Seroprevalence and mutations in the major hydrophilic region of hepatitis B virus among pregnant women in Huzhou, China.

BACKGROUND: Mother-to-child transmission (MTCT) is the main cause of hepatitis B virus (HBV) infections in China. However, there is a paucity of information on seroprevalence and mutations in HBV surface genes among pregnant women in Huzhou, China. METHODS: In this retrospective cross-sectional study, serum markers of 31 681 pregnant women were collected and analysed. The surface genes were amplified and directly sequenced. Mutations in the major hydrophilic region (MHR) were analysed in 171 randomly selected subjects. RESULTS: The seroprevalence of HBV infection was 3.32% (1053/31 681). The predominant HBV genotypes were B (57.4%) and C (42.6%). Pregnant women ≥30 y of age exhibited a higher hepatitis B surface antigen-positive rate than those <30 y of age. MHR mutations were found in 42.6% (72/169) of the subjects, several of which were escape mutations. The mutational frequencies in the a-determinant and first loop (AA124-137) were higher in genotype C than genotype B. Pregnant women with MHR mutations showed increased alanine transaminase, aspartate transaminase and gamma-glutamyl transpeptidase levels and decreased HBV loads. CONCLUSIONS: The HBV seroprevalence among pregnant women in Huzhou was intermediate. MHR mutations occur and the risk of MTCT still persists. Therefore, early screening, intervention and care for HBV-infected pregnant women should be strengthened to minimize or prevent MTCT of HBV.

Prevalence and genotype distribution of human papillomavirus infection in Huzhou City, eastern China, 2018-2019.

BACKGROUND: Human papillomavirus (HPV) infection is involved in cervical cancer development, and hence understanding its prevalence and genotype distribution is important. However, there are few reports on the prevalence and genotype distribution of HPV in the city of Huzhou in China. METHODS: In this retrospective cross-sectional study, 11,506 women who visited Huzhou Maternity & Child Health Care Hospital between January 2018 and October 2019 were enrolled. The results of HPV genotyping and cytology tests were analyzed. RESULTS: The overall prevalence of HPV infection was 15.5%. The rate of high-risk (HR) HPV infection (13.5%) was higher than that of single low-risk (LR) HPV infection (2.0%) (p<0.05). The five most common HPV genotypes were HPV52 (3.3%), 16 (1.9%), 58 (1.7%), 53 (1.5%), and 81 (1.2%). The infection rate of HPV peaked in women aged 16-24 and women aged ≥55. The infection rate of HPV58 or HPV81 appeared as a single peak in women aged ≥55. The rates of HR-HPV and LR-HPV infection were higher in subjects with abnormal cytology (p<0.05). CONCLUSIONS: HPV infection is high in Huzhou, and HPV53 and HPV81 are the prevalent genotypes. HPV infection rate is associated with age and cytology. Regional HPV surveillance is essential to optimize current HPV prevention and vaccine development.

Characterization of mutations in the reverse transcriptase region of hepatitis B virus in treated and untreated chronic hepatitis B patients.

BACKGROUND: The reverse transcriptase (RT) region of the hepatitis B virus (HBV) is the target of antiviral treatment. However, the discrepancy in RT mutations between nucleos(t)ide analogue (NA)-treated and -untreated chronic hepatitis B (CHB) patients is un clear. METHODS: Serum samples were collected from 119 NA-treated and 135 NA-untreated patients. The sampling time was decided by the clinician. Full-length HBV RT regions were amplified using nest polymerase chain reaction. The mutations within the RT region were analysed by direct sequencing. RESULTS: The incidence of RT mutations in treated patients was higher than that in untreated patients (p<0.05). The classic drug-resistant mutations were detected in 44.5% (53/119) of treated patients, which was significantly higher than in untreated patients (6.7% [9/135]) (p<0.05). The non-classical mutations showed their complexity and diversity in both patient groups. Multiple mutations (three or more) were more frequent in treated patients than in untreated patients (p<0.05). Several novel mutations might be related to NA resistance. CONCLUSIONS: The selection pressures of NAs accelerated the development of RT mutations, especially within the functional domain. Mutations in the RT region occurred not only at classical sites, but also at other non-classical sites, which might be related to drug resistance and/or viral replication. The biological function and fitness of HBV isolates harbouring these novel mutations need further in vitro and in vivo verification experiments.

Prevalence of Potential Resistance Related Variants Among Chinese Chronic Hepatitis B Patients Not Receiving Nucleos(T)ide Analogues.

BACKGROUND AND AIMS: Potential drug resistance (DR) related variants in the hepatitis B virus (HBV) reverse transcriptase (RT) region may be associated with the effectiveness of antiviral drugs and disease progression. The aim of this study was to investigate the prevalence and clinical characteristics of potential DR-related variants in Chinese CHB patients not receiving nucleos(t)ide analogues (NAs). PATIENTS AND METHODS: Two hundred and six untreated CHB patients from Huzhou Central Hospital in eastern China were recruited for this study. The serum DNA was extracted and the HBV RT region was amplified using nest polymerase chain reaction (nest-PCR). The 42 potential DR-related variants were analyzed by direct sequencing. RESULTS: Among these CHB patients, HBV genotype B and genotype C were identified in 121 (58.7%) and 85 (41.3%) patients, respectively. Potential DR-related variants were detected in 42.7% (88/206) of patients. Primary and secondary DR variants were found in 7.3% (15/206) of patients, including rtL80I/V, rtI169T, rtV173L rtL180M, rtA181T/V, rtM204I/V, and rtN236T. The variants at rt53, rt82, rt221, rt233, rt237, and rt256 were specific for genotype B, and those at rt38, rt84, rt126, rt139, rt153, rt191, rt214, rt238, and rt242 were specific for genotype C. Moreover, the variation frequency in the A-B interdomain (3.96%) was significantly higher than that in the functional domains (1.17%) and non-A-B interdomains (1.11%). Multivariate logistic regression analysis showed that lower HBV-DNA load (<106 IU/mL) was an independent factor associated with potential DR-related variants in untreated CHB patients (P <0.05). CONCLUSION: Potential DR-related variants were frequent and complex in untreated Chinese CHB patients. Furthermore, the variants may contribute to decreased serum HBV-DNA loads. However, the effects of potential DR-related variants on the antiviral therapy and liver disease progression require further study.

Monitoring of genotypic resistance profile in chronic hepatitis B patients receiving nucleos(t)ide analogues in Huzhou, China.

INTRODUCTION: Antiviral drug-resistance patterns of hepatitis B virus (HBV) mutants are complex and currently partly understood. The aim of this study was to monitor the genotypic resistance profile in patients with chronic hepatitis B (CHB) receiving nucleos(t)ide analogues (NAs) in Huzhou, eastern China. METHODOLOGY: Serum samples of 139 CHB patients undergoing NA treatment were obtained from Huzhou Central Hospital. The full-length HBV reverse transcriptase regions were amplified and sequenced. The NA resistance mutation positions, including rtL80, rtI169, rtV173, rtL180, rtA181, rtT184, rtA194, rtS202, rtM204, rtI233, rtN236, and rtM250 were analyzed. RESULTS: Genotypic resistance mutations were detected in 41.72% (58/139) of patients with CHB. Drug resistance mutations were detected at positions rt80, rt173, rt180, rt181, rt194, rt202, rt204, rt236, and rt250, but were not observed at positions rt169, rt184, and rt233. The prevalence of mutations at rtM204 was 54.44% in 90 patients who were treated with lamivudine (LAM) or telbivudine (LDT). RtN236 mutations were detected in 7.14% (2/28) of the patients receiving adefovir (ADV) therapy. Additionally, rtA181 mutations were observed in 4 patients with LAM, ADV, and LDT-based therapy, but not in those patients treated with entecavir (ETV). Among patients who harbored rtM204 combination mutations, rtM204I and rtM204V were significantly associated with rtL80I/V and rtL180M, respectively. CONCLUSIONS: The mutation patterns of NA-resistant HBV are complicated in CHB patients in the current clinical setting. Thus, it is necessary to persistently monitor the resistance mutations of HBV for optimizing antiviral therapy strategy and for preventing an outbreak of clinical resistance.

[Variability of Reverse Transcriptase Gene and S Gene in Lamivudine-treated Chronic Hepatitis B Patients].

We wished to undertake molecular characterization of the reverse transcriptase (RT) gene and overlapping surface (S) gene in lamivudine-treated patients with chronic infection with the hepatitis B virus (HBV). Sequencing analyses of the HBV RT/S gene of isolates from 25 chronic hepatitis B (CHB) patients with the YMDD mutation and 30 treatment-naïve CHB patients were undertaken. In patients with the YMDD mutation, rtM2041 was the major type of mutation (20/25, 80%). rtL80I was present in most of the patients with rtM204I (14/20, 70%). rtL180M coexisted with rtM204V (5/5, 100%). Patients with the YMDD mutation had a significantly higher prevalence of mutation of the RT gene than treatment-naïve CHB patients (P < 0.05). Classical primary resistance and secondary/compensatory mutations were detected at only five sites (rtL80, rtV173, rtL180, rtM204, rtM250) in CHB patients with the YMDD mutation. The frequency of nucleos(t)ide analog resistance (NAr) mutation within the RT gene in patients with the YMDD mutation was significantly higher than that in treatment-naïve patients (P < 0.05). Amino-acid mutations within the RT gene were also associated with other types of NAr in patients with the YMDD mutation. The rate of amino-acid variants within the S gene region was significantly higher in patients with the YMDD mutation than that in treatment-naïve patients (P < 0.05). sM133L and sG145R variants were also present in patients with the YMDD mutation. These observations suggest that CHB patients with the YMDD mutation also have NAr mutations related to other NA drugs, which might lead to cross-resistance in CHB patients. Variants present in the S gene region could cause changes in the antigenicity of HBsAg, which could result in a false-negative diagnosis of HBsAg and immune in escape of the HBV.

Development of hybrid-type modified chitosan derivative nanoparticles for the intracellular delivery of midkine-siRNA in hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most of the patients with HCC lose the surgical opportunity at the time of diagnosis. Some novel therapeutic modalities, like gene therapy, are promising for the treatment of HCC. However, the success of gene therapy depends on two aspects: efficient gene materials and gene delivery vectors. The present study was to develop new chitosan-based nanoparticles for a midkine-siRNA (anti-HCC gene drug) delivery. METHODS: The novel gene delivery vector (MixNCH) was synthesized by hybrid-type modification of chitosan with 2-chloroethylamine hydrochloride and N, N-dimethyl-2-chloroethylamine hydrochloride. The chemical structure of MixNCH was characterized by FT-IR and 1HNMR. The cytotoxicity of MixNCH was determined by MTS assay. The gene condensation ability and size, zeta potential and morphology of MixNCH/MK-siRNA nanoparticles were measured. The in vitro transfection and gene knockdown efficiency of midkine by MixNCH/MK-siRNA nanoparticles was detected by qRT-PCR and Western blotting. Gene knockdown effect at the molecule level on the proliferation of HepG2 in vitro was determined by MTS assay. RESULTS: MixNCH was successfully acquired by aminoalkylation modification of chitosan. The MixNCH could condense MK-siRNA well above the weight ratio of 3. The average size of MixNCH/MK-siRNA nanoparticles was 100-200 nm, and the surface charge was about +5 mV. Morphologically, MixNCH/MK-siRNA nanoparticles were in regular spherical shape with no aggregation. Regarding to the in vitro transfection of nanoparticles, the MixNCH/MK-siRNA nanoparticles reduced MK mRNA level to 14.03%+/-4.03%, which were comparable to Biotrans (8.94%+/-3.77%). MixNCH/MK-siRNA effectively inhibited the proliferation of HepG2 in vitro. CONCLUSION: MixNCH/MK-siRNA nanoparticles could be effective for the treatment of hepatocellular carcinoma.

[Development of EV71, CA16 and other enterovirus vrial real-time qualitative PCR diagnostic kit].

OBJECTIVE: A novel multiplex real-time RT-PCR kit was developed to detect EV71, CoxA16 and other human enteroviruses simultaneously with an internal amplification control to avoids false negatives, which used for hand, foot and mouth disease in the clinical diagnosis and epidemiological surveillance. METHODS: Design specific primers and probes of EV71, CA16, other intestinal virus and internal amplification control, improve the extraction method of virus nucleic acid. Optimization the detection system of real-time quantitative PCR. Research the products of the accuracy, stability, precision, amplification efficiency and detection of linear range. RESULTS: The primers and probes had high spicificity. The Viral RNA extraction effect of this Kit is as same as that of QIAamp Viral RNA mini Kit (QIAGEN company), but less reagent cost. The optimal concentrations of primers and probes are 0.2 micromol/L for all the upstream and downstream primers, 0.06 micromol/L for probes of other human enteroviruse, 0.08 micromol/L for probes of EV71 and CA16 respectively. The kit has good stability, accuracy and precision. The amplification efficiencies of EV71, CoxA16 and other human enteroviruses are 106% ,101% and 105% and the detection of linear range is from 10(9) copies/microl-10(2) copies/microl. CONCLUSION: The novel multiplex real-time RT-PCR kit for detecting EV71, CoxA16 and other human enteroviruses simultaneously with an internal amplification control has good stability, accuracy, precision and amplification efficiencies. So it has great value in clinical application.

Evaluation of a new tuberculosis-related interferon gamma release assay for tuberculosis infection diagnosis in Huzhou, eastern China.

OBJECTIVE: To compare the performance of a new tuberculosis-related interferon gamma release assay (TB-IGRA) with that of QuantiFERON-TB Gold In-Tube (QFT-GIT) for TB infection diagnosis in China. MATERIALS AND METHODS: A total of 458 active TB patients and 378 healthy individuals were enrolled. Among the 458 active TB patients, 395 had pulmonary TB and 63 had extra-pulmonary TB. The blood samples were collected from the active TB patients and health controls; then TB-IGRA and QFT-GIT were used to detect interferon gamma (IFN-γ) levels. RESULTS: The sensitivity, specificity, positive predictive value, and negative predictive value in TB infection diagnosis for active TB by the TB-IGRA were 83.4%, 94.2%, 94.5%, and 82.4%, respectively. For QFT-GIT, the sensitivity, specificity, positive predictive value, and negative predictive value in TB infection diagnosis for active TB were 81.4%, 97.1%, 97.1%, and 81.2%, respectively. CONCLUSIONS: TB-IGRA had a high sensitivity and specificity for TB infection; it could be comparable with the QFT-GIT assay. It might be a powerful assisting tool for TB infection diagnosis in the Chinese clinical setting.

Hospital-based prevalence of high-risk cervical HPV types infecting the general population and female sex workers in Huzhou, China.

OBJECTIVE: To evaluate the prevalence of high-risk HPV types among female sex workers and the general population in Huzhou, China. METHODS: Cervical samples from the general population (n=292) and female sex workers (n=288) in Huzhou, China, were collected between April, 2008, and October, 2009. Demographic, behavioral, and clinical data were obtained by interview. Genotyping of the HPV L1 gene was done via a PGMY09/PGMY11 PCR-based assay, and the cervical samples were subjected to cytology analysis. RESULTS: The prevalence of HPV was higher among female sex workers (66.7%) than among the general population (19.2%). Among female sex workers, HPV-16 (28.8%) was the most prevalent, followed by HPV-58 (24.0%) and HPV-52 (20.8%). The percentage of cervical abnormalities was higher among female sex workers (20.8%) than among the general population (4.8%). Multivariate analysis showed that education level and condom use during coitus were significantly associated with HPV infection (P<0.05). HPV-16, HPV-58 and HPV-52 were the dominant types, and were significantly associated with abnormal cervical cytology (P<0.05). CONCLUSION: Female sex workers in Huzhou, China, were found to have a greater probability of being infected with high-risk HPV, and novel vaccines against HPV-58 and HPV-52 should be developed. Using condoms could reduce the risk of infection.

Midkine mRNA level in peripheral blood mononuclear cells is a novel biomarker for primary non-small cell lung cancer: a prospective study.

BACKGROUND AND OBJECTIVES: Midkine (MK) mRNA was highly expressed in various human cancer tissues and cells. The present study aimed to investigate whether MK mRNA level in peripheral blood mononuclear cells (PBMC) could serve as a diagnostic biomarker for patients having primary non-small cell lung cancer (NSCLC). METHODS: MK mRNA level in PBMC from 87 patients with primary NSCLC, 35 patients with lung benign lesion (LEL), and 30 healthy volunteers was analyzed by real-time quantitative RT-PCR. The levels of serum carcinoembryonic antigen, carbohydrate antigen 125 (CA125), and neuron-specific enolase were detected by chemiluminescent microparticle enzyme immunoassay. RESULTS: PBMC MK mRNA level was significantly higher in patients with primary NSCLC than that in other groups (P < 0.001), while there was no significant difference between LEL patients and healthy volunteers (P > 0.05). Higher MK mRNA level was correlated with clinical stages (P = 0.026), differentiation (P = 0.025), and lymph node metastasis (P = 0.022) of NSCLC. Using a cutoff of 0.0063, the sensitivity and specificity of MK mRNA levels to differentiate between patients with NSCLC and patients with LEL were 57.47 and 93.33 %,and it were 56.32 and 93.33 % for patients with NSCLC and healthy volunteers, respectively. Furthermore, multivariate analysis indicated that PBMC MK mRNA level above the cutoff value presented a chance of 11-fold higher for NSCLC occurrence. CONCLUSIONS: MK mRNA level in PBMC may be a potential non-invasive molecular marker for the diagnosis of primary NSCLC.

Mutations in hepatitis B virus DNA from patients with coexisting HBsAg and anti-HBs.

BACKGROUND: The serological markers with coexistence of hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti-HBs) of hepatitis B virus (HBV) infection were rare pattern. The virological significance, immune response and clinical outcome of these patients remain largely unknown. OBJECTIVES: This research explores the relationship between this serological profile and HBV genome variants. STUDY DESIGN: We studied 35 patients both carrying HBsAg and anti-HBs (group I), and 70 patients with HBsAg positive but anti-HBs negative (group II, served as control). The HBV genome sequences were obtained by direct sequencing of polymerase chain reaction (PCR) products. RESULTS: The amino acid (aa) variation within major hydrophilic region (MHR), especially in the first loop (aa124-137) of "a" determinant in group I is significantly higher than those in group II. The aa variation of cytotoxic lymphocyte (CTL) epitope in HBsAg (aa87-aa95) in group I is also significantly higher than that in group II. Interestingly, the basal core promoter (BCP) double mutations (A1762T/G1764A) in group I is significantly higher than those in group II as well. CONCLUSIONS: In patients with HBV infection, the coexistence of HBsAg and anti-HBs is associated with an increased aa variability in several key areas of HBV genome. The molecular characteristic of HBV in HBsAg and anti-HBs positive patients is distinct and worth further studies.

Preparation and preliminary characterization of rabbit monoclonal antibodies against human midkine.

We prepared rabbit monoclonal antibodies that target human midkine (MK). The MK gene was amplified by PCR from the plasmid pEGFP-MK and subcloned into the prokaryotic expression vector pGEX-1λT to generate an N-terminally glutathione S-transferase (GST)-tagged fusion protein construct. Expression of the GST-MK fusion protein was achieved by IPTG induction in Escherichia coli cells. The expressed protein was purified using the GST system. After verifying purification, the fusion protein was used to immunize rabbits to prepare monoclonal antibodies against human MK by the rabbit hybridoma technique. The hybridomas generated were screened by an enzyme-link immunoassay (ELISA) for specificity, which was further characterized by Western blotting and ELISA. SDS-PAGE analysis showed that the purified protein corresponds to the calculated molecular weight. The GST-MK fusion protein was prepared. At least one hybridoma cell line secreting anti-MK MAb was obtained. Western blotting analysis confirmed the identity of the MAb. The titer of the MAbs measured by an indirect ELISA was 1:64,000. The affinity constant, which was measured by a non-competitive ELISA, was found to be 3.0 × 10(9) M(-1). Western blotting and immunohistochemistry analysis showed that the produced MAbs bind to the MK protein in cancerous tissues. The GST-MK fusion protein was successfully expressed and purified. The MAbs against MK were subsequently prepared, which should further aid research and the application of MK MAbs in clinical settings.

[Expression characteristics of nuclear factor kappa B in hepatocellular carcinoma tissues].

OBJECTIVE: To investigate the expression characteristics of nuclear factor kappa B (NF-kB) in hepatocellular carcinoma (HCC) tissues and its correlation with tumor necrosis factor alpha (TNF alpha) and clinical pathological features. METHODS: Thirty liver specimens from HCC patients were collected by self-control method. The localization and expression of NF-kappaB in HCC and their surrounding tissues were detected by immunohistochemistry and enzyme linked immunosorbent assay (ELISA), respectively. And the levels of TNF alpha in these tissues were analyzed by ELISA. RESULTS: The expressed NF-kappaB was localized in nucleus and cytoplasm in HCC, whereas only in cytoplasm in the surrounding tissues. The expression level and density of NF-kappaB in HCC tissues were obviously higher than those in the surrounding tissues (P < 0.01), which was positively correlated with increased TNF alpha in HCC tissues (r = 0.964, P < 0.01). No positive correlation was found between NF-kappaB expression and histological differentiation grade, number of tumor, size of tumor, and HBsAg positive (P > 0.05). CONCLUSION: The expression and localization of NF-kappaB in HCC tissues are obviously different from those in the surrounding normal liver tissues, and the level of nucleoprotein NF-kappaB in HCC tissues is correlated with expressed TNF alpha, suggesting that TNF alpha can activate NF-kB, the activated NF-kB then translocates to the nucleus and plays important role in the carcinogenesis of HCC.

Antisense oligonucleotide targeting p53 increased apoptosis of MCF-7 cells induced by ionizing radiation.

AIM: To investigate the effect of antisense compounds (AS) targeting human p53 mRNA on radiosensitivity of MCF-7 cells. METHODS: Western blotting and RTPCR were used to analyze the protein content and mRNA level. Additionally, cell proliferation, cell cycle and cell apoptosis were all analyzed in irradiated or sham-irradiated cells. RESULTS: Among the five antisense compounds (AS), AS3 was identified to efficiently inhibit p53 mRNA level and protein content. Interestingly, AS3 transfer has little effect on cell proliferation in DU-145 cells (mutant p53) after ionizing radiation (IR). In contrast, a marked increase of cell apoptosis and growth inhibition were observed in MCF-7 cells (wild-type p53), suggesting that AS3 can increase radiosensitivity of MCF-7 cells. Additionally, it was also observed that the transfection of AS3 decreased the fraction of G1 phase cells, and increased the proportion of S phase cells compared to untreated cells 24 h after IR in MCF-7 cell lines. CONCLUSION: AS3 transfection increases MCF-7 cell apoptosis induced by 5 Gy-radiation, and this mechanism may be closely associated with abrogation of G1 phase arrest.