The Treatment of Macrolide-Resistant Mycoplasma Pneumoniae Pneumonia in Children

Objective: To evaluate the ecacy and safety of levooxacin in children with macrolide-resistant Mycoplasma pneumoniae pneumonia. Methods: We retrospectively enrolled six conrmed cases of refractory Mycoplasma pneumoniae pneumonia (n=6) who were admitted in the pediatric respiratory ward of Shandong provincial hospital Aliated to Shandong rst Medical University between January 1st, 2020and February 29th, 2020. Levooxacin was given to the patients through the intravenous or oral route as per the following dosages :< 5 years, 8-10 mg/kg q12hours; > 5 years, 8-10 mg/kg, qd for ten days. The clinical data were collected and analyzed. Results: The average age of the enrolled cases was six years and nine months (range, four years, and seven months to eleven years and seven months). All cases were found to be drug-resistant and were treated with azithromycin combined with antibacterial drugs. Levooxacin was used in the patient’s refractory to macrolide antibiotics. The temperature of ve cases returned to normal 1 to 2 days after treatment with levooxacin, and the imaging of the four cases showed expected improvements. The gastrointestinal symptoms, neurological manifestations, joint symptoms, blood parameters, liver and kidney functions, and exercise conditions of the children were closely monitored. The follow-up time of the patients ranged from one week to ve months. No drug-related adverse reactions were observed in patients during treatment or during follow up. Conclusions: The clinical symptoms and imaging signicantly improved after treatment with levooxacin, and no drug-related adverse reactions were observed. Levooxacin proved to be an effective and safe drug in the treatment of children with macrolide-resistant mycoplasma pneumonia.


Introduction
Mycoplasma pneumoniae (MP) is a common pathogen that causes community-acquired pneumonia in children. In recent years, the resistance of MP to macrolide antibiotics has increased signi cantly. At the same time, the number of children with refractory Mycoplasma pneumoniae pneumonia (MPP) is increasing year after year. The health systems are facing signi cant challenges in carrying out the diagnosis and treatment of MPP in children. As a third-generation uoroquinolone, levo oxacin is a broad-spectrum antibiotic and has a wide distribution in the tissues of the body.
Levo oxacin is suitable for treating infectious diseases in various systems. However, the use of uoroquinolones is limited in children due to arthropathy issues in weight-bearing joints. At present, few studies have investigated the use of this drug in the pediatric population in China. To provide a reference for evaluating the e cacy and safety of levo oxacin in the pediatric population, we collected the clinical data of six children who were diagnosed with macrolide-resistant MPP and treated with levo oxacin in the department of Provincial hospital a liated to Shandong rst medical university.

General information
A total of six Cases ( ve males, one female) were admitted in the pediatric respiratory ward of Provincial hospital a liated to Shandong rst medical university from January 1st, 2020 to February 29th, 2020. These children had a mean age of six years and nine months and an age range of four years and seven months to eleven years and seven months. On admission, all the children tested positive for MP RNA and DNA. The mutational sites (2063A > G or 2064A > G) associated with drug resistance to macrolides antibiotics were present. According to the "Guide for the management of communityacquired pneumonia 2013 Edition" [1] , if the clinical signs aggravate, fever persists, and pulmonary imaging ndings aggravate even after treating the patients with macrolide antibiotics for seven days or more, the patients are diagnosed with refractory MPP. In our study, all six children were diagnosed with refractory MPP. Further, one child had pulmonary embolism; one child had a streptococcal infection, and two children had bronchial asthma. One child underwent ventilatorassisted respiratory therapy due to severe hypoxemia. Despite having pleural effusion, the ve children demonstrated good recovery (Table 1).

Etiological examination
MP RNA detection, bacterial culture, and antimicrobial susceptibility tests were carried out before treatment. MP RNA was detected using Simultaneous Ampli cation and Testing (SAT), and MP DNA was detected using Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). All specimens were aseptically collected. A total of nine respiratory tract specimens (deep sputum, alveolar lavage uid) were collected, and four specimens were collected from blood culture.

Levo oxacin treatment
In these children, the course of infection was 21-46 days before levo oxacin treatment, with an average of 31 days. They were given conventional azithromycin treatment for ve days. After three days of discontinuation, either their body temperature was still unstable, or they had enlarged pulmonary consolidation and (or) atelectasis; Further, the children were given azithromycin for an extended course (7 days), with overall treatment time from 3 weeks to 4 weeks. Other antibiotics, immunoglobulin, methylprednisolone, and other drugs were also used.
Three children were administered beroptic bronchoscopy. The remaining children did not undergo beroptic bronchoscopy due to the 2019 novel corona virus situation. All the children were given a su cient course of anti-infective treatment. The family members of the patients agreed and signed the informed consent forms. The application was then submitted to the Department of Pharmacy of the hospital.
The levo oxacin treatment modes were as follows: two children were given intravenous drip; two children were initially given intravenous drip but later changed to oral administration after signs of improvement while another two patients were given oral administration. Levo oxacin was produced by rst three pharmaceutical (Beijing) Co. Intravenous dosage was 500 mg / bag, while the oral dosage was 500 mg /tablet. According to the expert consensus for the children's application in accordance with uoroquinolone antibiotic drugs: children < 5 years were administered with 8-10 mg/kg/dose q12h (up to 750 mg/d) while the children > 5 years were administered with 8-10 mg/kg/dose qd, either intravenously or orally. The total course of treatment was ten days. During the levo oxacin treatment, other antibiotics, including azithromycin, were discontinued.

Observation of curative effect after treatment with levo oxacin
Before levo oxacin treatment, the body temperature of ve children (Case 1-5) was unstable. The highest body temperature uctuated between 37.6-38.8 ℃. After levo oxacin treatment, the body temperature became normal in 1-2 days. The fever did not recur during the hospitalization. The chest radiographs of four children showed signi cant postmedication improvements. However, the case 6 was critical with severe hypoxemia at the beginning of the illness and had undergone invasive ventilator-assisted breathing. This child took multiple beroptic bronchoscopies. The endoscopic mucosal erosion was severe. Ulcers appeared on carina and the walls of right main bronchus and other tubes Many plastic phlegm plugs were seen blocking the ori ce. The medial and lateral branches of the right middle lobe were almost occluded. The imaging improvements were not satisfactory. After ten days of treatment with levo oxacin, improvements were observed in the chest radiograph as compared with the one before treatment (Figure 1). Bronchoscopy ndings showed that the mucosal ulcers recovered well. The medical condition of these six children improved, and the children were subsequently discharged.

Investigation side effects of levo oxacin
The adverse reactions of levo oxacin include abnormalities of the cardiovascular system such as prolonged QT interval, ventricular arrhythmia; central nervous system abnormalities such as dizziness, anxiety, insomnia, and seizures; peripheral neuropathy; skeletal muscle diseases such as arthralgia, myalgia, muscle weakness, and tendon rupture, and other abnormalities such as liver toxicity, digestive tract discomfort, allergic reactions, leucopenia, and granulocytopenia. During the course of treatment, we closely monitored signs and symptoms of the gastrointestinal tract and nervous system and changes in the skin, joints, blood parameters, and liver and kidney functions. The six children treated with levo oxacin did not experience any symptoms of nausea, vomiting, dizziness, or other discomforts. Further, no skin rashes, mental disorders, or convulsions were observed. During ve months of follow-up, the children did not complain of any discomfort, including joint pain. Although two children had liver function abnormalities before medication, their liver function returned to normal after receiving hepatica. The rest of the children did not experience any abnormal liver or kidney function or a signi cant decrease in cell count.

Discussion
As one of the common community-acquired pneumonia pathogens, MP is one of the smallest microorganisms among bacteria and viruses. MPP accounts for 10-40% of community-acquired pneumonia cases [2] . Since MP lacks a cell wall, MP is resistant to cell wall targeting antibiotics [3] . Macrolides, tetracyclines, and quinolones are effective drugs for pediatric patients with MPP. In China, macrolide antibiotics are currently the rst choice for the treatment of children with MPP. Fluoroquinolones, including levo oxacin, have not been used extensively to treat children because of their safety concerns in the pediatric population. Therefore, levo oxacin is not approved for use in children.
Macrolide-resistant MPP was rst reported by a Japanese scholar in 2001. The problem of macrolide resistance in MP is a global concern. The rate of MP resistance in macrolide antibiotics is 3.5%~13.2% in the US [4] [5] and 90% or more in china [6]. of 2617 cause a low-level resistance [7] [8] . Cao B et al. [9] analyzed more than 20 reviews of MP resistance from 2000 to 2015 and summarized the order of point mutation frequency: A2063G, A2064G, A2063T, A2063C, A1290G, C2617A, and A2067G.Out of these mutations, A2063G and A2064G account for about 80%-90% of the cases.
As compared with patients infected with macrolide-sensitive MP, patients infected with macrolide-resistant MP are reported to have a longer duration of symptoms and longer course of antibiotic treatment. [10] But macrolide-resistant MP does not necessarily cause more severe illness as compared with macrolide-sensitive MP. Some children who were infected with macrolide-resistant MP also demonstrated satisfactory results when treated with conventional therapy. These observations may be due to the following factors: the anti-in ammatory effects of macrolides, the self-limiting nature of MP infection, and the different degrees of resistance to different macrolides. Zhuang Yuan [11] reported that due to immune, occur in the extra pulmonary system, including skin and mucous membranes, blood system, and central nervous system. Therefore, it is very important to timely select appropriate drugs to treat children infected with macrolide-resistant MP.
Morozumi et al. [12] gave tetracycline and quinolones to children with MP infection who were not effective in prolonging the application of macrolide antibiotics and achieved satisfactory results. Diana et al. [13] suggested that quinolones should be used to treat MPP in children who are refractory to macrolide antibiotics.
The helicase and topoisomerase IV of MP DNA. Were the target enzymes of quinolones. The quinolones inhibit MP by targeting protein synthesis. Levo oxacin is the third generation of quinolones. As compared with the rst and second generation of quinolones, levo oxacin has the advantage of being a wide spectrum antibiotic with strong antibacterial activity, good tissue permeability, low toxicity, and less adverse effects. However, levo oxacin is reported to cause articular cartilage abnormalities, leading to limb dysfunction in some juvenile animals [14] . These observations suggest that children Binz [15] reviewed and analyzed the clinical studies and concluded that there is no clear correlation between the application of uoroquinolones and musculoskeletal adverse events. The latest ve-year follow-up data showed that the incidence of musculoskeletal adverse events is low, and the events reverse after discontinuing the drug. Rosavonaet al. [16] conducted a systematic review and meta-analysis that involved eight studies and 23166 patients. The ndings demonstrated that uoroquinolones do not cause musculoskeletal diseases in minors, and these drugs should not be banned for children suffering from speci c infections. LIU et al. [17] analyzed ve randomized controlled trials (RCTs) involving1968 patients in the levo oxacin group and 1640 patients in the control group. Out of these ve studies, two studies showed that osteoarticular event rates were not statistically signi cant between the levo oxacin group and the control group. As per the other three studies, adverse events of bones and joints were not observed in both the groups during treatment and followup. These observations suggest that the incidence of levo oxacin-induced adverse events in bones and joints in children is low, and most of these adverse events can be attended during follow-up.
Pharmaceutical experts created the "Expert consensus on the application of uoroquinolone antibacterial drugs in children" [18] to standardize the application of these drugs in pediatrics. The "Expert consensus on diagnosis and treatment of mycoplasma pneumoniae pneumonia in children (2015)" recommends that children infected with macrolide-sensitive MP should be treated with macrolide antibiotics, and other antibiotics should be considered for MP resistant to macrolide antibiotics. The 6 cases in the present study were diagnosed with refractory MPP, and the mutations associated with drug resistance were present. After the conventional course of azithromycin treatment, the patients were still febrile. The course of treatment was extended to seven days. Three children were assisted by beroptic bronchoscopy. Although methylprednisolone and immunoglobulin were also administered simultaneously, the six children still had a recurrent fever and/or progressed imaging. Among them, one child was complicated with pulmonary embolism, and ve cases had massive consolidation hydrothorax of the lung. The clinicians adjusted the therapy to levo oxacin, and comprehensively evaluated the condition of the children. After treating the children with levo oxacin, the body temperature returned to normal, and the imaging also improved to varying degrees. The follow-up time of the patients after levo oxacin treatment ranged from one week to ve months. There were no drug-related adverse reactions during the course of treatment or follow-up. This study is limited in terms of the small number of included cases and short follow-up time.
The pharmacokinetic analysis of azithromycin by Zheng et al. [19] [20] highlighted that the rst 24-48 hours are crucial for the success of antimicrobial treatment and that the effective drug concentration is critical since it can lead to treatment failure or increased toxicity. The reduction in C-reactive protein (CRP) and neutrophil count in children with an azithromycin trough concentration of > 0.25 mg/L was signi cantly higher than that in children with an azithromycin trough concentration of < 0.25 mg/L. The azithromycin loading dose of 15 mg/kg reaches the effective target concentration within 24-48 hours. If this dose is followed by a maintenance dose of 10 mg/kg, the area under concentration-time curve over 24 h (fAUC) to the MIC 90 (fAUC/MIC) can be maintained above 50%. In this study, the mean clearance of azithromycin in children aged two to twelve years was 1.288 liters/h/kg. This value is in accordance with a previous pharmacokinetic study of intravenous azithromycin that reported average clearance (CL) values of 1.062 liters/h/kg in 7 children aged 2 to 6 years and 0.960 liters/h/kg in 8 children aged 6 to 12 years. The related risks of overdose for the proposed dosing regimens were 5.8% and 3.8%, respectively, for pediatric patients with normal and impaired liver function. In case the 15% dose reduction is not performed in children with ALT of 40, the probability of overdose increases from 3.8-6.3%. At present, the conventional dose of azithromycin is 10 mg/kg. The hypothesis that whether some children suffer from insu cient effective drug concentration that leads to poor therapeutic effect still needs further con rmatory studies.

Conclusions
The clinical symptoms and imaging signi cantly improved after treatment with levo oxacin, Levo oxacin proved to be an effective and safe drug in the treatment of children with macrolide-resistant mycoplasma pneumonia. Levo oxacin in children is an off-label drug, and the child's condition and treatment risk should be fully evaluated before considering levo oxacin as a potential treatment option. Before the administration of levo oxacin, clinicians should communicate with the family members of patients; evaluate the risks and bene ts of the drug, and choose the most bene cial option for the patient. Imaging changes in case 2 A) 7 days before levo oxacin B) 7 days after levo oxacin C) 1month after levo oxacin