Mutation Detection and Accurate Diagnosis of Extensively Drug-Resistant Tuberculosis: Report from a Tertiary Care Center in India

Abstract

We screened and spoligotyped 150 consecutive phenotypically confirmed extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) isolates (January 2008 to March 2009) for rifampin, isoniazid, fluoroquinolone, and aminoglycoside resistance targeting rpoB, inhA, katG, gyrA, gyrB, and rrs. Mutations predominant among XDR-TB were S315T (katG) (100% of isolates), S531L (rpoB) (97% of isolates), D94G (gyrA) (53% of isolates), and A1401G (rrs) (71% of isolates). Spoligotyping revealed 62% of the isolates to be Beijing.

The worldwide emergence of extensively drug resistant Mycobacterium tuberculosis (XDR-TB) is a major setback to tuberculosis (TB) control (10, 11, 12, 15). XDR-TB, defined as multidrug resistant (MDR) M. tuberculosis (i.e., resistant to rifampin and isoniazid) with additional resistance to any fluoroquinolone (FQ) and to any one of three s-line injectables, i.e., capreomycin, kanamycin, or amikacin (20).

The mycobacteriology laboratory of P. D. Hinduja National Hospital (PDHNH), a tertiary care center in central Mumbai, received 7,482 clinical specimens for TB culture using a mycobacterial growth indicator tube (MGIT) and Lowenstein-Jensen medium (LJ) from January 2008 to March 2009. Our laboratory has a referral bias toward nonresponders, as we mainly receive treatment failures and complicated TB cases and drug susceptibility testing (DST) is performed only on request. A total of 3,899 samples were positive for the Mycobacterium tuberculosis complex, and 2,522 patients requested DST, of which 1,640 (65%) were MDR and 150 (9.1%) were XDR-TB by the MGIT (19). We studied mutations targeting the genes conferring resistance to drugs included in the definition of XDR-TB (inhA, katG, rpoB, and gyrA [1, 5, 8, 10, 14, 22, 27] and gyrB and rrs [2, 16, 28]) and fingerprinted these patients' isolates by spoligotyping (6). DNA extraction from pure culture was performed by the cetyl trimethyl ammonium bromide (CTAB-NaCl) method (23). A single tube touchdown multiplex PCR for three target genes associated with resistance to isoniazid (inhA and katG) and rifampin (rpoB) was done and screened for mutations using an in-house reverse line blot hybridization (RLBH) assay (21). The screening of katG resulted in the identification of G315C (100%), the S315T mutation in katG (Table 1) conferring high-level resistance to isoniazid with an altered catalase-peroxidase activity (24). Twenty-one (14%) of these isolates showed the C15T mutation in the promoter region of the inhA gene in addition to the katG mutation. The promoter mutation was also seen among XDR-TB isolates in Portugal (18) and China (25). The S531L mutation in rpoB was more prevalent, observed in 146 (97%) of these isolates, whereas the H526Y mutation was observed only in 4 (3%) of the isolates. A previous study from this center (21) on MDR isolates revealed an array of point mutations from position 511 to position 531 in rpoB; however, the S531L mutation in rpoB dominated among XDR-TB isolates. This is similar to what was found in a study from Portugal (18), where the S531L mutation dominated in XDR-TB.

Table 1.

Frequency of mutations in the various genes observed among our 150 XDR-TB isolates

Drug(s)	Gene(s)	Mutation(s) seen	Amino acid change	No. (%) of isolates
Isoniazid	katG	G315C	S315T	150 (100)
Isoniazid	inhA	C-15T		21 (14)
Rifampin	rpoB	C531T	S531L	146 (97)
Rifampin	rpoB	A526G	H526Y	4 (3)
Fluoroquinolones
Ofloxacin	gyrA	C90T	A90V	37 (25)
Ofloxacin, moxifloxacin	gyrA	T91C	S91P	2 (1)
Ofloxacin, moxifloxacin	gyrA	A94G	D94G	79 (53)
Ofloxacin, moxifloxacin	gyrA	A94C	D94A	18 (12)
Ofloxacin, moxifloxacin	gyrA	G94A	D94N	14 (9)
Aminoglycosides
Kanamycin, amikacin	rrs	A1401G		106 (71)
Kanamycin, amikacin, capreomycin	rrs	G1484T		42 (28)
Capreomycin (n = 42)	tlyA, rrs	A205G plus G1484T	K69E	13 (31)
Capreomycin (n = 42)	tlyA, rrs	Deletion of T at nt 674 plus G1484T	Frameshift	3 (7)

Novel primers were designed to amplify a 350-bp region of gyrA, a 550-bp region of gyrB for fluoroquinolone resistance, and a 500-bp region of rrs (codons 1401 to 1484) for aminoglycoside resistance. The initial 50 amplified products were analyzed for bidirectional sequencing, and after mutation confirmation, 100 subsequent products were sent for unidirectional sequencing (Chromous Biotech Pvt., Ltd., Bangalore, India). DNA sequencing of the gyrA gene showed the presence of the D94G mutation in 79 (53%) isolates phenotypically resistant to ofloxacin and moxifloxacin, while the A90V mutation was observed in 37 (25%) isolates that were phenotypically sensitive to moxifloxacin but resistant to ofloxacin. This needs to be verified with the MIC at higher drug concentrations for ofloxacin (13). The D94G mutation at the 94th codon showed a high frequency, i.e., 53% among our isolates. Other mutations at the 94th codon, D94A and D94N, were found in 12% and 9% of the isolates, respectively. Other studies (18, 25) also show similar mutations in gyrA. All isolates were sequenced for gyrB mutations, but only one isolate phenotypically resistant to both ofloxacin and moxifloxacin showed an N510D mutation in gyrB in addition to the D94G mutation in gyrA. Sequencing of the rrs gene revealed the presence of the A1401G mutation in 106 (71%) isolates that were phenotypically resistant to kanamycin and amikacin. Cross-resistance between the two aminoglycosides kanamycin and amikacin is believed to exist (2, 16, 26, 28). Forty-two (28%) isolates phenotypically resistant to capreomycin in addition to kanamycin and amikacin showed the presence of a G1484T mutation, whereas other studies (18, 25) have reported several point mutations in the rrs gene in addition to the A1401G and G1484T mutations.

For capreomycin-resistant isolates, the tlyA gene (17) that was split in two parts (tlyA-1 [500 bp] and tlyA-2 [300 bp]) was amplified using novel primers. The amplified products were sent for bidirectional sequencing. Of the 42 capreomycin-resistant isolates sequenced for tlyA in addition to rrs, 13 (31%) isolates showed A205G (K69E) and 3 isolates showed deletion of T at nucleotide (nt) 674, resulting in a frameshift mutation. The remaining 26 isolates showed the G1484T mutation in the rrs gene only, with no tlyA mutation. Two isolates that were phenotypically resistant to kanamycin, amikacin, and capreomycin did not show any mutation in the rrs gene. This could be due to mutations in other hot spot regions that were not targeted in this study.

Fingerprinting of all isolates was performed by spoligotyping (6), and the pattern was compared to that in an international spoligotyping database (SpolDB4) (7, 29). The spoligotype pattern revealed that the Beijing genotype was predominant among XDR-TB isolates, which accounted for 62% of all the isolates, followed by the Central Asian (CAS) genotype (14%), and the T families, i.e., T1 (13%), East African-Indian family 5 (EAI5) (7%), and EAI 3 (2%). There were three single isolates that each belonged to family 35 (as assigned by the Spotclust database, which uses a computer algorithm based on studies from SpolDB3 [9]), T4, and the Haarlem genotype (Table 2). The sharing of a single spoligotype by 62% of the isolates suggests an important role for transmission of a dominant resistant clone. Spoligotyping studies from Mumbai have shown other genotypes, i.e., CAS and Manu1, to be predominant (4), whereas a previous study from our center (3) has reported a high proportion of Beijing strains among resistant cases. However, the results of the present study should not be overinterpreted, as sampling bias cannot be ruled out, due to the nonresponders referred to our center. Epidemiological associations between patients could not be ascertained, as patients were not residents from the same locality or workplace, and 119/150 (79%) were on second-line drugs for durations of more than 1 year.

Table 2.

Spoligotyping pattern of the 150 XDR-TB isolates

Serial no.	Octal code	Spoligotype	No. (%) of isolates with the genotype
1	000000000003771	Beijing	94 (62)
2	703777740003771	CAS	21 (14)
3	377777777760771	T1	19 (13)
4	737777777413371	EAI5	10 (6.7)
5	477777777413071	EAI3	3 (2)
6	703600000000771	Family 35	1 (0.6)
7	757777774020771	Haarlem1	1 (0.6)
8	777760057760771	T4	1 (0.6)

Thus, mutational analysis could conclusively predict 100% resistance to isoniazid, rifampin, and fluoroquinolone and 98% resistance to aminoglycosides among our XDR-TB cohort.