Background::Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.Methods::The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 +) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed. Results::The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10-unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10-unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts. Conclusions::This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.

Objective:To investigate the molecular mechanism of sorafenib against hepatocellular carcinoma.Methods:Sorafenib efficacy was screened and verified by the hepatocellular carcinoma patient-derived tumor xenograft (PDX) model. Veterinary B-mode ultrasonography and in vivo confocal laser scanning microscopy were used to observe PDX angiogenesis. Immunohistochemistry was used to observe the expression of proliferation and angiogenesis-related proteins in PDX tissue. Real-time quantitative PCR technology was used to observe the RUNX3 gene in PDX tissues. SPSS 17.0 statistical software was used for statistical analysis.Results:Four cases of PDX were used to screen the efficacy of sorafenib. PDX1 had a significant response to sorafenib, with an inhibition rate of 68.07%. Compared with the control group, sorafenib had significantly inhibited PDX1 relative tumor volume (5.76±2.14 vs. 11.71±2.87, P<0.05). Cell division index (39.50±7.72 vs. 67.10±9.14, P<0.05) and Ki67 expression (288.6±43.40 vs. 531.70±55.60, P<0.05) were significantly decreased. Veterinary B-mode ultrasonography showed evident blood flow signals in PDX1 tumors. In vivo confocal laser scanning microscopy results showed that sorafenib had significantly reduced the total vessel length (1573.00±236.21 vs. 2675.03±162.00, P<0.05) and area (11 145.33±1931.97 vs. 20 105.37±885.93, P<0.05)) of PDX1 tumors. Immunohistochemical results showed that sorafenib had significantly down-regulated the protein expressions of CD34 (27.55±3.76 vs. 45.47±5.57, P<0.05), VEGF (16.33±2.86 vs. 22.77±3.20, P<0.05) and MVD (38.75±6.01 vs. 55.50±8.61, P<0.05). Real-time PCR results showed that sorafenib had significantly up-regulated RUNX3 gene expression (2.14±0.71 vs. 1.00±0.36, P<0.05). However, there was a negative correlation between the expression of RUNX3 gene and the ratio of VEGF-positive cells in sorafenib group ( R2=0.509 7). Conclusion:Sorafenib may inhibit the PDX angiogenesis and the growth of hepatocellular carcinoma by regulating the RUNX3-VEGF pathway.

Objective:To explore the clinical features and survival of newly diagnosed follicular lymphoma (FL) patients with diffuse large B-cell lymphoma (DLBCL) component.Methods:1845 newly diagnosed FL patients aged ≥ 18 years with grades 1-3a in 11 medical centers in China from 2000 to 2020 were included, and patients with DLBCL component were screened. The clinical data and survival data of the patients were retrospectively analyzed, and the prognostic factors were screened by univariate and multivariate analysis.Results:146 patients (7.9% ) with newly diagnosed FL had DLBCL component. The median age was 56 (25-83) years, 79 males (54.1% ) . The pathology of 127 patients showed the proportion of DLBCL component. Patients were divided into two groups according to whether the proportion of DLBCL component was ≥ 50% . The study found that patients with DLBCL component ≥ 50% had higher grade 3 ratio (94.3% vs 91.9% , P=0.010) , Ki-67 index ≥ 70% ratio (58.5% vs 32.9% , P=0.013) and PET-CT SUVmax ≥ 13 ratio (72.4% vs 46.3% , P=0.030) than patients with DLBCL component<50% . All patients received CHOP or CHOP like ± rituximab chemotherapy. The overall response rate (ORR) was 88.2% , and the complete response (CR) rate was 76.4% . In the groups with different proportions of DLBCL component, there was no significant difference in the remission rate after induction treatment and the incidence of disease progression within 2 years after initiation of treatment (POD24) ( P<0.05) . The overall estimated 5-year progression free survival (PFS) rate was 58.9% , and the 5-year overall survival (OS) rate was 90.4% . The 5-year OS rate of POD24 patients was lower than that of non POD24 patients (70.3% vs 98.5% , P<0.001) . Compared with non maintenance treatment of rituximab, maintenance treatment of rituximab could not benefit the 5-year PFS rate (57.7% vs 58.8% , P=0.543) , and the 5-year OS rate had a benefit trend, but the difference was not statistically significant (100% vs 87.8% , P=0.082) . Multivariate analysis showed that failure to reach CR after induction treatment was an independent risk factor for PFS ( P=0.006) , while LDH higher than normal was an independent risk factor for OS ( P=0.031) . Conclusion:FL patients with DLBCL component ≥50% have more invasive clinical and pathological features. CHOP/CHOP like ± rituximab regimen can improve the clinical efficacy of patients. Rituximab maintenance therapy can not benefit the PFS and OS of patients. Failure to reach CR after induction therapy was the independent unfavorable factor for PFS.

Objective: To investigate the genetic characteristics of Lamivudine-resistant mutation patterns and HBV S gene mutants in patients with chronic hepatitis disease of different disease progression. Methods: Blood samples of LAM-resistant patients with chronic hepatitis disease were collected. HBV RT gene nucleotide sequences were obtained, and then differences in drug-resistant mutation patterns, drug susceptibility and HBV S gene mutants characteristics between the two groups were analyzed. Results: Forty-seven chronic hepatitis B (CHB) patients and 16 HBV-related liver cirrhosis (LC)/HBV-related hepatocellular carcinoma (HCC) patients were included in this study. M204I single point mutation and L180M+ M204I/V were the most common pattern during patients with chronic hepatitis disease (35/63, 55.56%). The numbers of resistant to three nucleos(t)ide analogues in LC/HCC group was higher than CHB group’s (62.50% vs 34.04%, P=0.046). In HBV S gene, more immune associated HBsAg-escape mutations were detected in LC/HCC group than that in CHB group (62.50% vs 31.91%, P=0.031). I126T/V and G145A (for LCC/HCC group, 60%), I126S/T and S117T (for CHB group, 46.67%) were showed as the most common form for HBsAg escape mutations in the two groups. The two groups both detected RT mutations concomitantly with stop codon mutations in S gene (rtA181T/sW172* and rtM204I/sW196*). Conclusions Different characteristics in Lamivudine-resistant mutations and associated HBV S gene mutants were found in patients with chronic hepatitis disease of different disease progression, and LC/HCC patients exhibit more multi-drug resistant variants and immune associated HBsAg-escape mutants than CHB patients.

Objective To evaluate the repeatability of HISCL HBsAg kits and the correlation comparing with Roche kits.Methods A total of 580 serum samples were collected,HBsAg levels were detected by HISCL HBsAg kits and Elecsys HBsAg Ⅱ Quant kits,respectively.Data were analyzed,and the specificity,sensitivity and anti-jamming capability of HISCL kits were assessed by comparing with Roche kits.Results The intra-assay coefficients of variation (CV) of HISCL HBsAg kits was ＜ 5.0％,and Interassay CV was ≤5.5％.238 samples were repeated testing with HISCL HBsAg kits and the coincidence was 100％.Within them,the repeating detection results of 174 positive samples by HISCL HBsAg kits had agood correlation (r =0.999,P ＜0.01).122 cases of normal control were detected negative by both kits,and the coincidence was 100％.The correlation of HBsAg results from 287 HBsAg positive samples detected by the two kits was r =0.996,P ＜ 0.01.For the samples containing suspicious interferences,the results of the two kits were similar(pregnant women and infants r =0.992,P =0.000;antiviral drugs r =0.994,P =0.000;various virus or syphilis r =0.995,P =0.000;HBV gene mutation r =0.998,P =0.000;antinuclear antibodies and rheumatoid factor r =0.997,P =0.000).Conclusions HISCL HBsAg kits have a good repeatability and a excellent correlation with Elecsys HBsAg Ⅱ Quant kits.

Objective To investigate the relationship between hepatitis B virus (HBV) infection and cytokeratin 18 (CK18) phosphorylation.Methods Liver tissues were taken from 21 chronic hepatitis B (CHB) patients by liver biopsy and 14 healthy controls were collected.Immunofluorescence double staining and Western blotting were used to detect CK18 pSer33 and CK18 pSer52 phosphorylation.HepG2 cell line was transfected with either 1.3 mer HBV vector or empty control vector.CK18 pSer33 or CK18 pSer52 phosphorylation were tested using immunofluorescence double staining and Western blotting.Total mRNA was extracted from the cells.The expressions of cell division cycle 2 (cdc2) and protein kinase Cε (pKCε) were analyzed by realtime polymerase chain reaction relative quantification.Statistical analyzee were performed by using two-independent samples t test.Results Compared to healthy controls,phosphorylation of CK18 pSer33 in HBsAg stained hepatocytes was significantly higher in CHB patients (t=6.618,P=0.000).However,no difference was found in phosphorylation of pSer52 (t=2.429,P=0.051).Phosphorylation of CK18 pSer33 and expression of cdc2 mRNA in HBV transfected HepG2 were significantly higher in empty vector transfected HepG2 (t=5.365,P=0.006),while no difference was found in phosphorylation of pSer52 and expression of pKCε mRNA (t=1.098,P=0.334).Conclusion HBV infection is significantly associated with phosphorylation of CK18 pSer33.

Objective To describe the clinical characteristics,CD4+ and CD8+ T cell counts as well as human immunodeficiency virus (HIV) RNA of acute HIV infection in men who have sex with men,and their correlations with the disease progression.Methods One hundred cases of acute HIV infection were followed up.Nuclear acid sequence-based amplification (NASBA) was used for plasma HIV RNA screening.Flow cytometry was served to test the CD4+ and CD8+ T cell counts.Hepatitis B surface antigen (HBsAg) and anti-hepatits C virus (HCV) antibody were detected using enzyme-linked immunosorbent assay.Rapid plasma reagin was applied to screen for Treponema pallidum antibody.Antibody positive specimens were tested with Treponema pallidum particle assay for validation.The positive results were identified as infection.Results Ninety-six cases were aged between 20 and 50 years old.Among 100 cases,9 were HBsAg-positive; 4 were anti-HCV positive; 40 were co-infected with syphilis.During the follow-up period,the median CD4+ T cell counts in the 1st and 3rd month were 510/μL and 499/μL,respectively.The median HIV RNA in 1st,3rd,6th and 24th month were 4.37,4.00,4.31 and 4.43 lg copy/mL,respectively.CD8+ T cell counts did not show significant change during the study.Among 38 rapid progressors,the initial mean CD4+ T cell counts was (358.0± 134.6)/μL,which was significantly lower than that of the non-rapid progressors with a mean CD4+ T cell counts of (559.2±203.4)/μL.Meanwhile,the initial mean HIV RNA of the rapid progressors was 4.71 lg copy/ mL,while that of the non rapid progressor was 4.18 lg copy/mL.The initial CD8+ T cell counts of the rapid progressors and non rapid progressors were 1 250.1/μL and 1 247.2/μL,respectively.Conclusions Acute HIV-1 infected men who have sex with men tend to be young.The initial CD4+ T cell counts and HIV RNA during acute infection could be used to predict the disease progress.

Objective To establish a mini-pool nucleic acid testing (NAT) assay using multiplex RT-nested PCR for the detection of HIV RNA, and apply it in screening for acute HIV infection among MSM. Methods Frozen EDTA plasma samples collected between Oct. 2008 and Mar. 2009 from 3 HIV infectors during window-period, a total of 30 HIV chronically infected individuals and 97 healthy subjects were used to develop the NAT assay. Plasma samples from 10 cases were pooled into one tube and centrifuged at high speed for the collection of viruses. HIV RNA was extracted. Two pairs of primers were designed according to two conserved regions of HIV RNA ( HXB2 nt 5783-nt 6228 and nt 1235-nt 2012).Multiplex RT-PCR and nested PCR were performed. Individual NAT-reactive samples were confirmed by commercially available NAT assays. The sensitivity and performance efficacy were also evaluated. The assay was then applied to 1 005 plasma specimens from MSM with negative or uncertain HIV antibody test results.These were collected in the same period as the other samples. Results ( 1 ) Two fragments of HIV were amplified successfully with the low detection limit of 162 copies/ml plasma; (2) Results of the mini-pool HIV NAT validation with samples from 3 HIV infectors during window-period were consistent with the expected values; (3) All 30 plasma samples from MSM with positive HIV antibody, which were tested by multiplex RT nested PCR, were found to be HIV RNA positive; (4) One out of 1 005 plasma samples was found to be HIV RNA positive, for this case acute infection was followed-up and sero-conversion was found. Conclusion Mini-pool NAT has good sensitivity, and may be applied to screening HIV RNA among MSM during window-period.

Objective To study the effects of self-made Chinese sherbal gargle on AIDS related oral lesion.Methods 353 hospitalized AIDS patients from June 2007 to December 2009 were divided randomly into the experimental group(179 patients)and the control group(174 patients).ALL the patients were treated with systemic anti-viral therapy while the experimental group was mouthwashed by self-made herbal gargle and the control group with normal saline solution.The incidence of new oral lesion and the changes of the originM lesion were observed.Results The incidence of new oral lesion in the experimental group was obviously lower than that of the control group.The cure rate and effective rate of original oral lesion were much higher than the control group.Conclusions Self-made herbal gargle shows good effect in preventing and treating the AIDS related oral lesion.