Genotype Analysis And Virulence Characteristics Of Cryptococcus Gattii Species Complex In Guangxi,Southern China

Objective The highly virulent,highly pathogenic and more resistant to antifungal drugs of C.gattii species complex have become a therapeutic problem.To initiate a better understanding of the population infection characteristic and pathogenicity of C.gattii species complex in Guangxi,southern China,the current study aimed to characterize C.gattii species complex isolates genetically and phenotypically.At the same time,the evolutionary relationship between Guangxi clinical isolates and global strains was elucidated.Methods(1)The clinical strains of Cryptococcus spp.isolated from patients with clinically confirmed cryptococcosis between 2014 and 2018,which were transferred onto CGB medium to screen out C.gattii species complex.(2)For a retrospective analysis of patients with C.gattii species complex infection,the data of the patients was collected using a standardized form that was based entirely on the medical reports of each patient.(3)Genomic DNA was extracted with the Nu Clean Plant Genomic DNA Kit.Based on the ISHAM consensus multi-locus sequence typing(MLST)scheme,seven loci(CAP59,GPD1,LAC1,IGS1,PLB1,SOD1 and URA5)were amplified and sequenced.Mating types were determined by using specific primers targeting the MFα and MFa pheromone genes.(4)Based on the seven loci sequence of MLST method,the phylogenetic tree was constructed by MEGA7 software,and principal component analysis was performed with the R package adegenet to explore the genetic relationships and geographic patterns of the strains.(5)The growth rate at 37℃,relative capsule size,melanin production capacity and mating test were constructed in vitro to observe and compare the virulence characteristic of clinical isolates of C.gattii species complex.(6)In vitro antifungal susceptibility test was performed by the broth microdilution method following the CLSI M27-A3 guidelines.Results(1)Among the one hundred and twenty clinical strains of Cryptococcus,eleven isolates had a positive reaction and turned the CGB medium blue,with a separation rate of C.gattii species complex was about 9%(11/120).(2)There are ten cryptococcal meningitis(CM)patients and one pulmonary cryptococcosis(PC)patient.Most of the patients have been significantly relieved after the cryptococcosis induction period treatment using combination therapy with two(AMB and FLC)or three(AMB,FLC and 5FC)antifungal drugs,while one patient died(GX0147)after giving up treatment because of no significant improvement.No patient had travel record to the C.gattii species complex outbreaks epidemic areas,but most of them had a history of contact with cryptococcal environmental exposure,such as Eucalyptus trees and pigeon droppings.(3)MLST analysis of the eleven isolates indicated that five isolates were C.gattii s.s.(AFLP4/VGI),and the other six isolates belonged to C.deuterogattii(AFLP6/VGII).All of the clinical isolates analyzed were MATα.(4)Although no highly virulent strains similar to AFLP6A/VGIIa and AFLP6C/VGIIc gene subtypes have been found in the outbreak areas of C.deuterogattii,such as Canada and the United States,evolutionary analysis and principal component analysis indicated that some strains of AFLP6/VGII in this study,as well as the highly virulent AFLP6A/VGIIa and AFLP6C/VGIIc were originated and evolved from the Brazilian C.deuterogattii in South America.(5)In vitro virulence test showed that all clinical isolates of C.gattii species complex can grow normally at 37℃.All of the strains could induce capsule formation in vitro,but the data revealed no significant difference in the capsule size between GX0104 and R265.Compared with the reference strains,some strains had greater melanin production at 30°C than 37°C.Three C.deuterogattii strains(GX0104,GX0105 and GX0147)were able to undergo sexual reproduction in vitro,and basidia and basidiospores were formed during sexual reproduction.(6)Antifungal susceptibility testing indicated that strains in this study are not resistant to antifungal drugs because their MIC values were within the susceptible range.Conclusion In summary,the results of physiological and biochemical characteristics research and mating experiments of some strains can produce more infected propagules show that there may be highly virulent and pathogenic of AFLP6/VGII genotype in Guangxi,southern China,and it has the potential risk of causing an outbreak of infection,which should be widely concerned and highly vigilant.Chinese clinical strains were genetically diverse,thus sexual reproduction/recombination could be one of the causes of the genetic diversity among the Chinese C.gattii species complex.Although none of the strains demonstrated resistance to these drugs,other factors may complicate the therapeutic outcomes of these patients.Due to the small sample size in this study,and C.gattii species complex has not been isolated from the ecological research,the molecular epidemiological characteristics and dissemination of C.gattii species complex in this area need to be further discussed in combination with environmental distribution in addition to the clinical study of expanding sample size.