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Role of phenotypic and genotypic investigations for the identification of non-tuberculous mycobacteria. Enrico Tortoli Florence – Italy Presented at 34° ICAAC – Chicago, September 14, 2003. 1990. 1995. 2000. The rise of mycobacterial species. The rise of mycobacterial species.
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Role of phenotypic and genotypic investigations for the identification of non-tuberculous mycobacteria Enrico Tortoli Florence – Italy Presented at 34° ICAAC – Chicago, September 14, 2003
1990 1995 2000 The rise of mycobacterial species
The rise of mycobacterial species • 94 officially recognized nontuberculous mycobacterial (NTM) species • On average 3 new species/yr described in the last 14 years • A large number of “new” mycobacterial sequences are piling up in GenBank
10 Slow growers
10 Slow growers
10 Slow growers
10 Rapid growers
10 Rapid growers
NTM, frequently raised criticisms • NTN differentiation is unnecessary, only their distinction from M. tuberculosis complex is of importance • The NTM are rarely clinically significant • In the cases in which NTM are clinically significant their identification at species level is of little use for the clinician
NTM, frequently raised criticisms. Replay 1 • NTN differentiation is unnecessary, only their distinction from M. tuberculosis complex is of importance • The identification of “new” NTM is essential to enlarge the current poor knowledge of such organisms and, first of all, of their clinical relevance
NTM, frequently raised criticisms. Replay 2 • The NTM are rarely clinically significant • Among NTM isolated from human samples, for 5 species only the involvement in disease has never been proved
NTM, frequently raised criticisms. Replay 3 • In the cases in which NTM are clinically significant their identification at species level is of little use for the clinician • Several NTM differ for virulence • Some NTM are preferentially involved in specific pathologies • Various NTM are characterized by different susceptibility pattern
Phenotypic identification methods • Conventional approach • Biochemical tests • Cultural tests • Selective inhibition tests • Lipid approach • Thin layer chromatography • Gas-chromatography • High performance liquid chromatography
Phenotypic identification methods • Conventional approach • Biochemical tests • Cultural tests • Selective inhibition tests • Lipid approach • Thin layer chromatography • Gas-chromatography • High performance liquid chromatography
Phenotypic identification methods • Conventional approach • Biochemical tests • Cultural tests • Selective inhibition tests • Lipid approach • Thin layer chromatography • Gas-chromatography • High performance liquid chromatography
10 Genotypic identification methods • Nucleic acid probes • PCR restriction analysis • Genetic sequencing of conserved regions
Limits of conventional methods • Conventional approach worked apparently well until the number of species remained limited • It run into problems with the expansion of the number of species • Need of larger panels of tests • Still insufficient information available for many species • Previously disregarded problems emerged • Low reproducibility of several tests • Biological variability of the strains • Misidentification of strains belonging to new species as variants of well established ones • Time consumption
Limitations of lipid methods • Culture-dependent • TLC and GLC • Large clusters of species exist sharing the same pattern • HPLC • Emergence, among new mycobacteria, of species with similar profiles (rapid growers)
Limitations of genetic methods • DNA probes • Highly reliable but available for common species only • PRA • Unidentified strains with patterns overlapping to the ones of well defined species • Species with multiple patterns (up to 8) • Genetic sequencing (16S rDNA) • Species with overlapping sequences • Species with multiple sequevars • Poor quality control of major databases
identification + identification new sequence Identification for diagnostic purpose DNA probe 16S rDNA sequence
Description of new species, minimal standard • Major biochemical and cultural features • Lipid structure • TLC • GLC • HPLC • Full 16S rDNA sequence
Conclusions • Genetic sequencing of 16S rDNA is at present the only method that, even when used alone, provides reliable identifications • Identifications emerging from PRA of 65kD hsp are satisfactory but many are the species whose pattern has been not yet determined • Among lipid investigations the HPLC is the one with higher discriminatory power, species do exist whose identification cannot be determined • With conventional tests a correct identification is achievable for “classical” mycobacteria only; selected tests may help in resolving ambiguities due to overlapping of genetic characters