| Dermatophytes is a filamentous fungi that parasite on human and animal keratin tissue (including skin, hair to nails) and has the ability of causing infection. Currently it has been reported there are approximately 45 species of dermatophytes, and about 20 species have pathogenic effects on humans. According to morphological characteristics of colonies and macromolecules spores, dermatophytes can be divided into three genera:Trichophyton, Microsporum and Epidermophyton. Tinea pedis, tinea manum, tinea corporis,tinea pedis, onychomycosis and other superficial infection caused by dermatophyte fungi epidemic throughout the world and has a very high incidence and prevalence.Trichophyton ruburm is clinically the most important pathogenic dermatophytes, which has reported that more than half of tinea corporis and 90% of tinea cruris and tinea are caused by T. ruburm. In recent years, studies have shown that in accompany with the wide application of immunosuppressants and corticosteroids and trauma. radiotherapy, long-term topical corticosteroid drugs are predisposing factors of deep infection caused by T. ruburm. Kerion, abscesses and granuloma and subcutaneous tissue infections are infected by T. ruburm. Now at home and abroad the researches of the T. rubrum granuloma strains mostly are single case reports, focused on the classification of T. ruburm, specific clinical manifestation, predisposing factors and pathogens of disease sources. There is no study about the T. rubrum granuloma strains. The first part of the experiment intends to do biological characteristics and genotyping research on T. rubrum strains isolated from patient with dermatophytes granuloma in our department, which were compared with T. rubrum strains isolated from patient with superficial infection. We mean to learn the microbiological characteristics of the T. rubrum granuloma strains, lay the foundation for further study of its pathogenesis.Deep infection caused by T. rubrum not only need systemic antifungal therapy, and the treatment time is long and easy to relapse, seriously affecting the quality of life of patients. In order to better treat dermatophytosis, the animal model of dermatophytosis has played a key role in assessing the efficacy of the antibacterial drugs. Now most of the animal model of dermatophyte is infected by Trichophyton mentagrophytes. and T. rubrum is an anthropophilic fungus, is the first challenge faced by a researcher who wants to study the disease in an animal model. Animal models using T. rubrum as the etiologic agent are scarce, and therefore little is known about the fungus-host interactions. In recent years, domestic scholars has established an experimental guinea pigs models of T. rubrum with the intervention of corticosteroid. But there is no related research about deep infection in animal models caused by T. rubrum. The second part of the experiment tend to using the T. rubrum strains isolated from patient with granuloma tissues, using the footpad injection method to construct an animal model of granulomatous T. rubrum, which can provide experimental basis for future study of exploring the infection pathogenesis, immune pathological process and dermatophytosis treatment efficacy evaluation. 1 A study on the morphological features and sequences of ribosomal genes in the T. rubrum isolated strains from the deep and superficial infection1.1 ObjectiveTo investigate the morphological features and sequences of ribosomal genes in the T. rubrum isolated strains from the deep and superficial infection.1.2 MethodsSource of strain:3 cases of T. rubrum induced granuloma from April 2013-May 2015 and 11 cases of superficial infection on April of 2015 were collected in Dermatology clinic in Jinling hospital. Standard strains of T. rubrum ATCCMYA4438 and T. mentagrophytes CMCC(F)T5a (presented by the Ministry of Health medical microbiology bacteria (viruses) Collection Manager fungal Center Institute of Dermatology, Chinese Academy of Medical Sciences).(1) Clinical isolates were purified and incubated on potato dextrose agar medium and placed cultured for 10 days at 27℃ incubator. Using traditional culture methods and molecular biology of the isolates were identified.(2) All the isolates were inoculated on potato dextrose agar medium,28℃ incubated for 7 d. Take the spores of T. rubrum and obtain a homogeneous bacteria suspension with saline, and adjusting spore concentration with hemocytometer plate of 1×107CFU/ml.(3) The granuloma strains and the standard strains ATCCMYA4438 suspension were inoculated 10μ L of bacterial suspension in potato dextrose agar plate with pipette, inoculated 18 per plant, were placed in 25℃,27℃,35℃,37℃,39℃, 41℃ all three. Superficial infection isolates bacterial suspension were inoculated 10 μL of bacterial suspension in 6 PDA plate with pipette,were placed in 27℃,37℃ at all three. Comparison of the size of the daily changes in colony morphology were observed in three weeks, take pictures and record the measurements outermost diameter of the colonies for 14 days and 21 days. Experiment was repeated three times and averaged.(4) According to the M38-A2 program, using standard clinical and laboratory trace amount of liquid dilution method to do in vitro susceptibility testing.4 kinds of antifungal agents are voriconazole, itraconazole, terbinafine, and ketoconazole.(5) 15 granuloma strains and superficial strains were typed by PCR amplification of tandem repetitive elements (TRSs) TRS-1 and tandem repetitive elements (TRSs) TRS-2 from the ribosomal DNA nontranscribed spacer region(NTS).(6) Applying SPSS20 software, a randomized block design information analysis of variance and paired samples t-test analysis were statistically analyzed of the points of the colony diameter values.1.3 ResultThree clinical isolates of patients with granuloma were identificated by traditional culture and molecular biologicalion and 30 clinical isolates from the patients with superficial infection were identified as T. rubrum by the traditional identification methods. The colony diameter of the standard strain ATCC 4438 at 25℃,27℃ is greater than that at 35℃,37 ℃ culture conditions (P<0.05), and the colony diameter of the three granuloma strains make no difference (P> 0.05) at 35℃, 37℃,27℃ three temperature conditions, and the colony diameter of the 11 superficial infection isolates at 27℃ culture conditions are greater than that at 37℃ cultured conditions (P<0.05). Granuloma strains and superficial strains have no difference in sensitivity to voriconazole, itraconazole, tenafine and ketoconazole. 15 granuloma strains and superficial strains were classified into 4 genotypes of TRS-1 and 2 genotypes of TRS-2.The sequence length of the TRS-1 region in NJT001 is 441bp, have one base difference from AF222889 in GenBank. The sequence length of the TRS-2 region in NJT001 is 508bp, have three base difference from AF222890 and AF222887 in GenBank. The sequence length of the TRS-1 region in NJT002 is 605bp, have one base difference from AF222887 in GenBank. The sequence length of the TRS-2 region in NJT002 is 509bp, have three bases difference from AF222890 and AF222887 in GenBank. The sequence length of the TRS-1 region in NJT003 is 898bp, have 19 bases difference from JX431933 in GenBank. The sequence length of the TRS-2 region in NJT003 is 508bp, have two bases difference from AF222890 and AF222887 in GenBank.1.4 ConclusionIdentification of T. rubrum granuloma strains requires pathology and molecular biology. T. rubrum granuloma strains in this article are more heat-resistant than the superficial infection isolates. Granuloma strains and superficial strains have no difference in sensitivity to voriconazole, itraconazole, tenafine and ketoconazole and there is no resistant-isolates. There is no difference from strain genotyping between granuloma strains and superficial strains.2. Establishing a granuloma animal model infected by Trichophytonrubrum2.1 ObjectiveSuperficial infection caused by T. rubrum strains can also cause deep infection, but relevant animal model has not been reported yet. To construct an granuloma animal model infected by T. rubrum.2.2 MethodSource of strain:3 cases ofT. rubrum induced granuloma from April 2013-May 2015and 2 cases of superficial infection on April of 2015 were collected in Dermatology clinic in Jinling hospital. Standard strains of T. rubrum ATCCMYA4438 (presented by the Ministry of Health medical microbiology bacteria (viruses) Collection Manager fungal Center Institute of Dermatology. Chinese Academy of Medical Sciences).(1) The general level of health BalB/C 52 mice were randomly divided into 13 cages.the temperature of the rat room was adjusted to 22 ℃, the lights turn off the lights 12h a day, and the food and sterile water were given demanded. Four mice were placed in a cage, numbered 1-13. The cage 13 mice were divided into three groups, group 1 was the control, glucocorticoid treatment was given, but not inject the T. rubrum. The second group was the group of PBS bacterial suspension, the 2-7 cage followed by inoculation NJT001. NJT002. NJT003. Superficial 1. Superficial2 and ATCCMYA4438 strains with the PBS bacterial suspension. The third group wasthe experimental group, including 8-13cages. followed by inoculation NJT001, NJT002. NJT003, Superficial 1, Superficial 2 and ATCCMYA4438 strains with the mucin bacterial suspension.(2) In addition to the control group, before the vaccination 7 days, once daily administration of dexamethasone sodium phosphate injection 25mg/(kg-d) intraperitoneal injection of the BalB/C mice. The test strains made of sterile PBS and sterile mucin solution (5g/100mL) mycelium paragraph (including conidia) suspension adjusted hyphal segments/spore concentration of 1× 108/ml.(3) Before the inoculation, the footpad of mice were routine disinfected. With 1ml syringe of above bacterial suspension to a footpad of mice injected 0.2ml bacterial suspension, and the other as a negative control. Blank control group was injected with 0.2ml saline.(4) All animals were given dexamethasone sodium phosphate injection 25 mg/(kg·d) by intraperitoneal injection once a day for a total of 21 times.The vaccinated footpad were observed daily about the swelling of the skin and the ulceration change. After 21 days, direct microscopic examination, fungal culture and histopathology were verified the infection results3. ResultApplying the appropriate dose of glucocorticoids, the mice inoculated with the T. rubrum granuloma strains with mucin suspension were examined after 21 days. Direct microscopic examination, fungal culture and histopathology results of tissue verify the infection. But the mice inoculated with the T. rubrum tinea strains with mucin suspension were examined after 21 days and showed the negative result.4. ConclusionUsing the clinical isolates of T. rubrum granuloma strains in our department, after applying the mucin and glucocorticoids interventions can build rubrum granuloma mice model. |
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