Resistance to Second-Line Anti-TB Drugs in Cambodia: A Phenotypic and Genetic Study

Figures & data

Box 1 Definition of Drug-Resistant Categories Based on Phenotypic DST Profiles

First-line drug resistance (FLD-R)*: Resistance to any first-line drugs (FLD) tested including INH, RIF, EMB and STM. Second-line drug resistance (SLD-R): Resistance to any second-line drugs tested including fluoroquinolones (Ofx) and second-line injectable drugs (Am, Km and Cm). Multidrug-resistance (MDR): Resistance to at least two of the most effective anti-TB drugs, INH and RIF. Non-MDR: Resistance to one or more FLD but not to INH and RIF at the same time. Quadruple drug resistance (QDR): Resistance to all four first line drugs tested including INH, RIF, EMB and STM. Fluoroquinolone resistance (FQ-R): Phenotypic resistance to any fluoroquinolone (ie Ofx in this study). Second-line injectable drug (SLID) resistance: Phenotypic resistance to at least one of three SLID including Am, Km and Cm. Extensively drug-resistant (XDR): MDR isolates simultaneously resistant to any fluoroquinolone (ie Ofx in this study) and at least to one SLID (Am, Km and Cm). Pre-extensively drug-resistant (pre-XDR): MDR isolates resistant either to any FQ (ie Ofx in this study) or one SLID (Am, Km and Cm).

Note: *Phenotypic DST is not available for pyrazinamide.

Abbreviations: Am, amikacin; Cm, capreomycin; DST, Drug susceptibility testing; EMB, ethambutol; INH, isoniazid; Km, kanamycin; Ofx, ofloxacin; R, resistance; RIF, rifampicin; STM, streptomycin.

Table 1 Demographic and Clinical Data, Phenotype and Mutations Associated with Anti-TB Drug Resistance of the 37 Studied Isolates

Isolates ID	Registration Date	Sex	Age	Household Location^a	TB Treatment History	Smear Result	Phenotypic Resistance		Drug Resistance Mutations (Genes)^b
							FLD	SLD	H (katG; inhA)	R (rpoB)	S (rpsL; rrs-F1)	E (embB)	Z (pncA)	FQ (gyrA; gyrB)	SLID (rrs-F2)
DR_001	2016	M	28	KH-01	Re-treatment	POS	HRSE	Ofx, Am, Km	S315T; WT	Q513-F514ins	L43R; WT	D328Y	>200bp del	D94G; WT	c1489t
DR_002	2016	F	23	KH-12	Re-treatment	POS	HRS	Ofx, Km	WT; c-15t and S94A	L511P & H526Q	L88R; WT	WT	A171E	A90V; WT	WT
DR_003	2017	F	51	KH-20	Re-treatment	POS	HRSE	Ofx	S315T; WT	S531L & L635P	L43R; WT	M306V	WT	D94A; WT	WT
DR_004	2017	M	50	KH-25	Re-treatment	POS	HRSE	Ofx	S315T; WT	S531L	L43R; WT	F330V	L19P	D94G; WT	WT
DR_007	2012	M	67	KH-03	Re-treatment	NA	HRS	Ofx	WT; c-15t and S94A	L511P & H526Q	L88R; WT	WT	A171E	A90V & WT; WT	WT
DRC_003	2015	F	30	KH-12	Re-treatment	NEG	HRS	Ofx	WT; c-15t and S94A	L511P & H526Q	L88R; WT	G406A	A171E	A90V; WT	WT
DRC_010	2015	M	40	KH-07	HIV/TB	POS	HRS	Am, Km	S315T; WT	H526R	L43R; WT	WT	WT	WT; WT	a1401g
DRC_016	2015	M	65	KH-07	Re-treatment	POS	S	Susceptible	G560S; WT	WT	L88R; WT	WT (L355L)	S104R	WT; S413A	WT
DRC_021	2015	M	25	KH-14	Re-treatment	POS	HRSE	Ofx	S315T; WT	H526D	WT; a514c	M306I	A171V	A90V; WT	WT
DRC_040	2015	M	41	KH-12	Re-treatment	POS	R	Ofx	WT; WT	H526Y	WT; WT	WT	WT	D94G; WT	WT
DRC_060	2014	M	70	KH-14	Re-treatment	NA	HS	Susceptible	WT; c-15t	WT	WT; c905a	WT	WT	WT; WT	g1177a
DRC_065	2014	F	32	KH-12	Re-treatment	POS	HS	Ofx	S315T; WT	I572F	L88T; WT	Q497R	H71Y	S91P; WT	WT
DRC_083	2014	M	52	KH-10	Re-treatment	POS	HRSE	Ofx	W477L & A479P; WT	S531L	WT; a514c	WT	Y103C & WT	D94G; WT	WT
DRC_098	2014	M	53	KH-12	Re-treatment	POS	HRS	Ofx	S315T; WT	S531L	L43R; WT	Q497L	V131G	D94G (S104S); WT	WT
DRC_109	2014	M	27	KH-20	Re-treatment	POS	R	Ofx	WT; WT	S531L	WT; WT	WT (L355L)	WT	G88A; WT	WT
DRC_121	2014	M	46	KH-12	Re-treatment	POS	HRSE	Ofx	S315T; WT	S531L	L88R; WT	WT	L35P	D94G; WT	WT
DRC_123	2014	F	23	KH-08	Re-treatment	NEG	S	Ofx	WT; WT	WT	L43R; WT	WT	WT	D94G; WT	WT
DRC_131	2013	F	63	KH-03	Re-treatment	POS	HR	Susceptible	WT; WT	S531L	WT; WT	WT (D534D)	WT	WT; I601L	WT
DRC_137	2013	F	76	KH-12	Re-Treatment	NEG	HRS	Ofx	S315T; WT	S531L	L43R; WT	WT	a-11g	D94G; A644D	WT
DRC_138	2013	M	27	KH-09	Re-Treatment	NEG	HRS	Susceptible	S315T; WT	D516V	L43R; WT	WT	WT	H87Q & D94G (I92I & S104S); A644D	WT
DRC_141	2013	M	71	KH-21	Re-treatment	NEG	H	Susceptible	F720S; c-15t	WT	WT; WT	WT	WT	WT; G512R	WT
DRC_146	2013	F	28	KH-10	Re-treatment	POS	HR	Susceptible	S315N; WT	H526D & M736T	WT; WT	D354A	a-11g	WT; G407C	WT
DRC_150	2013	M	50	KH-14	Re-treatment	POS	S	Ofx	WT; WT	WT	L43R; WT	WT	WT	D94G; WT	WT
DRC_177	2016	M	48	KH-08	Re-treatment	NEG	HRE	Ofx	S315T; WT	L511R & D516Y	WT; WT	M306L	V7G	D94G; WT	WT
DRC_195	2016	F	51	KH-12	Re-treatment	POS	HRE	Ofx	S315T; WT	L511R & D516Y	WT; WT	M306L	V7G	D94G; WT	WT
DRC_236	2012	M	30	KH-21	HIV/TB	POS	HR	Am, Km, Cm	S315T; WT	S531L	WT; WT	WT	WT	WT; WT	a1401g
DRC_251	2012	M	44	KH-12	Re-treatment	POS	HRSE	Ofx	S315T; WT	S531L	WT; a514c	M306V	D8E	D94G; WT	WT
DRI_008	2015	NA	NA	KH-12	NA	NEG	HS	Susceptible	S315T; WT	WT	L43R; WT	WT	WT	WT; A644D	WT
DRI_012	2014	F	43	KH-20	Re-treatment	POS	HR	Ofx	Deletion; WT	H526D	WT; WT	WT	WT	A90V; WT	WT
DRI_020	2014	M	53	KH-12	Re-treatment	POS	HRS	Ofx	S315T; WT	S531L	L43R; WT	Q497L	V131G	D94G (S104S); WT	WT
DRI_021	2014	M	52	KH-12	NA	NEG	S	Ofx	WT; WT	WT	L88R; WT	WT	WT	D94G; WT	WT
DRI_036	2013	F	80	KH-03	Re-treatment	NA	HR	Susceptible	S315T; WT	WT	WT; WT	WT	WT	WT; A432P	WT
DRI_062	2013	M	54	KH-03	Re-treatment	NA	H	Susceptible	S315T; WT	WT	WT; WT	WT	WT	WT; K600T	WT
DRI_070	2012	F	23	KH-03	Re-treatment	NA	HS	Susceptible	S315T; WT	WT	L43R; WT	WT	WT	WT; A644D	WT
DRI_151	2017	M	46	KH-12	Re-treatment	POS	HRSE	Ofx, Am, Km, Cm	S315T; WT	S531L	L88R; WT	F330S	T47P	D94G; WT	a1401g
DRI_154	2016	M	77	KH-12	Re-treatment	NA	H	Susceptible	WT; c-15t	WT	WT; WT	WT	WT	WT; WT	1204 ins a
DRI_189	2017	F	57	KH-07	Re-treatment	POS	HR	Susceptible	W149R; WT	S531L	WT; WT	WT	WT	D7N; A644D	WT

Notes: ^aPrimary subdivision codes of Cambodia: KH-01, Banteay Meanchey; KH-03, Kampong Cham; KH-07, Kampot; KH-08, Kandal; KH-09, Koh Kong; KH-10, Kratie; KH-12, Phnom Penh; KH-14, Prey Veng; KH-20, Svay Rieng; KH-21, Takeo; KH-25, Tbong Khmum; ^bSynonymous mutation is indicated by brackets.

Abbreviations: Am, amikacin; Cm, capreomycin; E, ethambutol; F, female; FQ, fluoroquinolones; H, isoniazid; Km, kanamycin; M, male; NA, not available; NEG, negative; Ofx, ofloxacin; POS, positive; R, rifampicin; S, streptomycin; TB, tuberculosis; WT, wild type (absence of non-synonymous mutation); Z, pyrazinamide.

Figure 1 Mycobacterium tuberculosis isolate collection and analytic methods.

Abbreviations: Am, amikacin; Cm, capreomycin; FLD, first-line drugs; FQ, fluoroquinolones; Km, kanamycin; M.tb, Mycobacterium tuberculosis; MDR, multidrug-resistant; Ofx, ofloxacin; SLID, second-line injectable drugs; TB, tuberculosis.

Table 2 Mutation Patterns in Genes Involved in Resistance to First- and Second-Line Drugs

Drug	Gene	Mutation Patterns in Drug Resistant Isolates^a	Mutation Patterns in Drug Susceptible Isolates^a
RIF	rpoB	D516V; H526D; H526R; H526Y; S531L; L511P & H526Q; H526D & M736T; L511R & D516Y; Q513-F514ins; S531L & L635P	I572F
INH	katG	W149R; S315T; S315N; W477L & A479P; Gene deletion	G560S
	inhA gene and promoter region	c-15t; c-15t and S94A
	katG + inhA	F720S + c-15t
EMB	embB	M306I; M306L; M306V; D328Y; F330S; F330V	D354A; G406A; Q497R; Q497L
STM	rpsL	K43R; K88R; K88T
	rrs-F1^b	a514c; c905a
Ofx	gyrA	G88A, A90V, S91P, D94A, D94G,
	gyrB		G407C^c, S413A^c, A432P^c, G512R^c, K600T^c, I601L^c, A644D^c
	gyrA + gyrB	D94G + A644D^a	D7Na + A644D^c, H87Q & D94G + A644D^c
SLID (Am, Km or Cm)	rrs-F2^d	a1401g, a1401g, c1489t	g1177a, 1204 ins a
PZA^e	pncA gene and promoter region	a-11g; V7G; D8E; L19P; L35P; T47P; H71Y; Y103C; S104R; V131G; A171E; A171V; partial pncA gene deletion

Notes: ^aDrug susceptibility patterns were obtained using the BACTEC MGIT 960 method used according to the standard critical antibiotic concentrations recommended by the WHO: RIF (1.0 µg/mL), INH (0.1 µg/mL), EMB (5.0 µg/mL), STM (0.1 µg/mL), Ofx (2.0 μg/mL); Am (1.0 μg/mL); Km (2.5 μg/mL), Cm (2.5 μg/mL); ^bSequencing targeting Loops 530 and 915 of the rrs gene; ^cMutation outside quinolone resistance-determining regions; ^dSequencing targeting the 1400–1500 regions of rrs gene; ^eSusceptibility to PZA is not available.

Abbreviations: Am, amikacin; Cm, capreomycin; EMB, ethambutol; INH, isoniazid; Km, kanamycin; Ofx, ofloxacin; PZA, pyrazinamide; RIF, rifampicin; SLID, second-line injectable agents; STM, streptomycin.

Table 3 Proportion of Resistance Phenotypes Among Beijing and Non-Beijing Genotypes

Phenotypic Drug Resistance	Beijing, n (%)^a	Non-Beijing, n (%)^a	Total, n (%)^a
XDR-TB	3 (100.0)	0	3
FQ-resistant; pre-XDR-TB	11 (78.6)	3 (21.4)	14
FQ-resistant; neither XDR- nor pre-XDR-TB	4 (66.7)	2 (33.3)	6
SLID-resistant; pre-XDR-TB	1 (50.0)	1 (50.0)	2
Total	19 (76.0)	6 (24.0)	25

Note: ^aProportion calculated by row.

Abbreviations: FQ, fluoroquinolone; SLID, second-line injectable drugs.

Figure 2 Phylogenetic analysis of spoligotyping and 24 loci MIRU-VNTR results of 23 Mycobacterium tuberculosis isolates resistant to SLD.

Abbreviations: Am, amikacin; Cm, capreomycin; Canetti: Mycobacterium canetti used as Outgroup; E, ethambutol; FLD, first-line drugs; H, isoniazid; Km, kanamycin; Ofx, ofloxacin; R, rifampicin; S, streptomycin; SLD, second-line drugs.