Etiology And Pathogenesis Analysis On Sporotrichosis

BackgroundSporotrichosis is a deep fungal infection caused by Sporothrix schenckii complex.The pathogen of the disease is a dimorphic fungus,which can produce different morphological characteristics under different temperature conditions.It grows as mycelium at room temperature in the natural environment,when infecting mammalian hosts,it presents a yeast-like phase,which can adapt to human body temperature（37 C）.Sporotrichosis is globally distributed,with a maximum incidence of 2.5%in tropical and subtropical regions.Northeast China and shandong province are high incidence areas.Sporotrichosis is an opportunistic infection.Due to the differences in host immunity and pathogen virulence,individuals infected with sporotrichosis present different clinical phenotypes:fixed cutaneous sporotrichosis,lymphocutaneous sporotrichosis,disseminated cutaneous sporotrichosis,and extracutaneous sporotrichosis.Studies have confirmed that the genetic predisposition of the host plays an important role in the body’s resistance to pathogen infection.The genetic defects of the primary immune system can affect the immune status of the host and lead to different clinical outcomes.Therefore,exploring the genetic predisposition of the host and interpreting the nature of infection differences among different individuals from the genetic level has become a hot topic in current research.Previous studies have confirmed that CARD9,MBL2,MASP2 and other gene defects are associated with susceptibility to a variety of fungal diseases,such as phaeohyphomycosis and invasive aspergillosis,however,whether sporotrichosis is associated with congenital genetic defects is unknown.The morphology,quantity,and virulence factors of the pathogen also affect the occurrence and prognosis of the disease.Sporothrix schenckii was considered as a single species in the past.Recent molecular studies have demonstrated that Sporothrix schenckii is a complex of six important species（S.albicans,S.brasiliensis,S.mexicana,S.globosa,S.luriei and S.schenckii sensu strict）with significant differences in geographical distribution,degree of virulence,disease patterns,and response to therapy.It is important to clarify the type and virulence of pathogen for the diagnosis,treatment and prognosis of the disease.However,there are few studies on the genotyping of Sporothrix schenckii complex in China.Therefore,genotyping of Sporothrix schenckii complex isolated from different regions in China is helpful to understand the current situation of pathogens in China and further explain the mechanism of different types of Sporothrix in the occurrence and development of diseases,which is of great significance for clinical diagnosis and treatment.Based on the above research background,this study is mainly divided into the following three parts:Part 1 Clinical Characterization of 61 cases of sporotrichosisTo analyze the clinical data of sporotrichosis in our hospital,and to summarize the regional clinical features of the disease.MethodsThe clinical data of 61 patients diagnosed with sporotrichosis by fungal culture in our hospital were retrospectively analyzed.Clinical characteristics,including the patient’s gender,age,onset time,residence,course of disease,history of trauma,clinical classification,skin lesions and location,treatment,fungal examination and pathological examination results were analyzed.ResultsThere were 39 males（64%）and 22 females（36%）.The age distribution ranged from 31 to 87 years with an average age of 62.81 years.This disease is more common in winter and spring and the average disease course was 2.5 months.There were 21（34%）cases with definite trauma history.35（57%）fixed cutaneous sporotrichosis,21（35%）Lymphocutaneous Sporotrichosis,five（8%）disseminated cutaneous sporotrichosis.The skin lesions were distributed in 23 cases（38%）of the face,17 cases（28%）of the hand,15 cases（24%）of the upper extremity,one case（2%）of the limbs and back,one case（2%）of the face and lower extremity,and four cases（6%）of the lower extremity.The average treatment time was 3 months.Spores were observed in five cases（8%）by direct microscopic examination.Fungal spores were found in 33（54%）histopathological specimens,28（46%）cases showed no fungal structure in infectious granuloma.ConclusionsAll 61 patients with sporotrichosis were from shandong province.The clinical features are high incidence in winter and spring.Elderly men are susceptible to infection and are more common in exposed parts such as the face and upper limbs.Fixed cutaneous and lymphocutaneous forms are more common.Part 2 Studies on genetic predisposition of the hostObjectiveTo explore the correlation between CARD9,MBL2,MASP2 genes and sporotrichosis,and to verify the functional defects of the discovered genetic defects.MethodsDNA was extracted from the peripheral blood of 61 patients with sporotrichosis and 300 controls.CARD9,MBL2 and MASP2 genes were amplified and sequenced by Sanger sequencing.The gene mutation in the samples was analyzed and compared between the case group and the control group by fisher exact test.Immunohistochemistry and enzyme-linked immunosorbent assay（ELISA）were used to preliminarily analyze the expression of genes in patients and controls.ResultsWe identified 3 variants（c.25G>A,p.Glu9Lys;c.820₈21insG,p.Asp274Glyfs;c.1118G>C,p.Arg373Pro）of CARD9 in patients with the carrier rate of 4.9%（3 out of 61 cases）,which was significantly different from our controls（0/300,0%）and East Asians in Exome Aggregation Consortium（ExAC）database（4/4291,0.09%）（P=4.63×10-3;P=8.81 ×10-5 respectively,Fisher’s Test）.We identified one novel mutation c.25G>A.MASP-2 sequencing in sporotrichosis patients revealed one novel mutation in exon 4:c.477₄78insTGCCACAACCAC（CHNH156₁59）.No mutations were detected in the other matched controls（0/300,P=0.169,Fisher’s exact test）and in the East Asian population（0/4290,Exome Aggregation Consortium database,P=0.014,Fisher’s exact test）.In addition,four variants of MASP2（c.464 A>G,p.H155R;c.1111 G>T,p.D371 Y;c.1130 T>C,p.V377A;c.1479 C>T,p.S493=）and one variant（c.161G>A,p.G54D）of MBL2 were identified in patient group and there were no significant differences in the allele frequencies and genotype frequencies between the patients and controls（P>0.05,Fisher’s Test）.Immunohistochemical results:CARD9 was highly expressed in the skin tissue of the case group compared with controls.There was no significant difference in the expression level between the mutation group and the non-mutation group.ELISA results:sporotrichosis patients had decreased levels of MBL and MASP2 in their serum samples compared to controls（mean value 304 vs 365 ng/ml P=0.002;mean value 244 vs 396 ng/ml P=0.022 T test,respectively）.The mutation in MASP2 did not affect protein expression.ConclusionsThe occurrence ratio of the detected mutations in patients with sporotrichosis is much higher than that of normal people.Preliminary studies showed that the gene mutation did not affect the protein expression,but whether the mutation affected the protein function needs further experimental exploration.Part 3 Phenotypic characteristics,genotyping and drug sensitivity of Sporothrix schenckii complexObjectiveTo study physiological,molecular characters and antifungal susceptibilities of Sporothrix schenckii complex in Shandong.Methods（1）Phenotypic characteristics:All isolated strains were inoculated at Sabouraud Dextrose Agar medium（SDA）27℃,After activation,hyphae and yeast transformation experiments were performed on Brain Heart Infusion Agar medium（BHI）37℃.The assimilation of three sugars（glucose,sucrose and raffinose）by different strains was tested by Yeast Nitrogen Base（YNB）liquid medium.（2）Genotyping of Sporothrix schenckii complex:DNA was extracted from 61 strains of isolates and Sanger sequencing was used for sequence analysis.PCR was performed using universal primers of ITS,CAL and β-tubulin genes.The PCR products were electrophoresed and then purified and sequenced.The sequencing results were compared in NCBI database and CBS database,and MEGA5.0 molecular evolution analysis software was used to construct the evolutionary tree.（3）Drug sensitivity of Sporothrix schenckii complex:The 61 strains were transferred to Potato Dextrose Agar（PDA）medium and BHI medium respectively to obtain mycelium phase and yeast phase colonies,and the in vitro drug sensitivity of the two forms was compared.The in vitro sensitivity of mycelium phase and yeast phase to anidulafungin,micafungin,caspofungin,5-flucytosine,posaconazole,voriconazole,itraconazole,fluconazole,amphotericin B and terbinafine was tested by SensititreTM YeastOneTM and microdilution method.The minimum inhibitory concentration（MIC）values of mycelium phase and yeast phase were calculated,and the geometric mean（GM）MIC50 and MIC90 were calculated.SPSS 19.0 software was used to conduct non-parametric rank sum test for MIC values,and P<0.05 was considered statistically significant.ResultsAll strains were capable of hyphae and yeast transformation and can assimilate glucose and sucrose,but not cottonseed sugar.All the 61 strains were S.globosa.In vitro antifungal susceptibilities test showed that the mycelium phase and yeast phase were the most sensitive to terbinafine,followed by itraconazole,and the least sensitive to fluconazole.The yeast phase of the same strain was more sensitive to terbifenafine,itraconazole,voriconazole,posaconazole,micafen,arnifene and 5-fluorouracil,compared with the mycelium（P<0.05）.However,fluconazole and amphotericin B had no significant difference in mycelium phase and yeast phase.ConclusionsAll strains of Sporothrix schenckii complex in our hospital were S.globosa.The mycelium phase and yeast phase were the most sensitive to terbinafine,followed by itraconazole,and the least sensitive to fluconazole.The yeast phase of the same strain is more sensitive than that of the mycelium.