1. Ximena Gonzalo and Francis Drobniewski, Department of Infectious Diseases, Imperial College, The Commonwealth Building, The Hammersmith Hospital, Du Cane Road, London W12 0NN.
Thionamide Testing: issues regarding quality of results and its implications for the diagnostic laboratory
Introduction and Purpose
Thionamides are second line anti TB agents widely used in the management of MDR and XDRTB.1 They are particularly valuable in CNS infection as they cross the blood-brain barrier.2 Resistance emerges quickly and cross resistance between ethionamide (ETH) and prothionamide (PTH) is believed to be complete.3 However, when testing in vitro using current WHO recommeded critical concentrations,4 this is not always observed. As part of international efforts to standardise second line testing, an assessment of accuracy of current methods was performed.
Methods
Second line susceptibilities were set up in the MGIT 960® (BD Diagnostics, Sparks, MD) at the National Mycobacterium Reference Laboratory, London.5 Ethionamide was purchased from Sigma-Aldrich. Prothionamide was kindly provided by Fatol. All those that showed discrepant results were repeated. A structured questionnaire was distributed amongst Tuberculosis laboratories in Europe to determine drug concentrations and methodologies used.
Results
Between 01/07/2012 and 31/12/2013, 308 second and third line susceptibility tests were performed as part of the routine workflow. 43 (13.96%) had discrepant results for ETH and PTH and were repeated following current standard operating procedures. Reproducibility as a pair was 37%, ETH 56% and PTH 81%. The most frequent pattern was ETH resistant/PTH susceptible.
12 laboratories answered the questionnaire. 8 laboratories tested ETH; 2 tested PTH and 2 tested both (Table 1).
Solvents used to dissolve the drugs varied as well. DMSO, ethylene-glycol and methanol were used.
Conclusions
PTH results are more reproducible than those of ETH. This may be partially explained by the well-known solubility issues related to these compounds that are more apparent for ETH since the stock solutions for the latter are more concentrated.
Testing in liquid medium had already been reported to have less sensitivity than solid media.6 Ethionamide MGIT testing does not correlate very well with the proportion method (PM) in LJ which suggests that the current critical concentrations (CC) are not appropriate.7 When compared with results using 7H10 media , discrepancies were also frequent.8,9
The current critical concentration is very close to the ecological cut-off. It has been suggested that in those circumstances, the introduction of the intermediate category may compensate for methodological variations.9 However, it is always difficult to interpret such a category in the clinical setting.
Even though these two drugs are reported to have 100% cross resistance, it is not unusual to see one result as (S) while the other is resistant (R). Reproducibility of each drug is problematic.
Laboratories only using MGIT and/or testing only ETH may be overcalling drug resistance.
Some of these errors may be overcome using molecular (inhA).
Significant differences in methodologies were found accross the centres. This may affect the final real concentration of drug in the stock solutions compromising the accuracy of the assays. Quality control programmes are needed.
References
1. Anon. Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis: 2011 Update - PubMed - NCBI. Available at: Accessed November 23, 2015.
2. Donald PR. Cerebrospinal fluid concentrations of antituberculosis agents in adults and children. Tuberculosis (Edinb) 2010; 90: 279–92. Available at: Accessed November 23, 2015.
3. Anon. Prothionamide. Tuberculosis (Edinb) 2008; 88: 139–40. Available at: Accessed November 23, 2015.
4. World Health Organization. Guidelines for surveillance of drug resistance in tuberculosis.
5. Rüsch-Gerdes S, Pfyffer GE, Casal M, Chadwick M, Siddiqi S, Ru 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.
6. Barreto AMW, Araújo JBM, de Melo Medeiros RF, de Souza Caldas PC. Evaluation of indirect susceptibility testing of Mycobacterium tuberculosis to the first- and second-line, and alternative drugs by the newer MB/BacT system. Mem Inst Oswaldo Cruz 2003; 98: 827–30. Available at:
7. Zhao L, Xia Q, Lin N, et al. Evaluation of BACTEC MGIT 960 system for the second-line drugs susceptibility testing of Mycobacterium tuberculosis in China. J Microbiol Methods 2012; 91: 212–4. Available at: Accessed March 27, 2014.
8. Steadham JE, Stall SK, Simmank JL. Use of the BACTEC system for drug susceptibility testing of Mycobacterium tuberculosis, M. kansasii, and M. avium complex. Diagn Microbiol Infect Dis 1985; 3: 33–40. Available at:
9. Schön T, Juréen P, Chryssanthou E, et al. Wild-type distributions of seven oral second-line drugs against Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2011; 15: 502–9. Available at: Accessed March 27, 2014.