JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2004, p. 5512–5516
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 42, No. 12
Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis
Isolates from India and Mexico by a Molecular Beacon Assay
Mandira Varma-Basil,1,2† Hiyam El-Hajj,3Roberto Colangeli,1Manzour Hernando Hazbo ´n,1
Sujeet Kumar,2Mridula Bose,2Miriam Bobadilla-del-Valle,4Lourdes Garcı ´a Garcı ´a,4
Araceli Herna ´ndez,4Fred Russell Kramer,3Jose Sifuentes Osornio,4
Alfredo Ponce-de-Leo ´n,4and David Alland1*
Department of Medicine, Division of Infectious Disease, New Jersey Medical School, The University of Medicine and Dentistry
of New Jersey,1and Department of Molecular Genetics, The Public Health Research Institute,3Newark, New Jersey;
Instituto Nacional de Ciencias Me ´dicas y Nutricio ´n Salvador Zubira ´n, Mexico City, Mexico4;
and Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India2
Received 15 April 2004/Returned for modification 21 May 2004/Accepted 2 August 2004
We assessed the performance of a rapid, single-well, real-time PCR assay for the detection of rifampin-
resistant Mycobacterium tuberculosis by using clinical isolates from north India and Mexico, regions with a high
incidence of tuberculosis. The assay uses five differently colored molecular beacons to determine if a short
region of the M. tuberculosis rpoB gene contains mutations that predict rifampin resistance in most isolates.
Until now, the assay had not been sufficiently tested on samples from countries with a high incidence of
tuberculosis. In the present study, the assay detected mutations in 16 out of 16 rifampin-resistant isolates from
north India (100%) and in 55 of 64 rifampin-resistant isolates from Mexico (86%) compared to results with
standard susceptibility testing. The assay did not detect mutations (a finding predictive of rifampin suscep-
tibility) in 37 out of 37 rifampin-susceptible isolates from India (100%) and 125 out of 126 rifampin-susceptible
isolates from Mexico (99%). DNA sequencing revealed that none of the nine rifampin-resistant isolates from
Mexico, which were misidentified as rifampin susceptible by the molecular beacon assay, contained a mutation
in the region targeted by the molecular beacons. The one rifampin-susceptible isolate from Mexico that
appeared to be rifampin resistant by the molecular beacon assay contained an S531W mutation, which is
usually associated with rifampin resistance. Of the rifampin-resistant isolates that were correctly identified in
the molecular beacon assay, one contained a novel L530A mutation and another contained a novel deletion
between codons 511 and 514. Overall, the molecular beacon assay appears to have sufficient sensitivity (89%)
and specificity (99%) for use in countries with a high prevalence of tuberculosis.
Multidrug-resistant tuberculosis (MDR-TB) is an increasing
problem worldwide (13). MDR-TB is associated with signifi-
cant mortality (12, 23) and has resulted in serious institutional
outbreaks (5). Rapid diagnostic assays for MDR-TB should
address these problems by enabling early isolation and treat-
ment of patients with this disease (9, 17). Rifampin resistance
is an excellent marker for multidrug-resistant Mycobacterium
tuberculosis, as 90% of rifampin-resistant M. tuberculosis
strains are also isoniazid resistant and, hence, are classified as
multidrug resistant (20). Rifampin resistance is also amenable
to detection by rapid genotypic assays, because approximately
95% of all rifampin-resistant strains contain mutations local-
ized in an 81-bp core region of the bacterial RNA polymerase
gene, rpoB (11, 17). Moreover, virtually all mutations that
occur in this region result in rifampin resistance. By contrast,
nearly all rifampin-susceptible M. tuberculosis isolates have the
same wild-type nucleotide sequence in this region (11, 17, 19).
Various molecular methods have been developed to rapidly
detect mutations in the M. tuberculosis rpoB core region, in-
cluding the line probe assay (3), single-strand conformational
polymorphism (SSCP) PCR (2, 20), and real-time PCR (6, 21,
22). Researchers developed a molecular beacon-based real-
time PCR assay for this purpose (14, 15) and later converted
this method into a multicolor, single-tube assay format (4). The
single-well molecular beacon assay used five molecular bea-
cons, each hybridizing to a different target segment within the
rpoB core region and each labeled with a differently colored
fluorophore. Each molecular beacon was designed to be so
specific that it could not bind to its target if the target sequence
differed from the wild-type M. tuberculosis rpoB sequence by
even a single nucleotide substitution (10). Because molecular
beacons fluoresce only when they are bound to their targets
(24), the absence of fluorescence from any fluorophore in the
assay indicates the presence of a mutation and thus predicts
rifampin resistance (4). The assay thus has the advantage that
it can detect unknown mutations in the rpoB region. The assay
was simple, rapid, specific, and highly sensitive in tests on
isolates of M. tuberculosis from New York City and Madrid
(15). It also correctly predicted that 11 clinical sputum samples
collected in Rio de Janeiro (Brazil) were rifampin susceptible
(4). However, the ability of the assay to detect rpoB mutations
in countries with a high incidence of tuberculosis, where dif-
ferent mutations could cause rifampin resistance, had not been
tested. Here we assess the suitability of the single-tube molec-
ular beacon assay to detect mutations in the rpoB gene of
* Corresponding author. Mailing address: Division of Infectious
Disease, New Jersey Medical School, 185 South Orange Ave., MSB
A920C, Newark, NJ 07103. Phone: (973) 972-2179. Fax: (973) 972-
0713. E-mail: email@example.com.
† Present address: Department of Microbiology, Vallabhbhai Patel
Chest Institute, University of Delhi, Delhi 110007, India.
clinical M. tuberculosis isolates from the high-incidence coun-
tries India and Mexico.
MATERIALS AND METHODS
M. tuberculosis isolates. A total of 243 isolates of M. tuberculosis from patients
from north India and Mexico were tested for mutations associated with resis-
tance to rifampin by the molecular beacon assay. Thirty-seven rifampin-suscep-
tible and 16 rifampin-resistant isolates were obtained from 53 patients with
tuberculosis in the outpatient department of Respiratory Medicine at the Vall-
abhbhai Patel Chest Institute in Delhi, India, between January 2001 and January
2002. The Vallabhbhai Patel Chest Institute serves as a referral center for
patients with respiratory diseases in north India. A large number (33%) of these
patients had histories of previous treatment at the time of collection of their
sputa. The isolates were biochemically characterized with nitrate reduction,
niacin production, catalase (7), and BACTEC NAP tests (Becton Dickinson
Microbiology Systems, Sparks, Md.). All 53 isolates were also characterized by
IS6110 fingerprinting (1). Another 190 isolates of M. tuberculosis were obtained
from the Instituto Nacional de Ciencias Me ´dicas y Nutricio ´n Salvador Zubira ´n,
in Mexico City, Mexico, which serves as a reference center for patients with
tuberculosis. The Mexican isolates were obtained from three different geograph-
ical regions (Mexico City, Huauchinango, and Orizaba) between 1995 and 2002
and were identified by BACTEC (Becton Dickinson) and the NAP test (Accu-
Probe). Fifty-nine of the isolates had been characterized by IS6110 typing (25).
Of the 190 Mexican isolates, 64 were rifampin resistant. No isolate from either
country was rifampin monoresistant.
Susceptibility testing. The susceptibility of the Delhi M. tuberculosis isolates to
isoniazid, rifampin, ethambutol, and streptomycin was tested by the standard
proportion method (7). Resistance was defined as greater than 1% growth in the
presence of 0.2 ?g of isoniazid/ml, 1 ?g of rifampin/ml, 5 ?g of ethambutol/ml,
and 2 ?g of streptomycin/ml (7). The susceptibility of the Mexican isolates to the
primary antituberculosis drugs was determined by the 460 TB BACTEC system
(Becton Dickinson) at the Instituto Nacional Ciencias Me ´dicas y Nutricio ´n, as
described previously (2).
Sample preparation for PCR. The M. tuberculosis reference strain H37Rv was
used as a positive control in the molecular beacon assays. Genomic DNA was
extracted from H37Rv and clinical M. tuberculosis isolates by treatment with
cetyltrimethylammonium bromide (CTAB) (Sigma, St. Louis, Mo.) in the pres-
ence of 0.7 M sodium chloride, as described previously (25).
Oligonucleotide sequences. The nucleotide sequences of the molecular bea-
cons and primers used in this study have been described previously (4). Five
molecular beacons were designed so that they hybridized to different segments of
the wild-type sequence of the M. tuberculosis rpoB core region. Each 15- to
20-bp-long probe sequence was selected so that the probe-target hybrid was just
strong enough to overcome the strength of the hairpin stem under assay condi-
tions. The five molecular beacons were labeled with five differently colored
fluorophores so that they could be well distinguished from each other in a single
reaction as described previously (4).
Assay conditions. PCR was performed in 96-well microtiter plates (Applied
Biosystems, Foster City, Calif.) as described previously (4). Fluorescence was
measured in every well during the annealing step throughout the course of each
reaction. The spectral data were automatically analyzed by the computer pro-
gram controlling the spectrofluorometric thermal cycler to determine the fluo-
rescence intensity contributed by each of the differently colored molecular bea-
cons. The presence of a mutation within the 81-bp rpoB core region was detected
by the absence of a characteristic rising fluorescence signal from one of the five
molecular beacons in the assay. Conversely, M. tuberculosis isolates were pre-
dicted to be rifampin susceptible when all five of the molecular beacons in the
assay generated a characteristic rising signal.
DNA sequencing and SSCP analysis. The sequences of rpoB core region
amplicons were analyzed in a subset of the study isolates by automated DNA
sequencing with an Applied Biosystems 3100 capillary sequencer using the prim-
ers described above. Fourteen isolates from Mexico had been previously ana-
lyzed by SSCP PCR, as described previously (2).
Molecular beacon assay results. The results of the molecu-
lar beacon assays are summarized in Table 1. Typical assay
results are shown in Fig. 1 and 2. Overall, the sensitivity of the
assay in both populations was 89%, specificity was 99%, posi-
tive predictive value was 99%, and negative predictive value
was 95%. Fluorescence signals in all five molecular beacons
developed in all 37 of the rifampin-susceptible isolates from
Delhi, India (specificity, 100%), including 5 rifampin-suscepti-
ble isolates that had previously been misidentified as being
rifampin resistant by the standard proportion method. The
rifampin susceptibility of these five isolates was confirmed by
repeat susceptibility testing after the results of the molecular
beacon assays were known. All 16 of the rifampin-resistant
isolates from Delhi produced a flat signal for at least one of the
molecular beacon probes (sensitivity, 100%) (Fig. 1). Probe E
most commonly detected a mutation, followed by probes B, D,
and A (Table 2). None of the isolates from Delhi appeared to
contain a mutation in the region of probe C. Two molecular
beacons failed to fluoresce in each of three isolates from Delhi
(Fig. 2). In one isolate, both probe A and probe D failed to
fluoresce; in a second isolate, probe B and probe E failed to
fluoresce; and in a third isolate, probe A and probe B failed to
fluoresce. These results suggested that the three isolates con-
tained more than one mutation in the rpoB core region.
Fluorescence signals in all five molecular beacons developed
in 124 of the 125 rifampin-susceptible Mexican isolates (spec-
ificity, 99%). A fluorescence signal failed to develop with probe
E in one of the Mexican isolates identified as susceptible to
rifampin by BACTEC analysis. Fifty-five of the 64 rifampin-
resistant isolates from Mexico presented a flat signal for at
least one of the molecular beacon probes, while fluorescence
developed in all five molecular beacons in nine of the rifampin-
resistant isolates (sensitivity, 86%). Probe E was again the
most common molecular beacon with a flat signal, followed by
probe D (Table 2). None of the isolates from Mexico gave a
negative signal with probes A and C or a negative signal with
more than one molecular beacon simultaneously.
DNA sequencing. The rpoB core region was sequenced in the
three Indian isolates that gave a single negative signal with
probe A. All three isolates were found to have the L511P
(CTG3CCG) mutation (Table 3). The Delhi isolates with two
TABLE 1. Comparison of molecular beacon assay results with those of phenotypic susceptibility testing
Total no. of
53 100 (16/16)
Total 24389 (71/80) 99 (162/163)9995
aAbility of the molecular beacon assay to detect resistance, expressed as a percentage (number of isolates resistant by both methods/number resistant by phenotypic
bAbility of the molecular beacon assay to detect susceptibility, expressed as a percentage (number of isolates susceptible by both methods/number susceptible by
phenotypic susceptibility testing).
VOL. 42, 2004DETECTION OF RIFAMPIN RESISTANCE IN INDIA AND MEXICO5513
negative signals each were also sequenced. One contained a
novel deletion between codons 511 and 514 (wild-type se-
quence TGAGCCAAT was deleted). The second isolate con-
tained both the L511P (CTG3CCG) and the H526Q
FIG. 1. Typical real-time PCR results for selected rifampin-suscep-
tible and rifampin-resistant M. tuberculosis isolates. (A) A rifampin-
susceptible isolate in which all five differently colored molecular bea-
cons hybridized to the rpoB core region. (B to E) Rifampin-resistant
isolates in which either (B) probe A, (C) probe B, (D) probe D, or (E)
probe E failed to fluoresce. None of the isolates had a flat signal for
probe C. The fluorescence of each molecular beacon is indicated as
follows: E, probe A; ‚, probe B; ■, probe C; Œ, probe D; and ?, probe
FIG. 2. Detection of double mutations or deletions. No fluores-
cence increase was observed for two differently colored probes in three
of the isolates, suggesting the presence of multiple mutations.
(A) Probes A and D failed to fluoresce; (B) probes B and E failed to
fluoresce; and (C) probes and A and B failed to fluoresce. The fluo-
rescence of each molecular beacon is indicated as follows: E, probe A;
Œ, probe B; ■, probe C; ?, probe D; and ‚, probe E.
TABLE 2. Distribution of assay results obtained with M.
tuberculosis clinical isolates in which at least one flat fluorescence
signal was observed
No. of positive detections of fluorescence signal
aDouble mutations were observed in two isolates (flat signals for probes A-D
bDouble mutations were observed in two isolates (flat signals for probes A-B
5514VARMA-BASIL ET AL. J. CLIN. MICROBIOL.
(CAC3CAG) mutations, and the third isolate contained both
the N516A (GAC3GCC) and the L533P (CTG3CCG) mu-
tations. The mutations in each isolate were consistent with the
probes that failed to give a positive signal. The isolates from
Delhi and Mexico that gave a flat signal in probe E were also
sequenced. The most common mutations in this group were
concentrated in codon 531 (Table 3). The most common mu-
tation at this codon was S531L (TCG3TTG), which occurred
in 7 out of 9 (78%) and 23 out of 36 (64%) of the Delhi and
Mexican isolates, respectively. The second most common mu-
tation was S531W (TCG3TGG), which occurred in 1 out of 9
(11%) and 8 out of 36 (22%) of the Delhi and Mexican iso-
lates, respectively. A mutation that has not previously been
identified, L530A, was found in a Mexican isolate that gave a
flat signal in probe E.
Nine of the rifampin-resistant isolates from Mexico gave
positive fluorescence signals in all five probes, suggesting that
their rpoB core region was a wild-type DNA sequence. No
mutations were seen by DNA sequencing in the rpoB core
region in eight of these isolates; the ninth isolate had an I572F
(ATC3TTC) mutation located outside of the core region tar-
geted by the five molecular beacons. One Mexican isolate that
was susceptible to rifampin showed a negative fluorescence
signal for probe E. DNA sequencing of this isolate showed that
an S531W mutation (normally strongly associated with ri-
fampin resistance) was present. Unfortunately, this isolate had
lost viability during freezing storage and rifampin susceptibility
could not be retested.
Comparison of the molecular beacon assay to single-strand
conformational polymorphism analysis. Twelve rifampin-re-
sistant and two rifampin-susceptible Mexican isolates that were
investigated in this study had previously been characterized by
single-strand conformational polymorphism PCR (2). The
SSCP PCR assay identified 4 of the 12 rifampin-resistant iso-
lates as rifampin-resistant rpoB mutants, while the molecular
beacon assay correctly identified 11 of these isolates as ri-
fampin resistant. One rifampin-resistant isolate was identified
as susceptible by both SSCP PCR and by the molecular beacon
assay. DNA sequencing of this isolate revealed the existence of
an I572F mutation outside the rpoB core region. Of the two
rifampin-susceptible isolates, both were identified as suscepti-
ble by SSCP PCR. In contrast, one of the susceptible isolates
was identified as rifampin resistant by the molecular beacon
assay. This isolate, already described above, contained an
S531W mutation in the rpoB core region. Thus, it is likely that
this isolate was actually rifampin resistant and was correctly
identified by the molecular beacon assay.
Correlation between IS6110 type and molecular beacon as-
say results. IS6110 typing was carried out to confirm that the
isolates of M. tuberculosis tested represented a diverse popu-
lation. Many of the isolates studied (79% of the Indian strains
and 38% of the Mexican strains) had unique banding patterns.
Eleven of the Indian isolates and 28 of the Mexican isolates
were identified as belonging to 5 and 14 clusters, respectively.
However, the clusters were not associated with any specific
mutation in the rpoB gene. These results suggest that the
isolates studied were genetically unrelated and most likely de-
veloped rifampin resistance independently.
This study demonstrates that the molecular beacon assay
effectively detects rifampin resistance in clinical M. tuberculosis
isolates from countries with a high incidence of tuberculosis.
Other advantages of this assay include the single-well format,
the ability to combine PCR and post-PCR analysis into a single
step, and the virtual elimination of cross-contamination con-
ferred by the ability to perform assays in closed tubes. The
assay detected mutations in the rpoB core region targeted by
the molecular beacons every time a mutation was present.
Conversely, the assay identified the core region as containing
the wild-type sequence every time that mutations were absent
from this region. The main limitation of the assay is that it is
unable to detect rifampin resistance caused by mutations out-
side of the rpoB core region. Indeed, the assay had a sensitivity
of 100% for the isolates from Delhi, but it had a sensitivity of
86% for isolates from Mexico, where it mistakenly identified 9
out of 64 rifampin-resistant isolates as being rifampin suscep-
tible because these isolates did not have mutations in the rpoB
core region. Screening assays should be highly specific, and the
molecular beacon assay had an overall specificity of 99%. The
assay identified 37 out of 37 rifampin-susceptible Indian iso-
lates as being rifampin susceptible, and it identified 125 of 126
rifampin-susceptible Mexican isolates as being rifampin sus-
ceptible. Furthermore, the sole rifampin-susceptible isolate
that was misidentified as being rifampin resistant contained an
S531W mutation that has previously been shown to be associ-
ated with rifampin resistance (11, 16). Thus, it is likely that this
susceptible isolate was, in fact, rifampin resistant. Unfortu-
nately, this sample was not viable for repeat susceptibility test-
ing. A potential problem of the assay is that it would be expected
TABLE 3. Mutations detected in the rpoB core region from isolates with flat fluorescence signals for probe A or E
No. of mutant isolates
aA fourth isolate with a negative signal for probe A had a deletion between codons 511 and 514.
VOL. 42, 2004DETECTION OF RIFAMPIN RESISTANCE IN INDIA AND MEXICO5515
mutations are exceedingly rare in M. tuberculosis (19), thus, this
problem does not have an important effect on specificity. In fact,
it is worth noting that in the present study, the assay correctly
identified five rifampin-susceptible isolates from India that had
been initially classified as rifampin resistant by conventional sus-
ceptibility testing (but later confirmed to be susceptible by repeat
testing). This observation suggests that the specificity of the mo-
lecular beacon assay may sometimes be higher than that of con-
ventional susceptibility testing.
We found that four isolates from Delhi gave negative signals
with probe A. Three of these isolates had mutations in codon
511, and the fourth had a novel deletion spanning codons 511
to 514. Mutations at codon 511 have been found by other
workers in India (8). No rifampin-resistant isolate from Mexico
contained a mutation in this region. This difference could re-
flect regional strain variations or differences in host factors.
None of the 243 isolates showed a negative fluorescence signal
with probe C, which targets rpoB codons 518 to 522. Earlier
studies from India have reported mutations in codon 518, but
only when they were accompanied by mutations in codon 531
(8). Other studies have reported mutations at codons 518, 521,
and 522 at frequencies of only 0.8, 1.5, and 3%, respectively
(18). Thus, it is possible that probe C could be omitted from
future assays without a major effect on assay sensitivity. We
also compared the results from the molecular beacon assays to
results obtained with SSCP PCR in 14 isolates from Mexico
City. Our results show that the molecular beacon assays were
much more sensitive in detecting rifampin resistance in this
group of isolates.
In summary, the molecular beacon assay was as effective at
detecting mutations associated with rifampin resistance in M.
tuberculosis isolates from northern India and Mexico as has
previously been reported for isolates from the United States
and Spain (15). The assay also identified rifampin-susceptible
isolates that had previously been misidentified as resistant,
further supporting the utility of a genetic approach to suscep-
tibility testing. The assay was also more effective than SSCP
PCR at detecting rifampin-resistant isolates. The assay is not
dependent on probes hybridizing to specific mutant codons;
hence, new and unknown mutations arising in a population,
such as those identified in this study, can be easily detected
with the same set of probes. With real-time instruments be-
coming more affordable, we anticipate that the assay can be-
come economically feasible for developing countries in the
near future. Ultimately, this assay will enable more rapid di-
agnosis, earlier treatment, and prompt implementation of in-
fection control procedures to reduce the morbidity, mortality,
and the spread of drug-resistant tuberculosis.
This work was supported by National Institutes of Health grants
AI-46669 and EB-00277. M.V.-B. received an Overseas Associateship
from the Department of Biotechnology of the Indian government.
D.A. and F.K. are among a group of coinvestigators who hold patents
in molecular beacons technology, and they receive income from licensees.
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