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Multidrug-resistant and heteroresistant Mycobacterium tuberculosis and associated gene mutations in Ethiopia

Open AccessPublished:June 25, 2015DOI:https://doi.org/10.1016/j.ijid.2015.06.013

      Highlights

      • The prevalence of multidrug-resistant tuberculosis (MDR-TB) was 36.3% in Amhara National Regional State, Ethiopia indicating that MDR-TB is increasing with time.
      • Unlike the high prevalence of MDR-TB, the prevalence of rifampicin and isoniazid heteroresistance was lower at 4.7% and 1.13% respectively.
      • Ser531Leu substitution and Ser315Thr1 substitution were the highest gene mutations for rifampicin and isoniazid, respectively.

      Summary

      Background

      The prevalence of multidrug-resistant tuberculosis (TB) among new and retreatment cases in 2011 in Ethiopia was 2.7% and 17.9%, respectively. However, data on heteroresistance and gene mutation profiles of Mycobacterium tuberculosis were not documented.

      Methods

      A cross-sectional study was conducted on 413 TB-positive clinical specimens submitted between 2012 and 2014 to Bahir Dar Regional Laboratory Center for confirmation of multidrug resistance. Resistance determining genes were analyzed using a line probe assay.

      Results

      Of 413 M. tuberculosis isolates, 150 (36.3%) were multidrug-resistant, 19 (4.6%) were resistant only to rifampicin, and 26 (6.3%) were resistant to isoniazid. Of 169 rifampicin-resistant and 176 isoniazid-resistant isolates, only eight (4.7%) showed rifampicin heteroresistance and only two (1.13%) showed isoniazid heteroresistance. Failing of the rpoB WT8 gene with corresponding hybridization of rpoB MUT3 (S531L substitution) accounted for 85 (50.3%) rifampicin-resistant mutations. Among 176 isoniazid-resistant isolates, 155 (88.1%) strains had the Ser315Thr1 substitution.

      Conclusions

      The prevalence of multidrug-resistant M. tuberculosis was high in the study area. Ser531Leu and Ser315Thr1 substitutions were the highest gene mutations for rifampicin and isoniazid, respectively.

      Keywords

      1. Introduction

      Multidrug-resistant tuberculosis (MDR-TB) is caused by strains of Mycobacterium tuberculosis that are resistant to isoniazid (INH) and rifampicin (RMP).

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      Despite the availability of highly efficacious treatment for decades, tuberculosis (TB) remains a major global health problem.

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      Globally, 3.5% of MDR-TB has been reported in new TB cases and 20.5% in previously treated TB cases.

      World Health Organization. Global tuberculosis report. WHO/HTM/TB/2014.08. Geneva, Switzerland: WHO; 2014.

      World Health Organization. Global tuberculosis control report. WHO/HTM/TB/2013. Geneva, Switzerland: WHO; 2013.

      In Ethiopia in 2011, the prevalence of MDR-TB among new and retreatment cases was reported to be 2.7% and 17.9%, respectively.
      • Lemma S.H.
      Proceedings of the 9th TB-research annual conference (TRAC): 21⿿23 March.
      An erratic drug supply, suboptimal physician prescription, poor patient adherence,
      • Zhang Y.
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      initial high bacterial population, and monotherapy have been associated with the emergence of resistance.
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      Some patients with TB harbor mixed populations of drug-susceptible and resistant organisms, a phenomenon that is referred to as heteroresistance. Therefore, heteroresistant strains are precursors for full resistance.
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      The genetic background of M. tuberculosis related to INH resistance is complex. However, mutations in several genes, including katG (catalase peroxidase coding genes),
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      ahpC, inhA, kasA, and ndh, have all been associated with INH resistance.
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      Between 50% and 95% of INH-resistant strains contain mutations in codon 315 of the katG gene.
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      • Yew W.
      Mechanisms of drug resistance in Mycobacterium tuberculosis [State of the art series]. Drug-resistant tuberculosis.
      Furthermore, 20% and 35% of INH-resistant strains contain mutations in the inhA regulatory region.
      • Barnard M.
      • Parsons L.
      • Miotto P.
      • Cirillo D.
      • Feldmann K.
      • Gutierrez C.
      • et al.
      Molecular detection of drug resistant tuberculosis by line probe assay. Laboratory manual for resource limited settings.
      • Da Silva A.
      • Palomino C.
      Molecular basis and mechanisms of drug resistance in M. tuberculosis: classical and new drugs.
      The most common inhA mutation occurs in its promoter region (C15T) and this is frequently associated with monoresistance.
      • Barnard M.
      • Parsons L.
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      • Cirillo D.
      • Feldmann K.
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      • et al.
      Molecular detection of drug resistant tuberculosis by line probe assay. Laboratory manual for resource limited settings.
      • Da Silva A.
      • Palomino C.
      Molecular basis and mechanisms of drug resistance in M. tuberculosis: classical and new drugs.
      Strains bearing mutations in the coding region of inhA show low-level resistance.
      • Gillespie H.
      Evolution of drug resistance in M. tuberculosis. Clinical and molecular perspective.
      • Jiao W.
      • Mokrousov I.
      • Sun Z.
      • Li M.
      • Liu J.
      • Narvskaya O.
      • et al.
      Molecular characteristics of rifampin and isoniazid resistant M. tuberculosis strains from Beijing.
      Mutations in the RNA polymerase β subunit (rpoB) gene have been found in about 96% of RMP-resistant M. tuberculosis isolates.
      • Da Silva A.
      • Palomino C.
      Molecular basis and mechanisms of drug resistance in M. tuberculosis: classical and new drugs.
      • Jiao W.
      • Mokrousov I.
      • Sun Z.
      • Li M.
      • Liu J.
      • Narvskaya O.
      • et al.
      Molecular characteristics of rifampin and isoniazid resistant M. tuberculosis strains from Beijing.
      • Yue J.
      • Shi W.
      • Xie J.
      • Li Y.
      • Zeng E.
      • Wang H.
      Mutations in the rpoB gene of multidrug-resistant M. tuberculosis isolates from China.
      Mutations in codons 531 and 526 are the most frequently reported mutations.
      • Da Silva A.
      • Palomino C.
      Molecular basis and mechanisms of drug resistance in M. tuberculosis: classical and new drugs.
      • Jiao W.
      • Mokrousov I.
      • Sun Z.
      • Li M.
      • Liu J.
      • Narvskaya O.
      • et al.
      Molecular characteristics of rifampin and isoniazid resistant M. tuberculosis strains from Beijing.
      • Yue J.
      • Shi W.
      • Xie J.
      • Li Y.
      • Zeng E.
      • Wang H.
      Mutations in the rpoB gene of multidrug-resistant M. tuberculosis isolates from China.
      Information on the current prevalence of MDR-TB, heteroresistance, and drug resistance mutations has not been documented in Amhara National Regional State (ANRS), Ethiopia. This study was conducted to determine the prevalence of MDR-TB, heteroresistance, and gene mutations to RMP and INH among presumptive MDR-TB cases in ANRS, Ethiopia.

      2. Materials and methods

      2.1 Study design and sampling technique

      A cross-sectional study was conducted between May 2012 and February 2014. During the study period, 856 presumptive MDR-TB cases (sputum and extrapulmonary (peritoneal fluid, tissue, lymph node aspirate, and pus specimens)) were referred to the Bahir Dar Regional Health Research Laboratory Center (BRHRLC). However, only 413 (48.2%) of these clinical samples were TB-positive. This study included the 413 M. tuberculosis isolates for gene mutation analysis.

      2.2 Specimen processing

      The clinical samples were processed using the N-acetyl-l-cysteine NaOH (NALC-NaOH) method. The processed samples were suspended in 1.0 ml sterile phosphate buffer (pH 7.0) and then 100 μl of resuspended pellet was inoculated onto two Lowenstein⿿Jensen (LJ) medium slants. Smears for microscopic examination were stained using the Ziehl⿿Neelsen (ZN) method.
      DNA was extracted from samples that were smear- and/or culture-positive using GenoLyse chemical methods. From the extracted DNA, 5 μl was used directly for PCR amplification. Master mix preparation, DNA addition, amplification, hybridization, and interpretation were performed as recommended by the manufacturer (Hain Lifescience GmbH, Nehren, Germany).
      • Barnard M.
      • Parsons L.
      • Miotto P.
      • Cirillo D.
      • Feldmann K.
      • Gutierrez C.
      • et al.
      Molecular detection of drug resistant tuberculosis by line probe assay. Laboratory manual for resource limited settings.

      Hain Lifescience. GenoTypeMTBDRplus VER2.0. Instructions for use. IFU-304A-02. Nehren, Germany: Hain Lifescience; 2011.

      Resistance determining genes were analyzed using a line probe assay (LPA).

      2.3 LPA interpretation

      Susceptibility to anti-TB drugs was defined as hybridization (presence of a band) to all the wild-type (WT) probes and no hybridization (absence of a band) to the mutant probes. The absence of hybridization of any WT and/or hybridization of any mutant gene indicates resistance to the respective drugs. Hybridization of WT and mutant genes indicates heteroresistance or a mixed infection (Figure 1).
      Figure thumbnail gr1
      Figure 1Line probe assay strips showing controls and rifampicin (RMP) and isoniazid (INH) resistance-associated banding patterns; Ethiopia, 2015.

      2.4 Statistical analysis

      All data were entered, cleared, and analyzed using IBM SPSS Statistics for Windows, version 20.0 (IBM Corp. Armonk, NY, USA). Descriptive statistics were used to visualize differences within the data. Binary logistic regression was used to assess possible factors associated with MDR-TB and heteroresistance. Gene mutations were analyzed manually.

      2.5 Data quality assurance

      DNA extraction positive (H37Rv) and negative controls and master mix controls were used. Fifty isolates were characterized at the national TB laboratory using the BACTEC MGIT (Mycobacteria Growth Indicator Tube) 960 TB system (BD Diagnostics, USA). Lot-to lot quality assurance systems were in place to verify the quality of the commercial kit. All procedures were done using standard operating procedures.

      2.6 Ethical considerations

      Ethical clearance was obtained from the Amhara Regional Health Bureau Research Ethics Review Committee and official permission was obtained from BRHRLC.

      3. Results

      3.1 Multidrug-resistant tuberculosis

      A total of 413 TB-positive patients were included in the study. Of these, 229 (55.4%) were males. The mean age of participants was 34.5 years and 211 (51.1%) were in the 19⿿34 years age group. Moreover, 342 (82.8%) patients were retreatment TB cases and 71 (17.2%) were newly identified TB cases. With regard to their TB history, 160 (38.7%) were relapse cases, 132 (32%) were failure cases, and 32 (7.7%) were defaulters (Table 1).
      Table 1Demographic characteristics and profile of presumptive MDR-TB cases (N = 413); Ethiopia, 2015
      VariablesNumberPercentage
      SexMale22955.4
      Female18444.6
      Age, years⿤18225.3
      19⿿3421151.1
      35⿿459723.5
      >458320.1
      TB historyNew cases7117.2
      Retreatment cases34282.8
      Treatment historyNew7117.2
      Relapse16038.7
      Failure13232
      Defaulter327.7
      Other184.4
      Total413100
      MDR, multidrug-resistant; TB, tuberculosis.
      Of the 413M. tuberculosis cases, 150 (36.3%) were MDR-TB (RMP+INH), 19 (4.6%) were resistant only to RMP, and 26 (6.3%) were resistant to INH.
      Considering possible associated variables such as gender, age, type of TB, and history of TB treatment, none was significantly associated with MDR-TB or with RMP or INH resistance (data not shown).

      3.2 Heteroresistance

      Of 169 RMP-resistant and 176 INH-resistant M. tuberculosis isolates, eight (4.7%) showed RMP heteroresistance and two (1.13%) showed INH heteroresistance. The possible variables such as gender, age, type of TB, and history of TB treatment were not significantly associated with heteroresistance (data not shown).

      3.3 RMP and INH resistance-associated gene mutations

      Of the 413 isolates, 169 (40.9%) were resistant to RMP. Failing of the rpoB WT8 gene with corresponding hybridization of rpoB MUT3 (Ser531Leu substitution) accounted for 85 (50.3%) mutations. Moreover, failing of WT7 with the appearance of mutants 2A and 2B (H526Y and H526D substitutions, respectively) shared 14.8% of RMP-resistant gene mutation. There was no failing of the rpoB WT1, 5, or 6 genes and no hybridization of the rpoB MUT1 probe (Table 2).
      Table 2Frequency and pattern of rpoB, katG, and inhA mutations of Mycobacterium tuberculosis; Ethiopia, 2015
      rpoB genekatG geneinhA genen (%)
      Failing WT probeMutationLocation of codons of WT/mutant/geneFailing WT probeMutationLocation of codons of WT/mutant/geneFailing WT probeMutationAnalyzed nucleic acid position
      MDR (n = 150)
      WT2 and 3No510⿿513WTMUT1S315T1NoNoNo9 (6)
      WT2, 3, and 4Unknown510⿿520WTMUT1S315T1NoNoNo3 (2)
      WT3 and 4Unknown513⿿519WTMUT1S315T1NoNoNo20 (13.3)
      WT3Unknown514⿿515WTMUT1S315T1NoNoNo1 (0.67)
      WT2No511⿿512NoNoNoWT1MUT1C15T1 (0.67)
      WT7MUT2AH526YWTMUT1S315T1NoNoNo14 (9.3)
      One patient had RMP heteroresistance.
      WT7MUT2BH526DWTMUT1S315T1NoNoNo5 (3.3)
      One patient had INH heteroresistance.
      WT7Unknown526WTMUT1S315T1NoNoNo14 (9.3)
      WT8MUT3S531LWTMUT1S315T1NoNoNo60 (40)
      Five patients had RMP heteroresistance.
      WT8Unknown531⿿533WTMUT1S315T1NoNoNo6 (4)
      WT8MUT3S531LWTNoDele, 315NoNoNo11 (7.3)
      One patient had RMP heteroresistance.
      WT8MUT3S531LNoNoNoWT1MUT1C15T1 (0.67)
      WT8MUT3S531LWTMUT1S315T1WT1MUT1C15T3 (2)
      One patient had INH heteroresistance.
      WT8MUT3S531LWTMUT2S315T2NoNoNo1 (0.67)
      WT8Unknown531⿿533WTNoDele, 315NoNoNo1 (0.67)
      RMP-MR (n = 19)
      WT7MUT2AH526YNoNoNoNoNoNo4 (21)
      WT7MUT2BH526DNoNoNoNoNoNo2 (10.5)
      NoMUT2AH526YNoNoNoNoNoNo1 (5.3)
      One patient had RMP heteroresistance.
      WT8MUT3S531LNoNoNoNoNoNo9 (47.4)
      WT8Unknown531⿿533NoNoNoNoNoNo3 (15.8)
      INH-MR (n = 26)
      NoNoNoWTMUT1S315T1NoNoNo19 (73.1)
      NoNoNoWTNoDele, 315NoNoNo4 (15.4)
      NoNoNoNoNoNoWT1MUT1C15T3 (11.5)
      C, cysteine; D, aspartate; Dele, Deletion; H, histidine; INH, isoniazid; L, leucine; LPA, line probe assay; MDR, multidrug-resistant; MR, Monoresistant; MUT, mutant; RMP, rifampicin; S, serine; T, threonine; WT, wild-type; Y, tyrosine.
      No = no failing of the WT gene, or no appearance of mutant gene; Unknown = there were two or more mutant genes but the LPA did not have a probe for these mutant genes.
      a One patient had RMP heteroresistance.
      b One patient had INH heteroresistance.
      c Five patients had RMP heteroresistance.
      Of 176 INH-resistant strains, 155 (88.1%) were due to failing of the katG WT gene with hybridization of the katG MUT probe (Ser315Thr1 substitution). Of the rest, five (2.8%) were due to failing at the inhA WT1 gene (C15T substitution). Moreover, three (1.7%) INH resistance cases were due to mutations from both Ser315Thr1 and C15T substitutions (Table 2).
      Based on gene mutation analysis, 60 (40%) MDR cases were due to Ser531Leu:Ser315Thr1 substitution. Moreover, the combination mutation of failing at WT3, 4 of RMP and Ser315Thr1 substitution of INH accounted for 20 (13.3%) MDR-TB. H526Y and Ser315Thr1 substitutions were MDR-TB mutations in 14 (9.3%) (Table 2). Out of 26 cases of RMP monoresistance, nine (47.4%) were due to Ser531Leu substitution and four (21%) to H526Y substitution. Nineteen (73.1%) of the INH monoresistance cases were due to Ser315Thr1 substitution and three (11.5%) to C15T substitution (Table 2).

      4. Discussion

      4.1 Multidrug-resistant tuberculosis

      This study showed that MDR-TB is a serious public health problem in Ethiopia. A high prevalence of MDR-TB with lower heteroresistance might indicate a high transmission rate of primary resistant TB in this area.
      The prevalence of MDR-TB in this study was higher than the drug resistance survey in Ethiopia.
      • Lemma S.H.
      Proceedings of the 9th TB-research annual conference (TRAC): 21⿿23 March.
      The reason for this might be differences in study population; the present study population comprised presumptive MDR-TB cases, but the Ethiopia drug resistance survey involved smear-positive TB cases. Furthermore, the prevalence is higher than those reported from Germany (4%), Iran (12.2%), China (5.6%), in World Health Organization (WHO) reports, and Swaziland.

      World Health Organization. Global tuberculosis control report. WHO/HTM/TB/2013. Geneva, Switzerland: WHO; 2013.

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      The reason for this might again be differences in study population, but might also be related to the time of the study, geography, and methodological differences.

      4.2 Heteroresistance

      The prevalence of RMP heteroresistance (mixed infection) was 1.9% (n = 8). Only two (0.5%) cases of INH heteroresistance were detected in this study. The proportion of heteroresistance in this study is lower than that reported in studies conducted in Uzbekistan and India.
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      The LPA is capable of detecting the presence of heteroresistance since the strips contain both WT and mutant probes (Figure 2A : bands 12 and 16).
      Figure thumbnail gr2
      Figure 2Line probe assay strips showing the heteroresistant population of Mycobacterium tuberculosis; Ethiopia, 2015.
      Source: Barnard M, Parsons L, Miotto P, Cirillo D, Feldmann K, Gutierrez C, Somoskovi A. Molecular detection of drug-resistant tuberculosis by line probe assay. Laboratory manual for resource-limited settings (http://www.finddiagnostics.org).

      4.3 RMP and INH resistance-associated gene mutations

      The assessment of gene mutations showed that codon 531 of the rpoB gene and codon 315 of the katG gene accounted for 50.3% and 88.1% of RMP and INH resistance, respectively. This finding is in agreement with those of studies done in China, Sweden, Turkey, Ethiopia, and India.
      • Jiao W.
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      • Sun Z.
      • Li M.
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      However, the level of Ser531Leu substitution was lower than reported in studies done in Brazil, Pakistan, China, and Nepal.
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      • Farooqi J.Q.
      • Khan E.
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      • Ali A.
      • Hasan Z.
      • Hasan R.
      Line probe assay for detection of rifampicin and isoniazid resistant tuberculosis in Pakistan.
      • Zhang L.
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      • Duo L.
      • Wang T.
      • Song X.
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      • Poudel A.
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      • Suzuki H.
      • Pandey B.D.
      • Maharjan B.
      • et al.
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      This might be due to differences in the strain and lineage of isolates. The mutation frequency of INH resistance due to C15T substitution in this study was 1.9% (n = 8); however, studies done in Pakistan, China, and Nepal have shown a higher prevalence of C15T substitution.
      • Farooqi J.Q.
      • Khan E.
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      • Hasan R.
      Line probe assay for detection of rifampicin and isoniazid resistant tuberculosis in Pakistan.
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      • Ye Y.
      • Duo L.
      • Wang T.
      • Song X.
      Application of Genotype MTBDRplus in rapid detection of the M. tuberculosis complex as well as its resistance to isoniazid and rifampin in a high volume laboratory in southern China.
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      • Maharjan B.
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      This study showed a high prevalence of MDR-TB and a lower prevalence of heteroresistant M. tuberculosis. Ser531Leu and Ser315Thr1 substitutions were the highest gene mutations for rifampicin and isoniazid, respectively.

      Acknowledgements

      We are grateful for the collaboration of the Cooperative Network of Tropical Diseases Research Center (Red de Investigación cooperative de Enfermedades Tropicales-RICET, Project RD12/0018/0001, Spain that enables the research.
      We express our deep appreciation to BRHRLC, the Ethiopian Public Health Institute-TB Research Team, and Mr Blessing T. Marondera (TB Laboratory Expert Consultant-Ethiopia, FIND Diagnostics/ExpandTB Project) for their help and advice on the quality assurance part of the study. Our deepest appreciation goes to Dr Elizabeth Thomas, American Society of Microbiology Lab Cap consultant for editing the language. We also thank the study participants.
      Conflict of interest: We the authors declare that we have no competing interests.

      Appendix A. Supplementary data

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