Microarray-based genotyping and detection of drug-resistant HBV mutations from 620 Chinese patients with chronic HBV infection

Background: Research has shown that hepatitis B virus (HBV) genotypes are closely linked to the clinical manifestations, treatment, and prognosis of the disease. Objective: To study the association between genotype and drug-resistant HBV mutations in 620 Chinese patients with chronic HBV infection. Methods: HBV DNA levels were determined using real-time quantitative PCR in plasma samples. Microarrays were performed for the simultaneous detection of HBV genotypes (HBV/B, C, and D) and drug-resistance-related hotspot mutations. A portion of the samples analyzed using microarrays was selected randomly and the data were confirmed using direct DNA sequencing. Results: Most samples were genotype C (471/620; 76.0%), followed by genotype B (149/620; 24.0%). Among the 620 patient samples, 17 (2.7%) had nucleotide analogs (NA) resistance-related mutations. Of these, nine and eight patients carried lamivudine (LAM)-/telbivudine (LdT)-resistance mutations (rtL180M, rtM204I/V) and adefovir (ADV)-resistance mutations (rtA181T/V, rtN236T), respectively. No patients had both lamivudine (LAM)- and either ade-fovir (ADV) or entecavir (ETV) resistance mutations. Additionally, out of the 620 patient samples, 64.0% (397/620) were also detected with the precore stop-codon mutation (G1896A) by microarray assay. Conclusion: The results of the current study revealed that the prevalence of nucleotide analogs (NA)-resistance in Chinese hospitalized HBV-positive patients was so low that intensive nucleotide analogs (NA)-resistance testing before nucleotide analog (NA) treatment might not be required. In addition, the present study suggests that chronic HBV patients with genotype C were infected with fitter viruses and had an increased prevalence of nucleotide analogs (NA)-resistance mutations compared to genotype B virus. .

Long-Term Outcomes and Dynamics of Mutants Associated with Lamivudine-Adefovir Rescue Therapy in Patients with Lamivudine-Resistant Chronic Hepatitis B

BACKGROUND/AIMS: To investigate the association between the baseline profiles and dynamics of hepatitis B virus (HBV) DNA polymerase gene mutations and the long-term virological response of lamivudine (LAM)-adefovir (ADV) combination therapy in patients with LAM-resistant chronic hepatitis B. METHODS: Seventy-five patients who received LAM-ADV combination therapy for more than 12 months were analyzed. Restriction fragment mass polymorphism assays were used to detect and monitor the dynamics of LAM- and ADV-resistant mutations. RESULTS: The median duration of LAM-ADV combination therapy was 26 months (range, 12 to 58 months). The baseline mutation profiles, rtM204I (p=0.992), rtM204I/V (p=0.177), and rtL180M (p=0.051), were not correlated with the cumulative virological response, and the baseline HBV DNA level (p=0.032) was the only independent predictive factor for cumulative virological response. Tests for LAM- and ADV-resistant mutations were performed in 12 suboptimal responders in weeks 48 and 96. The population of rtM204 mutants persisted or increased in 8 of 12 patients, and rtA181T mutants newly emerged as a minor population in four patients until 96 weeks. Nevertheless, the viral loads progressively decreased during rescue therapy, and these dynamics did not correlate with virological response. CONCLUSIONS: The baseline profile and dynamics of LAM-resistant mutations during LAM-ADV combination therapy are not associated with a virological response.

Chemometric optimization for simultaneous determination of anti-retroviral drugs by RP-HPLC

This work deals with multiple response simultaneous optimization using the Derringer's desirability function for the development of a reversed phase HPLC method for the simultaneous determination of lamivudine, tenofovir and efavirenz in commercial pharmaceutical preparations. Twenty experiments, taking the capacity factor of the first peak, resolution between the second and third peaks and the retention time of the third peak as the responses with three important variables as organic phase composition, buffer molarity, and flow rate, were used to design mathematical models. The experimental responses were fitted into a second order polynomial and the three responses were simultaneously optimized to predict the optimum conditions for the effective separation of the studied compounds. The optimum assay conditions were: methanol-triethylamine buffer [pH 3.0; 15.3 mM] [35:65%v/v] as the mobile phase and at a flow rate of 1.19 ml/min. While using this optimum condition, a baseline separation with a minimum resolution of 2.0 and a run time of less than 6 min was achieved. The method showed a good agreement between the experimental data and predictive value throughout the studied parameter space. The optimized assay condition was validated according to the International Conference on Harmonization guidelines to confirm specificity, linearity, accuracy, and precision.

Antiviral therapy against chronic hepatitis B in Brazil: high rates of lamivudine resistance mutations and correlation with HBV genotypes

The effectiveness of antiviral treatments of chronic hepatitis B has been poorly studied in Brazil. Here, hepatitis B virus (HBV) DNA positivity, drug resistance mutations and their association with HBV genotypes were evaluated in chronically HBV-infected patients under different drug regimens in Brazil. The study involved 129 patients under interferon or nucleos(t)ide analogue therapy for a median treatment time of 12 months. One hundred and five (81%) of these patients were treated with lamivudine (LAM), either in monotherapy or in combination with newer drugs, such as entecavir (ETV) or tenofovir (TDF). High (37.5-100%) rates of HBV DNA positivity were observed with all but one drug regimen (LAM + ETV). However, patients that were treated with ETV alone, TDF alone or with LAM combination therapies had a mean viral load that was 3-4 log lower than patients treated with LAM monotherapy. Of the patients treated with LAM, 47% developed resistance mutations. HBV genotypes A (59.1%), D (30.3%) and F (9.1%) were found. There was no association between the presence of LAM resistance mutations and genotypes, HBeAg status or treatment duration. Nevertheless, the rtM204V mutation was observed more frequently (12/13, 92%) in genotype A than in the others (p = 0.023). Six out of nine isolates that contained the rtM204I mutation belonged to genotype D and half of them displayed a single mutation. Genotype D isolates with the rtM204V variant preferentially displayed a triple mutation, while genotype A preferentially displayed a double mutation (p = 0.04).

In vitro plant regeneration system for common bean (Phaseolus vulgaris): effect of N6-benzylaminopurine and adenine sulphate / Sistema de regeneración de plantas in vitro para el frijol común (Phaseolus vulgaris): efecto del frl sulfato N6-bencilaminopurina y adenina

A method for regeneration of the commercially important common bean (Phaseolus vulgaris ) using N6-benzylaminopurine(BAP) and adenine sulphate (AS) was established. Embryogenic axes of the Costa Rican common bean cultivars Bribrí, Brunca, Guaymí, Huetar and Telire were cultured on Murashige and Skoog medium supplemented with 100 mgl-1 myo-inositol, 1 mgl-1 thiamine, 30 gl-1 sucrose, BAP (0, 5 and 10 mgl-1), AS (0, 20 and 40 mgl-1) and 8 gl-1 agar. Regardless of the concentration of BAP and AS in the induction medium, the number of shoots and leaves differed significantly among the common bean cultivars evaluated. The higher average of shoots was obtained for Brunca > Telire > Bribrí > Guaymí > Huetar. Moreover, independently of the cultivar, the induction medium supplemented with 5 mgl-1 BAP and 20 or 40 mgl-1 AS resulted in the higher average of shoots formation. Culture of Bribrí, Brunca, Guaymí, Huetar and Telire embryogenic axes on induction medium supplemented with different BAP and AS resulted in a differential response. Successful acclimatization of common bean in vitro plants were achieved in the greenhouse, and plants appeared morphologically normal. The regeneration system developed in this investigation for this important crop could be a useful tool for the genetic modification through mutagenesis or genetic transformation.

A case of severe hypophosphatemia related to adefovir dipivoxil treatment in a patient with liver cirrhosis related to hepatitis B virus / 대한간학회지

Adefovir dipivoxyl (ADV) effectively suppresses hepatitis B virus (HBV) replication but exhibits nephrotoxicity with severe hypophosphatemia when administered at a high dosage. This is the first report of severe hypophosphatemic osteomalacia induced by ADV at 10 mg/day. A 42-year-old man with HBV-related chronic liver disease presented with generalized bone pain, especially in the left ankle. He had been taking ADV for more than 1.5 years following a clinical breakthrough due to lamivudine-resistant HBV. Aggravating severe hypophosphatemia and elevated serum alkaline phosphatase levels with high bone fraction had been noted after 6 months of ADV therapy. Bone densitometry, simple bone X-rays, and a whole-body bone scan demonstrated osteoporosis and multiple areas with hot uptake, especially in the left ankle. All the image findings and symptoms improved after correcting the hypophosphatemia.

Clinical Outcomes after Discontinuation of Lamivudine in Chronic Hepatitis B Patients with Lamivudine Resistant HBV Mutant / 대한간학회지

BACKGROUND/AIMS: The therapeutic strategies of applying adefovir for treating lamivudine resistant HBV mutants are controversial. Thus, we observed the clinical outcomes after discontinuation of lamivudine to establish the timing to initiate adefovir therapy. METHODS: Fifty chronic hepatitis B (CHB) patients with lamivudine resistant HBV mutants who had received lamivudine for more than 12 months were included in the study. We investigated the clinical outcomes at 6 months after the end of treatment (EOT). We compared the serial clinical outcomes among respective groups based on serum ALT at the EOT and the clinical characteristics of patients with or without acute exacerbation (AE) and the HBeAg loss. We also investigated the predictive parameters of AE and HBeAg loss. RESULTS: Fifteen patients (30%) had experienced AE at 6 months after the EOT. Four patients received antiviral agents because of their hepatic decompensation. Patients with AE had higher serum ALT values and lower HBV DNA titers at EOT compared with those patients without AE. Serum ALT at the EOT was the predictive parameter of AE. Eight patients (21.6%) had newly developed HBeAg loss at 6 months after EOT. The total bilirubin at EOT was the predictive parameter of HBeAg loss. CONCLUSIONS: CHB patients with lamivudine resistant HBV mutants had favorable clinical outcomes at 6 months after EOT. Therefore, we can consider observing the clinical courses after discontinuation of lamivudine and it is not always required to overlap the adefovir for treating lamivudine resistant HBV mutants except for the treatment of patients with a high risk of developing decompensation.

Adefovir Dipivoxil Alone or in Combination with Ongoing Lamivudine in Patients with Decompensated Liver Disease and Lamivudine-resistant Hepatitis B Virus

The purpose of this prospective study was to evaluate the efficacy and safety of adefovir dipivoxil with or without ongoing lamivudine in decompensated lamivudine-resistant chronic hepatitis B patients. Forty-six hepatitis B e antigen (HBeAg)-positive patients with decompensated liver function and lamivudine-resistant hepatitis B virus (HBV) were assigned to adefovir dipivoxil monotherapy (n=18) or combination therapy with ongoing lamivudine (n=28) according to their own preference. After 24 weeks of treatment, 83% of monotherapy and 86% of combination therapy showed serum HBV DNA below detection limit (<0.5 pg/mL). Alanine aminotransferase (ALT) normalized in 78% and 82% respectively. Median Child-Pugh-Turcotte (CPT) score or Model for End-Stage Liver Disease (MELD) score reduced significantly by 3 or 5 point in monotherapy and 2 or 2 point in combination therapy respectively. There were no significant differences in rate of undetectable serum HBV DNA, median change of ALT and median reduction of CPT or MELD scores between the two groups. In conclusion, both adefovir dipivoxil monotherapy and combination therapy with ongoing lamivudine result in comparable virologic, biochemical, and clinical improvements in HBeAg-positive patients with decompensated liver function and lamivudine-resistant HBV. Combination with lamivudine showed no additional benefit over monotherapy during 24 weeks of treatment in these patients.

Therapeutic Effect of Adefovir Dipivoxil on Recurrent or de novo Infection of Hepatitis B Virus after Liver Transplantation: A Preliminary Report / 대한소화기학회지

BACKGROUND/AIMS: Anti-viral therapy using hepatitis B immune globulin and lamivudine could not prevent HBV recurrence after liver transplantation (LT) completely. Adefovir dipivoxil is a acyclic nucleotide phosphate analogue and known to have potent anti-HBV effect. In this study, we analyzed the therapeutic effect of adefovir for recurrent or de novo HBV infection after LT. METHODS: From December 2002 to October 2004, adefovir was administered in 12 post-LT patients of HBV infection (11 recurrent and 1 de novo infection). In these patients, lamivudine and other combined therapies were used before the introduction of adefovir. Thereafter, adefovir combined with lamivudine was administered to all patients. RESULTS: The duration of adefovir administration was 5.5-18 (median, 15.5) months. The median values of serum AST and ALT levels were significantly reduced from 86+/-80 IU/L and 140+/-103 IU/L, respectively before the adefovir administration to 42+/-19 IU/L and 38+/-33 IU/L after 2 months of administration. This trend of improved liver function persisted throughout the follow-up period. HBeAg seroconversion was achieved in 4 of 10 patients (40%) and HBsAg seroconversion was observed in 1 of 10 patients (10%). HBV DNA levels have decreased to undetectable levels by hybridization assay in 6 of 7 patients within the first 2 months of therapy. Nephrotoxicity and hypophosphatemia were not found in all of these patients. CONCLUSIONS: Based on this preliminary result, adefovir dipivoxil seems to be an effective and safe antiviral agent leading to viral inhibition and clinical improvement in post-LT patients with recurrent or de novo HBV infection.

Effect of phytohormone pretreatment on metabolic changes in Vigna radiata under salt stress.

Efficiency of pretreatment as foliar spray of indole-3-acetic acid, gibberellic acid and kinetin (6-furfuryl aminopurine) each ranging from 10(-7) to 10(-5) M in restoring the metabolic alterations imposed by NaCl salinity (E.C. value 4.0 m mhos/cm) was investigated in Vigna radiata (L.) Wilczek. Application of NaCl resulted in about 7% and 9% decrease in phenol content in mung bean leaf and root respectively. In leaf, NaCl caused 40% increase in polyphenol oxidase enzyme activity over the control set. This effect was accentuated in root, where salinity caused 200% increase in the enzyme activity. In leaf and root of mung-bean plant, ascorbic acid content decreased about 29% and 31% respectively under salinity stress as compared with control. Ascorbic acid oxidase enzyme activity increased under stress by about 55% and 23% respectively in leaf and root. It was noted that all the three growth regulators used in the present study were able to overcome to variable extents the adverse effects of stress imposed by NaCl solution.

Effect of IP3 and ryanodine treatments on the development of bovine parthenogenetic and reconstructed embryos

For parthenogenetic activation as a model system of nuclear transfer, microinjection and electroporation as activation treatments in bovine metaphase II oocytes were administered to each of three groups as follows: control group (treatments with Ca2+, Mg2+ -free PBS+100 micro M EGTA), IP3 group (control+25 micro M IP3) and IP3+ ryanodine group (control+25 micro M IP3+10 mM ryanodine). In experiments using microinjection, no significant differences were observed between any of the developmental stages of the electroporation experiment. For electroporation, cleavage rates were significantly higher in the IP3+ryanodine group than in the IP3 or control group (85.6% vs 73.7% or 67.6%, respectively). In the subsequent stages of embryonic development, such as morula and blastocyst formation, the IP3 and ryanodine group exhibited significantly higher rates of morula fomation than the IP3 or control groups (40.6% vs 24.2% or 16.7%, respectively). Similarly, the rate of blastocyst formation in the IP3+ryanodine group was significantly higher than the control group (16.3% vs 6.9%) but did not differ significantly from the IP3 group (16.3% vs 9.5%). In nuclear transfer, activation was performed at 30 hpm by microinjection and elecroporation with 25 micro M IP3+ 10 mM ryanodine followed by 6-DMAP treatment. No significant differences were observed at any stage of embryonic development and none of the embryos activated by electroporation reached either the morula or blastocyst stage. However, 3.8% and 1.9% of embryos activated by microinjection sucessfully developed to the morula and blastocyst stages, respectively. In conclusion, activation treatments using IP3 and ryanodine are able to support the development of bovine parthenogenetic and reconstructed embryos.

Estudio fase II de la administración secuencial de zidovudina (AZT), zalcitabina (ddC) y didanosina (ddI) a pacientes con infección avanzada por el virus de la inmunodeficiencia humana tipo 1 (VIH-1) / Study of administration consecutive periods of zidorudine (AZT), zalcitabine (ddC) and didanosine (ddI) infected individuals by HIV-1

Se estudiaron prospectivamente 18 pacientes infectados por VIH-1 y tratados con AZT (100 mg. tid durante 30 días), alternando con ddC (0.375 mg. tid durante 30 días) y con ddl (100 mg. tid durante 30 días) por periódos de 12 a 28 semanas, media 20. Doce pacientes recibieron este tratamiento después de otros antirretrovirales y 6 lo recibieron desde el diagnóstico. Antes y después de AZT/ddC/ddI, la media de células CD4 cambió de 211 a 256/ul (p=.06), y la media de peso de 68.2 a 67.4 kg. (p=.05). Las cifras de hemoglobina se incrementaron significativamente de 14.8 a 15.4 gr/dl (p=.02); este efecto fue más notable en pacientes previamente expuestos a AZT. En 6 de 12 casos desapareció la antigenemia p24 y en 2 de 5 disminuyeron los niveles de beta2-microglobulina sérica. En ningún paciente hubo hiperamilasemia como efecto adverso del ddl y en uno se instaló neuropatía periférica sensorial reversible como efecto colateral de ddC; en seis que habían recibido AZT cesaron los efectos adversos como intolerancia gástrica, cefalea y fiebre. La administración combinada secuencial de AZT/ddC/ddl fue bien tolerada y disminuyó la toxicidad de AZT