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 Table of Contents  
Year : 2016  |  Volume : 5  |  Issue : 2  |  Page : 142-147

Genotyping of mutations detected with GeneXpert

1 Laboratoire National de Référence de la Tuberculose, Institut Pasteur de Côte d'Ivoire, 01 BP 490 Abidjan 01, Cote d'Ivoire
2 Supranational Laboratory for Tuberculosis, Milan, Italy

Date of Web Publication9-Feb-2017

Correspondence Address:
K N'guessan Kouassi
Laboratoire National de Référence de la Tuberculose, Institut Pasteur de Côte d'Ivoire, 01 BP 490 Abidjan 01
Cote d'Ivoire
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Source of Support: None, Conflict of Interest: None

DOI: 10.1016/j.ijmyco.2016.01.001

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Objective/background: Tuberculosis remains an important cause of mortality worldwide. Previous tuberculosis treatment is a strong determinant of multi-drug resistant tuberculosis. The study objective was to describe the mutations detected of Mycobacterium tuberculosis (MTB) complex clinical strains screened with GeneXpert isolated from previously treated patients in Côte d'Ivoire.
Sputum collected and decontaminated by the n-acetyl-l-cysteine method was used to perform Ziehl–Neelsen staining, GeneXpert MTB/rifampicin, and culture on Lowenstein–Jensen medium. Drug susceptibility testing (DST) for first-line drugs was performed in a Bactec 960 Automated System. After strain identification by antigen MPT64 detection, DNA extraction, and genotyping with MTBDRplus assay was performed and interpreted. The strains muted in rpoB without a specific protein identified and were sequenced.
Mutant sequences were detected in 60 sputum samples with GeneXpert MTB/rifampicin of which 55 were confirmed multi-drug resistant MTB strains after DST. The most frequent mutations responsible for rifampin resistance were detected with MTBDRplus assay for 49 (81.7%) clinical strains, while sequencing was required for 11 (18.3%). H526Q mutation, L533P, and D516V associated respectively with L533P, A532A, and S522L, and were observed for three relapse cases. For these cases, GeneXpert and sequencing results were concordant. Discrepancies between GeneXpert and mycobacteria growth indicator tube-DST for rifampin were observed for three strains, on which D516Y, H526C, and L533P were identified.
Conclusion: In the setting of a high prevalence of drug resistance, characterization of the genetic basis of MTB strains resistant to rifampin could be screened first with MTBDRplus.

Keywords: Molecular assays, Resistance, Rifampin, Tuberculosis

How to cite this article:
Kouassi K N, Riccardo A, Christian C D, André G, Férilaha C, Hortense SA, Jean-marc A, Maria CD, Mireille D. Genotyping of mutations detected with GeneXpert. Int J Mycobacteriol 2016;5:142-7

How to cite this URL:
Kouassi K N, Riccardo A, Christian C D, André G, Férilaha C, Hortense SA, Jean-marc A, Maria CD, Mireille D. Genotyping of mutations detected with GeneXpert. Int J Mycobacteriol [serial online] 2016 [cited 2019 Aug 21];5:142-7. Available from: http://www.ijmyco.org/text.asp?2016/5/2/142/199922

  Introduction Top

Tuberculosis remains an important cause of morbidity and mortality worldwide [1]. The development of multidrug-resistant tuberculosis (MDR-TB), defined as resistance to at least isoniazid (INH) and rifampin (RMP) [2] represents a public health concern and threatens global TB control programs [3]. A variety of reasons may explain the emergence of Mycobacterium tuberculosis (MTB) resistant strains (interruption of treatment, inappropriate treatment, poor monitoring of patients under treatment, etc.). The World Health Organization (WHO) report 2013 estimated that MDR-TB cases among new and previously treated cases were 3.6% and 20.2%, respectively [1]. Sub-Saharan Africa shows the highest incidence of TB with 1.3 million new cases per year [1]. As in many Sub-Saharan African countries, TB is a major health problem in Côte d'Ivoire. In fact, 25,299TB cases were notified in 2013: that represents an estimated incidence of 106/100,000 population. The MDR-TB cases are estimated to be 2.5% and 53.2%, respectively, for new and previously treated cases [4],[5]. RMP and INH are the cornerstone current TB treatment. As RMP resistance is a good surrogate marker for multidrug resistance (MDR) [6],[7], the rapid detection of the mutations in a specific genomic region (RMP resistance-determining region) can contribute to a fast diagnosis of MDR-TB, allowing the initiation of an appropriated treatment. Several methods for rapid drug resistance detection have been validated [8]. GeneXpert MTB/rifampicin (RIF) assay endorsed by WHO [9] is one of the most used in routine analysis to confirm active TB cases and to detect mutations conferring RMP resistance.

This study objective was to characterize the mutations of MTB complex clinical strains screened with GeneXpert (Cepheid, Inc., Sunnyvale, CA, USA).

  Materials and methods Top

Sputum samples from 63 pulmonary TB cases smear positive (failure, relapse, defaulter) were recruited in the Reference Regional Center for TB and in the Teaching Hospital of Cocody and Treichville.

For each patient enrolled, two sputum samples were collected and transported at 4°C in an icebox to the National Reference Tuberculosis Laboratory at Institut Pasteur de Côte d'Ivoire.

Manipulations of infectious clinical specimens were performed in a Class II safety cabinet in a BLS3 laboratory. According to the WHO standard recommended procedures [10] (reference), sputum samples were decontaminated with N-acetyl-l-cystein, 4% NaOH-2.9% citrate (final concentration of NAOH 1%), followed by an incubation period at room temperature of 15min. Sputum samples were centrifuged at 3000g for 20min. The supernatant of concentrated sputum were carefully eliminated. The pellet was resuspended with 2mL of sterile phosphate buffer.

The GeneXpert MTB/RIF assay was performed according to manufacturer instructions. The GeneXpert MTB/RIF assay definition files version 4.4a was used. Data analysis for RMP-resistance detection was reported with cycle threshold differences superior to 4.5 as suggested by the manufacturer.

One hundred microliters of resuspended pellet were used to perform a smear which was stained by the Ziehl–Neelsen method. Two hundred microliters of suspension were inoculated on two Lowenstein–Jensen tubes and incubated at 37°C for 6weeks. Positive cultures were examined for acid fast bacilli detection using Ziehl–Neelsen staining. For diagnosis of MTB complex, detection of MPT64 was done in accordance with the manufacturer's procedure SB (Standard Diagnostics, Seoul, South Korea). Mycobacterium Growth Indicator Tube 960 automated drug susceptibility testing (MGIT-DST) was performed for first- and second-line drugs according to the Becton Dickinson product and procedure manual (BD Biosciences, Sparks, MD, USA). SIRE kit procedure (MGIT, Becton-Dickinson) was used for first-line drugs. The critical concentration of each drug was 1.0μg/mL, 0.1μg/mL, 1.0μg/mL, and 5μg/mL respectively for streptomycin, INH, RMP, and ethambutol. MGIT tubes inoculated were incubated in the automated MGIT 960 for 12days.

For each DST validated, 100mL of the negative control were transferred in 1,500μL eppendorf tube containing Tris-ethylenediaminetetraacetic acid. Bacterial suspension was resuspended by vortexing and was inactivated at 95°C for 20min. MTB complex strains inactivated were incubated in ultrasonic bath for 15min and centrifuged at 13,000g for 5min. Supernatant containing DNA was transferred into a fresh tube. For DNA extraction, a negative control was included in each run.

With DNA extracted, Genotype MTBDRplus assay version 2.0 (Hain Lifescience, Nehren, Germany) was performed as recommended by the manufacturer. The amplification mixture contained 35μL of primer-nucleotide Mix B, 10μL of Mix A (5μL 10× polymerase chain reaction buffer, 2μL of MgCl2, 3μL of molecular water, 1 unit of thermostable Taq DNA polymerase), and 5μL of extracted chromosomal DNA solution.

Amplification parameters used were: 15min of denaturation at 95°C, followed by 20 cycles of 30s at 95°C, and 2min at 65°C, followed by 30 additional cycles of 25s at 95°C, 40s at 53°C, and 40s at 70°C, ending with a final extension step of 8min at 70°C (1 cycle).

Hybridization and detection were performed with a TwinCubator (Hain Lifesciences GmbH, Germany) semi-automated washing and shaking device according to the manufacturer's instructions and using the reagents provided with the kit. Twenty microliters of denaturation solution was mixed to 20μL of amplified sample. Mixed solution was incubated at room temperature for 5min. One milliliter of prewarmed hybridization buffer was added before the membrane strips were placed and shaken in the hybridization solution for 30min at 45°C. After two washing steps, a colorimetric detection of the hybridized amplicons was obtained by the addition of the streptavidin alkalinephosphatase conjugate.

An internal quality control process with positive and negative controls was implemented during the study. An interpretable MTBDRplus assay was defined as a test strip with all control markers positive, including results of the markers for positive control (H37Rv strain), and negative control for DNA extraction and for mix preparation.

Strains with a mutation in the rpoB gene without specific protein detected were inactivated in ethanol (70%) and sent to Supranational Laboratory of Milan (Italy). Sequencing of the rpoB gene was performed. After sequence analysis and interpretation, the results obtained were sent to the National Reference Tuberculosis Laboratory located at Institut Pasteur de Côte d'Ivoire.

Data were entered in Microsoft Office Excel 2013. The gold standard for RMP susceptibility was MGIT 960 automated DST. Descriptive analysis was done based on data.

  Results Top

Sixty-three patients were recruited in different areas (North, West, South, East, Center) of Côte d'Ivoire. Of the 63 patients enrolled, 27 did not live in the Abidjan District. The mean age of patients was 32.6±11years. The youngest and oldest patients were aged 15years and 73years, respectively. Failure cases were 44, of which nine were co-infected (TB/human immunodeficiency virus-1). In this group the number of male patients was twice as high as women patients. Direct examination after Ziehl–Neelsen staining was negative for two clinical failure cases.

The second group was composed by 19 patients, of whom 17 and two were relapse cases and defaulters, respectively. Of the six women, one case of co-infection (TB/human immunodeficiency virus-1) was identified. The presence of acid fast bacilli was detected in the sputum of 18 patients ([[Table 1]]). Of the 63 patients enrolled, 32 (88.9%) and 23 (85.2%) living in the Abidjan District and outside, respectively, were infected by MTB strains resistant to at least to RMP and INH ([Table 2]).
Table 1: Socio-demographic, Clinical, and Bacteriologic Characteristics of Patients Recruited.

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Table 2: Description of results from tests performed and origin of the patients.

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Of the 63 GeneXpert MTB/RIF assays performed, curve amplification analyzed revealed that probes B did not bind to the mutant sequence for 24 sputum samples. Mutations detected interpreted as resistant to RMP was confirmed for 23 isolates with MGIT RMP susceptibility testing. Resistance to INH was also confirmed for these 23 clinical isolates ([Table 2]).

Among the three sputum samples for which five probes were bound to wildtype, a mutation in the rpoB gene was detected for one. This last molecular result and DST in liquid medium (RMP) were concordant ([Table 2]).

For one sputum sample, none of the five probes of GeneXpert MTB/RIF assay bound to a wildtype sequence. Culture performed on Lowenstein–Jensen was positive and permitted to identify a strain of MTB complex. The clinical strain was pan-susceptible for drugs tested ([Table 2]).

In total, discrepancies between GeneXpert MTB/RIF assay and MGIT RMP susceptibility testing were observed for four (6.3%) samples ([Table 2]).

Among the patients included, hybridization of DNA extracted from the strains showed a D516V, H526D, H526Y, and S531L mutation in the rpoB gene for 23, six, nine, and 11 patients, respectively ([Table 3]). Sequencing of the rpoB gene realized on eight strains revealed an insertion 1542TTC (codon 514), several punctual mutations at codon 526, and two mutations at the codon 533 ([Table 4]).
Table 3: Mutations described and drug susceptibility testing results for failure cases.

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Table 4: RpoB Gene Sequencing Results.

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Nine strains with a S531L mutation in the rpoB gene associated with a S315T mutation in the katG gene were observed. For one of them, phenotypic and molecular results were discordant ([Table 3]).

Discrepancies between phenotypic and molecular methods were also observed for three others cases. For two of them, mutant sequences did not hybridize with the probes 7 and 3–4, respectively ([Table 3]).

A clinical strain with mutant sequences detected using GeneXpert MTB/RIF assay (probe E) and MTBDRplus assay (probe 8) was interpreted susceptible with a phenotypic method ([Table 2] and [Table 3]). Sequencing of the rpoB gene revealed a L533P mutation ([Table 4]).

Sequencing of the rpoB gene permitted the detection of D516Y and H526C mutations. Of the 11 strains sequenced, three had a double mutation. For one of them, probe E did not bind to the mutant sequence ([Table 4]).

Hybridization by MTBDRplus assay permitted the characterization of 49/60 (81.7%) of the mutations occurring in the rpoB gene, while sequencing was required for 11 (18.3%) ([Table 3] and [Table 4]). Probes B and D did not bind to mutant sequences of one strain ([Table 2]). MTBDRplus assay revealed an absence of hybridization with probe 7. Specific proteins excreted were not detected. The sequencing of the rpoB gene detected a H526G mutation ([Table 4]).

  Discussion Top

As a major public health problem, TB stands in the way of development. Also, a major emphasis has been given on the rapid diagnosis of TB, notably MDR-TB. GeneXpert MTB/RIF is one of the molecular methods most used because the tool is sensitive, specific, and relatively easy to perform [11],[12],[13].

Discordant results between genotypic and phenotypic methods concerning RMP sensitivity were described [14]. In this study, four cases were reported of which one had a S531L mutation.

This mutation is one of the most frequently described and is well correlated with RMP phenotypic resistance [15],[16]. Here, discrepancies between phenotypic and molecular methods may be due either to technical errors that occurred in the strain suspension preparation before inoculation of MGIT or a problem with dysgonic strains and the gold standard used [14],[17].

The three other (D516Y, H526C, and L533P) mutations in the rpoB gene give frequent discrepancies between genotypic and phenotypic methods [17]. Nevertheless, for some authors, these mutations are concordant with phenotypic resistance to RMP [18],[19]. The three clinical strains were isolated from failure cases after a 2RHZE/2RH regimen. That strongly suggests of drug resistance.

The katG S315T mutation induces a high level resistance to INH and it is well correlated with phenotypic resistance [20],[21]. For one clinical strain carrying the rpoB H526Y mutation associated with the katG S315T mutation, DST for INH was interpreted susceptible ([Table 3]). This situation might be explained by the slow growth of MTB strains in the tube containing INH comparatively to other tubes inoculated.

For characterization of resistance detected using real-time polymerase chain reaction, we performed MTBDRplus assay on strains. Of the 60 strains with a mutation in the rpoB gene, 81.7% of them had a punctual mutation identified with this method. These mutations are the most described encoding for RMP resistance [15],[16]. For 18.3%, although the method has permitted the detection of the presence of a mutant sequence, a specific protein was not identified. Sequencing of the rpoB gene was required. The GeneXpert MTB/RIF and MTBDRplus screen the same fragment of 81bp of the rpoB gene. In fact, the mutations detected by the two methods are localized on the same position. This is clearly demonstrated with the three strains for which a double mutation has been detected in the rpoB gene by sequencing comparatively to GeneXpert MTB/RIF probes analysis ([Table 3]). In fact, for GeneXpert MTB/RIF probes B, C, D, and E screen the same position respectively for MTBDRplus the probes 3+4, 5+6, 7, and 8. For the silent mutation (A532A) associated with rpoB L533P, codon 532 is closed to 533 and the same probe E that did not bind to the mutant sequence detects them. This is the main reason why GeneXpert MTB/RIF has detected three double mutations. Unfortunately, probes C and D did not bind to the mutant sequence. Hybridization and sequencing revealed a mutant sequence only for the probe D characterized with H526G mutation ([Table 3]). This may be due to contamination that occurred during the inoculation of the cartridge. A mixed infection with a slowing growth strain may also explain this observation [22],[23]. Indeed, DST was performed from cultures obtained on Lowenstein–Jensen.

Contrary to the other mutations interpreted as resistant to RMP with GeneXpert MTB/RIF, for the sample of one relapse case, the five probes were bound to a mutant sequence. Analysis of the amplification curves showed that the earliest and latest threshold were at the 18 cycles and 29 cycles, respectively, for probe A and B. GeneXpert has permitted the identification of a hetero-resistance case that was not confirmed by MDTBRplus. Indeed, a D516V mutation was clearly detected. This difference might be in relation with the samples used for the assays (sputum for GeneXpert and strain for genotyping).

Previous treatment for TB is a strong determinant of MDR-TB [24],[25]. Of the 60 previously treated patients with a mutation in the rpoB gene, phenotypic results permitted the confirmation of 55 MDR-TB cases. Patients were recruited in different regions of the Côte d'Ivoire and most of them excreted bacilli. They represent an important way of MDR-TB strain dissemination in communities, particularly in low-income countries where there is not an adequate structure for their care. Therefore, directly observed treatment represents one of the best ways to treat and prevent propagation of resistant strains in communities.

In summary, MDR strains were identified in most of the previously treated patients. MTBDRplus assay has permitted the characterization of the genetic basis of RMP resistance for 81.7% of patients screened with GeneXpert, while for 18.3% of patients sequencing was required.

  Conflicts of interest Top

None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

  Acknowledgments Top

We thank Dr. Kouakou Jacquemin (Director of the National TB Control Program), Head physician of Tuberculosis Centers of Côte d'Ivoire, and their colleagues, Kekeletso Kao (Expand-TB) and Pepfar's Team of Côte d'Ivoire for their contributions.

  References Top

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  [Table 1], [Table 2], [Table 3], [Table 4]

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