First report of molecular detection of fluoroquinolone resistance-associated gyrA mutations in multidrug-resistant clinical Mycobacterium tuberculosis isolates in Kuwait
BMC Research Notes
First report of molecular detection of fluoroquinolone resistance-associated gyrA mutations in multidrug-resistant clinical Mycobacterium tuberculosis isolates in Kuwait
Noura M Al-Mutairi 0
Suhail Ahmad 0
Eiman Mokaddas 0
0 Department of Microbiology, Faculty of Medicine, Kuwait University , Kuwait
Background: Nearly 5% of all Mycobacterium tuberculosis strains worldwide are resistant at least to rifampicin and isoniazid (multidrug-resistant tuberculosis, MDR-TB). Inclusion of a fluoroquinolone and an injectable agent (kanamycin, amikacin or capreomycin) in multidrug therapy is crucial for proper treatment of MDR-TB. The incidence of MDR-TB in Kuwait is ~1%. MDR-TB strains additionally resistant to fluoroquinolones and injectable agents are defined as extensively drug-resistant (XDR-TB) strains and have been detected in >55 countries. Infections with XDR-TB strains have very poor prognosis. This study detected the occurrence of gyrA mutations associated with fluoroquinolone resistance among MDR-TB strains in Kuwait. Findings: Direct DNA sequencing of quinolone resistance-determining region of gyrA gene was performed to detect fluoroquinolone resistance-associated mutations in 85 MDR-TB strains isolated from 55 TB patients and 25 pansusceptible M. tuberculosis strains. For isolates exhibiting gyrA mutations, 3'-end of rrs (16S rRNA) was sequenced for the detection of XDR-TB. Fingerprinting of fluoroquinolone resistant MDR-TB strains was performed by detecting mutations in three (81 bp hot-spot, N-terminal and cluster II) regions of rpoB, katG codon 315 and inhA-regulatory region, polymorphisms at gyrA codon 95 and katG codon 463 by DNA sequencing and by doublerepetitive-element PCR for determining strain relatedness. None of the pansusceptible but six of 85 MDR-TB strains contained gyrA mutations. Only gyrA codon 94 was mutated in all six (D94A in one and D94G in five) strains. Three of six mutant strains were recovered from the same patient while three other strains represented individual patient isolates. Fingerprinting studies identified all individual patient isolates as epidemiologically distinct strains. All six strains with a gyrA mutation contained wild-type rrs sequence. Conclusions: Although fluoroquinolones are generally not used for chemotherapy of TB and drug susceptibility testing for second-line drugs is not carried out in Kuwait, four of 55 (7%) individual patient MDR-TB strains contained mutations in gyrA gene. The data advocate routine drug susceptibility testing for this important second-line drug for proper management of MDR-TB in Kuwait. Lack of mutations in 3'-end of rrs gene that confer resistance to injectable agents reduce the likelihood of occurrence of XDR-TB, at present, in Kuwait.
M; tuberculosis Fluoroquinolone resistance; gyrA mutations; Kuwait
Tuberculosis (TB), causing nearly 9 million active
disease cases and two million deaths worldwide every year,
is a major public health issue . Increasing resistance
of Mycobacterium tuberculosis strains to most-effective
(first-line) anti-TB drugs and strong association of
human immunodeficiency virus (HIV) pandemic with
active TB disease are the two major contributors to the
current global burden of TB [1-3]. Incomplete/improper
treatment of TB patients leads to evolution of
drugresistant M. tuberculosis strains due to chromosomal
mutations in genes encoding drug targets . Sequential
accumulation of mutations in target genes generate
multidrug-resistant (resistant at least to rifampicin and
isoniazid) M. tuberculosis (MDR-TB) and extensively
drugresistant (additionally resistant to fluoroquinolones and
an injectable anti-TB agent such as kanamycin, amikacin
or capreomycin) M. tuberculosis (XDR-TB) strains [4,5].
While proper treatment of drug-susceptible TB has
≥95% cure rate, effective treatment of MDR-TB is
difficult in developing countries as it is heavily dependent
on rapid diagnosis, supervised aggressive therapy with
several (5-6) expensive, toxic and less efficacious
(second-line) drugs for 18-24 months [4-7]. Inclusion of a
fluoroquinolone and an injectable agent in multidrug
treatment regimens have a more favorable outcome in
the treatment of MDR-TB [4-8]. Treatment of XDR-TB
is far more difficult even in developed countries while in
developing countries, XDR-TB is virtually an untreatable
disease [8-10]. The prognosis of XDR-TB in
HIV-coinfected TB patients is extremely poor, with fatality rates
varying from ~30% in developed countries to nearly
100% in developing countries [8-11]. Hence, all efforts
should be made to successfully cure the existing
MDRTB cases to avoid the emergence of XDR-TB [4,6,8,12].
Fluoroquinolones (FQs), particularly new generation
compounds (such as moxifloxacin and gatifloxacin) and
injectable aminoglycosides (kanamycin and amikacin) and
cyclic peptide (capreomycin) have excellent bactericidal
activity against M. tuberculosis and are crucial for proper
management of MDR-TB patients [4,8,12,13]. Widespread
emergence of MDR-TB has accelerated development of
rapid drug susceptibility testing (DST) procedures for
these important second-line drugs to help design effective
treatment strategies. The cellular target of FQs in M.
tuberculosis is DNA gyrase, a type II topoisomerase
consisting of two A and two B subunits encoded by gyrA and
gyrB genes, respectively . Mutations in a small region
of gyrA, called quinolone resistance-determining region
(QRDR) and, less frequently, in gyrB are the primary
mechanism of FQ resistance in M. tuberculosis [14-16].
Analysis of QRDR alone by genotypic tests has been
suggested as sufficient for rapid identification of vast majority
of FQ-resistant M. tuberculosis strains as additional
targeting of gyrB did not enhance the sensitivity significantly
[16,17]. The molecular basis of resistance of M.
tuberculosis strains to injectable agents such as aminoglycosides
(kanamycin and amikacin) and cyclic peptide
(capreomycin) has also been determined [4,18,19]. Nearly 90% to
95% of M. tuberculosis strains resistant to one or more of
the injectable agents contain mutations near the 3’-end of
rrs (16S rRNA) gene involving nucleotide positions A1401,
C1402, and G1484 [4,17-19].
The rate of MDR-TB is quite low (~1%) and FQs and
injectable agents are rarely used for chemotherapy of
TB in Kuwait . This study was carried out to detect
the occurrence of gyrA mutations associated with
fluoroquinolone resistance among MDR-TB strains. The
3’end of rrs gene was also sequenced among isolates
containing gyrA mutations to detect the occurrence of
XDR-TB in Kuwait.
Bacterial isolates and susceptibility testing
A total of 4926 M. tuberculosis strains were isolated
from TB patients during the study period (January 2001
to June 2008) at National Tuberculosis Reference
Laboratory in Kuwait. A total of 110 M. tuberculosis
isolates were analyzed in this study. These included 85
MDR-TB strains isolated from 55 TB patients
(representing all available MDR M. tuberculosis strains).
Twenty-five drug-susceptible M. tuberculosis strains
(isolated from 25 patients) were also included to ensure
that fluoroquinolone resistance-conferring mutations in
the gyrA gene are not found in pansusceptible M.
tuberculosis strains. M. tuberculosis H37Rv was used as a
control. Isolation and identification of M. tuberculosis
isolates was performed by using MGIT 960 system
(Becton Dickinson) and multiplex PCR as described
previously [20,21]. The phenotypic DST against first-line
drugs (isoniazid, rifampicin, ethambutol, and
streptomycin) was carried out by BACTEC 460 TB system as
described in detail previously .
DNA extraction for molecular assays
One ml of MGIT 960 culture of reference or clinical M.
tuberculosis isolate was heated with 40 mg Chelex-100
(Sigma-Aldrich) at 95°C for 20 min and then centrifuged
at 12,000 × g for 15 min . For a PCR, 2 μl of
supernatant was used as a source of DNA.
Detection of mutations in QRDR of the gyrA gene by DNA
The QRDR of the gyrA gene was amplified by
touchdown PCR by using GYRAF (5’-
CGCAGCTACATCGACTATGCGATG-3’) and GYRAR (5’-GGGATGAA
ATCGATGTCTCCTCG-3’) as amplification primers
and the reaction and thermal cycling conditions as
described in detail previously . The 400 bp
amplicons were purified by using PCR product purification
kit (Qiagen) and sequenced by using cycle DNA
sequencing kit (DTCS CEQ2000, Beckman-Coulter) and
GYRAFS (5’-CGGGTGCTCTATGCAATGTTC-3’) or
GYRARS (5’- GGCTTCGGTGTACCTCATCGCC-3’) as
internal sequencing primer. Although PCR products
were purified, internal primers were used for sequencing
of PCR amplicons to avoid interference from trace
amounts of primer dimers, if still present. The detailed
methodology of DNA sequencing was same as described
in detail previously . Nucleotide and amino acid
sequences of the amplified products were compared
with corresponding sequences from susceptible strain
M. tuberculosis H37Rv using BLAST. DNA sequencing
of QRDR also revealed the polymorphism (S95 or T95)
that occurs at gyrA codon 95 among M. tuberculosis
Fingerprinting of MDR-TB strains carrying gyrA mutation
Fingerprinting of MDR-TB strains carrying gyrA
mutation was carried out by direct DNA sequencing of
hotspot, N-terminal and cluster II regions of rpoB gene,
inhA-regulatory region and katG codon 315 to detect
mutations conferring resistance to rifampicin and
isoniazid, as described in detail elsewhere . The genetic
group  of the isolates was determined by detecting
the polymorphism at gyrA codon 95 (described above)
and at katG codon 463 (L463 + T95, Group I; R463 +
T95, Group II and R463 + S95, Group III). The
presence of R463/L463 at katG codon 463 was detected by
PCR amplification with KATG463F and KATG463R
primers followed by restriction digestion with Nci I, to
generate RFLP patterns, as described previously .
Further fingerprinting of the isolates was carried out by
double-repetitive-element (DRE)-PCR and isolates
yielding unique patters were classified as genotypically
distinct strains .
Detection of mutations at 3’-end of rrs gene by DNA
The 3’-end of rrs (16S rRNA) gene that is mutated in
nearly 90% to 95% of M. tuberculosis strains exhibiting
resistance to injectable agents (kanamycin, amikacin and
capreomycin) [17-19] was also amplified by touchdown
PCR by using 16S3F
(5’-GCGATGCCGCGAGGTTAAGCGAA-3’) and 16S3R (5’-CCAACAGTGTGTT
GGTGGCCAA-3’) as amplification primers and the
reaction and thermal cycling conditions described
previously . The 403 bp amplicons were purified and
sequenced by using 16S3FS (5’-ATCCTTAAAAGC
CGGTCTCAGT-3’) or 16S3RS (5’- CTCCTTAGAAA
GGAGGTGATCCA-3’) as internal sequencing primer.
Again, internal primers were used for sequencing to
avoid interference from trace amounts of primer dimers,
if still present along with of PCR amplicons of rrs gene.
The detailed DNA sequencing protocol has been
described in detail previously . Nucleotide and
amino acid sequences of the amplified products were
again compared with corresponding sequences from
susceptible strain M. tuberculosis H37Rv using BLAST.
Nucleotide sequence accession numbers
The DNA sequencing data reported in this study have
been deposited in EMBL under accession numbers
Phenotypic DST data for 85 MDR-TB strains showed
that 21 (25%) isolates were resistant to isoniazid and
rifampicin only while seven (8%), 23 (27%) and 34 (40%)
isolates were additionally resistant to streptomycin,
ethambutol and to streptomycin and ethambutol,
respectively (Table 1). Of the 55 patients yielding
MDRTB strains, only two were Kuwaiti nationals while the
remaining 53 patients were expatriate workers or their
family members. The countries of origin for the 53
expatriate patients included India (n = 24), Philippines
(n = 10), Egypt (n = 7), Bangladesh (n = 2), Indonesia (n
= 2), Syria (n = 2), Iraq (n = 2), Ethiopia (n = 1), Nepal
(n = 1), Nigeria (n = 1) and Pakistan (n = 1). The date
of arrival in Kuwait was not available for the expatriate
patients. All M. tuberculosis isolates were recovered
from HIV-seronegative adult TB patients between the
ages of 21 to 65 years and were resistant to the
indicated drugs on first isolation; however, information on
prior treatment of expatriate patients with anti-TB
drugs was not available. The 25 M. tuberculosis isolates
were susceptible to all first-line drugs (pansusceptible
Table 1 Resistance patterns and presence of
fluoroquinolone resistance-associated gyrA mutations in
110 M. tuberculosis strains tested.
Resistance pattern of
M. tuberculosis isolatea
aH, isoniazid; R, rifampicin, S, streptomycin; E, ethambutol
*3 of 4 M. tuberculosis isolates were recovered from the same TB patient
All 110 isolates were identified as M. tuberculosis by a
multiplex PCR that yielded 2 DNA fragments of ~473
bp and ~235 bp derived from oxyR and rpoB genes,
respectively, as expected (data from seven selected
MDR-TB strains are shown in Figure 1). The PCR
amplification of QRDR of gyrA gene from reference
strain M. tuberculosis H37Rv as well as from all 25
pansusceptible and 85 MDR M. tuberculosis strains yielded
an expected amplicon of ~400 bp. The DNA sequencing
data of all 25 pansusceptible M. tuberculosis strains
showed complete concordance with wild-type gyrA
sequence from reference strain except for polymorphism
(S95 or T95) at gyrA codon 95, which is not associated
with fluoroquinolone resistance . The DNA
sequencing data from 85 MDR-TB strains showed nucleotide
(and amino acid) changes in QRDR of the gyrA gene in
six isolates while the remaining 79 strains contained
wild-type sequences except for gyrA codon 95 (Table 1).
Among the six MDR-TB strains, nucleotide changes
were detected only at gyrA codon 94, with five strains
containing GAC94GGC (D94G) mutation and one
isolate containing GAC94GCC (D94A) mutation.
Interestingly, FQ resistance in MDR-TB strains was associated
with additional resistance to ethambutol. Thus, all
MDR-TB strains with gyrA mutations were resistant to
three or all four first-line drugs (Table 1). Three of six
FQ-resistant strains were isolated from the same patient
within a period of two months while the remaining
three isolates were recovered from three separate TB
Figure 1 Species-specific identification of M. tuberculosis
isolates by multiplex PCR. Representative agarose gel of
amplicons of multiplex PCR from 7 selected multidrug-resistant M.
tuberculosis isolates (lanes 1-7) showing M. tuberculosis-specific
amplification of two fragments of 473 bp and 235 bp (marked by
arrows) from oxyR and rpoB genes, respectively. Lane M is 100 bp
DNA ladder and the position of migration of 100 and 600 bp
fragments are marked.
patients. Thus, four of 55 (7%) individual patient
MDRTB strains contained mutations in QRDR of the gyrA
The year of isolation and results of fingerprinting
studies for the four individual patient MDR-TB strains
containing FQ resistance-associated gyrA mutations are
presented in Table 2. The isolates were recovered over a
four-year period (2004 to 2008) from male expatriate
patients originating from India (n = 3) and Bangladesh
(n = 1). Three patients developed pulmonary TB while
the fourth patient had extrapulmonary form of the
disease. Fingerprinting of the isolates based on specific
mutations in hot-spot, N-terminal and cluster II regions
of rpoB, katG codon 315 and inhA-regulatory region
and genetic group analyses showed that all four isolates
were distinct strains (Table 2). All four isolates also
yielded unique DRE-PCR patterns. The two repeat
isolates recovered from one patient (no. 3) exhibited the
same fingerprinting patterns as the first isolate from this
patient (data not shown).
Kuwait, an Arabian Gulf country with nearly 25 cases
per 100, 000 population is a low TB incidence country
. However, it has a large expatriate population
originating from TB endemic countries of South-Southeast
Asia. Nearly 600 patients are diagnosed with
mycobacterial infections every year. Nearly 95% of mycobacterial
infections are caused by M. tuberculosis (TB) while the
remaining 5% are caused by non-tuberculous
mycobacteria . The non-tuberculous mycobacterial infections
are more common among Kuwaiti patients, however,
~80% of TB patients are expatriate workers or their
family members [20,29]. This is despite the fact that all
expatriates are screened for TB and HIV on entry and
are allowed to stay in Kuwait only if they exhibit no
obvious signs (suspected lesions on chest radiograph) of
active TB disease or previous exposure to active disease.
Furthermore, all non-infectious TB cases are sent back
(after anti-TB therapy of 4-8 weeks, if required) to their
respective countries for further management . While
global proportion of MDR-TB has been estimated to be
~5.3% among all cases [2,3], only ~1% of M. tuberculosis
isolates in Kuwait are MDR-TB strains and nearly all of
these strains are isolated from expatriate patients .
The incidence of extrapulmonary TB in Kuwait is
relatively high, accounting for 44% of all TB cases and
majority of pulmonary TB patients have cavitary lesions
in the upper lobes [20,28]. These findings together with
the low incidence of TB in Kuwait are consistent with
observations that majority of active TB disease cases in
foreign-born persons occur as a result of reactivation of
prior (latent) infection, usually within the first few years
of their migration . Previous fingerprinting studies
Table 2 Patient’s demographic data and fingerprinting data for the four individual patient multidrug-resistant M.
tuberculosis strains containing a mutation in quinolone resistance-determining region (QRDR) of gyrA gene.
aH, isoniazid; R, rifampicin, S, streptomycin; E, ethambutol.
bAll four were male patients and all individual patient isolates contained AGC315ACC mutation in katG codon 315 and yielded unique patterns in DRE-PCR.
cThree (hot-spot, N-terminal and cluster II) regions of rpoB gene were sequenced to detect rifampicin resistance-conferring mutations and codon numbering
system of rpoB gene from Escherichia coli is used .
dInhA-RR, inhA regulatory region sequencing data.
eGenetic groups were assigned based on polymorphisms at katG codon 463 and gyrA codon 95(L463/T95, Group I; R463/S95, Group III)
fThis isolate either contained a mutation in other regions of the rpoB gene or in other genes involved in rifampicin resistance .
*Two repeat isolates recovered from this patient exhibited identical patterns.
have also shown that vast majority of MDR-TB cases
among expatriate patients in Kuwait have unique
patterns [31,32]. Since fingerprinting patterns are highly
variable in countries that have a low incidence of active
TB disease and immigrants originating from high
incidence countries , these observations also support
reactivation of previously acquired infection as the
major mechanism for active TB disease in most patients
The FQs play an important role in the treatment of
MDR-TB since their inclusion in therapy regimens
improves treatment outcome. Resistance of MDR-TB
strains to FQs is associated with poor treatment
outcome and is also one of two key defining conditions of
XDR-TB [4,7-10,12]. Thus, there is a pressing need for
rapid DST of MDR-TB strains against FQs to improve
clinical management. However, like many other
countries , routine DST for second-line drugs has not
been instituted in the National Tuberculosis Control
Program in Kuwait, mainly due to low rate of MDR-TB
. Before planning for routine phenotypic and/or
genotypic DST for FQs and other second-line drugs in
Kuwait, this study was carried out to detect the
occurrence of gyrA mutations associated with FQ resistance
among MDR-TB strains.
Our data showed that four of 55 (7%) individual
patient MDR-TB isolates in Kuwait contained mutations
in QRDR of the gyrA gene. Interestingly, all MDR-TB
strains with a gyrA mutation were additionally resistant
to ethambutol with/without additional resistance to
streptomycin. Previous studies have also noted an
association of FQ resistance in M. tuberculosis strains with
additional resistance to several first-line drugs [15,17].
All four individual patient isolates contained a mutation
at gyrA codon 94 which was also the most frequently
mutated codon among FQ-resistant M. tuberculosis
strains in several previous studies [15,17,34-37]. Three
of four individual patient isolates with gyrA mutation
contained D94G mutation while the fourth isolate
contained D94A mutation. Studies from Germany, Taiwan
and Russia have also reported that D94G is the most
common mutation observed in FQ-resistant M.
tuberculosis strains [15,17,35] while FQ-resistant strains from
Japan contained D94A as the most frequent mutation
. Although phenotypic drug susceptibility testing for
FQs was not performed in this study, the level of
resistance in FQ-resistant M. tuberculosis strains could be
inferred based on the nature and kind of gyrA mutations
detected. Previous studies have shown that M.
tuberculosis strains containing D94G mutation in the gyrA gene
exhibit minimum inhibitory concentration (MIC) of ≥4
μg/ml while those with D94A mutation have MIC of 2
μg/ml for levofloxacin [37,38]. Based on these
observations, it may be inferred that three of four FQ-resistant
M. tuberculosis strains from Kuwait were highly
resistant to fluoroquinolones.
Three of four patients infected with M. tuberculosis
strains containing gyrA mutation originated from India
while the fourth patient was from Bangladesh. Since
FQs are frequently used for other bacterial infections, it
is probable that many expatriate patients were exposed
to these agents in their respective native countries.
Recent data have shown that 24% and 9% of MDR-TB
strains from India and Bangladesh, respectively, are
resistant to FQs . The MDR-TB strains containing
gyrA mutations were cultured at the time of clinical
diagnosis before anti-TB treatment was initiated
(primary resistance) and not during treatment during a
fiveyear period and were genotypically distinct strains.
These observations together with the low incidence of
active TB disease and low rates of active transmission of
infection in Kuwait [20,28-30] imply that the four
expatriate patients did not acquire the infection recently
but were most likely latently infected with FQ-resistant
strains. A limitation of the present study is that
phenotypic DST for FQs was not performed. Previous studies
from several geographical locations have shown that
nearly 70% to 100% of FQ-resistant M. tuberculosis
strains contain mutations in QRDR of the gyrA gene
while FQ resistance in other isolates is associated with
mutations outside of QRDR of gyrA gene or due to gyrB
gene mutations [15,17,34-38]. It is, therefore, probable
that phenotypic DST may have identified few (one or
two) additional FQ-resistant MDR-TB strains in Kuwait.
Thus, phenotypic DST should be performed as an
important preliminary tool to determine the real
prevalence of FQ resistance in Kuwait.
The absence of mutations in 3’-end of rrs gene, which
confer resistance to kanamycin, amikacin or
capreomycin [17-19], among the six FQ-resistant MDR-TB strains
is reassuring as it decreases the possibility of the
presence of XDR-TB in Kuwait. This is most likely due to
very infrequent use of these injectable agents for the
treatment of TB in Kuwait.
The detection of fluoroquinolone resistance-associated
mutations in gyrA gene in four of 55 (7%) individual
patient MDR-TB strains strongly suggest the need for
routine drug susceptibility testing for this important
second-line drug for proper management of MDR-TB in
Kuwait. However, lack of mutations in 3’-end of rrs
gene that confer resistance to injectable agents is
encouraging and rules out, at least for now, the presence
of XDR-TB in Kuwait.
1. World Health Organization: Global tuberculosis control: WHO report 2010 . WHO/HTM/TB/2010 .7. Geneva , Switzerland: WHO ; 2010 .
2. World Health Organization: Anti-tuberculosis drug resistance in the world: fourth Global report . WHO/HTM/TB/2008 .394. Geneva, Switzerland: WHO ; 2008 .
3. Wright A , Zignol M , Van Deun A , Falzon D , Gerdes SR , Feldman K , Hoffner S , Drobniewski F , Barrera L , van Soolingen D , Boulabhal F , Paramasivan CN , Kam KM , Mitarai S , Nunn P , Raviglione M , for the Global Project on Anti-Tuberculosis Drug Resistance Surveillance: Epidemiology of antituberculosis drug resistance 2002-2007: an updated analysis of the Global Project on Anti-Tuberculosis Drug Resistance Surveillance . Lancet 2009 , 373 : 1861 - 1873 .
4. Ahmad S , Mokaddas E : Recent advances in the diagnosis and treatment of multidrug-resistant tuberculosis . Resp Med 2009 , 103 : 1777 - 1790 .
5. World Health Organization: Multidrug and extensively drug-resistant TB (M/XDR-TB): 2010 global report on surveillance and response . WHO/ HTM/TB/2010 .3. Geneva , Switzerland: WHO ; 2010 .
6. Mitnick CD , Castro KG , Harrington M , Sacks LV , Burman W : Randomized trials to optimize treatment of multidrug-resistant tuberculosis . PLoS Med 2007 , 4 :article no. e292.
7. Orenstein EW , Basu S , Shah NS , Andrews JR , Friedland GH , Moll AP , Gandhi NR , Galvani AP : Treatment outcomes among patients with multidrug-resistant tuberculosis: systematic review and meta-analysis . Lancet Infect Dis 2009 , 9 : 153 - 161 .
8. Ahmad S , Mokaddas E : New challenges in the treatment of tuberculosis in the 21st century . Treat Strat Resp 2011 , 1 : 100 - 110 .
9. Mitnick CD , Shin SS , Seung GY , Rich ML , Atwood SS , Furin JJ , Fitzmaurice GM , Alcantara Viru FA , Appleton SC , Bayona JN , Bonilla CA , Chalco K , Choi S , Franke MF , Fraser HS , Guerra D , Hurtado RM , Jazayeri D , Joseph K , Llaro K , Mestanza L , Mukherjee JS , Muñoz M , Palacios E , Sanchez E , Sloutsky A , Becerra MC : Comprehensive treatment of extensively drug-resistant tuberculosis . N Engl J Med 2008 , 359 : 563 - 574 .
10. Jacobson KR , Tierney DB , Jeon CY , Mitnick CD , Murray MB : Treatment outcomes among patients with extensively drug-resistant tuberculosis: systematic review and meta-analysis . Clin Infect Dis 2010 , 51 : 6 - 14 .
11. Gandhi NR , Moll A , Sturn AW , Pawinski R , Govender T , Lalloo U , Zeller K , Andrews J , Friedland G : Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa . Lancet 2006 , 368 : 1554 - 1556 .
12. Kliman K , Altraja A : Predictors of poor treatment outcome in multi and extensively drug-resistant pulmonary TB . Eur Respir J 2009 , 33 : 1085 - 1094 .
13. American Thoracic Society , CDC, Infectious Disease Society of America: Treatment of tuberculosis . Morb Mort Weekly Rep 2003 , 52 : 1 - 77 .
14. Ginsburg AS , Grosset JH , Bishai WR : Fluoroquinolones, tuberculosis, and resistance. Lancet Infect Dis 2003 , 3 : 432 - 442 .
15. Wang JY , Lee LN , Lai HC , Wang SK , Jan IS , Yu CJ , Hsueh PR , Yang PC : Fluoroquinolones resistance in Mycobacterium tuberculosis isolates: associated genetic mutations and relationship to antimicrobial exposure . J Antimicrob Chemother 2007 , 59 : 860 - 865 .
16. Chang KC , Yew WW , Chan RCY : Rapid assays for fluoroquinolone resistance in Mycobacterium tuberculosis: a systematic review and metaanalysis . J Antimicrob Chemother 2010 , 65 : 1551 - 1561 .
17. Hillemann D , Rusch-Gerdes S , Richter E : Feasibility of the GenoType MTBDRsl assay for fluoroquinolone, amikacin-capreomycin and ethambutol resistance testing of Mycobacterium tuberculosis strains and clinical specimens . J Clin Microbiol 2009 , 47 : 1767 - 1772 .
18. Maus CE , Pilkaytis BB , Shinnick TM : Molecular analysis of cross-resistance to capreomycin, kanamycin, amikacin and viomycin in Mycobacterium tuberculosis . Antimicrob Agents Chemother 2005 , 49 : 3192 - 3197 .
19. Jugheli L , Bzekalava N , de Rijk P , Fissette K , Portaels F , Rigouts L : High level cross-resistance between kanamycin, amikacin and capreomycin among Mycobacterium tuberculosis isolates from Georgia and a close relation with mutations in the rrs gene . Antimicrob Agents Chemother 2009 , 53 : 5064 - 5068 .
20. Mokaddas E , Ahmad S , Samir I : Secular trends in susceptibility patterns of Mycobacterium tuberculosis isolates in Kuwait , 1996 - 2005 . Int J Tuberc Lung Dis 2008 , 12 : 319 - 325 .
21. Mokaddas E , Ahmad S : Development and evaluation of a multiplex PCR for rapid detection and differentiation of Mycobacterium tuberculosis complex members from non-tuberculous mycobacteria . Jpn J Infect Dis 2007 , 60 : 140 - 144 .
22. Jaber AA , Ahmad S , Mokaddas E : Minor contribution of mutations at iniA codon 501 and embC-embA intergenic region in ethambutol-resistant clinical Mycobacterium tuberculosis isolates in Kuwait . Ann Clin Microbiol Antimicrob 2009 , 8 : 2 .
23. Ahmad S , Mokaddas E , Jaber AA : Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCR-RFLP targeting embB codons 306 and 497 and iniA codon 501 mutations . Mol Cell Probes 2004 , 18 : 299 - 306 .
24. Sreevatsan S , Pan X , Stockbauer KE , Connell ND , Kreiswirth BN , Whittam TS , Musser JM : Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination . Proc Natl Acad Sci USA 1997 , 94 : 9869 - 9874 .
25. Al-Mutairi N , Ahmad S , Mokaddas E : Performance comparison of four methods for rapid detection of multidrug-resistant Mycobacterium tuberculosis strains . Int J Tuberc Lung Dis 2011 , 15 : 110 - 115 .
26. Ahmad S , Jaber AA , Mokaddas E : Frequency of embB codon 306 mutations in ethambutol-susceptible and -resistant clinical Mycobacterium tuberculosis isolates in Kuwait . Tuberculosis 2007 , 87 : 123 - 129 .
27. Mokaddas E , Ahmad S , Abal AT : Molecular fingerprinting of isoniazidresistant Mycobacterium tuberculosis isolates from Chest Diseases Hospital in Kuwait . Microbiol Immunol 2002 , 46 : 767 - 771 .
28. Behbehani N , Abal A , Al-Shami A , Enarson DA : Epidemiology of tuberculosis in Kuwait from 1965 to 1999 . Int J Tuberc Lung Dis 2002 , 6 : 465 - 469 .
29. Mokaddas E , Ahmad S : Species spectrum of nontuberculous mycobacteria isolated from clinical specimens in Kuwait . Curr Microbiol 2008 , 56 : 413 - 417 .
30. Ahmad S : New approaches in the diagnosis and treatment of latent tuberculosis infection . Resp Res 2010 , 11 : 169 .
31. Ahmad S , Mokaddas E , Fares E : Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait and Dubai . Diagn Microbiol Infect Dis 2002 , 44 : 245 - 252 .
32. Ahmad S , Mokaddas E : The occurrence of rare rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis isolates from Kuwait . Int J Antimicrob Agents 2005 , 26 : 205 - 212 .
33. Van Soolingen D : Molecular epidemiology of tuberculosis and other mycobacterial infections: main methodologies and achievements . J Intern Med 2001 , 249 : 1 - 26 .
34. Shi R , Zhang J , Li C , Kazumi Y , Sugawara I : Emergence of ofloxacin resistance in Mycobacterium tuberculosis clinical isolates from China as determined by gyrA mutation analysis using denaturing high-pressure liquid chromatography and DNA sequencing . J Clin Microbiol 2006 , 44 : 4566 - 4568 .
35. Mokrousov I , Otten T , Manicheva O , Potapova Y , Vishnevsky B , Narvskaya O , Rastogi N : Molecular characterization of ofloxacin-resistant Mycobacterium tuberculosis strains from Russia . Antimicrob Agents Chemother 2008 , 52 : 2937 - 2939 .
36. van Doorn HR , An DD , de Jong MD , Lan NTN , Hoa DV , Quy HT , Chau NVV , Duy PM , Tho DQ , Chinh NT , Farrar JJ , Caws M : Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR . Int J Tuberc Lung Dis 2008 , 12 : 736 - 742 .
37. Ando H , Mitarai S , Kondo Y , Suetake T , Kato S , Mori T , Kirikae T : Evaluation of a line probe assay for the rapid detection of gyrA mutations associated with fluoroquinolone resistance in multidrug-resistant Mycobacterium tuberculosis . J Med Microbiol 2011 , 60 : 184 - 188 .
38. Giannoni F , Iona E , Sementilli F , Brunori L , Pardini M , Migliori GB , Orefici G , Fattorini L : Evaluation of a line probe assay for the rapid identification of gyrA mutations in Mycobacterium tuberculosis . Antimicrob Agents Chemother 2005 , 49 : 2928 - 2933 .