| Background:Cryptococcus neoformans is an important environmental pathogenic fungus.It has no close symbiotic relationships with the host but has high pathogenicity and infection rate,which is due to the excellent adaptability to the changeable ecological environment.It can cause cryptococcal meningitis with high mortality.According to the statistics,it infects one million people and causes 0.6 million people’s death every year in the world.As conditional pathogenic fungus,Cryptococcus neoformans tend to invade people with immune deficiency,especially those with HIV infection.It is reported that cryptococcal meningitis is responsible for 13%-44% of HIV deaths worldwide.According to China’s special national conditions,HIV patients are mainly admitted to local infectious disease hospitals or public health centers.So far,the epidemiological data of Cryptococcus neoformans reported in China are all from the affiliated hospitals of domestic universities and all of them are non-HIV patients,while the research on Cryptococcus neoformans with HIV in China is rarely reported.Therefore,in order to enrich the epidemiological data of HIV associated cryptococcosis in Chongqing and China,it is necessary to study the genetic diversity,phenotype and drug sensitivity of Cryptococcus neoformans infected with HIV in Chongqing,which will provide a theoretical basis for the prevention and control of HIV and the diagnosis of Cryptococcus neoformans.Objective:This study is aimed at showing the clinical characteristics and treatment plan of 58 cases with HIV-related cryptococcosis in Chongqing,and investigating the molecular types of Cryptococcus neoformans and the drug sensitivity information of five common antifungal drugs,so as to provide a scientific basis for improving the diagnosis and treatment of HIV-related cryptococcosis in Chongqing and even in China.Methods:1.To analyze and summarize the clinical data of 58 patients with HIV-related cryptococcal meningitis in Chongqing,and 171 strains of Cryptococcus were collected from clinical samples of CSF and blood.2.In order to determine whether 171 clinical isolates are the Cryptococcus neoformans,the traditional phenotypic identification methods such as ink staining and detection of Cryptococcus neoformans antigen were used to identify the related strains to the level of species or bacterial complex.3.To understand the genotype of 171 strains of Cryptococcus neoformans,the genome of related strains was extracted,and PCR amplification was carried out using serotype and mating-specific primers.Finally,the composition of clinical strains of Cryptococcus neoformans is determined.4.The MLST classification system recommended by Isham was used to determine the sequence type(ST)classification.5.In vitro antifungal susceptibility of 171 clinical isolates was determined.6.Phylogenetic analysis was carried out on the serial sequences of 7 housekeeping genes of the isolates and Cryptococcus strains reported in China(one representative strain for each ST)using MEGA version 7.0 software.Results:1.Among the 58 patients,46 were male and 12 were female;the age distribution ranged from 11 to 75 years old;the main source of the strain was cerebrospinal fluid(n =143;83.6%),followed by blood(n = 27;15.8%),and one strain was isolated from pleural fluid.2.The positive rate of ink staining was 64.91%,and the positive rate of Cryptococcus neoformans antigen detection was 100%.3.Among 171 clinical strains,167 were serotypes A,mating type α.there are four AD heterozygotes,which were AaDα.4.A total of five ST types were isolated from 171 clinical strains,there were 146 strains that belong to ST5(85.38%),11 strains that belong to ST31(6.43%),1 strain that belongs to ST91(0.58%),1 strain that belongs to ST15(0.58%),and 1 strain that belongs to ST79(0.58%),11 strains have not been identified,ST15 and ST79 have not been reported in other regions of China.5.Analysis of strain resistance revealed that there was one strain that is resistant to amphotericin B,two strains that are resistant to 5-fluorocytosine,one strain that is resistant to voriconazole,eight strains that are resistant to itraconazole,and no strains that is resistant to two or three drugs at the same time.6.The strains isolated from AIDS patients and the strains isolated in this study are all in cluster I,although ST79 is in cluster I,it is far from other strains in cluster I;Cluster II only contains one strain,which is VGI genotype and ST332;The strains of cluster III belong to C.gattii,and these strains were isolated from non AIDS patients.7.Most of the patients were single type infection,but six patients showed mixed infection.Conclusion:1.In Chongqing,the number of HIV patients with cryptococcal meningitis should not be ignored,and the clinical manifestations are not typical,which is easy to cause missed diagnosis and misdiagnosis.Making full use of the clinical methods,such as ink staining,latex agglutination test,combined with molecular detection,is a feasible scheme to improve the level of early diagnosis.2.In Chongqing,VNI was the main molecular type of clinical isolates,and ST5 was the main ST type,α-mating type accounted for the majority of the mating type,which was consistent with the results reported in China,such as Shanghai and Beijing.It is worth noting that four AD heterozygous strains(VNIII,AaDα)were found in this study,which was rare in domestic cryptococcosis patients,suggesting that the population of Cryptococcus infection strains combined with HIV patients in Chongqing may have higher genetic polymorphism.3.In this study,most of the patients were single type infection,but six patients showed mixed infection.4.The clinical strains in Chongqing were resistant to common antifungal drugs amphotericin B,5-fluorocytosine,itraconazole and voriconazole,especially itraconazole resistant strains. |
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