A novel general and efficient technique for dissociating antigen in circulating immune complexes.

Circulating immune complexes (CICs) are produced during the immune response. It is more clinically important to establish a general and efficient CICs dissociation technique for the detection of antigens for CICs other than the detection of free antigens in the serum. Polyethylene glycol (PEG) two-precipitation separation and glycine-HCl as a buffer system were employed to develop a general and efficient buffer dissociation technique to separate CICs from serum and dissociate antigens from CICs. The measurement value of new PEG two-precipitation separation technique was higher than traditional PEG precipitation separation technique. There were slight differences in the dissociation conditions of HCV Core-IC, HIV P24-IC, Ins-IC and TG-IC as compared to HBsAg-IC. The detection of antigens in HBsAg-IC, HCV Core-IC, HIV P24-IC, Ins-IC and TG-IC with this technique was superior to that with HCl Dissociation, Trypsin Digestion or Immune Complex Transfer technique. PEG two-precipitation dissociation technique may reduce macromolecular protein and the adhesion of free antigens during the co-precipitation, which increases the efficiency of separation and precipitation of CICs. This technique also avoids the damage of reagents to antigens, assuring the repeatability, reliability and validity. Thus, this technique is application in samples negative or positive for free antigens.

Clinical Characteristics and Correlation Analysis of Subjects with Chronic Hepatitis B Virus (HBV) Infection and Sustained Low Levels of Hepatitis B Surface Antigen (HBsAg).

BACKGROUND The aim of this study was to investigate the clinical characteristics of individuals with chronic hepatitis B virus (HBV) infection with persistent low levels of hepatitis B surface antigen (HBsAg) and to undertake a correlation analysis of the clinical characteristics. MATERIAL AND METHODS The study included 1,204 subjects with chronic HBV infection. Serum HBsAg, HBV envelope antigen (HBeAg), and HBV core antigen (HBcAg) levels were measured using the chemiluminescent microparticle immunoassay (CMIA) and the neutralization test. HBV DNA was measured using real-time fluorescence quantitative polymerase chain reaction (RT-FQ-PCR). RESULTS There were 1,023 subjects in the high-level HBsAg group (HBsAg level ≥10 IU/mL) and 181 subjects in the low-level HBsAg group (HBsAg level <10 IU/mL). In the low-level HBsAg group, the main serological pattern (93.37%) was HBsAg and HBeAg and HBcAg-positive (HBV-M2), and the asymptomatic carrier (ASC) status was 98.34%. The low-level HBsAg group had a lower HBV DNA-positive rate compared with the high-level HBsAg group (40.33% vs. 75.07%), with a normal distribution across all age groups (P>0.05). The low-level HBsAg group included an older age group. A low-level of HBsAg was positively correlated with a low level of replication of HBV DNA (r=0.452). CONCLUSIONS The findings of this study showed that individuals with chronic HBV infection and sustained low-levels of HBsAg were an older population and had a lower level of replicating HBV DNA when compared with individuals with high levels of HBsAg, and the majority (93.7%) were also HBsAg and HBeAg and HBcAg-positive.

Analysis of S gene characteristic sequences and changes in properties of protein expression in HBV ASCs with low-level HBsAg.

Objective: We detected the serum HBsAg immune complex (HBsAg-CIC) and sequenced the HBV S gene in these patients to reveal the association between sustained low-level expression of HBsAg and mutated S gene sequence characteristics, protein function changes, and HBsAg immune complex formation. Methods: A total of 204 samples were collected and divided into high-level (n = 60, HBsAg level >10 IU/ml) and low-level (n = 144, HBsAg level ≤ 10 IU/ml) HBsAg groups. The clinical and epidemiological data of the two groups were statistically compared. According to different serological patterns and genotypes, the HBsAg-CIC results of the high-level and low-level HBsAg groups were divided into different subgroups, and then the HBsAg-CIC positive rates among different subgroups were compared. We sequenced the S gene of HBV from the two groups and identified the relevant mutations in the MHR of the S gene. In addition, we compared the changes in HBsAg protein properties and functions after hot spot mutation in the MHR of the S gene. Results: Comparing the positive rates of HBsAg-CIC under different serological patterns and genotypes in the two groups, the HBsAg-CIC positive rate was higher in the low-level HBsAg group. Moreover, there was weak correlation between HBsAg-CIC and HBsAg or HBV DNA in both groups (r = 0.32, 0.27, 0.41, 0.48; P < 0.05). Sequencing of S gene in the two groups, showed that the hot-spot mutations were T126A, M133L/T/S, and F134L/T/I in MHR of S gene of genotype B, and hot-spot mutations were Q101R and I126S/T in MHR of S gene of genotype C. Additionally, the positive rate of MHR mutation in the S gene from HBsAg-CIC positive patients was higher in the low-level HBsAg group. Conclusion: The host immune process of clearing HBV seems to have multiple site mutations in MHR, which changes the physicochemical properties and functions of HBsAg and intensifies the formation of HBsAg-CIC, thus avoiding the effective recognition of HBsAg by the host and resulting in immune tolerance between the host and HBV, which may be one of the formation mechanisms of sustained low-level expression of HBsAg in the serum of HBV-infected persons.

Clinical characteristics and association analysis of persistent low-level HBsAg expression in a physical examination population with HBV infection.

Certain patients with hepatitis B virus (HBV) infection present with persistently low levels of serum hepatitis B surface antigen (HBsAg) and have been indicated to have low rates of HBV nucleic acid replication. To explore the serological and molecular epidemiological characteristics of HBV population with low-level HBsAg in the present study, associated serum markers and virologic genotype detection were performed accordingly. Determination of HBV markers was performed using a chemiluminescence immunoassay from which 2,544 out of 45,256 adults who underwent routine health examination were tested positive for HBsAg. HBV DNA was detected by real-time fluorescent quantitative PCR. The patients were divided into low-level and high-level groups, according to their HBsAg levels (cut-off value, 10 IU/ml). The prevalence and levels of HBsAg positivity and HBV DNA in patients with HBV infection were analyzed by age, sex, serological pattern and clinical type. The fibrosis status of patients with low-level HBsAg was assessed by determining the aspartate aminotransferase-to-platelet ratio (APRI), and sequencing was employed to determine serotypes and genotypes. HBV-infected patients with low-level HBsAg (<10 IU/ml) accounted for 15.41% of the 2,544 HBsAg-positive patients, and the prevalence of HBsAg positivity exhibited a tendency to increase with age. The male-to-female ratio was ~1.9:1, and the average age was 54.98±16.28 years among HBV-infected patients with low-level HBsAg. The major serological pattern and clinical types were HBsAg/antibody against hepatitis Be antigen (anti-HBe)/antibody against hepatitis B core antigen (anti-HBc)-positive (94.90%) and chronic asymptomatic (ASC) (97.95%), respectively. HBV DNA exhibited a low-level of replication and the prevalence of HBV DNA positivity assessed by the routine method and by the enrichment method was 27.74% (97/392) and 45.92% (180/392), respectively. No significant differences among the age groups were identified in the different HBsAg level groups (P>0.05). The prevalence of HBV DNA positivity was associated with HBsAg only in patients with serological pattern HBV-M2 (HBsAg/anti-HBe/anti-HBc-positive) in the low-level HBsAg group (odds ratio: 1.30; 95% CI: 1.15-1.47; P<0.05). The APRI had no association with age, HBsAg, HBV DNA level or liver function index in ASC patients in the low-level HBsAg group (P>0.05). The prevalence of the serotype adw and genotype B was 85.53 and 89.47%, respectively. Further improvement in the systematic study of populations with low-level HBsAg has important clinical and epidemiological significance for improving the detection of HBV serological markers, elucidating the mechanisms leading to low-level HBsAg, overcoming immune tolerance to eliminate HBV infection and preventing HBV transmission.

Analysis of clinical characteristics and S gene sequences in chronic asymptomatic HBV carriers with low-level HBsAg.

BACKGROUND: During the natural hepatitis B virus (HBV) infection process, some infected subjects are characterized by a sustained low serum HBV surface antigen (HBsAg) expression level. Most members in this population are chronic asymptomatic HBV carriers (ASCs). To elucidate the mechanism underlying low-level HBsAg expression in ASCs, we sequenced the HBV S gene in these patients to reveal specific sequence characteristics. METHODS: Overall, 1308 cases of chronic ASCs were grouped according to their HBsAg serum expression levels (10 IU/mL). The clinical characteristics of the population were analysed in detail. The HBV S gene was sequenced from 276 ASC cases with low-level HBsAg expression. Additionally, 100 of 1032 ASC cases with high-level HBsAg expression were randomly selected for HBV S gene sequencing based on age matching according to the low-level HBsAg group. A comparative analysis was conducted with the HBV S gene sequences from ASCs with low HBsAg expression and the HBV reference S gene sequences from ASCs with high HBsAg expression. RESULTS: The population with low-level HBsAg expression displayed the following primary clinical characteristics: mostly chronic asymptomatic HBV carriers, older age (mean age 55.09 years), HBsAg/anti-HBe/anti-HBc (core) positivity as the main serological pattern (97.1%), low HBV DNA replication (1.32 ± 1.60 log10 IU/mL), a low HBV-DNA positive rate (45.65%) and primarily genotype B (82.54%) and serotype adw (84.13%). The comparative analysis of the HBV S gene sequences from ASCs with low-level HBsAg showed significant mutations (including co-mutations) on both sides of the main hydrophilic region (MHR). CONCLUSION: Significant mutations in multiple regions and at multiple sites (including co-mutations) on both sides of the MHR may be one cause of the low HBsAg expression level in this population.

[Corrigendum] Sequence analysis of the Pre­S gene in chronic asymptomatic HBV carriers with low­level HBsAg.

After the publication of the article, the authors realize that the grant funding number for the support that they received from Natural Science Foundation of Zhejiang Province appeared incorrectly in the Funding section of the paper: This should have been featured as grant no. LY15H200001 (i.e., with an 'L' at the start of the number). The authors regret that this error was not corrected prior to the publication of their paper, and apologize to the funders for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 42: 2689-2699, 2018; DOI:10.3892/ijmm.2018.3831].

Establishment and evaluation of a general dissociation technique for antibodies in circulating immune complexes.

This study aimed to establish a general and efficient dissociation technique for detecting antibodies in circulating immune complexes (CICs) in serum and to evaluate its clinical application. CICs were efficiently separated from specimens using polyethylene glycol double-precipitation. The best conditions for anti-HBs dissociation from HBsAg-ICs were a pH of 1.80, incubation at 15 °C for 5-10 min, and detection within 10 min after neutralization. The mean dissociation rate, reproducibility, mean dissociation recovery rate and specificity of the new technique were 64.3%, < 5.97, 95.4 and 100%, respectively. They had a favourable linear relationship (r = 0.9932), and the stability of the reagents exceeded 24 months, except the CIC antibody dissociation reagent (> 12 months). Conditions for the dissociation of other CICs tested were similar, but there were differences in the rate of antibody dissociation. Different HBV-M patterns had significantly different levels and rates of antibody dissociation from HBsAg-IC (P < 0.05), and the detection rates of the corresponding antibodies in HCV, core-anti-HCV core antibody (HCV-ICs), HIV P24-anti-HIV P24 antibody (HIV-ICs), insulin-anti-insulin antibody (INS-ICs) and thyroid globulin-anti-thyroid globulin antibody CICs (TG-ICs) were 34.8, 66.7, 20 and 14.3%, respectively. These data suggest that our CIC antibody dissociation technique is a good general pretreatment technique for the detection of antibodies after the precipitation, separation and dissociation of multiple CICs.

Prevalence and drug resistance characteristics of carbapenem-resistant Enterobacteriaceae in Hangzhou, China.

With the abuse of antimicrobial agents in developing countries, increasing number of carbapenem-resistant Enterobacteriaceae (CRE) attracted considerable public concern. A retrospective study was conducted based on 242 CRE strains from a tertiary hospital in Hangzhou, China to investigate prevalence and drug resistance characteristics of CRE in southeast China. Bacterial species were identified. Antimicrobial susceptibility was examined by broth microdilution method or epsilometer test. Resistant ß-lactamase genes were identified by polymerase chain reaction and sequencing. Genotypes were investigated by phylogenetic analysis. Klebsiella pneumoniae and Escherichia coli were the most prevalent types of species, with occurrence in 71.9% and 21.9% of the strains, respectively. All strains exhibited high resistance (> 70%) against ß-lactam antibiotics, ciprofloxacin, trimethoprim-sulfamethoxazole, and nitrofurantoin but exhibited low resistance against tigecycline (0.8%) and minocycline (8.3%). A total of 123 strains harbored more than two kinds of ß-lactamase genes. blaKPC-2, blaSHV-11, blaTEM-1, and blaCTX-M-14 were the predominant genotypes, with detection rates of 60.3%, 61.6%, 43.4%, and 16.5%, respectively, and were highly identical with reference sequences in different countries, indicating potential horizontal dissemination. IMP-4 was the most frequent class B metallo-lactamases in this study. In conclusion, continuous surveillance and effective prevention should be emphasized to reduce spread of CRE.

Sequence analysis of the Pre-S gene in chronic asymptomatic HBV carriers with low-level HBsAg.

In a hepatitis B virus (HBV)­infected population, persistently low expression levels of serum HBV serum antigen (HBsAg) are present, particularly in chronic asymptomatic HBV carriers (ASCs). The present study sequenced the HBV Pre­S gene, and aimed to elucidate its features in ASCs with low HBsAg expression compared with in the established HBV Pre­S reference gene sequences from ASCs with high HBsAg expression. A total of 1,308 ASCs were grouped according to HBsAg serum levels (cut­off value, 10 IU/ml), and clinical characteristics were analyzed in detail. The HBV Pre­S gene was sequenced in 276 ASCs with low­level HBsAg; in addition, 100 of the remaining 1,032 ASCs with high­level HBsAg were randomly selected for HBV Pre­S gene sequencing on the basis of age matching with the low­level HBsAg group. Comparative analysis of the gene sequences from these groups was subsequently conducted. The major clinical features of the population with low­level HBsAg were as follows: Most were ASCs with chronic HBV infection; 97.1% were HBsAg/anti­HBe/anti­HBc­positive; 82.54% carried the B genotype; and 84.13% displayed the adw serotype. The results indicated that there were novel and meaningful mutations, including co­mutations, at numerous loci and sites in the Pre­S gene, as well as deletion mutations in the Pre­S2 gene. These mutations in the Pre­S1 and Pre­S2 gene fragments accounted for 65.38% (68/104) of the 104 B genotype cases in the low­level HBsAg group and 90.91% (20/22) of the 22 C genotype cases in the low­level HBsAg group, respectively. In conclusion, Pre­S gene mutations may be associated with HBV replication defects, which may be the cause of the observed low expression levels of HBsAg.

An epidemiological survey of HBV infection and low-level HBsAg in military camps in eastern China.

This study aimed to investigate hepatitis B virus (HBV) infection in military personnel in eastern China, which will provide a basis for the prevention of HBV infection.A total of 15,508 soldiers and 2386 officers were recruited from military camps in eastern China. The markers, deoxyribonucleic acid, serotypes, and genotypes of HBV in serum were detected and analyzed.Hepatitis B surface antigen (HBsAg) positive rate was 0.44% in soldiers, in whom the low-level HBsAg accounted for 88.24%. The HBsAg positive rate was 1.72% in officers in whom the low-level HBsAg accounted for 12.20%. There were significant differences in the prevalence of high-level and low-level HBsAg, HBV serotypes, HBV DNA positive rate, and mean log HBV DNA between officers and soldiers (P < .05). Compared with the conventional method for HBV DNA extraction, the enrichment method for HBV DNA extraction could significantly improve the positive rate and quantification of HBV DNA by real-time fluorescence quantitative polymerase chain reaction (P < .05). Sequencing of S gene in HBV was used for the determination of serotype and genotype of HBV. The sequencing success rate was significantly different between soldiers and officers (P < .05) as well as between high-level HBsAg group and low-level HBsAg group (P < .05). Significant difference was also observed in the genotype distribution between soldiers and officers (P < .05).HBV infection displays a low prevalence and a low epidemic state, and the prevalence of low-level HBsAg is higher in soldiers. We should pay attention to improve the quality of conscription examination as well as emphasize the surveillance, prevention, and protection of HBV infection in military officers.

The effects of local variation in light availability on pollinator visitation, pollen and resource limitation of female reproduction in Hosta ventricosa.

BACKGROUND: Light availability may have direct effects on reproduction through resource availability, and indirect effects on female reproduction by influencing plant-pollinator interactions. Floral display size, pollinator visitation per flower, resource and pollen limitation of fruit and seed production were quantified in a forested patch and an adjacent open patch of two populations of the perennial herb Hosta ventricosa. RESULTS: Plants in the open patch produced significantly larger floral displays than those in the forested patch in both populations. Floral display size had a positive effect on pollinator visitation rate per flower in one population, but no effect in the other. Plants in the open patch received approximately 8-11 times more visitation rates and produced significantly more fruit and seeds per flower than those in the forested patch. However, supplemental pollination resulted in significantly more fruit and seed production per flower compared to natural pollination in the forested patch but not in the open patch in one population, and did not enhance fruit and seed production in either the forested or the open patch in the other. In both populations, supplementally pollinated plants in the open patch produced significantly more fruit and seeds per flower than supplementally pollinated plants in the forested patch. CONCLUSIONS: In H. ventricosa, local variation in light conditions could affect pollinator activity and influence female reproduction through resource availability; however differences in the degree of pollen limitation between local habitats were found to be population-specific.

Epidemiology and transmission characteristics of human adenovirus type 7 caused acute respiratory disease outbreak in military trainees in East China.

BACKGROUND: Human adenovirus type 7 (HAdV7) is globally attracting great concern as its high morbidity and severity in respiratory diseases, especially in Asia. OBJECTIVE: To investigate the clinical and epidemiologic characteristics of HAdV7 infection outbreak in East China. METHODS: The clinical samples were collected from the patients of an ARD outbreak in East Chinafor the detection of causative pathogens by multiplex PCR. The molecular type of human adenovirus isolates were identified by sequencing and homologous comparison based on their hexon genes. The spatiotemporal dynamics of global HAdV7 was investigated using the phylogenetic and phylogeographic analyses. Total 67 referenced HAdV7 hexon sequences (>800 bp) from GenBank were selected for constructing the maximum likelihood tree by MEGA 5.1.0, grouped according to the tree topology for the further migration analysis by PAUP* 4.0 and MigraPhyla 1.0 b to understand the transmission patterns of HAdV7 in global epidemics. RESULTS: The results showed HAdV7 as the causative pathogen in this outbreak, and the outbreak strains had the hexon sequences highly identical with the isolates in Shaanxi (2012). The origin of HAdV7 was inferred as California, meanwhile a total of 21 migration routes were acquired. HAdV7 in this outbreak was statistically proven dispersed from Shaanxi province (2012). CONCLUSIONS: The analyses of epidemiology and transmission pattern of HAdV7 would not only enrich the molecular biological basic database but also provide theoretical basis for HAdV7 prevention and control strategy.