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P Shawn Mitchell

Publications and source records attributed to P Shawn Mitchell.

13 recordsLinked to original sources
PubMed2006

Detection of HIV-1 proviral DNA with the AMPLICOR HIV-1 DNA Test, version 1.5, following sample processing by the MagNA Pure LC instrument.

BACKGROUND: The AMPLICOR HIV-1 DNA Test, version 1.5 (AMP HIV-1 DNA 1.5), is a new commercially available PCR assay for the detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in human whole blood. OBJECTIVE: This study evaluates the performance characteristics of the assay following automated sample processing by the MagNA Pure LC instrument (MP). STUDY DESIGN: Analytical sensitivity and reproducibility were assessed by testing replicate HIV-1 DNA dilution panels over 5 days. Clinical sensitivity and specificity were studied among 28 HIV-1 DNA-positive clinical specimens, 60 specimens from healthy blood donors, and 63 specimens from HIV-1-seropositive patients with HIV-1 RNA plasma levels ranging from <50 to >100,000 copies/mL. RESULTS: Following MP sample processing, the assay yielded an analytical sensitivity (95% detection rate) of 66.3 copies/mL (95% CI, 50.7-106.8), with clinical sensitivity and specificity of 100%. CONCLUSIONS: MP is a reliable, labor-saving platform capable of processing specimens for AMP HIV-1 DNA 1.5. When combined with MP sample processing, AMP HIV-1 DNA 1.5 is a sensitive and reproducible assay for the detection of HIV-1 DNA in clinical whole blood specimens. However, the current AMP HIV-1 DNA 1.5 kit configuration may result in inefficient utilization of reagents.

Adolescent↗
PubMed2006

Evaluation of the invader assay for genotyping hepatitis C virus.

The Invader 1.0 assay (Invader HCV Genotyping Assay, version 1.0; Third Wave Technologies, Inc., Madison, WI) has been developed for the rapid differentiation of hepatitis C virus (HCV) genotypes 1 to 6 based on sequence variation within the HCV 5' noncoding (NC) region. In the present study, we evaluated the compatibility of Invader 1.0 with the COBAS MONITOR (COBAS AMPLICOR HCV MONITOR Test, version 2.0; Roche Molecular Systems, Inc., Branchburg, NJ), COBAS AMPLICOR (COBAS AMPLICOR Hepatitis C Virus Test, version 2.0; Roche Molecular Systems, Inc.), and COBAS TaqMan (COBAS TaqMan HCV Test; Roche Molecular Systems, Inc.) assays. The minimum HCV RNA titers required for successful HCV genotyping (>/=90% success rate) were 1,000 IU/ml for COBAS MONITOR, 100 IU/ml for COBAS AMPLICOR, and 10 IU/ml for COBAS TaqMan. Invader 1.0 results obtained from unpurified COBAS TaqMan amplification products of 111 retrospectively selected clinical serum specimens (genotypes 1 to 6, with virus titers ranging from 15.1 to 2.1 x 10(7) IU/ml) showed 98% concordance with results obtained from the TRUGENE HCV 5' NC Genotyping Kit (Bayer HealthCare LLC, Tarrytown, NY), used in conjunction with COBAS AMPLICOR. Although the assay is sensitive, accurate, and easy to perform, additional optimization of the Invader 1.0 interpretive software (Invader Data Analysis Worksheet) may be necessary to reduce potential misidentification of HCV genotypes in low-titer specimens. In summary, Invader 1.0 is compatible with a variety of commercially available PCR-based HCV 5' NC region amplification assays and is suitable for routine HCV genotyping in clinical laboratories.

5' Untranslated Regions↗
PubMed2006

Quantification of hepatitis B virus (HBV) DNA with a TaqMan HBV analyte-specific reagent following sample processing with the MagNA pure LC instrument.

TaqMan hepatitis B virus (HBV) analyte-specific reagent (ASR; Roche Molecular Systems, Inc., Branchburg, NJ) is designed for the quantification of HBV DNA in serum or plasma. The performance characteristics of TaqMan HBV ASR following automated sample processing with the MagNA Pure LC instrument (MP; Roche Applied Science, Indianapolis, IN) were evaluated in this study. Analytical sensitivity and precision were assessed with commercially available HBV standards, while clinical serum specimens from HBsAg-seropositive patients and healthy blood donors were used to determine clinical sensitivity, specificity, and correlation with other commercially available assays. Analytical studies yielded a limit of detection of 2.4 IU/ml, with good linearity and correlation (R(2) = 0.9958) with expected HBV DNA titers over a wide range (6.0 x 10(0) to 2.1 x 10(8) IU/ml). Clinical sensitivity and specificity of the assay combined with automated sample processing were both 100%. Comparison of TaqMan HBV ASR and VERSANT HBV DNA 3.0 assay (bDNA; Bayer HealthCare LLC, Tarrytown, NY) results among clinical specimens yielded good correlation (R(2) = 0.9237), with a mean difference in titer of -0.213 log(10) IU/ml (95% confidence interval, -0.678 to 1.10 log(10) IU/ml). The overall test failure rate was 2.0% among 204 clinical serum specimens tested. Total time required for MP sample processing and automated postelution handling of 24 samples was 224 min, with 57 min of actual hands-on time. MP is a reliable, labor-saving platform suitable for use with TaqMan HBV ASR, providing sensitive and accurate quantification of HBV DNA levels over a range of 8 log(10) IU/ml.

DNA, Viral↗
PubMed2005

Evaluation of the MagNA Pure LC used with the TRUGENE HBV Genotyping Kit.

BACKGROUND: The current manual sample processing method recommended for use with the TRUGENE HBV Genotyping Kit (TRUGENE HBV; Bayer HealthCare LLC, Tarrytown, NY) is labor-intensive and may be prone to specimen cross-contamination. Recent evaluations of the MagNA Pure LC (MP; Roche Applied Science, Indianapolis, IN) suggest that it is suitable for automated, contamination-free extraction and purification of viral nucleic acids from large-volume (1.0 mL) serum or plasma specimens. OBJECTIVES: We evaluated the MP Total Nucleic Acid Isolation Kit--Large Volume (Roche Applied Science) in conjunction with TRUGENE HBV to establish the analytical sensitivity (threshold titer) of the assay, in HBV DNA International Units (IU)/mL, for obtaining consistent, interpretable sequence data from TRUGENE HBV. STUDY DESIGN: HBV analytical standards, prepared as 10 replicates (1.0 mL each) at each of the following concentrations: 200, 1000, 5000, and 10,000 IU/mL, were processed by MP and analyzed by TRUGENE HBV according to manufacturer's instructions. Performance of TRUGENE HBV used in conjunction with MP sample processing was evaluated further using 22 clinical serum specimens containing low titers of HBV DNA. RESULTS: All replicates of HBV analytical standards at 1000, 5000, and 10,000 IU/mL yielded interpretable TRUGENE HBV sequences, whereas interpretable sequences were obtained in 90% (9 of 10) of the replicates at 200 IU/mL. TRUGENE HBV sequences were interpretable in 86% (19 of 22) of the clinical specimens studied. CONCLUSIONS: MP sample processing is efficient and suitable for use with TRUGENE HBV. When combined with MP sample processing, TRUGENE HBV yielded interpretable sequences from HBV analytical standards and clinical serum specimens with HBV DNA titers of > or =200 IU/mL.

DNA, Viral↗
PubMed2005

Evaluation of the COBAS TaqMan HCV test with automated sample processing using the MagNA pure LC instrument.

The COBAS TaqMan HCV Test (TaqMan HCV; Roche Molecular Systems Inc., Branchburg, N.J.) for hepatitis C virus (HCV) performed on the COBAS TaqMan 48 Analyzer (Roche Molecular Systems) currently relies on a manual sample processing method. Implementation of an automated sample processing method would facilitate the clinical use of this test. In this study, we evaluated the performance characteristics of TaqMan HCV following automated sample processing by the MagNA Pure LC instrument (MP; Roche Applied Science, Indianapolis, Ind.). The analytical sensitivity of TaqMan HCV following sample processing by MP was 8.1 IU/ml (95% confidence interval, 6.1 to 15.2). The assay showed good linearity (R(2) = 0.99) across a wide range of HCV RNA levels (25 to 5 x 10(6) IU/ml), with coefficients of variation ranging from 10% to 46%. Among 83 clinical specimens, the sensitivity and specificity of TaqMan HCV were 100% and 95%, respectively, when compared to the COBAS AMPLICOR hepatitis C virus test, version 2.0 (COBAS AMPLICOR; Roche Molecular Systems), with TaqMan HCV detecting two more HCV RNA-positive specimens than COBAS AMPLICOR. Both specimens were confirmed to be HCV RNA positive by the VERSANT HCV RNA qualitative test (Bayer HealthCare LLC, Tarrytown, N.Y.). There was also strong correlation (R(2) = 0.95) and good agreement between the results from TaqMan HCV and the VERSANT HCV RNA 3.0 assay (bDNA) (Bayer HealthCare LLC) among a group of 93 clinical specimens. The MP is a versatile, labor-saving sample processing platform suitable for reliable performance of TaqMan HCV.

Automation↗
PubMed2003

Evaluation of the TRUGENE HCV 5'NC genotyping kit with the new GeneLibrarian module 3.1.2 for genotyping of hepatitis C virus from clinical specimens.

The TRUGENE HCV 5'NC genotyping kit (GeneLibrarian modules 3.1.1 and 3.1.2) and VERSANT HCV genotyping assay were compared by using 96 hepatitis C virus (HCV) RNA-positive patient specimens, including HCV genotypes 1, 2, 3, 4, 5, 6, and 10. The TRUGENE HCV 5'NC genotyping kit (GeneLibrarian module 3.1.2) yielded the most accurate genotyping results.

5' Untranslated Regions↗
PubMed2003

Evaluation of the MagNA pure LC instrument for extraction of hepatitis C virus RNA for the COBAS AMPLICOR Hepatitis C Virus Test, version 2.0.

The COBAS AMPLICOR system has played a major role in the transition of molecular diagnostics from research to routine clinical laboratory use by automating the nucleic acid amplification and detection processes. However, sample preparation remains a labor-intensive portion of the procedure. In this study, we evaluated the performance of the COBAS AMPLICOR Hepatitis C Virus Test, version 2.0 (Roche Molecular Systems, Branchburg, N.J.) following manual hepatitis C virus (HCV) RNA extraction versus automated extraction with the MagNA Pure LC instrument (Roche Applied Science, Indianapolis, Ind.). Parallel replicate testing was performed with standard dilutions of 100, 75, 60, and 0 HCV IU/ml and 153 clinical specimens. An analytical sensitivity of 75 IU/ml was achieved with either the manual or the standard-volume (200 microl) automated extraction methodologies (25 of 26 [96.2%]; 95% confidence interval [95% CI], 80.4 to 99.9), whereas the clinical sensitivity and specificity were both 100% with either extraction method. A large-volume (1 ml) automated extraction method was also evaluated with standard dilutions of 40, 25, 10, and 0 IU/ml and the same 153 clinical specimens. The analytical sensitivity of the COBAS AMPLICOR assay with the large-volume extraction method was 25 HCV IU/ml (26 of 26 [100%]; 95% CI, 86.8 to 100), whereas the clinical sensitivity and specificity were both 100%. The MagNA Pure LC instrument is a versatile, labor-saving platform capable of integration with minimal modification of the existing assay procedure. The increased sensitivity of the COBAS AMPLICOR Hepatitis C Virus Test, version 2.0 performed in conjunction with large-volume HCV RNA extraction may be important in HCV diagnostic testing as new therapeutic strategies evolve.

Hepacivirus↗
PubMed2003

Clinical spectrum and laboratory characteristics associated with detection of herpes simplex virus DNA in cerebrospinal fluid.

OBJECTIVE: To determine the clinical, neurologic, and laboratory characteristics of patients with herpes simplex virus (HSV) type 1 (HSV-1) or HSV type 2 (HSV-2) DNA detected in cerebrospinal fluid (CSF) with use of polymerase chain reaction. PATIENTS AND METHODS: Clinical, laboratory, and demographic data were determined from 249 CSF specimens (collected from 247 patients >10 years of age) that tested positive for HSV-1 or HSV-2 DNA at the Mayo Clinic from January 1999 to August 2000. RESULTS: The median age of the 200 patients whose age was available was 70 years vs 40 years for those with HSV-1 or HSV-2 DNA in CSF, respectively. Detailed data were available for 39 and 78 patients with positive polymerase chain reaction results for HSV-1 and HSV-2, respectively. Of those with HSV-1 DNA detected in CSF, 89% had encephalitis, whereas most patients with HSV-2 DNA detected in CSF had findings compatible with meningitis. Only 5 (7%) of 69 patients in whom HSV-2 was detected in CSF had genital lesions at presentation, and none of the assessable patients with HSV-2 who had recurrent meningitis had active genital lesions at presentation. CONCLUSION: The vast majority (82%) of patients with HSV-2 detected in CSF had no history of genital herpes and no lesions at the time of presentation. Polymerase chain reaction assays designed to detect HSV in CSF should detect HSV-1 and HSV-2 and differentiate between HSV-1 and HSV-2.

Academic Medical Centers↗
PubMed2002

Comparison of line probe assay and DNA sequencing of 5' untranslated region for genotyping hepatitis C virus: description of novel line probe patterns.

We compared a commercial line probe assay (INNO-LiPA HCV II, Innogenetics, N.V., Ghent, Belgium, distributed by Bayer Diagnostics) to an in-house 5' untranslated region direct DNA sequencing method for genotyping hepatitis C virus (HCV). Initial evaluation demonstrated that the INNO-LiPA HCV II assay and sequencing assay assigned the same genotype for 110/132 (83.3%) patient specimens (98 subtype and 12 genotype only identifications). Following the initial evaluation, the INNO-LiPA HCV II assay was used routinely to genotype HCV from patient specimens submitted to our laboratory for genotyping (n = 1,739). During this second part of the study, novel line probe patterns have been noted and interpreted using the in-house direct sequencing assay. Reactivity at bands 1, 2, 3, 4, 5 and 8 (n = 4) or 1, 2, 3, 4, 6 and 7 (n = 2) represented HCV genotype 1. Reactivity at bands 1, 2, 5 and 9 (n = 1) represented HCV genotype 2. Reactivity at bands 1, 2, 5, 9 and 16 (n = 1) represented HCV genotype 4. Reactivity at bands 1, 2, 5, 9, 10, 11 (weak band) and 12 (n = 118) most likely represented HCV genotype 2b. This information should be of use to INNO-LiPA HCV II assay users.

5' Untranslated Regions↗
PubMed2002

A simple PDMS-based electro-fluidic interface for microchip electrophoretic separations.

High voltage electrodes for electrophoresis have been integrated into a polymer layer that can be reversibly bound to glass microchips for electrophoretic separations. By using the liquid precursor to the polymer polydimethylsiloxane (PDMS), platinum electrodes and reservoirs can be positioned prior to solidification, providing a simple and flexible method for electrode interface construction. Field strengths up to 875 V cm(-1) over an 8 cm separation channel can be applied to the system without any loss in performance of the interface. The interface can function as an electro-fluidic interface between the high voltage power supply and the separation channel and, when reversibly sealed to an etched glass plate, functions as a cover plate establishing a hybrid PDMS-glass microchip in which the electrodes are directly integrated onto the device. The versatility of this approach is not only demonstrated by separating DNA fragments in a novel buffer sieving matrix, but also with the molecular diagnostic analysis of a variety of DNA samples for Duschenne Muscular Dystrophy and cytomegalovirus (CMV) infection, using both microchip interface configurations.

Cytomegalovirus Infections↗
PubMed2002

Multiplex LightCycler PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal specimens.

A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9 B. pertussis and 3 B. parapertussis) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS481) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens (n = 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different (P = 0.0654). The insertion sequence IS481 is also present in Bordetella holmesii; specimens containing B. holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of B. pertussis and B. parapertussis in nasopharyngeal specimens.

Bordetella↗
PubMed2000

Varicella-zoster virus detection by polymerase chain reaction using bronchoalveolar lavage specimens.

We report the use of polymerase chain reaction (PCR) techniques from a bronchoalveolar lavage (BAL) specimen for the successful early diagnosis in a case of atypical but severe varicella-zoster virus (VZV) pneumonitis. Atypical presentations of disseminated VZV frequently prolong the time required for accurate diagnosis, resulting in increased morbidity, or mortality. Although further investigation is necessary to determine the sensitivity and positive predictive value of this test, PCR analysis of bronchoscopic specimens may expedite the diagnosis of disseminated VZV.

Aged↗