De Novo Superinfection of Hepatitis B Virus in an Anti-HBs Positive Patient with Recurrent Hepatitis C Following Liver Transplantation.

A 60-year-old woman with end stage liver cirrhosis caused by genotype 2 hepatitis C virus (HCV) infection received an orthotopic liver transplantation (OLT). The patient was negative for the hepatitis B surface antigen (HBsAg) and positive for the anti-hepatitis B surface antibody (anti-HBs) prior to and one and a half months following the OLT. Due to reactivation of hepatitis C, treatment with interferon-alpha and Ribavirin started two months following the OLT and resulted in a sustained virological response. We performed a liver biopsy because a biochemical response was not achieved. Surprisingly, liver pathology showed HBsAg-positive hepatocytes with a lobular hepatitis feature, which had been negative in the liver biopsy specimen obtained one and a half months post-OLT. High titers of both HBsAg and HBeAg were detected, while anti-HBs antibodies were not found. Tests for IgM anti-hepatitis B core antibody and anti-delta virus antibodies were negative. The serum HBV DNA titer was over 1×10(7) copies/mL. A sequencing analysis showed no mutation in the "a" determinant region, but revealed a mixture of wild and mutant strains at an overlapping region of the S and P genes (S codon 213 (Leu/Ile); P codons 221 (Phe/Tyr) and 222 (Ala/Thr)). These findings suggest that de novo hepatitis B can develop in patients with HCV infection during the post-OLT period despite the presence of protective anti-HBs.

A YIDD Mutation in a Case of Recurrent Hepatitis B after Liver Transplantation Induced by an S-escape Mutant.

A 47-year-old woman underwent orthotopic liver transplantation (OLT) for hepatitis B virus (HBV)-related end-stage liver cirrhosis. The patient received hepatitis B immunoglobulin prophylaxis after OLT. Despite the protective level of the serum anti-hepatitis-B surface antibody, HBV recurred at 22 months post-OLT and induced subacute hepatic failure. The pre-OLT HBV genome contained a complex mutation pattern in overlapping frame regions of the surface (S) and polymerase (P) genes, which is the same mutation pattern as seen in post-OLT HBV DNA. G145R and K141R mutations in the "a" determinant were detected only in the post-OLT sample. Clevudine (30 mg once daily) was administered for recurrent hepatitis B. Hepatitis B was reactivated with a flare-up, and a M204I mutation (YIDD mutant type) appeared with a higher viral load at 9 months after clevudine treatment. We report here a case of a YIDD mutation that developed in recurrent hepatitis B after OLT induced by an S-escape mutant.

Evolution of hepatitis B virus mutation during entecavir rescue therapy in patients with antiviral resistance to lamivudine and adefovir.

BACKGROUND: The efficacy of entecavir (ETV) monotherapy in treatment-experienced patients with chronic hepatitis B (CHB) is debatable. METHODS: A total of 22 hepatitis B e antigen (HBeAg)-positive CHB patients who had shown viral breakthrough or suboptimal response with lamivudine (3TC) and adefovir disoproxil (ADV) therapy were treated with 1.0 mg of ETV. Clinical and virological parameters were monitored every 3 months. Restriction fragment mass polymorphism assays were used to detect antiviral resistance. RESULTS: During 3TC and ADV therapy, 11 patients had rtM204V/I mutations, 2 had rtA181V/T or rtN236T, 7 had both and 2 had no 3TC- or ADV-related mutations. After switching to ETV monotherapy, the median change in serum hepatitis B virus (HBV) DNA level was -2.1 log(10) copies/ml. Virological response (HBV DNA<300 copies/ml) was achieved in 1 of 18 patients with pre-existing rt204 mutations, whereas it was achieved in all 4 patients without pre-existing rt204 mutations regardless of the presence of rt181 or rt236 mutations. Changes in mutational patterns during ETV therapy showed that rt204 mutations persisted or re-emerged. Relative abundances of rtM204V/I mutations in total viral populations gradually increased under ETV rescue, whereas those with rtA181V/T and rtN236T mutations decreased. ETV resistance mutations (rtL180M+rtT184I/L[rtS202G]+rtM204V) were detected in five patients with pre-existing rt204 mutations. CONCLUSIONS: ETV monotherapy resulted in a limited virological response in patients who had previously failed 3TC and ADV rescue therapy. The limited efficacy might be associated with residual or reselected rtM204V/I mutations leading to ETV resistance. Combination treatment including potent antiviral agents should be recommended for patients with pre-existing rtM204V/I mutations.

High-resolution human papillomavirus genotyping by MALDI-TOF mass spectrometry.

We describe a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS)-based assay for human papillomavirus (HPV) genotyping--the restriction fragment mass polymorphism (RFMP) assay, which is based on mass measurement of genotype-specific oligonucleotide fragments generated by TypeIIS restriction endonuclease cleavage after recognition sites have been introduced by PCR amplification. The use of a TypeIIS restriction enzyme makes the RFMP assay independent of sequence and applicable to a wide variety of HPV genotypes, because these enzymes have cleavage sites at a fixed distance from their recognition sites. After PCR amplification, samples are subjected to restriction enzyme digestion with FokI and BtsCI and desalting using Oasis purification plates, followed by analysis by MALDI-TOF MS. Overall, the protocol is simple, takes approximately 4-4.5 h and can accurately detect and identify at least 74 different HPV genotypes.

A simple and accurate SNP scoring strategy based on typeIIS restriction endonuclease cleavage and matrix-assisted laser desorption/ionization mass spectrometry.

BACKGROUND: We describe the development of a novel matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-based single nucleotide polymorphism (SNP) scoring strategy, termed Restriction Fragment Mass Polymorphism (RFMP) that is suitable for genotyping variations in a simple, accurate, and high-throughput manner. The assay is based on polymerase chain reaction (PCR) amplification and mass measurement of oligonucleotides containing a polymorphic base, to which a typeIIS restriction endonuclease recognition was introduced by PCR amplification. Enzymatic cleavage of the products leads to excision of oligonucleotide fragments representing base variation of the polymorphic site whose masses were determined by MALDI-TOF MS. RESULTS: The assay represents an improvement over previous methods because it relies on the direct mass determination of PCR products rather than on an indirect analysis, where a base-extended or fluorescent report tag is interpreted. The RFMP strategy is simple and straightforward, requiring one restriction digestion reaction following target amplification in a single vessel. With this technology, genotypes are generated with a high call rate (99.6%) and high accuracy (99.8%) as determined by independent sequencing. CONCLUSION: The simplicity, accuracy and amenability to high-throughput screening analysis should make the RFMP assay suitable for large-scale genotype association study as well as clinical genotyping in laboratories.

Polymorphisms in the DNA repair gene XRCC1 associated with basal cell carcinoma and squamous cell carcinoma of the skin in a Korean population.

DNA in most cells is regularly damaged by endogenous and exogenous mutagens. Unrepaired damage can result in apoptosis or may lead to unregulated cell growth and cancer. Inheritance of genetic variants at one or more loci results in reduced DNA repair capacity. This hospital-based case-control study examined whether polymorphisms in the DNA repair gene X-ray repair cross-complementing groups 1 (XRCC1) (Arg194Trp[C > T], Arg280His[G > A] and Arg399Gln[G > A]) play a role in susceptibility to skin cancer. We genotyped these polymorphisms for 212 histopathologically confirmed skin cancer cases (n = 114 basal cell carcinoma, n = 98 squamous cell carcinoma) and 207 age- and sex-matched healthy control cases in Korea. We found that individuals with the Arg/Gln and Arg/Gln + Gln/Gln genotypes at XRCC1 Arg399Gln(G > A) had an approximately 2-fold increased risk of basal cell carcinoma compared to individuals with the Arg/Arg genotype (adjusted odds ratio [AOR] = 2.812, 95% confidence interval [CI] 1.32-5.98, and AOR = 2.324, 95% CI 1.11-4.86). However, we observed that the 194Trp allele of the Arg194Trp(C > T) polymorphism was inversely associated with squamous cell carcinoma risk (Trp/Trp, AOR = 0.06, 95% CI 0.006-0.63). Our data suggest that the Arg194Trp and Arg399Gln polymorphisms may be differentially associated with skin cancer risk.