Abbott M2000 Research Articles

Evaluation of a dried blood spot HIV-1 RNA program for early infant diagnosis and viral load monitoring at rural and remote healthcare facilities

To assess technical and operational performance of a dried blood spot (DBS)-based HIV-1 RNA service for remote healthcare facilities in a low-income country. A method comparison and operational evaluation of DBS RNA against conventional tests for early infant diagnosis of HIV and HIV RNA quantitation under field conditions in Tanzania. DBSs were prepared and plasma was frozen at -80 degrees C. DBSs were mailed and plasma couriered to a central laboratory for testing using the Abbott m2000 system. Infant diagnosis DBSs were also tested for HIV-1 DNA by ROCHE COBAS AmpliPrep/COBAS TaqMan System. Results of DBS RNA were compared with conventional tests; program performance was described. Among 176 infant diagnosis participants, using a threshold of at least 1000 copies/ml, sensitivity and specificity of DBS versus plasma RNA were 1.00 and 0.99, and of DBS RNA versus DBS DNA were 0.97 and 1.00. Among 137 viral load monitoring participants, when plasma and DBS RNA were compared, r value was 0.9709; r value was 0.9675 for at least 5000 copies/ml but was 0.7301 for less than 5000 copies/ml. The highest plasma RNA value at which DBS RNA was not detected was 2084 copies/ml. Median (range) turnaround time from sample collection to result receipt at sites was 23 (4-69) days. The Tanzania mail service successfully transmitted all DBS and results between sites and the central laboratory. Under program conditions in Tanzania, DBS provided HIV-1 RNA results comparable to conventional methods to remote healthcare facilities. DBS RNA testing is an alternative to liquid plasma for HIV-1 RNA services in remote areas.

Evaluation of the Abbott m2000 rt RealTi me™ HIV-1 assay with manual sample preparation compared with the ROCHE COBAS ® AmpliPrep™/AMPLICOR™ HIV-1 MONITOR ® v1.5 using specimens from East Africa

The performance of the Abbott m2000 rt RealTi me™ HIV-1 assay (RealTi me HIV-1) with manual sample preparation was compared against the ROCHE COBAS ® AmpliPrep™/AMPLICOR™ HIV-1 MONITOR ® Test v1.5 (CAP/CA HIV-1) using samples collected from 100 donors infected with HIV and 20 donors not infected with HIV in northern Tanzania where HIV-1 subtypes A, C, D, and their recombinant forms predominate. The RealTi me HIV-1 appeared to have more within-run variability at high HIV-1 RNA concentrations, but total assay variability over the dynamic range tested was within the manufacturer's claim of <0.3 SD copies/mL. Accuracy studies showed 100% concordance for positive and negative values. When continuous values were examined, CAP/CA HIV-1 yielded higher values than the RealTi me HIV-1 at higher nominal HIV-1 RNA concentrations. The RealTi me HIV-1 assay showed excellent linearity between 2.5 and 7.0 log copies/mL. Of negative samples, 100% showed negative results, and >95% of samples with nominal concentrations of 40 copies/mL were detected at ≥40 copies/mL by RealTi me HIV-1. Manual sample preparation may contribute to higher total assay variability. This study suggests that the Abbott m2000 rt RealTi me HIV-1 assay with manual sample preparation is an acceptable and feasible alternative to the conventional ROCHE COBAS AmpliPrep/AMLICOR HIV-1 Monitor v1.5 assay and that the RealTi me HIV-1 assay performs well on samples from East Africa.

Pitfalls of internet-accessible diagnostic tests: inadequate performance of a CE-marked Chlamydia test for home use

Objectives: To evaluate the performance of a Conformitée Européenne (CE)-marked home test for Chlamydia trachomatis (CT) that is available over the internet. Methods: A total of 231 eligible women attending...

Performance evaluation of the QIAGEN EZ1 DSP Virus Kit with Abbott RealTi me 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 QIAGEŃs EZ1 DSP Virus Kit on the BioRobot EZ1 DSP was evaluated in combination with the Abbott RealTi me HIV-1, HCV, and HBV assays, followed by thermalcycling and detection on the Abbott m2000 rt 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 m2000 sp 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 RealTi me HIV-1, HCV, and HBV assays.

New variant chlamydia: Limitations of diagnostic tests and importance of epidemiologic surveillance

Introduction: The introduction of nucleic acid amplifi cation tests for the diagnosis of Chlamydia trachomatis infections in the 1990s represented the fi rst wholesale, practical substitution of traditional diagnostic methods in microbiology with this new technology. Prior to this, Chlamydia identifi cation in clinical specimens was often not routinely available or, if available, was dependent on tests with poor sensitivity. In addition, nucleic acid amplifi cation tests provide effi ciency of scale through batch testing, and they have proven to be nearly as reliable on urine specimens and self-collected vaginal specimens as they are on cervical and urethral specimens. Not surprisingly, with the introduction of reliable nucleic acid amplifi cation tests and expansion of Chlamydia testing services, the numbers of cases of genital C. trachomatis infections in the United States began to soar. Between 1990 and 2005, overall rates of Chlamydia infection in the United States went from ~ 150/100,000 population to ~ 340/100,000 population [1]. Several types of nucleic acid amplifi cation tests have been commercially introduced to detect Chlamydia. These include polymerase chain reaction (PCR), ligase chain reaction (LCR), and transcription-mediated amplifi cation (TMA). By their nature, the reliability of these tests depends on the selection of the appropriate target gene sequence and the conservation of that sequence over time. Some testing systems, including the Abbott m2000 (Abbott Diagnostics, Chicago, IL, USA) and the Cobas Amplicor/TaqMan48 (Roche Diagnostics, Basel, Switzerland), use the chlamydial cryptic plasmid as their target for amplifi cation. As the name implies, the actual function of this plasmid is unclear, but it provides several copies per cell and therefore results in increased sensitivity when compared with those tests that target chromosomal DNA [2]. However, what happens if and when, through random mutation, variant organisms appear that are lacking the sequence in question? It has long been recognized that plasmid-free strains of C. trachomatis exist in nature [3]. This did not seem to have any impact at the time on test system design. Apparently, however, now the clinical “chicken has come home to roost.” In Sweden, after a decade of rising C. trachomatis rates, it was observed that at least in one southern county in 2005 the rates declined by 25%. Rather than accept this as evidence of successful surveillance and disease management, investigators assumed there was a good chance that the diagnostic tests they were using were either faulty or that a change had occurred in the target for amplifi cation. After some study, a 377-bp deletion was found in the cryptic plasmid target region of a strain that initially had not been detected by routine testing. Had this occurred in the setting of anything less than effective surveillance, and the noted observation of an unexpected decline in prevalence after years of rising rates, it may not have come to light. Several studies and reports recently have highlighted this microbiologic phenomenon.

Update on the new variant of Chlamydia trachomatis: Prevalence and diagnostics

In 2006, a new variant of Chlamydia trachomatis (nvCT) was discovered in Sweden [1]. Due to a deletion in the target sequence for PCR amplification, nvCT has escaped detection by the nucleic acid amplification tests Abbott m2000 (Abbott Laboratories) and Cobas Amplicor/TaqMan48 (Roche Diagnostics) which together were used for 65% of all chlamydia tests in Sweden. The third test commonly used in Sweden, ProbeTec ET (Becton Dickinson [BD]), does detect the new variant because it uses a different DNA target sequence in the cryptic plasmid. The nvCT is spread all over Sweden and in early 2007, it accounted for between 20% and 65% of all chlamydia cases in Swedish counties where Abbott/Roche test systems were used [2]. In the four counties that used the BD system, the proportion of nvCT was only between 7% and 20% in the same time period. Ongoing studies are now following the spread of the nvCT in several counties.

A RealTi me 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 RealTi me 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 m2000 sp™ 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 m2000 rt™ 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 25 copies/mL with 1.0 mL of plasma, 39 copies/mL with 0.6 mL of plasma, 65 copies/mL with 0.5 mL of plasma, and 119 copies/mL with 0.2 mL of plasma.

Frequency of very low HCV viremia detected by a highly sensitive HCV-RNA assay

Background Only limited data is available on the frequency and clinical significance of very low hepatitis C viremia (<600 IU/ml) determined by novel sensitive methods for HCV quantification. Study design We evaluated the new Abbott m2000 RealTime PCR assay in 3213 consecutive anti-HCV-positive sera as well as in 50 HCV-recovered patients with sustained virological response to standard antiviral therapy. Results The assay showed a linear range between 10 1 IU/ml and 10 7 IU/ml for HCV genotypes 1–6. An HCV viremia below 600 IU/ml was detected more often with the m2000 RealTime PCR assay than with the Cobas Amplicor™ assay in viremic sera (7.1% versus 1.8%). Seventy-seven cases with HCV levels below 100 IU/ml not related to ongoing antiviral therapy were identified. An HCV-RNA of less than 12 IU/ml was found in nine of the 50 SVR patients. Two patients had a viral load of 34 IU/ml and 84 IU/ml, respectively, one of those showed persistently elevated ALT levels over a period of 5 years after the end of antiviral treatment. Conclusion An HCV viremia below 600 IU/ml can be detected in almost every 40th anti-HCV-positive sera using real-time PCR based assays. Low persisting HCV-RNA in patients after antiviral therapy may be associated with mild liver inflammation in single cases.

Experiences with the new genetic variant of Chlamydia trachomatis in Örebro county, Sweden – proportion, characteristics and effective diagnostic solution in an emergent situation

A Chlamydia trachomatis variant that contains a 377 bp deletion in the cryptic plasmid was recently reported in Sweden. This deletion includes the targets for Cobas Amplicor, Cobas TaqMan48, and Abbott m2000. We examined the proportion and characteristics of this variant in Orebro county, Sweden and developed an effective diagnostic solution. In total, 2,401 consecutive C. trachomatis culture samples and 536 PCR samples from symptomatic and asymptomatic patients and screened females were included. Culture, Cobas Amplicor, and LightMix 480HT were used for diagnosis. A mutant-specific PCR, plasmid sequencing, omp1 sequencing and multilocus sequence typing (MLST) were used to identify and characterise mutants. In total, 162 (6.7%) of the cultured samples were positive for C. trachomatis. However, 61 (38%) of those were negative when using Cobas Amplicor, and 60 of these were subsequently confirmed as the new variant. 13 of these mutant isolates were further characterised genetically, and all were of identical genotype E and the unique MLST sequence type: 21, 19, 1, 2, 1. Of all culture-positive samples, 161 of 162 were positive in the LightMix 480HT assay. The single negative sample was only weakly positive in culture, and negative in all PCRs. Of the 536 PCR samples, 37 were positive in both Cobas Amplicor and LightMix 480HT, 13 were only positive in LightMix 480HT (mutants), and two were only positive in Cobas Amplicor. Mutated C. trachomatis were prevalent in Orebro county in the period from October 2006 to February 2007, and it appeared to be a single clone. LightMix 480HT seemed sensitive, specific, and enabled high throughput diagnostics. However, rare low positive samples may be false-negative. Frequent surveillance and evaluations of diagnostic methods worldwide are crucial.

Evaluation of performance across the dynamic range of the Abbott RealTi me™ HIV-1 assay as compared to VERSANT HIV-1 RNA 3.0 and AMPLICOR HIV-1 MONITOR v1.5 using serial dilutions of 39 group M and O viruses

Performance of the Abbott m2000 instrument system and the Abbott RealTi me™ 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. RealTi me HIV-1, bDNA, and Monitor v1.5 tests quantified 87%, 78%, and 81% of samples, respectively. RealTi me HIV-1 detected an additional 31 samples at <40 copies/mL. For group M, RealTi me 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 ∼2 log 10 copies/mL in bDNA relative to the RealTi me HIV-1 assay. Sequence analysis revealed that RealTi me HIV-1 primer/probe binding sites are highly conserved and exhibit fewer nucleotide mismatches relative to Monitor v1.5. The automated m2000 system and RealTi me 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.

Performance of the automated Abbott RealTi me™ HIV-1 assay on a genetically diverse panel of specimens from Brazil

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 m2000 sp™ automated sample preparation system. Viral loads measured using the RealTi me™ HIV-1 (RealTi me HIV-1) assay and the Abbott m2000 rt™ 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 RealTi me 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 RealTi me HIV-1 assay and m2000 system offer the advantages of automation while providing reliable quantification of diverse HIV strains.