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 Table of Contents  
Year : 2017  |  Volume : 6  |  Issue : 2  |  Page : 167-170

Phenotypic low-level isoniazid resistance as a marker to predict ethionamide resistance in Mycobacterium tuberculosis

Department of Pathology and Laboratory Medicine, Aga Khan University Hospital, Karachi, Pakistan

Date of Web Publication19-May-2017

Correspondence Address:
Rumina Hasan
Aga Khan University Hospital, Stadium Road, Karachi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmy.ijmy_34_17

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Background: Tuberculosis is one of the most prevalent diseases in Pakistan. Pakistan has the highest burden of MDR-TB in the Eastern Mediterranean region. Ethionamide is an anti-tuberculous drug frequently used to treat MDR-TB. Its drug susceptibility testing is not easily available in resource limited settings. Since it acts on the same target protein as isoniazid (inhA protein encoded by inhA gene), we sought to find out if phenotypic isoniazid resistance can be a marker of ethionamide resistance. Materials and Methods: This was a retrospective observational study conducted at the Aga Khan University hospital section of microbiology. Data was retrieved between 2011 to 2014 for all culture positive MTB strains. All culture positive MTB isolates with susceptibilities to isoniazid and ethionamide recorded were included in the study. Isoniazid and ethionamide susceptibilities were performed using agar proportion method on Middlebrook 7H10 agar. Rate of Ethionamide resistance between low-level isoniazid resistant, high level isoniazid resistant and isoniazid sensitive MTB was compared. Results: A total of 11,274 isolates were included in the study. A statistically significant association (P < 0.001) was found between Ethionamide resistance and low-level isoniazid resistance (26.6%) as compared to high-level isoniazid resistance (8.85%) and isoniazid sensitivity (0.71%) in MTB strains. However this association was not seen in XDR-TB strains. Conclusion: Low level isoniazid resistance may be used as marker for phenotypic ethionamide resistance and hence guide clinicians' choice of antituberculous agent for MDR-TB in Pakistan. Further studies involving detection of genotypic association of isoniazid and ethionamide susceptibilities are needed before a final conclusion can be derived.

Keywords: Ethionamide, isoniazid, Mycobacterium tuberculosis

How to cite this article:
Qamar S, Farooqi JQ, Jabeen K, Hasan R. Phenotypic low-level isoniazid resistance as a marker to predict ethionamide resistance in Mycobacterium tuberculosis. Int J Mycobacteriol 2017;6:167-70

How to cite this URL:
Qamar S, Farooqi JQ, Jabeen K, Hasan R. Phenotypic low-level isoniazid resistance as a marker to predict ethionamide resistance in Mycobacterium tuberculosis. Int J Mycobacteriol [serial online] 2017 [cited 2021 Jan 18];6:167-70. Available from: https://www.ijmyco.org/text.asp?2017/6/2/167/206602

  Introduction Top

Antituberculosis (TB) drug resistance is a major public health problem. Ethionamide is a Group 4 anti-TB antibiotic. It is a structural analog of isoniazid and acts on the same target as isoniazid, i.e., inhA protein. The association of mutations in the promoter region inhA gene with isoniazid resistance is well established.[1] Cross-resistance between isoniazid and ethionamide has been documented in other regions.[2],[3]

In this study, we aimed to seek if low-level isoniazid resistance could be used as a surrogate marker to predict ethionamide resistance and whether it can guide clinicians' choice of selecting antituberculosis therapies to manage multidrug-resistant TB (MDR-TB) patients.

  Material and Methods Top

This was a retrospective study conducted in the Microbiology Section of Aga Khan University Hospital clinical laboratories. The AKUH mycobacteriology laboratory is a WHO Supranational Reference Laboratory for TB and receives samples from across the country. Laboratory data of samples received from 2011 to 2014 were retrieved, and all culture-positive Mycobacterium tuberculosis (MTB) isolates with susceptibility testing performed for isoniazid and ethionamide were included in the study. Drug susceptibilities were performed using agar proportion method on Middlebrook agar 7H10 using isoniazid in concentration of 0.2 μg/ml and 1 μg/ml and ethionamide 5 μg/ml. Previous studies have shown that although testing two different concentrations of isoniazid does not alter the overall susceptibility results; it can help to predict cross-resistance between isoniazid and its structural analogs.[4] Sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of isoniazid resistance for determining ethionamide resistance were calculated. Subgroup analysis to determine the rate of ethionamide resistance in low- and high-level isoniazid-resistant MTB strains was also calculated. Exemption to ethical approval was granted by the Ethical Review Committee, Aga Khan University.

  Results Top

During the study period, records of 17,184 strains were recovered from the laboratory database. Among these, isoniazid and ethionamide susceptibility data were available for 11,274 MTB strains which were included in the analysis, whereas 5910 strains had incomplete information and were excluded from the study. Strains from all across Pakistan were included; 3080 isolates from Karachi, 1437 from Lahore, 1187 from Peshawar, 145 from Islamabad, and 505 from Quetta.

Out of 11,274 strains, 5939 were isoniazid resistant (including both low level and high level) and 5335 were isoniazid sensitive. Ethionamide resistance was seen in 629 (5.57%) strains [Table 1].
Table 1: Frequency of ethionamide resistance in lowand high-level isoniazid.resistant and isoniazid-sensitive Mycobacterium tuberculosis strains

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Sensitivity, specificity, NPV, and PPV of isoniazid resistance as a marker of ethionamide resistance were 93.80%, 49.76%, 99.27%, and 9.93% with a significant P < 0.001. As inhA mutation is associated with low-level isoniazid resistance, we further divided the isoniazid-resistant MTB strains into low- and high-level isoniazid-resistant groups and compared the rate of ethionamide resistance. Our findings showed ethionamide resistance to be 26.6% in low-level isoniazid-resistant MTB strains as compared to 8.85% in high-level isoniazid-resistant MTB strains [Figure 1].
Figure 1: Flow diagram representing total number of MTB strains included in the study and their devision into different sub groups.

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A subgroup analysis was performed by further dividing low- and high-level isoniazid-resistant MTB strains into non-MDR, MDR, preextensively drug-resistant (XDR), and XDR groups. Among strains with low-level isoniazid-resistant (non-MDR, MDR, and pre-XDR) rate of ethionamide resistance was 26%–28% [Table 2]. Moreover, low-level isoniazid resistance was significantly associated with ethionamide resistance (P < 0.001) among all groups except XDR-TB strains.
Table 2: Comparison of ethionamide resistance between different classes of isoniazid-resistant Mycobacterium tuberculosis-C

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  Discussion Top

TB is a global health concern. According to the WHO report 2015, the prevalence of TB in Pakistan is about 361 cases/100,000.[5] It currently ranks fifth among the high TB burden countries, and although studies have shown nonuniformity in drug resistance rates among different regions in a country,[6] Pakistan still has the fourth highest prevalence of MDR-TB globally.[7] MDR-TB is caused by MTB resistant to at least isoniazid and rifampicin (RIF). Agents used to treat MDR-TB include second-line drugs; amikacin, kanamycin, capreomycin, fluoroquinolones, cycloserine, and thioamides: ethionamide and prothionamide.[8]

Ethionamide is readily available in Pakistan and considering its low cost and oral route of administration is one of the frequently used agents to treat MDR-TB. It is available as a pro-drug which undergoes activation by ethA enzyme to convert into its active form. It functions by disrupting the mycobacterial cell wall. Ethionamide is structurally related to isoniazid and both drugs act on the same target site, i.e., inhA protein. inhA protein, encoded by inhA gene, is a nicotinamine adenine dineucleotide-dependent enoyl-acyl carrier protein, responsible for fatty acid chain elongation and mycolic acid synthesis. Association of ethionamide resistance with inhA promoter and gene mutations has been already been explored previously.[9],[10] Our findings are consistent with previous studies which have shown a higher rate of ethionamide resistance among MTB strains with low-level isoniazid resistance and inhA mutation.[11],[12] High incidence of ethionamide resistance in MTB strains showing mutations in inhA promoter and inhA gene has also been reported from a study in India.[13]

Genetic diversity among MTB exists, and the distribution of resistance gene may alter in different geographic areas.[14] Molecular tests are recommended by the WHO which can be used to rapidly detect mutations associated with resistance in MTB. These include Xpert MTB/RIF and line probe assays (LPAs).[15] Currently approved Xpert MTB/RIF only detects mutation in the rpoB; however, LPAs can detect mutations in rpoB along with katG and inhA newer version of LPA (MTBDRsl) for second-line drugs has also been approved by the WHO which can detect resistance in fluoroquinolones and injectable anti-TB drugs by detecting mutations in gyrA/gyrB and rrs/eis, respectively, but does not detect mutations associated with ethionamide resistance. Previous studies have employed LPAs as a rapid means of detecting inhA mutation and associated phenotypic ethionamide resistance.[16] Knowledge regarding the presence or absence of inhA mutation using rapid molecular test such as LPA can be useful in enabling clinicians to decide in a timely manner whether to prescribe ethionamide for the treatment MDR-TB. Since LPAs are increasingly being used as a rapid test for the detection of MDR-TB, our results suggest that in this population, detection of inhA mutations on LPA increases the possibility of ethionamide resistance and highlights the need for drug sensitivity testing. Ethionamide resistance is not only attributed due to mutations in inhA gene but also due to ethA and ethR gene mutations:[17],[18] the ethA protein is itself regulated by ethR protein encoded by the ethR gene. There is thus also a need to explore the presence or absence of these mutations in MTB strains from our region, and to study the extent to which ethionamide resistance in our population is attributed to inhA or ethA/ethR.

Our study is limited by its retrospective nature and a single-center setting. However, the study was conducted on a large sample size and included strains from across the country. Mutations associated with isoniazid and ethionamide resistance were not explored, and studies evaluating the prevalence of inhA, ethA, and eth R mutations in addition to inhA are needed to better understand the association between ethionamide resistance and low-level isoniazid resistance in this population.

  Conclusion Top

In our population, low-level isoniazid resistance may predict phenotypic ethionamide resistance and hence guide clinicians. However, molecular studies detecting inhA and other mutations are further needed before definitive conclusions can be drawn.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Kim SY, Park YJ, Kim WI, Lee SH, Ludgerus Chang C, Kang SJ, et al. Molecular analysis of isoniazid resistance in Mycobacterium tuberculosis isolates recovered from South Korea. Diagn Microbiol Infect Dis 2003;47:497-502.  Back to cited text no. 1
Schaaf HS, Victor TC, Venter A, Brittle W, Jordaan AM, Hesseling AC, et al. Ethionamide cross- and co-resistance in children with isoniazid-resistant tuberculosis. Int J Tuberc Lung Dis 2009;13:1355-9.  Back to cited text no. 2
Zhang Y, Yew WW. Mechanisms of drug resistance in Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2009;13:1320-30.  Back to cited text no. 3
Lakshmi R, Ramachandran R, Sundar AS, Rahman F, Kumar V. Stratifying low level isoniazid resistance using additional intermediate drug concentration. Int J Mycobacteriol 2014;3:152-4.  Back to cited text no. 4
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Global Tuberculosis Report; 2016. Available from: http://www.who.int/tb/publications/global_report/en/. [Last accessed 2016 Dec 12].  Back to cited text no. 5
Rosales-Klintz S, Jureen P, Zalutskayae A, Skrahina A, Xu B, Hu Y, et al. Drug resistance-related mutations in multidrug-resistant Mycobacterium tuberculosis isolates from diverse geographical regions. Int J Mycobacteriol 2012;1:124-30.  Back to cited text no. 6
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World Health Organisation Stop Tuberculosis Program. Available from: http://www.emro.who.int/pak/programmes/stop-tuberculosis.html. [Last accessed 2016 Dec 12].  Back to cited text no. 7
WHO. Treatment of Tuberculosis Guidelines. Geneva: WHO; 2010. p. 30. Available from: http://www.who.int/tb. [Last accessed 2016 Dec 12].  Back to cited text no. 8
Machado D, Perdigão 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. 9
Banerjee A, Dubnau E, Quemard A, Balasubramanian V, Um KS, Wilson T, et al. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 1994;263:227-30.  Back to cited text no. 10
Morlock GP, Metchock B, Sikes D, Crawford JT, Cooksey RC. ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuberculosis isolates. Antimicrob Agents Chemother 2003;47:3799-805.  Back to cited text no. 11
Larsen MH, Vilchèze C, Kremer L, Besra GS, Parsons L, Salfinger M, et al. Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis. Mol Microbiol 2002;46:453-66.  Back to cited text no. 12
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
Das D, Dwibedi B, Kar SK. Low levels of anti TB drug resistance in Rayagada district of Odisha, India. Int J Mycobacteriol 2014;3:76-8.  Back to cited text no. 14
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Available from: http://www.who.int/tb/features_archive/xpert_rapid_tb_test/en/. [Last accessed 2017 Jan 18].  Back to cited text no. 16
Engohang-Ndong J, Baillat D, Aumercier M, Bellefontaine F, Besra GS, Locht C, et al. EthR, a repressor of the TetR/CamR family implicated in ethionamide resistance in mycobacteria, octamerizes cooperatively on its operator. Mol Microbiol 2004;51:175-88.  Back to cited text no. 17
Boonaiam S, Chaiprasert A, Prammananan T, Leechawengwongs M. Genotypic analysis of genes associated with isoniazid and ethionamide resistance in MDR-TB isolates from Thailand. Clin Microbiol Infect 2010;16:396-9.  Back to cited text no. 18


  [Figure 1]

  [Table 1], [Table 2]

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