Mycoplasma pneumoniae pneumonia (MPP) is a type of the community-acquired pneumonia (CAP), with M. pneumoniae being the primary cause of lower respiratory tract infection (LRTI) in children. MPP typically manifests as a benign, self-limiting condition associated with a positive prognosis. However, infrequent complications in other organs can pose significant health risks. Globally, M. pneumoniae epidemics occur every 3–7 years, with varying incidence rates (1–5). Recent report indicates a sharp rise in M. pneumoniae cases in several regions of China since June 2023 (6). Meanwhile, an increase of the incidence of infections with M. pneumoniae has also observed in 2023/2024 in Europe (7).These M. pneumoniae infection often leads to more severe clinical symptoms, which can not only impact the health of patients but also inflicts an economic burden on their families, often correlating with the severity of the infection and the effectiveness of antibiotic treatment.

After infection, M. pneumoniae adheres to the host's respiratory epithelium and produces numerous cytotoxic proteins, protecting itself from removal by mucociliary escalator mechanisms while damaging the host's pseudostratified epithelium (8). A recent study demonstrated that Club Cell Secretory Protein (CC16) plays an important role in the airway remodeling and pulmonary epithelium damage during respiratory infection with M. pneumoniae (9). Reports also indicate that the pathogenesis of M. pneumoniae infection is closely related to the stimulation of macrophages by this pathogen via toll-like receptors, which release immunomodulatory and inflammatory cytokines and chemokines (10). The severity of M. pneumoniae infection is related to the host immune system's response to the infection (11).

Conventionally, macrolides have been the preferred choice of antimicrobials for treating M. pneumoniae infections in children (11). However, recent observations indicate that despite treatment with macrolides for over 7 days, some cases still exhibit worsening clinical symptoms like persistent fever and aggravated lung imaging, leading to refractory MPP (RMPP) (12). Although macrolides are effective in treating RMPP, alternative treatments are required in cases of prolonged fever and hospitalization. The increasing prevalence of macrolide-resistant M. pneumoniae compromises treatment effectiveness. Additionally, excessive immune response, co-infections, and host conditions contribute to lung injury in MPP, further affecting the efficacy of macrolide therapy (13, 14). Timely and appropriate treatment is crucial to prevent complications like necrotizing pneumonia, bronchiolitis obliterans, thrombosis, and even death (15). Therefore, predicting macrolide responsiveness and susceptibility in the early stages of M. pneumoniae infection is essential.

Predicting treatment response at admission has recently become increasingly challenging. Although distinguishing between RMPP and MSMP has recently garnered attention, obtaining records of the gradually worsening syndrome or chest x-ray is difficult. Additionally, clinicians often switch treatments within 7 days of initial macrolide use for MPP patients. To confirm macrolide resistance, the drug resistance gene detection is required. However, drug resistance gene detection is not a routine test item and can be costly. Based on an actual treatment course, macrolide-unresponsive Mycoplasma pneumoniae pneumonia (MUMPP) is characterized by a persistent fever of 38.0℃ or higher, lasting for 72 h or more following macrolide therapy. Despite its frequency in clinical settings, few studies have characterized this condition. Patients with MUMPP face a higher risk of progressing to RMPP and/or severe clinical outcomes compared to MSMP patients, making it a critical issue in clinical practice (16). Although some reports indicate that switching to secondary treatments does not improve therapeutic efficacy compared to prolonged macrolide use in children with MUMPP (17), early administration of tetracycline antibiotics may effectively prevent the progression of MUMPP to RMPP and/or mitigate severe clinical outcomes (16).

In this study, patients were categorized into two distinct groups based on the duration of fever after macrolide administration: MUMPP (persistent fever of ≥38.0°C for ≥72 h after macrolide administration) and MSMP (fever subsided within 3 days). This study sought to compare and identify the differences in clinical and radiological characteristics between these two groups. Additionally, we evaluated potential markers such as IL-6, SF, D-dimer, and LDH for predicting MUMPP. This distinction may assist physicians in administering safe and effective treatment strategies, enhancing the efficacy of early-stage MPP management. Ultimately, this research aims to determine the distinguishing features of MUMPP from MSMP, thereby contributing to the advancement of MPP management strategies.

The recent surge in MUMPP incidence, particularly in children, is concerning and linked to macrolides resistant Mycoplasma pneumoniae strains infection and potentially life-threatening conditions (14, 18). Beyond increased bacterial load, an excessive host immune response among patients with MUMPP may lead to lung injury (13, 14). Without proper treatment, these patients may experience poor outcomes and prolonged disease courses, with numerous complications during the acute stage and potential long-term sequelae such as post-infectious bronchiolitis obliterans, bronchial asthma, unilateral transparent lung, and bronchiectasis (3, 19). Therefore, analyzing predictive factors for MUMPP and establishing early detection and timely treatment is crucial to prevent complications and sequelae.

Consistent with other studies, the median age of MPP patients was 7 years, with no statistically significant gender difference. M. pneumoniae incidence peaks in the summer and fall seasons in America (20) and between August and January in China (21). Similarly, more than half of MPP patients in our study were detected in the fourth quarter. Longer median hospital stay (8.97 days) often burdens medical resources and clinical staff, especially during epidemics. The clinical symptoms associated with M. pneumoniae CAP in multivariable analysis resemble those of respiratory viral illnesses (22), making it challenging to distinguish M. pneumoniae from other pathogens. However, related syndromes such as catarrhal syndrome were less common in our study (27.3%). The median BMI was lower in the MUMPP group, suggesting that smaller individuals may be less sensitive to macrolides and prone to severe syndromes. However, BMI showed no significant difference between the groups, highlighting the need for a larger patient sample in future studies.

Compared to the MSMP group, the MUMPP group exhibited a greater proportion of severe clinical syndromes, characterized by significantly longer fever duration, prolonged hospital stays, and more pronounced symptoms, including a higher percentage of shortness of breath (23.61%) and SpO₂ < 94% (29.17%). Furthermore, the ratio of SMPP ones was significantly higher in the MUMPP group than in the MSMP group (52.78% vs. 34.83%; p < 0.05), consistent with prior research (23). Although no patients required ICU admission with mechanical ventilation, a larger proportion of MUMPP patients required oxygen therapy, particularly heated and humidified high-flow oxygen support.

M. pneumoniae is known to induce a wide range of extrapulmonary manifestations affecting nearly every organ system, potentially leading to more severe medical complications than pneumonia (24). In our study, the MUMPP group exhibited a significantly higher proportion of extrapulmonary manifestations compared to the MSMP group. Gastrointestinal symptoms were the most prevalent extrapulmonary manifestation, particularly in the MUMPP group. However, MP-associated myocarditis, encephalitis, and other extrapulmonary complications were not observed, possibly due to the limited number of MPP patients included. In clinical practice, the possibility of MUMPP should be considered when similar intrapulmonary and extrapulmonary manifestations are observed in children.

Following M. pneumoniae infection, disruptions in innate and adaptive immunity lead to excessive inflammation in the lungs and throughout the body (25). In turn, the cytokines and chemokines released during these hyperinflammatory responses further amplify the inflammatory cascade (26). In our study, we assessed several serological markers to predict MUMPP. Our analysis revealed that the MUMPP group had significantly higher levels of N%, IL-6, SF, CRP, PCT, AST, D-dimer, and LDH compared to the MSMP group. Additionally, the neutrophil-to-lymphocyte ratio (NLR) and LDH-to-albumin ratio (LAR) were significantly higher in the MUMPP group. These findings suggest that excessive inflammatory and immune responses play crucial roles in the pathogenesis of MUMPP, consistent with previous studies (27, 28).

Ferritin is induced by activated macrophages, which produce tumor necrosis factor (TNF)-α (29). Serum ferritin (SF) often exhibits a non-specific increase in various infectious or inflammatory disorders, and its level can effectively reflect the body's immune defense ability (30). Previous studies have reported that the SF level rises progressively in children with MPP as their disease worsens (31). In our study, ROC curve analysis revealed that SF could be used as a predictor of MUMPP. SF had the largest AUC (0.7002) among all serum markers. Its predictive value for MUMPP was 104.1ug/L, with high specificity (90.32%) but low sensitivity (39.022%).

M. pneumoniae infection and abnormal immune response lead to systemic inflammation, vascular endothelial injury, subcutaneous collagen exposure, and vasoconstriction. This disrupts the balance of blood coagulation and anticoagulation, resulting in a hypercoagulable state and elevated D-dimer levels. Elevated D-dimer levels in children with MPP are linked to hypercoagulability and vascular endothelial dysfunction, correlating with disease severity (32, 33). Consistent with previous studies (34), our research shows that D-dimer is a predictive factor for MUMPP. Elevated D-dimer levels, especially >0.435 mg/L, contribute to the early diagnosis of MUMPP.

Lactic dehydrogenase (LDH), a non-specific inflammatory biomarker of tissue damage, is present in all tissue cytoplasm. Its release into the serum is linked to cell dissolution or membrane damage, serving as an important indicator of infection severity and inflammatory disease (35, 36). Previous studies suggest that LDH is a predictor of severity and a marker of glucocorticoid therapy efficacy in MPP patients (28, 37, 38). In the present study, we observed elevated LDH and decreased albumin levels, with the LDH to albumin ratio (LAR) having a higher AUC than LDH alone (0.6528 vs. 0.6218). The LAR is widely used as an indicator of tissue damage, nutritional status, and systemic inflammatory response. Previous studies have reported high LDH and low albumin levels in SMPP patients, with LAR assisting clinicians in evaluating the progression of severe M. pneumoniae infection (39). Our study identified an LAR cut-off of 8.476 for predicting MUMPP, with 86.59% sensitivity and 43.66% specificity.

Neutrophils, which participate in the first line of defense against infection, play a crucial role in the immune response to M. pneumoniae (40). Their numbers increase in peripheral blood (41), bronchoalveolar lavage fluid (25), and lung tissue (42) after M. pneumoniae infection. However, excessive inflammation may cause lymphocytes apoptosis, thereby reduce their numbers (43). This phenomenon has been observed in MPP patients (44). The neutrophil-to-lymphocyte ratio (NLR) in peripheral blood is a simple, rapid and widely available indicator that has been reported to be associated with poor prognosis in idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (45), and COVID-2019 (46). Research indicates that NLR at admission can predict the prognosis of MPP (47). One retrospective study (48) concluded that an NLR >3.92 might be a valuable predictor value for RMPP in children over 6 years old. Our study also found NLR has predictive value for MUMPP, with an NLR >3.212 at admission indicating MUMPP with a sensitivity and specificity of 81.48% and 48.53%, respectively.

Despite observing some differences, certain indicators such as CRP, a commonly used marker for the early evaluation and identification of MPP with severe complications (33, 49), demonstrated low predictive ability for MUMPP due to their combined low specificity and sensitivity. This may be attributed to their nonspecific nature and reflection of whole-body inflammation levels.

These findings indicate that MUMPP exhibits more severe pulmonary inflammatory reaction or tissue damage. A robust cellular immune response leads to severe ciliary dysfunction, reduced airway immune function, and impaired ciliary mucus clearance. This results in large-scale infiltration, atelectasis, and mucus plug formation, hindering the recovery lung inflammation (49, 50). In a study of 393 hospitalized children with MPP, lobar or segmental consolidation was the most common radiological finding (37%) (51). Pneumonia involving two or more lobes is more common in macrolide-refractory MPP patients (52, 53). Our study found unilateral infiltrates more frequent in the MSMP group and bilateral infiltrates more frequent in the MUMPP group, with a higher incidence of lobar pneumonia in the MUMPP group, consistent with previous studies (23).

The early assessment of MPP severity through imaging strategies is crucial to prevent adverse outcomes in clinical practice. Kim et al. (2021) demonstrated that children with MPP and pleural effusion had more severe pneumonia lesions and poorer treatment outcomes, leading to prolonged resolution of lung abnormalities (54). Our study found a higher proportion of pleural effusion in the MUMPP group compared to the MSMP group. Radiologic findings of lobar pneumonia and pleural effusion in the MUMPP group suggest severe illness due to macrolide resistance, higher M. pneumoniae burden, severe host reactions, or other refractory response factors. Therefore, in M. pneumoniae patients, the possibility of an ensuing refractory response to macrolides should be considered if bilateral infiltrates, pleural effusion, or lobar pneumonia are detected on chest radiography during the initial hospitalization period.

Clinical practice guidelines recommend macrolide antibiotic treatment for patients with LRTIs compatible with atypical pathogens like M. pneumoniae (55). Physicians often prescribe macrolides without positive microbiology results (56). In our study, almost all patients received antibiotics prior to admission, with macrolides being the most frequent outpatient antibiotic. Despite receiving macrolides earlier than the MSMP group, MUMPP patients were more likely to progress to SMPP and require flexible bronchoscopy. Previous study also suggests that early macrolide treatment or resistance does not necessarily mitigate the clinical severity of MPP (57). Therefore, early macrolide treatment may not prevent severe MPP manifestations.

Secondary treatment agents like tetracyclines, fluoroquinolones, and corticosteroids have been reported to improve MUMPP prognosis. Doxycycline (DXY), a tetracycline, is effective against both macrolide-susceptible and -resistant strains (58). In our study, 70.83% of MUMPP patients switched to DXY, a significantly higher proportion than the MSMP group (p < 0.05). Patients who switched antibiotics tended to have severe symptoms, resulting in a longer TTD in the MUMPP group compared to the MSMP group (median 24 h vs. 6 h). However, the average TTD with DXY was significantly shorter than those with prolonged macrolide use (17). Notably, no patients received glucocorticoids when administered DXY, and previous studies have also reported lower glucocorticoid use in the DXY group compared to the azithromycin group (59).

Japanese guidelines recommend switching to a tetracycline antibiotic if defervescence is not achieved within 48 h of macrolide therapy (60). Early oral doxycycline treatment can quickly improve clinical symptoms and promote the resolution of pulmonary inflammation (58, 61). Furthermore, the early use of minocycline may offer better economic and clinical benefits, even without test results for drug resistance genes (16). Our study showed that early use of DXC could significantly improve the duration of fever. These findings underscore the importance of promptly initiating appropriate second-line agents in treating MUMPP.

Therefore, identifying the risk factors of MUMPP is of great significant, especially in settings without possibility of testing mutations in the 23S ribosomal RNA gene. Children with MUMPP were more likely to experience prolonged fever, intrapulmonary or extrapulmonary complications, severe lung imaging changes, and elevated inflammatory markers (SF, IL-6, D-dimer, LAR, NLR). If such manifestations are observed in children, MUMPP should be highly considered, and early use of secondary agents should be contemplated. The indicators included in our study are easily obtainable and conducive to clinical use.