Performance of the G4 Xpert(R) MTB/RIF assay for the detection of Mycobacterium tuberculosis and rifampin resistance: a retrospective case-control study of analytical and clinical samples from high- and low-tuberculosis prevalence settings

BACKGROUND: The Xpert(R) MTB/RIF (Xpert) assay is a rapid PCR-based assay for the detection of Mycobacterium tuberculosis complex DNA (MTBc) and mutations associated with rifampin resistance (RIF). An updated version introduced in 2011, the G4 Xpert, included modifications to probe B and updated analytic software. METHODS: An analytical study was performed to assess Xpert detection of mutations associated with rifampin resistance in rifampin-susceptible and -resistant isolates. A clinical study was performed in which specimens from US and non-US persons suspected of tuberculosis (TB) were tested to determine Xpert performance characteristics. All specimens underwent smear microscopy, mycobacterial culture, conventional drug-susceptibility testing and Xpert testing; DNA from isolates with discordant rifampin resistance results was sequenced. RESULTS: Among 191 laboratory-prepared isolates in the analytical study, Xpert sensitivity for detection of rifampin resistance associated mutations was 97.7% and specificity was 90.8%, which increased to 99.0% after DNA sequencing analysis of the discordant samples. Of the 1,096 subjects in the four clinical studies, 49% were from the US. Overall, Xpert detected MTBc in 439 of 468 culture-positive specimens for a sensitivity of 93.8% (95% confidence interval [CI]: 91.2%-95.7%) and did not detect MTBc in 620 of 628 culture-negative specimens for a specificity of 98.7% (95% CI: 97.5%-99.4%). Sensitivity was 99.7% among smear-positive cases, and 76.1% among smear-negative cases. Non-determinate MTBc detection and false-positive RIF resistance results were low (1.2 and 0.9%, respectively). CONCLUSIONS: The updated Xpert assay retained the high sensitivity and specificity of the previous assay versions and demonstrated low rates of non-determinate and RIF resistance false positive results

Estimation of D-Arabinose by Gas Chromatography/Mass Spectrometry as Surrogate for Mycobacterial Lipoarabinomannan in Human Urine.

Globally, tuberculosis is slowly declining each year and it is estimated that 37 million lives were saved between 2000 and 2013 through effective diagnosis and treatment. Currently, diagnosis relies on demonstration of the bacteria, Mycobacterium tuberculosis (Mtb), in clinical specimens by serial sputum microscopy, culture and molecular testing. Commercial immunoassay lateral flow kits developed to detect Mtb lipoglycan lipoarabinomannan (LAM) in urine as a marker of active TB exhibit poor sensitivity, especially in immunocompetent individuals, perhaps due to low abundance of the analyte. Our present study was designed to develop methods to validate the presence of LAM in a quantitative fashion in human urine samples obtained from culture-confirmed TB patients. Herein we describe, a consolidated approach for isolating LAM from the urine and quantifying D-arabinose as a proxy for LAM, using Gas Chromatography/Mass Spectrometry. 298 urine samples obtained from a repository were rigorously analyzed and shown to contain varying amounts of LAM-equivalent ranging between ~10-40 ng/mL. To further substantiate that D-arabinose detected in the samples originated from LAM, tuberculostearic acid, the unique 10-methyloctadecanoic acid present at the phosphatidylinositol end of LAM was also analyzed in a set of samples and found to be present confirming that the D-arabinose was indeed derived from LAM. Among the 144 samples from culture-negative TB suspects, 30 showed presence of D-arabinose suggesting another source of the analyte, such as disseminated TB or from non-tuberculosis mycobacterium. Our work validates that LAM is present in the urine samples of culture-positive patients in small but readily detectable amounts. The study further substantiates LAM in urine as a powerful biomarker for active tuberculosis

Protocol for derivatization of D-Arabinose in urinary LAM and the corresponding mass fragmentation pattern.

<p>Protocol for derivatization of D-Arabinose in urinary LAM and the corresponding mass fragmentation pattern.</p

FIND urine sample purification and D-arabinose analysis.

<p>IS = Internal Standard; HIV = Human Immunodeficiency Virus; Pneu = Pneumonia; Atyp = Atypical TB,</p

Differences in peak patterns and retention times in GC/MS chromatogram for D- and L- arabinose.

<p>Differences in peak patterns and retention times in GC/MS chromatogram for D- and L- arabinose.</p

Summary of results for the GC/MS based urinary LAM detection on clinical samples.

<p>Summary of results for the GC/MS based urinary LAM detection on clinical samples.</p

Representative schematic structure of ManLAM, Insert in the Blue box show residues adapted as strategic surrogates for LAM.

<p>Representative schematic structure of ManLAM, Insert in the Blue box show residues adapted as strategic surrogates for LAM.</p

Protocol for TBSA detection by GC/MS.

<p>Protocol for TBSA detection by GC/MS.</p

TBSA Based LAM Estimation of 29 Urine Samples (TBSSMC -, D-Arabinose/LAM positive).

<p>TBSA Based LAM Estimation of 29 Urine Samples (TBSSMC -, D-Arabinose/LAM positive).</p

D-Arabinose estimation of Non-Endemic-Urine (NEU).

<p>1) Internal standard, 2) D-arabinose in NEU before purification; 3) D-arabinose not detected in NEU after purification.</p