Isoniazid and rifampicin heteroresistant Mycobacterium tuberculosis isolated from tuberculous meningitis patients in India

Highlights

• Timely diagnosis, treatment after drug susceptibility testing is vital for successful outcome.

• Heteroresistant isolates are difficult to detect and are preliminary stage to full resistance.

• Prevalence of Heteroresistant Mycobacterium tuberculosis was 4%, 1% to INH and rifampicin respectively by Genotype MTBDR assay.

Abstract

Background

Heteroresistant Mycobacterium tuberculosis (mixture of susceptible and resistant subpopulations) is thought to be a preliminary stage to full resistance and timely detection, initiation of correct treatment is vital for successful anti tubercular therapy. The aim of this study was to detect multi drug resistant (MDR) and heteroresistant M. tuberculosis with the associated gene mutations from patients of tuberculous meningitis.

Methods

A total of 197 M. tuberculosis isolates from 478 patients of TBM were isolated from July 2012 to July 2015 and subjected to drug susceptibility testing (DST) by BACTEC MGIT and Genotype MTBDR line probe assay (LPA). Heteroresistance was defined as presence of both WT and mutant genes in LPA.

Results

Of 197 M. tuberculosis isolates, 11 (5.6%) were MDR, 23 (11.6%), 1 (0.5%) were mono resistant to isoniazid (INH) and rifampicin (RMP) respectively. Heteroresistance was detected in 8 (4%), 2 (1%) isolates to INH and RMP respectively. INH heteroresistant strains had WT bands with mutation band S315T1 whereas RMP heteroresistant strains had WT bands with mutation band S531L.

Conclusion

The prevalence of MDR M. tuberculosis was 5.6% in TBM patients with the most common mutation being ΔWT band with S315T1 for INH and ΔWT band with S531T for RMP. MGIT DST was found to be more sensitive for detecting overall resistance in M. tuberculosis but inclusion of LPA not only reduced time for early initiation of appropriate treatment but also enabled detection of heteroresistance in 8 (4%), 2 (1%) isolates for INH and RMP respectively.

Introduction

Tuberculous meningitis (TBM) is a devastating complication of tuberculosis and timely diagnosis and treatment with effective drugs are the key factors for successful management of these patients. Existence of multi drug-resistant (MDR) Mycobacterium tuberculosis is well recognized from both pulmonary and extrapulmonary tuberculosis and is posing a great threat to tuberculosis control programs worldwide.1, 2 Besides existence of MDR M. tuberculosis, patients with tuberculosis may also harbor both drug susceptible and resistant M. tuberculosis subpopulations together, representing a phenomenon called heteroresistance.³ Heteroresistant M. tuberculosis is considered a preliminary stage to full resistance and failure to detect these bacteria may lead to treatment failure or spread of drug resistant bacteria to other patients.3, 4

Heteroresistance can occur due to two mechanisms, one being acquisition of both susceptible and resistant bacterial populations from a partially treated patient or slow evolution of resistant clones from susceptible clones.5, 6 The prevalence of heteroresistance is largely unknown with few reports of heteroresistance to isoniazid (INH), rifampicin (RMP), ethambutol (EMB), streptomycin (STM) and flouroquinolones from pulmonary isolates.3, 4, 5, 6, 7, 8, 9, 10 This heteroresistance is dependent upon local epidemiology and is particularly common in regions with high rates of tuberculosis, especially drug resistant tuberculosis.⁴ India accounts for more than 25% of the world's incident cases of tuberculosis with an estimated 35,400 MDR TB patients among incident TB cases in India.²

Numerous methods are available for drug susceptibility testing of M. tuberculosis isolates but detection of heteroresistant M. tuberculosis is in itself problematic as both the phenotypic and genotypic methods have their own limitations for detecting these strains.3, 5, 8, 9, 10 A number of genotypic methods have become available for early detection of drug resistance but little is known if they can also detect heteroresistance. Recently, Genotype MTBDR line probe assay (LPA) has been proposed to be capable of detecting the presence of heteroresistance since the strips contain both WT and mutant probes.3, 10 This study was conducted to determine the prevalence of multi drug resistant and heteroresistance M. tuberculosis isolated from patients of TBM using Phenotypic Mycobacterial growth indicator tubes (MGIT) in BACTEC 960 and LPA.