RESUMO
Mitochondrial DNA m.3243A > G mutation causes mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and its associated multi-organ disorders, including diabetes. To clarify associations between m.3243A > G organ heteroplasmy and clinical phenotypes, including the age at death, we combined genetic and pathological examinations from seven unreported and 36 literature cases of autopsied subjects. Clinical characteristics of subjects were as follows: male, 13; female, 28; unknown, 2; the age at death, 36.9 ± 20.2 [4-82] years; BMI, 16.0 ± 2.9 [13.0-22.3]; diabetes, N = 21 (49%), diabetes onset age 38.6 ± 14.2 years; deafness, N = 27 (63%); stroke-like episodes (StLEp), N = 25 (58%); congestive heart failure (CHF), N = 15 (35%); CHF onset age, 51.3 ± 14.5 years. Causes of death (N = 32) were as follows: cardiac, N = 13 (41%); infection, N = 8 (25%); StLEp, N = 4 (13%); gastrointestinal, N = 4 (13%); renal, N = 2 (6%); hepatic, N = 1 (2%). High and low heteroplasmies were confirmed in non-regenerative and regenerative organs, respectively. Heteroplasmy of the liver, spleen, leukocytes, and kidney for all subjects was significantly associated with the age at death. Furthermore, the age at death was related to juvenile-onset (any m.3243A > G-related symptoms appeared before 20) and stroke-like episodes. Multiple linear regression analysis with the age at death as an objective variable showed the significant contribution of liver heteroplasty and juvenile-onset to the age at death. m.3243A > G organ heteroplasmy levels, particularly hepatic heteroplasmy, are significantly associated with the age at death in deceased cases.
Assuntos
Diabetes Mellitus , Síndrome MELAS , Acidente Vascular Cerebral , Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Pré-Escolar , Criança , Adolescente , Adulto Jovem , Idoso de 80 Anos ou mais , Heteroplasmia , DNA Mitocondrial/genética , Mutação , Acidente Vascular Cerebral/complicações , Fígado/patologia , Síndrome MELAS/genéticaRESUMO
A 15-year-old boy was referred to our hospital with elevated hepatobiliary enzyme levels and jaundice. Magnetic resonance cholangiopancreatography performed at the previous medical facility revealed a stricture of the intrahepatic and extrahepatic bile duct. Computed tomography showed dilatation and wall thickness of the intrahepatic bile ducts. Primary sclerosing cholangitis or cholangiocarcinoma was suspected. Endoscopic retrograde cholangiopancreatography (ERCP) showed stricture in the intrahepatic and extrahepatic bile duct. On admission, the eosinophil count in the peripheral blood was normal; however, rapid hypereosinophilia in the peripheral blood was observed after admission, leading us to suspect eosinophilic cholangitis (EC). A bile duct biopsy showed inflammatory cells and eosinophil infiltration during a second ERCP. The patient was diagnosed with EC based on histopathology.
RESUMO
PURPOSE: Patients with epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer with BIM deletion polymorphism may have a limited response to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, some results of previous reports are discordant. It is necessary to evaluate the relationship between BIM polymorphism and the efficacy of EGFR-TKIs. METHODS: We retrospectively analyzed patients treated with EGFR-TKIs. We collected serum samples from patients before EGFR-TKI administration. We analyzed BIM deletion polymorphism and BIM single nucleotide polymorphism in exon 5 c465C > T by the Invader® assay. RESULTS: BIM deletion polymorphism was identified in 27 of 194 patients (13.9%). BIM single nucleotide polymorphism was identified in 29 of 194 patients (14.9%). The overall response ratio was 81.5% in patients with BIM deletion polymorphism, 89.7% with BIM single nucleotide polymorphism, and 83.6% with BIM wild type. Median progression-free survival was 10.3 months with BIM deletion polymorphism, 8.5 months with BIM single nucleotide polymorphism, and 10.4 months with BIM wild type. Overall survival was 38.4 months with BIM deletion polymorphism, 29.1 months with BIM single nucleotide polymorphism, and 31.6 months with BIM wild type. There were no significant differences between the groups in overall response ratio, progression-free survival, and overall survival. CONCLUSIONS: BIM polymorphism does not affect EGFR-TKI efficacy.
Assuntos
Proteína 11 Semelhante a Bcl-2/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Idoso , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Cloridrato de Erlotinib/farmacologia , Cloridrato de Erlotinib/uso terapêutico , Éxons/genética , Feminino , Mutação com Ganho de Função , Gefitinibe/farmacologia , Gefitinibe/uso terapêutico , Humanos , Japão/epidemiologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Intervalo Livre de Progressão , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinonas/farmacologia , Quinazolinonas/uso terapêutico , Estudos Retrospectivos , Deleção de SequênciaRESUMO
Deletion polymorphism of BCL-2-like protein 11 (BIM) is specifically found in East Asia. To explain some epidemiological discrepancies between Asian and Western countries, we analyzed a silent single nucleotide polymorphism (SNP) in exon 5 (c465C > T) and a deletion site (2903 bp) in intron 2 in 77 patients with follicular lymphoma by the Q-invader method using PCR. In females, 5-year progression-free survivals (PFS) were 20.0% in the BIM deletion group, 66.7% in the SNP group and 81.5% in the wild-type (WT) group (p = .0012). In the WT group, 5-year PFS was 40.4% in males (p = .0448 vs. female PFS). This tendency was strengthened in patients receiving rituximab (26.9% vs. 84.2%, p = .006). Superior PFS in the WT females in Japan was comparable with the results of cohort studies in the United States and Sweden. Favorable prognosis in Japanese females may be masked by the BIM deletion polymorphism.
Assuntos
Proteína 11 Semelhante a Bcl-2/genética , Biomarcadores Tumorais , Linfoma Folicular/genética , Linfoma Folicular/mortalidade , Polimorfismo Genético , Deleção de Sequência , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Feminino , Humanos , Japão/epidemiologia , Estimativa de Kaplan-Meier , Linfoma Folicular/diagnóstico , Linfoma Folicular/epidemiologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Polimorfismo de Nucleotídeo Único , Prognóstico , Fatores SexuaisRESUMO
BACKGROUND: Oncogenic human papillomavirus (HPV) infection, particularly multiple HPV types, is recognized as a necessary cause of anal cancer. However, a limited number of studies have reported the prevalence of anal HPV infection in Asia. We determined the prevalence, genotypes, and risk factors for anal HPV infection in Japanese HIV-positive men who have sex with men (MSM), heterosexual men, and women. METHODS: This cross-sectional study included 421 HIV-positive patients. At enrollment, we collected data on smoking, alcohol, co-morbidities, drugs, CD4 cell counts, HIV RNA levels, highly active anti-retroviral therapy (HAART) duration, sexually transmitted infections (STIs), and serological screening (syphilis, hepatitis B virus, Chlamydia trachomatis, Entamoeba histolytica). Anal swabs were collected for oncogenic HPV genotyping. RESULTS: Oncogenic HPV rate was 75.9% in MSM, 20.6% in heterosexual men, and 19.2% in women. HPV 16/18 types were detected in 34.9% of MSM, 17.7% of heterosexual men, and 11.5% of women. Multiple oncogenic HPV (≥2 oncogenic types) rate was 54.6% in MSM, 8.8% in heterosexual men, and 0% in women. In univariate analysis, younger age, male sex, MSM, CD4 <100, HIV viral load >50,000, no administration of HAART, and having ≥2 sexually transmitted infections (STIs) were significantly associated with oncogenic HPV infection, whereas higher smoking index and corticosteroid use were marginally associated with oncogenic HPV infection. In multivariate analysis, younger age (OR, 0.98 [0.96-0.99]), MSM (OR, 5.85 [2.33-14.71]), CD4 <100 (OR, 2.24 [1.00-5.01]), and having ≥2 STIs (OR, 2.81 [1.72-4.61]) were independently associated with oncogenic HPV infection. These 4 variables were also significant risk factors for multiple oncogenic HPV infection. CONCLUSIONS: Among Japanese HIV-infected patients, approximately two-thirds of MSM, one-fifth of heterosexual men, and one-fifth of women have anal oncogenic HPV infection. Younger age, MSM, ≥2 STIs, and immunosuppression confer a higher risk of infection with oncogenic HPV and multiple oncogenic types.
Assuntos
Doenças do Ânus/epidemiologia , Soropositividade para HIV/epidemiologia , Papillomavirus Humano 16 , Papillomavirus Humano 18 , Infecções por Papillomavirus/epidemiologia , Adulto , Fatores Etários , Doenças do Ânus/virologia , Estudos Transversais , Feminino , Soropositividade para HIV/tratamento farmacológico , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/virologia , Fatores SexuaisRESUMO
Hepatitis C virus (HCV) is a major worldwide public health problem, and mutations at amino acids 70 and 91 in the genotype 1b core region predict the effectiveness of combination therapy with peginterferon and ribavirin. An assay based on the Q-Invader technology was developed to determine the relative ratios of the mutant to wild-type virus with high sensitivity. The assay detected a minor type plasmid that constituted only 1% of a mixture of plasmids containing wild-type and mutant sequences. The calculated ratios agreed with those of the template DNA. A total of 123 serum samples of HCV in Japan were examined with the Q-Invader assay. The Q-Invader assay detected all of the mutations that were detected by direct sequencing and even some mutants that direct sequencing could not. PCR with mutant specific primers confirmed those mutations found by the Q-Invader assay and not by direct sequencing. The Q-Invader assay, thus, is a useful tool for detecting mutations at positions 70 and 91 in the HCV-1b core region.
Assuntos
Substituição de Aminoácidos , DNA Viral/sangue , Hepacivirus/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas do Core Viral/genética , Antivirais/uso terapêutico , Sequência de Bases , DNA Viral/genética , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Interferon-alfa/uso terapêutico , Mutação , Plasmídeos/genética , Plasmídeos/isolamento & purificação , Polietilenoglicóis/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Ribavirina/uso terapêutico , Análise de Sequência de DNARESUMO
When quantifying periodontopathic bacteria, it is important to use a convenient method that does not produce false negative results. The Invader assay is a convenient method because it does not involve gene amplification. The purpose of this study was to evaluate the validity of the Invader assay to quantify periodontopathic bacteria. The Invader technology was applied in quantifying five periodontopathic bacteria (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola). The Invader assay produced a linear quantitative detection range over concentrations spanning seven exponential values, with a detection limit of 10(3.7) copies/tube and intra-day and inter-day variance of 0.1% to 4.7% and 0.1% to 3.4%, respectively, in quantifying five periodontopathic bacteria. We compared the results of the Invader assay with those of real-time polymerase chain reaction (PCR) performed for quantifying five periodontopathic bacteria in 22 patients with periodontitis. Among the Invader-detectable bacterial strains of each species, significant correlations were observed in the counts of concerned bacterial species between these two methods, with correlation coefficients ranging from 0.757 to 0.996. This study validated repeatability and reproducibility of the Invader assay in quantifying periodontopathic bacteria and demonstrated consistent agreement between the Invader assay and real-time PCR in quantifying periodontopathic bacteria.
Assuntos
Bactérias/classificação , Técnicas de Genotipagem/métodos , Tipagem Molecular/métodos , Periodontite/microbiologia , Saliva/microbiologia , Adulto , Bactérias/genética , Bactérias/isolamento & purificação , DNA Bacteriano/análise , Feminino , Humanos , Limite de Detecção , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos TestesRESUMO
Drug resistance is a serious complication in the treatment of chronic myeloid leukemia (CML). The most common and best-characterized mechanism of secondary imatinib resistance in CML is the development of kinase domain mutations in the BCR-ABL gene. Second-generation tyrosine kinase inhibitors, such as dasatinib or nilotinib, overcome most of these mutations, but they are not effective against the T315I mutant. To determine whether these mutations contribute to clinical resistance, it is necessary to monitor the ratio of the mutant and wild-type forms. Here, we developed a polymerase chain reaction (PCR)-Invader assay for comparative quantitative analysis (qPI assay) of BCR-ABL transcripts with the T315I mutant clone. T315I ratios were calculated for the wild-type and mutant fold-over-zero (FOZ) values. In examination with 2 kinds of plasmids containing wild-type or T315I mutant PCR amplicons, mutant FOZ values were detected down to 1% of the total. The results of 12 serial samples from 2 patients (case A: Philadelphia-positive acute lymphoblastic leukemia and case B: CML) with the T315I mutant clone were compared with those of direct sequencing or 2 kinds of allele-specific oligonucleotide (ASO)-PCR. All samples showed the T315I mutation by qPI assay and ASO-PCR, and 10 samples showed it by direct sequencing. Significant correlation (correlation coefficient; r2 = 0.951) was noted between the qPI assay and quantitative ASO-PCR to analyze T315I mutant ratios. Thus, the qPI assay is a useful method for evaluating the T315I mutant clone in BCR-ABL transcripts.
Assuntos
Análise Mutacional de DNA/métodos , Genes abl , Mutação Puntual , Reação em Cadeia da Polimerase/métodos , Idoso , Antineoplásicos/uso terapêutico , Sequência de Bases , Benzamidas , Análise Mutacional de DNA/estatística & dados numéricos , DNA de Neoplasias/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Mesilato de Imatinib , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Leucemia Mieloide de Fase Crônica/genética , Masculino , Pessoa de Meia-Idade , Sondas de Oligonucleotídeos/genética , Piperazinas/uso terapêutico , Reação em Cadeia da Polimerase/estatística & dados numéricos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pirimidinas/uso terapêutico , RNA Neoplásico/genética , Pesquisa Translacional BiomédicaRESUMO
BCR-ABL1 kinase domain mutations were evaluated in 60 imatinib-resistant patients with Philadelphia-positive (Ph(+)) leukemia using PCR-Invader assay and direct sequencing. In chronic myelogenous leukemia (CML)--chronic phase (CP), 5 had P-loop mutations and 3 had T315I mutations. CML-CP patients with high Sokal score showed significantly higher incidence of mutations. P-loop mutations were associated with higher risk of disease progression. In CML-advanced phase, P-loop mutations and T315I mutation were associated with significantly shorter survival. In Ph(+) acute lymphoblastic leukemia, overall survival was poor irrespective of mutational status. The PCR-Invader assay is useful for screening of mutations and prediction of prognosis.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação , Piperazinas/uso terapêutico , Reação em Cadeia da Polimerase/métodos , Pirimidinas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Benzamidas , Análise Mutacional de DNA/métodos , Feminino , Proteínas de Fusão bcr-abl/análise , Proteínas de Fusão bcr-abl/química , Testes Genéticos/métodos , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Fosfotransferases/química , Fosfotransferases/genética , Prognóstico , Estrutura Terciária de Proteína/genética , Análise de Sobrevida , Adulto JovemRESUMO
Early detection of resistant mutations of hepatitis B virus (HBV) is important for patients on nucleos(t)ide analog therapy. An assay based on the PCR-Invader technology was developed to detect resistant mutations with high sensitivity. The assay specifically detects mutations at codons 180, 181, 184, 202, 204, and 250 of the HBV polymerase reverse transcriptase domain. These mutations result in resistance to lamivudine and entecavir. In mixtures of plasmids containing wild-type and resistant mutants, fold-over-zero values for resistant mutations were detected in 2% of the total. Seventy-five serum samples from patients, whose treatment had been switched from lamivudine to entecavir, were examined by the PCR-Invader assay and direct sequencing. The PCR-Invader assay detected all resistant mutations that were detected by direct sequencing and even detected the presence of mutants that direct sequencing could not. Cloning sequencing confirmed those mutations found by the PCR-Invader assay and not by direct sequencing. The PCR-Invader assay is a useful tool for the early detection of drug-resistant mutations.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Hepatite B/virologia , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase/métodos , DNA Viral/genética , Guanina/análogos & derivados , Guanina/farmacologia , Vírus da Hepatite B/isolamento & purificação , Humanos , Lamivudina/farmacologia , Testes de Sensibilidade Microbiana/métodos , DNA Polimerase Dirigida por RNA/genética , Sensibilidade e Especificidade , Proteínas Virais/genéticaRESUMO
Human papillomavirus (HPV) is associated with several cervical diseases. A simple, rapid, cost-effective assay for identifying viral genotypes would greatly aid efforts for early detection and disease prevention. A real-time polymerase chain reaction monitoring Invader reaction assay (Q-Invader assay) was developed for genotyping and comparative quantitative analysis of 14 high-risk HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 67, and 68). A total of 131 cervical samples containing HPV in Japan were examined by Q-Invader assay, and the results were compared with those from sequencing with consensus and genotype-specific primers. Genotypes determined by Q-Invader agreed with those of sequencing in all samples. Coinfections with multiple high-risk genotypes were correctly identified by Q-Invader assay in 27 samples. In addition, the relative ratios of the genotypes were determined. Thus, Q-Invader assay is a useful tool for genotyping and comparative quantitative analysis of high-risk HPV types.
Assuntos
Alphapapillomavirus/genética , Infecções por Papillomavirus/microbiologia , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , Humanos , Sensibilidade e EspecificidadeRESUMO
The Invader PLUS technology is a sensitive, rapid method for the detection and quantification of nucleic acid. While the original technology is based on the amplification by polymerase chain reaction (PCR) of the target sequence followed by its detection using the Invader technology, the current modification allows simultaneous PCR amplification and Invader reaction. The PCR primers and the Invader probes are designed to operate at the same temperature. This allows simpler design and faster results. This technology has been applied for the quantification of six periodontitis-related bacteria (Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Toreponema denticola, Tannerella forsythensis and Fusobacterium nucleatum). Direct comparison of this modified Invader PLUS with real-time PCR demonstrated similar linear range. Furthermore, testing of 64 volunteers showed a good correlation between both technologies with correlation factors r2 spanning between 0.827 and 0.987. We demonstrated here that the proposed improvement of the Invader PLUS allows the detection and quantification of DNA sequences using a simple design and protocol that can be implemented in clinical testing.
Assuntos
Bactérias/isolamento & purificação , DNA Bacteriano/análise , Técnicas Genéticas , Periodontite/microbiologia , Bactérias/química , Bactérias/genética , DNA Bacteriano/genética , HumanosRESUMO
We recently developed an Invader assay combined with reverse transcriptase polymerase-chain-reaction in order to quantify T315I bcr-abl transcripts. Using this assay, we serially monitored T315I bcr-abl transcripts in chronic myeloid leukemia (CML) patients whose bcr-abl transcripts were still detectable at 6 months after starting imatinib therapy. Although, we continued to monitor bcr-abl transcripts in 14 CML patients (13 chronic phases and 1 accelerated phase) for up to 12 months, there were no patients who were apparently resistant to imatinib due to the T315I mutation. In contrast, in a case of Philadelphia chromosome-positive acute lymphoid leukemia being treated with chemotherapy including imatinib, we monitored both wild-type and T315I bcr-abl transcripts, and found increased levels of T315I transcripts during relapse (0% at the time of diagnosis and 54.8% at relapse). Thus, our new approach could be a useful tool to study the kinetics of mutant clones and the pharmacokinetics of drug resistance with regard to the T315I mutation.
Assuntos
Proteínas de Fusão bcr-abl/análise , Proteínas de Fusão bcr-abl/genética , Técnicas Genéticas , Adulto , Idoso , Feminino , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Pessoa de Meia-Idade , Mutação/genéticaRESUMO
With its broad effective range for fluorescence detection, real-time PCR is one of the most valuable techniques for quantitation in molecular biology. A modified real-time PCR assay is described for determining viral load. The assay uses fluorescence to measure the number of PCR amplicons by monitoring the Invader reaction in four steps in the thermal cycle. The Invader reaction with its cleavase was performed at moderate temperature after the amplicon was denatured at a high temperature. The method was as effective as real-time PCR with a TaqMan probe in determining the quantity of virus in samples of human papillomavirus type 16. Importantly, the assay allows the use of a common probe for multiple reactions. Thus, this method is a rapid inexpensive assay with a common fluorescence probe that does not depend on the conformation of the target DNAs.
Assuntos
Endodesoxirribonucleases/metabolismo , Papillomavirus Humano 16 , Infecções por Papillomavirus , Reação em Cadeia da Polimerase/métodos , Neoplasias do Colo do Útero , Carga Viral , Colo do Útero/virologia , DNA Viral/análise , DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 16/fisiologia , Humanos , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/virologiaRESUMO
Hepatitis B virus is a worldwide public health problem. A simple and effective test to identify viral genotypes would greatly aid efforts to understand and control the spread of this disease. A serial invasive signal amplification reaction assay (PCR-Invader assay) was developed for distinguishing the known eight genotypes (A-H) and four subgenotypes (Aa, Ae, Ba, Bj) of hepatitis B virus (HBV). The preS/S and core regions were amplified by multiplex PCR and delivered to 12 wells containing genotype-specific Invader probes. By observing the fluorescence patterns in the wells, HBV sub/genotypes can be assigned. A total of 505 serum samples containing HBV/HBsAg in Japan was examined by PCR-Invader and compared the results with those from ELISA assays with monoclonal antibodies against epitopes on gene products of the preS2 region and with a genotype-specific probe assay (GSPA) based on the preS1 region. Genotypes determined by the PCR-Invader agreed with those of the ELISA method in 98.2% of cases and with the GSPA method in 97.1% of cases. Co-infection with two distinct genotypes was correctly identified by the PCR-Invader in four serum samples, as determined by GSPA. Thus, the PCR-Invader assay is a useful tool for detecting the 10 known HBV sub/genotypes.
Assuntos
Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Hepatite B/virologia , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Ensaio de Imunoadsorção Enzimática , Corantes Fluorescentes/análise , Genótipo , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Humanos , Técnicas de Sonda Molecular , Dados de Sequência Molecular , Soro/virologiaRESUMO
BACKGROUND/AIMS: Liver negative-strand hepatitis C virus (HCV) RNA is the most direct indicator of active viral replication but has only been examined in a few semiquantitative studies. METHODS: Positive- and negative-strand HCV RNA in the right (R) and left (L) liver lobes was quantified by rTth-based strand-specific real-time polymerase chain reaction for 48 chronic hepatitis C patients. RESULTS: Close correlations between lobes were seen for positive- and negative-strand amounts (r = 0.950; P < 0.001 and r = 0.920; P < 0.001, respectively). The ratio of negative to positive strands (median, 0.14 for R and 0.13 for L) varied by 2 log directly in relation to HCV replication assessed by liver negative strands but had no relation to liver positive strands and circulating HCV. Only negative-strand quantitation was inversely correlated with age (r = -0.322; P = 0.026 for R and r = -0.340; P = 0.018 for L), while liver tissues with hepatitis B virus DNA contained larger amounts of each strand. In 27 patients treated with enhanced interferon monotherapy, the amounts of liver negative strands (<4 log copies/100 ng RNA) were the only independent predictor of a sustained virologic response. CONCLUSIONS: Negative-strand quantitation is uniform in the liver and bears distinct relevance to the disease.
Assuntos
Hepacivirus/fisiologia , Hepatite C Crônica/virologia , RNA Viral/análise , Replicação Viral/genética , Adulto , Idoso , Biópsia/métodos , Progressão da Doença , Feminino , Hepatite C Crônica/patologia , Humanos , Laparoscopia , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
ABSTRACTS: BACKGROUND: The carcinogenesis of colorectal cancer has been accepted by a model for a cascade of genetic alterations, named the adenoma-carcinoma sequence. In order to elucidate the carcinogenesis of the colorectal cancer more clearly, the genetic abnormalies of the non-neoplastic mucosal epithelium of the colon and rectum should be investigated. It has been speculated that colonic Paneth cell metaplasia (PaM) is one of the pre-neoplastic mucosa of colonic cancer. Therefore, we studied the propria mucosa of the right colon with PaM from the standpoints of the frequency of the K-ras codon 12 mutations (K-ras), which is initial genetic abnormality in colorectal cancer, and the loss of heterozygosity of microsatellite markers (LOH-MS), which has a relationship to development of colorectal cancer. METHODS: Fifty-two regions with PaM histopathologically from 12 surgically resected right colon specimens were studied. DNA extraction of the colonic mucosa with PaM was obtained using a microdissection method, and the frequency of the K-ras of PaM was investigated by enriched polymerase chain reaction-enzyme linked mini-sequence assay, and the frequency of the LOH-MS (D2S123, D17S250 and D5S346) of PaM was examined by high resolution fluorescenced labeled PCR primers. RESULTS: K-ras mutation was detected in fifteen regions among 52 PaM (28.9%). All mutations were a single mutation and GGT changed to AGT in eleven and GAT in four. LOH-MS were detected in twenty-one regions among 52 PaM (40.4%) (D2S123: 35.4%, 17/48 regions, D17S250: 13.7%, 7/51 regions, and D5S346: 0%, 0/52 regions). No K-ras mutations and LOH-MS were detected in the controls (Colorectal mucosa with no PaM). CONCLUSIONS: Colonic mucosa with Paneth cell metaplasia may be one of the pre-neoplastic mucosa in the development of the colonic epithelial neoplasia.