INT J TUBERC LUNG DIS 12(11):1306–1312

© 2008 The Union

Comparison of MAS-PCR and GenoType MTBDR assay for the

detection of rifampicin-resistant Mycobacterium tuberculosis

D. Q. Tho,* D. T. M. Ha,*† P. M. Duy,* N. T. N. Lan,† D. V. Hoa,† N. V. V. Chau,‡ J. Farrar,*§ M. Caws*§

* Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, † Pham Ngoc Thach Hospital
for Tuberculosis and Lung Diseases, Ho Chi Minh City, ‡ Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam;
§ Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, UK

SUMMARY

SETTING: Pham Ngoc Thach Hospital for Tuberculosis
and Lung Diseases, the tertiary referral hospital for tu-
berculosis (TB) in Southern Vietnam.
O B J E C T I V E : To develop and evaluate a simple, rapid
and accurate multiplex allele specific polymerase chain
reaction (MAS-PCR) test to detect rifampicin (RMP) re-
sistance point mutations at codons 516, 526 or 531 in
the rpoB gene of Mycobacterium tuberculosis.
D E S I G N : The novel MAS-PCR was compared with the
commercial M. tuberculosis Drug Resistance (MTBDR)
test in 104 RMP-resistant and 50 RMP-susceptible rou-
tine isolates, defined by conventional 1% phenotypic
susceptibility testing.
R E S U LT S : The sensitivity of the MAS-PCR and MTBDR
tests was respectively 83.7% (95%CI 75.1–90.2) and
93.3% (95%CI 86.6–97.3). Both tests were 100% spe-
cific. The negative predictive value was 74.6% (95%CI
65.3–83.1) for the MAS-PCR and 87.7% (95%CI 80.0–
93.6) for the MTBDR test.
C O N C L U S I O N : The MTBDR test, although more sensi-
tive, is currently prohibitively expensive in resource-poor,
high-burden settings. The MAS-PCR described here
presents a less laborious economic alternative. A suscep-
tible result returned by either test cannot be used to ex-
clude multidrug-resistant TB.
K E Y W O R D S : tuberculosis; rifampicin; resistance; PCR;
MTBDR

ONE THIRD of the world’s population is infected
with Mycobacterium tuberculosis, 5–10% of whom
will develop active disease. Each year the disease kills
approximately 1.7 million people worldwide, 1.4 mil-
lion of whom (72%) are in 22 high-burden countries.1
Standard tuberculosis (TB) treatment regimens for
fully susceptible TB require multidrug regimens over a
minimum of 6 months. The rise of multidrug-resistant
tuberculosis (MDR-TB), defined as M. tuberculosis
resistant to at least isoniazid (INH) and rifampicin
(RMP), threatens the success of TB control programmes
in many regions of the world.2–4 It has been demon-
strated that MDR-TB can successfully transmit within
a community;5,6 early detection of infectious cases is
therefore vital to interrupt chains of transmission.7,8
MDR-TB is difficult to treat, with treatment dura-
tions of 18 months or longer, using more toxic and less
potent second-line drugs.9,10 Furthermore, the cost of
treating a patient with MDR-TB is many times the
cost of treating of a patient with fully susceptible TB,
even with the help of the Green Light Committee,
which has negotiated significant reductions in costs
of second-line drugs for government programmes.9,11
Detecting drug resistance, particularly MDR-TB,
before the instigation of inappropriate chemotherapy
reduces morbidity, mortality, economic costs and un-
necessary treatment with ineffective drugs. Conven-
tional phenotypic drug susceptibility testing (DST)
only returns results after several weeks of culture in
Mycobacterial Growth Indicator Tube or Löwenstein-
Jensen (LJ) media.
Two commercial molecular kits12 for MDR-TB are
currently available: the INNO-LiPA RMP TB test (In-
nogenetics, Zwijnaarde, Belgium)13 and the Geno-
Type MTBDR assay kits (Hain Lifesciences, Nehren,
Germany).14 These kits perform well in research set-
tings15–20 but, at over US$50 per test strip, are too ex-
pensive to be used routinely in developing countries
such as Vietnam. It is necessary to develop low-cost
techniques that are accurate, simple and rapid for the
identification of drug-resistant TB.
The authors of the present study21 and others22,23
have shown that RMP resistance is a surrogate marker
of MDR in some settings where RMP monoresistance
is rare. A molecular technique for the rapid detection
of RMP resistance can therefore be used to screen for

Correspondence to: Dau Quang Tho, Oxford University Clinical Research Unit, Hospital of Tropical Disease, 190 Ben
Ham Tu, District 5, Ho Chi Minh City, Vietnam. Tel: (+84) 8 923 9211. Fax: (+84) 8 835 3904. e-mail: dauquangtho@
yahoo.com
Article submitted 11 January 2008. Final version accepted 26 June 2008.
 MAS-PCR for RMP-resistant TB 1307

MDR-TB. As RMP monoresistance occurs more fre-
quently in human immunodeficiency virus (HIV) pos-
itive TB cases,24 in areas of high HIV prevalence RMP
resistance may not be applicable as a surrogate marker
of MDR-TB.
Resistance to RMP is conferred by single-step mu-
tation in the 81 base pair (bp) RMP resistance deter-
mining region (RRDR) of the rpoB gene which encodes
the beta subunit of the RNA polymerase. Single-base
substitutions, insertions or deletions in the rpoB gene
can lead to RMP resistance.25
Studies in the literature have used various molecular
mutation detection techniques to identify MDR-TB,
such as real-time polymerase chain reaction (PCR),26,27
single strain conformation polymorphism28 or sequenc-
ing to detect mutated codons.29,30 However, these
techniques require expensive equipment and techni-
cal expertise.
To minimise post-amplification processing, we used
three multiplex allele specific (MAS) PCR reactions for
each isolate, with two outer primers as positive con-
trol for each sample and one inner primer to detect
the mutation. In a previous study in Vietnam, we dem-
onstrated that the mutations in RRDR of the rpoB
gene are located mostly at three hot spot codons 531
(43%), 526 (31%) and 516 (15%), concurring with
prevalence reported worldwide.23,25
The MAS-PCR was compared with the commer-
cial reverse-hybridisation MTBDR test in a collection
of 104 RMP-resistant isolates with known RRDR se-
quence and 50 RMP-susceptible isolates. The MTBDR
test also detects INH resistance-conferring mutations
in the katG gene. Phenotypic DST results were con-
sidered the gold standard.
METHODS
Samples
One hundred and four consecutive isolates routinely
identified as RMP-resistant (102 [98.1%] of which
were MDR) and 50 RMP-susceptible isolates were
collected at Pham Ngoc Thach Hospital for TB and
Lung Diseases (PNT), Ho Chi Minh City, Vietnam,
between January and March 2005. RMP-resistant iso-
lates were sequenced in the RRDR of the rpoB gene,
as previously described.21 All 154 isolates were used
to evaluate the test described here by comparing it
with the commercial MTBDR test against the pheno-
typic DST results.
Drug susceptibility testing
DST was performed at PNT, a World Health Organiza-
tion (WHO) international reference laboratory, for
INH (0.2 μg/ml) and RMP (40 μg/ml) on LJ media
with the standard 1% proportion method according
to the WHO protocol.
DNA extraction
DNA extraction was performed using the cetyl tri-
methyl ammonium bromide (CTAB) method after cul-
ture on LJ. The purified DNA was dissolved in tris-
EDTA (TE) buffer, quantified, diluted to the final 15 ng/
μl working concentration and stored at −20°C.31
Sequencing
One hundred and four isolates were sequenced in the
RRDR and N-terminal of the rpoB gene with primers
TB146F and TB146R (Table 1), as previously de-
scribed.21 Half the volume of the CEQ Quick Start kit
(Beckman Coulter, Fullerton, CA, USA) was used to
sequence from both strands of a 350-bp fragment con-
taining an 81-bp sequence of RRDR (from position
760990 to 761339, H37Rv, Blast NC_000962.2).
GenoType MTBDR
The GenoType MTBDR test was performed accord-
ing to the manufacturer’s protocol. Briefly, 15 ng of
sample DNA was used as a template to amplify rpoB
and katG target sequences with biotin-labelled prim-
ers. The amplification product was analysed by reverse
hybridisation to membrane bound probes. Colori-
metric detection gave purple bands, read by eye. Neg-
ative controls (water) were included with each run.
MAS-PCR
Three separate PCR reactions were performed with
the same outer primers to amplify the RRDR of rpoB,
and one of three inner primers targeted to wild type

Table 1 Primers for sequencing and MAS-PCR

Temperature Concentration Target in Product length

Primer Sequence (5′–3′) °C nM rpoB gene bp
RPOBF GGG AGC GGA TGA CCA CCC A 68 125 Flank RRDR 350
RPOBR30 GCG GTA CGG CGT TTC GAT GAA C 68* 125*
TB146-F CTT CTC CGG GTC GAT GTC GTT G 64 300 N-terminal region 365
TB146-R32 CGC GCT TGT CGA CGT CAA ACT C 64 300
RIF2R GAC AGC GGG TTG TTC TGG T 68 300 Codon 516 139†
RIF3F CCG CTG TCG GGG TAG ACC CA 68 400 Codon 526 219‡
RIF4R CCG CCG GGC CCC ATC GCC G 68 300 Codon 531 184†
* The temperature and concentration for sequencing with RPOBR + RPOBF were respectively 65°C and 150 nM.
† Amplicon generated with primer RPOBF.
‡ Amplicon generated with primer RPOBR.

MAS-PCR = multiplex allele specific polymerase chain reaction; bp = base pair; RRDR = resistance determining region.
1308 The International Journal of Tuberculosis and Lung Disease

Figure 1 Amplicon profiles in MAS-PCR of isolates carrying mutations at each site. * a, codon
516; b, codon 526; c, codon 531. Samples: 1, mutation at codon 516; 2, mutation at codon 526;
3, mutation at codon 531; 4, codon 516 and 526 mutated; 5, wild type control sample; 6, nega-
tive control. M = 100 bp ladder; MAS-PCR = multiplex allele specific polymerase chain reaction;
bp = base pair.

at hot spot codons 516, 526 and 531 (Table 1). The
results were read with two bands for wild type isolates
and only one band for mutant isolates (Figure 1).
The 350 bp fragment of the rpoB gene was ampli-
fied using outer primers RPOBF (5′-GGGAGCGGA
TGACCACCCA-3′) and RPOBR (5′-GCGGTACGG
CGTTTCGATGAAC-3′). The inner primers used to
detect mutations at codon 516, 526 and 531 were
RIF2R (5′-GACAGCGGGTTGTTCTGGT-3′), RIF3F
(5′-CCGCTGTCGGGGTAGACCCA-3′) and RIF4R
(5′-CCGCCGGGCCCCATCGCCG-3′), respectively
(Table 1). The primers were designed using Primer
Express version 2.0 software (Applied Biosystems Inc,
Foster City, CA, USA).
Each PCR reaction contained 0.75 U Taq DNA
polymerase (Bioline, Taunton, MA, USA), 0.2 mM
of each dNTP (Roche, Basel, Switzerland) and 125–
400 nM of primers (Table 1) and 2 mM MgCl2. Fifteen
nanograms of genomic DNA was added. All of the re-
actions were amplified under the same thermocycling
programme: 95°C for 1 min, followed by 35 cycles of
95°C for 10 s, 68°C for 20 s, 72°C for 20 s and a final
step of 72°C for 5 min. A negative control (water) and
a positive control for each of the three target codons
was included with each batch processed. PCR products
were analysed by electrophoresis on 1% agarose gel
at 150V for 30 min.
All sensitivities and specificities were calculated in
comparison with conventional 1% phenotypic DST
as gold standard. Discrepant results were evaluated by
analysis of sequencing data.
RESULTS
The results of MAS-PCR, sequencing and the com-
mercial MTBDR test are summarised and shown in
Table 2. Sequencing showed that among 104 RMP-
resistant isolates, 92 isolates (88.5%) had mutations

Table 2 Comparison between MAS-PCR test and commercial MTBDR test in

104 RMP-resistant isolates
MTBDR test MAS-PCR RMP test
Isolates Resistant Susceptible Resistant Susceptible
Mutation n n n n n
S531L 46 46 0 45 1
H526D/F/L /R /S/Y 28 28 0 28 0
D516V 9 9 0 8 1
D516V, H526Q 1 1 0 1 0
D516A, H526N 1 1 0 1 0
L511P, H526N 1 1 0 1 0
Q513K, H526D 1 1 0 1 0
D516G, 522::SGL 1 1 0 1 0
D516A, L533P 1 1 0 1 0
Q513K /L 3 3 0 0 3
D516G, L533P 2 2 0 0 2
L511P, D516G 1 1 0 0 1
L511P, S512G 1 1 0 0 1
L533P 2 1 1 0 2
514::F 1 0 1 0 1
V146F 1 0 1 0 1
WT 4 0 4 0 4
Total, n (%) 104 97 (93.27) 7 (6.7) 87 (83.65) 17 (16.3)
MAS-PCR = multiplex allele specific polymerase chain reaction; MTBDR = GenoType MTBDR assay; RMP = rifampicin.
 MAS-PCR for RMP-resistant TB 1309

Table 3 Sensitivity of MAS-PCR and MTBDR with reference to 1% conventional phenotypic

drug susceptibility testing as gold standard
MAS-PCR MTBDR
Resistant Susceptible Resistant Susceptible
Conventional DST n (%) n (%) n (%) n (%)
RMP
Resistant (n = 104) 87 (83.65) 17 97 (93.3) 7 (6.7)
Susceptible (n = 50) 0 50 (100) 0 50 (100)
INH
Resistant (n = 132) NA NA 109 (82.6) 23 (17.4)
Susceptible (n = 22) NA NA 0 22 (100)
MAS-PCR = multiplex allele specific polymerase chain reaction; MTBDR = GenoType MTBDR assay; DST = drug sus-
ceptibility testing; RMP = rifampicin; INH = isoniazid; NA = not applicable.

located at the three target codons: 531 (43%), 526
(31%), 516 (15%). Eight (7.7%) had mutations at
other codons in the RRDR of the rpoB gene. Four
(3.8%) had no mutation in the regions that were
sequenced.
Using the MTBDR test, 97 isolates (93.3%) were
detected as resistant to RMP. Of the seven resistant
isolates returning a susceptible result, four had no mu-
tations in RRDR (Table 2), two had mutations outside
the location of the primers (L533P and V146F) and
the remaining isolate had three nucleotides inserted at
the end of a probe (514::F); thus, hybridisation still
occurred, returning a wild type result. The sensitivity
and the specificity of MTBDR for RMP resistance
were respectively 93.3% (95% confidence interval [CI]
86.6–97.3) and 100%. The positive predictive value
(PPV) was 100%, with a negative predictive value
(NPV) of 87.7% (95%CI 80.0–93.6).
MTBDR also detects mutations at katG315 that
are related to INH resistance. Of 154 isolates tested,
132 isolates were resistant (102 RMP-resistant and
30 RMP-susceptible) and 22 isolates were susceptible
to INH by DST. MTBDR detected INH resistance in
109/132 (82.6%) INH-resistant isolates, all of which
carried mutations at codon 315, of which 98 (72.2%)
were katGS315T mutants. The sensitivity and speci-
ficity of MTBDR for INH resistance were respectively
82.6% (95%CI 75.0–88.6) and 100% (Table 3). The
PPV and NPV for INH resistance were respectively
100% and 48.9% (95%CI 38.9–59.2). Of 154 isolates
in this study, 102 (66%) were MDR; the PPV and NPV
for MDR-TB in this sample set were respectively 100%
and 70.3% (95%CI 60.0–78.8).
MAS-PCR detected 87/104 (83.7%) RMP-resistant
isolates. Of the 17 discrepant results, four of the iso-
lates carried no mutations in any sequenced region,
eight had mutations in the rpoB gene but outside the
three target codons (Q513K/L ×3, L533P ×2, 514::F
×1, V146F ×1 and L511P/S512G ×1) (Table 2), two
showed evidence of a mixed population of resistant
and susceptible isolates on sequencing with double
peaks at a single allele (Figure 2), which would be de-
fined as wild type by MAS-PCR due to the generation
of the wild-type band. The remaining three isolates
had a D516G mutation, which primer RIF2R hybri-
dises weakly, generating a weak wild-type band and
returning a false-susceptible result.
The specificity of the MAS-PCR test was 100%,
with no susceptible isolates returning a false-positive
resistant result (Table 3). Sensitivity was 83.7%
(95%CI 75.1–90.2). The PPV and NPV for RMP re-
sistance in this sample set were respectively 100%
and 74.6% (95%CI 65.3–83.1).
DISCUSSION
Neither molecular test was 100% sensitive for the de-
tection of RMP-resistant TB; however, both tests are

Figure 2 Double peak in the sequencing trace from sample DQ786 showing a heterogeneous
population of susceptible strain (wild type, D516) and resistant mutant (D516V).
1310 The International Journal of Tuberculosis and Lung Disease
100% specific and therefore useful as rapid detection
tests for positive identification of RMP resistance and
by proxy for MDR-TB. A negative result from either
test cannot be used to rule out MDR-TB. The com-
mercial MTBDR test is more sensitive (93.3% vs.
83.7%) and allows the direct detection of INH-
resistant TB, directly confirming the diagnosis of MDR-
TB in the majority of cases (n/N = 80/102, 78.4%).
The MTBDR test used in this study was only able to
detect INH resistance mutations at katG315. The new
version of this test, MTBDRplus,33 has additional
probes for mutations at inhA-15 (the second most
common INH resistance mutation) and for additional
regions of the rpoB RRDR. Sensitivity for MDR-TB
detection should therefore be higher with the MTB-
DRplus test. The test format is user-friendly and inter-
pretation of the test is simple. However, at more than
US$50 per test strip, routine use of this kit in develop-
ing settings such as Vietnam is not currently feasible.
The MTBDRplus test strips are one of the technolo-
gies under evaluation in the Foundation for Innova-
tive New Diagnostics (FIND) portfolio, which aims
to evaluate novel diagnostic technologies for poverty-
related diseases and promote uptake of those that are
effective through public-private partnerships.* If suc-
cessful, the FIND demonstration projects for MTBDR
should lead to wider availability at significantly lower
costs (approximately US$5/test).
The MAS-PCR test evaluated here offers several ad-
vantages over the MTBDR test strips in a high-volume
setting. First, the cost for a batch of 10 samples is
around US$16 using reagents from local suppliers
without bulk discounts (US$1.6 per sample), exclud-
ing salary and capital equipment costs. The equipment
required is basic PCR equipment—a thermocycler, gel
electrophoresis equipment and ultraviolet transillu-
minator—which is now available in many reference
TB laboratories. There is minimal post-amplification
processing involved, considerably less hands-on time
and the MAS-PCR does not require adherence to a
strict time protocol for post-amplification process-
ing, which increases flexibility to incorporate the test
into a routine diagnostic laboratory schedule. After
DNA extraction, the MAS-PCR can be completed in
2 h, whereas the MTBDR test requires 6 h of post-
amplification processing (including PCR, electropho-
resis and hybridisation).
The performance of the MAS-PCR test in routine
use as an MDR detection tool should be evaluated
both on cultures and directly on clinical samples. How-
ever, molecular tests are only appropriate diagnostic
tools in laboratories that have sufficient resources and
expertise to implement quality control and assurance,
particularly regarding contamination.
There were 13 discrepant cases between sequenc-
ing and rpoB MAS-PCR. Eight of these isolates have
* www.finddiagnostics.org
mutations outside the three hot spot codons 516, 526
and 531. Three had a D516G mutation, which is not
detected by this test. The two remaining isolates prob-
ably contained a heterogeneous population of suscep-
tible and resistant isolates as they had a double peak
at a resistance allele on the sequencing trace. These iso-
lates were defined as wild type by the MAS-PCR test
due to generation of the wild-type band. The MTBDR
test is able to detect the resistant population in these
isolates, as hybridisation with the resistant band will
occur. It is likely that for clinical purposes these iso-
lates should be treated as resistant because a resistant
population is emerging. However, it is possible that
the patient may be successfully treated on a standard
regimen, particularly if the organism remains INH-
susceptible, and therefore the clinical implications of
such results remain unclear.
Molecular tests will not have 100% accuracy until
all the relevant mutations have been described. A sus-
ceptible result returned by a molecular test therefore
cannot yet be used to exclude RMP resistance. Ideally
DST should be performed on every patient at the time
of diagnosis; however, in high-volume developing set-
tings where this is not feasible, priority should be
given to cases where the index of suspicion is high,
such as HIV-positive patients, MDR contact cases and
retreatment/relapse cases, where a susceptible result
returned by molecular screening methods should be
confirmed by DST or other phenotypic methods. How-
ever, with high specificity (100%), molecular tech-
niques allow the early detection of MDR cases, poten-
tially reducing morbidity, mortality and interrupting
chains of transmission when combined with effective
MDR treatment programmes.
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RÉSUMÉ
le test commercial MTBDR (M. tuberculosis Drug Re-
sistance) dans des isolats de routine résistants à la RMP
(n = 104) ou sensibles à la RMP (n = 50) en fonction des
tests conventionnels phénotypiques de sensibilité à 1%.
R É S U LTAT S : La sensibilité du test MAS-PCR a été de
83,7% (IC95% 75,1–90,2) et celle du test MTBDR de
93,3% (IC95% 86,6–97,3). La spécificité a été de 100%
pour les deux tests. La valeur prédictive négative a été de
74,6% (IC95% 65,3–83,1) pour le test MAS-PCR et de
87,7% (IC95% 80,0–93,6) pour le test MTBDR.
1312 The International Journal of Tuberculosis and Lung Disease
C O N C L U S I O N : Le test MTBDR, bien que plus sensible,
est actuellement beaucoup trop coûteux dans les con-
textes à faibles ressources et à haut fardeau de TB et
pour cette raison le MAS-PCR décrit ici représente une
MARCO DE REFERENCIA : El hospital de tuberculosis
(TB) y enfermedades respiratorias Pham Ngoc Thach,
tercer hospital de referencia de TB en Vietnam del Sur.
O B J E T I V O : Elaborar y evaluar una prueba de reacción
en cadena de la polimerasa (PCR) con amplificación
múltiple (multiplex) de alelos específicos (MAS-PCR),
que sea sencilla rápida y precisa, con el fin de detectar
mutaciones puntuales en los codones 516, 526 o 531 del
gen rpoB de Mycobacterium tuberculosis, que se aso-
cian con la resistencia a rifampicina (RMP).
M É T O D O S : La nueva MAS-PCR se comparó con la
prueba comercial de genotipo M. tuberculosis drug re-
sistance (MTBDR), en 104 aislados clínicos recogidos
en forma sistemática, resistentes a RMP y en 50 aislados
sensibles, definidos mediante el método convencional
fenotípico de susceptibilidad del 1%.
alternative moins difficile et économique. Un résultat de
sensibilité provenant de l’un ou l’autre de ces tests ne
peut pas permettre d’exclure une TB multirésistante.
RESUMEN
R E S U LTA D O S : La sensibilidad de la MAS-PCR fue
83,7% (IC95% 75,1–90,2) y la sensibilidad de la prueba
MTBDR fue 93,3% (IC95% 86,6–97,3). Ambas prue-
bas tuvieron una especificidad de 100%. El valor de pre-
dicción negativa fue 74,6% (IC95% 65,3–83,1) para la
MAS-PCR y 87,7% (IC95% 80,0–93,6) para la prueba
MTBDR.
C O N C L U S I O N E S : Si bien la prueba de genotipo
MTBDR es más sensible, su costo es excesivo en la actu-
alidad en medios con escasos recursos y alta carga de
morbilidad ; la MAS-PCR descrita en el presente estudio
constituye una opción económica y menos dispendiosa.
Un resultado de susceptibilidad obtenido con cualquiera
de estos métodos no se puede utilizar con el fin de excluir
la TB multidrogorresistente.