Establishment And Preliminary Application Of Multiplex PCR Method For Common Superficial Mycosis Pathogens

Superficial fungal infections are the most widespread type of fungal pathogens in nature.In recent decades,the acceleration of urbanization and globalization has led to a continuous increase in infections caused by superficial fungi worldwide.The pathogenic fungi of superficial mycoses specifically invade the skin and other superficial tissues,causing severe itching and disabling damage to patients,leading to social embarrassment and significantly affecting personal quality of life.In patients with diabetes or immune deficiencies,superficial fungal infections can progress to deeper invasive infections with severe consequences.Rapid and accurate identification of common superficial fungal pathogens in clinical settings is crucial for the prevention,diagnosis,and treatment of superficial fungal infections.This study aims to establish a multiplex fluorescence quantitative PCR method for common superficial fungal pathogens,firstly using next-generation sequencing(NGS)technology to investigate the distribution of clinical superficial fungal pathogens and,in combination with the reported distribution of superficial fungal pathogens in various regions,identify the eight most common superficial fungal pathogens.A multiplex fluorescence quantitative PCR method was then established for these eight pathogens,and the method was evaluated.Finally,clinical samples of non-diabetic/diabetic populations infected with superficial fungi were analyzed.The main research content is as follows:By targeting amplification and enrichment of the ITS region of clinical samples of superficial fungal infections in Kunming,Yunnan,and performing amplicon sequencing,the results showed that the high abundance genera of superficial fungi were Trichophyton,Microsporum,Candida,and Malassezia.The top five species of superficial fungi were Candida albicans,Microsporum gypseum,Trichophyton rubrum,Malassezia furfur,and Trichophyton mentagrophytes,which were consistent with the dominant fungal species reported in Yunnan for superficial fungal infections.Based on the fungal abundance results of superficial fungi in the Kunming area of Yunnan Province studied by NGS,combined with the distribution of superficial fungal pathogens in Kunming and other areas that have been reported,8 common superficial fungal pathogens were identified:Trichophyton rubrum,Microsporum canis,Malassezia,Epidermophyton floccosum,Trichophyton mentagrophytes,Microsporum gypseum,Candida albicans,and Microsporum audouinii.A multiplex Taq Man probe-based fluorescence quantitative PCR method was established and validated for these pathogens.Results showed that the designed primer and probe sets had good specificity and high sensitivity for pathogen detection in single fluorescent quantitative PCR validation.Based on the viral genus classification,the 8 superficial fungi were divided into three groups after adjustment and validation:Group A included Trichophyton rubrum,Epidermophyton floccosum,and Microsporum canis;Group B included Microsporum gypseum and Trichophyton mentagrophytes;Group C included Candida albicans,Malassezia,and Microsporum audouinii.The specificity verification of the multiplex fluorescence quantitative PCR showed no cross-reactivity among the 8 superficial fungi primer and probe sets,indicating strong specificity.The sensitivity verification showed that the detection limit of superficial fungi in Group A was 1.03×10~1copies/μl for Trichophyton rubrum,1.04×10~1copies/μl for Epidermophyton floccosum,and 1.02×10~1copies/μl for Microsporum canis;the detection limits in Group B were 3.33×10~1copies/μl for Trichophyton mentagrophytes and 3.34×10~1copies/μl for Microsporum gypseum;and the detection limits in Group C were 7.15×10~1copies/μl for Candida albicans,7.3×10~1copies/μl for Malassezia,and 7.27×10~1copies/μl for Microsporum audouinii,indicating high sensitivity.The repeatability of the multiplex fluorescence quantitative PCR method was evaluated for intrabatch and interbatch,and the maximum variation coefficient(CV)of Ct values for the three superficial fungal groups was less than 5%,indicating good repeatability.Clinical sample verification results demonstrated that the multiplex fluorescence quantitative PCR method had good accuracy for pathogen detection.The last 45 samples of superficial fungal infections from non-diabetic patients and 55 samples from diabetic patients were collected from clinical settings.A common superficial fungal multiplex fluorescent quantitative PCR method was used to detect clinical samples from both groups.The results showed that 43 out of 45non-diabetic samples and 52 out of 55 diabetic samples were positive for superficial fungal infections.Among the non-diabetic samples,36 cases were positive for 5different fungal pathogens,including 7 cases of Candida albicans,8 cases of Microsporum gypseum,13 cases of Malassezia,4 cases of Trichophyton rubrum,and4 cases of Trichophyton mentagrophytes.7 samples(16.28%)had mixed infections.Among the diabetic samples,36 cases were positive for 5 different fungal pathogens,including 19 cases of Candida albicans,11 cases of Microsporum gypseum,2 cases of Malassezia,3 cases of Trichophyton rubrum,and 1 case of Trichophyton mentagrophytes.16 samples(30.77%)had mixed infections.The superficial fungal infections in the two groups of patients showed no significant difference in different gender groups,but there were significant differences in different age groups.Malassezia infections were mainly concentrated in the 0-19 age group in non-diabetic patients,consistent with the age characteristics of superficial fungal infections reported.Candida albicans and Microsporum gypseum infections were mainly concentrated in the 50-59 age group in diabetic patients,and Candida albicans was mainly found in patients aged 70-79,consistent with the reported cases of superficial fungal infections in diabetic patients.To sum up,this paper has established a multi fluorescent quantitative PCR method for common superficial fungal pathogens with good specificity,sensitivity and repeatability,and then analyzed the epidemiological characteristics of diabetes/non diabetes population infected with superficial mycosis.The research results provide an important technical method for the control of superficial mycosis and clinical diagnosis and treatment,To provide an important scientific basis for further in-depth study on the epidemic characteristics of pathogenic spectrum of superficial mycosis in diabetes/non diabetes population.