Ultrasensitive Amplification Refractory Mutation System Real-Time PCR (ARMS RT-PCR) Assay for Detection of Minority Hepatitis B Virus-Resistant Strains in the Era of Personalized Medicine

Resistance to antiviral treatment for chronic hepatitis B virus (HBV) has been associated with mutations in the HBV polymerase region. This study aimed at developing an ultrasensitive method for quantifying viral populations with all major HBV resistance-associated mutations, combining the amplification refractory mutation system real-time PCR (ARMS RT-PCR) with a molecular beacon using a LightCycler. The discriminatory ability of this method, the ARMS RT-PCR with molecular beacon assay, was 0.01 to 0.25% for the different HBV resistance-associated mutations, as determined by laboratory-synthesized wild-type (WT) and mutant (Mut) target sequences. The assay showed 100% sensitivity for the detection of mutant variants A181V, T184A, and N236T in samples from 41 chronically HBV-infected patients under antiviral therapy who had developed resistance-associated mutations detected by direct PCR Sanger sequencing. The ratio of mutant to wild-type viral populations (the Mut/WT ratio) was >1% in 38 (63.3%) of 60 samples from chronically HBV-infected nucleos(t)ide analogue-naive patients; combinations of mutations were also detected in half of these samples. The ARMS RT-PCR with molecular beacon assay achieved high sensitivity and discriminatory ability compared to the gold standard of direct PCR Sanger sequencing in identifying resistant viral populations in chronically HBV-infected patients receiving antiviral therapy. Apart from the dominant clones, other quasispecies were also quantified. In samples from chronically HBV-infected nucleos(t)ide analogue-naive patients, the assay proved to be a useful tool in detecting minor variant populations before the initiation of the treatment. These observations need further evaluation with prospective studies before they can be implemented in daily practice.     

Impact of interferon-alpha therapy on liver fibrosis progression in patients with HBeAg-negative chronic hepatitis B

The possible effect of interferon-alpha (IFNa) on liver fibrosis progression has not been adequately studied in chronic hepatitis B. We evaluated 147 patients with HBeAg-negative chronic hepatitis B who had > or =2 liver biopsies and had been treated with IFNa (n = 120) or had remained untreated (n = 27). The median interval between the two biopsies was 24 (12-160) months. All biopsies were scored blindly by a single liver histopathologist according to the classification of Ishak et al. (J Hepatol 1995; 22: 696-699). IFNa induced sustained biochemical response in 30, initial response and subsequent relapse in 57 and no response in 33 patients. Fibrosis improved in 17.5% of treated (sustained responders: 40%, relapsers: 9%, nonresponders: 12%) and 4% of untreated patients and worsened in 34% (sustained responders: 7%, relapsers: 40%, nonresponders: 48%) and 70% of cases, respectively (P = 0.002). The annual rate of fibrosis progression was worse in the untreated (0.427 +/- 0.119) than in treated patients (0.067 +/- 0.052, P = 0.001). However, the fibrosis progression rate in the untreated patients was not significantly different than the net fibrosis progression rate (after subtraction of IFNa duration) in nonresponders or relapsers. In multivariate analysis, worse fibrosis progression rate was associated with older age (P = 0.010), worse baseline grading score (P < 0.001), lower baseline fibrosis (P = 0.035) and the type of response to IFNa (P = 0.032). In conclusion, in HBeAg-negative chronic hepatitis B, IFNa significantly reduces the rate of fibrosis progression, but such an effect is mainly observed in patients with sustained biochemical responses. In relapsers and nonresponders, fibrosis benefit equals the treatment period. The strongest factor associated with fibrosis progression is the change in necroinflammatory activity.     

Serotype distribution and antimicrobial susceptibility of <Emphasis Type="Italic"> Streptococcus pneumoniae </Emphasis>causing invasive infections and acute otitis media in children

A prospective study was conducted to determine the serotypes and antibiotic resistance patterns of pneumococcal isolates from children with invasive pneumococcal disease (IPD) and acute otitis media (AOM). From October 2001 to May 2002, 65 children with IPD (28 bacteraemic pneumonia, 24 bacteraemia without focus, 7 meningitis, 6 other infections) and 78 with AOM were identified. The most common serotypes causing IPD were 14 (32.3%), 6B (20.0%), 1 (18.5%) and 19F (7.7%) whereas the predominant serotypes causing AOM were 19F (35.9%), 14 (16.7%) and 23F (9.1%). Sixty-nine percent of IPD and 70.5% of AOM were caused by vaccine serotypes. The vaccine serotypes were more commonly encountered in meningitis cases and in children younger than 2 years of age. Intermediate resistance to penicillin was observed in 6 of 65 (9.2%) IPD isolates, one of which was intermediately resistant to cefotaxime (1.6%), whereas none exhibited high-level resistance to penicillin or other beta-lactam antibiotics. A higher proportion of antimicrobial resistance was noted in AOM isolates; 29 of 78 (37.4%) exhibited intermediate resistance and 8 (10.2%) high level resistance to penicillin, four of which had intermediate resistance to cefotaxime. Significant resistance was also noted to erythromycin; 38.5% of IPD and 48.7% of AOM isolates were resistant. Multidrug resistance was observed in one IPD and in eight AOM isolates. Conclusion:these findings have implications in the potential use of 7-valent conjugate vaccine in our region.Abbreviations AOM acute otitis media - IPD invasive pneumococcal disease - MIC minimal inhibitory concentration     

Prediction of treatment-related HBsAg loss in HBeAG-negative chronic hepatitis B: a clue from serum HBsAg levels

BACKGROUND: Quantification of HBsAg in serum may be of clinical importance in predicting HBsAg seroconversion and complete response to treatment. METHODS: Serum HBsAg was quantified by ADVIA Centaur in 63 patients with HBeAg-negative chronic hepatitis B (CHBe-). A total of 42 had received interferon-alpha2b (IFN-alpha2b) (median 12.1 months; 19 sustained responders including 12 HBsAg-seroconvertors; 23 non-sustained responders) and 21 were on lamivudine (LAM) (median 33.0 months). Measurements were done at baseline, during and at the end of treatment, and during and at the end of follow-up. RESULTS: Baseline median [interquartile range (IQR)] HBsAg levels in all patients were 3286 (1602-7458) IU/ml, not different between IFN- and LAM-treated (P = 0.139). IFN significantly depressed HBsAg in all patients except IFN non-responders, but HBsAg decline persisted only in sustained responders. Low pretreatment HBsAg level was the only significant prognostic variable of HBsAg seroconversion by multivariate analysis. LAM treatment also suppressed HBsAg levels but at a significantly slower rate compared with IFN (P = 0.022). The median (IQR) estimated time to HBsAg undetectability (ETU-HBsAg), derived from best curve fitting, was 127 (87.6-263.5) months for LAM virological responders and 65.3 (36.3-95.0) months for IFN sustained responders (P = 0.002). In 12 HBsAg seroconvertors, ETU-HBsAg was similar to the real time of HBsAg loss (P = 0.525) and seroconversion (0.056). CONCLUSIONS: In CHBe-, IFN induces a sharper decrease in serum HBsAg compared with LAM and low pretreatment levels are significantly associated with HBsAg serocon-version. Serial HBsAg measurements may be useful for prediction of HBsAg loss and our data suggest that to achieve this, 5.4 years of sustained response to IFN or 10.6 years of effective LAM therapy are probably needed.     

Oral ganciclovir treatment in chronic hepatitis B virus infection: a pilot study.

BACKGROUND/AIMS: Ganciclovir is a nucleoside analogue with an excellent safety record; it is effective against cytomegalovirus infection in both its intravenous and its oral form. Intravenous administration of ganciclovir is active against hepatitis B virus but the efficacy of oral ganciclovir is unknown. The aim of this study was to evaluate the tolerability and primary efficacy in reducing HBV DNA levels and liver enzymes of 2 dosing schedules of oral ganciclovir in HBeAg-positive and -negative patients with chronic hepatitis B. METHODS: Oral ganciclovir was administered to 15 consecutive patients with active chronic hepatitis B (age 43+/-12 years; 73% males; seven HBeAg-positive and eight negative; no cirrhosis) in a pilot, phase I, open-label study. Before treatment, all patients were screened for 8 weeks to ascertain the persistence of biochemical and virological activity, and then randomized to receive 3 g (eight patients), or 6 g (seven patients) of oral ganciclovir daily for 8 weeks; following therapy, they were closely observed for 8 more weeks. RESULTS: Baseline HBV DNA declined by 99% or 2 log10 at the end of treatment (405.0 vs 3.9 MEq/ml, respectively) and in four patients serum HBV DNA became undetectable by the Monitor assay. Oral ganciclovir suppressed HBV equally well in HBeAg-positive and -negative patients, and the 3- and 6-g daily dose regimens were equally effective. The pre-treatment viral load, however, was a determinant of response, with patients in the lowest quartile of baseline HBV DNA levels responding significantly better than those in the upper quartile (3.0 vs 0.6 log10 respectively, p=0.002). Serum alanine aminotransferase levels became normal or declined in most patients. Oral ganciclovir was very well tolerated. Within 8 weeks after stopping medication, a relapse in serum HBV DNA levels occurred in nine of the 15 patients (60%). CONCLUSIONS: These findings suggest that ganciclovir administered orally at a dose of 3 g can achieve sufficient suppression of HBV replication. This dose, and even higher doses, are well tolerated and could be used as an alternative or in combination with other antivirals for the treatment of chronic hepatitis B.     

Interferon treatment with or without oral ganciclovir in HBeAg-negative chronic hepatitis B: a randomized study

The treatment of HBeAg-negative chronic hepatitis B with alpha interferon alone is unsatisfactory. We evaluated the efficacy of combined administration of interferon-a2a (IFN) with oral ganciclovir, a nucleoside analogue. Forty patients with hepatitis B virus (HBV)-DNA-positive/HBeAg-negative chronic hepatitis B, were randomized to receive 4.5 MU IFN thrice weekly, subcutaneously, alone or in combination with 3 g ganciclovir per os daily for 26 weeks and followed for 12 months after treatment. Mean serum HBV-DNA levels decreased by 4.0 log10 in the combination group (from 5 x 106 to 4.8 x 102 copies/ml) and by 2.2 log10 in the interferon group (from 8 x 106 to 4.8 x 104 copies/ml) by quantitative polymerase chain reaction (PCR). HBV-DNA became undetectable in 11 of 20 (55%) and in three of 20 (15%) patients in the two groups, respectively (P=0.02). The alanine aminotransferase levels became normal in all patients receiving combination therapy, compared to 75% of those in the interferon group. After cessation of therapy, HBV-DNA increased or reappeared in all patients with 85% also relapsing biochemically. One year after treatment, three patients in each group (15%) remained in sustained biochemical remission with very low serum HBV-DNA levels (median 15 700 copies/ml). We conclude that, in HBeAg-negative chronic hepatitis B, 6-month combination therapy with oral ganciclovir and IFN is associated with complete biochemical remission in all treated patients and a 4 log10 decrease in serum HBV-DNA levels. The end of treatment efficacy of this combination scheme is far super- ior to that of IFN monotherapy but sustained responses are few. Further studies are warranted to evaluate the efficacy of prolonged combination schemes with nucleoside analogues and IFN, compared to IFN monotherapy.     

Outcome of hepatitis B e antigen-negative chronic hepatitis B on long-term nucleos(t)ide analog therapy starting with lamivudine

We determined the clinical outcome of hepatitis e antigen (HBeAg)-negative chronic hepatitis B patients treated with long-term nucleos(t)ide analog therapy starting with lamivudine. We evaluated 201 such patients treated for 3.8 +/- 1.4 years and 2 historical similar cohorts: 1 treated with interferon-alfa (n = 209) and 1 untreated (n = 195). Virological or biochemical remission rate at 48 months under lamivudine was 34% or 36%, respectively, whereas adefovir was administered in 79 patients with virological-biochemical breakthroughs or no response. Of the lamivudine-treated patients, 4 died, 1 underwent a transplantation, and another 8 developed major events, all having advanced fibrosis at baseline and all but 1 having experienced breakthroughs or no response. At 5 years, survival was 96%, and major event-free survival was 93%. The major event-free survival was significantly better in patients with than in those without virological remission under lamivudine. At the end of follow-up, both survival and major event-free survival were independently associated with type of and response to treatment, being significantly better in patients under long-term antiviral therapy or interferon sustained responders than in interferon non-sustained responders or untreated cases (5-year survival: 96% or 98% vs. 88% or 90%, respectively). In conclusion, in HBeAg-negative chronic hepatitis B, long-term nucleos(t)ide analog therapy starting with lamivudine significantly improves survival and reduces the risk of major complications, compared with interferon non-sustained responders or untreated patients. In such patients with advanced fibrosis, close follow-up for lamivudine resistance and prompt onset of additional antiviral therapy is required or the ab initio use of agent(s) with low resistance rates should be considered.     

Severe variable decelerations and fetal pulse oximetry during the second stage of labor

OBJECTIVES: The aim of the study was to investigate the usefulness of fetal pulse oximetry in cases of severe variable decelerations in the second stage of labor. METHODS: It is a prospective study including 58 patients. Thirty-eight patients (group A) had a normal uncomplicated labor and 20 patients (group B) developed severe variable decelerations during the second stage of labor. All patients were primiparous with normal pregnancies and had electronic fetal monitoring of labor in conjunction with fetal pulse oximetry. An estimation of fetal pH and base deficit was performed at delivery in all patients. RESULTS: There was no statistically significant difference in relation to maternal age and gestational age between the two groups. Group A patients did not delivered neonates with metabolic acidosis. Six out of 20 (group B) patients delivered neonates with a pH <7.10 despite a fetal pulse oximetry reading of >30%. CONCLUSIONS: It appears that fetal pulse oximetry is not capable of detecting pre-acidotic or acidotic fetuses during the second stage of labor in patients with severe variable decelerations and the management of such patients should be supported by fetal scalp pH when indicated or otherwise the obstetrician should expedite delivery either with assisted operative delivery or cesarean section. Fetal heart rate monitoring was introduced into clinical practice over 30 years ago. It continues to be the predominant method of intrapartum fetal surveillance despite worries about its accuracy and efficacy.