Longitudinal profiles of highly sensitive hepatitis B surface antigen levels: re-evaluation of HBsAg seroclearance.

BACKGROUND & AIMS: Serologic profiles after hepatitis B surface antigen (HBsAg) seroclearance in chronic hepatitis B (CHB) have not been well-studied. METHODS: We employed a highly sensitive HBsAg (hs-HBsAg) assay (lower detection limit 0.5 mIU/ml), 100 times more sensitive than conventional HBsAg measurements. CHB patients achieving HBsAg seroclearance defined by conventional assays were followed up for serum hs-HBsAg, HBV DNA and antibody to HBsAg (anti-HBs) levels at 0 months, 6-12 months and 3-5 years after HBsAg seroclearance. Factors associated with hs-HBsAg detectability were determined. RESULTS: One hundred and nine patients were recruited; 94 (86.2%) were followed up to years 3-5; and 25 patients (22.9%) were on nucleoside analogue therapy for a median duration of 6.0 (range 1.5-12.7) years before HBsAg seroclearance. Detectable hs-HBsAg was noted in 88 (80.7%), 60 (55.0%) and 20 (21.3%) patients at 0 months, 6-12 months and 3-5 years respectively. At years 3-5, genotype B patients, when compared to genotype C patients, had a higher anti-HBs positive rate (63.2% and 41.1% respectively, P = 0.036). Serum anti-HBs positivity, when compared to persistent anti-HBs negativity, was associated with a lower rate of hs-HBsAg detection (7.4% and 40% respectively, P < 0.001). Multivariate analysis showed anti-HBs negativity at years 3-5 to be independently associated with persistently positive hs-HBsAg (P = 0.007, odds ratio 7.1, 95% confidence interval 1.7-29.3). CONCLUSION: Serum hs-HBsAg could detect HBsAg presence in a substantial proportion of CHB after HBsAg seroclearance defined by conventional assays, especially among anti-HBs negative individuals. Serum hs-HBsAg could potentially assist differentiating HBsAg-negative CHB from individuals with only past HBV exposure without carrier state.

The shufflon of Salmonella enterica serovar Typhi regulates type IVB pilus-mediated bacterial self-association.

Previously, it was shown that type IVB pili encoded by the Salmonella enterica serovar Typhi pil operon are used to facilitate bacterial entry into human intestinal epithelial cells in vitro and that such entry is inhibited by purified prepilin (pre-PilS) protein (X.-L. Zhang, I. S. M. Tsui, C. M. C. Yip, A. W. Y. Fung, D. K.-H. Wong, X. Dai, Y. Yang, J. Hackett, and C. Morris, Infect. Immun. 68:3067-3073, 2000). The pil operon concludes with a simple shufflon, and a recombinase gene product (Rci) inverts DNA in the C-terminal region of the pilV gene to allow synthesis of two distinct PilV proteins, PilV1 and PilV2, which are presumptive minor pilus proteins. We show here that the type IVB pili mediate bacterial self-association, but only when the PilV1 and PilV2 proteins are not expressed. This may be achieved in wild-type serovar Typhi by rapid DNA inversion activity of the shufflon. We show that the inversion activity inhibits the expression of genes inserted between the 19-bp inverted repeats used for Rci-mediated recombination and that the activity of Rci increases when DNA is supercoiled. The data suggest that serovar Typhi self-associates under conditions (such as low oxygen tension in the gut) that favor DNA supercoiling. These results explain (i) the function of the serovar Typhi shufflon and (ii) why there are only two possible shufflon states, in contrast to the many possible states of other shufflon systems. The data further indicate that a very early step in serovar Typhi pathogenesis may be type IVB pilus-mediated self-association of bacteria in the anaerobic human small intestine prior to invasion of the human gut epithelium. The suggested type IVB pilus-dependent step in typhoid fever pathogenesis may partially explain the enhanced invasiveness of serovar Typhi for humans.

Identification of O-antigen polymerase transcription and translation start signals and visualization of the protein in Salmonella enterica serovar Typhimurium.

The wzy/rfc gene, encoding the O-antigen polymerase, of Salmonella enterica serovar Typhimurium has been previously cloned and sequenced. In the present work, the wzy transcriptional startpoint was initially identified by primer extension. Next, wzy promoter strength in Escherichia coli K-12 was measured, and was found to be greater than that of the induced lac promoter. To define the Wzy translational startpoint, DNA including the wzy promoter and the putative first five residues of the Wzy protein was fused to the N-terminus of glutathione-S-transferase, and the fusion protein purified by affinity chromatography. N-terminal amino acid sequencing yielded the Wzy translational startpoint. Next, the Wzy protein was C-terminally tagged with the FLAG peptide, and immunoblotting of an S. typhimurium strain expressing a low-copy wzy-FLAG gene (five copies per cell) localized the intact Wzy protein in the cytoplasmic membrane of S. typhimurium cells. The Wzy protein was not well-expressed from a multi-copy wzy-FLAG+ plasmid in S. typhimurium, or in E. coli K-12.