Mycobacterial Diseases: Past, Present and Future
Moderator: Gary W. Procop
Section 4 -
Molecular Diagnostics for Mycobacterial Infections
Gary W. Procop
The diagnosis of tuberculosis has always been a challenge to the clinician and the laboratorian
alike. The clinical diagnosis of tuberculosis may be foremost in the differential diagnosis when signs,
symptoms, and exposure history are typical. However, atypical presentations have sundry manifestations
wherein the possibility of tuberculosis may not be strongly suspected. For example, patients with remote
histories of exposure and solitary skeletal lesions may not be initially suspected to have skeletal
tuberculosis, until histopathology, often at the time of frozen section analysis, demonstrates
necrotizing granulomas.  Similarly, tuberculous meningitis may not be suspected upon the initial
presentation of the patient.  In addition, tuberculosis in the immunocompromised host poses a
The isolation and characterization of the Koch's bacillus, demonstrated that culture-based methods
could be used to demonstrate the presence of this microorganism in the respiratory, urine, or tissue
specimens of infected patients. These methods have been refined over the many decades, and the
phenotypic profiles of M. tuberculosis and the non-tuberculosis mycobacteria
are well described. Although these methods have served the medical community for many years, and
continue to do so, they are time-consuming – given the slow growth of mycobacteria. In addition,
atypical reactions do occur and sometimes time-consuming tests need to be repeated which significantly
delays the time to identification.
The first major advance in the molecular identification of M.
tuberculosis and other mycobacteria was the development and wide spread use of signal-generating
hybridization probes that targeted species or complex-specific signature sequences in the ribosomal RNA
of the mycobacterial cells.
This approach proved feasible, since there are many mycobacteria
present in a positive culture and many copies of rRNA in each individual mycobacterial cell (i.e.,
abundant target for probe hybridization). This approach became the standard for rapid mycobacterial
identification and remains so in many institutions. Although useful, there are limitations with this
approach. These assays have sensitivity limitations that preclude there use directly on clinical
specimens. In addition, they only provide a single "yes/no" answer. This is excellent if the isolate is
identified (i.e., a "yes" is obtained). Otherwise, the laboratorian only knows what it is not, and has
to utilize either additional probes, which are expensive, or employ slower, traditional phenotypic
When Sanger or dideoxynucleotide sequencing by termination became more user friendly and
cost-effective, then this technology began to be used for the identification of cultivated mycobacteria.
Hundreds of nucleotides of sequence may be generated by this method, following a "broad-range" PCR that
amplifies the DNA segment of interest from the mycobacterium to be identified.
for such a PCR assay is that the primers used should amplify all clinically important mycobacteria,
whereas the sequences that are between the primers afford the differentiation of species or
clinically-important complexes. Not surprisingly, the 16S rDNA gene is commonly used for this purpose,
but other genetic targets, such as the rRNA polymerase gene (rpoB) have also
been demonstrated to be useful for this purpose. More recently, a newer sequencing chemistry –
sequencing by synthesis – or pyrosequencing – has been demonstrate to achieve similar differentiation,
although only a limited (30-50) nucleotide sequences are typically generated. 
DNA microarrays may also be used following a broad-range PCR.
These technologies effectively
achieve the simultaneous hybridization of multiple probes (even hundreds) in a single reaction. The
signals may generate a fluorescent or, more recently, an electrical signal. The traditional limitations
of these technologies have been the cost and the amount of data generated. These have been demonstrated
to be able to identify the common, clinically-relevant mycobacteria, as well as the genetic determinants
of resistance to rifampin and kanamycin. More recently, limited microarray have been designed, which use
a smaller number of probes and therefore generate less data. These limitations in the size of the
microarray will also have an influence on cost.
The approaches heretofore described have had a marked influence on the time to identification once a
mycobacterium species is recognized in culture, but the another approach has been to directly
characterize clinical specimens for the presence or the absence of M.
These approaches, when used in conjunction with clinical skills
and judgment, may optimize the use of respiratory isolation and direct therapy. Many M. tuberculosis complex-specific nucleic acid amplification assays have been
developed, two of which have FDA approval. Additionally, assays have been developed that recognize M. tuberculosis complex, as well as some of the more commonly encountered
Molecular technologies and techniques have evolved significantly since the time when the first
hybridization probes were used for the identification of mycobacteria. These methods, although often
expensive, afford the possibility to rapidly identify cultivated mycobacteria, and to characterize
clinical specimens with a reasonable degree of certainty for the presence or absence of M. tuberculosis. Regardless of the power of these techniques, they remain adjunct
tools and are not (yet) replacements for the acid fast stain and culture.
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