INT J TUBERC LUNG DIS 9(2):216–219 © 2005 IUATLD
SHORT COMMUNICATION
Second-line drug susceptibilities of Thai multidrug-resistant Mycobacterium tuberculosis isolates T. Prammananan, W. Arjratanakool, A. Chaiprasert, N. Tingtoy, M. Leechawengwong, N. Asawapokee, A. Leelarasamee, C. Dhiraputra Drug-Resistant Tuberculosis Research Fund, Siriraj Foundation, Faculty of Medicine, Siriraj Hospital, Bangkok, Thailand SUMMARY
The emergence of multidrug-resistant tuberculosis (MDRTB) is increasing and is exacerbated by the human immunodeficiency virus (HIV) epidemic. The standard short-course regimen used for the treatment of tuberculosis is likely to be ineffective against MDR-TB, leading to the need for second-line drugs. In such situations, drug susceptibility testing (DST) is necessary to select an appropriate treatment regimen. In this study, DST of 99
MDR-TB strains isolated in Thailand was performed using a drug-impregnated disc method. The results showed that 94.95% of the strains were susceptible to amikacin and kanamycin, 90.91% to ciprofloxacin and ofloxacin, 85.86% to para-aminosalicylic acid, and 78.79% to ethionamide. K E Y W O R D S : MDR-TB; second-line drugs; susceptibility
TUBERCULOSIS still constitutes a public health crisis, especially in the developing world. It has been exacerbated by the human immunodeficiency virus (HIV) epidemic and the ineffectiveness of case finding and treatment, resulting in the emergence of multidrugresistant tuberculosis (MDR-TB), defined as resistance to the most powerful anti-tuberculosis drugs, rifampicin (RMP) and isoniazid (INH). A staggering 1.9 million people around the globe die of tuberculosis each year; another 1.9 billion are infected with Mycobacterium tuberculosis and are at risk for active disease.1 It is estimated that between 1998 and 2030 there will be 225 million new cases of TB and 79 million deaths attributable to the disease.2 As the emergence of drug-resistant M. tuberculosis strains and even of MDR strains is increasingly reported from many countries,3,4 it is essential to perform drug susceptibility testing (DST) to prescribe an effective treatment regimen. Mycobacterial susceptibility testing is therefore important for appropriate patient management and should be done on initial isolates of M. tuberculosis from all patients and on clinically significant isolates of certain non-tuberculous mycobacteria (NTM). Although the first-line antituberculosis drugs, RMP, INH, ethambutol (EMB), pyrazinamide and streptomycin (SM) were discovered several decades ago, they are still used in standard short-course regimens for the treatment of tuberculo-
sis today. However, this regimen is likely to be ineffective for treating patients infected with MDR-TB, leading to the use of less effective, more toxic second-line drugs such as para-aminosalicylic acid (PAS), ethionamide (ETH), cycloserine, aminoglycosides and the fluoroquinolones for treatment of MDR-TB. In this report, DST of 99 MDR-TB strains isolated in the Molecular Mycology and Mycobacteriology Laboratory, Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Thailand, from March 2001 to September 2002, was performed against a single critical concentration of eight second-line drugs: amikacin (AMK), kanamycin (KM), azithromycin (AZM), clarithromycin (CM), ciprofloxacin (CFX), ofloxacin (OFX), ETH and PAS. All drugs were purchased from Becton Dickinson (Sparks, MD, USA). DST was carried out using the drug-impregnated disc method on Middlebrook 7H10 (Difco, Detroit, MI, USA) supplemented with 10% oleic acid-albumindextrose-catalase (OADC) (BBL, Becton Dickinson, USA) as recommended by the US Centers for Disease Control and Prevention (CDC).5 Briefly, the appropriate drug discs were dispensed aseptically into the centre of individual quadrants of sterile plastic dishes. Exactly 5.0 ml each of sterile, tempered (52C), complete M7H-10 medium was pipetted over the discs and the plates were left overnight at room temperature
Correspondence to: Dr Angkana Chaiprasert, Division of Mycology and Mycobacteriology, Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. Tel: (66) 024198256. Fax: (66) 024182094. e-mail:
[email protected] Article submitted 20 May 2003. Final version accepted 26 May 2004.
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Figure Antimicrobial susceptibility testing of MDR-TB against second-line drugs using the drugimpregnated disc method on Middlebrook 7H10 supplemented with 10% OADC. Resistance was reported when the colonies on drug containing quadrant 1% compared to the control quadrant. The MDR-TB strain in this Figure is resistant to azithromycin and clarithromycin. AMK amikacin; AZM azithromycin; CFX ciprofloxacin; CM clarithromycin; ETH ethionamide; KM kanamycin; OFX ofloxacin; PAS -amino-salicylic acid; MDR-TB multidrug resistant tuberculosis (resistance to at least isoniazid and rifampicin); OADC oleic acid-albumin-dextrosecatalase. The number indicates drug concentration in g of each disc.
to permit the drug to diffuse uniformly. The inoculum was prepared by suspending the M. tuberculosis cells in Middlebrook 7H9 with the turbidity adjusted to match that of MacFarland No. 1 standard with sterile distilled water. The cell suspension was diluted to 102 and 104 in sterile distilled water; these two dilutions were inoculated onto each quadrant and onto a quadrant containing drug-free medium which was used as a control. The plate was incubated at 37C until colonies appeared on the control quadrant (approximately 2–4 weeks). Resistance was reported when the colonies on the drug-containing quadrant appeared 1% compared to the drug-free control quadrant (Figure).
RESULTS AND DISCUSSION Among 99 MDR-TB strains, 22 were isolated from patients who had never been previously treated, 49 from previously treated patients, and 28 from pa-
Table
tients whose medical history was not available. The history of HIV status was not complete. From the results of 71 isolates from patients whose history was known, it was suggested that the MDR-TB rate among new cases was 30.9%, whereas it was 69.1% among previously treated cases. In addition, these MDR-TB isolates also showed resistance to either SM or EMB or both. Unexpectedly, the resistance rate among new cases to SM and EMB was 75% and 43.75%, respectively, whereas it was lower among previously treated cases (53.8% and 38.5% for SM and EMB, respectively). As shown in the Table, AMK and KM were the most effective drugs against MDR-TB from both new cases and previously treated cases; 94 of 99 MDR-TB strains (94.95%) were susceptible to these drugs, at a concentration of 6 g/ml in vitro. All resistant isolates were from previously treated patients. In two of five patients whose isolates revealed resistance to both aminoglycosides and fluoroquinolones, the strains showed sensitive phenotypes to either
Susceptibility of MDR-TB against second-line drugs Susceptible strains
Second-line drugs Amikacin Kanamycin Azithromycin Clarithromycin Ciprofloxacin Ofloxacin Ethionamide PAS
Concentration (g/ml)
New cases (n 22) n (%)
Previously treated cases (n 49) n (%)
6.0 6.0 3.0 3.0 1.0 1.0 5.0 2.0
22 (100) 22 (100) 0 0 22 (100) 22 (100) 17 (77.27) 20 (90.91)
44 (89.79) 44 (89.79) 1 (2.04) 5 (10.2) 43 (87.76) 43 (87.76) 36 (73.47) 42 (85.71)
History of treatment not available (n 28) n (%)
Total (n 99) n (%)
28 (100) 28 (100) 0 3 (10.7) 25 (89.28) 25 (89.28) 25 (89.28) 23 (82.14)
94 (94.95) 94 (94.95) 1 (1.01) 8 (8.08) 90 (90.91) 90 (90.91) 78 (78.79) 85 (85.86)
MDR-TB multidrug resistant tuberculosis (resistance to at least isoniazid and rifampin); PAS -amino salicylic acid.
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The International Journal of Tuberculosis and Lung Disease
ETH or PAS. AMK and KM are aminoglycoside antibiotics that bind to 16S rRNA as their target in a small ribosomal subunit and subsequently inhibit protein synthesis. A single point mutation at the drug binding site confers resistance to these drugs.6 Of 99 MDR-TB strains, 90 (90.91%) were sensitive to the fluoroquinolones CFX and OFX, at a concentration of 1 g/ml. This showed a 7% decrease from the 97.9% sensitivity to OFX performed during the last 4 years with 822 clinical isolates of M. tuberculosis in Bangkok, Thailand (data not shown). These drugs were also active against primary MDR-TB strains. Of nine patients infected with fluoroquinolone-resistant strains, three were newly diagnosed patients. This might result from the wide use of these drugs for treatment of many bacterial infections. Fluoroquinolones are broad-spectrum antibiotics that are widely available and have a favourable toxicity profile. They bind to DNA gyrase and inhibit bacterial replication. The resistant phenotype, resulting from mutations at gyrA, a gene encoding subunit A of the gyrase enzyme, predictably emerges when the drug is used alone.7,8 PAS, a drug used in the early chemotherapy era, and ETH, a derivative of isonicotinic acid, are less potent against MDR-TB than aminoglycosides and fluoroquinolones. Whereas 78.79% of MDR-TB isolates were sensitive to ETH, 85.86% were susceptible to PAS. In contrast, almost all MDR-TB isolates were resistant to AZM and CM as expected, as M. tuberculosis is naturally resistant to macrolide antibiotics. These results are in accordance with those reported by Rastogi et al., where aminoglycosides and fluoroquinolones were shown to be the most active drugs against MDR-TB.9 Typically used methods for DST of M. tuberculosis are based on the ability of the isolate to grow on agar or in broth containing a single critical concentration of one drug. The critical concentration of a drug represents the lowest concentration that inhibits wild strains but does not inhibit isolates from patients who are not responding clinically to therapy. In 1995, the National Committee for Clinical Laboratory Standards (NCCLS) proposed guidelines for mycobacterial susceptibility testing, document M24-T, which is a tentative standard that addressed only M. tuberculosis.10 The second edition of this tentative standard was recently published (NCCLS document M24-A), including revised guidelines for the testing of M. tuberculosis complex (MTBC) and proposed in addition recommendations for the testing of certain NTM and aerobic actinomycetes.11 In this document recommended drug concentrations for testing using the agar proportion method are given. Conventional DST with solid media such as Löwenstein-Jensen or Middlebrook 7H10 agar requires 3 or more weeks, and for some classical second-line drugs as well as newer antimicrobial agents, appropriate critical drug concentrations have not been fully established. For this reason,
a rapid method of susceptibility testing was developed to report the results of DST within 28 days of receipt of a specimen.12 The recommended rapid method of testing involved the use of commercial broth-based systems, such as the radiometric BACTEC 460TB system (Becton Dickinson), and the nonradiometric BACTEC MGIT 960 (Becton Dickinson) and ESP II (Trek Diagnostics Inc, Westlake, OH, USA) systems, which have been cleared by the US Food and Drug Administration (FDA). For drugs that have not been cleared by the FDA for testing with commercial systems, the standard agar proportion method should be used. Although the World Health Organization (WHO) recommended a multifaceted strategy known as DOTS, which includes standardised supervised short-course chemotherapy (SCC), it is likely to be ineffective against MDR-TB. Responding to this situation, the modified DOTS programme, termed DOTS-Plus, which requires DST to be performed on initial isolates, was established and evaluated for cost, effectiveness and feasibility by a working group of the Global Partnership to Stop TB. The evaluation of the results will be published elsewhere. Under DOTSPlus, DST will provide susceptibility results from which an individualised treatment regimen can be selected. It should be useful if DOTS-Plus is implemented in tuberculosis endemic countries with a high prevalence of MDR-TB. Acknowledgements This work is dedicated to Her 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 80th birthday. We would like to thank Dr Jane Hardy for her grammatical correction of the manuscript.
References 1 Dye C, Scheele S, Dolin P, Pathania V, Raviglione M C, for the WHO Global Surveillance and Monitoring Project. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. JAMA 1994; 282: 677–686. 2 Murray C J, Salomon J A. Modeling the impact of global tuberculosis strategies. Proc Natl Acad Sci USA 1998; 338: 13881–13886. 3 Pablos-Méndez A, Raviglione M C, Laszlo A, et al. Global surveillance for antituberculosis-drug resistance, 1994–1997. N Engl J Med 1998; 338: 1641–1649. 4 Bastian I, Rigouts L, Van Deun A, Portaels F. Directly observed treatment, short-course strategy and multidrug-resistant tuberculosis: are any modifications required? Bull World Health Organ 2000; 78: 238–251. 5 Kent P T, Kubica G P. Public health mycobacteriology. A guide for the level III laboratory. Atlanta, GA: US Department of Health and Human Services, Centers for Disease Control and Prevention, 1985. 6 Boettger E C. Resistance to drug targeting protein biosynthesis in mycobacteria. Trends Microbiol 1994; 2: 416–421. 7 Cambau E, Sougakoff W, Besson M, Truffot-Pernot C, Grosset J, Jarlier V. Selection of gyrA mutant of Mycobacterium tuber-
Second-line drug susceptibilities of MDR-TB
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10 National Committee for Clinical Laboratory Standards. Antimycobacterial susceptibility testing for Mycobacterium tuberculosis. Tentative standard M24-T. Wayne, PA: NCCLS, 1995. 11 National Committee for Clinical Laboratory Standards. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes; approved standard. NCCLS document M24-A.Wayne, PA: NCCLS, 2003. 12 Tenover F C, Crawford J T, Huebner R E, et al. The resurgence of tuberculosis: is your laboratory ready? J Clin Microbiol 1993; 31: 767–770.
RÉSUMÉ
L’émergence de tuberculose à germes multirésistants (TBMR) augmente et est accrue par l’épidémie du virus de l’immunodéficience humaine (VIH). Le régime standard de courte durée utilisé pour le traitement de la tuberculose est susceptible d’être inefficace à l’égard de TB-MR et entraîne la necessité de médicaments de seconde ligne. Dans ces conditions, des tests de sensibilité sont nécessaires pour sélectionner un régime de traitement appro-
prié. Dans cette étude en Thaïlande, nous avons pratiqué un test de sensibilité aux médicaments sur 99 souches TB-MR en utilisant une méthode d’imprégnation des disques par les médicaments. Il ressort que 94,95% des TB-MR étaient sensibles à l’amikacine et à la kanamycine, 90,91% à la ciprofloxacine et à l’ofloxacine, 85,86% à l’acide para-aminosalicylique et 78,79% à l’éthionamide. RESUMEN
La frecuencia de tuberculosis multirresistente a los medicamentos (MDR-TB) está aumentando y es exacerbada por la epidemia del virus de la inmunodeficiencia humana (VIH). El régimen estándar de corta duración utilizado para el tratamiento de la tuberculosis es susceptible de ser ineficaz contra la MDR-TB, lo que lleva a la utilización de medicamentos de segunda línea. En esta situación, las pruebas de sensibilidad son necesarias para seleccionar el régimen de tratamiento adecuado. En este
estudio se efectuaron pruebas de sensibilidad en 99 cepas de MDR-TB aisladas en Tailandia, utilizando el método de disco impregnado con medicamento. Los resultados mostraron que en un 94,95%, las cepas MDR-TB eran sensibles a amikacina y kanamicina, en un 90,91% eran sensibles a la ciprofloxacina y ofloxacina, en un 85,86% eran sensibles al acido para-aminosalicílico y en un 78,79% eran sensibles a la etionamida.
