Hepatitis B virus genetic diversity and its impact on diagnostic assays

Summary.  Hepatitis B virus (HBV) circulates in blood as closely related, but genetically diverse molecules called quasispecies. During replication, HBV production may approach 10¹¹ molecules/day, although during peak activity this rate may increase 100–1000 times. Generally, DNA polymerases have excellent fidelity in reading DNA templates because they are associated with an exonuclease which removes incorrectly added nucleotides. However, the HBV-DNA polymerase lacks fidelity and proofreading function partly because exonuclease activity is either absent or deficient. Thus, the HBV genome and especially the envelope gene, is mutated with unusually high frequency. These mutations can affect more than one open reading frame because of overlapping genes. The S gene contains an exposed major hydrophilic region (residues 110–155), which encompasses the 'a' determinant that is important for inducing immunity. Nucleotide substitutions in this region are common and result in reduced binding or failure to detect hepatitis B surface antigen (HBsAg) in diagnostic assays. Adaptive immunity also depends on the recognition of HBsAg by specific antibody and variants pose a threat if they interfere with binding to antibody. Finally, genomic hypervariability allows HBV to escape selection pressures imposed by antiviral therapies, vaccines and the host immune system, and is responsible for creating genotypes, subgenotypes and subtypes.     

提上睑肌三阶梯手术矫正上睑下垂的临床应用

目的 探讨提上睑肌三阶梯手术矫正上睑下垂的适应范围及效果。方法 根据提上睑肌肌力和上睑下垂的程度，将患者分为三组。A组为提上睑肌肌力好，上睑呈轻度下垂者。单纯采用提上睑肌徙前术矫正。B组为提上睑肌有一定的肌力，上睑呈中度下垂者。在提上睑肌徙前术的基础上。行提上睑肌缩短术矫正。C组为提上睑肌肌力差，上睑呈重度下垂者。在提上睑肌徙前、缩短术的基础上行提上睑肌反折悬吊术矫正。结果 87例患者术后随访6～30个月，上睑缘位于角膜缘下1mm．眼睑弧度自然，两眼对称，眼睑闭合自如，无暴露性角膜炎及结膜脱垂等并发症，效果满意。结论 提上睑肌徙前、缩短、反折悬吊三阶梯手术，可广泛适用于矫正不同程度的上睑下垂。     

Progression-free interval in ovarian cancer and predictive value of an ex vivo chemoresponse assay

The study objective was to determine the effectiveness of a phenotypic chemoresponse assay in predicting response to chemotherapy measured by progression-free interval (PFI) in a retrospective series of ovarian cancer patients whose tumor specimens had been tested with the ChemoFx assay. A statistically significant correlation between assay prediction of response and PFI was observed in 256 cases with an exact or partial match between drug(s) assayed and received. In 135 cases with an exact match, the hazard ratio for progression of the resistant group was 2.9 (confidence interval [CI]: 1.4-6.3; P < 0.01) compared to the sensitive group and 1.7 (CI: 1.2-2.5) for the intermediate compared to the sensitive group. The median PFI for patients treated with drugs assayed as resistant was 9 months, 14 months for those with drugs assayed as intermediately sensitive, and PFI had not been achieved for those with drugs assayed as sensitive. These data indicate that the ChemoFx assay is predictive of PFI in ovarian cancer. As the majority of ovarian cancers display different degrees of response to different chemotherapy agents ex vivo, the incorporation of assay information into treatment selection has the potential to improve clinical outcomes in ovarian cancer patients.     

Assay of cytomegalovirus susceptibility to ganciclovir in renal and heart transplant recipients

Ganciclovir (GCV) prophylaxis or pre-emptive therapy significantly reduce the rate of cytomegalovirus (CMV) disease and viremia, but increase the potential for emergence of ganciclovir-resistant CMV strains. The inhibitor concentration at 50% (IC(50)) of GCV from 156 CMV isolates from 59 renal or heart transplant recipients was calculated by means of a rapid phenotypic susceptibility assay. Twenty-seven strains were from 14 patients undergoing GCV therapy. The IC(50) was higher in patients under the prophylaxis regimen. One CMV strain, from a heart transplant recipient, became GCV-resistant after 1 month of therapy (IC(50)=13.7 micromol/l). These data, together with clinical and virological markers, suggested that a switch to foscarnet was necessary, and good evolution was observed. Thus, assay of CMV susceptibility to GCV could be helpful in clinical management.     

管碟法测定Nisin效价

对管碟法测定Nisin效价的条件进行了研究,考察了几个参数的影响,得出了Nisin测定的最佳条件:90mm培养皿中培养基加量为15mL,Na2HPO4·12H2O质量浓度1g/dL,菌悬液浓度109CFU/mL,琼脂质量浓度1g/dL,培养基pH值7.0,牛津杯中样品加液量100μL.在此条件下,Nisin效价在5～100IU/mL,其对数值与抑菌圈直径有较好的线性关系.     

斑点免疫金渗滤法的改进

目的 :改进斑点免疫金渗滤法的渗滤装置 ,降低成本 ,使之更适用于现场操作。方法 :用自制的圆形渗滤片替代塑料渗滤盒 ,并比较二者的效果。结果 :自制的渗滤片体积更小 ,成本更低 ,为一次性使用材料 ;检测抗体的效果与塑料渗滤盒法一致。结论 :自制的渗滤片优于常用的塑料渗滤盒     

固相板酶免法测定人血清游离三碘甲状腺原氨酸

目的 :探讨游离三碘甲状腺原氨酸 (FT3)的酶免疫测定法对于诊断甲状腺功能亢进和监测甲状腺激素补充治疗效果及预后情况的重要意义。方法 :采用聚苯乙烯96孔板作为固相载体 ,利用亲和纯化的兔抗T3 抗体和T3 - β-半乳糖苷酶标记物 ,建立了固相板竞争法。结果 :可测范围为0.48～45.0ng/L ,灵敏度0.13ng/L ,平均批内变异系数为 (n=20)6.4 % ,平均批间变异系数为 (n=8)12.4% ,正常参考范围4.14±0.97ng/L。正常人与甲亢病人能明显分开。结论 :酶免法测定血清FT3,其灵敏度、精密度均达到放免水平 ,且可避免同位素污染     

Using Computer Simulated Results of a Bulk Drug Substance Assay to Determine Acceptance Criteria for Method Validation

Purpose. To determine the statistical variability expected for a well designed HPLC assay of a bulk drug substance (BDS). The results are used to develop appropriate acceptance criteria for a method validation protocol as well as to evaluate the level of uncertainty expected for assay results using a variety of sampling/injection schemes. Methods. Computer simulation was used to generate a large quantity of data and the variability of the mock results was evaluated. Error propagation was also calculated, whenever possible, to confirm results obtained from the simulations. Results. Protocol acceptance criteria were developed that were consistent with the expected variability for data resulting from the execution of the validation protocol. In certain cases simulations provided the only avenue of obtaining results that could not otherwise be readily determined. Conclusions. Computer modeling can be used to obtain suitable acceptance criteria for validation results which are consistent with method variability. This is particularly significant in the case of linearity where it has been difficult to develop acceptance criteria based on anything other than analyst intuition and experience. Assay simulations clearly demonstrated that the variability expected for a typical BDS assay is large relative to the average specification range and therefore little insight about relative purity can be gained comparing individual passing assay results.     

Fumonisins as a possible contributory risk factor for primary liver cancer: A 3-year study of corn harvested in Haimen, China, by HPLC and ELISA

Employing HPLC fluorometry, gas-liquid chromatography (GLC) and a novel enzymelinked immunosorbent assay (ELISA) based on a monoclonal antibody, 40 corn samples, each collected in 1993 from agricultural stocks for human consumption in Haimen (Jiangsu County) and Penlai (Shandong Province), high- and low-risk areas for primary liver cancer (PLC) in China, respectively, were analysed for fumonisins (FBs), aflatoxins (AFs) and trichothecenes. Levels and positive rates of FBs and deoxynivalenol (DON) were significantly higher in Haimen than in Penlai. ELISA of the 40 corn samples harvested in the two areas in 1994 revealed that FB contamination levels and rates in these areas were comparable to those observed in 1993 in Haimen. ELISA analysis of 1993 and 1994 products revealed a wide occurrence of AFB₁ but the positive rates as well as levels were not significantly different between these areas. ELISA of the same sample number of corn harvested in 1995 revealed that FB contamination in Haimen was significantly higher than in Penlai. These 3-yearly surveys of corn samples (240 in total) demonstrated that corn harvested in Haimen was highly contaminated with FBs and that the contamination level, as well as positive rate in 1993 and 1995, were 10–50-fold higher than those in Penlai, suggesting FBs as a risk factor for promotion of PLC in endemic areas, along with the trichothecene DON. Co-contamination with AFs, potent hepatocarcinogens, was assumed to play an important role in the initiation of hepatocarcinogenesis.