Comparison of the clinical performance of restriction fragment mass polymorphism (RFMP) and Roche linear array HPV test assays for HPV detection and genotyping.

BACKGROUND: The need for accurate genotyping of human papillomavirus (HPV) infections is becoming increasingly important as HPV is the primary cause of cervical cancer worldwide. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based restriction fragment mass polymorphism (RFMP) assay provides accurate, broad-spectrum, high-throughput genotyping of HPV. OBJECTIVES: We evaluated the clinical performance of the RFMP assay compared to a commercially available Roche linear array HPV genotyping test (LA) for detecting and genotyping of HPV. STUDY DESIGN: The RFMP assay and the LA were compared for detecting and genotyping HPV among a cohort of 244 liquid-based cytology samples. RESULTS: Overall, 216 specimens (93.1%, κ = 0.86) generated concordant results for the presence or absence of high-risk HPV (HR-HPV) by the two assays. The RFMP assay and the LA assay generated concordant, compatible, and discordant genotyping results for 79.3, 9.9, and 10.8%, respectively. The diagnostic sensitivity and specificity of RFMP and LA for the cervical lesions of squamous cell carcinoma (SCC) were similar, at 92.9 and 85.0% (RFMP) and 92.9 and 83.8% (LA), respectively. In addition, the odds ratio for SCC with HR-HPV positivity estimated by the RFMP assay (73.7, 95% CI: 8.9-3173.3) was higher than the LA assay (67.0, 95% CI: 8.2-2887.0). CONCLUSIONS: The RFMP and the LA assays were highly comparable with regard to detection and genotyping analysis of HPV. The sensitivity and specificity of RFMP assay for the detection of HR-HPV in various levels of cervical lesions seems to be valuable in the monitoring of HPV-associated cervical cancer.

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.

Emergence of adefovir-resistant mutants after reversion to YMDD wild-type in lamivudine-resistant patients receiving adefovir monotherapy.

BACKGROUND: To evaluate the effect of reversion to YMDD wild-type on emergence of adefovir (ADV)-resistant mutation and antiviral activity of ADV in lamivudine (LAM)- resistant patients. METHODS: We determined YMDD mutations and ADV-resistant mutations before and every 3 months during ADV monotherapy in 33 LAM-resistant patients using the restriction fragment mass polymorphism (RFMP) method. RESULTS: Reversion to pure YMDD wild-type hepatitis B virus (HBV) occurred in 6% (2/33), 9% (3/33), 20% (4/20) and 35% (6/17) of patients after 12, 24, 36 and 48 weeks, respectively. Five (29%) patients were found to have pure YMDD mutants at 48 weeks of therapy. Among 33 patients, 4 (12%) patients developed ADV-resistant mutations at 48 weeks of therapy. Adefovir-resistant mutants emerged in all patients after reversion to YMDD wild-type HBV. The mean serum HBV reductions, evaluated at 24 weeks of therapy, were not different between patients with and without reversion to YMDD wild-type HBV (-3.1 log(10) copies/mL vs-3.4 log(10) copies/mL, P > 0.05). CONCLUSIONS: ADV-resistant mutations emerged after reversion to YMDD wild-type in LAM-resistant patients who received ADV monotherapy. Thus, ADV add-on therapy may be necessary to reduce the incidence of developing ADV resistance in patients with LAM resistance.

Association of lamivudine-resistant mutational patterns with the antiviral effect of adefovir in patients with chronic hepatitis B.

Adefovir has a potent antiviral activity as a rescue treatment against lamivudine-resistant strains. The aim of this study was to assess the patterns of lamivudine-resistant mutations and their influence on the virologic response to adefovir rescue therapy in patients with lamivudine-resistant chronic hepatitis B. Sixty-seven patients with lamivudine-resistant chronic hepatitis B were treated with adefovir monotherapy. Baseline blood samples were analyzed for lamivudine-resistant mutations via restriction fragment mass polymorphism. Virologic responses, ALT normalization and loss of HBeAg were assessed. Serum HBV DNA levels were measured using real-time PCR at baseline and 24 weeks of adefovir therapy. Of the 67 patients with chronic hepatitis B, 65 patients (97%) had lamivudine-resistant mutations in the YMDD motif [27 (41%) rtM204I, 22 (34%) rtM204V, and 16 (25%) rtM204I/V]. In addition to the YMDD mutations, the rtL180M, rtL80I, and rtV173L mutations were also present in 78%, 43%, and 11% of patients, respectively. The rtM204V mutation always accompanied rtL180M, and rtL80I was always observed in conjunction with rtM204I. Decrease in mean serum HBV did not differ between patients carrying the rtM204I versus rtM204V mutant at week 24 (-3.3 vs. -3.3 log(10) copies/ml, respectively; P = 0.303). The presence of the rtL180M, rtL80I, and rtV173L did not significantly affect viral load reduction during adefovir administration. These results demonstrate that the rtL80I mutant is co-selected with rtM204I as a compensatory mutation in the same manner as rtL180M with rtM204V, and that adefovir shows similar antiviral efficacy against all of the evaluated patterns of lamivudine-resistant HBV mutations.

Selection of precore mutants during lamivudine treatment in patients with chronic hepatitis B.

BACKGROUND/AIMS: Evolution of precore genes can occur during lamivudine therapy in HBV infection. This study investigated the changes in precore regions in patients treated with lamivudine and the pattern during relapse. METHODOLOGY: The sequences of codon 28 in precore region in serial samples of 16 patients with HBV (11 HBeAg-positive and 5 HBeAg-negative) treated with lamivudine were analyzed by restriction fragment mass polymorphism. RESULTS: Among 9 patients who had wild-type virus, the wild-type virus was replaced by A1896 during relapse after initial treatment in 2 patients, and a pure population with A1896 selected during relapse in all 4 patients with mixed infection. In 5 patients with A1896 during relapse, 3 patients initially reverted to wild-type and later selected A1896, and 2 patients maintained A1896 during lamivudine retreatment. In 8 patients showing HBeAg negative reactivation, 3 patients showed A1896 and 5 patients showed wildtype virus. CONCLUSIONS: Lamivudine therapy induced initial reversion from precore mutants to wild-type virus, but precore mutants reappeared in patients infected with precore mutants. In some patients infected by wildtype HBV, wild-type HBV was replaced by precore mutants, resulting in a flare-up of hepatitis after cessation of lamivudine administration, and HBeAg negativity did not always correspond to the presence of precore mutants.

[Factors affecting initial virologic response and emergence of resistant mutants after adefovir treatment in lamivudine-resistant chronic hepatitis B patients].

BACKGROUND/AIMS: Adefovir dipivoxil (adefovir) effectively inhibits both wild-type and lamivudine-resistant hepatitis B virus (HBV) replication. The development of adefovir resistance is both delayed and infrequent compared with lamivudine resistance. The aim of this study was to characterize the serologic, biochemical, and virologic response to adefovir, and to explore the factors affecting initial virologic response (IVR, defined as a decrease in serum HBV below 4 log10copies/mL after 6 month of treatment) and adefovir resistance in lamivudine resistant HBV-infected patients. METHODS: This study population comprised 76 patients with lamivudine-resistance who had received adefovir for more than 12 months between March 2004 and December 2006. The adefovir-resistant mutant was assayed at 6 months and 12 months during adefovir administration. Restriction-fragment mass polymorphism analysis was used for detecting YMDD and adefovir mutants. RESULTS: After adefovir administration, an IVR was observed in 31% of the patients with lamivudine resistance. Factors associated with an IVR were HBeAg negativity (P=0.04) and the presence of liver cirrhosis (P=0.04). Age, sex, pretreatment levels of alanine aminotransferase and aspartate aminotransferase, pretreatment HBV DNA levels, presence of precore mutation, and type of YMDD mutants were not related to an IVR during adefovir treatment. The prevalence of adefovir resistance was 5% and 13% at 6 months and 12 months after therapy, respectively. Mixed infection of the precore mutant was a risk factors for the emergence of adefovir resistance (P=0.01). CONCLUSIONS: Lamivudine-resistant HBV patients exhibiting HBeAg negativity and liver cirrhosis were more likely to achieve an IVR after adefovir therapy. Adefovir resistance was associated with mixed infection of the precore mutant.

[Association between CCR5 promoter polymorphisms and hepatitis B virus infection].

BACKGROUND/AIMS: Immunogenetic factors may play a role in determining the susceptibility of an individual to viral infection. CCR5 promoter polymorphisms are known to be associated with HIV infection. However, there has been no report on the association between CCR5 promoter polymorphism and HBV infection. Therefore, we investigated the relationship between the CCR5 promoter polymorphism and HBV infection. METHODS: A total of 377 patients were classified into two groups according to their HBV infection status: (1) the spontaneous clearance group (SC); HBsAg (-), anti-HBc (+), anti-HBs (+) (2) the chronic HBsAg (+) carrier group (CC); HBsAg (+), anti-HBc (+), anti-HBs (-). CCR5 polymorphisms were detected by employing matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)- based SNP scoring assay, termed Restriction Fragment Mass Polymorphism (RFMP), which exploits the differences in molecular masses between the common allele and rare allele bases of interest. RESULTS: We found that the genotype frequencies of CCR5 A59029G significantly differed between the SC group (n=138) and CC group (n=239) (P<0.05). The CCR5 59029A allelic genotype was associated with an increased risks of chronic infection rather than spontaneous clearance (P=0.002), and the presence of the CCR5 59029G allele was significantly associated with the spontaneous clearance of HBV (P=0.001). Strong linkage disequilibrium between the CCR5-59029 and the CCR5-59353 polymorphic variants was identified. None of the 377 subjects had the CCR5-32 bp deletion mutation. CONCLUSIONS: The CCR5 promoter polymorphisms at position 59029 might play a role in the clearance of HBV infection. This primary experimental evidence needs further studies to clarify the clinical usefulness of CCR5 promoter polymorphisms as a target for the screening or treatment of HBV infection.