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INT J TUBERC LUNG DIS 7(9):899–902 © 2003 IUATLD

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Drug susceptibility testing of Mycobacterium tuberculosis to fluoroquinolones: first experience with a quality control panel in the Nordic-Baltic collaboration I. S. Johansen,* A. R. Larsen,† P. Sandven,‡ B. Petrini,§ H. Soini,¶ K. Levina,# A. Sosnovskaja,** G. Skenders,†† S. Hoffner‡‡ * International Reference Laboratory of Mycobacteriology, † Department of Microbiological R&D, Statens Serum Institut, Copenhagen, Denmark; ‡ Division of Infectious Diseases Control, Institute of Public Health, Oslo, Norway; § Department of Clinical Microbiology, Karolinska Institute and Hospital, Stockholm, Sweden; ¶ Mycobacterial Reference Laboratory, National Public Health Institute, Turku, Finland; # Mycobacteriology Laboratory, Kivimae Hospital, Tallinn, Estonia; ** National Tuberculosis Reference Laboratory, National Tuberculosis and Lung Diseases Hospital, Vilnius, Lithuania; †† State Centre of Tuberculosis and Lung Diseases, Riga, Latvia; ‡‡ Department of Bacteriology, Swedish Institute for Infectious Diseases Control, Solna, Sweden SUMMARY

In the first attempt to establish a quality assurance programme for susceptibility testing of Mycobacterium tuberculosis to fluoroquinolones, 20 strains with different fluoroquinolone susceptibility patterns were distributed by the Supranational Reference Laboratory in Stockholm to the other mycobacterial reference laboratories of the Nordic and Baltic countries. Susceptibility testing to fluoroquinolones was performed according to routine procedures in each laboratory. Results were compared to sequence analysis of the gyrA gene and minimal inhibitory concentration determination. Most laboratories

found identical susceptibility patterns. The two resistant strains were correctly identified by all laboratories, but three laboratories each falsely reported one susceptible strain as resistant. These results indicate that the participating laboratories yield reliable results in detection of fluoroquinolone-resistant strains, although the need for a standardised quality assurance programme for drug susceptibility testing for fluoroquinolones is stressed by the strains falsely reported as resistant. K E Y W O R D S : second-line drugs; MDR-TB; quality assurance

THE INCREASING THREAT of multidrug-resistant tuberculosis (MDR-TB) has been documented in two worldwide surveys conducted by the World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (IUATLD).1,2 Resistance to first-line drugs is widespread, with MDR-TB ‘hot-spots’ in many parts of the world. The spread of MDR-TB can be prevented only if patients with drug-resistant tuberculosis are identified and treated with a combination of effective drugs. The first important step in achieving this goal is for microbiological laboratories to be able to perform reliable drug susceptibility testing with both first- and secondline drugs.3 Standard methods for testing first-line drugs have been established and an external quality assurance programme has been organised by the WHO.4 No such programmes exist for second-line drugs, despite the fact that effective treatment of patients with MDR-TB has become increasingly important.5,6 Various drug susceptibility testing methods for second-line drugs have been proposed,7–9 but

there is no internationally accepted reference method. Standardisation and improvement of drug susceptibility testing procedures for second-line anti-tuberculous drugs are therefore urgently needed. This is emphasised by the fact that expensive second-line drugs should become widely available as a result of the Green Light Committee collaboration. The aim of this study was to evaluate the ability of the participating laboratories to perform susceptibility testing for fluoroquinolone (FQ) drugs, and to commence the elaboration of a consensus protocol for the test. The Nordic-Baltic Network for Mycobacteriology In 1998, a Nordic-Baltic Network for Mycobacteriology was initiated by the supranational reference laboratory (SRL) in Sweden. The network includes national mycobacterial reference laboratories from Denmark, Estonia, Finland, Latvia, Lithuania, Norway and Sweden. This group has previously developed guidelines for routine diagnostic procedures, quality control and regulations for correct and safe work with mycobacte-

Correspondence to: Isik S Johansen, International Reference Laboratory of Mycobacteriology, Statens Serum Institut, 5 Artillerivej, 2300 Copenhagen, Denmark. Tel: (45) 326 83703. Fax: (45) 326 83871. e-mail: [email protected] Article submitted 15 November 2002. Final version accepted 6 March 2003.

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ria. The laboratories included also participate in the quality assurance programme for drug susceptibility testing of Mycobacterium tuberculosis in the WHO/ IUATLD supranational reference laboratory network. M. tuberculosis strains Each laboratory submitted a number of M. tuberculosis strains to the SRL in Sweden. Of these, 20 strains with different susceptibility patterns to FQ were selected. The strains were subcultured on LöwensteinJensen (LJ) slants, coded, and a panel of the 20 strains was sent to each study site. Determination of minimal inhibitory concentration (MIC) The MIC value of ciprofloxacin (CIP) was determined for all 20 strains at the SRL using the Bactec 460 system (Becton Dickinson, Sparks, MD, USA). The bacterial inoculum was prepared according to the manufacturer’s recommendations.9 The strains were tested for susceptibility to CIP in two-fold dilutions from 1 to 8 g/ml based on previous studies10,11 and described elsewhere.12 The growth control vial was prepared with a bacterial dilution of 1:100. The final concentration in all drug-containing vials was approximately 106 cfu/ml. The MIC was defined as the lowest concentration of the drug that inhibited more than 99% of bacterial growth. Determination of FQ-resistant gene FQs inhibit DNA synthesis by binding to the enzyme DNA gyrase. A number of specific mutations in a fragment of the gyrA gene, encoding the A subunit of DNAgyrase, have been found to cause FQ resistance in M. tuberculosis.13 All the strains were examined for such mutations, and the results were compared to the MIC values. A small amount of several colonies taken from the LJ media were resuspended in 1.2 ml TE buffer (10 mM Tris HCl, 1 mM EDTA pH 8), boiled for 10 min at 100C, and centrifuged for 5 min at 13 000 rpm; 2.5 l of the lysate was used for polymerase chain reaction (PCR) amplification of the gyrA genes. A 320-bp region of the gyrA gene was amplified from each isolate in a GeneAmp2400 Thermo cycler (Perkin Elmer, Boston, MA), as previously described.13 The amplicons were purified using QIAquick® spin columns (Quiagen Inc, Valencia, CA) according to the manufacturer’s instructions, and sequenced in both directions using the PCR primers and the Big Dye cycle sequencing kit (Applied Biosystems, Foster City CA). The amplicons were sequenced with an automatic sequencer ABI 377, and edited and aligned with the Seq.Ed program (Applied Biosystems). Drug susceptibility testing Each of the nine laboratories taking part in this study performed susceptibility testing for CIP and/or ofloxacin (OFL) using their respective routine procedures. Eight laboratories used the Bactec 460 radiometric sys-

tem and one laboratory used a narrow-range MIC method on 7H10 agar with drug concentrations of 2, 4 and 6 g/ml. A MIC value of 4 g/ml was considered resistant.14 The critical drug concentration used for the Bactec 460 method was 2 g/ml for the both antibiotics. Each laboratory made their own drug solutions according to the product insert. Both antibiotics were dissolved in sterile distilled water. OFL could also be dissolved in a 0.1 N NaOH solution. Thereafter the stock solutions were sterile filtered and approximately 20% of the initial filtered volume was discarded. The remaining volume was kept in small aliquots at 70C until use. The Bactec 460 method was performed according to the manufacturer’s recommendations for second-line drugs, as described in detail elsewhere.9 All results were registered as resistant (R) or sensitive (S) and forwarded to the SRL. Two isolates (no. 10 and 18) were reported resistant by all laboratories, irrespective of which method was used or which FQ drug was tested (Table). The resistant phenotypes were confirmed by sequence analysis of the FQ resistance region of gyrA, which showed that both of the isolates carried an A➝G substitution in position 2586 (Genbank: L27512) of the gyrA fragment, causing an amino acid change of aspartic acid to glycine. This mutation is known to mediate FQ resistance among others.13 The MICs were found to be 8 g/ml for both resistant strains. The remaining 18 isolates were reported to be sensitive by six laboratories (1, 2, 3, 4, 7 and SRL), while discrepant results were reported by three laboratories (Table): laboratories 5 and 6 (isolate 5) and laboratory 8 (isolate 11). All the isolates had a wild-type sequence in the analysed region of gyrA. The MICs of the susceptible isolates were 1 g/ml. An active quality assurance programme is essential in the clinical mycobacteriology laboratory in order to achieve accurate, reliable results. For first-line drugs susceptibility testing methods are standardised and an external quality assurance programme is well established. This is, however, not the case for secondline drugs. With the increasing use of these drugs for the treatment of MDR patients, standardisation of methods and the introduction of quality assurance programmes are urgently needed. The WHO has recently launched recommendations for drug susceptibility testing of selected second-line drugs using LJ slants as the standard medium.15 However, this method is both labour intensive and time consuming. The present study shows that the Bactec 460 radiometric system can provide comparable results within 1 week, as previously reported in the initial studies of MIC values of OFL and CIP using this system.10,11 A large multicenter study comprising second-line drugs later confirmed these results.8 The breakpoint concentration for OFL and CIP used in our study was in full agreement with all of these studies, i.e., 2 g/ml. Several reports have shown that mutations in the

Susceptibility testing of M. tuberculosis to fluoroquinolones

Table

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Results of fluoroquinolone quality assurance panel in the Nordic-Baltic collaboration Laboratory number, testing methods, antibiotics 1 MIC*

Isolate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

2 Bactec

3 Bactec

4 Bactec

5 Bactec

6 Bactec

7 Bactec

8 Bactec

SRL Bactec

CIP

OFL

CIP

OFL

CIP

OFL

CIP

OFL

OFL

OFL

CIP

CIP

S S S S S S S S S R S S S S S S S R S S







S S S S R S S S S R S S S S S S S R S S

S S S S R S S S S R S S S S S S S R S S


S S S S S S S S S R R S S S S S S R S S


* Narrow-range MIC method. SRL supranational reference laboratory; MIC minimum inhibitory concentration; CIP ciprofloxacin; OFL ofloxacin; S sensitive; R resistant.

gene encoding the gyrase A enzyme are responsible for FQ resistance, correlating with an increase of up to 30fold in MIC values.16 In our study, the sequence analysis of the gyrA gene revealed mutations only in the two strains yielding high MIC values. All laboratories reported these strains as resistant, and thus no very major error (false susceptible) was observed in our study, in line with Pfyffer et al.8 As reported by others, FQ resistance is relatively rare. We were therefore only able to include two resistant strains (10%) in our study. However, this correlates well with the percentage of FQresistant strains included in a previous study (8%).8 Furthermore a Dutch clinical outcome study on patients with MDR-TB revealed only 2.3% FQ resistance.5 Quality assurance programmes for first-line drugs have reported discrepancies for streptomycin and ethambutol, whereas no major problems were found for isoniazid and rifampicin.4,17,18 Similar problems should be expected initially for second-line drugs including FQ. We found three major errors (false resistant) that could have been caused by drug preparation or errors in the interpretation of the results. Furthermore, contamination by other bacteria or fungi in the antibiotic containing vial was a possibility. Confronted with the errors the laboratories retested the cultures with discrepant results and found that they were sensitive. Laboratory 5 reported over-inoculation and laboratory 6 reported misinterpretation of the initial result to be the cause of discrepancy. In order to optimise methods used for FQ susceptibility testing, and identify the step responsible for discrepant results in each laboratory, we recommend that resistant results should be verified by re-testing. In addition, microscopy and subculturing on blood plate

should be carried out in order to reveal possible contamination. These discrepancies warrant extended quality control studies where more resistant strains are included. Such studies will improve the reproducibility of susceptibility testing, as seen with first-line drugs.4,18 We suggest that standardised stock solutions of second-line drugs should be made commercially available in order to minimise laboratory errors, as in the case of first-line drugs. The task of standardisation of susceptibility testing of second-line drugs could initially be performed by the supranational reference laboratories, such that each laboratory would provide guidelines for certain drugs (genetic analysis, MIC value and method). Finally, the laboratories could provide blinded test panels containing well-characterised strains with different susceptibility patterns, and distribute them to the national laboratories. Acknowledgements The authors wish to thank Dr Bettina Lundgren, Vibeke Østergaard Thomsen and Annika Kruuner for their valuable comments on the study. This study was supported by the Swedish Baltic Sea Grant.

References 1 Pablos-Mendez A, Raviglione M C, Laszlo A, et al. Global surveillance for antituberculosis-drug resistance, 1994–1997. World Health Organization–International Union Against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance. N Engl J Med 1998; 338: 1641–1649. 2 Espinal M A, Laszlo A, Simonsen L, et al. Global trends in resistance to antituberculosis drugs. World Health Organization– International Union Against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance. N Engl J Med 2001; 344: 1294–1303.

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3 World Health Organization. Guidelines for establishing DOTS-PLUS pilot projects for the management of multidrugresistant tuberculosis (MDR-TB). Geneva: WHO, 2000. 4 Laszlo A, Rahman M, Raviglione M, Bustreo F. Quality assurance programme for drug susceptibility testing of Mycobacterium tuberculosis in the WHO/IUATLD Supranational Laboratory Network: first round of proficiency testing. Int J Tuberc Lung Dis 1997; 1: 231–238. 5 Geerligs W A, van Altena R, de Lange W C M, van Soolingen D, van der Werf T S. Multidrug-resistant tuberculosis: longterm treatment outcome in the Netherlands. Int J Tuberc Lung Dis 2000; 4: 758–764. 6 Tahaoglu K, Torun T, Sevim T, et al. The treatment of multidrug-resistant tuberculosis in Turkey. N Engl J Med 2001; 345: 170–174. 7 Inderlied C B, Salfinger M. Antimicrobial agents and susceptibility tests: Mycobacterium. In: Murray P R, Baron E J, Pfaller M A, Tenover F C, Yolken R H, eds. Manual of Clinical Microbiology. 6th ed. Washington: ASM, 1995: pp 1385–1404. 8 Pfyffer G E, Bonato D A, Ebrahimzadeh A, et al. Multicenter laboratory validation of susceptibility testing of Mycobacterium tuberculosis against classical second-line and newer antimicrobial drugs by using the radiometric BACTEC 460 technique and the proportion method with solid media. J Clin Microbiol 1999; 37: 3179–3186. 9 Siddiqi S H. BACTEC 460 TB SYSTEM. Product and Procedure Manual. Sparks, MD: Becton Dickinson Microbiology Systems, 1996. 10 Heifets L B, Lindholm-Levy P J. Bacteriostatic and bactericidal activity of ciprofloxacin and ofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex. Tubercle 1987; 68: 267–276. 11 Chen C H, Shih J F, Lindholm-Levy P J, Heifets L B. Minimal in-

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hibitory concentrations of rifabutin, ciprofloxacin, and ofloxacin against Mycobacterium tuberculosis isolated before treatment of patients in Taiwan. Am Rev Respir Dis 1989; 140: 987–989. Hoffner S E, Gezelius L, Olsson-Liljequist B. In-vitro activity of fluorinated quinolones and macrolides against drug-resistant Mycobacterium tuberculosis. J Antimicrob Chemother 1997; 40: 885–888. Takiff H E, Salazar L, Guerrero C, et al. Cloning and nucleotide sequence of Mycobacterium tuberculosis GyrA and GyrB genes and detection of quinolone resistance mutations. Antimicrob Agents Chemother 1994; 38: 773–780. Gorzynski E A, Gutman S I, Allen W. Comparative antimycobacterial activities of difloxacin, temafloxacin, enoxacin, pefloxacin, reference fluoroquinolones, and a new macrolide, clarithromycin. Antimicrob Agents Chemother 1989; 33: 591–592. World Health Organization. Guidelines for drug susceptibility testing for second-line anti-tuberculosis drugs for DOTSPLUS. Geneva: WHO, 2001. Kocagoz T, Hackbarth C J, Unsal I, Rosenberg E Y, Nikaido H, Chambers H F. Gyrase mutations in laboratory-selected, fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra. Antimicrob Agents Chemother 1996; 40: 1768–1774. Migliori G B, Ambrosetti M, Fattorini L, et al. Surveillance of anti-tuberculosis drug resistance: results of the 1998/1999 proficiency testing in Italy. SMIRA (Italian Multicentre Study on Antituberculosis Drug Resistance) Study Group. Int J Tuberc Lung Dis 2000; 4: 940–946. Laszlo A, Rahman M, Espinal M, Raviglione, M. Quality assurance programme for drug susceptibility testing of Mycobacterium tuberculosis in the WHO/IUATLD supranational reference laboratory network: five rounds of proficiency testing, 1994–1998. Int J Tuberc Lung Dis 2002; 6: 748–756.

RÉSUMÉ

Au cours du premier essai d’établissement d’un programme de contrôle de qualité pour les tests de sensibilité de Mycobacterium tuberculosis aux fluoroquinolones, 20 souches avec différents types de sensibilité à la fluoroquinolone ont été distribuées à partir du laboratoire supranational de référence de Stockholm vers les autres laboratoires de référence en mycobactériologie dans les régions du Nord et la région baltique. Les tests de sensibilité aux fluoroquinolones ont été pratiqués dans chaque laboratoire selon les procédures de routine. Les résultats ont été comparés à une analyse séquentielle du gène gyrA et à la détermination de la concentration mi-

nimale inhibitrice. La plupart des laboratoires ont trouvé des types de sensibilité identiques. Les deux souches résistantes ont été identifiées correctement par tous les laboratoires, mais trois laboratoires ont chacun considéré erronément une souche sensible comme étant résistante. Ces résultats indiquent que les laboratoires participants obtiennent des résultats fiables dans la détection des souches résistantes à la fluoroquinolone, bien que la nécessité d’un programme de contrôle de qualité standardisé pour les tests de sensibilité médicamenteuse aux fluoroquinolones ressorte de l’existence de souches signalées par erreur comme résistantes. RESUMEN

En un primer intento de establecer un programa de control de calidad para los tests de sensibilidad de Mycobacterium tuberculosis a las fluoroquinolonas, se distribuyeron 20 cepas con diferentes tipos de sensibilidad a la fluoroquinolona, a partir del laboratorio supranacional de referencia de Estocolmo, a otros laboratorios de referencia de micobacteriología de las regiones nórdicas y bálticas. Los tests de sensibilidad a la fluoroquinolona fueron realizados según los procedimientos de rutina de cada laboratorio. Los resultados fueron comparados a un análisis secuencial del gen gyrA y a la determinación de la con-

centración inhibitoria mínima. La mayoría de los laboratorios encontraron tipos de sensibilidad idénticos. Las dos cepas resistentes fueron identificadas correctamente por todos los laboratorios, pero tres laboratorios informaron, falsamente, cada uno una cepa sensible como resistente. Estos resultados indican que los laboratorios participantes producen resultados fiables para la detección de la resistencia a la fluoroquinolona, aunque se hace necesario un programa de control de calidad estandarizado para los tests de sensibilidad a la fluoroquinolona, vista la existencia de cepas informadas falsamente como resistentes.