本文選題：結(jié)核 + 復(fù)治肺結(jié)核患者��； 參考：《中國(guó)疾病預(yù)防控制中心》2015年碩士論文

【摘要】：目的：基于專科醫(yī)院數(shù)據(jù),了解初復(fù)治肺結(jié)核患者的耐藥譜及流行情況。闡明結(jié)核分枝桿菌耐藥相關(guān)基因的突變頻率和分子機(jī)制,并在多位點(diǎn)基因測(cè)序的基礎(chǔ)上分析耐藥相關(guān)基因的遺傳多樣性。方法：研究納入2013年6月到2014年12月來自北京胸科醫(yī)院的結(jié)核分枝桿菌臨床分離株,培養(yǎng)后通過MGIT 960液體藥敏實(shí)驗(yàn)對(duì)15種抗結(jié)核藥物,包括異煙肼(Isoniazid, INH)、利福平(Rifampicin, RIF)、鏈霉素(Streptomycin, STR)、乙胺丁醇(Ethambuto, EMB)、卡那霉素(Kanamycin, KAN)、阿米卡星(Amikacin、AMK)、卷曲霉(Capreomycin, CPM)、氧氟沙星(Ofloxacin, OFX)、左氧氟沙星(Levofloxacin, LFX)、莫西沙星(Moxifloxacin、MFX)、對(duì)氨基水楊酸(Paza-aminosalicylate、PAS)、丙硫異煙胺(Protionamide、PTO)、利奈唑胺(Linezolid, LZD)、乙硫異煙胺(Ethionamide, ETH)、吡嗪酰胺(Pyrazinamide, PZA)進(jìn)行耐藥表型實(shí)驗(yàn),并對(duì)耐藥相關(guān)基因進(jìn)行序列測(cè)定和分析。結(jié)果：本研究共納入500例臨床樣本,菌種鑒定71例為NTM,12例分離培養(yǎng)污染,剩余417例結(jié)核分枝桿菌臨床分離株進(jìn)行四種一線抗結(jié)核藥物的耐藥表型檢測(cè),其總耐藥率為47.2%(192/417),MDR耐藥率為28.2%(120/417)。其中復(fù)治患者的總耐藥率和MDR耐藥率分別為66.3%(122/184)和47.3%(87/184),初治患者分別為32.2%(75/233)和14.2%(33/233),復(fù)治患者的總耐藥率和MDR耐藥率均顯著高于初治患者(P0.001)。完成一線藥敏后,篩選100例復(fù)治肺結(jié)核患者和50例初治肺結(jié)核患者樣本進(jìn)行后續(xù)11種抗結(jié)核藥物的耐藥表型實(shí)驗(yàn)(包括PZA、AMK、KAN、CPM、 OFX、LFX、MFX、PAS、PTO、LZD、ETO),有5例在藥敏實(shí)驗(yàn)過程中發(fā)生污染,剩余初治患者樣本48例,復(fù)治患者樣本97例。其中PZA、AMK、 KAN、CPM、OFX、LFX和MFX對(duì)于復(fù)治患者的耐藥率分別為34.0%(33/97)、13.4(13/97)、18.6%(18/97)、29.9%(29/97)、39.2%(38/97),33.0%(32/97)和35.1%(34/97),而初治患者的耐藥率分別為12.5%(6/48)、0.0(0/48)、4.2%(2/48)、6.3%(3/48)、8.3%(4/48),12.5%(6/48)和12.5%(6/48)。復(fù)治患者對(duì)于這幾種藥物的耐藥率顯著高于初治患者(P0.05)。而PAS、PTO、LZD和ETO這4種藥物在初、復(fù)治患者的耐藥率之間無統(tǒng)計(jì)學(xué)差異。對(duì)已知15種藥物耐藥表型檢測(cè)結(jié)果的結(jié)核分枝桿菌就耐藥相關(guān)基因進(jìn)行測(cè)序,結(jié)果顯示對(duì)于INH表型耐藥的菌株中katG的突變率為89.3%(50/56),以S315T突變?yōu)橹�；inhA的突變率為10.7%(6/56),以啟動(dòng)子區(qū)-15(C→T)突變?yōu)橹�。這兩個(gè)位點(diǎn)可作為INH耐藥的快速診斷的分子標(biāo)記。oxyR-ahpC間區(qū)的突變率為7.1%,且所有突變位點(diǎn)均未在INH敏感的菌株中發(fā)現(xiàn),提示該區(qū)間可能與INH耐藥相關(guān)。katG的R463L位點(diǎn)與accD6的D229G位點(diǎn)的突變,在耐藥和敏感株中突變比率相似,提示這兩個(gè)位點(diǎn)可能與INH耐藥無關(guān)。對(duì)于RIF, rpoB的突變率為85.9%,是RIF耐藥主要機(jī)制。rpoB突變中以531和526為主要突變位點(diǎn),可用于RIF耐藥的快速診斷。對(duì)于STR,結(jié)核分枝桿菌rpsL和rrs的突變是鏈霉素耐藥的主要分子機(jī)制。rpsL的突變率為56.1%,在STR耐藥株中只發(fā)現(xiàn)K43R和K88R兩個(gè)突變位點(diǎn),該位點(diǎn)可用于STR的快速診斷。gidB的E92D+A205D聯(lián)合突變出現(xiàn)在94.7%的結(jié)核分枝桿菌中,提示此突變可能與北京家族基因型相關(guān)。對(duì)于EMB,結(jié)核分枝桿菌EMB耐藥發(fā)生與embB突變有關(guān)。研究中embB的突變率為50%,而306位點(diǎn)突變率可達(dá)到35%。該位點(diǎn)可用于EMB耐藥的快速診斷的分子標(biāo)記。結(jié)核分枝桿菌embB 297-497密碼子區(qū)可能是EMB耐藥決定區(qū)。二線抗結(jié)核藥物包括喹諾酮類(Fluoroquinolone, FQNs)和注射類(Injectable Drugs, INJs)。對(duì)于FQNs,其耐藥的發(fā)生主要與gyrA突變有關(guān),本研究中g(shù)yrA的突變率達(dá)到77.8%,可用于FQNs耐藥的快速診斷,其主要突變發(fā)生在是94和90位點(diǎn)。另外研究中顯示gyrB可能與FQNS耐藥沒有相關(guān)性。對(duì)于INJs,結(jié)核分枝桿菌tlyA顯示與CPM耐藥相關(guān),與AMK耐藥無關(guān)。rrsA1401G和C1402T突變顯示可能與AMK和CPM交叉耐藥有關(guān)。對(duì)于PZA,在耐藥菌株中pncA突變存在高度多樣性且分布廣泛,其突變的突變率為69.7%。提示與PZA耐藥相關(guān)。對(duì)于PAS,其耐藥發(fā)生可能與thyA突變有關(guān)。在本研究25例PAS耐藥菌株中,thyA的突變率為12%,各突變位點(diǎn)都僅有1例發(fā)生,無法確定特異性耐藥位點(diǎn)。對(duì)于ETH,其耐藥相關(guān)基因ethA的突變率為34.2%。突變類型存在高度多樣性,基本分布在1-1267堿基之間。通過多位點(diǎn)測(cè)序結(jié)果進(jìn)行遺傳多樣性發(fā)現(xiàn),MDR和XDR菌株dN/dS率大于1,顯示耐藥相關(guān)基因存在被正向選擇驅(qū)動(dòng)的進(jìn)化。且MDR和XDR菌株的核苷酸多樣性明顯高于敏感株,不同組菌株間存在統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論：�？漆t(yī)院就診肺結(jié)核患者耐藥率較高,且復(fù)治肺結(jié)核患者中耐藥率明顯高于初治患者。全面分析了常見抗結(jié)核藥物的耐藥相關(guān)基因的突變頻率和分子特征,為基于分子生物學(xué)快速診斷技術(shù)的應(yīng)用提供參考,并初步探討了耐藥相關(guān)基因的遺傳多樣性和選擇強(qiáng)度。
[Abstract]:Objective: to understand the drug resistance spectrum and epidemic situation of the patients with primary and treatment pulmonary tuberculosis based on the data of specialized hospital. To clarify the mutation frequency and molecular mechanism of the resistance related genes of Mycobacterium tuberculosis, and to analyze the genetic diversity of the resistance related genes on the basis of the sequencing of multilocus genes. From the clinical isolates of Mycobacterium tuberculosis in Beijing Thoracic Hospital, 15 anti tuberculosis drugs, including isoniazid (Isoniazid, INH), Li Fuping (Rifampicin, RIF), Streptomycin, STR, ethambutol (Ethambuto, EMB), kanamycin (Kanamycin, KAN), and Amikacin (Amikacin,), were tested through the MGIT 960 liquid drug sensitivity test. Capreomycin (CPM), ofloxacin (Ofloxacin, OFX), Levofloxacin, LFX, moxifloxacin (Moxifloxacin, MFX), p-aminosalicylic acid (Paza-aminosalicylate, PAS), propylene thioisoamine (Protionamide, PTO), linezolid, ethyl thiazinamide, and pyrazinamide. A total of 500 clinical samples were included in this study. In this study, 71 cases were identified as NTM, 12 cases were isolated and cultured, and the remaining 417 Mycobacterium tuberculosis clinical isolates were tested for the drug resistant phenotype of four first-line anti tuberculosis drugs, the total resistance rate was 47.2% (192/417), M The drug resistance rate of DR was 28.2% (120/417). The total resistance rate and MDR resistance rate of the retreated patients were 66.3% (122/184) and 47.3% (87/184) respectively. The initial treatment patients were 32.2% (75/233) and 14.2% (33/233) respectively. The total drug resistance rate and the MDR resistance rate of the retreated patients were significantly higher than those of the first treated patients (P0.001). After completing the first-line drug sensitivity, 100 cases of retreated pulmonary tuberculosis patients were screened. PZA, AMK, KAN, CPM, OFX, LFX, MFX, PAS, PTO, LZD, ETO) were carried out in 50 cases of first treated tuberculosis patients (including AMK, OFX, LFX, MFX, PAS, PTO, LZD, ETO). There were 5 cases in the drug sensitivity test, 48 cases of residual treatment and 97 cases of retreated patients. The resistance rates were 34% (33/97), 13.4 (13/97), 18.6% (18/97), 29.9% (29/97), 39.2% (38/97), 33% (32/97) and 35.1% (34/97), while the drug resistance rates of the first treated patients were 12.5% (6/48), 0 (0/48), 4.2% (2/48), 6.3% (3/48), 8.3% (4/48), 12.5% (4/48), and 12.5%. The drug resistance rate of these retreated patients was significantly higher than that of the first treated patients. .05). There was no statistical difference between the 4 drugs of PAS, PTO, LZD and ETO. The resistance related genes of Mycobacterium tuberculosis with known 15 drug resistance phenotypes were sequenced. The results showed that the mutation rate of katG in the strains resistant to INH phenotypes was 89.3% (50/56), and inhA was dominated by S315T mutation; inhA. The mutation rate was 10.7% (6/56) and the main mutation was -15 (C to T) in the promoter region. The two loci could be used as a molecular marker for the rapid diagnosis of INH resistance. The mutation rate was 7.1%, and all the mutation sites were not found in the INH sensitive strain, suggesting that the interval could be associated with R463L loci of.KatG associated with INH resistance and accD6 D229G. Point mutation is similar in resistance and sensitive strain, suggesting that these two loci may not be related to INH resistance. For RIF, the mutation rate of rpoB is 85.9%, and the main mechanism of RIF resistance is 531 and 526 as the main mutation site, which can be used for the rapid diagnosis of RIF resistance. For STR, the mutation of Mycobacterium tuberculosis rpsL and RRS is the chain. The mutation rate of.RpsL, the main molecular mechanism of mycophenin resistance, was 56.1%. Only the two mutation sites of K43R and K88R were found in the STR resistant strains. This site could be used for the E92D+A205D joint mutation of STR in the rapid diagnosis of.GidB in 94.7% Mycobacterium tuberculosis, suggesting that the mutation may be related to the genotype of the Beijing family. For EMB, the Mycobacterium tuberculosis is a member of the Mycobacterium tuberculosis. EMB resistance is associated with embB mutation. The mutation rate of embB in the study is 50%, and the mutation rate of the 306 loci can reach 35%., a molecular marker for rapid diagnosis of EMB resistance. The embB 297-497 codon area of Mycobacterium tuberculosis may be the EMB resistant region. The second line anti tuberculosis drugs include quinolones (Fluoroquinolone, FQNs), and Injection class (Injectable Drugs, INJs). For FQNs, the occurrence of drug resistance is mainly related to gyrA mutation. In this study, the mutation rate of gyrA is 77.8%, which can be used for the rapid diagnosis of FQNs resistance. The main mutation occurs at the 94 and 90 loci. In addition, the study shows that gyrB may not be associated with FQNS resistance. INJs, tlyA Mycobacterium tuberculosis It was found to be associated with CPM resistance..rrsA1401G and C1402T mutations associated with AMK resistance were associated with AMK and CPM cross resistance. For PZA, there was a high diversity and wide distribution of pncA mutations in resistant strains. The mutation rate of the mutant was associated with PZA resistance. For PAS, the occurrence of drug resistance may be associated with thyA mutations. In the study of 25 PAS resistant strains, the mutation rate of thyA was 12%, and only 1 of the mutation sites were occurring, and the specific resistance loci were not determined. For ETH, the mutation rate of the drug resistance related gene ethA was highly diverse in the 34.2%. mutation type and was basically distributed among the 1-1267 bases. Genetic diversity was carried out by the results of multipoint sequencing. The dN/dS rate of MDR and XDR was more than 1, indicating that the resistance related genes were driven by positive selection, and the nucleotide diversity of MDR and XDR strains was significantly higher than that of the sensitive strains, and there was a statistical difference between the different groups (P0.05). Conclusion: the drug resistance rate of the patients with pulmonary tuberculosis in the hospital was higher and the drug resistance rate was retreated in the patients with pulmonary tuberculosis. The mutation frequency and molecular characteristics of resistance related genes of common anti tuberculosis drugs were analyzed, and the genetic diversity and selection intensity of resistance related genes were preliminarily discussed.

【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R52;R446.5

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