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Journal of Clinical Microbiology, October 2001, p. 3736-3739, Vol. 39, No. 10
Department of Respiratory Medicine, School of
Medicine, Semmelweis University,1 and
Korányi National Institute for Tuberculosis and
Respiratory Medicine,4 Budapest, Prodia
Laboratory for Mycobacteria, Jósa Hospital,
Nyíregyháza,3
Borsod-Abaúj-Zeplén County Bureau of Public Health
and Medical Officers, Miskolc,5 and
Laboratory for Mycobacteria, School of Medicine, University of
Debrecen, Debrecen,6 Hungary, and
Wadsworth Center, New York State Department of
Health,2 Department of Medicine, Albany
Medical College,7 and Department of
Biomedical Sciences, School of Public Health, State University of
New York at Albany,8 Albany, New York
Received 24 January 2001/Returned for modification 2 May
2001/Accepted 30 May 2001
Two regions of rpoB associated with rifampin
resistance were sequenced in 29 rifampin-resistant (determined by the
proportion method) isolates of Mycobacterium
tuberculosis obtained from patients from three counties in
Hungary. Of the 29 resistant strains, 27 had a mutation in either the
81-bp region (26 strains) or the N-terminal region (1 strain), while
the other 2 strains had no mutations in either region. The locations
and frequencies of the mutations differed from those previously
reported. The most common mutation in this study, D516V, was found in
38% of the Hungarian strains, a frequency 2 to 10 times higher than
that found in studies from other countries. These same 29 isolates were
also evaluated with the Inno-LiPA Rif. TB test (LiPA), a reverse
hybridization assay for the rapid detection of rifampin resistance.
Although LiPA detected the presence of an rpoB mutation
in 26 of the resistant isolates, the type of mutation could not be
determined in 4 isolates because the mutations present were not among
those included on the LiPA strip. In addition, a silent mutation in one
of the rifampin-susceptible control strains was interpreted as rifampin
resistant by LiPA. These findings demonstrate the importance of
validating this rapid molecular test by comparison with DNA sequence
results in each geographic location before incorporating the test into
routine diagnostic work.
The recent worldwide increase
in the incidence of drug-resistant strains of Mycobacterium
tuberculosis has highlighted the need for faster and more accurate
detection of resistance to rifampin (RMP), one of the most important
antituberculosis drugs (17). RMP is most effective in
killing actively metabolizing M. tuberculosis, and resistance to RMP often results in high clinical relapse rates (5, 15). Because of the prolonged turnaround time for
conventional susceptibility testing, patients infected with
drug-resistant tuberculosis may be inadequately treated and thus remain
infectious for longer periods than those infected with susceptible strains.
Based on collective observations that mutations resulting in an amino
acid change within the 81-bp core region of the RNA polymerase
Therefore, the aim of the present study was to determine the drug
resistance profile of 29 RMP-resistant M. tuberculosis isolates obtained in East Hungary and to detect
and identify mutations present in the rpoB gene. Two
molecular assays were used. In the first, two regions of
rpoB that have been associated with RMP resistance were
amplified by PCR and the DNA sequence was determined. The results of
the DNA sequencing were then compared with results from a commercially
available rapid test, the PCR-based reverse hybridization line probe
assay (Inno-LiPA Rif. TB Test [LiPA]; Innogenetics N.V., Ghent, Belgium).
After 20 years of decline, the incidence of pulmonary tuberculosis in
Hungary increased by 18.1% between 1990 and 1999 (rising from 31.0 to
36.6 per 100,000 inhabitants) (1). In addition, East
Hungary (Borsod-Abaúj-Zemplén, Hajdú-Bihar,
and Szabolcs-Szatmár-Bereg counties) is the part of the country
with the highest incidence of tuberculosis generally (38.7, 51.5, and
56.7 per 100,000 inhabitants, respectively) and drug-resistant
tuberculosis specifically (1). In 1999, these three
counties collectively reported 888 of the 3,912 (22.7%)
tuberculosis cases in Hungary. The 29 RMP-resistant isolates examined
in this study were isolated from patients in these three counties
during 1999 (1).
The M. tuberculosis H37Rv ATCC 27294 strain and
six clinical M. tuberculosis isolates that were
pansusceptible for all four first-line antituberculosis drugs were used
as controls. All cultures were identified by means of the AccuProbe
culture identification test (Gen-Probe Inc., San Diego, Calif.) and
conventional biochemical tests (11, 14).
Susceptibility testing of all 36 isolates for isoniazid (INH), RMP,
ethambutol (EMB), and streptomycin (SM) was carried out by the
proportion method on Löwenstein-Jensen medium as described by
Canetti et al. (2). The critical concentrations for INH, RMP, EMB, and SM were 0.2, 40, 1.0, and 10 µg/ml, respectively. Of
the 29 RMP-resistant isolates, only 2 (6.9%) were resistant to RMP
alone. Twenty-six (89.7%) were also resistant to INH (and thus
classified as multidrug resistant), 18 (62.1%) were also resistant to
EMB, and 9 (31.0%) were also resistant to SM (Table 1). In all, 20 of the 29 (70.0%) were
resistant to at least three of the four first-line drugs.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3736-3739.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular Characterization of Rifampin-Resistant
Isolates of Mycobacterium tuberculosis from Hungary by
DNA Sequencing and the Line Probe Assay
ABSTRACT
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-subunit (rpoB) gene are found in more than 96% of RMP-resistant M. tuberculosis strains, several
molecular methods have been developed for the rapid (24- to 48-h)
detection of mutations in this region (3, 10, 16, 18, 24, 25,
28-30). In addition, other studies revealed that mutations
associated with RMP resistance can also occur in other regions of the
rpoB gene, although less frequently (6, 7, 23).
It has also been shown elsewhere that the information provided by these
molecular tests can serve as a molecular epidemiological marker since
the relative frequency of the alleles associated with resistance can vary geographically (10, 20).
TABLE 1.
Resistance patterns of RMP-resistant M. tuberculosis isolates from Hungary by the proportion method,
the line probe assay, and DNA sequencing of the 81-bp region of the
rpoB genea
On the day of detection of growth index 999, 200-µl aliquots from Bactec 12B subcultures of the susceptible control and RMP-resistant isolates were incubated at 80°C for 1 h to heat kill the mycobacterial cells. Using primers rpo95 (5'-CCACCCAGGACGTGGAGGCGATCACACCG-3') and rpo397 (5'-GTCAACCCGTTCGGGTTCATCGAAACG-3') flanking the 81-bp region of rpoB (9), a 329-bp product was generated from all 36 isolates. The same primers were used for DNA sequencing of both strands using the automated Applied Biosystems 377 DNA sequencer (Applied Biosystems, Foster City, Calif.). A recent study demonstrated that, in some RMP-resistant strains with the wild-type sequence in the 81-bp region, a V146F mutation was found in the N-terminal region (7). In order to detect the presence of this mutation, amplification and sequencing were performed using primers Tb176-f (5'-CTTCTCCGGGTCGATGTCGTTG-3') and Tb176-r (5'-CGCGCTTGTCGACGTCAAACTC-3') as described previously (7). A 365-bp product was generated and sequenced using the same primers.
The heat-killed samples were also used for production of a biotinylated 256-bp fragment of the rpoB gene using the LiPA kit according to the instructions of the manufacturer (Innogenetics). The biotin-labeled PCR product was denatured and hybridized to a strip with 10 specific oligonucleotide probes (19 to 23 bases long). One probe is specific for the M. tuberculosis complex (TB-P), while five partially overlapping wild-type probes (S1 to S5) encompass the region of the rpoB gene encoding amino acids 509 to 534. Four other probes are specific for the most common mutations, D516V, H526Y, H526D, and S531L (probes R2, R4a, R4b, and R5, respectively) (Innogenetics). Hybridized PCR product was detected, and the LiPA results were evaluated as described elsewhere (4).
In contrast with previous reports (Table
2) (8, 13, 19, 20, 22, 26, 27, 30,
31), the frequency of occurrence of particular mutations was
different in the isolates from East Hungary, with 11 (37.9%) isolates
carrying the less common D516V mutation. Nine (31.0%) isolates had an
S531L mutation, and two (6.9%) isolates had an H526D mutation. These
mutations were also correctly detected in the LiPA. In addition, DNA
sequencing identified a double mutation (S509T and D516V) and a
deletion (deletion 522-525) that have not been reported in the
literature before (32). In these two cases, the LiPA was
unable to detect the correct type of the mutation. However, it
indicated the presence of the genetic alteration (Table 1). The LiPA
also did not reveal the type of mutation in two additional strains with
rare mutation patterns (Q513K and Q513P) (Table 1). Moreover, the test
provided a false-resistant result with a pansusceptible control strain
with a silent mutation (R529R) (Table 1).
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The LiPA has been reported to be an easy-to-use test for the rapid detection of RMP resistance. The test is available in a kit format and, therefore, especially useful for routine work in clinical laboratories that are not capable of carrying out DNA sequencing (4, 21). In the present study, LiPA was able to detect a genetic alteration in 26 (89.7%) of the 29 RMP-resistant strains and to identify the particular mutation in 22 strains (75.9%) (Table 1).
The LiPA can provide the type of mutation for only the four most common mutations of the rpoB gene (S531L, H526Y, H526D, and D516V), while in cases of other mutations it indicates only the presence of a genetic alteration. Our findings suggest that in a geographic area such as East Hungary where less common or novel mutations of the rpoB gene occur more frequently, the interpretation of the LiPA results may be more difficult. In such an environment, the characterization of the type of the mutation (and the resultant amino acid change) by DNA sequencing is indispensable in order to avoid the report of any false RMP resistance results.
Although more than 96% of the RMP-resistant strains have a mutation within the 81-bp core region of the rpoB gene, a recent study revealed that a mutation associated with RMP resistance can also occur in other regions of the gene, although less frequently (7). The present study revealed three RMP-resistant isolates where neither the DNA sequencing of the 81-bp region nor the LiPA detected any mutations (Table 1). However, the DNA sequencing for the detection of the V146F mutation was positive in one of these three isolates (strain 28, Table 1). All the other study isolates had the wild-type sequence in this region. The two isolates with no mutations in either region indicate that at present the confirmation of molecular results by conventional tests is still warranted. In addition, since the routinely applied DNA sequencing methods usually examine only the 81-bp region of the rpoB gene (32), in cases where resistance is demonstrated in conventional susceptibility testing but no mutation is found we also suggest screening for the V146F mutation. If this assay also fails to detect a mutation, then other rare mutations of the rpoB gene, heteroresistance (a mixture of susceptible and resistant strains), or, less likely, another mechanism of resistance may be involved (6, 12, 23).
In conclusion, this study demonstrated that frequencies of particular mutations in RMP-resistant M. tuberculosis isolates from East Hungary are different from those that have been reported for isolates from other geographic areas (Table 2). DNA sequencing of the two regions of the rpoB gene identified mutations in 27 (93.1%) of the investigated 29 RMP-resistant isolates, and the LiPA identified mutations in 26 isolates (89.7%). However, the rapid LiPA was unable to determine the type of mutation in 4 of the 26 strains because these isolates contained unique mutations not included on the test strip. In addition, the one isolate that contained a V146F mutation in the N-terminal region of the rpoB gene was falsely interpreted as RMP susceptible in the LiPA. Finally, the LiPA gave a false-resistant result with one of the RMP-susceptible control strains that contained a silent mutation. These findings demonstrate the importance of validating molecular tests for the detection of RMP resistance using DNA sequence analysis and thus determining the frequencies of particular mutations in the test region before introducing the assay into routine clinical service.
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ACKNOWLEDGMENTS |
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This study was supported in part by grant F-23350 from the Hungarian Scientific Research Fund. Á. Somoskövi was supported by grants 1D43TW00915 and 2D43TW00233 from the Fogarty International Center, National Institutes of Health, Bethesda, Md.
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FOOTNOTES |
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* Corresponding author. Mailing address: Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, NY 12201-0509. Phone: (518) 474-2196. Fax: (518) 474-6964. E-mail: somoskov{at}wadsworth.org or medve{at}pulm.sote.hu.
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Journal of Clinical Microbiology, October 2001, p. 3736-3739, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3736-3739.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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