Comparative Clinical Evaluation of NeoPlex RB-8 with Seeplex PneumoBacter ACE for Simultaneous Detection of Eight Respiratory Bacterial Pathogens.

There are several convenient and accurate molecular assays to detect respiratory bacterial infection. The NeoPlex RB-8 detection kit (NeoPlex RB-8) is a new multiplex real-time PCR assay that simultaneously detects Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Haemophilus influenzae, Bordetella pertussis, Bordetella parapertussis, and Moraxella catarrhalis in a single test. This study compared the clinical concordance of NeoPlex RB-8 with another method, Seeplex PneumoBacter ACE detection assay (Seeplex PB ACE), which simultaneously detects S. pneumoniae, M. pneumoniae, C. pneumoniae, L. pneumophila, H. influenzae, and B. pertussis We tested 2,137 nasopharyngeal swab and sputum specimens using both assays. For discordant Bordetella parapertussis and M. catarrhalis specimens, we also performed bidirectional sequencing. For S. pneumoniae, M. pneumoniae, C. pneumoniae, L. pneumophila, H. influenzae, and B. pertussis, which are detected by both NeoPlex RB-8 and Seeplex PB ACE, the positive and negative agreement between the two assays ranged from 91.7 to 100% (κ = 0.918 to 1). S. pneumoniae and H. influenzae were the most discordant targets and measured with higher sensitivity and specificity by NeoPlex RB-8 than Seeplex PB ACE. For Bordetella parapertussis and M. catarrhalis, which are not detected by Seeplex PB ACE, NeoPlex RB-8 sensitivity and specificity were >99%. Overall, NeoPlex RB-8 was highly comparable to Seeplex PB ACE, but NeoPlex RB-8 was more clinically accurate, with higher throughput and more convenience.

Triticum aestivum ethanolic extract improves non-alcoholic fatty liver disease in mice fed a choline-deficient or high-fat diet.

BACKGROUND: Although non-alcoholic fatty liver disease (NAFLD) has become more prevalent with the rapid increase of obesity worldwide, no specific treatment has been developed. Several studies have shown that wheatgrass extract Triticum aestivum (TA) improves lipid metabolism. In the present study, we evaluated the efficacy of GM-T (an ethanolic TA extract) in a murine NAFLD model. Mice were separated into 12 groups (n = 10): two groups of normal diet, choline-deficient diet (CDD) or high-fat diet (HFD) with vehicle, CCD or HFD with silymarin (400 mg kg-1 day-1 ), and CCD or HFD with GM-T (100, 200 or 400 mg kg-1 day-1 ). The study was performed for 8 weeks for the CDD groups and 12 weeks for the HFD groups. RESULTS: In the CDD-fed mice, GM-T improved serum liver enzyme activities and liver inflammation score compared to vehicle. In the HFD-fed mice, GM-T improved blood lipid profiles, liver inflammation score, steatosis score and obesity compared to vehicle. CONCLUSION: The present study demonstrated that GM-T effectively improved NAFLD in mice via a mechanism that improved insulin resistance and lipid metabolism, suggesting the possibility of a functional dietary supplement to improve liver health, overall metabolic syndrome and obesity. © 2018 Society of Chemical Industry.

Effects of probiotics for the treatment of atopic dermatitis: a meta-analysis of randomized controlled trials.

BACKGROUND: The effects of probiotics on the treatment of atopic dermatitis (AD) are inconclusive. OBJECTIVE: To determine the clinical effect of probiotics in the management of AD overall and in different age groups. METHODS: A comprehensive search of databases through December 2013 was performed. For this meta-analysis, randomized controlled trials measuring the treatment effects of probiotics or synbiotics in patients diagnosed with AD were included. The primary outcome was a difference in Scoring of Atopic Dermatitis (SCORAD) values between the treatment and placebo groups overall and in different age populations. RESULTS: Twenty-five randomized controlled trials (n = 1,599) were available for this meta-analysis. Significant differences in SCORAD values favoring probiotics over the control were observed overall (mean -4.51, 95% confidence interval -6.78 to -2.24), in children 1 to 18 years old (-5.74, 95% confidence interval -7.27 to -4.20), and in adults (-8.26, 95% confidence interval -13.28 to -3.25). However, the effectiveness of probiotics in infants (<1 year old) with AD was not proved. The effect of synbiotic use was not significantly different from that of probiotic use. Treatment with a mixture of different bacterial species or of Lactobacillus species showed greater benefit than did treatment with Bifidobacterium species alone. CONCLUSION: The overall result of this meta-analysis suggests that probiotics could be an option for the treatment of AD, especially for moderate to severe AD in children and adults. However, no evidence was found supporting the beneficial role of probiotics in infants.

Comparison of digene hybrid capture 2, GeneMatrix PapilloScreen, and a PCR sequencing assay in detecting high-risk and probable high-risk oncogenic HPV genotypes in specimens from Korean women.

Most cervical cancers are caused by 15 high-risk (HR) and three probable high-risk (pHR) oncogenic types of human papillomavirus (HPV). However, current commercial HR HPV screening test products do not include pHR HPV genotypes. Recently, PapilloScreen has been developed to detect the 15 HR and three pHR HPV types. In this study, we evaluated the concordance levels and clinical performance of Hybrid Capture 2 (HC2), PapilloScreen, and a PCR sequencing assay in detecting HR and pHR HPV. The PapilloScreen (96.8 %) and PCR sequencing assay (96.8 %) demonstrated higher sensitivity than HC2 (80.7 %) for detecting HR and pHR HPV. The three assays showed similar specificities and positive or negative predictive values. The concordance levels were 86.5 % (κ = 0.68) and 86.5 % (κ = 0.67) between HC2 and PapilloScreen and between HC2 and PCR sequencing, respectively. A near-perfect concordance was observed between PapilloScreen and PCR sequencing (97.8 %, κ = 0.95). Overall, the agreement between the three assays suggests that the results obtained by the HC2 assay are more often discordant (12.6 %) than the PCR-based tests. In conclusion, PapilloScreen is highly sensitive for detecting high-grade CIN or cervical cancer. The PapilloScreen assay should be considered an accurate and sensitive method for detecting HR and pHR HPV infections and an epidemiological tool for prevalence studies as well as early diagnosis and intervention in HR and pHR HPV infections.

Chimeric adenovirus-based herpes zoster vaccine with the tPA signal peptide elicits a robust T-cell immune response.

Herpes zoster (HZ), or shingles, is caused by reactivation of the varicella-zoster virus (VZV), which remains latent in the sensory ganglia until immunity wanes with age. The representative HZ vaccine, Shingrix is efficacious but causes side effects due to vaccine adjuvants. Therefore, the development of highly efficacious vaccines with minimal side effects is required. We developed chimeric adenovirus vector (ChimAd)-based HZ vaccine candidates encoding the VZV glycoprotein E (gE). These candidates include ChimAd-tPAgE, in which the signal peptide is replaced with tissue plasminogen activator (tPA), and ChimAd-WTgE, which retains the original signal peptide. C57BL/6 mice were immunized with VZV-vaccine candidates, and cellular and humoral immune responses were evaluated using interferon-γ ELISPOT and ELISA. The ChimAd-based HZ vaccines induced high levels of gE-specific antibodies and cell-mediated immunity. ChimAd-tPAgE (optimal dose: 1 × 107 IFU) elicited a more robust gE-specific T-cell response than Shingrix and Zostavax, showing potential as HZ prophylactic vaccines.

Multidrug-resistant hepatitis B virus resulting from sequential monotherapy with lamivudine, adefovir, and entecavir: clonal evolution during lamivudine plus adefovir therapy.

Whether multidrug-resistant (MDR) hepatitis B virus (HBV) harbors mutations co-located in the same HBV clones that confer reduced sensitivity to antiviral therapy remains uncertain. This study investigated the evolution of MDR HBV strains developed from sequential monotherapy with lamivudine (LAM), adefovir (ADV), and entecavir (ETV) during LAM plus ADV salvage therapy. Sera were obtained from six patients who had developed sequential resistance to LAM, ADV, and ETV before and during LAM plus ADV therapy. The HBV genomes from each patient were amplified, cloned, and sequenced. Among 6 sets of 20 clones obtained before salvage therapy, all clones harbored the rtM204V mutation, and ETV-resistant mutations were detected with the rtM204V in 108 clones. The rtA181 mutation was not detected at baseline, but emerged in five patients during therapy. Among 9 sets of 20 clones obtained during salvage therapy, 39 clones harbored rtA181T/V ± rtN236T mutations, which were detected in the absence of rtM204 and ETV-resistant mutations in 37 clones (94.9%). Only two clones (5.1%) harbored both rtA181T/V and ETV-resistant mutations. The rtA181T/V mutation emerged after reversion from ETV-resistant mutants to wild-type HBV. Five patients achieved a partial virologic response to LAM plus ADV therapy. In conclusion, the majority of MDR mutations existed in different genomes. Suboptimal response to LAM plus ADV therapy may not result from the co-localization of MDR HBV mutations in the same genome, but instead the low antiviral potency of these drugs. Thus, more potent antiviral drug combinations may be an effective salvage therapy for patients infected with MDR HBV.

Mutational complex genotype of the hepatitis B virus X /precore regions as a novel predictive marker for hepatocellular carcinoma.

This study explored the combined effect of number and pattern of mutations in the X/precore regions of the hepatitis B virus (HBV) genome, mutational complex genotype (MCG), on hepatocellular carcinoma (HCC) development. Sequence variations were determined by direct sequencing and multiplex restriction fragment mass polymorphism analysis in 150 age-, sex- and hepatitis B e antigen (HBeAg) status-matched patients with and without HCC. In addition, a longitudinal study and an external validation of MCG were conducted. All were HBV subgenotype C2. Eight high-frequency mutations (G1613A, C1653T, T1753V, A1762T, G1764A, A1846T, G1896A and G1899A) were significantly associated with HCC. Whereas C1653T, T1753V, G1764A and A1846T were independent mutational factors for HCC, the significance of these individual mutations was negligible when analyzed with all clinico-virological variables. The total number of mutations was the only independent viral factor for HCC, irrespective of HBeAg status. There was a significant dose-risk relationship between the number of mutations and HCC, in which high risks for HCC were associated with mutation numbers ≥ 6. Pattern analysis of the mutations revealed disparity in distribution among the top seven high-risk mutation combination patterns, which accounted for 40 and 2.7% of HCC and non-HCC cases, respectively. The predictive accuracy of the high-risk mutations for HCC was similar to that of α-fetoprotein. Longitudinal and external validation studies also supported the association of mutation number with HCC development. MCG in the HBV X/precore regions is a risk indicator for HCC, and might serve as a new guide to the HCC screening scheme for chronic HBV carriers.

Adefovir-based combination therapy with entecavir or lamivudine for patients with entecavir-refractory chronic hepatitis B.

This study evaluated the efficacy of adefovir (ADV) plus lamivudine (LAM) or ADV add-on therapy for patients with entecavir (ETV)-refractory hepatitis B infection. Twenty-nine ETV-resistant and 8 patients with suboptimal response to ETV were enrolled. Twenty-seven patients received ADV + LAM therapy and 10 patients received ADV + ETV therapy for >24 weeks. In 29 patients who were ETV-resistant, the mean reduction in HBV DNA levels at 24 weeks was not different between the ADV + LAM and ADV + ETV groups (-1.98 log(10) IU/ml vs. -2.16 log(10) IU/ml; P = 0.792). Primary non-response was observed in 52.2% (12/23) of ADV + LAM group and 33.3% (2/6) of ADV + ETV group (P = 0.651). Initial virologic response (IVR) was observed in 17.4% (4/23) of ADV + LAM group and 33.3% (2/6) of ETV + ADV group (P = 0.362). In eight patients with suboptimal response to ETV, the ADV + ETV group had a greater reduction in HBV DNA at 24 and 48 weeks than the ADV + LAM group (-2.29 log(10) IU/ml vs. -0.09 log(10) IU/ml and -2.04 log(10) IU/ml vs. -0.72 log(10) IU/ml; P = 0.020 and P = 0.012, respectively). Primary non-response and IVR did not significantly differ between the two groups [100% (4/4) vs. 50% (2/4) and 0% (0/4) vs. 50% (2/4); P = 0.429 and P = 0.429, respectively]. The antiviral efficacies of ADV-based therapy with ETV or LAM for patients with ETV-resistant hepatitis B were limited and did not differ between the two groups. However, adding ADV to ETV may be more effective than ADV + LAM therapy in patients with suboptimal virologic response to ETV.

Analytical and clinical performances of a restriction fragment mass polymorphism assay for detection and genotyping of a wide spectrum of human papillomaviruses.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry-based restriction fragment mass polymorphism (RFMP) assay was adapted to human papillomavirus (HPV) genotyping. The analytical sensitivity and the clinical utility were evaluated by testing defined HPV genome equivalents and a total of 426 specimens composed of normal cytology, atypical squamous cells of undetermined significance, low grade squamous intraepithelial lesion, high grade squamous intraepithelial lesion and invasive squamous cell carcinoma. The RFMP assay was able to detect 38.4-114.6 genomic equivalents of a wide variety of HPV types. The RFMP assay detected 34 different HPV genotypes in cervical samples of which 8% were found to be multiple-type infections. The high-risk HPV positivity rate according to the histological diagnosis was 7.9% (8/101), 31.7% (38/120), 50% (55/110), 86% (37/43), 96.2% (50/52) in normal, atypical squamous cells of undetermined significance, low grade squamous intraepithelial lesion, high grade squamous intraepithelial lesion and squamous cell carcinoma subgroups, respectively. Diagnostic sensitivities/specificities for the cervical lesions of squamous cell carcinoma and high grade squamous intraepithelial lesion or worse histology were found to be 96.2%/92.1% and 91.6%/92.1%, respectively. The sensitivity, accuracy, wide range of genotype identification and high-throughput capacity with cost-effectiveness of the test consumables make the RFMP assay suitable for mass screening and monitoring of HPV-associated cervical cancer.

Analysis of TPOX short tandem repeat locus with matrix-associated laser desorption/ionization time-of-flight-based restriction fragment mass polymorphism assay.

Short tandem repeat (STR) loci are routinely analyzed by capillary electrophoresis. However, this method has several disadvantages, including long operational time, low throughput, and inaccuracy. As a result of the introduction of matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI), mass spectrometry has become an alternative method for genotyping polymorphic STR loci. Here we established a restriction fragment mass polymorphism (RFMP) assay for genotyping STR locus, TPOX, by typeIIS restriction endonuclease cleavage of polymerase chain reaction (PCR) amplicon followed by MALDI-TOF mass spectrometry. The resulting TPOX genotypes from this assay were in good agreement with the results from direct DNA sequencing and GeneScan assays. Our results showed that the RFMP assay is an accurate and high-throughput method for analyzing long DNA fragments such as STR markers. Further research with multiple STR loci may allow this assay to be used for diverse applications such as forensics, paternity tests, and detection of genetic disorders.

A purified inactivated vaccine derived from Vero cell-adapted zika virus elicits protection in mice.

The Zika virus (ZIKV) outbreak in 2015-2016 raised public health concerns and created a pressing need for vaccine development. However, no vaccine has been developed and most of the ones under development use a single serotype of ZIKV. In this study, we established a Vero cell-adapted ZIKV strain (GMZ-002) and developed a purified inactivated virus (PIV) vaccine. GMZ-002 presented significantly increased productivity in Vero cells, and IFNAR1-blocked C57BL/6 mice administered two doses of the PIV were fully protected against lethal challenge. Vaccine efficacy was illustrated by the high level of serum neutralizing antibodies and strong innate immune response, along with an absence of detectable viremia in vaccinated mice. Furthermore, anti-sera neutralized both African and Asian genetic lineages of the virus in vitro. Our results suggest that GMZ-002 PIV elicited robust and persistent protective immunity, and therefore represents a promising vaccine candidate for ZIKV.

[Genotypic resistance to entecavir in chronic hepatitis B patients].

BACKGROUND/AIMS: The prevalence and clinical characteristics of entecavir (ETV) resistance is not well known. The aim of this study was to determine the frequency of genotypic resistance in nonresponders and virologic breakthrough (VBT) patients. METHODS: The medical records of 76 chronic hepatitis B patients treated for a least 6 months from October 2006 to October 2008 were reviewed retrospectively. We divided patients into two groups: nucleoside analogue (NA)-naive patients (n=38) and LAM experienced patients (n=38). NA-naive and LAM experienced patients received ETV at 0.5 and 1.0 mg/day, respectively. The virologic response and VBT were investigated in both groups. We used the multiplex restriction fragment mass polymorphism (RFMP) method to test genotypic resistance at the rtI169, rtT184, rtS202, rtM204, and rtM250 sites. RESULTS: Age, gender, serum ALT, and HBV DNA level before treatment did not differ between the groups. Neither VBT nor nonresponse was observed in the NA-naive group, whereas VBT and nonresponse were observed in three patients each in the lamivudine (LAM)-experienced group; all six patients had YMDD mutation at study enrollment, all three patients with VBT had genotypic resistance to ETV, but the three nonresponse patients did not have genotypic resistance to ETV. CONCLUSIONS: We suspect that VBT is mostly associated with genotypic resistance to ETV. However, nonresponse might be associated with the continuance or reselection of the YMDD mutant in LAM-experienced patients.

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.

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.

Efficacy of gemcitabine in patients with non-small cell lung cancer according to promoter polymorphisms of the ribonucleotide reductase M1 gene.

PURPOSE: High ribonucleotide reductase M1 (RRM1) expression in resected lung cancers has been associated with better clinical outcomes. However, gemcitabine-treated patients with high tumoral RRM1 expression generally evidence poor prognoses due to the decreased efficacy of gemcitabine therapy. This study was designed in accordance with the hypothesis that polymorphisms (-37 and -524) of the RRM1 promoter gene sequence, which regulate RRM1 expression, could influence the efficacy and prognosis of lung cancer patients treated with gemcitabine-based chemotherapy. EXPERIMENTAL DESIGN: A retrospective dataset of 97 patients with advanced non-small cell lung cancer treated with gemcitabine regimens as a first-line treatment was studied in this work. The allelotyping of RRM1 promoter polymorphisms was conducted via real-time PCR using genomic DNA obtained from peripheral WBC. RESULTS: The RRM1 promoter allelotype was RR37CC-R524TT in 58 patients, RR37AC-RR524CT in 29 patients, and other allelotypes in 10 patients. The response rate for gemcitabine-containing chemotherapy was 49.5%. The response rate was significantly higher in the RR37AC-RR524CT group (65.5%) compared with the group containing other allelotypes (42.6%; P = 0.039). Overall survival and progression-free survival did not differ significantly by allelotype. CONCLUSIONS: We detected significant differences in response rates to gemcitabine-based chemotherapy according to the allelotypes of the RRM1 promoter sequence, which could be determined using the germline DNA. Further functional and clinical studies will be required before this can be used as a predictive marker.

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.

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.

Identification of hepatitis C virus genotype 6 in Korean patients by analysis of 5' untranslated region using a matrix assisted laser desorption/ionization time of flight-based assay, restriction fragment mass polymorphism.

Previous surveys of the prevalence of hepatitis C virus (HCV) in Korea have identified types 1 and 2, but little has been said of other genotypes and viral subtypes. In this study, HCV genotypes in Korea were investigated using Restriction Fragment Mass Polymorphism (RFMP) assay, a sensitive and specific method for genotyping based on MALDI-TOF mass spectrometry. A total of 1,043 independent serum samples from HCV-infected patients were analyzed. Of interest, 15 subjects (1.4%) were determined to contain HCV genotype 6 and 46 subjects (4.4%) contained mixed genotypes with the most prevalent genotypes being HCV 1b and 2a/c (45.0% and 35.4%, respectively). The 15 subjects with HCV genotype 6 comprised eight cases of subtype 6c, including one case of mixed infection with 1b, three cases of HCV 6a, and six cases of unassigned subtypes. Sequencing corroborated the identity of genotype 6 from 13 subjects, while the line probe assay (LiPA) mis-identified them as genotype 1b. The majority (7/9) of the genotype 6 patients enrolled for interferon/ribavirin therapy, achieved a sustained virologic response. The ability of the RFMP assay to differentiate various HCV genotypes should enable better analysis of the relationship between HCV genotype and disease prognosis.

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.