Evaluation of GENECUBE Mycoplasma for the detection of macrolide-resistant Mycoplasma pneumoniae

Abstract

Introduction. Resistance against macrolide antibiotics in Mycoplasma pneumoniae is becoming non-negligible in terms of both appropriate therapy and diagnostic stewardship. Molecular methods have attractive features for the identification of Mycoplasma pneumoniae as well as its resistance-associated mutations of 23S ribosomal RNA (rRNA). Hypothesis/Gap Statement. The automated molecular diagnostic sytem can identify macrolide-resistant M. pneumoniae . Aim. To assess the performance of an automated molecular diagnostic system, GENECUBE Mycoplasma, in the detection of macrolide resistance-associated mutations. Methodology. To evaluate whether the system can distinguish mutant from wild-type 23S rRNA, synthetic oligonucleotides mimicking known mutations (high-level macrolide resistance, mutation in positions 2063 and 2064; low-level macrolide resistance, mutation in position 2067) were assayed. To evaluate clinical oropharyngeal samples, purified nucleic acids were obtained from M. pneumoniae -positive samples by using the GENECUBE system from nine hospitals. After confirmation by re-evaluation of M. pneumoniae positivity, Sanger-based sequencing of 23S rRNA and mutant typing using GENECUBE Mycoplasma were performed. Results. The system reproducibly identified all synthetic oligonucleotides associated with high-level macrolide resistance. Detection errors were only observed for A2067G (in 2 of the 10 measurements). The point mutation in 23S rRNA was detected in 67 (26.9 %) of 249 confirmed M. pneumoniae -positive clinical samples. The mutations at positions 2063, 2064 and 2617 were observed in 65 (97.0 %), 2 (3.0 %) and 0 (0.0 %) of the 67 samples, respectively. The mutations at positions 2063 and 2064 were A2063G and A2064G, respectively. The results from mutant typing using GENECUBE Mycoplasma were in full agreement with the results from sequence-based typing. Conclusion. GENECUBE Mycoplasma is a reliable test for the identification of clinically significant macrolide-resistant M. pneumoniae .

Point-of-care molecular diagnosis of Mycoplasma pneumoniae including macrolide sensitivity using quenching probe polymerase chain reaction

“…Commercial molecular assays for detection of M . pneumoniae are listed in Table 3 [ 19 , 39 – 49 ]. The major features of the Smart Gene ® system and other assays are summarized as follows.…”

A Prospective Evaluation of the Analytical Performance of GENECUBE® HQ SARS-CoV-2 and GENECUBE® FLU A/B

“…The system automatically prepares reaction mixtures and amplifies and detects target genes in a short time and can analyze up to eight samples and four items at the same time in a single run. GENECUBE ® has been applied for several pathogens, including Mycobacterium tuberculosis [8], M. avium, M. intracellulare, Neisseria gonorrhoeae [9], Chlamydia trachomatis [9], Mycoplasma pneumoniae [7,10], Staphylococcus aureus (nuc and mecA) [11], and the toxin gene of Clostridioides difficile [12].…”

A prospective evaluation of the analytical performance of GENECUBE® HQ SARS-CoV-2 and GENECUBE® FLU A/B

“…The system automatically prepares reaction mixtures and amplifies and detects target genes in a short time, which, in a single run, can analyze up to eight samples and four items at the same time. GENECUBE ® has been applied for several pathogens, including Mycobacterium tuberculosis [8], M. avium, M. intracellulare, Neisseria gonorrhoeae [9], Chlamydia trachomatis [9], Mycoplasma pneumoniae [7,10], Staphylococcus aureus (nuc and mecA) [11] and the toxin gene of Clostridiodes difficile [12].…”