JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2011, p. 34503457 Vol. 49, No.

10
0095-1137/11/$12.00 doi:10.1128/JCM.01068-11
Copyright 2011, American Society for Microbiology. All Rights Reserved.
Rapid Detection of Isoniazid, Rifampin, and Ooxacin Resistance in
Mycobacterium tuberculosis Clinical Isolates Using
High-Resolution Melting Analysis

Xiaoyou Chen,
1
Fanrong Kong,
2
Qinning Wang,
2
Chuanyou Li,
1
Jianyuan Zhang,
1
and Gwendolyn L. Gilbert
2,3
*
Department of Tuberculosis, Beijing Chest Hospital, Capital Medical University, Beijing, China
1
; Centre for
Infectious Diseases and Microbiology-Public Health (CIDM-PH), Institute of Clinical Pathology and
Medical Research (ICPMR), Westmead, New South Wales, Australia
2
; and
Sydney Institute for Emerging Infectious Diseases and Biosecurity,
University of Sydney, Sydney, NSW 2006, Australia
3
Received 11 May 2011/Returned for modication 29 July 2011/Accepted 4 August 2011
A high-resolution melting analysis (HRMA) assay was developed to detect isoniazid, rifampin, and ooxacin
resistance in Mycobacterium tuberculosis by targeting resistance-associated mutations in the katG, mabA-inhA
promoter, rpoB, and gyrA genes. A set of 28 (17 drug-resistant and 11 fully susceptible) clinical M. tuberculosis
isolates was selected for development and evaluation of HRMA. PCR amplicons from the katG, mabA-inhA
promoter, rpoB, and gyrA genes of all 28 isolates were sequenced. HRMA results matched well with 18
mutations, identied by sequencing, in 17 drug-resistant isolates and the absence of mutations in 11 suscep-
tible isolates. Among 87 additional isolates with known resistance phenotypes, HRMA identied katG and/or
mabA-inhA promoter mutations in 66 of 69 (95.7%) isoniazid-resistant isolates, rpoB mutations in 51 of 54
(94.4%) rifampin-resistant isolates, and gyrA mutations in all of 41 (100%) ooxacin-resistant isolates. All
mutations within the HRMA primer target regions were detected as variant HRMA proles. The corresponding
specicities were 97.8%, 100%, and 98.6%, respectively. Most false-positive results were due to synonymous
mutations, which did not affect susceptibility. HRMA is a rapid, sensitive method for detection of drug
resistance in M. tuberculosis which could be used routinely for screening isolates in countries with a high
prevalence of tuberculosis and drug resistance or in individual isolates when drug resistance is suspected.
One-third of the worlds population is reportedly infected
with Mycobacterium tuberculosis, causing high mortality and
morbidity. Eight million to nine million new tuberculosis (TB)
cases and about 2 million deaths are reported each year (9, 18,
28). Multidrug-resistant TB (MDR-TB) is dened as resis-
tance of the isolate to at least isoniazid (INH) and rifampin
(RIF); extensively drug-resistant TB (XDR-TB) is MDR-TB in
which the isolate has additional resistance to any of uoro-
quinolones and at least one of three injectable second-line
antituberculosis drugs used in TB treatment, namely, capreo-
mycin, kanamycin, and amikacin (14, 21). MDR-TB and
XDR-TB are major obstacles to the control of TB worldwide.
More than 2.5 million patients were diagnosed with active TB
in 116 countries and regions in 2006; of them, 4.8% had
MDR-TB (3). About one in six (15%) patients with MDR-TB
had previously received treatment for TB for at least 1 month
(18), and at least 8.0% had XDR-TB, according to incomplete
data collected from 37 countries between 2002 and 2007 (41).
Drug susceptibility testing using molecular techniques can en-
hance the identication of drug-resistant M. tuberculosis. Several
scanning methods are available for detection of drug resistance
mutations, including denaturing gradient gel electrophoresis,
conformation-sensitive gel electrophoresis, temperature gradi-
ent capillary electrophoresis, denaturing high-performance liq-
uid chromatography (dHPLC), and high-density oligonucleo-
tide arrays (15, 20, 22, 30, 31, 33). These methods vary in
sensitivity and generally either are labor-intensive or require
sophisticated instrument to perform analyses. PCR-based DNA
sequencing of drug resistance-related genes is probably the
most rapid and specic method for the identication of muta-
tions. However, due to the high cost of sequencing and the
expertise required, it is not widely available, especially in the
countries that are the most severely threatened by TB, where
large numbers of samples need to be tested.
High-resolution melting analysis (HRMA) is a PCR-based
method for detection of DNA sequence variation by demon-
strating uorescence changes in the melting of the amplied
double-stranded DNA amplicon (35). The length, GC content,
and sequence complementarities of the DNA fragments con-
tribute to the melting characteristics of double-stranded DNA
(29). This technique has successfully been applied in mutation
scanning, single nucleotide polymorphism (SNP) genotyping,
and identication of many bacterial species, including screen-
ing for RIF and INH resistance in M. tuberculosis (6, 19, 23
25). With its simple and fast work ow, HRMA is easy to
perform and can be used for a large number of samples in a
short time (35). It is a good candidate tool for mutation scan-
ning (8).
* Corresponding author. Mailing address: Centre for Infectious Dis-
eases and Microbiology-Public Health (CIDM-PH), Institute of Clin-
ical Pathology and Medical Research (ICPMR), Westmead, New
South Wales, Australia. Phone: (612) 9845 6238. Fax: (612) 9893 8659.
E-mail: [email protected].
Xiaoyou Chen, Fanrong Kong, and Qinning Wang had similar
contributions to the work and so would be seen as co-rst authors.

Published ahead of print on 10 August 2011.

3450
In this study, the usefulness of HRMA for screening for
resistance in M. tuberculosis was evaluated by targeting the
genes rpoB, gyrA, and katG and the mabA-inhA promoter re-
gion, in all of which mutations are associated with drug resis-
tance (26). An HRMA protocol was developed and validated,
using clinical M. tuberculosis isolates with known phenotypic
susceptibility proles, for the rapid identication of MDR-TB.
MATERIALS AND METHODS
M. tuberculosis clinical isolates and drug susceptibility testing. A total of 115
clinical M. tuberculosis isolates isolated from patients attending the Department
of Tuberculosis, Beijing Chest Hospital, Capital Medical University, Bei-
jing,China, were tested. They had been identied as M. tuberculosis by either
the p-nitro--acetyl-amino--hydroxypropiophenone (NAP) test or the BD
ProbeTec ET (CTB) assay (Becton Dickinson Microbiology Systems, MD). Pheno-
typic drug susceptibility testing was performed by the absolute concentration
method (4, 32). Isolates were chosen to represent a variety of different suscep-
tibility proles and included 32 fully susceptible isolates; 45 INH- and RIF-
resistant isolates (MDR), of which 30 were also uoroquinolone (ooxacin
[OFLX]) resistant; 21, 6, and 5 isolates monoresistant to INH, RIF, and OFLX,
respectively; three INH- and OFLX-resistant isolates; and three RIF- and
OFLX-resistant isolates.
DNA extraction. Stored isolates were subcultured on Lowenstein-Jensen solid
medium and incubated at 37C for 2 to 4 weeks. Genomic DNA was extracted as
described previously (39).
HRMA primer design. We designed four pairs of primers for HRMA, one
each to amplify katG, rpoB, gyrA, and the promoter region of mabA-inhA,
specically targeting sites at which mutations associated with drug resistance are
found, namely, for katG, the 315 site (INH resistance); for the mabA-inhA
promoter, the 8 and 15 sites (INH resistance); for rpoB, the 81-bp RIF
resistance determining region (RRDR); and for gyrA, a region including several
resistance-associated sites (OFLX resistance) (26). Another four pairs of primers
were designed outside each HRMA primer region to amplify extended regions of
each gene for sequence analysis (Table 1).
Real-time PCR and high-resolution melting analysis. The PCR mixture was
prepared using a Qiagen HotStar Taq system, including 2.0 l 10 PCR buffer,
1.5 mM MgCl
2,
0.25 mM deoxynucleoside triphosphates, 0.125 M each primer,
1 U HotStar Taq polymerase, 1.0 l dimethyl sulfoxide, 0.2 l LC Green Plus dye
(Idaho Technology Inc., Salt Lake City, UT), 100 to 150 ng of DNA template,
and water, which was added to give a total volume of 20 l. PCR amplication
was performed on the Rotor-Gene 6000 apparatus (Corbett Research Pty. Ltd.,
Australia) with an initial denaturation at 95C for 15 min, followed by 40 cycles
of 94C for 30 s, 60C for 30 s, and 72C for 45 s and a nal elongation at 72C
for 10 min. A nontemplate control containing sterile distilled water was included
in the experiment.
For HRMA, a second hold was set at 50C for 60 s to allow reassociation of
DNA. The melt analysis was performed from 78 to 88C with uorescence data
acquisition at 0.1C increments with a hold of 2 s at each step. The HRMA curve
was analyzed using Rotor-Gene, version 1.7 (Build 65), software (Corbett Life
Science). The difference temperature plots were generated by converting the
wild-type melting prole to a horizontal line and normalizing the melting proles
of the examined samples against the wild-type prole.
DNA sequencing of katG, rpoB, and gyrA genes and mabA-inhA promoter. PCR
amplicons produced from the katG, rpoB, and gyrA genes and the mabA-inhA
promoter using the sequencing primers (Table 1) were puried using an
ExoSAP-IT PCR product cleanup kit (USB Corporation, Cleveland, OH) according
to the manufacturers instructions. The puried PCR products were sequenced
using forward and reverse primers on an ABI Prism 3100 genetic analyzer
(Applied Biosystems, Foster City, CA) using BigDye Terminator chemistry (ver-
sion 3.1). Sequences were analyzed by the Australia National Genomic Infor-
mation Service (ANGIS) (http://biomanager.info/).
RESULTS
HRMA optimization. Seventeen isolates with various drug
resistance phenotypes and 11 wild-type (fully sensitive) isolates
were selected for the development and initial evaluation of the
HRMA assay (Table 2). All four target genes of all 28 isolates
were sequenced prior to HRMA, and a total of 18 sequence
TABLE 1. PCR primers designed for and used in this study
Primer use and primer
a
Primer sequence (53)
Amplicon
size (bp)
Annealing
temp (C)
HRM temp
range (C)
Nucleotide
positions
b
GenBank
accession no.
HRMA
gyrA-F GGTGCTCTATGCAATGTTCG 234 63 8696 2467 to 24861 L27512.1
gyrA-R GCTTCGGTGTACCTCATCG 2700 to 2682
katG-F GCGGTCACACTTTCGGTAA 235 64 8595 2781 to 27991 X68081.1
katG-R GGTGTTCGTCCATACGACCT 2950 to 2931
rpoB-F CGCGATCAAGGAGTTCTTC 118 65 8494 2339 to 2357 L27989.1
rpoB-R TGACAGACCGCCGGGCCC 2456 to 2439
mabA-inhA-promoter-F GTCACACCGACAAACGTCAC 190 64 8292 100 to 119 U66801.1
mabA-inhA-promoter-R CTCCGGTAACCAGGACTGAA 296 to 271
Sequencing
gyrA-FS ACAGACACGACGTTGCCGC 672 69.97 NA
c
2309 to 2327 L27512.1
gyrA-RS GCCTTTAACCCGCCCCATGAC 72.09 NA 2980 to 2960
katG-FS TGCAGATGGGGCTGATCTACG 596 69.44 NA 2646 to 2666 X68081.1
katG-RS ACCCATGTCTCGGTGGATCAG 68.43 NA 3241 to 3221
rpoB-FS TGGTCCGCTTGCACGAGGGTCAGA 758 79.19 NA 2098 to 2121 L27989.1
rpoB-RS CAGGAAGGGAATCATCGCGG 70.87 NA 2855 to 2836
mabA-inhA-promoter-FS ACATACCTGCTGCGCAAT 400 61.9 NA 7 to 24 U66801.1
mabA-inhA-promoter-RS TCACATTCGACGCCAAAC 63.45 NA 405 to 388
a
F, forward primer; R, reverse primer; S, sequencing primer.
b
Nucleotide position is relative to the transcriptional start site of each gene.
c
NA, not applicable.
VOL. 49, 2011 RAPID DETECTION OF RESISTANCE IN M. TUBERCULOSIS 3451
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3452 CHEN ET AL. J. CLIN. MICROBIOL.
variants were identied, including katG (n 3), the mabA-
inhA promoter (n 3), rpoB (n 6), and gyrA (n 6), in the
17 drug-resistant isolates only. With one exception, sequence
variants demonstrated SNPs known to be associated with re-
sistance to the corresponding antibiotic. The exception was at
position 514 in rpoB of a RIF-resistant isolate (isolate 4533), in
which a duplication of TTC, which encodes a Phe (phenylala-
nine) insertion, was identied (Table 2).
HRMA curve proles were obtained for these 28 isolates.
All 44 targets in the 11 fully susceptible isolates, which had no
mutations, were correctly identied by HRMA to be wild type.
All 17 resistant isolates, which had mutations at one or more
sites, demonstrated variant melting curves for one or more
genes. Variant melting curves could be differentiated in the
normalized graphs (Fig. 1A to D) but were particularly clear in
the difference graphs (Fig. 1a to d). One INH-resistant isolate
and two RIF-resistant isolates had wild-type HRMA curves for
the katG gene and the mabA-inhA promoter (pattern K) and
the rpoB gene (patterns F and L), respectively, which were
consistent with sequencing results (Table 2). On the basis of
the different prole combinations for the four targets, there
were seven HRMA patterns identied in 17 selected isolates
with various drug resistance proles and one HRMA prole in
11 fully sensitive isolates (Table 2).
Sensitivity and specicity of HRMA. Another 87 isolates
produced 13 HRMA patterns, including the 7 HRMA proles
produced by 28 isolates tested in the preliminary evaluation.
Most of the 460 individual HRMA results were consistent with
their resistance phenotypes. There were seven exceptions (Ta-
ble 3): two isolates with pattern K (including one identied in
the preliminary evaluation), which were wild type by HRMA
for both katG and the mabA-inhA promoter but INH resistant;
one with pattern M, which was wild type for all HRMA pat-
terns but INH/RIF resistant; two with pattern F (one INH
susceptible and one RIF resistant), which had variant katG
proles and wild-type proles for all other HRMA patterns;
one with pattern E, which had a variant gyrA prole but which
was OFLX susceptible; and one with pattern L, which had a
wild-type prole for rpoB but which was RIF resistant. The
isolates which were resistant to all three drugs (INH, RIF, and
OFLX) demonstrated three HRMA patterns (in addition to
the two identied in the preliminary evaluation) (Table 3).
Two HRMA patterns were identied among the 32 fully
susceptible isolates; 31 (including 11 in the preliminary evalu-
ation) had wild-type proles for all four genes tested (pattern
M), as expected, but one had a katG variant (pattern F). Apart
from this one false-positive result, HRMA correctly identied
all (31 of 32; 96.9%) fully susceptible isolates.
Overall, variant proles were detected by HRMA for katG
and/or the mabA-inhA promoter in 66 of 69 (95.7%) INH-
resistant isolates: in rpoB for 51 of 54 (94.4%) RIF-resistant
isolates and in gyrA for all of 41 (100%) OFLX-resistant iso-
lates. The sensitivities and specicities of the HRMA are given
in Table 4.
Sequencing results. Amplicons from all four targets of all 87
isolates were sequenced, using primers targeting sites up-
stream and downstream of the HRMA forward and reverse
primer sites, respectively (Table 1). All HRMA results that
correctly predicted resistant or susceptible phenotypes were
conrmed by sequencing; i.e., no sequence mutations were
found in amplicons giving wild-type HRMA results, and vari-
ous sequence mutations were identied in those with variant
HRMA results. Sequencing results for HRMA proles that did
not correctly predict phenotypes were as follows: (i) the RIF-
resistant isolate with a wild-type HRMA prole in pattern F
had no mutations in the rpoB HRMA target region, but a
mutation at T480I (ACC 3ATC), outside the RRDR of rpoB,
was identied; (ii) the INH-susceptible isolate with a variant
katG HRMA prole in pattern F had a synonymous mutation
in katG, which did not change the amino acid sequence; (iii)
the OFLX-susceptible isolate with a gyrA HRMA variant in
pattern E had a synonymous mutation in the gyrA gene at
position 94 (GAC 3AAC); (iv) the INH/RIF-resistant isolate
with wild-type HRMA proles for all four targets (pattern M)
had a mutation at Y337C (TAC 3 TGC) of katG, which was
outside the HRMA detection region, but no mutations in rpoB;
and (v) no mutations were identied in two INH-resistant
isolates with HRMA pattern K (wild-type HRMA proles for
katG and the mabA-inhA promoter) or in a RIF-resistant iso-
late with pattern L (wild-type HRMA prole for rpoB).
The phenotypes of isolates with HRMA and/or sequencing
results inconsistent with the original phenotype were con-
rmed by retesting. These results suggest that genes other than
those targeted in this study contribute to the resistance in these
isolates.
Sequencing identied additional mutations outside the
HRMA target regions which did not affect the HRMA prole
or phenotypic resistance. For example, four OFLX-susceptible
isolates with wild-type HRMA proles had synonymous muta-
tions in gyrA at position 197 (CTG 3 CTA, three isolates) or
171 (CCC 3CCA, one isolate). All 115 clinical isolates tested
in this study exhibited a naturally occurring polymorphism
(AGC to ACC) at codon 95 in gyrA, which was not associated
with phenotypic resistance.
DISCUSSION
INH resistance is associated with gene mutations in one or
more of katG, inhA, the mabA-inhA promoter, kasA, oxyR, and
ahpC (26). It is reported that 50 to 70% of INH-resistant
isolates have katG mutations and that another 15 to 20% have
mabA-inhA promoter mutations. RIF resistance is due to mis-
sense mutations or, less commonly, small in-frame deletions or
insertions in or around the 81-bp RRDR of rpoB in 95% of
cases (26, 37); in the other 5%, mutations in other regions of
rpoB, such as V146F (1), or other genes, such as Rv2629 (e.g.,
mutation at A191C) (38), are responsible. Mutations in gyrA,
clustered in a short 40-amino-acid region known as the quin-
olone resistance-determining region (QRDR) (43), are re-
sponsible for 42% to 85% of the cases of OFLX resistance in
clinical isolates (11, 16, 26, 34, 42).
In this study, all 18 mutations, identied by sequencing of
four target genes in 17 INH-, RIF-, and/or OFLX-resistant
isolates, produced clearly distinguishable melting curves by
HRMA. Among all 115 isolates, INH, RIF, and OFLX resis-
tances were detected by HRMA with specicities of 97.8%,
100%, and 98.6%, respectively, and sensitivities of 95.7%,
94.4%, and 100%, respectively. However, the sensitivity was
100% for mutations within the target regions of the genes
studied. These results are consistent with those of the most
VOL. 49, 2011 RAPID DETECTION OF RESISTANCE IN M. TUBERCULOSIS 3453
FIG. 1. Normalized and temperature-shifted difference plots for mutant discrimination by HRMA in katG (A and a, respectively), mabA-inhA
promoter (B and b, respectively), rpoB (C and c, respectively), and gyrA (D and d, respectively). Wild-type proles are shown in black, and variant
proles are shown in color. Numbers in parentheses are numbers of isolates tested.
3454
recent studies, in which sensitivities have ranged from 84.1% to
87% for INH resistance and from 91% to 98.6% for RIF
resistance and the specicities have ranged from 98% to 100%
(7, 27). Only a few discrepancies between HRMA proles and
drug resistance phenotypes were observed in this study. Of all
83 isolates with single or multiple drug resistances, 5 (6.0%)
had wild-type HRMA proles and no mutations in the relevant
HRMA target regions were identied by sequencing, suggest-
ing other resistance mechanisms, which may include mutations
in other genes, such as kasA and ahpC for INH resistance, as
suggested elsewhere (26).
Duplex melting is now generally monitored using intercalat-
ing dyes such as LC Green (as in this study), rather than SYBR
green, which is biased against low-temperature-melting species
and does not detect heteroduplexes because it is redistributed
during melting (13, 40). LC Green is a new type of uorescent
DNA dye which binds to double-stranded but not single-
stranded DNA and can detect heteroduplexes during homo-
geneous melting curve analysis. It has successfully been used in
HRMA due to its ability to saturate available binding sites and
PCR products at a concentration compatible with PCR prod-
ucts. This means that it does not inhibit amplication or redis-
tribute as the amplicon melts. Its use allows homogeneous
genotyping without uorescently labeled probes and allele-
specic or real-time PCR instruments.
HRMA is a simple, closed-tube system, which not only re-
duces the risk of contamination but also increases sample
throughput because there is no requirement for physical sep-
aration of DNA molecules (36). HRMA provides a sensitive,
homogeneous scanning method using controlled heating at a
high rate and high data density. Variants are easily identied
because they distort the melting curve shape compared to that
TABLE 3. HRMA patterns, based on proles of four genes/region associated with antibiotic resistance, in 115 M. tuberculosis clinical isolates
HRMA
pattern
No. of isolates HRMA prole
a
Phenotype
b
Evaluation
set
Test set katG
mabA-inhA
promoter
rpoB gyrA INH RIF OFLX
A 3 4 V V V V R R R
B 1 V V V WT R R S
C 2 19 V WT V V R R R
D 1 7 V WT V WT R R S
E 3 V WT WT V R S R
1 V WT WT V R S S
F 17 V WT WT WT R S S
1 V WT WT WT S S S
1 V WT WT WT R R S
G 2 WT V V V R R R
H 2 WT V V WT R R S
I 3 WT V WT WT R S S
J 1 1 WT WT V V S R R
K 3 3 WT WT V WT S R S
1 1 WT WT V WT R R S
L 4 1 WT WT WT V S S R
1 WT WT WT V S R R
M 11 20 WT WT WT WT S S S
1 WT WT WT WT R R S
Total 28 87
a
V, variant; WT, wild type.
b
INH, isoniazid; RIF, rifampin; OFLX, ooxacin (uoroquinolone); S, susceptible; R, resistant.
TABLE 4. Sensitivities and specicities of HRMA in comparison with drug susceptibility testing result between three anti-MTB drugs
Drug Target
HRMA prole Drug resistance phenotype
Sensitivity
a
(%) Specicity
b
(%)
No. of
isolates
% variant
No. of
isolates
% resistant

Isoniazid katG 60 55 52.2 69 46 60.0 95.7 (87.599.0) 97.8 (87.699.9)
mabA-inhA promoter 15 100 13.0
Either or both 67 48 58.3
Rifampin rpoB 51 64 44.3 54 61 47.0 94.4 (84.398.7) 100 (94.9100)
Ooxacin gyrA 42 73 36.5 41 74 35.6 100 (92.6100) 98.6 (92.099.9)
a
Sensitivity is dened as the (number of drug-resistant isolates with mutations)/(number of drug-resistant isolates with mutations number of drug-resistant isolates
without mutations). Data in parentheses are 95% condence intervals, which were calculated with the free software available from http://www.measuringusability.com
/wald.htm and the adjusted Wald method.
b
Specicity is dened as the (number of drug-susceptible isolates without mutations)/(number of drug-susceptible isolates without mutations number of
drug-susceptible isolates with mutations). Data in parentheses are 95% condence intervals.
VOL. 49, 2011 RAPID DETECTION OF RESISTANCE IN M. TUBERCULOSIS 3455
for the wild type (10). Only two primers and one PCR per
target and a melting instrument are required. Reagent costs for
genotyping by amplicon melting are low because only a PCR
system and a generic dye are needed. No probes or specialized
reagents are required (17).
According to the binary combinations of base groups, SNPs
are divided into four classes: class 1 (C/T or G/A), class 2 (C/A
or G/T), class 3 (C/G), and class 4 (T/A) (17). Class 1 and 2
SNPs can be easily distinguished due to melting temperature
(T
m
) differences mostly of 0.8 to 1.4C. However, T
m
differ-
ences are usually less than 0.4C for class 3 and 4 SNPs (17),
making it more difcult to differentiate them. In our study,
mutations in katG at 315G 3 C, the mabA-inhA promoter at
15C 3G, and rpoB at 526C 3G are class 3 SNPs and so are
difcult to distinguish from wild type in normalized HRMA
graphs (Fig. 1A to C). However, they can easily be distin-
guished by temperature-shifted difference plots (Fig. 1a to c),
which should be used when SNPs are analyzed by HRMA.
The distinct advantage of genotypic drug susceptibility as-
says over phenotypic assays lies in the reduced turnaround
time. The PCR-based methods need only hours to complete
rather than days to weeks with phenotypic tests. Molecular
techniques based on PCR amplication of genes involved in
resistance mechanisms, followed by the detection of key mu-
tations associated with resistance, provide faster results (5).
Getting an early drug susceptibility result is clinically critical
for patients. It allows the more timely implementation of an
appropriate TB treatment regimen and, potentially, better out-
comes. It also allows surveillance of antibiotic resistance rates,
which are relevant to TB control and recommendations for
initial or empirical therapy. The prevalence of primary uoro-
quinolone-resistant TB is currently still low worldwide. How-
ever, extensive use of uoroquinolones for treatment of bac-
terial infections could result in increased rates of primary
uoroquinolone-resistant TB, especially in those countries
where there is a high burden of TB (2, 12).
In summary, this is the rst description of an HRMA assay
for four different drug resistance-related genes in M. tubercu-
losis. It is a novel, rapid, and sensitive method for screening for
gene mutations associated with M. tuberculosis drug resistance
and could be used routinely at low cost, which would be suit-
able for those laboratories in economically less developed
countries where the prevalence of TB is high.
ACKNOWLEDGMENT
This work was supported by the Scientic Program for the Returned
Scholar from Overseas in Beijing, China (major project 20080007).
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