The Epidemiological Analysis Of Fungal Infection And Study On Detection Methods Of Main Pathogenic Fungi

In recent years, with the increasing of fungal infections and the expanding of infection species, it's difficult to diagnose early due to the lack of specific symptoms and signs caused by fungal infection. Moreover, the limited antifungal agents as well as its considerable side effect and the serious problem of fungal resistance in clinical treatment lead to difficulties in treatment and high mortality. Therefore, it's an effective way of reducing the mortality of fungal infection by understanding the common characteristics and epidemiology of pathogenic fungi and establishing rapid, sensitive and specific methods for detection.Currently, the methods of fungal culture used for detection are time-consuming, laborious and have certein subjectivity and other shortcomings. Non-culture of fungi includes methods of serology, histopathology and molecular biology and so on. There are some difficulties in cloning antibodies and high false positive rate in serology. In addition, samples collected difficultly and traumatic defects exist in histopathology. PCR based on molecular biology techniques greatly promotes the development of fungal detection methods, such as microsatellites (also known as Simple sequence repeats, SSR), real-time fluorescence quantitative PCR (Real-time PCR), AuNPs (gold nanoparticles) labeling and DNA hybridization technology, but choosing the target genes is essential to the identification of fungi.Fungal mitochondrial DNA strictly abides by the maternal inheritance and different sections of mitochondrial DNA sequence are at different mutation rate. These characteristicses make mitochondria use for the species identification and classification. Among of them, the mitochondrial cytochrome C oxidase subunitâ… andâ…¡(COâ… , COâ…¡) genes have been widely used for phylogenetic analysis in birds, butterflies, fish and so on. And the COâ… gene has been selected as the standard gene of animal barcode. COâ…¡gene has become a primary molecular marker of phylogenetic analysis in insect because of a certain intraspecies stability and interspecies variability, but COâ… and COâ…¡genes are rarely used for identification and classification of fungi.Specimens and relevant information from patients of infecting Candida were collected to make an epidemiological analysis and discuss the drug susceptibility of the corresponding isolated pathogens. Moreover, three detection methods for common clinical Candida and Aspergillus, including SSR-PCR, Real-time PCR and PCR-AuNPs labeling-microplate hybridization, have been established in order to provide technical support on improving detection of pathogenic fungi in the study.1. The epidemiological analysis of fungal infection and study on drug susceptibilityIn this study, samples collected from infecting Candida were isolated, identified and made epidemiological analysis. The results showed that the samples were mainly from the respiratory medicine (61.2%). And fungi isolated from the sputum (82.8%) was the highest. Among the isolated pathogen, the percentage of Candida albicans was at most (72.2%), followed by Candida tropicalis (12.5%), Candida glabrata (8.6%), Candida krusei (2.4%) and others (4.3%). Moreover, patients over 60 years old with fungal infections accounted for 83.7%.Drug susceptibility analysis showed that the sensitivity of C.albicans to amphotericin B, fluconazole, 5-fluorocytosine and itraconazole were 100%, 92.7%, 91.4%, 64.2%, respectively. Only itraconazole showed resistance (1.4%), but its drug dependence sensitivity was up to 34.4%. In the isolated non-C.albicans, 1 case of C.tropicalis and 2 cases of C.glabrata were resistant to FCZ and 5 cases of C.krusei were resistant to FCZ naturally. The remaining non-C.albicans were susceptible to the four drugs.2. The establishment of SSR-PCR detection method on CandidaThe method of SSR-PCR for detecting Candida had been established using three microsatellite single primers. The reaction system and conditions were optimized through orthogonal design and SSR-PCR was used to identify the 209 strains of Candida which were isolated from hospital. The results showed that there were different fragment length polymorphisms of C.albicans, C.tropicalis, C.glabrata and C.krusei, which revealed good specificity. The bright, clear and stable polymorphic bands were obtained through SSR-PCR, which also showed good repeatability. The SSR-PCR method showed a good polymorphism and was very simple and convenient. The genotype of Candida isolated from hospital was analyzed, too.3. The establishment of Real-time PCR detection methodUniversal primers for filamentous fungi were first designed after blasting COâ… and COâ…¡genes sequences from filamentous fungi and yeasts in GenBank. A fragment of about 610bp was obtained using COI gene primers for 86 strains of 33 species in 15 genera, and a fragment of about 600bp was obtained using COâ…¡gene primers for 113 strains of 38 species in 15 genera. According to the blast for the sequences of COâ… gene of 19 species of Aspergillus and COâ…¡gene sequences of 23 species of Aspergillus, the sequences of COâ…¡gene showed certain interspecies variability and high intrapecies stability. Therefore, COâ…¡gene was used for the detection of filamentous fungi in the subsequent study.In this study, COâ…¡gene sequences of 23 species of Aspergillus and Candida were blasted, the specific primers were designed for detection of Real-time PCR method. Plasmids contained target genes of A.fumigatus and C.albicans were constructed as standard substances, then standard substances were serial diluted to render Real-time PCR standard curves. According to the measured sample Ct values, quantitative analysis of the initial templates were proceeded and determined the sensitivity concentration of detection was 1×102copies/μl. With C.albicans, C.glabrata, A.fumigatus, A.niger and other fungi, the results of Real-time PCR showed that only A.fumigatus and C.albicans were amplified, the other strains were not amplified, and melting curves were with a single peak, indicating that primers were with high specificity. Repetitive experimental results showed that the coefficient of variation was less than 3%, indicating that the method had good repeatability. These results showed that the method with specificity and high sensitivity could be a quantitative analysis of target DNA.4. The establishment of PCR-AuNPs labeling-microplate hybridization methodIn this study, obtained COâ…¡gene sequences on 23 species of Aspergillus were compared, then species-specific probes of A.fumigatus, A.niger and A.terreus by the 5 'end of the thiol were designed and labeled with AuNPs to establish PCR-AuNPs labeling-microplate hybridization method. The combination of the PCR amplification, AuNPs labeling and amplification of silver staining signal, and hybridization technology improve the specificity and sensitivity of detection. Biotin-labeled universal primers of filamentous fungi were used to amplify A.fumigatus, A.niger, A.terreus, C.albicans and other fungi including 10 species in 5 genera. Hybridization results of amplified products with probes showed that there were obvious reactivities between three Aspergillus species specific probes and corresponding target DNA. The Mean A₆₃₀±SD values of A.fumigatus probes were among 1.632±0.066; MeanA₆₃₀±SD values of A.niger probes were among 1.378±0.039; MeanA₆₃₀±SD value of A.terreus probes were among 1.436±0.048. There were no cross-reactions between Aspergillus species probes and other tested fungi, so these probes with good specificity can be used for distinguishing from the three common Aspergillus. In this study, A. fumigatus DNA were serial diluted for hybridization reaction and the results showed that the sensitivity is 0.01pg/μl of target DNA; there was linear correlation between target DNA concentration in the 0.01pg/μl ¹000pg/μl and absorbance. So the method could be used for semi-quantitative analysis of target DNA. A range of different concentrations of A. fumigatus templates were selected for repeat tests and coefficients of variation was less than 3%, indicating that the method has preferable repeatability. In addition, the signal of detection are magnified to 10⁶ times by the use of silver enhancement staining, and the results can be directly observed or made an objective evaluation on basis of absorbance measured. The detection method can detect multiple samples as well and provide the foundation for high-throughput detection of clinical samples.In conclusion, the study investigated the species and distribution of clinical Candida infections by epidemiological analysis and discussed the reasonable use of antifungal agents according to drug susceptibility test, which are conducive to the treatment and prevention of fungal infections. In the study, three detection methods including SSR-PCR, Real-time PCR and PCR-AuNPs labeling-microplate hybridization have already been established. These methods can provide a theoretical basis and technical support for the detection of pathogenic fungi.