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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~ Acknowledgements
 ~  References
 ~  Article Figures
 ~  Article Tables

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BRIEF COMMUNICATION
Year : 2014  |  Volume : 32  |  Issue : 3  |  Page : 318-322
 

Evaluation of the GenoType MTBDR assay for detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex isolates


1 Department of Microbiology, Faculty of Medicine, Akdeniz University, Antalya, Turkey
2 Department of Microbiology, Faculty of Medicine, Eskisehir Osmangazi University, Eskisehir, Turkey
3 Department of Microbiology, Faculty of Medicine, Istanbul University, Istanbul, Turkey

Date of Submission26-Jul-2013
Date of Acceptance10-Oct-2013
Date of Web Publication10-Jul-2014

Correspondence Address:
I Saglik
Department of Microbiology, Faculty of Medicine, Akdeniz University, Antalya
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.136587

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 ~ Abstract 

Detection of drug resistance plays a critical role in tuberculosis treatment. The aim of this study was to evaluate the performance of GenoType Mycobacteria Drug Resistance (MTBDR) assay (Hain Lifescience, Germany) and to compare it with radiometric BACTEC 460 TB system (Becton Dickinson, USA) for the detection of rifampicin (RIF) and isoniazid (INH) resistance in 84 Mycobacterium tuberculosis complex (MTBC) isolates. RIF resistance was identified in 6 of 7 (85.7%) isolates and INH resistance was identified in 8 of 14 (57.1%) isolates by the GenoType MTBDR assay. Compared with BACTEC system, the sensitivity, specificity, positive predictive value and negative predictive values were 85.7%, 98.7%, 85.7% and 98.7% for RIF resistance; and 57.1%, 100%, 100% and 92.1% for INH resistance, respectively. GenoType MTBDR assay is reliable when tested specimen is resistant to the tested drugs. Although test was more successful in the detection of RIF resistance, it exhibited low sensitivity for the detection of INH resistance.


Keywords: Drug resistance, isoniazid, mycobacterium tuberculosis complex, rifampicin


How to cite this article:
Saglik I, Oz Y, Kiraz N. Evaluation of the GenoType MTBDR assay for detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex isolates. Indian J Med Microbiol 2014;32:318-22

How to cite this URL:
Saglik I, Oz Y, Kiraz N. Evaluation of the GenoType MTBDR assay for detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex isolates. Indian J Med Microbiol [serial online] 2014 [cited 2020 Apr 7];32:318-22. Available from: http://www.ijmm.org/text.asp?2014/32/3/318/136587



 ~ Introduction Top


Multidrug-resistant-tuberculosis (MDR-TB), defined as the resistance to at least isoniazid (INH) and rifampicin (RIF), is mostly associated with high mortality rates. Rapid identification of MDR-TB strains is important for early initiation of appropriate therapy. Conventional drug susceptibility testing (DST) is a time-consuming process, relatively inexpensive and does not require any sophisticated equipment. Inappropriate choice of treatment regimen, wait time for the conventional DST may result in death for drug-resistant-TB cases. The radiometric BACTEC 460 TB DST is a rapid method with significantly low turn-around time and is more sensitive than conventional DST. However, with BACTEC 460TB DST may take 5-12 days to obtain results. Nevertheless, it is widely recognised as a reference method and many alternative systems have been often measured against it in the past two decades. [1],[2]

Molecular assays that are available today allow the prediction of drug resistance in clinical Mycobacterium tuberculosis (MTB) isolates within one working day. One of such molecular assay, GenoType Mycobacteria Drug Resistance (MTBDR) (Hain Lifescience GmbH, Nehren, Germany), is a commercial assay, which use a DNA strip technology and a reverse hybridisation method for a line probe assay. The MTBDR assay (first version introduced in 2004) identifies mutations in the rpoB gene as well as mutations in the katG gene for high-level INH resistance. [3] The GenoType MTBDR plus, the second-generation assay, also detects mutations in the inhA gene that confers resistance to low-levels of INH. [3],[4],[5]

The objects of this study are to evaluate the performance of GenoType MTBDR assay and to compare GenoType MTBDR assay with radiometric BACTEC 460 TB system for the detection of RIF and INH resistance in Mycobacterium tuberculosis complex (MTBC) isolates.


 ~ Materials and Methods Top


This study includes 84 clinical MTBC strains (22 female and 62 male, mean age 39.7 [17-71 years]), one sputum sample per patient isolated from sputum specimens of the patients in the Medical Faculty of Eskisehir Osmangazi University (Central Anatolia Region), Eskisehir, Turkey, in 2007. Seventy patients were newly diagnosed, whereas 14 patients were receiving anti-TB therapy. The strains were isolated from sputum specimens by BACTEC 460 liquid culture system (Becton Dickinson, Sparks, MD, USA). DST for INH and RIF was performed with the modified proportion method in the BACTEC system (Becton Dickinson, Sparks, MD, USA) according to the manufacturer's instructions. Differentiation of MTBC and non-TB mycobacteria was performed by conventional tests and the BACTEC 460 p-nitro-a-acetylamino-b-hydroxypropiophenone (NAP) test. Additionally, the BACTEC DST was applied for all strains as a routine laboratory step. The GenoType MTBDR assay (Hain Lifescience GmbH, Nehren, Germany) was performed according

to the manufacturer's instructions. For the isolation of DNA, 750 μl of actively growing liquid cultures was used. Following the extraction, 5 μl of the exracts and 1.25 units/reaction Taq Gold Polymerase (Applied Biosystems, USA) were used for polymerase chain reaction (PCR) amplification. The PCR products were denatured and were hybridised to a strip with specific oligonucleotide probes in an automated washing and shaking device (Automated Line Probe Assay System; Auto-LiPA, Innogenetics). Each strip contained 17 probes including five amplification and hybridisation controls (conjugate control [CC]; amplification control [UC; 23S rRNA]; MTBC specific control [23S rRNA]; rpoB [rpoB] and katG [katG] amplification control) to verify the test procedures, as shown in [Figure 1]. The standard strain M. tuberculosis H37Ra (ATCC 25177), which is susceptible to RIF and INH, was used as negative control throughout the study. Two isolates tested for RIF resistance and six isolates tested for INH resistance (one of them was the identical strain), which had incompatible results, were repeated using the MTBDR assay. Additionally, two invalid results, which had weakly intensity control bands, were repeated using more extracted samples by the MTBDR assay. The results of the test were evaluated according to the manufacturer's instructions.


 ~ Results Top


In this study, the INH and RIF resistance of MTBC isolates in sputum specimens of 84 patients were evaluated by GenoType MTBDR assay and BACTEC DST system. The presence of MTBC in the sample was confirmed by the MTBC-specific probe in all samples. The percentages of resistant isolates for RIF and INH were 8.3 (n = 7) and 16.6 (n = 14), respectively, by the BACTEC system. Sixty-nine isolates (82.1%) were susceptible to both anti-TB agents by the BACTEC system and MTBDR assay. Five isolates (6%) were resistance to both anti-TB agents by the BACTEC system and MTBDR assay. RIF resistance was correctly identified in six of seven isolates (85.7%) by the MTBDR assay [Table 1]. INH resistance was observed in 8 of 14 (57.1%) isolates by the MTBDR assay. Six INH resistant isolates were found as INH susceptible by the MTBDR assay [Table 2]. All of the INH-resistant isolates had katG S315T1 mutant probe hybridisation positivity (together with the loss of hybridisation band katG WT). We observed the loss of hybridisation band with rpoB wild-type 5 (WT5) probe for three isolates (hybridisation positivity was associated with S531L mutant probe band [MUT3] for two of them) and rpoB WT2 probe for other three isolates in the hybridisation patterns of RIF resistant isolates [Table 3] and [Figure 1]. One of the RIF resistant isolates was identified as RIF susceptible by the MTBDR assay. Although one isolate was found to be resistant by MTBDR assay, it was detected as susceptible by BACTEC system.
Table 1: Comparison of Genotype MTBDR test results with BACTEC 460 TB system data for RIF


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Table 2: Comparison of Genotype MTBDR test results with BACTEC 460 TB system data for INH


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Table 3: Genotype MTBDR test results for patterns


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Figure 1: MTBDR assay patterns: controls; conjugate (CC), amplification(UC), MTBC (Tub) and rpoB. rpoB wild-type probes (WT 1-5) and mutant probes (MUT 1,2A,2B,3); katG control; katG wild-type probe (WT); katG mutation probes (T1 and T2). Lane 1, katG WT deletion, katG T1 (S315T1); lane 2, rpoB WT2 and katG WT deletion, katG T1 (S315T1); lane 3 and 4, rpoB WT2 and katG WT deletion, katG T1 (S315T1); lane 5, rpoB WT1 and katG WT deletion, katG T1 (S315T1); lane 6, katG WT deletion, katG T1 (S315T1); lane 7, rpoB WT5 deletion; lane 8 and 9, rpoB WT5 deletion, rpoB MUT3 (S531L), katG WT deletion, katG T1 (S315T1)

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 ~ Discussion Top


In comparison to the BACTEC DST system, the MTBDR is an accurate and time-efficient method for the detection of resistance, it provides up with the MTBC detection within approximately one working day. However, proper laboratory design, standard biosafety procedures and quality control to avoid cross-contamination are required for the MTBDR, which is similar to other nucleic acid amplification assays. The BACTEC DST system is cheaper than the MTBDR but it is not easy to perform requiring high standards of biosafety. The use of radioactive materials, with the need for disposal of radioactive waste, represented the major disadvantage of the BACTEC system. [2]

The GenoType MTBDR assay detects resistance based on reverse hybridisation method. Another commercially available molecular test based on the reverse hybridisation principle is LiPA Rif-TB test (Innogenetics, Ghent, Belgium). This test has been shown to be highly sensitive and specific in the detection of RIF-resistant MTBC isolates. Although, this test identifies strains of the RIF resistant MTBC, it cannot evaluate the mutations leading to INH resistance. [6],[7],[8] In contrast, the GenoType MTBDR assay rapidly detects not only MTBC, but also the most common mutations associated with RIF and INH resistance in these isolates. RIF resistance is often considered as a marker for MDR-TB isolates. INH is an anti-TB drug that is used for both prophylaxis and treatment of MTBC infections. Therefore, the determination of the susceptibilities against these two drugs is critical. [9] Studies about DNA sequencing have proved that approximately 95% of RIF-resistant strains have a mutation within the 81-bp hotspot region of the rpoB gene. When gene mutations causing RIF resistance occur in a limited region, better results can be obtained by molecular methods developed for the determination of RIF resistance. [7],[8],[9],[10],[11],[12] Our study suggested a similar result where MTBDR test detected RIF resistance for six of seven isolates successfully (sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV) were 85.7%, 98.7%, 85.7% and 98.7%, respectively). Many previous studies have also found that sensitivity (82-99%) and specificity (95-100%) of MTBDR assay for the detection of RIF resistance to be high and the most common mutation area is codon 531 on the rpoB gene region. [3],[8],[9],[10],[11],[12],[13],[14] One isolate was found to be susceptible to RIF by BACTEC system, whereas MTBDR assay suggested that it is resistant to RIF. This isolate had rpoB WT1 hybridisation band loss. Previous studies indicate that the correlation between phenotypic resistance and genotypic mutations is not absolute. In addition, resistance mutation may be associated with variable phenotypic expression of drug resistance (low, moderate or high-level). Silent mutations may occur at the genetic level but no change in the drug susceptibility pattern. [8],[15] MTBDR assay could incorrectly identify silent or neutral mutations that are phenotypically susceptible. Nevertheless such mutations are considered as an insignificant problem. [6],[11],[13] We believe that, there is no phenotypic resistance in this isolate or there is low-level phenotypic resistance than the level of BACTEC DST, which could be detected (final drug concentration was 2.0 μg/ml for RIF).

Unlike RIF resistance mutations, mutations causing INH resistance are not limited to a specific gene region. It is reported that mutations on the position codon 315 of the katG gene are responsible for INH resistance in 60% of the cases worldwide. Additionally, INH resistance is caused by mutations in the inhA regulatory and coding region, and the ahpC-oxyR, ndh, and kasA genes.[9-14,16] Previous studies demonstrated that, MTBDR assay is not as successful for the detection of INH resistance as for the detection of RIF resistance. In general, high specificity (100%) and low sensitivity (67-88%) values have been observed. [3] In this study, MTBDR assay could detect the INH resistance in 8 (57.1%) of 14 isolates and it had low sensitivity and high specificity (sensitivity, specificity, PPV and NPV; 57.1%, 100%, 100% and 92.1%). The most frequent mutation causing INH resistance is observed to be in the katG S315T1 region. [3],[9],[10],[11],[12],[13],[14] However, due to the spread of resistant strains in the community, the incidence of mutations that cause resistance varies among different regions in the World. Previous studies in our country have shown that mutations at codon 315 were 60-77% of the INH-resistant isolates. The most frequent mutation pattern at codon 315 was Ser315Thr. [11],[17],[18] Our study also detects a similar result as all isolates with INH resistant had katG S315T1 mutant probe positivity. Ninety percent of the RIF-resistant MTB isolates in the Turkish patients were found to carry a mutation in rpoB gene region and codons possessing point mutations were 516 (7-24%), 526 (14-19%) and 531 (48-64%). [10],[11],[17],[18],[19],[20] We observed that the most common mutation causing RIF resistance was in rpoB S531L mutant probe region in this study. The rate of mutations identified is substantially similar with the results of other studies carried out in Turkey. Presumably, the six isolates (with resistance INH could not be detected with the MTBDR) carry a mutation in other genomic regions, such as fabG-inhA or other gene regions. However, some studies in our region could not demonstrate the existence of genotypic resistance as a part of the phenotypically resistant strains. [19],[20]

The MTBDR assay could not detect RIF resistance for one isolate (this isolate also had INH resistance that could not be detected by the

MTBDR assay). It is known that marginal or incomplete amplification could cause especially more than one negative signal. [11] Probably, the gene mutations causing these resistance may be out of the target region of the MTBDR assay. DNA sequencing is the gold standard method, which is detection of mutations to be responsible from drug resistance. Unfortunately it could not be performed in this study.

There are several studies showing increased sensitivity (from 67% to 91%) by GenoType MTBDR plus assay, which is the next version of Genotype MTBDR assay. The GenoType MTBDR plus assay has been found highly successful the detection of the RIF resistance. Although the sensitivity of MTBDR plus assay has increased for the detection of INH resistance, it is not successful as much as for the detection of RIF resistance. [4],[5],[21],[22],[23] Brossier et al., reported that the GenoType MTBDR plus assay has increased the detection of the low level INH resistance, however, it could not detect some of isolates with high level INH resistance. [4]

The cost of the MTBDR is relatively high and it may be expensive to be routinely used throughout the world. Thus, use of the MTBDR is more likely for patients with a high likelihood of MDRTB (e.g. patients with treatment failure or relapse from high incidence areas and/or previously treated patients). [1] Cost-effectiveness and cost-benefit of MTBDR are dependent on screening and diagnostic algorithms in different epidemiological settings. Furthermore, the MTBDR assay can be used on isolates from both solid and liquid culture and it can be applied directly on smear-positive pulmonary specimens. [9] Moreover, the World Health Organisation (WHO) recommended the use of molecular line probe assays for rapid diagnosis of MDR-TB in low and middle income settings. WHO also provides funds on molecular line probe assays for developing countries with high TB incidence. [24],[25]


 ~ Conclusion Top


GenoType MTBDR assay is a practical method that can be easily applied even in basic molecular laboratories. This test allows rapid and specific detection of the most frequent mutations leading to RIF and INH resistance in clinical MTBC isolates using strips. However, tests have been found more successful in the detection of RIF resistance. An important disadvantage of GenoType MTBDR assay is that it cannot detect the mutations in different gene regions because it can only detect the known major mutations in specific gene regions . In spite of the advances in molecular methods, confirmation of the results is still required with conventional methods in laboratories that use molecular tests for the detection of RIF and INH resistance.


 ~ Acknowledgements Top


This work was supported by the Research Projects Commission of Eskisehir Osmangazi University (number: 11023).

 
 ~ References Top

1.Migliori GB, Matteelli A, Cirillo D, Pai M. Diagnosis of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis: Current standards and challenges. Can J Infect Dis Med Microbiol 2008;19:169-72.  Back to cited text no. 1
    
2.Cruciani M, Scarparo C, Malena M, Bosco O, Serpelloni G, Mengoli C. Meta-analysis of BACTEC MGIT 960 and BACTEC 460 TB, with or without solid media, for detection of mycobacteria. J Clin Microbiol 2004;42:2321-5.  Back to cited text no. 2
    
3.Ling DI, Zwerling AA, Pai M. GenoType MTBDR assays for the diagnosis of multidrug-resistant tuberculosis: A meta-analysis. Eur Respir J 2008;32:1165-74.  Back to cited text no. 3
    
4.Brossier F, Veziris N, Jarlier V, Sougakoff W. Performance of MTBDR plus for detecting high/low levels of Mycobacterium tuberculosis resistance to isoniazid. Int J Tuberc Lung Dis 2009;13:260-5.  Back to cited text no. 4
    
5.Miotto P, Piana F, Cirillo DM, Migliori GB. Genotype MTBDRplus: A further step toward rapid identification of drug-resistant Mycobacterium tuberculosis. J Clin Microbiol 2008;46:393-4.  Back to cited text no. 5
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23.Friedrich SO, Venter A, Kayigire XA, Dawson R, Donald PR, Diacon AH. Suitability of Xpert MTB/RIF and genotype MTBDRplus for patient selection for a tuberculosis clinical trial. J Clin Microbiol 2011;49:2827-31.  Back to cited text no. 23
    
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