Pattern of InhA and KatG mutations in isoniazid monoresistant Mycobacterium tuberculosis isolates.

AIMS AND
OBJECTIVES: The aim of the study is to detect the pattern of genetic mutation, i.e., Inh A or Kat G or both (Inh A and kat G) in isoniazid (INH) monoresistant mycobacteria and to correlate with the pattern in multidrug-resistant (MDR) isolates.
MATERIALS AND METHODS: In this study, a quantitative research approach was used. The research design was descriptive observational study. The study was conducted at the Department of Respiratory Medicine, JLN Medical College, Ajmer, Rajasthan, and Intermediate Referral Laboratory, State TB Demonstration Centre, Ajmer. A total of 298 samples found to have resistant strains of Mycobacterium tuberculosis were enrolled with purposive sampling.
RESULTS: The mean age of patients was 40.27 ± 13.82 years. There were 250 (83.9%) males, while 48 (16.1%) were females. One hundred ninety-two (64.4%) were resistant for INH only, while the rest were resistant to both INH as well as rifampicin (MDR-tuberculosis). The most common mutation in INH monoresistance was kat G (125; 65.1%) as compared to inh A (54; 28.1%) and both inh A and kat G (13; 6.7%). Among kat G mutations, the most common gene pattern was the absence of WT (S315T) and the presence of MUT1 (S315T1).
CONCLUSION: Knowledge about mutation patterns of different INH resistant strains is important in the present era where there is a provision of separate regimens for INH monoresistant TB. Since these mutations are very closely related to high- or low-degree resistance to INH, the therapeutic regimens cannot be generalized.

INTRODUCTION

Drug resistance in tuberculosis (TB) is a major public health challenge in developing countries, and the emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis strains has become a major obstacle in the management of TB.[1] Isoniazid (INH) is one of the most potent antimycobacterial agents available for the treatment of TB and has both bactericidal and sterilizing actions. It inhibits mycolic acid biosynthesis of M. tuberculosis. However, the resistance to INH is most common among all first-line anti-TB drugs.[2] As far as monoresistance is concerned, resistance to INH (7.2%) exceeds other first-line anti-TB drugs (6.85% for streptomycin, 1.6% for ethambutol, and 4.6% for rifampicin).[3]

The results from studies conducted at Chennai and Bangalore have found an MDR-TB incidence of 12% in re-treatment cases and around 1%–3% in new cases.[4] According to the global annual surveys conducted by the World Health Organization (WHO), an estimated 490,000 cases of MDR-TB occurred among 10.4 million new active TB cases in 2016.[5] Three countries accounted for almost half of the world's cases of MDR/RR-TB: India (24%), China (13%), and the Russian Federation (10%). Globally, 3.5% of new TB cases and 18% of previously treated cases had MDR/rifampicin-resistant TB.[6]

The genetic background of M. tuberculosis related to INH resistance is very complex. Mutations in several genes, including KatG (catalase-peroxidase coding genes), InhA, AhpC, and KasA, have all been associated with INH resistance. Two molecular mechanisms have been shown to be the main cause of INH resistance, i.e., mutations in KatG and InhA. From 50% to 95% of INH-resistant strains contain mutations in codon 315 (WT1) of the KatG gene. Furthermore, 20%–35% of INH-resistant strains contain mutations in the InhA regulatory region.[78] The inhA gene encodes an enoyl-acyl carrier protein reductase involved in the fatty acid synthesis.[9]

The recognition of INH-resistance patterns and the frequency of katG and inhA mutations in different geographic areas may help to guide decision-making about standardization of treatment regimens or individualized treatment, as in the case of INH monoresistance or in the case of MDR-TB or extensively drug-resistant (XDR)-TB.[10] As mutations in katG, particularly at codon 315, confer high-level INH resistance, even high-dose INH is ineffective for the treatment of M. tuberculosis with this mutation profile. Mutations in inhA, on the other hand, may respond to high doses of INH.[11]

There is no documented study on molecular characterization of INH resistance with reference to monoresistance in the Western part of India. Majority of studies are from out of the country and very few studies are reported from India. Hence, this was of interest to analyze the KatG gene and InhA mutations in INH monoresistant isolates in this part of the country and to compare the pattern with other national and internationally available data.

MATERIALS AND METHODS

In this study, a quantitative research approach was adopted with a descriptive observational study. The study was conducted at Intermediate Referral Laboratory, Ajmer, and Respiratory Medicine Department, JLN Medical College, Ajmer, Rajasthan. Permission to use the data was obtained from the Intermediate Reference Laboratory, Ajmer, and approval was obtained from the institutional ethical committee, JLN Medical College, Ajmer. A total of 298 samples reported to be resistant to INH with or without RMP were enrolled through purposive sampling. The study duration was from August 1, 2017, to October 30, 2018.

The method used to detect the INH resistance was genotype MDR TB plus, version 2, Hain Lifesciences Nehren, Germany (first-line Line Probe Assay), made available under the Revised National Tuberculosis Control Program. The nucleotide sequences of the katG and inhA gene were recorded for each of the samples.

Data analysis was done with the use of statistical software SPSS 17.0 (IBM SPSS Version17.0, Chicago).

RESULTS

A total of 298 consecutive samples found to have either INH monoresistant or MDR strains were included in the study and were analyzed.

Figure 1 depicts that around two-third of the study population, that is, 192 (64.4%) were resistant for INH, while the rest were 106 (35.6) resistant to both inh And rifampicin (MDR-TB).

Figure 1

Total number of isolates registered for the study

The mean age of the patients was 40.27 ± 13.82 years. There were 250 males (83.9%), while only 48 (16.1%) were females. Table 1 shows that 163 (65.2%) of the males and 29 (60.4%) of the females were resistant to INH alone, while the rest were resistant to both INH and RMP. No significant difference was observed for INH resistance or MDR-TB between the two genders. Among INH-resistant patients, 73 (38.0%) were between the age group of 31–45 years and 44 (22.9%) were between the age group of 15–30 years, while in MDR patients, 41 (38.7%) were between the age group of 15–30 years. This difference was found to be statistically significant (P = 0.03).

Table 1

Association of type of resistance to INH or H&R with demographic variables of patients (n=298)

Demographic Variables	H Monoresistant		H & R Resistant (MDR)		P
	n	%	n	%
Sex
Male	163	65.2	87	34.8	0.51NS
Female	29	60.4	19	49.6
Age (in years)
15-30	44	22.9	41	38.7
31-45	73	38.0	35	33.0	0.03*
46-60	61	31.8	25	23.6
>60	14	7.3	5	4.7

NB: *= Significant at P<0.05, NS=Non-significant P>0.05

The most common mutation in INH resistant strains was in katG gene (125, 65.1%) followed by inhA gene (54, 28.1%). In 13 (6.7%) patients, both inhA and katG gene mutations were observed [Figure 2 and Table 2].

Figure 2

Pattern of genes in Isoniazid(H) Momoresistance Tuberculosis Patients (N=192)

Table 2

Pattern of gene mutations in INH Monoresistant mycobacterium TB strains (n=196)

inhA n (%)	katG n (%)	Both n (%)
58 (28.1%)	125 (65.1%)	13 (6.7%)

Comparing the pattern of gene mutations in INH monoresistant strains revealed that of 54 isolates with only inhA gene mutation, WT1 pattern was absent in 48 (88.9%) strains, while MUT1 pattern was present in 37 (68.5%) strains. KatG gene mutation patterns were observed in different sequences. WT was absent in 97.6% of patients, MUT1 was seen in 117 (93.6%) patients, while in only 2 (1.6%) of patients, MUT2 resistance was observed [Table 3].

Table 3

Analysis of patterns of gene mutations of InhA and katG in Isoniazide (H) Monoresistant Tuberculosis Patients (n=179)

Pattern of Gene Mutations	Locus
	WT1(-)		WT2 (-)		MUT1 (+)		MUT2 (+)		MUT3A(+)		MUT3B(+)
	n	%	n	%	n	%	n	%	n	%	n	%
InhAGene (58)	48	82.7	9	15.5	37	63.8	0	0.0	5	8.6	1	1.7
katG Gene (125)	122	97.6	0	0	117	93.6	2	1.6	0	0	0	0

The occurrence of mutation at various loci of katG and InhA genes was also very similar for MDRTB isolates as that of INH monoresistant strains [Table 4].

Table 4

Occurrence of mutations in inhA and katG genes in MDR TB Strains

inhA	n=5
WT1(-)	5 (100%)
WT2(-)	1 (20%)
MUT1(+)	4 (80%)
MUT2(+)	0 (0%)
MUT3A(+)	1 (20%)
MUT3B(+)	0 (0%)
katG	n=96
WT(-)	90 (93.8%)
MUT1(+)	85 (88.5%)
MUT2(+)	1 (1%)

The overall incidence of occurrence of inhA, katG, and combined pattern of mutation did not differ in MDR TB [Table 5], and katG was the most common mutation observed for both the groups.

Table 5

Line probe assay based band pattern analysis of all drug-resistant (H & HR) Mycobacterium tuberculosis strains (n=298)

INH monoresistant n=192	MDR TB n=106	Total n=298	Percentage
inhA 54 (28.1%)	inhA 5 (4.7%)	59	19.7
katG 125 (65.1%)	katG 96 (90.5%)	221	74.1
Both 13 (6.7%)	Both 5 (4.7%)	18	6.0

DISCUSSION

In the present study, of 298 patients, the mean age of the study population was 40.27 ± 13.82 years. One hundred ninety-three (64.7%) of 298 patients were in the age group of 15–45 years. Eighty-six (28.2%) patients followed by 19 (6.4%) patients were in the age group 45–60 years and more than 60 years, respectively. The mean age reported in other studies was 28.43 ± 14.32 years[12] and 26 years.[13] Thus, all these studies revealed similar age distribution among drug-resistant patients and clearly suggest that drug-resistant TB affects younger and economically productive age groups.

In the present study, most of the patients were male 250 (83.9%), while 48 (16.1%) were female. The male-to-female ratio was 5:1. Similar gender distribution was reported by Vilegas et al. (2016), i.e., 82.1% were male and 17.9% were female.[14] Surkova et al., Belarus, also reported similar gender distribution (934 patients; 660 were males [70.67 ± 1.5%] and 274 were females [29.33 ± 1.5%]) (P < 0.001).[15]

Of 298 drug-resistant isolates, 191 (64.4%) were INH monoresistant, while 107 (35.6%) isolates were resistant to both INH and rifampicin. The INH monoresistance group comprised 163 (84.89%) males and 29 (15.1%) females, while in Multi drug resistance patients, there were 87 (82.07%) males and 19 (17.2%) females.

In the present study, we observed that the most common mutation in INH monoresistant patients was in katG gene (125, 65.1%), followed by inhA gene which was observed in 54 (28.1%) patients, whereas in 13 (6.7%) patients, both inhA and katG gene mutations were observed. Almost similar results were reported by Yao et al. They observed that of 50 INH monoresistant patients, 41 (82%) had KatG mutations and 9 (18%) had inhA mutations.[16] Huyen et al. also reported that 75.3% had mutations in katG and 28.2% had mutations in the inhA promoter region.[17] A study by Kigozi et al. similarly observed that 80% had katG mutations and 6% had inhA mutations.[18] Tavakkoli and Nazemi also reported that in 17.24% and 82.76% of the strains, inhA genes and katG genes, respectively, were responsible for INH resistance.[19] Niehaus et al. found that 33.1% of 924 isolates had an inhA mutation with or without a katG mutation, representing 30.3% of those with MDR-TB, 47.2% of those with pre-XDR-TB, and 82.8% of those with XDR-TB.[20] Alagappan et al.investigated INH resistance mutations in M. tuberculosis in codon katG and in the promoter region of the inhA gene. One thousand eight hundred and twenty-one (11.8%) of 15,438 INH-resistant strains had detectable mutations: 71.0% in katG315 and 29.0% in the inhA promoter region.[21] Isakova et al. showed that 91.2% of strains were with katG gene mutations, whereas 7% of specimens had inhA gene and 2 more specimens (1.8%) had ahpC gene mutations.[22] Jagielski et al. also observed that 85.2% of MDR patients had katG mutations. About 3.7% of the patients of 54 had single inhA mutations, and the rest of the patients, i.e., 11.1%, had both the mutations.[23] Comparative results are depicted in the following Table 6.

Table 6

Comparative results of mutation patterns reported from different parts of world

Study/authors	Country	Number of patients (n)	InhA mutation	KatG mutation	Both
C. Alagappan et al. (2018)	India	1821	528 (29)	1297 (71)	-
J. Isakova et al. (2018)	Russia	114	8 (7)	104 (91.2)	-
Z. Tavakkoli, A. Nazem et al. (2018)	Brazil	116	20 (17.24)	96 (82.6)	-
V. R. Bollela et al. (2016)	Brazil	22	9 (40.9)	12 (54.5)	1 (4.5)
	Mozambique	38	4 (10.5)	32 (84.2)	2 (4.2)
Abraham J. Nieshaus et al. (2015)	South Africa	603	99 (16.41)	435 (72.13)	69 (11.44)
T. Jagieiski et al. (2015)	Poland	54	2 (3.7)	46 (88.2)	6 (11.1)
N. Shubladze et al. (2013)	Georgia	678	143 (22.6)	535 (84.3)	-
T. Luo et al. (2009)	China	242	24 (9.9)	216 (89.3)	02 (0.8)
Elis R Dalla Costa et al. (2009)	South America	224	43 (20)	181 (80)	-
Present study (2019)	India	298	59 (19.7)	221 (74.1)	18 (6)

Patterns observed in the present study are almost similar to that reported by others

It has been endorsed by the WHO that treating INH-resistance strains with standard anti-TB regimens for new patients resulted in 11% treatment failure and 10% relapses as compared to 2% and 5%, respectively, for drug-susceptible TB. Acquired drug resistance was also higher (8%) as compared to 1% in drug-susceptible TB. Acquisition of MDR-TB may thus be a serious consequence of the inadequate treatment of INH-resistant TB.

Furthermore, mutations in the katG or inhA promoter area are relevant to the clinicians.[24] The presence of mutations in katG alone or in combination with inhA signifies a high degree of resistance to INH. The addition of even high doses of INH for these patients is unlikely to increase the effectiveness of a regime. A mutation limited only to inhA, on the contrary, is usually associated with a low degree of INH resistance, and these individuals are likely to be benefitted with high doses of INH (10–15 mg/kg/day).[3]

In addition, mutations in katG genes have been most frequently associated with rpoB gene mutations, making katG mutation a better predictor of MDR-TB.[25] Our study also supports this hypothesis; katG mutation was observed in 96 isolates of 106 isolates (90.5%). Therefore, a knowledge about these gene mutations among treating physicians and the value of reporting these mutations by the laboratories cannot be overemphasized.

Our study had limitations in terms of differences in gene mutation patterns among recurrent TB patients and those without any history of anti-TB treatment in the past, as these data were not available for all those patients registered for the study.

CONCLUSION

The study concludes that the most common mutation in INH monoresistance is katG 125 (65.1%) as compare to inhA 54 (28.1%) and both inhA and katG 13 (6.7%). Among katG mutations, the most common gene pattern is the absence of WT (S315T) and the presence of MUT1 (S315T1), whereas among inhA mutations, the most common gene pattern is the absence of WT1 (C15T) and the presence of MUT1. For MDR-TB patients, the most common gene pattern is the absence of WT1 (C15T) and the presence of MUT1 for inhA, while it was the absence of WT (S315T) and the presence of MUT1 (S315T1) for katG. Our findings are very similar to other reports from India as well as from other countries. katG also happened to be the most common gene mutant associated with MDR-TB strains.

Since these mutations are very closely related to high- or low-degree resistance to INH and as the therapeutic regimen differs for the two, it is important to know about the gene pattern in each of these patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.