Frequency of first and second-line drug resistance-associated mutations among resistant <i>Mycobacterium tuberculosis</i> clinical isolates from São Paulo, Brazil

Matsui, Tania; Pinhata, Juliana Maíra Watanabe; Rabello, Michelle Christiane da Silva; Brandão, Angela Pires; Ferrazoli, Lucilaine; Leão, Sylvia Cardoso; Viana-Niero, Cristina; Oliveira, Rosangela Siqueira de

doi:10.1590/0074-02760200055

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ORIGINAL ARTICLE • Mem. Inst. Oswaldo Cruz 115 • 2020 • https://doi.org/10.1590/0074-02760200055 copy

Frequency of first and second-line drug resistance-associated mutations among resistant Mycobacterium tuberculosis clinical isolates from São Paulo, Brazil

Authorship SCIMAGO INSTITUTIONS RANKINGS

BACKGROUND

Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) has increased considerably worldwide.

OBJECTIVES

Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution.

METHODS

Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB).

FINDINGS

Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression.

MAIN CONCLUSIONS

The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.

Key words:
MDR-TB; pre-XDR-TB; XDR-TB; gyrA; gyrB; rrs

Characteristics	MDR-TB (n = 90)				pre-XDR-TB (n = 11)				XDR-TB (n = 5)
Characteristics	2016	2017	n	%	2016	2017	n	%	2016	2017	n	%
Gender (n = 106)
Male	35	30	65	72.2	4	1	5	45.5	2	-	2	40.0
Female	12	13	25	27.8	5	1	6	54.5	2	1	3	60.0
Ethnicity (n = 99)
White	16	19	35	42.2	3	1	4	36.4	3	1	4	80.0
Brown	21	14	35	42.2	4	1	5	45.5	1	-	1	20.0
Black	6	7	13	15.6	2	-	2	18.1	-	-	-	-
No information	4	3	7		-	-	-		-	-	-
Age (years) (n = 105)
15-29	11	10	21	23.6	3	-	3	27.3	-	1	1	20.0
30-59	31	22	53	59.6	5	2	7	63.6	3	-	3	60.0
≥ 60	5	10	15	16.8	1	-	1	9.1	1	-	1	20.0
No information	-	1	1		-	-	-		-	-	-
Mean ± SD	44.76 ± 15.10				39.45 ± 12.93				45 ± 13.81
Education level (years) (n = 86)
1 a 7	22	13	35	48,6	4	1	5	45.5	3	-		3	60.0
8 a 14	15	21	36	50.0	3	1	4	36.4	1	1		2	40.0
15 or more	-	1	1	1.4	2	-	2	18.1	-	-		-	-
No information	10	8	18						-	-		-
TB presentation (n = 104)
Pulmonary	44	40	84	95.4	9				-	-		-	2	11	100.0	4	1	5	100.0
Extrapulmonary	2	-	2	2.3	-	-	-		-	-		-	-
Pulmonary + Extrapulmonary	-	2	2	2.3	-	-	-		-	-		-	-
No information	1	1	2		-	-	-		-	-		-
HIV status (n = 99)
Negative	36	35	71	83.5	7	-	-		-	-		-	1	8	80.0	2	1	3	75.0
Positive	5	4	9	10.6	-	-	-	-	-	-		-	-
Not done	3	2	5	5.9	2	-	2	20.0	1	-		1	25.0
No information	3	2	5		-	1	1		1	-		1
Comorbidities (n = 102)
Alcohol abuse	2	1	3	3.4	-	1	1		1	-		1	-	-	-	-	-	-	-
Drug addiction	3	1	4	4.6	-	1	1	10.0	-	-		-	-
Smoking	9	7	16	18.4	1	-	1	10.0	-	-		-	-
Diabetes mellitus	4	6	10	11.5	1	-	1	10.0	-	-		-	-
Alcohol abuse, diabetes mellitus, drug addiction, smoking	17	6	23	26.4	2	-	2	20.0	2	-		2	40.0
None	8	14	22	25.3	4	-	4	40.0	2	1		3	60.0
Others	3	6	9	10.4	-	1	1	10.0	-	-		-	-
No information	1	2	3		1	-	1		-	-		-
Previous TB (n = 104)
No	20	15	35	39.8	3	-	1		-	-		-	2	5	45.5	-	1	1	20.0
Yes	26	27	53	60.2	6	-	6	54.5	4	-		4	80.0
No information	1	1	2		-	-	-		-	-		-
Treatment group (n = 104)
New case	20	15	35	39.8	3	-	-		-	-		-	2	5	45.5	-	1	1	20.0
Relapse	8	13	21	23.9	-	-	-	-	-	-		-	-
Retreatment	18	14	32	36.3	6	-	6	54.5	4	0		4	80.0
No information	1	1	2		-	-	-		-	-		-
Treatment outcome (n = 92)
Successful	29	30	59	76.6	4	-	-		-	-		-	1	5	50.0	2	1	3	60.0
Unsuccessful	11	7	18	23.4	4	1	5	50.0	2	-		2	40.0
No information	7	6	13		1	-	1		-	-		-
Death (n = 98)
No	38	35	73	88.0	5	-	1		-	-		-	2	7	63.6	3	1	4	80.0
Yes	6	4	10	12.0	3	-	4	36.4	1	-		1	20.0
No information	3	4	7		1	-	-		-	-		-

Instituto Oswaldo Cruz, Ministério da Saúde Av. Brasil, 4365 - Pavilhão Mourisco, Manguinhos, 21040-900 Rio de Janeiro RJ Brazil, Tel.: (55 21) 2562-1222, Fax: (55 21) 2562 1220 - Rio de Janeiro - RJ - Brazil
E-mail: memorias@fiocruz.br

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[1] + Corresponding author: cristina.viana@unifesp.br

Sequencing/target	BD BACTEC^TM MGIT^TM 960
	Rifampicin		Isoniazid
	Susceptible N (%)	Resistant N (%)	Susceptible N (%)	Resistant N (%)
Presence of mutation in rpoB	1 (0.65)	152 (97.4)	NA	NA
Absence of mutation in rpoB	1 (0.65)	2 (1.3)	NA	NA
Presence of mutation in katG	NA	NA	0	106 (67.9)
Presence of fabG-inhA mutation	NA	NA	0	20 (12.8)
Presence of mutation in katG and fabG-inhA	NA	NA	0	9 (5.8)
Absence of mutation in katG and fabG-inhA	NA	NA	0	21 (13.5)
Total	156 (100)		156 (100)

Mutation in rpoB gene/ Codon and amino acid change	Mutation in katG gene/ Codon and amino acid change	fabG-inhA region position and mutation	Number of isolates	%
Ser531Leu (TCG => TTG)	Ser315Thr (AGC => ACC)	NM	65	41.67
Ser531Leu (TCG => TTG)	NM	C -15 T	19	12.18
Ser531Leu (TCG => TTG)	NM	NM	15	9.62
Ser531Trp (TCG => TGG)	Ser315Thr (AGC => ACC)	NM	6	3.85
Ser531Leu (TCG => TTG)	Ser315Asn (AGC => AAC), Ala506Thr (GCC => ACC)	C -15 T	4	2.56
Ser531Trp (TCG => TGG)	Ser315Asn (AGC => AAC)	NM	2	1.28
Ser531Leu (TCG => TTG)	Asp259Gly (GAC => GGC)	NM	2	1.28
Ser531Leu (TCG => TTG)	Ser315Gly (AGC => GGC)	C -15 T	1	0.64
Ser531Leu (TCG => TTG)	Ser315Asn (AGC => AAC)	NM	1	0.64
Ser531Leu (TCG => TTG), Gln490Arg (CAG => CGG)	NM	C -15 T	1	0.64
Ser531Leu (TCG => TTG), Ile569Val (ATC => GTC)	Ser315Thr (AGC => ACC)	NM	1	0.64
His526Tyr (CAC => TAC)	Ser315Thr (AGC => ACC)	NM	4	2.56
His526Leu (CAC => CTC)	Ser315Thr (AGC => ACC)	G -17 T	3	1.92
His526Leu (CAC => CTC)	Ser315Thr (AGC => ACC)	NM	2	1.28
His526Cys (CAC => TGC)	Ser315Thr (AGC => ACC)	NM	2	1.28
His526Asp (CAC => GAC)	NM	NM	1	0.64
His526Asn (CAC => AAC)	NM	NM	1	0.64
His526Arg (CAC => CGC)	Ser315Thr (AGC => ACC)	NM	1	0.64
His526Asn (CAC => AAC), Phe505Leu (TTC => TTA)	Ser315Thr (AGC => ACC)	C -15 T	1	0.64
Asp516Val (GAC => GTC)	Ser315Thr (AGC => ACC)	NM	12	7.70
Asp516Tyr (GAC => TAC)	Ser315Thr (AGC => ACC)	NM	1	0.64
Asp516Tyr (GAC => TAC)	NM	NM	1	0.64
Asp516Ile (GAC => ATC)	Ser315Thr (AGC => ACC)	NM	1	0.64
Gln513Glu (CAA => GAA)	Ser315Thr (AGC => ACC)	NM	1	0.64
Ser522Leu (TCG => TTG)	Ser315Thr (AGC => ACC), Gly273Gly (GGT => GGC)	NM	1	0.64
Leu533Pro (CTG => CCG)	Ser315Thr (AGC => ACC)	NM	1	0.64
Ile572Tyr (ATC => TAC)	Ser315Thr (AGC => ACC)	NM	1	0.64
codon 518 deletion (AAC => del)	Asp381Asn (GAC => AAC)	NM	1	0.64
518 and 517 Codons deleted Asp516Asn (GAC => AAC) Met515Gln (ATG => CAG)	Ser315Thr (AGC => ACC)	NM	1	0.64
NM	NM	NM	3	1.92
Total			156	100,00

Resistance profile	Mutations Codon and amino acid change			Number of isolates	%
Resistance profile	gyrA	gyrB	rrs	Number of isolates	%
Pre-XDR	Ala90Val (GCG => GTG),	NM	NM	4	14.8
	Ser91Pro (TCG => CCG),	NM	NM	4	14.8
	Asp94Gly (GAC => GGC),	NM	NM	11	40.7
	Asp94Asn (GAC => AAC),	NM	NM	3	11.1
	Asp94Asn (GAC => AAC),	Lys526Lys (AAG => AAA)	NM	1	3.7
	NM^#	Asn499Asp (AAC => GAC)	NM	2	7.4
	NM^#	NM	NM	2*	7.4
	Total			27	100
XDR	Gly88Ala (GGC => GCC)	NM	A1401G	1	7.1
	Asp94Gly (GAC => GGC),	NM	A1401G	5	35.7
	NM^#	Asp449Val (GAT => GTT)	A1401G	4	28.6
	Asp94Gly (GAC => GGC, NM^#	NM	NM	2	14.3
	NM^#	Asn499Asp (AAC => GAC)	NM	2	14.3
	Total			14	100