INT J TUBERC LUNG DIS 18(8):961–963 Q 2014 The Union http://dx.doi.org/10.5588/ijtld.13.0197
Second-line drug susceptibilities of multidrug-resistant tuberculosis strains isolated in Thailand: an update A. Chaiprasert,*† S. Srimuang,† N. Tingtoy,* N. Makhao,† P. Sirirudeeporn,† N. Tomnongdee,† O. Theankeaw,† S. Charoensook,† M. Leechawengwongs,†‡ T. Prammananan†§ *Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, †Drug-Resistant Tuberculosis Research Fund, Siriraj Foundation, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, ‡ Vichaiyut Hospital, Bangkok, §TB Research Laboratory, National Centre for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, Thailand SUMMARY
The increasing incidence of multidrug-resistant tuberculosis (MDR-TB) and the emergence of extensively drug-resistant tuberculosis (XDR-TB) globally hamper the successful treatment and effective control of TB. Information on second-line drug susceptibility, which is of utmost importance for patient care, is still limited. This study demonstrates the susceptibilities of 1447 strains of MDR-TB, including 58 XDR-TB strains, isolated from Siriraj Hospital, Bangkok, Thailand, to
aminoglycosides, fluoroquinolones, ethionamide (ETH), para-aminosalicylic acid (PAS) and linezolid. Results revealed that 93–94% of the MDR-TB strains were susceptible to aminoglycosides, 85–98% to fluoroquinolones, 78% to ETH, 85% to PAS and 99% to linezolid. K E Y W O R D S : tuberculosis; resistance; anti-tuberculosis drug
THE EMERGENCE of multidrug-resistant tuberculosis (MDR-TB) hinders the successful treatment and effective control of TB. Recent surveillance of antituberculosis drug resistance from 80 countries and eight territories in 2007–2010 revealed that MDR-TB was found in 0–28.9% and 0–65.1% of new and previously treated cases, respectively. In three former Soviet Union countries and South Africa, more than 10% of MDR-TB cases had extensively drug-resistant (XDR) TB. Globally, MDR-TB was observed in 3.4% of all new TB cases and 19.8% of previously treated TB cases.1 Second-line anti-tuberculosis drug susceptibility testing (DST) is not usually performed in routine mycobacteriological laboratories. In 2010, only 16% of the TB patients estimated to have MDRTB were diagnosed and given treatment.2 In the future, as more and more patients with MDR-TB are diagnosed and started on treatment using second-line drugs, collection and analysis of data on second-line resistance will be of utmost importance for optimal patient care. This study surveyed the susceptibility of MDR- and XDR-TB strains against second-line anti-tuberculosis drugs. A total of 26 140 sputum samples were sent from 288 hospitals in 46 provinces of Thailand to the Drug-Resistant Tuberculosis Research Laboratory,
Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand, between 2003 and 2011. MDR- and XDR-TB strains isolated from these samples were included in the study and second-line DST was conducted. The study was approved by the Siriraj Ethics Committee, Mahidol University, Bangkok, Thailand (certificate of approval no. Si 208/2005). DST was carried out using the disc elution method on Middlebrook 7H10 (Difco, Detroit, MI, USA) supplemented with 10% oleic acid-albumin dextrosecatalase (OADC) (BBL, BD, Sparks, MD, USA), as recommended by the US Centers for Disease Control and Prevention (CDC; Atlanta, GA, USA).3 Briefly, the appropriate drug discs purchased from BBL (Difco) were dispensed aseptically into the centre of individual quadrants of sterile plastic dishes; 5.0 ml each of sterile tempered (528C), complete M7H-10 medium was pipetted over the discs and the plates were left overnight at room temperature to permit the drug to diffuse uniformly. The inoculum was prepared by suspending the M. tuberculosis cells in Middlebrook 7H9; the turbidity was adjusted to match that of MacFarland No. 1 standard. The cell suspension was diluted to 102 and 104 in sterile distilled water; these two dilutions were inoculated
Correspondence to: Therdsak Prammananan, TB Research Laboratory, National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathum Thani 12120, Thailand. Tel: (þ66) 2 419 8256. Fax: (þ66) 2 418 2094. email:
[email protected] Article submitted 10 March 2013. Final version accepted 17 March 2014.
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Table 1 Resistance of multidrug-resistant strains to second-line drugs Resistant strains, n Second-line drug Amikacin (n ¼ 1271) Kanamycin (n ¼ 1272) Ofloxacin (n ¼ 1271) Levofloxacin (n ¼ 1128) Moxifloxacin (n ¼ 1148) Gatifloxacin (n ¼ 1127) Linezolid (n ¼ 1129) Ethionamide (n ¼ 1255) PAS (n ¼ 1267)
Concentration mg/l 6.0 6.0 2.0 2.0 2.0 2.0 6.0 5.0 2.0
New cases n (%) 17 18 38 17 13 3 1 78 46
Previously treated cases n (%)
(4.6) (4.8) (10.2) (5.0) (3.8) (0.9) (0.3) (21.0) (12.4)
25 28 62 29 23 7 5 88 61
Treatment history not available n (%)
(6.2) (6.9) (15.4) (8.3) (6.4) (2.0) (1.4) (22.2) (15.0)
34 (6.9) 36 (7.3) 91(18.3) 51 (11.6) 41 (9.2) 15 (3.4) 2 (0.5) 106 (21.7) 82 (16.7)
Total n (%) 76 82 191 97 77 25 8 272 189
(6.0) (6.4) (15.0) (8.6) (6.7) (2.2) (0.7) (21.7) (14.9)
PAS ¼ para-aminosalicylic acid.
onto each quadrant and onto a quadrant containing drug-free medium, which was used as a control. The plate was incubated at 378C until colonies appeared on the control quadrant (approximately 2–4 weeks). Resistance was reported when the colonies on the drug-containing quadrant were 71% compared to the drug-free control quadrant. For quality control, M. tuberculosis H37Rv American Type Culture Collection 27294 was used as control. This strain was used each time a DST batch was set up. If any resistance was observed in the control strain, the results in that batch were not interpreted.
RESULTS AND DISCUSSION Of the 26 140 smear-positive sputum samples sent to our laboratory during 2003–2011, 16 533 strains were isolated and identified as M. tuberculosis. Of these, 1447 (8.8%) strains were classified as MDRTB and 58 (4%) as XDR-TB. The patients’ treatment history was known (n ¼ 13 060); MDR-TB strains were detected in respectively 3.8% and 26.3% of new cases and previously treated cases. The results of the second-line DST are shown in Table 1. The MDR-TB strains among both new and previously treated cases tested showed the least resistance to linezolid; 0.3–1.4% of MDR-TB strains were resistant to this drug. The majority of the MDRTB strains were resistant to ethionamide (ETH), whereas they were less resistant to aminoglycosides, such as amikacin (AMK) and kanamycin (KM), and fluoroquinolones (except for ofloxacin [OFX]). Among the fluoroquinolones tested, most MDR-TB strains were more resistant to OFX than the others, at approximately 15% resistance. Susceptibility of XDR-TB against second-line drugs is shown in Table 2. The tested strains showed the least resistance to linezolid, at 14%. Compared to previous reports in 20054 and 2011,5 no significant difference in drug resistance trends was observed with regard to resistance to any of the drugs except OFX. Resistance to OFX increased slightly, from 9% to 15%, over 10 years. This is within the
range of fluoroquinolone resistance (0–38%) reported by the World Health Organization (WHO),2 and similar to that (15.2% OFX and levofloxacin resistance) reported from Taiwan,6 but higher than reported from South Africa7 and India8 (4.8% and 9.0% OFX resistance, respectively). This might be due to the fact that OFX is a broad-spectrum antibiotic that has been used widely, for many years, for the treatment of bacterial infections. Among aminoglycosides, resistance to AMK and KM was found in 5.9% and 6.4%, respectively, compared with 2.7% for KM reported from South Africa.7 Moxifloxacin (MFX), one of the key components of the new TB drug regimen, showed approximately 7% resistance. This suggests that DST should be conducted for MFX to determine the resistance background of the bacteria in each setting before implementing the new anti-tuberculosis regimen, particularly MDR-TB. Due to the heterogeneity in patterns, these results cannot be used to guide the development of standardised regimens. These remain important, however, for guiding clinicians in developing an initial empirical regimen for previously treated MDR-TB patiens pending DST results. Acknowledgements This work is dedicated to Her Late Royal Highness Princess Galyani Vadhana Krom Luang Naradhiwas Rajanagarindra, Patron of the Drug-Resistant Tuberculosis Research Fund, Siriraj Foundation, on the occasion of HRH’s 89th birthday. The authors thank I Duangmanee for her assistance with the database. The study was supported by the Drug-Resistant Tuberculosis Research Fund, Mahidol University, Bangkok, Thailand.
Table 2 Resistance pattern of 58 extensively drug-resistant strains Drug Ethionamide Para-aminosalicylic acid Levofloxacin Moxifloxacin Gatifloxacin Linezolid
Resistant strains n (%) 20 47 43 35 18 8
(34.5) (81.0) (74.1) (60.3) (31.0) (13.8)
SLD susceptibilities of MDR-TB strains
Conflict of interest: none declared.
References 1 Zignol M, van Gemert W, Falzon D, et al. Surveillance of antituberculosis drug resistance in the world: an updated analysis, 2007–2010. Bull World Health Organ 2012; 90: 111–119D. 2 World Health Organization. Global tuberculosis control. WHO report 2011. WHO/HTM/TB/2011.16. Geneva, Switzerland: WHO, 2011. 3 Kent P T, Kubica G P. Public health microbiology, a guide for the level III laboratory. Atlanta, GA, USA: Department of Health and Human Services, Centers for Disease Control and Prevention, 1985. 4 Prammananan T, Arjratanakool W, Chaiprasert A, et al. Secondline drug susceptibilities of Thai multidrug-resistant Mycobacterium tuberculosis isolates. Int J Tuberc Lung Dis 2005; 9: 216– 219.
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5 Prammananan T, Chaiprasert A, Leechawengwongs M. 8-years experience of fluoroquinolone susceptibility testing of multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital, Thailand. Int J Antimicrob Agents 2011; 37: 84–85. 6 Huang T S, Kunin C M, Shin-Jung Lee S, Chen Y S, Tu H Z, Liu Y C. Trends in fluoroquinolone resistance of Mycobacterium tuberculosis complex in a Taiwanese medical centre: 1995–2003. J Antimicrob Chemother 2005; 56: 1508–1562. 7 Said H M, Kock M M, Ismail N A, et al. Molecular characterization and second-line antituberculosis drug resistance patterns of multidrug-resistant Mycobacterium tuberculosis isolates from the northern region of South Africa. J Clin Microbiol 2012; 50: 2857–2862. 8 Sherma S K, George N, Kadhiravan T, Saha P K, Mishra H K, Hanif M. Prevalence of extensively drug-resistant tuberculosis among patients with multidrug-resistant tuberculosis: a retrospective hospital-based study. Indian J Med Res 2009; 130: 392– 395.
SLD susceptibilities of MDR-TB strains
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RESUME
L’incidence croissante de la tuberculose multir´esistante (TB-MDR) et l’´emergence de tuberculose ultrar´esistante (TB-XDR) entrave dans le monde entier la r´eussite du traitement et de la lutte contre la TB. Les informations relatives a` la sensibilit´e aux m´edicaments de deuxi`eme intention sont tr`es importantes en mati`ere de prise en charge du patient, mais restent limit´ees. Cette e´ tude a mis en e´ vidence la pharmaco-sensibilit´e de 1447 souches
de TB-MDR, dont 58 souches de TB-XDR, isol´ees au Siriraj Hospital, en Tha¨ılande, aux aminosides, aux fluoroquinolones, a` l’´ethionamide (ETH), a` l’acide paraaminosalicylique (PAS) et au linezolide. Les r´esultats ont montr´e que 93% a` 94% des souches de TB-MDR e´ taient sensibles aux aminosides, 85% a` 98% aux fluoroquinolones, 78% a` l’ETH, 85% au PAS et 99% au linezolide. RESUMEN
La progresi on ´ de la incidencia de tuberculosis multidrogorresistente (TB-MDR) y la aparicion ´ de tuberculosis extremadamente drogorresistente (TBXDR) obstaculizan a escala mundial la eficacia del tratamiento y el control de la TB. Pese a la importancia crucial de la informacion ´ sobre la sensibilidad a los medicamentos de segunda l´ınea en el tratamiento de los pacientes, su utilizacion ´ es limitada. En el presente estudio se evaluo´ la sensibilidad de 1447 cepas de
Mycobacterium tuberculosis MDR, entre ellos 58 cepas TB-XDR, aisladas en el hospital Siriraj en Tailandia, a los aminoglucosidos, ´ las fluoroquinolonas, etionamida (ETH), el a´cido p-aminosalic´ılico (PAS) y a linezolid. Los resultados revelaron que de 93% a 94% de las cepas TB-MDR eran sensibles a los aminoglucosidos, ´ de 85% a 98% a las fluoroquinolonas, 78% a ETH, 85% a PAS y 99% a linezolid.
