Comparison of Pyrosequencing, Sanger Sequencing, and Melting Curve Analysis for Detection of Low-Frequency Macrolide-Resistant Mycoplasma pneumoniae Quasispecies in Respiratory Specimens

Abstract

Macrolide-resistant Mycoplasma pneumoniae (MRMP) is emerging worldwide and has been associated with treatment failure. In this study, we used pyrosequencing to detect low-frequency MRMP quasispecies in respiratory specimens, and we compared the findings with those obtained by Sanger sequencing and SimpleProbe PCR coupled with a melting 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 the M. pneumoniae-positive specimens, respectively. The A-to-G transition at position 2063 was the only mutation identified. Pyrosequencing identified A2063G MRMP 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 quasispecies by Sanger sequencing or SimpleProbe PCR. The specimens that could be detected by SimpleProbe PCR and Sanger sequencing had higher frequencies of MRMP quasispecies (51% to 100%) than those that could not be detected by those two methods (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 with MRMP identified by Sanger sequencing more often required a switch from macrolides to an alternative M. 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.

Emergence of Macrolide-Resistant Mycoplasma pneumoniae in Hong Kong Is Linked to Increasing Macrolide Resistance in Multilocus Variable-Number Tandem-Repeat Analysis Type 4-5-7-2

“…In nine patients, no information on the syndromic diagnosis was available. Clinical features and macrolide resistance genotyping results for 101 of the patients were reported previously (7,16). Nucleic acid extracts from the 257 patients with positive M. pneumoniae results were retrospectively retrieved for further testing.…”

“…This retrospective study was conducted in a health care region in Hong Kong comprising one university-affiliated hospital with 1,600 beds, three extended-care hospitals with a total of 1,600 beds, and one pediatric hospital with 160 beds. A diagnostic PCR assay for M. pneumoniae was provided as a routine service for inpatients by a clinical microbiology laboratory (7,16). Testing was initiated by clinicians, mainly in patients with features suspected to be due to M. pneumoniae pneumonia (2,17).…”

“…Nasopharyngeal aspirate samples were collected in viral transport medium (18). Sputum and other respiratory specimens were collected using standard techniques (16). Patients were included if their respiratory specimens were obtained for M. pneumoniae testing by PCR between January 2011 and December 2014.…”

“…Nucleic acid extraction was performed by using the NucliSENS easyMAG extraction system (bioMérieux, France) and stored at Ϫ80°C, as described previously (16). All testing was performed on nucleic acid extracted from the clinical specimens.…”

“…Real-time quantitative (qPCR) was conducted to detect M. pneumoniae using TaqMan universal PCR master mix (Applied Biosystems) in a StepOnePlus instrument (Applied Biosystems, Foster City, CA), as previously described (16). A series of 6 log 10 dilutions equivalent to 10 to 1 ϫ 10 6 copies per reaction mixture were prepared from a plasmid (pC-RII-TOPO vector; Invitrogen, CA) containing the corresponding target bacterial sequence to generate calibration curves; these were run in parallel with the test specimens.…”