Development and validation of a PCR test for rapid molecular diagnosis of macrolide-resistant Mycoplasma pneumoniae

Objective.

To develop and conduct the first stage of validation for a PCR test that identifies key mutations in the V domain of the 23S rRNA gene of M. pneumoniae (A2058G, A2059G, and A2062C). This test allows for the identification of macrolide-resistant M. pneumoniae strains in samples of native biological material.

Materials and Methods.

The study included a panel of M. pneumoniae DNA samples (n = 107) from the collection of the DeMaRes multicenter study, as well as native material (n = 52). This included oropharyngeal swabs (n = 45), tracheal aspiration (n = 4), and bronchial lavage (n = 3), which were obtained from March to April 2025 at G.N. Speranskiy Children Hospital No 9, Moscow. Nucleic acids were extracted from the native material using a RIBO-prep kit. The mutations in the 23S rRNA gene of M. pneumoniae was preliminarily determined using Sanger sequencing techniques and a modified PCR technique, which involved quenching fluorescence of the probe with primer. The concentration of control plasmids in order to determine the analytical sensitivity of the developed test was carried out using digital drip PCR.

Results.

The PCR test demonstrated 100% diagnostic sensitivity and specificity in detecting A2058G, A2059G, and A2062C mutations as well as wild-type M. pneumoniae DNA. Analytical sensitivity for all targets was 1 × 103 copies/ml. Among clinical samples with M. pneumoniae presence, the A2058G mutation prevailed at 35,7%, with A2059G and A2062C being less common. Results were consistent with sequencing data. The developed PCR test allows for the direct identification of the pathogen and M. pneumoniae’s resistance markers to macrolides in biological material. The results demonstrate its high diagnostic capabilities and suggest that it will be successful in laboratory diagnostics.

Conclusions.

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