Effect of temperature on storage of ethionamide during susceptibility testing

Journal of Microbiology andLakshmi 128 Infectious R, et Diseases al. Stability / of ethionamide during DST procedures 2013; 3 (3): 128-132 JMID doi: 10.5799/ahinjs.02.2013.03.0095 ORIGINAL ARTICLE Effect of temperature on storage of ethionamide during susceptibility testing Rajagopalan Lakshmi, Ranjani Ramachandran, Syam Sundar A, Baskaran M, Anandan M, Thiyagarajan V, Vanaja Kumar Department of Bacteriology, National Institute for Research in Tuberculosis, Chetpet, Chennai 600 031, India ABSTRACT Objective: Ethionamide being thermolabile in nature, effect of temperature on the drug and its stability in liquid and solid media during susceptibility testing procedures was assessed to understand the inconsistency in DST formats. Methods: Working solution of ethionamide and Lowenstein- Jensen (LJ) media incorporated with ethionamide were preincubated at 4°C and 37°C prior to DST methods was incubated till 4 weeks at different temperatures and utilized for DST in MGIT 960. Results: Degradation of ethionamide working solution was observed at 37°C after 3 weeks of incubation. Ethionamide incorporated LJ media can be stored without compromise on susceptibility up to 5 weeks. But, a week of prior incubation at 37°C has deleterious effect on the DST profile. Ethionamide was found to degrade at 37°C after different time points when stored as solution or as LJ media. Conclusion: Use of DST formats that provide results within 2 weeks can be recommended for ethionamide susceptibility testing. J Microbiol Infect Dis 2013; 3(3): 128-132 Key words: Degradation; Ethionamide; LJ medium; MGIT 960; Mycobacterium tuberculosis. Ethionamide depolama sıcaklığının duyarlılık testine etkisi ÖZET Amaç: Ethionamide ısıya duyarlı yapısı nedeniyle, ilaç duyarlılık testinde (DST) ısının etkisini ve sıvı veya katı ortamdaki dayanıklılık durumunun araştırılması amaçlanmıştır. Yöntemler: Ethionamide çalışma solüsyonu ve Lowenstein-Jensen (LJ) besiyeri hazırlanarak 4°C ve 37°C bekletildi ve ardından DST yöntemiyle 4 haftaya kadar MGIT 960 besiyeri ile test edildi. Bulgular: Ethionamide çalışma solusyonunda 37°C’de 3 haftalık inkübasyonun ardından bozunma olduğu gözlenmiştir. Ethionamide LJ ortamında beş haftaya kadar etkinliğinde bozulma olmadan saklanabilir. Fakat 37°C de bir hafta daha bekletilmesi ilaç duyarlılık profiline zararlı etkili olabilir. Ethionamide 37°C bekletilmesiyle farklı zaman aralıklarında sıvı ya da LJ ortamında bekletilmesiyle etkinliğinde değişmeler olduğu saptandı. Sonuç: DST ortamında ethionamide için iki haftaya kadar olan testlerin değerlendirilmesi önerilir. Anahtar kelimeler: Bozunma, Etionamide, LJ besiyeri, MGIT 960, Mycobacterium tuberculosis INTRODUCTION Ethionamide (ETH) though used in the treatment of multi drug resistant tuberculosis (MDR-TB) patients in the country for more than a decade; ambiguity in laboratory susceptibility results stills remains.1 Drug susceptibility testing (DST) for ETH pose a challenge as there arises inconsistency between testing formats for reasons unclear; mode of action, interpretation of resistance and potency of drug in the medium.2 Potency of the drug is a measure of purity of the drug in a formulation or in the complex used to stabilize the drug.3 On the other hand; potency also indicates effectiveness of the drug during testing procedures. If potency of the drug is maintained till completion of the test, the interpretation is considered valid and reproducible.4 Effort is underway to understand probable reasons for discrepancy within phenotypic testing methods. Several studies have dealt upon degradation of anti-tuberculosis drugs.5-7 Several anti-tuberculosis drugs except ka- Correspondence: Vanaja Kumar, Department of Bacteriology, National Institute for Research in Tuberculosis, Chetput, Chennai 600 031, India. Email: Received: 07.03.2013, Accepted: 11.07.2013 and Infectious Diseases 2013, All rights Vol reserved J Microbiol Infect DisCopyright © Journal of Microbiology www.jmidonline.org 3, No 3, September 2013 Lakshmi R, et al. Stability of ethionamide during DST procedures namycin and capreomycin was found to deteriorate even as early as 1 week and ethionamide after 2 weeks of incubation at 37°C.8 Degradation of drugs during combined therapy was reported especially in rifampicin when used with isoniazid. Isoniazid acts as catalyst triggering the degradation of rifampicin in acidic medium.5 Recovery of drug was also measured when mixed with food and juices in order to assess the effect of food components in maintaining stability of the drug.7 Loss in potency was indicated in the data sheet of certain drugs such as streptomycin dihydro-sulfate wherein around 50% loss is observed with varying pH and temperature. But the drug as solution is stable when stored at -20°C for more than a year.9 Therefore, effect of temperature on storage of ethionamide in solution and in combination with egg based solid Lowenstein- Jensen (LJ) media and was undertaken to determine a probable cause for discrepancy between susceptibility testing formats. METHODS Effect of temperature on ethionamide used in liquid culture systems (MGIT960) Stock concentration of 10,000µg/ml of ethionamide drug was prepared by dissolving 10mg of ETH powder (Sigma, USA) in 10ml of triethylene glycol (Merck). Working concentrations (2.5, 5.0, 7.5 and 10µg/ ml) were prepared from stock by diluting with sterile distilled water. One microliter aliquots of each concentration were stored at -80°C, 4°C and 37°C for a maximum of 4 weeks and tested once weekly. Drug susceptibility testing was performed weekly once in MGIT 960 system following manufacturer’s instructions for 5 strains inclusive of H37Rv, 3 strains sensitive and one strain resistant to ETH.10 Susceptibility results were recorded once the instrument provided a printed report. To minimize clonal variation, strains used in the experiment were sub cultured from primary MGIT 960 positive tube into fresh tube and used weekly. Effect of temperature on ethionamide drug used in solid LJ media Drug susceptibility testing of Mycobacterium tuberculosis isolates for ETH was performed according to standard procedure.11-12 Strains used in MGIT 960 were used in solid LJ medium. Strains were sub-cultured from original slope and one slope of each strain was tested every week for DST. Drug media at the specified concentrations of ETH were prepared in a single lot to restrict batch variation. Prepared media was pre-incubated at two different temperatures viz. 4°C and 37°C for a minimum of 1 J Microbiol Infect Dis 129 week to a maximum of 6 weeks before performing DST procedures. Susceptibility testing was done once weekly by proportion sensitivity testing (PST) and conventional minimum inhibitory concentration (MIC) methods. Conventional MIC method was performed using 4mg moist weight of culture, whereas for PST method, 1 mg of culture with three serial decreasing 1 in 10 fold dilutions were used. Briefly, one- third loopful of (...truncated)