Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus

Santos, Paula Fernanda Gonçalves dos; Costa, Elis Regina Dalla; Ramalho, Daniela M; Rossetti, Maria Lucia; Barcellos, Regina Bones; Nunes, Luciana de Souza; Esteves, Leonardo Souza; Rodenbusch, Rodrigo; Anthony, Richard M; Bergval, Indra; Sengstake, Sarah; Viveiros, Miguel; Kritski, Afrânio; Oliveira, Martha M

doi:10.1590/0074-02760160376

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ARTICLES • Mem. Inst. Oswaldo Cruz 112 (6) • June 2017 • https://doi.org/10.1590/0074-02760160376 copy

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype^®MTBDRplus

Authorship SCIMAGO INSTITUTIONS RANKINGS

BACKGROUND

To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed.

OBJECTIVE

To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr).

METHOD

96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays.

RESULTS

With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)].

CONCLUSION

Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

tuberculosis; MLPA; GenoTypeMTBDR^®plus; isoniazid resistance; rifampicin resistance; multidrug resistance

Probe	Target-specific sequence	Length (bp)	Target or information provided
embB-306	GTCGGACGACGGCTACATCCTGGGCATggcccgagtcgc cgaccacgccggctac	142	EMB resistance marker
katG-315	caccggaaccggtaaggacgcgatcaccaCCGGCATCGAGGTCGT ATGGACGAACACCCC	160	INH resistance marker
inhA-15	CGATTTCGGCCCGGCCGCGGCGAGATgataggttgtcggg gtgactgccacagcc	178	INH resistance marker
16S rRNA	CACGGGATGCATGTCTTGTGGTGGAAAgcgctttagcggt gtgggatgagcccgcggc	202	16S rRNA gene, MTBC specific
rpoB-176	cacgttcatcatcaacgggaccgagcgtgtggtgTTCAGCCAGCTGGT GCGGTCGCCC	229	RIF resistance marker
rpoB-531	GTTGACCCACAAGCGCCGACTGTTggcgctggggcccggcg gtctgtcacgt	256	RIF resistance marker
rpoB-526G	caacccgctgtcggggttgaccGACAAGCGCCGACTGTCGGCG CTGGGGCC	265	RIF resistance marker
rpoB-526T	caacccgctgtcggggttgaccTACAAGCGCCGACTGTCGGCG CTGGGGCC	274	RIF resistance marker
rpoB-522	agccaattcatggaccagaacaacccgctgtTGGGGTTGACCCACA AGCGCCGAC	283	RIF resistance marker
katG-463	GATTGCCAGCCTTAAGAGCCAGATCCGggcatcgggatt gactgtctcacagctagtttcgacc	319	Genotype marker, specific for PGG two and three
gyrA-95	GCACGGCGACGCGTCGATCTACGACACcctggtgcgcat ggcccagccctgg	328	Genotype marker, specific for PGG one and two
mutT2-58	CCCGAGAGCTCGCCGAAGAACTGCgactcgaggtcgccga cctcgcggtggg	355	Genotype marker, specific for Beijing two
mutT4-48	CGACCCCGGCAACGGCGAAGGggtcccggtcccgctcacctcg tcgcgggt	364	Genotype marker, specific for Beijing one, two and three
ogt-12	CGCACCATCGATAGCCCCATCGGAccattaaccctggccggg catggctcggtgttga	373	Genotype marker, specific for Beijing two
ogt-15	taccgcaccatcgatagccccatcgggccattaaGCCTGGCCGGGCAT GGCTCGGTGTTGA	382	Genotype marker, specific for Haarlem
ogt-37	cctgcggatgctcgagcagacgtatgagccaagccTCACACACTGGAC ACCCGACCCC	391	Genotype marker, specific for Beijing three and four
TbD1	GCGGTCGCGGGATTCAGCGTCTATcggttgcacggcatcttc ggctcgcacgaca	418	Absent in modern Mycobacterium tuberculosis strains

N^o of samples	DST		Sequencing			GenoType MTBDRplus (A)			MLPA (B)

	INH	RIF	katG	inhA	rpoB	katG	inhA	rpoB	katG	inhA	rpoB
79	S	S	WT	WT	WT	WT	WT	WT	WT	WT	WT
4	R	R	S315T	WT	S531L	S315T	WT	S531L	S315T	WT	S531L
4	R	S	S315T	WT	WT	S315T	WT	WT	S315T	WT	WT
1	R	R	S315T	WT	D516V/H526Y/S531L	WT	WT	H526Y/S531L	WT	WT	S531L
1	R	R	S315T	WT	D516V/S531L	S315T	WT	D516V	S315T	WT	WT
1	R	R	S315T	WT	D516V/S531L	S315T	WT	D516V	S315T	WT	S531L
1	R	S	WT	(-15C →T)	WT	WT	WT	WT	WT	(-15C →T)	WT
1	S	R	WT	WT	S531L	WT	WT	S531L	WT	WT	S531L
1	S	S	WT	WT	WT	S315T	WT	WT	WT	WT	WT
1	S	S	WT	WT	WT	S315T	WT	D516V	WT	WT	WT
1	R	S	S315T	WT	WT	S315T	WT	WT	S315T	WT	WT
1	R	S	S315T	WT	WT	S315T	WT	WT	S315T	WT	WT

Methods	INH		Assessment characteristic (%)			RIF		Assessment characteristic (%)

	Nº of strains (%)					Nº of strains (%)

	Resistant (n = 14)	Susceptible (n = 82)	Sensitivity	Specificity	Kappa (95% CI )	Resistant (n = 8)	Susceptible (n = 88)	Sensitivity	Specificity	Kappa (95% CI)
MTBDRplus (A)
Mutant	12	2	85.7 (59 - 97)	97.5 (91 - 99)	0.83 (0.75 - 0.91)	8	1	100 (63 - 100)	98.8 (96 - 100)	0.93 (0.88 - 0.98)
WT	2	80	85.7 (59 - 97)	97.5 (91 - 99)	0.83 (0.75 - 0.91)	0	87	100 (63 - 100)	98.8 (96 - 100)	0.93 (0.88 - 0.98)
MLPA (B)
Mutant	13	0	92.8 (66 - 99)	100 (95 - 100)	0.95 (0.91 - 0.100)	7	0	85.7 (51 - 99)	100 (99 - 100)	0.92 (0.87 - 0.98)
WT	1	82	92.8 (66 - 99)	100 (95 - 100)	0.95 (0.91 - 0.100)	1	88	85.7 (51 - 99)	100 (99 - 100)	0.92 (0.87 - 0.98)

Methods	INH		Assessment characteristic (%)			RIF		Assessment characteristic (%)

	Nº of strains (%)					Nº of strains (%)

	Resistant (n = 14)	Susceptible (n = 82)	Sensitivity (95% CI)	Specificity (95% CI)	Kappa (95% CI)	Resistant (n = 8)	Susceptible (n = 88)	Sensitivity (95% CI)	Specificity (95% CI)	Kappa (95% CI)
MTBDRplus (A)
Mutant	12	2	85.7 (59 - 97)	97.5 (91 - 99)	0.83 (0.75 - 0.91)	8	1	100 (63 - 100)	98.8 (96 - 100)	0.93 (0.88 - 0.98)
WT	2	80	85.7 (59 - 97)	97.5 (91 - 99)	0.83 (0.75 - 0.91)	0	87	100 (63 - 100)	98.8 (96 - 100)	0.93 (0.88 - 0.98)
MLPA (B)
Mutant	13	0	92.8 (66 - 99)	100 (95 - 100)	0.95 (0.91 - 1.00)	7	0	87.5 (51 - 99)	100 (99 - 100)	0.92 (0.87 - 0.98)
WT	1	82	92.8 (66 - 99)	100 (95 - 100)	0.95 (0.91 - 1.00)	1	88	87.5 (51 - 99)	100 (99 - 100)	0.92 (0.87 - 0.98)

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
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[1] + Corresponding author: dallacostaer@gmail.com