發(fā)布時間：2018-12-13 19:19

【摘要】：背景 近年來，侵襲性真菌感染發(fā)病率不斷上升，以念珠菌感染為主，其中白念珠菌為主。但由于預防性使用抗真菌藥物，非白念珠菌尤其是對氟康唑天然耐藥的克柔念珠菌感染出現上升趨勢，但白念珠菌感染仍占優(yōu)勢。隨著白念珠菌和克柔念珠菌對唑類抗真菌藥物的耐藥現象越來越嚴重，深入研究其耐藥機制迫在眉睫。 目的 調查本院住院患者侵襲性真菌感染的病原學特征及相關危險因素。了解白念珠菌和克柔念珠菌對常用抗真菌藥物的敏感性，對白念珠菌耐藥菌株和敏感菌株ERG3基因擴增、測序、生物信息學分析，明確白念珠菌ERG3基因突變與抗真菌藥物耐藥之間的關系。方法 收集2006-2010年山西醫(yī)科大學第二臨床醫(yī)學院452例住院患者無菌體液標本，使用沙氏培養(yǎng)基、玉米吐溫80瓊脂培養(yǎng)基、CHROMagar念珠菌顯色培養(yǎng)基和API 20C AUX鑒定系統(tǒng)進行念珠菌的分離和鑒定。從中分別選取35株白念珠菌和20株克柔念珠菌進行體外藥物敏感性試驗，確立耐藥菌株和敏感菌株。對白念珠菌ERG3基因進行PCR擴增、測序，最后應用BLAST軟件進行比對分析。 結果 (1)檢出的163株真菌中，，白念珠菌54.61%，非白念珠菌42.94%，霉菌2.45%。其中非白念珠菌從高到低依次為克柔念珠菌(16.57%)、光滑念珠菌(14.72%)、熱帶念珠菌(4.91%)、近平滑念珠菌(4.29%)和葡萄牙念珠菌(2.45%)；Logistic多因素回歸分析結果顯示：性別、病程、體溫、預防性使用抗真菌藥物、免疫抑制劑、有創(chuàng)傷的檢查和治療、患者預后和地域因素是住院患者無菌體液標本中侵襲性真菌感染的獨立危險因素。 (2)從中選取的35株白念珠菌體外藥物敏感性試驗結果顯示：白念珠菌對5-氟胞嘧啶和兩性霉素B敏感性為100%，對伊曲康唑、伏立康唑和氟康唑有不同程度的耐藥，耐藥率分別是17.14%、11.43%、8.57%，而且存在交叉耐藥和多重耐藥情況；20株克柔念珠菌體外藥物敏感性試驗結果顯示：克柔念珠菌對伏立康唑和兩性霉素B有較高的敏感性，5-氟胞嘧啶、氟康唑和伊曲康唑有不同程度的耐藥，耐藥率分別是20.00%、50.00%、45.00%，也有交叉耐藥和多重耐藥現象。 (3)本研究顯示白念珠菌臨床耐藥菌株和敏感菌株的ERG3基因均存在突變位點，共檢出9個點突變，其中耐藥菌株A17號同時存在1個同義突變和4個錯義突變。A17號菌株同義突變位點為1080bp。突變前堿基所在的密碼子為GGC，所編碼的氨基酸為甘氨酸，未引起氨基酸的改變。4個錯義突變位點分別為I340L、V374G、P902L、V1076A，突變發(fā)生氨基酸置換。第100位堿基由A突變?yōu)镃，導致第340位氨基酸由異亮氨酸轉變?yōu)榱涟彼�；�?34位堿基由T突變?yōu)镚，導致第374位氨基酸由纈氨酸轉變?yōu)楦拾彼�，�?62位堿基由C突變?yōu)門，導致第902位氨基酸由脯氨酸轉變?yōu)榱涟彼�；�?36位堿基由T突變?yōu)镃，導致第1076位氨基酸由纈氨酸轉變?yōu)楸彼幔@4個點突變在其它文獻中尚未見報道，是本實驗新發(fā)現的突變位點。其余菌株均為同義突變，突變位點分別是602bp、795bp、969bp、1080bp、1110bp，未引起氨基酸改變。 結論 (1)白念珠菌感染仍占優(yōu)勢，但非白念珠菌感染明顯上升，尤其是對氟康唑天然耐藥的克柔念珠菌，臨床醫(yī)師應重視發(fā)生的這一流行病學變化。 (2)體外藥敏試驗結果提示：白念珠菌對唑類藥物有不同程度的耐藥，并且對伊曲康唑的耐藥率高于氟康唑，但大多數菌株是敏感菌株，所以唑類抗真菌藥物仍然可以用于臨床上白念珠菌感染的治療。 (3)白念珠菌部分耐藥菌株存在麥角甾醇合成途徑中酶編碼基因ERG3的錯義突變，并且為多位點突變，但突變的數目和分布模式沒有明顯的規(guī)律性，所以ERG3基因突變在耐藥機制中的作用尚需進一步證實。實驗中發(fā)生的錯義突變是單獨突變還是同時突變才可以導致耐藥，也需要進一步研究。
[Abstract]:Background In recent years, the incidence of invasive fungal infection has been increasing, and the infection of candidiasis is the main, of which Candida albicans is the main.. However, as a result of the upward trend in the prophylactic use of antifungal drugs, non-Candida albicans, especially in the case of a natural drug-resistant gram of Candida, the infection of Candida albicans is still dominant With the increasing of the resistance of Candida albicans and C. g. C. to the anti-fungal drugs, it is urgent to study the mechanism of drug resistance.. Objective To investigate the etiological characteristics and related risk of invasive fungal infection in the hospital of our hospital Factors: To understand the sensitivity of Candida albicans and C. canalis to the common antifungal drugs, the amplification, sequencing and bioinformatics analysis of the drug-resistant strains of Candida albicans and the sensitive strain ERG3, and to clarify the relationship between the ERG3 gene mutation of the Candida albicans and the drug resistance of the antifungal drugs relationship Methods: 452 cases of sterile body fluid from the second clinical medical school of Shanxi Medical University in 2006-2010 were collected, and the isolation of Candida was carried out using the medium of Shahlet, the agar medium of Tween 80, the chromogenic medium of CHROMagar and the API 20C AUX identification system. and respectively selecting 35 strains of Candida albicans and 20 strains of Candida albicans to carry out in-vitro drug sensitivity test, and establishing a drug-resistant strain and a sensitivity. and performing PCR amplification and sequencing on the candida albicans ERG3 gene, and finally using the BLAST software to perform PCR amplification, sequencing and final application of the BLAST software to perform PCR amplification, sequencing, p-point Results (1) Among the 163 fungi detected, the Candida albicans was 54. 61%, and the non-candida albicans was 42. 94%. 2.45% of Candida, of which Candida albicans (16.57%), Candida glabrata (14. 72%), Candida tropicalis (4.91%), Candida glabrata (4.29%) and Candida utilis (2.45%) were in order from high to low, and the results of logistic multi-factor regression analysis showed that gender, The course of course, body temperature, preventive use of antifungal drugs, immunosuppressants, and trauma and treatment, the patient's prognosis and regional factors are the only one of the invasive fungal infections in the sterile body fluid specimen of the patient. The results of the in vitro drug sensitivity test of 35 strains of Candida albicans in vitro showed that the sensitivity of Candida albicans to 5-fluorophonate and amphotericin B was 100%, and the drug-resistance rate was 17.14%, respectively. 11. 43%, 8. 57%, and there were cross-resistance and multiple drug-resistance. The results of the in vitro drug sensitivity test of 20 strains of Candida utilis showed that the sensitivity, 5-fluorophonate, fluconazole and Idaikang were more sensitive to the test results of the in vitro drug sensitivity test of 20 strains of Candida utilis. In the same degree, the resistance rate was 20. 00%, 50. 00%, 45. 00%, respectively. (3) There was a mutation site in the ERG3 gene of the clinical drug-resistant strain of Candida albicans and the sensitive strain, 9 point mutations were detected, among which there were 1 synonymous mutation at the same time of the drug-resistant strain A17. and 4 missense mutations. It is 1080bp. The codon of the pre-mutation base is GGC, the encoded amino acid is glycine, and the change of amino acid is not caused. The four missense mutation sites are I340L, V374G, P902L, V1076A, and mutation. The amino acid substitution occurs. The 100-th base is changed from A to C, resulting in the transformation of the 340-th amino acid from isoleucine to leucine; the 134-th base is changed from T to G, leading to the conversion of the amino acid at position 374 to glycine, and the second base The mutation of the base from C to T, leading to the transformation of the amino acid at position 902 into leucine by the proline; the base of the 836 is changed from T to C, resulting in the 1076 amino acid being converted to alanine by the amino acid, the four point mutations have not been reported in other documents, which is the new experiment The other strains were the same. The mutation sites were 602bp, 795bp, 969bp, 1080bp and 1110bp, respectively. cause Conclusion (1) The infection of Candida albicans is still dominant, but the infection of non-Candida albicans is obviously increased, especially in the case of the natural drug-resistant Candida, the clinician should attach importance to the occurrence. This epidemiological change. (2) The results of in vitro drug sensitivity test suggest that Candida albicans has a different degree of resistance to the drug, and the drug resistance rate of the drug is higher than that of Flukang, but most of the strains are sensitive strains, so the anti-fungal drugs can still be used for clinical application. The treatment of Candida albicans infection. (3) the missense mutation of the enzyme-encoding gene ERG3 in the part of the drug-resistant strain of the Candida albicans, and the mutation of the multi-position point, but the number and the distribution pattern of the mutation have no obvious regularity, so the gene mutation of the ERG3 is in the drug-resistant mechanism. The role in the experiment still needs to be further confirmed. The missense mutation that occurs in the experiment is a single mutation or a simultaneous mutation.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R756

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