ReviewMolecular diagnostic tools in mycobacteriology
Introduction
Tuberculosis (TB) remains a major public health challenge worldwide as one third of the world population is exposed at some stage to Mycobacterium tuberculosis and TB is the first cause of death due to a single infectious agent in adults (Raviglione et al., 1995). It is estimated that nearly 1 billion people will be newly infected with TB between 2000 and 2020 and, furthermore, two hundred million people will develop disease and 35 million will die from TB within this period (WHO, 2001, Amdekar, 2005). Increasing movement of populations towards Europe and the United States in the last two decades has brought TB to the foreground of public health concern (CDC, 2006). Early diagnosis, together with adequate therapy and prevention measures against further transmission are essential for TB control. In addition, the incidence of infections by nontuberculous mycobacteria (NTM) has increased significantly over the same period, mainly due to the AIDS epidemic and the increase in the size of immunodeficient population. As treatment and infection control measures vary according to the aetiologic species, rapid and accurate identification to the species level is highly relevant.
The conventional methodology, which includes specimen treatment, microscopic examination for acid-fast bacilli, isolation with the use of solid and/or liquid culture, and the classic differentiation with biochemical tests (Fig. 1), is slow and takes several weeks. Over the last few years, new molecular methods have been introduced, including PCR-Restriction Fragment Length Polymorphism, real-time PCR, DNA sequencing, DNA strip assays as mycobacterial diagnostic tools (Fig. 1), leading to considerable improvement of both speed and accuracy of identification. Moreover, new species have been detected, the medical importance of which is under constant evaluation.
The prevalence of TB is further complicated by the appearance of strains with multidrug resistance (MDR) in almost 3% of all newly diagnosed patients (Dye et al., 2002). The conventional phenotypic methods for assessing drug resistance are slow and in order to avoid delays in both therapy and prevention of MDR transmission, various genotypic methods based on line probe assays, DNA sequencing or real-time PCR, have been proposed for detection of the mutations associated with resistance to anti-tuberculosis drugs.
The aim of the present report is to review the molecular methods used in mycobacterial diagnostics and to assess their diagnostic usefulness in a modern clinical mycobacteriology laboratory.
Section snippets
Direct detection of mycobacteria in clinical specimens
Several molecular techniques have been developed for direct detection of mycobacteria from clinical samples. These are based on amplification of unique mycobacterial DNA or RNA target fragments by PCR. The available in-house and commercial assays include:
Identification of mycobacterial species from culture by molecular methods
For many decades, the identification of mycobacterial isolates was performed on the basis of biochemical reactions and phenotypic characteristics, which are time-consuming and often give ambiguous results. The molecular methods for mycobacterial identification are now providing rapid and accurate results. Several methodologies have been used.
Molecular methods for detecting drug resistance in mycobacterial strains
Over the last few years, increasing resistance rates of M. tuberculosis have been observed in many parts of the world (Dye et al., 2002, WHO, 2000, Neonakis et al., 2007a). Problems with inadequate treatment and compliance are the usual causes of drug resistance development (Sharma and Mohan, 2004). Knowledge of the susceptibility pattern of the isolate is crucial for successful therapy. Although novel, alternative methods for phenotypic drug susceptibility testing have been proposed [MODS-
Molecular epidemiological methods
Regarding mycobacteria, molecular epidemiology is of critical significance. A series of new molecular methods try to associate specific genetic markers with the virulence of the strains, the underlying resistance mechanisms, the pathogenesis and the transmission dynamics. All this information is of great relevance for disease control efforts. The major methods used today are:
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