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Introduction: First Line-Line Probe Assay (FLLPA), a molecular test helps in diagnosing resistance of two First line ATT drugs-Isoniazid and Rifampicin. The genes causing Isoniazid resistance are enoyl acyl carrier protein (acp), reductase (Inh A), catalase–peroxidase (Kat G), alkyl hydroperoxide reductase (ahpC), oxidative stress regulator (oxyR), β-ketocyl acyl carrier protein synthase (KasA). FLLPA will be identifying only Inh A and Kat G genes for Isoniazid.
Aims and Objectives: The study endeavor to understand the gene patterns determining Isoniazid resistance due to deletion of Kat G and Inh A.
Methods: The study was an observational study, which took place in Intermediate Reference laboratory at Hyderabad for duration of three months i.e. May 2018 to July 2018. All the samples processed in First line -Line Probe Assay (FLLPA) found to be resistance due to either Kat G or Inh A gene were recorded, and the gene patterns were studied by applying appropriate statistics.
Results: The total tests performed which detected Isoniazid resistance are 185 of which 123 (66.5%) were resistance due to Kat G gene and 55 (29.7%) due to Inh A. Seven samples (3.8%) showed Resistance to both. There were 3 cases in which the resistance due to Kat G, all loci i.e. Kat G Locus Control, Wild type I and Kat G Mut 1 & 2 were absent. In one case of Isoniazid resistance due to Inh A there were loci, wild type 2 and Mut2 were present while rest of all loci were absent. There were few rare interpretations which implied Isoniazid Resistance.
Conclusion: The study showed most of the Isoniazid resistance was due to deletion of Kat G gene indicating high level resistance.
Keywords: Isoniazid, Monoresistance, FL-LPA, Kat G, Inh A
INTRODUCTION
Tuberculosis, caused by Mycobacterium tuberculosis, poses many challenges to community in terms of treatment, adherence and adverse drug reactions. Drug resistance is a serious problem itself posing many difficulties for physicians to tailor the regimen. There is immense need for study of wild types of genes to the growing needs of management of drug resistant TB. There were many changes in programmatic management of drug resistant TB.
Line Probe Assay and CBNAAT (Cartridge based Nucleic Acid Amplification Test) are two molecular technologies which diagnose TB and also detect resistance. CBNAAT detects resistance for Rifampicin whereas Line Probe Assay helps in diagnosing resistance for two drugs i.e. Isoniazidand Rifampicin. These two molecular tests have revolutionalized the diagnosis of drug resistant TB.
Isoniazid monoresistance accounts 16 % of all notified TB cases. There was no particular treatment in the previous guidelines. The genes causing Isoniazid resistance are enoyl acyl carrier Protein(acp), reductase (Inh A), catalase–peroxidase (Kat G), alkyl hydroperoxide reductase (ahpC), oxidative stress regulator (oxy R), β-Ketocylacyl carrier protein synthase (Kas A). Of all the above-mentioned genes FLLPA will be identifying only for Inh A and Kat G genes for Isoniazid.
First Line-Line Probe Assay which is a molecular technology which detects resistance of Isoniazid and Rifampicin. The use of this genotypic test has hastened to know the status of resistance of Isoniazid and Rifampicin, helping the treating physician to tailor the treatment regimen.
Isoniazid monoresistance posed another challenge in management of DRTB. There were many regimens followed in different countries. There was a clear mention of management in Isoniazid Monoresistance in Technical Operation Guidelines in 2016 under Revised National Tuberculosis Control Program(RNTCP) in India
The most common pattern will guide the management of Isoniazid Monoresistance Drug management, especially determining the use of other first Line drugs.
OBJECTIVE OF STUDY
To study the gene patterns in determining Isoniazid resistance by FLLPA.
METHODS
The study was an observational study which took place in Intermediate Reference Laboratory (IRL), Hyderabad, Telangana, India for period of 3 months (May 2018 to July 2018).
All the consecutive samples which were processed in FL-LPA and found to be resistance to Isoniazid only due to either Kat G or Inh A gene were noted, and the gene patterns were studied. All the data was recorded in Microsoft Excel and appropriate statistics applied.
Inclusion criteria
1) All the consecutive Pulmonary samples processed with FL-LPA and found resistance to Isoniazid.
2) All the samples collected were irrespective of HIV status and age of the patients.
Exclusion criteria
1) All extra pulmonary samples were not included in study.
2) All samples which are smear negative concentration and which are processed in Liquid culture and then subjected to FLLPA.
RESULTS
The total tests performed which detected Isoniazid resistance were 185 during the period May 2018 to July 2018. Of which 123 (66.5%) were resistance due to Kat G gene and 55 (29.7%) due to Inh A. Seven Tests (3.8%) showed Resistance to both (Table 1).
There were four cases in which the resistance was due to Kat G, all loci i.e. Kat G locus control, Wild type I and Kat G Mut 1 & 2 were absent which prompted resistance to Isoniazid.
There were 119 cases were mutation was expressed in Mut 1 and two in Mut 2 which indicated that high dose Isoniazid cannot be used. Regimen has to be designed in such a way that Isoniazid shall not be included.
In 106 cases of Isoniazid resistance to Kat G, the expression was - locus control was present, Wild Types were absent, Mut 1 present and Mut 2 present. This clearly indicated that Isoniazid cannot be used even at higher dose and regimen has to be tailored accordingly.
There was one case where in resistance to Isoniazid inferred as absence in locus control and mutation but presence of wild type1 was expressed.
Expression of presence of locus control and all other (wild type, Mut1 & Mut2) were absent in five cases.
There was expression of both Mut 1 and Mut 2 in one case.
All the above instances discouraged the use of Isoniazid even at higher doses. There were 55 cases of Isoniazid resistance due to Inh A (Table 2).
Locus control was present in all cases. Wild type I was absent in 40 cases and in 2 cases wild Type II were not seen. Mut were expressed in 50 cases, Mut II and Mut 3a in only one case. Expression of Mut 3b was not seen in all cases.
In one case, expression of locus control, wild type I, wild type II, Mut I were seen, and rest were not seen.
In one case, Inh A, wild type II, and Mut 2 are seen and rest were absent. In all above cases, where resistance due to Isoniazid was due to Inh A gene, high dose of Isoniazid could help.
There were 7 cases where there was resistance expression both due Inh A and Kat G gene. The expressed gene patterns were as follows (Table 3 and Figure 1).
DISCUSSION
Isoniazid was first synthesized in Prague in 1912 and is critically important first line drug. The prodrug isoniazid inhibits mycolic acid synthesis and mycobacterial cell wall formation [1]. Isoniazid monoresistance is estimated to be about 16-22% in National Drug Resistance survey 2016. Resistance is most frequently caused by mutations in genes coding for a bacterial catalase-peroxidase enzyme or a enoyl-acyl carrier protein reductase, although multiple other genes are implicated in isoniazid resistance [2].
First Line LPA is molecular tests which gives information on resistance of both Isoniazid and Rifampicin. Genes that cause Isoniazid resistance in FLLPA are Kat G and Inh A. Kat G resistance indicates high level resistance and Inh A indicates low level resistance to Isoniazid. Inh A gene also gives information whether to use Ethionamaide (Eto), a second line drug for TB or not. The samples collected were processed for decontamination and subjected to FLLPA after smear reading showed positive. LPA includes the step and reading is done expression of bands.
Reasons for false-negative LPA may include the following: reagents not equilibrated to room temperature; addition of insufficient reagents, improper mixing of reagents, addition of reagents in incorrect amounts; improper immersion of strips in the reagents during incubation; improper washing of strips and improper sampling, storage, transport or preparation of specimen [3]. Table 4 enables us to interpret the FLLPA result.
In the study we have observed that resistance was more due to Kat G rather than Inh A.
Locus Control for Kat G was absent for four cases where we needed to infer that the particular sample was resistant, which programmatically was considered Isoniazid resistance.
Most of the times in Kat G resistance was mostly due to expression of MUT1.MUT2 expression was noticed very low.
In following instances (Table 5), Isoniazid resistance has to be inferred.
In special cases of InhA resistance, the following was inferred (Table 6).
In one typical case where Isoniazid resistance was due to both Kat G and InhA, where resistance was inferred as follows (Table 7).
Resistance to Isoniazid is prevalent with substantial geographical variation [3].
As observed in many studies conducted by Charan et al. [4] our study too showed that Kat G resistance of Isoniazid was mainly due to absence of Wild type (S315T).
Increasing the dose of Isoniazid in resistance due to Kat G may not be helpful. Hence in all guidelines it was mentioned to remove the drug once resistance is noticed [5].
In many studies conducted in world on Isoniazid resistance the results were as follows (Table 8) and similar results were reported in our study too.
The presence of mutations in Kat G alone or in combination with Inh A 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 Inh A, 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) [10].
CONCLUSION
The above study concludes that most common mutation in Isoniazid monoresistance is Kat G. Among Kat G mutations, absence of Wild type (C15T) and presence of MUT 1(S315T1) is observed more frequently. Ethionamide is also not effective in Inh A mutation of Isoniazid monoresistance. In order to design the regimen, gene patterns are important to study.
CONFLICT OF INTERESTS
None declared.
1. Donnell M (2018) Isoniazid Monoresistance: A precursor to multidrug-resistant tuberculosis? Ann Am Thorac Soc Mar 15: 306-307.
2. Farooqi JQ (2012) Line probe assay for detection of Rifampicin and Isoniazid Resistant Tuberculosis in Pakistan”. J Pak Med Assoc 62: 767‐772.
3. Desikan P (2017) Line probe assay for detection of Mycobacterium tuberculosis complex. Indian J Med Res 145: 70-73.
4. Charan AS, Gupta N, Dixit R (2020) Pattern of InhA and KatG mutations in isoniazid monoresistant Mycobacterium tuberculosis isolates. Lung India 37: 227231.
5. Tiemersma EW, van der Werf MJ, Borgdorff MW, Williams BG, Nagelkerke NJ (2011) Natural history of tuberculosis: Duration and fatality of untreated pulmonary tuberculosis in HIV negative patients: A systematic review. PLoS One 6: e17601.
6. Yao C, Zhu T, Li Y, Zhang L, Zhang B, et al. (2010) Detection of rpoB, katG and inhA gene mutations in Mycobacterium tuberculosis clinical isolates from Chongqing as determined by microarray. Clin Microbiol Infect 16: 1639-1643.
7. Huyen MN, Cobelens FG, Buu TN, Lan NT, Dung NH, et al. (2013) Epidemiology of isoniazid resistance mutations and their effect on tuberculosis treatment outcomes. Antimicrob Agents Chemothe 57: 3620-3627.
8. Alagappan C, Shivekar SS, Brammacharry U, Kapalamurthy CVR, Sakkaravarthy A, et al. (2018) Prevalence of mutations in genes associated with isoniazid resistance in Mycobacterium tuberculosis isolates from re-treated smear-positive pulmonary tuberculosis patients: A meta-analysis. J Glob Antimicrob Resist 14: 253-259.
9. Abraham JN, Mlisana K, Gandhi NR, Mathema B, James CM (2015) High Prevalence of inhA Promoter Mutations among Patients with Drug-Resistant Tuberculosis in KwaZulu-Natal, South Africa. PLOS One 10: e0135003.
10. World Health Organization (2018) WHO treatment guidelines for isoniazid resistant tuberculosis: Supplement to the WHO treatment guidelines for drug resistant tuberculosis. Available online at: https://www.who.int/tb/publications/2018/WHO_guidelines_isoniazid_resistant_TB/en/
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