role of phenotypic and genotypic investigations for the identification of non-tuberculous...
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Role of phenotypic and genotypic investigations for the Role of phenotypic and genotypic investigations for the identification of non-tuberculous mycobacteriaidentification of non-tuberculous mycobacteria
Enrico TortoliFlorence – Italy
Presented at 34° ICAAC – Chicago, September 14, 2003
The rise of mycobacterial speciesThe rise of mycobacterial species
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1995
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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
The rise of mycobacterial speciesThe rise of mycobacterial species
Slow growersSlow growers
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Slow growersSlow growers
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Slow growersSlow growers
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Rapid growersRapid growers
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Rapid growersRapid growers
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NTM, frequently raised criticismsNTM, frequently raised criticisms
NTN differentiation is unnecessary, only their distinction from M. tuberculosis complex is of importance
The NTM are rarely clinically significantIn 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 1NTM, 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
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 2NTM, frequently raised criticisms. Replay 2
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
NTM, frequently raised criticisms. Replay 3NTM, frequently raised criticisms. Replay 3
Phenotypic identification methodsPhenotypic identification methods
Conventional approach– Biochemical tests
– Cultural tests
– Selective inhibition tests
Lipid approach– Thin layer chromatography
– Gas-chromatography
– High performance liquid chromatography
Phenotypic identification methodsPhenotypic identification methods
Conventional approach– Biochemical tests
– Cultural tests
– Selective inhibition tests
Lipid approach– Thin layer chromatography
– Gas-chromatography
– High performance liquid chromatography
Phenotypic identification methodsPhenotypic identification methods
Conventional approach– Biochemical tests
– Cultural tests
– Selective inhibition tests
Lipid approach– Thin layer chromatography
– Gas-chromatography
– High performance liquid chromatography
Nucleic acid probesPCR restriction
analysisGenetic sequencing of
conserved regions
Genotypic identification methodsGenotypic identification methods
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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
Limits of conventional methodsLimits of conventional methods
Culture-dependentTLC 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 lipid methodsLimitations of lipid 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
Limitations of genetic methodsLimitations of genetic methods
Identification for diagnostic purposeIdentification for diagnostic purpose
DNA probe
16S rDNAsequence
+ identification
identification
new sequence
Major biochemical and cultural featuresLipid structure
– TLC
– GLC
– HPLC
Full 16S rDNA sequence
Description of new species, minimal standardDescription of new species, minimal standard
ConclusionsConclusions
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
