RESUMO
Successful pre-emptive anti-cytomegalovirus (CMV) therapy relies on sensitive, specific and reproducible tests for CMV detection. Real-time polymerase chain reaction (PCR) for CMV-DNA provides a superior reproducibility and sensitivity than pp65-antigenemia. Evaluation of a novel commercial real-time PCR for CMV-DNA associated with a fully automated DNA extraction from whole blood (WB) was performed, studying the correlation with pp65-antigenemia in guiding pre-emptive therapy. Analytical evaluation showed that PCR correctly quantitated CMV from 500 to 500,000copies/ml with a close correlation with expected values (r=0.999). Clinical evaluation on 375 consecutive WB samples from 48 infected patients (18 pre-emptively treated for pp65 values >/=50 positive cells) showed that according to pp65-antigenemia of 0, 1-10, 11-49 and >/=50 positive cells, median DNA levels were 3.7, 3.9, 4.6 and 5.6 log(10)copies/ml, respectively. According to existing pre-emptive treatment criteria based on pp65-antigenemia, receiver-operating curve analysis indicated 5.3log/ml (200,000genomes/ml) as the best CMV-DNA level to discriminate between patients requiring pre-emptive therapy and those who did not (positive predictive value: 91%; negative predictive value: 74%; sensitivity and specificity: 68 and 93%). In conclusion, real-time PCR provides reliable results for monitoring the developing of CMV infection, allowing for the definition of CMV-DNA thresholds associated with infection progress.
Assuntos
Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/virologia , Citomegalovirus/isolamento & purificação , DNA Viral/sangue , Reação em Cadeia da Polimerase/métodos , Carga Viral/métodos , Antivirais/uso terapêutico , Humanos , Fosfoproteínas/imunologia , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Estatística como Assunto , Proteínas da Matriz Viral/imunologiaRESUMO
Performances of the new automatic system COBAS AmpliPrep/COBAS TaqMan 48 (CAP/CTM) (Roche, Branchburg, NJ) for HBV DNA extraction and real-time PCR quantification were assessed and compared with the endpoint PCR COBAS AMPLICOR HBV Monitor (CAHBM, Roche). Analytical evaluation with proficiency panels from UK National External Quality Assessment Scheme (UK NEQAS) over a 1-year period of distribution showed that CAP/CTM correctly measured HBV DNA levels with a close correlation between expected and observed values (r=0.995). Clinical evaluation as tested with samples from 11 HBsAg-positive patients undergoing antiviral therapy (71 serial specimens of plasma), demonstrated excellent correlation with CAHBM (r=0.958, mean difference in quantitation: 0.14 log, IU/ml), but CAP/CTM detected longer period of residual viremia. HBV DNA reduction was much higher in the combination schedule (Lamivudine+Adefovir), than in Adefovir monotherapy (5.1 vs. 3.5 logs). In conclusion, CAP/CTM allows for an accurate and standardized quantification of HBV DNA in high through-put laboratories. Due to it high sensitivity, it may further improve the detection of emerging drug resistance strains and the assessment of antiviral therapy.
Assuntos
Antivirais/uso terapêutico , DNA Viral/sangue , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Reação em Cadeia da Polimerase/métodos , Adenina/análogos & derivados , Adenina/uso terapêutico , Automação , DNA Viral/análise , DNA Viral/isolamento & purificação , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/genética , Humanos , Lamivudina/uso terapêutico , Organofosfonatos/uso terapêutico , Taq PolimeraseRESUMO
Diagnosis and monitoring of HCV infection relies on sensitive and accurate HCV RNA detection and quantitation. The performance of the COBAS AmpliPrep/COBAS TaqMan 48 (CAP/CTM) (Roche, Branchburg, NJ), a fully automated, real-time PCR HCV RNA quantitative test was assessed and compared with the branched-DNA (bDNA) assay. Clinical evaluation on 576 specimens obtained from patients with chronic hepatitis C showed a good correlation (r = 0.893) between the two test, but the CAP/CTM scored higher HCV RNA titers than the bDNA across all viral genotypes. The mean bDNA versus CAP/CTM log10 IU/ml differences were -0.49, -0.4, -0.54, -0.26 for genotype 1a, 1b, 2a/2c, 3a, and 4, respectively. These differences reached statistical significance for genotypes 1b, 2a/c, and 3a. The ability of the CAP/CTM to monitor patients undergoing antiviral therapy and correctly identify the weeks 4 and 12 rapid and early virological responses was confirmed. The broader dynamic range of the CAP/CTM compared with the bDNA allowed for a better definition of viral kinetics. In conclusion, the CAP/CTM appears as a reliable and user-friendly assay to monitor HCV viremia during treatment of patients with chronic hepatitis. Its high sensitivity and wide dynamic range may help a better definition of viral load changes during antiviral therapy.
Assuntos
Ensaio de Amplificação de Sinal de DNA Ramificado/métodos , Hepacivirus/isolamento & purificação , Hepatite C Crônica/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/sangue , Carga Viral/métodos , Humanos , Kit de Reagentes para Diagnóstico , RobóticaRESUMO
Success in antiviral therapy for chronic hepatitis B is supported by highly sensitive PCR-based assays for hepatitis B virus (HBV) DNA. Nucleic acid extraction from biologic specimens is technically demanding, and reliable PCR results depend on it. The performances of the fully automatic system COBAS AmpliPrep-COBAS TaqMan 48 (CAP-CTM; Roche, Branchburg, NJ) for HBV DNA extraction and real-time PCR quantification were assessed and compared to the endpoint PCR COBAS AMPLICOR HBV monitor (CAHBM; Roche). Analytical evaluation with a proficiency panel showed that CAP-CTM quantitated HBV DNA levels in one single run over a wide dynamic range (7 logs) with a close correlation between expected and observed values (r = 0.976, interassay variability below 5%). Clinical evaluation, as tested with samples from 92 HBsAg-positive patients, demonstrated excellent correlation with CAHBM (r = 0.966, mean difference in quantitation = 0.36 log(10) IU/ml). CAP-CTM detected 10% more viremic patients and longer periods of residual viremia in those on therapy. In lamivudine (LAM)-resistant patients, the reduction of HBV DNA after 12 months of Adefovir (ADF) was higher in the combination (LAM+ADF) schedule than in ADF monotherapy (5.1 logs versus 3.5 logs), suggesting a benefit in continuing LAM. CAP-CTM detected HBV DNA in liver biopsy samples from 15% of HBsAg-negative, anti-HBcAg-positive graft donors with no HBV DNA in plasma. The amount of intrahepatic HBV DNA was significantly lower in occult HBV infection than in overt disease. CAP-CTM can improve the management of HBV infection and the assessment of antiviral therapy and drug resistance, supporting further insights in the emerging area of occult HBV infection.
Assuntos
DNA Viral/sangue , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/virologia , Reação em Cadeia da Polimerase/métodos , Viremia/virologia , Adenina/análogos & derivados , Adenina/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Automação , Biópsia , DNA Viral/análise , DNA Viral/isolamento & purificação , Farmacorresistência Viral , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Humanos , Lamivudina/farmacologia , Lamivudina/uso terapêutico , Fígado/virologia , Técnicas de Amplificação de Ácido Nucleico , Organofosfonatos/uso terapêutico , Sensibilidade e Especificidade , Taq PolimeraseRESUMO
The polymerase chain reaction (PCR) for cytomegalovirus (CMV) DNA quantitation provides sensitive and specific data for detecting CMV as well as monitoring the infection and determining the appropriate antiviral strategy. A recently introduced real-time PCR assay for CMV DNA quantitation was applied on 158 peripheral blood leukocytes (PBLs) from 32 liver-transplanted patients with CMV asymptomatic infection and correlated with a commercial quantitative end-point PCR (COBAS AMPLICOR CMV Monitor) and CMV pp65 antigenemia. A good correlation was found between real-time PCR and pp65 antigen test (r2 = 0.691) and the two PCR assays (r2 = 0.761). Real-time PCR data were higher in pre-emptive treated patients (>20 pp65 + positive cells, median CMV DNA value: 3.8 log(10) copies/500,000 PBLs) than in not-treated ones (2.9 logs). According to pp65 levels of 0, 1-10, 11-20, 21-50, 51-100, and >100 positive cells/200,000 PBLs, median CMV DNA by real-time PCR was 2.6, 3.0, 3.6, 4.0, 4.2, and 4.8 logs, respectively, (CMV DNA levels by COBAS AMPLICOR: 2.8, 2.9, 3.8, 3.7, 3.9, and 4.0 logs). For samples with >20 pp65 + cells, real-time PCR gave significantly higher values than in groups with <20 pp65 + cells, whereas the COBAS AMPLICOR results showed a slower progression rate. Dilutions of CMV AD169 strain were used to probe real-time PCR reproducibility (between and intra-assay variability <2%) and sensitivity (100% detection rate at 10 copies/reaction, 28.5% with end-point PCR). In conclusion, real-time PCR significantly improves the study of CMV DNA dynamics due to a more reliable quantitation of CMV DNA for moderate and high DNA level compared to end-point PCR with better sensitivity and specificity. Real-time PCR provides more precise information for evaluating infection progress and assessing antiviral response, simplifying and accelerating the process of producing a reliable quantitation of CMV DNA for clinical purposes.
Assuntos
Citomegalovirus/genética , Citomegalovirus/isolamento & purificação , DNA Viral/sangue , DNA Viral/genética , Transplante de Órgãos , Reação em Cadeia da Polimerase/métodos , Antígenos Virais/sangue , Citomegalovirus/imunologia , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/etiologia , Infecções por Citomegalovirus/virologia , Humanos , Imunoensaio/métodos , Imunoensaio/estatística & dados numéricos , Leucócitos/virologia , Transplante de Fígado/efeitos adversos , Transplante de Órgãos/efeitos adversos , Fosfoproteínas/sangue , Fosfoproteínas/imunologia , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sensibilidade e Especificidade , Proteínas da Matriz Viral/sangue , Proteínas da Matriz Viral/imunologia , Virologia/métodos , Virologia/estatística & dados numéricosRESUMO
Cytomegalovirus (CMV) infection is an important cause of morbidity in solid organ recipients. Early markers to identify the progress of the infection and patients at high risk are required in order to apply a strategy of pre-emptive therapy. The efficacy of pre-emptive therapy relies on accurate laboratory tests to monitor CMV infection. The evaluation of CMV DNA kinetics by the polymerase chain reaction (PCR) is widely used for the management of CMV infection but markers predicting the progression of the infection and standardization of the technique are essential for the clinical interpretation of PCR results. A commercially available PCR system, the COBAS AMPLICOR Monitor (Roche Diagnostics, Brachburg, NJ), was used for the quantitation of CMV DNA in weekly blood samples (n = 504) from 47 solid organ recipients in the first 6 months after transplantation. PCR results were evaluated according to the development of clinical disease in order to find a DNA threshold and time points predicting the progression of CMV infection. Week 4 from transplantation was the earliest time point to note a significant difference between those patients who eventually developed CMV disease (n = 30) and those who remained asymptomatically infected (n = 17). At week 4, viral loads were significantly higher in patients who developed CMV disease than in asymptomatic infections (median value: 4 log(10)/10(6) leukocytes vs. 2.8, P < 0.0001). At week 4, a DNA level >/=4 log(10)/10(6) leukocytes was associated with a 45.37 odds ratio for CMV disease. Any increase >/=1 log from the first DNA detection to week 4 correlated with the clinical progression of CMV infection (odds ratio 1.74). In those patients who were treated with anti-CMV therapy, a >97% reduction of the baseline viral load was associated with a complete therapeutic success. In conclusion, CMV infection is a highly dynamic process and the quantitation of CMV DNA by PCR is a powerful marker to control successfully the infection, but a strict follow-up of the recipient and standardized PCR tests are mandatory for the best management of the infection.