Mycoplasma Pneumoniae (MP) is an important pathogenic microorganism causing community-acquired pneumonia in children. Owing to its independent survival between bacteria and viruses, its drug resistance rate has risen sharply in recent years, with an annual increase in incidence (1). Previous clinical experience often suggests that MP pneumonia (MPP) can be self-limiting. However, due to the incomplete development of children's tissues, organs, and immune system functions, MP infection can progress quickly and easily, leading to severe pneumonia, extrapulmonary complications (2), and multiple organ failure (3). Furthermore, multiple factors, such as local signs of wheezing and dyspnea combined with systemic infectious poisoning symptoms, cause children to have poor prognoses and high disability and mortality rates. The corresponding sequelae can affect children's daily academic performance, life, and quality of life even if they survive (4, 5). Therefore, early screening and precise assessment of the harm caused by MP infection provide crucial objective clinical predictive guidance for the treatment, prognosis, and well-being of infected children.

In previous studies, serological methods were mainly used to diagnose MP infection, and the detection of immunoglobulin M (IgM) antibodies in the acute phase or the increased immunoglobulin G antibody titers in the acute and recovery phases were the main indicators of current infection. However, for infants and young or immunocompromised children, the sensitivity of serological methods is low because IgM antibodies are associated with their immune status, and antibodies are not often detected (6). Real-time fluorescence quantitative polymerase chain reaction (PCR) has been used to detect the load of MP DNA in the bronchoalveolar lavage fluid (BALF) of children in the control, non-MPP and MPP groups compared with the positivity rate of MP antibody in a single serum. The false-positive and false-negative rates detected using BALF as a sample were significantly lower than those detected using serum, suggesting that using BALF as a test specimen provides higher accuracy. The same group of data also suggests that MP DNA in BALF has a positive rate of 88% in the early stage of the disease (1–7 days), which is significantly higher than the 9% positive rate of serum MP antibody within the same period, indicating that using BALF as a specimen provides a higher degree of accuracy in the early diagnosis of MPP (7). Notably, some researchers have also demonstrated that the detection rate of MP infection can be significantly improved by using the BALF MP DNA test, and the BALF MP DNA results are not affected by the duration of the disease (8).

Quantitative fluorescence detection of MP DNA can quantify the level of MP DNA in the body and accurately reflect the infection and replication of MP in a child's body. Clinicians can also infer the severity of a child's condition based on the copy number of MP DNA and control the medication dosage to achieve the desired therapeutic effect (9). Furthermore, local MP DNA load can objectively reflect the balance between the pathogen and host immune clearance capacity (10). There are currently few studies on the correlation between local MP DNA load in the respiratory tract and clinical severity in children with MP globally. Similar studies have been conducted in China. However, the specimen types are mostly sputum and nasopharyngeal swabs (11–14). Studies have shown a higher false-negative rate of MPP due to the predominantly dry cough, low secretion, and sputum in the early stage of the disease, and poor sputum compliance in pediatric patients, leading to the mixing of saliva in the specimen (15). The upper respiratory tract of the healthy population also carries MP, leading to a higher false-positive rate of the specimen compared with the specimen from the BALF (16). Therefore, BALF can be obtained directly from the diseased tracheobronchial mucosa compared with serology, sputum, and nasopharyngeal swabs as test samples to confirm the diagnosis of MPP. This can minimize the contamination rate of the specimen and is more objective, direct, and independent of the disease duration. However, there has been no clear conclusion regarding using BALF as a sampling specimen in children (7, 17). Therefore, it is necessary to conduct more in-depth and detailed research on the relationship between MP DNA load and clinical severity in the BALF of children infected with MP to obtain more objective conclusions.

This study aimed to analyze MP DNA load and genotyping data, clinical manifestations, relevant laboratory data, imaging, and extrapulmonary complications of infected children to explore the relationship between MP load and the severity of children's clinical conditions and provide a basis for monitoring and predicting MPP.

MP is one of the most important pathogens causing human respiratory infections, with widespread population susceptibility, periodic cluster epidemics occurring every 3–7 years, and infection rates as high as 50%–80% during peak epidemiologic periods or in limited spaces, with preschool and school-age children having the highest rates of infection. The disease has a slow onset and can be latent in the respiratory tract of infected patients, causing a prolonged disease course and the clinical symptoms after infection are diverse, with heavy symptoms and light signs leading to a high tendency to underestimate the disease upon imaging; therefore, it is particularly essential to correctly understand its clinical features and treatment methods.

Notably, several studies have confirmed that severe MP infections and refractory cases are significantly associated with MP pathogenicity and individual host immune response (3, 5). Therefore, studying the relationship between MP pathogenicity and the degree of host inflammatory and immune response status is essential to improve the treatment rate and quality of survival in patients with severe and/or refractory MP infections. Waites et al. (24) suggested that the local MP load responds to the balance between pathogen and host immune clearance; therefore, effective access to the biological properties of in situ MP infection is critical to achieving this theoretical study. This study aimed to analyze the association between MP DNA load in the BALF and MP typing, laboratory indicators, imaging manifestations, and clinical phenotypes to provide a basis for clinical disease assessment and MPP diagnostic protocols.

Previous studies have found that Mpn13, Mpn14, Mpn15, and Mpn16 strains exhibiting 4-5-7-2 are mostly P1-I type strains, whereas strains exhibiting 3-5-6-2 are mostly P1-II type strains. In this study, 128 and 37 cases, respectively, supported this hypothesis (25). However, the relationship between MP genotype and DNA load has rarely been reported. Nilsson et al. (10) found a correlation between the clinical severity of MP infection and DNA load but found no significant correlation with its subtype. However, their study performed only one type of typing, PCR-RFLP, for MP P1 adhesion protein, and most of the study population was selected from young and middle-aged people. The relationship between the MP genotype and DNA load in children needs further exploration, considering that children are not a reduced version of adults and have different development and morbidity types. In the 165 cases of BALF successfully cultured in this study, the typing results showed no statistical difference in the distribution of P1 and MLVA genotypes among the different MP DNA load samples, which is consistent with the report of Nilsson et al., who confirmed no significant association between MP subtypes and DNA load from the perspective of MP infection in adults.

MP invading the respiratory tract can directly activate the host inflammatory response, causing increased secretion of multiple inflammatory mediators, resulting in organismal damage and participation in developing severe and refractory MPP (3, 5). This study found that the CRP, PCT, and AST levels in children in the high-load group were significantly higher than those of children in the low-load group. A linear trend correlation analysis using Spearman's correlation coefficients also showed a positive correlation between MP DNA load and these serum biochemical indices, suggesting that MP DNA load is correlated with inflammatory response. Previous studies have confirmed that CRP, a non-specific systemic inflammatory response protein, reflects the severity of acute temporal pneumonia and extrapulmonary tissue damage (24, 26). PCT is a calcitonin precursor secreted by the thyroid gland. It is present in small amounts in an organism's normal state, and its level increases linearly when infected (27). Notably, some studies have suggested that both inflammatory mediators are closely associated with MPP and are critical biomarkers for measuring MP infection status and predicting the occurrence of severe and refractory MPP (28, 29). Serum CRP and PCT levels are lower after MP infection than after bacterial infection; however, they help predict severe MPP (28). The present study found that CRP and PCT levels were higher in children in the high-load group than in those in the low-load group, and the difference was statistically significant. MP DNA load was positively correlated with inflammatory response. The positive correlation between MP DNA load and inflammatory response suggests that in clinical practice, anti-inflammatory therapy should be strengthened early in patients with a high load during infection to avoid the excessive release of inflammatory mediators or autoimmune imbalance leading to disease exacerbation and drug resistance. Among the other biochemical indicators, increased AST was significant, associated with an increased rate of digestive system comorbidity; however, alanine aminotransferase (ALT), which reflects the characteristics of liver function, did not suggest specific changes. This was analyzed because AST can be detected in various body tissues and is not tissue-specific; for example, mildly hemolyzed erythrocytes, myocardial infarction, and cardiac arrhythmia can cause abnormal increases in AST. Therefore, this study aimed to improve other relevant experimental data to clarify their application in diagnosing MP infections.

This study showed that children in the low-load group had significantly higher cough duration, wheezing frequency, and shortness of breath than those in the high-load group, and the WBC and MONO counts were significantly increased in the low-load group. This may be because the increase in WBC and MONO counts in the low-load group and lesions are mainly concentrated in the airway, which may promote the accumulation of airway epithelial cells and the release of several inflammatory factors, which trigger airway hyperresponsiveness and lead to shortness of breath and wheezing in children.

Children in the high-load group had longer hospital stays and higher maximum fever temperatures, which resulted in a higher incidence of chills. The Spearman correlation coefficients showed the same trend, suggesting that this may be associated with the difficulty in clearing overloaded MP in the body and the persistent immune imbalance in children. In clinical practice, it is necessary to extend the period of relevant drug therapy and strengthen immune-related treatments. Local high MP load infection can directly damage bronchial epithelial cells, resulting in epithelial cell necrosis, reduced or even absent cilia, and impaired secretion drainage, which aggravates poor local toxin clearance, forming a vicious circle, leading to bronchial mucosal erosion, secretion obstruction, plastic sputum formation, and local ischemia and hypoxia, resulting in epithelial cell repair dysfunction, end small vessel fibrosis, ischemic and hypoxic injury of small airways, irreversible fibrotic lesions and airway obstruction. This leads to pulmonary atelectasis, bronchial occlusion, increased local inflammatory response, severe damage to lung tissue, and peripheral respiratory failure, which can significantly increase invasive operations such as tracheal intubation and mechanical ventilation and seriously affect prognosis (17, 30).

In addition to pulmonary inflammation, MP infection can cause multi-organ damage through immune cascade reactions mediated by multi-pathway mechanisms, such as multisystem lesions in the skin and digestive, urinary, hematological, neurological, and circulatory systems (31, 32), and in severe cases, acute pericardial tamponade, hemolytic uremic syndrome, and ischemic stroke (33, 34). This is due to the presence of partially common antigens between MP antigens and specific body tissues (heart, liver, lung, brain, kidney, and smooth muscle), which can induce the body to produce autoantibodies such as Immunoglobulins G and E, inducing autoimmune reactions and causing damage to body tissues with the same antigenic structure, which can lead to damage to other target organs outside the respiratory tract and extrapulmonary complications in multiple systems (35–37).

In this study, children in the high-load group were more likely to have imaging changes, such as pleural effusion, and the incidence of respiratory infections and extrapulmonary complications was higher than that of children in the low-load group. MP infection causes a double blow to extrapulmonary organs by direct damage and an indirect immune response, causing multi-organ damage to the primary lung and extrapulmonary tissues. Therefore, we hypothesized that a high MP load could predict the severity of airway and extrapulmonary damage in children and provide data to support early disease intervention.

In conclusion, MP DNA load was not significantly correlated with MP typing but was significantly correlated with clinical manifestations, laboratory parameters, and imaging characteristics in children. Therefore, monitoring MP load can provide reasonable, economical, and efficient reference suggestions for the diagnosis and severity assessment of children with MP infection, which can help monitor and predict the disease and guide the rational use of clinical drugs to effectively improve children's prognosis, survival rate, and quality of life.