Development of the Abbott RealTime ZIKA assay for the qualitative detection of Zika virus RNA from serum, plasma, urine, and whole blood specimens using the m2000 system.

Zika virus is an arthropod-borne flavivirus that has rapidly developed into a world-wide concern. Discovered in 1947, the virus was relatively obscure until an outbreak occurred in 2007 in the Yap islands and spread eventually to the Americas in 2015. Only 20% of patients infected with Zika virus develop symptoms. However, there can be serious consequences of infection including birth defects in developing fetuses and links to Guillain-Barré syndrome. The swift rise in infections has necessitated the development of diagnostic tests for both the detection of viral RNA and the presence of virus-specific antibodies. Abbott has developed a dual target RT-PCR assay for the detection of Zika virus RNA within serum, plasma, whole blood, and urine using the automated m2000 system for sample extraction to result reporting. The Abbott RealTime ZIKA assay has a limit of detection of 30 copies per mL in serum, 40 copies per mL in plasma and urine, and 120 copies per mL in whole blood and demonstrates high specificity against challenges from closely related infectious agents.

Commutability of Cytomegalovirus WHO International Standard in Different Matrices.

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.

Sequence conservation of the region targeted by the Abbott RealTime HCV viral load assay.

The Abbott RealTime (RT) HCV assay targets the 5' untranslated region (UTR) of the HCV genome. Here, we analyzed the sequence variability of the assay target regions from 1,092 specimens. Thermodynamic modeling of the percentage of primers/probes bound at the assay annealing temperature was performed to assess the potential effect of sequence variability. An analysis of this large data set revealed that the primer and probe binding sites of the RealTime HCV viral load assay are highly conserved and that naturally occurring sequence polymorphisms are not expected to discernibly impact assay performance.

Analytical characterization of an assay designed to detect and identify diverse agents of disseminated viral infection.

BACKGROUND: Diverse viruses often reactivate in or infect cancer patients, patients with immunocompromising infections or genetic conditions, and transplant recipients undergoing immunosuppressive therapy. These infections can disseminate, leading to death, transplant rejection, and other severe outcomes. OBJECTIVES: To develop and characterize an assay capable of inclusive and accurate identification of diverse potentially disseminating viruses directly from plasma specimens. STUDY DESIGN: We developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to simultaneously detect and identify adenovirus, enterovirus, polyomaviruses JC and BK, parvovirus B19, HSV-1, HSV-2, VZV, EBV, CMV, and herpesviruses 6-8 in plasma specimens. The assay performance was characterized analytically, and the results from clinical plasma samples were compared to the results obtained from single-analyte real time PCR tests currently used in clinical practice. RESULTS: The assay demonstrated sensitivity and specificity to diverse strains of the targeted viral families and robustness to interfering substances and potentially cross reacting organisms. The assay yielded 94% sensitivity when testing clinical plasma samples previously identified as positive using standard-of-care real-time PCR tests for a single target virus (available samples included positive samples for 11 viruses targeted by the assay). CONCLUSIONS: The assay functioned as designed, providing simultaneous broad-spectrum detection and identification of diverse agents of disseminated viral infection. Among 156 clinical samples tested, 37 detections were made in addition to the detections matching the initial clinical positive results.

Multi-center evaluation of the Abbott RealTime HCV assay for monitoring patients undergoing antiviral therapy for chronic hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) RNA monitoring during antiviral therapy is essential for early prediction of treatment success and failure to peginterferon alfa/ribavirin (PEG-IFN/RBV) therapy. OBJECTIVES: In this multi-center study we assessed the clinical utility of the Abbott RealTime HCV assay for monitoring patients undergoing antiviral therapy for chronic infection with HCV genotypes (GT) 1-3. STUDY DESIGN: We analyzed serum from 361 patients with chronic hepatitis C who had been treated with PEG-IFN/RBV. The predictive value of rapid virologic response (RVR), partial (≥2log(10) decline) and complete (HCV-RNA undetectable) early virologic response (pEVR/cEVR) based on RealTime HCV for achieving sustained virologic response was evaluated. In addition, the utility of RealTime HCV to tailor treatment duration according to individual virologic responses was studied in a subset of 136 GT 1 patients and compared to the reference tests, Versant HCV Quantitative 3.0 (bDNA) and Qualitative (TMA) assay. RESULTS: At week 4 of therapy, patients with RVR had a 100% and 93.5% probability to achieve an SVR in GT 1 and GT 2/3 patients, respectively. At week 12, patients who did not achieve a pEVR had a 97.2% and 100% probability of not achieving an SVR. In addition, assignment of GT 1 patients to abbreviated or extended treatment durations based on low baseline HCV-RNA (<800,000IU/mL) and RVR or pEVR was highly concordant between RealTime HCV and bDNA/TMA assays (97.8% and 91.9%, respectively). CONCLUSIONS: The RealTime HCV assay is suitable for monitoring virologic response to PEG-INF/RBV therapy and tailoring treatment duration accordingly.

Changes in hepatitis C viral load during first 14 days can predict the undetectable time point of serum viral load by pegylated interferon and ribavirin therapy.

AIM: In the treatment of chronic hepatitis C, pegylated interferon (PEG-IFN) and ribavirin combination therapy must be continued for an adequate duration to improve the rate of sustained virological response. We attempted to predict the time point at which serum hepatitis C virus (HCV) RNA are undetectable during combination therapy. METHODS: Patients with HCV genotype 1b were enrolled in a model preparation (n = 35) and a validation group (n = 70). All patients received PEG-IFN-α-2b/ribavirin combination therapy for at least 48 weeks, and serological samples were screened a minimum of 17 times during the therapy. Serum HCV RNA were measured by the Abbott RealTime HCV assay. Using the HCV dynamics model described by Neumann et al., we used multiple linear regression analysis to select factors that affected the undetectable time point. RESULTS: Difference in viral load between weeks 1 and 2 was the only predictive factor for the undetectable time point of serum HCV RNA (r(2) = 0.67, P < 0.0005), and we derived the following prediction equation: undetectable time point (week) = 13.495 × (viral load at day 14 [log IU/mL] - viral load at day 7 [log IU/mL]) + 25.456. The equation was applicable to the validation group. CONCLUSION: We created a formula for predicting the undetectable time point from viral load measurements early in PEG-IFN-α-2b/ribavirin combination therapy. An early response reflects sensitivity to therapy, and the estimation of an undetectable time point would be useful for determining the optimal duration of treatment for chronic hepatitis C patients.

International standards and reference materials for quantitative molecular infectious disease testing.

The utility of quantitative molecular diagnostics for patient management depends on the ability to relate patient results to prior results or to absolute values in clinical practice guidelines. To do this, those results need to be comparable across time and methods, either by producing the same value across methods and test versions or by using reliable and stable conversions. Universally available standards and reference materials specific to quantitative molecular technologies are critical to this process but are few in number. This review describes recent history in the establishment of international standards for nucleic acid test development, organizations involved in current efforts, and future issues and initiatives.

Principles and analytical performance of Abbott RealTime High Risk HPV test.

BACKGROUND: Abbott RealTime High Risk (HR) HPV is a new automated, qualitative real-time PCR test for detection of DNA from 14 high-risk human papillomavirus (HPV) types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) in cervical specimens. The test can also differentiate between HPV 16, HPV 18 and non-HPV 16/18 types in a single reaction. OBJECTIVES: This article describes the principles of assay design and the analytical performance of Abbott RealTime HR HPV. STUDY DESIGN: The analytical performance characteristics of Abbott RealTime HR HPV were evaluated in terms of its sensitivity for each of the 14 high-risk types included in the test, specificity (cross-reactivity), potential for interference by substances that may be present in cervical specimens, and reproducibility. RESULTS: Abbott RealTime HR HPV provided sensitive detection of the 14 high-risk HPV types included in the test. It was also highly specific to the HPV types targeted by the test and did not show cross-reactivity with 15 low-risk HPV types tested, or non-specific reactivity with other common microorganisms that may be present in the female anogenital tract. Test results were not impacted by potential interfering substances evaluated in the study. The test generated highly reproducible results in an in-house study and in studies carried out at 13 external evaluation sites. CONCLUSIONS: Abbott RealTime HR HPV demonstrated a robust analytical performance with reproducible and reliable results.

Performance evaluation of the QIAGEN EZ1 DSP Virus Kit with Abbott RealTime HIV-1, HBV and HCV assays.

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.

A RealTime HIV-1 viral load assay for automated quantitation of HIV-1 RNA in genetically diverse group M subtypes A-H, group O and group N samples.

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.

Clinical performance of the LCx HCV RNA quantitative assay.

This study was conducted to assess the performance of the Abbott laboratories LCx HCV RNA Quantitative Assay (LCx assay) in the clinical setting. Four clinical laboratories measured LCx assay precision, specificity, and linearity. In addition, a method comparison was conducted between the LCx assay and the Roche HCV Amplicor Monitor, version 2.0 (Roche Monitor 2.0) and the Bayer VERSANT HCV RNA 3.0 Assay (Bayer bDNA 3.0) quantitative assays. For precision, the observed LCx assay intra-assay standard deviation (S.D.) was 0.060-0.117 log IU/ml, the inter-assay S.D. was 0.083-0.133 log IU/ml, the inter-lot S.D. was 0.105-0.177 log IU/ml, the inter-site S.D. was 0.099-0.190 log IU/ml, and the total S.D. was 0.113-0.190 log IU/ml. The specificity of the LCx assay was 99.4% (542/545; 95% CI, 98.4-99.9%). For linearity, the mean pooled LCx assay results were linear (r=0.994) over the range of the panel (2.54-5.15 log IU/ml). A method comparison demonstrated a correlation coefficient of 0.881 between the LCx assay and Roche Monitor 2.0, 0.872 between the LCx assay and Bayer bDNA 3.0, and 0.870 between Roche Monitor 2.0 and Bayer bDNA 3.0. The mean LCx assay result was 0.04 log IU/ml (95% CI, -0.08, 0.01) lower than the mean Roche Monitor 2.0 result, but 0.57 log IU/ml (95% CI, 0.53, 0.61) higher than the mean Bayer bDNA 3.0 result. The mean Roche Monitor 2.0 result was 0.60 log IU/ml (95% CI, 0.56, 0.65) higher than the mean Bayer bDNA 3.0 result. The LCx assay quantitated genotypes 1-4 with statistical equivalency. The vast majority (98.9%, 278/281) of paired LCx assay-Roche Monitor 2.0 specimen results were within 1 log IU/ml. Similarly, 86.6% (240/277) of paired LCx assay and Bayer bDNA 3.0 specimen results were within 1 log, as were 85.6% (237/277) of paired Roche Monitor 2.0 and Bayer specimen results. These data demonstrate that the LCx assay may be used for quantitation of HCV RNA in HCV-infected individuals.

Performance attributes of the LCx HCV RNA quantitative assay.

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.

Molecular beacons as diagnostic tools: technology and applications.

Molecular beacons are single-stranded, fluorophore-labeled nucleic acid probes that are capable of generating a fluorescent signal in the presence of target, but are dark in the absence of target. Molecular beacons allow multiplex detection of PCR products in real time in a homogeneous assay format. Real time detection is inherently quantitative and affords a greater dynamic range than end-point detection methods. Reactions in a homogeneous assay format are sealed before amplification takes place, providing improved contamination control. A single cycler/reader instrument, coupled with automated sample preparation, results in higher throughput and greater ease of use. A multiplex qualitative assay that detects Chlamydia trachomatis and Neisseria gonorrhoeae, along with an internal control, has been developed. High specificity is achieved through careful selection of primers, probes and assay conditions. Quantitative HIV, HCV, and HBV viral load assays, with sensitivities of 50 copies/ml, 20 IU/ml, and 50 copies/ml, respectively, are achievable. The viral load assays are designed to quantitate all subtype and genotype specimens equivalently. A molecular beacon assay has been designed to detect a single nucleotide polymorphism in the beta2 adrenergic receptor gene.