Comparative Evaluation of Acid-fast Staining for the Detection of Mycobacterium Fortuitum

Objective] Fluorescent staining is of paramount importance, not only for confi rming the presence of mycobacteria in a given specimen but also for providing an estimated growth quantifi cation. In this study, for rapidly growing Mycobacterium fortuitum, we evaluated the effectiveness of a rapid fl uorescent staining method employing auraminerhodamine (AR) fl uorescent stain and acridine-orange (AO) fl uorescent stain compared to that of the standard ZiehlNeelsen (ZN) stain currently in use in our laboratory. [Method] We evaluated the acid-fast nature of M.fortuitum strain ATCC6841 and 42 clinical isolates from each patient diagnosed at NHO Kinki-chuo Chest Medical Center. These isolates were preliminarily identifi ed as M.fortuitum using DNA-DNA hybridization (DDH Mycobacteria; Kyokuto Pharmaceutical, Tokyo, Japan). These isolates were further identifi ed by comparative sequence analysis of the ITS regions and the partial 16S rRNA gene. [Results] A total of 26 M.fortuitum strains (61.9%) demonstrated the lack of an acid-fast nature by AR staining, and slightly fewer demonstrated the same by AO staining. Sequence analysis of these 42 clinical isolates led to the identifi cation of 35 M.fortuitum subsp. acetamidolyticum isolates (83.3%) and 7 closely M.fortuitum isolates. [Discussion] This work reported the loss of the acid-fast nature of specifi c M.fortuitum strains. It is likely that both the specifi c cell envelope of M.fortuitum and the staining mechanics could have been responsible for the loss of the acid-fast nature since the 2 different fl uorescent stains yielded the same results. M.fortuitum is a mycobacterium species that does not stain with the commonly used fl uorescence microscopy technique. Therefore, we suggested the use of an identifi cation scheme for these organisms that employs ZN staining and the study of cultural characteristics (growth rate, temperature, and pigment production).