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Sapovirus (SaV) is one of the pathogens related to acute gastroenteritis (AGE) in adults and children worldwide. This study reported the diversity of SaV genotypes in children with AGE in Japan from July 2014 to June 2017. Of a total of 2259 stool samples tested by using reverse transcription-PCR method and further analyzed by nucleotide sequencing, 114 (5.0%) were positive for SaV and GI.1 (83.3%) was the most predominant genotype, followed by GII.1, GIV.1, GI.2, GI.3, and GII.3 genotypes. Monthly distribution analysis demonstrated two epidemic peaks from July to December 2015 and February to May 2017. However, no detection peak was observed in 2014 and 2016. Phylogenetic analysis of the complete VP1 nucleotide sequences of these GI.1 strains revealed two major clusters of GI.1 and each of which contained GI.1 strains of both 2015 and 2017. This study suggests that the continuous surveillance of SaV is needed to monitor high genetic diversity in Japanese children with AGE.

Introduction. Macrolides could be a potential alternative treatment for Treponema pallidum infections in patients; however, macrolide-resistant T. pallidum is spreading rapidly worldwide. Hypothesis/Gap Statement. There are presently no alternatives to serological tests for syphilis that can be used to evaluate therapeutic effects due to the fact that T. pallidum cannot be cultured in vitro. Aim. In this study, we constructed a method for rapidly identifying T. pallidum and confirming macrolide resistance by using loop-mediated isothermal amplification (LAMP) with peptide nucleic acids (PNAs). Methodology. A set of LAMP primers was designed to span nucleotide positions 2058 and 2059 in 23S rRNA. A PNA clamping probe was also designed to be complementary to the wild-type sequence (A2058/A2059) and positioned to interfere with both the annealing of the 3′ end of the backward inner primer and the concomitant extension. Prior to the LAMP assay, swab samples from suspected syphilitic lesions were boiled for DNA extraction. Results. The assay had an equivalent detection limit of 1.0×101 copies/reaction and showed specificity against 38 pathogens. In the presence of a 4 µM PNA probe, LAMP amplified up to 1.0×101 copies/reaction using plasmids harbouring the complementary mutant sequences (A2058G or A2059G), whereas amplification was completely blocked for the wild-type sequence up to a concentration of 1.0×103 copies/reaction. For the 66 PCR-positive clinical specimens, the overall detection rate via LAMP was 93.9 % (62/66). Amplification was successful for all 53 mutant samples and was incomplete for all nine WT samples by the PNA-mediated LAMP assays. Conclusion. We developed a PNA-mediated LAMP method that enabled us to rapidly identify T. pallidum and determine its macrolide susceptibility via a culture-independent protocol.

Background Enteric caliciviruses, including noroviruses (NoVs) and sapoviruses (SaVs) are the most significant pathogens associated with waterborne and foodborne outbreaks of nonbacterial acute gastroenteritis in humans worldwide. Methods In this study, 126 environmental water samples collected from 6 different sources in Chiang Mai, Thailand from November 2016 to July 2018 were examined for the presence of genogroups I, II, IV (GI, GII, GIV) NoVs and SaVs by using RT-nested PCR assays, genome sequencing, and phylogenetic analysis, Results Forty out of 126 (31.7%) water samples were positive for one or more caliciviruses throughout the years of study with high prevalence in winter. Among 126 tested specimens, 34 (27.0%), 30 (23.8%), 3 (2.4%), and 2 (1.6%) were positive for NoV GI, GII, GIV, and SaV, respectively. For NoV GI, 6 different genotypes were identified with the most predominant of GI.1 genotype (17 strains). In addition, 6 different genotypes of GII were detected with high prevalence of GII.17 (12 strains) and GII.2 (11 strains). It was interesting to note that our study reported the detection of NoV GIV for the first time in water samples in Thailand, and all were GIV.1 genotype. For SaV detection, only 2 water samples were positive for SaV GI. Conclusions The data revealed heterogeneity and highly dynamic distribution of NoV GI, GII, GIV, and SaV in environmental water in Chiang Mai, Thailand, during the study period of 2016–2018.

Species A rotaviruses are a major cause of acute gastroenteritis in infants and young children worldwide. Reassortment is a common phenomenon due to the segmented nature of the rotavirus genome. The complete coding sequences of a species A rotavirus strain isolated from the feces of a child with acute gastroenteritis in Japan in 2018 were determined using an unbiased viral metagenomics approach. The genetic analysis revealed that the rotavirus strain had an unusual genomic constellation (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1), suggesting reassortment of a genotype 1 with a genotype 2 rotavirus, from which the NSP4-encoding gene was acquired.

The threat of predation is a driving force in the evolution of animals. We have previously reported that Xenopus laevis enhanced their tail muscles and increased their swimming speeds in the presence of Japanese larval salamander predators. Herein, we investigated the induced gene expression changes in the brains of tadpoles under the threat of predation using 3′-tag digital gene expression profiling. We found that many muscle genes were expressed after 24 h of exposure to predation. Ingenuity pathway analysis further showed that after 24 h of a predation threat, various signal transduction genes were stimulated, such as those affecting the actin cytoskeleton and CREB pathways, and that these might increase microtubule dynamics, axonogenesis, cognition, and memory. To verify the increase in microtubule dynamics, DiI was inserted through the tadpole nostrils. Extension of the axons was clearly observed from the nostril to the diencephalon and was significantly increased (P ≤ 0.0001) after 24 h of exposure to predation, compared with that of the control. The dynamic changes in the signal transductions appeared to bring about new connections in the neural networks, as suggested by the microtubule dynamics. These connections may result in improved memory and cognition abilities, and subsequently increase survivability.

The roles of interleukin-22 (IL-22) in carcinogenesis have been proposed in various neoplasms. Increased expression of IL-22 has been observed in oral squamous cell carcinoma (OSCC) lesions as well as in other cancers. OSCC is still associated with poor prognosis and a high mortality rate because of its invasiveness and frequent lymph node metastasis. In the present study, we investigated the effects of IL-22 on OSCC cells. The human OSCC cell lines Ca9-22 and SAS were stimulated with IL-22 (1-10 ng/mL), and their migration abilities were examined using a cell scratch assay. A Matrigel invasion assay was performed to evaluate the invasion abilities of OSCC cells. Signal transducer and activator of transcription 3 (STAT3) phosphorylation, matrix metalloproteinase (MMP) and epithelial-mesenchymal transition (EMT)-related genes and proteins were also examined. IL-22 treatment promoted the migration and invasion abilities of OSCC cells without increasing their viability. IL-22 stimulation also induced STAT3 phosphorylation, MMP-9 activity and EMT-related genes and proteins. Our findings suggest that IL-22 has possible roles in the development of OSCC.

Background Diversity in group A rotavirus (RVA) strains after introduction of RV-vaccines remains an emerging concern worldwide. In this study, we investigated the prevalence and distribution of RVA genotypes in Japanese children with acute gastroenteritis (AGE) from 2015 to 2018. In addition, a comparison of the genotypes in pre-vaccination (2006–2012) and post-vaccination (2012–2018) periods was conducted to understand the impact of these vaccines on genotype distribution. Methods Fecal samples were collected regularly from outpatient clinics in six localities: Hokkaido, Tokyo, Shizuoka, Osaka, Kyoto, and Saga. RVA were screened and genotyped by RT-PCR and sequence-based genotyping. Results During the period 2015–2018, RVA was detected in 307 (19.7%) samples out of 1557 specimens: 29.9% (95% CI: 25.8% to 34.3%), 17.9% (95% CI: 14.7% to 21.5%), and 13% (95% CI: 10.3% to 16.0%) were detected RVA-positive in 2015–2016, 2016–2017 and 2017–2018, respectively. The average detection of RVA in pre-vaccination (2006–2012) and post-vaccination (2012–2018) era remained almost similar (18%-20%). The G2P[4]I2 (52.1%, 95% CI: 43.5%-60.6%) remained the most common genotype in 2015–2016, whereas G8P[8]I2 (55.9%, 95% CI: 45.2%-66.2%) dominated in 2016–2017. In 2017–2018, G9P[8]I2 (42.0%, 95% CI: 30.5%-53.9%) prevailed, followed by G9P[8]I1 (23.0%, 95% CI: 14.0%-34.2%). The detection rate of some common genotypes of pre-vaccination era like G1P[8] and G3P[8] has been reduced after introduction of RV-vaccine, whereas genotypes that were sporadic before the introduction of vaccines like G2P[4], G2P[8], G9P[8] and G8P[8] were emerged/reemerged in post-vaccination period. Conclusions Our study presented the diversity in circulating RVA genotypes in Japan before and after introduction of RV-vaccines. Sudden emergence of DS-1-like (I2) unusual strains in post-vaccination era remains alarming. Continuous monitoring of RVA genotypes is therefore indispensable to refine future vaccine strategy.

Despite efforts to develop effective treatments and vaccines, Mycobacterium tuberculosis (Mtb), particularly pulmonary Mtb, continues to provide major health challenges worldwide. To improve immunization against the persistent health challenge of Mtb infection, we have studied the CD8+ T cell response to Bacillus Calmette-Guerin (BCG) and recombinant BCG (rBCG) in mice. Here, we generated CD8+ T cells with an rBCG-based vaccine encoding the Ag85B protein of M. kansasii, termed rBCG-Mkan85B, followed by boosting with plasmid DNA expressing the Ag85B gene (DNA-Mkan85B). We identified two MHC-I (H2-Kd )-restricted epitopes that induce cross-reactive responses to Mtb and other related mycobacteria in both BALB/c (H2d ) and CB6F1 (H2b/d ) mice. The H2-Kd -restricted peptide epitopes elicited polyfunctional CD8+ T cell responses that were also highly cross-reactive with those of other proteins of the Ag85 complex. Tetramer staining indicated that the two H2-Kd -restricted epitopes elicit distinct CD8+ T cell populations, a result explained by the X-ray structure of the two peptide/H2-Kd complexes. These results suggest that rBCG-Mkan85B vector-based immunization and DNA-Mkan85B boost may enhance CD8+ T cell response to Mtb, and might help to overcome the limited effectiveness of the current BCG in eliciting tuberculosis immunity.

Current apheresis or blood purification practices depend primarily on polymer systems that enable filtering and adsorbing pathogenic substances in blood: the latter consist of base polymer-based carriers and active ligands. However, some ceramic materials, such as silica, titania, and hydroxyapatite, are known to be blood compatible. The research on their use for apheresis is very small in comparison to the polymer systems, but the ceramic-based materials have been steadily discussed until now. This article aims to provide some feasible ideas and strategies together with their microstructures for further apheresis applications. After blood clotting pathways and apheresis practice, several blood-compatible materials in the form of particles, coatings (layers), and macrospheres are introduced. In addition, the materials for stent applications are also mentioned because the highest blood compatibility is demanded. Examples of bilirubin, b2-microglobulin (B2M), bovine serum albumin (BSA), and lysozyme (LYZ) adsorption on titania gel particles, Zn-substituted hydroxyapatite, and so-gel derived silica macrospheres are presented.

Reports of invasive disease due to Streptococcus pneumoniae have declined since the introduction of pneumococcal conjugate vaccines (PCV7 and PCV13). The incidence of invasive diseases due to S. pneumoniae that are not addressed by the vaccines, however, has increased in children and adults, creating a global public health problem. Previously, we established the loop-mediated isothermal amplification (LAMP) method for a PCV13 serotype-specific assay. In the current study, we developed a rapid, simple, and cost-effective assay to detect serotypes in the 23-valent pneumococcal polysaccharide vaccine (PPSV23) using the LAMP method. In this study, LAMP primer sets for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F of S. pneumoniae were developed. The reactivity, specificity, and sensitivity of LAMP assays were determined and compared to those of conventional PCR. The feasibility of LAMP assays in clinical application in patients with invasive pneumococcal diseases was validated by defining the detection limit of the LAMP assay with bacterial genomic DNA-spiked blood specimens. The specificity of each LAMP assay was determined using 44 serotypes of pneumococcal strains. Their sensitivity was 100 copies per reaction versus 103 to 106 copies per reaction for PCR assays. Using DNA-spiked blood specimens, excluding the LAMP assay that targeted serotype 22F (103 copies per reaction), the limit of detection of the LAMP assay was similar to that with purified DNA as the template (102 copies per reaction), compared with 103 to >106 copies per reaction for PCR assays. In conclusion, a rapid and simple LAMP-based PPSV23-targeted serotype detection assay was developed for use in many countries. This study is the first report of a LAMP-based assay for identification of PPSV23 serotypes. Further evaluation of this assay is needed through surveillance and vaccine efficacy studies.

The microbiome in the female reproductive tract (FRT) plays a critical role in modulating the immune system. The mucosal epithelial barrier reliably distinguishes commensal symbionts from pathogens. However, disruption of the barrier causes dysbiosis, excessive inflammation, and infections, potentially resulting in reproductive complications. In this chapter, we provide an overview of the interaction between the epithelial barrier function and the microbiota, focusing on the lower FRT and Lactobacillus, a major dominant genus in the vagina. Information about evolution and probiotics is also provided to promote an in-depth understanding of the topic.

BACKGROUND Acute gastroenteritis is the most common cause of illness and death in infants and young children worldwide. Rotaviruses (RVs) are the major viruses that cause acute gastroenteritis in young children, especially in developing countries in Asia and Africa. METHODS The presence of rotavirus antigens in sera of four unvaccinated pediatric patients, aged between 4 and 6 years with severe diarrhea and dehydration, were detected by using three immunochromatographic (IC) kits. In addition, the presence of anti-rotavirus IgG, IgA, and IgM antibodies and their concentrations in patient sera were also determined by enzyme immunoassay (EIA). RESULTS All three kits could detect rotavirus antigen in patient sera with different intensity of the test lines. When patient sera were pretreated with anti-VP6 rotavirus mouse monoclonal antibody prior to testing, the rotavirus positive test lines disappeared, suggesting that all patient sera contained VP6 protein antigen of rotavirus. Assessment of antibody concentration in these patient sera revealed that all patient sera contained IgG, IgA, and IgM antibodies against rotavirus antigen at different concentrations. CONCLUSIONS The sensitivity of rotavirus protein detection in the patient sera of one IC kit brand was comparable to those of the EIA, suggesting this IC kit could be an alternative screening method for rapid diagnosis of rotavirus infection.

Species A rotaviruses (RVs) are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years. Currently available RV vaccines were adapted from wild-type RV strains by serial passage of cultured cells or by reassortment between human and animal RV strains. These traditional methods require large-scale screening and genotyping to obtain vaccine candidates. Reverse genetics is a tractable, rapid, and reproducible approach to generating recombinant RV vaccine candidates carrying any VP4 and VP7 genes that provide selected antigenicity. Here, we developed a vaccine platform by generating recombinant RVs carrying VP4 (P[4] and P[8]), VP7 (G1, G2, G3, G8, and G9), and/or VP6 genes cloned from human RV clinical samples using the simian RV SA11 strain (G3P[2]) as a backbone. Neutralization assays using monoclonal antibodies and murine antisera revealed that recombinant VP4 and VP7 monoreassortant viruses exhibited altered antigenicity. However, replication of VP4 monoreassortant viruses was severely impaired. Generation of recombinant RVs harboring a chimeric VP4 protein for SA11 and human RV gene components revealed that the VP8* fragment was responsible for efficient infectivity of recombinant RVs. Although this system must be improved because the yield of vaccine viruses directly affects vaccine manufacturing costs, reverse genetics requires less time than traditional methods and enables rapid production of safe and effective vaccine candidates. IMPORTANCE Although vaccines have reduced global RV-associated hospitalization and mortality over the past decade, the multisegmented genome of RVs allows reassortment of VP4 and VP7 genes from different RV species and strains. The evolutionary dynamics of novel RV genotypes and their constellations have led to great genomic and antigenic diversity. The reverse genetics system is a powerful tool for manipulating RV genes, thereby controlling viral antigenicity, growth capacity, and pathogenicity. Here, we generated recombinant simian RVs (strain SA11) carrying heterologous VP4 and VP7 genes cloned from clinical isolates and showed that VP4- or VP7-substituted chimeric viruses can be used for antigenic characterization of RV outer capsid proteins and as improved seed viruses for vaccine production.

Introduction Recent studies have shown that the endometrium possesses unique microbiomes, including Lactobacillus. However, the roles of these microbes are currently unknown, especially in placentation and the early stage of pregnancy. Methods The immortalized human first-trimester trophoblast cell line HTR-8/SVneo was cultured in the presence or absence of Lactobacillus crispatus. Invasive and migrative activities were directly evaluated using an optical microscope and a time-lapse imaging system. Protein levels of the invasion-related protein matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 were evaluated using ELISA. Results Matrigel invasion of HTR-8/SVneo cells was significantly increased by L. crispatus, though migration was not affected. The culture supernatant of L. crispatus also promoted invasion. Additionally, levels of the active forms of MMP-1 and MMP-2 in the cell culture medium were upregulated by L. crispatus treatment, but that of MMP-9 was not changed. Discussion L. crispatus promotes trophoblast invasion with an increase in MMP-1 and MMP-2 activation. Our results might explain why Lactobacillus dominance in the endometrium seems beneficial for implantation. Nevertheless, further research is required to determine whether the promotion of trophoblast invasion by L. cripatus is favorable for successful placentation at the early stage of pregnancy.

At the end of 2019, a new coronavirus disease, COVID‐19, emerged and quickly spread around the world. Severe acute respiratory syndrome Coronavirus 2 (SARS‐CoV‐2), the causative virus of this disease, belongs to the β‐coronavirus family, together with SARS and middle east respiratory syndrome, and has similar biological characteristics to these viruses. For obstetricians, the susceptibility and prognoses of pregnant women and the effects of the infection on the fetus have been the focus of attention; however, at present, the seriousness of the disease in pregnant women is not apparent, and COVID‐19 does not increase the rate of miscarriage, stillbirth, preterm labor or teratogenicity. Even so, carriers might transmit SARS‐CoV‐2 to pregnant women. Thus, we must keep in mind that all medical personnel must understand and maintain standard precautions in their clinical and laboratory practices.

Group A rotavirus is a leading cause of severe acute gastroenteritis worldwide. In this study, the first complete coding sequences of 11 RNA segments of human group A rotavirus G12P[8] in Japan were determined by an unbiased viral metagenomics. Its genomic constellation (VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes) was identified as G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. When performing the genetic analysis, we discovered an intergenotypic recombination event in the pig group A rotavirus G12P[8] strain BUW-14-A008. The novel recombination was found between two different genotypes G12 and G3 in the VP7 gene, and P[8] and P[13] in the VP4 gene.

Cow milk is a nourishing food containing numerous essential nutrients. In Japan, the consumption of cow milk is thought to enhance resistance to exhaustion-related diseases. Although several nutrients in cow milk, such as lactoferrin, are thought to modulate immune cells, the mechanisms remain unclear. Recently, the immunoregulatory functions of food-derived microRNAs or exosomes have been reported. Therefore, we studied the effects of exosomes derived from cow milk (CM-Exs) on immune cells in the present study. We obtained blood samples from healthy adult donors with the approval of the ethics committee. Peripheral blood mononuclear cells (PBMCs) were stimulated with CM-Exs in the absence or presence of interleukin-2 (IL-2) and IL-12. Cell surface markers and intracellular cytokine production were analysed by flow cytometry. CM-Ex stimulation enhanced the expression of CD69 on NK cells. Although CM-Ex stimulation alone did not induce interferon-γ (IFN-γ) production by NK cells or γδT cells, simultaneous stimulation with CM-Ex, IL-2 and IL-12 significantly enhanced IFN-γ production. In conclusion, cow milk consumption alone may not activate immune cells; however, CM-Exs could enhance immune cells under inflammatory conditions.

Introduction Recently, increased frequencies of carbapenemase-producing Enterobacteriaceae have been reported worldwide. Among multiple genetic subtypes, oxacillinase (OXA)-48 β-lactamase-producing strains have been associated with inbound infection because they have been detected predominantly in patients who traveled outside of Japan. However, a recent case report of OXA-48 β-lactamase-producing Enterobacteriaceae suggested the latent spread of domestic infections. Due to a lack of specific inhibitors, culture-based detection of OXA-48 β-lactamase-producing bacteria is difficult. Thus, DNA-based detection methods, including PCR, direct sequencing and loop-mediated isothermal amplification (LAMP), have been employed. Among these methods, LAMP detection is more favorable than other methods because of its technical simplicity and low cost. Methods We designed novel LAMP primers to detect OXA-48 β-lactamase-producing bacteria and investigated their possible clinical applications with bacterial genome-spiked human materials (cerebrospinal fluid, blood, feces, urine, and sputum). We evaluated the specificity of the LAMP primers using 37 bacterial strains: 8 standard, 9 reference, and 20 clinical Gram-negative strains. Results Our LAMP primers detected 10 copies of the OXA-48 type β-lactamase gene and exhibited no cross reactivity with other β-lactamase genes. Sensitivity was not influenced in any clinical sample, in contrast to PCR detection, which was strongly inhibited by substances in fecal samples. Conclusions These results suggest the superior performance of LAMP compared with conventional PCR for detecting the OXA-48 type β-lactamase gene in various clinical samples.

Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS (S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis, S. peroris, S. infantis, S. australis, S. parasanguinis, S. sinensis, S. sanguinis, S. gordonii, and S. cristatus) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner "What's in My Pot?" (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes (n = 514), clinical isolates (n = 31), and reference strains (n = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates (S. pneumoniae = 8, S. mitis = 17 and S. oralis = 6) and 4 reference strains (S. pneumoniae = 1, S. mitis = 1, S. oralis = 1, and S. pseudopneumoniae = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except S. infantis, S. australis, S. peroris, and S. sinensis. The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys.

One-year surveillance for enteric viruses in raw sewage was conducted in Kansai area, central part of Japan from July 2015 to June 2016. The raw sewage was collected monthly from an inlet polluted pool and was concentrated by polyethylene glycol (PEG) precipitation. Twelve sewage samples were screened for nineteen kinds of enteric viruses by using RT-PCR method and further analyzed by nucleotide sequencing. Twelve enteric viruses were found in the investigative sewage samples. Rotavirus A and norovirus GI and GII with several genotypes were detected all year round. Interestingly, norovirus GII.17 (Kawasaki-like strain) and rotavirus G2 that caused the outbreaks in Japan last epidemic season were also found in sewage. Moreover, adenovirus, astrovirus, sapovirus, bocavirus, human parechovirus, enterovirus, Aichi virus, Saffold virus and salivirus were also detected. Enterovirus D68 was detected only in the same month as those of enterovirus D68 outbreak in Japan. The rotavirus B and C, hepatitis A and E viruses, human cosavirus, bufavirus and rosavirus were not detected in this surveillance. The study provides the information on the enteric viruses contaminated in raw sewage, which is valuable for risk assessment. Our results imply that the viruses detected in sewage may be associated with infections in the Japanese population.

In children, the incidence of pneumococcal meningitis has decreased since the introduction of pneumococcal conjugate vaccine (PCV7 and PCV13). However, since the introduction of the vaccine, developed countries have seen the emergence of non-PCV13 serotypes. However, invasive pneumococcal disease (IPD) caused by PCV13-targeted serotypes still represents an important public health problem in resource-limited countries. To develop a rapid, simple, and cost-effective assay to detect serotypes of Streptococcus pneumoniae, we developed a novel loop-mediated isothermal amplification (LAMP) assay based on the sequences available for the 13 capsular types that are included in PCV13: 1, 3, 4, 5, 6 A, 6B, 7 F, 9 V, 14, 18 C, 19 A, 19 F, and 23 F. We evaluated test reactivity, specificity, sensitivity and performance, and compared the results between established LAMP and conventional PCR assays. To support its clinical use, the detection limits of the LAMP assay were evaluated using bacterial genomic DNA-spiked cerebrospinal fluid (CSF) and blood specimens. We confirmed the specificity of the LAMP assay using 41 serotypes of pneumococcal strains. The sensitivity of the LAMP assay was 10 to 100 copies per reaction, compared to 10 to 104 copies per reaction for PCR assays. The detection limits of the LAMP assay were comparable when using DNA-spiked CSF and blood specimens, as compared to using purified DNA as the template. In conclusion, a rapid and simple LAMP-based pneumococcal serotyping method has been developed. This is the first report of a LAMP method for a PCV13 serotype-specific identification assay, which could be a promising step to facilitate epidemiological studies of pneumococcal serotyping.

Some outbreaks caused by ebolaviruses have been associated with wildlife mortalities in the past. Here, we discuss the possible roles played by animal carcasses during an ebolavirus outbreak. Corpses of wild animals that died due to ebolavirus infection or other reasons might be eaten by vertebrates and invertebrates, spreading live ebolaviruses to other animals, including humans. To prevent and contain an ebolavirus outbreak, not only potential reservoirs but also all organisms with a high likelihood of virus exposure need to be investigated.

This study describes the detection and molecular characterization of two rare G8P[14] and G3P[3] rotavirus strains, which were collected from children with acute gastroenteritis in 2014 in Japan. Among 247 rotaviruses, one G8P[14] (strain 12,597) and one G3P[3] (strain 12,638) rotaviruses were detected. The genotypes of 11 gene segments of these two rotavirus strains (RVA/Human-wt/JPN/12597/2014/G8P[14] and RVA/Human-wt/JPN/12638/2014/G3P[3]) were characterized. The genotype constellation of strain 12,597 was assigned to G8-P[14]-I2-R2-C2-M2-A3-N2-T9-E2-H3, and this strain possessed a rare T9 genotype of NSP3 gene which has never been reported previously in combination with G8 genotype of VP7 gene. Molecular characterization and phylogenetic analysis suggested that the strain 12,597 had the consensus G8P[14] backbone that originated from the rotaviruses of animal origins such as cows, deer, dogs, and cats. The genotype constellation of strain 12,638 was identified as G3-P[3]-I3-R3-C3-M3-A9-N2-T3-E3-H6. The VP7 and VP4 genotypes of strain 12,638 was similar to those of the Cat97-like strains, but the VP1, VP2, and VP3 were closely related to those of the AU-1-like strain. Interestingly, the NSP1 to NSP3 genes shared highest identities with those of a bat rotavirus (RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3] strain). These findings indicated that the strain 12,638 was an intra-genotype reassortant strain among the AU-1-like strains, the Cat97-like strains and the bat strain. Interestingly, the strains 12,597 and 12,638 possessed the same N2 genotype of NSP2 gene. The results of this study support the possible roles of interspecies transmission and multiple reassortment events for generating the genetic diversity of rotavirus in human.

Rotavirus vaccines have been available in Japan since 2011. This study conducted to monitor the trend of group A rotavirus (RVA) genotypes 3 years after vaccine introduction. A total of, 539 fecal samples were collected from children with acute gastroenteritis in six regions during July 2014-June 2015. Among them, 178 samples (33.0%) were positive for RVA. The most predominant genotype was G1P[8] (35.9%) followed by G2P[4] (26.4%), G9P[8] (21.3%), G3P[8] (4.5%), and G3P[9] (4.5%). The detection rate of G2P[4] was increased soon after vaccine introduction. Sequence analyses of VP7 and VP4 genes of the representative G2P[4] strains were found to be clustered in sub-lineage IVa of lineage IV. It is noteworthy that one amino acid substitution in the antigenic epitope (Q114P) of VP4 gene was found in representative G2P[4] strains of the current study. However, it is unclear whether the change in antigenic epitope is due to the effect of vaccination or due to natural variation, warranting further continuous monitoring of rotavirus evolution after vaccine introduction.

The incidence of infections with nontuberculous mycobacteria (NTM) has been increasing worldwide. The emergence of multidrug-resistant NTM is a serious clinical concern, and a vaccine for NTM has not yet been developed. We previously developed a new recombinant Bacillus Calmette–Guérin (rBCG) vaccine encoding the antigen 85B (Ag85B) protein of Mycobacterium kansasii—termed rBCG-Mkan85B—which was used together with a booster immunization with plasmid DNA expressing the same M. kansasii Ag85B gene (DNA-Mkan85B). We reported that rBCG-Mkan85B/DNA-Mkan85B prime–boost immunization elicited various NTM strain-specific CD4+ and CD8+ T cells and induced Mycobacterium&nbsp, tuberculosis-specific immunity. In this study, to investigate the protective effect against M. kansasii infection, we challenged mice vaccinated with a rBCG-Mkan85B or rBCG-Mkan85B/DNA-Mkan85B prime–boost strategy with virulent M. kansasii. Although BCG and rBCG-Mkan85B immunization each suppressed the growth of M. kansasii in the mouse lungs, the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination reduced the bacterial burden more significantly. Moreover, the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination induced antigen-specific CD4+ and CD8+ T cells. Our data suggest that rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination effectively enhances antigen-specific T cells. Our novel rBCG could be a potential alternative to clinical BCG for preventing various NTM infections.

Parechovirus A (PeV-A), previously known as human parechovirus, is a common pathogen in children that can cause respiratory and gastrointestinal diseases as well as severe neurological disease. Take advantage of our previous findings on the genetic diversity of PeV-A circulating in Japanese children with acute gastroenteritis (AGE), this study was conducted to investigate the genetic diversity of PeV-A isolated from children with AGE in Japan as well as their clinical symptoms. Of 1070 stool samples collected from Japanese infants and children with AGE during the 2-year period from July 2016 to June 2018, 76 were positive for PeV-A by multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) and were subjected to genotyping based on viral protein 1 (VP1) sequences. Five different PeV-A genotypes including PeV-A1B, −A2, −A3, −A4, and -A6 were detected with predominant of PeV-A1 clade B genotype. This study revealed a high genetic diversity of PeV-A circulating in Japanese infants and children with AGE and the PeV-A2, a rare genotype, was detected for the first time in Japan in patients with AGE. The clinical symptoms observed in these patients included diarrhea, vomiting, fever, cough, rhinorrhea, and dehydration.

One prominent cause of implant failure is infection; therefore, research is focusing on developing surface coatings that render the surface resistant to colonization by micro-organisms. Permanently attached coatings of antimicrobial molecules are of particular interest because of the reduced cytoxicity and lower risk of developing resistance compared to controlled release coatings. In this study, we focus on the chemical grafting of bioactive molecules on titanium. To concentrate the molecules at the metallic implant surface, we propose electrophoretic deposition (EPD) applying alternating current (AC) signals with an asymmetrical wave shape. We show that for the model molecule bovine serum albumin (BSA), as well as for the clinically relevant antifungal lipopeptide caspofungin (CASP), the deposition yield is drastically improved by superimposing a DC offset in the direction of the high-amplitude peak of the AC signal. Additionally, in order to produce immobilized CASP coatings, this experimental AC/DC-EPD method is combined with an established surface activation protocol. Principle component analysis (PCA) of time-of-flight secondary ion mass spectrometry (ToF-SIMS) data confirm the immobilization of CASP with higher yield as compared to a diffusion-controlled process, and higher purity than the clinical CASP starting suspensions. Scratch testing data indicate good coating adhesion. Importantly, the coatings remain active against the fungal pathogen C. albicans as shown by in vitro biofilm experiments. In summary, this paper delivers a proof-of-concept for the application of AC-EPD as a fast grafting tool for antimicrobial molecules without compromising their activities.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2018 there were nine themed workshops, four of which are summarized in this report. These workshops discussed new knowledge and technological innovations in the following areas of research: 1) viviparity in ocean-living species; 2) placental imaging; 3) epigenetics; and 4) extracellular vesicles in pregnancy.

Infections caused by multidrug-resistant Pseudomonas aeruginosa in hospitalized patients are often fatal, and nosocomial infections caused by Guiana extended-spectrum (GES) β-lactamase-producing strains are of growing concern. Several genotypes of the GES β-lactamase gene (blaGES) include a single missense mutation, a change from G to A at nucleotide position 493 (G493A) that changes glycine to serine; the mutant enzyme exhibits carbapenemase activity. Rapid and reliable identification of drug-resistance is important in clinical settings; however, culture methods remain the gold standard. Conventional and real-time PCR cannot identify carbapenemase-producing genotypes, and direct DNA sequencing is essential. We established a novel loop-mediated isothermal amplification (LAMP) method to detect various genotypes of blaGES and another LAMP method to discriminate carbapenemase genotypes of blaGES. We evaluated the two assays using clinical P. aeruginosa strains. Two primer sets targeting blaGES (GES-LAMP) and the point mutation (Carba-GES-LAMP) were designed and evaluated for specificity and sensitivity. The detection limit of the GES-LAMP method was assessed using purified DNA and DNA-spiked clinical samples (urine, sputum, and blood). To determine the clinical usefulness of the methods, we used different (genotypically and phenotypically) P. aeruginosa clinical isolates, collected from diverse geographical locations between 2003 and 2012. The novel LAMP assay targeting blaGES was highly specific. The detection limit was 10 DNA copies per reaction; the assay was 10-fold more sensitive than conventional PCR. The LAMP assay detected blaGES with high sensitivity in all DNA-spiked samples; PCR did not detect blaGES in blood samples. The GES-LAMP method correctly detected the 5 isolates containing blaGES among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two blaGES positive organisms with carbapenemase activity. To the best of our knowledge, this is the first report of the GES β-lactamase gene detection assay using the LAMP method. Our new assays effectively detect blaGES and critical unique mutations.

Multiplex RT-PCR method using five sets of panel primers was developed for the detection of diarrheal viruses, including rotavirus A, B, and C, adenovirus, astrovirus, norovirus GI and GII, sapovirus, Aichi virus, parechovirus, enterovirus, cosavirus, bocavirus, and Saffold virus. The sensitivity of the method was evaluated and tested with 751 fecal specimens collected from Japanese children with acute diarrhea. Several kinds of viruses were detected in 528 out of 751 (70.3%) fecal specimens. Mixed-infection with different viruses in clinical specimens could also be effectively detected. The method proved to be reliable with highly sensitive and specific and useful for routine diagnosis. J. Med. Virol. 89:818-824, 2017. © 2016 Wiley Periodicals, Inc.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that diminishes tissue destruction during inflammation. A recent report revealed high levels of SLPI expression in the oral carcinoma cell. In addition, overexpression of SLPI up-regulates metastasis in lung carcinoma cells. On the other hand, matrix metalloproteinases (MMPs) are proteinases that participate in extracellular matrix degradation. SLPI and MMPs are involved as accelerators of the tumor invasion process; however, their exact roles are not fully understood. Understanding the mechanism of tumor invasion requires models that take the effect of microenvironmental factors into account. In one such in vitro model, different carcinoma cells have been shown to invade myoma tissue in highly distinct patterns. We have used this myoma model, as it provides a more natural stroma-like environment, to investigate the role of SLPI in tumor invasion. Our results indicate that the model provides a relevant matrix for tumor invasion studies, and that SLPI is important for the invasion of oral carcinoma Ca9-22 cells in conjunction with MMPs. Furthermore, using bioinformatics analysis, we have identified candidates as key molecules involved in SLPI-mediated tumor invasion.

A number of molecular epidemiological studies reported the detection of enteric viruses in asymptomatic children. The role of these viruses in an asymptomatic infection remains unclear. This study investigated the enteric viruses in the stool samples collected from children without diarrhea. Stool samples were collected during June to October 2016, from 227 children who lived in Matlab, Bangladesh. Seventeen enteric viruses, including rotavirus A, B, and C (RVA, RVB, and RVC), norovirus GI (NoV GI), norovirus GII (NoV GII), sapovirus (SaV), adenovirus (AdV), human astrovirus (HAstV), Aichivirus (AiV), human parechovirus (HPeV), enterovirus (EV), human bocavirus (HBoV), Saffold virus (SAFV), human cosavirus (HCoSV), bufavirus (BufV), salivirus (SalV), and rosavirus (RoV), were investigated by RT-PCR method. One hundred and eighty-two (80.2%; 182/227) samples were positive for some of these viruses, and 19.8% (45/227) were negative. Among the positive samples, 46.7% (85/182) were a single infection, and 53.3% (97/182) were coinfection with multiple viruses. The HCoSV was the most prevalent virus (41.4%), followed by EV (32.2%), NoV GII (25.6%), HPeV (8.8%), RVA (6.2%), AdV (5.7%), AiV (5.3%), SAFV (4.4%), and SaV (2.6%). Each of NoV GI, HAstV, HBoV, and BufV was detected at 0.4%. However, RVB, RVC, SalV, and RoV were not detected in this study. Phylogenetic analysis showed that diverse HCoSV species and genotypes were circulating in Bangladesh, and four strains of species A are proposed to be new genotypes. The data indicated that non-diarrheal Bangladeshi children were asymptomatically infected with wide varieties of enteric viruses.

AIMS To investigate the presence of noroviruses in recycled water and sewage sludge obtained from a wastewater treatment plant in Bangkok Metropolitan Region, Thailand. METHODS AND RESULTS Twenty-seven recycled water and twenty-three sewage sludge samples were tested for the presence of norovirus genogroup (G)I and GII using RT-nested PCR. Molecular characterization of noroviruses was undertaken by DNA sequencing and phylogenetic analysis. The level of the RNA genome of the noroviruses was determined using quantitative real-time RT-PCR. Noroviruses were detected in 44·4% of recycled water samples and 73·9% of sewage sludge samples. Norovirus GI.2 and GII.4 were identified in recycled water samples at levels of 2·19 × 101 and 3·26 × 104 RNA copies per litre. Six different genotypes of GI (GI.1, GI.2, GI.5a, GI.5b, GI.6b and GI.7) and GII.17 were identified in sewage sludge samples at levels ranging from 1·99 × 101 -1·43 × 105 RNA copies per gram wet weight. Four recombinant norovirus strains were detected in sewage sludge samples, namely GII.P16-GII.2, GII.P16-GII.4, GII.P16-GII.13 and GII.P21-GII.13. CONCLUSIONS These findings provide evidence that noroviruses may be spread to the community and environment via the use of recycled water for plant areas, and sewage sludge for land application. SIGNIFICANCE AND IMPACT OF THE STUDY This is the first study demonstrating recombinant norovirus strains in sewage sludge samples. The presence of noroviruses in recycled water and sewage sludge contributes to a health risk of environmental exposure.

Problem The functions of vaginal lactobacilli in susceptibility to infectious diseases as regards epithelial barrier integrity and wound healing remain incompletely understood. Method of study Lactobacillus crispatus, one of the most common Lactobacillus species in the vagina and among the most protective against sexually transmitted infections, was cocultured with an immortalized human vaginal epithelial cell line (MS74), and a scratch assay was performed to evaluate re-epithelialization. The concentration of vascular endothelial growth factor A (VEGF) was measured using enzyme-linked immunosorbent assay (ELISA). An immunofluorescence assay was performed to locate the expression of VEGF and VEGF receptor (VEGFR) 1 and 2. The effects of the bacterial supernatant of L. crispatus were also evaluated. Results Lactobacillus crispatus significantly accelerated re-epithelialization of MS74 cells, accompanied by an increase in VEGF concentration. In contrast, heat-killed L. crispatus did not show this effect. The bacterial supernatant of L. crispatus also induced re-epithelialization. The immunoreactivity of VEGF was higher at the scratched edge, whereas VEGFR1 and 2 stained site-independently. Recombinant VEGF induced cell migration in a dose-dependent manner. The bacterial supernatant of L. crispatus also significantly accelerated re-epithelialization in MS74 cells and increased the concentration of VEGF in the culture 24 hours after the scratch. Conclusion These results may enhance our knowledge of the importance of L. crispatus in the healing of damaged vaginal epithelium and protection against the consequent risk of pathogenic infections, such as human immunodeficiency virus (HIV), and improve our understanding of vaginal epithelial barrier integrity maintenance by this bacterium.

Global burden of acute viral gastroenteritis remains high, particularly in developing countries including Bangladesh. Sewage water (SW) is an important node to monitor enteric pathogens both in the environment and among the population. Analysis of SW in Dhaka city deems crucially important because a large number of urban-city dwellers live in Dhaka city, the capital of Bangladesh, under a constant threat of precarious sewerage system. In this study, we collected raw SW from five locations of Dhaka city every month from June 2016 to May 2017. It was concentrated with polyethylene glycol (PEG) and investigated for three major enteric viruses, rotavirus A (RVA), norovirus GII (NoV GII) and adenovirus (AdV) using polymerase chain reaction (PCR). Most of these SW samples collected from both hospitals and non-hospital areas yielded enteric viruses: 76% samples were positive for AdV, followed by 53% NoV GII and 38% RVA. Viral load was determined as much as 1 × 107 copies/ml for RVA and 3.5 × 103 copies/ml for NoV GII. Importantly, NoV GII and AdV that can affect people of all ages were predominated during monsoon also when SW overflows and spreads over a wide and crowded area. Genotypes G1, G2, G3, G8, and G9 for RVA, GII.4 for NoV, and type 41 for AdV were detected representing the current profile of circulating genotypes in the population. This study provides the first evidence of distribution of major diarrheal viruses in SW in Dhaka city which is alarming showing grave risk of impending outbreaks through exposure.

Background Helicobacter pylori eradication is imperative to prevent gastric cancer before the development of gastric atrophy and/or intestinal metaplasia. However, there is no clear consensus on the optimal age for H. pylori eradication, and changes in the gut microbiota after eradication and their potential future impacts remain unknown. Our aim was to assess gut microbiome changes after H. pylori eradication in children. Methods Changes in the gut microbiota before and after H. pylori eradication was prospectively investigated in 8 students without any underlying diseases by 16 s rRNA genes using next-generation sequencing. A total of 24 stool samples were collected, and operational taxonomic unit analysis was performed. As secondary analyses, alpha and beta diversity were evaluated. Furthermore, pre-treatment structures in the microbiome were compared with structures 1 week and 2 months after treatment. Results Alpha diversity analysis showed that both richness and evenness were recovered to pre-treatment levels at 2 months after eradication therapy. There were almost no differences in bacterial species abundance between pre- and post-treatment samples in beta diversity analysis. Although the relative abundance of Bacteroidetes tended to increase and Actinobacteria significantly decreased immediately after eradication, the taxonomic composition was similar to that pre-eradication at 2 months post-eradication. However, 2 students showed significant structural changes in their relative abundances at the phylum level. Conclusions This study suggests a limited impact of H. pylori eradication therapy on the gut microbiome in healthy mid-teenagers. However, we should continuously monitor the effects of changes to the gut microbiome caused by antibiotic use.

Objectives The effect of Helicobacter pylori eradication on the gut microbiota of teenagers is unknown; hence, this study aimed to assess changes in the gut microbiome after H. pylori eradication therapy in teenagers. Materials and methods Changes in gut microbiota before and after H. pylori eradication were prospectively investigated in eight students without any underlying diseases, via next-generation sequencing of 16S rDNA. Twenty-four stool samples were collected, and operational taxonomic unit analysis was performed. As secondary analyses, alpha and beta diversity were evaluated. Furthermore, pre-treatment microbiome compositions were compared with those 1 week and 2 months after treatment. Results Alpha diversity analysis revealed that both species richness and evenness were recovered to pre-treatment levels at 2 months after eradication therapy. Slight but non-significant differences were observed in bacterial species abundance between pre- and post-treatment samples, upon beta diversity analysis. Although the relative abundance of Bacteroidetes tended to increase and that of Actinobacteria significantly decreased immediately after eradication therapy, the taxonomic composition was similar to that before treatment and at 2 months post-eradication. However, two students showed significant changes in the gut microbiota in relative abundances at the level of the phylum, class, and order. Conclusions Although H. pylori eradication therapy caused short-term dysbiosis, microbial diversity was restored in healthy teenagers. However, as the relative abundance of gut microbiota in some cases remained altered, the effect of H. pylori eradication therapy on the gut microbiome should be continuously monitored.

Rubella virus (RuV), which belongs to the family Togaviridae and genus Rubivirus, causes systemic infection in children and young adults and congenital rubella syndrome in developing fetuses if the infection occurs during pregnancy. The mechanisms of fetal infection by RuV are not completely understood. Myelin oligodendrocyte glycoprotein (MOG) is reported to be a cellular receptor for RuV; however, it is mainly expressed in the central nervous system. Therefore, it is thought that other receptors are also responsible for virus entry into susceptible cells. In this study, we found that first-trimester trophoblast cells were resistant to RuV. In addition, we showed that HaCaT cells (an immortalized keratinocyte cell line) that did not express MOG on their surface were infected with RuV. This finding is one of the first demonstrations of MOG-independent RuV infection of susceptible host cells and suggests that it is important to continue searching for alternative RuV receptors. In addition, this study reports the resistance of first-trimester trophoblast cells to RuV and suggests that utilizing an epithelial–mesenchymal transition approach to study the mechanisms of transplacental vertical RuV infection.

Acute gastroenteritis has been recognized as one of the most common diseases in humans and continues to be a major public health problem worldwide. Several groups of viruses have been reported as the causative agents of acute gastroenteritis, including rotavirus, norovirus, sapovirus, human astrovirus, adenovirus, and an increasing number of others which have been reported more recently. The epidemiology, prevalence, seasonality, and outbreaks of these viruses have been reviewed in a number of studies conducted in Japan over three decades. Rotavirus and norovirus were the two most common viruses detected almost equally in children under 5 years of age who were suffering from acute gastroenteritis. Like many other countries, the main rotavirus strains circulating in pediatric patients in Japan are G1P[8], G2P[4], G3P[8], and G9P[8]. Norovirus GII.4 was involved in most outbreaks in Japan and found to be associated with the emergence of new variants Sydney_2012. The classic human astrovirus, MLB, and VA clades astroviruses were also commonly found in pediatric patients with acute diarrhea. The sapovirus and adenovirus have been identified as the minor viral causative agents for acute gastroenteritis in Japan.

This study investigated the genetic variability of astroviruses in children with acute gastroenteritis in Japan in 2012 to 2013. A total of 330 fecal specimens collected from children with diarrhea were tested for astroviruses using RT-PCR and nucleotide sequencing techniques. Among the tested specimens, 54 (16.4%) were positive for astrovirus. Three different phylogenetic clades were detected. MLB was the most predominant clade (35 samples), followed by classic astrovirus (17 samples), and VA astrovirus (2 samples). Two types of MLB were identified (MLB1 and MLB2), while classic astroviruses were HAstV1 and HAstV4. Two VA astroviruses were identified as VA2.

Acute gastroenteritis continues to be a major public health problem worldwide. A wide variety of viruses associated with diarrhea disease is being reported continually. This study investigated the epidemiological situation of viruses that cause diarrhea in Japanese pediatric patients. This study enrolled a total of 2,381 fecal specimens collected between 2009 and 2013 from Japanese children with acute gastroenteritis. There is currently a 70.4% prevalence of viruses causing diarrhea among these Japanese pediatric outpatients. Norovirus was detected in 39.3% of the patients, whereas the prevalence of rotavirus, human parechovirus, enterovirus, adenovirus, sapovirus, astrovirus, and Aichi virus was 20.1, 6.6, 6.1, 5.6, 4.8, 2.3, and 0.1%, respectively. Co-infections were observed at the prevalence rates of 13.4 and 0.5% for double infections and triple infections, respectively. Mixed viral infections were found commonly in Japanese outpatients, and the norovirus seemed to play a major role in co-infections. Viral diarrhea cases were detected mostly in children younger than 3 years of age. The norovirus and rotavirus can be detected throughout the year, with a peak during the cold and dry seasons, whereas other common viruses are found during no specific season. Surveillance data revealed that a wide variety of viruses has caused diarrhea to circulate currently in Japanese pediatric outpatients, with very high detection rates; and norovirus and rotavirus are the most important pathogens. The data obtained from this study are valuable for compiling the overall picture of several viruses that causes diarrhea and associates with acute gastroenteritis in the Japanese pediatric population.

Genetic variability makes influenza virus to escape the immunity and causes yearly epidemics. Monitoring those changes is necessary for vaccine selection. In addition, H3N2 viruses were considered to be seeded from Southeast Asia before spreading globally. This study described the molecular epidemiology of influenza A during the post-pandemic season 2010-2011 in Vietnam. Nasopharyngeal samples were collected from children with respiratory infections at Children's Hospital 2, Ho Chi Minh City. The HA, NA, M genes were amplified, sequenced and analyzed. Thirty-five of 1,082 (3.2%) patients were positive for influenza A, including 14 pandemic H1N1 2009 (H1N1pdm09) and 21 H3N2 infections. H3N2 was dominant in the rainy season (May-October 2010) while H1N1pdm09 was dominant in the dry season (November 2010-April 2011). Phylogenetic analysis showed that Vietnamese H1N1pdm09 sequences in 2010-2011 formed the distinct cluster, with other contemporary Asian and 2012-American sequences, suggesting a possible common ancestor. All were oseltamivir-sensitive except two strains carrying S247N and D199N in NA which reduced the neuraminidase inhibitor susceptibility. The Vietnamese H3N2 viruses in mid-2010 belonged to the emerging subclade Perth10/2010, which then spread worldwide in 2011. The Vietnamese influenza viruses were well matched with the Southern Hemisphere vaccine formulation. Mutations at antigenic sites were also identified in these viruses. Surveillance of influenza viruses in tropical countries is important not only for development of their prevention and control strategies but also for earlier identification of the newly emerged strains that may be selected for future vaccine.

Since human norovirus is non-cultivable, murine norovirus and feline calicivirus have been used as surrogates. In this study, the virucidal effects of ethanol-based sanitizers with different concentrations of additives (malic acid/sodium malate, glycerin-fatty acid ester) against murine norovirus and feline calicivirus F4 were examined. The ethanol-based sanitizers at pH 7 showed sufficient virucidal effects, but glycerin-fatty acid ester included in ethanol-based sanitizers at pH 4 or 6 reduced the virucidal effects against murine norovirus. The ethanol-based sanitizers containing malic acid/sodium malate inactivated feline calicivirus F4 in shorter time, but there is no difference between ethanol-based sanitizers with and without glycerin-fatty acid ester. Traditionally, feline calicivirus has been used for long time as a surrogate virus for human norovirus. However, this study suggested that murine norovirus and feline calicivirus F4 had different sensitivity with the additive components of ethanol-based sanitizers. Therefore, using feline calicivirus alone as a surrogate for human norovirus may not be sufficient to evaluate the virucidal effect of sanitizers on food-borne infections caused by human norovirus. Sanitizers having virucidal effects against at least both murine norovirus and feline calicivirus may be more suitable to inactivate human norovirus.

Of 972 stool samples collected from infants and children with acute gastroenteritis in six different regions (Hokkaido, Tokyo, Shizuoka, Kyoto, Osaka, and Saga) of Japan during 2-year period from July 2014 to June 2016, 63 samples (6.5%) determined to be positive for enterovirus by multiplex RT-PCR were subjected to genotype determination based on the partial VP1 region using the CODEHOP method. Sixty-two strains were succeeded in genotyping and seventeen EV types were identified. The majority of the studied strains belonged to EV-A (30 of 62; 48.4%) and EV-B (31 of 62, 50%) species, and the remaining strain was of the EV-D species. The most frequently detected type was Coxsackievirus A5 (CV-A5) in 2014–2015 while was CV-B5 in 2015–2016. This study provides an insight into the genetic diversity of EV with the predominance of EV-A and EV-B species in Japanese infants and children with acute gastroenteritis during 2014–2016.

Problem Ureaplasma species occasionally cause chorioamnionitis and premature labor. We developed a novel assay employing a loop-mediated isothermal amplification (LAMP) method to detect Ureaplasma parvum and Ureaplasma urealyticum. Method of study Loop-mediated isothermal amplification primers were designed to amplify Ureaplasma-specific ureaseB genes. Four U. parvum strains, 5 U. urealyticum strains and 14 reference bacterial species were evaluated. Forty-six vaginal swab samples were analyzed by LAMP, culture, and PCR. Results Our LAMP primers were specific to each species and had no cross-reaction. Of 46 clinical specimens, the sensitivity, specificity, and positive and negative predictive values of the LAMP method were 100% (12/12), 100% (34/34), 100% (12/12), and 100% (34/34), respectively, whereas those of PCR were 66.7% (8/12), 100% (34/34), 100% (8/8), and 89.5% (34/38), respectively, compared to culture-based detection. Conclusion The LAMP detection method outperformed the culture and PCR methods. Early detection enables appropriate antibiotic selection for improved prenatal outcomes.