Species Identification Of Superficial Fungal Diseases And Antifungal Effect Of Nitrogen-Doped Carbon Nanozyme Combined With NIR

Background Superficial fungal disease refers to fungal infections of the skin and mucous membranes,hair and fingernails,mainly caused by Dermatophyte and Yeast,but the distribution of species in different regions and seasons is different,and Species vary in different infection sites.Since the species of different infection sites may be different,It's still unknown whether the species of infection in different sites of the same patient are the same?Is there an autoinfection?In recent years,with the abuse of antibiotics,immunosuppressant drugs,anti-tumor drugs,and the factors of chronic diseases and chronic infections,the incidence of clinical fungal infections,especially superficial fungal diseases has gradually increased.At the same time,it is also difficult to cure superficial fungal diseases due to the increase of drug-resistant fungi.Nanozymes are a class of nanomaterials with enzyme-like activity,and their antibacterial activity is a hotspot research in the global.Carbon materials have attracted attention because of their good biocompatibility.They can be doped with other materials to adjust their enzymatic activity.Moreover,carbon materials have good photothermal effects and can absorb light energy and convert it into thermal energy.Studies have reported that carbon nanozymes have an inhibitory effect on bacteria,but less research about their inhibitory effect on fungi.Nitrogen-doped carbon nanozyme(N-Carbon nanozyme)not only has a variety of enzyme activities,but also has a photothermal effect.So the effect of N-Carbon nanozyme combined with Near Infrared(NIR)on fungi needs to be explored.Objective To identify and analyze the main strains of clinical superficial fungal diseases and the distribution of species in different body sites;to investigate whether there are differences in the fungal species in different infected body sites of the same patient.To explore the antifungal effect of N-Carbon nanozyme combined with NIR on Candida albicans(C.albicans)and Trichophyton rubrum(T.rubrum)in vitro,and provide a new strategy for clinical treatment of superficial fungal diseases.Method1.55 cases of patients diagnosed as superficial fungal diseases in the department of dermatology,clinical medical college of Yangzhou University from April 2019 to October 2019 were enrolled,Fungal detection under microscopy and fungal culture of the samples taken from skin lesions were performed,and followed by isolated strain purification.2.DNA of filamentous fungi were extracted by benzyl chloride;ITS1 and ITS2segments were amplified by using universal primers and genes sequencing were conducted.Fingerprint analysis by using simple repeat sequence oligonucleotide(GACA)4 as primers for polymerase chain reaction(PCR)were performed;Saccharomyces were identified directly by mass spectrometer assay.3.N-Carbon nanozyme was prepared by pyrolysis method,and characteristics were observed under scanning and transmission electron microscopy.4.Measure the photothermal conversion effect of N-Carbon nanozyme under 808nm NIR irradiation.5.Using the plate counting method,the optimal material concentration and optimal light parameters of N-Carbon nanozyme combined with 808nm NIR against Candida albicans were screened according to the number of colony forming units(CFU).6.Taking C.albicans and T.rubrum as study objects,in vitro antifungal experiments of N-Carbon nanozyme combined with 808nm NIR were performed.The experimental groups were designed as:(I)blank control group:the fungus untreated;(II)light group:the fungus was exposed to 2.5W/cm~2NIR for 8 min;(III)material group:the fungus was treated with 250?g/ml N-carbon nanozyme for 30min;(IV)the experimental group:the fungus was treated with 250?g/ml N-carbon nanozyme combined with 2.5W/cm~2 NIR for 8 min.7.The antifungal activity was observed by SYTO9/PI live/dead staining.The antifungal activity of C.albicans was also studied by scanning electron microscopy and plate counting method,and determined by the number of colony forming units(CFU).Result1.55 patients with superficial fungal diseases,including 25 cases of tinea cruris(33.3%),21 cases of tinea pedis(28%),14 cases of tinea corporis(18.7%),12 cases of onychomycosis(16%),and 3 cases of tinea manuum(4%).Positive fungal detections under microscopy were found in 63 cases(84%),and 71 strains of fungi were isolated by culture(94.7%).20 of them had infections in two sites of the body.2.Filamentous fungi were sequenced after amplification of ITS1 and ITS2 segments.Among them,63 strains of T.rubrum and 5 strains of Microsporum canis;1 strain of C.albicans,1 strain of Candida smoothia and 1 strain of Candida famata was detected by mass spectrometer.Of the 20 patients with two-site infections,18 were infected with the same species-T.rubrum.In the fingerprint,the Microsporum canis contains 800kb and1200kb bands,and the T.rubrum contains 600kb,950kb and 1050kb bands.3.N-Carbon nanozyme can convert light energy into thermal energy under NIR irradiation,and its light-to-heat conversion rate is concentration-dependent and has a positive correlation with the intensity of power.4.According to the result of counting the colony forming units(CFU)by the plate method,the reaction conditions were set to:N-Carbon nanozyme 250?g/ml;NIR2.5W/cm~2,8min.5.In vitro experiments against C.albicans,no significant difference was found among the light group,the material group and the blank control group.In the experimental group of C.albicans,it was observed that at least 99.9%of C.albicans were killed by plate counting;The SYTO9/PI live/dead staining showed most of the C.albicans were stained as red fluorescence,indicating a high fungal killing power.In addition,the biological scanning electron microscope revealed that the cell morphology of C.albicans distorted obviously.6.In vitro experiments against T.rubrum,although a certain degree of red fluorescence can be seen in the light group and the material group by usingthe SYTO9/PI live/dead staining method,the red fluorescence signal of the experimental group significantly increased,indicating that most of the T.rubrum were killed.Conclusion1.Superficial fungal diseases are mostly caused by Dermatophytes.The fungal infection in different sites of the same patient mostly caused by the same strain,considering the possibility of autologous infection.2.The molecular identification of filamentous fungi by combining ITS1 and ITS2segments can basically identify species.At the same time,through PCR fingerprint analysis,the same species usually display same bands fingerprint,which can help batch identification.3.N-Carbon nanozyme combined with NIR can significantly inhibit C.albicans and T.rubrum.