First Detection of Mycobacterium tuberculosis Clinical Isolates Harboring I491F Borderline Resistance rpoB Mutation in Myanmar

LETTER

Myanmar is one of the high-burden countries for tuberculosis (TB), HIV-associated TB, and multidrug-resistant/rifampin (RIF)-resistant TB (MDR/RR-TB). The World Health Organization (WHO) estimated the TB incidence at 308 per 100,000 population, laboratory-confirmed MDR/RR-TB at 2,368 cases in 2020, and the proportion of MDR/RR-TB among new and previously treated patients at 4.9 and 20%, respectively, in 2018 (1, 2). Currently, GeneXpert MTB/RIF is the main routine diagnostic test for TB and RR-TB. Detection of initial RIF resistance is critical for clinical algorithms (3).

The WHO-endorsed molecular drug susceptibility tests (mDSTs) GeneXpert MTB/RIF and GenoType MTBDRplus detect mutations in the 81-bp RIF resistance-determining region (RRDR) of the rpoB gene, which are associated with over 95% of RIF resistance (4, 5). I491F, an rpoB mutation located outside the RRDR, is not detectable by routine mDSTs (6). It is also a low-level resistance mutation that is undetectable by the phenotypic DST (pDST) Bactec mycobacterial growth indicator tube (MGIT) (7). This mutation has been previously reported in many countries, with a prevalence ranging from 0.5% to 30% of RIF-resistant strains (8–10). Recently, it accounted for up to 56% of RR-TB in Eswatini (2, 11).

In this study, we performed retrospective whole-genome sequencing (WGS) on a subset of Mycobacterium tuberculosis (MTB) isolates from pulmonary TB patients who enrolled in an observational cohort study on treatment response biomarkers during 2020 to 2022. Of 129 MTB isolates, 35 isolates consisting of 23 from patients who completed drug-susceptible TB (DS-TB) treatment and 12 from patients who completed MDR-TB treatment were selected for WGS. DNA was extracted from MTB by the cetyltrimethylammonium bromide method (12). WGS was conducted using the Illumina MiSeq platform as described previously (13, 14). Phylogenetic and mutation analysis were done with the PhyResSE version 1.0 and SAM-TB online platforms (15, 16). Variant callings were analyzed using the reference strain (H37Rv).

On analysis of 35 MTB genomes, the I491F rpoB mutation was detected in 3 (8.6%) isolates from DS-TB patients (Table 1). All three isolates were separate strains, although two belonged to the same overarching lineage (see Fig. S1 in the supplemental material). These patients remained sputum smear and culture positive at the 5-month follow-up and were designated DS-TB treatment failure cases. The GeneXpert MTB/RIF, Bactec-MGIT, and GenoType MTBDRplus tests were carried out at 5 months but failed to detect RIF resistance, although they identified resistance to isoniazid and other anti-TB drugs.

TABLE 1.

Demographic characteristics, phenotypic and molecular drug susceptibility, drug resistance mutations, lineage, and drug resistance patterns of three Mycobacterium tuberculosis isolates harboring the I491F rpoB mutation^a

Sr. no.	Case ID	Age (yr)	Sex	Result(s) from:						Drug resistance patterns
				MGIT pDST	mDST		WGS
					GeneXpert MTB/RIF	GenoType MTBDRplus	Anti-TB drug	Resistance mutations (target gene/mutation)	Lineage
1	DSH026	22	M	Susceptible to RIF, FQs and SLIDs; resistant to INH, EMB, PZA and SM	MTB detected; RR not detected		SM	rpsL (K43R)	Beijing (Lineage 2)	MDR and resistance to all other first-line drugs
						katG (S315T)	INH	katG (S315T)
						rpoB (WT)	RIF	rpoB (I491F)
							EMB	embB (M306I)
							PZA	pncA (Y103H)
2	IRTB003	33	M	Susceptible to RIF, PZA, SM FQ and SLIDs; resistant to INH and EMB	MTB detected; RR not detected	inhA (C-15T)	INH	fabG1-inhA (C-15T)	East African Indian (Lineage 1)	MDR and EMB resistance
						rpoB (WT)	RIF	rpoB (I491F)
							EMB	embB (M306V)
3	IRTB006	65	M	Susceptible to RIF; resistant to INH, EMB, PZA, SM, FQ, and SLIDs	MTB detected; RR not detected		SM	rpsL (K43R)	Beijing (Lineage 2)	Pre-XDR and resistance to all other first-line drugs and SLIDs
						katG (S315T)	INH	katG (S315T)
						rpoB (WT)	RIF	rpoB (I491F)
							EMB	embB (M306V)
							PZA	pncA (T103C)
							FQ	gyrA (A90V)
							SLIDs	rrs (1401G)

^aGeneral abbreviations: M, male; F, female; MGIT, mycobacterial growth indicator tube; WT, wild type; pDST, phenotypic drug susceptibility test; mDSTs, molecular drug susceptibility tests; WGS, whole-genome sequencing; MTB, Mycobacterium tuberculosis; RR, rifampin resistance; MDR, multidrug resistance; Pre-XDR, pre-extensively drug resistant (MDR plus FQ resistance). Abbreviations for first-line anti-TB drugs: SM, streptomycin; INH, isoniazid; RIF, rifampin; EMB, ethambutol; PZA, pyrazinamide. Abbreviations for second-line anti-TB drugs (according to old definition): FQ, fluoroquinolones (levofloxacin, moxifloxaxin, and ofloxacin); SLID, second-line injectable drugs (amikacin, kanamycin, and capreomycin).

In a recent Myanmar study, the I491F mutation was not detected among 309 MTB isolates collected during 2016 to 2018 (17). Our study is the first to report the I491F rpoB mutation among Myanmar MTB isolates. The WHO recommended that seven borderline resistance rpoB mutations, including I491F, need to be treated with MDR-TB treatment (18). In our study, all three patients with the I491F rpoB mutation missed MDR-TB treatment and had poor treatment outcomes. MDR/RR-TB strains with borderline resistance mutations missed by routine diagnostic assays can result in ineffective treatment, continued amplification, and concurrent silent transmission in the community. Further research is needed to ascertain the prevalence and clinical impact of these strains in Myanmar.

Ethics approval was obtained from the University of Public Health, Yangon, Myanmar (UPH-IRB-UPH 2019/Research/37).

Data availability.

We have submitted our sequences to NCBI SRA under accession no. SAMN31143431, SAMN28463015, and SAMN28463016.