This retrospective study included 281 children who were admitted to the Department of Pediatrics of Renhe Hospital affiliated with Three Gorges University between September and December 2023 and who were diagnosed with MP infection by multiplex PCR combined with NGS. All 227 patients with resistance had MP samples positive for mutations at the A2063G locus. The remaining 54 patients were excluded from the common mutation loci of A2063G, A2064G, A2067G, and C2617G and were considered to have NMRMP, with a macrolide resistance rate of 80.8%. This finding is consistent with those of other regional studies, in which the A2063G mutation in the V region of the 23S rRNA gene was the most common, followed by A2064G, which were associated with high levels of resistance, as evidenced by prolonged length of hospitalization, days of fever and cough, and course of antibiotics [7]. The sex composition of the children in both groups was balanced, with no significant differences in age, ranging from 7 months to 12 years (median, 7 years). However, the number of patients > 5 years of age who have a more developed immune system was significantly higher than that of younger children; thus, MP infection may stimulate an excessive inflammatory response and the development of refractory MPP (RMPP). However, mycoplasma resistance does not necessarily always lead to RMPP, but it is also associated with factors such as anti-MP drug selection, GC and gamma globulin use, and autoimmune status [8]. Immunodeficient children infected with MP have relatively mild lung damage and were excluded from this study [9]. Resistance rates vary considerably by region depending on the prevalent MP genotypes, with resistance rates of 3%, 8.6%, 0%, and 3.3% reported in Europe, North America, South America, and Oceania, respectively [10]. Therapeutic choices for MRMP also alter macrolide resistance rates. Tolfloxacin has been approved for pediatric use in Japan since 2010, and macrolide resistance rates decreased by 59.3% and 43.6% in 2014 and 2015, respectively [11, 12]. In addition, limitations exist regarding testing and statistical methods, with a few methods differentiating based on a child's response to macrolide therapy, a method that lacks accuracy [13]. The traditional method involves MP culture, which requires substantial training, is time-consuming, and is not conducive to early diagnosis and treatment. In contrast, molecular biology methods are better suited to rapid diagnosis and clarification of gene mutations [14]. In this study, a third method, tNGS, was applied, using Jinqi Rui biological reagents to detect almost all drug-resistant genotypes of endemic MP, minimizing group selection errors. A total of 107 respiratory pathogens, including MP, could be detected simultaneously. The mechanism of MP resistance to macrolides involves a mutation in the V region of the 23S rRNA gene that reduces the binding of this class of antibiotics, thus decreasing their ability to inhibit MP protein synthesis, potentially leading to less manageable respiratory damage and inflammatory responses. Consistent with the results of this study, previous studies have reported longer fever and hospitalization in children with MRMP [15]. However, macrolides remain the first choice for children with MP in China; only when infections do not respond to macrolides treatment are patients switched to tetracycline or quinolone alternatives, as appropriate. This is because of the potential risks associated with such antimicrobials, with tetracyclines potentially leading to the yellowing of teeth and enamel hypoplasia and quinolones demonstrating a risk of cartilage damage in young animals and tendon rupture in humans. In the present study, azithromycin anti-MP therapy was preferred for most children in the A2063G group, which showed better healing, which may be related to the active GC and fiberoptic bronchoscopy treatment in this group. GC use can alleviate local inflammation and modulate the immune response with a dose-dependent efficacy [16]. In the present study, the A2063G group had a higher rate of hormone use and a longer time required for discontinuation compared with the NMRMP group (P < 0.05); however, the difference from the single-treatment dose was not significant. Some children with solid lung lesions and bronchial plastination were treated with fiberoptic bronchoscopy; however, the microscopic diagnosis rates did not differ significantly between the two groups (Table 4). Wu et al. showed that timely fiberoptic bronchoscopy is effective in reducing complications and sequelae [17]. Early refinement of fiberoptic bronchoscopy and the removal of bronchial plastination are effective treatment options [18]. In young children and those in whom alternative drug therapy is risky, macrolides combined with GC and fiberoptic bronchoscopy may be considered. Moreover, the rate of MRMP-resistant mutations is not 100%, and these may coexist with NMRMP. Depending on the disease severity and the need for alternative anti-MP therapy, individualized treatment plans should be developed.
In the present study, we aimed to identify laboratory indicators that could predict drug-resistant mutations in MP. We observed a higher percentage of monocytes in the A2063G group (P = 0.02), consistent with the results reported by Wang et al., who observed that the development of MPP was associated with an increased proportion of non-classical and intermediate monocytes that can migrate from the peripheral blood to the site of inflammation, differentiate into dendritic cells and macrophages, and secrete pro-inflammatory cytokines that respond to an increased risk of lung tissue damage [19, 20]. In contrast to previous studies, the other laboratory test results in the present study showed no statistically significant differences between the groups (Table 2), possibly due to selection bias caused by regional restrictions. Our imaging statistics showed a higher probability of infection in the right versus both lungs in both groups, with no significant difference between right and left lung infections. This observation may occur due to direct MP damage and an abnormal immune response in the host. Children with early presentation or a mild immune response present with a unilateral lung infection, whereas those with a longer course of disease or a more mature immune system progress to bilateral lung infection [21]. The prevalence rates of solid lung lesions, effusion, and fiberoptic bronchoscopy treatment did not differ significantly between the two groups. Imaging and fiberoptic bronchoscopy results reflect the severity of pneumonia in children; as the participants were selected from the pediatric inpatient unit, and children with less severe conditions were treated in the outpatient clinic, the NMRMP group may have presented with a more severe condition but fewer hospitalizations in the same time period compared to the A2063G group. Some of the children in the A2063G group had pleural hypertrophy, alveoli, and emphysema, which were not observed in the NMRMP group. Statistically significant differences may have been observed if the sample size was larger.
Logistic regression analysis was performed to investigate the possible predictors of drug-resistant gene mutations in MP further. The results of the univariate analysis revealed significant differences between the A2063G and NMRMP groups in the number of days of fever before admission, mixed infections, number of days and doses of hormone use, and percentage of monocyte counts in laboratory tests, suggesting that these indices may be related to the occurrence of the A2063G mutation in the MP resistance gene. The results of further multifactorial logistic regression suggested that mixed infection was an independent risk factor for the A2063G mutation (OR = 12.950, P < 0.001), consistent with the findings reported by Zhou et al. [22] that coinfection of MRMP with both viruses and bacteria resulted in a more severe condition for children, and that antibiotics should be used as appropriate in the case of coinfections with other respiratory tract pathogens. However, the mechanism of action of other respiratory pathogens on mutations in the MP resistance locus is not clear, and further experiments are needed. Hormone dose was a protective factor in the present study (OR = 0.475, P < 0.05). The severity of MP infection is related to the host immune response, and cytokines such as interleukin-2, 10, and 11 and other immune cells are involved in disease progression [23]. Thus, the early administration of GC downregulates relevant cell-mediated immune responses, mitigates disease severity, shortens the duration of macrolide use, and reduces the risk of MP resistance mutations [24]. However, during long-term use of GC, children should be monitored for changes in blood glucose level, blood pressure, and electrolyte levels. Additionally, clinicians should be alert for side effects such as gastrointestinal bleeding and monitor growth, development, and cortisol secretion in these patients.
The strength of this retrospective study is the statistical determination of the prevalence of MP in children in this region (western Hubei province in east-central China), as most of the relevant data in China have been reported in North China. Second, the use of multiplex PCR combined with NGS to analyze the trend of macrolide resistance and mixed infection of respiratory pathogens in children with MP, improving the accuracy of disease diagnosis and guiding targeted use of antibiotics. The limitations of this study include selection bias, which may require larger samples and prospective experiments for further confirmation in future studies. Moreover, the children's information was obtained from the inpatient department in the region, which was not included in the study due to incomplete outpatient data. Finally, the results were regional and may not be applicable to other regions. More clinical cases should be included in future studies to validate and improve the model further.
In summary, our study found that A2063G is the most common mutation site for macrolide resistance genes. These children have longer days of fever and hospital stay before admission, significantly higher percentages of monocytes, higher rates of glucocorticoid use, and longer days of discontinuation compared to children without A2063G. Through logistic regression analysis, we found that mixed infection with other respiratory pathogens is an independent risk factor for the development of resistance genes, and the use of hormone dosage is a protective factor. In this study, children under 8 years old with MRMP were treated with a combination of macrolide drugs, GC, and fiberoptic bronchoscopy, which showed good therapeutic effects and helped them avoid the risks of using drugs beyond the instructions, providing new ideas for the treatment of MRMP. At the same time, data on the prevalence trend of Mycoplasma in central China has been supplemented, providing reference data for other regions.