Rifampin Drug Resistance Tests for

Tuberculosis: Challenging the Gold

Standard
Armand Van Deun, Kya J. M. Aung, Valentin Bola, Rossin
Lebeke, Mohamed Anwar Hossain, Willem Bram de Rijk,
Leen Rigouts, Aysel Gumusboga, Gabriela Torrea and
Bouke C. de Jong
J. Clin. Microbiol. 2013, 51(8):2633. DOI:
10.1128/JCM.00553-13.
Published Ahead of Print 12 June 2013.

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Rifampin Drug Resistance Tests for Tuberculosis: Challenging the
Gold Standard
Armand Van Deun,a,b Kya J. M. Aung,c Valentin Bola,d Rossin Lebeke,d Mohamed Anwar Hossain,c Willem Bram de Rijk,a
Leen Rigouts,a Aysel Gumusboga,a Gabriela Torrea,a Bouke C. de Jonga
Mycobacteriology Unit, Institute of Tropical Medicine, Antwerp, Belgiuma; International Union Against Tuberculosis and Lung Disease, Paris, Franceb; Damien Foundation
Bangladesh, Banani, Dhaka, Bangladeshc; Coordination Provinciale Lpre/Tuberculose, Kinshasa, Democratic Republic of Congod

The rapid diagnosis of rifampin resistance is hampered by a reported insufficient specificity of molecular techniques for detec-
tion of rpoB mutations. Our objective for this study was to document the prevalence and prognostic value of rpoB mutations
with unclear phenotypic resistance. The study design entailed sequencing directly from sputum of first failure or relapse patients
without phenotypic selection and comparison of the standard retreatment regimen outcome, according to the mutation present.
We found that among all rpoB mutations, the best-documented disputed rifampin resistance mutations (511Pro, 516Tyr,

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526Asn, 526Leu, 533Pro, and 572Phe) made up 13.1% and 10.6% of all mutations in strains from Bangladesh and Kinshasa, re-
spectively. Except for the 511Pro and 526Asn mutations, most of these strains with disputed mutations tested rifampin resistant
in routine Lwenstein-Jensen medium proportion method drug susceptibility testing (DST; 78.7%), but significantly less than
those with common, undisputed mutations (96.3%). With 63% of patients experiencing failure or relapse in both groups, there
was no difference in outcome of first-line retreatment between patients carrying a strain with disputed versus common muta-
tions. We conclude that rifampin resistance that is difficult to detect by the gold standard, phenotypic DST, is clinically and epi-
demiologically highly relevant. Sensitivity rather than specificity is imperfect with any rifampin DST method. Even at a low prev-
alence of rifampin resistance, a rifampin-resistant result issued by a competent laboratory may not warrant confirmation,
although the absence of a necessity for confirmation needs to be confirmed for molecular results among new cases. However, a
result of rifampin susceptibility should be questioned when suspicion is very high, and further DST using a different system (i.e.,
genotypic after phenotypic testing) would be fully justified.

P rompt diagnosis of multidrug-resistant tuberculosis (MDR-TB)

has been the main obstacle to its correct management and
control. This problem would seem to have been solved with the
now been confirmed for a larger number and range of mutated
strains (6).
What remains unclear is the importance of such discordant
development of molecular techniques applicable also in high- strains with disputed rpoB mutations in terms of the relative
prevalence, low-income settings, such as the Genotype MTBDR- frequency and impact on outcome of rifampin-based standard
Plus and Gene Xpert MTB/RIF assays. However, though very therapy. They are commonly considered to be very rare (7), and
rapid and highly sensitive, these tests are not considered highly their MICs can be below the conventional critical concentration
specific for the diagnosis of rifampin resistance (1). From calcula- (8). For this reason, they are often considered susceptible, despite
tions based on this experimentally imperfect specificity, the World reports on adverse treatment outcome (9). Moreover, their prev-
Health Organization (WHO) has thus recommended that an alence may be underestimated because very few large molecular
Xpert result indicating resistance must be confirmed by another surveys have been performed without phenotypic DST preselec-
technique when rifampin resistance prevalence is below 15% (2). tion. Their low MICs and in our own experience often also pro-
Obviously, this requirement reverses the gains made in the early nounced fitness loss make them difficult to grow and thus to de-
and rapid diagnosis of MDR-TB in most settings. tect in phenotypic DST. Moreover, the critical concentrations
used in DST have not been established to detect each and every
Phenotypic TB drug susceptibility testing (DST) is the gold
clinically resistant strain (10, 11).
standard and has hitherto not been questioned, although we are
In this report we describe the distribution of rpoB mutations
well aware of important differences between the various tech-
found by DNA sequencing directly from sputum in systematic
niques, particularly for some drugs, e.g., ethambutol (3). Tradi-
samples from two populations of first retreatment cases. We also
tionally considered to be highly accurate and reliable, rifampin
analyzed standardized first-line drug retreatment outcome by
DST was also found not to be that straightforward in the rounds of mutation, comparing the clinical prognostic value of pheno-
proficiency testing among the supranational TB reference labora-
tories (SRL) (4). Strains yielding highly discordant results in these
high-profile laboratories carried specific rpoB mutations, and re- Received 26 February 2013 Returned for modification 26 March 2013
sults were shown to depend on method and details of the tech- Accepted 7 April 2013
nique used (5). The Bactec 460 radiometric and Mycobacteria Published ahead of print 12 June 2013
Growth Indicator Tube (MGIT 960) automated DST methods Address correspondence to Armand Van Deun, avdeun@itg.be.
systematically classified strains as susceptible that were usually Copyright 2013, American Society for Microbiology. All Rights Reserved.
resistant by the absolute concentration or proportion method on doi:10.1128/JCM.00553-13
Lwenstein-Jensen (LJ) medium as well as agar medium. This has

August 2013 Volume 51 Number 8 Journal of Clinical Microbiology p. 26332640 jcm.asm.org 2633
Van Deun et al.

typic and genotypic DST. Based on our results, we challenge surveillance, performed without ethics review or informed consent. On
consideration of phenotypic methods as the gold standard for some stored specimens we conducted retrospective laboratory tests. Anal-
rifampin DST. ysis was done on deidentified results.

RESULTS
MATERIALS AND METHODS
Patients and specimens. Since the mid-1990s, strains from Bangladesh, Of the 1,018 Bangladesh sputum samples from retreatment pa-
or sputa transported in cetylpyridinium chloride (CPC) from Kinshasa tients that were tested, 108 failed to amplify, 28 contained DNA
(Democratic Republic of Congo), were tested by DST on solid medium at from mycobacteria other than M. tuberculosis (nontuberculosis
the Antwerp SRL for drug resistance surveillance through systematic sam- mycobacteria), and 882 (86.6%) yielded a TB-specific rpoB ampli-
pling of smear-defined recurrences (failure and relapse/reinfection) after con. For 1,390 Kinshasa samples tested, these figures were, respec-
primary treatment (category 1) (12). For a number of years, a portion of tively, 102, 15, and 1,273 (91.6%). Core region and other previ-
the sputum samples were sent as ethanol-preserved samples for rpoB se- ously described resistance-conferring mutations were not
quencing, in the context of retrospective studies on acquired rifampin detected from 707 (80.2%) Bangladesh and 1,019 (80.1%) Kin-
resistance (13). These systematic sample data were used to determine the shasa sequences.
mutation prevalence rates reported here.
Table 1 shows the mutations detected, their frequencies, and
For the second part of the study, analysis regarding the impact of
particular mutations on treatment outcome, all Bangladesh first-line re- growth on culture. There were 35 different alleles among 175 mu-

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treatment episodes (category 2) were included for which an rpoB sequence tations from Bangladesh, versus 30 alleles from 254 mutations
at the start was recorded in the laboratory database, so that not only those from Kinshasa, and only 17 alleles occurred in both populations.
included in the acquired resistance studies above were part of the analysis. The 531Leu substitution made up half or more of both series,
Reference laboratory tests. Primary culture and first-line DST on LJ followed by 7 to 10% each for 526Asp and 526Tyr for Bangladesh
with final reading at 6 weeks were performed using standard methods, as and 516Val for Kinshasa. Together, these four mutations repre-
previously described (14). Rapid, direct DST in liquid medium on micros- sented two-thirds of either population. The disputed resistance
copy slides was performed at the local laboratories, with rifampin critical group accounted for 13.1% in Bangladesh and 10.6% in Kinshasa.
concentrations of 0.5 and 1.0 g/ml and reading at 10 to 14 days (15). Among the remaining were 16 (5.7%/2.4%, Bangladesh/
Sequencing was performed later and independently of phenotypic
Kinshasa) double mutations and 16 (4.6%/3.1%) highly unusual
DST. DNA extracts from clinical specimens were prepared using the
automated Boom extraction method (16, 17). Primers for amplification of mutations. Some of the latter had not been described previously
the rpoB gene covered codons 176 to 672, including all areas from which (508Asn, 516Gln, 518Ser, 523Glu, and INS511) or were reported
rifampin resistance mutations have been described and not only the ri- only as part of a double mutation (535Ser, 536Ser).
fampin resistance-determining region (RRDR; codons 507 to 533) (18). Overall recovery in culture was very low from Kinshasa sam-
All RRDR mutations, plus others previously reported for the rpoB gene (7, ples (177/251; 70.5%), which is at least partly explained by often
19), were considered potentially relevant for rifampin resistance and kept very long transport delays. For Bangladesh samples, culture posi-
for analysis. tivity did not seem to be less frequent in the disputed resistance
Data and analysis of treatment outcome impact. Individual Bangla- group (139/167; 91%), but lower sensitivity of cultures appeared
desh patient data were captured using Epi-Info 6.04d (Centers for Disease to be associated with specific mutations (516Val and 533Pro; 60 to
Control and Prevention, Atlanta, GA). Epidata (www.epidata.dk) was
70% [P 0.29]). All but 1 of 16 double mutation samples grew,
used for the reference laboratory database and for data analysis. For mul-
tiple isolates, a single rifampin sequence and/or phenotypic DST result but only 4/16 with unusual mutations grew, which was signifi-
was kept per treatment episode, with a mutation or resistance result taking cantly lower than for the other groups (P 0.001). From each
precedence in case of discordance. Unique treatment episode identifiers study site, there was also one sample with a triple mutation, but
allowed linkage of both databases to assess the impact of initial rpoB mu- none of these yielded culture growth.
tations on treatment outcome, after a visual identity check using other Table 2 shows M. tuberculosis isolates that tested resistant to
variables. rifampin on LJ at the standard 40-g/ml critical concentration,
Mutations were identified by rpoB codon number (Escherichia coli sorted by rpoB mutation found in the sputum, for both popula-
numbering) and amino acid substitution. Based on published data re- tions together. Altogether, 295/315 (93.3%) of the strains tested
garding the MIC (ratio to the critical DST concentration, 1 to 8) and phenotypically resistant. Also, disputed mutations were usually
frequent discordant results in studies, as well as DST proficiency testing
found resistant (32/38 [84.2%], 95% confidence interval [CI],
rounds among the SRLs (48, 2026), the following mutations were clas-
sified as conferring disputed resistance: 511Pro, 516Tyr, 526Asn, 68.1% to 93.4%), except 511Pro and 526Asn (1/3 and 2/4 only).
526Leu, 526Ser, 533Pro, and 572Phe. The most common, high resistance The difference was just significant with the undisputed group
mutations were grouped as undisputed resistance: 526Arg, 526Asp, (220/230; 95.5% CI, 91.6% to 97.7%; P 0.02).
526Tyr, and 531Leu. Several other mutations shown below in the tables Table 3 shows an analysis of phenotypic DST results by rpoB
might belong to one of these groups as well but had been described too mutation, for the LJ proportion method as well as local rapid slide
rarely to be classified beyond doubt. Multiple mutations are presented as DST. This series was expanded to include all Bangladesh isolates in
such, with each mutation shown in alphabetical order. For 14 cases of our database with a mutation, and we assumed that the same
heteroresistance with mutant as well as wild-type DNA, only the mutation mutation was present in subsequent isolates from the same patient
is shown. treatment episode. Of 894 tests, 91.9% (95% CI, 89.9% to 93.6%)
The usual TB control program definitions, based on smear micros-
was resistant on LJ, versus 96.4% (95% CI, 90.5% to 98.8%) of 111
copy, were applied (27). Posttreatment follow-up was passive, with con-
tinuous update of individual electronic treatment records. Recurrence- on slide DST (statistically nonsignificant, P 0.055). Of the dis-
free cure was defined as cure or treatment completion without a smear- puted resistance mutation strains, only 78.7% and 84.2% were
positive recurrence registered at any time. found resistant on LJ and slide DST, mainly caused by half the
Ethical considerations. This study retrospectively used specimens 511Pro and an occasional 516Tyr or 526Asn strain testing suscep-
and data collected in the course of routine patient care and resistance tible. On LJ, the difference was significantly different from the

2634 jcm.asm.org Journal of Clinical Microbiology

 Rifampin Gold Standard

TABLE 1 rpoB mutations found in first-recurrence sputum samples from Bangladesh and Kinshasa: relative frequency and growth on culture
Bangladesh Kinshasa
Frequency Positive cultures Frequency Positive cultures
Mutation(s) in rpoB sequence [n (%)] [no. positive/no. testeda (%)] [n (%)] [no. positive/no. testeda (%)]
508Asn 1 (0.4) 0/1
508Ile 1 (0.6) 0/1
509Thr&526Leu 1 (0.6) 1/1
511Arg&516Gly 1 (0.4) 1/1
511Pro 3 (1.7) 3/3
511Pro&512Cys 1 (0.6) 1/1
511Val 1 (0.4) 1/1
513Glu&516Phe 2 (0.8) 2/2
513Glu&516Val 1 (0.4) 1/1
513Lys 1 (0.6) 1/1 1 (0.4) 1/1
513Lys&526Asp 2 (1.1) 2/2
513Pro 2 (0.8) 2/2

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514Leu&516Val&531Leu 1 (0.6) 0/1
515Ile 1 (0.4) 0/1
515Ile&533Pro 1 (0.4) 1/1
515Thr&516Gly 1 (0.6) 1/1
516Asn 1 (0.6) 0/1
516Gln 1 (0.4) 0/1
516Lys&526Asn 1 (0.6) 1/1
516Phe 2 (1.1) 2/2 2 (0.8) 1/2
516Phe&531Leu 1 (0.6) 1/1
516Tyr 4 (2.3) 4/4 5 (2.0) 4/5
516Tyr&533Pro 1 (0.6%) 1/1
516Val 6 (3.4) 3/5 18 (7.1) 11/18 (61)
518Ser 1 (0.6) 0/1
522Gln 8 (3.1) 8/8
522Leu 2 (1.1) 1/2 1 (0.4) 0/1
523Glu 1 (0.6) 0/1 1 (0.4) 0/1
524Trp&525Pro&DEL526_527 1 (0.4) 0/1
526Arg 5 (2.9) 3/4 2 (0.8) 2/2
526Arg&531Glu 1 (0.6) 1/1
526Asn 2 (1.1) 2/2 4 (1.6) 2/4
526Asn&533Pro 1 (0.4) 0/1
526Asn&572Leu 1 (0.6) 1/1
526Asp 12 (6.9) 11/12 (86) 5 (2.0) 4/5
526Leu 5 (2.9) 4/4 6 (2.4) 4/6
526Tyr 17 (9.7) 16/17 (94) 6 (2.4) 5/6
531Gln 1 (0.6) 0/1
531Leu 82 (46.9) 66/77 (86) 160 (63.0) 113/157 (72)
531Phe 1 (0.6) 0/1
531Trp 2 (1.1) 2/2 1 (0.4) 1/1
533Pro 7 (4.0) 5/7 7 (2.8) 5/7
535Ser 1 (0.6) 0/1 4 (1.6) 3/4
536Ser 1 (0.6) 0/1
572Phe 2 (1.1) 2/2 5 (2.0) 3/5
DEL509_511 3 (1.2) 1/3
DEL513_515 3 (1.7) 3/3 2 (0.8) 0/1
INS511 1 (0.6) 1/1

Total no. of isolates with a mutation(s) 175 (100.0) 139/167 (83.2) 254 (100.0) 177/251 (70.5)
No. with disputed resistance 23 (13.1) 20/22 (90.9) 27 (10.6) 18/27 (66.7)
No. with undisputed resistance 116 (66.3) 96/110 (87.3) 173 (68.1) 124/170 (72.9)
No. of isolates with double mutations 10 (5.7) 10/10 (100.0) 6 (2.4) 5/6
No. of isolates with unusual mutation(s) 8 (4.6) 1/8 8 (3.1) 3/8
a
Percentages are shown only if the denominator was at least 10.

undisputed group (96.3% resistant; CI, 94.2% to 97.7%). Double 345 treatment episodes, average success without recorded relapse
mutation strains always tested resistant in both systems. was 21% (CI, 17.3% to 26.2%), versus 63% (CI, 58.1% to 68.5%)
The outcomes for category 2 standardized retreatment under failure or relapse recurrence, with a 4.2 ratio of failures to relapses.
field conditions are shown by rpoB mutation in Table 4. For the These proportions barely differed between the disputed and un-

August 2013 Volume 51 Number 8 jcm.asm.org 2635

Van Deun et al.

TABLE 2 Phenotypic rifampin resistance of M. tuberculosis isolates with ing only the more commonly described of these disputed
particular rpoB mutations (Bangladesh and Kinshasa series combined) mutations (511Pro, 516Tyr, 526Asn, 526Leu, 533Pro, and
detected using the LJ proportion method at a critical concentration of 572Phe), we found that they made up over 10% of all rpoB muta-
40 g/ml
tions among failure and relapse cases from Bangladesh as well as
No. of isolates: Kinshasa. A systematic series from Hong Kong with over 3,000
rpoB sequence % resistant
mutation(s) Tested Resistant (95% CI)a isolates screened by molecular technique without phenotypic DST
509Thr&526Leu 1 1
preselection found 21% prevalence among 89 RRDR mutated
511Arg&516Gly 1 1 strains, counting only 511Pro, 526Leu, and 533Pro (28). How-
511Pro 3 1 ever, this series must have consisted largely of new cases. We are
511Pro&512Cys 1 1 not aware of other large series screened without conventional DST
511Val 1 0 preselection, which leads to systematic underestimation of their
513Glu&516Phe 2 2 prevalence. However, just 533Pro, the most easily detected dis-
513Glu&516Val 1 1 puted mutation, occurs at a rather constant frequency of 3 to 6%,
513Lys 2 2 according to many reports (29). There are reasons to believe that
513Lys&526Asp 2 2 511Pro and 516Tyr might be detected at similar frequencies, if
513Pro 2 2
they were not so easily missed in phenotypic DST, while 572Phe
515Ile&533Pro 1 1

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515Thr&516Gly 1 1
also would be reported more frequently if molecular DST did not
516Lys&526Asn 1 1 target only the RRDR (codons 507 to 533). Moreover, there is a
516Phe 3 3 whole range of rarely reported and thus ill-known mutations that
516Phe&531Leu 1 1 might very well also belong to the disputed group.
516Tyr 8 7 Although our SRL is known to declare such strains most often
516Tyr&533Pro 1 1 resistant in the phenotypic DST proficiency testing rounds (4), it
516Val 14 14 100 (73.2100.0) also missed about 8% of all, or 20% of disputed, rifampin resis-
522Gln 8 8 tance in routine work. Feuerriegel et al. reported that in a system-
522Leu 1 1 atic series of Sierra Leone retreatment case isolates retested by
526Arg 5 5
DNA sequencing, 5/21 (24%) rpoB mutations were found among
526Arg&531Glu 1 1
526Asn 4 2
strains classified as rifampin susceptible by phenotypic DST. All
526Asn&572Leu 1 1 but one strain originally classified as resistant carried the undis-
526Asp 15 15 100 (74.7100.0) puted 531Leu or 526Arg or Tyr mutations, while among the sus-
526Leu 8 8 ceptible isolates there were three 516Tyr and one each of the
526Tyr 21 20 95 (74.199.8) 511Pro and 533Pro mutations. Those authors confirmed these
531Leu 179 170 95 (90.497.5) disputed strains to be susceptible and concluded there was 94%
531Trp 3 3 specificity with DNA sequencing because repeat testing with the
533Pro 10 9 90 (54.199.5) MGIT 960 system confirmed their MICs were below the conven-
535Ser 3 0 tional 1.0-g/ml breakpoint (10).
572Phe 5 5
In our view, the conventional gold standard, i.e., phenotypic
DEL509_511 1 1
DEL513_515 3 3
DST, fails for these strains. We have previously shown that rapid
INS511 1 0 phenotypic DST, specifically automated MGIT, classifies many
Wildtype 944 56 6 (4.57.7) rpoB mutated strains as susceptible (or fails to yield a valid result),
Any mutation 315 294 93.3 (89.895.7) while such strains usually test resistant by other methods in vari-
Disputed resistance 38 32 84.2 (68.193.4) ous proficient laboratories (5), and most have clearly raised MICs
Undisputed resistance 230 220 95.5 (91.697.7) (6). They may be so hard to detect because of fitness loss and
Double mutations 15 15 100 (74.7100.0) slower growth in the presence of the drug compared to the drug-
Unusual mutations 4 0 free controls, particularly if the resistance level is relatively low
a
Percentages are shown only if the denominator (total number of isolates) was at least (30). Indirect laboratory evidence that the disputed and other very
10. rare, little-known mutations (i.e., 516Gly, 515Thr) do confer
some degree of resistance can be inferred from their overrepresen-
tation in double and triple mutations. These occurred at levels of
disputed groups, with exactly the same percentage of recurrence several percent in our series, far too frequent to have come up
and hardly more relapse-free registered cures (27% of 70 versus simultaneously. The fitness cost of each mutation must thus have
20% of 214 episodes; nonsignificant). There might have been been less determinant than the continued selective pressure for
more relapses relative to failures in the disputed group (ratio, 2.4), further increased resistance levels, implying that resistance due to
particularly with the 511Pro and 533Pro mutations. All five un- the first mutation was indeed borderline. At the same time, the
usual mutations within the RRDR with treatment outcome avail- first mutation conferred sufficient resistance to allow the bacilli to
able were recorded as relapse-free cures. replicate during rifampin treatment. This explains why no double
mutation was composed of two undisputed mutations, but at
DISCUSSION most one, which we hypothesize to have arisen last. We would
Our results show that rpoB mutations that result in rifampin re- even postulate that the clinical significance of very rare single mu-
sistance that is regularly or even systematically missed by stan- tations can be deducted from their occurrence in multiple muta-
dard, WHO-endorsed DST methods are not uncommon. Select- tions, such as the 535Ser or 536Ser mutations in our series, even

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 Rifampin Gold Standard

TABLE 3 Rifampin resistance observed with routine phenotypic DST, by rpoB mutation
DST on LJ Slide DST
No. rifampin % resistant No. rifampin
rpoB mutation(s) No. tested resistant (95% CI)a No. tested resistant % (95% CI)a
509Arg&526Gln 1 1
509Thr&526Leu 3 3 1 1
511Arg&531Leu 2 2
511Pro 30 14 47 4 2
511Pro&515Leu 3 3
511Pro&515Val 1 1 1 1
511Pro&516Val 1 1
511Pro&526Arg 2 2
511Pro&526Leu 1 1
511Pro&531Leu 7 7
512Arg&516Gly 2 2
513Glu 2 2

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513Lys 14 14 100 2 2
513Lys&526Asp 2 2
513Pro 4 3
515Arg&516Val 1 1
515Ile&516Tyr 4 4
515Thr&516Gly 7 7 1 1
516Lys&526Asn 3 3 1 1
516Phe 16 15 94
516Phe&531Leu 1 1
516Tyr 34 30 88 5 5
516Val 57 56 98 4 4
522Gln 1 1
522Leu 13 11 85
526Arg 13 13 100 1 1
526Arg&531Glu 1 1
526Asn 5 3
526Asp 31 31 100 3 3
526Cys 4 3
526Gly 1 1
526Leu 27 26 96 1 1
526Pro 3 2
526Tyr 83 79 95 16 16
531Gly 2 1 1 1
531Leu 392 377 96 56 55
531Phe 1 1
531Trp 16 14 88
533Pro 67 55 82 8 7
572Leu&526Asn 3 3 1 1
572Phe 15 12 80 1 1
DEL513_515 6 5 3 3
DEL517 2 2
INS511 1 0
INS512_513 2 1
INS513_514 5 5 1 1
INS514 2 0
Any mutation 894 822 91.9 (89.993.6) 111 107 96.4 (90.598.8)
Disputed resistance 112 81 78.7 (71.884.3) 16 19 84.2 (59.595.8)
Undisputed resistance 558 535 96.3 (94.297.7) 78 77 98.7 (91.999.9)
Double mutations 45 45 100.0 (90.2100) 5 5
a
Percentages are shown only if the denominator was at least 10.

though this could not be concluded from their MICs or the clinical adverse outcomes for standard first-line retreatment in an excel-
outcomes. lent TB control project, our data showed that these disputed mu-
DST should predict what clinicians can expect as action tations have exactly the same poor clinical prognosis as the most
from the drug, whatever the method used, or MIC or mutation frequent undisputed mutations. A few reports had already sug-
type found. Based on almost two-thirds of bacteriologically gested that such strains have clinical relevance. Williamson

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Van Deun et al.

TABLE 4 Outcome of category 2 cases (first-line retreatment regimen), by rpoB mutation detected at time of prime treatment failure or relapsea
Relapse-free cure FL and RLb
No. of
a
Mutations(s) in rpoB sequence episodes n % (95% CI) No. reported % (95% CI)a FL/RL ratio
508Ile 1 1 0
509Thr&526Leu 1 1 0
511Arg&531Leu 1 0 1 Only FL
511Pro 21 3 14 15 71 1.1
511Pro&512Cys 1 0 1 Only FL
511Pro&515Leu 1 0 1 Only FL
511Pro&516Val 1 0 1 Only FL
513Glu 1 0 1 Only FL
513Lys 3 1 1 Only FL
513Lys&526Asp 1 0 0
513Pro 1 0 1 Only FL
515Thr&516Gly 2 1 1 Only FL
516Phe 5 1 4 1.0

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516Phe&531Leu 1 0 0
516Tyr 7 2 4 Only FL
516Val 15 1 7 12 80 5.0
516Val&531Leu 1 0 0
518Ser 1 1 0
522Leu 4 1 3 Only FL
523Glu 1 1 0
526Arg 7 2 6 Only FL
526Arg&531Glu 1 0 1 Only FL
526Asn 5 3 2 Only FL
526Asp 17 4 24 8 47 1.0
526Cys 2 0 2 Only FL
526Leu 10 2 20 7 70 2.5
526Tyr 31 9 29 19 61 18.0
531Leu 159 27 17 101 64 6.0
531Trp 6 1 5 4.0
533Pro 20 7 35 12 60 1.4
535Ser 1 1 0
536Ser 1 1 0
572Phe 5 2 2 Only FL
DEL513_515 3 0 3 2.0
INS512_513 3 0 3 Only RL
INS513_514 3 1 2 1.0

Any mutation 345 74 21 (17.326.2) 219 63 (58.168.5) 4.2

Disputed resistance 70 19 27 (17.539.3) 44 63 (50.473.9) 2.4
Undisputed resistance 214 42 20 (14.725.7) 134 63 (55.769.0) 5.8
Double mutations 11 2 18 (3.252.2) 6 55 (24.681.9) Only FL
Unusual mutations 5 5 0
a
Percentages are shown only if the denominator was at least 10.
b
FL, failure; RL, relapse.

reported four cases from New Zealand, retrospectively detected infectiousness resulting from delayed diagnosis of MDR-TB be-
by using Xpert, among MGIT-DST susceptible cases, among cause of susceptible DST results, followed by prolonged, treat-
whom three had failed treatment while one was found postmor- ment with low effectiveness and repeated relapse. In our own ex-
tem. All cases represented disputed mutations, single or combined perience, patients afterwards documented to have had strains with
(511Pro&515Ile, 526Asn&532Val, 516Tyr, 526Leu) (9). van Ingen these mutations were started on curative MDR treatment only
reported a small outbreak with a 516Tyr strain from Holland (31). after several first-line retreatment relapses, or they were errone-
That these strains can be highly meaningful on the population ously switched back from effective MDR to first-line treatment
level was suggested also by the report of Ioerger et al. Their ge- upon receipt of a susceptible DST result, with an adverse outcome.
nome analysis of MDR and extremely drug-resistant strains from Because of this failure of the phenotypic gold standard, molec-
the KwaZulu-Natal outbreak showed the high transmissibility of a ular rifampin DST techniques thus perform with higher specificity
516Tyr as well as a 516Gly&533Pro mutant strain (32). It is pos- than generally believed. The original publication introducing
sible that the very high HIV prevalence in this population facili- GeneXpert for TB concluded a 98.1% specificity for rifampin re-
tated transmission. However, it is also conceivable that patients sistance, although sequencing had shown RRDR mutations in all
with such disputed resistant strains have a prolonged period of nine discordant phenotypically susceptible strains (33). Six be-

2638 jcm.asm.org Journal of Clinical Microbiology

 Rifampin Gold Standard

longed to the disputed mutations (four 511Pro, one 516Tyr, and ically highly relevant and occurs too frequently to continue to be
one 533Pro), one mutation belonged to the undisputed (526Tyr), ignored. The underlying rpoB mutations are readily detected by
one to the unusual mutations (DEL518; MDR in our laboratory), gene sequencing, which should be used to correct the phenotypic
and one represented a silent mutation (514PheTTT [C. Boehme gold standard when evaluating the performance characteristics of
and S. Ruesch-Gerdes, personal communication]). These se- rifampin resistance diagnostic tests. The problem of any rifampin
quence results were not accepted for resolution of discrepancies DST method is not imperfect specificity but suboptimal sensitiv-
because of doubts regarding the significance of the mutations ity. The predictive value of an Xpert or LPA resistance result may
(34), but our data indicate that the Xpert rifampin specificity in be very high also when there is low prevalence, but this requires
that study was in fact 99.8% if we kept only the silent mutation as further study that includes new cases. A susceptible result should
a false resistance result. Other reports on Xpert false rifampin be questioned when suspicion is very high, and further DST using
resistance results have mentioned mutations found by sequencing a different system (i.e., genotypic after phenotypic) would be fully
(35). True-false resistance without any mutation detected by se- justified.
quencing has been documented occasionally for earlier versions of
the MTB/RIF cartridge, and possibly more with extrapulmonary ACKNOWLEDGMENT
tuberculosis (36, 37). This work was generously supported by a grant from the Damien Foun-
Based on testing of mainly retreatment cases, silent RRDR mu- dation Belgium.

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tations occur at below 0.5% frequency in our laboratory. Also,
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