Inconsistent Results with the Xpert-MTB/Rif Assay in Detection of Mycobacterium

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1 JCM Accepts, published online ahead of print on 12 July 2013 J. Clin. Microbiol. doi: /jcm Copyright 2013, American Society for Microbiology. All Rights Reserved Inconsistent Results with the Xpert-MTB/Rif Assay in Detection of Mycobacterium tuberculosis with an rpob Mutation Associated with Low Level of Rifampin Resistance: Diagnostic Implications Akos Somoskovi 1, 2, #, Vanessa Deggim 1, Diana Ciardo 3, Guido V. Bloemberg 1 1 Institut für Medizinische Mikrobiologie Universität Zürich, Gloriastrasse 30-32, Zurich, CH 8006 Switzerland; 2 Nationales Zentrum für Mykobakterien, Universität Zürich, Gloriastrasse 30-32, Zurich, CH 8006 Switzerland; 3 Viollier AG, Spalenring , Basel, CH 4002 Switzerland Running title: False rifampin susceptibility with Xpert-MTB/Rif # Corresponding author: Akos Somoskovi, M.D., Ph.D., D.Sc. Swiss National Reference Center for Mycobacteria Institute for Medical Microbiology, University of Zurich Gloriastrasse 30-32, Zurich, CH 8006 Switzerland Phone: Cell Phone: asomoskoevi@imm.uzh.ch

2 Abstract: Xpert-MTB/Rif is one of the most frequently used molecular screening test for multidrugresistant tuberculosis worldwide. We report false negative results of the assay in the presence of rpob Leu533Pro that is associated with low level of phenotypic rifampin resistance. Accurate and timely confirmation of rifampin results with Xpert-MTB/Rif is imperative. Downloaded from on December 31, 2018 by guest 2

3 The accurate diagnosis of drug resistant and multidrug-resistant tuberculosis (MDR-TB) is imperative to initiate adequate treatment, to avoid transmission of the disease and to prevent development of further drug resistance. Due to its worldwide roll out and rapid implementation the Xpert-MTB/Rif assay (Cepheid, USA) has become one of the most frequently used molecular screening test for tuberculosis (TB) and MDR-TB in both resource poor and resource rich countries (1). Recently, a 67 years old Swiss-born 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 presence of M. tuberculosis complex (MTBC) in two sputa (Table 1; sputa no. 1 and 2). In addition, these specimens showed indeterminate and determinate rifampin (RMP) resistance, respectively (Table 1, sputum no. 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 on false RMP resistance with the assay (2, 3) the clinician cast doubt about the accuracy of Xpert- MTB/Rif RMP results. Therefore, an additional four sputa were submitted to the Swiss National Reference Center for Mycobacteria for rapid confirmation before initiating MDR-TB therapy. Further patient testing revealed HIV positivity. The four additional sputum specimens (Table 1, no. 3-6) were acid-fast smear positive by the Ziehl-Neelsen method (4) and Xpert-MTB/Rif testing detected the presence of MTBC. Surprisingly, all samples were scored RMP susceptible with 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 was performed as described previously (3, 5), which revealed an rpob Leu (CTG)533Pro(CCG) and a katg His(CAG)315Gln(CAC) mutation respectively in all four specimens. 3

4 Growth detection and quantitative phenotypic drug susceptibility testing (DST) for first and second line antituberculosis drugs using the MGIT 960 system and EpiCenter software with the TB exist module (Becton-Dickinson Microbiology Systems, Sparks, MD, USA) was performed as described earlier (6). Quantitative DST identified resistance to RMP at 0.5 μg/ml and susceptibility at 1.0, 4.0 and 20 μg/ml, and resistance to rifabutin (RFB) at 0.1 μg/ml and susceptibility at 0.4 and 2.0 μg/ml concentrations. DST for INH showed resistance at 0.1 μg/ml and 1.0 μg/ml, and intermediate resistance at 3.0 and 10.0 μg/ml. No drug resistance was identified by conventional DST for other first and second line drugs. Direct molecular results were confirmed by DNA sequencing of the rpob and katg genes of the culture isolates and gave concordant results. Previously, in vitro experiments indicated that Xpert-MTB/Rif cannot detect Leu533Pro unless 100% of the DNA population was mutant (7). PCR followed DNA sequencing directly from the sputa or cultures did not show double peaks (potential signs of heteroresistance) at the corresponding Leu533Pro mutant position in the sequence electropherograms. Therefore, we have no indications from molecular testing that a heteroresistance population was present in the patient. Moreover, the clinical information also argues against heteroresistance since resistance in patients with newly diagnosed TB (such as our case) is usually associated with a high proportion of drug resistant population compared to patients with acquired drug resistance who were previously treated for tuberculosis already (8) Detailed analysis of the Xpert-MTB/Rif assay parameters revealed that rpob Probe-E (encompassing codon 533) was hybridizing significantly less and showed markedly lower than 5 ΔCt max values (the Xpert-MTB/Rif software is applying a ΔCt max cut-off > 5 for the automated 4

5 detection of RMP resistance) (9) than the other four probes, but that did not result in RMP resistance identification by the Xpert software (software version 4.3) (Table 1). The bacterial load of the different specimens was variable, which may have resulted in variable Probe-E hybridization and lack of detecting mutation Leu533Pro (Table 1). These findings may warrant the thorough revision of Probe-E sequence length, and its hybridization and software detection parameters. Our report clearly shows that Xpert-MTB/Rif can be false RMP susceptible in the presence of mutation Leu533Pro in clinical specimens with different bacterial loads. Since bacterial load of patients may vary in subsequent specimens this non-reproducibility may generate serious diagnostic confusion and may significantly impact assay predictive values especially in low incidence settings. RMP resistant tuberculosis has a major impact on patient outcome and removal of RMP is a very significant alteration to the standard treatment. Reliable molecular detection of rpob mutations Leu533Pro, Leu511Pro, Asp516Tyr and His526Leu has important diagnostic consequences since these mutations can be associated with treatment failure using standard regimens that containing 600 mg/day RMP (2, 10, 11). Such rpob mutant strains can be missed by conventional DST since they are resistant to lower RMP concentrations that are not routinely tested in many laboratories (10). Although rpob Leu533Pro, Leu511Pro, Asp516Tyr and His526Leu mutations are less common their prevalence may reach 10-22% (12).However, it has also been shown that screening for RMP resistance alone is not always a reliable proxy for the presumptive diagnosis of MDR-TB neither in low nor in high prevalence settings indicating the need for rapid INH susceptibility screening in addition to RMP (13, 14). 5

6 In line with these observations, our results underline that accurate and timely confirmation of RMP resistant or non-reproducible Xpert-MTB/Rif results and rapid molecular screening for INH resistance are imperative, especially in a low incidence setting when clinical suspicion argues against MDR-TB or in peripheral settings where staff may be less experienced in test interpretation, critically reviewing assay parameters, and troubleshooting. Correct and rapid identification of mutations can also provide valuable preliminary information on the level of drug resistance and can direct what additional non routine drug concentrations and second line drugs can be included in subsequent conventional DST. Finally, the present report shows that the definitive diagnosis of MDR-TB may not be based on a single test but should always be established on a thorough evaluation and verification of all clinical and laboratory findings. Acknowledgement: The authors wish to thank E.C. Böttger for his valuable comments on the manuscript. 6

7 109 References: Weyer K, Mirzayev F, Migliori G, Van Gemert W, D'Ambrosio L, Zignol M, Floyd K, Centis R, Cirillo D, Tortoli E, Gilpin C, Iragena J, Falzon D, Raviglione M Rapid molecular TB diagnosis: evidence, policy-making and global implementation of Xpert(R)MTB/RIF. Eur. Respir. J. 42: Williamson DA, Basu I, Bower J, Freeman JT, Henderson G, Roberts SA An evaluation of the Xpert MTB/RIF assay and detection of false-positive rifampicin resistance in Mycobacterium tuberculosis. Diagn. Microbiol. Infect. Dis. 74: Zbinden A, Keller PM, Bloemberg GV Rapid molecular detection of tuberculosis. N. Engl. J. Med. 364:183; author reply Kent P, Kubica G Public health mycobacteriology: A guide for the level III laboratory. Centers for Disease Control and Prevention, Atlanta, Georgia. 5. Telenti A, Imboden P, Marchesi F, Schmidheini T, Bodmer T Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis. Antimicrob. Agents Chemother. 37: Springer B, Lucke K, Calligaris-Maibach R, Ritter C, Bottger EC Quantitative drug susceptibility testing of Mycobacterium tuberculosis by use of MGIT 960 and EpiCenter instrumentation. J. Clin. Microbiol. 47: Blakemore R, Story E, Helb D, Kop J, Banada P, Owens MR, Chakravorty S, Jones M, Alland D Evaluation of the analytical performance of the Xpert MTB/RIF assay. J. Clin. Microbiol. 48:

8 Lipsitch M, Levin BR Population dynamics of tuberculosis treatment: mathematical models of the roles of non-compliance and bacterial heterogeneity in the evolution of drug resistance. Int. J. Tuberc. Lung Dis. 2: Scott LE, McCarthy K, Gous N, Nduna M, Van Rie A, Sanne I, Venter WF, Duse A, Stevens W Comparison of Xpert MTB/RIF with other nucleic acid technologies for diagnosing pulmonary tuberculosis in a high HIV prevalence setting: a prospective study. PLoS Med. 8:e Horne DJ, Pinto LM, Arentz M, Lin SY, Desmond E, Flores LL, Steingart KR, Minion J Diagnostic accuracy and reproducibility of WHO-endorsed phenotypic drug susceptibility testing methods for first-line and second-line antituberculosis drugs. J. Clin. Microbiol. 51: Williamson DA, Roberts SA, Bower JE, Vaughan R, Newton S, Lowe O, Lewis CA, Freeman JT Clinical failures associated with rpob mutations in phenotypically occult multidrug-resistant Mycobacterium tuberculosis. Int. J. Tuberc. Lung Dis. 16: Van Deun A, Barrera L, Bastian I, Fattorini L, Hoffmann H, Kam KM, Rigouts L, Rusch- Gerdes S, Wright A Mycobacterium tuberculosis strains with highly discordant rifampin susceptibility test results. J. Clin. Microbiol. 47: Kurbatova EV, Cavanaugh JS, Shah NS, Wright A, Kim H, Metchock B, Van Deun A, Barrera L, Boulahbal F, Richter E, Martin-Casabona N, Arias F, Zemanova I, Drobniewski F, Santos Silva A, Coulter C, Lumb R, Cegielski JP Rifampicin-resistant 8

9 Mycobacterium tuberculosis: susceptibility to isoniazid and other anti-tuberculosis drugs. Int. J. Tuberc. Lung Dis. 16: Mukinda FK, Theron D, van der Spuy GD, Jacobson KR, Roscher M, Streicher EM, Musekiwa A, Coetzee GJ, Victor TC, Marais BJ, Nachega JB, Warren RM, Schaaf HS Rise in rifampicin-monoresistant tuberculosis in Western Cape, South Africa. Int. J. Tuberc. Lung Dis. 16: Downloaded from on December 31, 2018 by guest 9

10 Table 1. Xpert-MTB/Rif results and assay parameters of six sputum specimens tested at the primary laboratory (sputa no. 1 and 2; grey) and at the Swiss National Reference Center for Mycobacteria (sputa no. 3-6). Sample repeated MTBC detected/ bacterial yes/ very low yes/ low yes/ high yes/ medium yes/ medium yes/ medium yes/ medium load RMP Resistance indeterminate yes yes no no no no Cartridge Type G3 and software version 4.3 yes yes yes yes yes yes yes Ct value Probe A Probe B Probe C Probe D Probe E a > SPC End Point Probe A Probe B Probe C Probe D Probe E a SPC ΔCt max value b > a Probe E Ct and End Point values are markedly different in all 6 specimens compared to Probes A-D but the testing software (Cartridge G3, Version 4.3) of the assay flagged RMP resistance only in two initial specimens with high and low bacterial load in contrast to other specimens with very low or medium bacterial load indicating an analytical problem of the software. b The software is applying a ΔCt max cut-off > 5 for the automated detection of RMP resistance. Abbreviations: MTBC = M. tuberculosis complex; RMP = rifampin; SPC = sample processing control; Ct = crossing point.