6957	C. gattii	αB	VGI	C. gattii	VGI	2.159	1.821 (VGIII)
IUM 94-6315	C. gattii	αB	VGI	C. gattii	VGI	2.040	1.943 (VGIV)
IPf 189	C. gattii	αB	VGIII	C. gattii	VGIII	2.199	2.199
WM 137	C. gattii	αC	VGIII	C. gattii	VGIII	2.438	2.058
NIMHg 155	C. gattii	αC	VGIV	C. gattii	VGIV	2.240	2.188
WM 779	C. gattii	αC	VGIV	C. gattii	VGIV	2.495	2.294
NIMH 103	C. gattii	αC	VGIV	C. gattii	VGIV	2.145	2.064

^aLog(score) values resulting from the second match gave correct identification, with the exception of four C. gattii isolates, for which the corresponding molecular types are indicated in parentheses.

^bIUM, Università degli Studi di Milano, Milan, Italy.

^cCR, Università Cattolica del Sacro Cuore, Rome, Italy.

^dThe only isolate with discordant results.

^eWM, Westmead Millennium Institute, Sydney, Australia.

^fIP, Institute Pasteur, Paris, France.

^gNIMH, National Institute of Mental Health and Neurosciences, Bangalore, Karnataka, India.

Fig 1.

Cluster analysis of MALDI-TOF MS spectra of selected reference or challenge isolates of C. neoformans and C. gattii. Distance is displayed in relative units.

Our results confirm the role of MALDI-TOF for species level differentiation of clinical fungi (11, 17, 24, 36; for a review, see reference 31). In addition, the current data mirror what has already been demonstrated in certain bacteria (13, 32, 34, 37) and Cryptococcus (25) and show that MALDI-TOF MS has the potential to differentiate not only between two closely related (sibling) species, C. neoformans and C. gattii, but also to discriminate C. neoformans at the subspecies level (i.e., to discriminate C. neoformans var. neoformans from C. neoformans var. grubii or, in this study, also from the AD hybrid). Furthermore, here we show for the first time and with a good level of reliability that MALDI-TOF can be applied for the rapid recognition of cryptococcal genotypes and, by extension, for fungal strain typing (unpublished data).

Misidentification of molecular genotypes within the C. neoformans-C. gattii complex has epidemiological and, more importantly, clinical repercussions (33). For example, C. gattii VGII was considered to be rare until it had been linked with the Vancouver Island outbreak of cryptococcosis (19), whose range has dramatically expanded into the Pacific Northwest of the United States (5). Differences exist among the molecular types of C. neoformans and C. gattii with regard to the in vitro susceptibility to antifungal agents, especially azoles (8, 18). Also, the emergence of highly virulent C. gattii strains (6, 7, 23) has positioned causative species, genotype, and geographic origin as important considerations when deciding on treatment options for cryptococcosis.

Whereas MLST or AFLP is usually employed for the molecular subtyping of Cryptococcus species (27), the use of a single target (i.e., the intergenic spacer) or “serotype-associated” allele (i.e., CAP59) through DNA sequencing or PCR amplification effectively distinguishes the C. neoformans varieties and C. gattii (12, 22). Although some inconsistency occurs with these genomic techniques, this limitation is minimal compared to those of conventional serotyping or biochemical methods. Thus, McTaggart et al. (26) developed a rapid identification algorithm that incorporates commercial biochemical tests, differential media, and DNA sequence analysis to distinguish clinically relevant Cryptococcus species. In contrast, the large spectrum of proteins detected by MALDI-TOF MS should enable it to discriminate between closely related species and to classify organisms at the subspecies level (29). Our study demonstrates the applicability of this approach to Cryptococcus by virtue of complexity at the species, variety, hybrid, serotype, and genotype levels.

In conclusion, MALDI-TOF MS has the potential to become a useful tool for the routine identification and typing of pathogenic fungi, providing the clinician with timely and reliable results.