Real-world efficacy of daclatasvir and asunaprevir with respect to resistance-associated substitutions

AIM To investigate daclatasvir(DCV) and asunaprevir(ASV) efficacy in hepatitis C(HCV) patients, with respect to resistance-associated substitutions(RASs).METHODS A total of 392 HCV-infected patients from multiple centers were included in this study. We evaluated their clinical courses and sustained virologic responses(SVR) according to pretreatment factors(gender, age, history of interferon-based regimens, platelet counts, level of viremia, pretreatment NA5A:L31, and Y93 substitutions). We also analyzed the pretreatment and post-treatment major RASs of NS3:D168, NS5A:L31 and Y93 substitutions using a direct-sequencing method in 17 patients who were unable to achieve SVR at 12 wk after treatment completion(SVR12).RESULTS The overall SVR12 rate was 88.3%. Thirty-one patients discontinued treatment before 24 wk because of adverse events, 23 of whom achieved SVR12. There were no significant differences in SVR12 rates with respect to gender, age, history of interferon-based regimens, and platelet counts. The SVR12 rate in patients with viral loads of ≥ 6.0 log IU/m L was significantly lower than those with viral loads of < 6.0 log IU/m L(P < 0.001). The SVR12 rate in patients with Y93 substitution-positive was significantly lower than those with Y93 substitution-negative(P < 0.001). The L31 substitution-positive group showed a lower SVR12 rate than the L31 substitution-negative group, but the difference was not statistically significant. Seventeen patients who did not achieve SVR12 and had available pretreatment and post-treatment sera had additional RASs in NS3:D168, NS5:L31, and Y93 substitution at treatment failure.CONCLUSION Combination of DCV and ASV is associated with a high SVR rate. Baseline RASs should be thoroughly assessed to avoid additional RASs after treatment failure.

直接抗病毒药物治疗失败的慢性丙型肝炎患者临床特点及基因型分析

目的探索直接抗病毒药物(DAA)治疗失败的慢性丙型肝炎(CHC)患者的临床特点及基因型特征。方法选取2021年9月至2022年12月咸阳市第一人民医院DAA治疗失败的患者28例,同时期DAA治疗成功者100例。酶法检测肝功能、PCR-反向杂交法检测HCV基因型、Sanger测序法检测耐药相关替代突变(RAS)片段,分析DAA观察组患者临床特征及基因型特点。结果失败组和有效组HCV RNA>105 IU/mL分别为23例(82.1%)、34例(34.0%),差异有统计学意义(χ^(2)=20.526,P=0.001)。DAA治疗失败组男性患者TBil、DBil分别为14.61(10.98,20.78)μmol/L、4.94(3.08,7.48)μmol/L,均高于女性患者的6.65(4.90,8.40)μmol/L、2.50(1.78,3.07)μmol/L(Z=-2.018和-2.456,均P=0.01)。纳入患者共检测出3种HCV基因型,分别为1b、2a及3b,有26例未测出基因型,失败组HCV 1b、2a及3b基因型分别为11例(39.3%)、6例(21.4%)及5例(17.8%),有效组分别为56例(56.0%)、15例(15.0%)及9例(9.0%)。失败组检测出NS5A和NS5B2种基因片段,但是RAS发生率不同。结论DAA治疗失败者会受性别、病毒载量、基因型以及RAS种类影响。

Analysis of the effect of HCV resistance-associated substitutions on the short-term efficacy of DAA after single administration in three phase Ib clinical trials

As crucial factors in hepatitis C virus(HCV)management,resistance-associated substitutions(RASs)are associated with the treatment outcome of some direct-acting antiviral(DAA)-based regimens.In this study,we mainly analyzed the impact of baseline Y93 H or Y93 Y/H on the short-term efficacy after single administration of NS5 A inhibitors in three phaseⅠb clinical trials(yimitasvir phosphate,KW-136 and fopitasvir),and analyzed the prevalence of baseline RASs and treatment-emergent RASs.A total of 94 treatment-naive HCV genotype(GT)-1 b(n=63)and GT-2 a(n=31)Chinese patients were enrolled in three phase lb clinical trials.We investigated RASs in 77 patients with next generation or Sanger sequencing.In the 7-day trial of yimitasvir phosphate,the mean maximum HCV RNA decrease of patients with baseline Y93 H or Y93 Y/H was lower than that of patients without the mutation in the 30 mg and 200 mg cohorts(0.83 vs.2.45 log10 IU/mL and 1.92 vs.2.63 log10 IU/mL).In the3-day trial of KW-136,the mean maximum HCV RNA decrease in patients with baseline Y93 H or Y93 Y/H was lower than that of patients without the mutation in the 30,60 and 120 mg cohorts(1.58 vs.2.89 log10 IU/mL,3.16 vs.4.09 log10 IU/mL and3.00 vs.5.04 log10 IU/mL,respectively).In the 3-day trial of fopitasvir,only 30 mg group had baseline Y93 H or Y93 Y/H,and the average maximum HCV RNA decrease of patients with baseline Y93 H or Y93 Y/H was lower than that of patients without the mutation(1.45 vs.3.59 log10 IU/mL).In the three trials,baseline RASs were observed in 54 patients(70.1%;54/77).The most prevalent baseline RASs were Y93 H and Y93 Y/H(18.2%;14/77),followed by L3 IM(16.9%;13/77).The most common RASs after single administration of DAA were Y93 H and Y93 Y/H.Our data could provide reference for future clinical treatment and clinical trial.

Efficacy of direct-acting antiviral treatment for chronic hepatitis C: A single hospital experience

AIM To evaluate the efficacy of direct-acting antivirals(DAAs) in Kanto Rosai Hospital. METHODS All patients with hepatitis C virus(HCV) who underwent DAA prescription were enrolled in this study. The present study was a single center retrospective analysis using patients infected with HCV genotype 1 or 2. Resistance analysis was performed by using direct sequencing and cycleave PCR in genotype 1 patients treated with interferon(IFN)-free DAA. The primary endpoint was sustained virologic response at 12 wk after therapy(SVR12).RESULTS A total of 117 patients participated in the study, including 135 with genotype 1 and 42 with genotype 2. Of the 135 patients with genotype 1, 16 received protease inhibitor + IFN + ribavirin and all achieved SVR. Of the 119 patients who received IFN-free DAA(in different combinations), 102 achieved SVR and 9 failed(7/9 were on daclatasvir/asunaprevir and 2/9 on ledipasvir/sofosbuvir). Efficacy analysis was done only for 43 patients who received daclatasvir/asunaprevir. From this analysis, Y93 resistance-associated substitutions were significantly correlated with SVR.CONCLUSION The SVR rate was 98% for genotype 1 and 100% for genotype 2. However, caution is needed for HCV NS5 A resistance-associated substitutions that are selected by HCV NS5 A inhibitors because cerebrovascular adverse events are induced by some DAA drugs.

Retreatment of patients with treatment failure of directacting antivirals: Focus on hepatitis C virus genotype 1b

The recent development of direct-acting antiviral agents(DAAs) against hepatitis C virus(HCV) infection could lead to higher sustained virological response(SVR) rates, with shorter treatment durations and fewer adverse events compared with regimens that include interferon. However, a relatively small proportion of patients cannot achieve SVR in the first treatment, including DAAs with or without peginterferon and/or ribavirin. Although retreatment with a combination of DAAs should be conducted for these patients, it is more difficult to achieve SVR when retreating these patients because of resistance-associated substitutions(RASs) or treatment-emergent substitutions. In Japan, HCV genotype 1 b(GT1 b) is founded in 70% of HCVinfected individuals. In this minireview, we summarize the retreatment regimens and their SVR rates for HCV GT1 b. It is important to avoid drugs that target the regions targeted by initial drugs, but next-generation combinations of DAAs, such as sofosbuvir/velpatasvir/voxilaprevir for 12 wk or glecaprevir/pibrentasvir for 12 wk, are proposed to be potential solution for the HCV GT1 b-infected patients with treatment failure, mainly on a basis of targeting distinctive regions. Clinicians should follow the new information and resources for DAAs and select the proper combination of DAAs for the retreatment of HCV GT1 b-infected patients with treatment failure.

雪旺细胞对骨组织工程支架材料复合成骨细胞修复兔桡骨缺损的影响

【目的】探讨雪旺细胞对骨组织工程支架材料复合成骨细胞修复兔桡骨缺损的影响。【方法】分离培养免骨髓间充质干细胞（BMSCs），诱导分化为成骨细胞（IOBs），并从乳兔中提取雪旺细胞（SCs），S-100免疫荧光染色鉴定。采用新西兰大白兔建立桡骨缺损模型，随机平均分成5组：支架材料组（B组）在骨缺损处植入纳米壳聚糖／羟基磷灰石多孔支架；IO＆／支架材料组（C组）在骨缺损处植入种植10＆的支架材料；SCs＋支架材料组（D组）骨缺损处植入种植SCs的支架材料，IOBs／SCs／支架材料组（E组）：骨缺损处植入同时种植IOBs和SCs的支架材料；空白对照组（A组）在骨缺损处不做任何处理。各组在术后8周评估各组大白兔桡骨缺损部位新生骨的形成情况。【结果】术后8周x射线检测结果显示：B组、C纽、D组骨缺损部分愈合，C组、D组优于B组，E组骨缺损完全愈合，而A组骨缺损未愈合。HE染色结果显示：8周时同B组或c组或D组比较，E组成骨细胞和骨小梁数量最多，骨修复最快；免疫组化检测结果显示：脑源性神经生长因子（BI）NF）和神经生长因子（NGF）在E组中的表达较其他组别明显增多。【结论]SCs能够显著促进工程支架材料复合成骨细胞修复兔桡骨缺损，同神经营养因子BDNF和NGF表达紧密相关。

Exploring the hepatitis C virus genome using single molecule realtime sequencing

Single molecular real-time(SMRT)sequencing,also called third-generation sequencing,is a novel sequencing technique capable of generating extremely long contiguous sequence reads.While conventional short-read sequencing cannot evaluate the linkage of nucleotide substitutions distant from one another,SMRT sequencing can directly demonstrate linkage of nucleotide changes over a span of more than 20 kbp,and thus can be applied to directly examine the haplotypes of viruses or bacteria whose genome structures are changing in real time.In addition,an error correction method(circular consensus sequencing)has been established and repeated sequencing of a single-molecule DNA template can result in extremely high accuracy.The advantages of long read sequencing enable accurate determination of the haplotypes of individual viral clones.SMRT sequencing has been applied in various studies of viral genomes including determination of the full-length contiguous genome sequence of hepatitis C virus(HCV),targeted deep sequencing of the HCV NS5A gene,and assessment of heterogeneity among viral populations.Recently,the emergence of multi-drug resistant HCV viruses has become a significant clinical issue and has been also demonstrated using SMRT sequencing.In this review,we introduce the novel third-generation PacBio RSII/Sequel systems,compare them with conventional next-generation sequencers,and summarize previous studies in which SMRT sequencing technology has been applied for HCV genome analysis.We also refer to another long-read sequencing platform,nanopore sequencing technology,and discuss the advantages,limitations and future perspectives in using these thirdgeneration sequencers for HCV genome analysis.

New real-time-PCR method to identify single point mutations in hepatitis C virus

AIM To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus(HCV).METHODS In patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80 K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. Light Cycler methods, based on real-time PCR with sequencespecific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest. RESULTS Using nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10%(mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80 K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80 K was detected in 14.6% of G1 a patients and 0% of G1 b in our setting. CONCLUSION A fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single point mutation in highly variable genomes.

Direct-acting Antiviral Regimens for Patients with Chronic Infection of Hepatitis C Virus Genotype 3 in China

Hepatitis C virus(HCV)genotype(GT)3 infection is associated with a more rapid hepatic disease progression than the other genotypes.Hence,early HCV clearance slows down the disease progression and is important for improving prognosis in GT3-infected patients.Nevertheless,compared with other genotypes,GT3 is difficult-to-treat with direct-acting antivirals,especially in the presence of cirrhosis.Current guidelines recommend several regimens which have been proven to be effective in GT3-infected patients from the Western world(North America,Europe,and Oceania).In China,GT3 infection comprises 8.7–11.7%of the 10 million patients infected with HCV and has strikingly different characteristics from that in Western countries.Unlike the Western countries,where GT3a is the predominant subtype,GT3a and 3b each affect roughly half of Chinese GT3-infected patients,with 94–96%of the GT3b-infected patients carrying A30K+L31M double NS5A resistance-associated substitutions.Phase 3 clinical trials including GT3b-infected patients have suggested that GT3b infection is difficult to cure,making the regimen choice for GT3b-infected patients an urgent clinical gap to be filled.This review includes discussions on the epidemiology of HCV GT3 in China,recommendations from guidelines,and clinical data from both Western countries and China.The aim is to provide knowledge that will elucidate the challenges in treating Chinese GT3-infected patients and propose potential solutions and future research directions.

当前社会保障突出问题的理论探讨

当前社会保障制度中的突出问题是资金来源不足。为了保证充足的资金来源 ,以职工工资总额计算的总缴费率必须等于失业率、离退休率、综合患病率等项之和。以国有资产切块建立社会保障基金的归还欠帐思路有诸多欠妥之处。通过“发债、征税、收租”的统筹解决办法有望建立起一个稳定、规范和有效的社会保障制度。