A Multisite Assessment of the Quantitative Capabilities of the Xpert MTB/RIF Assay

Abstract

Rationale: The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy. Objectives: Evaluate bacterial load quantitation by Xpert MTB/RIF compared with conventional quantitative methods. Methods: Xpert MTB/RIF results were compared with smear-microscopy, semiquantiative solid culture, and time-to-detection in liquid culture for 741 patients and 2,008 samples tested in a multisite clinical trial. An internal control real-time polymerase chain reaction was evaluated for its ability to identify inaccurate quantitative Xpert MTB/RIF results. Measurements and Main Results: Assays with an internal control Ct greater than 34 were likely to be inaccurately quantitated; this represented 15% of M. tuberculosis-positive tests. Excluding these, decreasing M. tuberculosis Ct was associated with increasing smear microscopy grade for smears of concentrated sputum pellets (r s ¼ 20.77) and directly from sputum (r s ¼ 20.71). A Ct cutoff of approximately 27.7 best predicted smear-positive status. The association between M. tuberculosis Ct and time-to-detection in liquid culture (r s ¼ 0.68) and semiquantitative colony counts (r s ¼ 20.56) was weaker than smear. Tests of paired same-patient sputum showed that highviscosity sputum samples contained 332 more M. tuberculosis than nonviscous samples. Comparisons between the grade of the acid-fast bacilli smear and Xpert MTB/RIF quantitative data across study sites enabled us to identify a site outlier in microscopy. Conclusions: Xpert MTB/RIF quantitation offers a new, standardized approach to measuring bacterial burden in the sputum of patients with tuberculosis.Keywords: tuberculosis; molecular diagnostics; diagnostic techniques and procedures; diagnosis; clinical trial Measurements of bacterial load have long played an important role in tuberculosis diagnostics. Semiquantitative or quantitative measures of the number of Mycobacterium tuberculosis bacilli present within a clinical sample have been clinically useful for determining disease severity, assessing transmission risk, or monitoring therapy (1-3). Quantitative readouts have also aided in the investigation of potentially false-positive results (4-6).The Xpert MTB/RIF (Cepheid, Sunnyvale, CA) assay simultaneously detects the presence of M. tuberculosis and its susceptibility to the important first-line drug rifampin in less than 2 hours (7). The assay is contained in a small plastic cartridge, and it is run on the GeneXpert platform (Cepheid), a diagnostic The Xpert MTB/RIF assay is a rapid diagnostic test for tuberculosis and rifampin resistance. A number of studies have examined the sensitivity and specificity of the assay in various clinical settings and with various sample types. The test can also provide an estimate of the bacterial load present in the s...

Exploring Alternative Biomaterials for Diagnosis of Pulmonary Tuberculosis in HIV-Negative Patients by Use of the GeneXpert MTB/RIF Assay

“…We also examined all of the Xpert assays (whether positive or negative for M. tuberculosis) for evidence of partial PCR inhibition. A sample was considered to show evidence of partial PCR inhibition when the cycle threshold (C T ) of the Xpert assay's internal control reaction was greater than 34 (24). Assays with internal control C T values of Ͼ34 were observed in 0 EBC samples, 1 (3.8%) saliva sample, 3 (12.5%) blood samples, 5 (19.2%) urine samples, 8 direct (3.3%) sputum samples, and 11 (4.2%) concentrated sputum samples (P value of inhibition in direct sputum samples versus EBC samples is 0.6, versus saliva samples is 0.1, versus blood samples is 0.07, versus urine samples is 0.001, and versus sputum pellets is 0.64).…”

“…It has been shown that the Xpert assay is equally sensitive when performed with either type of sputum sample (19). However, the assay also provides quantitative information in the form of a cycle threshold (C T ) that has the potential to be used to monitor bacterial load (24). The quantitative information provided by these two types of sputum samples have never been rigorously compared.…”

Performance of Xpert MTB/RIF Assay in Diagnosis of Pleural Tuberculosis by Use of Pleural Fluid Samples

“…However, on its utility in pleural TB, only a few studies with small sample size have been carried out; most of these were from low-TB-burden countries (6)(7)(8), and none were from India.…”

Multicenter Feasibility Study To Assess External Quality Assessment Panels for Xpert MTB/RIF Assay in South Africa

“…The increased variation (increased SD) in the C T values of the CDC and GLI panels (due to some Xpert MTB/RIF tests reporting delayed probe hybridization and some reporting dropout probes with no hybridization) may be attributed to the use of MGIT bulk stock, which may have increased clumping of bacterial cells compared to that of the bulk stock manufactured in the single-cell format used in the NHLS product or other factors inherent to their specific preparation. Minimal variations may, however, be more acceptable in an EQA program monitoring RIF resistance rates using differences in probe dropout or probe delayed hybridization as well as differences in C T values across the reported semiquantitative categories, since studies have already described the potential use of the Xpert assay for patient monitoring using C T values (9,15).…”

“…Resistance to rifampin (RIF) is determined by delayed or drop-out hybridization of the molecular beacon probes (5 probes span the rpoB target, and 1 probe hybridizes to the internal control). Delayed hybridization occurs when there is a Ͼ4 C T difference (for assay version G4) between any probes and dropout is reported when the C T is zero (9). The mean C T value and the SD of the C T for each probe (A to E) of each panel were calculated.…”