- Selection of identification method and drug resistance of Chinese C. gattii strains
C. neoformans and C. gattii are two important fungal pathogens for humans and animals. Both of them are round with capsule and positive urease test. These routine laboratory identification methods are unable to distinguish the two species [7]. Although there were several reports about infection of C. gattii from China [8-10], the number of C. gattii infections may have been underestimated because most laboratories utilize ink staining and biochemical methods, like VITEK 2 compact, for routine identificatio. CGB agar was recommended to identify C. gattii from C. neoformans due to its convenience and low cost [7], but it took longer time to get the result. Molecular methods would be faster and more accurate for differentiation of the two pathogenic Cryptococci species. However, they are not available in all clinical laboratories, where an accurate identificaction of species should be made in order to install the proper treatment. In this study, eight strains of C. gattii were not only correctly identified but also grouped by MALDI-TOF MS with Bruker software 3.0. Of course, eight strains was not enough and more strains should be studied to obtain the spectrum difference between groups of C. gattii.
The results of antifungal susceptibility test by two kits were in good agreement, except that MICs of Fluconazole were 2~4 times higher by Yeast one than by ATB fungus 3. All the C.gattii isolates were susceptible to 5-Flucytosine, Amphotericin B, Itraconazole, Voriconazole and Posaconazole, only one strain was resistant to Fluconazole with MIC of 128 by Yeast one. However, interpretation of MICs of antifungal agents including Fluconazole for category of “susceptibility” or “resistance” was hampered by the lack of clinical breakpoints for C. gattii. Some previous studies have shown that C. gattii may be less susceptible to antifungal agents compared with C. neoformans. In a study from Taiwan, C. gattii was less susceptible to 5‑Flucytosine and amphotericin B, and in Spain, MICs of Fluconazole, Voriconazole and Posaconazole to C. gattii were significantly higher [11-13]. The correlation between susceptibility profile and genotype of C. gattii has rarely been studied [14], our data indicated that antifungal agents exhibited higher MICs against isolates of genotype VGII than genotype VGI, which agreed with the data of Hagen et al and Lockhart et al. [14-17]. However, Clinical relevance between MIC breakpoint and epidemiological cut-off value (ECV) based on MIC distributions of wild-type strains has currently been studied on both C. neoformans and C. gattii isolates from Europe, USA, Australia, Brazil, Canada, India and South Africa. ECVs of Amphotericin B (0.5~1μg/ml), 5-Flucytosine (4~16μg/ml), Fluconazole (8~32μg/ml) and other azoles varied similarly by molecular type for both C. neoformans and C. gattii [15, 16].
- Molecular and epidemical characteristics of Chinese C. gattii strains
Up to now, four genotypes of C. gattii have been detected, they were VGI, VGII, VGIII and VGIV. Lockhart and colleagues had reported that VGII and VGIII were the most-frequently identified isolates in America, VGIV was almost exclusively in Africa, and VGI predominated in Europe, Australia and Asia [18]. C. gattii, as an important pathogen, caused outbreak in British Columbia, Canada and the Pacific Northwest, the United States. But 8 strains in our study were pathogens causing sporadic infections according to the strain origination and their isolation time.
- Patient information and clinical characteristics
All the patients in our study had no recent travel history, the C. gattii infection occurred regionally and domestically. They were immunocompetent young male adults with age range of 21~60 years old, which was considered as the reason for increased exposure to environmental sources and increased chance for infection [19].. Most patients had headache and fever, three patients showed neck stiffness, positive Kernig’s sign and meningeal lesions,meningeal enhancement or lacunar infarction by brain MRI. Most of them also demonstrated irregular nodule, consolidation and mass by lung CT, which was consistent with the research made by Ngamskulrungroj who indicated that C. gattii could cause fatal lung infection [20].
It was reported that host response varied based on the genotype of the organism and concomitant illnesses [21]. There was also study which revealed that C. gattii VGII strains were more virulent than VGI strains and VGIIa were even more virulent than VGIIb independent of their clinical or environmental origin [22]. In our study, all patients were treated by Fluconazole plus amphotericin B, most of them improved effectively without severe neurological sequelae. This might be explained by the fact that these patients infected by C. gattii VGI more than C. gattii VGII on the one hand, and on the other hand, two of them were infected with C. gattii VGII which showed sequence mutations in the gene location of RAS1 and FTR1 compared with reference strains of R265 and R272 respectively. Whether the mutations were relevant to virulence decrease, further work needs to be done.
- Analysis of differentially expressed protein in two genotypes of C. gattii.
Our study showed significant difference in protein expression spectra between VGI and VGII. GO is a very important tool of biological information. By establishing a set of controlled words with dynamic form, the attributes of genes and gene products in organisms can be described comprehensively. KEGG is the main public database with which the most important biochemical metabolic pathways and signal transduction pathways can be determined. Our results suggested that the differential protein of C. gattii VGI and VG II was mainly located on the organelles associated with the Golgi body, which meant energy metabolism of C. gattii might be involved in the difference of pathogenesis mechanism for different genotype of C. gattii. Study on the secretory protein and the secretory vesicles of the two genotypes of C. gattii would be beneficial to the understanding of the pathogenesis of C. gattii.