Pathology Patterns Reviews
Tuberculosis
Advances in Laboratory Diagnosis and Drug Susceptibility Testing
Quentin Eichbaum, MD, PhD, MPH,1 and Eric J. Rubin, MD, PhD2
Key Words: Susceptibility; Rapid culture; Molecular beacons; Nested PCR; Ligation; Spoligotyping; Strains
DOI: 10.1309/XX1NBANX8JKMTUQN
Abstract
Laboratory diagnosis of mycobacterial infection is
complicated by the fastidious growth requirements of
the bacillus organism. Delays in diagnosis can impede
effective treatment and surveillance of the disease.
Control of Mycobacterium tuberculosis has also been
aggravated by the emergence of multidrug-resistant
tuberculosis, and enhanced methods of antibiotic
susceptibility testing are needed. Molecular laboratory
techniques and advances in rapid culture methods have
led to the development of diagnostic and drug
susceptibility tests that are rapid and reliable. This
article describes recent innovations in the molecular
diagnosis and antibiotic susceptibility testing of M
tuberculosis.
© American Society for Clinical Pathology
Magnitude of the Clinical Problem
Despite rapid advances in molecular diagnosis and drug
therapies, tuberculosis (TB) remains among the leading
causes of death in the world today. The disease affects more
than one third of the world’s population, with about 8 million
new cases and approximately 2 million TB deaths a year.1,2
Disconcertingly, 95% of the disease burden is borne by
developing countries whose health care systems and
economies are poorly prepared to cope with the epidemic. In
the United States, the use of antituberculosis drugs, improved
standards of living, and better infection control measures
contributed to the decline of the disease by the early 1980s.
Since the mid-1980s, however, the emergence of the HIV
epidemic, together with associated factors such as deterio-
rating health infrastructures and a growing vulnerable immi-
grant population, have resulted in a persistent challenge.3,4
The increasing number of infections with multidrug-resistant
strains of Mycobacterium tuberculosis also has been
alarming, with some areas of the world estimated to have
multidrug resistance rates as high as 36%.5 All isolates of M
tuberculosis now must be tested for antibiotic resistance.5
M tuberculosis grows very slowly, and, therefore, diag-
nosis by culture is a long process. In some cases, appropriate
therapy has not been recognized until after the death of
patients due to progressive disease. Devising enhanced and
cost-effective methods for rapidly and reliably diagnosing TB
and for determining the antibiotic susceptibility of isolates
will be critical determinants for establishing effective control
and surveillance measures for the disease and for devising
efficacious treatment regimens. This article reviews some of
the challenges we face in diagnosing M tuberculosis and
describes recent advances in molecular diagnosis and antibi-
otic susceptibility testing for the disease.
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Eichbaum and Rubin / TUBERCULOSIS
Diagnosis: Clinical and Laboratory
Clinical Test: The Tuberculin/Mantoux Test
Clinically, the most common test for M tuberculosis
infection is the tuberculin or Mantoux test. The goal of the test
is to distinguish TB-infected patients from those who have
never been infected. The test does not discriminate between
patients with active infection (with multiplying organisms and
clinical symptoms) and silent infection (with no clear clinical
symptoms). Moreover, some patients previously have been
vaccinated against TB using the attenuated strain Mycobac-
terium bovis BCG. Because much of the world’s population
receives this vaccine (despite its rather poor ability to protect
against adult TB), some will have persistently false-positive
purified protein derivative (PPD) test results, which compro-
mises the value of this clinical test and continues to spur the
search for sensitive and specific laboratory tests.
A recently developed variation of the tuberculin PPD
assay is the Quantiferon-TB assay (Cellestis, Melbourne,
Australia). This is a quantitative in vitro diagnostic assay for
the detection of M tuberculosis in humans with a low to high
risk of infection. Undiluted whole blood is stimulated with
PPD and incubated overnight, and plasma samples are assayed
for interferon-gamma production using a single-step enzyme-
linked immunosorbent assay. One antibody attaches the inter-
feron to the plate, and the other quantitates the interferon via
an enzyme colorimetric assay. The assay provides a more
objectively controlled and quantifiable assay than the skin
tuberculin test, has a lower rate of false-positive results than
the skin tuberculin test, and yields results within 24 hours.
Laboratory Tests: Inadequacy of Conventional Methods
Laboratory diagnosis of TB has been a challenge in part
because of the fastidious growth requirements of these slow-
growing bacilli. Conventional methods of diagnosing M
tuberculosis are slow because they rely on bacterial growth
in culture and on conventional plate methods for identifying
species. Nevertheless, some conventional methods are useful
for distinguishing tuberculous from nontuberculous
mycobacteria and others for differentiating species such as
Mycobacterium bovis from M tuberculosis.5,6
Acid-Fast Staining
The acid-fast bacillus (AFB) “smear” using direct
microscopic examination remains the initial step for evalua-
tion of TB. The method is inexpensive and fairly rapid. It is
the only laboratory diagnostic test for TB used in most of the
world. Results should be available within 24 hours of spec-
imen submission. Three types of staining procedures are
used: Ziehl-Neelsen, fluorochrome, and Kinyoun. For organ-
isms recovered from culture, the classic Ziehl-Neelsen
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(carbolfuchsin) stain is preferred because it permits
enhanced visualization of the morphologic features of the
organism and has superior specificity for identification of M
tuberculosis. For screening, the fluorochrome dye rhodamine
(or rhodamine-auramine) is used instead of the conventional
Ziehl-Neelsen or Kinyoun procedures because the bright
fluorescence of stained bacteria under UV microscopy
increases the sensitivity of detection at relatively low micro-
scopic power. The Kinyoun procedure is a stain done without
heating, unlike the Ziehl-Neelsen that requires heating of the
microscope slide for enhanced stain penetration.
An advantage of the AFB stain is that the number of
infecting bacilli can be estimated, and, therefore, the extent
of a patient’s infectiousness can be assessed for clinical and
epidemiologic outcomes. However, the approximately 100
different species of the genus Mycobacterium all appear
similar on AFB staining, and factors such as the staining
method, specimen type, and culture method can influence
the sensitivity.7 The AFB smear without culture cannot
distinguish between tuberculous and nontuberculous
mycobacteria and is most useful for diagnosing pulmonary
rather than extrapulmonary TB (which is on the increase
owing to AIDS). Moreover, the test has poor sensitivity
(ranging from 20% to 80%8), even though its specificity for
microscopic identification is relatively high (90%-100%).7,9
Traditional Culture Methods
Identification of mycobacteria traditionally has relied on
culture on solid media. Culture is far more sensitive than smear
microscopy alone and often is necessary for precise species
identification and for drug susceptibility testing. The sensitivity
of culture is 80% to 85%, and the specificity is 98%.10 Isolates
are plated on a variety of solid media (synthetic, such as
Middlebrook 7H11 agar, and egg-based, such as Löwenstein-
Jensen slants). After cultures, a variety of methods can be used
to differentiate the mycobacteria. These include looking for
pigmentation (that may or may not require exposure to light to
develop) and biochemical testing. One of the major criteria
used to identify isolates is the time required for growth. Fast
growers, a group that includes Mycobacterium fortuitum and
Mycobacterium chelonae, generally will grow in less than 1
week. However, M tuberculosis requires from 3 to 8 weeks to
form a colony on these media. During this time, patients may
remain untreated and can continue to serve as sources of new
infection. Thus, more rapid and reliable tests would greatly aid
in the treatment and control of disease.
Methods for Rapid Diagnosis
A laboratory-based strategy for more effectively dealing
with the ongoing epidemic of TB (and multidrug-resistant
© American Society for Clinical Pathology

TB) should focus on generating tests capable of the
following: (1) rapid diagnosis, including species identifica-
tion; (2) increased sensitivity and specificity; (3) rapid
antibiotic susceptibility testing; and (4) rapid and definitive
strain typing.
Recent advances in molecular biology have ushered in
numerous exciting improvements in microbial diagnostics
and antibiotic susceptibility testing. For several infectious
diseases, this has resulted in more rapid and accurate diag-
nosis with impressive treatment outcomes. Assays that previ-
ously took days or even weeks to complete can now
frequently be done with improved sensitivity and specificity
in less than a day. We review some of the advances and chal-
lenges in the molecular and biochemical diagnosis of M
tuberculosis, as well as new methods of testing susceptibility
to first-line antimycobacterial drugs.
Culture: Rapid Methods
The introduction of semiautomated liquid culture
systems substantially reduced turnaround times from 3 to 8
weeks to 7 to 12 days for identification of positive
cultures.11 Moreover, the sensitivity of culture is greater
than that of the acid-fast smear. Liquid culture requires only
10 to 100 bacilli per milliliter of sputum for generation of a
positive culture, whereas AFB microscopic detection
requires 5,000 to 10,000 bacilli per milliliter.12 A study
comparing AFB staining and culture methods against clin-
ical criteria as the “gold standard” for TB diagnosis yielded
a sensitivity of 53.7% for AFB staining and 87.5% for
culture methods.13 Both methods, however, showed speci-
ficities of greater than 99%.
In addition to continuous growth monitoring systems
(BACTEC 9000MB [Becton Dickinson, Franklin Lakes, NJ]
and ESP Culture System II [TREK Diagnostic Systems,
West Lake, OH]), there are 3 other commonly used liquid
culture methods available: (1) BACTEC 460TB, a radio-
metric method using culture media containing carbon 14
(14C)-labeled palmitic acid that is metabolized to measurable
14CO ; (2) the SeptiChek AFB System, a biphasic culture
2
detection system that includes both liquid and solid modified
Middlebrook media; and (3) the Mycobacteria Growth Indi-
cator Tube (MGIT) method (which measures mycobacterial
growth as a function of oxygen consumption as indicated by
an increase in measurable fluorescence). Positive cultures
obtained by these methods are verified by AFB staining and
species identification methods (see “Species Identification”).
An automated system using the MGIT technique (MGIT
960 system) was developed recently. This system permits
rapid throughput of specimens at 4 times that of the semiau-
tomated BACTEC 460TB system. The MGIT system also
does not use radioactivity and has fewer manual labor
requirements.14
© American Society for Clinical Pathology
Pathology Patterns Reviews
Molecular Approaches
Molecular methods are routinely used to distinguish
between tuberculous and nontuberculous species of
mycobacteria. In addition, identification of mycobacterial
nucleic acids is being used for strain typing of mycobacteria
to determine epidemiologic patterns relevant to the surveil-
lance and prevention of infection. These approaches also are
proving to be rapid, reliable methods of identifying bacteria
directly from smear-positive clinical specimens and might
prove useful in some smear-negative cases.15
The sensitivity of nucleic acid probe technology is
enhanced by a variety of amplification techniques. These
techniques fall mostly into the following 4 categories: (1)
target amplification systems; (2) signal amplification
systems; (3) probe amplification systems; and (4) automated
systems of target preparation, amplification, and detection.
Amplification of the Molecular Target
Polymerase Chain Reaction (PCR) Methods.—Amplifi-
cations using the polymerase chain reaction (PCR) may be
achieved by a variety of techniques that vary in their sensi-
tivity, specificity, and ease of use. These methods are as
follows: (1) standard DNA PCR amplification; (2) nested,
heminested, and balanced heminested PCR; and (3) ribo-
somal RNA (rRNA) amplification. Identification of M tuber-
culosis may be from single-copy or repeat sequences of
DNA or RNA. Repetitive nucleic acid sequences have the
advantage of serving as “preamplified” sequences, thereby
increasing the sensitivity of the PCR assay. Mutation of 1
sequence of the repeat sequences would not yield a false-
negative result, owing to amplification of the remaining
intact sequences. This would reduce the overall risk of false-
negative results compared with single-copy sequences in
which such mutations may yield false-negative results.
One method of standard DNA PCR amplification is the
Roche Amplicor Mycobacterium Tuberculosis Test, a
method used for direct detection of M tuberculosis complex
in respiratory specimens. The system uses deoxyuridine
triphosphate (dUTP) as a substrate, as well as an enzyme
(uracil-N-glycosylase) to destroy carryover from previous
PCR runs. The dUTP nucleotides present from previous runs
are destroyed preferentially during the first denaturation
cycle before the start of thermal cycling. The method
prevents contaminating carryover from previous PCR runs,
thereby reducing the chances of false-positive results and
improving the specificity of the assay.
The Roche Amplicor system performs very well on
AFB smear–positive specimens (sensitivity, 95%-96%;
specificity, 100%) and is readily capable of differentiating
patients with TB from those infected with nontuberculous
species. For AFB smear–negative patients, the sensitivity is
about half (sensitivity, 48%-53%; specificity, 96%-99%).16
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One complication of amplification techniques lies in
coinfection of samples with other organisms that may go
undetected. For instance, if the PCR assay is directed at
detection of Mycobacterium avium complex, coinfection
with M tuberculosis may be missed. To overcome this
problem, multiplex PCR techniques using genus-specific
primer pairs for detecting various species within the same
assay are being developed.
By comparison with standard PCR using a single primer
set, nested PCR uses an additional set of primers to improve
sensitivity of the amplified product. In the standard 2-step
nested PCR technique, a set of outer primers initially ampli-
fies a DNA sequence followed by a second round of PCR in
which a set of inner primers amplifies a shorter amplicon
contained (nested) within the stretch of DNA amplified
during the first round ❚Figure 1A❚.
Compared with conventional PCR using a single set of
primers, improvements in sensitivity of up to 1,000-fold can
be achieved in detection of mycobacteria.17,18 Disadvantages
of this nested method of amplification include the additional
manipulations required, the greater length of time the assay
requires, and the increased risks of contamination. To over-
come these risks and impediments, single-tube nested PCR
with a uracil-N-glycosylase–dUTP system has been used
with some success (see the preceding discussion about the
Roche Amplicor PCR method).19 However, genomes such as
that of M tuberculosis, which have a high G + C content,
pose a problem in the optimal placement of nested primers

A B C

A C B
C B
B A CA

PCR PCR
PCR
1st Round 1st Round 1st Round

A CA

C
D B B
A

2nd Round 2nd Round 2nd Round

A C

C C
CA

C
C
❚Figure 1❚ Nested, heminested, and balanced heminested polymerase chain reaction (PCR). A, In the standard 2-step nested
PCR method, an outer set of primers initially amplifies a DNA sequence. This is followed by a second round of PCR in which a
set of inner primers amplifies a shorter amplicon contained (nested) within the stretch of DNA amplified during the first round.
B, In heminested PCR, a set of outer primers (A and B) anneals to the DNA strands under optimal conditions, one primer (A)
being more concentrated than the other (B). The more concentrated primer participates in a second round of amplification with
another inner primer (C), while the other outer primer (B) is depleted owing to its low concentration, as depicted by the smaller
arrow. C, In balanced heminested PCR, initially only the outer primers (B and CA) hybridize to the DNA, whereas the inner
primer (C) cannot hybridize due to its low melting temperature. These outer primers have equal concentrations, which results in
symmetric amplification. One outer primer (CA) contains the same sequence as the inner primer (C), and thus serves as an
annealing template for this inner primer during a second round of amplification when a lower annealing temperature permits
this inner primer to bind. The inner primer also has a higher concentration and can anneal to both strands, leading to a more
balanced and efficient amplification than is possible in the standard heminested PCR method.

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owing to their shortage of unique sequences. Amplifications
may become asymmetric and the overall reaction, therefore,
less efficient.
Heminested PCR attempts to overcome primer place-
ment incompatibilities by manipulating the concentrations of
primers in a reaction that uses 2 outer primers but only 1
inner (nested) primer ❚Figure 1B❚. During the first round of
amplification, temperature constraints permit only the outer
primers to anneal. One of the outer primers is more concen-
trated than the other since it also participates in the next
round of amplification with the single inner primer, whereas
the other outer primer is depleted owing to its low concentra-
tion. The method is, nevertheless, not optimally efficient
because it results in asymmetric amplification.
Balanced heminested PCR overcomes the problem of
asymmetric amplification. In this method, the original outer
primer that participates in both rounds of amplification in the
standard heminested technique is replaced with another
primer containing the same sequence as the opposite inner
primer at the 5' end. In this manner, a target is created for the
inner primers of both strands, leading to balanced, highly
efficient amplifications with minimum risk of cross-contami-
nation ❚Figure 1C❚. The balanced heminested method of
PCR is particularly useful for the detection of M tuberculosis
in smear-negative specimens.18 One study showed the sensi-
tivity of the assay improved by 15% (to 75%), whereas the
specificity was identical to that of standard PCR (100%).
Thus, the balanced heminested PCR method is one of the
most sensitive assays for the detection of M tuberculosis in
smear-negative specimens.
M tuberculosis contains a large number of copies of the
rRNA 16S subunit that can serve as a source of “preampli-
fied” target for in vitro amplification via reverse transcrip-
tase–PCR. The multiplicity of these copies serves to increase
the sensitivity of the assay. Because stretches of this 16S
rRNA sequence are highly variable among species, they also
can serve as targets differentiating species of mycobacteria
through sequencing of the amplified product. This method of
species identification has been found to have the advantages
of accuracy, speed, and versatility over some of the more
conventional methods such as restriction fragment enzyme
digestion (see “Nucleic Acid Sequencing” under “Species
Identification”).20,21
Target Amplification by Non-PCR Methods: Restriction
Fragment Enzyme Digestion.—The development of non-
PCR amplification techniques has been fueled by proprietary
claims on PCR enzymes (held by Roche). This has led to a
search for methods of amplification that do not use any of
the proprietary PCR systems. Transcription-mediated ampli-
fication (TMA) assays involve the conversion of rRNA
substrates to single-stranded DNA substrates that then are
transcribed into RNA amplicons.
© American Society for Clinical Pathology
Pathology Patterns Reviews
Gen-Probe’s Mycobacterium Tuberculosis Direct Test is
such a TMA test in which mycobacterial rRNA targets are
converted to DNA that then is transcribed to RNA ampli-
cons. The amplicons are detected through complementary
binding with a single-stranded DNA probe containing a
chemiluminescent label. Stable hybrid RNA-DNA molecules
are bound selectively by the acridinium ester reporter mole-
cule, which is itself “excited” to yield measurable chemilu-
minescence. The amplification reaction occurs at a single
temperature and in a single liquid phase that does not require
separation of products. Furthermore, the abundance of rRNA
inside M tuberculosis serves as a preamplified target that
enhances the sensitivity of the assay. The assay is performed
in a single tube with minimal carryover contamination risks
and does not entail purification of nucleic acids. Moreover, it
can be completed within about 5 hours and also is capable of
detecting related members of the M tuberculosis complex.22
The Mycobacterium Tuberculosis Direct Test has been
evaluated in a number of studies.23-26 Sensitivity ranges
between 86% and 98% and specificity between 96.8% and
100%. Yet, the test is prone to discrepancies resulting from the
quality of the specimen, the burden of bacilli, and the presence
of inhibitors in the amplification reaction. The test must always
be performed in conjunction with mycobacterial culture.
Amplification of the Signal Detection System
Recent methods of detecting amplified products have
used colorimetry, chemiluminescence, digoxigenin, and
branched DNA (bDNA). The most advanced detection
systems combine nonradioactive methods with automation
and the ability to detect amplified sequences from multiple
samples in microtiter plates at the same time.
Chemiluminescence and Colorimetry (via Digoxigenin
Incorporation).—An assay developed by Fiss et al26 amplified
a region of the mycobacterial 65-kd heat shock protein while
incorporating digoxigenin-labeled uridine triphosphate into the
amplified product. In this method, target DNA fixed to
membranes is detected through hybridization to DNA probes
conjugated with alkaline phosphatase, and the signal is ampli-
fied by chemiluminescence. This sensitive chemiluminescence
assay obviates the need for radioactive signal amplification
and has a fast turnaround time of 1 to 2 days. The assay can
detect as few as 100 mycobacterial colony-forming units. The
authors also developed a reverse dot blot assay in which
amplified DNA product fixed to membranes is detected with
an antidigoxigenin antibody conjugated with alkaline phos-
phatase before signal amplification with chemiluminescence.
The assay is capable of effectively distinguishing M tubercu-
losis isolates from M avium complex isolates.
A similar digoxigenin assay developed by Wilson et al27
uses a colorimetric enzyme detection system. A first round of
amplification uses unlabeled outer primers, followed by a second
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amplification round with inner (nested) primers, each labeled
with biotin or digoxigenin. The amplified product incorporates
both biotin and digoxigenin. The biotin moiety is used to attach
the product to an avidin-coated microtiter plate, and the digoxi-
genin moiety reacts with an alkaline phosphatase–conjugated
antibody. Addition of enzyme substrate permits detection and
quantitation of the amplified product.
Branched DNA-Mediated Alkaline Phosphatase Detec-
tion.—Another new method of signal amplification (success-
fully used for determination of viral loads of HIV and
hepatitis C virus but as yet not applied to the detection of
mycobacteria) involves the use of bDNA. 28 The probe
(binding target DNA or RNA) contains synthetic branched
DNA molecules with typically 15 branched arms of DNA,
each of which is capable of binding 3 alkaline phosphatase
molecules (to give a total of 45 alkaline phosphatase mole-
cules per bDNA molecule). Amplification is achieved by
incubation with a chemiluminescence substrate to yield a
highly amplified signal directly proportional to the amount
of target nucleic acid.
Amplification of the Molecular Probe: Ligase Chain
Reaction
A commercially available probe amplification assay for
detection of M tuberculosis in clinical specimens is based on
the ligase chain reaction (LCx assay, Abbott Laboratories,
Chicago, IL). This test involves a high-temperature DNA
denaturation step, followed by a cooling step in which 2 sets
of adjacent complementary oligonucleotides anneal to the
single-stranded target molecules and then themselves ligate
❚Figure 2❚. The ligation product from this cycle then serves
as the template for the next cycle’s ligation reaction. Repeti-
tions of this cycle lead to an exponential amplification of
ligation products.
Several studies have compared the LCx assay with other
assays for the detection of M tuberculosis.29-31 Garrino et al29
compared the LCx assay with direct microscopy and stan-
dard microbiologic culture. The sensitivity of the LCx assay
was 98% for microscope smear-positive specimens but only
27% for smear-negative samples. Only 84.6% of patients
with positive culture results also had positive results by this

G G AC TT AG C TA G G AC TT AG C TA
T G
DNA denaturation
and annealing of Mismatched
oligonucleotides oligonucleotides—
A no annealing
C
C C T G A AT C G AT
C C T G A AT C G AT

Thermostabile
ligase—ligates No ligation
oligonucleotides

Denaturation,
annealing, and
ligation cycle
repeated 30 times

❚Figure 2❚ Ligase chain reaction involves the denaturation of DNA followed by annealing of 2 sets of adjacent primers to each
strand of DNA. The primers themselves then ligate, and the new ligation product created serves as a template for the
polymerase chain reaction. The cycle then repeats itself, resulting in exponential amplification of the original segment of DNA. If
the adjacent sets of primers are mismatched, complementary binding to the DNA strands will not take place and the primers
will not be ligated and cannot serve as the template for the next amplification cycle.

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ligase chain reaction method. Although the specificity of the
assay (100%) was equivalent to that of culture (100%) and
acid-fast staining (99%), its sensitivity (78%) was lower than
that of direct microscopy for acid-fast staining. Thus, the test
may be of limited usefulness in populations with low TB
prevalence (such as in the United States).
Other studies have concluded that the sensitivity of the
LCx assay is at least as good as that of other direct amplifica-
tion tests for detecting M tuberculosis in respiratory tract
specimens.31 Although LCx reliability was greatest for smear-
positive samples, it also has some benefit for the diagnosis of
smear-negative samples in patients in whom there is a high
clinical suspicion of pulmonary TB. A study comparing 3
nucleic acid amplification methods (COBAS AMPLICOR
PCR, Gen-Probe Mycobacterium Tuberculosis Direct Test,
and LCx) found no significant overall differences in the sensi-
tivities and specificities of the 3 tests applied to combined
smear-positive and smear-negative samples. All the same,
some have cautioned that these molecular tests should be
used only as adjuncts to smears and culture.32
Automated Systems of Target Preparation, Amplification,
and Detection
The continued drive for more rapid, more cost-effective,
and less labor-intensive methods of molecular testing has led
to the development of systems that automate the steps of
target preparation, amplification, and detection.
The first system to automate both the amplification and
detection steps of PCR testing was the COBAS
AMPLICOR instrument.33 This is a bench-top instrument
intended for routine PCR diagnostic testing and capable of
up to 50 detection results per hour following the initial
amplification step. An improvement on this system is the
COBAS TaqMan that similarly automates the PCR amplifi-
cation and detection steps but provides real-time quantita-
tive PCR detection within a period of minutes using fluoro-
genic reporter molecules.
The COBAS AmpliPrep system automates specimen
preparation. Target nucleic acid is released from the microbe
and captured with specific oligonucleotides that become
attached to magnetic beads via a biotin-streptavidin binding
reaction.34 The attached target then is automatically purified
and concentrated by the machine. So far, the method has
been used for preparation of hepatitis C virus and HIV speci-
mens for which it has reduced hands-on time by 76%
compared with manual specimen preparation methods.35
Recently, Gen-Probe developed a high-volume multiple-
assay instrument (TIGRIS) with automated nucleic acid
purification, amplification, and detection systems to replace
the manual and semiautomatic tasks of the TMA system.34,35
Presumably, Gen-Probe’s Mycobacterium Tuberculosis
Direct Test eventually will be run on this instrument.
© American Society for Clinical Pathology
Pathology Patterns Reviews
Serologic Methods
A number of serologic assays for the detection of M
tuberculosis have been developed with the objective of
improving time to diagnosis. While several of these diag-
nostic assays are more rapid than conventional assays, none
are as yet sufficiently accurate for routine use, nor have these
tests proved to be more sensitive for the detection of M tuber-
culosis in smear-negative samples of patients, where they
presumably would find their best applicability. The tests also
are as yet incapable of distinguishing between active and
latent TB infection, and they distinguish only poorly between
M tuberculosis and nontuberculous mycobacterial infections.
They remain relatively costly and require additional personnel
training, which likely places them outside the financial reach
of many developing countries where they would be most
needed. Chan et al36 comprehensively reviewed current sero-
logic tests for TB and concluded that they offer little advan-
tage over conventional and molecular testing.
Amplification From Histologic Specimens
In patients in whom the diagnosis of TB depends on
tissue examination rather than isolation of the mycobac-
terium from bodily secretions, amplification of mycobacte-
rial DNA provides a potentially sensitive and rapid method
of detection. This is particularly true for surgical specimens
found unexpectedly to contain AFB on histopathologic
examination and when tissue is no longer available for
culture. Human tissue samples for histopathologic examina-
tion are stored most commonly as formalin-fixed, paraffin-
embedded blocks. Tissue-preserving substances in the blocks
preclude subsequent culturing of organisms for diagnostic
purposes. Although tissue preservation can alter the physical
and chemical properties of DNA, PCR can still be performed
on DNA retrieved from such fixed tissue. Moreover, DNA
remains generally stable with time, and sequences of M
tuberculosis have been retrieved from archeological speci-
mens, such as from a mummified woman who died about
900 years ago.37
Nevertheless, fixation can affect the sensitivity and
specificity of PCR assays. Factors that have been shown to
affect PCR amplification include the type of fixative used,
buffering of the formalin, duration of fixation, and the size of
the amplified product.38,39 Several studies have successfully
applied PCR techniques to detect M tuberculosis DNA in
formalin-fixed tissues.40,41 Fixation in 10% neutral-buffered
formalin for up to 7 days has little effect on assay sensitivity.
Rish et al42 detected as few as 9 mycobacterial organisms in
a 5-µm section of tissue. Salian et al43 compared PCR ampli-
fication of mycobacterial DNA in a variety of fixed tissue
types obtained from various clinical laboratories within and
outside the United States from patients with clinically diag-
nosed TB. On positive cultures, PCR amplification of M
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Eichbaum and Rubin / TUBERCULOSIS
tuberculosis DNA (IS6110 segment) was 100% sensitive and
93% specific. By comparison with the clinical diagnosis of
TB, PCR was 73.6% sensitive and 100% specific.
Marchetti and coworkers44 compared the sensitivities
and specificities of 4 nested PCR assays amplifying different
M tuberculosis genomic DNA sequences from formalin-
fixed, paraffin-embedded tissues. The highest sensitivities
(80%-87%) using different primer sets were obtained with
the highly repetitive IS6110 sequence. However the overall
efficacy of PCR depended strongly on parameters such as
DNA concentration, target DNA size, and the repetitiveness
of the amplified sequence.
A method using magnetic beads (Dynabeads, Dynal)
instead of the conventional ethanol-chloroform method for
extraction of DNA from tissues can be used to obtain nucleic
acid from formalin-fixed, paraffin wax–embedded tissues
and frozen tissues.45 The method requires less than 2 hours
to prepare DNA for PCR from either paraffin or frozen
sections. Magnetic bead extraction conceivably might be
adapted for extracting antigens of interest by linking the
requisite specific antibodies to the beads and directly
extracting the relevant antigens from fixed tissues or body
fluid samples.
Diagnostic PCR amplification of M tuberculosis from
fixed tissue sections should be done in conjunction with
other laboratory and clinical data to reach an accurate diag-
nosis of TB and obviate false-negative results. Marchetti et
al44 pointed out that M tuberculosis DNA has the capacity to
remain in tissue specimens for many years in the absence of
any viable organisms, as has been confirmed by the recovery
of such DNA from archeological specimens, as well as from
patients who have fully recovered after treatment for
pulmonary TB. Despite these caveats, PCR has the potential
to increase the accuracy of diagnosis of M tuberculosis infec-
tion in tissue specimens and is of especial value in infections
associated with a granulomatous response.
Species Identification
Although most of the effort for enhanced diagnosis of
mycobacteria has focused on M tuberculosis, identification
of other clinically important species cannot be ignored, espe-
cially since the species may dictate the regimen of antimicro-
bial therapy. This is particularly true for nontuberculous
mycobacteria. As of now, nucleic acid amplification methods
(discussed in the preceding sections) are the most rapid and
reliable methods of identifying members of the M tubercu-
losis complex.
Nucleic Acid Sequencing
Mycobacterial species may be identified by species-
specific sequences. The small 16S subunit of rRNA
containing various conserved, variable, and hypervariable
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stretches of nucleic acid has proved particularly useful for
providing species differentiation. Comparison of unique and
more conserved regions of nucleic acid with this rRNA
subunit using a biotinylated primer for single-stranded, solid-
phase sequencing has permitted accurate and rapid species
identification.
Identification by nucleic acid sequencing usually can be
completed within 1 working day and has the advantages over
conventional biochemical techniques of versatility, accuracy,
and speed. Not only does the method permit accurate identi-
fication in a routine microbiology laboratory of known
mycobacteria isolates but it also permits identification of
previously unidentified species.
The genus Mycobacterium has been shown to be more
complex than anticipated, containing several newly charac-
terized pathogens such as Mycobacterium interjectum,
Mycobacterium conspicuum, and Mycobacterium inter-
medium. Standard biochemical techniques of identification
underestimated the complexity of this genus, and these
biochemical assays themselves were subject to considerable
interassay variability. The newer 16S rRNA sequencing
method has permitted identification of some mycobacteria
whose fastidious growth patterns made biochemical charac-
terization impossible.
Gas-Liquid Chromatography
Gas-liquid chromatography (GLC) analyzes the short-
chained fatty acids in the mycobacterial cell wall for species
identification. The mycobacterial identification system based
on this method was adapted to an identification system
developed by Microbial ID Inc (MIDI, Newark, DE). The
MIDI database contains descriptions of more than 30
species, and the system correctly identifies about 80% of all
isolates.46 This system correctly identified 77% of M tuber-
culosis and 83% of M avium complex isolates. Although
many organisms could be identified to the species level, a
few still required a limited number of additional biochemical
tests for confirmation. Thus, this GLC system of identifica-
tion is best used in association with other biochemical tests
and examination of colonial morphologic features. Optimal
application of this GLC system for mycobacterial species
identification is best achieved in combination with nucleic
acid probe testing.
High-Performance Liquid Chromatography Methods
The number of mycolic acids contained in the wall of
each species of mycobacteria is constant and can be used
for species identification using high-performance liquid
chromatography (HPLC). Saponification of the mycolic
acids yields long-chain fatty acids that can be extracted
into organic solvent and analyzed by HPLC where they
yield distinct identifiable peaks. Identification of
© American Society for Clinical Pathology

mycobacteria by this method is based on the number of
these chromatographic peaks and their height and position
(retention time). These are recorded in a computer-based
reference library available from the Centers for Disease
Control and Prevention, where this method was developed
during the 1970s.
Although the method has a high specificity for the iden-
tification of certain mycobacterial species, some of the
commercially available HPLC systems have exhibited incon-
sistencies in identification. Overall, the system seems best
suited for identification of M tuberculosis complex and M
avium complex by fluorescence detection of mycolic acids
directly from BACTEC 12B vials that exhibit acid-fast smear
positivity. The method most likely will be restricted to refer-
ence laboratories owing to the cost and the technical exper-
tise required.
Rapid Antibiotic Susceptibility Testing
With the alarming increase in the number of drug-resis-
tant cases of TB, rapid and effective methods of drug suscep-
tibility testing have assumed renewed importance. Wenger
and colleagues47 showed that the clinical outcome and the
control of TB transmission are influenced strongly by the
time taken to obtain drug susceptibility results. The Centers
for Disease Control and Prevention has recommended that
the most rapid methods be used for testing all isolates of M
tuberculosis and that results of these tests should be available
within 30 days of specimen receipt.
Conventional Susceptibility Testing
Conventionally, susceptibility of mycobacteria to
antibiotics has been tested by plating liquid cultures of each
organism onto antibiotic-impregnated and control agar.
Effective susceptibility is gauged as growth inhibition of
99% of the organisms. The critical concentration of the
antimycobacterial drug is the concentration of drug
required to inhibit growth above the 1% threshold of the
tested population of bacilli. Generally, an isolate of M
tuberculosis is tested against 5 antimicrobials, referred to as
first-line or primary drugs. Testing for susceptibility against
all 10 to 12 available drugs can require 2 to 3 months,
whereas susceptibility profiles against the first-line drugs
usually are available within 4 to 7 days from the time the
culture becomes positive.
Conventionally, 3 methods of mycobacterial susceptibility
testing have been used48: The method of absolute concentra-
tion expresses resistance as the lowest concentration of drug
that inhibits all growth (minimum inhibitory concentration) in
a standardized inoculum compared with a similar drug-free
inoculum. The resistance ratio method compares growth of
© American Society for Clinical Pathology
Pathology Patterns Reviews
test organisms at 3 different concentrations of antibiotic with
growth of a control wild-type strain. The proportion method
compares, at several dilutions of standardized inoculum, the
number of colonies on each antibiotic plate with growth on
control drug-free plates. The extent of growth in the presence
or absence of drug is expressed as a percentage, with more
than 1% considered clinically resistant.
New Approaches
Established, more rapid tests of susceptibility testing
include the BACTEC radiometric method, the E-test,49 and
the redox-indicator-dye method,50 which take an average of
1 to 2 weeks to complete. The BACTEC system assesses
antibiotic susceptibility radiometrically by measuring the
amount of carbon dioxide produced from a 14C isotope
added to the antibiotic-containing and the control media. A
relative increase over time in radioactivity incorporated into
carbon dioxide indicates resistance to the antibiotic. The
test has been used with isoniazid, rifampin, ethambutol,
pyrazinamide, and streptomycin. Although it can be
completed in about 10 days, the assay has the drawback of
involving radioactivity and of being relatively expensive.
By comparison, more recent molecular methods, such as
flow cytometry51 and genotypic assays,52 and nonradio-
metric phenotypic (or viability) assays can be completed in
just a few days.
Genotypic Assays
Genotypic assays for antibiotic susceptibility testing are
based on genetic mutations that are correlated with the resis-
tance pattern. These assays have the advantage of being very
rapid. However, their use is limited by the available number
of polymorphisms responsible for antimicrobial resistance.
Antibiotic resistance can be limited to a fairly small area of a
single gene (eg, rifampin resistance) or can be spread over
portions of many genes and their regulatory regions (eg,
isoniazid resistance). The value of sequence-based methods
for antibiotic susceptibility testing is dependent on the
number and location of mutations mediating resistance.
Molecular Beacon Sequence Analysis.—One method,
molecular beacon sequence analysis, is capable of distin-
guishing single nucleotide substitutions, as well as inser-
tions in bacterial DNA. The method entails the production
of a fluorescent signal dependent on the formation of a
sequence-specific hybrid molecule formed between the
probe and target DNA and performed in the setting of a real-
time PCR assay.
The sequence-dependent fluorogenic reporter molecules
(called molecular beacons) are single-stranded nucleic acid
molecules with a stem-and-loop structure ❚Figure 3❚. The
loop portion serves as a probe hybridizing to a complemen-
tary target nucleic acid sequence. The stem is formed by the
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Eichbaum and Rubin / TUBERCULOSIS

annealing of complementary arm sequences on either Phenotypic (Viability) Assays

side of the probe sequence. One arm has a fluorescent
moiety attached, the other a nonfluorescent quencher
molecule. In the absence of a target molecule, the
quencher-fluorescent hybrid remains intact and prevents
emission by the fluorophore. On encountering a target
molecule, a stable probe-target hybrid is formed that
undergoes a conformational change separating the
quencher arm from the fluorescence arm, thereby permit-
ting the fluorophore to fluoresce.
The strengths of the method lie in the specific target
binding achieved, the option of using different colored fluo-
rophores, and the use of hermetically sealed PCR tubes that
obviate cross-contamination and are readily amenable to
automation. Telenti et al53 used the method successfully to
detect genotypic mutations associated with resistance to
rifampin in M tuberculosis. They confirmed that 96% of
rifampin-resistant M tuberculosis strains had mutations in an
81-base-pair (bp) core region of the rpoB gene.
Phenotypic assays for testing antibiotic susceptibility are
based on assessing mycobacterial viability in the presence of
effective antibiotics. Viability can be assessed by quanti-
tating adenosine triphosphate stores, by ascertaining the
ability of mycobacteria to support bacteriophage replication,
or by synthesis of the reporter gene product, firefly
luciferase.54-56
MGIT Assay.—Rapid liquid culture methods have been
adapted for susceptibility testing and have reduced the turn-
around times from several weeks to 6 to 12 days after receipt
of the specimen.11,57 The MGIT method, which measures
oxygen consumption through a UV indicator source (see
“Culture: Rapid Methods”), has been adapted for suscepti-
bility testing by assessing the viability of mycobacteria in the
presence and absence of antibiotics.58
Mycobacteriophage Plaque Assays.—The “phage-
amplified biologically assay,” developed by Wilson et al,55
uses mycobacteriophages (viruses that infect mycobacteria)

Q
Loop

+
Stem Target RNA

Q F

Molecular beacon Q F
with quenching
of fluorescent label
Initial hybridization F
with target RNA

No fluorescence due to quenching

Fluorescence following
unraveling of molecular
beacon and separation
of quencher molecule

❚Figure 3❚ Molecular beacon sequence analysis uses nucleic acid hybridization probes called molecular beacons in the setting of
a sensitive real-time polymerase chain reaction (PCR) assay. These molecules are single-stranded, nucleic acid structures that
produce a fluorescent signal on binding to their target DNA sequence. The molecules have a stem-and-loop structure. The loop
portion contains the specific hybridization sequence complementary to the target sequence, whereas the stem is formed from
the terminal complementary sequences of the loop. One arm of the stem has a fluorescent (F) reporter molecule attached and
the other a nonfluorescent quencher (Q) molecule. The quencher molecule forms a hybrid with the fluorescent molecule and
prevents fluorescence, while the stem remains intact. On encountering and hybridizing to a target sequence, the loop structure
opens, thereby also unraveling the stem. The quencher and fluorescent arms become separated, thereby permitting the
fluorophore to fluoresce. The molecular beacons can serve as primers in a real-time PCR. Several differently colored
fluorescence molecules can be used simultaneously in the same reaction to detect different target sequences. The assay is
capable of distinguishing single nucleotide substitutions as well as insertions in bacterial DNA. In the context of
antimycobacterial resistance testing, absence of any one of the fluorescent colors indicates resistance.

S12 Am J Clin Pathol 2002;118(Suppl 1):S3-S17 © American Society for Clinical Pathology
S12 DOI: 10.1092/XX1NBANX8JKMTUQN

to distinguish viable mycobacteria resistant to antibiotics
from those susceptible to antibiotic action. Only viable
mycobacteria can become infected with the mycobacterio-
phage, replicate, and eventually lyse the mycobacteria,
whereas dead bacilli cannot ❚Figure 4❚.
To perform this method, a lawn of mycobacteria is
initially incubated with the test antibiotic before being
infected with mycobacteriophage. A phagicidal chemical
then is used to kill the remaining noninfecting extracellular
phages. Only viable mycobacteria that are resistant to the
antibiotic can offer the infecting mycobacteriophages protec-
tion from inactivation. The protected mycobacteriophages
begin replicating and eventually lyse the viable host bacilli.
Lysis is discernible as clear areas (or plaques) on a lawn of
mycobacteria. Thus, the number of plaques is related directly
to the degree of antibiotic resistance. The assay takes an
average of 2 to 3 days to complete compared with 10 days
for conventional assays such as the resistance ratio method.
Eltringham and colleagues59 extended this method of
susceptibility testing to include ethambutol, pyrazinamide,
streptomycin, and ciprofloxacin. The method showed sensitiv-
ities between 90% and 100% and specificities between 70%
and 100% for the drugs tested. The test is easy to perform and
is a low-cost, reliable method requiring little training to screen
for antimycobacterial resistance in isolates of M tuberculosis.
Luciferase Assay.—A luciferase assay developed by
Riska et al60 uses a genetically engineered bacteriophage
instead of the wild-type phage of the phage-amplified
biologically assay. This method uses a lytic bacteriophage
that has been engineered to contain the gene for luciferase.
Under appropriate conditions, luciferase will emit light and,
thus, serve as a signal amplifier. The emitted light can be
detected easily using photographic film and can be adapted
for use in the low-budget settings of developing countries
using a custom-made light-tight aluminum box (the “Bronx
Box”), which houses a 96-well plate and instant photography
film. This method permits rapid discrimination between
strains susceptible or resistant to the antituberculosis agents
isoniazid and rifampin,60 with sensitivities and specificities
of virtually 100%. Susceptibility testing by this method also
can be done for ethambutol and streptomycin, but with
considerably lower sensitivity and specificity.
Strain Typing
Recent work suggests that strains of M tuberculosis
differ in virulence. For instance, strains with large genomic
deletions might be less virulent than those with point muta-
tions or small deletions.61 The typing of strains of M tuber-
culosis has become an important tool for surveillance,
control, and prevention of TB. Strain typing has been used
© American Society for Clinical Pathology
Pathology Patterns Reviews
Pure culture + phage Pure culture + phage + antibiotic
Addition of phagicide to
lyse extracellular phage
Plates with rapid grower
Mycobacterium smegmatis
Plaques No plaques
❚Figure 4❚ Phage-amplified biologically (PhaB) assay. The
PhaB assay makes use of bacteriophages infecting
mycobacteria (mycobacteriophages) to test for mycobacteria
resistant to specific antibiotics. Mycobacteria initially are
incubated with or without antibiotic and then infected with
mycobacteriophage. Extracellular phages are neutralized with
a “phagicidal” chemical, and the samples are further
incubated to permit replication of intracellular phages. Only
mycobacteria not susceptible to the antibiotic can offer the
phage protection from the phagicidal chemical. The
protected phages replicate and eventually lyse the
mycobacteriophage, leaving areas of clear lysis (plaques) on a
lawn of mycobacteria (rapid growing Mycobacterium
smegmatis). The number of these plaques is directly related
to the degree of antibiotic resistance.
effectively to monitor epidemiologic outbreaks and, on a
smaller scale, to determine sources of cross-contamination in
laboratories.61 These methods of typing also have permitted
a better understanding of the biology of TB infection by
showing, for example, that patients who recover from TB
may nevertheless remain susceptible to a second infection.62
Restriction Fragment Length Polymorphism
Restriction fragment length polymorphism (RFLP) has
been the gold standard method for strain typing of mycobac-
teria. The method entails the enzymatic digestion of a partic-
ular DNA element that produces fragments that can be sepa-
rated by electrophoresis. The fragments are transferred to a
nylon membrane, and a chemiluminescence-labeled DNA
probe is used to detect, by autoradiography, the location and
copy number of the specific relevant sequence.
With regard to strain typing of mycobacteria, an RFLP
fingerprinting method based on the repetitive DNA IS6110
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Eichbaum and Rubin / TUBERCULOSIS
sequence has proved valuable. The method is based on the
detection of the copy number and location of the genetic
element IS6110, an insertion element present in the direct
repeat (DR) locus of most M tuberculosis strains. Epidemio-
logically, comparison of IS6110 RFLP profiles causing
disease within communities has led to identification of iden-
tical isolates and permitted tracking of disease between indi-
viduals.63 Disadvantages of this RFLP method of strain
typing include the lengthy time required for culture and the
poor ability to discriminate among strains that possess only 1
or 2 copies of IS6110.64
Ligation-Mediated PCR
Ligation-mediated PCR is a method in which effective
DNA amplification depends on creation of a ligation product
between 2 specific primers ❚Figure 5❚. Enzymatic cleavage of
the relevant mycobacterial DNA is followed by primer
extension using a gene-specific oligonucleotide. Adapter (or
linker) molecules are added to create blunt DNA ends. PCR
then is performed using one primer located in the
“adapter/linker” region and another in the gene sequence.
A ligation-mediated PCR method for strain typing of M
tuberculosis involves the amplification of sequences flanking
the IS6110 repetitive sequence. The method uses one primer
located within the IS6110 RFLP sequence and a second located
in the linker region ligated to restriction enzyme–digested
Enzyme restriction (Sal I)
Gene sequence (IS6110)
Primer extension
Adapter/linker
addition
PCR with primers
in adapter/linker
region and specific
gene sequence
❚Figure 5❚ Ligation-mediated polymerase chain reaction
(PCR) entails the following steps: (1) specific enzymatic
cleavage of DNA followed by (2) primer extension with a
gene-specific oligonucleotide using DNA polymerase; (3)
ligation to the DNA of an adapter or of polynucleotide linker
molecules to create known blunt ends that (4) are subjected
to PCR using primers, one of which is located in the
adapter/linker region and the other in the specific gene
sequence. For mycobacterial strain typing, the restriction
fragment length polymorphism repetitive DNA element
IS6110 is used in the PCR with one primer located in a SalI
digested linker region and the other within the IS6110 region.
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S14 DOI: 10.1092/XX1NBANX8JKMTUQN
genomic DNA.65 The method has been shown to yield well-
resolved bands in analyzable and reproducible patterns that
are useful epidemiologic tools. The method is of particular
value as a rapid method of fingerprinting M tuberculosis
and has been found to have better discrimination than
spoligotyping.66
Spoligotyping
Spoligotyping (spacer oligotyping) is a rapid and
economical method for typing M tuberculosis strains using
spacer markers located in the polymorphic chromosomal DR
locus ❚Figure 6❚. The DR locus contains a variable number of
short 36-bp direct repeats interspersed with nonrepetitive
spacer sequences that vary in size from 35 to 41 bp. 67
Mycobacterial strains differ in the number of these DR
repeats contained in their genomes, as well as in the size of
the nonrepetitive spacers.
The spoligotyping technique entails PCR amplification
of the DR region of each strain, followed by hybridization to
a membrane containing covalently associated oligonu-
cleotides that correspond to the various spacer sequences.
Because strain isolates of mycobacteria differ in the number
of these spacer sequences, they can be distinguished on the
basis of the patterns of hybridization signals they produce. An
advantage of spoligotyping is that it uses an initial PCR
amplification step and, therefore, can be applied directly on
clinical samples without the time-consuming step of culture.68
The method initially was used to differentiate M bovis from
M tuberculosis, but computers now permit extensive simulta-
neous analysis of different molecular strain patterns. This
advance has yielded important epidemiologic information.69
Sequence Analysis
Strains of mycobacteria are shown to be very similar by
genomic sequencing. However, comparative genomic studies
that have examined the complete DNA sequence of M tuber-
culosis have yielded information about regions of the
genome that are deleted in certain strains of M tuberculosis
complex.70 Molecular amplification procedures were applied
by Parsons et al 71 to study the presence or absence of
specific genomic regions in 88 members of the M tubercu-
losis complex. The study demonstrated that Mycobacterium
microti and M bovis BCG each have unique genotypes,
whereas Mycobacterium africanum was the most diverse
genetically. Thus, sequence analysis has been of some
benefit in distinguishing species and strains of mycobacteria.
Microarray Comparative Genomics
DNA microarray analysis not only has been able to deter-
mine strain differences but also has yielded important
epidemiologic information about virulence patterns. Kato-
Maeda et al61 used oligonucleotide microarrays to detect
© American Society for Clinical Pathology
 Pathology Patterns Reviews

small-scale deletions among 19 clinically and epidemiologi- Spacer Oligonucleotides

cally well-characterized strains of M tuberculosis. The study 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 22 23 24 25 26 27 28 29 30 31 32 37 38 39 40 41 42 43
H37Rv

found that the likelihood of strains causing pulmonary cavita- 20 21 33 34 35 36

BCG

1 H37Rv
tion decreased as the extent of genomic deletion increased, 2 Centro
3 BCG

suggesting that accumulation of mutations in M tuberculosis 4

5
6
decreases the organism’s pathogenicity. The investigators 7
8
9
concluded that deletions tend to occur in regions where the 10
11

genes are no longer essential for survival, whereas regions that 12

13
14

are never deleted are most likely critical for survival. 15

16

Strains
17
18
19
20
21
22
23
24
Conclusions 25
26
27
28
The alarming spread of multidrug-resistant TB has set a 29
30
31
new tone to the search for effective control of tubercular 32
33
34
disease. Whereas molecular methods have begun to trans- 35
36

form the mycobacteriology laboratory in developed nations, 37

38
39
M bovis
significant challenges, exacerbated by limited resources, 40
41

remain to be surmounted in developing countries, which bear

95% of the disease burden.
The World Health Organization has estimated a sputum-
smear positive detection rate as low as 35%. This low detec-
tion rate is due in part to a lack of necessary medical and
laboratory resources in endemic areas and to the inadequacy
and slowness of conventional methods of diagnosis and
susceptibility testing. Rapid molecular methods of diagnosis
and drug susceptibility testing offer the best opportunity for
successful control, especially if the methods also are afford-
able and applicable in developing countries. Currently, many
of the methods are costly and require considerable expertise
and sophisticated equipment. But these obstacles are not
insurmountable. Developing effective, relatively low cost
molecular tools for diagnosing TB should be attainable if
one considers the successful generation of similar diagnostic
tools for diseases such as malaria and HIV and AIDS.
So far, a drawback of several molecular methods has
been their continued reliance on culture to ensure optimal
isolation rates for test sensitivity and specificity and to estab-
lish archival reference cultures.72 How much more sensitive
these molecular assays are than culture methods is not easy
to ascertain, although several tests on the market have been
evaluated with sensitivities of more than 90%. Evaluations
are, however, often performed in expert laboratories under
ideal conditions, and performance can decrease markedly in
less well-equipped laboratories.72
In any case, development of more sensitive molecular
methods that can be performed without the reliance on
cultures will improve the speed of diagnosis and drug
susceptibility testing and, ultimately, also should be afford-
able and applicable in the resource-poor settings of the
developing world where they are currently most needed.
❚Figure 6❚ Spoligotyping is a method of strain typing that takes
advantage of variability in the polymorphic chromosomal direct
repeat (DR) locus of different strains of mycobacteria. The DR
locus of each strain contains a variable number of 36-base-pair
(bp) DR elements interspersed with spacer sequences that
also vary in size between 35 and 41 bp according to the strain.
The DR locus of a particular myco-bacterial strain is amplified
by polymerase chain reaction, and the amplification product is
hybridized to a membrane containing the covalently bound
associated oligonucleotides that correspond to the various
strain-specific spacer sequences. Positive binding (dark spots)
at a specific position on the membrane indicates the presence
of the strain-specific spacer and, thus, identifies the specific
strain (numbered on the right). Courtesy of Isogen Bioscience
BV, Maarssen, the Netherlands.
From the Departments of 1Pathology, Division of Laboratory
Medicine, Massachusetts General Hospital and Harvard Medical
School, and 2Immunology and Infectious Diseases, Harvard
School of Public Health, Boston, MA.
Address reprint requests to Dr Eichbaum: Dept of Pathology,
Division of Laboratory Medicine, Massachusetts General Hospital,
Warren 2, Harvard Medical School, 55 Fruit St, Boston, MA 02114.
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