Quantification of complete viral particles in inactivated avian influenza virus antigen by high performance size exclusion chromatography coupled with multi-angle laser light scattering / 生物工程学报

We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R2=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.

Advances in quantification of lentiviral vectors / 生物工程学报

Immunotherapy is becoming an effective and less invasive strategy that can be applied to the treatment of various malignancies. Lentiviral vectors (LVs) have shown great potential in immunotherapy as they can stably integrate relatively large foreign DNA, and effectively transduce dividing and non-dividing cells. Clinical application needs high quality LVs, and therefore strict quality control of the final products is necessary to ensure their purity, efficacy and safety. The quantitative detection of LVs is among the key parts of product development and quality control. In this paper, the existing methods for quantitative detection of LVs are summarized, including fluorescence activated cell sorter (FACS), P24 enzyme-linked immuno sorbent assay (P24 ELISA), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing(TRPS) and virus counter(VC).Their advantages and disadvantages are listed, and future development and challenges are discussed.

Emergency management practice for COVID-19 in designated hospitals of Hangzhou / 中华医院管理杂志

At present, we are fighting against the outbreak of COVID-19 in China.For the purposes of diagnosis and treatment of these patients, Hangzhou Xixi Hospital, as a designated hospital, made available the wards quickly, initiated the management system of public health emergencies, and established a " tolerate admission-strict discharge" patients management program. Meanwhile, the hospital has established an emergency supply and coordinated distribution mechanism for medical protection materials, and a full-system and multi-model training system, ensuring smooth progress of the diagnosis and treatment work.

Emergency management practice of novel coronavirus pneumonia in designated hospitals / 中华医院管理杂志

At present, we are fighting against the outbreak of novel coronavirus pneumonia (NCP) in China. For the purposes of diagnosis and treatment of NCP patients, Hangzhou Xixi Hospital, as a designated hospital, make available the wards quickly, initiated the management system of public health emergencies, and established a "tolerate admission- strict discharge" patients management program. Meanwhile, the hospital has established an emergency supply and coordinated distribution mechanism for medical protection materials, and a full-system and multi-model training system, ensuring smooth progress of the diagnosis and treatment work.

Vaccine pretreatment for quantification of 146S antigen in foot-and-mouth disease vaccines by high performance size exclusion chromatography / 生物工程学报

We developed a pre-treatment method to remove interfering substances during quantification of 146S antigens in foot-and-mouth disease (FMD) vaccines by high performance size exclusion chromatography (HPSEC). Three methods, including ultracentrifugation, PEG precipitation and nuclease digestion, were optimized and compared for removal efficiency of the interfering impurities in FMD vaccines. Under optimized conditions, the 146S contents in two batches of FMD vaccines were determined to be 7.1 and 7.6 μg/mL by ultracentrifugation, 9.7 and 10.4 μg/mL by PEG precipitation, and 10.5 and 10.4 μg/mL by nuclease digestion. The optimal condition for nuclease digestion using Benzonase determined by response surface method was as follows: appending Benzonase into 200 μL of antigen phase to a final concentration of 421 U/mL and incubating at 25.1 °C for 1.29 h. This method has advantages including efficient removal of the interfering impurities, fast processing speed, and mild operating conditions. Then 12 bathes of FMD vaccines with different serotypes produced by 4 manufacturers were tested to verify the established treatment method. Results showed the method was applicable to various FMD vaccines with good reproducibility (RSD<5.3%, n=3). The developed method removed interference from impurities during quantification of 146S, and therefore would broaden the application of HPSEC in vaccine quality control and ensure the accuracy and reliability.

Using size-exclusion chromatography to quantify the 146S antigen in inactivated foot-and-mouth disease vaccine / 生物工程学报

The aim of this study is to quantify the 146S antigen in foot-and-mouth disease virus (FMDV) inactivated vaccine by size-exclusion chromatography (SEC). The analysis was performed on a TSKgel G4000SWXL column (7.8 mm×30 cm), with a pH 7.2 buffer salt system as the mobile phase. The flow rate was 0.6 mL/min, the injection volume was 100 μL and the detection wavelength was 259 nm. The calibration curve was established by using purified inactivated FMDV (serotype O) 146S antigen; 3 batches of vaccine formulated by inactivated antigen solution were tested to verify the accuracy, reproducibility, specificity and tolerability of the method. At last 16 batches of vaccine were determined by the SEC method. Results showed a good linearity between peak area and concentration of 146S antigen in the range between 0.56 and 67.42 μg/mL (R2=0.996, n=10), and the average recovery rate of 146S antigen in the 3 batches of vaccine formulated in lab were 93.6% (RSD=2.7%, n=3), 102.3% (RSD=2.6%, n=3), and 95.5% (RSD=5.1%, n=3). The method was proved accurate and reliable with good reproducibility (RSD=0.5%, n=6), and applied to determine 16 batches of the commercial FMDV vaccine. According to the above results, the SEC method is high effective for 146S antigen quantify in the inactivated FMDV vaccine and would provide strong support for the vaccine quality control.

Expression of T lymphocytes and activated subsets in patients with HIV/AIDS and early latent syphilis / 中华临床感染病杂志

ObjectiveTo investigate the expression of T lymphocytes and activated subsets in patients with HIV/AIDS and early latent syphilis. MethodsT lymphocytes and activated subsets ( HLADR+ CD3 +/CD3 + , HLA-DR+ CD4 +/CD4 + and HLA-DR+ CD8 +/CD8 + ) as well as rapid plasma reagin (RPR) and treponema pallidum particle agglutination (TPPA) test were detected by flow cytometry in 78 patients with HIV/AIDS, 66 patients with HIV/AIDS and early latent syphilis, and 30 healthy subjects. SPSS 13.0 was used for statistical analysis, and t (for normal distribution) or Mann-Whitney U (for skew distribution) tests were performed to compare between the groups. ResultsThe absolute counts of CD4+ T cells in patients with HIV/AIDS and early latent syphilis were significantly higher than those in HIV/AIDS patients ( t = 2. 041 and 2. 223, P ＜ 0.05 ), but no difference in the counts of CD3 + T cells and CD8 + T cells was observed (tcD3 =0. 362 and 0.692, tcD8 =0.043 and 0.617, P＞0.05). HLA-DR+ CD4 +/CD4 +level in AIDS plus syphilis group was much higher than that in HIV plus syphilis group ( t = 2. 647, P ＜ 0. 05 ), but no difference was observed in HLA-DR+ CD3 +/CD3 + and HLA-DR+ CDs +/CDs + ( t = 1. 112 and 0. 093, P ＞ 0.05). ConclusionsImmune function in patients with HIV/AIDS and early latent syphilis may be enhanced temporarily.

Correlation of T-lymphocytes expressing HLA-DR antigen with serum HBV DNA and HBeAg levels in chronic hepatitis B / 中华临床感染病杂志

Objective To investigate the correlation of T-lymphocyte expressing HLA-DR with serum HBV DNA and HBeAg contents in chronic hepatitis B. Methods Totally 134 chronic hepatitis B patients and 36 healthy blood donors were enrolled in the study. The T-lymphocytes (CD3 + HLA-DR + ,CD4 + HLA-DR+ and CD8 + HLA-DR+ T) expressing HLA-DR were detected by flow cytometry, the serum HBV viral loads were detected by the real-time quantitative PCR and HBeAg was detected by chemiluminescence method. According to serum HBV DNA viral loads patients were defined as HBV DNA negative (≤ 103 copies/mL), low (＞ 103 - 105 copies/mL), medium (＞ 105 - 107 copies/mL) and high groups (＞ 107 - 109 copies/mL) ; according to serum HBeAg levels, patients were defined as HBeAg negative (≤1 PEIU/mL), low (＞ 1 - 100 PEIU/mL), medium (＞ 100-1 000 PEIU/mL) and high groups (＞ 1 000-10 000 PEIU/mL). T test and one-way ANOVA were performed. Results With HBV DNA loads, HBeAg levels increased, the percentage of CD3 + HLA-DR + , CD4 + HLA-DR + and CD8 + HLA-DR +decreased, especially CD8 + HLA-DR +. Compared with HBV DNA negative group, the percentages of CD3 +HLA-DR + , CD4 + HLA-DR + and CD8 + HLA-DR + were significantly reduced in high group (t = 3. 686,4. 592 and 3. 216, P ＜ 0. 0l); the percentages of CD4 + HLA-DR + and CD8 + HLA-DR + were also reduced in medium group (t = 3. 761 and 2.862, P ＜ 0.01); while in low group, only the percentage of CD8 + HLA-DR + was reduced (t = 2.215, P ＜ 0.05). Compared with HBeAg negative group, the percentages of CD3 +HLA-DR+, CD4 + HLA-DR+ and CD8 + HLA-DR+ were significantly reduced in medium and high groups (thigher =3. 144, 2.222 and 4.035; tmiddle =3.311, 2.362 and 3.374, P ＜0.05), while in the low group,only the percentage of CD8+HLA-DR+ was reduced (t=2.029, P＜0. 05). Conclusion The combined measurement of HBV DNA, HBeAg and T-lymphocytes expressing HLA-DR in chronic hepatitis B patients may not only help to evaluate the immune status of patients, but also can predict the disease progression and clinical outcomes.

Risk Factors and Infection Characteristics in Patients with Chronic Severe Hepatitis B / 中华医院感染学杂志

OBJECTIVE To analyze the characteristics of nosocomial infections,risk factors and prevention measures in patients with chronic severe hepatitis B.METHODS A retrospective review of the medical records of 354 patients with chronic severe hepatitis B admitted between Jan 2006 and Dec 2006 was performed.RESULTS The incidence of nosocomial infection in patients with chronic severe hepatitis B was 16.67% and mainly infection sites consisted of abdominal cavity(40.32%),and upper respiratory tract(22.58%).The most common infection(47.46%) was occurred during the period of hospitalization 15-30 days after and the most commonly pathogens were Gram-negative bacilli(68.75%).The infection risk factors were associated with invasive operation,hypoalbuminemia,endotoxemia,advanced age,antibiotics application and decrease in cell immune function.CONCLUSIONS It is important for the patients with chronic severe hepatitis B to strengthen management on related risk factors in order to prevent nosocomial infection effectively.

Detection of Chlamydia trachomatis and Ureaplasma urealyticum by Fluorescence Quantitative PCR / 中华医院感染学杂志

OBJECTIVE To discuss the infection rate of Chlamydia trachomatis(Ct) and Ureaplasma urealyticum(Uu) in patients with non-gonococcal infection.METHODS Fluorescence quantitative PCR method was used on 1025 cases and 30 cases of NGU patients for Ct and Uu detection.RESULTS Of 1025 NGU patients,positive Ct alone accounted for 156 cases,the positive rate was 15.22%.505 cases were separate Uu,the positive rate was 49.27%.Ct,Uu mixed in 217 cases,the positive rate was 21.17%.The detection rate was 85.66%.Uu infection rate in women was more than that in men(?2 = 104.56 P0.05).of control group,the Ct Uu Results negative.CONCLUSIONS In NGH patients,Uu is most common pathgen in man and woman.To diagnosis of NGU,Uu and Ct should be followed by Ct infection rate but no gender tested at the same time to avoid missed diagnosis.