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Identification of Mycobacteria
High Performance Liquid Chromatography (HPLC)
This test is the primary identification
test at TDSHS for all mycobacteria. Identification of mycobacteria by
HPLC is species specific and is based on the separation of mycolic
acids contained in the cell wall of the bacteria. The range of
speciation and the sensitivity of the test are greater than that of
genetic probe (without amplification) and conventional biochemical
testing. HPLC testing also can be performed directly on a clinical
specimen with a positive smear regardless of the source of the
specimen. For culture confirmation, pure viable cultures on solid or in
liquid media should be submitted for testing.
A limited number of Mycobacterium
species can be identified by the use of commercially available DNA
probes. The GenProbe AccuProbe system is a nucleic acid hybridization
test that utilizes a highly specific and sensitive, single stranded DNA
probe. The probe is labeled with a chemiluminescent acridinium ester
and is complimentary to the ribosomal RNA (rRNA) of the organism.
During cell lysis, the rRNA is released and the labeled DNA probe
combines with the target rRNA to form a stable DNA:RNA hybrid. A
selection reagent destroys all unbound probe. The hybrids are detected
and measured in a GenProbe luminometer. A positive result is a
numerical reading that is equal to or greater than an established
TDSHS currently uses DNA probes specific for M. tuberculosis complex, M. avium complex, M. kansasii, and M. gordonae.
The use of DNA probes provides a rapid identification for these
mycobacterial species in a few hours versus weeks when conventional
methods are employed. Pure, viable cultures on solid media should be
submitted for testing and should be less than four weeks old.
Conventional Biochemical Tests
Once the isolate has been placed in a
subgroup based on rate of growth and pigmentation, identification to
the species or complex level may be initiated. Key biochemical tests
useful for identifying the suspected species are selected and the
organism’s biochemical profile is elucidated. The profile is compared
to the reaction patterns of known species. If an unknown organism
matches any one pattern exactly, the identification may be considered
definitive for that species.
Susceptibility Testing of Mycobacteria
The BACTEC procedure for susceptibility testing of M. tuberculosis is based on mycobacterial utilization of 14C - labeled palmitic acid as a sole carbon source in a liquid medium. During respiration, the organism produces 14CO2. The amount of 14CO2
is a reflection of the rate and amount of growth occurring in the
medium-containing vial and is numerically expressed as the "Growth
Index"(GI). When an antituberculous drug is added to the medium,
inhibition of growth occurs if the test organism is susceptible. This
suppression is detected by either a decrease or a very small increase
in the daily GI relative to the drug-free control. However, if the
organism is resistant, little or no suppression of growth occurs.
Three primary antituberculous drugs are tested by TDSHS in BACTEC: isoniazid, rifampin, and ethambutol. If a strain of M. tuberculosis
is resistant to a primary drug, then these secondary drugs are tested
in BACTEC: PZA, streptomycin, ethionamide, kanamycin, rifabutin, and
ofloxacin. The BACTEC method of susceptibility testing offers an
advantage over conventional testing because results are reportable
generally in 4-6 days versus 3 weeks. Acceptable cultures for
susceptibility testing are pure, viable cultures in liquid or on solid
media that have not been subcultured excessively.
Agar Proportion Method
The agar proportion susceptibility test
is based on a comparison of the number of microbial colonies growing on
the surface of drug-containing agar relative to the number of colonies
growing on the drug-free control. Antituberculous drugs are
incorporated into cooled Middlebrook 7H10 agar. The agar is then poured
into quadrant petri plates. One quadrant in each plate is reserved for
the control. The organism is reported as resistant to a particular drug
if the number of colonies on the drug quadrant is equal to or greater
than 1% of the number of colonies growing on the drug-free control.
The TDSHS panel of drugs for agar proportion susceptibility testing of M. tuberculosis
contains isoniazid, rifampin, ethambutol, streptomycin, rifabutin,
ethionamide, kanamycin, capreomycin, and ofloxacin. Rifampin
susceptibility for M. kansasii is also performed by TDSHS.