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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 3  |  Issue : 3  |  Page : 182-188

Detection of genetic mutations in inh A and kat G for isoniazid and its association with rifampicin resistance in tuberculosis confirmed by line probe assay: Its rationality for isoniazid prophylaxis empirical or drug susceptibility testing guided


State TB Demonstration Cum Training Centre, Intermediate Reference Laboratory, Kolkata, West Bengal, India

Date of Submission24-Jun-2019
Date of Decision24-Jul-2019
Date of Acceptance28-Jul-2019
Date of Web Publication10-Sep-2019

Correspondence Address:
Dr. Prasanta Kumar Das
State TB Demonstration Cum Training Centre, Intermediate Reference Laboratory, Kolkata - 700 010, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_89_19

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  Abstract 


Background: The preventive treatment of tuberculosis (TB) is practiced worldwide to execute effective control on its spread. The need for a streamlined definitely structured guideline is realized worldwide. Although there are several guidelines, proper rationale to the use of antibiotics as an effective prophylaxis is yet an area to be discussed. The practice of using an anti-tubercular drug without seeing the resistance pattern and its rationality as a prophylaxis is at the question. Involvement of inhA and kat G in conferring isoniazid (INH) resistance among the rifampicin (RMP) resistant as well as sensitive cases were observed and compared to find out any correlation between resistance of INH and RMP. Moreover, the treatment of drug-resistant TB with the second line of anti-tubercular drugs has been a difficult affair. The revised national TB program advocates INH prophylaxis to the pediatric population who are at an augmented risk of getting the infection from close contacts, without knowing the resistant pattern of the strain of contacts. The study projects the inhA and kat G status among the RMP-resistant adult and pediatric cases. The genetic patterns of INH resistance and their different mutations among isolates of the cases obtained from the adult and pediatric age group of RMP resistance are taken under the study. The point and periodic prevalence is worked out and considered in terms of evaluating the prophylaxis among the probable contacts of the source cases. The guidelines of managing of contacts of multidrug-resistant (MDR) TB and extensively drug-resistant TB patients have little scientific evidence to support and there is lack of national guidelines owing to the discrepancies as is strongly felt.[1] Pediatric contact groups are likely to have latent TB infection (LTBI) that might follow an active TB disease development. Contacts of MDR-TB are considered as LTBI cases. These cases are subjected to preventive therapy.[2] Most of the public health program follows the WHO guideline advocating the use of INH as a prophylaxis irrespective of the resistant status of the source strain. Various national programs advocated administering of a conventional drug as a preventive therapy without actually knowing the susceptibility pattern of the infecting strain of the source case.[3] The selection of a preventive regimen with single or multiple drugs ideally to be based on considering the availability and the bactericidal activity of the primarily infecting strain. Drug-resistant TB among the new and retreatment cases was about 5% in accordance to the surveillance done.[4] The overall proportion of MDR-TB, defined as TB resistant to at least INH and RMP, with or without resistance to other first-line drugs, was 5.3%, ranging from 0% to 35% of reported TB cases.[4] The percentage among the retreatment cases showed a steady rise over the years. The retreatment cases contributed about 20% of MDR-TB among the high TB burden nation.[4] In 2009, only 30,000 (7%) of the 440,000 estimated MDR-TB cases globally were notified, and of them, only 11,000 (3%) were put on treatment known to be consistent with international guidelines.[5] A systematic analysis of the nature of the resistance conferred toward INH may contribute in understanding the common as well as rare genetic alterations. INH resistance is classified as high and low-level resistance based on the minimum inhibitory concentrations. It has been found that the high- and low-level resistance toward INH is associated with the kat G and inh A genes, respectively. Treatment protocols are availed in accordance. The INH resistance is determined in all tubercular cases adult and pediatric irrespective of RMP resistance status, it is found that 92.7% are INH resistant by kat G among RMP-resistant cases. Whereas 17.8% of INH resistance by katG are even among RMP sensitive cases. This relates to a community threat even more after the implication of INH prophylaxis as a part of national program supported by the WHO, and a challenging threat of rising trend of INH resistance is apprehended. Study Design: The study was undertaken at the Intermediate reference laboratory under state TB demonstration cum training center Kolkata, India under revised national TB control program, from April 2012 to December 2016. The study was done among the 442 RMP-resistant isolates recovered from retreatment cases in the year 2012 as well as 627 from newly diagnosed RMP sensitive cases from 2013 to 2016 the study involves a probe into the point and periodic prevalence of INH among the RMP resistant as well as sensitive cases, the most common and rare patterns of genetic mutation that has led to the INH resistance due to inh A as well as kat G among the RMP resistant and sensitive cases. The common and rare codons along with their change in amino acid sequences involved in conferring the resistance were looked for. Methods: The detection of the patterns of genetic mutation is done by the Genotype MTBDR plus V2 kits.(Hains Life Sciences) based on DNA strip technology (Line Probe Assay).[6] Results: Of 442 cases 410 cases showed INH resistance. Out of which exclusive involvement with inhA was seen in 32 cases. Nineteen cases showed involvement of both inh A and kat G. resistance 359 cases accounting to 87.5% showed exclusive involvement of kat G thereby conferring high degree of resistance. The higher percentage of kat G-induced INH resistance among the RMP resistant strains depicts the spread of a higher degree of INH resistance. The question remains how effective would be the INH prophylaxis in pediatric age group in this scenario. The analysis has shown that only around 14.93% of cases reflected exclusive mutations at around 15 and 16 regions of the inhA gene. The presence of both the Wild and mutant gene segments simultaneously (WT and MUT3) accounted to a percentage of 60.3% among the inh A-dependent INH resistance.

Keywords: Contact cases, intermediate reference laboratory Kolkata, isoniazid prophylaxis, Isoniazid resistance determining region mutation, katG inhA gene, Mycobacterium tuberculosis complex insert (key words) retreatment preventive treatment, state tuberculosis demonstration, and training center, tuberculosis labs


How to cite this article:
Das PK, Ganguly SB, Mandal B. Detection of genetic mutations in inh A and kat G for isoniazid and its association with rifampicin resistance in tuberculosis confirmed by line probe assay: Its rationality for isoniazid prophylaxis empirical or drug susceptibility testing guided. Biomed Biotechnol Res J 2019;3:182-8

How to cite this URL:
Das PK, Ganguly SB, Mandal B. Detection of genetic mutations in inh A and kat G for isoniazid and its association with rifampicin resistance in tuberculosis confirmed by line probe assay: Its rationality for isoniazid prophylaxis empirical or drug susceptibility testing guided. Biomed Biotechnol Res J [serial online] 2019 [cited 2019 Nov 20];3:182-8. Available from: http://www.bmbtrj.org/text.asp?2019/3/3/182/266570




  Introduction Top


Tuberculosis (TB) still remains the greatest killer disease of all times. The drug resistance has worsened the situation increasing the resistance towards the first line of drugs.

As evident the scenario at district in India for drug-resistant TB among the new and retreatment cases was about 5% in accordance to the surveillance done in the year 2000 and 2001.

In 1994, in 35 countries, the first WHO-IUATLD anti TB drug resistance surveillance was carried out. The study showed primary and acquired multidrug resistance as 1.4% and 13%, respectively. An estimate of 4,89,139 cases of multidrug-resistant (MDR) TB emerged in 2006.[7]

In July 2008, the US Centers for Disease Control and Prevention during the MDR-TB outbreak in Micronesia, it was found out of the 232 contacts to 15 developed the TB. On evaluation, it was found that 66.7% of these patients carried the resistant strains. The preventive regimen with a resistant drug thus raises a fundamental question over the control of the disease.[2]

The percentage among the retreatment cases showed a steady rise over the years. The retreatment cases contributed about 15%–20% among the high Tb burden nations.[8] About 3% of the MDR cases.

A systematic analysis of the nature of the resistance conferred may contribute in understanding the epidemiology of transmission of strains among the population. The study involves a probe into the most common patterns of genetic mutations in inh A that has led to the isoniazid (INH) resistance among the retreatment cases.

The majority among the inh A dependant INH mutations were seen to be occurring at the 15th codon of the inh A promoter region. The lower percentage of inh A-dependent mutations with respect to the evaluated studies may prompt in a pear pressure for prolonged retreatment regimen might have converting several of low dose drug-resistant strains to high dose drug-resistant strains.

Moreover, in high burden country with a high prevalence of the resistant strain, the situation aggravates using a resistant drugs for a prophylaxis. If the spread of the primary infection from the source case is by a strain resistant to, using that drug may be a futile exercise. Most of the countries have been practicing INH prophylaxis for children, contact, and Latent tubercular infective cases.

The study explores the percentage prevalence of the INH resistance among the rifampicin (RMP)-resistant source cases whose contacts are empirically subjected to INH.


  Methods Top


The study involve retrospective multidimensional analysis of the Line probe Assay data of the samples coming to intermediate Reference Laboratory, Kolkata from April 2012 to June 2014. The proposal was reviewed by Research and Ethical committee of Intermediate Reference Laboratory, Kolkata, India. the Ethics committee Approval number STDC/IRL – FL LPA 011/ 2012 dated 5th April 2012. Reversed hybridization of the oligonucleotide probes to the designated gene segments of the rpo B gene was done for 442 isolates by. Line probe assay (LPA) was done using the MTBDR Genotype Version 2. Reproducibility of LPA results was very high with 98.1% concordance of results between the two laboratories.[6] The band pattern can be seen by the available Line probe assay cellulose nitrate filter strip and detected accordingly [Figure 1].
Figure 1: Isoniazid resistance region of the inhA gene: inhA WT 1-2: inhA wild type probes. The numbers specify the position of the amino acids (codons listed in the table). The codons for which mutation probes were designed are highlighted[6]

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Smear-positive samples were subjected to the LPA directly after subsequent extraction of the genome and multiplex PCR of the specific gene segments using biotinylated primers.[9] The utility studies showed testing for INH by LPA accorded to a sensitivity of 92.7% and specificity of 98.33%.[10]

The extraction of the bacterial genome was done using Lysis buffer. The multiplex PCR cycle involved 15 min at 95°C (1 cycle) for denaturation, 30 s 95°C and 2 min 65°C (20 cycles) for anealation. 25 s 95°C, 40 s 50°C, 40 s 70°C (30 cycles) for amplification and 8 min 70°C (1 cycle) for the extension.[6]

The targeted genetic segment was the inh A promoter region. Codon 8, 15, and 16 was looked for.

Among majority of cases considering all possible combinations, it was found that combination exhibiting the Presence of MUT1 and absence of Wild Type 1 (WT1) contributed to the most of the inh A dependant low-level resistance conferred (about 67.3%) involving the codon 15 and 16 of the promoter region of inh A gene.


  Results, Interpretation and Discussion Top


A total of 442 RMP-resistant cases and 627 RMP sensitive were studied out of which 52 Mycobacterium TB complex strains showed resistance toward INH due to the involvement of inh A exclusively for RMP-resistant cases, and only 7 cases of inhA involvement conferring INH resistance in RMP sensitive cases.

Genetic pattern analysis of these isolates was done by reversed hybridization technology using oligonucleotide probes covering the inh A promoter region covering codon 8–16 and katG region covering at the 315 codon.

Inh A involvement

Among the resistance only due to the absence of WT band (s) with no positive Mutation band absence of WT1, WT2 contributed to 15% and 3.8% of the cases.

This involves the point mutations, sequencing of the strains would specify the point mutations that have occurred in these regions. Absences of more than one WT bands were shown in 4% of cases. These involved mutations at the 15 and 16 codons that facilitated change of amino acids from cysteine to threonine and Alanine to Glycine, respectively.

Among the resistance due to the development of positive Mutation band(s) and absence of corresponding WT band(s). The presence of MUT1 with the absence of WT1 showed 67.3%, which is by far the largest pattern of exhibited resistance occurring as a result of point mutations in inhA. The conversion of cysteine to threonine at the codon15 in the promoter region was found to be maximally involved in conferring the low-level resistance.

The presence of MUT 2 along with the absence of WT1 showed the second-most common pattern. The change of alanine to Glycine at the codon 16 of the inhA promoter region accounted for 7.6% only.

The most common among the absence of a single WT segments were WT1 and presence of MUT1 which was around 67.1%. The point mutations involved codons C15T and A16G.

Among the cohort showing resistance due to presence of mutation band(s) whereas corresponding WT band is also positive MUT1 Pos along with WT 1 pos contributed in 2% of the cases (With confidence interval ± 2.52 at 95% Confidence limit).[11]

Inh A codes for the Enoyl-acyl carrier protein (ACP) reductase of the type II fatty acid syntase (FAS-II) system. The enzyme helps in the biosynthesis of mycolic acids. The predominant component of mycobacterial cell walls.[12]

Enoyl ACP Reductase Catalyzes the NADH-dependent reduction of the double bond of 2-trans-enoyl-[acyl-carrier protein], a fatty acid elongation step cycle of the FAS-II pathway.[13] It is seen the exclusive point Mutation of the 15th base pair promoter sequence of inhA that converts cysteine residue to threonine contributes the most among the inhA conferred resistance toward INH.

The presence of the most common pattern was the presence of MUT1 with the absence of WT1. The pattern contributes most to the inh A dependant INH resistance isolates among retreatment cases. Percentage contribution of Band Patterns of INH resistance due to inh A.

Involvement of kat G and inhA with respect to rifampicin susceptibility

For kat G the most common pattern was seen as Mut1 (Pos) and WT (NEG) where a point mutations has occurred in codon 315 [Table 1]. It has been observed that the inh A resistance pattern is seen in 11.7% and kat G resistance is 90% among the RMP-resistant isolates however inhA resistance pattern is seen only in 1.1% and kat G in 17.8% among the RMP sensitive isolates isolated from the TB patients [Table 2], [Table 3], [Table 4], [Table 5]. Here, it is to be noted that MUT1 involves a 15th region of inh A promoter gene.[6] On analyzing the data for katG resistance pattern, majority (88.7%) have resistance due to positive MUT 1 and negative WT 1 whereas 4.3% have Positive MUT1 and Positive WT 1 [Table 2] and [Table 3].
Table 1: Mutation in the katG gene and the corresponding wild type and mutation bands

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Table 2: Patterns contributing to the resistance

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Table 3: Patterns of kat G contributing to the resistance in rifampicin resistance cases

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Table 4: Patterns of inh A contributing to the resistance in rifampicin sensitive cases

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Table 5: Patterns of kat G contributing to the resistance in rifampicin sensitive cases

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Among the inh A resistance pattern 67.3% have resistance due to positive MUT 1 and negative WT 1 whereas 1.9% have Positive MUT1 and Positive WT 1. It is known that inh A confers low resistance toward INH and its found in 21% of the MDR cases.[14] In the study, it was observed only 11.7% among the retreatment MDR cases had INH resistance due to mutation in the inh A.

Change in Inh A WT1 at the 15 position involves transiltion of C-15 T. Similarly transitions from A-16G and T8C are shown and 16th and 8th position [Table 6].
Table 6: Mutation in the inhA promoter region and the corresponding wild type and mutation bands[6]

Click here to view


The presence of the inh A-induced resistance below the evaluated observation among the retreatment cases may prompt conversion of high-level resistance due to kat G owing to the long retreatment protocols due to the dearth of availability of second-line drugs before 2009.

The majority of the inh A resistance in the study occurred due to the mutations in the codon 15 and 16 of the inhA promoter gene. Sequencing would definitely reveal that whether any rare mutations prevail beyond this zone.

Out of the 442 cases 410 cases (92.7%) showed resistance to INH with the involvement of katG among RMP-resistant cases however of the 627 RMP sensitive cases 112 cases (17.8%) showing resistance to INH with the involvement of katG.

Isoniazid as a prophylaxis

It was seen that prophylaxis by INH for 36 months in HIV infected contacts was more effective than 6 months of prophylaxis.[15] The study by Martinson et al. showed the importance of combination drugs during prophylaxis.[16]

Though some studies indicated long-term prevention, the chance of not developing the disease in lifetime cannot be ruled out.[17]

Preventive therapy is well tolerated in latent TB infection (LTBI) patients undergoing renal transplantation.[18]

Based on the studies, the WHO recommends INH as a preventive therapy for HIV-infected LTBI cases topic.[19]

There are publications to corroborate the efficacies of the drug when used as a prophylaxis. However, most of the studies are based on the clinical outcomes and results of the baseline diagnostic tests with varied analytical sensitivities. The exact quantum of infection with the exact strain responding to the treatment is lacking in all most all the studies, thereby raising a question on the rationale of its use. The study explores the point and periodic prevalence of the INH-resistant strains. The pattern of resistance is shown owing to their point mutations.


  Conclusion Top


The band Pattern analysis for inh A-related mutations showed the majority of the mutations to be occurring at the 15th codon of the inh A promoter region. In only one rare occasion presence of both mutant and wild type segments are found. This might be a case of mixed population. The lower percentage of inh A dependant mutations with respect to the evaluated studies may be a result of the prolonged retreatment schedules. The study was done among the 442 RMP-resistant isolates recovered from retreatment cases in the year 2012 as well as 627 from newly diagnosed RMP sensitive cases from a period of 2013-2016.

These cases were on retreatment schedule since long as no second-line drugs were available before 2012. A pear pressure for prolonged retreatment regimen might have converted several of low-dose resistant strains to high-dose resistant strains.

The study revealed an alarming 92.7% percentage of resistance among the RMP-resistant cases. Moreover, kat G-induced resistance was found to be on the rise 87.5 Percentage which suggested the increasing resistance towards INH. India accounts for an Annual rate of transmitting infection 1.5, failure of preventive treatment adherence would worsen the situation rendering the increase in INH resistance among the population.

Out of 442 cases, 410 cases showed INH resistance. Out of which resistance due to inh A was seen in 32 cases, 19 cases showed resistance due to mutations in both inh A as well as kat G. 359 cases accounting to 87.5% showed exclusive involvement of kat G. As it is evident that kat G confers a higher degree of INH resistance, the higher percentage of kat G-induced INH resistance among the RMP-resistant strains depicts the spread of a higher degree of INH resistance being the driver for RMP resistance.[20] A similar study for RMP resistance is carried over showing the conversion of sensitive strain to resistant ones among heterogeneous strains for a patient who are put on anti-tubercular drugs.[21]

Initially, it was thought that in MDR-TB, exposure to both INH and RMP results in the mutations that confer resistance to each drug. However,in vitro analysis suggested that preexisting INH resistance allele has a role in influencing the subsequent rpo B mutations.[22],[23] This has been corroborated in our study when around 92.7 percent of the RMP-resistant strains shown to have an existing high degree of INH resistance by kat G.

Seconding the observations of drug-resistant clinical Mycobacterium tuberculosis isolates have shown that specific INH resistance allele are more frequently associated with resistance to other drugs.[24],[25]

The question remains how effective would be the INH prophylaxis in pediatric age group contacts of the cases in this scenario. The rise of the high level of resistance may suggest the irrational empirical use of the drug as a prophylaxis.

The preventive treatment of Tub by a drug which accounts for substantial spread of its resistance needs further introspection. The efficacies studies are based on the duration of 10–15 years. The chances of disease development in a lifetime among the subjects given by the conventional means of preventive needs are to be reviewed. A wise rationale would be administering a drug whose sensitivity is prevalent in most of the isolated strains from the source cases. Preventive therapy based on the Drug sensitivity of the source cases would be more judicious to fight out the latent infections among the children and contacts.

Due to high INH resistance in cases where RMP resistance is present the INH prophylaxis in contacts is even worsening the situation as baseline 92.7% of INH resistance is noted through katG in our extensive study.

To achieve the target of control of TB cases globally not only treatment to be focussed on Universal Drug Sensitivity Testing and early initiation of treatment based on the sensitivity, but also care to be taken for prophylaxis of drugs to contacts of established cases, and if resource permits selection of drugs for prophylaxis should be also drug sensitivity guided.

Acknowledgment

We are thankful to all Medical officers and Technical officers, RNTCP West Bengal, India for providing technical inputs and all patients and staffs contributing for their dedication.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Quémard A, Sacchettini JC, Dessen A, Vilcheze C, Bittman R, Jacobs WR Jr., et al. Enzymatic characterization of the target for isoniazid in Mycobacterium tuberculosis. Biochemistry 1995;34:8235-41.  Back to cited text no. 13
    
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Cavusoglu C, Turhan A, Akinci P, Soyler I. Evaluation of the genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates. J Clin Microbiol 2006;44:2338-42.  Back to cited text no. 14
    
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Samandari T, Agizew TB, Nyirenda S, Tedla Z, Sibanda T, Shang N, et al. 6-month versus 36-month isoniazid preventive treatment for tuberculosis in adults with HIV infection in Botswana: A randomised, double-blind, placebo-controlled trial. Lancet 2011;377:1588-98.  Back to cited text no. 15
    
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