Diagnostic Implications of Inconsistent Results Obtained with the Xpert MTB/Rif Assay in Detection of Mycobacterium tuberculosis Isolates with an rpoB Mutation Associated with Low-Level Rifampin Resistance

Abstract

c Xpert-MTB/Rif is one of the most frequently used molecular screening tests for multidrug-resistant tuberculosis worldwide. We report false-negative assay results in the presence of rpoB Leu533Pro, which is associated with low-level phenotypic rifampin resistance. Accurate and timely confirmation of rifampin susceptibility results obtained with Xpert-MTB/Rif is imperative.T he accurate diagnosis of drug-resistant and multidrug-resistant (MDR) tuberculosis (TB) is imperative to initiate adequate treatment, to avoid transmission of the disease, and to prevent the development of further drug resistance. Because of its worldwide rollout and rapid implementation, the Xpert-MTB/Rif assay (Cepheid) has become one of the most frequently used molecular screening tests for TB and MDR TB in both resource-poor and resource-rich countries (1). Recently, a 67-year-old Swissborn male patient was admitted to a secondary-care hospital in Switzerland with clinical and radiologic suspicion of pulmonary TB. Rapid testing by Xpert-MTB/Rif showed the presence of Mycobacterium tuberculosis complex in two sputum samples (Table 1; samples 1 and 2). In addition, these specimens showed indeterminate and definite rifampin (RMP) resistance, respectively (Table 1, samples 1 and 2). Since the patient had no history of TB and was from Switzerland (a low MDR TB incidence setting), based on previous reports of false RMP resistance assay results (2, 3), the clinician doubted the accuracy of the Xpert-MTB/ Rif RMP results. Therefore, an additional four sputum samples were submitted to the Swiss National Reference Center for Mycobacteria for rapid confirmation before initiation of MDR TB therapy. Further patient testing revealed HIV positivity.The four additional sputum specimens (Table 1, samples 3 to 6) were acid-fast smear positive by the Ziehl-Neelsen method (4), and Xpert-MTB/Rif testing detected the presence of M. tuberculosis complex. Surprisingly, all of the samples were scored RMP susceptible by the molecular assay (Table 1). In order to resolve the discrepant Xpert-MTB/Rif results and to rapidly confirm or rule out MDR TB, direct rpoB sequencing of the 81-bp core region and additional molecular screening for isoniazid (INH) resistance were performed as described previously (3, 5) and revealed rpoB Leu(CTG)533Pro(CCG) and katG Ser(AGC)315Thr(ACC) mutations, respectively, in all four specimens. Growth detection and quantitative phenotypic drug susceptibility testing (DST) for firstand second-line antituberculosis drugs with the MGIT 960 system and EpiCenter software with the TB eXiST module (Becton, Dickinson Microbiology Systems, Sparks, MD) were performed as described earlier (6). Quantitative DST identified resistance to RMP at 0.5 g/ml and susceptibility at 1.0, 4.0, and 20 g/ml, and resistance to rifabutin at 0.1 g/ml and susceptibility at 0.4 and 2.0 g/ml. DST for INH showed resistance at 0.1 and 1.0 g/ml and intermediate resistance at 3.0 and 10.0 g/ml. No drug resistance was identified by conventional DST for other first-and second...

Clinical implications of the global multidrug-resistant tuberculosis epidemic

“…18 Furthermore, the test has been shown to give false negative results in the presence of particular rpoB mutations that are associated with low-level rifampicin resistance. 19 …”

Comparison of Three Commercial Molecular Assays for Detection of Rifampin and Isoniazid Resistance among Mycobacterium tuberculosis Isolates in a High-HIV-Prevalence Setting

“…When evaluations were repeated using the most recent release of the version 2.0 assay as well as the GXP (G4 cartridge) assay, these mutations were detected by the MTBDRplus version 2.0 assay in codon 530 to 533 of the rpoB region (missing wild-type 8 band with no corresponding mutation band) and by the GXP (G4 cartridge) assay (probe E not binding). This mutation has been associated with discordant genotypic and phenotypic susceptibility results, notably when the widely employed broth-based phenotypic susceptibility testing methods are utilized; hence, the L533P mutation was thought to be a silent mutation, with no impact on the efficacy of rifampin (10), and has also been shown to be missed by the G3 cartridge (11,12). Upon a retrospective review of our laboratory records, these two isolates had tested as rifampin susceptible with the MGIT 960 system.…”

Evaluation of the AID TB Resistance Line Probe Assay for Rapid Detection of Genetic Alterations Associated with Drug Resistance in Mycobacterium tuberculosis Strains

“…One of the few commercially available tests is the Genotype MTBDRplus assay (Hain Lifescience GmbH, Germany), which was endorsed by the World Health Organization (WHO) in 2008 and was recently extended for the detection of second-line drug resistance mutations (Genotype MTBDRsl) (4)(5)(6)(7)(8). The more recently endorsed Xpert MTB/RIF assay (Cepheid, USA) facilitates rapid detection of the M. tuberculosis complex (9) but is limited to detection of rifampin resistance mutations in rpoB (10,11). Similarly, the INNO-LiPA RIF.TB line probe assay (Fujirebio Inc., Ghent, Belgium) is limited to the detection of rpoB mutations associated with rifampin resistance but also is able to detect the emergence of rifampin-resistant populations due to the presence of wild-type (wt) and mutant probes (12).…”