We found that more hsp65 fragment differences than rpoB fragment (data not shown) may explain

the size differences with highly variable sequence for species identification but difficult interpretation in hsp65 PRA. Some sub-types of NTM species are relevant to clinical management, such as the M. kansasii and MAC. M. kansasii type 1 is the most common type associated with human disease [26–28] because of its high pathogenicity. However, M. kansasii types 3–7 are most often isolated from the environment and rarely from humans, and have no significant role in clinical management [26, 27]. MAC can be divided into M. avium subsp. avium and M. intracellulare because drug sensitivity test and clinical SN-38 in vitro outcomes are different between these two sub-types [29, 30]. It is important to identify NTM to the sub-type level both for epidemiologic data and for differentiating potentially pathogenic sub-types [26, 27]. Combined rpoB DPRA and hsp65 PRA with capillary electrophoresis provides precise species identification and overcomes problems associated with discrimination by hsp65 PRA band sizes. This combined method takes

Sapitinib mw around 2–3 days to complete in the laboratory once clinical isolates have been received. However, the identification algorithm has some limitations. First, it could not discriminate M. intermedium type 1 from M. intracellulare type 3, and second, not every hospital laboratory will be equipped with the appropriate Cepharanthine equipment for this method. Conclusion In conclusion, the novel flow chart and identification algorithm obtained by combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can easily differentiate MTC from NTM and identify mycobacterial species to the sub-type level, which is helpful for clinical management. The results are complementary

to 16 S rDNA sequencing, and the effective algorithm provides rapid and accurate mycobacterial species identification. Methods Mycobacterial isolates Fourteen mycobacterial reference strains including one MTC and 13 NTM strains and 376 clinical respiratory specimens, including sputum, broncho-alveolar lavage, and aspirated secretion from endotracheal tubes, were Quisinostat mw collected from January to July 2007 from Taichung Veterans General Hospital (Taichung, Taiwan). The respiratory specimens were digested by a N-acetyl-l-cysteine-NaOH decontamination procedure, centrifugal concentration, and sputum dissolving agents [31]. The processed specimens or the concentrated specimens were inoculated into MGIT culture tubes and incubated in the BACTEC 960 instrument at 37°C until a positive signal appeared. Positive BACTEC cultures were smeared on glass slides and Kinyoun staining was used to screen for AFB [31]. Mycobacteria in the positive BACTEC cultures were isolated and identified by conventional methods [12, 13] .