the media for susceptibility testing. No multicenter trials have been conducted evaluating the utility of the media in low, medium, or high prevalence areas [20].

Molecular Tests

Molecular assays based on amplification techniques targeting insertion element IS 6110, 16 S rRNA gene, internal transcribed spacer gene, or the hsp65 gene that can detect or confirm the presence of mycobacteria in clinical samples include routine, nested, real-time PCR, and PCR combined with enzyme restriction analysis (PCR-REA). Mycobacteria identification is confirmed by species-specific probes and/or their distinct enzymatic profile or patterns [21, 22].

Nucleic Acid Hybridization Probes

Nucleic acid probes allow rapid identification of select, common mycobacteria species. Probes can be employed for direct detection of mycobacteria in smear positive or highly suspicious tissue samples. Acridinium ester-labeled DNA probes complementary to the16S rRNA mycobacteria gene (AccuProbe; Gen-Probe Inc, San Diego, CA) are available for confirmation of M. tuberculosis complex, M. avium complex, M. kansaii, and M. gordonae. Probes are added to sonicated colonies, form a DNA–rRNA hybrid and are detected by a luminometer. Turnaround time is about 2 h. Sensitivity varies depending on the species. Comparison with Bactec, AccuProbes sensitivity was >85–100 and 100% specificity. Turnaround time is 2 h [21, 22].

Line Probes

Several line probe assays have been developed for the detection of mycobacteria species targeting either the 16S-23S rRNA internal spacer region (INNO LiPA Mycobacteria v2, Innogenetics, Ghent, Belgium) or the 23S rRNA gene (GenoType Mycobacteria MTBC, GenoType Mycobacterium CM, GenoType AS, GenoType LepraeDR, Hain Lifescience, Nehren, Germany) [21, 22].

The assays are based on the reverse hybridization of biotinylated PCR products to their complementary probes immobilized as parallel lines on a membrane strip. Detection and identification is via colorimetric detection using an automated instrument. Seventeen of the most frequently encountered mycobacteria species, including M. tuberculosis complex, M. avium, M. intracellulare, M. chelonae, M. gordonae,

M. smegmatis, M. fortuitum complex, and M. marinum are identified with the InnoLiPA kit, while the combination of the first three Hain Lifescience kits (GenoType MTBC, CM, AS) can identify M. tuberculosis and 30 different nontuberculous mycobacteria including the most frequently species isolated from mycobacterial keratitis. Colonies growing on solid or liquid media can be used with these two

systems. Turnaround time is 6 h. The M. leprae kit can confirm the presence of M. leprae as well as resistance to dapsone, ofloxacin, and rifampicin [21, 22].

DNA Sequencing

DNA sequencing is considered the “gold standard” for definitive identification of mycobacteria species. The procedure includes amplification with universal mycobacterial primers (6S rRNA gene or the hsp65 gene) followed by product (DNA) sequencing in an automated sequencer. Sequences are then compared with a database with known mycobacteria sequences [21, 22].

FISH (Fluorescent In Situ Hybridization) Assay

PNA-FISH-Fluorescent in situ hybridization tests using peptide nucleic acid coupled to a fluorescent probe are available for the detection of Mycobacteria tuberculosis, M. leprae, and nontuberculous mycobacteria from clinical samples, cultures, tissues, and paraffin sections [23]. It is a rapid and accurate technique for species identification and direct detection in tissues [24].

DNA Microarray

Hybridization of species-specific probes on a DNA chip allows for rapid identification of M. tuberculosis and other mycobacteria species. This technique is currently restricted to reference or research laboratories. Due to expense, this technique is currently restricted to reference or research laboratories [21, 22].

Pyrosequencing technology is a unique technique based on nucleic acid sequencing by addition and detection of released pyrophosphate during synthesis [25]. Tuohy et al. used it as a tool for the identification of mycobacterial species and documented it as a rapid and acceptable method for identifying the most frequent mycobacterial species recovered from clinical samples. Galor et al. used it to identify a case of Nocardia keratitis [26]. It could provide a rapid method for identification of unusual ocular pathogens including mycobacteria species [25].

Pulse Field Gel Electrophoresis (PFGE)

Pulse field gel electrophoresis is a molecular typing method that can characterize mycobacterial species associated with outbreaks. It has been instrumental in confirming LASIK outbreaks in the United States and Brazil [4, 27, 28]. It is timeconsuming and requires molecular expertise.

The polymerase chain reaction and other molecular diagnostics are ideal for the detection and/or confirmation of mycobacteria species in ocular samples due to