Takeshi Tajima

Simultaneous Detection of Pathogens in Clinical Samples from Patients with Community-Acquired Pneumonia by Real-Time PCR with Pathogen-Specific Molecular Beacon Probes

ABSTRACT In this study, real-time PCR with pathogen-specific molecular beacons (MB) and primers was evaluated for prediction of community-acquired pneumonia (CAP) causative agents, detecting six main CAP agents, Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Streptococcus pyogenes, simultaneously. The PCR assay was evaluated for fresh clinical specimens from infants and children (n = 389) and from adults (n = 40). The MB probes and primers are both pathogen specific, namely, the lytA gene for S. pneumoniae, the mip gene for L. pneumophila, and 16S rRNA genes for the remaining four organisms. DNA extraction of clinical specimens was performed with a commercially available EXTRAGEN II kit, and amplification was performed with Stratagene Mx3000P. The limit of detection for these pathogens ranged from 2 copies to 18 copies. The whole process from DNA extraction to the analysis was finished in less than 2 h. The obtained sensitivity and specificity of this real-time PCR study relative to those of conventional cultures were as follows: 96.2% and 93.2% for S. pneumoniae, 95.8% and 95.4% for H. influenzae, 100% and 100% for S. pyogenes, and 100% and 95.4% for M. pneumoniae, respectively. The sensitivity and specificity for M. pneumoniae relative to those of a serologic assay were 90.2% and 97.9%, respectively. In six clinical samples of C. pneumoniae, the real-time PCR gave positive predictable values, and in those cases, elevation of the titer value was also observed. In conclusion, we demonstrated that a real-time PCR assay with pathogen-specific MB is useful in identifying CAP causative agents rapidly and in examining the clinical course of empirical chemotherapy in a timely manner, supporting conventional culture methods.

Rapid Effectiveness of Minocycline or Doxycycline Against Macrolide-Resistant Mycoplasma pneumoniae Infection in a 2011 Outbreak Among Japanese Children

BACKGROUND Mycoplasma pneumoniae is a major pathogen causing community-acquired pneumonia in children and young adults. Outbreaks typically occur at intervals of several years. In 2011, a widespread outbreak was associated with macrolide-resistant M. pneumoniae (MRMP) in Japanese children, often those of school age. METHODS Two hundred fifty-eight children were diagnosed with M. pneumoniae-associated pneumonia based on chest radiography, real-time polymerase chain reaction (PCR), and antibody titers between January and December 2011. Mycoplasma pneumoniae cultures obtained from nasopharyngeal samples using appropriate broth were subjected to real-time PCR, by which decreases in M. pneumoniae in patients treated with minocycline (MIN), doxycycline (DOX), or tosufloxacin (TFX) were calculated. Mutations of the 23S ribosomal RNA gene that confer high resistance to macrolides in M. pneumoniae were identified by DNA sequencing. RESULTS Among 202 M. pneumoniae isolates from M. pneumoniae-associated pneumonia patients, 176 (87.1%) were MRMP. Macrolide-resistant M. pneumoniae infection was significantly related to school age (P < .01) and initial administration of macrolides (P < .01). Minocycline or DOX (n = 125) or TFX or levofloxacin (n = 15) was used for definitive treatment of MRMP patients. Minocycline or DOX was significantly more effective than TFX (P ≤ .05) in achieving defervescence within 24 hours and in decreasing numbers of M. pneumoniae DNA copies 3 days after initiation. CONCLUSIONS 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.

Emergence of Macrolide-Resistant Mycoplasma pneumoniae with a 23S rRNA Gene Mutation

ABSTRACT 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 MIC90s in the following order: 0.00195 μg/ml for azithromycin and telithromycin, 0.0078 μg/ml for clarithromycin, 0.0156 μg/ml for erythromycin, 0.0625 μg/ml for sitafloxacin, 0.5 μg/ml for minocycline, and 1 μg/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 μg/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 described previously (A. Cousin-Allery, A. Charron, B. D. Barbeyrac, G. Fremy, J. S. Jensen, H. Renaudin, and C. Bebear, Epidemiol. Infect. 124:103-111, 2000). These findings suggest that excessive usage of MLs acts as a trigger to select mutations on the corresponding 23S rRNA gene with the resultant occurrence of ML-resistant M. pneumoniae. Monitoring ML susceptibilities for M. pneumoniae is necessary in the future.

Single genotype of measles virus is dominant whereas several genotypes of mumps virus are co-circulating.

We have reported that in Japan measles virus strains have been classified into three distinct different genotypes (C1, D3 and D5) under the new international genotype classification since 1984. Similarly, mumps virus strains have been divided into two genotypes with three subtypes (B1, B2, B3, and D) under the proposed international classification since 1976. To differentiate these genotypes we developed a restriction fragment length polymorphism assay in the hemagglutinin (H) region for measles virus and in the hemagglutinin‐neuraminidase (HN) region for mumps virus to facilitate the expanded molecular epidemiology. In the Sapporo 1995/1996 measles outbreak, all 26 strains were classified as D5. Among 32 samples from patients with measles from 1994 to 1997 in Tokyo, 28 were identified as D5 and four were D3; these D3 strains were ascertained as a same hospital acquired infection. Among 45 strains obtained in the Tokyo 1999 outbreak, 38 were D3 and the remaining seven were D5. The dominant genotype of measles in Tokyo has replaced from D5 to D3 similar to the Chicago1/89 strain. We obtained 220 samples from patients with mumps from 1993 to 1997 and they were classified into one strain of B1, 14 strains of B2, 151 strains of B3, and 54 strains of D. Therefore, we suggest that two or three subtypes of mumps virus are co‐circulating with a different geographic pattern in genotype distribution, whereas a single measles virus genotype is dominantly observed, showing different epidemiological patterns. J. Med. Virol. 62:278–285, 2000.

Rapid optimization of antimicrobial chemotherapy given to pediatric patients with community-acquired pneumonia using PCR techniques with serology and standard culture

Children (n = 117; mean age 2.4 ± 2.9 years) were diagnosed as having community-acquired pneumonia (CAP) using clinical symptoms, chest X-rays, and hematological data. The causative pathogen was determined using real-time polymerase chain reaction (PCR) (6 bacteria), multiple reverse transcription-PCR (MPCR; 11 viruses), bacterial culture, and serology. The initial chemotherapy was evaluated based on the pathogens identified using PCR. We found 27 viral cases (23.1%), 25 bacterial cases (21.4%), 45 mixed infections with virus and bacteria (38.5%), 10 Mycoplasma pneumoniae (8.5%), 7 mixed infections with M. pneumoniae and another pathogen (6.0%), 1 Chlamydophila pneumoniae (0.9%), and 2 unknown pathogens (1.7%). Streptococcus pneumoniae and Haemophilus influenzae accounted for 58 (49.5%) and 27 (23.0%) of the cases, respectively. The median values (50%) of the white blood cell count (WBC) and C-reactive protein (CRP) using the box-and-whisker and plot method, respectively, were 11.7 × 103 mm−3 and 1.4 mg/dl in viral infections, 15.6 × 103 mm−3 and 4.8 mg/dl in mixed infections with virus and bacteria, 17.8 × 103 mm−3 and 6.3 mg/dl in bacterial infections, 6.7 × 103 mm−3 and 1.4 mg/dl in M. pneumoniae infections, and 21.5 × 103 mm−3 and 6.4 mg/dl in mixed infections with M. pneumoniae and other bacterial infections. Sulbactam/ampicillin (n = 61), carbapenems (n = 12), and ceftriaxone (n = 7) were selected for the patients suspected of having bacterial infections alone or mixed infections with bacterial and viruses in accordance with our criteria defined tentatively. For those with M. pneumoniae and C. pneumoniae infections, azithromycin or minocycline was initially used. Treatments averaged 3–5 days. The empirical chemotherapy was improper in 9.4% of cases in relation to the etiologic agents finally identified. We conclude that rapid and comprehensive identification using PCR can provide optimal antimicrobial chemotherapy for CAP patients.

Pandemic (H1N1) 2009-associated Pneumonia in Children, Japan

To describe clinical aspects of pandemic (H1N1) 2009 virus–associated pneumonia in children, we studied 80 such children, including 17 (21%) with complications, who were admitted to 5 hospitals in Japan during August–November 2009 after a mean of 2.9 symptomatic days. All enrolled patients recovered (median hospitalization 6 days). Timely access to hospitals may have contributed to favorable outcomes.

Killing kinetics of minocycline, doxycycline and tosufloxacin against macrolide-resistant Mycoplasma pneumoniae

Macrolide-resistant Mycoplasma pneumoniae (MRMP) has emerged and is increasing worldwide. In a 2011 outbreak of MRMP infections in Japan, symptoms failed to improve in many patients who initially received macrolides; the therapeutic agent was then changed to minocycline (MIN), doxycycline (DOX) or tosufloxacin (TFX). In this study, the bactericidal effects of these three agents against MRMP were evaluated. Time-kill kinetics against MRMP and macrolide-susceptible M. pneumoniae (MSMP) were determined for 5 days at concentrations corresponding to the respective minimum inhibitory concentration (MIC) and 2 × MIC, i.e. 1 µg/mL and 2 µg/mL for MIN, 0.5 µg/mL and 1 µg/mL for DOX, and 0.5 µg/mL and 1 µg/mL for TFX. The post-antibiotic effects (PAE) of these agents in culture against MRMP were also examined based on their pharmacokinetic parameters in children. Following exposure of MRMP and MSMP to up to twice the respective MICs of MIN, DOX and TFX, viable cells initially numbering 106 CFU/mL had decreased similarly to 103 CFU/mL after 4 days. Clarithromycin and azithromycin showed good bactericidal action against MSMP but not against MRMP. PAEs against MRMP appeared superior with MIN and DOX compared with TFX. In infection with M. pneumoniae having a generation time exceeding 6 h, a therapeutic agent must be selected in consideration of pharmacokinetic parameters, not MICs alone.

Evaluation of new immunochromatographic assay kit for adenovirus detection in throat swab: Comparison with culture and real-time PCR results

A new immunochromatographic (IC) assay kit, BD Veritor System Adeno was evaluated to comparing with commercial available kit, BD Adeno Examan, cell culture, and real-time PCR using throat swab samples. Specimens were collected from 146 pediatric patients between July 2011 and January 2012. Mean age of patients was 4 years (8 months-15 years old). Patients were diagnosed with pharyngitis (n = 67), tonsillitis (n = 45), pharyngoconjunctival fever (n = 26), upper respiratory tract infection (n = 6), conjunctivitis (n = 1), or bronchitis (n = 1). Thirty-one of the patients (21.2%) had more than one disease. Among all samples, 61 (41.8%) were positive for adenovirus with BD Veritor System Adeno; 68 (46.6%) with BD Adeno Examan; 63 (43.2%) with real-time PCR; and 65 (44.5%) with cell culture. Serotype 3 (n = 41; 63.1%) was predominant among the 65 adenovirus isolates, followed by serotype 2 (n = 12; 18.5%), 1 (n = 6; 9.2%), 5 (n = 4; 6.2%), and 4 (n = 2; 3.1%). Relative sensitivity and specificity of BD Veritor System Adeno, BD Adeno Examan, and real-time PCR were 93.8% and 98.7%, 96.9% and 93.8%, and 96.9% and 100%, respectively. Positive predictive and negative predictive values for these methods were 98.4% and 95.1%, 92.6% and 97.4%, and 100% and 97.6%, respectively. The sensitivity and specificity of real-time PCR was greater than that of IC assay kits. However, IC assay kits also showed high sensitivity and specificity appropriate for clinical use.

Immunochromatography test for rapid diagnosis of Mycoplasma pneumoniae infection

The sensitivity and specificity of a new rapid Mycoplasma pneumoniae antigen immunochromatography (IC) test, DK‐MP‐001, were determined using particle agglutination (PA) antibody response and loop‐mediated isothermal amplification (LAMP) gene detection as the gold standard. Of 165 patients, 59 were diagnosed with M. pneumoniae infection based on a ≥fourfold rise of serum PA antibody during the course of the illness. Of the first visit swabs, 60 were positive for M. pneumoniae on LAMP, and 49 were positive for M. pneumoniae antigen on IC test. Compared with PA antibody and LAMP, the sensitivity/specificity of the IC test were 81.4% (48/59) and 99.1% (105/106); and 81.7% (49/60) and 100% (105/105), respectively. IC test detected antigen in pharyngeal swabs more sensitively than in nasal swabs for the same subjects (P < 0.05). The IC test performs well enough to be used with pharyngeal swabs at the first examination.