Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs Among Mycobacterium tuberculosis by a Reverse Dot Blot Hybridization Assay

Figures & data

Table 1 Sequences, Amplicon Sizes and Concentrations of Primers Used in Multiplex PCRs

Drug	Gene/Region	Primer	Sequence (5ʹ→3ʹ)	Amplicon Size (bp)	Final Concentration
FQs	gyrA	gyrA-F	GGGTGCTCTATGCAATGTTCG	314	0.28 μM
		gyrA-R	GCCGTCGTAGTTAGGGATGA
KN/AMK/CPM	rrs 1400 region	rrs-F	AACGCTGCGGTGAATACG	253	0.26 μM
		rrs-R	CGCCCACTACAGACAAGAAC
KN	eis promoter	eis-F	TGCCAGACACTGTCGTCGTAA	482	0.5 μM
		eis-R	GCTCATGCAAGGTGGTAGCG

Abbreviations: FQs, fluoroquinolones; KN, kanamycin; AMK, amikacin; CPM, capreomycin.

Table 2 List of Oligonucleotide Probes Used in This RDBH Assay

Drug	Gene	Genotype	Type	Sequence of Probes (5ʹ→3ʹ)	GC%
FQs	gyrA	90-94	Wild	C GCG TCG ATC TAC GAC A	58.8
		90 GCG-GTG	Mutant	TC GTA GAT CGA CAC GTC G	58.8
		91 TCG-CCG	Mutant	C GCG CCG ATC TAC GAC A	64.7
		94 GAC-GGC	Mutant	GT GCC GTA GAT CGA CGC	64.7
		94 GAC-GCC	Mutant	G TCG ATC TAC GCC ACC CTG	63.2
		94 GAC-TAC	Mutant	ATC TAC TAC ACC CTG GTG C	52.6
		94 GAC-AAC	Mutant	C GCG TCG ATC TAC AAC AC	55.6
		94 GAC-CAC	Mutant	C GCG TCG ATC TAC CAC AC	61.1
KN/AMK/CPM	rrs	1392–1407	Wild	CCGCCCGTCACGTCAT	68.8
		1401 A-G	Mutant	CCGTCGCGTCATGAAAGT	55.6
KN	eis promoter	(−17)-(0)	Wild	TGCCACAGTCGGATTCTG	55.6
		(−10) G-A	Mutant	ATGCCACAATCGGATTCTG	47.4
		(−14) C-T	Mutant	GGCATATGCTACAGTCGGA	52.6

Note: Nucleotides with an underline were the mutant that the probe aimed to detect.

Abbreviations: FQs, fluoroquinolones; KN, kanamycin; AMK, amikacin; CPM, capreomycin.

Table 3 Drug Susceptibility Patterns of 170 Clinical M. tuberculosis Isolates

Susceptibility or Resistance	Number of Strains
Fully susceptible	57
R-monoresistant	10
O-monoresistant	3
Over all poly-resistant	7
RO	1
OE	1
RSO	1
HSO	3
HSCKO	1
MDR except XDR	67
HR	7
HRO	4
HRS	2
HREO	6
HRSO	9
HRSEO	39
Over all XDR	26
HROK	2
HROC	1
HREOC	1
HRSOK	2
HRSEOK	3
HRSOCKA	1
HREOCKA	1
HRSEOCK	4
HRSEOCKA	11

Abbreviations: H, isoniazid; R, rifampicin; S, streptomycin; E, ethambutol; O, ofloxacin; C, capreomycin; K, kanamycin; A, amikacin; MDR, multi-drug resistant; XDR, extensively drug resistant.

Table 4 Distribution of Mutations in Genes and Regions Associated with Ofloxacin, Kanamycin, Amikacin, Capreomycin and Second-Line Injectable Drug Resistance in the Drug Resistant M. tuberculosis Isolates

Phenotype Resistance (No. of Isolates)	Genes/Regions	Mutation Types		No. of Resistant Isolates with Mutation
		nt Change	AA Change
Over all OFX (94)	gyrA	GGC88GCC	Gly88Ala	2
		GCG90GTG	Ala90Val	11
		GCG90GTG, TCG91CCG	Ala90Val, Ser91Pro	1
		GCG90GTG, GAC94GCC	Ala90Val, Asp94Ala	1
		TCG91CCG	Ser91Pro	8
		TCG91CCG, GAC94GCC	Ser91Pro, Asp94Ala	1
		GAC94GCC	Asp94Ala	11
		GAC94GGC	Asp94Gly	18
		GAC94CAC	Asp94His	3
		GAC94TAC	Asp94Tyr	3
		GAC94AAC	Asp94Asn	2
		GAC94GTC	Asp94Val	2
		WT		31
Over all KN (25)	eis promoter	−10G-A	–	2
		−14C-T	–	1
		−29 deleted G	–	1
	rrs 1400 region	1401 A-G	–	11
		1484 G-T	–	1
		WT*		9
Over all AMK (13)	rrs 1400 region	1401A-G	–	8
		1484G-T	–	1
		WT		4
Over all CPM (20)	rrs 1400 region	1401A-G	–	11
		1484G-T	–	1
		WT		8
SLIDs (27)	rrs 1400 region	1401A-G	–	11
		1484G-T	–	1
	eis promoter	−10G-A	–	2
		−29 deleted G	–	1
		−14C-T	–	1
		WT*		11

Note: *Means that isolates showed wild type both in rrs 1400 region and eis promoter.

Abbreviations: nt, nucleotide; AA, amino acid; OFX, ofloxacin; KN, kanamycin; AMK, amikacin; CPM, capreomycin; SLID means that the isolates were resistant to at least one of kanamycin, amikacin and capreomycin; WT, wild type.

Table 5 Distributions of Mutations in rrs 1400 Region and eis Promoter by Sequencing in 27 Second-Line Injectable Drug Resistant M. tuberculosis Isolates According to the Resistance Patterns*

Phenotypic Resistance Patterns (No. of Isolates)	Genes/Regions	nt Change	No. of Resistant Isolates with Mutation
AMK+KN+CPM (13)	rrs 1400 region	1401A- G	8
		1484G-T	1
		WT	4
	eis promoter	WT	13
KN+CPM (5)	rrs 1400 region	1401A-G	2
		WT	3
	eis promoter	WT	5
Mono-KN (7)	rrs 1400 region	1401A-G	1
		WT	6
	eis promoter	−10G-A	2
		−29 deleted G	1
		−14C-T	1
		WT	3
Mono-CPM (2)	rrs 1400 region	WT	2
	eis promoter	WT	2

Note: *The resistance patterns only included the resistance of the isolates to kanamycin, amikacin and capreomycin.

Abbreviations: nt, nucleotide; KN, kanamycin; AMK, amikacin; CPM, capreomycin; WT, wild type.

Figure 1 The hybridized image detected with the reverse dot blot hybridization assay for ofloxacin and second-line injectable drug resistance conferring mutations.

Notes: Lane 1: H37Rv reference strain; lane 2: Negative control; lanes 3 to 40: Mycobacterium tuberculosis clinical isolates. The sequence of probe gyrA 90-94 WT1 was equal to that of gyrA 90-94 WT2. The results of the RDBH assay, phenotypic drug susceptibility testing and sequencing of each sample are shown in Supplemental.

Table 6 Sensitivity, Specificity, PPV, NPV and Agreement of the RDBH Assay Compared with Phenotypic DST Among M. tuberculosis

Drugs	Phenotypic DST	RDBH Assay
		R	S	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)	Concordance (%)	Kappa value
OFX	R	60	34	63.8	100.0	100.0	69.1	80.0	0.61
	S	0	76
KN	R	15	10	60.0	100.0	100.0	93.5	94.1	0.72
	S	0	145
AMK	R	8	5	61.5	98.1	72.7	96.9	95.3	0.64
	S	3	154
CPM	R	10	10	50.0	99.3	90.9	93.7	93.5	0.61
	S	1	149
SLIDs*	R	15	12	55.6	100.0	100.0	92.2	92.9	0.68
	S	0	143
XDR	Yes	11	15	42.3	100.0	100.0	90.6	91.2	0.55
	No	0	144

Note: *Isolates resistant to at least one of kanamycin, amikacin and capreomycin were defined as SLID resistant, in contrast, some were defined as SLID susceptible.

Abbreviations: S, susceptible; R, resistant; PPV, positive predictive value; NPV, negative predictive value; OFX, ofloxacin; KN, kanamycin; AMK, amikacin; CPM, capreomycin; DST, drug susceptibility testing; SLIDs, second-line injectable drugs; XDR, extensively drug-resistant.

Table 7 Sensitivity, Specificity, PPV, NPV and Agreement of the RDBH Assay Compared with Sequencing Among M. tuberculosis Isolates

Drugs	Sequencing	RDBH Assay
		M	W	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)	Concordance	Kappa value
OFX	M	60	3	95.2	100.0	100.0	97.3	98.2	0.96
	W	0	107
SLIDs/KN^a	M	15	1	93.8	100.0	100.0	99.4	99.4	0.97
	W	0	154
AMK/CPM^b	M	11	1	91.6	100.0	100.0	98.8	99.4	0.87
	W	0	158
XDR	Yes	11	3	84.6	100.0	100.0	98.1	98.2	0.87
	No	0	156

Notes: ^aMeans that the genotypic susceptibility or resistance profiles of second-line injectable drugs or kanamycin were both defined by mutations in rrs 1400 region and eis promoter acquired by sequencing; ^bMeans that the genotypic susceptibility or resistance profiles of amikaxin or capreomycin were both defined by mutations in rrs 1400 region acquired by sequencing.

Abbreviations: M, mutatant; W, wild type; PPV, positive predictive value; NPV, negative predicte value; OFX, ofloxacin; KN, kanamycin; AMK, amikacin; CPM, capreomycin; SLIDs, second-line injectable drugs; XDR, extensively drug-resistant.