Gene Sequencing for Routine Verification of Pyrazinamide Resistance in Mycobacterium tuberculosis: a Role for pncA but Not rpsA.
ABSTRACT Pyrazinamide (PZA) is an important component of first-line therapy for the treatment of tuberculosis. Here, we evaluate targeted gene sequencing as a supplement to phenotypic PZA susceptibility testing of Mycobacterium tuberculosis. Routine sequencing of pncA, but not rpsA, is effective for verification of PZA susceptibility results.
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ABSTRACT: Standard culture-based testing of the susceptibility of Mycobacterium tuberculosis to pyrazinamide is difficult to perform. This systematic review with meta-analyses evaluated the roles of molecular assays targeting pncA and of pyrazinamidase assays. PubMed and Embase were searched for relevant publications in English. Sensitivity and specificity were estimated in bivariate random-effects models. Of 128 articles identified, 73 sets of data involving culture isolates were initially included in meta-analyses. Summary estimates of sensitivity and specificity, respectively, were 87% and 93% for PCR-DNA sequencing (n = 29), 75% and 95% for PCR-single-stranded conformation polymorphism (SSCP) (n = 5), 96% and 97% for a mixture of other molecular assays (n = 6), and 89% and 97% for pyrazinamidase assays using the Wayne method (n = 33). The median prevalence (range) of pyrazinamide resistance was 51% (31% to 89%) in multidrug-resistant M. tuberculosis isolates and 5% (0% to 9%) in non-multidrug-resistant isolates. Excluding studies with possibly considerable false resistance in the reference assay gave the following estimates of sensitivity and specificity, respectively: 92% and 93% for PCR-DNA sequencing (n = 20), 98% and 96% for other molecular assays (n = 5), and 91% and 97% for the Wayne assay (n = 27). The Wayne assay had significant funnel plot asymmetry, so the test performance might have been overestimated. Considering the prevalence of pyrazinamide resistance in different clinical settings, PCR-DNA sequencing, and possibly other molecular assays targeting pncA, can detect pyrazinamide resistance in multidrug-resistant M. tuberculosis isolates, with predictive values largely exceeding 90%, and rule out pyrazinamide resistance in non-multidrug-resistant isolates, with predictive values exceeding 99%. Molecular assays are probably the way forward for detecting pyrazinamide resistance.Antimicrobial Agents and Chemotherapy 07/2011; 55(10):4499-505. · 4.57 Impact Factor
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ABSTRACT: India has the highest estimated burden of tuberculosis in the world, accounting for 21% of all tuberculosis cases world-wide. However, due to lack of systematic analysis using multiple markers the available information on the genomic diversity of Mycobacterium tuberculosis in India is limited. Thus, 65 M. tuberculosis isolates from New Delhi, India were analyzed by spoligotyping, MIRU-VNTR, large deletion PCR typing and single nucleotide polymorphism analysis (SNP). The Central Asian (CAS) 1 _DELHI sub-lineage was the most prevalent sub-lineage comprising 46.2% (n = 30) of all isolates, with shared-type (ST) 26 being the most dominant genotype comprising 24.6% (n = 16) of all isolates. Other sub-lineages observed were: East-African Indian (EAI)-5 (9.2%, n = 6), EAI6_BGD1 (6.2%, n = 4), EAI3_IND, CAS and T1 with 6.2% each (n = 4 each), Beijing (4.6%, n = 3), CAS2 (3.1%, n = 2), and X1 and X2 with 1 isolate each. Genotyping results from five isolates (7.7%) did not match any existing spoligopatterns, and one isolate, ST124, belonged to an undefined lineage. Twenty-six percent of the isolates belonged to the TbD1+ PGG1 genogroup. SNP analysis of the pncA gene revealed a CAS-lineage specific silent mutation, S65S, which was observed for all CAS-lineage isolates (except two ST26 isolates) and in 1 orphan. Mutations in the pncA gene, conferring resistance to pyrazinamide, were observed in 15.4% of all isolates. Collectively, mutations in the rpoB gene, the katG gene and in both rpoB and katG genes, conferring resistance to rifampicin and isoniazid, respectively, were more frequent in CAS1_DELHI isolates compared to non-CAS_DELHI isolates (OR: 3.1, CI95% [1.11, 8.70], P = 0.045). The increased frequency of drug-resistance could not be linked to the patients' history of previous anti-tuberculosis treatment (OR: 1.156, CI95% [0.40, 3.36], P = 0.79). Fifty-six percent of all new tuberculosis patients had mutations in either the katG gene or the rpoB gene, or in both katG and rpoB genes. CAS1_DELHI isolates circulating in New Delhi, India have a high frequency of mutations in the rpoB and katG genes. A silent mutation (S65S) in the pncA gene can be used as a putative genetic marker for CAS-lineage isolates.PLoS ONE 02/2009; 4(2):e4540. · 3.73 Impact Factor
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ABSTRACT: Testing for susceptibility to pyrazinamide (PZA) and analysis of the pncA gene sequences of 423 Mycobacterium tuberculosis complex isolates have revealed a unique silent nucleotide substitution that enables the rapid identification of "M. canettii" (proposed name). Moreover, the lack of a defined mutation within the pncA gene strongly suggests that an alternative mechanism is responsible for PZA resistance. Our results indicate that DNA sequencing of the pncA gene has the potential to shorten the turnaround time and increase the accuracy of PZA susceptibility testing of the M. tuberculosis complex.Journal of Clinical Microbiology 03/2007; 45(2):595-9. · 4.07 Impact Factor
Gene Sequencing for Routine Verification of Pyrazinamide Resistance
in Mycobacterium tuberculosis: a Role for pncA but Not rpsA
David C. Alexander,a,bJennifer H. Ma,aJennifer L. Guthrie,aJoanne Blair,aPam Chedore,aand Frances B. Jamiesona,b
Public Health Ontario, Public Health Laboratories, Toronto, Ontario, Canada,aand University of Toronto, Department of Laboratory Medicine and Pathobiology, Toronto,
geted gene sequencing as a supplement to phenotypic PZA susceptibility testing of Mycobacterium tuberculosis. Routine se-
priate tuberculosis (TB) therapy and help prevent the emergence
and spread of drug-resistant Mycobacterium tuberculosis strains.
Pyrazinamide (PZA) is a front-line drug for the treatment of TB.
Administered during the 2-month, intensive phase of the stan-
dard short-course regimen, PZA is effective primarily against
slowly replicating bacilli and, thus, complements the activities of
isoniazid (INH) and rifampin (RIF), which are bactericidal for
rapidly replicating organisms.
PZA is a prodrug. Conversion to the active form, pyrazinoic
acid (POA), is mediated by the pyrazinamidase (PZase) encoded
by the pncA gene. It is well established that mutations in pncA can
mulation of POA (6). However, some PZA-resistant (PZAr)
strains have wild-type pncA (pncAWT) alleles. In such strains, re-
sistance has been proposed to result from altered PZA uptake,
9). Recently, it has been demonstrated that POA binding to the
30S ribosomal protein S1 inhibits the trans-translation activity
required for efficient protein synthesis (7). Mutations in rpsA,
which encodes the S1 protein, result in altered POA binding and
can mediate PZA resistance in pncAWTstrains.
is challenging (2). PZase activity and the intracellular accumula-
tion of POA increase with decreasing pH, but Mycobacterium tu-
Laboratory Standards Institute (CLSI) recommends the Bactec
460TB radiometric system with Bactec 460TB PZA test medium
(BD Diagnostics Systems, Sparks, MD) as the reference method
for phenotypic PZA susceptibility testing (1, 4). However, the
460TB system has been discontinued and the 460TB PZA test
medium is no longer being manufactured. Many laboratories, in-
cluding Public Health Ontario (PHO), have adopted the Bactec
MGIT 960 (BT960) platform for PZA testing. At our large public
health laboratory, the switch to BT960-based testing was accom-
strains that were PZArby the BT960 method but PZA-susceptible
(PZAs) according to the reference 460TB method (3). To ensure
accurate susceptibility results, confirmatory testing of potential
PZArisolates is necessary. A phenotypic strategy, involving a sec-
ond round of BT960-based testing, can be effective but does not
resolve all cases (3, 8), and repeat testing requires an additional 5
to 7 days to complete (1). In contrast, confirmatory testing using
have investigated the utility of targeted gene sequencing for rapid
verification of PZArresults.
To assess the utility of sequencing, archived DNA from 141
were from the PHO strain collection and were originally isolated
spoligotype, 12-locus mycobacterial interspersed repetitive-unit–
variable-number tandem-repeat (MIRU-VNTR) pattern, and
first-line drug susceptibility profiles of each isolate were deter-
the supplemental material). Review of the original PZA suscepti-
that 77 isolates were PZA sensitive (PZAs) and 64 were PZA resis-
tant (PZAr). For the current study, the pncA gene was amplified
using primers pnc1 (5=-GGCGTCATGGACCCTATATC-3=) and
pnc2 (5=-CAACAGTTCATCCCGGTTC-3=) (5). The 670-bp am-
plicon encompassed the complete pncA coding region and 80 bp
of upstream DNA. Individual PCRs were prepared with HotStar
cycler (G-Storm Ltd.; Somerset, United Kingdom) under the fol-
extension (72°C for 1 min), and a final extension at 72°C for 10
cycle sequencing (Life Technologies, CA) with the Applied Bio-
systems 3730xl DNA analyzer (Life Technologies). All PZAs
strains were wild type for pncA, although a previously recognized
variant allele (C195T, Ser65) was observed in a subset of East
African Indian lineage isolates (10). In contrast, pncA mutations,
including insertions, deletions, nonsynonymous changes, and
1; see also Table S1 in the supplemental material for additional
Received 5 March 2012 Returned for modification 16 April 2012
Accepted 7 August 2012
Published ahead of print 15 August 2012
Address correspondence to Frances B. Jamieson, email@example.com.
Supplemental material for this article may be found at http://jcm.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jcm.asm.org Journal of Clinical Microbiologyp. 3726–3728 November 2012 Volume 50 Number 11
details). Eleven isolates were phenotypically PZArbut genotypi-
cally pncAWT. Of these discordant isolates, 5 were monoresistant
PZArand 6 were resistant to multiple first-line drugs. Eight of the
discordant isolates were successfully resurrected from frozen
stocks, and first-line susceptibility testing was repeated using the
BT960 method (3). In contrast to the original 460TB results,
BT960-based testing indicated that 5 of these strains were PZAs
and, thus, was concordant with pncA sequencing. A cause for the
discrepant BT960 and 460TB findings was not able to be deter-
mined, but the results indicate that the “gold standard” 460TB
method is not infallible. Of the three remaining discordant iso-
lates, one was PZA monoresistant and two were multidrug-resis-
from the resurrected strains, and upon resequencing, pncA muta-
tions were identified in both MDR-TB isolates. No mutations
were found in the final isolate, and this one monoresistant strain
remained phenotypically PZArand genotypically pncAWT.
To examine the potential contribution of rpsA mutations to
PZA resistance, 13 PZAsstrains and the 11 isolates initially con-
sidered discordant were analyzed (Table 2). To obtain full-length
rpsA sequences, five overlapping amplicons were generated by
PCR using the following primers: rspA-1F, 5=-ATGCCGAGTCC
CACCGTC-3=; rspA-1R, 5=-ACCCTTGACGACCTCGATGA-3=;
rspA-2F, 5=-AAACGCGCGCAGTACG-3=; rspA-2R, 5=-GTGAC
CTCGTCACCAACCTG-3=; rspA-3F, 5=-GACGGTCTGGTGCA
rspA-4F, 5=-ATGGCTTGAGGGATTCGAAAA-3=; rspA-4R, 5=-A
GCTTGAG-3=; and rspA-M5R, 5=-CTACTGGCCGACGACTGA
T-3=. PCR and sequencing conditions were identical to those
described for pncA except that a longer extension time (72°C
for 1.5 min) was used. A synonymous rpsA mutation (A636C,
indicate that this is not a lineage-specific mutation. One isolate
also harbored a second synonymous change (G960A, Leu320).
Nonsynonymous mutations were present in two strains. G1318A
(Ala440Thr)wasobservedinthe Mycobacteriumbovis BCGrefer-
ence strain and is conserved among publicly available genomes of
M. bovis and M. bovis BCG. PZA resistance in M. bovis is a recog-
nized phenomenon that is traditionally attributed to the pncA
A169G (His57Asp) mutation. The impact of the RpsA Ala440Thr
?Ala438, has been characterized and found to impair POA bind-
PZAr/pncAWTstrains bearing dual RpsA Thr5Ser and Asp123Ala
similar mutation, rpsA A364G (Lys122Glu), was present in one
the Thr5Ser, Asp123Ala, or Lys122Glu mutations impact POA
binding or simply represent regions of RpsA that tolerate amino
acid substitutions. Our phenotypic findings suggest that RpsA
may also be innocuous. However, sequence comparison indicates
that Thr5Ser is present in the RpsA ortholog of Mycobacterium
avium. Similarly, the RpsA ortholog of Mycobacterium canettii
strain CIPT 140010059 exhibits 2 changes, Thr5Ala and
Thr210Ala, relative to M. tuberculosis H37Rv. Strains of the M.
avium complex and M. canettii isolates are considered PZAreven
though they are genotypically pncAWTand exhibit PZase activity
(9, 11). The Thr5Ser/Thr5Ala variants may explain this phenom-
Although rpsA may contribute to PZA resistance, rpsA se-
TABLE 1 Phenotypic and genotypic characteristics of pyrazinamide-
resistant clinical isolatesa
No. of isolates with each drug resistance
Total no. of
isolatesPZA only Polyc
5 (1) 1 (0)3 (0)9 (1)
aValues in parentheses are the numbers of strains remaining after repeat testing of
discordant isolates. A total of 8 strains were excluded. Five strains were reclassified as
PZA sensitive, and three were not able to be retested.
bDrug susceptibility testing was performed on the 460TB (all 64 isolates) and BT960
(11 discordant isolates) platforms.
cStrains that are resistant to (at least) PZA and INH but not RIF.
dStrains that are resistant to (at least) PZA, INH, and RIF.
eXDR, extensively drug resistant.
fC418A and an 8-bp deletion were observed in all 3 isolates.
TABLE 2 Genotypic characterization of rpsA in clinical and reference isolatesa
No. of isolates with:
Total no. of
Synonymous changeNonsynonymous change
460TB resistant andb:
M. bovis BCG11
aThe susceptibility profiles of eight strains were retested.
bIncludes 11 discordant isolates initially considered PZA resistant and pncAWT.
cStrain is a double mutant (A636C and G960A) and counted only once.
pncA and rpsA Sequencing for PZA Testing
November 2012 Volume 50 Number 11jcm.asm.org 3727
no phenotypically informative mutations were identified. In con-
all but four PZArisolates and no PZAsstrains. This indicates that,
for Mycobacterium tuberculosis, DNA sequencing of pncA but not
rpsA is a robust tool for routine and rapid verification of PZA
We acknowledge the excellent work of the Public Health Ontario TB and
Mycobacteriology Laboratory (PHL-Toronto) staff responsible for the
initial isolation, cultivation, and susceptibility testing of the clinical iso-
lates used in the study.
1. Becton Dickinson and Company. 2002. Bactec MGIT 960 PZA kit for the
antimycobacterial susceptibility testing of Mycobacterium tuberculosis.
Becton Dickinson and Company, Sparks, MD.
2. Chang KC, Yew WW, Zhang Y. 2011. Pyrazinamide susceptibility testing
in Mycobacterium tuberculosis: a systematic review with meta-analyses.
Antimicrob. Agents Chemother. 55:4499–4505.
3. Chedore P, Bertucci L, Wolfe J, Sharma M, Jamieson F. 2010. Potential
for erroneous results indicating resistance when using the Bactec MGIT
960 system for testing susceptibility of Mycobacterium tuberculosis to pyr-
azinamide. J. Clin. Microbiol. 48:300–301.
4. CLSI. 2011. Susceptibility testing of mycobacteria, nocardiae, and other
aerobic actinomycetes; approved standard—second edition. CLSI docu-
ment M24-A2. Clinical and Laboratory Standards Institute, Wayne, PA.
5. Nguyen D, et al. 2003. Widespread pyrazinamide-resistant Mycobacte-
rium tuberculosis family in a low-incidence setting. J. Clin. Microbiol.
6. Scorpio A, Zhang Y. 1996. Mutations in pncA, a gene encoding pyrazi-
namidase/nicotinamidase, cause resistance to the antituberculous drug
pyrazinamide in tubercle bacillus. Nat. Med. 2:662–667.
7. Shi W, et al. 2011. Pyrazinamide inhibits trans-translation in Mycobac-
terium tuberculosis. Science 333:1630–1632.
8. Simons SO, et al. 2012. Validation of pncA gene sequencing in combina-
tion with the mycobacterial growth indicator tube method to test suscep-
tibility of Mycobacterium tuberculosis to pyrazinamide. J. Clin. Microbiol.
9. Somoskovi A, et al. 2007. Sequencing of the pncA gene in members of the
Mycobacterium tuberculosis complex has important diagnostic applica-
tions: identification of a species-specific pncA mutation in “Mycobacte-
rium canettii” and the reliable and rapid predictor of pyrazinamide resis-
tance. J. Clin. Microbiol. 45:595–599.
10. Stavrum R, Myneedu VP, Arora VK, Ahmed N, Grewal HM. 2009.
In-depth molecular characterization of Mycobacterium tuberculosis from
New Delhi—predominance of drug resistant isolates of the ‘modern’
(TbD1) type. PLoS One 4:e4540. doi:10.1371/journal.pone.0004540.
11. Sun Z, Scorpio A, Zhang Y. 1997. The pncA gene from naturally pyrazi-
fers pyrazinamide susceptibility to resistant M. tuberculosis complex or-
ganisms. Microbiology 143:3367–3373.
Alexander et al.
jcm.asm.org Journal of Clinical Microbiology