|
 
 |
| ORIGINAL ARTICLE |
|
| Year : 2020 | Volume
: 9
| Issue : 4 | Page : 368-372 |
|
Characterization of Mycobacterium tuberculosis heteroresistance by genotyping
Lida Jouca de Assis Figueredo1, Isabela Neves de Almeida1, Claudio José Augusto2, Valéria Martins Soares3, Philip Noel Suffys4, Wânia da Silva Carvalho5, Silvana Spíndola de Miranda1
1 Mycobacteria Research Laboratory, Department of Internal Medicine, Faculty of Medicine, Federal University of Minas Gerais, Minas Gerais, Brazil
2 Mycobacteria Laboratory, Central Laboratory of Public Health of Minas Gerais, Ezequiel Dias Foundation, Minas Gerais, Brazil
3 Júlia Kubitschek Hospital, Hospital Federation of the State of Minas Gerais, Minas Gerais, Brazil
4 Oswaldo Cruz Institute,Molecular Biology Laboratory Applied to Mycobacteria, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil
5 Department of Social Pharmacy, Faculty of Pharmacy, Federal University of Minas Gerais, Minas Gerais, Brazil
| Date of Submission | 30-Jul-2020 |
| Date of Decision | 04-Aug-2020 |
| Date of Acceptance | 10-Aug-2020 |
| Date of Web Publication | 15-Dec-2020 |
Correspondence Address:
Silvana Spíndola de Miranda
Avenida Professor Alfredo Balena, 190 - Santa Efigenia, Belo Horizonte 30130-100, Minas Gerais
Brazil
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ijmy.ijmy_132_20
Background: Heteroresistance is the coexistence of susceptible and resistant strains in the same individual, considered the preliminary step for total resistance, and can stem from mixed infection or clonal heterogeneity. The aim of this study was to evaluate the heteroresistance of Mycobacterium tuberculosis to rifampicin and isoniazid and its characterization. Method: GenoType MTBDRplus®; Sanger sequencing of the rpoB, katG, and inhA genes; and Mycobacterial Interspersed Repetitive Unit – Variable Number Tandem Repeat (MIRU-VNTR) were performed. Results: In a total of 654 isolates, 530 were resistant, 124 were susceptible, and 29 were heteroresistant to a first-line drug. GenoType MTBDRplus® detected heteroresistance in the rpoB gene in 26/29 (89.6%), as compared to 5/29 (17.2%) in the katG gene and 2/29 (6.8%) in the inhA gene. Four isolates showed heteroresistance in these genes. The Sanger sequencing detected heteroresistance in the rpoB gene in 7/29 (24.1%), as compared to 3/29 (10.3%) in the katG gene. In one isolate, heteroresistance was concomitant in both the rpoB and katG genes. MIRU-VNTR detected mixed infection in three heteroresistant isolates, while four isolates showed clonal heterogeneity. Conclusions: GenoType MTBDRplus® detected more cases of heteroresistance when compared to sequencing. It was also possible to characterize mixed infection and clonal heterogeneity by MIRU-VNTR.
Keywords: Heteroresistance, mixed infection, multidrug-resistant tuberculosis, Mycobacterium tuberculosis
How to cite this article:
Assis Figueredo LJ, de Almeida IN, Augusto CJ, Soares VM, Suffys PN, Silva Carvalho Wd, de Miranda SS. Characterization of Mycobacterium tuberculosis heteroresistance by genotyping. Int J Mycobacteriol 2020;9:368-72 |
How to cite this URL:
Assis Figueredo LJ, de Almeida IN, Augusto CJ, Soares VM, Suffys PN, Silva Carvalho Wd, de Miranda SS. Characterization of Mycobacterium tuberculosis heteroresistance by genotyping. Int J Mycobacteriol [serial online] 2020 [cited 2021 Jan 25];9:368-72. Available from: https://www.ijmyco.org/text.asp?2020/9/4/368/303438 |
| Introduction | |  |
In 2018, the incidence of tuberculosis (TB) in the world was 132/100,000 inhabitants, and the multidrug-resistant TB (MDR-TB)/resistant to rifampicin TB (RR-TB) incidence was 484,000/population. It was estimated that 3.4% of new TB cases and 18% of retreatment cases had MDR-TB/RR-TB.[1]
In Brazil, in the same year, it was estimated that there was an incidence of 1.2/100,000 inhabitants in reported cases of MDR-TB/RR-TB, with 1.5% among new cases and 8% among previously treated cases. Of this total, only 1119 MDR-TB/RR-TB cases and 26 extensively drug-resistant TB cases were confirmed in the laboratory.[1]
Rapid acquired resistance may be caused by late diagnosis, inadequate treatment regimen, or treatment abandonment, as well as by heteroresistance, which is the coexistence of susceptible and resistant bacteria in the same patient.[2],[3]
Among the mechanisms that explain heteroresistance, a mixed infection is one of the most common, defined by the presence of strains with different patterns in two or more loci,[4] or clonal heterogeneity due to the division of a single lineage into susceptible and resistant clones due to biological evolution.[5] The diagnosis of mixed infection is laborious and requires the strains to be individually identified from the original culture.[6]
The identification of Mycobacterium tuberculosis heteroresistance is underestimated, and GenoType MTBDRplus® (GenoType) and Sanger sequencing have been described as genotypic methods that can aid in this detection,[7],[8],[9] while the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) is commonly used to detect mixed infection and clonal heterogeneity.[4]
The present study, therefore, sought to evaluate the heteroresistance of M. tuberculosis to rifampicin (RIF) and isoniazid (INH) using GenoType and sequencing, as well as the characterization of strains by MIRU-VNTR.
| Method | | |
Sample collection
The present study was performed in the Mycobacteria Research Laboratory of the School of Medicine of Federal University of Minas Gerais (FUMG) and included 654 clinical isolates of M. tuberculosis from the Júlia Kubitschek Hospital, a tertiary reference center; FUMG Clinical Hospital (FUMG), a high-complexity hospital; and the Mycobacterial Laboratory of the Ezequiel Dias Foundation/Central Laboratory of Public Health of Minas Gerais, from 2008 to 2017.
The treatment history and outcome of patients were obtained from the Notifiable Diseases Information System.
Drug susceptibility testing
Drug susceptibility testing was performed by the BACTEC™ MGIT™ 960 Systems by Becton Dickinson® (MGIT-960) at the Mycobacterial Laboratory of the Ezequiel Dias Foundation, as described by Siddiqi and Rüsch-Gerdes[10]
GenotypeMTBDRplus®
Molecular tests were performed in the Laboratory of Molecular Biology and Public Health, FUMG School of Pharmacy. The DNA was extracted from the colonies of clinical isolates in a solid Lowenstein–Jensen medium, using the GenoLyse® method, according to the manufacturer's instructions,[11] and a 5 μL of DNA was used for amplification by multiplex polymerase chain reaction (PCR) and reverse hybridization on the DNA strip.[11],[12] The hybridized strip for all wild-type (WT) probes and nonhybridization for mutation probes proved to be susceptible. The absence of hybridization in WT probes or hybridization of any mutant probe indicates resistance, whereas the hybridization in WT and mutation probes indicates heteroresistance.[8],[9]
Sanger sequencing
The sequencing of the rpoB, katG, and inhA genes was performed using the DNA extracted by GenoLyse,®;[11] using primers as described by Dalla Costa et al.,[13] according to the following protocol: buffer 10x, MgCl2 50 μM, dNTP 2.5 μM, primer forward and reverse 20 pmol, Taq 5U, and 5 μL of DNA. PCR products were purified with polyethylene glycol 8000, and the Big Dye Terminator Kit v3.1, forward and reverse primer at 5 pmol, and 1 μL of the purified PCR product, was used.[13],[14] The sequencing analysis was performed using the Applied Biosystems SeqScape® v2.5, Foster City, California, United States, and heteroresistance was determined when the sequencing simultaneously presented WT and mutation sequences.
Characterization of Mycobacterium tuberculosis heteroresistance by mycobacterial interspersed repetitive unit-variable number tandem repeat
The M. tuberculosis molecular characterization was based on 24 different loci containing MIRU-VNTR in fragments generated by manual monoplex PCR, as described by Supply et al.[15] Lineage identification of M. tuberculosis isolates was carried out using tree-based identification tools on the MIRUVNTRplus database (https://www.miru-vntrplus.org)[16] to analyze the presence of mixed infection or clonal heterogeneity in heteroresistant isolates. The mixed infection was defined by the presence of strains with different patterns in two or more loci, and clonal heterogeneity was determined when two distinct alleles in only one locus were identified.
Ethics
This study was approved by the FUMG Research Ethics Committee, logged under protocol number 122.941 (CAAE 06611912.8.0000.5149).
| Results | | |
Drug susceptibility testing
Of the 654 M. tuberculosis isolates, MGIT-960 identified 530/654 (81%) as susceptible and 124/654 (19%) as resistant, with 110/124 (88.7%) MDR-TB.
Genotype® MTBDRplus
Of the 654 isolates, 520/654 (79.5%) were susceptible and 134/654 (20.5%) were resistant when the GenoType was applied. A divergence was found in ten isolates, when compared to the MGIT; the results are shown in [Table 1].
In this study, 84.3% (113/134) of the resistant's isolates were RIF resistant with mutations in the rpoB gene. By contrast, 90.6% (106/134) of the INH-resistant isolates presented mutations in the katG gene and 30.8% (36/134) in the inhA gene, while the concomitant mutations in the katG and inhA genes occurred in 21.3% (25/106) of the isolates.
Heteroresistance was observed in 4.4% (29/654) of the isolates. Heteroresistance to RIF was detected in codons S531 L and H526Y of the rpoB gene, while the INH heteroresistance was detected in codon C315T of the katG gene and in codon C15T of the inhA gene. In three isolates, heteroresistance occurred concomitantly in rpoB and katG genes and in one isolate in the rpoB, katG, and inhA genes [Table 2].
Sanger sequencing
The coexistence of WT sequences and mutations was detected by Sanger sequencing in 34.5% (10/29) of the isolates, which, upon applying the GenoType, were detected as heteroresistant. The heteroresistance to RIF was detected in codons S531 L and S522A of the rpoB gene, while heteroresistance to INH was identified in codons C315T, G299C, and W293 L of the katG gene. In one isolate, the heteroresistance was simultaneous in the rpoB and katG genes [Table 3].
Characterization of Mycobacterium tuberculosis heteroresistance
MIRU-VNTR 24 loci was performed on the 29 isolates to distinguish whether heteroresistance originally occurred from mixed infection or clonal heterogeneity. Among the isolates, 3/29 (10.3%) presented mixed infection with two distinct alleles in two or more loci, confirming the presence of two different strains, and 4/29 (13.8%) isolates presented two distinct alleles in only one locus, confirming clonal heterogeneity [Table 4]. | Table 4: Characterization of heteroresistance using mycobacterial interspersed repetitive unit-variable number tandem repeat (n=7)
Click here to view |
Another parameter analyzed in this study was the relationship of mixed infection and clonal heterogeneity with outcomes. Of the three patients with mixed infection, all were TB-MDR when analyzed by the MGIT-960, two with an outcome of abandonment and one with a cure.
Among the four isolates with clonal heterogeneity, two were TB-MDR and two were susceptible, when analyzed by the MGIT-960. Of these, three had a history of abandonment and one of cure [Table 5]. | Table 5: Phenotypic test, outcome, and characterization of mixed infection and clonal heterogeneity using mycobacterial interspersed repetitive unit-variable number tandem repeat (n=7)
Click here to view |
Patients that presented heteroresistant isolates changed the treatment in 25/29 (86.2%) cases, while 22/25 (88%) used several schemes. The outcome was a cure in 15/29 (51.7%) of the cases.
| Discussion | | |
Drug susceptibility testing
The conflicting results found in this study between resistant MGIT-960 and susceptible GenoType, may be due to mutations in regions that were not covered by the probes in GenoType.[17] The discordant results, in which susceptible MGIT-960 and resistant GenoType are explained by heteroresistance, reflect the slow evolution of the bacteria of a profile susceptible to resistance, which is not uncommon in M. tuberculosis.[18]
There is also the transmission of susceptible and resistant bacteria from patients who have not adhered to the treatment. These patients tend to harbor mycobacteria in genotypically detectable but phenotypically undetectable proportions.[2]
Heteroresistance, when associated with mixed infections of M. tuberculosis, causes an increase in the rate of false-negative drug resistance tests obtained by phenotypic susceptibility tests.[19],[20],[21] Therefore, heteroresistance can interfere in both the diagnosis and the treatment, which is more difficult to detect when using the phenotypic method from the preliminary stage to the total resistance.[2],[3]
Molecular tests showed a low rate of heteroresistance, 29/654 (4.4%) when using GenoType and 10/654 (1.5%) when using Sanger sequencing. These results were similar to those reported in studies conducted in other regions, such as 3.7% (RIF) and 5.0% (INH) in Zimbabwe,[9] 1.4% in Italy,[22] and 1.9% in Finland and Russia.[23] However, there are studies that report higher rates, such as 14.3% in Uzbekistan[2] and 28.8% in India.[3] This discrepancy may be related to differences in detection methods or the prevalence of drug-resistant TB in each location.[2]
GenoType detected greater heteroresistance because the proportion of mycobacteria in the same isolate must be 95% susceptible and 5% resistant, as compared to Sanger sequencing, in which 50% is susceptible and 50% is resistant, as reported by Folkvardsen et al.[7]
In detecting heteroresistance, GenoType showed the absence of certain probes that were detected by Sanger sequencing, requiring the incorporation of additional probes.[8],[12],[24]
When heteroresistance is detected in GenoType or Sanger sequencing, the choice of appropriate treatment is complex. Therefore, it is necessary to interpret the genotype data together with the clinical data of the patients,[25] taking into account the locations of the mutations and determining whether or not they are related to low or moderate level of resistance through quantitative tests, such as the minimum inhibitory concentration.[26]
Characterization of Mycobacterium tuberculosis heteroresistance
Mixed infections and heteroresistance may be particularly common in regions with a high TB rate, especially MDR-TB,[3],[6],[27] and they can aid M. tuberculosis strains in the acquisition of mutations, facilitate the spread of drug-resistant strains, and increase the rate of treatment failure.[17],[24] MIRU-VNTR is an important tool and has been used to assess whether mixed infections contribute to heteroresistance.[27]
Heteroresistance caused by clonal heterogeneity infection occurs due to the segregation of susceptible and resistant organisms.[5] Numerous reports on selective pressure in inadequate treatments have been described in prior literature,[28] as well as in the present study, in which these patients showed a history of abandonment and several previous treatments.
| Conclusions | | |
GenoType MTBDRplus® detected more cases of heteroresistance when compared to sequencing, and it was possible to characterize mixed infection and clonal heterogeneity by MIRU-VNTR.
Acknowledgment
The authors wish to acknowledge Universidade Federal de Minas Gerais, the Minas Gerais State Research Support Foundation (APQ 03266-13/APQ 00094-12), and the National Council for Scientific and Technological Development (CNPq 446796/2014 and 310174/2017-7). We would also like to thank the Postgraduate Program in Health Sciences: Infectious Diseases and Tropical Medicine, the Universidade Federal de Minas Gerais School of Medicine; CAPES, for their support and encouragement in this work, the Ezequiel Dias Foundation, and the Brazilian TB Network (REDE TB).
Financial support and sponsorship
This study was financially supported by the Minas Gerais State Research Support Foundation (APQ 03266-13/APQ 00094-12) and the National Council for Scientific and Technological Development (CNPq 446796/2014 and 310174/2017-7). We would also like to thank the Postgraduate Program in Health Sciences: Infectious Diseases and Tropical Medicine, the Universidade Federal de Minas Gerais School of Medicine; Coordination for the Improvement of Higher Education Professionals – CAPES.
Conflicts of interest
There are no conflicts of interest.
| References | | |
| 1. | World Health Organization. WHO Global Report, Global Tuberculosis Report 2019. Geneva: World Health Organization; 2019.  |
| 2. | Hofmann-Thiel S, van Ingen J, Feldmann K, Turaev L, Uzakova GT, Murmusaeva G, et al. Mechanisms of heteroresistance to isoniazid and rifampin of Mycobacterium tuberculosis in Tashkent, Uzbekistan. Eur Respir J 2009;33:368-74. |
| 3. | Kumar P, Balooni V, Sharma BK, Kapil V, Sachdeva KS, Singh S. High degree of multi-drug resistance and hetero-resistance in pulmonary TB patients from Punjab state of India. Tuberculosis (Edinb) 2014;94:73-80. |
| 4. | Zheng C, Li S, Luo Z, Pi R, Sun H, He Q, et al. Mixed Infections and rifampin heteroresistance among Mycobacterium tuberculosis clinical isolates. J Clin Microbiol 2015;53:2138-47. |
| 5. | Zhang Z, Wang Y, Pang Y, Liu C. Comparison of different drug susceptibility test methods to detect rifampin heteroresistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2014;58:5632-5. |
| 6. | Lazzarini LC, Rosenfeld J, Huard RC, Hill V, Lapa e Silva JR, DeSalle R, et al. Mycobacterium tuberculosis spoligotypes that may derive from mixed strain infections are revealed by a novel computational approach. Infect Genet Evol 2012;12:798-806. |
| 7. | Folkvardsen DB, Thomsen Vø, Rigouts L, Rasmussen EM, Bang D, Bernaerts G, et al. Rifampin heteroresistance in Mycobacterium tuberculosis cultures as detected by phenotypic and genotypic drug susceptibility test methods. J Clin Microbiol 2013;51:4220-2. |
| 8. | Bedewi Omer Z, Mekonnen Y, Worku A, Zewde A, Medhin G, Mohammed T, et al. Evaluation of the GenoType MTBDRplus assay for detection of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates in central Ethiopia. Int J Mycobacteriol 2016;5:475-81. |
| 9. | Stephen S, Muzhizhizhi D, Dhibi N, Chidemo T, Samaneka W, Matubu TA, et al. Validation of the GenoType ® MTBDR plus Ver 2.0 assay for detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex isolates at UZCHS-CTRC TB research laboratory. Int J Mycobacteriol 2019;8:83-8. [ PUBMED] [Full text] |
| 10. | Siddiqi SH, Rüsch-Gerdes S. For BACTEC TM MGIT 960 TM TB System (Also applicable for Manual MGIT); 2006. |
| 11. | Hain Lifescience. GmbH, Germany. Genotype ® MTBDRplus VER 2.0; 2012. p. 1-14. |
| 12. | Idrees F, Irfan M, Jabeen K, Farooqi J, Hasan R. Diagnostic performance of GenoType ®MTBDRplus line probe assay in bronchoalveolar lavage for pulmonary tuberculosis diagnosis in sputum scarce and smearnegative patients. Int J Mycobacteriol 2017;6:122-6. [ PUBMED] [Full text] |
| 13. | Dalla Costa ER, Ribeiro MO, Silva MS, Arnold LS, Rostirolla DC, Cafrune PI, et al. Correlations of mutations in katG, oxyR-ahpC and inhA genes and in vitro susceptibility in Mycobacterium tuberculosis clinical strains segregated by spoligotype families from tuberculosis prevalent countries in South America. BMC Microbiol 2009;9:39. |
| 14. | Silva MS, Senna SG, Ribeiro MO, Valim AR, Telles MA, Kritski A, et al. Mutations in katG, inhA, and ahpC genes of Brazilian isoniazid-resistant isolates of Mycobacterium tuberculosis. J Clin Microbiol 2003;41:4471-4. |
| 15. | Supply P, Allix C, Lesjean S, Cardoso-Oelemann M, Rüsch-Gerdes S, Willery E, et al. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 2006;44:4498-510. |
| 16. | Allix-Béguec C, Harmsen D, Weniger T, Supply P, Niemann S. Evaluation and strategy for use of MIRU-VNTRplus, a multifunctional database for online analysis of genotyping data and phylogenetic identification of Mycobacterium tuberculosis complex isolates. J Clin Microbiol 2008;46:2692-9. |
| 17. | Lacoma A, Garcia-Sierra N, Prat C, Ruiz-Manzano J, Haba L, Rosés S, et al. GenoType MTBDRplus assay for molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis strains and clinical samples. J Clin Microbiol 2008;46:3660-7. |
| 18. | Rinder H, Mieskes KT, Löscher T. Heteroresistance in Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2001;5:339-45. |
| 19. | Shamputa IC, Jugheli L, Sadradze N, Willery E, Portaels F, Supply P, et al. Mixed infection and clonal representativeness of a single sputum sample in tuberculosis patients from a penitentiary hospital in Georgia. Respir Res 2006;7:99. |
| 20. | van Rie A, Victor TC, Richardson M, Johnson R, van der Spuy GD, Murray EJ, et al. Reinfection and mixed infection cause changing Mycobacterium tuberculosis drug-resistance patterns. Am J Respir Crit Care Med 2005;172:636-42. |
| 21. | Zetola NM, Shin SS, Tumedi KA, Moeti K, Ncube R, Nicol M, et al. Mixed Mycobacterium tuberculosis complex infections and false-negative results for rifampin resistance by GeneXpert MTB/RIF are associated with poor clinical outcomes. J Clin Microbiol 2014;52:2422-9. |
| 22. | Miotto P, Piana F, Penati V, Canducci F, Migliori GB, Cirillo DM. Use of genotype MTBDR assay for molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical strains isolated in Italy. J Clin Microbiol 2006;44:2485-91. |
| 23. | Mäkinen J, Marttila HJ, Marjamäki M, Viljanen MK, Soini H. Comparison of two commercially available DNA line probe assays for detection of multidrug-resistant Mycobacterium tuberculosis. J Clin Microbiol 2006;44:350-2. |
| 24. | Bolotin S, Alexander DC, Chedore P, Drews SJ, Jamieson F. Molecular characterization of drug-resistant Mycobacterium tuberculosis isolates from Ontario, Canada. J Antimicrob Chemother 2009;64:263-6. |
| 25. | Tolani MP, D'souza DT, Mistry NF. Drug resistance mutations and heteroresistance detected using the GenoType MTBDRplus assay and their implication for treatment outcomes in patients from Mumbai, India. BMC Infect Dis 2012;12:9. |
| 26. | Böttger EC. Chapter 14: Drug resistance in Mycobacterium tuberculosis: Molecular mechanisms and laboratory susceptibility testing. Donald PR, van Helden PD, editors. Antituberculosis Chemotherapy. Prog Respir Res 2011;40:128-44. |
| 27. | Cohen T, van Helden PD, Wilson D, Colijn C, McLaughlin MM, Abubakar I, et al. Mixed-strain Mycobacterium tuberculosis infections and the implications for tuberculosis treatment and control. Clin Microbiol Rev 2012;25:708-19. |
| 28. | Woodford N, Ellington MJ. The emergence of antibiotic resistance by mutation. Clin Microbiol Infect 2007;13:5-18. |
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
|
Search Pubmed for