Primary resistance to integrase inhibitors in Shenzhen.

OBJECTIVES: In recent years, integrase strand transfer inhibitor (INSTI)-containing regimens have been increasingly adopted in treatment for HIV/AIDS and promoted as non-occupational post-exposure prophylaxis in China. This study aims to describe the prevalence of resistance to integrase and drug resistance mutations (DRMs) among ART-naive patients in Shenzhen, China. METHODS: Serum samples and demographic information were collected from newly reported ART-naive patients in Shenzhen in 2020. The study sequenced the coding sequence of the HIV-1 integrase gene and determined the DRMs.​. RESULTS: In this study, 1682 newly reported cases were included and 1071 of them were successfully sequenced finally. The prevalence of primary drug resistance was 1.77%, with 19 samples showing varying degrees of resistance to INSTIs. The study detected six major DRMs in 16 individuals and eight accessory DRMs in 24 individuals. The prevalence of transmitted drug resistance (TDR) mutations was 1.21%, with five transmitted mutations detected in 13 individuals. The prevalence of drug resistance to raltegravir and elvitegravir was statistically higher than to bictegravir, cabotegravir and dolutegravir. CONCLUSIONS: The prevalence of INSTI resistance in Shenzhen in 2020 was relatively high. ​Continued surveillance for resistance to INSTIs is recommended and treatment regimens should be adopted based on the pattern of resistance to INSTIs. ​Dolutegravir or bictegravir is first recommended when considering INSTIs as treatment regimens.

Antibody response to the third dose of inactivated COVID-19 vaccine in people living with HIV (PLWH): A longitudinal cohort.

The immunogenicity induced by the third dose of inactivated coronavirus disease 2019 (COVID-19) vaccines in people living with HIV (PLWH) is unclear, and relevant literature is extremely scarce. It is important to add evidence on the humoral immune response induced by the third dose of inactivated COVID-19 vaccine in PLWH. We collected peripheral venous blood for spike receptor binding domain-protein specific immunoglobulin G (S-RBD-IgG) antibody tests at 28 days after the second dose (T1 ), 180 days after the second dose (T2 ) and 35 days after the third dose (T3 ) of inactivated COVID-19 vaccines in PLWH. The differences in S-RBD-IgG antibody levels and specific seroprevalence among T1 , T2 , and T3 time periods were analyzed, and the effects of age, vaccine brand, and CD4+ T cell count on the levels and specific seroprevalence of S-RBD-IgG antibody induced by the third dose in PLWH were examined. The third dose of inactivated COVID-19 vaccines induced strong S-RBD-IgG antibody responses in PLWH. The levels and specific seroprevalence of S-RBD-IgG antibody were significantly higher than those at 28 and 180 days after the second dose and were not affected by vaccine brand or CD4+ T cell count. Younger PLWH produced higher levels of S-RBD-IgG antibody. The third dose of inactivated COVID-19 vaccine showed good immunogenicity in PLWH. It is necessary to popularize the third dose in the PLWH population, especially PLWH who do not respond to two doses of inactivated COVID-19 vaccines. Meanwhile, the durability of the protection provided by the third dose in PLWH must be continuously monitored.

Increasing trends of overweight and obesity in treatment-naive people living with HIV in Shenzhen from 2014 to 2020: an emerging health concern.

Background: With the early initiation of antiretroviral therapy (ART) in China, the demographics of treatment-naïve people living with HIV (PLWH) are moving closer to those of the general population, which is characterized by a gradual increase in metabolic indicators. However, the epidemic trends of overweight and obesity over the past decade in treatment-naïve PLWH ready to initiate ART have not yet been investigated. Methods: A cross-sectional study was conducted, including 12,135 consecutive treatment-naïve PLWH ready to initiate ART in Shenzhen, using data retrieved from the China National Free Antiretroviral Treatment Program database from 2014 to 2020. The chi-square test was used to examine the trends of overweight and obesity between age groups, and multivariate logistic regression was used to identify the association of overweight and obesity with hyperglycemia and dyslipidemia. Results: During the 7-year study period, 12,135 treatment-naïve PLWH ready to initiate ART were included, among whom 1,837 (15.1%) were overweight and 388 (3.2%) were obese. The prevalence of overweight rose from 11.4 to 17.3% (Z = -4.58, P for trend <0.01) and that of obesity from 2.0% to 4.2% (Z = -6.45, P for trend <0.01) from 2014 to 2020. The annual prevalence of overweight was the highest in the age group of participants >35 years compared to prevalence in other age groups during the period 2014-2020. Compared with those who were not overweight or obese, PLWH who were overweight or obese were more likely to have hyperglycemia (aOR 1.84, 95% CI: 1.37-2.49 for overweight; aOR 2.68, 95% CI: 1.62-4.44 for obesity), higher ALT level (aOR 2.70, 95% CI: 2.33-3.13 for overweight; aOR 3.85, 95% CI: 2.93-5.05 for obesity), higher TG levels (aOR 1.89, 95% CI 1.63-2.19 for overweight; aOR 2.56, 95% CI 1.97-3.32 for obesity), and lower HDL levels (aOR 1.67, 95% CI 1.44-1.95 for overweight; aOR 2.06, 95% CI 1.54-2.77 for obesity). Conclusion: The prevalence of overweight and obesity in treatment-naive PLWH increased steadily from 2014 to 2020 in Shenzhen. Overweight and obese in treatment-naive PLWH ready to initiate ART were associated with dyslipidemia and hyperglycemia. Public health authorities should take proactive steps to address these issues by implementing targeted screening, intervention programs including lifestyle modifications, and integrated healthcare services.

Failure to seroconvert after three doses of inactivated COVID-19 vaccines in a patient co-infected with HBV and HIV: A case report.

In the global context of the COVID-19 pandemic, the overall benefits of getting any COVID-19 vaccine approved by the World Health Organization for emergency use outweigh the potential risks, even in people with weakened immune systems, including people living with HIV (PLWH). At present, there are no reports of HIV/hepatitis B virus (HBV) co-infected patients receiving a booster dose of the inactivated COVID-19 vaccine. Here, we describe a patient with HIV/HBV co-infection who did not seroconvert to three doses of the inactivated COVID-19 vaccine.

IgG Antibody Responses and Immune Persistence of Two Doses of BBIBP-CorV Vaccine or CoronaVac Vaccine in People Living with HIV (PLWH) in Shenzhen, China.

The purpose of this study was to preliminarily evaluate the immunogenicity and immune persistence of inactivated SARS-CoV-2 vaccines in PLWH in the real world. We collected blood samples from 132 PLWH aged 18−59 years who were vaccinated with two doses of BBIBP-CorV vaccine (Sinopharm) or CoronaVac vaccine (SinoVac) at 28 ± 7 days and 180 ± 20 days the after second dose, to detect the level of Spike receptor binding domain-protein specific IgG (S-RBD-IgG) by using chemiluminescence. We found that the BBIBP-CorV vaccine or the CoronaVac vaccine induced lower S-RBD-IgG antibody seropositivity rates and levels in PLWH than in healthy controls (HCs). The BBIBP-CorV vaccine or the CoronaVac vaccine induced lower humoral immune responses in PLWH, having lower CD4+T cell counts (<350 cells/µL) compared to PLWH, and having higher CD4+T cell counts (≥350 cells/µL) after a second dose of vaccination. The BBIBP-CorV vaccine or the CoronaVac vaccine induced lower S-RBD-IgG antibody levels in PLWH, having CD4+T cell counts ≥350 cells/µL compared to HCs. No negative effects were observed in terms of the CD4+T cell counts and HIV RNA viral load (VL) of PLWH after vaccination. Ninety-nine PLWH and eighty-three HCs completed a second blood collection for testing; we found a statistically significant decrease in the humoral immune response both in PLWH and HCs from 28 days to 180 days after a second dose of BBIBP-CorV vaccine or CoronaVac vaccine. The S-RBD-IgG antibody induced by the BBIBP-CorV vaccine or the CoronaVac vaccine declined faster in the PLWH population than in the healthy population, and two doses of the BBIBP-CorV vaccine or the CoronaVac vaccine may not be enough to provide PLWH with persistent immunity against SARS-CoV-2. It is necessary for PLWH to be prioritized for a third dose over the healthy population, but the immunogenicity of the third dose of the homologous or heterologous vaccine requires further study.

Dolutegravir Plus Lamivudine Dual-Drug Regimen in Treatment-Naive HIV-1-Infected Patients With High-Level Viral Load: Preliminary Data From the Real World.

BACKGROUND: Some inpatients with HIV-RNA ≥500,000 copies/mL in China need to use 2-drug regimen for some reasons, although limited data are available for dolutegravir plus lamivudine (3TC) in those patients with ultra-high viral loads. METHODS: We conducted a single-center retrospective-prospective study in China and enrolled 42 ART-naive HIV-infected inpatients who use a once-daily 2-drug regimen because of various reasons (drug interaction, renal impairment, age, and other related comorbidities).They were divided into 2 groups, low viral load group (baseline viral load <500,000 copies/mL, n = 20) and high viral load group (baseline viral load ≥500,000 copies/mL, n = 22). All patients were followed up for 48 weeks. RESULTS: The median of baseline viral load was 5.74 log10 copies/mL and CD4+ T-cell count was 59 cells/µL. At week 48, there was no significant difference (P = 0.598) in proportions of participants with HIV-1 RNA <50 copies/mL [90%, 95% confidence interval (CI) (75.6% to 104.4%) in low viral load groups vs 95.5%, 95% CI (86.0% to 104.9%) in high viral load groups]. No differences were found in mean increase of CD4+ T-cell count from baseline between 2 groups (218 ± 122 vs 265 ± 127 cells/µL, P = 0.245). There is no grade 3 or higher treatment-related adverse events and none discontinued treatment because of adverse events. CONCLUSIONS: The results of our study in real world support dolutegravir + 3TC dual regimen as a promising therapy option for treatment-naive HIV-infected patient with baseline viral load ≥500,000 copies/mL.

Higher Risk of Dyslipidemia With Coformulated Elvitegravir, Cobicistat, Emtricitabine, and Tenofovir Alafenamide than Efavirenz, Lamivudine, and Tenofovir Disoproxil Fumarate Among Antiretroviral-Naive People Living With HIV in China.

BACKGROUND: We aimed to examine the evolution of blood lipids and compare the risk of dyslipidemia between antiretroviral-naive people living with HIV who received tenofovir disoproxil fumarate (TDF), lamivudine (3TC), and efavirenz (EFV) (TDF + 3TC + EFV) and those who received coformulated elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide (E/C/F/TAF). METHODS: We retrospectively reviewed the medical records of 2343 antiretroviral-naive people living with HIV who initiated TDF + 3TC + EFV or E/C/F/TAF. A propensity score matching method was used to compare longitudinal changes of blood lipids between the 2 groups. RESULTS: By using 1:3 matching ratio, we included 253 and 91 matched patients in TDF + 3TC + EFV group and E/C/F/TAF group, respectively. The levels of total cholesterol, triglyceride, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol were higher in E/C/F/TAF group than those in TDF + 3TC + EFV group at 3, 6, 9, and 12 months (Wilcoxon test, all Ps < 0.05), except for high-density lipoprotein cholesterol at 9 and 12 months. The cumulative rates of hypercholesterolemia, hypertriglyceridemia, and high LDL-C in PLWH with normal lipid levels in E/C/F/TAF group were higher than those in TDF + 3TC + EFV group (hypercholesterolemia, 59.7% vs 21.5%, P < 0.001; hypertriglyceridemia, 69.5% vs 46.3%, P < 00.001; and high LDL-C, 41.5% vs 14.2%, P < 0.001). Multivariate analysis showed treatment with E/C/F/TAF was associated with a significantly higher risk of hypercholesterolemia [adjusted hazard ratio (HR), 4.12; 95% confidence interval (CI): 2.65 to 6.41], hypertriglyceridemia (adjusted HR, 1.69; 95% CI: 1.18 to 2.43), and high LDL-C (adjusted HR, 4.60; 95% CI: 2.66 to 7.97). CONCLUSIONS: We concluded that treatment with E/C/F/TAF resulted in higher risks of dyslipidemia compared with TDF + 3TC + EFV.

High population-attributable fractions of traditional risk factors for non-AIDS-defining diseases among people living with HIV in China: a cohort study.

Morbidity and mortality of non-AIDS-defining diseases (NADs) have become the increasing burden of people living with HIV (PLWH) with long-term antiretroviral therapy (ART). We aimed to quantify the contribution of modifiable risk factors to NADs. We included PLWHs starting ART at the Third People's Hospital of Shenzhen (China) from Jan 1, 2010 to Dec 31, 2017. We defined NAD outcomes of interest as cardiovascular disease (CVD), end-stage liver disease (ESLD), advanced renal disease (ARD), and non-AIDS-defining cancers (NADCs). We estimated incidence of outcomes and population-attributable fractions (PAFs) of modifiable traditional and HIV-related risk factors for each outcome. Overall, 8,301 participants (median age at study entry, 31 years) contributed 33,146 person-years of follow-up (PYFU). Incidence of CVD (362/100,000 PYFU) was the highest among outcomes, followed by that of ARD (270/100,000 PYFU), ESLD (213/100,000 PYFU), and NADC (152/100,000 PYFU). Totally, 34.14% of CVD was attributable to smoking, 7.98% to hypertension, and 6.44% to diabetes. For ESLD, 24.57% and 25.04% of it could be avoided if chronic hepatitis B and C virus infection, respectively, did not present. The leading PAFs for ARD were declined estimated glomerular filtration rate (eGFR) (39.68%) and low CD4 count (39.61%), followed by diabetes (10.19%). PAFs of hypertension, diabetes, and smoking for CVD, and declined eGFR and diabetes for ARD increased with age. The contribution of traditional risk factors for these NADs far outweighed the HIV-related risk factors. Individual-level interventions and population-level policy-making is needed to focus on these factors to prevent NADs in long-term management of HIV infection.

Evolution of blood lipids and risk factors of dyslipidemia among people living with human immunodeficiency virus who had received first-line antiretroviral regimens for 3 years in Shenzhen.

BACKGROUND: Lipid abnormalities are prevalent among people living with human immunodeficiency virus (HIV) (PLWH) and contribute to increasing risk of cardiovascular events. This study aims to investigate the incidence of dyslipidemia and its risk factors in PLWH after receiving different first-line free antiretroviral regimens. METHODS: PLWH who sought care at the Third People's Hospital of Shenzhen from January 2014 to December 2018 were included, and the baseline characteristics and clinical data during the follow-up were collected, including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). The risk factors of dyslipidemia after antiretroviral therapy were analyzed with the generalized estimating equation model. RESULTS: Among the 7623 PLWH included, the mean levels of TC, HDL-C and LDL-C were 4.23 ±â€Š0.85 mmol/L, 1.27 ±â€Š0.29 mmol/L and 2.54 ±â€Š0.65 mmol/L, respectively, and the median TG was 1.17 (IQR: 0.85-1.68) mmol/L. Compared with that in PLWH receiving tenofovir disoproxil fumarate (TDF) + lamivudine (3TC) + ritonavir-boosted lopinavir (LPV/r), zidovudine (AZT) + 3TC + efavirenz (EFV), and AZT + 3TC + LPV/r, the incidence of dyslipidemia was lower in PLWH receiving TDF + 3TC + EFV. In multivariate analysis, we found that the risks of elevations of TG, TC, and LDL-C were higher with TDF + 3TC + LPV/r (TG: odds ratio [OR] = 2.82, 95% confidence interval [CI]: 2.55-3.11, P < 0.001; TC: OR = 1.24, 95% CI: 1.14-1.35, P < 0.001; LDL: OR = 1.06, 95% CI: 1.00-1.12, P = 0.041), AZT + 3TC + EFV (TG: OR = 1.41, 95% CI: 1.28-1.55, P < 0.001; TC: OR = 1.43, 95% CI: 1.31-1.56, P < 0.001; LDL: OR = 1.18, 95% CI: 1.12-1.25, P < 0.001), and AZT + 3TC + LPV/r (TG: OR = 3.08, 95% CI: 2.65-3.59, P < 0.001; TC: OR = 2.40, 95% CI: 1.96-2.94, P < 0.001; LDL: OR = 1.52, 95% CI: 1.37-1.69, P < 0.001) than with TDF + 3TC + EFV, while treatment with TDF + 3TC + LPV/r was less likely to restore HDL-C levels compared with TDF + 3TC + EFV (OR = 0.95, 95% CI: 0.92-0.97, P < 0.001). In addition to antiretroviral regimens, antiretroviral therapy duration, older age, overweight, obesity and other traditional factors were also important risk factors for dyslipidemia. CONCLUSION: The incidence of dyslipidemia varies with different antiretroviral regimens, with TDF + 3TC + EFV having lower risk for dyslipidemia than the other first-line free antiretroviral regimens in China.

CD4+ T Cell Count, Sleep, Depression, and Anxiety in People Living With HIV: A Growth Curve Mixture Modeling.

We investigated changes in CD T cell counts related to sleep quality, depression, anxiety, and sociodemographic variables in heterogeneous groups of people living with HIV in a 6-month prospective study. Our longitudinal study involved 247 ambulatory patients living with HIV and using antiretroviral therapy. Sleep quality, anxiety, depression, and CD T cell counts were assessed three times at 3-month intervals. Growth curve mixture modeling was conducted to explore changes over time. A two-class mixture model with logarithmic change pattern fit the data best. For the majority of the sample (89.1%), anxiety, depression, and sleep quality did not change when CD T cells increased. For a small proportion of the sample (11.9%), sleep quality, anxiety, and depression deteriorated when CD T cells decreased. Marital status and alcohol use affected the classification significantly. Health care professionals should provide relevant services to people living with HIV with decreasing CD T cell counts.

[Association between CD4+CD25+Foxp3+ regulatory T cells and serum transforming growth factor beta 1 in patients with chronic hepatitis B].

OBJECTIVE: To investigate whether the CD4+CD25+Foxp3+ regulatory T cells are associated with serum TGF beta 1 in patients with hepatitis B. METHODS: Patients with chronic hepatitis B (CHB), chronic asymptomatic carriers (AsC), normal subjects (NS) and the resolved from HBV infection (Resolved) were recruited in this study. Flow cytometric analysis was used to detect the frequency and phenotype of peripheral CD4+CD25+Foxp3+ T cells, and Foxp3 gene expression were examined by real time PCR. Serum TGF beta 1 levels were measured by ELISA (enzyme-linked immunosorbent assay). RESULTS: Patients with CHB or AsC exhibited significantly higher frequency of CD4+CD25+Foxp3+ T cells compared to healthy controls. CD4+CD25+ T cells derived from patients with CHB and AsC expressed higher level of Foxp3-mRNA. Furthermore, the frequency of CD4+CD25+Foxp3+ regulatory T cells was correlated with serum HBV DNA copy numbers in patients with CHB and AsC. Our results indicated that the serum TGF beta was increased in CHB and AsC patients compared to control patients, and that serum TGF beta was correlated with the expression of Foxp3-mRNA and the frequency of CD4+CD25+Foxp3+ regulatory T cells in patients with CHB and AsC. CONCLUSIONS: The findings have important implication in the understanding of the role and mechanism of aberrant CD4+CD25+Foxp3+ regulatory T cells in the maintenance of chronicity in hepatitis B patients.

Interferon-γ upregulates Δ42PD1 expression on human monocytes via the PI3K/AKT pathway.

BACKGROUND: We recently identified a novel alternatively spliced isoform of human programmed cell death 1 (PD-1), named Δ42PD1, which contains a 42-base-pair in-frame deletion compared with the full-length PD-1. Δ42PD1 is likely constitutively expressed on human monocytes and down-regulated in patients infected with human immunodeficiency virus type 1 (HIV-1). The mechanism underlying the regulation of Δ42PD-1 expression in monocytes remains unknown. METHODS: By flow cytometry, we investigated the effect of Interferon-gamma (INF-γ) on the expression of Δ42PD1 in primary human monocytes as well as monocytic cell lines THP-1 and U937 cells. In addition, signaling pathway inhibitors and Δ42PD1-specific blocking antibody were used to explore the pathway involved in INF-γ-induced Δ42PD1 upregulation, and to elucidate the relationship between Δ42PD1 and TNF-α or IL-6 production by INF-γ primed monocytes in response to pre-fixed E. coli. Furthermore, we assessed T-cell proliferation, activation and cytokine production as enriched CD4+ T cells were co-cultured with THP-1 or U937 cells, with or without Δ42PD1-blocking antibody. RESULTS: Treatment of human peripheral blood mononuclear cells (PBMCs) with IFN-γ resulted in an approximately 4-fold increase in the expression of Δ42PD1 on monocytes. Similarly, IFN-γ upregulates Δ42PD1 expression on human monocytic cell lines THP-1 and U937, in a time- and dose-dependent manner. IFN-γ-induced Δ42PD1 upregulation was abolished by JAK inhibitors Ruxolitinib and Tasocitinib, PI3K inhibitor LY294002, and AKT inhibitor MK-2206, respectively, but not by STAT1 inhibitor and MAPK signaling pathway inhibitors. JAK, PI3K-AKT, and MAPK signaling inhibitors abolished effectively the production of TNF-α and IL-6 in INF-γ-primed monocytes in response to pre-fixed E. coli. In contrast, Δ42PD1-specific blocking antibody did not affect the IFN-γ-induced priming effect. Furthermore, the MFI ratio of Δ42PD1 to full-length PD-1 (PD-1 Δ/F ratio) was significantly and positively correlated with TNF-α (P = 0.0289, r = 0.6038) produced by circulating CD14+ monocytes in response to pre-fixed E. coli. Notably, Δ42PD1 blockage significantly inhibited CD4+ T-cells proliferation and cytokine production in the co-culture conditions. CONCLUSIONS: We demonstrated that IFN-γ increases Δ42PD1 expression on human monocytes via activating the PI3K/AKT signaling pathway downstream of JAKs, and that the PD-1 Δ/F ratio is a potential biomarker to predict the functional state of monocytes. Notably, we revealed the Δ42PD1 play a role in T-cell regulation, providing a novel potential approach to manipulate adaptive immune response.

Six-Year Immunologic Recovery and Virological Suppression of HIV Patients on LPV/r-Based Second-Line Antiretroviral Treatment: A Multi-Center Real-World Cohort Study in China.

The World Health Organization guidelines recommend lopinavir/ritonavir (LPV/r) as a second-line antiretroviral therapy (ART) for HIV-infected adults in middle-income and low-income countries as a protease inhibitor boost based on clinical trials; however, the real-world safety and efficacy remain unknown. Therefore, we conducted a large-scale, multicenter retrospective cohort study to evaluate the efficacy and safety of LPV/r-based ART among HIV-infected adults in China in whom first-line therapy failed. The data were obtained from a national database covering 17 clinics in China for six years of follow-up from 2009 to 2016. Failure of first-line treatment was determined according to a viral load at least 400 copies/ml at week 48, non-completers at week 48 for any reason, and those who switched ART before week 48 for any reason such as side effects. Treatment effectiveness was assessed by the rate of CD4+T cell recovery, defined as >500 cells/mm3, and the proportion of patients achieving viral suppression, defined as <400 or <50 copies/ml according to the methods used during treatment. Safety was assessed by rates of LPV/r-related adverse events (AEs), including lipid disorder, severe abnormal liver function, myelosuppression, and renal function. Between 2009 and 2016, 1196 participants (median, 36 years old; IQR, 30-43 years) were ultimately enrolled. All patients had been on LPV/r-based second-line ART treatment for more than one year after failure of any first-line ART regimen. Overall CD4+T cell counts increased from 138 cells/mm3 to 475 cells/mm3 and 37.2% of all participants reached CD4 recovery. Viral suppression rates dramatically increased at the end of the first year (<400 copies/ml, 88.8%; <50 copies/ml, 76.7%) and gradually increased during follow-up (<400 copies/ml, 95.8%; <50 copies/ml, 94.4%). The most frequently reported AEs were LPV/r-induced lipid disorders with no obvious increase on LDL-C at follow-up visits. This is the first real-world LPV/r-based second-line treatment study to cover such a large population in China. These results provide strong clinical evidence that LPV/r-based second-line ART is effective in increasing CD4+T cell counts and viral suppression rates with tolerable side effects in HIV-infected adults in China in whom first-line treatment had failed.

Hepatitis B virus (HBV) genotypes/subgenotypes in China: mutations in core promoter and precore/core and their clinical implications.

BACKGROUND: The association of hepatitis B virus (HBV) genotypes with clinical course of infection is increasingly recognized. OBJECTIVES: In order to investigate the genetic diversity of HBV and its clinical implications, 241 HBV-infected patients including 34 with hepatocellular carcinoma (HCC) were enrolled in this study. METHODS: HBV genotyping was performed with an ELISA assay. HBV subgenotypes were determined by PCR-RFLP. HBV core promoter/precore/core mutations were analyzed by direct sequencing. RESULTS: The overall prevalence of HBV/B and C was 65% and 33%, respectively. Among HBV/C, 42% were Cs/C1 and 58% were Ce/C2. The HBV/C1 was only found in the patients originating from Southern China (p=0.0001). Among HCC patients, HBV/C2 was only found in the elder age group (> or =51 years; p<0.05) and HBV/Ba was associated with young HCC patients (<35 years). Mutations associated with HCC were V1753 and T1762/A1764 (p<0.01). The prevalence of the V1753 was higher in HBV/C1 strains (p<0.04), A1898 was only found among HBV/C1 (p=0.056). T1762/A1764 was frequently demonstrated in both subgenotypes. The T1858 (90%) and A1896 (40%) mutations were most frequent in HBV/C2 (p<0.008). CONCLUSIONS: HBV/C1 and HBV/C2 have distinct geographic distributions in China. V1753 in addition to T1762/A1764 double mutation in the basal core promoter region seems to be associated with HCC development, especially in the patients with HBV/C1.

[Construction and application of a genechip method for detection of hepatitis B virus lamivudine-resistant mutants and basal core promotor/Pre-C mutants].

OBJECTIVE: The objective of this research is to construct a clinic-usable genechip method for detection of hepatitis B virus lamivudine-resistant mutants and basal core promotor/Pre-C mutants, compare this method with DNA sequencing to investigate this genechip's character (sensity, specificity, stability and practicability in clinic) and apply it in clinic. METHODS: This genechip detection method can detect the DNA and 8 mutative site of HBV, include 3 lamivudine-resistant mutation site(No. 180, 204, 207 site in DNA polymerase gene), 5 HBeAg escape-related mutation site (nt 1896, 1899, 1862, 1764,1762 site in BCP/Pre-C region).The results of genechip method was verified by DNA sequencing. RESULTS: In detecting HBV DNA, the results of genechip were agree with 100% of the results of DNA sequencing. In detecting HBV mutants, 251 sites (in 32 samples, 256 sites) showed the same results using both methods, and only 5 sites were not completely match (P > 0.05). In these 5 sites, genechip methods got multi-infection results, but sequencing got single-infection results. CONCLUSION: These results suggest that genechip method has the same positive rate and almost these same specificity with DNA sequencing method, and is better than DNA sequencing method in detecting multi-infected HBV strains. [Key words]

[To investigate pathogen of hand, foot and mouth disease in Shenzhen in 2008].

OBJECTIVE: To investigate EV71 and CA16 pathogen of HFMD in Shenzhen in 2008, and to provide the evidence for the prevention and treatment HFMD. METHOD: Using RT-PCR technology to detect the EV71 and CoxA16 genes of 307 samples HFMD; sequencing the purified PCR products from 14 samples. Using ClustalW2 online analysis software for sequence and phylogenetic analysis of enterovirus 71. RESULT: Percentage of positive EV71 from different samples is shown as follows respectively: positive EV71 from stool samples is 24.4% (75/307), from throat swab--7.8% (24/307), from peripheral blood--12.5% (1/8). Percentage of positive CoxA16 is shown as follows respectively: positive EV71 from stool samples is 13.8% (28/203), from throat swab-11.0% (20/181). Among all the 307 samples, three are positive for both EV71 and CoxA16. EV71 and CoxA16 are not detected in the samples of cerebrospinal fluid.Comparative analysis of nucleotide sequences of EV71 with those of strains BrCr and 11 deposited in GenBank demonstrated numerous disparities from 8 samples, but residue 595 from 2 samples and residue 658 from 1 sample are variable. The phylogenetic analysis based on VP1 region demonstrates that strains from 2 samples has the nearest genetic relationship with anhui strains, the farthest with BrCr and SHH02-6, SHZH02-40, SHZH03-58 strains, also strains from other 12 samples have the farthest genetic relationship with them. The genotypes A, B and C were classified as proposed by Brown et al. (1999). The EV71 from 14 samples were the member of genotype C. CONCLUSION: EV71 among the pathogen of HFMD in Shenzhen in 2008 was majority. These EV71 may belong to the same genegroup with Anhui predominant strains.

[Expression of recombinant VP1 protein of enterovirus 71 and development of serological assay for detection of EV71 infection].

OBJECTIVE: To obtain a recombinant purified Enterovirus 71 VPI protein and establishment of an early, rapid and accurate serological ELISA (enzyme-linked immunosorbent assay) for detection of EV71 infection. METHODS: VP1 gene was amplified by PCR and clonel into pET-21b (+) vector, the positive recombinant plasmid were transformed into E. coli BI21(DE3), and was induced with IPTG, the recombinant protein by SDS-PAGE and Western Blot assays. Finally, the recombinant purified VP1 protein was used as a coated antigen for detection of serum anti-IgM and IgG against EV71 by ELISA. RESULTS: The purified VP1 was obtained, and it can be recognized by sera of patients with EV71 infection associated with hand-foot-mouth disease. The A values of anti-EV71 IgM and IgG were significantly elevated as compared to healthy objects and HFMD patients without EV71 infection (P < 0.05). The sensitivity and specificity of IgM to EV71 were 73% and 77% compared with the RT-PCR results, respectively;and those of IgG being 82% and 83%, respectively. CONCLUSIONS: The recombinant protein VP1 was produced and purified, and it was proved to have a good antigenicity and could be used to develop a serological diagnosis kit for EV71 infection in the future.

[Immunosuppression induced by measles virus in adult patients is not related to CD4+ CD25+ regulatory T cell induction].

OBJECTIVE: To investigate of the relationship of the immunosuppression induced by Measles virus in adult patients and CD4+ CD25+ regulatory T cell. METHODS: Thirty-four patients with measles and 27 healthy control subjects were included in this study. The whole blood was collected and CD4+ CD25+ cell and FoxP3+ cell were analyzed by flow cytometry, and CD4+ CD25- and CD4+ CD25+ T lymphocytes were isolated from PBMCs of patients with measles or healthy donors, CD4+ CD25- T cells were cultured in absence or presence of anti-CD3, or BCG, or live attenuated MV. The cell culture supernatant was collected after 72 hours and the concentration of IFN-gamma and IL-10 was determined. RESULTS: Compared to healthy donors, we observed a reduction of the number of white blood cells and lymphocytes in patients with measles, but there was not significantly different in the frequency of CD4+ CD25+ T cells and CD4+ CD25high T cells within the total CD4+ population in the blood. Treg from both measles patients and healthy controls significantly inhibited IFN-gamma production by CD4+ CD25- T cells in response to anti-CD3 stimulation. CONCLUSION: Induction and expansion of Treg may not represent a mechanism involved in the establishment of immune suppression by MV.