The Epidemiology Of Sporotrichosis And Identification And Phenotypic Characteristics Of Sporothrix And In Vitro Susceptibility Against Antifungals And Traditional Chinese Medicines

PartⅠ The Epidemiological Characteristics of205Cases ofSporotrichosis in DalianObjective：To summarize and analyze the epidemicological characteristics of205cases of sporotrichosis in Dalian.Methods：A retrospective study of the clinical data of Sporotrichosis diagnosed inthe1stAffiliated Hospital of Dalian Medical University between1998and2011wasperformed.Results：The cases of sporotrichosis per year were15cases or so which werediagnosed in the1st Affiliated Hospital of Dalian Medical University in the last14years without a big increase. The results of this study showed higher incidence offemale, male and female incidence ratio was1:1.14. The age of sporotrichosis patientswas from3years old to83years old with a high percentage of people between40and69years old（74.7%）. A proportion of sporotrichosis occurred in chridren（10.7%）.Sporotrichosis had a high incidence in winter or spring.Most patients had a history oftrauma（62.4%）. The most common clinical presentation was fixed type (51.2%),followed by lymphocutaneous type (37.6%). We reported a case of disseminatedcutaneous sporotrichosis without immunodeficiency and other latent diseases.Thelesions on upper limbs were the most common（47.8%）, followed by on face（37.1%）.The lesions of fixed type usually occurred on face and upper limbs, the lesions oflymphocutaneous type were more commonly on upper limbs, and disseminated typepresented as multiple cutaneous lesions. The lesions on the face more often presented as fixed type (69.7％) than those on the limbs (44.1％). The patients came mainly from thecountryside of surrounding regions of Dalian, with Zhuanghe, Pulandian andWafangdian being areas of high-incidence.Conclusion： The incidence of sporotrichosis in Dalian had a steady trendcompared to outbreaks in Jilin province in recent years, which belonged to sporadiccases. The epidemiological characteristics of our cases were similar to those reported byother domestic authors. A case of disseminated cutaneous sporotrichosis in animmunocompetent patient was reported. PartⅡ Identification and Phylogenetic Characteristics of SporothrixIsolates in Northeast ChinaObjective：To perform molecular identification and phylogenetic analysis ofsporothrix isolates of clinical origin in Northeast China, to investigate phylogeneticspecies of the Sporothrix complex in these areas and to determine whether differentclinical forms are associated with different phylogenetic species and whether outbreaksin Jilin province is caused by more virulent species than sporadic cases in Heilongjiangand Liaoning provinces.Methods：Preliminary morphological identification was performed by macroscopicfeatures of colony, microscopic features of conidia, and temperature dimorphism. Thenthe phylogenetic tree of the CAL locus by Neighbor Joining—NJ was established for74sporothrix isolates of clinical origin in Northeast China and16isolates reported in theliterature.Results： All isolates were morphologically identified as S.schenckii sensulato.Then the sequences of partial calmodulin-encoding gene of our isolates werecompared by BLAST with sequences available from NCBI GenBank.The sequencesblast revealed all isolates had99%similarity with the type strain of S.globosa(AM116908) and100%similarity with the type strain of S.globosa (AM490354).Butsimilarity with the type strain of S.schenckii (AM117437) was only88%. Thephylogenetic tree of the CAL locus analyzed by Neighbor-Joining revealed sixwell-defined and supported groups, i.e. six phylogenetic species. All clinical isolatesoriginated in Northeast China in this study were clustered with group S. globosa (AM116908and AM399018). Furthermore, our results showed that S. globosa could befurther subdivided into two sub-clades,68of our isolates were clustered withAM116908.1and three previously published Chinese environmental isolates(AM399002, AM399005, AM399004), the other six isolates were clustered withAM399018.Conclusion：(1)S.globosa is the predominant pathogenic species in NortheastChina up to now. To our knowledge, this is the first report of phylogenetic analysis ofChinese clinical isolates of sporothrix.(2)Clinical forms of sporotrichosis were notdetermined by phylogenetic species of sporothrix.(3)Outbreaks of sporotrichosis inJilin province was caused by S.globosa.(4)S.globosa was divided into two highlysupported sub-clades. PartⅢ Phenotypic Characteristics andIn Vitro Susceptibility against Antifungalsand Traditional Chinese Medicines of S.globosaObjective：To summarize phenotypic characteristics of S.globosa in China so thatcorrelated checks can be carried out routinely in the lab for identification of speciesquickly, to understand in vitro drug susceptibility of S.globosa in China, and to exploretraditional Chinese medicines with anti-sporothrix activity and provide a new topicalmedication.Methods：(1)All isolates were subcultured on SDA, PDA, CMA, and OA platerespectively and incubated at30°C in the dark for21days, then growth rate andmorphology of colony were observed.(2)The microscopic features were determinedfrom slide cultures made on CMA after10days of incubation at30°C. Coverslips wereexamined under a light microscope.(3) All isolates were inoculated on PDA plate at30°C,35°C,37°C respectively in the dark for21days, then the colony diameters weremeasured.(4) Carbohydrate assimilation tests were performed in96-well microplatesand tested for dextrose, sucrose and raffinose.(5)According to the M38-A2techniqueand the microdilution method proposed by CLSI, the susceptibility tests were conductedfor4drugs (terbinafine, amphotericin B, itraconazole and caspofungin) against themycelial phase of74S.globosa isolates in China.(6)In vitro anti-sporothrix activity of eugenol, citral, cinnamaldehyde was explored according to the M38-A2technique andthe microdilution method.Results：(1) The macroscopic morphologies of all isolates were similar on any kindof medium, with many wrinkles on the surface of colony and short floss on the edge ofcolony. The colony on OA plate was the darkest, followed by CMA, and then PDA. Themelanin on SDA was the least.(2) Not only subhyaline, obovoid conidia but also thickwalled, dark brown, globose conidia were observed after incubation for10days at30℃on CMA.(3)After incubation respectively at30°C,35°C,37°C in the dark for3weeks,the best fungal growth was observed at30°C, the colonies attained a diameter of25to45mm with the mean colony diameter of34mm.Colony growth at35°C was slower thanthat observed at30°C, with12~15mm diameter and mean13mm diameter.At37°Cmost isolates showed restricted growth(up to mean9mm in diameter) with the exceptionof three isolates which were unable to grow at37°C.(4)Carbohydrate assimilation testspresented the following results: all isolates assimilated dextrose and sucrose, and wereunable to assimilate raffinose.(5)Against the mycelial phase of74S.globosa isolates,TRB showed the best in vitro antifungal activity with MICs of0.06-0.5μg/ml and ageometric mean (GM) MIC of0.1866μg/ml, followed by amphotericin B with MICs of2-8μg/ml and a geometric mean (GM) MIC of4.8252μg/ml. Both itraconazole andcaspofungin showed high MICs. The MICs of S.globosa isolates of different clinicaltypes for every kind of antifungal drug showed no statistical difference.(6)Eugenol,citral, cinnamaldehyde all showed anti-sporothrix activity with a geometric mean (GM)MIC of709.1481μg/ml for eugenol,509.0566μg/ml for citral, and437.8494μg/ml forcinnamaldehyde.Statistical analysis showed that in vitro anti-sporothrix activity of citraland cinnamaldehyde was stronger than that of Eugenol.Conclusion：(1) S.globosa produced globose to subglobose, brown to dark brownconidia and the colony diameter of S.globosa was less than50mm after21days ofincubation on PDA at30°C.(2)S.globosa assimilated sucrose, and was unable toassimilate raffinose.(3)S.globosa showed restricted growth or no growth at37°C.(4)Among the4antifungal drugs, TRB showed the best in vitro antifungal activity withthe lowest geometric mean MIC.There was no correlation between in vitro drugsusceptibility of S.globosa isolates and clinical forms of sporotrichosis.(5)In vitroanti-sporothrix activity of citral and cinnamaldehyde was stronger than that of Eugenol.