|Year : 2013 | Volume
| Issue : 4 | Page : 395-400
Mutation in katG315 is, possibly, a good prognostic marker for treatment with second-line drugs in multi-drug resistant tuberculosis: A preliminary study
Mohanad M Ahmed1, Suhad H Mohammed1, Hassan AA Nasurallah2
1 Department of Microbiology, College of Medicine, University of Kerbala, Kerbala, Iraq
2 Department of Internal Medicine, College of Medicine, University of Kerbala and Al-Hussein Medical City, Kerbala, Iraq
|Date of Submission||17-Feb-2013|
|Date of Acceptance||04-Jul-2013|
|Date of Web Publication||25-Sep-2013|
Mohanad M Ahmed
Department of Microbiology, College of Medicine, University of Kerbala, Kerbala
Source of Support: None, Conflict of Interest: None
The aim of this study was to explore baseline data, laboratory and molecular analyses to determine if any could serve as potential prognostic marker(s) for treatment response to second line tuberculosis regimens. Of a total number of 50 multi-drug resistant tuberculosis (MDR-TB) patients starting second-line drug MDR-TB treatment in Iraq, only 21 showed treatment adherence and thus, included in this study. Response to treatment was monitored for 11 months by sputum microscopy and culture. We explored baseline data, laboratory and molecular analyses to determine if any could serve as potential prognostic marker(s) for treatment response. Highly significant association (P = 0.019) was detected between mutations in katG315 codon and good response to second-line anti-TB drugs. Spoligotyping and mycobacterial interspersed repetitive unit variable number tandem repeat confirmed that katG315-mutatnt isolates were genotypically unrelated. The katG315 mutation is a potential prognostic marker for treatment response to second-line anti-tuberculosis drugs. One possible explanation of our results is that the katG315-mutants are sensitive to bacterial killing by "oxidative killing."
Keywords: KatG315, multi-drug resistant, second-line drugs, tuberculosis
|How to cite this article:|
Ahmed MM, Mohammed SH, Nasurallah HA. Mutation in katG315 is, possibly, a good prognostic marker for treatment with second-line drugs in multi-drug resistant tuberculosis: A preliminary study. Indian J Med Microbiol 2013;31:395-400
|How to cite this URL:|
Ahmed MM, Mohammed SH, Nasurallah HA. Mutation in katG315 is, possibly, a good prognostic marker for treatment with second-line drugs in multi-drug resistant tuberculosis: A preliminary study. Indian J Med Microbiol [serial online] 2013 [cited 2019 Jun 16];31:395-400. Available from: http://www.ijmm.org/text.asp?2013/31/4/395/118899
| ~ Introduction|| |
The most recent World Health Organization report showed that drug resistance tuberculosis (TB) is spreading and may be on the rise.  While, previous estimates demonstrated the prevalence of drug resistance at around 5%, a very recent study documented a prevalence up to 10 times higher in some places, where almost half of the patients with infectious disease are transmitting multi-drug resistant (MDR) strains of Mycobacterium tuberculosis.  MDR and extensive drug resistance (XDR) TB represent the major threat for TB control. 
MDR and XDR-TB treatment involve using second-line anti-TB drugs, which are less effective, more toxic and regimens takes longer periods (up to 24 months or more). Delayed detection of treatment failure during the course of those regimens may entails risks of transmitting extremely drug resistance strains and may contribute to the development of strains that would resist almost all known anti-TB drugs. Pre-treatment prognostic markers are needed as they could allow stratification of patients into groups with different treatment requirement and controlling measures. The only validated prognostic marker for the outcome of treatment with a second-line anti-TB drug regimen is the phenotypic drug susceptibility testing (DST) results prior to treatment.  Dalton, et al. (2012) assessed resistance to second-line anti-TB drugs in eight countries and found that previous treatment with second-line drugs was consistently the strongest risk factor for subsequently resistance to these drugs, which increased the risk of XDR-TB by more than four times.  In Iraq, as this is the first time a second-line regimens are being used, it is not possible to assess the previous treatment with second-line drugs as a prognostic marker.
In this study, we aimed to explore the baseline data of patients in addition to genotyping and molecular analyses for clues about possible risk factors for resistance or prognostic markers for response to treatment with a second-line anti-TB regimen.
| ~ Subjects and Methods|| |
On June, 2011, a total of 50 MDR-TB patients started treatment with second-line anti-TB drugs at the National Centre of Tuberculosis and Chest illnesses-National Tuberculosis Programme in Baghdad, Iraq. However, 29 patients showed irregular adherence to treatment and follow-up. Only 21 patients showed treatment adherence and thus, included in this study. According to the guidelines for the programmatic management of drug resistant, the treatment included an intensive phase of 8 months followed by continuation phase planned to continue for up to 16 months with monthly monitoring by smear microscopy and culture. 
Basic demographic data were collected and DST to four first-line anti-TB drugs was performed.  Spoligotyping and mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) were used to genotype the isolates. , Two independent allele-specific polymerase chain reaction (PCR) systems were used to detect the mutations in katG315 and inhAP-15 codons,  whereas, a single-step multiplex allele-specific PCR assay was used to detect mutations in the rpoB gene (codons 516,526, and 531). 
| ~ Results|| |
Detailed data and results are shown in [Table 1]. A comparison of spoligotyping results with the international spoligotyping database (SpolDB4) showed that 8 isolates belong to T1 sub lineage/group, 7 belong to CAS1-Delhi, 2 belong to H3, 2 belong to H4 (Ural-2), one belongs to the Turkey sub lineage. One did not matching any sub lineages in the international database (SpolDB4) and thus, is designated as unknown sub lineage. Genotyping by the 15 locus MIRU-VNTR method identified a single cluster containing 3 isolates belonging to Shared International Type-1144 (SIT-1144), whereas the rest of the isolates were unique.
|Table 1: Detailed results obtained including demographic, epidemiologic, drug‑resistance, and genotyping information on a total of 21 M. tuberculosis strains isolated from Iraqi patients before initiation of treatment with second‑line drugs|
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Regarding to treatment outcome, 'good response' to treatment with second-line drugs (determined by sputum and culture conversion to negative) was seen in 13 cases (61.9%). A total of 4 cases (19%) showed 'poor response' (determined by consistent smear microscopy and culture positivity along the study period. In addition, 4 (19%) cases showed conversion of smear microscopy and culture to negativity but later on, reverted to positivity.
We compared the available demographic data and results of DST, however, no association could be detected [results are summarized in [Table 2]. In addition, no specific association could be found between the M. tuberculosis genotypes with the responses to treatment with second-line drugs.
|Table 2: Correlation of age, gender and DST profile with response to treatment with second‑line anti‑tuberculosis drugs|
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Next, we compared the responses to treatment with the results of allele-specific PCR assays for mutations in rpoB, katG315, and inhAP-15. Surprisingly, we found a significant association between mutations in the katG315 codon and a 'good response' to treatment with second-line drugs (P = 0.019), where all isolates (n = 8) harbouring katG315 mutations were shown to respond well to treatment as shown in [Table 3]. It's worthy to mention that a 'good response' was seen in 13 cases and katG315 mutations were found in 8/13 (61.54%). However, no significant association was found with mutations in inhAP-15 or rpoB. Moreover, combined spoligotyping and MIRU-VNTR results have revealed that the isolates harbouring mutations in katG315 codons are unrelated because they are belong to different M. tuberculosis SITs.
|Table 3: Correlations of mutations in katG315, inhAP‑15 and rpoB with response to treatment with second‑line anti‑tuberculosis drugs|
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| ~ Discussion|| |
This preliminary reported, for the 1 st time, a significant association between katG315 mutations and 'good response' to treatment with second-line anti-TB drug. If these results are confirmed, then they could have significant implications for and applications in TB treatment and control. One potential argument of our results would be is that the isolates harbouring mutations in katG315 codons are represent a clone and consequently, the results generated thereof would represent characteristics of this clone rather than a general phenomenon. However, the combined spoligotyping and MIRU-VNTR results revealed that the isolates are unrelated and thus, our results seem not to be confined to a certain clone or sublineages. Indeed, the katG-mutant isolates in the current study belong to distinct M. tuberculosis sublineages that are globally distributed such CAS1-Delhi, H3 and T1. 
The M. tuberculosis katG gene encodes a dual function enzyme catalase-peroxidase, which confers sensitivity in M. tuberculosis to isoniazid (INH). Mutations in katG315 are associated with resistance to INH in 50-70% of strains.  INH-resistant clinical isolates of M. tuberculosis often lose the catalase and peroxidase enzyme encoded by katG, especially in high level resistance strains (minimal inhibitory concentration [MIC] >5 μg/ml) (low-level resistance strains [MIC <1 μg/ml] often still possess catalase activity). 
One potential mechanistic explanation for our results is could be suggested in the context of 'oxidative killing.' M. tuberculosis is a facultative intracellular bacterium and once phagocytosed, the organism resides in a vacuole. Within this vacuole, the organism is exposed several bactericidal mechanisms, including the production of reactive oxygen intermediates. However, M. tuberculosis has been shown to have a high resistance to killing by up to millimolar concentrations of H 2 O 2 . This resistance is believed to be mediated largely by the mycobacterial catalase-peroxidase protein encoded by the gene katG and to lesser extent by another gene product of ahpC. 
Inadequate use of INH in the treatment of TB infections may lead to selection of isoniazid-resistant katG-mutant strains (that have lost the katG activity). These strains, though resistant to isoniazid, are susceptible to oxidative stress. Manca et al. (1999) have shown that strains with no detectable katG expression or catalase activity are relatively sensitive to killing by exogenous H 2 O 2 .  Macrophages incubated for several days in the presence of aminoglycosides accumulate these drugs inside their cytoplasm and were found to trigger anti-bactericidal activities of the macrophages through upregulation of killing mechanisms including reactive oxygen intermediates.  Accordingly, we suggest a mechanistic explanation for our result. Favourable response to second-line treatment arises because second-line agents, aminoglycosides (possibly other drugs), activate macrophages to upregulate oxidative killing mechanisms at the site M. tuberculosis infection. Strains with a katG315-mutation are sensitive to 'oxidative killing' owing to diminished catalase activity thereby clearing infection. The katG315 mutants are sensitive to resulting in killing of bacteria by 'oxidative killing'.
| ~ Conclusion|| |
In conclusion, these preliminary data implicate katG315 mutation as a potential prognostic for treatment response to second-line anti-TB drugs; however, it needs to be confirmed in a larger study.
| ~ Acknowledgments|| |
The authors would like to thank Dr. Parrisa Farnia, Mycrobacterial Research Center, National Research Institute for Tuberculosis and Lung Diseases (NRITLD), Tehran, Iran for technical assistance.
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[Table 1], [Table 2], [Table 3]