重庆地区HIV阴性免疫力低下患者肺孢子感染分子流行病学初步研究

发布时间：2018-05-21 20:38

本文选题：肺孢子肺炎 + HIV阴性免疫力低下者　；参考：《重庆医科大学》2014年硕士论文

【摘要】：目的：这项研究的目的是通过检测重庆地区人类免疫缺陷病毒(Human immunodeficiency virus,HIV)阴性、免疫力低下患者痰液中的肺孢子（Pneumocystis carinii, PC）来探讨该类患者PC的感染率；通过比较不同检测方法对PC的检测效率，从而探讨实时荧光定量PCR（Real-time fluorescence quantitative PCR, r-PCR）在临床上对HIV阴性免疫力低下患者合并肺孢子肺炎(Pneumocystis carinii pneumonia, PCP)的诊断价值；通过对PC内转录间隔区(Internal transcribed spacer, ITS)基因进行序列分析来对重庆地区PC的基因多态性进行初步分析。方法：收集重庆地区71例HIV阴性免疫力低下患者的痰液标本，其中结缔组织疾病患者23例，肿瘤放化疗患者21例，血液系统疾病患者15例，肝移植术后患者7例，肾移植术后患者5例。以ITS基因为目的基因，分别运用六亚甲基四胺银（Gomort methenamine silver stain,GMS)染色法、巢式PCR法和r-PCR法检测标本中的PC，将两种PCR法检测均为阳性的标本的PCR产物送予基因序列测定，然后进行PCITS基因多态性分析。结果：（1）71名患者中，痰液标本经GMS染色后，仅有2例结果为阳性，，经巢式PCR检测后，25例结果为阳性，巢式PCR的检出率显著高于GMS染色法；PC感染率为35.21%，其中肿瘤放化疗后患者、血液系统疾病患者、肾移植患者、肝移植患者、结缔组织疾病患者感染率分别为33.33%、26.67%、40.00%、42.86%、39.13%，各病例组间感染率差异无显著性，但本组病例PC感染率显著高于同期国外报道，感染率同2009年国内报道值接近。（2）根据临床PCP诊断结果，巢式PCR假阳性率较高为36.00%，其特异度为83.01%，灵敏度为88.89%，阳性预测值为64.00%，当用103copes/ml为判断r-PCR结果阴阳性的阈值时，r-PCR的灵敏度（83.33%）同巢式PCR无显著差异性，但它的特异度（98.11%）和阳性预测值（93.75%）均显著高于巢式PCR；临床诊断PCP患者中r-PCR检测出PC的基因载量显著高于非临床诊断PCP患者。（3）通过对15例阳性标本的PCR反应物进行测序，获得15条ITS序列，其中ITS1共获得了4个基因型（B,M,E,K）和11条新序列，获得了ITS2的3个基因型（g,i,b）和12条新序列，5.8SrRNA得到了7条新序列，经过组合，得到了1个已知的ITS基因型（Bi）和14个新组合序列，而所有新序列与国外报道的原型比较，存在6~21个碱基差异，差异主要表现为碱基缺失和碱基替换。结论：重庆地区HIV阴性免疫力低下患者群PC感染率较高，临床医师应增强对该类患者是否合并PC感染的警觉性；在HIV阴性免疫力低下患者中，使用痰液为标本时，r-PCR比巢式PCR更有助于准确而快速地诊断PCP，并且可以区分PC定植与感染从而指导临床诊治；重庆区域中，PC-ITS基因具有基因多态性，且可能有新的基因分型。
[Abstract]:Objective: the aim of this study was to investigate the infection rate of human immunodeficiency virus (immunodeficiency virus) in the sputum of patients with low immunity by detecting the Pneumocystis carinii (PCS) in the sputum of patients with human immunodeficiency virus (immunodeficiency) in Chongqing area. By comparing the efficiency of different detection methods for PC, the diagnostic value of real-time fluorescent quantitative PCR(Real-time fluorescence quantitative PCR, r-PCR for Pneumocystis carinii pneumonia, PCP) in HIV negative immunocompromised patients with Pneumocystis was studied. The polymorphism of internal transcribed spacer, ITS) gene of PC in Chongqing was analyzed by sequence analysis of internal transcriptional spacer region of PC. Methods: sputum samples were collected from 71 patients with HIV negative immunosuppression in Chongqing, including 23 patients with connective tissue disease, 21 patients with tumor radiotherapy and chemotherapy, 15 patients with hematological diseases and 7 patients with liver transplantation. 5 cases after renal transplantation. The ITS gene was detected by Hexamethylenetetramine Gomort methenamine silver stencils staining method, nested PCR method and r-PCR method respectively. The PCR products of both positive samples detected by PCR method were sent to the gene sequence analysis. Then the polymorphism of PCITS gene was analyzed. Results only 2 sputum samples were positive after GMS staining, and 25 cases were positive after nested PCR examination. The positive rate of nested PCR was significantly higher than that of GMS staining (35.21%). The infection rates of patients with hematological diseases, renal transplantation, liver transplantation and connective tissue diseases were 33.33 and 26.676.67 respectively. There was no significant difference in infection rate among different groups, but the PC infection rate in this group was significantly higher than that reported abroad in the same period. The infection rate was close to the reported value in 2009. (2) according to the clinical PCP diagnosis results, The false positive rate of nested PCR was 36.00, the specificity was 83.01, the sensitivity was 88.89, and the positive predictive value was 64.00. The sensitivity of r-PCR was 83.33 when 103copes/ml was used as the threshold to judge the negative and positive results of r-PCR. But its specificity (98.11) and positive predictive value (93.755) were significantly higher than those of nested PCP. The gene load of PC detected by r-PCR in clinically diagnosed PCP patients was significantly higher than that in non-clinically diagnosed PCP patients. 15 ITS sequences were obtained, of which 4 genotypes were obtained from ITS1, 11 new sequences, 3 genotypes of ITS2 and 5. 8s rRNA, and 7 new sequences were obtained by combination. A known ITS genotype (Bibi) and 14 new combination sequences were obtained. Compared with the reported prototypes, there were 6 ~ 21 nucleotide differences in all the new sequences. The differences were mainly expressed as base deletion and base substitution. Conclusion: the infection rate of PC in HIV negative immunocompromised patients in Chongqing area is relatively high. Clinicians should enhance their awareness of whether these patients are complicated with PC infection, and in patients with HIV negative immunosuppression, Using sputum as a sample, r-PCR is more helpful than nested PCR in the accurate and rapid diagnosis of PCR, and it can distinguish between PC colonization and infection to guide clinical diagnosis and treatment. In Chongqing region, PC-ITS gene has gene polymorphism and may have new genotyping.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91

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