An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.

Comparison of COBAS AmpliPrep/COBAS TaqMan HCV Qualitative Test v2.0 with COBAS AMPLICOR Hepatitis C Virus Test v2.0 for the Qualitative Detection of Hepatitis C Virus RNA in Korean Clinical Samples / 임상검사와정도관리

BACKGROUND: We comparatively evaluated the performance of the conventional COBAS Amplicor HCV test v2.0 (CAM; Roche Molecular Systems, USA) and the newly developed COBAS AmpliPrep/COBAS TaqMan HCV test v2.0 (CAP/CTM; Roche Molecular Systems) for qualitative detection of hepatitis C virus (HCV) RNA in clinical samples. METHODS: Six hundred serum samples (100 HCV-positive, 500 HCV-negative, as determined by CAM) were selected and analysed using the new qualitative HCV RNA test, CAP/CTM qualitative test. Results were compared by confirmatory CAP/CTM quantitative test, which is a quantitative HCV RNA real-time polymerase chain reaction by Roche Molecular Systems, and anti-HCV test (Roche Diagnostics GmbH, Germany). Twenty-two additional serum samples, which gave a gray zone result by CAM, were selected for comparison. RESULTS: The two qualitative HCV RNA assays yielded concordant results for 586 of 600 tested samples (concordance rate, 97.7%; kappa coefficient, 0.92; 95% confidence interval [CI], 0.87 to 0.96; P<0.001). Upon re-testing by CAM, we found that the concordance rate increased to 98.2% (kappa coefficient, 0.93; 95% CI, 0.89 to 0.97; P<0.001). The additional 22 samples showing gray zone results for CAM were retested and were also tested by CAP/CTM. The results for 13 of these samples changed to negative and were now concordant with the CAP/CTM and confirmatory CAP/CTM quantitative results. For the remaining samples, the results were variable. For all the 22 samples, the results of the new CAP/CTM were in agreement with those obtained by confirmatory CAP/CTM quantitative test. CONCLUSIONS: The results of the two assays were in good agreement, with 97.7% concordance rate. However, CAP/CTM is more sensitive than CAM and showed no gray zone results. Therefore, it can be a more efficient and useful test for the qualitative detection of HCV RNA in clinical samples.

Utilización del ensayo Cobas Amplicor Monitor HIV-1 en el diagnóstico temprano de la infección por HIV en pediatría / Utility of the Cobas Amplicor Monitor HIV-1 assay in the early diagnosis of HIV pediatric infection

Dado que en Argentina el ensayo de HIV RNA cualitativo (QL) ha sido discontinuado, el objetivo del presente trabajo fue evaluar el ensayo de RNA cuantitativo (CV) COBAS AMPLICOR Monitor HIV-1 para su posible incorporación en el diagnóstico pediátrico de infección por HIV en niños expuestos perinatalmente y estimar la concordancia con QL. Fueron incluidas 214 muestras (134 pacientes), 174 muestras de 113 pacientes negativos y 40 correspondientes a 21 pacientes positivos con diagnóstico definitivo. Todas las muestras fueron estudiadas para CV usando el procedimiento estándar (ST, rango 400-750.000 copias/ml) y aquellas por debajo del rango lineal se procesaron por el método ultrasensible (US, rango 50-100.000 copias/ml). El ensayo de CV fue comparado con el de QL en un subgrupo de 93 muestras (78 negativas y 15 positivas). Las 174 muestras pertenecientes a niños HIV negativos tuvieron CV indetectables y entre las 40 muestras pertenecientes a niños HIV positivos, 37 evidenciaron CV detectables (2-5,88 log 10). En 4/37 muestras se detectaron CV entre 165-575 copias/ml, que en muestras posteriores evidenciaron altos valores de CV. El 83,7 % de las muestras resultaron con CV> a 4 log 10. La sensibilidad de la CV según grupo etario fue de 77,7 % < 15 días, 93,3 % entre 15-45 días y la especificidad general del 100 %. La concordancia global entre QL y CV fue del 97,8 %, siendo el ensayo de CV más sensible. Nuestros resultados sugieren la inclusión del ensayo COBAS AMPLICOR en el algoritmo diagnóstico, no presentando resultados falsos positivos.

Since in Argentina qualitative dHIV RNA (QL) test, has been discontinued, the objective of this study was to evaluate the quantitative RNA (VL) COBAS AMPLICOR monitor HIV-1, for its possible inclusion in the paediatric diagnosis of HIV infection and to evaluate the concordance between both assays. In this study 214 samples corresponding to 134 patients were included, 174 and 40 specimens from 113 HIV-negative and 21 HIV-positive patients respectively. All samples were studied for VL using the standard procedure (ST, range 400-750.000 copies/mal), and those below the linear range were further tested by the ultrasensitive method (US, range 50-100.000 copies/ml) QL and VL assays were compared among a subgroup of 93 samples (78 negative and 15 positive). All 174 samples from HIV negative children had undetectable VL. 37/40 samples from HIV-positive had detectable VL (2-5,88 log 10) 4/37 specimens had VL between 165-575 copies/ml, later samples from the corresponding patients showed high VL. 83,7 % of the samples had VL> 4 log 10. The sensitivities found were 77,7 % and 93,3 % among infants < 15 days and between 15-45 days of aged respectively. The general specificity of the COBAS AMPLICOR was 100 %. The concordance between QL and VL eas 97,8 %, showing the later one better sensitivity. Our results support the inclusion of COBAS AMPLICOR in the diagnosis algorithm, not showing false positive results.

Evaluation of the Real-Q HCV Quantification Kit / 대한임상미생물학회지

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.

Comparison of In-house PCR with Conventional Techniques and Cobas Amplicor M. tuberculosis(TM) Kit for Detection of Mycobacterium tuberculosis

PURPOSE: Polymerase chain reaction (PCR) assay, introduced as a fast and sensitive diagnostic method, is useful in detecting Mycobacterium tuberculosis. The purpose of this study was to evaluate the usefulness of in-house PCR assay in the detection of Mycobacterium tuberculosis by comparing PCR results with conventional diagnostic techniques and Cobas Amplicor M. tuberculosis(TM) kit. MATERIALS and METHODS: We retrospectively assessed the diagnostic yield of in-house PCR method employed for the amplification IS6110 sequences in 2,973 specimens. We also compared in-house PCR with Cobas Amplicor M. tuberculosis(TM) kit in 120 specimens collected from June to July 2006. Routine acid-fast stain (AFS) and culture assay were also performed and analyzed. RESULTS: Of 2,973 cases, 2,832 cases (95.3%) showed consistent results between in house PCR, AFS and culture methods, whereas 141 (4.7%) displayed inconsistent results. The sensitivities, specificities, and positive and negative predictive values of each method were as follows: 77.5%, 99.7%, 95.5%, and 98.0%, respectively for PCR; 49.2%, 100%, 100%, and 95.7%, respectively, for AFS method; and 80.7%, 100%, 100%, and 98.3%, respectively, for culture assay. Consistent results between PCR and Cobas Amplicor M. tuberculosis(TM) kit were shown in 109 cases (90.8%). The sensitivities, specificities, and positive and negative predictive values of each method were as follows: 81.3%, 98.9%, 96.3%, and 93.5% respectively for PCR and 71.9%, 100%, 100%, and 90.7%, respectively, for Cobas Amplicor(TM) kit. CONCLUSION: In-house PCR and Cobas Amplicor(TM) kit show high sensitivity and specificity, and are reliable tests in the diagnosis of tuberculosis.

HBV DNA Quantitation Using Real-time PCR / 대한진단검사의학회지

BACKGROUND: Accurate measurement of the concentration of hepatitis B virus (HBV) DNA in clinical samples is important for the appropriate treatment of patients and evaluation of their therapeutic responses. In addition, the concentration of HBV DNA in the serum of patients with chronic HBV infection has a very broad range. Real-time PCR is very sensitive and useful to detect HBV DNA in a wide range of concentrations. We designed new primers and probes for real-time PCR to detect HBV DNA. METHODS: Primers and probes specific for HBV were designed. EUROHEP HBV DNA standards (NIBSC, Hertfordshire, UK) with the HBV DNA concentration of 7.0 x 10(4) copies/mL was used to determine the calibration curve and efficacy for the real-time PCR assay. Sensitivity, dynamic range, and precision were evaluated. The correlation between the real-time PCR and Cobas Amplicor HBV Monitor(TM) assay in the measurement of serum HBV DNA concentrations in 52 patients with chronic HBV infection was evaluated. RESULTS: The sensitivity of the assay was approximately 6.08 x 10(2) copies/mL for HBV, and the quantitation was accurate and reproducible over a wide dynamic range from 6.1 x 0(2) to 6.5 x 10(9) copies/mL without any dilution of specimens. The assay showed low coefficients of variation of repeatability (3.7-24.9%) and reproducibility (7.8-24.7%). The results were found to correlate well with those obtained by Cobas Amplicor HBV Monitor(TM) kit. CONCLUSIONS: We provide a new in-house method for the measurement of serum HBV DNA using real-time PCR, which enables us to detect HBV DNA rapidly, sensitively, and accurately.

Clinical Evaluation of BDProbeTec ET Direct TB Assay for Detection of Mycobacterium tuberculosis Complex in Respiratory and Non-Respiratory Specimens / 대한임상병리학회지

BACKGROUND: Recently developed BDProbeTec ET direct TB assay (BDET; Becton Dickinson, Sparks, MD, USA) uses strand displacement amplification in which DNA segments of the IS6110 are amplified and detected by a fluorescent energy transfer. In this study, we evaluated the performance of the BDET for direct detection of Mycobacterium tuberculosis complex (MTBC) in respiratory and non-respiratory specimens by comparing results with those of COBAS AMPLICOR MTB assay (CAS: Roche, Basel, Switzerland) and Mycobacteria growth indicator tube (MGIT) in a clinical setting. METHODS: A total of 281 (152 respiratory and 129 non-respiratory) specimens collected from 253 patients were tested in parallel with three assays. The results were evaluated in combination with both MGIT cultures and clinical data set to the so-called "gold standard". RESULTS: Forty specimens (14.2%) were from patients with tuberculosis, and thirty specimens (10.7%) were culture positive for MTBC. The level of agreement between cultures, CAS, and BDET was 87.2%. The sensitivities, specificities, and the positive and negative predictive values for respiratory specimens were 69.2%, 100%, 100%, and 94.0%, respectively, for CAS, and 76.9%, 92.9%, 69.0%, and 95.1%, respectively, for BDET. The same values for non-respiratory specimens were 42.9%, 100%, 100%, and 93.5%, respectively, for CAS, and 100%, 93.0%, 63.6%, and 100%, respectively, for BDET. CAS was significantly more specific than BDET with both respiratory (P=0.002) and non-respiratory (P=0.004) specimens and BDET was far more sensitive than CAS for non-respiratory specimens (P=0.001). CONCLUSTIONS: Considering the low sensitivities of other available tests, BDET is a potentially excellent diagnostic tool for diagnosis of extra-pulmonary tuberculosis.

Performance of HCV and HIV-1 Nucleic Acid Amplification Test(NAT) in Korean Blood Donors / 대한수혈학회지

BACKGROUND: There is still risk of acquiring HCV and HIV by transfusion due to window phase. Screening for HCV and HIV-1 by nucleic acid amplification testing (NAT) may improve blood safety allowing detection during the preseroconversion window in donors. METHODS: We investigated NAT usefulness using COBAS AMPLICOR analyzer (Roche). The following sample population were tested:1) 15,552 HCV/HIV-1 seronegative random blood donor samples for HCV and HIV-1 NAT;2) 696 high ALT and 271 HCV EIA positive samples for HCV NAT;3) 1,152 HIV-1 EIA reactive samples for HIV-1 NAT. NAT was performed on pools of 24 donations according to the assay protocol. RESLUTS: Six pools showed initial reactive reactions in HCV NAT and one pool showed initial reactive reaction in HIV-1 NAT. But no donor sample was found repeatedly reactive by this assay. CONCLUSIONS: Although there were false positive reactions, specificity of the NAT assay was high enough for the assay to be applied as a blood screening test and implementation of this assay is expected to improve blood safety and be useful for blood products use.