Prevalence of nonalcoholic fatty liver disease and the related risk factors among healthy adults: A cross-sectional study in Chongqing, China.

Background: Epidemiological characteristics of nonalcoholic fatty liver disease (NAFLD) in Chongqing, a west-central city of China, remain unclear. The objective of this study was to investigate the prevalence of NAFLD and the related risk factors among healthy adults for physical examination in Chongqing. Methods: A total of 110,626 subjects were enrolled in the present study. Each of the participants underwent physical examination, laboratory measurements, and abdominal ultrasonography. The chi-square test was employed to compare differences in the NAFLD prevalence, and logistic regression analysis was used to estimate the odds ratio for risk factors of NAFLD. Results: The prevalence of NAFLD in individuals in the population of Chongqing was 28.5%, and the prevalence in men (38.1%) was significantly higher than that in women (13.6%) (OR = 2.44; 95% CI: 2.31-2.58). NAFLD was more common in men aged 51-60 years and women over 60 years. Approximately 79.1% of the people with obesity and 52.1% of the people with central obesity had NAFLD. The prevalence of NAFLD in people with hypertension and cholelithiasis was 48.9 and 38.4%, respectively. Logistic regression showed that gender, age, body max index (BMI), central obesity, hypertension, impaired fasting glucose/diabetes mellitus (DM), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), hyperuricemia (HUA), alanine transaminase (ALT), and cholelithiasis were independently associated with the presence of NAFLD. Conclusion: The prevalence of NAFLD among healthy adults in Chongqing was high. To improve the prevention and management of NAFLD, special attention should be paid to the factors associated with the presence of NAFLD, including higher BMI, higher waist circumference, higher blood glucose, hypertension, hypertriglyceridemia, hyperuricemia, cholelithiasis, and elevated ALT.

Analysis of the prevalence of drug-resistant hepatitis B virus in patients with antiviral therapy failure in a Chinese tertiary referral liver centre (2010-2014).

OBJECTIVES: To study the prevalence of drug-resistant HBV in patients with therapy failure in a Chinese tertiary referral liver centre. METHODS: 1223 HBV-infected patients who underwent genotypic resistance testing between 2010-2014 were studied. RESULTS: 3TC genotypic resistance was the most common (46.5%), followed by LdT resistant (46.2%), ETV intermediate (37.9%), ADV resistant (11.4%), TDF intermediate (11.4%) and ETV resistant (1.7%). The 3TC resistance rate increased from 39.8% in 2010 to 56.6% in 2013, before decreasing to 49.5% in 2014, evidence of a lagging effect of l-nucleoside consumption. M204I, N236T and L180M+M204V+V173L/S202G were the most common substitutions for l-nucleoside (3TC and LdT), ADV and ETV genotypic resistant phenotypes, respectively. 3TC-exposed patients showed a high multiple genetic resistance rate (3TC-resistant+LdT-resistant+ETV intermediate; 58.8%). Resistance rates to 3TC, LdT and ETV in HCC patients were significantly higher than in cirrhosis and CHB patients. Resistance rates to different drugs showed no statistical difference between genotype B and C patients, whilst some amino acid substitution showed genotype bias, e.g. N236T incidence in genotype B was significantly higher than in genotype C (43.2% vs. 5.9%; P<0.0001), and genotype C isolates had a significantly higher A181V/T incidence than genotype B (54.9% vs. 19.3%; P<0.0001). CONCLUSIONS: 3TC genotypic resistance was most common in this centre, whilst ETV had the lowest resistance rate. HBV genotypes had no impact on antiviral drug resistance, except for some drug resistance substitutions bias. Optional initial therapy and subsequent rescue treatment should be based on knowledge of nucleos(t)ide analogue resistance.

Deep sequencing analysis of HBV genotype shift and correlation with antiviral efficiency during adefovir dipivoxil therapy.

BACKGROUND: Viral genotype shift in chronic hepatitis B (CHB) patients during antiviral therapy has been reported, but the underlying mechanism remains elusive. METHODS: 38 CHB patients treated with ADV for one year were selected for studying genotype shift by both deep sequencing and Sanger sequencing method. RESULTS: Sanger sequencing method found that 7.9% patients showed mixed genotype before ADV therapy. In contrast, all 38 patients showed mixed genotype before ADV treatment by deep sequencing. 95.5% mixed genotype rate was also obtained from additional 200 treatment-naïve CHB patients. Of the 13 patients with genotype shift, the fraction of the minor genotype in 5 patients (38%) increased gradually during the course of ADV treatment. Furthermore, responses to ADV and HBeAg seroconversion were associated with the high rate of genotype shift, suggesting drug and immune pressure may be key factors to induce genotype shift. Interestingly, patients with genotype C had a significantly higher rate of genotype shift than genotype B. In genotype shift group, ADV treatment induced a marked enhancement of genotype B ratio accompanied by a reduction of genotype C ratio, suggesting genotype C may be more sensitive to ADV than genotype B. Moreover, patients with dominant genotype C may have a better therapeutic effect. Finally, genotype shifts was correlated with clinical improvement in terms of ALT. CONCLUSIONS: Our findings provided a rational explanation for genotype shift among ADV-treated CHB patients. The genotype and genotype shift might be associated with antiviral efficiency.

A novel baseline hepatitis B virus sequencing-based strategy for predicting adefovir antiviral response.

Adefovir dipivoxil (ADV) is used as first-line monotherapy or rescue therapy in chronic hepatitis B (CHB) patients. In this study, we sought to identify nucleotide changes in the reverse transcriptase (RT) of hepatitis B virus (HBV) at baseline and explore their predictive value for ADV antiviral response. Ultra-deep pyrosequencing (UDPS) was utilized to determine HBV genetic variability within the RT region at baseline and during a 48-week ADV therapy. According to the viral load at the end of ADV treatment, all patients were classified into responders (HBV DNA level reduction of ⩾ 3 log 10 IU/mL) and suboptimal responders (HBV DNA level reduction of <3 log 10 IU/mL). Based on UDPS data at baseline, we identified 11 nucleotide substitutions whose combination frequency was significantly associated with the antiviral response among 36 CHB patients in the study group. However, the baseline distribution and frequency of rt181 and rt236 substitutions known to confer ADV resistance was a poor predictor for the antiviral response. Compared with baseline serum HBeAg, HBV-DNA and ALT levels, the baseline HBV sequence-based model showed higher predictive accuracy for ADV response. In an independent cohort of 31 validation patients with CHB, the sequence-based model provided greater predictive potency than the HBeAg/HBV-DNA/ALT and the HBeAg/HBV-DNA/ALT/sequence combinations. Taken together, we confirm the presence of ADV resistance variants in treatment-naïve patients and firstly unravel the predictive value of the baseline mutations in the HBV RT region for ADV antiviral response.

Comparison of a novel real-time PCR assay with sequence analysis, reverse hybridization, and multiplex PCR for hepatitis B virus type B and C genotyping.

We compared a novel real-time genotyping and quantitative PCR (GQ-PCR) assay, direct sequence analysis, reverse hybridization, and multiplex PCR for genotyping hepatitis B virus (HBV) in 127 HBV-infected patients. We found that GQ-PCR had the highest concordance with sequence analysis and the highest detection rate for mixed genotype detecting.

Simultaneous genotyping and quantification of hepatitis B virus for genotypes B and C by real-time PCR assay.

Hepatitis B virus (HBV) is an important cause of human chronic liver diseases and is a major public health problem. Viral load and HBV genotype play critical roles in determining clinical outcomes and response to antiviral treatment in hepatitis B patients. Viral genotype detection and quantification assays are currently in use with different levels of effectiveness. In this study, the performance of a real-time genotyping and quantitative PCR (GQ-PCR)-based assay was evaluated. Through the use of genotype-specific primers and probes, this assay provides simultaneous identification and quantification of genotypes B and C in a single reaction. Our GQ-PCR correctly identified all predefined genotypes B and C, and no cross-reaction between genotypes B and C were observed. The GQ-PCR identified more cases of HBV infections with mixed genotypes B and C than direct sequencing did. Samples from 127 HBV-infected Chinese patients were genotyped with GQ-PCR, revealing 56.7% HBV as genotype B, 13.4% as genotype C, and 29.8% as mixed genotypes B and C. This assay provides a reliable, efficient, and cost-effective means for quantification of the B and C genotypes of HBV in single or mixed infections. This assay is suitable for sequential monitoring of viral load levels and for determining the relationship between the genotype viral load and stage of disease in Asians.

In vivo characterization of Streptococcus pneumoniae genes involved in the pathogenesis of meningitis by differential fluorescence induction.

OBJECTIVE: To further understand the pathogenesis of pneumococcal meningitis, and provide some target candidates for the development of drugs. METHODS: This study was performed at the Department of Laboratory Medicine, Key Laboratory of Diagnostic Medicine (Ministry of Education), Chongqing Medical University, Chongqing, China from March 2006 to December 2007. A promoter-trap library of Streptococcus pneumoniae TIGR4, reported by green fluorescent protein was constructed, and used to infect BALB/c mice (n=15) intranasally, to set up a meningitis model. The control group (n=5) were inoculated with sterile phosphate buffered saline. The bacteria containing the promoter fusions induced only in meningitis brain tissue, not in vitro were screened by differential fluorescence induction. The obtained bacteria were prepared to re-infect the mice and re-screened, as above. The sorted bacteria were spread on trypticase soy agar with 5% sheep blood agar plates containing chloramphenicol (2.5 g/mL), and were used for DNA cloning, sequencing, and bioinformatics analysis. RESULTS: A total of 52 genes were obtained. Bioinformatics analysis revealed that these in vivo induced genes were involved in functions such as, adherence, energy metabolism, nutrient substance transport, transcription regulation, DNA metabolism, as well as, cell wall synthesis. In addition, there were some genes encoding for some hypothetical proteins with unknown, or putative functions. CONCLUSION: Pneumococcal genes involved in meningitis identified in this study are potential targets to understand the pathogenesis of pneumococcal meningitis.

Enhanced protection against pneumococcal infection elicited by immunization with the combination of PspA, PspC, and ClpP.

Immunization with a combination of several virulence-associated proteins is one of the strategies of developing effective protein-based vaccines to enhance the protection against Streptococcus pneumoniae. In this study, we evaluated the protection effects against pneumococcal infection caused by S. pneumoniae TIGR4 in BALB/c mice immunized with either single pneumococcal surface protein A (PspA), pneumococcal surface protein C (PspC), the caseinolytic protease (ClpP) or their combinations. The median survival times for mice immunized with single antigen or their combinations were significantly longer than that for mice treated with adjuvant alone. Mice treated with a combination of three antigens survived significantly longer than those that received either single or two antigens. The highest survival rate of the various groups of mice was observed with the combination of three antigens, this survival rate was significantly different from those for mice that received either single antigen or the combinations of two antigens except the mixture of ClpP and PspA. In the experiment of passive immunization with hyperimmune serums containing their specific polyclonal antibodies (anti-PspA serum, anti-PspC serum, anti-ClpP serum), the median survival times for mice immunized with hyperimmune serums containing specific polyclonal antibodies were significantly longer than that for control mice, the treatment of serum containing only one single polyclonal antibody could not provide higher survival rate than control serum. However, the survival rates for mice treated with the serums containing combined polyclonal antibodies were significantly higher than those for mice treated with either control serum or anti-PspA serum alone. Immunization with the combination of three hyperimmune serums also provided the best protection against S. pneumoniae. Compared to mice treated with serum containing single polyclonal antibody, the survival rate for mice treated with serums containing three polyclonal antibodies was significantly higher but was not different from those for mice treated with serums containing two polyclonal antibodies. Our findings provided evidence that a mixture of PspA, PspC, and ClpP or their polyclonal antibodies could enhance the protection against pneumococcal infection acting a synergetic effect.

Assessment of a method for detecting serum HBV DNA with HBV DNA probe labelled directly by alkaline phosphatase.

OBJECTIVE: To assess a sensitive and specific technique for detecting serum HBV DNA with an HBV DNA probe labelled directly by alkaline phosphatase (AlkPhos Direc probe). METHODS: AlkPhos Direc probe was prepared with purified HBV DNA labelled directly by alkaline phosphatase. The probe and chemiluminescent substrate CDP-star for AP were used in hybridization assay. HBV DNA was detected by autoradiography on a film. The results of 80 samples were compared between the chemiluminescent dot blot hybridization assay with the AlkPhos Direc probe and another assay with the digoxigenin-labelled HBV DNA probe. The correlation of seventy-sample results of fluorescent quantitative HBV DNA PCR assay and dot blot hybridization assay with the AlkPhos Direc probe was analysed. RESULTS: The sensitivity of the AlkPhos Direc probe was 10 pg at least. The coincidence of the AlkPhos Direc probe was 100% compared with that of the digoxigenin-labelled HBV DNA probe. A correlation coefficient of HBV DNA quantitative results between fluorescent quantitative HBV DNA PCR assay and dot blot hybridization assay with the AlkPhos Direc probe was 0.98 (P<0.01). CONCLUSIONS: The method detecting HBV DNA in serum with the HBV DNA AlkPhos Direc probe is sensitive and specific. The results of the two assays with the AlkPhos Direc probe or with the digoxigenin-labelled HBV DNA probe are completely coincident. The correlation of HBV DNA quantitative results between fluorescent QPCR assay and dot blot hybridization assay with the AlkPhos Direc probe is satisfactory.

[Establishment and evaluation of the method for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase].

OBJECTIVE: To establish a sensitive and specific technique for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase (AlkPhos Direc probe). METHODS: The probe that purified HBV DNA sequence was labeled directly by alkaline phosphatase and chemiluminescent substrate CDP-star for AP was used in the hybridization assay. HBV DNA was detected by autoradiography on the film. The test compared the chemiluminescen dot blot hybridization assay for 80 samples with digoxigenin-labeled HBV DNA probe detective method. The correlation of 70 samples test results between fluorescent quantitative HBV DNA PCR method and dot blot hybridization assay by AlkPhos Direc probe was analysed. RESULTS: The sensitivity of the probe labeled directly by alkaline phosphatase was 10pg at least. The coincidence was 100% compared with digoxigenin-labeled HBV DNA probe detection. A correlation coefficient of HBV DNA quantitative results between fluorescent quantitative HBV DNA PCR (QPCR) method and dot blot hybridization assay by AlkPhos Direc probe was 0.98 (P<0.01). CONCLUSIONS: The method detecting HBV DNA in serum by HBV DNA AlkPhos Direc probe is sensitive and specific. The results between two methods with AlkPhos Direc and digoxigenin-labeled HBV DNA probe are coincident completely. The correlation of HBV DNA quantitative results between fluorescent QPCR method and dot blot hybridization assay by AlkPhos Direc probe is satisfactory.