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Extensively drug-resistant Mycobacterium tuberculosis strains isolated in Côte d’Ivoire
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ScienceDirect www.sciencedirect.com Médecine et maladies infectieuses 45 (2015) 303–305
Case report
Extensively drug-resistant Mycobacterium tuberculosis strains isolated in Côte d’Ivoire Isolement et identification de souches ultra-résistantes du complexe Mycobacterium tuberculosis, en Côte d’Ivoire K. N’guessan a,∗ , A.S. Bakayoko b , J.M. Ahui-Brou c , A.O. Kouakou d , B. Coulibaly e , A. Guei a , M. Dosso a a
Institut Pasteur de Côte d’Ivoire, 01 BP 490, Abidjan 01, Côte d’Ivoire Service de pneumologie, CHU de Treichville, Abidjan, Côte d’Ivoire c Service de pneumologie, CHU de Cocody, Abidjan, Côte d’Ivoire d Programme national de lutte contre la tuberculose, Abidjan Côte d’Ivoire e Centre antituberculeux de Yopougon, Abidjan, Côte d’Ivoire b
Received 3 March 2015; received in revised form 27 April 2015; accepted 18 May 2015 Available online 17 June 2015
Keywords: Mycobacterium tuberculosis complex; Drug susceptibility testing; Resistant genes Mots clés : Complexe Mycobacterium tuberculosis ; Test de sensibilité au médicament ; Gènes de résistance
1. First case study A 35-year-old male patient was treated with a 2RHZE/4RH regimen. The treatment was considered to have failed after direct examination of sputum smear at 6th month revealing the presence of acid-fast bacilli (AFB). The patient’s sputum samples were collected in recommended containers at the Centre antituberculeux de Yopougon (CAT), an intermediate tuberculosis center. The samples were transported in an icebox (4 ◦ C) to the National Tuberculosis Reference Laboratory (NTRL). The sputum samples were decontaminated with sodium hydroxide N-acetyl-l-cysteine for 15 min [1]. Ziehl-Neelsen staining with 500 l of pellet was performed. It revealed the presence of acid-fast bacilli (AFB) and quantified 2+. A MTBDRplus assay was performed directly on sputum samples, as recommended by the manufacturer. The molecular test allowed detecting D516V and S315T mutations respectively in the rpoB and katG gene.
∗
Corresponding author. E-mail address: [email protected] (K. N’guessan).
http://dx.doi.org/10.1016/j.medmal.2015.05.004 0399-077X/© 2015 Elsevier Masson SAS. All rights reserved.
The other biological investigations performed (clinical and biological symptoms, immunology, hematology, etc.) before initiating a regimen containing second-line drugs results were normal. The patient was then treated with a 4KmMfxPtoHCfzEZ/5MfxCfzEZ regimen. The treatment was re-evaluated every month. A sputum smear examination and culture on Lowenstein-Jensen were performed. After 6 months of treatment, the samples were still detected as positive for AFB. The sputum samples were decontaminated and 200 l of pellet were inoculated on Lowenstein-Jensen medium and incubated at 37 ◦ C. After 3 weeks of incubation, the isolates obtained on Lowenstein-Jensen medium were quantified 3+. The TB Ag MPT64 Rapid test (Standard Diagnostics, Seoul, South Korea) was used to identify Mycobacterium tuberculosis complex strains according to the manufacturer’s procedure. Mycobacterium growth indicator tube (MGIT 960) automated Drug susceptibility testing (DST) for first- and second-line drugs were performed on M. tuberculosis complex species. A standard protocol was used for Drug susceptibility testing according to the Becton-Dickinson product and procedure manual (BD, Biosciences, Sparks, Maryland, USA). MGIT-DST revealed that the isolated strains grew in presence of critical concentration of
304
K. N’guessan et al. / Médecine et maladies infectieuses 45 (2015) 303–305
Table 1 Patient’s socio demographic, clinical, and bacteriological data. Données socio-démographiques, cliniques et bactériologiques des patients. Data Age (years) Sex Occupation Clinical data
Bacteriological tests
Direct examination (Initial diagnosis)
Regimen Initiated Outcome Second line regimen
Identified risks factors
Bacteriological data
Culture susceptibility testing for first-line drugs
Susceptibility testing for second-line drugs
Sequencing
rpoB gene katG gene Promoter inhA Rifampin (1 g/ml) Isoniazid (0.1 g/ml) Streptomycin (1 g/ml) Ethambutol (5 g/ml) Ofloxacin (2 g/ml) Kanamycin (5 g/ml) Amikacin (1 g/ml) Ethionamide (5 g/ml) gyrA gene rrs gene eis promoter
MDR-TB treatment outcome
each drug tested except for streptomycin (Table 1). The patient died after 10 months of treatment for MDR-TB.
2. Second case study The second case was a failure of retreatment after 2RHZES/1RHZE/5RHE regimen followed in the same tuberculosis center. It concerned a 52-year-old male patient with a history of diabetes and HIV1 infection treated with a tenofovir/emtricidine/efavirenz combination. The patient’s sputum samples were sent to the Côte d’Ivoire Pasteur Institute in the same conditions of transfer and practices. A rapid molecular test with an MTBDRplus assay revealed D516V and S315T mutations respectively in the rpoB and katG gene. After clinical and biological investigations, he was treated with 8Z KmLfxPtoPAS/16ZLfxPtoPAS. The monthly monitoring during treatment revealed AFB in sputum after 14 months. Cultures on Lowenstein-Jensen medium were positive (3+) after 3 weeks of incubation at 37 ◦ C, and allowed identifying M. tuberculosis complex strains. The MGIT-DST results were positive at the 12th day for first-line drugs and at the 10th day for second-line drugs. The clinical isolates were resistant to all drugs tested (Table 1).
Patient no 2
35 Male Shopkeeper 2RHZE/4RH regimen
52 Male Electrician 2RHZES/1RHZE/5RHE regimen June 2012 3+ Failure 8Z KmLfxPtoPAS/16ZLfxPtoPAS Positive 1.35 g/ml 14 months of regimen for 2nd-line drugs 2+ D516V S315T No mutation 3+ Resistant Resistant Resistant Resistant Resistant Resistant Resistant Resistant D94G A1401G No mutation Failure
October 2013 2+ Failure 4KmMfxPtoHCfzEZ/5MfxCfzEZ Negative 0.8 g/ml 6 months of regimen for 2nd-line drugs
HIV Blood Glucose
Direct examination (monitoring of treatment) MTBDRplus assay
Patient no 1
2+ D516V S315T No mutation 3+ Resistant Resistant Susceptible Resistant Resistant Resistant Resistant Resistant D94G No mutation C14T Failure
The clinical strains of the 2 patients were inactivated in an ethanol solution at 70% and sent to the supranational reference laboratory network in Milan for confirmation of NRTBL results. The results of sequencing performed at the supranational laboratory allowed detecting D94G, A1401G and C14 T mutations respectively in the gyrA, rrs, and eis gene (Table 1). After the intensive phase of treatment for MDR-TB, cultures were positive again demonstrating the failure of long-term treatment for MDR-TB. A new regimen with bedaquiline was suggested. Drug-resistant tuberculosis is a major threat to TB control worldwide [2]. Multidrug-resistant tuberculosis (MDR-TB) is defined as tuberculosis caused by a strain resistant to isoniazid (INH) and rifampin (RMP) and extensively drug-resistant tuberculosis (XDR-TB) as multidrug-resistant with additional resistance to any fluoroquinolone and any second-line injectable drug (kanamycin, amikacin, or capreomycin) [3]. Health experts considered that the extensively drug-resistant tuberculosis outbreak that occurred in 2005–2006 in KwaZulu-Natal was underestimated [4]. Whether an emerging disease or an old health problem, XDR-TB is a global public health threat. Usually, few laboratories can perform the microbiological diagnosis in low-income countries, especially in Africa where tuberculosis is endemic.
K. N’guessan et al. / Médecine et maladies infectieuses 45 (2015) 303–305
Laboratory confirmation is essential to ensure a correct diagnosis and an appropriate treatment for most infectious diseases, and especially for the emergence of resistant M. tuberculosis strains. According to the 2014 World Health Organization report, approximately 9% of patients carrying MDR-TB presented with extensively drug-resistant tuberculosis [2]. The current strategy is based on rapid detection of mutations encoding resistance to rifampin and isoniazid. Consequently, it is not excluded that these 2 patients were initially infected by M. tuberculosis strains with additional resistance to any fluoroquinolone and at least 1 of the 3 following injectable drugs (capreomycin, kanamycin, and amikacin). A patient carrying XDR-TB, living in Côte d’Ivoire, was already diagnosed in France at the Percy Military hospital [5]. The 3 diagnosed XDR-TB cases revealed an inadequate management of tuberculosis [3]. The MGIT 960 automated liquid culture system is probably the most frequently used and best validated at this moment [6,7]. The BACTEC S.I.R.E. drug kit was used for first-line drugs susceptibility testing. A contamination of DST could be excluded since positive results were obtained in 12 days. Tubes containing second-line drugs were prepared by adding 100 l of each drug. The contamination of tubes prepared and inoculated could be excluded considering the results of automated MGIT-DST assays that were obtained after 10 days. The detection of clinical isolate growth in drug-containing tubes proved the phenotypic resistance to the drug considered, especially for second-line drugs [8]. Our results were confirmed by the Milan supranational laboratory, through sequencing of genes implicated in resistance (fluoroquinolones, aminoglycosides). The mutations detected in gyrA, rrs gene and in eis promoter region are implicated in phenotypic resistance [9,10]. Although, a new and appropriate regimen was initiated, the treatment failed and 1 of the 2 patients died. Given these results, identifying XDR-TB cases is very important for public health and is, in Côte d’Ivoire, a strong indicator for all healthcare providers fighting TB. Furthermore, the strategy used to fight TB must be pertinent and updated according to epidemiological data. Globally, the diagnosis has been improved and appropriate regimens must be accessible to decrease M. tuberculosis strain dissemination in the community. In this last case, direct observed treatment remains essential to fight tuberculosis. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
305
Authors contribution Pr. K. N’guessan wrote the article. Pr. A.S. Bakayoko managed MDR-TB cases and proofread the article. Dr. J.M Ahui-brou managed MDR-TB cases at the Yopougon CAT and reviewed the article and clinical data. Dr. A.O. Kouakou is a member of NTCP for MDR-TB cases. Dr. B. Coulibaly is the head of the Yopougon tuberculosis center. Mr. A. Guei is a biotechnologist and performed culture and DST. Pr. M. Dosso is the director of the Côte d’Ivoire Pasteur institute. Acknowledgements We thank Dr. Elisa Tagliani and Dr. Daniela Cirillo (SRL, Milan, Italy), Kekeletso Kao (Expand TB), and the PEPFAR Côte d’Ivoire team of for their contributions. References [1] Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. Atlanta: USDHHS, Centers for Disease Control; 1985. [2] World Health Organization. Global tuberculosis report 2014. Geneva 27, Switzerland: WHO/HTM/TB/2014.08; 2014. [3] Raviglione MC, Smith IM. XDR tuberculosis: implications for global public health. N Engl J Med 2007;356:656–9. [4] Singh JA, Upshur R, Padayatchi N. XDR-TB in South Africa: no time for denial or complacency. PLoS Med 2007;4:e50. [5] Bakayoko AS, Ahui BJM, Kone Z, Daix ATJ, Samake K, Domoua KMS, et al. Extensively drug resistant tuberculosis in Ivory Coast. Rev Pneumol Clin 2015 http://dx.doi.org/10.1016/j.pneumo.2014.12.009 [6] Rusch-Gerdes S, Pfyffer GE, Casal M, Chadwick M, Siddiqi S. Multicenter laboratory validation of the BACTEC MGIT 960 technique for testing susceptibilities of Mycobacterium tuberculosis to classical second-line drugs and newer antimicrobials. J Clin Microbiol 2006;44:688–92. [7] Ardito F, Posteraro B, Sanguinetti M, Zanetti S, Fadda G. Evaluation of BACTEC Mycobacteria Growth Indicator Tube (MGIT 960) automated system for drug susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 2001;39:4440–4. [8] Jakko VI, Sami S, Rina Z, Tridia VL, Miranda KA, Martin JB, et al. Comparative study on genotypic and phenotypic second-line drug resistance testing of Mycobacterium tuberculosis complex isolates. J Clin Microbiol 2010;48:2749–53. [9] Maruri F, Sterling TR, Kaiga AW, Blackman A, Van der Heijden YF, Mayer C, et al. A systematic review of gyrase mutations associated with fluoroquinolone-resistant Mycobacterium tuberculosis and a proposed gyrase numbering system. J Antimicrob Chemother 2012;67: 819–31. [10] Gikalo MB, Nosova EY, Krylova LY, Moroz AM. The role of eis mutations in the development of kanamycin resistance in Mycobacterium tuberculosis isolates from the Moscow region. J Antimicrob Chemother 2012;67:2107–9.
ScienceDirect www.sciencedirect.com Médecine et maladies infectieuses 45 (2015) 303–305
Case report
Extensively drug-resistant Mycobacterium tuberculosis strains isolated in Côte d’Ivoire Isolement et identification de souches ultra-résistantes du complexe Mycobacterium tuberculosis, en Côte d’Ivoire K. N’guessan a,∗ , A.S. Bakayoko b , J.M. Ahui-Brou c , A.O. Kouakou d , B. Coulibaly e , A. Guei a , M. Dosso a a
Institut Pasteur de Côte d’Ivoire, 01 BP 490, Abidjan 01, Côte d’Ivoire Service de pneumologie, CHU de Treichville, Abidjan, Côte d’Ivoire c Service de pneumologie, CHU de Cocody, Abidjan, Côte d’Ivoire d Programme national de lutte contre la tuberculose, Abidjan Côte d’Ivoire e Centre antituberculeux de Yopougon, Abidjan, Côte d’Ivoire b
Received 3 March 2015; received in revised form 27 April 2015; accepted 18 May 2015 Available online 17 June 2015
Keywords: Mycobacterium tuberculosis complex; Drug susceptibility testing; Resistant genes Mots clés : Complexe Mycobacterium tuberculosis ; Test de sensibilité au médicament ; Gènes de résistance
1. First case study A 35-year-old male patient was treated with a 2RHZE/4RH regimen. The treatment was considered to have failed after direct examination of sputum smear at 6th month revealing the presence of acid-fast bacilli (AFB). The patient’s sputum samples were collected in recommended containers at the Centre antituberculeux de Yopougon (CAT), an intermediate tuberculosis center. The samples were transported in an icebox (4 ◦ C) to the National Tuberculosis Reference Laboratory (NTRL). The sputum samples were decontaminated with sodium hydroxide N-acetyl-l-cysteine for 15 min [1]. Ziehl-Neelsen staining with 500 l of pellet was performed. It revealed the presence of acid-fast bacilli (AFB) and quantified 2+. A MTBDRplus assay was performed directly on sputum samples, as recommended by the manufacturer. The molecular test allowed detecting D516V and S315T mutations respectively in the rpoB and katG gene.
∗
Corresponding author. E-mail address: [email protected] (K. N’guessan).
http://dx.doi.org/10.1016/j.medmal.2015.05.004 0399-077X/© 2015 Elsevier Masson SAS. All rights reserved.
The other biological investigations performed (clinical and biological symptoms, immunology, hematology, etc.) before initiating a regimen containing second-line drugs results were normal. The patient was then treated with a 4KmMfxPtoHCfzEZ/5MfxCfzEZ regimen. The treatment was re-evaluated every month. A sputum smear examination and culture on Lowenstein-Jensen were performed. After 6 months of treatment, the samples were still detected as positive for AFB. The sputum samples were decontaminated and 200 l of pellet were inoculated on Lowenstein-Jensen medium and incubated at 37 ◦ C. After 3 weeks of incubation, the isolates obtained on Lowenstein-Jensen medium were quantified 3+. The TB Ag MPT64 Rapid test (Standard Diagnostics, Seoul, South Korea) was used to identify Mycobacterium tuberculosis complex strains according to the manufacturer’s procedure. Mycobacterium growth indicator tube (MGIT 960) automated Drug susceptibility testing (DST) for first- and second-line drugs were performed on M. tuberculosis complex species. A standard protocol was used for Drug susceptibility testing according to the Becton-Dickinson product and procedure manual (BD, Biosciences, Sparks, Maryland, USA). MGIT-DST revealed that the isolated strains grew in presence of critical concentration of
304
K. N’guessan et al. / Médecine et maladies infectieuses 45 (2015) 303–305
Table 1 Patient’s socio demographic, clinical, and bacteriological data. Données socio-démographiques, cliniques et bactériologiques des patients. Data Age (years) Sex Occupation Clinical data
Bacteriological tests
Direct examination (Initial diagnosis)
Regimen Initiated Outcome Second line regimen
Identified risks factors
Bacteriological data
Culture susceptibility testing for first-line drugs
Susceptibility testing for second-line drugs
Sequencing
rpoB gene katG gene Promoter inhA Rifampin (1 g/ml) Isoniazid (0.1 g/ml) Streptomycin (1 g/ml) Ethambutol (5 g/ml) Ofloxacin (2 g/ml) Kanamycin (5 g/ml) Amikacin (1 g/ml) Ethionamide (5 g/ml) gyrA gene rrs gene eis promoter
MDR-TB treatment outcome
each drug tested except for streptomycin (Table 1). The patient died after 10 months of treatment for MDR-TB.
2. Second case study The second case was a failure of retreatment after 2RHZES/1RHZE/5RHE regimen followed in the same tuberculosis center. It concerned a 52-year-old male patient with a history of diabetes and HIV1 infection treated with a tenofovir/emtricidine/efavirenz combination. The patient’s sputum samples were sent to the Côte d’Ivoire Pasteur Institute in the same conditions of transfer and practices. A rapid molecular test with an MTBDRplus assay revealed D516V and S315T mutations respectively in the rpoB and katG gene. After clinical and biological investigations, he was treated with 8Z KmLfxPtoPAS/16ZLfxPtoPAS. The monthly monitoring during treatment revealed AFB in sputum after 14 months. Cultures on Lowenstein-Jensen medium were positive (3+) after 3 weeks of incubation at 37 ◦ C, and allowed identifying M. tuberculosis complex strains. The MGIT-DST results were positive at the 12th day for first-line drugs and at the 10th day for second-line drugs. The clinical isolates were resistant to all drugs tested (Table 1).
Patient no 2
35 Male Shopkeeper 2RHZE/4RH regimen
52 Male Electrician 2RHZES/1RHZE/5RHE regimen June 2012 3+ Failure 8Z KmLfxPtoPAS/16ZLfxPtoPAS Positive 1.35 g/ml 14 months of regimen for 2nd-line drugs 2+ D516V S315T No mutation 3+ Resistant Resistant Resistant Resistant Resistant Resistant Resistant Resistant D94G A1401G No mutation Failure
October 2013 2+ Failure 4KmMfxPtoHCfzEZ/5MfxCfzEZ Negative 0.8 g/ml 6 months of regimen for 2nd-line drugs
HIV Blood Glucose
Direct examination (monitoring of treatment) MTBDRplus assay
Patient no 1
2+ D516V S315T No mutation 3+ Resistant Resistant Susceptible Resistant Resistant Resistant Resistant Resistant D94G No mutation C14T Failure
The clinical strains of the 2 patients were inactivated in an ethanol solution at 70% and sent to the supranational reference laboratory network in Milan for confirmation of NRTBL results. The results of sequencing performed at the supranational laboratory allowed detecting D94G, A1401G and C14 T mutations respectively in the gyrA, rrs, and eis gene (Table 1). After the intensive phase of treatment for MDR-TB, cultures were positive again demonstrating the failure of long-term treatment for MDR-TB. A new regimen with bedaquiline was suggested. Drug-resistant tuberculosis is a major threat to TB control worldwide [2]. Multidrug-resistant tuberculosis (MDR-TB) is defined as tuberculosis caused by a strain resistant to isoniazid (INH) and rifampin (RMP) and extensively drug-resistant tuberculosis (XDR-TB) as multidrug-resistant with additional resistance to any fluoroquinolone and any second-line injectable drug (kanamycin, amikacin, or capreomycin) [3]. Health experts considered that the extensively drug-resistant tuberculosis outbreak that occurred in 2005–2006 in KwaZulu-Natal was underestimated [4]. Whether an emerging disease or an old health problem, XDR-TB is a global public health threat. Usually, few laboratories can perform the microbiological diagnosis in low-income countries, especially in Africa where tuberculosis is endemic.
K. N’guessan et al. / Médecine et maladies infectieuses 45 (2015) 303–305
Laboratory confirmation is essential to ensure a correct diagnosis and an appropriate treatment for most infectious diseases, and especially for the emergence of resistant M. tuberculosis strains. According to the 2014 World Health Organization report, approximately 9% of patients carrying MDR-TB presented with extensively drug-resistant tuberculosis [2]. The current strategy is based on rapid detection of mutations encoding resistance to rifampin and isoniazid. Consequently, it is not excluded that these 2 patients were initially infected by M. tuberculosis strains with additional resistance to any fluoroquinolone and at least 1 of the 3 following injectable drugs (capreomycin, kanamycin, and amikacin). A patient carrying XDR-TB, living in Côte d’Ivoire, was already diagnosed in France at the Percy Military hospital [5]. The 3 diagnosed XDR-TB cases revealed an inadequate management of tuberculosis [3]. The MGIT 960 automated liquid culture system is probably the most frequently used and best validated at this moment [6,7]. The BACTEC S.I.R.E. drug kit was used for first-line drugs susceptibility testing. A contamination of DST could be excluded since positive results were obtained in 12 days. Tubes containing second-line drugs were prepared by adding 100 l of each drug. The contamination of tubes prepared and inoculated could be excluded considering the results of automated MGIT-DST assays that were obtained after 10 days. The detection of clinical isolate growth in drug-containing tubes proved the phenotypic resistance to the drug considered, especially for second-line drugs [8]. Our results were confirmed by the Milan supranational laboratory, through sequencing of genes implicated in resistance (fluoroquinolones, aminoglycosides). The mutations detected in gyrA, rrs gene and in eis promoter region are implicated in phenotypic resistance [9,10]. Although, a new and appropriate regimen was initiated, the treatment failed and 1 of the 2 patients died. Given these results, identifying XDR-TB cases is very important for public health and is, in Côte d’Ivoire, a strong indicator for all healthcare providers fighting TB. Furthermore, the strategy used to fight TB must be pertinent and updated according to epidemiological data. Globally, the diagnosis has been improved and appropriate regimens must be accessible to decrease M. tuberculosis strain dissemination in the community. In this last case, direct observed treatment remains essential to fight tuberculosis. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
305
Authors contribution Pr. K. N’guessan wrote the article. Pr. A.S. Bakayoko managed MDR-TB cases and proofread the article. Dr. J.M Ahui-brou managed MDR-TB cases at the Yopougon CAT and reviewed the article and clinical data. Dr. A.O. Kouakou is a member of NTCP for MDR-TB cases. Dr. B. Coulibaly is the head of the Yopougon tuberculosis center. Mr. A. Guei is a biotechnologist and performed culture and DST. Pr. M. Dosso is the director of the Côte d’Ivoire Pasteur institute. Acknowledgements We thank Dr. Elisa Tagliani and Dr. Daniela Cirillo (SRL, Milan, Italy), Kekeletso Kao (Expand TB), and the PEPFAR Côte d’Ivoire team of for their contributions. References [1] Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. Atlanta: USDHHS, Centers for Disease Control; 1985. [2] World Health Organization. Global tuberculosis report 2014. Geneva 27, Switzerland: WHO/HTM/TB/2014.08; 2014. [3] Raviglione MC, Smith IM. XDR tuberculosis: implications for global public health. N Engl J Med 2007;356:656–9. [4] Singh JA, Upshur R, Padayatchi N. XDR-TB in South Africa: no time for denial or complacency. PLoS Med 2007;4:e50. [5] Bakayoko AS, Ahui BJM, Kone Z, Daix ATJ, Samake K, Domoua KMS, et al. Extensively drug resistant tuberculosis in Ivory Coast. Rev Pneumol Clin 2015 http://dx.doi.org/10.1016/j.pneumo.2014.12.009 [6] Rusch-Gerdes S, Pfyffer GE, Casal M, Chadwick M, Siddiqi S. Multicenter laboratory validation of the BACTEC MGIT 960 technique for testing susceptibilities of Mycobacterium tuberculosis to classical second-line drugs and newer antimicrobials. J Clin Microbiol 2006;44:688–92. [7] Ardito F, Posteraro B, Sanguinetti M, Zanetti S, Fadda G. Evaluation of BACTEC Mycobacteria Growth Indicator Tube (MGIT 960) automated system for drug susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 2001;39:4440–4. [8] Jakko VI, Sami S, Rina Z, Tridia VL, Miranda KA, Martin JB, et al. Comparative study on genotypic and phenotypic second-line drug resistance testing of Mycobacterium tuberculosis complex isolates. J Clin Microbiol 2010;48:2749–53. [9] Maruri F, Sterling TR, Kaiga AW, Blackman A, Van der Heijden YF, Mayer C, et al. A systematic review of gyrase mutations associated with fluoroquinolone-resistant Mycobacterium tuberculosis and a proposed gyrase numbering system. J Antimicrob Chemother 2012;67: 819–31. [10] Gikalo MB, Nosova EY, Krylova LY, Moroz AM. The role of eis mutations in the development of kanamycin resistance in Mycobacterium tuberculosis isolates from the Moscow region. J Antimicrob Chemother 2012;67:2107–9.