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Original filename: 10.1016@j.meegid.2018.01.022.pdf
Title: Differences in rpoB, katG and inhA mutations between new and previously treated tuberculosis cases using the GenoType MTBDRplus assay
Author: Gerardo Alvarez-Uria

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Infection, Genetics and Evolution 59 (2018) 48–50

Contents lists available at ScienceDirect

Infection, Genetics and Evolution
journal homepage: www.elsevier.com/locate/meegid

Short communication

Differences in rpoB, katG and inhA mutations between new and previously
treated tuberculosis cases using the GenoType MTBDRplus assay

T



Gerardo Alvarez-Uria , Raghuprakash Reddy
a
b

Department of Infectious Diseases, Rural Development Trust Hospital, Bathalapalli, AP, India
Department of Microbiology, Rural Development Trust Hospital, Bathalapalli, AP, India.

A R T I C L E I N F O

A B S T R A C T

Keywords:
Rifampin
Isoniazid
Drug resistance
Mycobacterium tuberculosis
Reproductive fitness
Transmission
Therapy

Drug resistance in new cases reflects primary transmission of resistant strains, while drug resistance in previously treated patients is more likely to reflect acquired resistance during previous tuberculosis (TB) treatment.
In this study from a rural hospital in South India, we compared molecular differences between new and previously treated TB patients who had isoniazid or rifampicin resistance-conferring mutations using the GenoType
MTBDRplus assay. Out of 2112 TB patients, 245 (11.6%) had rpoB mutations and 338 (16%) had isoniazid
resistance-conferring mutations. Among patients with rpoB mutations, the proportion of new and previously
treated cases with no isoniazid resistance-conferring mutations was 41.2% and 26% (P-value = 0.02; risk ratio
[RR] 1.58, 95% confidence interval [CI] 1.09–2.31; risk difference [RD] 15.2%, 95% CI 18.2–28.6), respectively.
Among patients with isoniazid resistance-conferring mutations, the proportion of new and previously treated
cases with no rpoB mutations was 71.8% and 33.2% (P-value < 0.0001; RR 2.17, 95% CI 1.73–2.71; RD 38.7%,
95% CI 28.8–48.6), and the proportion with single inhA mutations (versus having katG mutations) was 33.1%
and 20.9% (P-value = 0.012; RR 1.58, 95% CI 1.11–2.27; RD 12.2%, 95% CI 2.57–21.8), respectively. The most
common resistance mutations were S531 L in the rpoB gene, S315T1 in the katG gene and C15T in the inhA gene,
and there were no significant differences between new and previously treated patients. In conclusion, new TB
cases were less likely to have combined isoniazid and rifampicin resistance-conferring mutations and, in cases
with isoniazid resistance, they were more likely to have single inhA mutations than katG mutations. Taking into
account that previous research has shown katG mutations precede mutations in the rpoB gene in most cases of
rifampicin resistant TB, our results suggest a negative epistatic association between katG and rpoB mutations.

According to the World Health Organization (WHO), drug resistance
is a major threat for global tuberculosis (TB) care and prevention. In
2016, 4.1% of new cases and 19% of previously treated cases had rifampicin resistant TB, and 7.3% of new cases and 14% of previously
treated cases had isoniazid resistant TB without concurrent rifampicin
resistance (World Health Organization, 2017). Drug resistance in new
cases reflects direct interpersonal transmission of drug-resistant strains,
while resistance in previously treated patients is more likely to reflect
acquired drug resistance during previous TB treatment, although primary transmitted resistance is also possible (Izu et al., 2013). In this
study, we aimed to identify molecular differences in isoniazid and rifampicin resistance-conferring mutations between new and previously
treated TB cases using the GenoType MTBDRplus assay.
The study was performed in the Rural Development Trust General
Hospital, a non-profit 325-bed hospital in Bathalapalli, a rural village in
Anantapur district, Andhra Pradesh, India.

We collected microbiological data of all samples processed from 1
July 2014 to 18 October 2017 with the GenoType MTBDRplus ver 2.0
assay after isolation of Mycobacterium tuberculosis from liquid culture.
Sample processing and interpretation were performed following manufacturer's instructions (Hain Lifescience, Nehren, Germany). The
MTBDRplus assay is a commercially available molecular line probe
assay endorsed by WHO with good diagnostic accuracy compared to
conventional drug susceptibility testing (Bai et al., 2016). The assay can
detect Mycobacterium tuberculosis complex and mutations conferring rifampicin resistance (in the hotspot 81 base-pair region of rpoB gene)
and isoniazid resistance (at codon 315 of the katG gene and in the inhA
promoter region) using two types of probes: mutation and wild-type
probes. Mutation probes detect the most common resistance mutations,
while wild-type probes comprise the most important resistance regions
in the respective genes. When a mutation is present, the amplicon
cannot bind to the corresponding wild-type probe and, hence, the


Corresponding author: Department of Infectious Diseases, Bathalapalli Rural Development Trust Hospital, Kadiri Road, Bathalapalli, 515661 Anantapur District, Andhra Pradesh,
India.
E-mail address: gerardouria@gmail.com (G. Alvarez-Uria).

https://doi.org/10.1016/j.meegid.2018.01.022
Received 13 December 2017; Accepted 25 January 2018
1567-1348/ © 2018 Elsevier B.V. All rights reserved.

Infection, Genetics and Evolution 59 (2018) 48–50

G. Alvarez-Uria, R. Reddy

95% CI 2.57–21.8), respectively. However, when patients with isoniazid resistance were segregated by presence of rpoB mutations, the
proportion of new and previously treated patients with katG mutations
was 82.5% and 83.2% (P-value = 0.917; RR 1.04, 95% CI 0.48–2.26;
RD 0.7%, 95% CI −12.7–14.1) in patients with rpoB mutations, and
60.8% and 70.8% (P-value = 0.188; RR 1.34, 95% CI 0.86–2.1; RD
10%, 95% CI −4.6–24.6) in patients without rpoB mutations, respectively.
Differences in rpoB, katG and inhA mutations are presented in
Table 2. The greatest difference was observed in the H526Y mutations
of the rpoB gen (0% in new patients vs. 9% in previously treated patients), but differences in rpoB mutations were only marginally signficant (P = 0.05). There were no signficant differences in katG and inhA
mutations.
With the expansion of the GeneXpert MTB/RIF assay to diagnose TB
in developing countries, isoniazid resistance is likely to be unknown to
most patients diagnosed with rifampicin resistant TB in high-TB burden
settings (World Health Organization, 2017). The WHO recommends the
use of isoniazid in the treatment of rifampicin resistant TB if isoniazid
resistance is unknown (World Health Organization and Global
Tuberculosis Programme, 2016). Our results indicate that adding isoniazid to rifampicin resistant TB therapy could be more beneficial in
new cases than in previously treated cases. Moreover, new TB cases
with isoniazid resistance had lower prevalence of rpoB mutations and
higher prevalence of single inhA mutations, suggesting that they are
more likely to respond to first-line TB therapy even in the presence of
isoniazid resistance-conferring mutations (Domínguez et al., 2016).
Drug resistance mutations occur randomly in TB bacilli. Drug
pressure selects drug resistance strains in individuals receiving inadequate TB treatment and accumulation of further resistance seems to
be a step-wise phenomenon (Izu et al., 2013). New research has demonstrated that katG S315T mutation precede rpoB mutations across all
TB lineages (Cohen et al., 2015; Manson et al., 2017). As previously
treated patients can transmit resistant strains at an early stage (when
the strain is only resistant to isoniazid) or at a later stage (when the
strain is both resistant to isoniazid and rifampicin), this could explain
the higher prevalence of combined rpoB/katG mutations in previously
treated cases among patients with isoniazid resistance. However, if katG
mutations precede rpoB mutations in most rifampicin resistant TB cases,
the lower prevalence of isoniazid resistance in new cases with rpoB
mutations observed in our study suggests that rifampicin-resistant
strains that lose isoniazid resistance-conferring mutations are more
capable to be transmitted to other individuals in the community than
strains with both rifampicin and isoniazid resistance. However, new
studies using whole genome techniques to investigate negative epistatic
interactions between isoniazid and rifampicin mutations are needed to
confirm this hypothesis.

absence of the wild-type probe hybridization indicates a mutations in
the respective region. A positive mutation probe is usually accompanied
by the absence of the corresponding wild-type probe, but both can be
present if the sample contains two or more TB strains because of mixed
culture or contamination (Seifert et al., 2016). For this study, samples
with simultaneous presence of wild-type and mutation probes were
considered as contaminations and excluded. Mutations in the inhA and
katG genes confer low- and high-level isoniazid resistance, respectively,
and inhA mutations affect susceptibility to ethionamide (Unissa et al.,
2016). To facilitate interpretation of the results, in patients with more
than one valid result, only the first sample was included. Patients were
defined as new cases if they have never had TB treatment or had previously received antituberculosis drugs for < 30 days, and as previously
treated cases otherwise. To investigate molecular differences between
new and previously treated cases, we followed a stepwise analysis: first
we described the proportion of patients with rpoB mutations who had
isoniazid resistance-conferring mutations, and vice versa; second we
described the proportion of patients with isoniazid resistance-conferring mutations who had katG mutations (versus having single inhA
mutations); and third, we analysed differences in rpoB, katG and inhA
mutations. The study was approved by the Ethics Committee of the
Rural Development Trust Hospital.
During the study period, 2337 samples were processed with the
MTBDRplus assay, 59 were considered as contaminations and 166
samples were excluded because they were repeated in the same patients
after a first valid report. In the remaining samples, the MTBDRplus
assay showed no mutations in 1700 patients, and rifampicin or isoniazid resistance-conferring mutations in 412 patients (rifampicin and
isoniazid resistance in 171, rifampicin mono-resistance in 74 and isoniazid mono-resistance in 167).
In the 412 patients with isoniazid or rifampicin resistance-conferring mutations, the median age was 38.6 years (interquartile range
30.1–50), 125 (30.3%) were female, 186 (45.2%) were HIV infected,
170 (41.3%) were new cases and 242 (58.7%) were previously treated
cases.
The proportion of new and previously treated cases with mutations
in rpoB, inhA and katG genes are presented in Table 1. Among 245
patients with rpoB mutations, the proportion of new and previously
treated cases with no isoniazid resistance-conferring mutations was
41.2% and 26% (P-value = 0.02; risk ratio [RR] 1.58, 95% confidence
interval [CI] 1.09–2.31; risk difference [RD] 15.2%, 95% CI 18.2–28.6),
respectively. Among 338 patients with isoniazid resistance-conferring
mutations, the proportion of new and previously treated cases with no
rpoB mutations was 71.8% and 33.2% (P-value < 0.0001; RR 2.17,
95% CI 1.73–2.71; RD 38.7%, 95% CI 28.8–48.6), and the proportion
with katG mutations (versus having single inhA mutations) was 66.9%
and 79.1% (P-value = 0.012; RR 1.58, 95% CI 1.11–2.27; RD 12.2%,

Table 1
Proportion of new and previously treated patients with isoniazid or rifampicin resistance-conferring mutations using the GenoType MTBDRplus assay.
Total

New

Previously treated

N (%)

N (%)

N (%)

Patients with rpoB mutations
No inhA or katG mutations
inhA
katG
inhA & katG

74 (30.2)
29 (11.84)
136 (55.51)
6 (2.45)

28 (41.18)
7 (10.29)
31 (45.59)
2 (2.94)

46 (25.99)
22 (12.43)
105 (59.32)
4 (2.26)

Patients with katG or inhA mutations
inhA
katG
inhA & katG
rpoB & inhA
rpoB & katG
rpoB & inhA & katG

59 (17.46)
106 (31.36)
2 (0.59)
29 (8.58)
136 (40.24)
6 (1.78)

40 (28.17)
62 (43.66)
0 (0)
7 (4.93)
31 (21.83)
2 (1.41)

19 (9.69)
44 (22.45)
2 (1.02)
22 (11.22)
105 (53.57)
4 (2.04)

49

Infection, Genetics and Evolution 59 (2018) 48–50

G. Alvarez-Uria, R. Reddy

Table 2
Rifampicin (rpoB) and isoniazid (katG and inhA) resistance-conferring mutation in new and previously treated cases using the MTBDRplus assay.

rpoB
D516V
H526Y
H526D
S531L
Only WTPA
katG
S315T1
S315T2
Only WTPA
inhA
C15T
A16G
T8C
T8A
Only WTPA

Total

New

Previously treated

N (%)

N (%)

N (%)

7 (2.86)
16 (6.53)
11 (4.49)
117 (47.76)
94 (38.37)

1 (1.47)
0 (0)
4 (5.88)
34 (50)
29 (42.65)

6 (3.39)
16 (9.04)
7 (3.95)
83 (46.89)
65 (36.72)

221 (88.4)
8 (3.2)
21 (8.4)

84 (88.42)
4 (4.21)
7 (7.37)

137 (88.39)
4 (2.58)
14 (9.03)

80 (83.33)
0 (0)
2 (2.08)
0 (0)
14 (14.58)

39 (79.59)
0 (0)
2 (4.08)
0 (0)
8 (16.33)

41 (87.23)
0 (0)
0 (0)
0 (0)
6 (12.77)

P-value

0.05

0.692

0.458

WTPA, wild type probe absence.

Competing interests

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ijtld.15.0221.
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prespecified elements to compare drug resistance in treatment-naïve and experienced
tuberculosis cases. PLoS Comput. Biol. 9, e1002973. http://dx.doi.org/10.1371/
journal.pcbi.1002973.
Manson, A.L., Cohen, K.A., Abeel, T., Desjardins, C.A., Armstrong, D.T., Barry, C.E.,
Brand, J., TBResist Global Genome Consortium, Chapman, S.B., Cho, S.-N.,
Gabrielian, A., Gomez, J., Jodals, A.M., Joloba, M., Jureen, P., Lee, J.S., Malinga, L.,
Maiga, M., Nordenberg, D., Noroc, E., Romancenco, E., Salazar, A., Ssengooba, W.,
Velayati, A.A., Winglee, K., Zalutskaya, A., Via, L.E., Cassell, G.H., Dorman, S.E.,
Ellner, J., Farnia, P., Galagan, J.E., Rosenthal, A., Crudu, V., Homorodean, D., Hsueh,
P.-R., Narayanan, S., Pym, A.S., Skrahina, A., Swaminathan, S., Van der Walt, M.,
Alland, D., Bishai, W.R., Cohen, T., Hoffner, S., Birren, B.W., Earl, A.M., 2017.
Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides
insights into the emergence and spread of multidrug resistance. Nat. Genet. 49,
395–402. http://dx.doi.org/10.1038/ng.3767.
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The authors declare that they have no competing interests.
Acknowledgements
We would like the thank Anil Kumar Krolagondi and Francis Isac
Kagola for their help.
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