Immunochromatography-based lateral flow assays using
monoclonal antibody against the MPB64 protein (Rv1980c or
RD2) secreted by the M. tuberculosis complex are reported to be a
reliable, rapid, and simple tool to identify the M. tuberculosis complex and differentiate its members from NTM in growth-positive
solid or liquid cultures. At present, three commercial tests are
available for routine clinical diagnostic use, the SD Bioline Ag
MPT64 Rapid assay (Standard Diagnostics, Kyonggido, South
Korea) (CE marked), the Capilia TB-Neo assay (Tauns, Numazu,
Japan) (CE marked), and the MGIT TBc Identification test
(Becton-Dickinson Instrument Systems, Sparks, MD, USA)
(CE marked).
1.15 Biochemical
Tests for Mycobacteria
Once a culture is obtained, biochemical testing for strain characterization, as well as for identification of genus and species, can start.
After determining whether a sample is an SGM or an RGM, there
are a number of biochemical tests that can narrow down the species
identification (see Tables 2 and 3). In addition, molecular methods
are becoming more critical as new closely related species are
discovered.
1.15.1 Nitrate and Niacin
Two of the most cost-effective and efficient biochemical methods
for identifying mycobacteria are nitrate reduction and niacin testing. Nitrate reduction will identify mycobacterium based on the
presence of nitrate reductase. Niacin plays a role in the oxidation–
reduction reactions that occur during metabolic syntheses of all
mycobacteria while all species of mycobacteria produce niacin,
M. tuberculosis accumulates the largest amount in the medium on
which it is growing (usually L-J).
1.15.2 Pyrazinamidase
This assay was initially used to differentiate M. tuberculosis var. bovis
from M. tuberculosis var. tuberculosis and the M. avium complex, as
well as M. marinum from M. kansasii [39]. Pyrazinamidase activity
was observed by inoculating pyrazinamidase agar with live culture.
However, based on Bergey’s manual [40] and the reclassification of
the M. tuberculosis complex [9], this test was removed from the
current testing scheme used at ATCC (see Table 3).
1.15.3 Pigmentation
Pigmentation can be useful in determining species identity. Inoculated L-J media can be wrapped in foil and incubated in darkness. If
a color other than buff or cream is observed, the isolate may be a
scotochromogen. If no pigment is observed, remove the foil and
allow the media to sit in light for 1 h. If a pigment develops, the
isolate is a photochromogen. If no pigment is observed in darkness
or light, the isolate is a nonchromogen. In addition, polysorbate
80 hydrolysis and urea hydrolysis can be implemented to identify
scotochromogen and nonphotochromogen Mycobacterium spp.
Culturing Mycobacteria
15
monoclonal antibody against the MPB64 protein (Rv1980c or
RD2) secreted by the M. tuberculosis complex are reported to be a
reliable, rapid, and simple tool to identify the M. tuberculosis complex and differentiate its members from NTM in growth-positive
solid or liquid cultures. At present, three commercial tests are
available for routine clinical diagnostic use, the SD Bioline Ag
MPT64 Rapid assay (Standard Diagnostics, Kyonggido, South
Korea) (CE marked), the Capilia TB-Neo assay (Tauns, Numazu,
Japan) (CE marked), and the MGIT TBc Identification test
(Becton-Dickinson Instrument Systems, Sparks, MD, USA)
(CE marked).
1.15 Biochemical
Tests for Mycobacteria
Once a culture is obtained, biochemical testing for strain characterization, as well as for identification of genus and species, can start.
After determining whether a sample is an SGM or an RGM, there
are a number of biochemical tests that can narrow down the species
identification (see Tables 2 and 3). In addition, molecular methods
are becoming more critical as new closely related species are
discovered.
1.15.1 Nitrate and Niacin
Two of the most cost-effective and efficient biochemical methods
for identifying mycobacteria are nitrate reduction and niacin testing. Nitrate reduction will identify mycobacterium based on the
presence of nitrate reductase. Niacin plays a role in the oxidation–
reduction reactions that occur during metabolic syntheses of all
mycobacteria while all species of mycobacteria produce niacin,
M. tuberculosis accumulates the largest amount in the medium on
which it is growing (usually L-J).
1.15.2 Pyrazinamidase
This assay was initially used to differentiate M. tuberculosis var. bovis
from M. tuberculosis var. tuberculosis and the M. avium complex, as
well as M. marinum from M. kansasii [39]. Pyrazinamidase activity
was observed by inoculating pyrazinamidase agar with live culture.
However, based on Bergey’s manual [40] and the reclassification of
the M. tuberculosis complex [9], this test was removed from the
current testing scheme used at ATCC (see Table 3).
1.15.3 Pigmentation
Pigmentation can be useful in determining species identity. Inoculated L-J media can be wrapped in foil and incubated in darkness. If
a color other than buff or cream is observed, the isolate may be a
scotochromogen. If no pigment is observed, remove the foil and
allow the media to sit in light for 1 h. If a pigment develops, the
isolate is a photochromogen. If no pigment is observed in darkness
or light, the isolate is a nonchromogen. In addition, polysorbate
80 hydrolysis and urea hydrolysis can be implemented to identify
scotochromogen and nonphotochromogen Mycobacterium spp.
Culturing Mycobacteria
15