Genotype Of 107 Strains Of Cryptococcus Neoformans/C.Gattii In China And Application Of The Diversilab System In Genotyping Cryptococcus Neoformans/C. Gattii

[Objectives] To analyze the genotype characteristics of 107 strains of Cryptococcus neoformans/C. gattii in China,84 from clinical samples and 23 from environmental sources. And to verify the feasibility of genotyping C. neoformans and C. gattii by the DiversiLab system.[Methods] Clinical data including sex, age, onset time of cryptococcosis, underlying diseases et al of the 76 cryptococcal patients between 2007 and 2013 from Peking Union Medical College Hospital and Beijing Ditan Hospital were retrospectively analyzed. Pigeon droppings were collected from the home (H1) that one of the systemic lupus erythematosus (SLE) patients in the study had visited to isolate C. neoformans. Pigeon droppings were also sampled from another home (H2) 10 km away from H1 as control. Conventional methods including CHORMagar medium、API 20C AUX and Vitek 2 Compact were used on the 84 cryptococcal strains of the patients above and 23 environmental strains of C. neoformans from pigeon droppings in H1 and H2. CGB medium was used to distinguish C. gattii from C. neoformans. Serotype and mating type of these strains were determined by specific primers for C. neoformans and C. gattii. ITS of rDNA were amplified and sequenced by consensus primers, ITS1 and ITS4. Restriction fragment length polymorphism of gene URA5 (URA5-RFLP) was performed to genotype these cryptococcal isolates initially. Then multi-locus sequence typing (MLST) was applied on them by amplifying separately their 7 MLST genes including CAP59, GPD1, IGS1, LAC1, PLB1, SOD1 and URA5. The PCR products of ITS and 7 genes of MLST were sequenced commercially from two directions. The molecular phylogenetic tree was constructed on the basis of concatenated seven MLST loci of tested isolates and cryptococcal reference strains using MEGA 6.06. Automated repetitive PCR was performed using Fungal Kits of the DiversiLab system on these tested isolates and reference strains of C. neoformans and C. gattii. The amplified fragments of rep-PCR were seperated by electrophoresis in a microfluidics DNA LabChip. Classification and typing of them were further analyzed respectively through the DiversiLab software V3.4.40. The DiversiLab system analysis was also performed on the and the two clinical C. neoformans (PU 66 and PU 112) simultaneously isolated from the cerebral spinal fluid (CSF) of the SLE patient to trace the source of two clinical strains.[Results] The DiversiLab system was first applied in the world by the study to genotype 116 strains of C. neoformans and C. gattii, including 82 clinical strains of C. neofromans, 2 clinical strains of C. gattii,23 environmental strains of C. neoformans and 9 reference strains of C. neoformans and C. gattii. Except for one strain of genotype VNB, identical results were obtained through the DiversiLab system compared to MLST and URA5-RFLP analyses on genotyping the other 115 strains of C. neoformans and C. gattii. First strain of genotype VNB of C. neoformans in China and other Asian countries was found by using MLST analysis in the study. One strain of C. gattii was first found in the northern region where belongs to temperate climate in China.All these 76 cryptococcal patients in the study came from 15 provinces and 3 municipalities respectively.20 patients were Human Immunodeficiency Virus (HIV) positive and fatality rate of them was thirty percent.10 were SLE patients and their fatality rate was forty percent.11 patients had no obvious underlying diseases and they were all alive.82 were C. neoformans and 2 were C. gattii in the 84 clinical cryptococcal isolates.1 was hybrid (serotype AD), the others were all serotype A in the 82 strains of C. neoformans. The hybrid isolate belonged to genotype VNIII, one was genotype VNB, and the other 80 isolates were genotype A, the most common genotype which accounted for 95%. MLST sequence type (ST) of 77 isolates of C. neoformans were ST 5 which accounted for 91.6%(77/84). ST 5 was the most common MLST ST, followed by ST 31, ST 63, ST 182, ST 265, ST 295, ST 296 and ST 332, one strain for each ST. ST 265, ST 295 and ST296 were new STs. Two strains of C. gattii in our study were serotype B/C, and they belonged to VGI and VGII, respectively. MLST STs of them were ST 182 and ST 332. ST 332 was novel ST. The mating type of hybrid in the study was a/a, and that of the other 83 strains of C. neoformans and C. gattii tested in the study were all mating type a.. All the 23 environmental strains were uniformly genotyped as C. neoformans var. grubii VNI, serotype A, mating type a. The two clinical strains and all the 11 strains from the pigeon droppings in H1 belonged to genotype VNIc, their MLST STs were ST 5 except that MLST ST of PU 112 was ST 265. The twelve C. neoformans from pigeon droppings in H2 belonged to genotype VNIb, and their MLST STs were ST 31 (n=11) and ST 297 (n=1). ST 297 was novel MLST ST. The phylogenetic tree based on ITS sequences showed that genotype VNI, VNII, VNB clustered together. Meanwhile, the phylogenetic tree constructed on the basis of IGS sequences revealed that PU 146 clustered together with WM 626, the reference strain of C. neoformans, genotype VNII. However, PU 146 was identified as genotype VNI of C. neoformans through URA5-RFLP and MLST analyses. The strain similarities by classification between top match strains in the database of yeast or mould of the DiversiLab system nowdays and tested isolates and reference strains of C. neoformans and C. gatttii were uniformly less than 80%. PU157 was identified as VNB by 7-locus MLST analysis, however, PU157 was assigned as VNII according to URA5-RFLP, while it was more likely classified as VNI by the DiversiLab system analysis. Moreover, though AD hybrids could not been processed by MLST, PU 43 (serotype AD, the clinical hybrid) and WM 628 (seotype AD, reference hybrid), together with the other 7 kinds of genotypes of C. neoformans and C. gattii could be distinguished by the DiversiLab system. There was an excellent agreement amongst the three methods including URA5-RFLP, MLST and DiversiLab analyses in genotyping the remainder 107 tested isolates and standard strains of C. neoformans and C. gattii. DiversiLab analysis showed that strain similarity between the two clinical strains (PU 66 and PU 112) was 96.7%, and there were several peaks differences between them. In relation to environmental samples, the highest strain similarity that was 99.3% was observed for P U66 and PU 70 (H1). However, none of the environmental isolates had similarity over 98.6% comparing to PU 112.[Conclusions] The DiversiLab system can be used in genotyping of C. neoformans and C. gattii rapidly and correctly; Genotype VNI of C. neoformans was the most common genotype of C. neoformanslC. gattii in China. However, the genotype characteristics of C. neoformans/C. gattii from clinical and environmental sources in China need to be investigated more extensively and more thoroughly; Genotype VNB and genotype VNII couldn’t be distinguished by URA5-RFLP; Sequencing of ITS and IGS could recognized C. gattii from C. neoformans, but could not identify the genotypes VNI, VNII and VNB; The fatality rates of immunocompromised patients, such as HIV and SLE, were higer when they were infected Cryptococcus, but the mechanism of cryptococcal infection occurred in the population with no obvious underlying diseases need further research; Genotype analysis should be performed on the dual isolates of C. neoformans/C. gattii with different morphology in the mixed infection from the same clinical sample.