Vancomycin-resistant enterococci (VRE) strains with the VanD phenotype and vanA genotype (VanD-vanA) have been reported in Asian countries. The VanD phenotype is characterized by low-level resistance to vancomycin and susceptibility or intermediate resistance to teicoplanin. We retrospectively determined the risk factors, clinical outcomes, and virulence factors for VanD-vanA VRE (20 patients colonized with Enterococcus faecium) compared to VanA-vanA VRE (20 patients colonized with E. faecium). Multiple VRE colonizations and recent glycopeptide use were related to the presence of the VanA phenotype. There were no significant differences between patients colonized with VanD-vanA VRE and VanA-vanA VRE for duration of hospital stay, duration of intensive care unit stay, or hospital mortality. The esp gene was identified from all enterococci, while 90% of VanD-vanA VRE isolates and 95% of VanA-vanA VRE isolates were positive for the hyl gene. VanA-vanA VRE was subsequently isolated from sequential samples in 8 of 20 patients (40%) with VanD-vanA VRE. All of these patients had received glycopeptides during the interval between sample collection, and 2 of 8 paired isolates (VanD-vanA VRE and VanA-vanA VRE) were closely related subtypes according to pulsed-field gel electrophoresis analysis. In conclusion, VanD-vanA VRE isolates might represent an unstable, heterogeneous population that can convert to the VanA phenotype after exposure to glycopeptides.

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Following the implementation of the national measles elimination program, the Republic of Korea declared in 2006 that measles had been eliminated. However in 2011, a measles outbreak was reported in Gyeongnam Province in the southeastern part of the country. We conducted active case-based surveillance and analyzed the data of cases reported in 2011 to identify the factors contributing to the reemergence of measles in this province. Of 41 confirmed measles cases reported in Korea, 32 were from within the Gyeongnam Province. Among cases identified in the outbreak, 97% had inadequate history of immunization, 28% were not immunized at the recommended ages, and 22% were infants aged 6–11 months. The outbreak involved transmission in 3 hospitals, 1 kindergarten, 1 day-care center, and 3 households. Molecular analysis of measles virus isolates from 11 cases revealed the same D9 genotype, which was the first to be discovered in Korea. In conclusion, inadequate immunization coverage, non-timely immunization, infants under 12 months of age, nosocomial transmission, and international importation may play important roles in the reemergence of measles in Korea during the attempted sustained elimination of the disease.

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Actinomyces naeslundii and Streptococcus gordonii are the predominant bacteria and initial colonizers of oral microflora. The binding of A. naeslundii and S. gordonii and the interaction between them on the salivary pellicle-coated tooth surface play an important role in the biofilm development. Recently, we reported that NOD/SCID.e2f1⁻ mice are a useful model for studying oral biofilm formation by Streptococcus mutans on the tooth surface. In this study, we aimed to determine whether NOD/SCID.e2f1⁻ mice can be used for studying oral colonization of A. naeslundii and S. gordonii. Colonization of A. naeslundii in mice fed with 1% sucrose water for 24 h before inoculation was higher than that among mice fed with sucrose water for 1 h. A. naeslundii colonization using mixed species-inoculation was lower than that using single-species inoculation 30–90 min after inoculation; however, the colonization was higher 120–180 min after inoculation. The mixed inoculation induced better colonization of S. gordonii than single-species inoculation 60–180 min after inoculation. Polyclonal and fluorescein isothiocyanate-labeled antibody stained bacteria showed better colonization of S. gordonii when a mixed culture is used in vivo. NOD/SCID.e2f1⁻ mice were useful for studying the initial colonization of A. naeslundii and S. gordonii. Long-term supply of sucrose water creates a favorable environment for the initial colonization of A. naeslundii that, in turn, supports the colonization of S. gordonii.

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A mass rabies vaccination of dogs has been conducted annually in Japan over the last 60 years. To assess both current levels of rabies virus-neutralizing antibody (VNA) in dogs and the rationale for current vaccination procedures, we used a rapid fluorescent focus inhibition test to determine VNA levels in 756 dogs that had visited animal hospitals in Japan. We found that 51.1% of the dogs that had received 1 rabies vaccination had protective VNA levels (≥0.5 IU/ml) with a geometric mean of 0.61 IU/ml. In contrast, 97.8% of the dogs that had been vaccinated at least twice had protective VNA levels with a geometric mean of 7.86 IU/ml. Furthermore, 97.9–100% of the dogs vaccinated at least twice retained protective VNA levels into the second year after the last vaccination. Although VNA levels in the dogs vaccinated at least twice tended to decline 2 years after the last vaccination, 78.9% retained protective VNA levels. Thus, the current rabies vaccination schedule provides adequate protection, but the registration system and vaccination schedule needs to be improved to ensure that increased numbers of dogs are vaccinated against rabies.

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The findings of this study revealed a 22% prevalence of Streptococcus pneumoniae among healthy children in Japan; the prevalence tended to increase with age. We identified attendance at day-care facilities and the presence of older siblings as the major risk factors associated with pneumococcal carriage. A significant decrease in pneumococcal colonization was observed among children during the summer; however, a seasonal variation was independent of various factors. Our findings suggest that prior immunization with heptavalent pneumococcal conjugate vaccine within the current recommended schedule is required for children exposed to these risk factors.

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To our knowledge, no one has conducted a multi-center trial evaluating the efficacy of antivenom and cepharanthine (CEP) for the treatment of mamushi (Gloydius blomhoffii) bites. Thus, we conducted a large-scale survey among tertiary care centers in Japan from November 2009 to October 2010 to evaluate the efficacy of antivenom and CEP for the treatment of mamushi bites. We divided the therapeutic interventions received by patients into 4 groups: CEP, antivenom, both CEP and antivenom, and neither CEP nor antivenom. We collected data on age, sex, comorbidities, laboratory measurements, length of hospital stay, and grades of mamushi bites (indication of bite severity ranged from I [mild] to V [severe]). We sent questionnaires to 219 tertiary care centers, of which 114 (52.1%) returned completed questionnaires. Two hundred and thirty-four cases of mamushi bites were reported. Among the severe cases (grades of mamushi bites III, IV, and V), patients administered antivenom had a significantly shorter length of hospital stay than those administered CEP (P = 0.024). In contrast, there was no significant difference in the length of hospital stay between mild cases (grades of mamushi bites I and II) (P = 0.77). Our results show that antivenom is effective in reducing the length of hospital stay in patients with severe mamushi bites.

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With the increase in global transportation, imported malaria has become a significant public health concern in Japan. In the present study, we retrospectively analyzed all imported malaria cases in Okinawa Prefecture from 1988 to 2012. In that period, 23 patients with imported malaria were admitted to the University of the Ryukyus Hospital. Malaria types observed included Plasmodium falciparum (14 cases), P. vivax (7 cases), combined P. falciparum and P. ovale (1 case), and combined P. vivax and P. malariae (1 case). All cases were resolved by anti-malarial treatment. The clinical data from these patients highlights the importance of collecting patient travel history and ensuring an adequate supply of both diagnostic test and drug treatments in Okinawa.

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We aimed at determining the characteristics of patients with primary and late latent syphilis who were non-reactive on initial screening by rapid plasma reagin (RPR) but reactive by treponemal tests. We collected the RPR test results of all primary and late latent syphilis patients in our hospital from December 2000 to March 2012. The characteristics of syphilis patients who were non-reactive by RPR testing were compared to those of reactive patients. Multiple logistic regression was used to identify factors associated with non-reactive RPR results. Among primary syphilis patients, 37 (16.5%) were non-reactive on initial RPR and were compared with the 187 reactive cases. Age >35 years was an independent factor associated with a non-reactive result in primary cases (odds ratio [OR], 95% confidence intervals [CI] = 3.55 [1.39–9.07]). Of the late latent patients, 61 (8.8%) were non-reactive by RPR and 636 were reactive. Age >34 years was also an independent factor associated with a non-reactive result in late latent cases (OR [95% CI] = 4.30 [2.28–8.12]). This study suggests that RPR testing alone is insufficient to diagnose primary and late latent infections, especially in middle-aged and elderly individuals. Syphilis detection was lower for patients with primary syphilis than for those with late latent syphilis based on the results of the RPR.

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Haemophilus influenzae type b vaccine conjugated with tetanus toxoid (HibT) was licensed for use in childhood immunization in Japan in 2007. As adsorbed diphtheria-tetanus-acellular pertussis (DTaP) combined with HibT vaccine has not been introduced in Japan, DTaP and HibT vaccines are injected at separate sites with a similar immunization schedule. There are various interfering or stimulatory effects between components of combined vaccines contained in DTaP and HibT vaccines. In this study, we investigated the effect of HibT containing combination vaccines on anti-tetanus potencies by using animal models (mouse, guinea pig, and rat). HibT vaccine and HibT components of imported DTaP-HibT vaccine alone showed comparable or higher anti-tetanus potency than DTaP vaccine and DTaP-containing components of combination vaccines. Mixing these components before injection resulted in potencies greater than the sum of individual potencies. Injecting individual components at separate sites in animals resulted in potency roughly equivalent to the sum of the individual potencies. These results provide useful information regarding the use of HibT-containing multivalent vaccines in childhood immunization.

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A mouse-human chimeric antibody that can neutralize botulinum neurotoxin serotype E (BoNT/E) was developed. Variable regions of heavy and light chains obtained using a mouse hybridoma clone (E9–4) cDNA, which was selected on the basis of neutralizing activity against BoNT/E, were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and a mouse-human chimeric antibody (EC94) was purified to examine binding and neutralizing activity against BoNT/E. EC94 exhibited the same levels of binding activities against BoNT/E as those of a parent mouse monoclonal antibody and neutralized more than 4,000 LD₅₀/mg antibody. This chimeric antibody seems to be a useful candidate for infant botulism in which the use of passive immunotherapy is not planned so as to avoid serious events such as anaphylactic shock. We designed shuffling chimeric antibodies with replacement of V_H or V_L of EC94 with that of a chimeric antibody (AC24) that possessed neutralizing activity against BoNT/A. These shuffling antibodies did not exhibit neutralizing activity against either BoNT/E or BoNT/A.

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Cryptococcus neoformans and Cryptococcus gattii are the causative agents of cryptococcosis. Despite its importance, our knowledge of the epidemiology of cryptococcosis in Japan remains limited. To establish an epidemiological database on cryptococcosis in Japan, we determined the genetic variability of 44 Japanese clinical isolates of C. neoformans (var. grubii: serotype A) by multilocus sequence typing (MLST). The strains were clinically isolated from 1992 to 2011 in 5 different areas of Japan (the Hokkaido region [n = 1], Kanto region [n = 32], Chubu region [n = 1], Kansai region [n = 1], and Kyushu region [n = 9]). According to the method recommended by the International Society for Human and Animal Mycology cryptococcal genotyping working group, 36 isolates (82%) were identified as sequence type (ST)46. The remaining strains belonged to ST45 (n = 1) and ST47 (n = 1), and 6 isolates belonged to novel independent STs. There was little geographic difference in the ST population. Our present data are still limited; however, because most clinical isolates showed the same MLST profile in Japan, applying the current MLST scheme for Cryptococcus may at times be insufficient for investigating the infection route among outbreak cases. To solve this problem, it may be necessary to investigate other gene loci or develop a novel method with greater discriminatory power. However, in cases in which a strain belongs to a minor ST, our data may serve as useful epidemiological information in Japan.

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To investigate the molecular epidemiology of human metapneumovirus (HMPV) infections in acute respiratory infections (ARI), we performed genetic analysis of the F gene in HMPV from patients with ARI in Fukui Prefecture from August 2005 to July 2011. HMPV was detected in 53 of 741 nasopharyngeal swabs (7.2%). Phylogenetic analysis helped us assign 31 strains to subgroup A2, 1 strain to subgroup B1, and 21 strains to subgroup B2. The prevalence of HMPV was peaked between January and June. A high degree of nucleotide identity was seen among subgroup A2 strains (95.6–100%) and subgroup B2 strains (97.5–100%). In addition, no positively selected sites (substitutions) were found in the F gene in these HMPV strains. The results suggest that the prevalent HMPV strains in Fukui were associated with various ARI in Japan during the investigation period.

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The ASAN Easy Test H. pylori is a newly developed rapid test for Helicobacter pylori that uses a stool antigen immunochromatographic assay (S-ICT). The aim of this study was to evaluate this kit for the diagnosis of H. pylori under various histological conditions. We enrolled 266 consecutive patients undergoing a routine health checkup and endoscopy with biopsy. H. pylori infection was confirmed if at least 2 out of the 3 following tests were positive: histology, real-time polymerase chain reaction (PCR), and the stool antigen test by enzyme immunoassay (S-EIA). Histological examination was performed using hematoxylin-eosin and silver staining. Real-time PCR was performed with a probe for the UreA gene as described previously. The S-EIA and the evaluated kit were used according to the manufacturers' instructions. Of the 266 patients, 209 were eligible for participation. The evaluation results were as follows: sensitivity, 84.5%; specificity, 96.2%; positive-predictive value, 95.6%; negative-predictive value, 86.4%; and accuracy, 90.4%. The performance of the kit was unaffected by histological findings such as atrophic gastritis, ulcers, and intestinal metaplasia. The newly developed S-ICT assay is a non-invasive rapid test for the diagnosis of H. pylori that exhibits good performance in routine health checkup patients.

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To improve the hemagglutinin (HA) antigen yield of influenza A(H1N1)pdm09 candidate vaccine viruses, we generated 7:1, 6:2, and 5:3 genetic reassortant viruses between wild-type (H1N1)pdm09 (A/California/7/2009) (Cal7) and a high-yielding master virus, A/Puerto Rico/8/34 (PR8). These viruses contained the HA; HA and neuraminidase (NA); and HA, NA, and M genes, respectively, derived from Cal7, on a PR8 backbone. The influence of the amino acid residue at position 223 in Cal7 HA on virus growth and HA antigen yield differed between these reassortant viruses. NIIDRG-7, a 7:1 virus possessing arginine at position 223, exhibited a 10-fold higher 50% egg infectious dose (EID₅₀) (10.0 log₁₀EID₅₀/ml) than the 5:3 and 6:2 viruses. It also had 1.5- to 3-fold higher protein (13.8 μg/ml of allantoic fluids) and HA antigen (4.1 μg/ml of allantoic fluids) yields than the 5:3 and 6:2 viruses, which possessed identical Cal7 HA proteins. However, the HA antigen yield of the other 7:1 virus, which possessed glutamine at position 223 was 60% of that of NIIDRG-7. In addition, a novel 6:2 virus possessing Cal7 HA and the NA of A/Wisconsin/10/98 (a triple reassortant swine-like H1N1 virus), produced 107% of the HA yield of NIIDRG-7. In this study, we showed that the balance between HA and NA in the influenza A(H1N1)pdm09 virus affects its protein and antigen yield.

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The objective of this study was to assess the distribution of Chlamydia trachomatis genovars in male sexually transmitted disease (STD) clinic patients from 1 province in eastern China (Jiangsu) and 3 provinces in southern China (Guangdong, Guangxi, and Hainan). Urine specimens (n = 140) were collected from 46 public STD clinics in the 4 provinces. Specimens that tested positive for C. trachomatis by polymerase chain reaction were genotyped for the infecting C. trachomatis strain by amplifying and sequencing the genovar-specific ompA gene, which encodes the chlamydial major outer-membrane protein. Nine C. trachomatis genovars were identified from 129 specimens; they consisted of the F (25.6%), E (17.1%), J (16.3%), D (15.5%), G (11.6%), K (5.4%), H (3.9%), I (1.6%), and B (0.8%) genovars. Nine genovars were found in specimens from Guangxi province, 6 were found in specimens from each of Guangdong and Hainan provinces, but only 5 were found in specimens from Jiangsu province. Genovar G/Ga was shown to be endemic among male patients in southern China.

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The aim of this study was to examine the link between Campylobacter jejuni isolates obtained from chicken meat (n = 7) and gastroenteritis patients (n = 744). In total, 751 isolates were subjected to Lior serotyping. All the isolates from chicken meats were serotyped as Lior serotype 76 (LIO76). Among 23 of the identified LIO76 strains, 13 strains (6 from chicken meat and 7 from clinical specimens) were indistinguishable by Penner serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis. These strains were isolated in 2 different Japanese prefectures in 2004–2005, suggesting that chicken meat is an etiological agent of Campylobacter gastroenteritis and that a diffuse outbreak occurred during this time. Therefore, a continuous surveillance program should be established in Japan in order to prevent Campylobacter gastroenteritis, especially large-scale food-borne outbreaks.

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