Evaluation of the Cepheid GeneXpert BCR-ABL Assay

Patients with chronic myeloid leukemia harbor the chromosomal translocation t(9;22), which corresponds to fusion of the BCR and ABL genes at the DNA level. The translated fusion product is an oncogenic protein with increased ABL tyrosine kinase activity causing cell transformation. To date, reverse transcriptase-polymerase chain reaction is considered the most sensitive method available for detecting low copy numbers of the BCR-ABL gene fusion. Recently, Cepheid introduced its GeneXpert-based assay for the identification of the BCR-ABL gene fusion in cells from blood samples. This system comprises a walk-away self-contained instrument that combines cartridge-based microfluidic sample preparation with reverse transcriptase-polymerase chain reaction-based fluorescent signal detection and BCR-ABL and ABL Ct (threshold cycle) determination. The difference between the BCR-ABL Ct and ABL Ct (ΔCt) is expected to represent the ratio of the two populations of mRNAs and ultimately the percentage of neoplastic cells present. We tested whether this BCR-ABL fusion detection system could be used as a clinical diagnostic tool for monitoring patients with minimal residual disease of chronic myelogenous leukemia. We report similar performance characteristics, including limit of detection, specificity, sensitivity, and precision, of this automated BCR-ABL fusion detection system to those of a manual TaqMan reverse transcriptase-polymerase chain reaction-based test

Analytical Performance Characteristics of the Cepheid GeneXpert Ebola Assay for the Detection of Ebola Virus

<div><p>Background</p><p>The recently developed Xpert® Ebola Assay is a novel nucleic acid amplification test for simplified detection of Ebola virus (EBOV) in whole blood and buccal swab samples. The assay targets sequences in two EBOV genes, lowering the risk for new variants to escape detection in the test. The objective of this report is to present analytical characteristics of the Xpert® Ebola Assay on whole blood samples.</p><p>Methods and Findings</p><p>This study evaluated the assay’s analytical sensitivity, analytical specificity, inclusivity and exclusivity performance in whole blood specimens. EBOV RNA, inactivated EBOV, and infectious EBOV were used as targets. The dynamic range of the assay, the inactivation of virus, and specimen stability were also evaluated. The lower limit of detection (LoD) for the assay using inactivated virus was estimated to be 73 copies/mL (95% CI: 51–97 copies/mL). The LoD for infectious virus was estimated to be 1 plaque-forming unit/mL, and for RNA to be 232 copies/mL (95% CI 163–302 copies/mL). The assay correctly identified five different Ebola viruses, Yambuku-Mayinga, Makona-C07, Yambuku-Ecran, Gabon-Ilembe, and Kikwit-956210, and correctly excluded all non-EBOV isolates tested. The conditions used by Xpert® Ebola for inactivation of infectious virus reduced EBOV titer by ≥6 logs.</p><p>Conclusion</p><p>In summary, we found the Xpert® Ebola Assay to have high analytical sensitivity and specificity for the detection of EBOV in whole blood. It offers ease of use, fast turnaround time, and remote monitoring. The test has an efficient viral inactivation protocol, fulfills inclusivity and exclusivity criteria, and has specimen stability characteristics consistent with the need for decentralized testing. The simplicity of the assay should enable testing in a wide variety of laboratory settings, including remote laboratories that are not capable of performing highly complex nucleic acid amplification tests, and during outbreaks where time to detection is critical.</p></div

Limit of Detection of the Xpert® Ebola Assay and Altona RealStar® Ebolavirus RT-PCR Kit 1.0 using Inactivated EBOV Yambuku-Mayinga.

<p>* One replicate was removed due to a manufacturing error.</p><p>NA, Not Applicable</p><p>Limit of Detection of the Xpert® Ebola Assay and Altona RealStar® Ebolavirus RT-PCR Kit 1.0 using Inactivated EBOV Yambuku-Mayinga.</p

Primer and probe sequences for the EBOV targets used in the Xpert® Ebola Assay.

<p>GP, Glycoprotien; NP, Nucleoprotein; HMBS, human hydroxymethilbilane synthase</p><p>Primer and probe sequences for the EBOV targets used in the Xpert® Ebola Assay.</p

Numbers of positive replicates per level in the Xpert® Ebola Assay for EBOV Makona-C07, and for EBOV Yambuku-Mayinga RNA, both in EDTA-whole blood.

<p>Numbers of positive replicates per level in the Xpert® Ebola Assay for EBOV Makona-C07, and for EBOV Yambuku-Mayinga RNA, both in EDTA-whole blood.</p

Analytical Reactivity and Specificity (Inclusivity and Exclusivity).

<p>LoD, Limit of Detection; PFU, Plaque Forming Unit</p><p>^aGenBank or ATCC number, if available</p><p>^bDr. Robert Tesh, University of Texas Medical Branch, Galveston, TX</p><p>^cNational Veterinary Service Laboratory, Ames, IA</p><p>^dCenters for Disease Control and Prevention, Atlanta, GA</p><p>^eDr. Donald Bouyer, University of Texas Medical Branch, Galveston, TX</p><p>^fDepartment of Defense, Critical Reagents Program</p><p>^gUniversity of California, Davis</p><p>^hRNA concentration of the stock material</p><p>Analytical Reactivity and Specificity (Inclusivity and Exclusivity).</p

Linearity of the Xpert® Ebola Assay and Altona RealStar® Ebolavirus RT-PCR Kit 1.0.

<p>Serial dilutions of inactivated EBOV Yambuku-Mayinga was spiked into EDTA-whole blood and tested in triplicate. Linear regression lines are shown for the Xpert® Ebola nucleoprotein (NP) and glycoprotein (GP) targets (NP: y = -3.44x+46.11, R² = 0.98; GP: y = -3.87x+51.45, R² = 0.87), and Altona RealStar® Ebolavirus (y = -4.51x+50.17, R² = 0.93).</p

Range finding analysis of the Xpert® Ebola Assay, Altona RealStar® Ebolavirus RT-PCR Kit 1.0, and BioFire Defense LLC FilmArray Biothreat-E Test® using Inactivated EBOV Yambuku-Mayinga.

<p>Range finding analysis of the Xpert® Ebola Assay, Altona RealStar® Ebolavirus RT-PCR Kit 1.0, and BioFire Defense LLC FilmArray Biothreat-E Test® using Inactivated EBOV Yambuku-Mayinga.</p