Multi Drug Resistant Tuberculosis in Bhutan: A Look into The Line Probe Assay Results

Background: Bhutan plans to end TB by 2030 in line with the WHO End TB by 2030 goal. In 2016 National tuberculosis reference laboratory (NTRL) has performed Line probe assay (LPA) on 506 TB isolates, a huge jump from 326 cases in 2015. Objectives: This review is aimed to understand the common mutations seen in the MDR isolates depicted by the line probe assay performed on the Mtb isolates from Bhutanese patients. Methods: Line probe assay (LPA) results available from February 2014 to December 2016 at NTRL, has been retrospectively reviewed for drug resistance mutations. The data were of specimens from both from both pulmonary as well as extra pulmonary sites on which LPA has been performed. Findings: Bhutan has seen an increase in the number of specimens referred in to NTRL for LPA and DST. Fifty-seven (14.4%) MDR was seen in 2014, 16% in 2017 and 10.7% in 2016. Mono resistance to isoniazid was found more frequently compared to rifampicin mono-resistance by over 5 times. From our observation, MDRTB in Bhutanese isolates were mainly from the most frequently observed mutation worldwide, i.e at nt530533 (WT8), nt513-517 (WT3), and S531L (MUT3) of rpoB gene and at nt315 (WT1) and S315T1 (MUT1) of katG gene. Occasional involvement of inhA gene was found at -15/-16 (WT1), -8 (WT2) and C15T (MUT1). More than 75% of the MDR cases were due to dual mutations in the rpoB and katG genes (71.9% in 2014, 86.5% in 2015 and 88.9% in 2016). Among all the 163 MDR isolates of the three consecutive years, the most frequent combination of mutation observed (127/163, 77.9%) was the loss of rpoB WT8 (530-533) with rpoB MUT3 (S531L) and the loss of KatG WT1 (315) with KatG MUT1 (S315T1). The loss of rpoB WT8 (530-533) accounted for 93.9% (153/163), closely followed by WT3 (513-517) with 82.2% (134/163). WT7 (526-529) composed of 4.3% (7/163 cases) of MDRTB. About 85% (138/163) of rpoB MUT3 (S531L) was detected followed by 3 cases each of MUT2A and 2B. Ninety eight percent of MDR had WT bands missing and 93% had katG MUT1 bands depicting mutations in S315T. katG MUT2, S315T2, was the most uncommon mutations with only a single case in 2014. No mutations or loss of ropB WT1, ropB WT2 and ropBWT6 have been detected in the MDR isolates, however in the RIF-MR no mutations were found from ropB WT1 through ropB WT6. Losses of ropB WT7 & 8 were found to be the most common finding among the Bhutanese isolates. Mutations in katG commonly composed of the loss of WT with or without MUT1 and 2. The most common involvement in inhA gene was the loss of WT1 and WT2 bands with or without MUT1 band. However, no mutations in katG and inhA were observed together at any location. Mutations in katG and inhA were however detected in MDR along with mutation in rpoB gene. Mutations in inhA, though less frequent in our MDR and INH Mono resistant isolates, it was reported to have a strong correlation with XDR TB in Western Cape and Eastern Cape Provinces, South Africa [1]. This is the first study where LPA depicted INH and RIF-drug-resistance-conferring mutations in MDR M. tuberculosis strains have been described. Main Conclusion: LPA has facilitated diagnosis of MR/MDR TB and guide appropriate therapy. However, in resource limited country like Bhutan LPA has contributed immensely towards proper treatment for the patients.


Introduction
Geographically, Bhutan is a tiny kingdom of 7,87,338 Bhutanese and an area of 38,394sq. kms [3]

Epidemiology and Emergence of MDR-TB in Bhutan
Despite high prevalence of tuberculosis in neighboring Asian countries, Bhutan is among the low TB burden countries. Worldwide drug resistance has evolved due to poor understanding among those infected of the importance of adherence to treatment, quality of treatment as well as administrative and programmatic lapses.
Moreover, other factors like social, demographical and geographical aspects at local or national levels, such as the development of the country play an enormous role in the epidemiology of tuberculosis in many countries alike [4]. Bhutan has a functional National Tuberculosis Control Program (NTCP) instituted in 1986 [5] and since then tuberculosis control activities have gradually been gearing up with awareness advocacies resulting in increased case detection. Tuberculosis in pediatrics, which is normally missed by many surveillances have however been well recognized in the country with excellent treatment outcome [6]. Furthermore, the rising HIV cases in the country, is a wakeup call for vigilance and renewed efforts to combat HIV-TB co-morbidities in the community. This review aims to present the mutations patterns occurring in the genes conferring mono-resistance to either INH or RIF (RR DR); or to both; MDR, from the band patterns deciphered from the hybridization strips of the LPA from the past 3 years data from the Bhutanese population. The findings showed that mutations in the rpoB, katG, and inhA genes are similar to those reported from other parts of the world. Since no proper phenotypic and genotypic study is available in other parts of Bhutan, we cannot rule out the possibility of the existence of similar MDR or pre-XDR/XDR strains within the country. Due to inadequate monitoring and a lack of proper treatment regimens, MDR-TB and XDR-TB remain major threats to the Bhutanese population.

Materials and Methods
Line probe assay (LPA) results available from February 2014 to December 2016 at NTRL, has been retrospectively assessed for drug resistance. The data were of specimens of both from both pulmonary as well as extra pulmonary sites.

Mycobacterium tuberculosis isolates
Sputum or extra pulmonary specimens which were Acid fast positive by Zeihl Neelson staining are referred to NTRL for DST from the district hospitals and the Basic health units across the country. For this study a total of 293 isolates with valid LPA test results were used for further analysis of their resistance patterns after omitting 1021isolates which were found to be sensitive to INH and RIF or resulted in invalid tests on LPA.     (Table 1). Data from solid culture DST was not available to complement the LPA findings. Figure 1

Am J Biomed Sci & Res
Copy@ Sonam Pelden In 2014 the second commonest combination was rpoB WTL + katG     of ropB gene were found to be the most common finding among the Bhutanese isolates. Mutations in katG commonly composed of the loss of WT with or without MUT1 and 2 ( Table 6). The most common involvement in inhA gene was the loss of WT1 and WT2 bands with or without MUT1 band.   isolates, it was reported to have a strong correlation with XDR TB in Western Cape and Eastern Cape Provinces, South Africa [1]. Many reported a low-level resistance associated with the mutations in the inhA which could be therapeutically overcome with high dose isoniazid [11,12]. India, the closest neighbor who is among the 30 countries of high TB burden countries in the world has reported 79,000 drug resistant Tb cases in 2015 among which 28,876 were MDR/RR-TB and 3048 were XDR-TB [13]. Free permeable border allows, intensive cross border trade and close interaction, with a high possibility of transmitting TB bacilli among the people. It would be interesting to delve into the epidemiological and genetic study to see associations between our strains thereby helping cross-border advocacy and refine control policies. However, it is also important to note that M. tb strains exhibit geographical variation. Patra et al. observed different mutation patterns in RRDR of rpoB studied at the same location depicting changes in mutation profiles [14]. Such rapid tests require designing of probes based on the knowledge of mutation profile in different geographical area, however the constantly changing resistance patterns may be a challenge in achieving a fool-proof test kit for a particular region. for the detection of rifampicin resistance and 85.7% and 100% for the detection of isoniazid resistance [17].
This is the first study where LPA depicted INH and RIF-drugresistance-conferring mutations in MDR M. tuberculosis strains have been described. Now with the shifting of focus from diagnosis to the prediction of possible resistances that would be acquired by the isolates [10], curbing of tuberculosis seems promising. However, in resource limited country like Bhutan LPA has contributed immensely towards proper treatment for the patients. Bhutan is rapidly developing with increased human mobility.

i.
No phenotypic DST data available for comparison and Loss of valuable data in the invalid results.
ii. Therefore low, intermediate or high-level resistance cannot be determined.
iii. LPA does not cover unidentified mutations in other genomic regions (like ahpC, kasA, furA).
iv. Data was available only for 3 years as LPA was only recently, therefore changes in the band patterns over many years cannot be compared.