Does the Capsule Interfere with Performance of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Cryptococcus neoformans and Cryptococcus gattii?

Abstract

We described the impact of the capsule size for Cryptococcus neoformans and Cryptococcus gattii identification at the species level by Bruker matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). After experimental capsule size modulation, we observed that reducing the capsule size resulted in improved identification by Bruker MALDI-TOF MS across all of the reference strains analyzed.

TEXT

Cryptococcus neoformans and Cryptococcus gattii are relevant species among the pathogenic basidiomycetous yeasts responsible for infection in humans (1). These organisms usually produce a polysaccharide capsule that acts as an important virulence factor against the host's defenses (2). Besides its epidemiological importance, species differentiation in this genus has major clinical relevance since patients with central nervous system (CNS) infection by C. gattii have a higher risk of neurological complications, need a more prolonged course of induction antifungal therapy, and have poorer prognoses than those with C. neoformans infections (3, 4).

Recently, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a precise technology for Cryptococcus species identification (ID), replacing conventional and time-consuming phenotypic methods and providing an alternative to expensive and labor-intensive molecular techniques (5, 6). However, during routine practice in our clinical microbiology laboratory, we observed that some Cryptococcus isolates with prominent capsule sizes had low discriminatory ID when using Bruker MALDI-TOF MS analysis. These isolates required the application of old phenotypic methods, which delayed the release of the final result. This led us to investigate the impact of cryptococcal capsule size in correct species ID by Bruker MALDI-TOF MS analysis.

For this purpose, reference strains of the eight genotypes of C. neoformans and C. gattii, WM148 (serotype A, VNI), WM626 (serotype A, VNII), WM 628 (serotype AD, VNIII), WM629 (serotype D, VNIV), WM179 (serotype B, VGI), WM178 (serotype B, VGII), WM161 (serotype B, VGIII), and WM779 (serotype C, VGIV), were subjected to capsule size modulation according to previously described methods (7, 8). Briefly, 2 ml of capsule growth-inducing medium (CGIM) (Sabouraud dextrose broth [BD, Franklin Lakes, NJ, USA] diluted 10 times with sterile water, pH 7.3) containing 2 × 10⁶ yeast cells was incubated at 37°C with shaking. In an attempt to increase the variability in capsule size, all strains were subjected to a prolonged incubation in CGIM (up to 28 days) and were evaluated simultaneously on days 2, 3, 7, 14, 21, and 28, giving a total of 48 capsule size measurements (six replicates for each strain of the two Cryptococcus species). Next, yeast cells collected from the CGIM were submitted to a progressive capsule reduction protocol with four consecutive initial seedings in Sabouraud dextrose agar (SDA; BD) and two more seedings in the capsule-reducing medium (CRM) (SDA plus 2.9% NaCl). During this reduction assay, strains were incubated at 30°C, and each seeding had its capsule size analysis after 48 h of incubation, giving a total of 48 capsule size measurements (six replicates for each strain of the two Cryptococcus species, with four from the SDA medium and two from the CRM). For the capsule size measurements, yeast cell suspensions were stained with India ink and examined in an optical microscope equipped with an AxioCam MRc digital camera and AxioVision release 4.8 software (Zeiss, Oberkochen, Germany). Different slide fields were randomly chosen, and 40 to 50 cells were measured to determine the mean value of the capsule sizes. Finally, the same yeast cell suspensions were analyzed by Bruker MALDI-TOF MS. For protein extraction, the suspension was washed twice with sterile water and was centrifuged at 13,000 rpm for 10 min; the pellet was resuspended in sterile water and mixed thoroughly. Subsequently, chemical extraction with ethanol and formic acid was carried out according to the manufacturer's instructions. After the extraction protocol, 1.2 μl of the supernatant was spotted on each well of the steel target plate and was air dried and overlaid with 1.2 μl of matrix solution (saturated solution of α-cyano-4-hydroxy cinnamic acid in organic solvent [50% acetonitrile and 2.5% trifluoroacetic acid]; Sigma-Aldrich, St. Louis, MO, USA). Mass spectra were generated with the microflex MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) and were compared to the main spectra (MSP) of C. neoformans and C. gattii from an extended database (Biotyper v3.1 [Bruker Daltonics] plus our in-house database with the MSPs of the aforementioned Cryptococcus reference strains). Mass spectrometry results were expressed in log-score (LS) values between 0 and 3.000, which is considered acceptable for species ID at values of >2.000 and for genus ID between values of 1.700 and 1.999. The Bruker MALDI-TOF MS results were correlated to the cryptococcal capsule size, and comparisons between groups were performed using Fisher's exact or chi-square tests for categorical variables and Mann-Whitney and Kruskal-Wallis tests for continuous nonparametric variables (SPSS 18.0; SPSS, Inc., Chicago, IL, USA). The P value was set to 0.05.

The mean capsule size of the 48 replicates with capsule induction was 9.42 ± 6.12 μm, with 4.91 ± 2.94 μm and 13.93 ± 5.05 μm for C. neoformans and C. gattii (P < 0.0001), respectively. Successful species ID was obtained in 10 (20.8%) of the capsule-induced replicates. Bruker MALDI-TOF MS analyses made with the four C. neoformans genotypes after capsule induction failed to obtain the correct species assignment in 14 (58.3%) of 24 replicates, whereas all of the 24 replicates from the C. gattii genotypes failed species ID after capsule induction (C. gattii versus C. neoformans, P = 0.005). Forty-two (87.5%) of the 48 replicates were identified correctly after capsule size reduction (P < 0.0001 versus after capsule size induction). Specifically, the mean capsule size of the 32 replicates from the SDA was 3.16 ± 2.34 μm, and 26 of these replicates had correct species ID (81.25%). The mean capsule size of the 16 replicates from the CRM was 0.98 ± 0.86 μm (P < 0.0001 versus the SDA replicates), and all of these replicates (16/16) had correct species ID. The correlation of capsule size and species ID by Bruker MALDI-TOF MS for the 48 replicates in each of the two experimental conditions performed with the Cryptococcus species is summarized in Table 1. The negative effect of the cryptococcal capsule in the mass spectrum quality is exemplified on Fig. 1. After stratifying the replicates according to their LS values, those with LS of >2 showed a mean capsule size of 2.46 ± 2.18 μm, whereas the mean capsule sizes of the replicates with LS values between 1.7 and 2 and <1.7 were 8.7 ± 5.41 μm and 11.91 ± 6.08 μm, respectively (P < 0.0001).

TABLE 1.

Capsule size and MALDI-TOF mass spectrometry species identification of the replicates of Cryptococcus neoformans and Cryptococcus gattii strains after capsule modulation

Strain (genotype)	After capsule induction		After capsule reduction
	Range of capsule size (μm)	No. of species identified/total no. of species (%)	Range of capsule size in SDA (μm)	No. of species identified in SDA/total no. of species (%)	Range of capsule size in CRM (μm)	No. of species identified in CRM/total no. of species (%)
Cryptococcus neoformansa
WM 148 (VNI)	0.8–1.91	6/6 (100)	0.16–0.48	4/4 (100)	0.16–0.16	2/2 (100)
WM 626 (VNII)	3.5–5.09	3/6 (50)	0.8–3.34	3/4 (75)	0.48–0.8	2/2 (100)
WM 628 (VNIII)	3.98–7.48	1/6 (16)	0.95–1.59	3/4 (75)	0.16–1.11	2/2 (100)
WM 629 (VNIV)	6.36–12.41	0/6 (0)	1.11–3.02	3/4 (75)	0.32–0.8	2/2 (100)
Cryptococcus gattiia						2/2 (100)
WM 179 (VGI)	8.27–18.46	0/6 (0)	3.34–4.3	3/4 (75)	0.8–1.99	2/2 (100)
WM 178 (VGII)	8.75–17.18	0/6 (0)	3.66–6.52	3/4 (75)	0.16–2.15	2/2 (100)
WM 161 (VGIII)	6.36–28.64	0/6 (0)	4.93–8.91	3/4 (75)	1.43–2.86	2/2 (100)
WM 779 (VGIV)	10.5–17.02	0/6 (0)	2.86–3.66	4/4 (100)	0.32–2.07	2/2 (100)

^aFourty-eight replicates were used for each of the two experimental conditions performed with the Cryptococcus species.

FIG 1.

Mass spectra (MS) of Cryptococcus gattii VGIII after capsule size modulation. (A) Poor quality MS with a log-score value of <2 from a replicate with a mean capsule size of 17.02 μm. (B) Higher quality MS from a replicate with a mean capsule size of 1.43 μm and showing a best match LS of 2.285.

MALDI-TOF MS is an emerging technology that is successfully replacing conventional phenotypic methods for microorganism identification with fast and accurate results (9). However, the published results of MALDI-TOF MS for C. neoformans and C. gattii ID (5, 6), based mainly on the analyses of strains from culture collections, may have overestimated its performance. Fresh specimens from patients can have larger capsules since capsule growth is an evading mechanism against the mammalian immune system (2, 10). As shown here, large capsules impair Cryptococcus species ID by Bruker MALDI-TOF MS. This might explain the recent reports of nonidentification or misidentifications of Cryptococcus clinical isolates in routine practice in parallel to limitations of the available databases (11–16). Similarly, other encapsulated virulent pathogens, such as Streptococcus pneumoniae, Haemophilus influenzae, and Klebsiella pneumoniae, were linked to nonidentification or misidentifications by MALDI-TOF MS (17). Some authors hypothesized that the capsule prevents efficient lysis, which results in poor spectral quality, while others addressed this issue by proposing removal of the extracellular matrix (e.g., capsular polysaccharide, melanin) through the improvement of the extraction protocol (e.g., washing steps, vortexing with beads) (17–18). However, modifications in the extraction protocol may interfere with the matching of spectra with the MSP database. While this remains a matter of debate, we propose that whenever fresh isolates with prominent capsules are not identified by Bruker MALDI-TOF MS, a single 48-h seeding in CRM should be carried out since it resulted in 100% identification. As previously reported (18), interspecies differences in capsule size were also observed in our study, in which C. gattii isolates often produced capsules larger than those of C. neoformans isolates, which resulted in a higher number of replicates without ID by Bruker MALDI-TOF MS after capsule induction. Considering that, the application of the capsule-reduction protocol might be particularly useful when there is clinical suspicion of C. gattii infection (e.g., nonimmunocompromised hosts). Our data should be further validated by analysis with a higher number of isolates in the routine clinical laboratory.

In conclusion, our results illustrate the negative impact of the cryptococcal capsule for ID by Bruker MALDI-TOF MS, with the species C. gattii being more susceptible to this phenomenon. Reducing the capsule size may improve mass spectra quality, and consequently, Cryptococcus species ID using this technology may be achieved.