No abstract

Among 380 Mycoplasma pneumoniae isolates from 3,678 pediatric patients with community-acquired pneumonia, 50 macrolide-resistant strains had an A2063G transition in domain V of the 23S rRNA, whereas 5 had an A2064G transition. These resistant strains increased rapidly from April 2002 to December 2006.For Mycoplasma pneumoniae, a major etiologic agent of lower respiratory tract infections acquired in the community, 14-membered ring macrolides (ML) generally are recognized as first-choice agents. In Japan, ML-resistant (ML r ) M. pneumoniae possessing a 23S rRNA mutation first was isolated from pediatric patients with community-acquired pneumonia (CAP) and bronchitis as reported in 2001 by Okazaki et al. (4). Patient symptoms appeared to be prolonged when isolates showed ML resistance (5).We subjected M. pneumoniae isolated from pediatric patients with CAP between 2002 and 2006 to susceptibility evaluation for eight agents, including ML. In strains showing ML resistance, the 23S rRNA gene was analyzed.Between April 2002 and December 2006, 3,678 clinical samples were sent to our laboratory from pediatricians affiliated with 10 institutions participating in the Acute Respiratory Diseases Study Group. All samples originating from pediatric patients diagnosed with pneumonia according to clinical symptoms and chest X-ray images were collected after informed consent was given by the patients and/or their parents or guardians.Immediately after receipt, the samples were suspended in 1.5 ml of pleuropneumonia-like organism (PPLO) broth (Difco, Detroit, MI). DNA then was extracted by using Extragen II (Tosoh, Tokyo, Japan) according to the manufacturer's protocol. Real-time PCR to detect M. pneumoniae was performed as described previously (2) using the extracted DNA. Culture of M. pneumoniae was carried out for PCR-positive samples using PPLO broth according to previously described methods (6).The MICs of eight agents for M. pneumoniae isolates were determined with microdilution methods using PPLO broth.These agents were erythromycin, clarithromycin, azithromycin, josamycin, rokitamycin, telithromycin, minocycline, and levofloxacin. M. pneumoniae M129 strain was used as a control.The full length of the 23S rRNA gene was sequenced by methods described previously (3) in 55 M. pneumoniae strains showing ML resistance.For patients with adequate clinical information, clinical courses of CAP caused by ML r M. pneumoniae (n ϭ 53) were compared to those of CAP with ML-susceptible (ML s ) M. pneumoniae (n ϭ 58). Variables compared included (i) the number of days from initiation of ML treatment until defervescence to 37°C and (ii) whether or not initial treatment with ML was changed later to another agent. Body temperature that exceeded 38°C at least once daily was defined as ongoing fever. Table 1 Table 2 shows the MIC range, MIC 50 , and MIC 90 for eight agents according to the presence or absence of a mutation of the 23S rRNA gene in the 380 M. pneumoniae isolates; 50 strains had an A2063G transition in domain V, and 5 strains had...

Macrolides are inappropriate as first-choice agents against MRMP in terms of shortening the clinical course and decreasing M. pneumoniae. Control and prevention of MRMP outbreaks in children require early decreases in M. pneumoniae as well as improvement of clinical findings.

The epidemiological monitoring of macrolide resistance in this species has become necessary in France and Europe, and will be made easier by using these PCR assays.

No abstract

We have developed a real-time reverse transcription-PCR (RT-PCR) method to detect 13 respiratory viruses: influenza virus A and B; respiratory syncytial virus (RSV) subgroup A and B; parainfluenza virus (PIV) 1, 2, and 3; adenovirus; rhinovirus (RV); enterovirus; coronavirus (OC43); human metapneumovirus (hMPV); and human bocavirus (HBoV). The new method for detection of these viruses was applied simultaneously with real-time PCR for the detection of six bacterial pathogens in clinical samples from 1700 pediatric patients with community-acquired pneumonia (CAP). Of all the patients, 32.5% were suspected to have single bacterial infections; 1.9%, multiple bacterial infections; 15.2%, coinfections of bacteria and viruses; 25.8%, single viral infections; and 2.1%, multiple viral infections. In the remaining 22.6%, the etiology was unknown. The breakdown of suspected causative pathogens was as follows: 24.4% were Streptococcus pneumoniae, 14.8% were Mycoplasma pneumoniae, 11.3% were Haemophilus influenzae, and 1.4% were Chlamydophila pneumoniae. The breakdown of viruses was as follows: 14.5% were RV, 9.4% were RSV, 7.4% were hMPV, 7.2% were PIV, and 2.9% were HBoV. The new method will contribute to advances in the accuracy of diagnosis and should also result in the appropriate use of antimicrobials.

To clarify year-to-year changes in capsular serotypes, resistance genotypes, and multilocus sequence types of Streptococcus pneumoniae, we compared isolates collected from patients with invasive pneumococcal disease before and after introductions of 7- and 13-valent pneumococcal conjugate vaccines (PCV7 and PVC13, respectively). From April 2010 through March 2017, we collected 2,856 isolates from children and adults throughout Japan. Proportions of PCV13 serotypes among children decreased from 89.0% in fiscal year 2010 to 12.1% in fiscal year 2016 and among adults from 74.1% to 36.2%. Although nonvaccine serotypes increased after introduction of PCV13, genotypic penicillin resistance decreased from 54.3% in 2010 to 11.2% in 2016 among children and from 32.4% to 15.5% among adults. However, genotypic penicillin resistance emerged in 9 nonvaccine serotypes, but not 15A and 35B. Multilocus sequence typing suggested that resistant strains among nonvaccine serotypes may have evolved from clonal complexes 156 and 81. A more broadly effective vaccine is needed.

A total of 195 Mycoplasma pneumoniae strains were isolated from 2,462 clinical specimens collected between April 2002 and March 2004 from pediatric outpatients with respiratory tract infections. Susceptibilities to six macrolide antibiotics (ML), telithromycin, minocycline, levofloxacin, and sitafloxacin were determined by the microdilution method using PPLO broth. A total of 183 M. pneumoniae isolates were susceptible to all agents and had excellent MIC 90 s in the following order: 0.00195 g/ml for azithromycin and telithromycin, 0.0078 g/ml for clarithromycin, 0.0156 g/ml for erythromycin, 0.0625 g/ml for sitafloxacin, 0.5 g/ml for minocycline, and 1 g/ml for levofloxacin. Notably, 12 ML-resistant M. pneumoniae strains were isolated from patients with pneumonia (10 strains) or acute bronchitis (2 strains). These strains showed resistance to ML with MICs of >1 g/ml, except to rokitamycin. Transition mutations of A2063G or A2064G, which correspond to A2058 and A2059 in Escherichia coli, in domain V on the 23S rRNA gene in 11 ML-resistant strains were identified. By pulsed-field gel electrophoresis typing, these strains were classified into groups I and Vb, as Mycoplasma pneumoniae is one of the main pathogens in respiratory tract infections (RTI) acquired in the community. In school-aged children and young adults with communityacquired pneumonia (CAP), M. pneumoniae accounts for as many as 10 to 30% of cases (4,6,13).In recent years, the clinical diagnosis for M. pneumoniae infection has relied on serological methods such as passive agglutination (Serodia-Myco II kit, Fujirebio, Tokyo, Japan) and complement fixation, even though a PCR method was partially applied (3,22,26). Accordingly, culture methods for this pathogen that require up to 1 week or more are rarely carried out in the laboratory. Thus, the current status of susceptibility to macrolide antibiotics (ML) used as the firstchoice agent for M. pneumoniae is unclear.In Japan, in parallel with the increase in usage of oral 14-membered ring ML (14-ML) and azithromycin for RTI, M. pneumoniae showing resistance to 14-ML has been isolated from clinical samples from pediatric outpatients with CAP (12, 16). We suspected an increase in cases with ML-resistant M. pneumoniae infection from prolonged clinical symptoms. In such cases, PCR positivity with rapid identification of M. pneumoniae persisted after the administration of clarithromycin or azithromycin for several days.We isolated causative M. pneumoniae by cultivation from clinical specimens collected from pediatric outpatients with RTI to determine susceptibilities to 10 oral antibiotics and identified a transition mutation on the 23S rRNA gene in ML-resistant isolates. MATERIALS AND METHODSMicroorganisms. M. pneumoniae was isolated from clinical specimens from patients with RTI. The specimens were collected from pediatric outpatients who visited 10 medical Japanese institutions participating in the study group on acute respiratory diseases (ARD). A total of 2,462 samples were sent to our laboratory (Kitasa...