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Vol. 26. Núm. S9.
Utilidad de la biología molecular en el diagnóstico microbiológico
Páginas 33-41 (julio 2008)
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Vol. 26. Núm. S9.
Utilidad de la biología molecular en el diagnóstico microbiológico
Páginas 33-41 (julio 2008)
Acceso a texto completo
Utilidad de la biología molecular en el diagnóstico microbiológico de las infecciones por micobacterias
Utility of molecular biology in the microbiological diagnosis of mycobacterial infections
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4876
José Domínguez, Silvia Blanco, Alicia Lacoma, Nerea García-Sierra, Cristina Prat, Vicente Ausina
Autor para correspondencia
vausina.germanstrias@gencat.net

Correspondencia: Servei de Microbiologia. Hospital Universitari Germans Trias i Pujol. Carretera del Canyet, s/n. 08916. Badalona. Barcelona. España.
Servei de Microbiologia. Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol. Badalona. Barcelona. España. CIBER Enfermedades Respiratorias. Instituto de Salud Carlos III. Madrid. España. Departament de Genètica i Microbiologia. Universitat Autònoma de Barcelona. Barcelona. España
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Las micobacterias constituyen un grupo de bacterias de gran interés en medicina, ya que, junto a especies telúricas y oportunistas, se hallan 2 especies (Mycobacterium tuberculosis y Mycobacterium leprae) de gran importancia en salud pública. A pesar de los esfuerzos realizados para su control, la tuberculosis (TB) sigue siendo en la actualidad uno de los problemas sanitarios de más trascendencia mundial.

En los últimos años, la micobacteriología ha experimentado importantes avances tecnológicos. A pesar de ello, el diagnóstico temprano de la infección por micobacterias y, especialmente de la TB, sigue recayendo en el examen microscópico de las muestras teñidas de manera adecuada. En la actualidad, éste sigue siendo el procedimiento más simple, de mejor coste-efectividad y rapidez para proporcionar al clínico una orientación diagnóstica preliminar.

El control efectivo de la TB se basa en la detección rápida de M. tuberculosis, seguido por la inmediata implementación del tratamiento antituberculoso adecuado. La emergencia de cepas resistentes a los fármacos antituberculosos agudiza la necesidad de disponer de métodos rápidos de detección de M. tuberculosis y de resistencias. La disponibilidad de métodos de epidemiología molecular de fácil implementación y estandarización, que nos permitan identificar casos relacionados, es fundamental para identificar brotes epidémicos que ayuden a controlar la propagación de la TB.

Aun reconociendo los evidentes progresos realizados en el diagnóstico molecular de las infecciones micobacterianas, las técnicas disponibles son todavía insuficientes. En esta revisión, describimos el estado actual de las principales técnicas moleculares para la detección directa de micobacterias en muestras clínicas, para su identificación, detección de resistencias a los principales fármacos antituberculosos y de epidemiología molecular. En cada caso, destacamos las ventajas y las limitaciones de ellas.

En un próximo futuro la micobacteriología clínica evolucionará, con bastante probabilidad, hacia la universalización de las técnicas genéticas aplicadas al diagnóstico directo y la detección de resistencias. La epidemiología molecular de la TB se realizará, en sus diferentes aplicaciones, con técnicas más rápidas y automatizadas que las actuales.

Palabras clave:
Micobacterias
Detección directa
Identificación
Detección de resistencias
Epidemiología molecular

Species within the Mycobacterium genus are of major medical interest, since, together with environmental and opportunistic species, there are two species (Mycobacterium tuberculosis and Mycobacterium leprae) that remain an important public health challenge. Despite efforts to control tuberculosis (TB), this disease remains one of the most prominent health problems worldwide. In the last few years, mycobacteriology has experienced major technological advances. Nevertheless, the early diagnosis of mycobacterial infection and, especially of TB, is still based on microscopic examination of properly stained samples. At present, this procedure is still the simplest, fastest and most cost-effective method for preliminary diagnostic guidance. Effective control of TB is based on rapid detection of M. tuberculosis, followed by immediate implementation of the appropriate antituberculosis therapy. Because of the emergence of multidrug resistant strains, the development of rapid diagnostic methods, both for identification of M. tuberculosis and susceptibility testing, has become a pressing need. The availability of molecular epidemiology methods that are easy to implement and standardized and that would allow identification of related cases is of key importance to identify epidemic outbreaks and control the spread of TB. Despite the evident progress in the molecular diagnosis of mycobacterial infections, the available techniques are still inadequate. In this review, we describe the state of the art of the main molecular techniques for direct detection of mycobacteria in clinical samples, their identification, detection of resistance to the most important antituberculosis agents, and molecular epidemiology. In each case, we describe the advantages and limitations of current techniques. In the near future, clinical mycobacteriology will probably evolve to the universal use of genetic techniques for direct diagnosis and detection of resistance. The molecular epidemiology of TB will be performed, in its various applications, by faster and more automated techniques than those currently available.

Key words:
Mycobacteria
Direct detection
Identification
Resistance detection
Molecular epidemiology
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Bibliografía
[1.]
C.D. DeAngelis, A. Flanagin.
Tuberculosis - a global problem requiring a global solution.
JAMA, 293 (2005), pp. 2793-2794
[2.]
CIDSA.
Diagnostic Standards and Classification of Tuberculosis in Adults and Children. This official statement of the American Thoracic Society and the Centers for Disease Control and Prevention was adopted by the ATS Board of Directors, July 1999. This statement was endorsed by the Council of the Infectious Disease Society of America, September 1999.
Am J Respir Crit Care Med, 161 (2000), pp. 1376-1395
[3.]
L.L. Flores, M. Pai, J.M. Colford Jr, L.W. Riley.
In-house nucleic acid amplification tests for the detection of Mycobacterium tuberculosis in sputum specimens: meta-analysis and meta-regression.
BMC Microbiol, 5 (2005), pp. 55
[4.]
F. Gamboa, G. Fernandez, E. Padilla, J.M. Manterola, J. Lonca, P.J. Cardona, et al.
Comparative evaluation of initial and new versions of the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test for direct detection of Mycobacterium tuberculosis in respiratory and nonrespiratory specimens.
J Clin Microbiol, 36 (1998), pp. 684-689
[5.]
C. Piersimoni, C. Scarparo, P. Piccoli, A. Rigon, G. Ruggiero, D. Nista, et al.
Performance assessment of two commercial amplification assays for direct detection of Mycobacterium tuberculosis complex from respiratory and extrapulmonary specimens.
J Clin Microbiol, 40 (2002), pp. 4138-4142
[6.]
P. Visca, P. De Mori, A. Festa, M.L. Montrone, M. Amicosante, L.P. Pucillo.
Evaluation of the BDProbeTec strand displacement amplification assay in comparison with the AMTD II direct test for rapid diagnosis of tuberculosis.
Clin Microbiol Infect, 10 (2004), pp. 332-334
[7.]
A.M. Middleton, P. Cullinan, R. Wilson, J.R. Kerr, M.V. Chadwick.
Interpreting the results of the amplified Mycobacterium tuberculosis direct test for detection of M. tuberculosis rRNA.
J Clin Microbiol, 41 (2003), pp. 2741-2743
[8.]
L. Alcala, M.J. Ruiz-Serrano, S. Hernangomez, M. Marin, D. Garcia de Viedma, R. San Juan, et al.
Evaluation of the upgraded amplified Mycobacterium tuberculosis direct test (gen-probe) for direct detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens.
Diagn Microbiol Infect Dis, 41 (2001), pp. 51-56
[9.]
F. Gamboa, J. Dominguez, E. Padilla, J.M. Manterola, E. Gazapo, J. Lonca, et al.
Rapid diagnosis of extrapulmonary tuberculosis by ligase chain reaction amplification.
J Clin Microbiol, 36 (1998), pp. 1324-1329
[10.]
V. Ausina, F. Gamboa, E. Gazapo, J.M. Manterola, J. Lonca, L. Matas, et al.
Evaluation of the semiautomated Abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis in respiratory specimens.
J Clin Microbiol, 35 (1997), pp. 1996-2002
[11.]
E. Padilla, J.M. Manterola, V. Gonzalez, C.G. Thornton, M.D. Quesada, M.D. Sanchez, et al.
Comparison of the sodium hydroxide specimen processing method with the C18-carboxypropylbetaine specimen processing method using independent specimens with auramine smear, the MB/BacT liquid culture system, and the COBAS AMPLICOR MTB test.
J Clin Microbiol, 43 (2005), pp. 6091-6097
[12.]
J.W. Moon, Y.S. Chang, S.K. Kim, Y.S. Kim, H.M. Lee, S.K. Kim, et al.
The clinical utility of polymerase chain reaction for the diagnosis of pleural tuberculosis.
Clin Infect Dis, 41 (2005), pp. 660-666
[13.]
W.H. Goessens, P. De Man, J.G. Koeleman, A. Luijendijk, R. Te Witt, H.P. Endtz, et al.
Comparison of the COBAS AMPLICOR MTB and BDProbeTec ET assays for detection of Mycobacterium tuberculosis in respiratory specimens.
J Clin Microbiol, 43 (2005), pp. 2563-2566
[14.]
S. Burggraf, U. Reischl, N. Malik, M. Bollwein, L. Naumann, B. Olgemoller.
Comparison of an internally controlled, large-volume LightCycler assay for detection of Mycobacterium tuberculosis in clinical samples with the COBAS AMPLICOR assay.
J Clin Microbiol, 43 (2005), pp. 1564-1569
[15.]
J.Y. Wang, L.N. Lee, H.L. Hsu, P.R. Hsueh, K.T. Luh.
Performance assessment of the DR. MTBC Screen assay and the BD ProbeTec ET system for direct detection of Mycobacterium tuberculosis in respiratory specimens.
J Clin Microbiol, 44 (2006), pp. 716-719
[16.]
J.Y. Wang, L.N. Lee, C.S. Chou, C.Y. Huang, S.K. Wang, H.C. Lai, et al.
Performance assessment of a nested-PCR assay (the RAPID BAP-MTB) and the BD ProbeTec ET system for detection of Mycobacterium tuberculosis in clinical specimens.
J Clin Microbiol, 42 (2004), pp. 4599-4603
[17.]
T.D. McHugh, C.F. Pope, C.L. Ling, S. Patel, O.J. Billington, R.D. Gosling, et al.
Prospective evaluation of BDProbeTec strand displacement amplification (SDA) system for diagnosis of tuberculosis in non-respiratory and respiratory samples.
J Med Microbiol, 53 (2004), pp. 1215-1219
[18.]
F. Perandin, G. Pinsi, C. Signorini, N. Manca.
Evaluation of INNO-LiPA assay for direct detection of mycobacteria in pulmonary and extrapulmonary specimens.
New Microbiol, 29 (2006), pp. 133-138
[19.]
F. Franco-Alvarez de Luna, P. Ruiz, J. Gutierrez, M. Casal.
Evaluation of the GenoType Mycobacteria Direct assay for detection of Mycobacterium tuberculosis complex and four atypical mycobacterial species in clinical samples.
J Clin Microbiol, 44 (2006), pp. 3025-3027
[20.]
D. Hillemann, J. Galle, E. Vollmer, E. Richter.
Real-time PCR assay for improved detection of Mycobacterium tuberculosis complex in paraffin-embedded tissues.
Int J Tuberc Lung Dis, 10 (2006), pp. 340-342
[21.]
W.C. Yam, K.Y. Yuen, S.Y. Kam, L.S. Yiu, K.S. Chan, C.C. Leung, et al.
Diagnostic application of genotypic identification of mycobacteria.
J Med Microbiol, 55 (2006), pp. 529-536
[22.]
E. Padilla, J.M. Manterola, O.F. Rasmussen, J. Lonca, J. Dominguez, L. Matas, et al.
Evaluation of a fluorescence hybridisation assay using peptide nucleic acid probes for identification and differentiation of tuberculous and non-tuberculous mycobacteria in liquid cultures.
Eur J Clin Microbiol Infect Dis, 19 (2000), pp. 140-145
[23.]
F. Trueba, M. Fabre, P. Saint-Blancard.
Rapid identification of Mycobacterium genavense with a new commercially available molecular test, INNOLiPA MYCOBACTERIA v2.
J Clin Microbiol, 42 (2004), pp. 4403-4404
[24.]
E. Padilla, V. Gonzalez, J.M. Manterola, A. Perez, M.D. Quesada, S. Gordillo, et al.
Comparative evaluation of the new version of the INNO-LiPA Mycobacteria and genotype Mycobacterium assays for identification of Mycobacterium species from MB/BacT liquid cultures artificially inoculated with mycobacterial strains.
J Clin Microbiol, 42 (2004), pp. 3083-3088
[25.]
E. Richter, S. Rusch-Gerdes, D. Hillemann.
Evaluation of the GenoType Mycobacterium Assay for identification of mycobacterial species from cultures.
J Clin Microbiol, 44 (2006), pp. 1769-1775
[26.]
Z. Gitti, I. Neonakis, G. Fanti, F. Kontos, S. Maraki, Y. Tselentis.
Use of the GenoType Mycobacterium CM and AS assays to analyze 76 nontuberculous mycobacterial isolates from Greece.
J Clin Microbiol, 44 (2006), pp. 2244-2246
[27.]
A. Telenti, F. Marchesi, M. Balz, F. Bally, E.C. Bottger, T. Bodmer.
Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis.
J Clin Microbiol, 31 (1993), pp. 175-178
[28.]
F. Brunello, M. Ligozzi, E. Cristelli, S. Bonora, E. Tortoli, R. Fontana.
Identification of 54 mycobacterial species by PCR-restriction fragment length polymorphism analysis of the hsp65 gene.
J Clin Microbiol, 39 (2001), pp. 2799-2806
[29.]
M.J. Espy, J.R. Uhl, L.M. Sloan, S.P. Buckwalter, M.F. Jones, E.A. Vetter, et al.
Real-time PCR in clinical microbiology: applications for routine laboratory testing.
Clin Microbiol Rev, 19 (2006), pp. 165-256
[30.]
S. Ramaswamy, J.M. Musser.
Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update.
Tuber Lung Dis, 79 (1998), pp. 3-29
[31.]
S.V. Ramaswamy, R. Reich, S.J. Dou, L. Jasperse, X. Pan, A. Wanger, et al.
Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium tuberculosis.
Antimicrob Agents Chemother, 47 (2003), pp. 1241-1250
[32.]
D. Van Soolingen, P.E. De Haas, H.R. Van Doorn, E. Kuijper, H. Rinder, M.W. Borgdorff.
Mutations at amino acid position 315 of the katG gene are associated with high-level resistance to isoniazid, other drug resistance, and successful transmission of Mycobacterium tuberculosis in the Netherlands.
J Infect Dis, 182 (2000), pp. 1788-1790
[33.]
F. Brossier, N. Veziris, C. Truffot-Pernot, V. Jarlier, W. Sougakoff.
Performance of the genotype MTBDR line probe assay for detection of resistance to rifampin and isoniazid in strains of Mycobacterium tuberculosis with low- and high-level resistance.
J Clin Microbiol, 44 (2006), pp. 3659-3664
[34.]
A. Telenti, N. Honore, C. Bernasconi, J. March, A. Ortega, B. Heym, et al.
Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level.
J Clin Microbiol, 35 (1997), pp. 719-723
[35.]
F. Gamboa, P.J. Cardona, J.M. Manterola, J. Lonca, L. Matas, E. Padilla, et al.
Evaluation of a commercial probe assay for detection of rifampin resistance in Mycobacterium tuberculosis directly from respiratory and nonrespiratory clinical samples.
Eur J Clin Microbiol Infect Dis, 17 (1998), pp. 189-192
[36.]
D. Hillemann, S. Rusch-Gerdes, E. Richter.
Evaluation of the GenoType MTBDRplus assay for rifampin and isoniazid susceptibility testing of Mycobacterium tuberculosis strains and clinical specimens.
J Clin Microbiol, 45 (2007), pp. 2635-2640
[37.]
L.M. Aragon, F. Navarro, V. Heiser, M. Garrigo, M. Espanol, P. Coll.
Rapid detection of specific gene mutations associated with isoniazid or rifampicin resistance in Mycobacterium tuberculosis clinical isolates using non-fluorescent low-density DNA microarrays.
J Antimicrob Chemother, 57 (2006), pp. 825-831
[38.]
M.J. Torres, A. Criado, J.C. Palomares, J. Aznar.
Use of real-time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance-associated mutations in Mycobacterium tuberculosis.
J Clin Microbiol, 38 (2000), pp. 3194-3199
[39.]
D. Garcia de Viedma, M. Del Sol Diaz Infantes, F. Lasala, F. Chaves, L. Alcala, E. Bouza.
New real-time PCR able to detect in a single tube multiple rifampin resistance mutations and high-level isoniazid resistance mutations in Mycobacterium tuberculosis.
J Clin Microbiol, 40 (2002), pp. 988-995
[40.]
B. Mathema, N.E. Kurepina, P.J. Bifani, B.N. Kreiswirth.
Molecular epidemiology of tuberculosis: current insights.
Clin Microbiol Rev, 19 (2006), pp. 658-685
[41.]
J.D. Van Embden, M.D. Cave, J.T. Crawford, J.W. Dale, K.D. Eisenach, B. Gicquel, et al.
Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology.
J Clin Microbiol, 31 (1993), pp. 406-409
[42.]
J. Kamerbeek, L. Schouls, A. Kolk, M. Van Agterveld, D. Van Soolingen, S. Kuijper, et al.
Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.
J Clin Microbiol, 35 (1997), pp. 907-914
[43.]
P. Supply, J. Magdalena, S. Himpens, C. Locht.
Identification of novel intergenic repetitive units in a mycobacterial two-component system operon.
Mol Microbiol, 26 (1997), pp. 991-1003
[44.]
N. Alonso-Rodriguez, M. Martinez-Lirola, M. Herranz, M. Sanchez-Benitez, P. Barroso, E. Bouza, et al.
Evaluation of the new advanced 15-loci MIRUVNTR genotyping tool in Mycobacterium tuberculosis molecular epidemiology studies.
BMC Microbiol, 8 (2008), pp. 34
[45.]
N. Thorne, J.T. Evans, E.G. Smith, P.M. Hawkey, S. Gharbia, C. Arnold.
An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110 restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting.
Clin Microbiol Infect, 13 (2007), pp. 964-970
[46.]
J.N. Goulding, J. Stanley, N. Saunders, C. Arnold.
Genome-sequence-based fluorescent amplified-fragment length polymorphism analysis of Mycobacterium tuberculosis.
J Clin Microbiol, 38 (2000), pp. 1121-1126
[47.]
Y. Kassama, M. Shemko, N. Shetty, Z. Fang, G. Macintire, V. Gant, et al.
An improved fluorescent amplified fragment length polymorphism method for typing Mycobacterium tuberculosis.
J Clin Microbiol, 44 (2006), pp. 288-289
[48.]
M.A. Diggle, S.C. Clarke.
Pyrosequencing: sequence typing at the speed of light.
Mol Biotechnol, 28 (2004), pp. 129-137
[49.]
C. Arnold, L. Westland, G. Mowat, A. Underwood, J. Magee, S. Gharbia.
Single-nucleotide polymorphism-based differentiation and drug resistance detection in Mycobacterium tuberculosis from isolates or directly from sputum.
Clin Microbiol Infect, 11 (2005), pp. 122-130
[50.]
D. Isola, M. Pardini, F. Varaine, S. Niemann, S. Rusch-Gerdes, L. Fattorini, et al.
A Pyrosequencing assay for rapid recognition of SNPs in Mycobacterium tuberculosis embB306 region.
J Microbiol Methods, 62 (2005), pp. 113-120
[51.]
J.R. Zhao, Y.J. Bai, Q.H. Zhang, Y. Wang, M. Luo, X.J. Yan.
Pyrosequencing-based approach for rapid detection of rifampin-resistant Mycobacterium tuberculosis.
Diagn Microbiol Infect Dis, 51 (2005), pp. 135-137
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