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Journal of Clinical Microbiology, October 2004, p. 4498-4502, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4498-4502.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Detection of Mutations Associated with Isoniazid and Rifampin Resistance in Mycobacterium tuberculosis Isolates from Samara Region, Russian Federation

V. Nikolayevsky,¹ T. Brown,¹ Y. Balabanova,^1,2 M. Ruddy,¹ I. Fedorin,² and F. Drobniewski^1*

Health Protection Agency Mycobacterium Reference Unit and Department of Infectious Diseases, King's College Hospital (Dulwich), London, United Kingdom,¹ Samara Tuberculosis Service, Samara City, Samara, Russian Federation²

Received 30 January 2004/ Returned for modification 29 March 2004/ Accepted 29 June 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
High incidence rates of isoniazid-, rifampin-, and multiple-drug-resistant tuberculosis have been reported in countries of the former Soviet Union (FSU). Genotypic (unlike phenotypic) drug resistance assays do not require viable cultures but require accurate knowledge of both the target gene and the mutations associated with resistance. For these assays to be clinically useful, they must be able to detect the range of mutations seen in isolates from the population of tuberculosis patients to which they are applied. Two novel macroarrays were applied to detect mutations associated with rifampin (rpoB) and isoniazid (katG and inhA) resistance. In a sample of 233 isolates from patients in Samara, central Russia, 46.5% of isolates possessed mutations in both the rpoB and the katG (or inhA) genes. Combined results from the macroarrays demonstrated concordance in 95.4 and 90.4% of phenotypically defined rifampin- and isoniazid-resistant isolates, respectively. The contribution of different mutations to resistance was comparable to that reported previously for non-FSU countries, with 90% of rifampin-resistant isolates and 93% of isoniazid resistant isolates due to rpoB531 and katG315 mutations, respectively. The percentage of phenotypically resistant rifampin isolates with no mutations in the rpoB codons 509 to 536 was 4.2%, which was similar to previous reports. Novel macroarrays offer a rapid, accurate, and relatively cheap system for the identification of rifampin-, isoniazid-, and multiple-drug-resistant Mycobacterium tuberculosis isolates.

Top Abstract Introduction Materials and Methods Results Discussion References

 
High tuberculosis (TB) incidence rates have been reported across many of the former Soviet Union countries, including Russia, Ukraine, and the Baltic states (11, 16, 25). In Russia, a TB incidence of 90.7 per 100,000 population was recorded in 2000, with a slight decrease in 2001 (17). Similarly high rates of drug resistance, particularly of multiple-drug-resistant tuberculosis (MDRTB) cases, have been reported in the same region, contributing to low cure rates for TB and compromising the effectiveness of TB programs. Rates of newly diagnosed cases of MDRTB as high as 14% have been reported in Estonia (11, 12), for example, but there is no comprehensive national data for Russia or the Ukraine. Some regions in Russia have reported high resistance rates. For example, in 2000, rates of primary and secondary resistance to at least one drug were reported to be 33 and 85%, respectively, in Archangel Oblast (20). High resistance rates, especially of MDRTB isolates, were reported in Samara and St. Petersburg (7, 13). Conversely, although large institutional outbreaks of drug-resistant TB and MDRTB have been reported in the United States (1, 14), the United Kingdom (5), and other European countries, the overall MDRTB incidence and prevalence in these areas have been low (8).

At present, the mainstay of laboratory diagnosis of pulmonary TB in Russia is based upon sputum microscopy and isolation of strains on solid culture media (usually Lowenstein-Jensen medium). Subsequently, drug susceptibility testing is performed on Lowenstein-Jensen medium with an absolute concentration method (21). In general, laboratory performance in Russia is compromised by a lack of adequate laboratory equipment and good-quality reagents and the use of nonstandardized methods. This poor performance applies particularly to the accuracy of drug susceptibility testing.

Molecular methods for mycobacterial detection, identification of organisms to the species level, and drug resistance analysis have been described extensively in recent years. At least 11 genes have been reported to be involved in the development of resistance to the main anti-TB drugs (18). The detection of TB isolates with resistance to rifampin or isoniazid and to the two drugs in combination (MDRTB) is of importance clinically and for public health TB control.

Resistance to rifampin has been associated in over 90% of cases with mutations in the 81-bp core region of the rpoB gene encoding the ß-subunit of RNA polymerase (4, 9), but the geographical distribution of certain types of gene mutations varies. Generally, single nucleotide substitutions in codons 531 and 526 are the most frequent mutations associated with rifampin resistance (15), but in some parts of the world, mutations in other codons contribute significantly to rifampin resistance development. For example, mutations in codons 511 and 516 were reported to be the most frequent in isolates in Mexico and Hungary, respectively (2, 3).

Isoniazid resistance occurs due to substitutions in the catalase-peroxidase katG gene in more than three-quarters of cases and, more rarely, due to mutations in inhA (less than 10%) and the ahpC gene (18, 24, 27).

The commercially available INNO-LiPA Rif.TB kit (Innogenetics, Ghent, Belgium) is widely used for the detection of rpoB mutations associated with rifampin resistance. Its application to clinical isolates shows a good correlation with phenotypically derived rifampin resistance (90 to 98%) (3, 10, 19, 23). Application of such commercial kits for screening purposes is limited to regions with high TB and MDRTB incidence rates due to the relatively high cost and inability to analyze isoniazid resistance. A first-generation noncommercial dot blot hybridization strategy based on the amplification of gene fragments known to confer rifampin and isoniazid resistance, followed by hybridization to oligonucleotide probes corresponding to normal and mutant genotypes, has been shown to be cost-effective and accurate: for 61 isolates, rifampin and isoniazid resistance was correctly predicted in 90 and 75% of isolates, respectively (22).

The aims of the study were (i) to determine the range and frequency of the codons involved in isoniazid, rifampin, and multiple drug resistance and (ii) to determine if a restricted number of genetic loci could be used to deduce phenotypic isoniazid, rifampin, and multiple drug resistance (MDRTB array) in Mycobacterium tuberculosis isolates from Samara, Russia, by use of directed oligonucleotide arrays. This study was undertaken as a collaborative project between the Samara Oblast TB Service in central Russia and the United Kingdom.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial isolates. A total of 233 clinical isolates of M. tuberculosis were cultured from the sputum of individual patients from Samara in the Russian Federation.

Bacteriological methods. All sputum specimens were cultured on Lowenstein-Jensen medium, and the identities of mycobacterial cultures were confirmed by a combination of growth conditions, macroscopic appearance (including pigmentation), microscopic appearance, biochemical characterization, and DNA hybridization (Accuprobe; Genprobe, San Diego, Calif.). Drug susceptibility testing was performed with the resistance ratio method on Lowenstein-Jensen medium (6).

Array analysis of loci associated with drug resistance. (i) PCR. Crude DNA extracts were prepared for PCR by heating cell suspensions with chloroform at 80°C for 20 min as previously described (26). PCR was carried out with 20-µl volumes containing 2 µl of 10x PCR buffer (Bioline, Ltd., London, United Kingdom), 0.5 U of Taq polymerase (Bioline), 0.5 µl of a 2 mM concentration of a deoxynucleoside triphosphate mixture (Bioline), 0.5 µl of a 20 µM concentration of a primer mix (containing the six 5'-biotin-labeled primers shown in Table 1), 15.5 µl of water, and 1 µl of DNA extract. Thermal cycling was performed on a Perkin-Elmer 9700 thermocycler with the following program: 1 cycle of 5 min at 95°C; 30 cycles of 30 s at 65°C and 60 s at 72°C; and 1 cycle of 5 min at 72°C. The presence of PCR products was confirmed by agarose gel electrophoresis.

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TABLE 1. Primers and probes designed and used in this study

(ii) Arrays. Oligonucleotide probes (Invitrogen, Paisley, United Kingdom) were designed, diluted to 20 µM in water, and applied to a nylon membrane (Osmonics, Minnetonka, Minn.) by use of a handheld arraying device (VP Scientific, San Diego, Calif.). Probes were UV cross-linked to the nylon membrane. Two arrays were used consecutively in this study. The first macroarray (an MDRTB screening array) was designed to detect mutant genotypes at katG315, the inhA locus, and the rpoB locus associated with rifampin resistance, as shown in Fig. 1, by use of the probes K315WTC, K315GC, TOMIWT, TOMIMUT1, MRURP3, MRURP6, MRURP9, MRURP12, MRURP17, and MRURP22 (Table 1). The second, an rpoB macroarray, was designed to detect and analyze mutations in rpoB, only in more detail, as shown in Fig. 1 (panel 2), and consisted of 27 probes, MRURP1 to MRURP27 (Table 1).

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FIG. 1. Macroarray design. (Panel 1) MDRTB screening array and examples of arrays hybridized with labeled PCR products. (A and B) Duplicate hybridized arrays showing WT rpoB, katG, and inhA genotypes, indicative of a rifampin- and isoniazid-susceptible isolate. Rifampin-sensitive probes, such as all rpoB probes (MRURP3, -6, -9, -12, -17, and -22), show hybridization and so have the WT phenotype; isoniazid-sensitive probes, such as K315WTC, show binding, indicating the WT K315GC genotype; and TOMIWT shows hybridization, indicating a WT inhA locus. (C and D) Duplicate arrays showing mutant rpoB (arrow labeled 1) and katG (arrow labeled 2) genotypes indicative of an MDRTB isolate. Rifampin-resistant probes, such as MRURP22, show absence of binding (indicating a mutation in codon 531); isoniazid-resistant probes, marked by an absence of binding to K315WTC and corresponding hybridization with K315GC, indicate the KG315 AGCACC genotype; hybridization to TOMIWT and an absence of binding to TOMIMUT1 indicate a WT inhA locus. (Panel 2) Diagram of the rpoB array in which numbers 1 to 27 represent WT rpoB probes MRURP1 to -27, respectively. A, orientation spot; B, color development control.

(iii) Reverse hybridization. Biotin-labeled PCR products were denatured by the addition of an equal volume of denaturation solution (0.4 M NaOH, 0.02 M EDTA) and incubation at room temperature for 15 min. A 20-µl aliquot of the denatured PCR was added to a polyethylene tube containing an array and 500 µl of hybridization solution (5x SSPE [1x SSPE is 0.18 M NaCl, 10 mM Na₂PO₄, and 1 mM EDTA {pH 7.7}]; 0.5% sodium dodecyl sulfate), which was agitated in a hybridization oven at 62°C for 30 min. Membranes were then washed twice in a wash solution (0.1 M Tris, 0.1 M NaCl [pH 7.5]) and once in a wash solution containing 0.1% blocking reagent (Roche, Lewes, United Kingdom) before being incubated for 30 min with a 1/100 dilution of streptavidin-alkaline phosphatase conjugate (BioGenex, San Ramon, Calif.). The membranes were then washed twice in the wash solution without blocking reagent before being incubated for 30 min with NBT-BCIP (45 µl of nitroblue tetrazolium [U.S. Biochemicals, Cleveland, Ohio] at a 75-mg/ml concentration in dimethylformamide and 5-bromo-4-chloro-3-indolyl phosphate [U.S. Biochemicals] at a 50-mg/ml concentration in dimethylformamide) in a light-proof container. The membranes were washed in water before being air dried, and the hybridization patterns were noted.

(iv) Interpretation. Hybridization to any of the probes directed towards rpoB is indicative of a wild-type (WT) genotype at that locus. Conversely, lack of hybridization with a given rpoB probe is indicative of a mutant genotype at that locus. The probes included in the MDRTB screening array each investigate a limited number of codons: MRURP3 for codons 509 to 516, MRURP6 for codons 512 to 519, MRURP9 for codons 515 to 522, MRURP12 for codons 518 to 525, MRU17 for codons 523 to 529, and MRURP22 for codons 528 to 534. Hybridization with K315WTC is indicative of a katG codon 315 WT genotype, whereas absence of such hybridization is indicative of a mutant genotype at or surrounding this locus. Absence of hybridization with K315WTC and hybridization with K315GC is indicative of the katG315 AGCACC genotype. Likewise, hybridization with TOMIWT is indicative of an inhA upstream WT genotype, whereas the absence of such hybridization is indicative of a mutant genotype at or surrounding this locus. The absence of hybridization with TOMIWT and the presence of hybridization with TOMIMUT1 are indicative of the inhA15 CT mutation.

DNA sequencing. The PCR products were generated by the method given above, except that single primer pairs (Table 1) were used, resulting in a single rpoB, katG, or inhA PCR product. These products were diluted 1/100 in purified water and sequenced with CEQ Quick Start sequencing kits and a CEQ 8000 instrument (Beckman Coulter, High Wycombe, United Kingdom) in accordance with the manufacturer's instructions. The PCR products were sequenced in both directions with the amplification primers given in Table 1.

Top Abstract Introduction Materials and Methods Results Discussion References

 
All cultures of M. tuberculosis from Samara used in this study were confirmed to be M. tuberculosis based on microscopic and phenotypic characteristics in the reference laboratories in Samara and London, United Kingdom. Multiplex PCR amplification of all 233 isolates was shown to be successful by agarose gel electrophoresis. These products were used in the reverse hybridization phase of the MDRTB screening array, and typical results are shown in Fig. 1. Isolates with WT genotypes at katG codon 315 and inhA15 were interpreted as being susceptible to isoniazid, whereas isolates with mutant genotypes at either or both of these loci were interpreted as being resistant to isoniazid. Similarly, isolates with a WT genotype at rpoB codons 509 to 534 were interpreted as being susceptible to rifampin, whereas isolates with a mutant genotype were interpreted as being resistant to rifampin. Taking the phenotypic results as the true results, 90.4% of isoniazid-resistant isolates and 79.3% of rifampin-resistant isolates were deemed to be resistant by the initial genotypic method; i.e., there was a reasonably high proportion of apparently false-negative rifampin resistance results. A second rpoB array with a higher resolution than that included in the MDRTB screening array was used to reanalyze 47 DNA extracts from isolates with phenotypic and genotypic rifampin susceptibility results that were discrepant. These data showed that 20 isolates did indeed harbor a mutation in rpoB where none was detected with the initial screening array. Conversely, 17 isolates did not appear to harbor a mutation where the screening array had suggested such a genotype. Each codon was investigated by multiple probes in the rpoB array, thus providing a more accurate analysis of the rpoB locus than that provided by the MDRTB screening array. A total of seven rifampin-resistant isolates showed no evidence of mutations by use of the two different macroarray systems. The rpoB loci from these isolates were sequenced, which revealed five to have WT genotypes and two to have mutant genotypes, one H526Y (CTC) and the other L533P (CCG). The five isolates that showed no evidence of mutation at their rpoB loci represented 4.2% of the total number of rifampin-resistant isolates in the collection.

In order to verify the performance of the probes used to analyze the loci associated with isoniazid resistance on the MDRTB screening array, these loci in six randomly selected isoniazid-resistant isolates and six isoniazid-susceptible isolates were sequenced. The sequencing results were concordant with the array results.

A comparative analysis of the combined genotype results (from both macroarrays and sequencing) for both isoniazid and rifampin and the phenotype results is given in Table 2. Genotypically, 46.5% of isolates were designated as MDRTB isolates, that is, they possessed mutations in the rpoB locus and katG or inhA. Overall, results for 38 of 233 isolates (16.3%) were discordant when the two drugs were considered together; 107 of 120 phenotypically determined MDRTB isolates (89.2%) were correctly identified genotypically.

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TABLE 2. Comparison of genotypic macroarray results for multiple-drug-resistant isolates with equivalent phenotypic resultsa

The distribution of mutations associated with isoniazid and rifampin resistance shown in this study is shown in Table 3. Mutations in katG codon 315 alone were seen in 93% of isolates containing mutations associated with isoniazid resistance, in 2% of strains with mutations at the inhA15 locus alone, and in 5% of strains with mutations at both loci. Mutations at the rpoB locus were defined by the MDRTB screening array as MRURP3, -6, -9, -12, -17, or -22 negative (i.e., nonbinding). Of isolates harboring mutations associated with rifampin resistance, 90% had mutations within the MRURP22 probe region, 4.2% had mutations within the MRURP17 probe region, and 6% had mutations distributed among the remaining probe regions. Mutations within codons 526 and 531 were analyzed with probes MRURP17 and MRURP22, respectively.

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TABLE 3. Distribution of mutations associated with isoniazid and rifampin resistance (MDRTB) seen in 233 isolates from Samara, Russia

Top Abstract Introduction Materials and Methods Results Discussion References

 
The safe phenotypic analysis of drug resistance requires a costly infrastructure and takes 1 to 2 weeks with an initial culture and at least 3 to 4 weeks with an initial specimen. Genotypic (unlike phenotypic) assays do not require viable cultures but require accurate knowledge of both the target gene and the mutations associated with resistance. For a genotypic method to be clinically useful, it needs to be accurate, reproducible, and able to detect the range of mutations seen in isolates from the patient population to which it is applied. Combining results from the two macroarray analyses revealed mutations consistent with resistance in 95.4% of cultures that were phenotypically rifampin resistant and in 90.4% of those that were phenotypically isoniazid resistant. These concordance values are higher than those previously reported for noncommercial molecular drug susceptibility analysis systems (22). This finding suggests that the inclusion of additional probes in an array increases system performance. Sequencing the rpoB and katG genes demonstrated the specificity of the macroarray approach.

The distribution of different mutations associated with rifampin and isoniazid resistance was found to be similar to that previously reported for the majority of non-former Soviet Union European countries (13, 15, 18), with a dominance of substitutions in rpoB codon 531 (up to 90% in the present study) and in katG codon 315 (93% in the present study). This finding differs from the results of the Hungarian study, which showed that mutations in codon 516 were more commonly associated with rifampin resistance (3). The percentage of phenotypically resistant strains with no detected mutations in the rpoB region associated with rifampin was 4.2% in this study, which is close to that reported previously (3, 19, 22, 23).

This study demonstrates the broad applicability of the use of macroarrays for the detection of mutations consistent with rifampin- and isoniazid-resistant TB isolates and MDRTB isolates. The prototype noncommercial arrays described here have demonstrated that analysis of a limited number of loci associated with rifampin and isoniazid resistance is an effective strategy for identifying rifampin and isoniazid resistance in Samara. This system is relatively simple and safe to use and in this preliminary analysis correlated well with phenotypic drug resistance testing. The specific genotypic detection assays of MDRTB isolates (i.e., with resistance to both isoniazid and rifampin) performed well in comparison with the combined phenotypic MDRTB analysis. The results of these analyses have been incorporated into the development of a new generation of arrays.

The implementation of the World Health Organization's "DOTS Plus" strategy in countries such as Russia will be greatly assisted by the use of inexpensive, reproducible, and safe methods of MDRTB isolate detection. Rapid MDRTB identification should lead to the earlier institution of appropriate chemotherapy, improving the probability of individual cure and survival, and in countries where MDRTB is relatively common, it may reduce transmission and thus lead to an improvement in public health.

 
We thank all the bacteriologists in Samara and the Mycobacterium Reference Unit who participated in the study.

This study was funded by the United Kingdom Department for International Development (CNTR 0034).

 
* Corresponding author. Mailing address: HPA Mycobacterium Reference Unit and Department of Infectious Diseases, King's College Hospital (Dulwich), East Dulwich Grove, London SE22 8QF, United Kingdom. Phone: 208 693 1312. Fax: 207 346 6477. E-mail: francis.drobniewski{at}kcl.ac.uk.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, October 2004, p. 4498-4502, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4498-4502.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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