|Year : 2020 | Volume
| Issue : 2 | Page : 212-215
Mutation in atpE and Rv0678 genes associated with bedaquline resistance among drug-resistant tuberculosis patients: A pilot study from a high-burden setting in Northern India
Binit Kumar Singh1, Manish Soneja1, Rohini Sharma2, Jigyasa Chaubey1, Parul Kodan1, Pankaj Jorwal1, Neeraj Nischal1, Shivani Chandra3, Ranjani Ramachandran4, Naveet Wig1
1 Department of Medicine, All India Institute of Medical Sciences, New Delhi, India
2 Department of Mycobacteriology, Laboratory Division, National Tuberculosis Institute, Bengaluru, Karnataka, India
3 Department of Public Health, RTL, Northern Region, World Health Organization, India
4 Department of Public Health, NPOTB and Laboratories, World Health Organization, India
|Date of Web Publication||29-May-2020|
Department of Medicine, All India Institute of Medical Sciences, New Delhi - 110 029
Source of Support: None, Conflict of Interest: None
Background: Mutations in atpE gene or transcriptional repressor Rv0678 gene associated with inhibition of adenosine 5′-triphosphate synthase and upregulation of efflux pumps, respectively, may potentially lead to in vitro resistance to bedaquiline. This is the first study from India, which looks at mutations associated with this novel drug. Methods: In 2019 (January to June), a total of 68 laboratory-confirmed pre-extensively drug-resistant tuberculosis (XDR-TB) (fluoroquinolone resistant [n = 52] and second-line injectables resistant [n = 12]) and 4 × DR-TB culture specimens were included. All specimens were evaluated for genetic analysis using predesigned primers of atpE and Rv0678 genes. Results: Among the pre-XDR-TB isolates (n = 64), there were no mutations found in either atpE or Rv0678. However, among the XDR-TB isolates (n = 4), one specimen (25%) was found to be associated with a mutation in atpE gene at position 49, resulting in the amino acid leucine replaced by proline (L-49-P). No mutations were observed with the Rv0678 gene. Conclusion: In this study, genetic analysis showed that only one-fourth XDR-TB isolates had a mutation in the atpE gene; there were no other mutations found in the Rv0678 gene. To the best of our knowledge, this novel mutation (L-49-P) in atpE gene is being reported for the first time in northern India.
Keywords: Bedaquline, drug susceptibility testing, Mycobacterium tuberculosis complex
|How to cite this article:|
Singh BK, Soneja M, Sharma R, Chaubey J, Kodan P, Jorwal P, Nischal N, Chandra S, Ramachandran R, Wig N. Mutation in atpE and Rv0678 genes associated with bedaquline resistance among drug-resistant tuberculosis patients: A pilot study from a high-burden setting in Northern India. Int J Mycobacteriol 2020;9:212-5
|How to cite this URL:|
Singh BK, Soneja M, Sharma R, Chaubey J, Kodan P, Jorwal P, Nischal N, Chandra S, Ramachandran R, Wig N. Mutation in atpE and Rv0678 genes associated with bedaquline resistance among drug-resistant tuberculosis patients: A pilot study from a high-burden setting in Northern India. Int J Mycobacteriol [serial online] 2020 [cited 2020 Jul 11];9:212-5. Available from: http://www.ijmyco.org/text.asp?2020/9/2/212/285226
| Introduction|| |
The incidence of multi-drug resistant tuberculosis (MDR-TB) and extensively drug-resistant TB (XDR-TB) has been increasing substantially. India (24%), China (13%), and the Russian Federation (10%) account almost half of the MDR/rifampicin (RIF)-resistant-TB cases. In our effort to fight against TB and plan and vision to End TB by 2035, drug-resistant TB is a major obstacle. The novel drug bedaquiline (BDQ) was introduced as a hope in this gloomy scenario to manage drug-resistant (DR) TB. It was approved in 2012 by the US Food and Drug Administration (FDA) for the treatment of multidrug-resistant (MDR) TB., The drug is presently available in India as a part of the condition access program for BDQ for the treatment of drug-resistant TB under the Revised National TB Control Programme Central TB Division Directorate General of Health Services, Ministry of Health and Family Welfare, Government of India.
BDQ represents the diarylquinolines group of antimycobacterial agents, with significant bactericidal activity in both replication and nonreplicating mycobacteria. The activity of ATP-synthase inhibited by BDQ results in the reduction of bacterial ATP synthesis. However, different mutation patterns already found in the atpE gene leads to resistance against BDQ. A low-level resistance associated with transcriptional receptor Rv0678 gene, an efflux pump-related MmpS5-MmpL5 genes have been associated with cross-resistant between clofazimine (CFZ) and BDQ in vitro. Although BDQ has shown the least cross-resistance with most pre-existing first- and second-line anti-TB drugs, mutations associated with Rv0678 were majorly linked with the mechanism of CFZ resistance., A recent study has reported the association of low-level BDQ resistance with pepQ gene with unknown mode of action.
In India, BDQ is available free of cost under the National TB program under condition access. It is administered along with the standard DR-TB regimen. Recommended dose is 400 mg once daily for 2 weeks, followed by 200 mg thrice weekly for 22 weeks. After 24 weeks of BDQ therapy, the MDR-TB regimen should be continued as per the national TB treatment guidelines., No study from India has looked into the resistance pattern of this drug. To the best of our knowledge, this is the first study from India, which looks at mutations in atpE and Rv0678 genes associated with BDQ drug and has important implications in the management of DR TB.
| Methods|| |
This study was conducted between January 2019 and June 2019 on 68 culture-positive Mycobacterium tuberculosis specimens (64 were pre-XDR-TB and four were XDR-TB) at Intermediate Reference Laboratory, All India Institute of Medical Sciences (AIIMS) Hospital, New Delhi, India and was approved by Ethics Committee of the AIIMS, New Delhi, India (Ref No. IEC-256/05.04.2019, RP-15/2019, Date of approval: 22.04.2019). In this study, we have selected patients who were not yet exposed to BDQ in their regimen.
All investigations associated with culture-positive specimens were processed in biosafety cabinet Class-II in a biosafety level III laboratory. The samples were processed according to the WHO guidelines by using the NALC-NaOH decontamination procedure (final NaOH concentration, 1%). The decontaminated and digested samples were neutralized using (phosphate-buffered saline; pH 6.8) and mixed properly.
Line probe assay
Line probe assay (LPA) methods ware applied to find out initial investigations. A GenoLyse kit (Hain Lifescience, Nehren, Germany) was used for DNA extraction from all specimens. The interpretation was performed for both Genotype MTBDRplus V.2 and Genotype MTBDRsl V.2 according to the manufacturer's instructions (Hain LifeScience GmbH, Nehren, Germany).
Conventional drug susceptibility testing on mycobacteria growth indicator tube-960
Indirect drug susceptibility testing (DST) was performed by using the final critical concentration of anti-TB drugs, i.e., 1.0 μg/ml of amikacin (AMK), 2.5 μg/ml of kanamycin (KAM) and capreomycin (CAP), 0.5 μg/ml and 2.0 μg/ml of moxifloxacin (MFX), 2.0 μg/ml ofloxacin (OFX) and 1.5 μg/ml levofloxacin (LFX). H37Rv strain was used as the reference standard for the phenotypic DST test. Due to the unavailability of BDQ and CFZ drugs, no DST was performed for the same in this study. However, a recent study has been successfully validated the role of mycobacteria growth indicator tube-960 for the DST of BDQ drug powder.
Extracted DNA was amplified using specific primers atpE and Rv0678 genes [Table 1]., Sequencing was performed on ABI Prism 3130 × l genetic analyzer (Applied Biosystem, Carlsbad, CA, USA) according to the manufacturer's instructions.
Sequence results were analyzed using BioEdit Software (Thomas Hall, Raleigh, NC, USA) and ClustalW 2.0.
| Results|| |
A total of 68 including culture isolates of pre-XDR (n = 64) and XDR-TB (n = 4) were selected for this study. Among pre-XDR-TB, 52 isolates were confirmed as fluoroquinolone (FLQ) resistant, while 12 isolates were found to have second-line injectables (SLI) resistant using as LPA as an initial method to identify resistance patterns in the present study. Four isolates were identified as XDR, i.e., resistant to both FLQ and SLI on LPA. In phenotypic DST, all 52 FLQ-resistant isolates were observed resistant to RIF, INH, LFX, OFX, MFX (0.5 μg/ml) drugs, while 12 SLI-resistant isolates were found to be resistant to RIF, INH, AMK, KAM drugs. In the case of XDR, all four isolates were found to be resistant to RIF, INH, LFX, OFX, MFX (0.5 μg/ml), AMK, KM, CAP drugs [Table 2].
|Table 2: Drug susceptibility pattern with mutation in atpE and Rv0678 (n=68)|
Click here to view
On performing DNA sequencing using targeted atpE and RV0678 gene, no mutations were observed in either FLQ mono-resistant (n = 52) or SLI mono-resistant (n = 12) isolates. In case of XDR-TB (n = 4) isolates, one (25%) isolate was associated with mutation in atpE gene at position 145 (CTG-145-CCA) which resulted in the change of the amino acid from leucine to proline (L-49-P); however, no mutation was observed with RV0678 gene [Table 2].
| Discussion|| |
In the global epidemic of DR-TB, the role of novel drugs like BDQ remains crucial. Recent trials also look into the potential role of the drug in shorter treatment regimens. The safety and side effect profile is currently being investigated as a part of the Phase III STREAM (Standardized Treatment Regimen of Anti-TB drugs for patients with MDR-TB) trial. While it is important to closely look at the clinical effects of the drug, it is equally important to identify mutations and keep a close watch to determine the future of this wonder drug.
In recent years, clinical failure cases of BDQ based treatment regimen has been reported form worldwide, and most of them were found to be resistant due to the mutation in the Rv0678 gene associated with efflux pump pathways., A study conducted in Belgium by Villellas et al., 2018 showed a high prevalence of mutation in the Rv0678 gene among CFZ or BDQ unexposed MDR-TB patients. In contrast, the present study did not find any mutations in the Rv0678 gene region of pre-XDR or XDR-TB isolates. However, mutations associated with atpE gene linked with BDQ drug target site were found in one sample. In sequencing, we have found a new type of mutation in atpE gene at position L-49-P, which has not been reported in previous studies. The clinical significance of this new mutation needs to be determined.
A relatively low burden of BDQ resistance in Indian scenario as in this study needs clinical correlation and a larger cohort to confirm the same. However, if the same is confirmed it may be an encouraging ray of light in efforts toward managing DR-TB epidemic. Close monitoring of this drug is important to decide its future and is an important step toward our commitment to eliminate TB.
A study of Tiberi et al., 2018 suggested that only one-third of CFZ-resistant strains were also found to be BDQ resistant, while all BDQ resistant were found to be resistant to CFZ. This could have important implications as BDQ resistance may act as a “surrogate marker” for CFZ resistance. However, in the present study, we did not perform CFZ DST to confirm the same.
| Conclusion|| |
This study shows a novel mutation in BDQ-associated atpE gene in XDR-TB isolates. No other mutations were found in the RV0678 gene either in XDR or pre-XDR-TB isolates. Although mutations in atpE and Rv0678 genes were found to be uncommon in this pilot study from a high disease burden country a larger sample set comprising diverse strains along with phenotypic correlation is the need of the hour to know the resistance pattern of this new drug.
The authors would like to thank state State TB Officer, National Capital Territory, Delhi, and Foundation for Innovative New Diagnostic for uninterrupted logistic support in patient care.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Diacon AH, Pym A, Grobusch MP, de los Rios JM, Gotuzzo E, Vasilyeva I, et al
. Multidrug-resistant tuberculosis and culture conversion with bedaquiline. N Engl J Med 2014;371:723-32.
Mirsaeidi M. After 40 years, new medicine for combating TB. Int J Mycobacteriol 2013;2:1-2. [Full text]
Koul A, Vranckx L, Dendouga N, Balemans W, Van den Wyngaert I, Vergauwen K, et al
. Diarylquinolines are bactericidal for dormant mycobacteria as a result of disturbed ATP homeostasis. J Biol Chem 2008;283:25273-80.
Segala E, Sougakoff W, Nevejans-Chauffour A, Jarlier V, Petrella S. New mutations in the mycobacterial ATP synthase: new insights into the binding of the diarylquinoline TMC207 to the ATP synthase C-ring structure. Antimicrob Agents Chemother 2012;56:2326-34.
Huitric E, Verhasselt P, Koul A, Andries K, Hoffner S, Andersson DI. Rates and mechanisms of resistance development in Mycobacterium tuberculosis
to a novel diarylquinoline ATP synthase inhibitor. Antimicrob Agents Chemother 2010;54:1022-8.
Somoskovi A, Bruderer V, Hömke R, Bloemberg GV, Böttger EC. A mutation associated with clofazimine and bedaquiline cross-resistance in MDR-TB following bedaquiline treatment. Eur Respir J 2015;45:554-7.
Hards K, Robson JR, Berney M, Shaw L, Bald D, Koul A, et al
. Bactericidal mode of action of bedaquiline. J Antimicrob Chemother 2015;70:2028-37.
Zhang S, Chen J, Cui P, Shi W, Zhang W, Zhang Y. Identification of novel mutations associated with clofazimine resistance in Mycobacterium tuberculosis
. J Antimicrob Chemother 2015;70:2507-10.
Almeida D, Ioerger T, Tyagi S, Li SY, Mdluli K, Andries K, et al
. Mutations in pepQ confer low-level resistance to bedaquiline and clofazimine in Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2016;60:4590-9.
Field SK. Bedaquiline for the treatment of multidrug-resistant tuberculosis: great promise or disappointment? Ther Adv Chronic Dis 2015;6:170-84.
Velayati AA, Farnia P, Masjedi MR. Totally drug-resistant tuberculosis (TDR-TB): A debate on global health communities. Int J Mycobacteriol 2013;2:71-2. [Full text]
World Health Organization. Companion Handbook to the WHO Guidelines for the Programmatic Management of Drug-Resistant Tuberculosis. World Health Organization; 2014. Available from: http://www.ncbi.nlm.nih.gov/books/NBK247420/
. [Last accessed on 2018 Nov 26].
Sivaramakrishnan G, Subramanyam B, Kumar MP, Golla R, Tripathy SP, Mondal R. Validation of bedaquiline drug-susceptibility testing by BACTEC MGIT 960 system for Mycobacterium tuberculosis
. Int J Mycobacteriol 2019;8:329-32.
] [Full text]
Villellas C, Coeck N, Meehan CJ, Lounis N, de Jong B, Rigouts L, et al
. Unexpected high prevalence of resistance-associated Rv0678 variants in MDR-TB patients without documented prior use of clofazimine or bedaquiline. J Antimicrob Chemother 2017;72:684-90.
Silva DR, Dalcolmo M, Tiberi S, Arbex MA, Munoz-Torrico M, Duarte R, et al
. New and repurposed drugs to treat multidrug-and extensively drug-resistant tuberculosis. J Bras Pneumol 2018;44:153-60.
Zimenkov DV, Nosova EY, Kulagina EV, Antonova OV, Arslanbaeva LR, Isakova AI, et al
. Examination of bedaquiline-and linezolid-resistant Mycobacterium tuberculosis
isolates from the Moscow region. J Antimicrob Chemother 2017;72:1901-6.
Bloemberg GV, Keller PM, Stucki D, Trauner A, Borrell S, Latshang T, et al
. Acquired Resistance to Bedaquiline and Delamanid in Therapy for Tuberculosis. N Engl J Med 2015;373:1986-8.
Hartkoorn RC, Uplekar S, Cole ST. Cross-resistance between clofazimine and bedaquiline through upregulation of MmpL5 in Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2014;58:2979-81.
Tiberi S, Cabibbe AM, Tomlins J, Cirillo DM, Migliori GB. Bedaquiline phenotypic and genotypic susceptibility testing, work in progress! EBioMedicine 2018;29:11-2.
[Table 1], [Table 2]