1 Departament de Microbiologia, Centre de Diagnòstic Biomèdic, Hospital Clinic-IDIBAPS; 2 Servei de Microbiologia, Hospital Universitari de Bellvitge; 3 Servei de Microbiologia, Hospital Universitari de la Santa Creu i Sant Pau; 4 Servei de Microbiologia, Hospital Universitari Germans Trias i Pujol; 5 Servei de Microbiologia, Laboratori de Referència de Catalunya, Barcelona, Spain
Received 1 December 2004; returned 2 February 2005; revised 17 March 2005; accepted 21 March 2005
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Abstract |
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Methods: Six pairs of fluorogenic 5' exonuclease probes (Taqman®), mutated and wild-type, were designed for six targets: codon 315 of katG, substitution C209T in the regulatory region of inhA, and codons 513, 516, 526 and 531 of rpoB.
Results: A total of 98 clinical samples harbouring resistant bacilli from 55 patients and 126 samples harbouring susceptible bacilli from 126 patients were processed. The isolates from samples were tested for drug susceptibility with the radiometric method and sequenced for the same genetic targets. Among the samples, 93 harboured isoniazid-resistant bacilli. According to the sequencing results, 30 had mutations in katG, 30 in inhA and 33 (35.4%) had no mutations in these targets. All 27 clinical specimens harbouring rifampicin-resistant bacilli showed mutations in rpoB. The detection threshold of this method in detecting target genes in serial dilutions of artificial samples was 1.5 x 103 cfu/mL. In clinical samples, the sensitivity ranged from 30.4 to 35.3% for smear-negative samples and from 95.1 to 99.2% for smear-positive samples, with a specificity of 100%. In this study, the overall sensitivity in detecting patients having the target mutations was 74.3%.
Conclusions: The main advantage of the described method is the possibility of detecting rifampicin and isoniazid resistance within 4872 h after sample collection, with a sensitivity of nearly 100% in smear-positive samples if the chosen target is responsible for the resistance.
Keywords: TB , drug resistance , detection method
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Introduction |
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Lack of treatment compliance and ineffective control and follow-up of the patients are the most important causes for the appearance of drug resistance. A recent global WHO/IUATLD project on antituberculosis drug resistance surveillance showed a prevalence of drug resistance among new cases of higher than 10% in more than 30 countries and it also identified 14 countries where the incidence of multidrug-resistant tuberculosis (MDR-TB) was more than 3%2 among new cases.
Resistance to isoniazid and rifampicin decreases the possibility of cure since there are limited effective alternative drugs. The drug susceptibility test on solid or liquid medium is the standard procedure to establish drug resistance for Mycobacterium tuberculosis. However, results are not available until 68 weeks after sample collection.3
M. tuberculosis drug resistance is mainly due to mutations in genes encoding drug target or drug converting enzymes. katG and the regulatory zone of inhA are the most frequently associated with isoniazid resistance.46 In 95% of the cases, resistance to rifampicin is associated with mutations in the rifampicin resistance-determining region of rpoB.4,6
In recent years, several molecular techniques, most of which are directed to detection in positive cultures, have been applied to detect mutations related to antituberculous drug resistance, e.g. the use of restriction enzymes or heminested amplification acting in the mutated zone,7,8 PCR-single strand conformational polymorphism,9 the line probe assay10,11 or EIA-hybridization.12 Methods based on fluorescent hybridization probes have recently been designed for real-time PCR equipment.1322 Few studies, most including only positive samples, have focused on direct detection in clinical samples.79,12,1721 The aim of this study was to develop and evaluate a method for rapid direct detection in smear-positive and -negative clinical samples, of the main mutations causing isoniazid and rifampicin resistance of M. tuberculosis using real-time amplification and fluorogenic 5' exonuclease probes (Taqman®).
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Materials and methods |
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This was a multicentre study including five university hospitals in the Barcelona area covering a population of around 2.5 million people.
Patients
Patients diagnosed with tuberculosis and with sputum samples that were culture-positive for M. tuberculosis were prospectively recruited over a 2 year period (May 1999 to May 2001) in all of the participating centres.
Samples and strains
Ninety-eight clinical samples from 55 patients harbouring isoniazid-resistant and/or rifampicin-resistant bacilli and 126 samples from patients harbouring susceptible bacilli, used as a control group, were collected at the time of clinical diagnosis and were processed following the standard procedures for staining and culture.3 Aliquots (1 mL) of decontaminated samples were frozen at 80°C until genetic studies were performed. For the direct detection protocol, one to three samples from all patients having resistance to isoniazid and/or rifampicin were processed.
All the isolates from study samples were also frozen at 80°C and recovered for sequencing procedures.
Susceptibility testing
Standard drug susceptibility testing of isolates was performed and interpreted in each laboratory using the radiometric method (Bactec®).3
Fluorogenic 5' exonuclease probes (Taqman®) and primer design
The following gene sequences were selected from GenBank: IS6110 used as an amplification control and the resistance genes katG, inhA and rpoB. According to the mutations characterized in the study area,23 the following zones were selected: codon 315 of katG, substitution C209T in the ribosome binding site (RBS) of inhA, and five codons of the M. tuberculosis rpoB: 507, 513, 516, 526 and 531. Using the software program Primer Express 1.5 (Applied Biosystems®), the fluorogenic 5' exonuclease probes (Taqman®) and the primers were designed. Two probes were designed for each mutation, one homologous to the susceptible DNA isolate (labelled with FAM fluorophore) and the other to the mutated DNA isolate (labelled with VIC fluorophore). In the case of rpoB, the probes were designed to cover all 27 codons. For IS6110, a unique probe was designed, labelled with FAM fluorophore (Table 1).
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From culture strains
A 0.5 McFarland suspension was made in 400 µL of TE buffer with 100 µL of crystal beads (Sigma®), vortexed for 2 min and heated at 95°C for 45 min. After 10 min of centrifugation at 13 000 rpm, the supernatant was kept at 20°C for the amplification procedure.
From clinical samples
A 1 mL aliquot of decontaminated sample was centrifuged at 13 000 rpm for 10 min, reconstituted with 200 µL of TE buffer and inactivated for 45 min at 95°C. DNA was purified using a silica gel column system (High Pure DNA Extraction KitRoche Diagnostics®) and kept at 20°C until use.
From artificial samples
The same protocol as that for the clinical samples was used, except that previous decontamination was omitted.
Amplification-hybridization assay
Assays were performed in a spectrofluorometric thermal cycler (ABI Prism 7700, Applied Biosystems®). The reactions were done in sealed wells on a microtitre plate (Applied Biosystems®). For each studied sample, eight amplification reactions were done (IS6110, codon 315 of katG, RBS of inhA and codons 507, 513, 516, 526 and 531 of rpoB; Table 1). The initial region of the sequence covering codon 507 was analysed using only a wild probe since it was not possible to design an operative mutant probe due to the G/C composition of the sequence. The pair of probes, wild-type and mutant, designed for each resistance codon were mixed in the same amplification reaction containing: 1x Master Mix solution (Applied Biosystems®), 500 nM of each primer, 200 nM of fluorogenic 5' exonuclease probes (Taqman®), 5 µL of sample and up to 25 µL of distilled water. The same program was used in all the amplification rounds: 2 min at 50°C, 10 min at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C.
Amplification result analysis
The analysis of the results was performed using Sequence Detection System software (Applied Biosystems®) and was based on two parameters: the cycle threshold (CT) in which the detection of fluorescence starts, and the cumulative fluorescence signal (CFS) of each probe at the end of the 40 amplification cycles. Any fluorescence signal detected before the 40th cycle was considered as DNA amplification detection following Applied Biosystems indications. The interpretation was as follows: fluorescence signal in IS6110 reaction indicated the presence of the M. tuberculosis complex, allowing analysis of the results of the resistance determinant genes. For each resistance determinant gene, the CFS of both probes was added and the signal percentage of each probe was compared with the other one as well as to the CT of each probe. A predominant signal percentage of one probe ( >70%) and earlier CT compared with the other probe, indicated specific hybridization.
Standardization of the technique
In order to analyse the specificity of the method, a collection of 13 non-tuberculous mycobacteria and 53 M. tuberculosis-resistant strains was used. The resistant strains were characterized in a previous study by our research group.24 Accordingly, the 53 strains were isoniazid-resistant, with 17 having a mutation in codon 315 of katG and 17 having a mutation in the RBS of inhA. The remaining 19 showed mutations in other zones or in other genes. Fifteen of these strains were also resistant to rifampicin.
In order to determine the amplification detection threshold for the sensitivity study, artificial samples were prepared as follows: sputum samples from three non-tuberculous patients were decontaminated and the resulting pellets were mixed. Three isolates of M. tuberculosis resistant to isoniazid were selected, one mutated in the inhA gene and two mutated in codon 315 of katG. One of these latter isolates was also resistant to rifampicin. Bacterial suspensions of each strain were adjusted to a turbidity equivalent to that of a 1 McFarland standard and serial dilutions up to 1 x 103 cfu/mL were made. An aliquot of 1 mL of sputum sample was mixed with 1 mL of bacterial suspension. Thereafter these were processed as a conventional sample. A total of six artificial samples were prepared for each strain, with final dilutions of 1.5 x 106, 1.5 x 105, 1.5 x 104, 7.5 x 103, 1.5 x 103 and 7.5 x 102.
Sequencing reaction
The isolates from the samples processed for direct detection were sequenced to determine the presence or absence of mutations studied in clinical samples. The same fragment used for direct detection was amplified for each gene. The amplification program was the following: 10 min at 95°C, and 40 cycles of 30 s at 95°C, 1 min at 60°C and 30 s at 72°C. The amplified DNA was purified by silica gel columns (DNA Extraction Kit; Gibco®), and the sequencing reaction was carried out using the Big Dye sequencing kit (Applied Biosystems®) on an automated sequencer (ABI Prism 3100 Applied Biosystems®).
Statistical analysis
The sensitivity and specificity of detecting each mutation and isoniazid and rifampicin resistance were calculated using Epiinfo V.6.
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Results |
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The resistant isolates of 55 patients were studied: 44 were only resistant to isoniazid, two were only resistant to rifampicin and nine were resistant to both drugs. Among the 53 resistant to isoniazid, 18 were mutated in codon 315 of katG and showed the most frequent mutation (AGCACC), 19 were mutated in the RBS of inhA showing the most frequent change (C
T at position 15) and 16 did not show mutations in the studied fragments. With respect to the 11 rifampicin-resistant isolates with mutations in rpoB, six were mutated in codon 531 (TCG
TTG), two isolates in codon 526 (CAC
GAC) and three isolates in codon 516, two having the most frequent change (GAC
GTC) and one showing a previously undescribed mutation (GAC
TTC).
Standardization of the technique
Specificity of the method
Among the 53 isolates with isoniazid resistance, the 19 without mutations in the studied genes showed a specific fluorescence signal and earlier CT with the wild-type probe. The remaining 34 isolates with mutations in the selected codons of katG and inhA showed a more intense fluorescence signal (CFS range between 74.5 and 100%) and earlier CT (range of 2.6115 cycles before) with the mutant probe than with the wild-type probe. The 15 isolates resistant to rifampicin also showed a specific fluorescence signal (CFS range between 75 and 100%) and earlier CT (2.415 cycles before) with one of the mutant probes (Table 2). Five isolates resistant to isoniazid and one resistant to rifampicin with a non-common mutation in the same codons, showed a weak fluorescence signal (ranged between 40 and 50%) but earlier CT (14 cycles before) with the mutant probe but better than with the wild-type probe.
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Sensitivity of the method
In the three series of artificial samples at dilutions of 1.5 x 106, 1.5 x 105, 1.5 x 104 and 7.5 x 103 cfu/mL, all the probes showed fluorescence. At a dilution of 1.5 x 103 cfu/mL, the probes were positive in two of the three samples. At a dilution of 7.5 x 102 cfu/mL, all the probes were negative. The detection threshold was established at 1.5 x 103 cfu/mL.
Clinical sample study
Ninety-eight samples harbouring resistant bacilli, corresponding to 55 patients and 126 samples harbouring susceptible bacilli from 126 patients were studied. Among the clinical specimens harbouring resistant bacilli, 93 were resistant to isoniazid and 27 to rifampicin. Most of the samples harbouring rifampicin-resistant bacilli, 22 out of 27, also contained bacilli resistant to isoniazid (Table 3).
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The sensitivity of hybridization with the probe pair from each studied gene for detecting their complementary sequence in clinical samples, ranged from 30.4 to 99.2% for smear-positive samples, and from 30.4 to 35.3% in smear-negative samples (Table 4).
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In this study, 52.7% (29/55) of the patients included were found to harbour bacilli resistant to isoniazid and/or rifampicin, with or without a known mutation. On inclusion of the patients with specific mutations, the sensitivity increased to 74.3%.
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Discussion |
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The strategy followed in this study was detecting isoniazid and rifampicin resistance in smear-positive and -negative specimens using a real-time PCR method based on fluorogenic 5' exonuclease probes (Taqman®) that could detect the most prevalent targets in our area.
The specificity of the proposed scheme was 100%, since all the results agreed with the previous drug susceptibility test and with the sequence analysis performed in the isolates from the samples studied. The yield of the sensitivity should be considered from two points of view: (i) the sensitivity of the pair of probes to detect their respective target, wild-type or mutant, which was similar for the resistance genes (Table 4), and (ii) the sensitivity for detecting cases with resistance to the studied drugs, independently of the mutation or genetic alteration causing them. The first approach depends on the strength of the technique design and the second depends on the proportion of positive and negative smear samples included. The simultaneous use of the wild-type and mutant probes in the same tube or plate-well directed to anneal in the same strand allowed them to operate competitively, increasing the specificity of the results. This also allowed control of the sensitivity, since one of the pairs of probes should hybridize to consider the sample interpretable. In this study, more than two-thirds of the patients with isoniazid-resistant isolates had mutations in the analysed targets, whereas all cases showing resistance to rifampicin had mutations in rpoB. Overall, in this study the pair of probes of each gene detected more than 70% of the samples with target resistance. In addition to resistance genes, we also tested the IS6110 for diagnosis with its sensitivity being higher than that of the resistance genes, probably because there are several copies of this target sequence.
In recent years, drug-resistant tuberculosis has emerged as a threat in different parts of the world,2 with multidrug-resistance being a major problem in low-income countries and in several areas of Eastern Europe.25 In our study area, the reported resistance in non-treated and previously treated patients was 5.7 and 20.5%, respectively.24
Several recent studies have shown that rifampicin resistance in up to 9598% of cases is caused by mutations in rpoB.6 However, isoniazid resistance involves several genes. Mutations in katG and inhA have been found in a large proportion of isoniazid-resistant isolates. Nonetheless, the proportion between both genes varies in different countries, ranging from katG mutations of rare occurrence2628 to a moderate 50%29,30 or a high proportion up to 7993%.7,31 In a recent study in our area, katG mutations accounted for 55% of isoniazid resistance cases with 32% occurring in inhA.23
Several methods for genetic detection of resistance to isoniazid and rifampicin have been developed during the last few years. These include sequence analysis, the line probe assay for detection of rifampicin,4,32 PCR-strand single conformational polymorphism,4,32 allele-specific PCR for isoniazid and rifampicin,7,8 RFLP-PCR for isoniazid,7 PCR-EIA12 and the detection of point mutations using real-time PCR.1517,1922
Very few studies in the literature have been published for direct detection of resistance in clinical samples.7,1721 With respect to rifampicin, Mokrousov et al.7 showed 82.8% sensitivity for 87 smear-positive samples using an allele-specific rpoB-PCR assay and Johansen et al.18 studied 60 samples detecting rifampicin resistance in 77.7% of smear-positive samples and in 41.6% of smear-negative samples, using InnoLipa Rif.TB. This latter report found a similar sensitivity to that shown in this study. There are two studies referring to isoniazid. Mokrousov et al.7 reported the use of allele-specific PCR and PCR-RFLP methods in a few smear-positive samples to detect mutations in codon 315 of katG. Using a real-time PCR methodology and minor groove binder (MGB) probes directed to codon 315 of katG, van Doorn et al.17 found a sensitivity of 90% in smear-positive samples and 65% in smear-negative samples. Likewise, using MGB probes directed to hybridize the wild-type codon 315 of katG and to cover the 81 bp area of rpoB, Wada et al.21 tested 27 sputum samples and found a sensitivity of 59.2%, which rose to 100% with the use of a nested PCR model. The methodology reported by van Doorn et al.17 and Wada et al.21 is similar to that used in this study, but was only directed to codon 315 of katG for the detection of isoniazid resistance. Ruíz et al.19 presented a method based on real-time PCR using FRET probes for detecting isoniazid and rifampicin resistance in the same genes as those used in our study. These authors showed a sensitivity ranging from 96.5 to 97.5%, analysing 205 smear-positive samples. Marin et al.20 also used FRET probes for the detection of 13 point mutations, including the 81 bp area of rpoB and codon 315 of katG. With the implementation of this strategy in smear-positive clinical samples, the authors achieved a sensitivity of 100%.
The main advantages of this study are the possibility to detect rifampicin and isoniazid resistance within 4872 h after sample collection, being close to 100% in smear-positive samples with an overall sensitivity of 75%, which is important from an epidemiological point of view. Moreover, the strategy used in the design is useful, since it covers the most frequent mutations in our area.
There are some limitations to the method described. The sensitivity in smear-negative samples should be improved. An additional limitation is that 30% of the isoniazid-resistant isolates in this study did not have genetic alterations in the target gene sequences. The prospective recruitment of cases in this study indicates that the observed distribution of the resistance mutations probably corresponds to our area. Therefore, the description of other genetic targets would improve the sensitivity.
In conclusion, this study discusses a method that is able to rapidly detect rifampicin and isoniazid resistance directly in clinical samples covering the most frequent targets in our geographical area. The strategy of application of this method should be decided in each centre or area, and should depend on several factors such as incidence of resistance in the general population and the presence of risk factors such as previous treatment or foreign-born patients who import M. tuberculosis infection from countries with a higher incidence of drug resistance.
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Acknowledgements |
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