This study was supported by the Minas Gerais State Research Support Foundation (FAPEMIG) protocol number 65/10 and the National Council
for Scientific and Technological Development [Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq)] protocol number
310174/2014-7-CNPQ.Universidade Federal de Minas Gerais. Faculdade de Medicina. Grupo de Pesquisa em Micobactérias. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Medicina Grupo de Pesquisa em Micobactérias. Belo Horizonte, MG, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade Federal de Minas Gerais. Faculdade de Medicina Grupo de Pesquisa em Micobactérias. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Faculdade de Medicina Grupo de Pesquisa em Micobactérias. Belo Horizonte, MG, Brasil.Universidade Federal do Rio de Janeiro. Programa Acadêmico de Tuberculose. Rio de Janeiro, RJ, Brasil.Universidade Federal de Minas Gerais. Faculdade de Farmácia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Consultoria e Apoio Técnico. Belo Horizonte, MG, Brasil.Introduction: This study aimed to evaluate a new commercial kit, Kit SIRE Nitratase-PlastLabor, for testing the drug susceptibility
of clinical Mycobacterium tuberculosis isolates. Methods: The accuracy of the Kit SIRE Nitratase was evaluated by examining
the susceptibility (streptomycin, isoniazid, rifampicin, and ethambutol) of 40 M. tuberculosis isolates, using the proportion method
with Lowenstein-Jensen medium or the BACTEC MGIT 960 system. Results: The detection accuracy for streptomycin, isoniazid,
rifampicin, and ethambutol was 95%, 97.5%, 100%, and 80%, respectively. Conclusions: The exceptional accuracy demonstrated
by Kit SIRE Nitratase for isoniazid and rifampicin makes the kit an attractive option for screening M. tuberculosis strain resistance

Almeida, Isabela Neves de

Carvalho, Wânia da Silva

Figueiredo, Jamilly dos Reis de

Figueredo, Lida Jouca de Assis

Kritski, Afrânio Lineu

Lopes, Maria Luiza

Mansur, Maria de Fátima Filardi Oliveira

Miranda, Silvana Spindola de

Revista da Sociedade Brasileira de Medicina Tropical

English

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550Rev Soc Bras Med Trop 50(4):550-553, July-August, 2017doi: 10.1590/0037-8682-0447-2016Short CommunicationCorresponding author: Profa. Silvana Spíndola de Miranda. e-mail: silvanaspindola@gmail.comReceived 20 October 2016Accepted 4 May 2017Evaluation of the Commercial Kit SIRE Nitratase for detecting resistant Mycobacterium tuberculosis in BrazilSilvana Spindola de Miranda[1], Isabela Neves de Almeida[2], Maria Luiza Lopes[3],  Jamilly dos Reis de Figueiredo[2], Lida Jouca de Assis Figueredo[2], Afrânio Lineu Kritski[4], Wânia da Silva Carvalho[5] and Maria de Fátima Filardi Oliveira Mansur[6][1]. Grupo de Pesquisa em Micobacterioses, Faculdade de Medicina, Universidade Federal de Minas Gerais, REDE-TB, Belo Horizonte, MG, Brasil.  [2]. Grupo de Pesquisa em Micobactérias, Faculdade de Medicina, Universidade Federal de Minas Gerais, REDE-TB, Belo Horizonte, MG, Brasil.  [3]. Serviço de Vigilância em Saúde, Instituto Evandro Chagas, Ministério da Saúde, Ananindeua, PA, Brasil. [4]. Programa Acadêmico de Tuberculose, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil. [5]. Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil. [6]. Consultoria e Apoio Técnico, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil. AbstractIntroduction: This study aimed to evaluate a new commercial kit, Kit SIRE Nitratase-PlastLabor, for testing the drug susceptibility of clinical Mycobacterium tuberculosis isolates. Methods: The accuracy of the Kit SIRE Nitratase was evaluated by examining the susceptibility (streptomycin, isoniazid, rifampicin, and ethambutol) of 40 M. tuberculosis isolates, using the proportion method with Lowenstein-Jensen medium or the BACTEC MGIT 960 system. Results: The detection accuracy for streptomycin, isoniazid, rifampicin, and ethambutol was 95%, 97.5%, 100%, and 80%, respectively. Conclusions: The exceptional accuracy demonstrated by Kit SIRE Nitratase for isoniazid and rifampicin makes the kit an attractive option for screening M. tuberculosis strain resistance. Keywords: Tuberculosis. MDR. Nitrate reductase.Tuberculosis (TB) is one of the most important global public health concerns. An increasing incidence of multidrug resistant-tuberculosis (MDR-TB) cases (simultaneous resistance to isoniazid and rifampicin) has resulted in treatment failure and these drug-resistant strains have been able to spread into the community at large. Recently, extensively drug-resistant strains (additionally resistant to aminoglycosides and quinolones) have also been reported1. In terms of public health, the most important measure for controlling these events is the early detection of MDR-TB so that timely and appropriate treatment can be adopted1. Two types of methods are commonly used to detect MDR-TB: 1) phenotypic methods, such as proportion method (PM)-based drug susceptibility tests (DST), which measure the 1% proportion of culture growth with solid medium containing antibiotics at a critical concentration2; these are laborious and require several weeks to yield results. 2) Molecular-based methods, such as GeneXpert MTB/Rif (Cepheid) and GenoType MDR plus (BioMeriéux) that detect mutations known to be associated with phenotypic resistance to drugs2. The commercial automated BACTEC MGIT 960 system (Becton Dickinson Diagnostic Systems, Sparks, MD, USA) uses liquid media and is an expensive system requiring specific equipment and technical support1. Since 2009, The World Health Organization (WHO) has recommended the use of the noncommercial nitrate reductase assay (NRA) for the rapid screening of patients suspected of having contracted MDR-TB3. The NRA uses the detection of nitrate reduction as an indication of growth; therefore, results can be obtained more rapidly than by the visual detection of colonies. Tests results are available in 7 days for most isolates, which is comparable to the time required by the BACTEC liquid medium method and much faster than the solid medium proportion method, which requires 3 to 4 weeks4. This method was previously reported as a useful tool for the rapid and accurate detection of drug resistance to first-line antituberculous drugs2,4. However, the use of noncommercial tests has been associated with low reproducibility and lack of quality control, as reported for the NRA. Adaptation of the NRA as a commercial test may overcome these limitations; industrial production has been demonstrated to control quality and reproducibility at all the steps of the manufacturing process of an in-vitro Diagnostic (IVD) Kit for commercialization5. Approximately 30% of DST were performed in Brazil in 2015. Of the 1900 reported cases of MDR-TB, 1197 had a laboratory diagnosis. Reasons for non-performance of DST include difficulties with equipment maintenance and/or the availability of human resources to meet the demand1,6. One possible solution   551Miranda SS et al. - Commercial Kit SIRE Nitratase validationDrugs PM and MGIT 960 Kit SIRE Nitratase Sensitivity (%) Specificity (%) Accuracy (%)Drug susceptibility of the strainsStrains (n)Drug-resistant strainsDrug-susceptible strainsStreptomycina Resistant 15 14 1 93.3 96.0 95.0Susceptible 25 1 24 95%CI:80.7-100.0 95%CI: 88.3-100.0 95%CI: 88.7-100.0Isoniazidb Resistant 19 18 1 100.0 95.5 97.5Susceptible 21 0 21 95%CI: 86.8-100.0 95%CI: 92.7-100.0Rifampicin Resistant 19 19 0 100.0 100.0 100.0Susceptible 21 0 21Ethambutolc Resistant 13 7 6 77.8 80.6 80.0Susceptible 27 2 25 95%CI:50.6-100.0 995%CI: 66.7-94.6 95%CI: 67.6-92.4  TABLE 1The Kit SIRE Nitratase results (n= 40)SIRE: streptomycin, isoniazid, rifampicin, and ethambutol; PM: proportion method; MGIT 960: BACTEC MGIT 960 system; 95% CI: Confidence interval.  a:Kappa = 0.89 (very good). b:Kappa = 0.95 (very good) . c:Kappa = 0.50 (moderate).to this problem is the newly formed partnership between the Brazilian Tuberculosis Research Network (REDE-TB), the Federal University of Minas Gerais (UFMG), and PlastLabor Brazilian Industry, the consortium that originally initiated industrial production of the first Brazilian commercial IVD DST – Kit SIRE Nitratase. Thus, the aim of this study was to assess the accuracy of the Kit SIRE Nitratase. A total of 40 Mycobacterium tuberculosis clinical isolates were selected for this study from the strain collection of each laboratory (the Mycobacteria Research Laboratory of the UFMG Medical School and the Evandro Chagas Institute of the Brazilian Ministry of Health). Susceptibility tests were performed at the Tuberculosis Reference Laboratories of Minas Gerais and Pará, Brazil. DST were performed using the 1% proportion method on Lowenstein-Jensen (LJ) medium2 (DIFCO; Becton Dickinson Diagnostic Systems) and later adapted to the commercial automated BACTEC MGIT 960 system7 (Becton Dickinson Diagnostic Systems) with liquid medium; these were considered the gold standard methods for comparison with the Kit SIRE Nitratase. The participating laboratories were subjected to external quality control performed by ControLab 2002-2012, Brazil. This company certifies technical competence, credibility, and operational capacity to carry out tests according to ISO 9001 certification. The commercial Kit SIRE Nitratase was used according to the protocol previously described by Ängeby et al.4; the critical concentrations of isoniazid, rifampicin, streptomycin, and ethambutol in the LJ medium were 0.2µg/mL, 40µg/mL, 4µg/mL, and 2µg/mL, respectively. The LJ medium was prepared according to manufacturer’s specifications (DIFCO; Becton Dickinson Diagnostic Systems) with the addition of potassium nitrate (final concentration of 1mg/mL). Tubes without drugs were used as the growth control. The company was accredited and validated by the Brazilian National Agency of Sanitary Surveillance [Agência Nacional de Vigilância Sanitária (ANVISA)].Inoculum turbidity was adjusted to McFarland tube nº 1 and diluted 1:10; 0.2mL of the 1:10 dilution was used to inoculate three drug-free tubes and 0.2mL of the undiluted suspension (McFarland tube nº 1) was inoculated into tubes containing drugs. The inoculum originated from the first isolate.The tubes were incubated at 37 °C for 7 days. The reading included the preparation of a color-developing reagent mixture consisting of one part 50% (vol/vol) concentrated HCl, two parts 0.2% (wt/vol) sulfanilamide, and two parts 0.1% (wt/vol) n-1-naphthylethylenediamine dihydrochloride. These reagents were mixed separately immediately prior to being added to the tubes to detect growth. Following 7 days of incubation, 0.5mL of the reagent mixture was added to one of the three drug-free tubes. If any color change occurred, the corresponding antibiotic-containing tubes were developed by adding the same color-developing reagent mixture. If no color change was observed in the drug-free control tube, this tube was discarded and the other two tubes were reincubated. This procedure was repeated at day 10, using the second growth control tube and, when necessary, at day 14, using the third growth control tube. A strain was considered resistant if the color change in the tube containing the drug was greater than or equal to the color in the 1:10-diluted growth control tube4,8. The M. tuberculosis H37Rv reference strain, susceptible to all anti-tuberculosis drugs, as well as M. tuberculosis ATCC 35822 (INH-resistant), ATCC 35838 (RIF-resistant), ATCC 35837 (ETM-resistant), and ATCC 35820 (STR-resistant) quality control strains were used as control strains. The ATCC strains were tested in triplicate using the same lot. The specificity, sensitivity, accuracy, and Kappa value were calculated according to Godoy and Braile9. The Kit SIRE Nitratase showed 100% accuracy for the ATCC strains (Table 1). As shown in Table 1, the accuracy for the 40 tested clinical M. tuberculosis isolates was high for rifampicin and isoniazid (100% and 97.5%, respectively), but 552Rev Soc Bras Med Trop 50(4):550-553, July-August, 2017relatively lower for streptomycin and ethambutol (95% and 80%) compared with the gold standard methods (1% proportion method on LJ and the BACTEC MGIT 960 system). The time required for obtaining results ranged from 7 to 10 days. The concordance (Kappa) between the gold standard and the Kit SIRE Nitratase was very good for streptomycin, isoniazid, and rifampicin and moderate for ethambutol. These discrepant results for ethambutol have been reported in previous studies8,10. The Kit SIRE Nitratase was easy to perform and provided an affordable alternative method for the rapid detection of resistance to first-line antituberculous drugs in the 40 clinical M. tuberculosis strains. The accuracy values determined in this study are similar to those reported by other studies using noncommercial NRA2,4,8,11. A recent meta-analysis11 comprised of 38 studies showed that the SIRE Nitratase method exhibits high sensitivity and specificity in identifying isoniazid and rifampicin resistance10. Both the sensitivity and specificity values for ethambutol (variation of 55%-75% and 75%, respectively) and streptomycin (variation of 92-100% and 78%-99%, respectively)11 were lower in the present study. Streptomycin and ethambutol have been established as drugs that are difficult to test by conventional methods12. The accuracy for streptomycin reported in previous studies2,8 is lower than that obtained in this study (85.3% vs. 95%). A number of factors, such as different growth kinetics, cross-contamination, strain growth difficulties, and minimum inhibitory concentrations approaching the critical concentration, may have caused these discrepant results13. Nevertheless, the results obtained herein are quite promising, especially in terms of the drugs that need to be assayed by routine DST, isoniazid and rifampicin, which are the two most effective anti-TB drugs currently available for clinical practice and are at the core of the WHO’s first-line anti-TB regimen3. Additionally, the analytical performance of the Kit SIRE Nitratase demonstrates that it could reduce the turnaround time for drug susceptibility testing if applied directly to sputum smear-positive samples, as previously described10. Thus, studies evaluating the direct application of the SIRE Nitratase assay to sputum smear positive samples are necessary and will be performed in the near future. The turnaround time for results in our study (7-10 days) is consistent with the meta-analysis of noncommercial NRA studies conducted by Coban et al.11, which found that the mean turnaround time for test results ranged from 5 to 14 days. This study has some limitations. We did not perform the reproducibility test in triplicate on three different days; however, this is a preliminary study and 100% accuracy was obtained using the ATCC strains. Another limitation is that the Kit SIRE Nitratase has a short shelf life (30 days) compared to the BACTEC MGIT 960 system in which the drug is incorporated into the medium at the time of use7.In conclusion, the SIRE Nitratase assay constitutes a straightforward and affordable tool for the detection of phenotypic TB drug resistance in low-resource and developing countries with limited laboratory facilities. Its high accuracy, low cost, ease of operation, and lack of requirement for sophisticated equipment make it a sensible option for TB laboratories with limited resources; however, the SIRE Nitratase assay should be carried out at multiple locations. In a study conducted in South Africa, the cost of phenotypic DST using BACTEC MGIT 960 ranged from $20.10-$42.3714. These gold-standards methods, especially the automated BACTEC MGIT 960 system recently adapted for the determination of personalized minimum inhibitory concentrations7, are crucial for national TB reference laboratories to provide reliable information regarding the resistance levels of MDR-TB isolates to first- and second-line drugs; however, a simple and affordable test needs to be implemented in the network of regional and local TB laboratories in Brazil in close connection with the national TB reference laboratories, to increase the national DST coverage. Given the low coverage of DST in Brazil, the incorporation of this test in the public health system in Brazil is predicated to have a positive clinical and economic impact on TB control, especially that focused on phenotypic confirmation/validation of MDR-TB suspicions based on molecular results obtained from the nation-wide implementation of GeneXpert MTB/RIF15. The excellent accuracy of the SIRE Nitratase assay for isoniazid and rifampicin makes it an attractive option for screening M. tuberculosis strain resistance. AcknowledgementsThe authors wish to thank the Minas Gerais State Research Support Foundation (FAPEMIG) and the National Council for Scientific and Technological Development (CNPq) for their financial support. We also wish to thank the Graduate Department of the UFMG Medical School; REDE-TB; the Evandro Chagas Institute, Brazilian Ministry of Health; PlastLabor; and all participating laboratory employees. Moreover, we thank Prof. Miguel Viveiros from the Instituto Higiene Medicina Tropical of Nova Lisboa University, Portugal, for reviewing this manuscript.Conflict of interestThe authors declare that no conflicts of interest exist. Financial supportThis study was supported by the Minas Gerais State Research Support Foundation (FAPEMIG) protocol number 65/10 and the National Council for Scientific and Technological Development [Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)] protocol number 310174/2014-7-CNPQ. REFERENCES 1. World Health Organization (WHO). Global tuberculosis report 2016. Available from: who.int/tb/publications/global_report/en/.2. Lemus D, Martin A, Montoro E, Portaels F, Palomino JC. Rapid alternative methods for detection of rifampicin resistance in Mycobacterium tuberculosis. J Antimicrob Chemother. 2004;54(1):130-3. 3. World Health Organization (WHO). Non-Commercial culture methods and Mycobacteriophage-Based assays for rapid screening of patients at risk of drug-resistant tuberculosis. 2009. Available from: http://www.who.int/tb/publications/2009.   5534. Ängeby KA, Klintz L, Hoffner SE. Rapid and inexpensive drug susceptibilitytesting of Mycobacterium tuberculosis with a nitrate reductase assay. J Clin Microbiol. 2002;40(2):553-5.5. Perkins MD. New diagnostic tools for tuberculosis. Int J Tuberc Lung Dis. 2000;4(12 Suppl 2):S182-8.6. Augusto CJ, Carvalho WS, Gonçalves AD, Ceccato MGB, Miranda SS. Características da tuberculose no estado de Minas Gerais entre 2002 e 2009. J Bras Pneumol. 2013;39(3):357-64.7. Fonseca LS, Vieira GBO, Sobral LF, Ribeiro EO, Marsico AG. Comparative evaluation under routine conditions of the nitrate reduction assay, the proportion assay and the MGIT 960 assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis. Mem Inst Oswaldo Cruz. 2012;107(1):142-4. 8. Mansur MFO, Carvalho WS, Silva RB, Cata Preta RG, Fernandes Junior LA, Miranda SS. Evaluation of the nitrate reductase assay for the rapid detection of resistance to first-line medications in Mycobacterium tuberculosis strains isolated from patients in a general hospital. J Bras Pneumol. 2012;38(2):210-3.9. Godoy MF, Braile DM. Cálculos estatísticos básicos para testes diagnósticos. Versão 1999. [Internet]. Brasil; 1999 [acesso em 2011 jan. 20]. Disponível em: www.braile.com.br/download/download.htm10. Musa HR, Ambroggi M, Souto A, Ängeby KA. Drug susceptibility testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on microscopy-positive sputum samples. J Clin Microbiol. 2005;43(7):3159-61.11. Coban AY, Deveci A, Sunter AT, Martin A. Nitrate Reductas Assay for Rapid Detection of Isoniazid, Rifampin, Ethambutol, and Streptomycin Resistance in Mycobacterium tuberculosis: a Systematic Review and Meta-Analysis. J Clin Microbiol. 2014;52(1):15-9.12. Laszlo A, Rahman M, Espinal M, Raviglione M, WHO/IUATLD Network of Supranational Reference Laboratories. Quality assurance programme for drug susceptibility testing of Mycobacterium tuberculosis in the WHO/IUATLD Supranational Reference Laboratory Network: five rounds of proficiency testing, 1994-1998. Int J Tuberc Lung Dis. 2002;6(9):748-56. 13. Association of Public Health Laboratories. TB Drug Susceptibility Testing. Expert Panel Meeting Summary Report. Atlanta: Association of Public Health Laboratories; 2007; 16p.14. Shah M, Chihota V, Coetzee G, Churchyard G, Dorman ES. Comparison of laboratory costs of rapid molecular tests and conventional diagnostics for detection of tuberculosis and drug-resistant tuberculosis in South Africa. BMC Infect Dis. 2013;13:352.15. Agência Nacional de Vigilância Sanitária (ANVISA). Boletim Brasileiro de Avaliação de Tecnologia em Saúde (BRATS) nº 16 (versão 1.1). XPERT® MTB/RIF no Diagnóstico da Tuberculose Pulmonar. BRATS 2011;16. Miranda SS et al. - Commercial Kit SIRE Nitratase validation

Evaluation of the commercial kit SIRE Nitratase for detecting resistant Mycobacterium tuberculosis in Brazil

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Evaluation of the commercial kit SIRE Nitratase for detecting resistant Mycobacterium tuberculosis in Brazil

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