Background
Tuberculosis (TB), a leading cause of death globally, with increasing rates of drug resistance is of concern. Timely diagnosis and treatment are the key elements of the effort to combat TB and reduce transmission by rendering infectious cases non-infectious.
Only 5% of the World Health Organization (WHO) estimated global multidrug resistant TB (MDRTB) case load of 440,000 is currently detected [
1,
2]. Detection by conventional drug susceptibility testing (DST) requires considerable resources of infrastructure and trained personnel. The WHO recommends the use of MGIT960 and line probe assays (LPAs) towards quicker MDR detection [
3] since phenotypic DST takes 4 to 6 weeks from the receipt of clinical samples.
Commercial and in-house systems for the rapid detection of rifampicin (RIF) resistant
Mycobacterium tuberculosis (
M. tb) take 5-8 hrs from the time of sample collection [
4,
5]. The GenoType MTBDR
plus is superior in that it detects mutations associated with both rifampicin and isoniazid resistance unlike the INNO-LiPA Rif.TB (Innogenetics, Belgium) which detects mutations only to the former. Unlike RIF resistance, in which 95% of isolates have mutations within an 81-bp region of the
rpoB gene encoding the RNA polymerase β subunit [
6], isoniazid (INH) resistance has been associated with mutations in several genes [
7,
8]. Furthermore, since the technique is polymerase chain reaction (PCR) based, it allows detection of low levels of resistant bacteria amidst a predominantly susceptible population, providing a more accurate representation of the susceptibility of the infecting bacteria [
9,
10]. Different mutations lead to varying degrees of resistance and influence bacterial ability to multiply [
11]. Studies have reported the potential use of sophisticated techniques such as sequencing to detect drug resistance mutations which can serve as epidemiological markers, since the relative frequency of alleles associated with resistance varies geographically [
12‐
14]. The application of the GenoType MTBDR
plus assay has been reported in other high burden settings such as Russia, South Africa and China, but has not yet been reported from our setting [
10,
15,
16].
Despite guidelines that advocate DST for patients failing any treatment regimen [
17], it is only performed in 0.5% of notified previously treated TB cases [
1]. The endemic setting of Mumbai with reports of increasing levels of MDRTB [
18‐
21] and a high case load would benefit from the introduction of molecular methods to detect resistance, overcoming the drawbacks of culture methods.
This study was therefore undertaken to evaluate the MTBDRplus for detection of MDR, defined as resistance to at least INH and RIF, in pulmonary TB (PTB) patients in Mumbai. The dual objectives thus encompassed determination of the nature and frequency of mutations associated with resistance and correlations, if any, between the type of mutation and treatment outcome of the patient. Additionally, the assay enabled the detection of heteroresistance to both INH and RIF.
Discussion
To the best of our knowledge this is the first study from this setting to investigate genotypic profiles in the rpoB, katG and inhA regions associated with DR using the MTBDRplus assay. Techniques which detect MDR mutations in new cases at onset or during therapy would enable rapid identification of MDR and facilitate the modification of regimens with improvement to programme practices.
Overall, the concordance between the methods for INH and RIF ranged from 86-97% and 94-99% respectively. Fifteen discordant results were obtained for INH resistance, of which 5 were phenotypically resistant but genotypically sensitive, possibly because the mutations lie outside the regions covered by the probes. Ten discordant results were Buddemeyer sensitive but MTBDR
plus resistant, wherein 5 were
inhA mutants, known to be associated with low level resistance [
27]. Of the remainder, 4 were onset isolates with
katG mutations, of which 2 showed a mixed profile. This may explain the phenotypic sensitivity despite the
katG mutations being associated with high level resistance [
27]. The remainder 2 profiles showed the
katG S315T1 mutation but were INH sensitive by the Buddemeyer. For one of these isolates, the interpretation of the drug susceptibility could have altered from sensitivity to resistance on extended incubation. For the second isolate, there was no indication of resistance and the discordance could reflect a technical anomaly in the phenotypic assay, or an "adaptation" by the strain which has prevented phenotypic expression of the
katG mutation.
Of 5 isolates that were MTBDR
plus RIF resistant but Buddemeyer sensitive, 4 lacked the WT8 band, including 3 which showed the MUT3 (S531L). Though associated with high level resistance, its detection at onset could imply a smaller proportion of resistant bacteria. Low level resistance which may remain undetected despite conventional DST has been previously reported [
28,
29].
Heteroresistance, reflecting the slow evolution of bacteria from a sensitive to resistant profile, is not uncommon in
M. tb [
30]. However our study is probably the first to report relatively high levels of heteroresistance in all 3 genes in an endemic setting. Two explanations are offered for this finding. Firstly, heteroresistance could have arisen due to transmission of both susceptible and resistant bacterial populations from drug resistant patients to previously untreated cases. An endemic setting like Mumbai would be prone to the prevalence of MDR strains, and hence new cases, even at onset, are likely to harbour resistant bacteria at proportions genotypically detectable but phenotypically undetectable, as suggested by the detection of mixed profiles in our onset isolates [
31]. Studies on heteroresistance have shown that phenotypic DST results corresponded to the mutated, i.e. resistant, organism [
31]. Secondly, the presence of exclusively sensitive bacteria at onset which gradually develop resistance during therapy, with incomplete elimination of the sensitive population by fifth month, would result in phenotypic resistance but with both forms remaining detectable genotypically. This possibility has been explained through mathematical modelling of the scenario in which MDR bacilli arise from a completely sensitive original infection [
32]. The second scenario may explain the higher occurrence of mixed patterns among our fifth month isolates in comparison to the onset isolates. The probability of any bias towards detection of heteroresistance has been reduced by excluding patients with various other likely contributory factors such as prior treatment and defaulting. DNA fingerprinting techniques such as Mycobacterial Interspersed Repetitive Units Variable Number Tandem repeats (MIRU VNTR) would help gauge whether multiple infections have contributed to the heteroresistance detected.
The detection of heteroresistance seems to support our finding of an association between a clean resistant profile for the katG and a poor outcome, since the presence of exclusively resistant strains is more likely to result in non responsiveness to treatment. Identification of hetereoresistance can be used to probe outcomes of smear examination based "cure" since microscopy may not be sensitive enough to detect a small focus of bacteria which have evolved from sensitivity to resistance during therapy.
INH resistance can develop through mutations in the
inhA open reading frame (ORF) (0-5%) or the promoter (8-20%). Between 40 to 95% of INH resistant isolates have mutations in
katG, 75-90% of which are in codon 315, with 10-25% in other loci [
33]. The frequency of the
katG S315T substitution in
M. tb strains varies globally in relation to the prevalence of TB: from 26-30% in regions with intermediate/low prevalence [
34] upto 91% of strains in Russia [
35]. Most reports reveal higher levels of
katG mutations in comparison to the
inhA mutations, viz. 73% and 22% [
36], 46% and 27% [
37], 64% and 42% [
10] respectively. However we detected nearly equivalent levels of
katG - 55% at onset and fifth month; and
inhA - 58% onset and 55% fifth month in concurrence with Lacoma et al. [
27]. We found dual mutations in
katG and
inhA in 12.5% and 9% of onset and fifth month isolates, comparable to the 3-13% reported elsewhere [
2,
15,
38].
Mutations in
katG 315 may be favoured because they decrease INH activation without abolishing catalase-peroxidase activity, reflecting its low fitness cost [
11,
33,
39‐
41]. The detection of equal proportions of
katG and
inhA mutations indicate that in our setting,
inhA may also have a low fitness cost. This may be a consequence of the extended occurrence of MDR allowing for acquisition of compensatory mutations such as in the
ahpC [
42]. Despite their equivalent levels,
katG but not
inhA mutations, were associated with treatment failure.
Our data identified associations between
katG 315 mutations and ethambutol resistance as well as poor outcome. It has been reported that the
iniBAC promoter is induced by cell wall biosynthesis inhibitors such as isoniazid and ethambutol [
43]. Overexpression of the
iniA gene confers a tolerance-like phenotype to INH and ETB. Furthermore, the
iniA gene product is an essential component of an MDR-like pump [
44]. Resistance to INH through
katG mutations might influence response to other drugs, allowing development of resistance to ethambutol [
45] and streptomycin [
46], leading to poor treatment response.
Of the 86 RIF resistant isolates, 70 showed a mutation in the 530-533 region of the
rpoB. However only 23 (27%) showed the specific S531L mutation as compared to other studies reporting 46-79% of strains with this mutation [
10,
27,
36,
37]. The low fitness cost of
rpoB S531L [
47] may account for its high frequency in these regions (i.e. South Africa, France, Spain) though its occurrence has also been reported to be as low as 30-31% in India and Hungary [
12,
48]. The lower proportion of the S531L compared to the D516V mutation indicates that the latter is also not associated with a fitness cost, at least in our setting.
Our study failed to find any association between a particular mutation and the occurrence of monoresistance or MDR. However, other studies have reported a significantly higher level of
katG and S531L mutations in MDR isolates compared to INH or RIF monoresistant isolates respectively [
10,
38]. It is likely that this difference is due to the relatively low occurrence of S531L and the equivalent proportions of
katG and
inhA mutations in our cohort.
The value of RIF as a surrogate MDR marker has been documented [
49] and further corroborated in our study. Despite their advantages, genotypic methods do not always identify phenotypically resistant strains [
38], highlighting the limitations of molecular testing and need for supplementation with culture or additional probes. Additionally resistance can be inferred from the absence of a wild type signal alone, without confirmation by a mutant probe signal (in 47% of our isolates) and may be due to a mutation in a region not associated with resistance [
29]. Such susceptible isolates would be called resistant leading to the unnecessary removal of RIF and/or INH from therapy. Moreover since a proportion of INH resistance, particularly in monoresistant isolates, could be due to resistance determinants other than
katG S315T and
inhA C15T, these isolates would also be indicated as susceptible. This highlights the need for the interpretation of genotypic data in conjunction with patient clinical status and the determination of mutations specific to certain geographical locales.
Competing interests
No author has a financial or other conflict of interest related to this work. None of the authors have an association that poses any conflict of interest. The funders had no part in the decision to publish the manuscript.
Authors' contributions
MPT and DTBD performed the assays, performed the analysis and drafted the manuscript. NFM provided inputs into data analysis and interpretation and editing of the manuscript. All authors read and approved the final manuscript.