{"references": ["WHO. Global Tuberculosis Report 2019. Geneva: World Health Organization; 2019.", "Huard RC, de Oliveira Lazzarini LC, Butler WR, van Soolingen D, Ho JL. PCR-Based Method to Differentiate the Subspecies of the Mycobacterium tuberculosis Complex on the Basis of Genomic Deletions. J Clin Microbiol. 2003; 41(4): p. 1637-1650.", "Clarridge JE, 3rd. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev. 2004; 17(4): p. 840-862.", "Kim BJ, Lee SH, Lyu MA, Kim SJ, Chae GT, Kim EC, et al. Identification of Mycobacterial Species by Comparative Sequence Analysis of the RNA Polymerase Gene (rpoB). J Clin Micorb. 1999; 37(6): p. 1714-1720.", "Huang CC, Chen JH, Hu ST, Chiou CS, Huang WC, Hsu JY, et al. Combined rpoB duplex PCR and hsp65 PCR restriction fragment length polymorphism with capillary electrophoresis as an effective algorithm for identification of Mycobacterial species from clinical isolates. BMC Microbiol. 2012; 12: p. 137.", "Jang MA, Koh WJ, Huh HJ, Kim SY, Jeon K, Ki C S, et al. Distribution of Nontuberculous Mycobacteria by Multigene Sequence Based Typing and Clinical Significance of Isolated Strains. J Clin Microb. 2014; 52(4): p. 1207-1212.", "Kim SY, Shin SJ, Lee NY, Koh WJ. First Case of Pulmonary Disease Caused by a Mycobacterium avium Complex Strain of Presumed Veterinary Origin in an Adult Human Patient. J Clin Microb. 2013; 51(6): p. 1993-1995.", "Phelippeau M, Osman DA, Musso D, Drancourt M. Epidemiology of Nontuberculous Mycobacteria in French Polynesia. J Clin Microbiol. 2015; 53(12): p. 3798-3804.", "Roychowdhury T, Mandal S, Bhattacharya A. Analysis of IS6110 insertion sites provide a glimpse into genome evolution of Mycobacterium tuberculosis. Sci Rep. 2015; 5:p. 12567.", "Barr\u00f3n H, Monteghirfo M, Rivera N. Diagn\u00f3stico molecular de Mycobacterium tuberculosis en biopsias pleurales embebidas en parafina. An Fac Med Lima. 2006; 67(1): p. 11-18.", "Thierry D, Cave MD, Eisenach KD, Crawford JT, Bates JH, Gicquel B, et al. IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res. 1990; 18(1): p. 188.", "Nghiem MN, Nguyen BV, Nguyen ST, Vo TT, Nong HV. A Simple, Single Triplex PCR of IS6110, IS1081, and 23S Ribosomal DNA Targets, Developed for Rapid Detection and Discrimination of Mycobacterium from Clinical Samples. J Microbiol Biotechnol. 2015; 25(5): p. 745-752.", "Phyu WE, Wah WA, Jong SL, Go-Eun C, Chulhum LC. Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods. J. Korean Med Sci. 2016; 31(11): p. 1673-1683.", "Zaczek A, Brzostek A, Wojtasik A, Sajduda A, Dziadek J. Comparison of ligation-mediated PCR methods in differentiation of Mycobacterium tuberculosis strains. Biomed Res Int. 2014;(782071).", "Espinosa de los Monteros LE, Gal\u00e1n JC, Guti\u00e9rrez M, Samper S, Garc\u00eda Mar\u00edn JF, Mart\u00edn C, et al. Allele-Specific PCR Method Based on pncA and oxyR Sequences for Distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: Intraspecific M. bovis pncA Sequence Polymorphism. J. Clin. Microbiol. 1998; 36(1): p. 239-242.", "Peres RL, Maciel EL, Morais CG, Ribeiro FC, Vinhas SA, Pinheiro C, et al. Comparison of two concentrations of NALC-NaOH for decontamination of sputum for mycobacterial culture. Int J Tuberc Lung Dis. 2009; 13(12): p. 1572-1575.", "Li\u00e9bana E, Aranaz A, Francis B, Cousins D. Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex. J Clin Microbiol. 1996; 34(4): p. 933-938.", "Shah DH BR, Bakshi CS, Singh RK. A multiplex-PCR for the diferentiation of Mycobacterium bovis and Mycobacterium tuberculosis. FEMS Microbiology Letters. 2002; 214(1): p. 39-43.", "Babafemi EO, Cherian BP, Banting L, Mills GA, Ngianga K2. Effectiveness of real-time polymerase chain reaction assay for the detection of Mycobacterium tuberculosis in pathological samples: a systematic review and meta-analysis. Syst. Rev. 2017; 6(1): p. 215.", "Guajardo-Lara C, Salda\u00f1a -Ram\u00edrez MI, Hern\u00e1ndez-Galv\u00e1n NN, Dimas-Adame MA, Valdovinos-Ch\u00e1vez S. MGIT y otros m\u00e9todos para diagnosticar tuberculosis en un sistema hospitalario privado con baja incidencia. Rev Med Inst Mex Seguro Soc. 2018; 56(2): p. 158-162.", "Maldonado-Bernal C, Ramos-Garibay A, Rios-Sarabia N, Serrano H, Carrera M, Navarrete-Franco G, et al. Nested Polymerase Chain Reaction and Cutaneous Tuberculosis. Am J Dermatopathol. 2018; 41(6): p. 428-435.", "Li, S., Liu, B., Peng, M., Chen, M., Yin, W., Tang, H., Ren, H. Diagnostic accuracy of Xpert MTB/RIF for tuberculosis detection in different regions with different endemic burden: A systematic review and meta-analysis. PLoS One. 2017; 12(7): p. e0180725.", "Altez-Fernandez C, Ortiz V, Mirzazadeh M, Zegarra L, Seas C, Ugarte-Gil C. Diagnostic accuracy of nucleic acid amplification tests (NAATs) in urine for genitourinary tuberculosis: a systematic review and meta-analysis. BMC Infect Dis. 2017; 17(1).", "Mendez-Samperio P. Diagnosis of Tuberculosis in HIV Co-infected Individuals: Current Status, Challenges and Opportunities for the Future. Scand J Immunol. 2017; 86(2): p. 76-82.", "Nathavitharana RR, Cudahy PG, Schumacher SG, Steingart KR, Pai M, Denkinger CM. Accuracy of line probe assays for the diagnosis of pulmonary and multidrug-resistant tuberculosis: a systematic review and meta-analysis. Eur Respir J. 2017; 49(1).", "Akkaya O, Kurtoglu MG. Comparison of Conventional and Molecular Methods Used for Diagnosis of Mycobacterium tuberculosis in Clinical Samples. Clin Lab. 2019; 65(10).", "Al-Mahbashi AA, Mukhtar MM, Mahgoub ES. Molecular typing of Mycobacterium spp. isolates from Yemeni tuberculosis patients. East Mediterr Health J. 2013; 19(11): p. 942-946.", "Shakeel K, Iram S, Akhtar M, Hussain S, Maryam H, Anwar A. Diagnostic validation of rapid molecular detection of Mycobacterium tuberculosis in pus samples by GeneXpert. J Pak Med Assoc. 2018; 68(1): p. 33-37.", "Dorronsoro I, Torroba L. Microbiolog\u00eda de la tuberculosis. An Sist Sanit Navar. 2007; 30(Supl 2): p. 67-84.", "OPS. Manual para el diagn\u00f3stico bacteriol\u00f3gico de la tuberculosis. Normas y Gu\u00eda T\u00e9cnica - Parte 2. Cultivo. Luc\u00eda Barrera CGM, editor. Washington: Organizaci\u00f3n Panamericana de la Salud; 2008.", "Rodr\u00edguez AJ, Palma S, Maestre JL, Saavedra D, Reyes T, Perovani M. Detecci\u00f3n de micobacterias en muestras cl\u00ednicas mediante la Reacci\u00f3n en Cadena de la Polimerasa. Rev Biomed. 2000; 11(4): p. 257-262.", "Hashimoto T, Suzuki K, Amitani R, Kuze F. Rapid detection of Mycobacterium tuberculosis in sputa by the amplification of IS6110. Intern Med. 1995; 34(7): p. 605-610.", "Kolk AH, Kox LF, van Leeuwen J, Kuijper S, Jansen HM. Clinical utility of the polymerase chain reaction in the diagnosis of extrapulmonary tuberculosis. Eur Respir J. 1988; 11(6): p. 1222-1226.", "Springer B, Stockman L, Teschner K, Roberts GD, B\u00f6tter EC. Two-laboratory collaborative study on identification of mycobacteria: molecular versusphenotypic methods. J Clinic Microbiol. 1996; 32(2): p. 296-303.", "Turenne CY, Tschetter L, Wolfe J, Kabani A. Necessity of Quality-Controlled 16S rRNA Gene Sequence Databases: Identifying Nontuberculous Mycobacterium Species. J Clinic Microbiol. 2002; 39(10): p. 3637\u20133648.", "Marques L\u00c9C, Frota CC, Quetz JDS, Bind\u00e1 AH, Mota RMS, Pontes MAA, et al. Evaluation of 16S rRNA qPCR for detection of Mycobacterium leprae DNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases. Pathog Glob Health. 2018; 112(2): p. 72-78.", "Ngan GH, Ng LM, Jureen R, Lin RT, Teo JW. Development of multiplex PCR assays based on the 16S-23S rRNA internal transcribed spacer for the detection of clinically relevant nontuberculous mycobacteria. Leet Appl Microbiol. 2011; 52(5): p. 546-554.", "Satilmis S, Ulger Toprak N, Iling C, Soyletir G. Evaluation of direct 16S rRNA PCR from clinical samples for bacterial detection in normally sterile body sites. J Infect Dev Ctres. 2019; 13(11): p. 978-983.", "Nieminen T, Pakarinen J, Tsitko I, Salkinoja -Salonen M, Breitenstein A, Ali-Vehmas T, et al. 16S rRNA targeted sandwich hybridization method for direct quantification of mycobacteria in soils. J Microbiol Methods. 2006; 67(1): p. 44-55.", "Puk K, Banach T, Wawrzyniak A, Adaszek L, Zi\u0119tek J, Winiarczyk S, et al. Detection of Mycobacterium marinum, M. peregrinum, M. fortuitum and M. abscessus in aquarium fish. J Fish Dis. 2018; 41(1): p. 153-156.", "Sinha P, Gupta A, Prakash P, Anupurba S, Tripathi R, Srivastava GN. Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32 kD alpha antigen encoding gene fragments. BMC Infect Dis. 2016; 16(123).", "Spositto FLE, Campanerut P AZ, Ghiraldi LD, Leite CQF, Hirata MH, R.D.C. H, et al. Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex. Braz J Microbiol. 2014; 45(3): p. 841-843.", "Merker M, Kohl TA, Niemann S, Supply P. The Evolution of Strain Typing in the Mycobacterium tuberculosis Complex. Adv Exp Med Biol. 2017; 1019: p. 43-78.", "Soto ME, Del Carmen \u00c1vila-Casado M, Huesca-G\u00f3mez C, Alarcon GV, Castrejon V, Soto V, et al. Detection of IS6110 and HupB gene sequences of Mycobacterium tuberculosis and bovis in the aortic tissue of patients with Takayasu's arteritis. BMC Infect Dis. 2012; 12(194).", "V\u00e1zquez-Marrufo G, Mar\u00edn-Hern\u00e1ndez D, Zavala-P\u00e1ramo MG, V\u00e1zquez-Narvaez G, Alvarez-Aguilar C, V\u00e1zquez-Garcidue\u00f1as MS. Hnju7 Genetic diversity among Mycobacterium tuberculosis isolates from Mexican patients. Can J Microbiol. 2008; 54(8): p. 610-618."]}

RESUMEN Antecedentes: La incidencia de tuberculosis (TB) en México aumenta debido, entre otras causas, a un diagnóstico inespecífico o retardado. Marcadores moleculares como los genes rpoB y 16S rRNA se han utilizado en la identificación del género Mycobacterium. La secuencia de inserción 6110 (IS6110) ha sido empleada como marcadorgenético en la genotipificación del complejo Mycobacterium tuberculosis (CMTB). Objetivo: Realizar un estudio retrospectivo para evaluar a los genes rpoB, 16S rRNA y la IS6110, en la identificación de aislados del género Mycobacterium y del CMTB. Métodos: Se analizaron 146 muestras clínicas. Empleando la reacción en cadena de la polimerasa (PCR) se amplificaron fragmentos de los genes rpoB, 16S rRNA e IS6110. La especie M. bovis fue identificada mediante un ensayo de PCR Multiplex. Resultados: En 65 muestras clínicas se determinó la presencia de micobacterias mediante amplificación de los genes 16S rRNA y rpoB. En 22/65 aislados se amplificaron únicamente los genes rpoB y/o 16S rRNA. La presencia del CMTB se confirmó en 43/65 aislados mediante la amplificación de la IS6110. La comparación de las secuencias obtenidas de los fragmentos amplificados de los genes rpoB, 16S rRNA y la IS6110 con las reportadas en el GenBank, demostró una homología de 98%-100% con especies de micobacterias pertenecientes al CMTB. Discusión: El uso de técnicas moleculares para el diagnóstico de enfermedades infecciosas permite obtener resultados precisos y en tiempos cortos. En el caso de las infecciones por micobacterias, el empleo de los marcadores genéticos rpoB, 16S rRNA y la IS6110 coadyuva al diagnóstico diferencial entre TB y micobacteriosis, lo que contribuye a orientar el tratamiento que se administre al paciente. ABSTRACT Background: Tuberculosis (TB) incidence in Mexico is increasing due to an unspecific or delayed diagnosis, among several other causes. Molecular markers such as rpoB and 16S rRNA genes have been used to identify the Mycobacterium genus. Insertion sequence 6110 (IS6110) has been used as a genetic marker in Mycobacterium tuberculosis complex (MTBC) genotyping. Objective: To perform a retrospective study to evaluate rpoB and 16S rRNA genes and IS6110 usefulness to identify the Mycobacterium genus and MTBC isolates. Methods: A total of 146 clinical samples were analyzed. Amplification products from rpoB, 16S rRNA and IS6110 were obtained by Polymerase Chain Reaction (PCR). M. bovis was identified using a Multiplex PCR assay. Results: Mycobacteria were identified in 65 clinical samples as shown by rpoB and 16S rRNA genes amplification. In 22 isolates (22/65) amplification products were obtained only for rpoB and/or 16S rRNA genes. MTBC presence was confirmed in 43/65 isolates as IS6110 amplification product was obtained. Sequencing products of rpoB, 16S rRNA and IS6110 showed 98%-100% homology with MTBC species reported in the GenBank database. Discussion: Inclusion of molecular techniques in infectious diseases diagnosis allows to get precise results in short time. In mycobacterial infections, molecular makers such as rpoB, 16S rRNA and IS6110, largely contribute to make a specific diagnosis and differentiate between TB and mycobacteriosis, leading to the best treatment administration to the patient.