RESUMO
BACKGROUND: Hepatitis C virus (HCV) RNA quantification is necessary for predicting the therapeutic response and assessing treatment results in patients with chronic HCV infection. Recently, real-time PCR technology for HCV RNA quantification displayed good linearity within the dynamic range. Thus, it is gradually replacing branched-DNA (bDNA) and PCR- hybridization assays. In this study, we evaluated the performance of the Real-QTM HCV quantification kit (biosewoom. Inc., Seoul, Korea) developed in Korea. METHODS: We evaluated the HCV quantification kit for detection limit, specificity, linearity, accuracy, and recovery rate of HCV RNA standard material. The results were analyzed for a correlation with those of Cobas Amplicor HCV Monitor 2.0. RESULTS: The HCV quantification kit showed a high recovery rate of HCV RNA standard material of various concentrations and amplication of HCV RNA equally in all genotypes. Hepatitis B virus and human immunodeficiency virus showed no cross-reactivity with HCV. Within-run and between-run coefficients of variation (CV) were 9.52~15.84% and 9.40~17.53%, respectively. Between-day coefficients of variation were 11.62~18.04%, and detection limit was 44 IU/mL. It showed a good correlation with Cobas Amplicor HCV Monitor 2.0 (R2=0.8954). CONCLUSION: The Real-Q HCV quantification kit showed a good specificity, sensitivity, linearity, and accuracy; therefore, we propose that it is fully adequate for monitoring antiviral therapy in patients with chronic HCV infection.
Assuntos
Humanos , Quimera , Genótipo , Hepacivirus , Vírus da Hepatite B , HIV , Limite de Detecção , Compostos Organotiofosforados , Reação em Cadeia da Polimerase em Tempo Real , RNA , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA. METHODS: We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients. RESULTS: The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4-19.5%. It detected CMV DNA in a linear range from 1 x 10(2) to 5 x 10(11) copies/mL (P<10(-13), R2=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (K=0.221, P<10(-6)). In comparison of quantitative results, the correlation between two tests was significant (r=0.45, P<10(-17)). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10(-3) and P<10(-7), respectively). CONCLUSIONS: The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.
Assuntos
Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Citomegalovirus/genética , Infecções por Citomegalovirus/diagnóstico , DNA Viral/sangue , Fosfoproteínas/análise , Reação em Cadeia da Polimerase/métodos , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Carga Viral/métodos , Proteínas da Matriz Viral/análiseRESUMO
BACKGROUND: Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA. METHODS: We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients. RESULTS: The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4-19.5%. It detected CMV DNA in a linear range from 1 x 10(2) to 5 x 10(11) copies/mL (P<10(-13), R2=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (K=0.221, P<10(-6)). In comparison of quantitative results, the correlation between two tests was significant (r=0.45, P<10(-17)). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10(-3) and P<10(-7), respectively). CONCLUSIONS: The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.
Assuntos
Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Citomegalovirus/genética , Infecções por Citomegalovirus/diagnóstico , DNA Viral/sangue , Fosfoproteínas/análise , Reação em Cadeia da Polimerase/métodos , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Carga Viral/métodos , Proteínas da Matriz Viral/análiseRESUMO
BACKGROUND: We evaluated the performance of a newly developed real-time polymerase chain reaction (PCR) method using TaqMan probe (TP) and internal control (IC) for quantitation of BK virus (BKV) DNA. METHODS: PCR primers and TP were targeted for the VP1 of BKV and 300 bp-region of VP1 was cloned to prepare a standard DNA. Threshold cycles (Ct) of IC was set at 33+/-3. The recovery rates, precision, linearity, and limit of detection (LOD) were measured using the standard DNA. To correlate TP with previous hybridization probe (HP) method, Ct of those were compared using 35 HP-positive and 15 HP-negative specimens, and the interpretation agreement was analyzed in 63 consecutive clinical specimens including 32 urines and 31 plasmas. Fifty-three53 specimens measured for IC were analyzed for positive rates and levels of BKV according to Ct of IC. RESULTS: The average recovery rate was 101.1% and intra-assay and inter-assay coefficiency variations were 0.017~0.059 and 0.036, respectively, with the specimens of 3 log/mL, and 0.041~0.063 and 0.045, respectively, with the specimens of 6 log/mL. LOD was 183 copies/mL and linearity range was 2.7 log- 12 log/mL. Ct of TP were correlated with those of HP with the function of y=0.8912x+0.3164 (R2=0.9062). Among 63 clinical specimens, 16 were positive in TP and 12 were positive in HP with an agreement of 90.4%. Ct of IC were over 36 in 31 specimens (22 urines and 9 plasmas), of which BKV DNA was much higher in 7 (22.5%) BKV-positive specimens (5.9+/-1.7 log/mL) than in 4 (18.1%) BKV-positive specimens (3.9+/-1.0 log/mL) of 22 having Ct of IC < or =36.; 5.9+/-1.7 vs. 3.9+/-1.0 log/mL. CONCLUSION: TP warrants to be a reliable method for quantification of BKV. IC seemed to be essential to differentiate false-negative results or underestimation of BKV in clinical specimens, especially in urine.
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Vírus BK , Células Clonais , DNA , Limite de Detecção , Plasma , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Hepatitis B virus (HBV) DNA quantification is important for the management of HBV infection and identification of the development of resistance. The susceptibility to contamination and more variable reproducibility of results with the conventional HBV DNA quantification method have raised the need of a more simple and accurate method for HBV DNA quantification. Real-time quantitative PCR assays recently introduced in the laboratory can meet these needs. In this study, we evaluated the performance of the Real-Q HBV Quantification kit developed in Korea. METHODS: We evaluated the recovery of DNA extraction, the interference of internal control, an analytical sensitivity, specificity, and reproducibility, a clinical specificity, and a reportable range of the Real-Q HBV Quantification kit. The quantification result was also compared to that obtained by the Digene Hybrid-Capture II. RESULTS: The mean percent recovery was 108.6% and there was no interference with the internal control on DNA extraction. None of HIV, hepatitis C virus, or cytomegalovirus showed a cross-reactivity with HBV. This assay detected HBV DNA in a linear range from 10(2) to 10(10) copies/mL, with the detection limit of 56 copies/mL. The assay exhibited a low within-run CV (coefficient of variation) (8.7-11.9%), between-run CV (10.5-14.7%), and between-day CV (13.2-21.4%). No HBV DNA was detected in any of 100 samples without HBV, resulting in a clinical specificity of 100%. The levels of HBV DNA showed a good correlation with those determined with Digene Hybrid-Capture II (R2=0.9827). CONCLUSIONS: The Real-Q HBV Quantification kit showed a good analytical sensitivity, specificity, and high reliability with a broad reportable range. This assay should be clinically useful in managing patients with HBV infection.
Assuntos
Humanos , Citomegalovirus , DNA , Hepacivirus , Vírus da Hepatite B , HIV , Coreia (Geográfico) , Limite de Detecção , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The BCR-ABL rearrangement, the molecular hallmark of chronic myeloid leukemia (CML) can be used as a marker to identify residual disease after therapy. So far, very limited data exists in Korea regarding the frequency of BCR-ABL fusion gene in patients with CML. The objective of this study was to identify the type of BCR-ABL fusion variants of CML patients in Korea. METHODS: We performed a two-step reverse transcription-polymerase chain reaction(RT- PCR) to detect BCR-ABL specific mRNA in 154 CML patients who were diagnosed by histologic examination and cytogenetics at our institute between January 1997 and November 2000. We used different primer set to amplify various breakpoints of BCR-ABL fusion gene. RESULTS: All patients showed at least one of BCR-ABL transcripts. One hundred and four of 154 patients (67.5%) represented b3a2 transcript that was most frequent transcript in our CML patients. 44 patients (28.6%) had b2a2 transcript and b3a2+b2a2 splicing variants were identified in 5 cases (3.25%). In addition, c3a2 variant which is very rare transcript was identified in a patient (0.65%). CONCLUSIONS: The RT-PCR assay could identify the presence of the BCR-ABL transcript in all patients with exquisite sensitivity. The frequency of BCR-ABL transcript was different from that of western countries but similar to that of eastern. Long-term follow up of CML patients with different variants are needed to determine the prognostic importance of each gene variant.
Assuntos
Humanos , Citogenética , Coreia (Geográfico) , Leucemia Mielogênica Crônica BCR-ABL Positiva , RNA MensageiroRESUMO
STI571 is an effective agent for the patients with chronic myeloid leukemia (CML). But, complete molecular response with STI571 is rarely reported in accelerated phase CML. Here we report a patient with accelerated phase CML who achieved complete molecular response with STI571. A 60-year old female patient visited emergency room with syncope. Her white blood cell count was 30,800/microliter (basophil 23%), hemoglobin 8.9g/dL, and platelet counts 2,748,000/microliter. Bone marrow was hypercellular with increase in megakaryocyte and basophils (15%). She was diagnosed as an accelerated phase CML. Seven days after stopping hydroxyurea, we used STI571 in a daily oral dose of 600mg. Generalized edema and skin rash were observed 15 days after treatment (all grade 1) and were controlled well with conservative management. Complete hematologic and cytogenetic responses were achieved after 1 month and 3 months of therapy with STI571 respectively. Complete molecular response was simultaneously proven by conventional reverse transcriptase PCR and real-time PCR analysis. The patient still remained in complete hematologic, cytogenetic, and molecular responses for 24 months. Treatment with STI571 was well tolerated and rapid hematologic improvement was observed. This case shows STI571 can induce complete molecular response as well as cytogenetic response in accelerated phase CML.
Assuntos
Feminino , Humanos , Pessoa de Meia-Idade , Basófilos , Medula Óssea , Citogenética , Edema , Serviço Hospitalar de Emergência , Exantema , Hidroxiureia , Leucemia Mielogênica Crônica BCR-ABL Positiva , Contagem de Leucócitos , Megacariócitos , Contagem de Plaquetas , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síncope , Mesilato de ImatinibRESUMO
BACKGROUND: STI571, a potent and specific inhibitor of the BCR-ABL tyrosine kinase, causes arrest of growth or apoptosis in leukemic cells that express BCR-ABL. We evaluated the therapeutic effects and clinical events after the STI571 treatment in advanced chronic myelogenous leukemia (CML). METHODS: STI571 was administered orally to 24 patients with CML in accelerated phase (AP) (N=17) or blast crisis (BC) (N=7) with a daily dose of 600mg. Adverse events were observed, and hemotologic, cytogenetic, and molecular responses were evaluated on 1 month of STI571 treatment. RESULTS: Hematologic responses were observed in 20 of 24 patients with higher complete hematologic responses in AP (35.3%) compared to BC (14.3%). Partial cytogenetic responses were observed in 2 cases. Fluorescence in situ hybridization showed significant decrease in the percentage of BCR-ABL positive cells, but all still remained above the upper limit of normal range at the time of analysis. No significant changes were observed in BCR-ABL transcripts after treatment by reverse transcription polymerase chain reaction (PCR) and real-time quantitative PCR. Non- hematologic adverse events after STI571 treatment were minimal, whilst hematologic ones were significant with higher frequency in BC rather than AP. CONCLUSION: STI571 induced rapid and significant hematologic responses in patients with advanced CML and adverse events were tolerable. The fact that no responses were achieved in some of these advanced cases underlies the importance of earlier treatment with STI571 to prolong the survival.