Molecular Characterization of High and Low Virulent Escherichia coli Clinical Strains Isolated from Patients with Urinary Tract Infections with or without Bacteremia in Southern Taiwan.

Objective: The most common extraintestinal pathogen and infection site is uropathogenic Escherichia coli (UPEC), which causes urinary tract infections (UTIs). UPEC is also a common pathogen in bloodstream infections; in severe cases, it can lead to death. Although host and bacterial virulence factors have been demonstrated to be associated with UTI pathogenesis, the role of the related contributing factors in UTI and urinary source bacteremia is not yet fully understood. This study aimed to compare and analyze the factors contributing to urinary bacteremia in patients with UTI. Methods: A total of 171 E. coli strains collected from patients with UTI and urinary source bacteremia at Chiayi Christian Hospital were used. Phylogenetic groups and virulence factors were determined using PCR. Drug resistance patterns were determined using the disk diffusion assay. Results: Previous studies have demonstrated that fimbriae and papGII may be associated with first-step infections and severe UTIs, respectively. As expected, highly virulent E. coli strains (belonging to the phylogenetic B2 and D groups) were dominant in the bacteremic UTI (90%) and UTI (86.27%) groups. However, our results showed that the UTI group had a significantly higher prevalence of sfa/focDE (belonging to the S and FIC fimbriae) than the bacteremic UTI group (29.4% vs 12.5%; p=0.008). In the bacteremic group, we found that sfa/focDE was only detected in highly virulent strains. The bacteremic UTI group had a significantly higher prevalence of papGII (belonging to P fimbriae) than the UTI group (55.8% vs 37.3%; p=0.026). In addition, the P fimbriae gene cluster, including papC, papEF, and papGII, was predominant in highly virulent strains. Notably, our results show that multidrug-resistant (MDR) strains were significantly less virulent than non MDR strains. Conclusion: Taken together, our results provide insights into the contributing factors in patients with UTI and urinary bacteremia.

Characterization of Hepatitis B Virus in Tenofovir-Treated and Untreated Chronically Infected Mothers and Their Immunoprophylaxis Failure Infants.

BACKGROUND: Maternal tenofovir disoproxil fumarate (TDF) therapy during late pregnancy can reduce mother-to-infant transmission of hepatitis B virus (HBV). We investigated HBV mutations associated with maternal TDF therapy and their role in infant immunonophylaxis failure (IPF). METHODS: Serum samples from untreated (n = 89) and TDF-treated (n = 68), highly viremic, chronically infected mothers and their infants were analyzed for HBV DNA by nested polymerase chain reaction (PCR) and direct sequencing. RESULTS: At delivery, compared with untreated mothers, TDF-treated mothers had a lower HBV DNA titer and a higher frequency of basal core promoter (BCP) gene mutations, but they had similar frequencies in pre-S/S and pre-core/core mutations. The 14 mothers harboring surface "a" determinant mutants did not transmit the mutants to their immunized infants. Such mutants were found in 3 of 13 IPF infants; the 13 mothers had wild-type hepatitis B surface antigen (HBsAg). In univariable analysis, maternal HBV DNA titer (odds ratio [OR]: 1.54; 95% confidence intervals [CI]: 1.02-2.33; P = .039), genotype C (OR: 4.18; 95% CI: 1.28-13.62; P = .018) and pre-S1 wild-type sequence (OR: 6.33; 95% CI: 1.85-21.68; P = .003) at delivery were associated with infant IPF. Multivariable analyses showed that maternal genotype C (OR: 3.71; 95% CI: 1.11-12.36; P = .033) and pre-S1 wild-type (OR: 6.34; 95% CI: 1.79-22.44; P = .004) were associated with infant IPF independently of maternal viremia. CONCLUSIONS: Along with high maternal HBV DNA titer at delivery, maternal genotype C and pre-S1 wild-type sequence were potential risk factors for infant IPF, although BCP mutations were not. The offspring of pregnant women harboring "a" determinant mutants as major strains seemed to be protected by immunoprophylaxis. CLINICAL TRIALS REGISTRATION: NCT01312012.

Hepatitis B virus X gene impacts on the innate immunity and immune-tolerant phase in chronic hepatitis B virus infection.

BACKGROUND AND AIMS: The immunologic features involved in the immune-tolerant phase of chronic hepatitis B (CHB) virus (HBV) infection are unclear. The hepatitis B virus X (HBx) protein disrupts IFN-ß induction by downregulating MAVS and may destroy subsequent HBV-specific adaptive immunity. We aimed to analyse the impacts of genetic variability of HBx in CHB patients on the immune-tolerant phase during long-term follow-up. METHODS: Children with CHB in the immune-tolerant phase were recruited and followed longitudinally. HBx gene sequencing of infecting HBV strains was performed, and the effects of HBx mutations on the immune-tolerant phase were assessed. Restoration of the host immune response to end the immune-tolerant phase was investigated by immunoblotting, immunostaining, ELISA and reporter assays of MAVS/IFN-ß signalling in liver cell lines, patient liver tissues and the HBV plasmid replication system. RESULTS: A total of 173 children (median age, 6.92 years) were recruited. Patients carrying HBx R87G, I127V and R87G + I127V double mutations exhibited higher cumulative incidences of immune-tolerant phase breakthrough (p = .011, p = .006 and p = .017 respectively). Cells transfected with HBx R87G and I127V mutants and pHBV1.3-B6.3 replicons containing the HBx R87G and I127V mutations exhibited statistically increased levels of IFN-ß, especially under poly(I:C) stimulation or Flag-MAVS cotransfection. HA-HBx wild-type interacted with Flag-MAVS and enhanced its ubiquitination, but this ability was diminished in the R87G and I127V mutants. CONCLUSIONS: HBx suppresses IFN-ß induction. R87G and I127V mutation restored IFN-ß production by preventing MAVS degradation, contributing to curtailing the HBV immune-tolerant phase in CHB patients.

Genetic variants of NTCP gene and hepatitis B vaccine failure in Taiwanese children of hepatitis B e antigen positive mothers.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) vaccine failure remains a hurdle to the global elimination of HBV infections in the vaccination era. We aimed to elucidate the relationships between HBV entry receptor sodium taurocholate co-transporting polypeptide (NTCP) and vaccine failure in children born to highly infectious mothers. METHODS: The genetic variants rs7154439, rs4646285, rs4646287, and rs2296651 were genotyped in 170 children with chronic HBV infections and 138 control children of mothers positive for hepatitis B e antigen (HBeAg). All children received hepatitis B immunoglobulin and complete HBV vaccination. Total RNAs from 82 adult non-tumor liver tissues were quantified for NTCP, type I interferons and interferon-induced transmembrane protein 3 (IFITM3) levels. RESULTS: A higher rate of the GA/AA genotype (28.3% vs. 15.3%, p = 0.006) of the genetic variant rs4646287 in intron 1 of the NTCP gene was detected in control children compared to the carrier children. The rs4646287 G > A genotype was associated with younger ages at which spontaneous HBeAg seroconversion occurred (10.8 ± 8.4 vs. 14.6 ± 8.7 years, p = 0.003) in chronic HBV-infected children. Unique correlation patterns of NTCP and innate immunity-related genes (type I interferons and IFITM3) were found in HBV-infected liver tissues with the rs4646287 G > A genotype. CONCLUSION: The rs4646287 G > A genotype of the NTCP gene may be associated with lower risk for HBV vaccine failure in children born to highly infectious mothers. The protective effect of rs4646287 G > A was also present in carrier children, evidenced by earlier spontaneous HBeAg seroconversion.

Occult Hepatitis B Virus Infection in Immunized Infants Born to Untreated and Tenofovir-Treated Highly Viremic Mothers.

Tenofovir disoproxil fumurate (TDF) therapy during late pregnancy in highly viremic mothers can reduce residual overt hepatitis B virus (HBV) infections of their infants that occur despite immunoprophylaxis.1,2 Occult HBV infection (OBI) has been defined as the presence of HBV DNA in liver or sera in subjects seronegative for hepatitis B surface antigen (HBsAg).3 OBI has been found in varying proportions of immunized infants born to HBsAg-positive mothers.4-6 We aimed to investigate the impact of maternal TDF therapy during pregnancy on vertically acquired OBI.

Occult hepatitis B virus and surface antigen mutant infection in healthy vaccinated cohorts and children with various forms of hepatitis and multiple transfusions.

BACKGROUND AND AIMS: Despite the success of universal infant immunization initiated in Taiwan in 1984, occult hepatitis B virus infection (OBI) and circulating surface antigen mutants remain potential obstacles for eventual eradication of HBV infection. METHODS: From 3299 apparently healthy, neonatally-vaccinated subjects (<30 years of age ) enrolled during 2014 serosurvey, we recruited all HBsAg-positive (n = 17), all HBsAg-negative but anti-HBc-positive (n = 132) and randomly selected HBsAg-negative and anti-HBc-negative subjects (n = 411). These recruited subjects and 81 HBsAg-negative children with various forms of hepatitis and multiple transfusions were analysed for serum HBV DNA. RESULTS: In healthy, HBsAg-negative subjects, OBI frequency was higher in anti-HBc-positive than anti-HBc-negative individuals (8/90[8.9%] vs 8/301[2.7%], P = 0.0192) aged <18-years, but was not different between anti-HBc-positive and anti-HBc-negative individuals (0/11[0%] vs 3/110[2.7%], P > 0.05) aged 18 to 30 years. OBI occurred more frequently in children of HBsAg-positive mothers than in children of HBsAg-negative mothers (10/101 [9.9%] vs 1/75 [1.3%], P = 0.025). The prevalence of surface 'a' determinant (aa110-160) mutants was 13.3% (2/15) in OBI subjects compared to 36.4% (4/11) in HBsAg-positive subjects (P > 0.05). OBI was found in 30% (3/10) of serologic 'non-A to E' viral hepatitis, 14.3% (3/21) of chronic hepatitis C and 2.0% (1/50) of multitransfused, thalassemic children. CONCLUSIONS: In this highly immunized population, surface antigen mutant infection is uncommon and has low contribution to OBI development. HBsAg screening plus highly sensitive HBV DNA assays are needed for assurance of blood supply safety. Multiple transfusions from HBsAg-negative blood donors rarely result in persistent HBV infection. HBV might be related to some of serologic 'non-A to E' viral hepatitis.

Universal infant immunization and occult hepatitis B virus infection in children and adolescents: a population-based study.

UNLABELLED: To determine whether universal infant immunization affects occult hepatitis B virus (HBV) infection (OBI), serum samples from hepatitis B surface antigen (HBsAg)-negative subjects <18 years enrolled during six sequential seroepidemiological surveys conducted between 1984 (just before universal infant immunization) and 2009 were analyzed. Study subjects were divided into unvaccinated cohorts (born before 1984) and vaccinated cohorts (born after 1984). HBV-DNA positivity was determined by positivity of nested polymerase chain reaction in at least two of three regions (pre-S, S, and pre-core/core genes). OBI frequency was lower in vaccinated than unvaccinated antibody to hepatitis B core antigen (anti-HBc)-negative subjects (0 of 392 [0%] vs. 4 of 218 [1.8%]; P = 0.007), tended to be higher in vaccinated than unvaccinated anti-HBc-positive subjects (16 of 334 [4.8%] vs. 3 of 181 [1.7%]; P = 0.072), and was higher in vaccinated than unvaccinated subjects seropositive for both antibody to hepatitis B surface antigen (anti-HBs) and anti-HBc (13 of 233 [5.6%] vs. 3 of 170 [1.8%]; P = 0.025). By using known anti-HBc seropositivity rate in children in our serosurveys, the estimated OBI frequency per 10(4) HBsAg-negative subjects declined from 160.7 in unvaccinated cohorts to 11.5 in vaccinated cohorts. In vaccinated cohorts, OBI frequency was higher in anti-HBc-positive subjects than in anti-HBc-negative subjects (16 of 334 [4.8%] vs. 0 of 392 [0%]; P < 0.001). Subjects with OBI had much lower viral load (P < 0.001) and a trend of higher mutation rates in "a" determinant of HBsAg than age-comparable, HBsAg-positive subjects. CONCLUSIONS: Reduction of OBI in immunized subjects complements the well-documented universal infant immunization-related benefit of markedly reduced overt HBV infection. Breakthrough infections in immunized subjects seem to associate with more occurrence of OBI than natural infections in unvaccinated subjects. In the postvaccination era, anti-HBc seropositivity is a useful marker for OBI screening in HBsAg-negative subjects, and a very-low-level viral replication and HBsAg expression is the major mechanism underlying OBI.

Long-term follow-up of children with postnatal immunoprophylaxis failure who were infected with hepatitis B virus surface antigen gene mutant.

BACKGROUND: The long-term evolution and outcomes of infection with a hepatitis B virus (HBV) surface antigen (HBsAg) gene mutant (hereafter, "HBsAg-mutant HBV") among immunized children remain unclear. METHODS: Ninety-five HBsAg-positive children aged 0.5-18.4 years (mean age, 5.8 years) who did not respond to postnatal immunoprophylaxis were prospectively followed for 1-22.8 years (mean follow-up, 8.5 years). Clinical, serologic, and virologic features were compared between 38 untreated HBV e antigen (HBeAg)-positive children carrying HBsAg-mutant HBV (group 1) and 49 children carrying wild-type HBV (group 2). HBsAg-mutant HBV was examined in 20 immunized children presenting with HBV-related hepatocellular carcinoma (HCC). RESULTS: The initial alanine aminotransferase (ALT) level, the maximal ALT level during the HBeAg-positive phase of HBV infection, the cumulative incidence of HBeAg seroconversion (P = .0018), and the rate of low serum HBV DNA load (defined as <10 copies/mL) at the last visit (P = .006) were higher in group 1 than in group 2. A higher frequency of HBV genotype C and a higher ALT level during surface mutant viremia were observed in codon 110-129 mutants than in codon 144-145 mutants. None of the 95 patients developed cirrhosis or HCC. HBsAg-mutant HBV was detected in 3 of 8 (38%) HBV DNA-positive children with HCC. CONCLUSIONS: HBeAg-positive immunized children carrying HBsAg-mutant HBV may develop hepatitis activity, HBeAg seroconversion, and a low viremic state earlier than those carrying wild-type HBV. Continuous monitoring of children with wild-type HBV infection and those with HBsAg-mutant HBV for possible development of HCC is needed.

Persistent elevation of hepatocyte growth factor activator inhibitors in cholangiopathies affects liver fibrosis and differentiation.

UNLABELLED: Alteration of cell surface proteolysis has been proposed to play a role in liver fibrosis, a grave complication of biliary atresia (BA). In this study we investigated the roles of hepatocyte growth factor activator inhibitor (HAI)-1 and -2 in the progression of BA. The expression levels of HAI-1 and -2 were significantly increased in BA livers compared with those in neonatal hepatitis and correlated with disease progression. In BA livers, HAI-1 and -2 were coexpressed in cells involved in ductular reactions. In other selective cholangiopathies, ductular cells positive for HAI-1 or HAI-2 also increased in number. Inflammatory cytokines, growth factors, and bile acids differentially up-regulated expression of HAI-1 and -2 transcripts in fetal liver cells and this induction could be antagonized by a cyclooxygenase-2 inhibitor. Conditioned media from cell lines stably overexpressing HAI-1 or HAI-2 enhanced the fibrogenic activity of portal fibroblasts and stellate cells, suggesting that both proteins might be involved in liver fibrosis. Because HAI-1 and -2 colocalized in ductular reactions sharing similar features to those observed during normal liver development, we sought to investigate the role of HAI-1 and -2 in cholangiopathies by exploring their functions in fetal liver cells. Knockdown of HAI-1 or HAI-2 promoted bidirectional differentiation of hepatoblast-derived cells. In addition, we showed that the hepatocyte growth factor activator, mitogen-activated protein kinase kinase 1, and phosphatidylinositol 3-kinase signaling pathways were involved in hepatic differentiation enhanced by HAI-2 knockdown. CONCLUSION: HAI-1 and -2 are overexpressed in the liver in cholangiopathies with ductular reactions and are possibly involved in liver fibrosis and hepatic differentiation; they could be investigated as disease markers and potential therapeutic targets.

Effects of maternal screening and universal immunization to prevent mother-to-infant transmission of HBV.

BACKGROUND & AIMS: Mother-to-infant transmission is the major cause of hepatitis B virus (HBV) infection among immunized children. There has been much debate about screening pregnant women and administering hepatitis B immunoglobulin (HBIG) to newborns. We analyzed the rate of HBV infection among children born to hepatitis B surface antigen (HBsAg)-positive mothers and whether HBIG administration reduces transmission. METHODS: We analyzed data from 2356 children born to HBsAg-positive mothers, identified through prenatal maternal screens. In addition to HBV vaccines, HBIG was given to all 583 children with hepatitis B e antigen (HBeAg)-positive mothers and to 723 of 1773 children with HBeAg-negative mothers. Serology tests for HBV were performed from 2007 to 2009, when children were 0.5-10 years old. RESULTS: A significantly greater percentage of children with HBeAg-positive mothers tested positive for antibodies against the hepatitis B core protein (16.76%) and HBsAg (9.26%) than children with HBeAg-negative mothers (1.58% and 0.29%, respectively; P < .0001 and <.001). Among the HBV-infected children, the rate of chronicity also was higher among children with HBeAg-positive mothers than children with HBeAg-negative mothers (54% vs 17%; P = .002). Similar rates of antibodies against the hepatitis B core protein (0.99% and 1.88%; P = .19) and HBsAg (0.14% and 0.29%; P = .65) were noted in children born to HBeAg-negative mothers who were or were not given HBIG. Infantile fulminant hepatitis developed in 1 of 1050 children who did not receive HBIG (.095%). CONCLUSIONS: Children born to HBeAg-positive mothers are at greatest risk for chronic HBV infection (9.26%), despite immunization. Administration of HBIG to infants born to HBeAg-negative mothers did not appear to reduce the rate of chronic HBV infection, but might prevent infantile fulminant hepatitis. Screening pregnant women for HBsAg and HBeAg might control mother-to-infant transmission of HBV.

No increase in prevalence of hepatitis B surface antigen mutant in a population of children and adolescents who were fully covered by universal infant immunization.

BACKGROUND: Mutants of the a determinant of hepatitis B surface antigen (HBsAg) can escape neutralization by vaccine-induced antibodies and prevail in an immunized population. METHODS: We evaluated the a mutants in a pediatric population surveyed in 2004 and compared these findings with the data of previous surveys. RESULTS: There were 38 children and 74 adolescents who were HBsAg positive, and serum hepatitis B virus (HBV) DNA was obtained and tested from 31 and 34 of them, respectively. The a mutants were found in 7 (22.6%) of 31 HBV DNA-positive children and in 7 (0.10%) of 7234 children, the entire population that was surveyed in 2004. After the beginning of universal immunization, the very low prevalence of mutants has remained unchanged for 20 years. More a mutants were found in immunized than in nonimmunized HBV DNA-positive children aged 1-4 years old (31% vs 4%, P = .016) but not in those children aged 5-12 years old. Approximately 68% of immunized, mutant-infected children had carrier mothers. More a mutants emerged in children immunized with plasma-derived vaccines than in those immunized with recombinant vaccines (14 of 5166 vs 3 of 4970, respectively; P = .04). HBV DNA levels were significantly lower in hepatitis B e antigen-positive sera containing the G145R mutant than were levels in sera containing wild-type virus. HBsAg-negative sera containing a mutants had very low HBV DNA levels. CONCLUSIONS: Less infectivity of G145R, recombinant vaccine use, and mutant loss with older age seem to decrease the a mutant prevalence in an immunized population over time.

Pre-S2 deletions of hepatitis B virus and hepatocellular carcinoma in children.

UNLABELLED: The cause of early oncogenesis in hepatitis B virus (HBV)-related childhood hepatocellular carcinoma (HCC) remains unclear. This study investigated whether pre-S deletion of HBV is related to childhood HCC. By using nested polymerase chain reaction, we compared the pre-S sequence of HBV from sera of children with HCC against control children with similar chronic HBV infection. The HBV in sera of children with HCC had a significantly higher rate of pre-S deletion than that of children with chronic HBV infection (p = 0.008). All except one of the pre-S deletions from the HCC group involved the pre-S2 region, whereas no pre-S2 deletion was found in the chronic HBV group (p = 0.003). There was a trend whereby genotype-C sera had a higher rate of pre-S2 deletion than genotype-B sera (p = 0.11). A multivariate logistic regression model revealed that pre-S deletion was an independent risk factor for HCC in children (odds ratio: 36.69, p = 0.015). In conclusion, pre-S2 deletion does not need to take decades to occur; its presence in nearly half of children with HCC, in contrast to its absence in children with chronic HBV infection, suggests a link between pre-S2 deletion and HCC development in children. ABBREVIATIONS: :

Interferon-alpha treatment in children and young adults with chronic hepatitis B: a long-term follow-up study in Taiwan.

BACKGROUND/AIMS: The short- and long-term benefits of interferon (IFN)-alpha therapy in young patients with chronic hepatitis B (CHB) acquiring infection perinatally or during early childhood have been questioned. METHODS: Twenty-one Taiwanese hepatitis B envelope antigen (HBeAg)-positive CHB patients aged 1.8-21.8 years (median 14.0 years) with alanine aminotransferase (ALT)>80 IU/L at entry were enrolled for IFN-alpha therapy. They received IFN-alpha therapy with a dose of 3 MU/m(2)/day three times a week for 24 weeks. A control group included untreated 21 CHB patients closely matched for gender, age, duration of ALT >80 IU/L and HBeAg status. All 42 patients were prospectively followed for 6.5-12.5 years after the end of therapy. RESULTS: The cumulative rate of virological response [anti-HBe seroconversion and serum hepatitis B virus (HBV)-DNA <10(5) copies/ml] was not different between the IFN-treated patients and control patients at 1 year (41 vs 44%) and at 6 years (88 vs 89%) after stopping treatment. Serum hepatitis B surface antigen loss occurred in two (9.5%) treated patients and in one (4.8%) control patient. Patients with a successful treatment response (anti-HBe seroconversion, HBV-DNA <10(2) copies/ml and ALT normalization at 1 year after stopping treatment) were younger than those without a successful response (P=0.03). A lower pretreatment serum HBV-DNA level (<2 x 10(8) copies/ml) is not only a significant factor to predict successful treatment response (P=0.008) but also has a beneficial effect on the long-term cumulative rate of virological response in IFN-treated patients (P=0.021), but not in control patients. Genotype difference or emergence of a precore stop codon mutant before treatment was not predictive for HBeAg clearance. CONCLUSION: For young CHB patients in Taiwan with infection occurring perinatally or in early childhood, the real advantage of IFN-alpha therapy was not observed. IFN-alpha therapy showed a beneficial effect on short- and long-term virological outcomes only in those with a lower pretreatment serum HBV-DNA level.

SEN virus infection in children in Taiwan: transmission route and role in blood transfusion and liver diseases.

BACKGROUND: SEN virus (SENV) is a newly discovered DNA virus. We conducted this study to evaluate potential modes of SENV transmission and the pathogenic effect of SENV on liver diseases in children. METHODS: Polymerase chain reaction was used to detect 2 SENV variant (SENV-D and SENV-H) DNA in sera from healthy individuals and diseased children. Nucleotide sequence of SENV was determined by direct sequencing. RESULTS: SENV infection was assessed in healthy individuals, including 50 newborns (sera collected from the umbilical cord), 24 infants, 46 preschool children (aged 1-6 years), 42 school children of an age before that of the first sexual experience (aged 7-12 years), 62 adolescents (13-18 years), 72 young adults (19-30 years) and 32 adults (>30 years). The prevalence of SENV-D and/or SENV-H (SENV-D/H) viremia in each group was 0%, 17%, 24%, 24%, 27%, 33% and 40%, respectively. The prevalence of SENV-D/H viremia in 18 children with non-A to E hepatitis, 64 thalassemic children, 80 children transfused during cardiac surgery, 30 children with chronic hepatitis B, 9 children with chronic hepatitis C and 32 infants with biliary atresia was 11%, 61%, 80%, 83%, 67% and 50%, respectively. SENV was found more frequently in all patient groups than in 174 age-matched controls (P < 0.01), with the exception of non-A to E hepatitis (11% versus 24% in the control group; P = 0.27). In 2 infants with proven intrauterine hepatitis B viral infection, identical SENV-D nucleotide sequence existed in both the maternal and neonate serum. Elevated alanine aminotransferase concentrations were rarely observed in children who acquired isolated SENV viremia because of transfusion for surgery. Infection with SENV in children with chronic hepatitis C virus or hepatitis B viral infection was not associated with higher peak alanine aminotransferase values. CONCLUSION: SENV is transmitted mainly via nonparenteral daily contact and frequently occurs early in life. Transfusion can significantly increase the rate of SENV viremia. SENV does not appear to cause hepatitis in children.