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Drug resistance mutations in polymerase region and related influencing factors in patients with hepatitis B virus infection
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摘要:
目的探讨江苏徐州地区HBV感染者逆转录聚合酶区（RT）基因耐药变异模式及与临床特征的关系,并分析影响耐药的相关因素。方法收集2014年5月-2019年4月于徐州医科大学附属医院感染科行HBV RT区检测的242例HBV感染者的临床资料,比较不同基因型、HBeAg状态患者的临床特征。既往有明确核苷（酸）类药物用药史的HBV感染者164例,其中发生已知耐药位点变异118例,未发生已知耐药位点变异46例,两组患者行影响HBV RT区耐药变异危险因素分析。计量资料采用独立样本t检验或Mann-Whitney U检验。计数资料组间比较采用χ2检验。采用logistic回归分析筛选影响患者耐药的危险因素。结果江苏徐州地区HBV感染者RT区耐药突变模式以rtL180M+rtM204I/V/S（25例,21. 2%）、rtA181T/V（16例,13. 6%）、rtM204I/V/S（15例,12. 7%）多见。242例患者中B基因型13例（5. 4%）,C基因型229例（94. 6%）,未发现其他基因型。B基因型感染者ALT水平高于C基因型,差异有统计学意义（U=-2. 096...
Abstract:Objective To investigate the pattern of drug resistance mutations of the genes in the reverse transcriptase( RT) region and its association with clinical features in patients with hepatitis B virus( HBV) infection in Xuzhou,Jiangsu,China,as well as the influencing factors for drug resistance. Methods A total of 242 patients with HBV infection who underwent the detection of the HBV RT region in Department of Infectious Diseases,The Affiliated Hospital of Xuzhou Medical University,from May 2014 to April 2019 were enrolled,and the clinical features were compared between the patients with different genotypes and HBeAg statuses. Of all patients,164 had a clear medication history of nucleos( t) ide analogues( NAs),among whom 118 had known mutations at drug resistance loci and 46 did not have such mutations,and the risk factors for drug resistance mutations in the HBV RT region were analyzed for the two groups. The independent samples t-test or the Mann-Whitney U test was used for comparison of continuous data between groups; the chi-square test was used for comparison of categorical data between groups; a logistic regression analysis was used to investigate the risk factors for drug resistance. Results Among the patients with HBV infection in Xuzhou,rtL180 M + rtM204 I/V/S was the most common pattern of drug resistance mutation in the RT region and was observed in 25 patients( 21. 2%),followed by rtA181 T/V in 16 patients( 13. 6%) and rtM204 I/V/S in 15 patients( 12. 7%). Of all 242 patients,13( 5. 4%) had genotype B and 229( 94. 6%) had genotype C,and no other genotypes were found. The patients with genotype B had a significantly higher level of alanine aminotransferase( ALT) than those with genotype C( U =-2. 096,P =0. 036). Compared with the HBeAg-positive group,the HBeAg-negative group had a significantly older age( t = 4. 580,P < 0. 001) and significantly lower HBV DNA load and HBs Ag level( t = 2. 145 and 3. 526,P = 0. 033 and 0. 001). The HBe Ag-negative group had a significantly longer course of disease and a significantly higher level of gamma-glutamyl transpeptidase( GGT) than the HBe Ag-positive group( U =-2. 561 and-2. 016,P = 0. 010 and 0. 044). Compared with the HBe Ag-positive group,the HBe Ag-negative group had a significantly lower proportion of patients with chronic hepatitis B and a significantly higher proportion of patients with liver cirrhosis or hepatocellular carcinoma( χ2= 20. 609,P < 0. 001). The multivariate logistic regression analysis showed that administration of lamivudine + adefovir dipivoxil( OR = 0. 080,95% CI: 0. 008-0. 748,P < 0. 05),administration of adefovir dipivoxil( OR = 5. 493,95% CI: 1. 377-21. 909,P < 0. 05),and improper drug withdrawal( OR = 5. 945,95% CI: 1. 921-18. 403,P < 0. 05) were independent risk factors for drug resistance in patients with HBV infection. Conclusion Most of the patients with HBV infection in Xuzhou are infected with HBV genotype C,with a complex and diverse pattern of drug resistance mutations. HBV DNA replication is active in HBe Ag-positive patients,and therefore,it is recommended to initially select antiviral drugs with high efficiency and low resistance and strengthen the compliance with antiviral therapy.
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Key words:
- hepatitis B virus /
- mutation antiviral agents /
- nucleoside(acid) analogues /
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急性胰腺炎(acute pamcreatitis，AP)是临床常见消化系统急腹症之一[1]，近年来，其发病率不断上升[2]。Yokoe等[3]研究显示，15%~20%的AP进展为重症急性胰腺炎(severe acute pancreatic, SAP)。脓毒症是病原微生物侵入血液引起的全身感染性疾病，据Sagana等[4]报道美国每年约有0.6%的人发生脓毒症。SAP病情凶险极易引发脓毒症，SAP一旦发生脓毒症不仅加重医疗费用负担、延长住院时间，还可能并发脓毒性休克，多器官功能障碍，病情进展甚至会引起死亡[5]，临床诊治极为困难。本研究回顾性分析SAP患者的临床资料，分析SAP患者并发脓毒症的相关因素，旨在为临床防治提供参考。
1. 资料与方法
1.1 研究对象
收集2007年1月—2020年3月贵州医科大学第三附属医院与黔南州人民医院收治的SAP患者临床资料。SAP诊断标准参考中华医学会制定的《急性胰腺炎诊治指南(2014)》[6]。脓毒症诊断标准参照国家卫生健康委颁发的《医院感染诊断标准(试行)》[7]。纳入标准：(1)年龄≥16周岁；(2)符合SAP诊断。剔除标准：(1)病历记录不全；(2)伴有恶性肿瘤晚期或使用糖皮质激素患者；(3)入院手术前已合并脓毒症者；(4)伴有其他部位原发性感染者。
1.2 研究方法
根据SAP是否发生脓毒症分为脓毒症与非脓毒症，记录每例患者年龄、性别、APACHEⅡ评分、血糖、血钙、血清总胆固醇、血清甘油三酯、血尿素氮、血清白蛋白、血清肌酐、胰腺坏死范围所占比例，以及入住ICU、低氧血症、深静脉置管、机械通气、预防性使用抗生素、血液净化、手术病灶坏死组织清除方式、留置导尿情况，血培养检出病原菌种类等临床资料。本研究所纳入SAP患者采取急诊手术清除病灶坏死组织，手术方式分为开腹与腹腔镜两种方式。
1.3 伦理学审查
本研究通过贵州医科大学第三附属医院伦理委员会审批，批号：2020-002，并经患者及家属知情同意。
1.4 统计学方法
采用SPSS 24.0软件进行数据分析。计量资料以x±s表示，两组间比较采用t检验，计数资料两组间比较采用χ2检验。多因素分析采用logistic回归分析。P < 0.05为差异有统计学意义。
2. 结果
2.1 一般资料
研究共纳入SAP患者178例，其中男106例、女72例, 年龄16~77岁，平均(49.69±14.77) 岁。发生脓毒症56例(31.46%)，其中男36例、女20例，平均(51.29±13.92)岁。
2.2 脓毒症菌种分布
在56例SAP并发脓毒症患者血培养中共分离出61株病原菌，其中革兰阳性菌14株，占22.95%，革兰阴性菌39株，占63.93%，真菌8株，占13.11%(表 1)。
表 1 SAP合并脓毒症患者的菌种构成比病原菌 株数(n=61) 构成比(%) 革兰阳性菌 14 22.95 表皮葡萄球菌 8 13.11 溶血葡萄球菌 4 6.56 粪肠球菌 2 3.28 革兰阴性菌 39 63.93 肺炎克雷伯菌 11 18.03 鲍曼不动杆菌 9 14.75 铜绿假单胞菌 9 14.75 大肠埃希菌 7 11.48 嗜麦芽窄食假单胞菌 2 3.28 阴沟肠杆菌 1 1.64 真菌 8 13.11 白色假丝酵母菌 5 8.20 光滑假丝酵母菌 2 3.28 热带假丝酵母菌 1 1.64 2.2 单因素分析
单因素分析显示，APACHEⅡ评分、血糖、血钙、血清总胆固醇、血清甘油三酯、血尿素氮、血清肌酐、血清白蛋白，以及入住ICU、低氧血症、深静脉置管、机械通气、手术方式、血液净化、留置导尿、胰腺坏死范围在脓毒症和非脓毒症患者间差异均有统计学意义(P值均 < 0.05)(表 2)。
表 2 SAP并发脓毒症的单因素分析因素 非脓毒症(n=122) 脓毒症(n=56) 统计值 P值 年龄(岁) 48.95±15.14 51.29±13.92 t=1.011 0.314 男/女(例) 70/52 36/20 χ2=0.761 0.383 APACHEⅡ评分(分) 24.35±5.86 27.71±5.56 t=3.683 < 0.001 入住ICU(例) 41 31 χ2=7.538 0.006 低氧血症(例) 36 31 χ2=10.926 0.001 深静脉置管(例) 82 46 χ2=4.235 0.040 机械通气(例) 44 30 χ2=4.842 0.028 血糖(mmol/L) 11.37±3.80 13.13±4.34 t=2.596 0.011 预防性使用抗生素(例) 51 21 χ2=0.295 0.587 手术方式(例) χ2=8.249 0.004 腹腔镜 43 8 开腹 79 48 血液净化(例) 83 29 χ2=4.343 0.037 留置导尿(例) 34 34 χ2=17.539 < 0.001 胰腺坏死范围(例) χ2=13.386 0.001 >50% 9 12 30%~50% 39 25 < 30% 74 19 血钙(mmol/L) 2.26±0.32 2.14±0.33 t=-2.144 0.034 血清总胆固醇(mmol/L) 6.13±2.26 7.03±2.20 t=2.498 0.014 血清甘油三酯(mmol/L) 2.02±1.12 2.59±1.23 t=2.946 0.004 血尿素氮(mmol/L) 7.13±2.52 9.05±4.56 t=2.951 0.004 血清肌酐(μmol/L) 116.46±46.78 147.87±67.31 t=3.160 0.002 血清白蛋白(g/L) 36.08±7.95 32.62±10.22 t=-2.246 0.027 2.3 多因素分析
将单因素分析中有统计学意义的指标纳入logistic多因素回归分析，结果显示，APACHEⅡ评分、低氧血症、血糖、胰腺坏死范围、血清肌酐是SAP并发脓毒症的独立危险因素，采用腹腔镜清除病灶坏死组织为SAP并发脓毒症的独立保护因素(P值均 < 0.05)(表 3)。
表 3 SAP并发脓毒症的多因素分析变量 B SE Wald P值 OR 95%CI APACHEⅡ评分(分) 1.909 0.574 11.063 0.001 6.748 2.191~20.788 入住ICU 0.994 0.652 2.321 0.128 2.701 0.752~9.700 低氧血症 1.219 0.568 4.607 0.032 3.383 1.112~10.293 深静脉置管 0.577 0.677 0.728 0.394 1.781 0.473~6.710 机械通气 0.750 0.560 1.794 0.180 2.118 0.706~6.350 血糖(mmol/L) 1.665 0.767 4.714 0.030 5.288 1.176~23.781 手术方式(腹腔镜) -1.387 0.682 4.133 0.042 0.250 0.066~0.951 血液净化 -0.185 0.554 0.112 0.738 0.831 0.280~2.463 留置导尿 0.636 0.559 1.293 0.256 1.889 0.631~5.651 胰腺坏死范围 1.709 0.640 7.130 0.008 5.523 1.575~19.360 血钙(mmol/L) -0.964 0.586 2.710 0.100 0.381 0.121~1.202 血清总胆固醇(mmol/L) 0.498 0.593 0.703 0.402 1.645 0.514~5.263 血清甘油三酯(mmol/L) 0.740 0.840 0.777 0.378 2.097 0.404~10.880 血尿素氮(mmol/L) 1.066 0.630 2.862 0.091 2.903 0.845~9.977 血清肌酐(μmol/L) 1.612 0.671 5.771 0.016 5.012 1.345~18.672 血清白蛋白(g/L) -0.719 0.705 1.041 0.308 0.487 0.122~1.939 3. 讨论
SAP是常见消化系统急症，后期继发感染性胰腺坏死的概率较高[8-9]。脓毒症是SAP的严重并发症之一，也是患者后期死亡的重要原因。本研究显示，在178例SAP患者中发生脓毒症56例(31.46%)，与陈莎燕等[10]报道结果相近，提示SAP患者并发脓毒症的概率较高，直接影响治疗的预后，临床应予以注意。本研究在56例SAP并发脓毒症患者血培养中共分离出61株病原菌，其中革兰阳性球菌14株，占22.95%，革兰阴性杆菌39株，占63.93%，真菌8株，占13.11%，与廖全凤等[11]研究结果相似，临床应根据其感染病原菌特点选用抗菌药物。
APACHE-Ⅱ评分是判断SAP严重程度与预后的重要评分系统，评分越高提示病情越严重，免疫功能越差，病原菌越易进入血液形成脓毒症[12]；SAP胰腺组织灌注不足，此时合并低氧血症可增加胰腺组织缺氧程度与坏死范围，导致胰腺感染增加并侵入血流[13]，同时SAP常伴发肺部感染等胰腺外感染，后者又可加重SAP患者的感染严重程度，严重者引起低氧血症与呼吸功能衰竭，甚至发生多器官功能衰竭风险[14]；胰腺的内分泌部分泌的胰岛素是调节血糖的重要激素，SAP胰腺坏死损伤胰岛导致胰岛素分泌不足，糖代谢紊乱，血糖升高，有利于病原菌入侵，同时其免疫功能下降，更利于病原菌入侵血流，增加脓毒症的发生[15]。肌酐是肌肉代谢产物, 肌肉中肌酸通过非酶脱水反应产生，再释放进入血液中，随尿排出体外。因此血清肌酐与人体肌肉总量密切相关，而不易受饮食等影响；再加上肌酐为小分子物质，通过肾小球滤过，很少被肾小管吸收，每日体内产生量几乎均随尿排出，不受尿量影响，因此，临床上将血清肌酐作为肾功能重要指标之一，血清肌酐升高提示肾功能受损[16]；本研究显示，血清肌酐与脓毒症密切相关，可能原因为肾功能不全时白细胞趋化性功能受损，淋巴细胞功能障碍，体内免疫球蛋白降低，免疫功能下降，容易发生脓毒症[17-18]。手术病灶清除引流是治疗感染性胰腺坏死的重要手段[19]；感染性胰腺坏死病灶清除引流手术的方式有开腹与微创手术两种，手术可清除炎性病灶减少感染，但是开放手术创伤大，可引起坏死炎性胰腺组织扩散，感染病原菌侵入血流，同时开腹手术破坏人体自然屏障，外界环境中的病原菌也易通过切口侵入引起脓毒症[20]，而微创手术方式既可清除炎性坏死组织，又可减少外界病原菌侵入，可减少脓毒症[21]。胰腺坏死程度越高提示感染性胰腺坏死病灶越大，产生的炎性坏死组织越多，对周围组织破坏也越大，病原菌越易侵入血流产生胰外感染[22]。多因素分析显示，APACHEⅡ评分、低氧血症、血糖、胰腺坏死程度高、血清肌酐等因素是SAP并发脓毒症的独立危险因素，采用微创手术方式清除病灶坏死组织为SAP并发脓毒症的独立保护因素。
总之，SAP并发脓毒症与多因素相关。控制血糖，保护肺肾等重器官功能，采用微创手术方式清除病灶坏死组织，注意重症、胰腺坏死程度高患者的救治是减少SAP并发脓毒症的重要措施。
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