Rapid identification of mycobacteria other than tuberculosis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract

Background: Mycobacteria other than tuberculosis (MOTT) may cause pulmonary infections and skin infections to both healthy individuals and immunocompromised patients. Some species of MOTT may cause pulmonary disease that is indistinguishable from tuberculosis. The identification of MOTT currently performing in clinical diagnostic laboratories is either by conventional methods, or by molecular methods. However, conventional methods are time consuming and the cost of molecular methods for each specimen is relatively high. Therefore, Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is introduced as a rapid and simple method for mycobacteria identification. The use of MALDI-TOF MS for MOTT identification would contribute to a better patient management in clinical diagnostic laboratories. Aim: The aim of this study is to evaluate the simplified protein extraction protocol for MOTT identification. The identification results from 16S ribosomal RNA (16S rRNA) sequencing and 65-kDa heat shock protein (hsp65) gene sequencing are set as the “gold standard” for MOTT identification, supported by conventional tests for further confirmation in this study. The turnaround time by MALDI-TOF MS for MOTT identification was also being compared to the one by molecular methods. Methods: Fifty clinical isolates were collected from Queen Mary Hospital, Hong Kong. All isolates, including both fast-growing MOTT and slow-growing MOTT were cultured on Löwenstein-Jensen (LJ) medium and incubated for 3 and 7 days respectively. A simplified protein extraction protocol developed by bioMérieux was used. The extracted protein was used for MALDI-TOF analysis by Microflex LT™ MALDI-TOF mass spectrometer. DNA of all clinical strains were extracted and identified by both partial 16S rRNA gene sequencing and hsp65 gene sequencing, supported with conventional tests. Results: A total of 50 clinical strains of MOTT were identified either to species-level or in group/complex by partial 16S rRNA sequencing and hsp65 gene sequencing. There were 28 fast-growing MOTT and 22 slow-growing MOTT in the study. For the partial 16S rRNA sequencing, there were 17 out of 50 (34%) isolates could obtain identification results to species-level or in group/complex. In contrast, all the 50 (100%) isolates could successfully be identified to species-level or in group/complex successfully by hsp65 gene sequencing. For the MOTT identification by MALDI-TOF, the sensitivity and the specificity were 80% and 100% respectively. The turnaround time of rapid MOTT identification by MALDI-TOF MS and by molecular methods was found to be around 3 hours and 2 days respectively. Conclusion: The MALDI-TOF MS with simplified extraction protocol for MOTT identification showed compatible results to molecular methods but with a shorter turnaround time. The use of MALDI-TOF MS for MOTT identification may contribute to better patient management in clinical diagnostic laboratories.