laboratories, these assays can be of value. These assays still provide a
set of characteristics to describe an isolate, reason why they are still
used as part of the quality control testing.
Novel technologies have been developed as accurate tools for
species ID. Matrix-Assisted Laser Desorption/Ionization-Time of
Flight mass spectrometry (MALDI-TOF MS) is being used with
increasing frequency to identify mycobacterial isolates. MALDITOF MS detects the abundance of proteins with specific mass-tocharge ratios, which is displayed as a spectrum. The spectral data are
then compared to a database to determine the likely identity of the
organism. There are currently two commercially available MALDITOF MS systems in the United States, the MALDI Biotyper (Bruker Daltonics, Billerica, MA) and the Vitek MS system (bioMe ´rieux, Durham, NC). The Vitek MS system has an FDA-cleared
database, which includes mycobacteria. Laboratories using the
MALDI Biotyper must build their own databases or rely on
research-use-only databases for their laboratory-developed protocols. Like 16S rRNA gene sequencing, the accuracy of MALDITOF MS is dependent on both the robustness of the database and
the quality of the spectra obtained. Obtaining quality spectra can be
difficult with mycobacteria due to their complex cell walls. Therefore, whole-cell preparations (i.e., applying a colony directly on a
target plate) do not work for mycobacteria. A pre-extraction step
must occur, usually involving bead beating or vortexing in ethanol,
formic acid, and acetonitrile; the extracted proteins are then
spotted onto the plate. Importantly, the extraction procedure
should be performed in a BSL-3 laboratory (or a BSL-2 laboratory
using BSL-3 practices) and verified for its ability to effectively kill
M. tuberculosis prior to moving the target plate to the mass
spectrometer.
High Performance Liquid Chromatography (HPLC) was first
proposed for the identification of mycobacteria by the CDC and
was offered as a standard test at the CDC Mycobacteriology Reference Laboratory in 1990 [37]. HPLC can identify RGM into
groups or complexes but is not specific enough to identify most
species. HPLC has been replaced in most laboratories by molecular
methods (e.g., rpoB gene sequencing and MALDI-TOF) for more
accurate species identification. A commercial HPLC system, the
Sherlock Mycobacteria Identification System (MYCO-LS) (MIDI
Inc., Newark, DE), is an FDA-cleared HPLC method for the
identification of mycobacteria. MYCO-LS identifies 25 species of
mycobacteria by the analysis of mycolic acids. Like the CDC HPLC
method, mycolic acids are extracted from unknown mycobacteria,
and the HPLC mycolic acid profile is then compared to a library of
reference strain profiles. The mycobacteria may be identified to the
species or to the species-complex group level. Several studies have
evaluated MYCO-LS and found that it is a rapid and accurate
alternative method for mycobacterial species identification; however, it has a limited library of mycobacteria [38].
14
Elizabeth Wallace et al.
set of characteristics to describe an isolate, reason why they are still
used as part of the quality control testing.
Novel technologies have been developed as accurate tools for
species ID. Matrix-Assisted Laser Desorption/Ionization-Time of
Flight mass spectrometry (MALDI-TOF MS) is being used with
increasing frequency to identify mycobacterial isolates. MALDITOF MS detects the abundance of proteins with specific mass-tocharge ratios, which is displayed as a spectrum. The spectral data are
then compared to a database to determine the likely identity of the
organism. There are currently two commercially available MALDITOF MS systems in the United States, the MALDI Biotyper (Bruker Daltonics, Billerica, MA) and the Vitek MS system (bioMe ´rieux, Durham, NC). The Vitek MS system has an FDA-cleared
database, which includes mycobacteria. Laboratories using the
MALDI Biotyper must build their own databases or rely on
research-use-only databases for their laboratory-developed protocols. Like 16S rRNA gene sequencing, the accuracy of MALDITOF MS is dependent on both the robustness of the database and
the quality of the spectra obtained. Obtaining quality spectra can be
difficult with mycobacteria due to their complex cell walls. Therefore, whole-cell preparations (i.e., applying a colony directly on a
target plate) do not work for mycobacteria. A pre-extraction step
must occur, usually involving bead beating or vortexing in ethanol,
formic acid, and acetonitrile; the extracted proteins are then
spotted onto the plate. Importantly, the extraction procedure
should be performed in a BSL-3 laboratory (or a BSL-2 laboratory
using BSL-3 practices) and verified for its ability to effectively kill
M. tuberculosis prior to moving the target plate to the mass
spectrometer.
High Performance Liquid Chromatography (HPLC) was first
proposed for the identification of mycobacteria by the CDC and
was offered as a standard test at the CDC Mycobacteriology Reference Laboratory in 1990 [37]. HPLC can identify RGM into
groups or complexes but is not specific enough to identify most
species. HPLC has been replaced in most laboratories by molecular
methods (e.g., rpoB gene sequencing and MALDI-TOF) for more
accurate species identification. A commercial HPLC system, the
Sherlock Mycobacteria Identification System (MYCO-LS) (MIDI
Inc., Newark, DE), is an FDA-cleared HPLC method for the
identification of mycobacteria. MYCO-LS identifies 25 species of
mycobacteria by the analysis of mycolic acids. Like the CDC HPLC
method, mycolic acids are extracted from unknown mycobacteria,
and the HPLC mycolic acid profile is then compared to a library of
reference strain profiles. The mycobacteria may be identified to the
species or to the species-complex group level. Several studies have
evaluated MYCO-LS and found that it is a rapid and accurate
alternative method for mycobacterial species identification; however, it has a limited library of mycobacteria [38].
14
Elizabeth Wallace et al.