1-磷酸鞘胺醇受体3参与巨噬细胞迁移及脓毒症诱导的心肌损伤

目的探讨巨噬细胞中1-磷酸鞘胺醇受体(sphingosine 1-phosphate receptor,S1PR)的表达及其作用,观察干预S1PR3(S1P3)对脂多糖诱导的心肌损伤的影响。方法传代培养小鼠Ana-1巨噬细胞,给予脂多糖(lipopolysaccharide,LPS,100 ng/ml)刺激或S1P3特异性抑制剂CAY-10444(10μmol/L)干预,细胞随机分为对照组、LPS组、CAY-10444组、CAY-10444预处理2h+LPS组,Transwell小室观测巨噬细胞迁移,蛋白免疫印迹检测巨噬细胞S1PR的表达,并检测p-Akt/Akt蛋白水平。在体实验,6~8周龄雄性C57/B6小鼠,随机分为对照组、LPS组、CAY-10444组、CAY-10444干预+LPS组,每组12只,LPS(10 mg/kg)腹腔注射,或CAY-10444 1 mg/kg于LPS诱导后30 min腹腔注射干预,24 h后取心脏组织HE染色观察病理改变,免疫组化染色观察巨噬细胞浸润程度以及炎症因子的表达情况,实时荧光定量PCR检测心肌损伤标记分子BNP、巨噬细胞表面分子F4/80、炎症因子TNF-α、IL-1β、IL-6的mRNA水平。结果与对照组比较,LPS诱导巨噬细胞大量迁移S1P3蛋白表达增加(P0.01),p-Akt Ser473/Akt表达上调(P0.01);与LPS组相比,S1P3抑制剂CAY-10444干预后再给予LPS刺激,巨噬细胞迁移被抑制(P0.01),p-Akt Ser473/Akt表达也降低(P0.01);在体实验,LPS诱导小鼠后BNP mRNA水平明显上调(P0.01),同时F4/80以及炎症因子TNF-α、IL-1β、IL-6的mRNA水平上调(P0.01),HE染色可见心肌损伤及炎细胞浸润,免疫组化染色法显示F4/80及炎症因子的大量阳性表达(P0.01);使用S1P3抑制剂后,与LPS组比较,心肌损伤减轻免疫组化中巨噬细胞减少(P0.01),炎症因子表达降低(P0.01),BNP mRNA水平降低(P0.01),F4/80以及TNF-α、IL-1β、IL-6的mRNA水平也明显降低(P0.01)。结论抑制巨噬细胞S1P3表达可抑制巨噬细胞的迁移并提示p-Akt/Akt与了这一过程,此外,S1P3抑制剂的干预可有效减轻LPS诱导的心肌损伤。     

老年2型糖尿病并发ACS患者血清GGT与慢血流-无复流的相关性及其对预后的影响

目的探讨老年2型糖尿病(T2DM)并发急性冠脉综合征(ACS)患者血清γ谷氨酰胺转肽酶(GGT)水平与经皮冠状动脉介入(PCI)治疗术后冠脉慢血流-无复流的相关性及其对预后的影响。方法选取188例诊断为ACS并行急诊PCI的老年T2DM患者,根据TIMI血流分级和校正的TIMI血流帧计数(c TFC)方法评价冠脉血流分为正常血流组(156例)和慢血流-无复流组(32例),分析GGT及其他危险因素与冠脉慢血流-无复流的相关性和主要不良心血管事件(MACE)的发生率。结果慢血流-无复流组的血清GGT水平高于正常血流组[(49±18)U/L vs.(31±13)U/L,P0.01]。相关分析结果显示,血清GGT与冠脉慢血流-无复流呈正相关(r=0.389,P0.01)。血清GGT与PCI术后冠脉慢血流-无复流、住院期间及术后12个月MACE独立相关(分别OR=1.093,95%CI:1.058~1.129,P0.01;OR=1.047,95%CI:1.012~1.082,P0.05及OR=1.058,95%CI:1.028~1.089,P0.01)。结论老年T2DM并发ACS患者血清GGT水平与冠脉慢血流-无复流相关,血清GGT可能是预测冠脉风险的评价指标。     

肠道微生态影响动脉粥样硬化发生发展的机制

动脉粥样硬化(AS)不仅是一种炎症性疾病,而且属于一种代谢性疾病。肠道微生态的改变可对AS的发生发展产生双面影响。一方面,肠道菌群紊乱可以通过影响机体的胆碱代谢、氧化应激、炎症反应等机制直接促进AS产生发展,此外,可通过导致AS危险因素肥胖、高脂血症、糖尿病等的产生这些间接机制促AS的进展。另一方面,益生菌及益生元的增加则可有效地降低肠道微生物内毒素产生、增强肠道屏障、减轻机体质量、缓解炎症反应、改善胰岛素抵抗,进而在AS的进展方面发挥重要作用。因此,合理调控机体肠道微生态环境成为AS防治的新型重要手段。     

HBV endemicity in Mexico is associated with HBV genotypes H and G

Hepatitis B virus(HBV)genotypes have distinct genetic and geographic diversity and may be associated with specific clinical characteristics,progression,severity of disease and antiviral response.Herein,we provide an updated overview of the endemicity of HBV genotypes H and G in Mexico.HBV genotype H is predominant among the Mexican population,but not in Central America.Its geographic distribution is related to a typical endemicity among the Mexicans which is characterized by a low hepatitis B surface antigen seroprevalence,apparently due to a rapid resolution of the infection,low viral loads and a high prevalence of occult B infection.During chronic infections,genotype H is detected in mixtures with other HBV genotypes and associated with other co-morbidities,such as obesity,alcoholism and co-infection with hepatitis C virus or human immunodeficiency virus.Hepatocellular carcinoma prevalence is low.Thus,antiviral therapy may differ significantly from the standard guidelines established worldwide.The high prevalence of HBV genotype G in the Americas,especially among the Mexican population,raises new questions regarding its geographic origin that will require further investigation.     

Harnessing the potential of gene editing technology using CRISPR in inflammatory bowel disease

The molecular scalpel of clustered regularly interspersed short palindromic repeats/CRISPR associated protein 9(CRISPR/Cas9) technology may be sharp enough to begin cutting the genes implicated in inflammatory bowel disease(IBD) and consequently decrease the 6.3 billion dollar annual financial healthcare burden in the treatment of IBD. For the past few years CRISPR technology has drastically revolutionized DNA engineering and biomedical research field. We are beginning to see its application in gene manipulation of sickle cell disease,human immunodeficiency virus resistant embryologic twin gene modification and IBD genes such as Gatm(Glycine amidinotransferase, mitochondrial),nucleotide-binding oligomerization domain-containing protein 2, KRT12 and other genes implicated in adaptive immune convergence pathways have been subjected to gene editing, however there are very few publications. Furthermore,since Crohn's disease and ulcerative colitis have shared disease susceptibility and share genetic gene profile, it is paramount and is more advantageous to use CRISPR technology to maximize impact. Although, currently CRISPR does have its limitations due to limited number of specific Cas enzymes, off-target activity,protospacer adjacent motifs and crossfire between different target sites. However,these limitations have given researchers further insight on how to augment and manipulate enzymes to enable precise gene excision and limit crossfire between target sites.     

Abdominal compartment syndrome:Often overlooked conditions in medical intensive care units

Intra-abdominal hypertension(IAH)and abdominal compartment syndrome are well recognized entities among surgical patients.Nevertheless,a number of prospective and retrospective observational studies have shown that IAH is prevalent in about half of the critically ill patients in the medical intensive care units(ICU)and has been widely recognized as an independent risk factor for mortality.It is alarming to note that many members of the critical care team in medical ICU are not aware of the consequences of untreated IAH and the delay in making the diagnosis leads to increased morbidity and mortality.Frequently it is underdiagnosed and undertreated in this patient population.Elevated intraabdominal pressure decreases the blood flow to the kidneys and other abdominal viscera and also results in reduced cardiac output and difficulties in ventilating the patient because of increased intrathoracic pressure.When intraabdominal hypertension is not promptly recognized and treated,it leads to abdominal compartment syndrome,multiorgan dysfunction syndrome and death.Large volume fluid resuscitation is very common in medical ICU patients presenting with sepsis,shock and other inflammatory conditions like pancreatitis and it is one of the major risk factors for the development of intra-abdominal hypertension.This article presents an overview of the epidemiology,definitions,risk factors,pathophysiology and management of IAH and abdominal compartment syndrome in critically ill medical ICU patients.     

Current status of endoscopic retrograde cholangiopancreatography in patients with surgically altered anatomy

Endoscopic retrograde cholangiopancreatography(ERCP)in patients with surgically altered anatomy must be performed by a highly experienced endoscopist.The challenges are accessing the afferent limb in different types of reconstruction,cannulating a papilla with a reverse orientation,and performing therapeutic interventions with uncommon endoscopic accessories.The development of endoscopic techniques has led to higher success rates in this group of patients.Device-assisted ERCP is the endoscopic procedure of choice for high success rates in short-limb reconstruction;however,these success rate is lower in long-limb reconstruction.ERCP assisted by endoscopic ultrasonography is now popular because it can be performed independent of the limb length;however,it must be performed by a highly experienced and skilled endoscopist.Stent deployment and small stone removal can be performed immediately after ERCP assisted by endoscopic ultrasonography,but the second session is needed for other difficult procedures such as cholangioscopy-guided electrohydraulic lithotripsy.Laparoscopic-assisted ERCP has an almost 100%success rate in longlimb reconstruction because of the use of a conventional side-view duodenoscope,which is compatible with standard accessories.This requires cooperation between the surgeon and endoscopist and is suitable in urgent situations requiring concomitant cholecystectomy.This review focuses on the advantages,disadvantages,and outcomes of various procedures that are suitable in different situations and reconstruction types.Emerging new techniques and their outcomes are also discussed.     

Loss of BRCA1 expression leads to worse survival in patients with gastric carcinoma

AIM: To investigate the expression deficiency of key molecular markers in the homologous recombination pathway. METHODS: Expression loss of breast cancer type 1 susceptibility protein (BRCA1), ataxia telangiectasia mutated (ATM), ATM-Rad3-related (ATR), mediator of DNA damage checkpoint protein 1 (MDC1) and meiotic recombination 11 (Mre11) were correlated with their clinicopathological parameters in gastric cancer (GC). One hundred and twenty treatment-naive GC samples were formalin-fixed and paraffin-embedded into tissue blocks. Two representative cores from each block were extracted and constructed into tissue microarrays. Expression levels of BRCA1, ATM, ATR, MDC1 and Mre11 were determined using immunohistochemical analysis, and correlated with clinical parameters, including age, gender, Lauren subtype, tumor grades, clinical stage and overall survival.RESULTS: Expression loss of BRCA1, ATM, ATR, MDC1, and Mre11 was found in 21.4%, 20.2%, 21.0%, 11.1% and 4.6%, respectively, of interpretable cases. BRCA1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 8.2% vs 31.7%, P = 0.001), higher tumor grade (Ⅰ/Ⅱ vs Ⅲ, 10.7% vs 20.5;Ⅰ/Ⅱ vs Ⅳ, 10.7% vs 54.5%, P = 0.047) and advanced clinical stage (Ⅰ/Ⅱ vs Ⅲ, 12.9% vs 16.9%;Ⅰ /Ⅱ vs Ⅳ, 12.9% vs 45.5%, P = 0.006). MDC1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 0% vs 19.7%, P = 0.001) and higher tumor grade (Ⅰ/Ⅱ vs Ⅲ, 0% vs 12%;Ⅰ/Ⅱ vs Ⅳ, 0% vs 30.8%, P = 0.012). In addition, the survival time of the patients with expression loss of BRCA1 was significantly shorter than those with positive expression of BRCA1 (2-year survival rate, 32.4% vs 62.8%, P = 0.015). No correlations were found between clinicopathological parameters and expression loss of ATM, ATR and Mre11. CONCLUSION: Our results support the hypothesis that homologous recombination deficiency plays an important role in the progression of gastric carcinoma. Loss of expression of BRCA1 and     

Assessment of chronic radiation proctopathy and radiofrequency ablation treatment follow-up with optical coherence tomography angiography: A pilot study

BACKGROUND Chronic radiation proctopathy(CRP) occurs as a result of pelvic radiation therapy and is associated with formation of abnormal vasculature that may lead to persistent rectal bleeding. While incidence is declining due to refinement of radiation delivery techniques, CRP remains one of the major complications of pelvic radiation therapy and significantly affects patient quality of life.Radiofrequency ablation(RFA) is an emerging treatment modality for eradicating abnormal vasculature associated with CRP. However, questions remain regarding CRP pathophysiology and optimal disease management.AIM To study feasibility of optical coherence tomography angiography(OCTA) for investigating subsurface vascular alterations in CRP and response to RFA treatment.METHODS Two patients with normal rectum and 8 patients referred for, or undergoing endoscopic RFA treatment for CRP were imaged with a prototype ultrahighspeed optical coherence tomography(OCT) system over 15 OCT/colonoscopy visits(2 normal patients, 5 RFA-na?ve patients, 8 RFA-follow-up visits). OCT and OCTA was performed by placing the OCT catheter onto the dentate line and rectum without endoscopic guidance. OCTA enabled depth-resolved microvasculature imaging using motion contrast from flowing blood, withoutrequiring injected dyes. OCTA features of normal and abnormal microvasculature were assessed in the mucosa and submucosa. Blinded reading of OCTA images was performed to assess the association of abnormal rectal microvasculature with CRP and RFA treatment, and rectal telangiectasia density endoscopic scoring.RESULTS OCTA/OCT images are intrinsically co-registered and enabled depth-resolved visualization of microvasculature in the mucosa and submucosa. OCTA visualized normal vascular patterns with regular honeycomb patterns vs abnormal vasculature with distorted honeycomb patterns and ectatic/tortuous microvasculature in the rectal mucosa. Normal arterioles and venules < 200 μm in diameter versus abnormal heterogenous enlarged arterioles and venules > 200μm in diameter were visualized in the rectal submucosa. Abnormal mucosal vasculature occurred in 0 of 2 normal patients and 3 of 5 RFA-na?ve patients,while abnormal submucosal vasculature occurred more often, in 1 of 2 normal patients and 5 of 5 RFA-na?ve patients. After RFA treatment, vascular abnormalities decreased, with abnormal mucosal vasculature observed in 0 of 8 RFA-follow-up visits and abnormal submucosal vasculature observed in only and 2 of 8 RFA-follow-up visits.CONCLUSION OCTA visualizes depth-resolved microvascular abnormalities in CRP, allowing assessment of superficial features which are endoscopically visible as well as deeper vasculature which cannot be seen endoscopically. OCTA/OCT of the rectum can be performed in conjunction with, or independently from endoscopy.Further studies are warranted to investigate if OCTA/OCT can elucidate pathophysiology of CRP or improve management.     

PNPLA3 I148M polymorphism and progressive liver disease

The 148 Isoleucine to Methionine protein variant(I148M)of patatin-like phospholipase domain-containing 3(PNPLA3),a protein is expressed in the liver and is involved in lipid metabolism,has recently been identified as a major determinant of liver fat content.Several studies confirmed that the I148M variant predisposes towards the full spectrum of liver damage associated with fatty liver:from simple steatosis to steatohepatitis and progressive fibrosis.Furthermore,the I148M variant represents a major determinant of progression of alcohol related steatohepatitis to cirrhosis,and to influence fibrogenesis and related clinical outcomes in chronic hepatitis C virus hepatitis,and possibly chronic hepatitis B virus hepatitis,hereditary hemochromatosis and primary sclerosing cholangitis.All in all,studies suggest that the I148M polymorphism may represent a general modifier of fibrogenesis in liver diseases.Remarkably,the effect of the I148M variant on fibrosis was independent of that on hepatic steatosis and inflammation,suggesting that it may affect both the quantity and quality of hepatic lipids and the biology of non-parenchymal liver cells besides hepatocytes,directly promoting fibrogenesis.Therefore,PNPLA3 is a key player in liver disease progression.Assessment of the I148M polymorphism will possibly inform clinical practice in the future,whereas the determination of the effect of the 148M variant will reveal mechanisms involved in hepatic fibrogenesis.     

YMDD variants of HBV DNA polymerase gene: Rapid detection and clinicopathoiogical analysis with long-term Iamivudine therapy after liver transplantation

AIM: To look for a rapid low-cost technique for the detection of HBV variants. METHODS: Two patients who underwent orthotopic liver transplantation (OLT) for HBV infection were treated with lamivudine (100 mg daily) and HBV infection recurred in the grafted livers. The patients were monitored intensively for liver enzymes, hepatitis B surface antigen (HBsAg) and HBV DNA in serum. Liver biopsy was performed regularly. HBV DNA in a conserved polymerase domain (the YMDD locus) was amplified from serum of each patient by PCR and sequenced. HBV genotypes were analyzed by restriction fragment length polymorphism (RFLP) of the PCR products generated from a fragment of the polymerase gene. RESULTS: YMDD wild-type HBV was detected in one patient by PCR-RFLP and DNA sequencing 19 mo after OLT, and YIDD mutant-type HBV in the other patient, 16 mo after OLT. CONCLUSION: PCR-RFLP assay is an accurate and simple method for genotyping lamivudine-resistant HBV variants.     

拉米夫定耐药患者乙型肝炎病毒聚合酶基因G743C和G743A点突变的检测及分析

目的 探讨拉米夫定耐药患者乙型肝炎病毒聚合酶(HBVP)基因点突变。方法 聚合酶链反应(PCR)扩增拉米夫定耐药患者的HBVP基因，产物经直接测序检测其YMDD变异；同时，用PCR-限制性片段长度多态性(PCR—RFLP)分析测序后的17例YIDD变异样本，先用3对引物扩增HBVP基因，再分别用3个限制性内切酶FokⅠ、SspⅠ、Alw441酶切扩增产物，酶切产物用8．0％聚丙烯酰胺凝胶电泳分析。结果 拉米夫定耐药患者HBVP基因PCR产物序列分析结果与GenBank的HBV标准株相比，发现16例存在HBVP基因G743C点突变和1例存在G743A点突变。其核苷酸序列由ATG变为ATC和ATA，YMDD基因序列发生变异，即由YMDD变异为YIDD；但是，PCRRFLP不能确定这17例样本存在YIDD变异。结论拉米夫定耐药患者存在HBVP基因G743C和G743A的点突变，同样引起YIDD变异；而PCR—RFLP法不能准确地检测出G743C和G743A的点突变；该发现对HBVP基因YMDD变异检测和临床均有指导意义。     

拉米夫定治疗前后乙型肝炎病毒P区基因的动态变化

目的研究拉米夫定治疗前后慢性乙型肝炎患者体内乙型肝炎病毒P基因区的变异情况。方法从5例慢性乙型肝炎患者拉米夫定治疗前和治疗12个月的血清标本中，扩增目的片段，阳性结果双酶切后克隆至JM105感受态细胞。每份标本随机挑取20个阳性克隆，以错配聚合酶链反应一限制性片段长度多态性分析法检测YMDD基因序列，出现变异者进行双向测序。结果5例慢性乙型肝炎患者在拉米夫定治疗12个月后，其中2例HBVDNA为阴性，2例HBVDNA转阴后又转阳，测序结果提示出现M5521变异；1例HBVDNA始终阳性，和治疗前一样存在D553G的变异。结论D553G变异可能是慢性乙型肝炎患者拉米夫定治疗无效的原因之一，拉米夫定治疗后出现的乙型肝炎病毒基因组P区YMDD变异是抗病毒药物诱导的结果。     

Rapid and high throughput detection of HBV YMDD mutants with fluorescence polarization

AIM: To develop a simple and rapid detection of HBV gene variants and prediction of lamivudine-resistance in patients.METHODS: Initially, plasmids harboring the wild-type or mutant HBV DNA fragments were used in a model system.The technique was then applied to clinical samples for an analysis of YMDD mutations. The sera were extracted from chronic hepatitis patients who had received lamivudine treatment for more than one year. P region gene of HBV was amplified by polymerase chain reaction. The excess primers and dNTPs in PCR products were removed by cleaning-up reagents. Template-directed dye-terminator incorporation reaction was performed and Rl10 or TAMRA labeled acyclo-terminator was added on the 3‘ end of TDI-primer specifically. Fluorescence polarization value was measured with Victor 2 multilabel counter and the genotypes of HBV were analyzed. RESULTS: The YMDD genotypes in recombined positive plasmid and 56 serum samples of HBV infected patients were analyzed by using our TDI-FP method and the specificity and sensitivity were confirmed by DNA sequencing. Five of 56 serum samples showed YVDD phenotype (9 %), including 1 YMDD and YVDD mixed infection. Four of 56 showed YIDDphenotype (7.1%). CONCLUSION: This is a simple, rapid, low cost and high throughput assay to detect HBV polymerase gene variants and suitable for large-scale screening and prediction of the lamivudine-resistance in clinical samples.     

Occult HBV infection and YMDD variants in hemodialysis patients with chronic HCV infection

BACKGROUND/AIMS: End-stage renal disease patients on chronic hemodialysis are at risk for both hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Although the prevalence is unknown in hemodialysis patients, occult HBV infection is frequent in subjects with chronic HCV infection. We aimed to investigate (1) the prevalence and clinical impact of occult HBV infection in hemodialysis patients with chronic HCV infection, and (2) the frequency of YMDD variants (tyrosine-methionine-aspartate-aspartate amino acid motif of HBV polymerase) in this setting. METHODS: Thirty-three anti-HCV and HCV-RNA-positive, HBsAg-negative hemodialysis patients (mean age 36.9+/-10.4 years, 22 male) were admitted to this study. HBV-DNA (Innogenetics kit) and HCV-RNA (Cobas Amplicor HCV kit) were investigated by polymerase chain reaction technique (PCR). YMDD mutation was studied in all HBV-DNA-positive patients by the BOOM method. RESULTS: HBV-DNA was detected in 12 of 33 patients (36.4%) by PCR. Their mean age was 33.0+/-9.0 years. Age, dialysis period (years) and biochemical parameters were not significantly different in patients with and without occult HBV infection. YMDD variants were identified in six of 12 (50%) patients with occult HBV infection. CONCLUSIONS: Occult HBV infection is frequent in hemodialysis patients with chronic HCV infection. YMDD variants are common in this setting.     

拉米夫定抗乙型肝炎病毒感染中YMDD变异类型及时间研究

目的 研究拉米夫定抗乙型肝炎病毒(HBV)感染中P基因发生YMDD变异的类型和出现时间。方法 取拉米夫定治疗过程中HBV DNA由阴性再次转为阳性33例患者及HBV DNA始终保持阳性达一年或一年以上2例患者的血清,经PCR扩增HBV的P基因,对扩增产物进行测序及用3个限制性内切酶分析HBVP基因的YMDD变异。 结果 14例出现YMDD变异,其中6例YVDD变异,4例YIDD变异,3例YI/VDD混合变异,1例YI/MDD变异。变异出现时间平均为(11.07±3.65)个月,最短5个月,最长7个月。其中YIDD为(10.00±1.41)个月,YVDD为(11.67±4.41)个月,YI/VDD为(13.33±3.31)个月,三种变异类型的出现时间差异无显著性(F=0.543,P>0.05)。3例YMDD变异后拉米夫定加量至200mg/d,未能消除变异病毒。 结论 拉米夫定抗HBV感染后变异有多种形式,包括YIDD、YVDD、YI/VDD、YI/MDD。变异发生时间一般在治疗后(11.07±3.65)个月。变异类型与用药时间无关。     

Usefulness of dried blood samples for quantification and molecular characterization of HBV-DNA

The purpose of this study was to assess the use of dried blood spot (DBS) samples for hepatitis B virus (HBV) DNA quantification, HBV genotyping, and detection of G1896A precore mutants and variants in the YMDD polymerase motif. We studied DBS and serum samples from 82 patients with chronic HBV infection (23 hepatitis B e antigen [HBeAg]-positive and 39 HBeAg-negative), 20 HBeAg-inactive carriers, and 15 HBeAg-negative patients under lamivudine therapy (selected from chronic HBV patients). DBS samples consisted of approximately 20 microL of blood applied to 5-mm paper disks. HBV DNA quantification and HBV precore mutant detection were done using real-time polymerase chain reaction, HBV genotyping using restriction fragment length polymorphism, and YMDD variant detection by Inno-lipa assay. DBS and serum results were compared. HBV DNA was detected in a range of 10(2)-10(8) copies/mL, with low intra-assay and inter-assay variation (<10%). Median DBS HBV DNA (copies/mL) was: 3.7 x 10(6) in HBeAg-positive, 6.2 x 10(5) in HBeAg-negative, and 5.5 x 10(2) in inactive carriers (P <.05). HBV DNA was positive in serum (median 5 x 10(3) copies/mL) but negative in DBS for five inactive carriers. The correlation coefficient between HBV DNA concentration in DBS versus serum samples was r(2) = 0.96 (P <.001). The sensitivity of HBV DNA detection in DBS samples was 1 log(10) lower than in serum samples. Concordance between DBS and serum for HBV genotyping, and for precore mutant and YMDD variant detection was optimal. DBS storage for 7 days at room temperature and 21 days at -20 degrees C revealed no decrease in HBV DNA levels or integrity. In conclusion, the DBS sample is useful for HBV DNA quantification, genotyping, and detection of precore mutant and YMDD variants. All four determinations can be completed with a single drop of dried blood.     

乙型肝炎病毒多聚酶变异与拉米夫定治疗反应

目的 探讨乙型肝炎病毒（HBV）多聚酶酪氨酸－蛋氨酸－天氧氨酸－天冬氨酸（YMDD）基序变异对拉米夫定抗病毒疗效的影响。方法 对19例拉米夫定治疗（100mg/d）48周时血清病毒核酸阳性的慢性乙型肝炎患者,采用聚合酶链反应（ PCR）产物直接测序技术,检测其血清中HBV多取和酶（YMDD）变异情况,并观察其血清HBV DNA和丙氨酸转氨酶（ALT）水平。结果 在检出YMDD变异株的10 例患者（4例为YV^550DD型,均伴L^526→M突变,6例为YI^550DD型）,至52周为止,共5例出现HBV DNA突发（分枝DNA信号扩增法）,其中2例伴ALT再高。追踪其治疗前血清均未发生上述变异。而在未检出YMDD变异的9例患者中,1例出现DNA突发后又阴转,而另1例治疗过程中HBV DNA未阴转、ALT始终波动于正常值上下的患者,发现多聚酶其他部位的变异^473→R,D^477→N,D^480→N,L^491→M)治疗前后相同。结论 乙型肝炎病毒多聚酶（ YMDD）变异与药物诱导相关,发生变异者较无变异者疗效降低;在拉米夫定治疗过程中,该变异的检测将有助于其疗效监测。     

乙型肝炎病毒前C基因区变异对YMDD变异毒株复制活性的影响

目的调查接受拉米夫定治疗的乙型肝炎病毒（HBV）感染者中HBV前C基因区终止密码变异（A1896，PC）和基本核心启动子变异（BCP，T1762／A1764）对YMDD变异毒株复制活性的影响。方法应用聚合酶链反应-限制性片断长度多态性（PCR—RFLP）法对197例接受拉米夫定治疗后发生YMDD耐药变异的患者进行HBV基因型、PC及BCP变异检测，并用实时荧光定量法对所有患者血清HBVDNA进行定量。结果197例YMDD变异株感染者中B基因型占51．8％，C基因型占48．2％；有61例（31．0％）发生PC变异，69例（35．0％）发生BCP变异；PC变异在B基因型中的发生率明显高于C基因型（X^2=8．433，P=0．004），而BCP变异在C基因型中的发生率显著高于B基因型（X^2=16．83，P〈O．001）；发生PC或BCP变异的HBV感染者，其血清中HBVDNA水平与野生株相比差异均无统计学意义。结论HBV前C基因区的PC或BCP变异与基因型具有相关性，但这两种变异对YMDD变异株感染者的血清病毒水平均无影响。     

YMDD motif mutations in chronic hepatitis B antiviral treatment naïve patients: a multi-center study

ObjectiveThis study aimed to determine the natural prevalence of variants of tyrosinemethionine- aspartic acid-aspartic acid (YMDD) motif in patients with chronic hepatitis B (CHB), and to explore its relation with demographic and clinical features, hepatitis B virus (HBV) genotypes, and HBV DNA levels.MethodsA total of 1,042 antiviral treatment naïve CHB patients (including with lamivudine [LAM]) in the past year were recruited from outpatient and inpatient departments of six centers from December 2008 to June 2010. YMDD variants were analyzed using the HBV drug resistance line probe assay (Inno-Lipa HBV-DR). HBV genotypes were detected with polymerase chain reaction (PCR) microcosmic nucleic acid cross-ELISA, and HBV deoxyribonucleic acid (DNA) was quantitated with real-time PCR. All serum samples underwent tests for HBV, HCV, and HDV with ELISA.ResultsYMDD variants were detected in 23.3% (243/1042) of CHB patients. YMDD mutation was accompanied by L180M mutation in 154 (76.9%) patients. Both wild-type HBV and YMDD variant HBV were present in 231 of 243 patients. Interestingly, 12 patients had only YIDD and/or YVDD variants without wild YMDD motif. In addition, 27.2% (98/359) of HbeAg-positive patients had YMDD mutations, which was higher than that in HbeAg-negative patients (21.2%, 145/683). The incidence of YMDD varied among patients with different HBV genotypes, but the difference was not significant. Moreover, the incidence of YMDD in patients with high HBV DNA level was significantly higher than that in those with low HBV DNA level.ConclusionMutation of YMDD motif was detectable at a high rate in CHB patients in this study. The incidence of YMDD may be correlated with HBeAg and HBV DNA level.