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Articles |
1
Department of Pathology, Baylor College of Medicine, Houston, TX 77030.
2
Mycobacterial Reference Laboratory, National Public
Health Institute, Kiinamyllynkatu 13, 20520 Turku, Finland.
3
Laboratory of Human Bacterial Pathogenesis, Rocky
Mountain Laboratories, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, 903 South 4th St., Hamilton,
MT 59840.
aAuthor for correspondence. Fax 406-363-9427; e-mail jmusser{at}niaid.nih.gov.
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Abstract |
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 Top Abstract IntroductionDirect Detection of Mycobacteria...Identification of Mycobacterial...Identification of Antibiotic...References |
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Introduction |
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TopAbstractIntroductionDirect Detection of Mycobacteria...Identification of Mycobacterial...Identification of Antibiotic...References |
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Although the initial diagnosis of mycobacterial disease often is based on clinical data, definitive diagnosis usually involves the isolation and identification of the infecting organism in the laboratory. The usual laboratory procedure for clinical specimens involves decontamination and digestion of the specimen, microscopic examination for the presence of acid-fast bacilli (AFB), isolation of the organism by culture, and identification and drug susceptibility testing of the recovered organism. Because of the slow growth rate of mycobacteria, isolation, identification, and drug susceptibility testing can take several weeks or longer.
During the past 10 years, several molecular methods have been developed for direct detection, identification, and susceptibility testing of mycobacteria. These methods can potentially reduce the diagnostic time from weeks to days. The aim of this review is to briefly describe the currently available methods and their impact on diagnosis and management of TB.
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Direct Detection of Mycobacteria from Specimens |
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TopAbstractIntroductionDirect Detection of Mycobacteria...Identification of Mycobacterial...Identification of Antibiotic...References |
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The Food and Drug Administration (FDA) has approved two NAA tests for direct detection of M. tuberculosis from clinical specimens. These are the Enhanced Mycobacterium tuberculosis Direct Test (E-MTD; Gen-Probe, San Diego, CA) and the Amplicor Mycobacterium tuberculosis Test (Amplicor; Roche Diagnostic Systems, Inc., Branchburg, NJ).
amplicor
The Amplicor test is based on the PCR. In this assay,
mycobacterial DNA is amplified with genus-specific primers formulated
on the basis of the 16S rRNA gene. After amplification, the amplicons
are denatured to form single strands and added to a microtiter plate
containing a bound, M. tuberculosis complex-specific
oligonucleotide probe. An avidin-horseradish peroxidase conjugate then
binds to the bound, biotin-labeled amplicons. The conjugate then reacts
with peroxide and 3,3',5,5'-tetramethylbenzidine in dimethylformamide
to form a color complex. The results are measured with a photometer.
False-positive results produced by carryover contamination are
prevented by the incorporation of dUTP coupled with
uracil-N-glycosylase restriction. After the clinical
specimen has been processed with the standard
N-acetyl-L-cysteine–NaOH
decontamination method, the Amplicor results are available in 
6.5 h.
An automated version of this test is available in Europe (Cobas
Amplicor).
The overall sensitivity of the Amplicor test (compared with culture)
for respiratory specimens is 79.4–91.9%, the specificity is
99.6–99.8%, the positive predictive value is 92.6–96.6%, and the
negative predictive value is 98.6–98.7% (Table 1
). However, the sensitivity for smear-negative specimens is
somewhat lower, 40.0–73.1% (3)(4)(5). Therefore, the Amplicor
test has been approved by the FDA only for direct detection of M.
tuberculosis in AFB smear-positive respiratory specimens. Chin et
al. (6) reported that the sensitivity of the Amplicor test
was similar to that of culture (58% vs 56%) for detecting M.
tuberculosis from respiratory specimens when the clinical case
definition of TB was used as the reference standard. However, Al
Zahrani et al. (7) reported that although the Amplicor test
had excellent specificity (100%), it was less sensitive than culture
(42% vs 73%) for diagnosis of minimal active pulmonary TB (patients
suspected of having TB but without spontaneous sputum or with
AFB-negative smears).
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e-mtd
The E-MTD test is based on the transcription-mediated
amplification system developed by Kwoh et al. (8). In this
assay, rRNA is released from the target cells by sonication, and a
promoter-primer binds to the rRNA target. Reverse transcriptase is then
used to copy rRNA to a cDNA-RNA hybrid. The initial RNA strand is
degraded, and a second primer binds to the cDNA and is extended,
leading to the formation of double-stranded cDNA, which is then
transcribed by DNA-directed RNA polymerase to produce more rRNA
molecules. The new transcripts serve as templates for reverse
transcription and further amplification. The RNA amplicons are detected
with an acridinium ester-labeled DNA probe in a solution hybridization
assay. Importantly, the amplification procedure is isothermal and the
reaction is performed in a single tube, which helps to reduce carryover
contamination. After standard decontamination of the clinical specimen,
the E-MTD test can be completed in 3.5 h.
The E-MTD test has been reported to perform well with both AFB smear-positive and smear-negative specimens. The overall sensitivity (compared with culture) for respiratory specimens is 90.9–95.2%, the specificity is 98.8–100%, the positive predictive value is 83.3–100%, and the negative predictive value is 98.4–99.6% (9)(10)(11). In a study where the performance of the E-MTD test was assessed based on the degree of clinical suspicion of TB, the greatest utility was documented for patients with an intermediate clinical suspicion of having TB (12). The E-MTD test is FDA-approved for detection of M. tuberculosis in both AFB smear-positive and smear-negative specimens.
Scarparo et al. (13) compared the performance of the E-MTD and the Cobas Amplicor tests with 486 respiratory and nonrespiratory specimens obtained from 323 patients. No significant differences were observed between the results of the assays. However, it was noted that although the turnaround time is shorter for the E-MTD test, the Amplicor test can be fully automated and has an internal control for monitoring amplification inhibitors.
clinical utility
The clinical utility of the commercial NAA tests has been
discussed in a recent review (14) and in guidelines
published by the FDA (15). In brief, it is recommended that
sputum specimens be collected on 3 different days for AFB smear and
culture. The NAA test should be performed on the first specimen
collected, the first smear-positive specimen, and additional specimens
if needed. If the first specimen is both AFB smear-positive and
NAA-positive, the patient can be presumed to have TB. However, if the
specimen is smear-positive but NAA-negative, a test for inhibitors
should be performed. This option is available in the Amplicor test, but
if the E-MTD test is used, a specimen to which M.
tuberculosis DNA has been added must be analyzed. If inhibitors
are not detected and additional specimens remain NAA-negative, the
patient can be presumed to have nontuberculous mycobacteria. However,
if inhibitors are detected, the NAA test does not offer any diagnostic
help. If a specimen is smear-negative and NAA-positive and the same
result is obtained with an additional specimen, the patient can be
presumed to have TB. In the case that all sputum specimens remain
smear-negative and NAA-negative, the patient can be presumed to be not
infectious; however, this does not exclude the possibility of active
TB, and clinical judgment must be used in decisions regarding TB
therapy (14)(15).
With respect to extrapulmonary TB, clinical diagnosis often is uncertain, and NAA tests could provide important information to the clinician. Although the currently available NAA tests have been approved for respiratory specimens only, they have also been used to test several different types of nonrespiratory specimens. In general, the performance of both tests with nonrespiratory specimens has been similar to their performance with respiratory specimens (13)(16)(17). NAA tests also appear to be useful for early identification of M. tuberculosis complex for all specimen types (except for blood) grown in liquid cultures (18)(19). However, the NAA tests must be validated in house if they are used for other than respiratory specimens.
In conclusion, the currently available NAA tests can enhance diagnostic speed, but they do not replace AFB smear or culture. Because the tests can only detect M. tuberculosis, cultures are still needed for identification of nontuberculous mycobacteria and for drug susceptibility testing. Because the tests cannot distinguish between live and dead organisms, they cannot be used to monitor TB therapy. Clinicians should interpret the NAA test results based on the clinical situation, and the tests should usually be performed at the request of the clinician.
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Identification of Mycobacterial Species from Culture |
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TopAbstractIntroductionDirect Detection of Mycobacteria...Identification of Mycobacterial...Identification of Antibiotic...References |
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dna probes
Commercial DNA probes (AccuProbe; Gen-Probe Inc.) have been
available for some time for identification of clinically important
mycobacterial species, including M. tuberculosis complex,
M. avium, M. intracellulare, M. avium
complex, M. kansasii, and M. gordonae. The tests
are based on species-specific DNA probes that hybridize with rRNA
released from bacteria. The probes are labeled with acridinium ester,
and results are measured with a luminometer. For culture-positive
specimens, the turnaround time for the method is 2 h. The method is
very easy to perform, and no special instrumentation is needed. The
probes have been evaluated extensively in clinical practice and are
rapid, sensitive, and specific (20)(21). The
probes can also be combined with the BACTEC or other liquid-based
culture systems to further decrease the time needed for species
identification (22)(23). However, probes are not
available for all pathogenic mycobacterial species, and those isolates
must be identified by other methods. In addition, the M.
tuberculosis complex probe cannot differentiate between the
members of this complex (M. tuberculosis, M.
bovis, M. bovis BCG, M. africanum, and
M. microti).
pcr-based sequencing
PCR-based sequencing has become the gold standard for
identification of mycobacterial species. The method consists of PCR
amplification of mycobacterial DNA with genus-specific primers and
sequencing of the amplicons. The organism is identified by comparison
of the nucleotide sequence with reference sequences. As commonly
practiced, only one sequencing reaction is needed for a definitive
identification. This method also allows for direct detection of
mycobacterial species that cannot be grown on conventional laboratory
culture media, and several previously unrecognized species have been
identified (24)(25).
The target most commonly used is the gene coding for the 16S rRNA. This gene is present in all bacterial species and contains both conserved and variable regions, making it an ideal target for taxonomic purposes. The 16S rRNA gene has been sequenced from a large number of mycobacterial species, and the identification method based on this gene has been evaluated extensively in diagnostic laboratories. Sequencing of two hypervariable regions of the 16S rRNA gene allows for identification of the majority of mycobacterial species. However, members of the M. tuberculosis complex cannot be distinguished. Similarly, an important pathogen, M. kansasii, has a sequence identical to that of a nonpathogenic species, M. gastri, and additional 16S rRNA gene regions need to be sequenced to differentiate M. marinum from M. ulcerans (26)(27).
Several other target genes have been characterized for this purpose. The genes coding for the 32-kDa protein (28), the 65-kDa heat shock protein (29), and the 16S-23S rRNA internal transcribed spacer (30) contain enough sequence diversity to distinguish all clinically important mycobacteria except for the members of the M. tuberculosis complex. These target genes also allow for differentiation of M. kansasii and M. gastri. In addition, because of the intraspecies variation observed in the 65-kDa protein gene, this target can also be used for distinguishing clones of certain mycobacterial species (31).
dna microarrays
High-density oligonucleotide arrays (DNA microarrays) offer the
possibility of rapid examination of large amounts of DNA sequences with
a single hybridization step. This approach has recently been applied to
simultaneous species identification and detection of mutations that
confer rifampin resistance in mycobacteria. This technique is based on
hybridization of fluorescently labeled PCR amplicons generated from
bacterial colonies to a DNA array containing nucleotide probes. The
bound amplicons emit a fluorescent signal that is detected with a
scanner. The probes used in this array are based on 82 unique 16S rRNA
sequences that allow for discrimination of 54 mycobacterial species and
51 sequences that contain unique rpoB gene mutations. The
method correctly identified 67 of the 70 isolates representing 27
mycobacterial species. All three M. szulgai isolates were
identified as M. malmoense because of an error in the probe
sequence. The turnaround time for this method when performed on culture
positive specimens was only 4 h (32)(33).
Molecular methods offer many advantages over conventional methods in the identification of mycobacterial species. The results are obtained rapidly, are reliable and reproducible, and even mixed or contaminated cultures can be analyzed. The probes are already widely used in clinical laboratories for the identification of the most common mycobacterial species. Because automatic DNA sequencers and the programs used for analyzing sequence data have become technically simpler, the PCR-based sequencing method is now being used in many mycobacterial reference laboratories as the routine method for species assignment. The DNA microarray method holds great promise for the future because it is easy to perform, it can be readily automated, and it allows for identification of a large number of mycobacterial species in one reaction.
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Identification of Antibiotic Resistance-associated Mutations |
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TopAbstractIntroductionDirect Detection of Mycobacteria...Identification of Mycobacterial...Identification of Antibiotic...References |
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After the primary culture results are available, conventional drug
susceptibility testing performed on solid medium takes 2–4 weeks.
When the radiometric BACTEC drug susceptibility testing system is used,
several weeks are needed to obtain results. Advances in molecular
biology have made it possible to investigate the genetic mechanisms of
drug resistance in M. tuberculosis and to develop methods
for rapid detection of mutations associated with resistance. The
currently available methods have mostly been developed for detection of
rifampin (RIF) resistance because the genetic basis of RIF resistance
in M. tuberculosis is fairly simple and well characterized,
whereas the molecular basis of resistance to other first-line TB drugs
is much more complex. Moreover, resistance to RIF can often be used as
a marker of multidrug-resistant TB.
genetic basis of resistance
The genetic basis of resistance to the five first-line TB drugs is
described here in brief. A detailed review of this topic has recently
been published (34). RIF is a semisynthetic derivative of
rifamycin that is used as a first-line TB drug. RIF binds to the ß
subunit of RNA polymerase, encoded by the rpoB gene, and
inhibits transcription initiation. Virtually all (96%) RIF-resistant
isolates have point mutations in an 81-bp region of this gene, and
these mutations are absent in susceptible isolates, making it an ideal
target for development of molecular drug susceptibility testing methods
(34).
In contrast to RIF, the genetic basis of resistance to other TB drugs
is more complex. Isoniazid (INH) is a synthetic, bactericidal agent
that is used only for treatment of TB because basically all other
bacteria are inherently resistant to INH. Alterations in a relatively
large number of genes have been associated with INH resistance, but
mutations in two genes, katG and inhA, are found
in 75–85% of INH-resistant M. tuberculosis isolates.
Streptomycin is an aminoglycoside antibiotic that inhibits protein
synthesis. Approximately 65–75% of streptomycin-resistant M.
tuberculosis isolates have mutations in the 16S rRNA gene or the
rpsL gene, which codes for the ribosomal protein S12. More
than 70% of the pyrazinamide-resistant M. tuberculosis
isolates have mutations in the pncA gene, which encodes for
pyrazinamidase, an enzyme that converts pyrazinamide to its active
form. Ethambutol inhibits the incorporation of essential mycolic acids
into the mycobacterial cell wall. Mutations in the embB gene
are associated with ethambutol resistance in 70% of resistant
isolates (34).
pcr-based sequencing
PCR-based sequencing is the main technique used to elucidate the
genetic mechanisms of drug resistance in M. tuberculosis. It
is the most direct and reliable method for studying mutations and
allows for detection of both previously recognized and unrecognized
mutations. Unfortunately, the method is not as readily applicable for
routine identification of drug resistance mutations as it is for
identification of mycobacterial species because many different genes
may be involved, as is the case in INH resistance, or the mutations may
be scattered in a large segment of the gene. This means that several
sequencing reactions need to be performed for each isolate. However,
for targets such as rpoB, where mutations associated with
RIF resistance are concentrated in a very short segment of the
gene, PCR-based sequencing is a useful technique (31).
line-probe assay
The Line Probe assay (LiPA; Inno-Genetics N.V., Zwijndrecht,
Belgium) has been developed for rapid detection of RIF resistance. The
test is based on the reverse hybridization method, and it consists of
PCR amplification of a segment of the rpoB gene and
denaturation and hybridization of the biotinylated PCR amplicons to
capture probes bound to a nitrocellulose strip. The bound amplicons are
then detected with alkaline phosphatase-conjugated streptavidin and
BCIP/NBT chromogen, producing a color reaction. The LiPA test strip
contains five probes for wild-type rpoB sequences and four
probes for specific rpoB mutations, in addition to a
conjugate control and M. tuberculosis control probes. The
interpretation of the banding pattern on the strip allows for
identification of M. tuberculosis complex and detection of
rpoB mutations. The test can be performed on M.
tuberculosis cultures or directly from clinical specimens. The
turnaround time for the test is <48 h. Overall concordance of the LiPA
test with phenotypic susceptibility testing and direct sequencing, when
performed from cultures, has been reported to be good, varying from
92.2% to 99.0% (35)(36)(37). Although the LiPA test only
detects 4 of the 35 distinct rpoB mutations, 75% of the
RIF-resistant clinical isolates carry 1 of the 4 mutations
(34), making the LiPA test a useful method for rapid
detection of RIF resistance. However, the test cannot be used for
detection of rare mutations.
dna microarrays
DNA microarray technology described for mycobacterial species
identification can also be used for rapid detection of mutations that
are associated with resistance to TB drugs. Gingeras et al.
(32) studied 44 RIF-resistant M. tuberculosis
isolates with a DNA array containing sequences from the rpoB
gene. A total of 40 isolates had a previously recognized mutation, one
new mutation was detected, and no rpoB mutations were found
in the remaining 3 isolates by either the DNA array or sequencing.
These results are in accordance with the data showing that 4% of
the RIF-resistant isolates have an unknown resistance mechanism
(34). In a study performed by Troesch et al.
(33), 15 RIF-resistant and 1 sensitive M.
tuberculosis isolate were analyzed with a similar DNA array. All
15 resistant isolates had mutations in the rpoB gene, and
the mutations were correctly detected with the DNA array. Mutation
types included single- and double-base substitutions and three- and
six-base deletions; in addition, one previously unrecognized mutation
was found. The results were completely concordant with sequencing
results (33). A DNA microarray for simultaneous detection of
various drug resistance determinants is currently being developed. It
contains sequences from the katG, inhA,
rpoB, rpsL, and gyrA (associated with
fluoroquinolone resistance) genes (32). This approach has
the potential of becoming the most effective and rapid method for
detection of drug resistance mutations in M. tuberculosis.
Despite the clear advantages that molecular methods offer for drug susceptibility testing, they all suffer from the problem that the genetic basis of resistance is not fully understood for any TB drug for all M. tuberculosis isolates. This means that detection of a mutation associated with resistance is clinically relevant, but a lack of mutations in the target gene does not necessarily mean that the organism is susceptible to the drug in question. The currently available molecular methods may aid in rapid detection of mutations associated with drug resistance, but the test results must always be confirmed by phenotypic methods.
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Footnotes |
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References |
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TopAbstractIntroductionDirect Detection of Mycobacteria...Identification of Mycobacterial...Identification of Antibiotic...References |
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