[Differentiation of species within the Mycobacterium tuberculosis complex by molecular techniques].
BACKGROUND
The Mycobacterium tuberculosis complex includes the following species: Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium bovis-BCG, Mycobacterium microti, Mycobacterium caprae, Mycobacterium pinnipedii, and Mycobacterium canettii. These species cause tuberculosis in humans and animals. Identification of mycobacterial strains has classically been performed by phenotype study. Over the last years, laboratories have developed several molecular techniques to differentiate between these species. The aim of this study is to evaluate these methods and develop a simple, fast, identification scheme.


METHODS
We analyzed 251 strains randomly obtained from the strains studied in 2004, and 797 strains received by the Reference Laboratory between 2005 and 2007. Phenotype characterization of 4183 strains isolated during that period was done by studying the colony morphology, characteristics in culture, nitrate reduction, niacin accumulation, and growth in the presence of thiophen-2-carboxylic acid hydrazide 10 microg/mL and pyrazinamide 50 microg/mL. The molecular identification scheme designed was as follows: 1) gyrB PCR-RFLP with RsaI, TaqI or SacII and hsp65 RFLP/PCR with HhaI., and 2) multiplex-PCR to determine the presence/absence of the RD9 and RD1 regions.


RESULTS
The results showed 100% agreement between phenotype study and the molecular scheme.


CONCLUSIONS
This molecular identification scheme is a simple and fast method, with 100% sensitivity and specificity, that can be implemented in most clinical laboratories at a low cost.
Herrera-León
Laura
L
Laboratorio de Referencia de Micobacterias, Servicio de Bacteriología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, España. lherrera@isciii.es
Pozuelo-Díaz
Rodolfo
R
Molina Moreno
Tamara
T
Valverde Cobacho
Azucena
A
Saiz Vega
Pilar
P
Jiménez Pajares
María Soledad
MS
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002264:Carboxylic Acids; D002417:Cattle; D018834:Chaperonin 60 / Q000235:genetics; D027081:DNA Gyrase / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D006801:Humans; D009161:Mycobacterium / Q000145:classification / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009163:Mycobacterium bovis / Q000145:classification / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009525:Niacin / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D011718:Pyrazinamide / Q000494:pharmacology; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013876:Thiophenes / Q000494:pharmacology; D014376:Tuberculosis / Q000382:microbiology*; D014380:Tuberculosis, Bovine / Q000382:microbiology*
D001426:Bacterial Proteins; D002264:Carboxylic Acids; D018834:Chaperonin 60; D004269:DNA, Bacterial; D009566:Nitrates; D013876:Thiophenes; C074119:heat-shock protein 65, Mycobacterium; D009525:Niacin; D011718:Pyrazinamide; C550746:2-thiophene carboxylic acid; D027081:DNA Gyrase