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Commutability of quantitative standards allows patient results to be compared across molecular diagnostic methods and laboratories. This is critical to establishing quantitative thresholds for use in clinical decision-making. A matrix effect associated with the 1st cytomegalovirus (CMV) WHO international standard (IS) was identified using the Abbott RealTime CMV assay. A commutability study was performed to compare the CMV WHO IS and patient specimens diluted in plasma and whole blood. Patient specimens showed similar CMV DNA quantitation values regardless of the diluent or extraction procedure used. The CMV WHO IS, on the other hand, exhibited a matrix effect. The CMV concentration reported for the WHO IS diluted in plasma was within the 95% prediction interval established with patient samples. In contrast, the reported DNA concentration of the CMV WHO IS diluted in whole blood was reduced approximately 0.4 log copies/ml, and values fell outside the 95% prediction interval. Calibrating the assay by using the CMV WHO IS diluted in whole blood would introduce a bias for CMV whole-blood quantitation; samples would be reported as having higher measured concentrations, by approximately 0.4 log IU/ml. Based on the commutability study with patient samples, the RealTime CMV assay was standardized based on the CMV WHO IS diluted in plasma. A revision of the instructions for use of the CMV WHO IS should be considered to alert users of the potential impact from the diluent matrix. The identification of a matrix effect with the CMV WHO IS underscores the importance of assessing commutability of the IS in order to achieve consistent results across methods.
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Infecciones por Citomegalovirus/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Carga Viral/normas , Humanos , Proyectos PilotoRESUMEN
The Abbott RealTime MTB assay is a nucleic acid amplification test (NAAT) for the detection of Mycobacterium tuberculosis complex DNA. The sample inactivation procedure used in the assay, consisting of one part sample treated with 3 parts inactivation reagent for 60 min, effectively reduced viscosity and inactivated M. tuberculosis in clinical specimens.
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Técnicas Bacteriológicas/métodos , Desinfección/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Manejo de Especímenes/métodos , Humanos , Viabilidad Microbiana , ViscosidadRESUMEN
Diagnostic laboratories are under increasing pressure to improve and expand their services. Greater flexibility in sample processing is a critical factor that can improve the time to results while reducing reagent waste, making laboratories more efficient and cost-effective. The introduction of the Abbott mPLUS feature, with the capacity for extended use of amplification reagents, significantly increases the flexibility of the m2000 platform and enables laboratories to customize their workflows based on sample arrival patterns. The flexibility in sample batch size offered by mPLUS enables significant reductions in processing times. For hepatitis B virus tests, a reduction in sample turnaround times of up to 30% (105 min) was observed for batches of 12 samples compared with those for batches of 24 samples; for Chlamydia trachomatis/Neisseria gonorrhoeae tests, the ability to run batches of 24 samples reduced the turnaround time by 83% (54 min) compared with that for batches of 48 samples. Excellent correlations between mPLUS and m2000 standard condition results were observed for all RealTime viral load assays evaluated in this study, with correlation r values of 0.998 for all assays tested. For the qualitative RealTime C. trachomatis/N. gonorrhoeae assay, the overall agreements between the two conditions tested were >98% for C. trachomatis and 100% for N. gonorrhoeae. Comparable precision results were observed for the two conditions tested for all RealTime assays. The enhanced mPLUS capability provides clinical laboratories with increased efficiencies to meet increasingly stringent turnaround time requirements without increased costs associated with discarding partially used amplification reagents.
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Infecciones Bacterianas/diagnóstico , Carga Bacteriana/métodos , Técnicas de Diagnóstico Molecular/métodos , Manejo de Especímenes/métodos , Carga Viral/métodos , Virosis/diagnóstico , Infecciones Bacterianas/microbiología , Humanos , Factores de Tiempo , Virosis/virologíaRESUMEN
HIV-1 viral load testing is essential to the management of HIV-1-infected patients, and proper specimen handling ensures accurate viral load (VL) results. This study was performed to (i) evaluate the effect of freezing plasma in situ in BD Vacutainer plasma preparation tubes (PPT) on the accuracy of HIV-1 viral load results using the Abbott RealTime HIV-1 assay and (ii) evaluate the effect of whole-blood storage in the PPT for 6 h at room temperature prior to centrifugation (PPT6H) rather than 2 h as specified in the PPT product insert. Of the 64 HIV-positive subjects evaluated, 29 had average viral load counts of >40 copies/ml in at least one of the tubes tested and 35 subjects had a result of either "undetected target" or "below the limit of quantification" (LOQ) for all or some of the tubes regardless of handling condition. For the 29 subjects with VLs that were >LOQ, the mean biases between plasma from Vacutainer K(2)EDTA tubes and plasma frozen in situ in PPT and between K(2)EDTA tube plasma and plasma from PPT6H (log(10) copies/ml) were 0.005 and -0.001, respectively, and r(2) was >0.92 for all correlations. We conclude that VLs determined from plasma frozen in situ in PPT are equivalent to VLs in K(2)EDTA tube plasma and can be used for accurate quantification of HIV-1 RNA in the Abbott RealTime HIV-1 assay. Furthermore specimens collected in PPT can be stored for 6 h at room temperature with no effect on viral load results as measured by the Abbott RealTime HIV-1 assay.
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Recolección de Muestras de Sangre/métodos , Infecciones por VIH/virología , VIH-1/genética , Carga Viral/métodos , Adulto , Análisis de Varianza , Progresión de la Enfermedad , Diseño de Investigaciones Epidemiológicas , Infecciones por VIH/diagnóstico , Humanos , Modelos Lineales , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Nucleic acid testing is essential for the detection and quantification of HCV RNA in the diagnosis of HCV infection and treatment monitoring. The Alinity m HCV assay was recently developed by Abbott Molecular for rapid detection and quantification of HCV RNA on the fully automated, continuous, random-access Alinity m analyzer. OBJECTIVES: Our study assessed the performance of the new Alinity m HCV assay for detection and quantification of HCV RNA in a large series of patient samples of various genotypes. This international, multicentric study evaluated the linearity, precision, and reproducibility of the Alinity m HCV assay and its performance in comparison to three other HCV assays currently used in clinical practice. RESULTS: The Alinity m HCV assay demonstrated high linearity (correlation coefficient râ¯=â¯1.00), precision (coefficients of variation [CV] 6.6-13.5 %) and reproducibility (CV 1.7-4.3 % across three control lots). At a concentration near the lower limit of detection, the Alinity m HCV assay exhibited >98 % detectability. The Alinity m HCV assay showed excellent correlation with comparator HCV assays in serum (nâ¯=â¯406) and plasma (nâ¯=â¯1401) samples (correlation coefficients ≥0.96, bias 0.01 to 0.14 Log10 IU/mL). More than 95 % of the quantified results with the Alinity m HCV assay were less than mean bias ± 1.96 SD different from those of the comparator assays. CONCLUSIONS: The newly developed Alinity m HCV assay is sensitive, reproducible, and accurately quantifies HCV RNA levels in serum and plasma samples from patients with chronic HCV infection, with no impact of HCV genotype on assay performance.
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Hepacivirus , Hepatitis C , Genotipo , Hepacivirus/genética , Humanos , ARN Viral , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Carga ViralRESUMEN
Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5'-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3'-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.
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Sondas de ADN/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Disparidad de Par Base , Secuencia de Bases , Colorantes Fluorescentes/química , Variación Genética , VIH-1/genética , TermodinámicaRESUMEN
INTRODUCTION: The Abbott RealTime MTB RIF/INH Resistance Assay (RT MTB RIF/INH) is an assay for the detection of rifampicin (RIF)- and/or isoniazid (INH)-resistant Mycobacterium tuberculosis (MTB). The assay can be used to test sputum, bronchial alveolar lavage, and N-Acetyl-L-Cysteine (NALC)/NaOH pellets prepared from these samples. The assay can be used in direct testing mode, or in reflex mode following a MTB positive result produced by its companion assay, Abbott RT MTB. METHODS: In this study, the direct testing mode was used to test paired sputum and NALC/NaOH pellets prepared from sputum collected from Bangladesh TB patients. One hundred and thirty two paired samples were tested. RESULTS: The RT MTB RIF/INH inhibition rate was 0%. One hundred and twenty-two paired samples had results above the assay limit of detection and were analyzed by comparing with results from phenotypic drug sensitivity testing, GeneXpert MTB/RIF (Xpert), and MTBDR plus (Hain). RT MTB RIF/INH results were in good agreement with those of GeneXpert and Hain. CONCLUSION: The ability of this assay to detect RIF and INH resistance may contribute to the global control of multidrug resistant tuberculosis.
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Performance of the Abbott m2000 instrument system and the Abbott RealTime HIV-1 assay was evaluated using a panel of 37 group M (subtypes A-D, F, G, CRF01_AE, CRF02_AG and unique recombinant forms) and 2 group O virus isolates. Testing was performed on 273 sample dilutions and compared to VERSANT HIV-1 RNA 3.0 (bDNA) and AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) test results. RealTime HIV-1, bDNA, and Monitor v1.5 tests quantified 87%, 78%, and 81% of samples, respectively. RealTime HIV-1 detected an additional 31 samples at < 40 copies/mL. For group M, RealTime HIV-1 dilution profiles and viral loads were highly correlated with bDNA and Monitor v1.5 values; 87% and 89% of values were within 0.5 log(10) copies/mL. In contrast, the group O viruses were not detected by Monitor v1.5 and were substantially underquantified by approximately 2 log(10) copies/mL in bDNA relative to the RealTime HIV-1 assay. Sequence analysis revealed that RealTime HIV-1 primer/probe binding sites are highly conserved and exhibit fewer nucleotide mismatches relative to Monitor v1.5. The automated m2000 system and RealTime HIV-1 assay offer the advantages of efficient sample processing and throughput with reduced "hands-on" time while providing improved sensitivity, expanded dynamic range and reliable quantification of genetically diverse HIV-1 strains.
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Ensayo de Amplificación de Señal de ADN Ramificado/métodos , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Carga Viral , Automatización , Estudios de Evaluación como Asunto , Variación Genética , VIH-1/clasificación , VIH-1/genética , Humanos , Juego de Reactivos para DiagnósticoRESUMEN
The Abbott RealTime HIV-1 assay is an automated test for monitoring HIV-1 viral load in plasma samples. The assay uses reverse transcription polymerase chain reaction (RT-PCR) technology with homogeneous real-time fluorescent detection. Automated sample preparation is performed on the m2000sp instrument where RNA is isolated using magnetic microparticle technology and dispensed to a PCR tray together with the amplification reagents. The PCR tray is then transferred to the Abbott m2000rt instrument for amplification and real-time detection. The assay utilizes two distinct sets of primers and probes for HIV-1 and for internal control (IC). The IC is processed along with each sample to control for sample recovery and inhibition. The HIV-1 primer and probe sequences are targeted to the integrase (IN) region of the polymerase (pol) gene. Due to the selection of a highly conserved target region and a novel, mismatch tolerant probe design, the assay can quantitate HIV-1 group M subtypes A-H, group O, and group N isolates. The assay provides high reproducibility and a wide dynamic range, allowing quantitation from 40 copies to 10 million copies of HIV-1 RNA per milliliter of plasma. HIV-1 RNA concentrations detected with 95% probability were 25copies/mL with 1.0mL of plasma, 39copies/mL with 0.6mL of plasma, 65copies/mL with 0.5mL of plasma, and 119copies/mL with 0.2mL of plasma.
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Infecciones por VIH/virología , VIH-1/aislamiento & purificación , ARN Viral/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Carga Viral/métodos , VIH-1/clasificación , VIH-1/genética , Humanos , ARN Viral/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: HIV RNA suppression is a key indicator for monitoring success of antiretroviral therapy. From a logistical perspective, viral load (VL) testing using Dried Blood Spots (DBS) is a promising alternative to plasma based VL testing in resource-limited settings. OBJECTIVES: To evaluate the analytical and clinical performance of the Abbott RealTime HIV-1 assay using a fully automated one-spot DBS sample protocol. STUDY DESIGN: Limit of detection (LOD), linearity, lower limit of quantitation (LLQ), upper limit of quantitation (ULQ), and precision were determined using serial dilutions of HIV-1 Virology Quality Assurance stock (VQA Rush University), or HIV-1-containing armored RNA, made in venous blood. To evaluate correlation, bias, and agreement, 497 HIV-1 positive adult clinical samples were collected from Ivory Coast, Uganda and South Africa. For each HIV-1 participant, DBS-fingerprick, DBS-venous and plasma sample results were compared. Correlation and bias values were obtained. The sensitivity and specificity were analyzed at a threshold of 1000 HIV-1 copies/mL generated using the standard plasma protocol. RESULTS: The Abbott HIV-1 DBS protocol had an LOD of 839 copies/mL, a linear range from 500 to 1×107 copies/mL, an LLQ of 839 copies/mL, a ULQ of 1×107 copies/mL, and an inter-assay SD of ≤0.30 log copies/mL for all tested levels within this range. With clinical samples, the correlation coefficient (r value) was 0.896 between DBS-fingerprick and plasma and 0.901 between DBS-venous and plasma, and the bias was -0.07 log copies/mL between DBS-fingerprick and plasma and -0.02 log copies/mL between DBS-venous and plasma. The sensitivity of DBS-fingerprick and DBS-venous was 93%, while the specificity of both DBS methods was 95%. CONCLUSION: The results demonstrated that the Abbott RealTime HIV-1 assay with DBS sample protocol is highly sensitive, specific and precise across a wide dynamic range and correlates well with plasma values. The Abbott RealTime HIV-1 assay with DBS sample protocol provides an alternative sample collection and transfer option in resource-limited settings and expands the utility of a viral load test to monitor HIV-1 ART treatment for infected patients.
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Pruebas con Sangre Seca , Infecciones por VIH/virología , VIH-1/fisiología , ARN Viral/sangre , Carga Viral/métodos , Adulto , Recolección de Muestras de Sangre/métodos , Côte d'Ivoire , Infecciones por VIH/diagnóstico , Infecciones por VIH/epidemiología , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Límite de Detección , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Pruebas Serológicas/métodos , Sudáfrica , Manejo de Especímenes , Carga Viral/instrumentación , Adulto JovenRESUMEN
The combination of automated sample preparation and real-time RT-PCR for measurement of HIV-1 viral load has the potential to significantly enhance throughput, reduce operator-associated error, and increase assay sensitivity and dynamic range. In this study, RNA was extracted from the plasma of 91 HIV-1 seropositive Brazilian blood donors using the Abbott m2000sp automated sample preparation system. Viral loads measured using the RealTime HIV-1 (RealTime HIV-1) assay and the Abbott m2000rt instrument were compared to values obtained in the LCx HIV RNA quantitative assay. Subtype was determined for 89 of 91 specimens by sequence/phylogenetic analysis of three genomic regions: gag p24, pol integrase, and env gp41. The panel included 69 subtype B, 1 C, 2 F, and 17 recombinant strains. Eighty-seven specimens were quantified by both assays. Two specimens were quantified only in RealTime HIV-1. Two additional specimens below the detection limit of both assays were also negative on PCR amplification. Viral load results were highly correlated, and good agreement was observed between assays with 90% of values within 0.5 log(10)copies/ml. The RealTime HIV-1 assay and m2000 system offer the advantages of automation while providing reliable quantification of diverse HIV strains.
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Automatización , Seropositividad para VIH/virología , VIH-1/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Carga Viral/métodos , Donantes de Sangre , Brasil , Proteína p24 del Núcleo del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , Integrasa de VIH/genética , VIH-1/genética , HumanosRESUMEN
Automated RNA extraction and quantitation of HIV-1 by real-time PCR offer potential advantages of efficient sample processing, improved sensitivity, expanded dynamic range and reduced contamination risk. In this study, plasma was collected from 100 HIV-1 infected patients visiting The Courtyard Clinic of St. George's Hospital in London, United Kingdom (UK). Viral loads measured using the automated Abbott RealTime HIV-1 assay (m2000sp sample preparation and m2000rt amplification and detection instruments) were compared to results obtained with Versant HIV-1 RNA 3.0 (bDNA), AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) and LCx HIV RNA Quantitative (LCx HIV) assays. Based on gag p24, pol integrase, and env gp41 sequences, the panel included 26 subtype A, 20 B, 27 C, 10 D, 1 CRF01_AE, 3 CRF02_AG and 13 recombinant viruses. RealTime HIV-1, bDNA, Monitor v1.5 and LCx HIV quantitated 82, 74, 82, and 83% of samples, respectively, with 82, 71, 69 and 80 of the 100 samples measured within the dynamic ranges. Viral loads were highly correlated with 99% of values within 1 log(10) copies/ml between tests. The automated m2000 system and RealTime HIV-1 assay can increase laboratory throughput, enhance overall efficiency and reduce operator-associated errors while providing reliable quantitation of genetically diverse strains of HIV-1.
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Infecciones por VIH/virología , VIH-1/aislamiento & purificación , ARN Viral/análisis , Carga Viral , Automatización , Genotipo , Humanos , Variaciones Dependientes del Observador , Plasma/virología , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , Estadística como AsuntoRESUMEN
Clinical management of drug-resistant tuberculosis patients continues to present significant challenges to global health. To tackle these challenges, the Abbott RealTime MTB RIF/INH Resistance assay was developed to accelerate the diagnosis of rifampicin and/or isoniazid resistant tuberculosis to within a day. This article summarizes the performance of the Abbott RealTime MTB RIF/INH Resistance assay; including reliability, analytical sensitivity, and clinical sensitivity/specificity as compared to Cepheid GeneXpert MTB/RIF version 1.0 and Hain MTBDRplus version 2.0. The limit of detection (LOD) of the Abbott RealTime MTB RIF/INH Resistance assay was determined to be 32 colony forming units/milliliter (cfu/mL) using the Mycobacterium tuberculosis (MTB) strain H37Rv cell line. For rifampicin resistance detection, the Abbott RealTime MTB RIF/INH Resistance assay demonstrated statistically equivalent clinical sensitivity and specificity as compared to Cepheid GeneXpert MTB/RIF. For isoniazid resistance detection, the assay demonstrated statistically equivalent clinical sensitivity and specificity as compared to Hain MTBDRplus. The performance data presented herein demonstrate that the Abbott RealTime MTB RIF/INH Resistance assay is a sensitive, robust, and reliable test for realtime simultaneous detection of first line anti-tuberculosis antibiotics rifampicin and isoniazid in patient specimens.
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Antituberculosos/farmacología , Isoniazida/farmacología , Mycobacterium tuberculosis/aislamiento & purificación , Rifampin/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Pulmonar/diagnóstico , Antituberculosos/uso terapéutico , Recuento de Colonia Microbiana , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Isoniazida/uso terapéutico , Pruebas de Sensibilidad Microbiana/métodos , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Rifampin/uso terapéutico , Sensibilidad y Especificidad , Manejo de Especímenes/métodos , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/microbiologíaRESUMEN
Nucleic acid amplification test (NAAT)-based assays provide fast and sensitive results compared to conventional TB tests. The performance of the new Abbott Molecular MTB assay for the qualitative detection of MTB complex using the automated m2000™ system or manual sample preparation is summarized in this paper. The assay detects eight MTB complex subspecies. The observed limit of detection (LOD) when used to test an MTB H37Rv panel was 2.45 colony forming units (cfu)/mL, while the claimed assay LOD with this MTB strain is 17 cfu/mL. No cross reactivity, or carryover were observed in the study. The clinical sensitivity of the assay was 93% overall; 99% in smear positive, culture positive specimens, and 81% in smear negative, culture positive samples. The clinical specificity was 97%. The inhibition rate in the study was 0.34%. The data suggest that Abbott RealTime MTB is a reliable, robust and sensitive assay for the molecular detection of MTB.
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Técnicas Bacteriológicas , ADN Bacteriano/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Mycobacterium tuberculosis/genética , Técnicas de Amplificación de Ácido Nucleico , Tuberculosis Pulmonar/diagnóstico , Recuento de Colonia Microbiana , Humanos , Límite de Detección , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/aislamiento & purificación , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Esputo/microbiología , Tuberculosis Pulmonar/microbiologíaRESUMEN
The LCx HCV RNA quantitative assay (Abbott Laboratories, North Chicago, IL) is designed to use competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and microparticle enzyme immunoassay (MEIA), in combination with a modified Qiagen sample preparation method, to measure the level of hepatitis C virus (HCV) in human plasma and serum. The assay provides quantitative results in international units (IU) of HCV RNA/ml, in copies of HCV RNA/ml, or their log (base 10) equivalents. A conversion study determined that 1IU equals 4.3 copies. The LCx HCV assay detected HCV RNA transcripts representative of genotypes 1-6 with near equal efficiency. The assay did not cross-react with high concentrations of 21 potentially cross-reactive microorganisms or with 100 HCV-negative specimens. The lower limit of detection was demonstrated to be 23IU/ml. The LCx assay had similar sensitivity to the Roche Amplicor HCV (version 2.0) qualitative assay when used to test panels containing 6, 12, 23, and 47IU/ml. The assay linear range was shown to extend from 23 to 2.3millionIU/ml. The intra-assay standard deviation (S.D.) was < or =0.066 logIU/ml for the four HCV positive samples tested, while for the same samples the observed inter-assay S.D. was < or =0.075 logIU/ml. The overall mean assay quantitation value for seven HCV-positive WHO-standardized Acrometrix NAP linearity panel members was within 0.06 logIU/ml of the mean assigned value. The assay was demonstrated to correlate acceptably against the Roche Amplicor HCV monitor test (version 2.0). These data suggest that the assay is standardized appropriately against the WHO standard across its linear range and can be used for quantitation of HCV. In addition, with a sensitivity of 23IU/ml, the assay can be used to determine if post-therapy viral clearance has occurred.
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Hepacivirus/genética , Hepacivirus/aislamiento & purificación , ARN Viral/sangre , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Virología/métodos , Reacciones Cruzadas , Genotipo , Hepacivirus/inmunología , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Técnicas para Inmunoenzimas/métodos , Técnicas para Inmunoenzimas/normas , Técnicas para Inmunoenzimas/estadística & datos numéricos , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , Sensibilidad y Especificidad , Viremia/tratamiento farmacológico , Viremia/virología , Virología/normas , Virología/estadística & datos numéricosRESUMEN
The potential association between xenotropic murine leukemia virus-related virus (XMRV) and prostate cancer and chronic fatigue syndrome (CFS) has been much debated. To help resolve the potential role of XMRV in human disease, it is critical to develop sensitive and accurate reverse transcriptase (RT)-PCR assays to screen for the virus. Single-round RT-PCR assays were developed on the automated m2000™ system for detection of the pol or env regions of XMRV in whole blood, plasma, urine cell pellets and urogenital swab samples. Assay performance was assessed by testing two blinded panels, one comprised of whole blood and the other of plasma spiked with serial dilutions of XMRV-infected tissue culture cells and supernatant, respectively, prepared by the Blood XMRV Scientific Research Working Group (SRWG). For both whole blood and plasma panel testing, the assays showed excellent specificity and sensitivity as compared to the other tests included in the SRWG phase I study. Analytical specificity of the assays was also evaluated. Neither pol nor env PCR assays detected a panel of potential cross-reactive microorganisms, although some cross-reaction was observed with mouse genomic DNA. Screening of 196 normal human blood donor plasma, 214 HIV-1 seropositive plasma, 20 formalin-fixed paraffin-embedded (FFPE) prostate cancer specimens, 4 FFPE benign prostate specimens, 400 urine pellets from prostate cancer patients, 166 urine pellets from non-prostate cancer patients, and 135 cervical swab specimens, detected no samples as unequivocally XMRV positive.
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Productos del Gen env/genética , Productos del Gen pol/genética , Tamizaje Masivo/métodos , Técnicas de Diagnóstico Molecular/métodos , Infecciones por Retroviridae/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/aislamiento & purificación , Anciano , Automatización/métodos , Sangre/virología , Femenino , Genitales/virología , Humanos , Masculino , Persona de Mediana Edad , Infecciones por Retroviridae/virología , Sensibilidad y Especificidad , Orina/virología , Virología/métodosRESUMEN
Abbott RealTime HIV-1 Qualitative is an in vitro real-time PCR assay for detecting HIV-1 nucleic acids in human plasma and dried blood spots (DBS). The assay was designed to be used in diagnosis of HIV-1 infections in pediatric and adult patients, with an emphasis on the applicability in resource-limited settings. Use of DBS facilitates specimen collection from remote areas and transportation to testing laboratories. Small sample input requirement facilitates testing of specimens with limited collection volume. The Abbott RealTime HIV-1 Qualitative assay is capable of detecting HIV-1 group M subtypes A-H, group O and group N samples. HIV-1 virus concentrations detected with 95% probability were 80 copies/mL of plasma using the plasma protocol, and 2469 copies/mL of whole blood using the DBS protocol. The assay detected HIV-1 infection in 13 seroconversion panels an average 10.5 days earlier than an HIV-1 antibody test and 4.9 days earlier than a p24 antigen test. For specimens collected from 6 weeks to 18 months old infants born to HIV-1 positive mothers, assay results using both the DBS and plasma protocols agreed well with the Roche Amplicor HIV-1 DNA Test version 1.5 (95.5% agreement for DBS and 97.8% agreement for plasma).
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Sangre/virología , Desecación , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Plasma/virología , Manejo de Especímenes/métodos , Carga Viral/métodos , Adulto , ADN Viral/genética , ADN Viral/aislamiento & purificación , VIH-1/genética , Humanos , Lactante , ARN Viral/genética , ARN Viral/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Automated sample preparation systems must meet the demands of routine diagnostics laboratories with regard to performance characteristics and compatibility with downstream assays. OBJECTIVES: In this study, the performance of QIAGEN EZ1 DSP Virus Kit on the BioRobot EZ1 DSP was evaluated in combination with the Abbott RealTime HIV-1, HCV, and HBV assays, followed by thermalcycling and detection on the Abbott m2000rt platform. STUDY DESIGN: The following performance characteristics were evaluated: linear range and precision, sensitivity, cross-contamination, effects of interfering substances and correlation. RESULTS: Linearity was observed within the tested ranges (for HIV-1: 2.0-6.0 log copies/ml, HCV: 1.3-6.9 log IU/ml, HBV: 1.6-7.6 log copies/ml). Excellent precision was obtained (inter-assay standard deviation for HIV-1: 0.06-0.17 log copies/ml (>2.17 log copies/ml), HCV: 0.05-0.11 log IU/ml (>2.09 log IU/ml), HBV: 0.03-0.07 log copies/ml (>2.55 log copies/ml)), with good sensitivity (95% hit rates for HIV-1: 50 copies/ml, HCV: 12.5 IU/ml, HBV: 10 IU/ml). No cross-contamination was observed, as well as no negative impact of elevated levels of various interfering substances. In addition, HCV and HBV viral load measurements after BioRobot EZ1 DSP extraction correlated well with those obtained after Abbott m2000sp extraction. CONCLUSIONS: This evaluation demonstrates that the QIAGEN EZ1 DSP Virus Kit provides an attractive solution for fully automated, low throughput sample preparation for use with the Abbott RealTime HIV-1, HCV, and HBV assays.
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Infecciones por VIH/diagnóstico , Hepatitis B/diagnóstico , Hepatitis C/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Manejo de Especímenes/métodos , Virología/métodos , VIH-1/aislamiento & purificación , Hepacivirus/aislamiento & purificación , Humanos , Orthohepadnavirus/aislamiento & purificación , Juego de Reactivos para Diagnóstico , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Abbott RealTime High Risk (HR) HPV is a recently developed test for the detection of 14 high-risk oncogenic HPV types combined with the ability to concurrently identify genotypes 16 and 18. OBJECTIVES: The clinical performance of the Abbott RealTime HR HPV test was evaluated in comparison with the Hybrid Capture 2 (HC2) test for the detection of cervical intraepithelial neoplasia 2 or worse (CIN2+). The relative accuracy of the Abbott RealTime HR HPV to detect high-risk HPV was also determined. STUDY DESIGN: Cervical specimens were collected from 702 patients with abnormal cytology who were referred for colposcopy, and were tested with liquid based cytology (LBC), Abbott RealTime HR HPV and HC2. Genotyping was done using the Linear Array (LA) method. Histological assessment was used as the gold standard for disease status. Clinical performance for detection of disease was evaluated for Abbott RealTime HR HPV in comparison with HC2 in the overall population and in each cytological grade. The relative accuracy for detection of high-risk HPV was assessed by concordance between the two tests and based on LA genotyping. RESULTS AND CONCLUSIONS: The Abbott RealTime HR HPV showed similar clinical performance for detection of CIN2+ when compared with HC2, for both the overall population and those with a cytological grade of atypical squamous cells of undetermined significance (ASC-US). The accuracy for detection of high-risk HPV was significantly higher with Abbott RealTime HR HPV than with HC2.
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Técnicas de Diagnóstico Molecular/métodos , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/diagnóstico , Displasia del Cuello del Útero/diagnóstico , Displasia del Cuello del Útero/prevención & control , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , Sensibilidad y Especificidad , Estados Unidos , Frotis Vaginal , Adulto JovenRESUMEN
BACKGROUND: High-risk human papillomavirus (HPV) is the causative agent of cervical cancer. Among the high-risk types, infection with HPV 16 and 18 is associated with significantly higher risk of disease progression, and consequently these two types together cause approximately 70% of invasive cervical cancer worldwide. Identification of HPV 16 and HPV 18 can provide valuable information for risk stratification and clinical management of patients infected with these two types in both ASC-US triage and primary screening in women over age 30. It may also be valuable in the assessment of HPV vaccine efficacy. Abbott RealTime High Risk (HR) HPV is a recently developed test for the detection of 14 high-risk HPV types with the ability to concurrently identify HPV 16 and 18. OBJECTIVE: To evaluate the clinical performance of Abbott RealTime HR HPV test. STUDY DESIGN: Abbott RealTime HR HPV was evaluated with 253 cervical specimens obtained from patients with CIN 3 and 340 specimens from patients with cervical cancer to determine clinical sensitivity of the test and the prevalence of types 16 and 18. Additionally, 757 cervical specimens obtained from women 30 years of age or older with normal cytology in a general screening population were tested to determine high-risk HPV positivity rate. RESULTS: The Abbott RealTime HR HPV test detected 97.2% (246/253) of CIN 3 specimens and 98.5% (335/340) of cancer specimens. HPV 16 was the most prevalent type in both CIN 3 (72.8%) and cancer specimens (64.5%). HPV 16 and 18 combined were detected in 78.9% of high-risk HPV positive CIN 3 and 84.8% of high-risk HPV positive cancer specimens. In specimens from women 30 years of age or older with normal cytology in a screening population, the HPV positivity rate was 6.5% (49/757). CONCLUSIONS: Abbott RealTime HR HPV is a highly sensitive test for detection of high-grade cervical disease and cancer. The HPV 16 and HPV 18 typing capability of the test offers the advantage of stratifying patients at greater risk of progression and may thus aid in better patient care and management.