Qingxian Cai

Comparison of three different HCV genotyping methods: Core, NS5B sequence analysis and line probe assay

To evaluate the capacity of Versant HCV genotype assay (LiPA) 2.0 to identify hepatitis C virus (HCV) genotypes, 110 serum samples were collected from chronic hepatitis C patients. Three methods were compared: core sequence analysis, NS5B sequence analysis and the INNO-LiPA assay. The result showed that 102 (92.7%) of the samples were amplified in either or both regions, of which 97 were amplified in the core region and 62 were amplified in the NS5B region. Correlation analysis showed that amplification rates of subgenomic regions were associated with viral loads. Basic local alignment search tool (BLAST) and phylogenetic analysis showed that the 102 samples were classified into 5 categories: subtype 1b, 2a, 3a, 3b and 6a at frequencies of 61.8% (63), 9.8% (10), 3.9% (4), 3.9% (4) and 20.6% (21), respectively. Compared with sequencing methods, 66.7% (68) of the 102 samples were identified completely by LiPA 2.0, whereas 19.6% (20) were assigned incompletely (indistinguishable or not identified subtype) and 13.7% (14) were misclassified. Of 21 genotype 6a samples, 11 were mistyped as 1b. In conclusion, LiPA 2.0 was not suitable for identifying HCV genotypes in the samples tested, whereas core sequence analysis remained an ideal method for genotyping HCV.

Prediction of response to pegylated-interferon-α and ribavirin therapy in Chinese patients infected with different hepatitis C virus genotype

BackgroundThe standard treatment for patients with chronic hepatitis C (CHC), pegylated interferon-α (PEG-IFN) plus ribavirin (RBV) does not provide a sustained virological response (SVR) in all patients. Genetic variations at the interleukin 28B (IL-28B) locus are important in predicting outcome following therapy in CHC patients.ResultsWe investigated the role of IL28B variations (rs8099917) in response to PEG-IFN-α/RBV treatment and evaluated its association with the risk of the null virological response (NVR) and relapse (REL) in different viral genotypes. We found that the overall distributions of the genotype among the SVR, NVR, and REL groups were significantly different (P<0.001). Patients with the TG genotype had an increased risk of NVR and REL (OR=6.45 95% CI =2.88–14.47, P<0.001 for NVR; OR=2.51, 95% CI =1.29–4.86, P=0.006 for REL, respectively), and patients with the GG genotype had a further increased risk of NVR and REL (OR=12.04, 95% CI =3.21–45.13, P<0.001 for NVR; ,OR=4.30, 95% CI =1.21–15.13, P=0.017 for REL, respectively). G variant genotypes (TG+GG) also had an increased risk of NVR and REL, and there was a significant trend for a dose-effect of G allele on the risk of NVR and REL (P<0.05). The SVR rate in TT higher than in TG+GG was more pronounced in those patients infected with non-G1 compared to the patients infected with G1. The treatment response did differ based on the rs8099917 genotype in patients with different viral genotypes, compared with patients infected with the non-G1, the G1 infected patients had an increased risk of NVR and REL (OR=2.03 95% CI =1.03–4.01, P=0.04 for NVR and OR=2.58, 95% CI =1.35–4.94, P=0.004 for REL, respectively). Moreover, multivariate regression analysis show that the rs8099917 G allele was the only independent factor significantly associated with a NVR and REL.ConclusionThis study suggests that host genetic polymorphisms rs8099917 in the vicinity of IL-28B is the most important predictor of treatment response of PEG-IFN-α/RBV for HCV patients in China.

Effect of drug resistance mutations on antiviral agents in HCV patients.

BACKGROUND Gene polymorphism of HCV is an important cause of drug resistance to direct-acting antivirals (DAAs). METHODS Nested PCR assays were performed to amplify the HCV viral regions of NS3, NS5A and NS5B. RESULTS Major resistant mutation A156S was found in 18.33% of patients with HCV-1b and 64.28% of patients with HCV-2a. HCV-6a patients had a Q80K mutation rate of 95.45%, while the mutation rate of V170I was up to 100%. Mutation frequency varied with the different genotypes of HCV. The proportion of four resistance mutations (M36L, Q80K, A156S, V170I) in different groups were statistically significant (P<0.05). Resistant mutation Q30R was detected in 116 (72.5%) samples with HCV-1b and -6a, L31M was found in 16 patients, including 12 with HCV-2a and 4 with HCV-6a, H58P was discovered in 42.5% (68/160) of patients with the genotypes Q30R, L31M and H58P; Y93C was found in 9individuals with only HCV-2a. In HCV NS5B sequences, only a few resistant variants were detected, including C316N and S282T. CONCLUSIONS Naturally occurring dominant resistance mutations to HCV DAAs pre-existed in treatment-naive patients in China. Mutation frequency and characteristics varied with the HCV genotype.

Prevalence of IL-28B and ITPA genotypes in Chinese Han population infected persistently with hepatitis C virus genotype 6 or HCV-1

The geographic distribution, demographics, epidemiology, host factors, and clinical characteristics of persistent HCV‐6 infection in China need further characterization. This multicenter study enrolled 63 patients with persistent HCV‐6 infection and 63 patients with persistent HCV‐1 infection as controls. Blood biochemistry, quantitation of HCV RNA levels, and identification of host IL‐28B genotypes (rs12979860, rs8099917, and rs12980275) and ITPA genotype (rs1127354) were performed to estimate potential variability in host factors that may affect response to treatment. The mean HCV‐6 RNA level (3.8E6 IU/ml) was significantly higher than that in patients infected with HCV‐1 (1.7E6 IU/ml; P < 0.001). Patients persistently infected with HCV‐6 had a high prevalence of IL‐28B rs12979860 CC genotype (92.1%), rs8099917 TT genotype (93.7%), and rs12980275 AA genotype (90.5%). Their prevalence in patients infected with HCV‐1 was only modestly lower (82.5%, 84.1%, and 82.5%, respectively; P > 0.05). The inosine triphosphate pyrophosphatase (ITPA) SNP rs1127354 CC genotype was present in 66.7% of patients infected with HCV‐6, comparable to that of patients infected with HCV‐1 (65.1%; P > 0.05). There were no differences in the liver function, proportion of hepatic cirrhosis patients or patients with increased serum glucose between these two groups. Persistent HCV‐6 infection in Chinese Han is found mainly in the southern China. Chinese Han with chronic HCV‐1 or HCV‐6 infection have IL‐28B genotypes, suggesting responsiveness to interferon‐based pharmacotherapy. Most patients (67%) possess the ITPA genotype associated with susceptibility to ribavirin‐induced hemolysis. The routes of transmission for HCV‐6 genotype were more diversified than HCV‐1 genotype. The outbreak of HCV‐6 infection through blood transfusion progressed faster than HCV‐1. J. Med. Virol. 85:1163–1169, 2013.

Naturally Occurring Resistance-Associated Variants to Hepatitis C Virus Direct-Acting Antiviral Agents in Treatment-Naive HCV Genotype 6a-Infected Patients

Background and Objective The direct-acting antiviral agents (DAAs) antiviral therapy has drastically improved the prognosis of hepatitis C virus (HCV) patients. However, the viral drug resistance-associated variants (RAVs) can limit the efficacy of DAAs. For the HCV-6a is not the predominant prevalent genotype; the data on the prevalence of naturally occurring RAVs in it is scarce. Our study aims to assess the prevalence of RAVs in treatment-naive HCV-6a patients. Methods Nested PCR assays were performed on 95 HCV-6a patients to amplify HCV viral regions of NS3, NS5A, and NS5B. Results In NS3/4A region, we detected Q80K in 95.5% isolates (84/88) and D168E in 2.3% isolates (2/88). In NS5A region, we detected Q30R in 93.2% isolates (82/88), L31M in 4.6% isolates (4/88), and H58P in 6.8% isolates (6/88). In NS5B region, we detected A15G in 2.3% isolates (2/88), S96T in 1.1% isolates (1/88), and S282T in 20.7% isolates (17/88) and we detected I482L in 100% isolates (4/4), V494A in 50% isolates (2/4), and V499A in 100% isolates (4/4). Conclusions RAVs to DAAs preexist in treatment-naive HCV-6a patients. Further studies should address the issue of the impact of RAVs in response to DAA therapies for HCV-6a patients.

Favorable Outcomes of Chinese HCV-Related Cirrhotic Patients with Sustained Virological Response after Pegylated Interferon Plus Ribavirin Treatment

Few studies have conducted follow-up investigations of the clinical course in HCV-related cirrhotic patients who achieved a sustained virological response (SVR) with pegylated interferon plus ribavirin treatment (PegIFN + RBV). We investigated the clinical course and laboratory data in a prospective cohort study enrolling HCV-related cirrhotic patients who received PegIFN + RBV between August 2008 and July 2013 in China. Complete blood counts, liver function tests, and HCV-RNA were serially examined. Liver-related complications were recorded. To detect hepatocellular carcinoma (HCC), alpha-fetoprotein assays, and ultrasound scans were repeated at 6-month intervals. Twenty-five patients were enrolled, including 8 patients with decompensation events before treatment. Eighteen patients achieved SVR with a mean follow-up period of 25.78 months. During the follow-up period, only one patient exhibited HCV-RNA positivity and no decompensation events were detected, but 4 patients developed HCC after SVR. APRI decreased more in patients with SVR than in patients with non-SVR (median, −1.33 versus 0.86, P < 0.001). The albumin levels and platelet counts significantly increased during the follow-up period after SVR (44.27 ± 4.09 versus 42.63 ± 4.37, P = 0.037 and 173.89 ± 87.36 versus 160.11 ± 77.97, P = 0.047). These data indicated that HCV-related cirrhotic patients with SVR after PegIFN + RBV may have a favorable clinical course and improvements in laboratory data. Moreover, HCC should be monitored.

24 versus 48 Weeks of Peginterferon Plus Ribavirin in Hepatitis C Virus Genotype 6 Chronically Infected Patients with a Rapid Virological Response: A Non-Inferiority Randomized Controlled Trial

Objectives The optimal treatment of hepatitis C virus (HCV) genotype 6 is unclear owing to its limited geographic distribution. Because of a high predictive value of rapid virological response (RVR) for sustained virological response (SVR), we conducted an open-label randomized controlled trial to compare 24- and 48-week peginterferon/ribavirin combination therapy for patients with HCV genotype 6 in Southern China who achieved an RVR. Methods and Findings Treatment-naive, non-cirrhotic patients with chronic hepatitis C genotype 6 were treated with pegylated interferon α-2a (180 μg/week) and ribavirin (800–1,200 mg, according to weight) for 4 weeks. Patients who achieved an RVR, which was defined as HCV RNA negativity at week 4 (<50 IU), were randomized to receive either an additional 20 or 44 weeks of treatment (24- and 48-week treatment groups, respectively). The primary outcome measure was SVR. From January 2011 to June 2014, 152(152/210, 72.4%) patients with HCV genotype 6a and RVR were randomized 1:1 to the 24- or 48-week treatment group. The SVR rates in the 24- and 48-week groups in the intention-to-treat analysis were 90.8% (69/76) and 88.2% (67/76), respectively; those in the per-protocol analysis were 95.7% (67/70) and 97.0% (64/66), respectively. More patients in the 48-week group had anemia (46.1% vs. 28.9%, P = 0.03), but other adverse events were comparable between the groups. The limitation of the present study was that only patients from Southern China were enrolled which may inhibit the extensive application of the findings. Conclusion Twenty-four weeks of peginterferon/ribavirin combination therapy was non-inferior to 48 weeks in patients with HCV genotype 6a in Southern China who achieved an RVR. Trial Registration ClinicalTrials.gov NCT01263860

Treatment of pegylated interferon-α2a in chronic hepatitis B patients demonstrating a spontaneous decline in HBV DNA after acute exacerbation.

BACKGROUND Acute exacerbation (AE) in chronic hepatitis B (CHB) is usually followed by a spontaneous decline in HBV DNA levels. The subsequent treatment is controversial. In this study, we evaluated the efficacy and safety of pegylated interferon-α2a (PEG-IFN-α2a) for such CHB patients. METHODS A total of 74 hepatitis B e antigen (HBeAg)-positive patients with a spontaneous HBV DNA decline (by >2 log10 IU•ml(-1), compared with baseline levels before antiviral treatment) after AE (alanine aminotransferase [ALT]: 10-30-fold the upper limit of normal [ULN], total bilirubin [TBIL]: 2-20 mg•dl(-1), prothrombin time activity >60%) were included. In total, 22 patients (group A) received PEG-IFN-α2a treatment (180 µg•kg(-1)•week(-1), when ALT was <10 ULN and TBIL<2 mg•dl(-1)) for 48 weeks, with 48 weeks of treatment-free follow-up. Twenty-one patients (group B) selected continual entecavir therapy. Thirty-one patients (group C, control group) received routine liver-protective drugs. RESULTS At week 96, virological response rates were 90.5%, 100% and 48%, and ALT normalization rates were 81%, 95% and 40% for groups A, B and C, respectively. HBeAg seroconversion rates were 71.4%, 45% and 32% in groups A, B and C, respectively. A high hepatitis B surface antigen (HBsAg) loss rate was observed in PEG-IFN-α2a-treated patients, while no entecavir-treated patients achieved HBsAg loss. Group A patients suffered from typical PEG-IFN therapy-related adverse events. No severe adverse event was observed in any groups. CONCLUSIONS PEG-IFN-α2a is effective and safe for treating CHB patients demonstrating a spontaneous decline in HBV DNA after AE, and yields an increased likelihood of HBsAg loss.

The effects of direct-acting antiviral agents on the frequency of myeloid-derived suppressor cells and natural killer cells in patients with chronic hepatitis C: LI et al.

Currently, hepatitis C antiviral therapy is entering a new era with the use of direct‐acting antiviral (DAA) agents. However, the precise immunological influences of DAA therapy in patients with chronic hepatitis C (CHC) are insufficiently understood. This study aimed to investigate the effects of DAA therapy on the frequency of myeloid‐derived suppressor cells (MDSCs), T lymphocytes, and natural killer (NK) cells in patients with CHC. Thirty‐two treatment‐naive CHC patients were treated with DAA therapy, and the frequency of immune cells was analyzed by flow cytometry at various time points during and after therapy. Sixteen healthy donors were recruited for comparison. DAA therapy decreased the frequency of MDSCs and monocytic MDSCs in patients with CHC to a normal level. DAA therapy also increased the CD8+ T and NK cell levels in patients with CHC. In addition, activation (NKp30 and NKp46) and inhibitory (NKG2A) receptors on NK cells were downregulated to yield an NK cell phenotype resembling that observed in the healthy controls. This study provides insight into the normalization of immune cell levels under DAA therapy and indicates that restoration of the immune system in patients with CHC strongly supports long‐term curative hepatitis C virus eradication.