Integrating the Xpert MTB/RIF Assay into a Diagnostic Workflow for Rapid Detection of Mycobacterium tuberculosis in a Low-Prevalence Area

Abstract

bThe Xpert MTB/RIF assay is a rapid and fully automated real-time PCR assay. The performance of the Xpert MTB/RIF assay as a primary screening test for urgent clinical specimens was evaluated during a 2-year period. The results showed that replacing smear microscopy with the Xpert MTB/RIF assay facilitates laboratory handling and improves the sensitivity and specificity of Mycobacterium tuberculosis detection. Rapid and accurate diagnosis of tuberculosis (TB) is indispensable to adequately manage the disease and control its transmission. Acid-fast bacilli (AFB) smear microscopy is an established low-cost screening procedure to identify patients with tuberculosis. However, the sensitivity of smear microscopy is low, varying between 22 and 80% (1). In general, the routine application of nucleic acid amplification techniques for detection of Mycobacterium tuberculosis results in accurate diagnosis of tuberculosis (2, 3, 4, 5, 6) but requires laborious processing time and dedicated biosafety conditions. The recently introduced Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) is a fully automated, walk-away real-time PCR-based assay with a time to result of approximately 2 h. To detect M. tuberculosis and mutations associated with resistance to rifampin (RIF), the 81-bp core region of the rpoB gene is amplified and probed with five overlapping molecular beacons (7). M. tuberculosis is identified when at least 2 probes give a positive signal within a predefined number of cycles. A RIF mutation is detected by lack of or delayed onset of fluorescence of at least one molecular beacon. Disadvantages of the Xpert MTB/RIF are its exceeding costs and a reduced sensitivity in comparison with other PCR M. tuberculosis assays (8, 9).The objective of the present study was to evaluate the feasibility of the Xpert MTB/RIF assay to replace direct smear microscopy as a primary screening test for urgent clinical specimens in a setting of low TB prevalence.The Institute of Medical Microbiology (University of Zurich) is a tertiary care diagnostic center that receives clinical specimens 7 days/week for AFB microscopy to urgently confirm or rule out TB in newly identified suspect cases. Such samples are marked as urgent cases and are not to be confused with regular samples submitted to the mycobacteriology laboratory. All respiratory and nonrespiratory specimens submitted for urgent smear microscopy between July 2010 and June 2012 were included in this study; ethical approval was not needed. Specimens were adjusted to 5 ml with sterile distilled water when the specimen volume was Ͻ5 ml. The Xpert MTB/RIF assay was performed following the manufacturer's instructions for respiratory specimens, using a 1-ml aliquot. Nonrespiratory specimens were tested similarly. The remaining 4 ml of all unprocessed specimen with the exception of cerebrospinal fluid (CSF) was homogenized and decontaminated using an equal volume of N-acetyl-L-cysteine (NALC)-2% NaOH for 15 min (10). After neutralization with phosphate buffer (67 mM, pH 6.8) and centrifugation, ...

Diagnostic accuracy of the Xpert® MTB/RIF cycle threshold level to predict smear positivity: a meta-analysis

“…Fourteen study teams provided anonymised individual-level data from 15 studies for the metaanalysis (Table 1). 21,25,27,29,[30][31][32][33][34][35][36][37][38][39] Descriptive systematic review on association between C T values and smear status Ten studies investigated the association between smear status and C T values ( Figure 1). 11,20,21,26,[33][34][35][36][37][38][39][40] Seven of the ten studies were conducted in sub-Saharan Africa, and HIV prevalence among study participants ranged from 3% to 60%.…”

“…Two studies did not provide information on HIV prevalence. 27,36 Overall studies were of moderate to good quality, although they did not always report on their efforts to minimise bias. Most, however, were consecutive or random collections of samples, and nearly all, except three, did report on the flow of patients in the study (Table 1).…”

“…37 Meta-analysis of individual-level data Individual-level data from 14 studies and one unpublished data set were included (Table 1; see Appendix for description of unpublished data set). 21,25,27,29,[30][31][32][33][34][35][36][37][38][39] Collectively this amounted to 7511 respiratory samples from 4447 patients. A total of 1406 patients had at least one culture-positive sample, 1212 of whom had at least one Xpertpositive sample, 636 had two Xpert-positive samples and 11 patients more than three.…”

Evaluation of the Cepheid Xpert MTB/RIF assay

“…On the other hand, silent mutations (i.e., mutations that do not change the amino acid sequence such as 514Phe (TTC/TTT)) in the RRDR have been found that generated false-positive results with Xpert MTB/RIF [84–86]. Early reports of false-positive rifampicin resistance in strains later found to be wild-type for rpoB led to modifications of the assay to improve performance [87–89]. False-positive results for rifampicin resistance could lead to an incorrect diagnosis of MDR TB and subject patients to longer and less-effective treatment with second-line drugs.…”

Performance of the Xpert MTB/RIF Assay on Nonrespiratory Specimens and Accuracy of This Assay for Detection of Rifampin Resistance in a Low-Prevalence Setting

“…ith interest, we read the paper "Integrating the Xpert MTB/ RIF Assay into a Diagnostic Workflow for Rapid Detection of Mycobacterium tuberculosis in a Low-Prevalence Area" by Deggim et al (1). Based on the failed detection of Mycobacterium tuberculosis complex (MTB) by the Xpert assay (GeneXpert; Cepheid, Sunnyvale, CA) in 3 of 4 culture-positive nonrespiratory specimens, the authors concluded that such specimens may not be suitable for Xpert testing, in particular for paucibacillary samples.…”