Use of Genotype MTBDR Assay for Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Strains Isol
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《微生物临床杂志》
Emerging Bacterial Pathogens, San Raffaele Scientific Institute, Milan, Italy
Istituto Villa Marelli, Milan, Italy
Vita-Salute San Raffaele University and Microbiology and Virology Laboratory, Milan, Italy
WHO Collaborating Centre for Tuberculosis and Lung Disease, S. Maugeri Foundation, IRCCS, Tradate, Italy
ABSTRACT
Mycobacterium tuberculosis is one of the leading causes of death worldwide, and multidrug-resistant tuberculosis (MDR-TB) is associated with a high case fatality rate. Rapid identification of resistant strains is crucial for the early administration of appropriate therapy, for prevention of development of further resistance, and to curtail the spread of MDR strains. The Genotype MTBDR (Hain Lifescience, Nehren, Germany) is a reverse hybridization line probe assay designed for the rapid detection of rpoB and katG gene mutations in clinical isolates. The ability of this technique to correctly identify resistant and MDR-TB strains was tested on 206 isolates from the Italian drug resistance surveillance system. This panel included the majority of MDR strains isolated in Italy in the past 3 years. The results of the test were compared to conventional drug susceptibility test performed on isolated strains and verified by sequencing the regions of interest of the bacterial genome. The rate of concordance between the results of the MTBDR and those obtained with "in vitro" sensitivity was 91.5% (130 of 142) for rifampin and 67.1% (116 of 173) for isoniazid. We also applied this test directly to a panel of 36 clinical specimens collected from patients with active TB. The MTBDR correctly identified the two cases of MDR-TB included in the panel. These results show that the MTBDR test is useful in the detection and management of tuberculosis when MDR disease is suspected.
INTRODUCTION
Drug-resistant Mycobacterium tuberculosis strains are a real threat to tuberculosis (TB) control worldwide, as strains resistant to major anti-TB drugs have emerged. Multidrug-resistant TB (MDR-TB; resistant at least to rifampin and isoniazid) requires prolonged and expensive chemotherapy, with low cure and high fatality rates (8). In several Eastern European countries economic crisis and health system weaknesses have led to an increase in the numbers of TB cases, together with the establishment of hot spots for MDR-TB. Laboratory surveillance of drug resistance and early identification of resistant strains are critical steps for the beginning of appropriate treatment. Any delay in resistant strain identification jeopardizes the efforts to control the transmission of the disease.
Rifampin (RIF) resistance is due to mutation in a relatively small fragment (81 bp) of the rpoB gene encoding for the -subunit of the RNA polymerase (12, 29, 35); isoniazid (INH) resistance is caused by mutations in one of several regions of the katG gene, the inhA regulatory and coding region, and the ahpC-oxyR, ndh, and kasA genes (3, 4, 18, 19, 26, 29, 32, 33, 36, 39). Analysis of strains collected in different countries shows different prevalences of the mutations (24).
Easy-to-perform, rapid, and cost-effective assays based on molecular techniques that are suitable for application in clinical mycobacteriology laboratories are necessary to evaluate the presence of genomic mutations conferring resistance. Detection of resistance by conventional methods is inadequate due to the slow growth rate of M. tuberculosis; in addition, direct detection of known mutations could be more reliable in predicting the response to therapy.
The Genotype MTBDR (Hain Lifescience, Nehren, Germany) is a new commercial and easy-to-perform assay developed for the detection of RIF and/or INH resistance in TB strains. The test is based on reverse hybridization between amplicons derived from a multiplex PCR and nitrocellulose-bound wild-type and mutated probes for the mutations of interest.
The aim of the present study was to evaluate Genotype MTBDR as a rapid diagnostic tool to detect rpoB and katG gene mutations that are associated with RIF and INH resistance, respectively, in TB isolates.
We tested 206 M. tuberculosis strains isolated in Italy during the past 3 years, including 139 MDR strains and 30 fully susceptible ones. A total of 69 strains were collected from Italian patients, and 137 were obtained from foreign-born people. In order to show that the test is suitable for the direct detection on clinical specimens, we also performed the test on respiratory samples collected from active TB patients.
MATERIALS AND METHODS
Strains analyzed. We analyzed 206 M. tuberculosis strains isolated in Italy from January 2002 to June 2005. This panel included 139 MDR, 30 fully susceptible, and 37 monoresistant or polyresistant (non-MDR) strains (Table 1). A total of 85% of the MDR strains were isolated from retreatment cases.
The study population represented more than 90% of the MDR strains isolated in Italy and collected during the SMIRA (for Italian Resistance Surveillance Study) project (23). A total of 69 strains were collected from native Italian patients, and 137 strains were from foreign-born people. Drug susceptibility testing for RIF and INH on TB-positive cultures was performed by using MGIT 9600 and/or radiometric Bactec 460 (Becton Dickinson Microbiology Systems, Cockeysville, MD) according to the manufacturer's instructions. The susceptibility of all strains included in the study was rechecked by a proportion method on Lwenstein-Jensen according to the SMIRA protocol (23).
Clinical specimens. Direct detection from clinical specimens was performed on a panel of 36 Amplified Mycobacterium Tuberculosis Direct test (MTD; bioMerieux, Marcy l'Etoile, France)-positive respiratory samples. A total of 32 of 36 isolates were classified as smear positive (ranging from scanty to "++"), and 4 of 36 isolates were classified as smear negative by smear microscopy performed after fluorochrome staining.
Genotypic characterization. Genomic bacterial DNA was extracted from M. tuberculosis cultures on Lwenstein-Jensen slants according to a standard protocol described elsewhere (37). The genomic regions involved in the resistant phenotype were amplified according to a previously described protocol (17) with some adjustments. Each amplification reaction contained 1.5 mM MgCl2, 0.3 mM concentrations of each deoxynucleoside triphosphate, 10 pmol of each primer, 2.5 U of HotStart Taq DNA Polymerase (QIAGEN), and 5 μl of lysate in 30 μl of distilled sterile water. Amplification was performed in a GeneAmp thermal cycler (Perkin-Elmer, Foster City, CA) beginning with 1 cycle at 95°C for 15 min (Taq activation step), followed by 30 cycles at 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min, followed by a final extension at 72°C for 15 min. We sequenced the hot-spot region of the rpoB gene with the primers RpoB_r (5'-GCGGTACGGCGTTTCGATGAAC-3'), RpoB_f (5'-GGGAGCGGATGACCACCCA-3') (17) and the N-terminal region with RpoB7R (5'-GACGGTGTCGCGCTTGTCGAC-3'), RpoB6F (5'-CGACGAGTGCAAAGACAAGGACA-3') (2). The region of the katG gene including the S315T mutation and the mabAF-inhA operon region were sequenced with the primers KatG_r (5'-CCGGCACCGGCGCCGTCCTTG-3'); KatG_f (5'-CGGCGCATGGCCATGAACGACGTC-3'); InhA_301Rev (5'-CGATCCCCCGGTTTCCTCCG-3'), designed with pDRAW32 version 1.1.90 (Acaclone); and MabAF (5'-CGAAGTGTGCTGAGTCACACCG-3') (13).
Direct sequencing of the PCR products was carried out with an ABI Prism 3100 capillary sequencer (Applied Biosystems, Foster City, CA) and an ABI Prism BigDye Terminator kit v. 2.1 (Applied Biosystems) according to the instructions provided by manufacturer.
Genotype MTBDR assay. The Genotype MTBDR assay is based on reverse hybridization between amplicons derived from a multiplex PCR and nitrocellulose-bound probes covering overlapping wild-type (WT) sequences of the hot-spot regions (rpoB WT 1-rpoB WT 5, katG WT), the four most frequent mutations for rpoB (rpoB MUT probes), and mutations at codon 315 in katG (katG MUT probes) (Fig. 1). The presence of a mutation is indicated by the lack of hybridization on one or more of WT probes with or without hybridization on the MUT probes. There are also a universal control (UC) that verifies the presence of amplicons derived from gram-positive bacteria with a high percentage of G+C genomic content and a specific control (TUB) for the M. tuberculosis complex isolates. After denaturation, the single-stranded biotin-labeled amplicons are hybridized to membrane-bound probes at 45°C for 30 min on a shaking water bath. The strips are washed for 15 min at 45°C according to the manufacturer's instructions, and hybridization is detected by the addition of a streptavidin-alkaline phosphatase conjugate, followed by an alkaline phosphatase substrate reaction. The results are available the same day with one-step PCR.
FIG. 1. Schematic representation of rpoB "hot-spot region" showing the positions of wild-type and mutated probes present in the strip. The average size of the probes is 20 bases. The arrowheads indicate the numbers and positions of omitted codons.
DNA from solid and liquid cultures was extracted by thermal lysis and sonication, and the Genotype MTBDR assay was performed in accordance with the manufacturer's instructions. In brief, from solid media three to five colonies were suspended in 1 ml of sterile distilled water, and mycobacteria were lysed by incubation at 95°C for 30 min and sonication for 15 min. Tubes were centrifuged at 13,000 rpm for 10 min at 4°C, and 5 μl of lysate was used for amplification with the provided biotinylated primers. The amplification was performed in a GeneAmp System 2400 (Perkin-Elmer) thermal cycler with a protocol consisting of 1 cycle at 95°C for 15 min (Taq activation cycle), 10 cycles of denaturation at 95°C for 30 s and primer annealing at 58°C for 2 min, followed by 20 cycles of denaturation at 95°C for 25 s, primer annealing at 53°C for 40 s, and extension at 70°C for 40 s, followed finally by a final extension at 70°C for 8 min.
The molecular weight of the amplicons was verified on a 2% agarose gel as reported in the manufacturer's technical manual. Hybridization and washing were performed with a fully automated machine (APOLLO 20/2TC; MATEC Medizintecnik GmbH, Munsingen, Germany) or manually using the TWINCUBATOR hybridization tray (HAIN Lifescience, Nehren, Germany). Strips were interpreted for susceptibility or resistance to RIF and INH according to the manufacturer's instructions.
Direct detection from clinical specimens. For direct detection from clinical specimens, 500 μl of decontaminated sample were centrifuged at 13,000 rpm for 15 min; the pellet was then resuspended in 75 μl of sterile distilled water and processed as described for liquid cultures. The amplification was performed in a GeneAmp system 2400 (Perkin-Elmer) thermal cycler with a protocol consisting of 1 cycle at 95°C for 15 min (Taq activation cycle), followed by 10 cycles of denaturation at 95°C for 30 s and primer annealing at 58°C for 2 min, 30 cycles of denaturation at 95°C for 25 s, primer annealing at 53°C for 40 s, and extension at 70°C for 40 s, followed finally by extension at 70°C for 8 min.
RESULTS
Strain analysis. We applied the Genotype MTBDR assay to 206 M. tuberculosis strains isolated in Italy within the SMIRA prospective study for resistance monitoring in Italy (11, 23). Conventional susceptibility testing on all strains included in the present study was performed by the MGIT 9600 automated system and confirmed by a proportion method on Lwenstein-Jensen according to the World Health Organization's recommended protocol. Based on conventional susceptibility testing, 139 strains were classified as MDR, 30 strains were susceptible to the four first line drugs tested (RIF, INH, streptomycin [STR], and ethambutol [EMB]), and 37 were monoresistant or polyresistant but non-MDR.
Samples were sequenced for the regions of interest of rpoB and katG genes to compare the strip results with the nucleotide sequence. In addition, a small region of the promoter of the inhA gene was sequenced to detect the –15 CT mutation in the mabAF-inhA operon, which was previously reported to be involved in INH and ethionamide resistance (13, 25).
Thirteen different MTBDR hybridization patterns corresponding to 17 aminoacidic substitutions in the rpoB hot-spot region were found. Figure 1 shows the positions of wild-type and mutated probes in the hot-spot region of rpoB. All of the mutations found in the hot-spot region of rpoB gene are reported in Fig. 2. In 103 strains the specific mutations could be identified directly by hybridization to the oligonucleotide targeting the mutated sequence, and in 30 strains they could be identified indirectly by the absence of specific wild-type signals (Table 2). Of the 142 RIF-resistant strains, 141 (99.3%) were recognized correctly by the MTBDR, with 91 showing a mutation in region 5, 20 showing a mutation in region 4, 4 showing a mutation in region 3, 10 showing a mutation in region 2, 3 showing a mutation in region 1, 2 showing a mutation in regions 1 and 2, 2 showing a mutation in regions 2 and 5, and 1 showing a mutation in regions 1, 2, and 4.
FIG. 2. Mutations found in the rpoB "hot-spot region" of 142 M. tuberculosis strains. The arrowheads indicate the numbers and positions of omitted codons.
As expected, the test recognized as wild type a strain that had a duplication at codon 514 (15, 16). In six cases the test was able to identify correctly the mutation in a subregion within hot-spot region but did not classify the amino acid substitution despite the presence of a specific probe targeting that mutation (three cases for H526D, one case for H526Y, and two cases for S531L).
Codon 531 was the most frequently affected, being mutated in 90 of the 142 RIF-resistant strains (63.3%) included in the study, with 87 strains showing the specific nucleotide exchange of TCG to TTG, resulting in the amino acid substitution of serine to leucine (one strain showing a concomitant mutation at codon 516). Five strains had mutations located in codons 531 to 533, but the test was not able to identify the amino acid substitution. Codon 526 was affected in 20 cases (14.1%), with the test able to identify the specific mutation (H526Y) in 9 cases; codon 516 was affected in 13 strains (9.2%). Two strains presented hybridization patterns showing mutation, together with the wild-type pattern for the same codon, suggesting the simultaneous presence of wild-type and mutated DNA sequences. In these strains the presence of wild-type and mutated sequences was confirmed by sequencing analysis.
Eight strains phenotypically resistant to RIF were classified as susceptible by the test due to the presence of a hybridization pattern typical of the wild-type sequences in the hot-spot region. These strains were confirmed as wild type for the hot-spot region by sequencing. Sequencing of the upstream 268-bp region (positions 361 to 628), where additional mutations have been reported in literature (2), identified a valine-to-phenylalanine mutation at codon 170 in three of these strains.
A total of 61 of the 64 strains phenotypically RIF susceptible were recognized correctly. Three strains phenotypically RIF susceptible were identified as resistant by the presence of mutation in codon 533 (two cases) and by mutations in codons 511 and 516 (one case). In all three cases the mutations were confirmed by sequencing.
Analysis of INH resistance (Table 3) showed that the serine-to-threonine mutation at codon 315 was found in 115 strains (66.5%) of the 173 INH-R by susceptibility testing but in none of the 33 susceptible strains. A total of 112 strains showed the mutation AGCACC; only in one case did we find the mutation AGCACA. In one strain mutation in codon 315 was indicated by the absence of the wild-type hybridization signal only; sequencing demonstrated that the mutation was S315N (not targeted by a probe on the strip). Interestingly, the katG codon 315 mutation was observed at a much higher frequency in MDR isolates (67.6%) than in INH-monoresistant isolates (37.5%). Two strains presented hybridization patterns showing mutation together with the wild type, indicating the presence both wild-type and mutated DNA sequences. Fifty-six strains (32.4%) phenotypically INH-R were recognized as wild type at codon 315, as confirmed by sequencing. In one resistant case it was not possible to obtain any hybridization pattern for the INH probes despite a readable pattern for the RIF probes. Sequencing of this strain showed an extensive number of mutations in the examined region.
Mutations in the mabAF-inhA operon have been previously associated with INH resistance (13). Our investigation on the promoter region of the mabAF-inhA operon of the INH-resistant strains showing a wild-type sequence on KatG revealed that one-third (20 of 56) of them are mutated in this region (–15 CT mutation).
Clinical specimen analysis. Thirty-six respiratory specimens collected from patients with a high clinical suspicion of active TB and positive for M. tuberculosis DNA as determined by MTD were evaluated for the presence of RIF- and INH-resistant M. tuberculosis by using the Genotype MTBDR kit.
The MTBDR assay simultaneously detected the presence of TB complex DNA and identified the presence of rpoB and/or katG gene mutations by hybridization with specific immobilized probes. The data obtained with the MTBDR assay were compared to "in vitro" susceptibility testing performed on the corresponding TB-positive cultures, and the results are reported in Table 4. All samples showed hybridization with the specific TB complex probe, confirming the MTD results; 29 of 36 samples showed no presence of mutation, whereas 2 showed a hybridization pattern suggestive of RIF resistance, one of them associated with the S315T katG mutation. Cultures from the two samples confirmed the presence of MDR strains. The presence of the S315T katG mutations was observed in an additional six cases; in four cases we observed a line probe profile consistent with a mixed infection with a sensitive and a resistant strain. Culture of the samples and sensitivity test performed on multiple colonies of the strain confirmed in this case the presence of an INH-resistant population together with a sensitive one.
The sensitivity and specificity of the test for detecting INH- and/or RIF-resistant strains from culture and clinical specimens are reported in Table 5.
DISCUSSION
We sought here to evaluate the potential contribution of a new test, Genotype MTBDR, to the timely laboratory detection of RIF- and INH-resistant strains, in particular when MDR disease is suspected.
RIF and INH are crucial drugs in tuberculosis treatment. RIF resistance is rarely observed as monoresistance and for this reason it is often considered a marker for MDR isolates. Published studies underline the relevance of molecular analysis indicating that isolates harboring mutations in codons 526 and 531 show both high-level resistance to RIF and cross-resistance between rifabutin and RIF (31, 40, 41 ). INH is the drug used for treatment of latent TB infection in close contacts of infectious cases. For these reasons it is critical for the clinician to know as soon as possible the susceptibility to these two key drugs. Several genotypic assays for resistance detection have been developed in the past few years (7, 10, 22, 27, 30, 34). Most of them requires equipment and skilled human resources that are not always available to routine clinical mycobacteriology laboratories.
The LiPA Rif-TB (Innogenetics, Ghent, Belgium) assay, a molecular method based on the reverse hybridization principle, became commercially available a few years ago. The test identifies strains as members of the M. tuberculosis complex, but it is restricted to the evaluation of the presence of mutations conferring RIF resistance.
The Genotype MTBDR test allows rapid and specific detection of most mutations conferring resistance not only to RIF but also to INH in M. tuberculosis isolates. This test combines the probes targeting the 81-bp "hot-spot" region in the rpoB gene, with two probes targeting two different mutations at position 315 of the katG gene. Mutations affecting this codon are responsible for INH resistance in ca. 60% of the cases worldwide (1, 20). The simultaneous detection of resistance to both RIF and INH allows an early diagnosis of an MDR case. Previous reports (5-7, 17, 21, 28) suggested that the frequency of particular mutations and the correlation of RIF resistance to the MDR phenotype could be different in selected populations.
As reported in Table 5, our results indicate that 99.3% (141 of 142) of the RIF-resistant strains investigated were correctly identified as RIF resistant by the test, and in 72.5% of the cases the mutation was identified by specific hybridization with a mutated probe. The MTBDR test was able to easily identify the presence of mutations in positions 526 and 531 and could be used as a predictor of MDR in 110 cases.
As expected, the S531L mutation was the most frequently observed in Italian MDR strains and was demonstrated in 84 cases (60.4%), followed by the H526Y in 9 cases (6.5%). A high frequency of the S531L mutation has also been reported in studies performed in other countries (9, 14, 20, 38).
A mutated codon 315 in katG was responsible for INH resistance in 67.1% (116 of 173) of the resistant strains included in the study, and all 116 strains were correctly detected by the LiPA assay.
Regarding the 139 MDR strains, the rpoB and katG probes selected in the test proved to be a sufficient tool to detect the relevant mutations conferring resistance to both RIF and INH of 97 of 139 of the cases, with a sensitivity of 69.8%. If we consider RIF resistance alone an accurate marker for MDR, the sensitivity of the test in MDR detection increases to 95.0%, with a specificity of 99.3%. The only false-negative case on the rpoB hot-spot region was due to the duplication of codon 514.
In three cases the MTBDR test was able to identify three additional MDR strains compared to the conventional susceptibility test. In all three cases the strains were collected from foreign-born, previously treated patients with known resistance to INH in one case and to INH, pyrazinamide (PZA), and STR in the other two. It is important to underline the capability of the MTBDR test to identify mutations conferring resistance to RIF in strains with a low level of resistance. As reported by Srivastava et al. (34), specific mutations in rpoB could be associated with low-level RIF resistance not detectable by a routine sensitivity test performed on Lwenstein-Jensen with a RIF concentration of 40 μg/ml. Importantly, a progression to a full MDR phenotype was observed from the same patients by sensitivity testing of serially collected isolates.
The main limitation of the test is low overall sensitivity for INH resistance. In fact, we were able to identify INH resistance only in 67% of the INH-resistant strains due to the fact that the test targets only the katG mutation at position 315. The addition of primers and probe targeting the –15 CT mabAF-inhA mutation will substantially increase the number of INH resistant strains identified by the test. This mutation is also linked to ethionamide resistance (25). An additional limitation that needs to be considered is the predicted detection limit of 5 to 10% mutant/wild-type ratio. The MTBDR is a reliable test when used on clinical isolates, but culture growth requires 2 to 4 weeks, and sensitivity results are not available at the beginning of therapy (15). Performing the test on selected clinical specimens may give critical information needed for appropriate treatment in a very short time and can be crucial for avoiding the transmission of drug-resistant M. tuberculosis strains. Indeed, the results are available in the same day. Considering the RIF resistance, the MTBDR profiles obtained from clinical samples were consistent with the results of sensitivity testing performed on the strains obtained from their culture. For both resistant and sensitive strains the concordance rate was 100%. For INH, we had a mixed pattern of sensitive and resistant population in six samples. The sensitivity test results for INH indicated resistance in five cases and sensitivity in one case.
The results of our study demonstrate that the rate of concordance of the Genotype MTBDR findings with those of traditional methods and sequencing data is high. For this reason this test can be successfully applied in a clinical laboratory setting when a rapid sensitivity testing is required for the correct management of patients or the contacts of resistant cases. However, because only some of the mutations are targeted, this molecular test cannot be considered, at the present time, as a full alternative to conventional susceptibility testing for RIF and INH, and the results obtained by molecular methods must be confirmed by phenotypic tests.
Due to the high prevalence of MDR cases in some areas in Europe and to the high numbers of immigrants from these regions, the additional cost of the test could be justified in all new cases or retreatments in which MDR-TB is suspected, and the results of the screening could allow appropriate management of the patients.
ACKNOWLEDGMENTS
This study was partially supported by Italian grant RF04/126 to D.M.C.
We thank P. Cavallerio, M. Sampaolo, and M. A. Grasso for technical assistance; E. Boeri for helpful discussions of the data; and Arnika s.r.l., Milan, Italy, for providing the automated instruments for the Genotype MTBDR assay.
FOOTNOTES
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Istituto Villa Marelli, Milan, Italy
Vita-Salute San Raffaele University and Microbiology and Virology Laboratory, Milan, Italy
WHO Collaborating Centre for Tuberculosis and Lung Disease, S. Maugeri Foundation, IRCCS, Tradate, Italy
ABSTRACT
Mycobacterium tuberculosis is one of the leading causes of death worldwide, and multidrug-resistant tuberculosis (MDR-TB) is associated with a high case fatality rate. Rapid identification of resistant strains is crucial for the early administration of appropriate therapy, for prevention of development of further resistance, and to curtail the spread of MDR strains. The Genotype MTBDR (Hain Lifescience, Nehren, Germany) is a reverse hybridization line probe assay designed for the rapid detection of rpoB and katG gene mutations in clinical isolates. The ability of this technique to correctly identify resistant and MDR-TB strains was tested on 206 isolates from the Italian drug resistance surveillance system. This panel included the majority of MDR strains isolated in Italy in the past 3 years. The results of the test were compared to conventional drug susceptibility test performed on isolated strains and verified by sequencing the regions of interest of the bacterial genome. The rate of concordance between the results of the MTBDR and those obtained with "in vitro" sensitivity was 91.5% (130 of 142) for rifampin and 67.1% (116 of 173) for isoniazid. We also applied this test directly to a panel of 36 clinical specimens collected from patients with active TB. The MTBDR correctly identified the two cases of MDR-TB included in the panel. These results show that the MTBDR test is useful in the detection and management of tuberculosis when MDR disease is suspected.
INTRODUCTION
Drug-resistant Mycobacterium tuberculosis strains are a real threat to tuberculosis (TB) control worldwide, as strains resistant to major anti-TB drugs have emerged. Multidrug-resistant TB (MDR-TB; resistant at least to rifampin and isoniazid) requires prolonged and expensive chemotherapy, with low cure and high fatality rates (8). In several Eastern European countries economic crisis and health system weaknesses have led to an increase in the numbers of TB cases, together with the establishment of hot spots for MDR-TB. Laboratory surveillance of drug resistance and early identification of resistant strains are critical steps for the beginning of appropriate treatment. Any delay in resistant strain identification jeopardizes the efforts to control the transmission of the disease.
Rifampin (RIF) resistance is due to mutation in a relatively small fragment (81 bp) of the rpoB gene encoding for the -subunit of the RNA polymerase (12, 29, 35); isoniazid (INH) resistance is caused by mutations in one of several regions of the katG gene, the inhA regulatory and coding region, and the ahpC-oxyR, ndh, and kasA genes (3, 4, 18, 19, 26, 29, 32, 33, 36, 39). Analysis of strains collected in different countries shows different prevalences of the mutations (24).
Easy-to-perform, rapid, and cost-effective assays based on molecular techniques that are suitable for application in clinical mycobacteriology laboratories are necessary to evaluate the presence of genomic mutations conferring resistance. Detection of resistance by conventional methods is inadequate due to the slow growth rate of M. tuberculosis; in addition, direct detection of known mutations could be more reliable in predicting the response to therapy.
The Genotype MTBDR (Hain Lifescience, Nehren, Germany) is a new commercial and easy-to-perform assay developed for the detection of RIF and/or INH resistance in TB strains. The test is based on reverse hybridization between amplicons derived from a multiplex PCR and nitrocellulose-bound wild-type and mutated probes for the mutations of interest.
The aim of the present study was to evaluate Genotype MTBDR as a rapid diagnostic tool to detect rpoB and katG gene mutations that are associated with RIF and INH resistance, respectively, in TB isolates.
We tested 206 M. tuberculosis strains isolated in Italy during the past 3 years, including 139 MDR strains and 30 fully susceptible ones. A total of 69 strains were collected from Italian patients, and 137 were obtained from foreign-born people. In order to show that the test is suitable for the direct detection on clinical specimens, we also performed the test on respiratory samples collected from active TB patients.
MATERIALS AND METHODS
Strains analyzed. We analyzed 206 M. tuberculosis strains isolated in Italy from January 2002 to June 2005. This panel included 139 MDR, 30 fully susceptible, and 37 monoresistant or polyresistant (non-MDR) strains (Table 1). A total of 85% of the MDR strains were isolated from retreatment cases.
The study population represented more than 90% of the MDR strains isolated in Italy and collected during the SMIRA (for Italian Resistance Surveillance Study) project (23). A total of 69 strains were collected from native Italian patients, and 137 strains were from foreign-born people. Drug susceptibility testing for RIF and INH on TB-positive cultures was performed by using MGIT 9600 and/or radiometric Bactec 460 (Becton Dickinson Microbiology Systems, Cockeysville, MD) according to the manufacturer's instructions. The susceptibility of all strains included in the study was rechecked by a proportion method on Lwenstein-Jensen according to the SMIRA protocol (23).
Clinical specimens. Direct detection from clinical specimens was performed on a panel of 36 Amplified Mycobacterium Tuberculosis Direct test (MTD; bioMerieux, Marcy l'Etoile, France)-positive respiratory samples. A total of 32 of 36 isolates were classified as smear positive (ranging from scanty to "++"), and 4 of 36 isolates were classified as smear negative by smear microscopy performed after fluorochrome staining.
Genotypic characterization. Genomic bacterial DNA was extracted from M. tuberculosis cultures on Lwenstein-Jensen slants according to a standard protocol described elsewhere (37). The genomic regions involved in the resistant phenotype were amplified according to a previously described protocol (17) with some adjustments. Each amplification reaction contained 1.5 mM MgCl2, 0.3 mM concentrations of each deoxynucleoside triphosphate, 10 pmol of each primer, 2.5 U of HotStart Taq DNA Polymerase (QIAGEN), and 5 μl of lysate in 30 μl of distilled sterile water. Amplification was performed in a GeneAmp thermal cycler (Perkin-Elmer, Foster City, CA) beginning with 1 cycle at 95°C for 15 min (Taq activation step), followed by 30 cycles at 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min, followed by a final extension at 72°C for 15 min. We sequenced the hot-spot region of the rpoB gene with the primers RpoB_r (5'-GCGGTACGGCGTTTCGATGAAC-3'), RpoB_f (5'-GGGAGCGGATGACCACCCA-3') (17) and the N-terminal region with RpoB7R (5'-GACGGTGTCGCGCTTGTCGAC-3'), RpoB6F (5'-CGACGAGTGCAAAGACAAGGACA-3') (2). The region of the katG gene including the S315T mutation and the mabAF-inhA operon region were sequenced with the primers KatG_r (5'-CCGGCACCGGCGCCGTCCTTG-3'); KatG_f (5'-CGGCGCATGGCCATGAACGACGTC-3'); InhA_301Rev (5'-CGATCCCCCGGTTTCCTCCG-3'), designed with pDRAW32 version 1.1.90 (Acaclone); and MabAF (5'-CGAAGTGTGCTGAGTCACACCG-3') (13).
Direct sequencing of the PCR products was carried out with an ABI Prism 3100 capillary sequencer (Applied Biosystems, Foster City, CA) and an ABI Prism BigDye Terminator kit v. 2.1 (Applied Biosystems) according to the instructions provided by manufacturer.
Genotype MTBDR assay. The Genotype MTBDR assay is based on reverse hybridization between amplicons derived from a multiplex PCR and nitrocellulose-bound probes covering overlapping wild-type (WT) sequences of the hot-spot regions (rpoB WT 1-rpoB WT 5, katG WT), the four most frequent mutations for rpoB (rpoB MUT probes), and mutations at codon 315 in katG (katG MUT probes) (Fig. 1). The presence of a mutation is indicated by the lack of hybridization on one or more of WT probes with or without hybridization on the MUT probes. There are also a universal control (UC) that verifies the presence of amplicons derived from gram-positive bacteria with a high percentage of G+C genomic content and a specific control (TUB) for the M. tuberculosis complex isolates. After denaturation, the single-stranded biotin-labeled amplicons are hybridized to membrane-bound probes at 45°C for 30 min on a shaking water bath. The strips are washed for 15 min at 45°C according to the manufacturer's instructions, and hybridization is detected by the addition of a streptavidin-alkaline phosphatase conjugate, followed by an alkaline phosphatase substrate reaction. The results are available the same day with one-step PCR.
FIG. 1. Schematic representation of rpoB "hot-spot region" showing the positions of wild-type and mutated probes present in the strip. The average size of the probes is 20 bases. The arrowheads indicate the numbers and positions of omitted codons.
DNA from solid and liquid cultures was extracted by thermal lysis and sonication, and the Genotype MTBDR assay was performed in accordance with the manufacturer's instructions. In brief, from solid media three to five colonies were suspended in 1 ml of sterile distilled water, and mycobacteria were lysed by incubation at 95°C for 30 min and sonication for 15 min. Tubes were centrifuged at 13,000 rpm for 10 min at 4°C, and 5 μl of lysate was used for amplification with the provided biotinylated primers. The amplification was performed in a GeneAmp System 2400 (Perkin-Elmer) thermal cycler with a protocol consisting of 1 cycle at 95°C for 15 min (Taq activation cycle), 10 cycles of denaturation at 95°C for 30 s and primer annealing at 58°C for 2 min, followed by 20 cycles of denaturation at 95°C for 25 s, primer annealing at 53°C for 40 s, and extension at 70°C for 40 s, followed finally by a final extension at 70°C for 8 min.
The molecular weight of the amplicons was verified on a 2% agarose gel as reported in the manufacturer's technical manual. Hybridization and washing were performed with a fully automated machine (APOLLO 20/2TC; MATEC Medizintecnik GmbH, Munsingen, Germany) or manually using the TWINCUBATOR hybridization tray (HAIN Lifescience, Nehren, Germany). Strips were interpreted for susceptibility or resistance to RIF and INH according to the manufacturer's instructions.
Direct detection from clinical specimens. For direct detection from clinical specimens, 500 μl of decontaminated sample were centrifuged at 13,000 rpm for 15 min; the pellet was then resuspended in 75 μl of sterile distilled water and processed as described for liquid cultures. The amplification was performed in a GeneAmp system 2400 (Perkin-Elmer) thermal cycler with a protocol consisting of 1 cycle at 95°C for 15 min (Taq activation cycle), followed by 10 cycles of denaturation at 95°C for 30 s and primer annealing at 58°C for 2 min, 30 cycles of denaturation at 95°C for 25 s, primer annealing at 53°C for 40 s, and extension at 70°C for 40 s, followed finally by extension at 70°C for 8 min.
RESULTS
Strain analysis. We applied the Genotype MTBDR assay to 206 M. tuberculosis strains isolated in Italy within the SMIRA prospective study for resistance monitoring in Italy (11, 23). Conventional susceptibility testing on all strains included in the present study was performed by the MGIT 9600 automated system and confirmed by a proportion method on Lwenstein-Jensen according to the World Health Organization's recommended protocol. Based on conventional susceptibility testing, 139 strains were classified as MDR, 30 strains were susceptible to the four first line drugs tested (RIF, INH, streptomycin [STR], and ethambutol [EMB]), and 37 were monoresistant or polyresistant but non-MDR.
Samples were sequenced for the regions of interest of rpoB and katG genes to compare the strip results with the nucleotide sequence. In addition, a small region of the promoter of the inhA gene was sequenced to detect the –15 CT mutation in the mabAF-inhA operon, which was previously reported to be involved in INH and ethionamide resistance (13, 25).
Thirteen different MTBDR hybridization patterns corresponding to 17 aminoacidic substitutions in the rpoB hot-spot region were found. Figure 1 shows the positions of wild-type and mutated probes in the hot-spot region of rpoB. All of the mutations found in the hot-spot region of rpoB gene are reported in Fig. 2. In 103 strains the specific mutations could be identified directly by hybridization to the oligonucleotide targeting the mutated sequence, and in 30 strains they could be identified indirectly by the absence of specific wild-type signals (Table 2). Of the 142 RIF-resistant strains, 141 (99.3%) were recognized correctly by the MTBDR, with 91 showing a mutation in region 5, 20 showing a mutation in region 4, 4 showing a mutation in region 3, 10 showing a mutation in region 2, 3 showing a mutation in region 1, 2 showing a mutation in regions 1 and 2, 2 showing a mutation in regions 2 and 5, and 1 showing a mutation in regions 1, 2, and 4.
FIG. 2. Mutations found in the rpoB "hot-spot region" of 142 M. tuberculosis strains. The arrowheads indicate the numbers and positions of omitted codons.
As expected, the test recognized as wild type a strain that had a duplication at codon 514 (15, 16). In six cases the test was able to identify correctly the mutation in a subregion within hot-spot region but did not classify the amino acid substitution despite the presence of a specific probe targeting that mutation (three cases for H526D, one case for H526Y, and two cases for S531L).
Codon 531 was the most frequently affected, being mutated in 90 of the 142 RIF-resistant strains (63.3%) included in the study, with 87 strains showing the specific nucleotide exchange of TCG to TTG, resulting in the amino acid substitution of serine to leucine (one strain showing a concomitant mutation at codon 516). Five strains had mutations located in codons 531 to 533, but the test was not able to identify the amino acid substitution. Codon 526 was affected in 20 cases (14.1%), with the test able to identify the specific mutation (H526Y) in 9 cases; codon 516 was affected in 13 strains (9.2%). Two strains presented hybridization patterns showing mutation, together with the wild-type pattern for the same codon, suggesting the simultaneous presence of wild-type and mutated DNA sequences. In these strains the presence of wild-type and mutated sequences was confirmed by sequencing analysis.
Eight strains phenotypically resistant to RIF were classified as susceptible by the test due to the presence of a hybridization pattern typical of the wild-type sequences in the hot-spot region. These strains were confirmed as wild type for the hot-spot region by sequencing. Sequencing of the upstream 268-bp region (positions 361 to 628), where additional mutations have been reported in literature (2), identified a valine-to-phenylalanine mutation at codon 170 in three of these strains.
A total of 61 of the 64 strains phenotypically RIF susceptible were recognized correctly. Three strains phenotypically RIF susceptible were identified as resistant by the presence of mutation in codon 533 (two cases) and by mutations in codons 511 and 516 (one case). In all three cases the mutations were confirmed by sequencing.
Analysis of INH resistance (Table 3) showed that the serine-to-threonine mutation at codon 315 was found in 115 strains (66.5%) of the 173 INH-R by susceptibility testing but in none of the 33 susceptible strains. A total of 112 strains showed the mutation AGCACC; only in one case did we find the mutation AGCACA. In one strain mutation in codon 315 was indicated by the absence of the wild-type hybridization signal only; sequencing demonstrated that the mutation was S315N (not targeted by a probe on the strip). Interestingly, the katG codon 315 mutation was observed at a much higher frequency in MDR isolates (67.6%) than in INH-monoresistant isolates (37.5%). Two strains presented hybridization patterns showing mutation together with the wild type, indicating the presence both wild-type and mutated DNA sequences. Fifty-six strains (32.4%) phenotypically INH-R were recognized as wild type at codon 315, as confirmed by sequencing. In one resistant case it was not possible to obtain any hybridization pattern for the INH probes despite a readable pattern for the RIF probes. Sequencing of this strain showed an extensive number of mutations in the examined region.
Mutations in the mabAF-inhA operon have been previously associated with INH resistance (13). Our investigation on the promoter region of the mabAF-inhA operon of the INH-resistant strains showing a wild-type sequence on KatG revealed that one-third (20 of 56) of them are mutated in this region (–15 CT mutation).
Clinical specimen analysis. Thirty-six respiratory specimens collected from patients with a high clinical suspicion of active TB and positive for M. tuberculosis DNA as determined by MTD were evaluated for the presence of RIF- and INH-resistant M. tuberculosis by using the Genotype MTBDR kit.
The MTBDR assay simultaneously detected the presence of TB complex DNA and identified the presence of rpoB and/or katG gene mutations by hybridization with specific immobilized probes. The data obtained with the MTBDR assay were compared to "in vitro" susceptibility testing performed on the corresponding TB-positive cultures, and the results are reported in Table 4. All samples showed hybridization with the specific TB complex probe, confirming the MTD results; 29 of 36 samples showed no presence of mutation, whereas 2 showed a hybridization pattern suggestive of RIF resistance, one of them associated with the S315T katG mutation. Cultures from the two samples confirmed the presence of MDR strains. The presence of the S315T katG mutations was observed in an additional six cases; in four cases we observed a line probe profile consistent with a mixed infection with a sensitive and a resistant strain. Culture of the samples and sensitivity test performed on multiple colonies of the strain confirmed in this case the presence of an INH-resistant population together with a sensitive one.
The sensitivity and specificity of the test for detecting INH- and/or RIF-resistant strains from culture and clinical specimens are reported in Table 5.
DISCUSSION
We sought here to evaluate the potential contribution of a new test, Genotype MTBDR, to the timely laboratory detection of RIF- and INH-resistant strains, in particular when MDR disease is suspected.
RIF and INH are crucial drugs in tuberculosis treatment. RIF resistance is rarely observed as monoresistance and for this reason it is often considered a marker for MDR isolates. Published studies underline the relevance of molecular analysis indicating that isolates harboring mutations in codons 526 and 531 show both high-level resistance to RIF and cross-resistance between rifabutin and RIF (31, 40, 41 ). INH is the drug used for treatment of latent TB infection in close contacts of infectious cases. For these reasons it is critical for the clinician to know as soon as possible the susceptibility to these two key drugs. Several genotypic assays for resistance detection have been developed in the past few years (7, 10, 22, 27, 30, 34). Most of them requires equipment and skilled human resources that are not always available to routine clinical mycobacteriology laboratories.
The LiPA Rif-TB (Innogenetics, Ghent, Belgium) assay, a molecular method based on the reverse hybridization principle, became commercially available a few years ago. The test identifies strains as members of the M. tuberculosis complex, but it is restricted to the evaluation of the presence of mutations conferring RIF resistance.
The Genotype MTBDR test allows rapid and specific detection of most mutations conferring resistance not only to RIF but also to INH in M. tuberculosis isolates. This test combines the probes targeting the 81-bp "hot-spot" region in the rpoB gene, with two probes targeting two different mutations at position 315 of the katG gene. Mutations affecting this codon are responsible for INH resistance in ca. 60% of the cases worldwide (1, 20). The simultaneous detection of resistance to both RIF and INH allows an early diagnosis of an MDR case. Previous reports (5-7, 17, 21, 28) suggested that the frequency of particular mutations and the correlation of RIF resistance to the MDR phenotype could be different in selected populations.
As reported in Table 5, our results indicate that 99.3% (141 of 142) of the RIF-resistant strains investigated were correctly identified as RIF resistant by the test, and in 72.5% of the cases the mutation was identified by specific hybridization with a mutated probe. The MTBDR test was able to easily identify the presence of mutations in positions 526 and 531 and could be used as a predictor of MDR in 110 cases.
As expected, the S531L mutation was the most frequently observed in Italian MDR strains and was demonstrated in 84 cases (60.4%), followed by the H526Y in 9 cases (6.5%). A high frequency of the S531L mutation has also been reported in studies performed in other countries (9, 14, 20, 38).
A mutated codon 315 in katG was responsible for INH resistance in 67.1% (116 of 173) of the resistant strains included in the study, and all 116 strains were correctly detected by the LiPA assay.
Regarding the 139 MDR strains, the rpoB and katG probes selected in the test proved to be a sufficient tool to detect the relevant mutations conferring resistance to both RIF and INH of 97 of 139 of the cases, with a sensitivity of 69.8%. If we consider RIF resistance alone an accurate marker for MDR, the sensitivity of the test in MDR detection increases to 95.0%, with a specificity of 99.3%. The only false-negative case on the rpoB hot-spot region was due to the duplication of codon 514.
In three cases the MTBDR test was able to identify three additional MDR strains compared to the conventional susceptibility test. In all three cases the strains were collected from foreign-born, previously treated patients with known resistance to INH in one case and to INH, pyrazinamide (PZA), and STR in the other two. It is important to underline the capability of the MTBDR test to identify mutations conferring resistance to RIF in strains with a low level of resistance. As reported by Srivastava et al. (34), specific mutations in rpoB could be associated with low-level RIF resistance not detectable by a routine sensitivity test performed on Lwenstein-Jensen with a RIF concentration of 40 μg/ml. Importantly, a progression to a full MDR phenotype was observed from the same patients by sensitivity testing of serially collected isolates.
The main limitation of the test is low overall sensitivity for INH resistance. In fact, we were able to identify INH resistance only in 67% of the INH-resistant strains due to the fact that the test targets only the katG mutation at position 315. The addition of primers and probe targeting the –15 CT mabAF-inhA mutation will substantially increase the number of INH resistant strains identified by the test. This mutation is also linked to ethionamide resistance (25). An additional limitation that needs to be considered is the predicted detection limit of 5 to 10% mutant/wild-type ratio. The MTBDR is a reliable test when used on clinical isolates, but culture growth requires 2 to 4 weeks, and sensitivity results are not available at the beginning of therapy (15). Performing the test on selected clinical specimens may give critical information needed for appropriate treatment in a very short time and can be crucial for avoiding the transmission of drug-resistant M. tuberculosis strains. Indeed, the results are available in the same day. Considering the RIF resistance, the MTBDR profiles obtained from clinical samples were consistent with the results of sensitivity testing performed on the strains obtained from their culture. For both resistant and sensitive strains the concordance rate was 100%. For INH, we had a mixed pattern of sensitive and resistant population in six samples. The sensitivity test results for INH indicated resistance in five cases and sensitivity in one case.
The results of our study demonstrate that the rate of concordance of the Genotype MTBDR findings with those of traditional methods and sequencing data is high. For this reason this test can be successfully applied in a clinical laboratory setting when a rapid sensitivity testing is required for the correct management of patients or the contacts of resistant cases. However, because only some of the mutations are targeted, this molecular test cannot be considered, at the present time, as a full alternative to conventional susceptibility testing for RIF and INH, and the results obtained by molecular methods must be confirmed by phenotypic tests.
Due to the high prevalence of MDR cases in some areas in Europe and to the high numbers of immigrants from these regions, the additional cost of the test could be justified in all new cases or retreatments in which MDR-TB is suspected, and the results of the screening could allow appropriate management of the patients.
ACKNOWLEDGMENTS
This study was partially supported by Italian grant RF04/126 to D.M.C.
We thank P. Cavallerio, M. Sampaolo, and M. A. Grasso for technical assistance; E. Boeri for helpful discussions of the data; and Arnika s.r.l., Milan, Italy, for providing the automated instruments for the Genotype MTBDR assay.
FOOTNOTES
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