信亮亮　李冰　榮義輝

101100　北京　通州區(qū)新華醫(yī)院消化內(nèi)科(信亮亮，李冰)；解放軍第三〇二醫(yī)院肝臟腫瘤診療與研究中心(榮義輝)

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·論著·

HBV合并HEV感染導(dǎo)致慢加急性肝衰竭HBV-PC區(qū)變異及臨床特征分析

信亮亮李冰榮義輝

101100北京通州區(qū)新華醫(yī)院消化內(nèi)科(信亮亮，李冰)；解放軍第三〇二醫(yī)院肝臟腫瘤診療與研究中心(榮義輝)

【摘要】目的分析乙型肝炎病毒合并戊型肝炎病毒導(dǎo)致的慢加急性肝衰竭的HBV-PC區(qū)變異及臨床特征。方法回顧性分析HBV感染的慢加急性肝衰竭(ACLF)患者69例，其中單獨(dú)HBV感染患者39例，HBV合并HEV感染患者30例；比較2組患者肝功能、HBV DNA水平、凝血功能、MELD評分以及預(yù)后情況；PCR擴(kuò)增HBV-PC區(qū)序列，測序與ACLF相關(guān)的變異位點(diǎn)A1762T、G1764A、C1766T、T1768A、G1896A、A1762T+G1764A、G1764A+C1766T+T1768A，比較兩組患者之間的差異。分析HBV合并HEV感染存活與死亡患者，Logistic回歸分析重疊感染患者預(yù)后相關(guān)因素。結(jié)果與單純 HBV感染組比較，合并HEV感染組患者的TBil[(216.4 ± 12.1) μmol/L對(364.2 ± 170.24) μmol/L]、肝性腦病發(fā)生率(17.9%對33.3%)、MELD評分(21.26 ± 6.65對28.26 ± 8.65 )均呈不同程度的升高；PTA [(33.3±22.4)%對(24.5±20.1)%]明顯降低，差異均有統(tǒng)計學(xué)意義(P<0.05)；2組患者HBV-PC變異分析比較位點(diǎn)A1762T(66.7%對76.7%)、G1764A(69.2%對80.0%)、A1762T+G1764A(59.0%對70.0%)和G1764A+C1766T+T1768A(2.6%對10.0%) 差異有統(tǒng)計學(xué)意義(P<0.05)。HBV合并HEV感染患者存活組與死亡組比較，MELD評分和肝性腦病發(fā)生率明顯升高，PTA明顯降低，差異均有統(tǒng)計學(xué)意義(P<0.05)； HBV-PC變異分析比較G1764A+C1766T+T1768A 三聯(lián)變異差異有統(tǒng)計學(xué)意義(P<0.05)；Logistic回歸分析顯示，TBil(P=0.006，OR=2.672)、PTA(P=0.036，OR=2.115)、MELD評分(P=0.003，OR=1.682)、肝性腦病并發(fā)癥(P=0.001，OR=3.631)和G1764A+C1766T+T1768A 三聯(lián)變異(P=0.043，OR=2.081)因素與預(yù)后有關(guān)。結(jié)論單獨(dú)HBV與合并HEB感染導(dǎo)致的ACLF患者病情更加嚴(yán)重，預(yù)后更差。TBil、MELD評分、肝性腦病并發(fā)癥和HBV-PC區(qū)G1764A+C1766T+T1768A 三聯(lián)變異發(fā)生率越高。PTA越低，ACLF患者預(yù)后越差。

【關(guān)鍵詞】乙型肝炎病毒；戊型肝炎病毒；慢加急性肝衰竭；HBV-PC區(qū)變異

HBV感染可引起多種臨床表現(xiàn)，包括無癥狀HBV攜帶狀態(tài)、輕中度和重度慢性乙型肝炎以及慢加急性肝衰竭(ACLF)等。在我國，乙型肝炎相關(guān)的ACLF患者占所有ACLF患者80%以上，致死率高達(dá)60%～80%[1]。研究顯示，病毒和宿主因素在HBV慢性感染的嚴(yán)重程度上可能起著一定作用，HBV 重疊感染其他病毒會使乙型肝炎癥狀加重，臨床以HBV合并HEV感染較為常見。 HBV基本核心啟動子(BCP)/前C區(qū)的突變可增加病毒復(fù)制力，影響HBeAg的表達(dá)和分泌，導(dǎo)致HBeAg陰性慢性乙型肝炎，從而影響病情進(jìn)展[2]。本研究回顧性分析69例ACLF患者，篩選出其中30例HBV合并HEV感染的ACLF患者，對其臨床資料和HBV-PC變異作比較分析。

資料和方法

一、一般資料

收集2011年6月至2014年6月北京通州區(qū)新華醫(yī)院住院的 ACLF患者69例，其中單獨(dú)HBV感染導(dǎo)致ACLF患者39例為對照組；HBV合并HEV感染導(dǎo)致的ACLF患者30例為觀察組。HEV感染和ACLF診斷符合2009年《戊型病毒性肝炎診療規(guī)范》[3]及《肝衰竭診治指南(2012年版)》標(biāo)準(zhǔn)[4]。排除妊娠期女性、藥物性肝損傷、自身免疫性肝病、合并HAV、HCV和HIV感染、肝硬化、肝癌等。

二、觀察指標(biāo)

以診斷患者為ACLF的時間點(diǎn)為基線，收集基線時患者肝功能、HBV DNA水平、凝血功能、MELD評分(MELD評分=3.8×ln[TBil(mg/dL)]+11.2×ln(INR)+9.6×ln[肌酐(mg/dL)]+6.4)。HBV合并HEV感染導(dǎo)致ACLF患者病情好轉(zhuǎn)且穩(wěn)定24周為存活組，病情惡化、死亡和轉(zhuǎn)院失訪的患者為死亡組。

三、患者血清HBV DNA的提取以及HBV-PC區(qū)PCR擴(kuò)增

血清中HBV DNA提取方法參照文獻(xiàn)[5]，套式PCR擴(kuò)增BCP/前C區(qū)基因片段，產(chǎn)物大小695 bp。 對PCR產(chǎn)物直接測序，結(jié)果用Vector NTI 10.0軟件進(jìn)行比對，重點(diǎn)分析5個位點(diǎn)的變異，包括：1762、1764、1766、1768 和1896位點(diǎn)。

四、統(tǒng)計學(xué)處理

結(jié)果

一、觀察組與對照組的臨床資料和HBV-PC變異率比較

兩組患者性別、年齡、白蛋白、ALT、HBV DNA載量差異無統(tǒng)計學(xué)意義，HBV-PC C1766T、T1768A和G1896A位點(diǎn)變異率差異無統(tǒng)計學(xué)意義(P>0.05)；與對照組相比，觀察組在TBil、PTA、MELD評分、合并肝性腦病和24周死亡率升高，HBV-PC A1762T、G1764A、A1762T+G1764A和G1764A+C1766T+T1768A位點(diǎn)變異率升高(P<0.05)。見表1。

二、觀察組中存活者與死亡者的臨床資料和HBV-PC變異率比較

存活與死亡患者資料單因素分析顯示，年齡、TBil、PTA、MELD評分、合并肝性腦病和HBV-PC三聯(lián)變異G1764A+C1766T+T1768A差異有統(tǒng)計學(xué)意義(P<0.05)；多因素Logistic回歸分析顯示，存活組與死亡組在TBil、PTA、MELD評分、合并肝性腦病和HBV-PC三聯(lián)變異G1764A+C1766T+T1768A等5種風(fēng)險因素與預(yù)后相關(guān)，見表2。

表1　兩組患者臨床資料和HBV-PC變異位點(diǎn)比較

表2　HBV合并HEV感染患者存活組與死亡組臨床資料比較

續(xù)表2

指標(biāo)存活組(n=14)死亡組(n=16)單因素P值多因素P值OR值(95%CI)ALT(IU/L) ≥311890.829 <31167MELD評分 ≥286110.0110.0031.682(4.464～1.161) <28851合并肝性腦病 有280.0070.0013.631(4.684～1.101) 無128A1762T 有10130.084 無43G1764A 有11130.076 無33A1762T+G1764A 有9120.068 無54G1764A+C1766T+T1768A 有030.0290.0432.08(3.165～1.061) 無1413G1896A 有9100.678 無56

討論

我國HBV感染人群約 9300萬例[4]，全球每年新發(fā)戊肝約3400 萬例，其中死亡30萬例[6]。由于各型嗜肝病毒之間沒有交叉免疫性， 在慢性乙型肝炎患者病程中仍可以重疊合并感染HEV。慢性乙型肝炎重疊急性戊型肝炎患者與單獨(dú)慢性乙型肝炎患者相比TBil、并發(fā)癥發(fā)生率、重型肝炎發(fā)生率和病死率均明顯升高， 而 ALT、Alb和 PTA均明顯降低[7]。已有研究提示PC 區(qū)突變可能是影響HBV感染疾病進(jìn)展的重要因素之一。本研究回顧性分析了HBV合并HEV感染導(dǎo)致 ACLF 患者臨床資料，發(fā)現(xiàn)與單純HBV感染導(dǎo)致ACLF患者比較，HBV合并HEV感染導(dǎo)致的ACLF患者TBil、MELD評分、合并肝性腦病發(fā)生率和病死率明顯升高，PTA則明顯降低；A1762T、G1764A、A1762T+G1764A和G1764A+C1766T+T1768A位點(diǎn)變異率升高。以上分析結(jié)果提示HBV合并感染HEV相關(guān)的ACLF患者病情更為嚴(yán)重，預(yù)后更差。

ACLF發(fā)病較為復(fù)雜，而慢性乙型肝炎合并HEV感染病情加重目前機(jī)制還不清楚。急性戊型肝炎的發(fā)病主要與細(xì)胞免疫有關(guān)，慢性乙型肝炎患者合并HEV感染后，HEV直接破壞并誘發(fā)免疫反應(yīng)對肝細(xì)胞造成損傷[8]；也會產(chǎn)生一些誘發(fā)因子，刺激免疫細(xì)胞，介導(dǎo)TNF-α、IL-6等已知與凋亡相關(guān)的細(xì)胞信號途徑，使肝細(xì)胞進(jìn)一步損傷[9]。HBV 基因變異導(dǎo)致的病毒生物學(xué)特性改變也是導(dǎo)致ACLF的重要因素之一[10]。慢性乙型肝炎患者合并HEV感染以后，可能加速了肝病的發(fā)展進(jìn)程，導(dǎo)致患者病情加重。而HEV分泌蛋白可能直接或間接與HBV調(diào)控區(qū)相互作用，使HBV向病程加重的方向選擇性變異。HBV合并HEV導(dǎo)致ACLF患者預(yù)后多因素分析顯示，存活者和死亡者TBil、PTA、MELD評分、合并肝性腦病和HBV-PC三聯(lián)變異G1764A+C1766T+T1768A等5種風(fēng)險因素與預(yù)后相關(guān)。研究表明，MELD評分可用于急性肝衰竭和ACLF的預(yù)后評估[11]。合并肝性腦病等并發(fā)癥在肝衰竭患者中經(jīng)常出現(xiàn)，是影響ACLF患者預(yù)后的重要因素，G1764A+C1766T+T1768A變異也有報道，該三聯(lián)變異是ACLF患者HBV序列特有，具有激活下游基因調(diào)控的功能[11-12]。

總之，HBV合并HEV感染導(dǎo)致的ACLF患者病情更重，預(yù)后更差，并且HBV-PC的調(diào)控區(qū)變異也向使ACLF加重的方向發(fā)展，但本研究樣本量較小，今后將在增大樣本量的基礎(chǔ)上進(jìn)一步分析。

參考文獻(xiàn)

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2 Chen MT, Billaud JN, Sallberg M, et al. A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen.Proc Natl Acad Sci USA, 2004, 101: 14913.

3 中國醫(yī)師協(xié)會感染科醫(yī)師分會. 戊型病毒性肝炎診療規(guī)范. 中華臨床感染病雜志, 2009, 2: 260-263.

4 中華醫(yī)學(xué)會感染病學(xué)分會肝衰竭與人工肝學(xué)組，中華醫(yī)學(xué)會肝病學(xué)分會重型肝炎與人工肝學(xué)組. 肝衰竭診治指南(2012年版). 中華臨床感染病雜志,2012, 5(6): 321-327.

5 Xu ZH, Ren XQ, Liu Y, et al. Association of hepatitis B virus mutations in basal core promoter and precore regions with severity of liver disease: an investigation of 793 Chinese patients with mild and severe chronic hepatitis B and acute-on-chronic liver failure. J Gastroenterol, 2011, 46: 391-400.

6 WHO. Viral hepatitis in the WHO south-east Asia region. India: regional office for south-east Asia, 2011.

7 Acharya SK, Sharma PK, Singh RA, et al. Hepatitis E virus(HEV) infection in patients with cirrhosis associated with rapid decompen sation and death.J Hepatol, 2007, 46: 387-394.

8 Wedemeyer H, Pischke S, Manns MP. Pathogenesis and treatment of hepatitis E virus infection. Gastroenterology,2012,142:1388.

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12丁劍波，羅曉嵐，田一梅，等. 慢性乙型重型肝炎預(yù)后影響因素 Logistic回歸分析.傳染病信息，2012，25：161-163.

13王耀，劉妍，許智慧，等.G1764A/C1766T/T1768A三聯(lián)突變對HBV核心啟動子活性的上調(diào)作用.解放軍醫(yī)學(xué)雜志，2010,35: 954-957.

(本文編輯：錢燕)

Analysis of HBV PC mutations in ACLF patients infected with HBV combined HEV and their clinical characteristics

XINLiang-liang,LIBing,RONGYi-hui.

ThedigestiveinternalmedicineofXin-HuahospitalinTongzhoudistrict,Beijing101100,China

【Abstract】ObjectiveTo study hepatitis B virus (HBV) basic core promoter and pre-C (PC) nucleotides mutations in patients coinfected with hepatitis E virus (HEV) related acute on chronic liver failure (ACLF) , and to analyze the clinical characteristics. MethodsSixty-nine cases with ACLF caused by HBV infection were retrospectively analyzed, of which 39 patients suffered single HBV infection, and the other 30 patients suffered HBV and HEV coinfection. Liver function, HBV DNA level, blood coagulation function, model for end stage liver disease (MELD) score and prognosis of the two groups were compared respectively. Polymerase chain reaction (PCR) was used for assessing HBV PC region amplification. Sequencing analysis was applied to detect reported ACLF-related mutation sites A1762T, G1764A, C1766T, T1768A, G1896A, A1762T + G1764A and G1764A + C1766T + T1768A for comparative analysis between the two groups. The survival rates and mortality of patients with HBV and HEV coinfection were evaluated for related prognostic factors by logistic regression analysis. ResultsIn the coinfection group, total bilirubin (TBiL), incidence of hepatic encephalopathy and levels of the MELD score were significantly increased (216.4 ± 12.1 vs 364.2 ± 170.24, 17.9% vs 33.3%, 21.26 ± 6.65 vs 28.26 ± 8.65, respectively) when compared with those in single infection group, while prothrombin time activity (PTA) was obviously decreased (33.3 ± 22.4 vs 24.5 ± 20.1) (P<0.05). The comparative analysis of HBV-PC mutation sites A1762T (66.7% vs 76.7%), G1764A (69.2% vs 80.0%), A1762T+G1764A (59.0% vs 70.0%) and G1764A+C1766T+T1768A (2.6% vs 10.0%) showed statistically differences between the two groups (P<0.05). Additionally, among HBV and HEV coinfection patients, the MELD score and incidence of hepatic encephalopathy in death group increased significantly when compared to those in survival group, but the PTA decreased obviously (P<0.05). The comparative analysis of HBV-PC mutation site G1764A + C1766T + T1768A triplet mutation also showed significant difference (P<0.05) between the two groups in patients with coinfection. The logistic regression analysis suggested that TBiL (P=0.006, OR=2.672), PTA (P=0.036, OR=2.115), MELD score (P=0.003, OR=1.682), hepatic encephalopathy incidence (P=0.001, OR=3.631) and G1764A + C1766T + T1768A triplet mutation (P=0.043, OR=0.043) were all related to the prognosis. Conclusion The prognosis is poor in ACLF patients caused by HBV and HEV coinfection, which could be indicated from high TBiL level, MELD score, incidence of hepatic encephalopathy and HBV-PC area G1764A+C1766T+T1768A triplet mutation rate. Furthermore, lower PTA level always predicts a worse prognosis.

【Key words】Hepatitis B virus; Hepatitis E virus; Acute-on-chronic liver failure; HBV-PC nucleotides mutations

(收稿日期：2015-11-01)

Corresponding author:XIN Liang-liang, Email: xinliangliangwz_1980@sina.com

通信作者：信亮亮，Email：xinliangliangwz_1980@sina.com