The cobas® 6800/8800 System: a new era of automation in molecular diagnostics.

INTRODUCTION: Molecular diagnostics is a key component of laboratory medicine. Here, the authors review key triggers of ever-increasing automation in nucleic acid amplification testing (NAAT) with a focus on specific automated Polymerase Chain Reaction (PCR) testing and platforms such as the recently launched cobas® 6800 and cobas® 8800 Systems. The benefits of such automation for different stakeholders including patients, clinicians, laboratory personnel, hospital administrators, payers, and manufacturers are described. Areas Covered: The authors describe how molecular diagnostics has achieved total laboratory automation over time, rivaling clinical chemistry to significantly improve testing efficiency. Finally, the authors discuss how advances in automation decrease the development time for new tests enabling clinicians to more readily provide test results. Expert Commentary: The advancements described enable complete diagnostic solutions whereby specific test results can be combined with relevant patient data sets to allow healthcare providers to deliver comprehensive clinical recommendations in multiple fields ranging from infectious disease to outbreak management and blood safety solutions.

Chronic Hepatitis B, C, and D.

Chronic hepatitis B, C, and D virus infections contribute significantly to the morbidity and mortality of immunocompromised individuals. To contextualize discussion of these infections in immunocompromised patients, this paper provides an overview of aspects of infection in normal hosts. It then describes differences in disease, diagnostic testing, and therapeutic management observed in immunocompromised patients.

Comparison of Three Different FDA-Approved Plasma HIV-1 RNA Assay Platforms Confirms the Virologic Failure Endpoint of 200 Copies per Milliliter Despite Improved Assay Sensitivity.

Discrepancies between HIV-1 RNA results assayed by different FDA-approved platforms have been reported. Plasma samples collected from 332 randomly selected clinical trial participants during the second year of antiretroviral treatment were assayed with three FDA-approved platforms: UltraSensitive Roche Amplicor Monitor, v1.5 (Monitor), the Abbott RealTime HIV-1 test on the m2000 system (Abbott), and the Roche TaqMan HIV-1 test, v2.0 (TaqMan). Samples from 61 additional participants with confirmed HIV-1 RNA levels of >50 copies/ml during trial follow-up were also included. Endpoints were HIV-1 RNA quantification of ≤50 copies/ml versus >50 copies/ml at an individual-sample level (primary) and determination of confirmed virologic failure (VF) from longitudinal samples. A total of 389 participants had results obtained from all assays on at least one sample (median = 6). Proportions of results of >50 copies/ml were 19% (Monitor), 22% (TaqMan), and 25% (Abbott). Despite indication of strong agreement (Cohen's kappa, 0.76 to 0.82), Abbott was more likely to detect HIV-1 RNA levels of >50 copies/ml than Monitor (matched-pair odds ratio [mOR] = 4.2; modified Obuchowski P < 0.001) and TaqMan (mOR = 2.1; P < 0.001); TaqMan was more likely than Monitor (mOR = 2.6; P < 0.001). Despite strong agreement in classifying VF across assay comparisons (kappa, 0.75 to 0.92), at a 50-copies/ml threshold, differences in the probability of VF classification (in the same direction as primary) were apparent (all McNemar's P < 0.007). At a 200-copies/ml VF threshold, no differences between assays were apparent (all P > 0.13). Despite strong agreement among assays, significant differences were observed with respect to detecting HIV-1 RNA levels of >50 copies/ml and identifying VF at the 50-copies/ml threshold. This has important implications for the definition of VF in clinical trials and clinical practice.

Evaluation of the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HCV Test, v2.0 and comparison to assays used in routine clinical practice in an international multicenter clinical trial: The ExPECT study.

BACKGROUND: The COBAS(®) AmpliPrep(®)/COBAS(®) TaqMan(®) HCV Test, v2.0 (CAP/CTM2) is used for HCV RNA viral load monitoring. OBJECTIVES: The performance of the CAP/CTM2 was compared to other widely used tests, including a manual version of the assay (the COBAS(®) TaqMan(®) HCV Test, v2.0 for use with the High Pure System, HPS/CTM2) predominantly used during phase III clinical trials for the new direct acting antiviral therapies. STUDY DESIGN: Low HCV RNA level comparisons were performed across tests (Abbott Realtime HCV Test, ART; COBAS(®) AmpliPrep(®)/COBAS(®) TaqMan(®) HCV Test, v1.0, CAP/CTM1; CAP/CTM2; and HPS/CTM2) using dilutions of the 2nd HCV WHO International Standard. Additionally, the clinical performance of the CAP/CTM2 was evaluated with 421 leftover HCV RNA-positive routine clinical samples. RESULTS: All quantifiable WHO dilutions were within ±0.3log10IU/mL of the expected results across tests and the analytical sensitivity resulted in a limit of detection of 12IU/mL (95% confidence interval, 10, 15). When clinical samples were tested the results for 87% (367 of 421) of all sample comparisons were within ±0.5log10IU/mL. When low viral load results (25-3500IU/mL) were compared, values obtained by the ART assay were significantly lower (p<0.0001) than those obtained with the CAP/CTM2. CONCLUSIONS: The new CAP/CTM2 showed good accuracy with comparable sensitivity to comparator assays. The new kit is well-suited for use in the routine diagnostic laboratory, especially for accurate monitoring of patients receiving triple therapy or interferone-free regimens.

Age-related differences in head impact exposure of 9-13 year old football players.

While more than two-thirds of football players are under the age of 14, little biomechanics research has been conducted to quantify head impact exposure for players in this age range. The objective of this study was to quantify head impact exposure and the influence of age for 9-13 year old football players. Kinematic data from over 4400 head impacts were collected from 25 youth football players on two teams for one season using in-helmet accelerometers. The 50th and 95th percentile magnitudes for linear and rotational head acceleration and the number of impacts sustained were determined for each player. Comparisons were made for each metric by team and by player age. Players on the 9-11 year old team sustained an average of 154 ± 126 impacts with a 95th percentile linear acceleration magnitude of 40 g. On the 10 to 13 year old team, players sustained an average of 193 ± 158 impacts with a 95th percentile linear acceleration magnitude of 47 g. Players on the older team sustained slightly more and higher magnitude impacts than those on the younger team. These differences in head impact exposure were not significant due to large variation between players within each team.

Molecular diagnostics in the management of chronic hepatitis C: key considerations in the era of new antiviral therapies.

Molecular tests that detect and/or quantify HCV RNA are important in the diagnosis and management of patients with chronic hepatitis C (CHC) undergoing anti-viral therapy. The primary goal of anti-HCV therapy is to achieve a sustained virologic response (SVR) defined as "undetectable" Hepatitis C Virus (HCV) RNA in the serum or plasma at 12 to 24 weeks following the end of treatment.

Seasonal variation of depressive symptoms in unipolar major depressive disorder.

OBJECTIVES: Retrospective and cross-sectional studies of seasonal variation of depressive symptoms in unipolar major depression have yielded conflicting results. We examined seasonal variation of mood symptoms in a long-term prospective cohort - the Collaborative Depression Study (CDS). METHODS: The sample included 298 CDS participants from five academic centers with a prospectively derived diagnosis of unipolar major depression who were followed for at least ten years of annual or semi-annual assessments. Generalized linear mixed models were utilized to investigate the presence of seasonal patterns. In a subset of 271 participants followed for at least 20 years, the stability of a winter depressive pattern was assessed across the first two decades of follow-up. RESULTS: A small increase in proportion of time depressed was found in the months surrounding the winter solstice, although the greatest symptom burden was seen in December through April with a peak in March. The relative burden of winter depressive symptoms in the first decade demonstrated no relationship to that of the second decade. The onset of new episodes was highest October through January, peaking in January. CONCLUSIONS: There exists a small but statistically significant peak in depressive symptoms from the month of the winter solstice to the month of the spring equinox. However, the predominance of winter depressive symptoms did not appear stable over the long-term course of illness.

Comparative performances of HIV-1 RNA load assays at low viral load levels: results of an international collaboration.

Low-level viremia during antiretroviral therapy and its accurate measurement are increasingly relevant. Here, we present an international collaboration of 4,221 paired blood plasma viral load (pVL) results from four commercial assays, emphasizing the data with low pVL. The assays compared were the Abbott RealTime assay, the Roche Amplicor assay, and the Roche TaqMan version 1 and version 2 assays. The correlation between the assays was 0.90 to 0.97. However, at a low pVL, the correlation fell to 0.45 to 0.85. The observed interassay concordance was higher when detectability was defined as 200 copies/ml than when it was defined as 50 copies/ml. A pVL of ∼100 to 125 copies/ml by the TaqMan version 1 and version 2 assays corresponded best to a 50-copies/ml threshold with the Amplicor assay. Correlation and concordance between the viral load assays were lower at a low pVL. Clear guidelines are needed on the clinical significance of low-level viremia.

Comparison of two quantitative real-time CMV-PCR tests calibrated against the 1st WHO international standard for viral load monitoring of renal transplant patients.

Cytomegalovirus (CMV) replication in organ transplant recipients is commonly diagnosed by quantitative PCR methods. However, there has been a poor inter-laboratory correlation of viral load values due to the lack of an international reference standard. In a recent study, the COBAS® AmpliPrep/COBAS® TaqMan® (CAP/CTM) CMV test calibrated to the 1st WHO CMV standard, showed good reproducibility in CMV load values across multiple laboratories. Fifty-seven follow-up plasma specimens from 10 kidney transplant recipients with CMV replication were examined using the new quantitative CAP/CTM CMV test and the "in-house" quantitative CMV real-time PCR method, also calibrated against the 1st WHO CMV standard for their clinical applicability for monitoring CMV load in renal transplant patients. By CAP/CTM CMV test 49/57 specimens were CMV-DNA positive compared to 44/57 by the "in-house" PCR test. The "in-house" PCR and CAP/CTM CMV test correlated well in monitoring individual kidney transplant patients. Conversion of the CMV-DNA copies to IUs made the results of the "in-house" PCR and CAP/CTM CMV test less uniform in analysis of the patient samples. In specimens of one patient, significant underquantification of CMV load with "in-house" PCR emerged during follow-up due to a point mutation in the "in-house" PCR primer sequence. The CAP/CTM CMV test was found suitable for diagnosing and monitoring CMV replication in renal transplant patients. Multicenter studies are needed to provide more information of the commutability of the 1st WHO CMV standard and to define the clinical thresholds.

Genome-wide copy number variation analysis of a Branchio-oto-renal syndrome cohort identifies a recombination hotspot and implicates new candidate genes.

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial arch anomalies, hearing loss and renal dysmorphology. Although haploinsufficiency of EYA1 and SIX1 are known to cause BOR, copy number variation analysis has only been performed on a limited number of BOR patients. In this study, we used high-resolution array-based comparative genomic hybridization on 32 BOR probands negative for coding-sequence and splice-site mutations in known BOR-causing genes to identify potential disease-causing genomic rearrangements. Of the >1,000 rare and novel copy number variants we identified, four were heterozygous deletions of EYA1 and several downstream genes that had nearly identical breakpoints associated with retroviral sequence blocks, suggesting that non-allelic homologous recombination seeded by this recombination hotspot is important in the pathogenesis of BOR. A different heterozygous deletion removing the last exon of EYA1 was identified in an additional proband. Thus, in total five probands (14 %) had deletions of all or part of EYA1. Using a novel disease-gene prioritization strategy that includes network analysis of genes associated with other deletions suggests that SHARPIN (Sipl1), FGF3 and the HOXA gene cluster may contribute to the pathogenesis of BOR.

Head impact exposure in youth football: elementary school ages 9-12 years and the effect of practice structure.

Head impact exposure in youth football has not been well-documented, despite children under the age of 14 accounting for 70% of all football players in the United States. The objective of this study was to quantify the head impact exposure of youth football players, age 9-12, for all practices and games over the course of single season. A total of 50 players (age = 11.0 ± 1.1 years) on three teams were equipped with helmet mounted accelerometer arrays, which monitored each impact players sustained during practices and games. During the season, 11,978 impacts were recorded for this age group. Players averaged 240 ± 147 impacts for the season with linear and rotational 95th percentile magnitudes of 43 ± 7 g and 2034 ± 361 rad/s(2). Overall, practice and game sessions involved similar impact frequencies and magnitudes. One of the three teams however, had substantially fewer impacts per practice and lower 95th percentile magnitudes in practices due to a concerted effort to limit contact in practices. The same team also participated in fewer practices, further reducing the number of impacts each player experienced in practice. Head impact exposures in games showed no statistical difference. While the acceleration magnitudes among 9-12 year old players tended to be lower than those reported for older players, some recorded high magnitude impacts were similar to those seen at the high school and college level. Head impact exposure in youth football may be appreciably reduced by limiting contact in practices. Further research is required to assess whether such a reduction in head impact exposure will result in a reduction in concussion incidence.

HCV RNA viral load assessments in the era of direct-acting antivirals.

Recent regulatory approvals of the NS3/4A protease inhibitors boceprevir and telaprevir launched a new therapeutic era for hepatitis C virus (HCV) genotype 1 infection. Decisions to shorten, extend, or stop treatment with these direct-acting antiviral (DAA) regimens require accurate quantification of serum HCV RNA levels. To effectively use DAA therapies, clinicians must understand performance characteristics of HCV RNA real-time PCR assays and the clinical significance of HCV RNA that is detectable below the lower limit of quantification. This review summarizes terms used to report HCV RNA viral load results, explains the analytical performance of the PCR assay used in the clinical trials of boceprevir and telaprevir, and compares currently available commercial assays.

CMV antigenemia and quantitative viral load assessments in hematopoietic stem cell transplant recipients.

BACKGROUND: Sensitive and reliable diagnostic tests are essential for the prevention of cytomegalovirus (CMV) disease after hematopoietic stem cell transplantation (HSCT). pp65 antigenemia and polymerase chain reaction (PCR) assays are commonly used to monitor CMV in HSCT recipients. However, there is considerable intra- and inter-laboratory variability in the results, which impact comparability and clinical practice. OBJECTIVES/STUDY DESIGN: Using 380 samples from 135 HSCT recipients, we compared the new FDA approved quantitative PCR assay, COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) CMV test (CAP/CTM CMV test) developed and standardized using the 1st WHO International Standard for CMV with pp65 antigenemia and COBAS(®) AMPLICOR MONITOR CMV tests. RESULTS: The median time between transplantation and testing samples was 57 days (range, 0-207 days). The median CMV load (log(10)) was 3.17 IU/mL (3.21 copies/mL). Among samples with detectable CMV load, 52% were negative by pp65 antigenemia. CMV loads were higher in pp65 antigenemia-positive than in negative samples. One pp65-antigenemia-positive cell per 100,000 leukocytes corresponded to a median CMV load of 1200 IU/mL. CMV loads determined by the CAP/CTM CMV test were slightly lower than the ones by the AMPLICOR MONITOR CMV test (-0.15 [95% CI, -0.18 to -0.13] copies/mL), but slope differences indicated only limited co-linearity. CONCLUSIONS: The CAP/CTM CMV test is more sensitive than pp65 antigenemia and the AMPLICOR MONITOR CMV test in HSCT recipients. The lower limit of quantification and co-linearity with the international WHO standard renders the CAP/CTM CMV test suitable for future clinical trials defining viral load thresholds of CMV therapy.

Second-generation Cobas AmpliPrep/Cobas TaqMan HCV quantitative test for viral load monitoring: a novel dual-probe assay design.

Hepatitis C virus (HCV) RNA viral load (VL) monitoring is a well-established diagnostic tool for the management of chronic hepatitis C patients. HCV RNA VL results are used to make treatment decisions with the goal of therapy to achieve an undetectable VL result. Therefore, a sensitive assay with high specificity in detecting and accurately quantifying HCV RNA across genotypes is critical. Additionally, a lower sample volume requirement is desirable for the laboratory and the patient. This study evaluated the performance characteristics of a second-generation real-time PCR assay, the Cobas AmpliPrep/Cobas TaqMan HCV quantitative test, version 2.0 (CAP/CTM HCV test, v2.0), designed with a novel dual-probe approach and an optimized automated extraction and amplification procedure. The new assay demonstrated a limit of detection and lower limit of quantification of 15 IU/ml across all HCV genotypes and was linear from 15 to 100,000,000 IU/ml with high accuracy (<0.2-log(10) difference) and precision (standard deviation of 0.04 to 0.22 log(10)). A specificity of 100% was demonstrated with 600 HCV-seronegative specimens without cross-reactivity or interference. Correlation to the Cobas AmpliPrep/Cobas TaqMan HCV test (version 1) was good (n = 412 genotype 1 to 6 samples, R(2) = 0.88; R(2) = 0.94 without 105 genotype 4 samples). Paired plasma and serum samples showed similar performance (n = 25, R(2) = 0.99). The sample input volume was reduced from 1 to 0.65 ml in the second version. The CAP/CTM HCV test, v2.0, demonstrated excellent performance and sensitivity across all HCV genotypes with a smaller sample volume. The new HCV RNA VL assay has performance characteristics that make it suitable for use with currently available direct-acting antiviral agents.

An international multicenter performance analysis of cytomegalovirus load tests.

BACKGROUND: Quantification of cytomegalovirus (CMV) load is central to the management of CMV infections in immunocompromised patients, but quantitative results currently differ significantly across methods and laboratories. METHODS: The COBAS AmpliPrep/COBAS TaqMan CMV Test (CAP/CTM CMV test), developed using the first World Health Organization CMV standard in the calibration process, was compared to local assays used by 5 laboratories at transplant centers in the United States and Europe. Blinded plasma panels (n = 90) spiked with 2.18-6.7 log(10) copies/mL and clinical plasma samples from immunocompromised patients (n = 660) were tested. RESULTS: Observed mean panel member concentrations by site and 95% confidence intervals (CIs) of the data combined across sites were narrower for CAP/CTM CMV test compared with local assays. The 95% CI in log(10) copies/mL of the combined data per panel member for CAP/CTM CMV test vs comparator assays was .17 vs 1.5 at 2.18 log(10) copies/mL; .14 vs .52 at 2.74 log(10) copies/mL; .16 vs .6 at 3.3 log(10) copies/mL; .2 vs 1.11 at 4.3 log(10) copies/mL; .21 vs 1.13 at 4.7 log(10) copies/mL; and .18 vs 1.4 at 6.7 log(10) copies/mL. In clinical specimens, constant and variable quantification differences between the CAP/CTM CMV test and comparator assays were observed. CONCLUSIONS: High interlaboratory agreement and precision of CAP/CTM CMV test results across 5 different laboratories over 4 orders of magnitude suggest that this assay could be valuable in prospective studies identifying clinical viral load thresholds for CMV treatment.

Comparison of three Roche hepatitis B virus viral load assay formats.

Two FDA-approved (in vitro diagnostic [IVD]) hepatitis B virus (HBV) viral load assays, the manual Cobas TaqMan HBV Test for use with the High Pure System (HP) and the automated Cobas AmpliPrep/Cobas TaqMan HBV Test v2.0 (CAP/CTM), were compared to a modified (not FDA-approved) version of the HP assay by automating the DNA extraction using the Total Nucleic Acid Isolation (TNAI) kit on the Cobas AmpliPrep. On average, CAP/CTM measurements were 0.08 log IU/ml higher than HP results (n = 206), and TNAI results were 0.17 log IU/ml higher than HP results (n = 166). The limit of detection (LOD), as determined by probit analysis using dilutions of the 2nd HBV international standard, was 10.2 IU/ml for CAP/CTM. The data sets for HP and TNAI were insufficient for probit analysis; however, there was 100% detection at ≥ 5 or ≥ 10 IU/ml for TNAI and HP, respectively. Linearity was demonstrated between 60 and 2,000,000 IU/ml, with slopes between 0.95 and 0.99 and R(2) values of >0.99 for all assays. Total precision (log percent coefficient of variance [CV]) was between 0.8% and 2.1% at 4.3 log IU/ml and between 1.4% and 4.9% at 2.3 log IU/ml. Correlation of samples, reproducibility, linearity, and LOD were acceptable and similar in all assays. The CAP/CTM assay and, to a lesser extent, the TNAI assay reduced hands-on time due to automation. There were no instances of contamination detected in negative samples during the course of the study, despite testing several samples up to 9.6 log IU/ml. The incidence of false-positive negative controls in HP and CAP/CTM clinical testing was <0.5% over 6 to 7 months of testing.

Evolution in the sensitivity of quantitative HIV-1 viral load tests.

Significant advancements in molecular diagnostics have been made since the inception and application of PCR-based technologies in clinical diagnostic laboratories and the management of HIV-1 infected patients. More recently, real-time PCR has improved the overall performance of assays used for detecting and quantifying HIV-1 RNA viral load in patients undergoing antiretroviral treatment. The effects of these changes and the interpretations of the HIV-1 viral load results are discussed in this review in the context of the different assays used, the viral dynamics of the HIV-1 virus, and the recent changes to HIV-1 treatment guidelines.

Molecular analysis of mitochondrial DNA point mutations by polymerase chain reaction.

Mitochondrial respiratory chain disorders are clinically and genetically heterogeneous. There are several mitochondrial DNA (mtDNA) point mutations responsible for common mitochondrial diseases such as mitochondrial encephalopathy, lactic acidosis, stroke-like events, myoclonic epilepsy and ragged red fibers, neuropathy, ataxia, retinitis pigmentosa, and Leber's hereditary optic neuropathy. As a result of the clinical overlap, it is usually necessary to analyze more than one mutation for a patient suspected of a mitochondrial disorder. Molecular diagnosis is often performed using polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis of the most likely point mutations. However, this method is time-consuming and often produces problems associated with incomplete restriction enzyme digestion. In addition, PCR/RFLP analysis may not be able to detect a low percentage of heteroplasmy. For a more effective method of diagnosing mtDNA disorders, we have developed a multiplex PCR/ allele-specific oligonucleotide (ASO) dot blot hybridization method to simultaneously analyze 11 point mutations. The PCR products from a DNA sample containing a homoplasmic wild-type or mutant mtDNA sequence will hybridize to either the wild-type or the mutant ASO probe. The PCR products of a heteroplasmic DNA sample will hybridize to both wild-type and mutant ASO probes. This PCR/ASO method allows the detection of low percentage mutant heteroplasmy.