Population Structure Characteristics, Colonization Factors And Antifungal Susceptibility Testing Of Candida Albicans In Patients With Cirrhosis

PART I POPULATION STRUCTURE CHARACTERISTICS OF Candida albicans COLONIZED IN PATIENTS WITH CIRRHOSISObjectives Genotype of Candida albicans isolates from patients with cirrhosis was identified by mutilocus sequence typing(MLST)in order to characterize population structure of C.albicans colonized in patients with cirrhosis.Methods 1.Three hundred and sixty-five fecal samples from 181 patients with cirrhosis(cirrhosis group)and 184 healthy individuals(control group),and 207 samples from other anatomic sites in these patients with cirrhosis(103 from oral,84 from hydrothoax or ascite and 20 from blood)were collected.Through culture in sarcopal agar plate medium,isolation and preliminary evaluation by CHROM agar,finally isolates were identified as C.albicans by internal transcribed spacer(ITS).All identified isolates were genotyped by MLST of 7 housekeeping genes combination in order to identify sequence type(ST),and diploid sequence type(DST)of all isolates was determined by combination of identified STs.2.The phylogenetic tree of all DSTs from faecal isolates was constructed by unweighted pair-group method with arithmetic means(UPGMA)using MEGA 7.0 version software.Difference of C.albicans population structure between cirrhosis group and control group wascompared and analyzed by ?2-test.Homology analysis was also utilized to identify the clone cluster(CC)of all DSTs by e BURST version3 software.3.The phylogenetic tree was constructed by UPGMA in order to analysis the population structure characteristics of C.albicans DSTs of isolates from other anatomic sites in these patients with cirrhosis.Difference between or among different genotypes of isolates from two or above anatomic sites in same patients was analyzed by checking nucleotide sequence.Results 1.There were 121 isolates identified by ITS,including 88 isolates from faeces(52 isolates from cirrhosis group and 36 isolates from control group)and 33 isolates from other anatomic sites of patients with cirrhosis(22 isolates from oral,8 isolates from hydrothoax or ascite,3isolates from blood).Among 121 isolates,97 DSTs were determined by MLST,including 60 new DSTs;174 STs,including 13 new STs.There were 94 variability polymorphism points in 7 housekeeping genes.There were 2.55 STs determined by every variability polymorphism loci in AAT1 a housekeeping gene;however,there were 1.18 STs determined by every variability polymorphism loci in MPIb housekeeping gene.2.According to UPGMA,88 isolates from faeces were distributed into 14 clades and 2 singletons.These isolates belonged to 23 CCs and 16 singletons by e BURST analysis.Among 88 isolates,there were 52 isolates from cirrhosis group,and 36 isolates from control group.Clade 8 was the major clade of C.albicans in cirrhosis group.In clade 8,isolates from cirrhosis group was significant higher than that from control group(22/52:3/36;?2=12.07,p=0.001<0.05).Clade 18 was the most common Clade among 36 isolates in the control group.There was no significant difference between control group and cirrhosis group in Clade 18(8/36: 10/52;?2=0.117,p=0.731>0.05).Among all Clade 18 isolates from faeces,there were 94.44%(17/18)isolates attributed to CC8 by e BURST analysis,andthe founding DST of CC8 was DST727.The detective rate of DSTs 1931 strains was highest(5/52,9.61%)in cirrhosis group,higher than that in control group(1/36,2.78%),but there was no significant difference of the detective rate between two groups(? 2=1.565,p=0.394>0.05).3.Thirty-three isolates from different anatomic sites of patients with cirrhosis were attributed to 8 Clades and 3 singletons.Among these isolates,Clade 8and Clade 18 were common Clades,11 and 8 isolates respectively.Among11 isolates from hydrothoax,ascite and blood,4 isolates belonged to Clade18 and 3 isolates belonged to Clade 8.C.albicans isolates of twenty-two patients with cirrhosis were detected not only from patients faeces but also from other different anatomic sites(from 16 patients' oral and faeces,3patients' faece and ascite,2 patients' faece and blood,a patient's oral,faeces and ascite).The genotypes of isolates from different anatomic sites in7 patients were same,but those in 15 patients were different.In these different genotypes,there were differences in 33 nucleotide sites by checking the nucleotide sequence,and loss of Heterozygosity(LOH)in 32 sites was presented.Conclusion1.The genotype of C.albicans shows diversity.Among 7house-keeping genes,AAT1 a possesses strong genotyping ability,but MPIb is more stable.2.Clade 8 is the special Clade of C.albicans colonized in patients with cirrhosis.CC8 in Clade 18 is the major CC of C.albicans colonized in the intestinal tract of local people.DST1931 may be the prevalent genotype of C.albicans colonized in the intestinal tract of patients with cirrhosis.3.Clade 8 and Clade 18 may be the two major Clades of system candidasis in local patients with cirrhosis.C.albicans colonized in gut is the source of system candidasis in patients with cirrhosis.LOH leads to the genotype difference of isolates from different anatomic sites in same patient with cirrhosis.Part II COLONIZATION FACTORS AND ANTIFUNGAL SUSCEPTIBILITY TESTING OF Candida albicans IN PATIENTS WITH CIRRHOSISObjective Colonization correlated factors were investigated and analyzed.The special genotype or clade of drug-resistant strains was analyzed by anti-fungal susceptibility testing.Overall results are expected to provide theoretical basis for the prevention from C.albicans infection.Methods1.The detective rates of C.albicans isolates from faeces of the two groups(cirrhosis group and control group)were compared by ?2test.2.The personal information obtained by questionnaire and severity evaluated by Child-pugh classification of 181 patients with cirrhosis were collected.Univariate analysis and logistic regression were used to analysis the colonization correlative factors of C.albicans in patients with cirrhosis.3.Anti-fungal susceptibility testing was performed among all isolates from patients with cirrhosis and healthy individuals by conventional dilution.Results1.The detective rate of C.albicans isolates from cirrhosis group was significantly higher than that from control group(52/181 :36/184,?2=4.188,p=0.041<0.05).2.Through univariate and logistic regression analysis,influence factors of C.albicans colonization in patients with cirrhosis included age,gender,economic situation,animal,alcoholic,pickled food,eating raw garlic,>7 days antibiotic treatment and severity of cirrhosis patients(P<0.05).There was no significant difference among different antibiotic to C.albicans colonization in patients with cirrhosis(OR:0.47,p=0.178>0.05).3.One hundred and twenty-one isolates were detected by anti-fungal susceptibility testing,showing 8 drug resistantisolates(including two isolates from different anatomic sites of same patients with cirrhosis).All 8 isolates were resistant to itraconazole,7isolates to voricinazole and 4 isolates to fluconazole.However,all 8isolates were susceptive to 5-fluorouracil.Five isolates(5/8,62.5%)resistant to antifungal agents belonged to Clade 18.All isolates from different anatomic sites of same patients with cirrhosis shared the same result of antifungal susceptibility.Conclusion 1.Compared with healthy individuals,patients with cirrhosis are easily colonized by C.albican.2.The risk factors of C.albicans colonized in patients with cirrhosis include increase of age,female,animal,alcohol intake,pickled food,>7 days antibiotic treatment and severe condition.However,well economic condition and eating raw garlic reduce the colonization risk of C.albicans in patients with cirrhosis.3.Clade 18 is the common drug resistant Clade.C.albicans isolates show resistant to azole antifungal agent frequently,but susceptive to5-fluorouracil,indicating local patients infected by C.albicans should avoid to use azole antifungal agent.Genotypes from different anatomic sites of same patients with cirrhosis may be different,but share same antifungal susceptibility,suggesting there is the same source of C.albicans isolates colonized in different anatomic sites in same patients with cirrhosis.