Curve Analysis for Detection of Low-Frequency Macrolide-Resistant Mycoplasma pneumoniae Quasispecies in Respiratory Specimens

Macrolide-resistantMycoplasma pneumoniae (MRMP) is emerging worldwide and has been associated with treatment failure. In this study, we used pyrosequencing to detect low-frequencyMRMPquasispecies in respiratory specimens, andwe compared the findings with those obtained by Sanger sequencing and SimpleProbe PCR coupled with amelting curve analysis (SimpleProbe PCR). Sanger sequencing, SimpleProbe PCR, and pyrosequencing were successfully performed for 96.7 % (88/91), 96.7 % (88/91), and 93.4 % (85/91) of theM. pneumoniae-positive specimens, respectively. The A-to-G transition at position 2063 was the only mutation identified. Pyrosequencing identified A2063GMRMP quasispecies populations in 78.8 % (67/88) of the specimens. Only 38.8 % (26/67) of these specimens with the A2063G quasispecies detected by pyrosequencing were found to be A2063G qua-sispecies by Sanger sequencing or SimpleProbe PCR. The specimens that could be detected by SimpleProbe PCR and Sanger se-quencing had higher frequencies of MRMP quasispecies (51 % to 100%) than those that could not be detected by those twometh-ods (1 % to 44%). SimpleProbe PCR correctly categorized all specimens that were identified as wild type or mutant by Sanger sequencing. The clinical characteristics of the patients were not significantly different when they were grouped by the presence or absence of MRMP quasispecies, while patients withMRMP identified by Sanger sequencing more often required a switch frommacrolides to an alternativeM. pneumoniae-targeted therapy. The clinical significance of mutant quasispecies should be investigated further with larger patient populations and with specimens obtained before and after macrolide therapy. Mycoplasma pneumoniae is a major cause of community-ac-quired pneumonia, especially for children and young adult