A Series Of Study About The Genotype Of Cryptococcus Neoformans And In Vitro Susceptibility, Molecular Diagnosis

BackgroundRecently, the development of molecular techniques has greatly improved ourunderstanding of genetic diversity of Cryptococcus species complex。Several differentmolecular typing methods have been used in epidemiological and genotypic analysis of theCryptococcus species complex, including random amplified polymorphic DNA (RAPD),PCR fingerprinting, restriction fragment length polymorphism (RFLP) analysis, AFLPanalysis，sequencing analysis of single locus and multi locus.In this study, we determined the genotype of61Cryptococcus neoformans andCryptococcus gattii Complex strains isolate from HIV-positive and HIV-negative patientsby using the IGS1gene and CAP59gene sequencing analysis. The study of correlation ofspecies,genotype and in vitro susceptibility, the clinical outcome will plays an importantrole to guide clinical therapy. In the study,Microdilution method M27-A2program wasused to test the antifugal susceptibilities of clinical isolates t amphotericinB,fluconazole,itraconazole and flucytosine and we try to find the relationship betweengenotype and MICs.Otherwise, how to diagnosis the cryptococcal infection timely and excisely ismeaningful to initiating antifungal therapy and improving clinical outcome. We built a newmolecular method based on IGS1gene to detect Cryptococus.spp more quickly by usingLAMP.Objects1. multi locus sequencing typing and genotype determination2To investigate the antifugal susceptibilities of clinical isolates to amphotericin B,fluconazole, itraconazole and flucytosine3. To analysis the correlation of genotype and MICs4, To build a new molecular method to detect Cryptoccus. Spp by using LAMP Methods1. multi locus sequencing typing and genotype determinationA total of61Chinese clinical Cryptococcus neoformans species complex isolateswere analysed by sequences of IGS1gene and Capsule59gene, all of the strains weredetermined their genotype.2To investigate the antifugal susceptibilities of clinical isolates to amphotericin B,fluconazole, itraconazole and flucytosineMicrodilution method M27-A2program was used to test the antifugal susceptibilitiesof clinical isolates t amphotericin B,fluconazole,itraconazole and flucytosine3. To analysis the correlation of genotype and MICsAnalysis of susceptibility data with origins, species and genotypes.4, To build a new molecular method to detect Cryptoccus. Spp by using LAMPBuild a new molecular method based on IGS1gene combining the LAMP technique.To examine the specificity of the new method by detecing the Cryptococcal spp and other48fungal species. Sensitivity was tested by10-fold DNA dilution series.Results1. multi locus sequencing typing and genotype determinationIn all the61strains isolated from Chinese HIV-Negative and-Positive patients,. VNI(73.77%) was the most represented genotype,5strains(8,20%) were VNII genotype,2strains(3,28%) were VNIII genotype, and the rest (14,75%)were VGI genotype2To investigate the antifugal susceptibilities of clinical isolates to amphotericin B,fluconazole, itraconazole and flucytosineThe MIC90and susceptibility ranges for total Cryptococcus species complex isolateswere as follows:1(0.0625~1) μg/ml for amphotericin B,4(0.125~16) μg/ml forfuconazole,0.25(0.0313~4) μg/ml for itraconazole, and4(0.125~8) μg/ml forflucytosine.3. To analysis the correlation of genotype and MICs(1)When the isolates were analyzed according to the origin of the patients the isolatesfrom HIV-negative patients showed lower geometric mean for fluconazole (1.1589/3.1228, p=0.001) and flucytosine (1.4038/2.9720, p<0.001), compared with those from AIDSpatients.(2)There were no significant differences in susceptibilities between the species for eachagent(3)Although the geometric mean MICs were different, it showed no statistically significantdifference (P>0.05) between genotype VNI and VGI4, To build a new molecular method to detect Cryptoccus. Spp by using LAMPThe experiment using LAMP primers for Cryptococcus neoformans IGS1gene did notshow cross-reactivity with other fungi, the specificity assay showed that positiveamplification is only obtained with the genus Crypotcoccus neoformans and Cryptococcusgattii complex. The detection limit was found to be0.002fg.Cnclusion1. The isolates of VNI genotype is the most majority of clinical Cryptococcusneoformans species complex isolates from Chinese HIV-Positive and HIV-Negativepatients。2，Most of the clinical Cryptococcus species showed excellent suscepibitily to theamphotericin B, fluconazole, itraconazole and flucytosine. Whereas there was a high rateof tolerance against amphotericin B (MICs range from0.5-1μg/ml) in these clinicalisolates. These data suggested that amphotericin B as first-line therapy for Cryptococcusmay lead to the tolerance or even resistance of pathogen over time although the explicitmechanism is not clear so far.3,As for fluconazole and flucytosine, Cryptococcus neoformans isolates from AIDSpatients were less susceptible than those from Non-AIDS patients.4,The study of correlations of species, genotypes and in vitro susceptibilities showed nosignificant clinical meaning.5,The LAMP technique based on Cryptococcus neoformans H99IGS1gene enablesspecific deciton of Cryptococcus neoformans and Cryptococcus gattii Complex. Themethod provides a powerful tool for rapid diagnosis in the clinical lab.