Clinical Evaluation of Macrolide-resistant Mycoplasma pneumonia e Infections in Pediatric Japanese Patients

Background: In recent years, an increase in Mycoplasma pneumonia e infections among children has become a social issue in Japan. Methods: During a 2-year collection period (2011-2012), we evaluated trends during the first and second halves of both years. Only patients with positive rapid antigen detection test results (ImmunoCard Mycoplasma : Meridian Bioscience, Inc Cincinnati, OH, USA) were included. The evaluation items included the patient number, sex, radiography findings, white blood cell (WBC) count, C-reactive protein (CRP) level, and clinical macrolide-resistance rate. Results: The patient number increased significantly during the latter halves of the years. The macrolide-resistance rate also increased during the same periods. A similar trend was observed with respect to radiography findings but not with respect to the WBC count and CRP


INTRODUCTION
Mycoplasma pneumoniae is the main causative pathogen of respiratory tract infections in children. Generally, macrolides are the first treatment agents used against M. pneumoniae infections in children. In recent years, however, macrolide-resistant M. pneumoniae has been reported to comprise more than 40% of all M. pneumoniae isolates from M. pneumoniae infections in Japanese children. This trend has been reported not only in Japan but also in other countries. Therefore, this increased incidence of macrolide-resistant M. pneumoniae infections required attention with respect to the treatment of mycoplasmal infections in children.

MATERIALS AND METHODS
We enrolled all pediatric patients with acute respiratory lower tract infections who visited our pediatric section between January 2011 and December 2012. The patient characteristics are listed in Table 1. Mycoplasma test results were excluded from the study. Findings suggestive of pneumonia that were visible on radiographs were classified into 3 grades as follows: 1, mild; 2, moderate; and 3, heavy and/or atelectasis. The white blood cell (WBC) counts and Creactive protein (CRP) levels were estimated using the blood laboratory findings.
Determinations of macrolide-resistant M. pneumoniae infections were made for cases in which no improvements with respect to clinical efficacy were observed during a 5-day period and/or the chest radiography findings indicated aggravation in response to macrolides therapy. Clarithromycin was the first-line macrolide therapy. Erythromycin, rokitamycin, or azithromycin was prescribed only when internal clarithromycin administration proved difficult. If the patient was determined to harbor a macrolide-resistant M. pneumoniae infection, we switched from macrolide therapy to minocycline (MINO) or tosufloxacin (TFLX). We prescribed MINO to patients who were at least 8 years of age and TFLX to patients who were younger than 8 years of age to avoid tooth discoloration.
Concomitant infections with other bacteria were possible in patients with Mycoplasma pneumoniae infections. We excluded patients with abnormally WBC counts and CRP levels.
We also excluded patients who had been treated with MINO or TFLX prior to macrolide therapy.
The study period was divided into 4 parts: Ⅰ, the first half of the year of 2011: Ⅱ, the latter half of 2011: Ⅲ , the first half of 2012; and Ⅳ, the latter half of 2012.
This single-institution study was conducted at the Department of Pediatrics, Tokyo Takanawa Hospital.
Consent was obtained from the parents' parents, who were provided with information about our treatment policy.

RESULTS
This clinical study included 189 patients. The patients' ages and genders are shown in Table  1. A statistically significant difference was observed with respect to the average age but not the sex.
The antimicrobial agents used in the study are listed in Table 2. The number of cases that required MINO or TFLX increased during the latter halves of the study years.
The WBC counts, CRP levels, and radiography grades are listed in Table 3. During the 4 study periods, a statistically significant difference was observed with respect to the radiography grades but not the WBC counts and CRP levels.
The number of macrolide-resistant cases and the resistance rate are shown in Table 4. The clinical macrolide-resistance rate increased by more than 70% during the latter halves of the 2 study years.   [3]. Furthermore, the incidence rates of macrolide-resistant M. pneumoniae infections also increased in other countries [4][5][6][7].
Until 2010, we generally prescribed macrolides to the pediatric patients with M. pneumoniae infections, and most patients were successfully cured without switching from macrolide therapy. However, during the latter period of 2011, many cases required swiching from macrolides to MINO or TFLX because of persistent fevers and coughing or abnormalities on the chest radiograph.
Matsubara et al. reported that the clinical efficacy of macrolides for the treatment of macrolide-resistant M. pneumoniae was significantly lower than the clinical efficacy for cases of macrolide-sensitive M. pneumoniae [8].
In the present study, however, more than 70% of the patients during the latter halves of the 2 study years required swiching to MINO or TFLX.
The administration of MINO to children less than 8 years of age is inappropriate because of the risk of tooth discoloration; therefore, MINO was only prescribed to patients who were at least 8 years of age. Accordingly, we prescribed TFLX to patients with clinical macrolideresistant M. pneumoniae infections who were aged less than 8 years. MINO and TFLX exhibited good antimycoplasmal activities against the clinical macrolide-resistant M. pneumoniae infections in our study.
Regarding problems associated with this study, it is initially difficult to diagnose macrolideresistant M. pneumoniae infections. Additionally, there were no apparent differences in the clinical symptoms, laboratory data, and radiography findings between the cases with macrolide-sensitive and resistant M. pneumoniae infections. Therefore, we performed a second evaluation of the clinical macrolide efficacy after a 3-day interval and consequently determined whether the patient required a switch from macrolide therapy. However, changes with respect to macrolide therapy for children with clinical macrolide-resistant M. pneumoniae infections are controversial.
M. pneumoniae infections are difficult to diagnose because there are no specific early-stage clinical, epidemiological, or laboratory observations. Similarly, the radiographic findings are not specific. Therefore, our diagnoses of M. pneumoniae infections were based on results obtained using the Meridian ImmunoCard Mycoplasma test, a card-based enzyme-linked immunosorbent assay designed to detect IgM antibodies against M. pneumoniae. Matas et al. reported that the Meridian ImmunoCard Mycoplasma test appeared to be a good screening assay for M. pneumoniae IgM titers in children [9]. However, this test cannot detect IgM antibodies at early stages and yields positive results for a long period after an infection has been resolved. Jacobs reported that IgM titers could be detected beginning at 7 days after the onset of symptoms in patients with primary M. pneumoniae infections [10].
Given the above information, we generally diagnosed M. pneumoniae infections based on the clinical symptoms, blood test results, radiography findings, and Meridian ImmunoCard Mycoplasma test results. Our diagnostic criteria for M. pneumoniae infection included prolonged coughing, abnormalities on the radiograph, and positive Meridian ImmunoCard Mycoplasma test results. In our study, the radiography findings were statistically significant with respect to the incidence of macrolide-resistant M. pneumoniae infections.
M. pneumoniae infections generally occur throughout the year. However, the results of our study demonstrated that the infection incidence increased during the latter halves of both years. The reason for this phenomenon is unclear, however, the number of patients increased along with the increased incidence of macrolide-resistant M. pneumoniae infections. Therefore, an etiological explanation for the increased incidence of macrolideresistant M. pneumoniae infections during the latter half of the year needs to be identified.
Specific point mutations were reported in all macrolide-resistant M. pneumoniae strains [3,11]. Morozumi et al. reported that these macrolide-resistant strains carried either an A2063G or an A2064G transition in domain V of the 23S rRNA gene and that the prevalence of these resistant strains had increased rapidly in Japan [3]. Akaike et al. reported that the macrolide-resistant strains exhibited high resistance to erythromycin, clarithromycin, and azithromycin. Conversely, TFLX exhibited potent antimycoplasmal activity [11]. In our study, the clinical efficacies of the antimicrobial agents displayed similar tendencies to those reported previously.

CONCLUTION
In conclusion, macrolides were previously considered an appropriate first-line agent for the treatment of M. pneumoniae infections in children. Therefore, it is important to monitor the incidence of macrolide-resistant M. pneumoniae infections in the community and to select appropriate therapeutic agents according to the macrolide-resistant or macrolide-sensitive status of the infectious M. pneumoniae strain.

CONSENT
The personal information that can identify an individual does not include it in this article.

ETHICAL APPROVAL
This study was performed with the permission of the Tokyo Takanawa Hospital Ethics Committee including the outside member consisting of school teachers and the lawyer. All authors hereby declare that all experiments have been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.