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COMMENTARY
Year : 2015  |  Volume : 142  |  Issue : 5  |  Page : 512-514

Dilemmas with ethionamide susceptibility testing of Mycobacterium tuberculosis: A microbiologist & physician's nightmare


1 Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110 007, India
2 Department of Respiratory Medicine, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110 007, India

Date of Web Publication9-Dec-2015

Correspondence Address:
Mandira Varma-Basil
Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110 007
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0971-5916.171272

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How to cite this article:
Varma-Basil M, Prasad R. Dilemmas with ethionamide susceptibility testing of Mycobacterium tuberculosis: A microbiologist & physician's nightmare. Indian J Med Res 2015;142:512-4

How to cite this URL:
Varma-Basil M, Prasad R. Dilemmas with ethionamide susceptibility testing of Mycobacterium tuberculosis: A microbiologist & physician's nightmare. Indian J Med Res [serial online] 2015 [cited 2018 Oct 23];142:512-4. Available from: http://www.ijmr.org.in/text.asp?2015/142/5/512/171272

Multidrug resistant tuberculosis (MDR-TB) has been a topic of growing importance in the last decade. Emergence of MDR-TB has complicated infections caused by human immunodeficiency virus (HIV) and the re-emergence of tuberculosis in the West. MDR-TB is a major therapeutic challenge as the organism is resistant to two key anti-tuberculosis drugs, isoniazid and rifampicin. Programmatic management of drug resistant tuberculosis using second line therapy has been standardized through the development of international guidelines [1] . In India, the Revised National Tuberculosis Control Programme (RNTCP) advocates second line regimen comprising of six drugs - kanamycin, levofloxacin, ethionamide, pyrazinamide, ethambutol and cycloserine for 6-9 months of intensive phase therapy and levofloxacin, ethionamide, ethambutol and cycloserine for the next 18 months of the continuation phase [2] . Second line therapy is more complicated than the first line therapy as it has reduced potency, severe side effects and reduced efficacy. Hence, RNTCP advocates that poor response to treatment should prompt an examination of the programmatic, clinical or microbiological causes [2] .

The lack of rapid, accurate and reliable drug susceptibility testing of the second line anti-tuberculosis drugs makes it difficult to examine the microbiological causes of poor response. In vitro results of drug susceptibility testing to the second line drugs also show poor clinical correlation leading the physicians to rely more on their clinical acumen than factual data. Empirical treatment is advocated, not taking into account the risk of medication to which the Mycobacterium tuberculosis isolate may be resistant. Pharmacokinetic variability whereby poor circulating drug concentrations may impair killing of isolates with borderline susceptibility is also not considered [3] . This may further amplify resistance. For a drug like ethionamide, where drug susceptibility pattern is difficult to determine and the therapy is empirical, development of resistance may be a serious cause of concern. Lakshmi et al[4] in this issue have thus taken up a crucial investigation in the wake of emergence of MDR-TB and extensively drug resistant tuberculosis (XDR-TB).

Testing for drug susceptibility to ethionamide, a crucial part of second line anti-tuberculosis therapy, is problematic as the difference characterizing drug resistance or susceptibility is small. Moreover, the minimum inhibitory concentration (MIC) is very near to the therapeutic index as commented by Lakshmi et al, in their article in this issue [4] . In addition, the drug is thermolabile, which makes drug susceptibility testing more difficult [5] . Due to these issues, there is poor clinical and laboratory correlation [5] .

Inspite of the importance of ethionamide in the treatment of MDR-TB, there are only a few reports on the drug susceptibility testing of this agent. The gold standard of drug susceptibility testing against anti-tuberculosis agents is the proportion method that uses a single critical concentration of drugs to determine resistance or susceptibility. Criteria for measuring resistance such as critical concentrations and critical proportions of drugs affect the treatment predictive value of the drug susceptibility assay. The World Health Organization (WHO) has defined critical concentrations for various second line drugs taking into account the in vitro criteria for resistance using representative clinical samples and recommended a concentration of 40 µg/ml for proportion method of drug susceptibility testing of ethionamide [6],[7] . Lakshmi et al[4] also used the critical concentration of 40 µg/ml to perform the drug susceptibility assay by proportion method. However, the thermolabile nature of ethionamide may play a role in reducing the potency of the drug during inspissation of the drug containing medium. Deterioration of the drug during prolonged incubation period required for M. tuberculosis can also be crucial. Technical errors, as mentioned by Lakshmi et al[4] like inoculum preparation, incubation and interpretation can add to erroneous results and should be minimized.

Various investigators have standardized the critical concentration of ethionamide to be used in assays other than the proportion method, such as MIC and MGIT960. Kim et al[8] used a critical concentration of 5 µg/ml for MGIT 960. Mpagama et al[3] used 5 µg/ml of ethionamide as the critical concentration to segregate borderline susceptible from resistant through MIC on MycoTB sensititer plates (Trek Diagnostics, Cleveland, Ohio, USA). Lakshmi et al[4] recommended a much higher concentration of 80 µg/ml for MIC on Lowenstein-Jensen (L-J) medium. The sensitivity of the MIC improved using 80 µg/ml as the cut-off. Though the MIC method has been observed to be more efficient than the proportion method [5] , it has not yet been recommended to drive therapeutic decisions. The need of the hour is to generate more data on ethionamide susceptibility profile of a specific geographical region, since it has been reported that geographical variation in strains also leads to a shift in the MIC value [9] . This would entail re-calibration of MIC values for each geographical region.

In future, molecular tests may help in overcoming the problems encountered during testing for ethionamide susceptibility. Structural analogues, ethionamide and isoniazid share the same molecular target, the NADH-dependent enoyl-acyl carrier protein reductase InhA, which is involved in the synthesis of mycolic acids. Hence, cross-resistance to isoniazid and ethionamide may be observed in clinical isolates. Ethionamide acts by inhibiting mycobacterial cell wall mycolic acid synthesis and requires activation by a prodrug activator, NADPH-specific flavin adenosine dinucleotide containing mono-oxygenase encoded by ethA [10] . The expression of EthA is negatively regulated by EthR, a regulator that interacts directly with the ethA promoter region. Ethionamide resistance has been shown to be associated with mutations at ethA and ethR [10],[11] .It has been reported that the presence of a mutation in the inhA regulatory region together with a mutation in the inhA coding region is associated with high-level resistance to ethionamide among the MDR-TB isolates [12] . In a study at Mumbai, Vadwai et al[13] studied 69 phenotypically determined ethionamide resistant isolates and found that inhA promoter mutation was associated with ethionamide resistance in 21 isolates. They concluded that inhA mutation could be considered as a marker for ethionamide resistance in India. However, since 69.5 per cent of the ethionamide resistant isolates did not have inhA promoter mutations, alternative mechanisms could not be ruled out [13] . Identifying the molecular basis of ethionamide resistance could be an important focus for future studies. Further studies are needed to address the basis for mechanisms of ethionamide susceptibility.

The future holds a lot of promises with newer molecular assays. Considering the problems faced due to phenotypic drug susceptibility profile, future assays for drug susceptibility may include a combination of molecular and conventional assays for second line drug susceptibility testing. What is also needed is better microbiological-clinical correlation for drug susceptibility profiles since there is a vast difference between in vitro and in vivo results. Bigger cohorts correlating the drug susceptibility profile with patient's clinical outcome are also needed to retard the emergence of extensively drug resistant tuberculosis.

 
   References Top

1.
World Health Organization. Guidelines for the programmatic management of drug-resistant tuberculosis-2011 update. Geneva, Switzerland: WHO; 2011. Available from: http://whqlibdoc.who.int/publications/2011/9789241501583_eng.pdf, accessed on September 16, 2014.  Back to cited text no. 1
    
2.
Central TB Division (CTD). Guidelines on Programmatic management of drug resistant TB (PMDT) in India. New Delhi: Directorate General of Health Services; 2012. Available from: http://www.tbcindia.nic.in/pdfs/Guidelines%20for%20PMDT%20in%20India%20-%20May%202012.pdf, accessed on September 16, 2014.   Back to cited text no. 2
    
3.
Mpagama SG, Houpt ER, Stroup S, Kumburu H, Gratz J, Kibiki GS, et al. Application of quantitative second-line drug susceptibility testing at a multidrug-resistant tuberculosis hospital in Tanzania. BMC Infect Dis 2013; 14 : 432.   Back to cited text no. 3
    
4.
Lakshmi R, Ramachandran R, r0 avi Kumar D, Shyam Sundar A, Radhika G, Rahman F, et al. Revisiting the susceptibility testing of Mycobacterium tuberculosis to ethionamide in solid culture medium. Indian J Med Res 2015; 142 : 538-42.  Back to cited text no. 4
    
5.
Mitchison DA. Drug resistance in tuberculosis. Eur r0 espir j 2005; 25 : 376-9.   Back to cited text no. 5
    
6.
Parsons LM, Somoskövi A, Gutierrez C, Lee E, Paramasivan CN, Abimiku A, et al. Laboratory diagnosis of tuberculosis in resource-poor countries: challenges and opportunities. Clin Microbiol Rev 2011; 24 : 314-50.  Back to cited text no. 6
    
7.
Kam KM, Sloutsky A, Yip CW, Bulled N, Seung KJ, Zignol M, et al. Determination of critical concentrations of second-line anti-tuberculosis drugs with clinical and microbiological relevance. Int J Tuberc Lung Dis 2010; 14 : 282-8.  Back to cited text no. 7
    
8.
Kim H, Seo M, Park YK, Yoo JI, Lee YS, Chung GT, et al. Evaluation of MGIT 960 System for the Second-line Drugs Susceptibility Testing of Mycobacterium tuberculosis. Tuberc Res Treat 2013; 2013 : 108401.  Back to cited text no. 8
    
9.
Lefford MJ, Mitchison DA. Comparison of methods for testing the sensitivity of Mycobacterium tuberculosis to ethionamide. Tubercle 1966; 47 : 250-61.  Back to cited text no. 9
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10.
Baulard AR, Betts JC, Engohang-Ndong J, Quan S, McAdam RA, Brennan PJ, et al. Activation of the pro-drug ethionamide is regulated in mycobacteria. J Biol Chem 2000; 275 : 28326-31.  Back to cited text no. 10
    
11.
DeBarber AE, Mdluli K, Bosman M, Bekker LG, Barry CE 3 rd . Ethionamide activation and sensitivity in multidrug-resistant Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2000; 97 : 9677-82.  Back to cited text no. 11
    
12.
Machado D, Perdigáo J, Ramos J, Couto I, Portugal I, Ritter C, et al. High-level resistance to isoniazid and ethionamide in multidrug-resistant Mycobacterium tuberculosis of the Lisboa family is associated with inhA double mutations. J Antimicrob Chemother 2013; 68 : 1728-32.  Back to cited text no. 12
    
13.
Vadwai V, Ajbani K, Jose M, Vineeth VP, Nikam C, Deshmukh M, et al. Can inhA mutation predict ethionamide resistance? Int J Tuberc Lung Dis 2013; 17 : 129-30.  Back to cited text no. 13
    




 

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