Eiichi Nakayama

Increased macrolide resistance of Mycoplasma pneumoniae in pediatric patients with community-acquired pneumonia.

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.

Comprehensive detection of causative pathogens using real-time PCR to diagnose pediatric community-acquired pneumonia

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.

Rapid detection of eight causative pathogens for the diagnosis of bacterial meningitis by real-time PCR

We aimed to detect causative pathogens in cerebrospinal fluid (CSF) collected from patients diagnosed with bacterial meningitis by real-time polymerase chain reaction (PCR). In addition to Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae described previously, five other pathogens, Neisseria meningitidis, Escherichia coli, Streptococcus agalactiae, Staphylococcus aureus, and Listeria monocytogenes, were targeted, based on a large-scale surveillance in Japan. Results in CSF from neonates and children (n = 150), and from adults (n = 18) analyzed by real-time PCR with molecular beacon probes were compared with those of conventional culturing. The total time from DNA extraction from CSF to PCR analysis was 1.5 h. The limit of detection for these pathogens ranged from 5 copies to 28 copies per tube. Nonspecific positive reactions were not recognized for 37 microorganisms in clinical isolates as a negative control. The pathogens were detected in 72.0% of the samples by real-time PCR, but in only 48.2% by culture, although the microorganisms were completely concordant. With the real-time PCR, the detection rate of H. influenzae from CSF was high, at 45.2%, followed by S. pneumoniae (21.4%), S. agalactiae (2.4%), E. coli (1.8%), L. monocytogenes (0.6%), and M. pneumoniae (0.6%). The detection rate with PCR was significantly better than that with cultures in patients with antibiotic administration (χ2 = 18.3182; P = 0.0000). In conclusion, detection with real-time PCR is useful for rapidly identifying the causative pathogens of meningitis and for examining the clinical course of chemotherapy.

Capsular Type and Antibiotic Resistance in Streptococcus agalactiae Isolates from Patients, Ranging from Newborns to the Elderly, with Invasive Infections

ABSTRACT Streptococcus agalactiae isolates (n = 189) from patients with invasive infections were analyzed for capsular type by PCR, for antimicrobial susceptibility, and for the presence of resistance genes. In contrast to the predominance of capsular type III in children, types Ib and V were most common among adults. All 45 levofloxacin-resistant strains had two amino acid substitutions, Ser81Leu in the gyrA gene and Ser79Phe in the parC gene, and showed similar pulsed-field gel electrophoresis patterns.

Distribution of emm type and antibiotic susceptibility of group A streptococci causing invasive and noninvasive disease.

To determine the prevalence of macrolide antibiotic and levofloxacin resistance in infections with Streptococcus pyogenes (group A streptococcus or GAS), strains were collected from 45 medical institutions in various parts of Japan between October 2003 and September 2006. Four hundred and eighty-two strains from patients with GAS infections were characterized genetically. Strains were classified into four groups according to the type of infection: invasive infections (n=74) including sepsis, cellulitis and toxic-shock-like syndrome; acute otitis media (AOM; n=23); abscess (n=53); and pharyngotonsillitis (n=332). Among all strains, 32 emm types were identified; emm1 was significantly more common in invasive infections (39.2 %) and AOM (43.5 %) than in abscesses (3.8 %) or pharyngotonsillitis (10.2 %). emm12 and emm4 each accounted for 23.5 % of pharyngotonsillitis cases. Susceptibility of GAS strains to eight beta-lactam agents was excellent, with MICs of 0.0005-0.063 mug ml(-1). Macrolide-resistant strains accounted for 16.2 % of all strains, while the percentages of strains possessing the resistance genes erm(A), erm(B) and mef(A) were 2.5 %, 6.2 % and 7.5 %, respectively. Although no strains with high resistance to levofloxacin were found, strains with an MIC of 2-4 mug ml(-1) (17.4 %) had amino acid substitutions at either Ser-79 or Asp-83 in ParC. These levofloxacin-intermediately resistant strains included 16 emm types, but macrolide-resistant strains were more likely than others to represent certain emm types.

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.

Pre-exposure immunization against rabies using Japanese rabies vaccine following the WHO recommended schedule

We examined the efficacy and safety of the Japanese purified chick embryo cell rabies vaccine (PCEC-K) when administered on days 0, 7, and 28, as recommended by the WHO. Post-vaccination serum samples were obtained from 53 human subjects, and rabies antibody titers were determined by a combination of enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody (NA) assay. By day 42 of the experiment, which was 2xa0weeks after the third dose, all subjects had developed NA titers of 0.5xa0IU/ml or higher. The geometric mean titers of ELISA antibody and NA were 3.8xa0EU/ml and 5.7xa0IU/ml, respectively. Overall, the vaccine was well tolerated by all subjects. These results suggest that PCEC-K used for pre-exposure immunization according to the WHO schedule is as immunogenic and effective as the current pre-exposure immunization regimen in Japan, which consists of vaccines administered on days 0, 28, and 180. An accelerated schedule would be of great advantage to Japanese travelers, who could complete the required three doses for primary immunization in 1xa0month.

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.

Immunogenicity of intradermal vaccination of Japanese rabies vaccine for preexposure immunization following WHO recommendation

In the present study, we evaluated the immunogenicity of intradermal vaccination of Japanese purified chick embryo cell rabies vaccine (PCEC-K) for preexposure immunization (PEI). A total of 39 healthy subjects were administered a single 0.1-ml dose of PCEC-K intradermally at the antebrachial region on days 0, 7, and 28. To assess immunogenicity, rabies neutralizing antibody (NA) titers were measured on days 7, 28, and 42 post vaccination. By day 42, all subjects developed NA titers ≥0.5xa0IU/ml (geometric mean titer, 2.7xa0IU/ml), a level that is considered protective. The vaccine was well tolerated; vaccinated subjects displayed minimal redness and pruritus. Although a 1.0-ml dose of PCEC-K administered subcutaneously is considered the standard method, the intradermal regimen using a 0.1-ml dose of PCEC-K is immunogenic, safe, and highly recommended for situations of vaccine shortage.