Detection of Drug Resistant Mycobacterium Tuberculosis Strains Using Kit SIRE Nitratase®: a Multicenter Study

The Commercial Kit SIRE Nitratase^® PlastLabor, is a drug susceptibility test kit used to detect Mycobacterium tuberculosis resistance to first-line TB treatment drugs. The present study aimed at evaluating its performance in a multicenter study.

(2) Methods:

To determine its accuracy, the proportion methods in Lowenstein Jensen medium or the BACTEC^TMMGIT^TM960 system was used as a gold standard.

(3) Results:

The study revealed that the respective accuracies of the kit with 190 M. tuberculosis clinical isolates, using the proportion methods in Lowenstein Jensen medium or BACTEC^TMMGIT^TM960 system as a gold standard, were 93.9% and 94.6%, 96.9% and 94.6%, 98.0% and 97.8%, and 98.0% and 98.9%, for streptomycin, isoniazid, rifampicin, and ethambutol, respectively.

(4) Conclusion:

Thus, the kit can rapidly screen resistance to streptomycin, isoniazid, rifampicin, and ethambutol. Additionally, it does not require sophisticated equipment; hence, it can be easily used in the laboratories of low and middle income countries.

Keywords:
tuberculosis; MDR; nitrate reductase; technology

Authorship

Silvana Spindola de Miranda ^**Correspondence: silvanaspindola@gmail.com; Tel.: +55-31-988217283

Federal University of Minas Gerais, Faculty of Medicine, Mycobacteria Research Laboratory, Belo Horizonte, Minas Gerais, BrazilFederal University of Minas GeraisBrazilBelo Horizonte, Minas Gerais, BrazilFederal University of Minas Gerais, Faculty of Medicine, Mycobacteria Research Laboratory, Belo Horizonte, Minas Gerais, Brazil

http://orcid.org/0000-0001-7245-4472

Isabela Neves de Almeida

http://orcid.org/0000-0001-6152-7648

Maria de Fátima Filardi Oliveira Mansur

http://orcid.org/0000-0002-2414-0273

Lida Jouca de Assis Figueredo

http://orcid.org/0000-0001-5355-0784

Wânia da Silva Carvalho

Federal University of Minas Gerais, Faculty of Pharmacy, Department of Social Pharmacy, Belo Horizonte, Minas Gerais, BrazilFederal University of Minas GeraisBrazilBelo Horizonte, Minas Gerais, BrazilFederal University of Minas Gerais, Faculty of Pharmacy, Department of Social Pharmacy, Belo Horizonte, Minas Gerais, Brazil

http://orcid.org/0000-0002-2575-6352

João Paulo Amaral Hadaad

Federal University of Minas Gerais, Veterinary School, Department of Preventive Veterinary Medicine, Belo Horizonte, Minas Gerais, BrazilFederal University of Minas GeraisBrazilBelo Horizonte, Minas Gerais, BrazilFederal University of Minas Gerais, Veterinary School, Department of Preventive Veterinary Medicine, Belo Horizonte, Minas Gerais, Brazil

http://orcid.org/0000-0003-2823-6288

Jaciara de Lourdes do Carmo Guimarães Diniz

Federal University of Rio Grande, Faculty of Medicine, Laboratory of Mycobacteria, Rio Grande, Rio Grande do Sul, BrazilFederal University of Rio GrandeBrazilRio Grande, Rio Grande do Sul, BrazilFederal University of Rio Grande, Faculty of Medicine, Laboratory of Mycobacteria, Rio Grande, Rio Grande do Sul, Brazil

http://orcid.org/0000-0001-6210-5658

Andrea von Groll

http://orcid.org/0000-0002-6727-372X

Pedro Almeida da Silva

http://orcid.org/0000-0003-1666-1295

Maria Luiza Lopes

Oswaldo Cruz Foundation, Evandro Chagas Institute, Ananideua, Pará, BrazilOswaldo Cruz FoundationBrazilAnanideua, Pará, BrazilOswaldo Cruz Foundation, Evandro Chagas Institute, Ananideua, Pará, Brazil

http://orcid.org/0000-0001-6894-1366

Marcelo Cordeiro dos Santos

Tropical Medicine Foundation Dr. Heitor Vieira Dourado, Manaus, Amazonas, BrazilTropical Medicine Foundation Dr. Heitor Vieira DouradoBrazilManaus, Amazonas, BrazilTropical Medicine Foundation Dr. Heitor Vieira Dourado, Manaus, Amazonas, Brazil

http://orcid.org/0000-0002-7140-7145

Alexandra Brito

http://orcid.org/0000-0002-9036-702X

Fernanda Carvalho de Queiroz Mello

Federal University of Rio de Janeiro, Institute of Chest Diseases, Clementino Fraga Filho University Hospital, Rio de Janeiro, Rio de Janeiro, BrazilFederal University of Rio de JaneiroBrazilRio de Janeiro, Rio de Janeiro, BrazilFederal University of Rio de Janeiro, Institute of Chest Diseases, Clementino Fraga Filho University Hospital, Rio de Janeiro, Rio de Janeiro, Brazil

http://orcid.org/0000-0003-3250-6738

Thiago da Silva Santos Malaquias

http://orcid.org/0000-0001-8783-8962

Julio Croda

Federal University of Grande Dourados, Faculty of Health Sciences, Dourados, Mato Grosso do Sul, BrazilFederal University of Grande DouradosBrazilDourados, Mato Grosso do Sul, BrazilFederal University of Grande Dourados, Faculty of Health Sciences, Dourados, Mato Grosso do Sul, Brazil

Oswaldo Cruz Foundation, Campo Grande, Mato Grosso do Sul, BrazilOswaldo Cruz FoundationBrazilCampo Grande, Mato Grosso do Sul, BrazilOswaldo Cruz Foundation, Campo Grande, Mato Grosso do Sul, Brazil

http://orcid.org/0000-0002-6665-6825

Juliana Maira Watanabe Pinhata

Adolfo Lutz Institute, Bacteriology Center, Tuberculosis and Mycobacteriosis Center, São Paulo, São Paulo, BrazilAdolfo Lutz InstituteBrazilSão Paulo, São Paulo, BrazilAdolfo Lutz Institute, Bacteriology Center, Tuberculosis and Mycobacteriosis Center, São Paulo, São Paulo, Brazil

http://orcid.org/0000-0001-5758-6688

Rosângela Siqueira de Oliveira

http://orcid.org/0000-0002-5188-8367

Erica Chimara

http://orcid.org/0000-0001-9574-8449

Maria Lúcia Rossetti

State Secretariat of Health of Rio Grande do Sul, State Center for Health Surveillance, Center for Scientific and Technological Development, Porto Alegre, Rio Grande do Sul, BrazilState Secretariat of Health of Rio Grande do SulBrazilPorto Alegre, Rio Grande do Sul, BrazilState Secretariat of Health of Rio Grande do Sul, State Center for Health Surveillance, Center for Scientific and Technological Development, Porto Alegre, Rio Grande do Sul, Brazil

http://orcid.org/0000-0002-9672-9394

Maria Laura Halon

http://orcid.org/0000-0001-5866-0827

Maria Cristina Lourenço

Oswaldo Cruz Foundation, National Institute of Infectology Evandro Chagas, Laboratory of Bacteriology and Bioassays of Rio de Janeiro, Rio de Janeiro, BrazilOswaldo Cruz FoundationBrazilRio de Janeiro, BrazilOswaldo Cruz Foundation, National Institute of Infectology Evandro Chagas, Laboratory of Bacteriology and Bioassays of Rio de Janeiro, Rio de Janeiro, Brazil

http://orcid.org/0000-0003-0382-9108

Reginalda Ferreira de Melo Medeiros

Sergio Arouca National Public Health School, Professor Hélio Fraga Reference Center, Rio de Janeiro, Rio de Janeiro, BrazilSergio Arouca National Public Health SchoolBrazilRio de Janeiro, Rio de Janeiro, BrazilSergio Arouca National Public Health School, Professor Hélio Fraga Reference Center, Rio de Janeiro, Rio de Janeiro, Brazil

http://orcid.org/0000-0001-9357-4932

Fátima Cristina Onofre Fandinho Montes

http://orcid.org/0000-0003-3896-3526

Diana Machado

NOVA University of Lisbon, Institute of Hygiene and Tropical Medicine, Medical Microbiology Unit, Global Health and Tropical Medicine, Lisboa, PortugalNOVA University of LisbonPortugalLisboa, Portugal NOVA University of Lisbon, Institute of Hygiene and Tropical Medicine, Medical Microbiology Unit, Global Health and Tropical Medicine, Lisboa, Portugal

http://orcid.org/0000-0002-6740-2632

Miguel Viveiros

http://orcid.org/0000-0001-9676-6251

Afrânio Lineu Kritski

http://orcid.org/0000-0002-5900-6007

*Correspondence: silvanaspindola@gmail.com; Tel.: +55-31-988217283

Conflicts of Interest:

The Kit SIRE Nitratase® is a commercial test develop after a consortium formed between the Brazilian Tuberculosis Research Network (REDE-TB) at UFMG and PlastLabor©. The company Plast labor the amount of R$ 9.684,44 for the transfer of distributed technology as follows: R$ 3.000 of extension bag (Silvana and Maria de Fátima); R$ 3.300,00 of Autonomous; R$ 1.500,00 of consumables; R$ 193,79 UFMG 2%, R$ 484,47 Unity 5%; R$ 484,47 of Departament 5%; R$ 726,71 of Fundep 7,5%. During the multicenter validation, Isabela Neves de Almeida has received R$ 10.000 in order to financial support for travel to meetings relevant to the study or other reasons.

SCIMAGO INSTITUTIONS RANKINGS

Oswaldo Cruz Foundation, Campo Grande, Mato Grosso do Sul, BrazilOswaldo Cruz FoundationBrazilCampo Grande, Mato Grosso do Sul, BrazilOswaldo Cruz Foundation, Campo Grande, Mato Grosso do Sul, Brazil

Federal University of Rio de Janeiro, Faculty of Medicine, Tuberculosis Research Center, Rio de Janeiro, Rio de Janeiro, BrazilFederal University of Rio de JaneiroBrazilRio de Janeiro, Rio de Janeiro, BrazilFederal University of Rio de Janeiro, Faculty of Medicine, Tuberculosis Research Center, Rio de Janeiro, Rio de Janeiro, Brazil

Figures | Tables

Figures (3)
Tables (4)

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Figure 1
The Kit SIRE Nitratase^® components

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Figure 2
The time to obtain results by Kit SIRE Nitratase^®

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Figure 3
Interpretation of results of DST by Kit SIRE Nitratase^®

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Table 1
Drug susceptibility testing using Kit SIRE Nitratase^®; sensitivity, specificity, and accuracy using the Lowenstein Jensen medium as a standard (n = 98)

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Table 2
Drug susceptibility testing using Kit SIRE Nitratase^®; sensitivity, specificity, and accuracy using the BACTEC^TM MGIT^TM 960 system as a standard (n = 92)

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Table 3
The agreement rates according to the site of Kit SIRE Nitratase^® use

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Table 4
Kit SIRE Nitratase^® repeatability test results with proficiency strains.

Figure 1 The Kit SIRE Nitratase^® components

Legend: (a) = Kit SIRE Nitratase^® complete; (b) = Control tubes (without drug); (c) = SIRE tubes: a LJ+KNO₃ medium with S (STR): streptomycin, I (INH): isoniazid, R (RIF): rifampicin, E (EMB): ethambutol, respectively.

Figure 2 The time to obtain results by Kit SIRE Nitratase^®

Legend: In 72% stands for 136 of 190 clinical isolates of M. tuberculosis; 21% stands for 40 of 190 clinical isolates of M. tuberculosis; 7% stands for 14 of 190 clinical isolates of M. tuberculosis.

Figure 3 Interpretation of results of DST by Kit SIRE Nitratase^®

Legend: A = Test of a susceptible strain. B = Test of a strain resistant to isoniazid and rifampicin. C = Test of a strain resistant to all tested drugs. C*: control tube (without drug); S (STR): streptomycin, I (INH): isoniazid, R (RIF): rifampicin, E (EMB): ethambutol. An isolate was considered resistant to the antibiotic at the critical concentration if the color change in the tube containing the drug was darker or equal to the color developed in the 1:10 growth control tube.

Table 1 Drug susceptibility testing using Kit SIRE Nitratase^®; sensitivity, specificity, and accuracy using the Lowenstein Jensen medium as a standard (n = 98)

Drug	Drug susceptibility profile by NRA	Proportion Method using Lowenstein Jensen		Sensitivity (%)	Specificity (%)	Accuracy (%)
		Resistant	Susceptible
Streptomycin^a	Resistant	33	6	100	90.8	93.9
	Susceptible	0	59
Isoniazid^b	Resistant	41	3	100	94.7	96.9
	Susceptible	0	54
Rifampicin^c	Resistant	28	0	93.3	100	98
	Susceptible	2	68
Ethambutol^d	Resistant	17	0	89.5	100	98
	Susceptible	2	79

Legend: a: Kappa = 0.87 (very good); b: Kappa = 0.94 (very good); c: Kappa = 0.95 (very good); d: Kappa = 0.93 (very good). Included sites: Pará/IEC, Manaus/FMT, IPEC/Fiocruz, IDT/UFRJ, and CDCT/RS.

Table 2 Drug susceptibility testing using Kit SIRE Nitratase^®; sensitivity, specificity, and accuracy using the BACTEC^TM MGIT^TM 960 system as a standard (n = 92)

Drug	Drug susceptibility profile by NRA	BACTEC MGIT 960		Sensitivity (%)	Specificity (%)	Accuracy (%)
		Resistant	Susceptible
Streptomycin^a	Resistant	33	5	100	91.5	94.6
	Susceptible	0	54
Isoniazid^b	Resistant	38	0	88.4	100	94.6
	Susceptible	5	49
Rifampicin^c	Resistant	33	0	94.3	100	97.8
	Susceptible	2	57
Ethambutol^d	Resistant	25	1	100	98.5	98.9
	Susceptible	0	66

Legend: a: Kappa = 0.89 (very good); b: Kappa = 0.89 (very good); c: Kappa = 0.95 (very good); d: Kappa = 0.97 (very good). Included sites: Adolfo Lutz/SP, CRPHF/Fiocruz, Dourados/UFGD, and IHMT/Lisboa.

Table 3 The agreement rates according to the site of Kit SIRE Nitratase^® use

Kappa Agreement values* Standard Method - Proportion Method using the BACTEC^TMMGIT^TM960 system
Site	S	I	R	E
IAL/SP	0.82	1.00	1.00	0.83
CRHF/RJ	0.80	0.92	0.91	0.78
LPCS/MS	0.69	0.89	0.64	-----^a
IHMT/PT	0.83	1.00	1.00	0.82
Kappa Agreement values* Standard Method - Proportion Method using the Lowenstein Jensen medium
Site	S	I	R	E
IEC/PA	1.00	1.00	0.83	0.51
IDT/RJ	0.91	1.00	1.00	0.78
IPEC/RJ	0.69	0.74	1.00	0.86
FMT/AM	0.73	0.76	1.00	-----^a
CDCT/RS	0.71	-----^b	1.00	0.53

Legend: IAL/SP, Instituto Adolfo Lutz, São Paulo; CRHF/RJ, Centro de Referência Hélio Fraga, Fundação Oswaldo Cruz, Rio de Janeiro; IDT/RJ, Instituto de Doença do Tórax da Universidade Federal do Rio de Janeiro, Rio de Janeiro; IPEC/RJ, Instituto Nacional de Doenças Infecciosas, Fundação Oswaldo Cruz, Rio de Janeiro; IEC/PA, Instituto Evandro Chagas, Fundação Oswaldo Cruz, Pará; FMT/AM, Fundação de Medicina Tropical, Amazonas; CDCT/RS, Centro de Desenvolvimento Científico e Tecnológico, Centro Estadual de Vigilância em Saúde, Secretaria Estadual da Saúde do Rio Grande do Sul, Rio Grande do Sul; LPCS/MS, Laboratório de Pesquisa em Ciências da Saúde, Faculdade de Medicina da Universidade Federal da Grande Dourados, Mato Grosso do Sul. IHMT/PT, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Portugal.
*Kappa <0.20 (poor); 0.21-0.40 (weak); 0.41-0.60 (moderate); 0.61-0.80 (good); > 0.80 (very good). S (STR), streptomycin; I (INH), isoniazid; R (RIF), rifampicin; E (EMB), ethambutol.
^a
, Kappa not calculated (strains susceptible only to ethambutol).
^b
, Kappa not calculated (strains resistant only to isoniazid).

Table 4 Kit SIRE Nitratase^® repeatability test results with proficiency strains.

Drug	Triple Kappa	Interpretation of triple Kappa*
S	0.83	very good
I	0.81	very good
R	0.88	very good
E	0.30	weak

Legend:*Kappa < 0.20 (poor); 0.21-0.40 (weak); 0.41-0.60 (moderate); 0.61-0.80 (good); >0.80 (very good); S (STR), streptomycin; I (INH), isoniazid; R (RIF), rifampicin; E (EMB), ethambutol.

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Detection of Drug Resistant Mycobacterium Tuberculosis Strains Using Kit SIRE Nitratase®: a Multicenter Study

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[1] *Correspondence: silvanaspindola@gmail.com; Tel.: +55-31-988217283