Mycoplasma pneumoniae is a common etiologic agent of community-acquired pneumonia (CAP) among children. Although M. pneumoniae infection often causes a mild and self-limiting disease, pneumonia develops in ≈10%–20% of pediatric patients (1). First-line therapies for M. pneumoniae infection are based on macrolides, a group of antimicrobial drugs widely used in outpatient settings because of their high oral bioavailability. However, overuse and indiscriminate use of macrolides have contributed to the emergence of macrolide-resistant M. pneumoniae (MRMP). Point mutations in the V region of the M. pneumoniae 23S rRNA gene have been associated with macrolide resistance (2). In recent years, prevalence of MRMP has increased and is very high in Asia (13.6%–100%) (2–4). During spring/summer 2023, hundreds of children with CAP were admitted daily to each of the major hospitals in Hanoi, Vietnam. M. pneumoniae has emerged as the major pathogen detected in approximately one third of patients with CAP (5). We analyzed the mutations in the 23S rRNA gene of M. pneumoniae isolated from nasopharyngeal samples of pediatric CAP patients during the 2023 outbreak in Vinmec Times City Hospital, Hanoi.

During May 1–July 31, 2024, the real-time PCR Allplex Respiratory Panel 4 detected M. pneumoniae in 411 (26.1%) of 1,578 nasopharyngeal samples from children with suspected CAP. Among M. pneumoniae–positive samples with a cycle threshold <30, we randomly selected 13 samples from 13 patients for gene sequencing. We amplified the DNA sequence of the 748-bp region (nt 1963–2710) of the 23S rRNA gene containing all known MRMP mutations by using MRMP-F1 (5′-CGTCCCGCTTGAATGGTGTA-3′) and MRMP-R1 (5′-GGCGCTACAACTGGAGCATA-3′). We sequenced the amplicons according to the Sanger sequencing method by using a BigDye Terminator v3.1 Cycle Sequencing Kit and Applied Biosystems 3500 Dx Genetic Analyzer instrument (both Thermo Fisher Scientific, https://www.thermofisher.com). We assembled the generated sequence data and analyzed them for variations by comparing with the reference M. pneumoniae strain M129 23S ribosomal RNA gene (GenBank accession no. NR_077056.1), using BLAST (http://blast.ncbi.nlm.nih.gov). We used ClustalW to perform multiple alignments (6). Subsequently, we constructed the phylogenetic tree according to the maximum-likelihood method with bootstrap analysis (n = 500) by using MEGA11 software (https://www.megasoftware.net). The 2-dimensional secondary structure of the 23S rRNA gene was predicted by the R2DT tool (RNAcentral) according to an SA_LSU_3D template provided by RiboVision (7).

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Figure. Phylogenetic tree and location of mutations for Mycoplasma pneumoniaestrains identified in pediatric patients hospitalized with community-acquired pneumonia, Hanoi, Vietnam, spring/summer 2023. A) Maximum-likelihood phylogenetic analysis of the domain...

Of the 13 samples, 6 (46.2%) showed single-nucleotide variation from the type strain sequence in the V region of the 23S rRNA gene. A known A2063G mutation was detected in 4 samples, and a novel variant C2353T was found in 2 samples (Figure, panel A).

The known MRMP mutation A2063G is the most prevalent mutation reported to date compared with other infrequent mutations (e.g., A2063T/C, A2064G, A2067G, A1290G, and C2617A) (2,8). Mutations at site 2063 are also associated with a high level of macrolide resistance (9,10). The National Institutes of Health databases showed no recorded evidence for the sequences containing the C2353T variant observed in our study (Figure, panel B). We hypothesize that under selection pressure during CAP treatment with macrolides, C2353T mutants have emerged with macrolide resistance. Previous reports have shown that different mutations can lead to different levels of macrolide affinity as well as MIC elevation (8). Demonstration of MRMP by culture and MIC is not regularly done in clinical practice; thus, rapid detection of MRMP mutation may provide useful information for guiding antimicrobial drug therapy.

Clinical nonresponse to initial macrolide treatment was experienced by 3 (50%) of the 6 patients with the novel or known mutation and 2 (28.6%) of the 7 without (Table; Appendix Table). Other respiratory bacteria were co-detected in approximately two thirds of patients in both groups, which might also affect clinical characteristics.

In summary, we detected the novel C2353T variant and known A2063G mutations in the 23S rRNA gene in nearly half of the pediatric patients with M. pneumoniae–associated CAP in Vinmec Times City Hospital during the 2023 outbreak in northern Vietnam. Our findings are consistent with those of other studies regarding the rising prevalence of MRMP in Southeast Asia. Our study findings may indicate circulation of different MRMP variants in Vietnam or emergence of new MRMP variants during the recent M. pneumoniae-associated CAP outbreak among children.

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