Neutralization tests have been routinely used for the identification of human adenovirus C species (HAdV-C) in Japan until 2007. The aim of this study was to clarify the serological cross-reactivity of antiserum that has been used exclusively in Japan and to describe the first identification of HAdV type 57 (HAdV-57) in Japan. Anti-HAdV serum to HAdV-1, 2, 5, and 6 was quantitatively evaluated for cross-reactivity to the HAdV-57 isolates. Anti-HAdV-6 serum neutralized HAdV-57 with a concentration that was 32 to 64-fold higher than what was necessary to neutralize homologous HAdV-6. HAdV-1, 2, and 5 strains were not neutralized by anti HAdV-6 serum. Furthermore, 28 HAdV-6 strains isolated from 6,476 clinical samples were re-examined for HAdVs detected in the Shimane Prefecture of Japan from 2005 to 2014. These 28 strains were re-examined by PCR-sequencing techniques using the penton, hexon, and fiber regions. 3 isolates were determined to be HAdV-57. These data show that HAdV-57 had already invaded Japan as early as 2005, and that HAdV-57 strains were misidentified as HAdV-6.

Geno2Pheno (coreceptor), a genotypic tropism test, demonstrates excellent agreement with the phenotypic tropism test for subtype B and some other subtypes. However, potential X4-overcalling for CRF01_AE might occur with the present version. To confirm X4 overcalling for AE and to optimize the algorithm for use with AE, we compared the tropism of 22 AE samples by both genotypic and phenotypic methods. The env V3 region was analyzed by bulk sequencing, and tropism was evaluated using the Geno2Pheno algorithm. PhenXR, a phenotypic tropism test, was performed in parallel to determine chemokine receptor preferences. A high X4-overcalling for select samples and a low rate of R5-concordant samples (9.1%) were observed for AE with the current version of Geno2Pheno (coreceptor). On the other hand, the new version, namely, Geno2Pheno (Sanger), showed a high concordance rate of 81.8%, with PhenXR. Because majority of the samples were selected based on discrepancies in the genotypic tropism calls between the present version Geno2Pheno (coreceptor) (FPR<10%) and the new version Geno2Pheno (Sanger) (X4-risk<36), it remains to be determined whether the new version provides improved R5-calls for the AE sequences in general or only in this setting. Further clinical validation studies are warranted.

Tsutsugamushi disease and Japanese spotted fever are representative rickettsioses in Japan, and are caused by infection with Orientia tsutsugamushi and Rickettsia japonica, respectively. For molecular-based diagnosis, conventional PCR assays, which independently amplify respective rickettsial DNA, are usually used; however, this approach is time-consuming. Here, we describe a new duplex real-time PCR assay for the simultaneous detection of O. tsutsugamushi and spotted fever group rickettsiae, and its evaluation using several PCR conditions in 6 public health laboratories. The detection limit of the assay was estimated to be 10² copies and the sensitivity was almost identical to that of 3 conventional PCR methods. A total of 317 febrile patients were selected as clinically suspected or confirmed cases of rickettsioses. The detection efficiency of this assay for O. tsutsugamushi from blood or skin (eschar) specimens appeared to be almost the same as that of the conventional PCR method, even when performed in different laboratories, whereas the efficiency for spotted fever group rickettsiae tended to be higher than that of the 2 traditional double PCR assays. Our duplex real-time PCR is thus a powerful tool for the rapid diagnosis of rickettsioses, especially at the acute stage of infection.

Comprehensive analysis of bacterial DNA has enhanced our understanding of the maternal microbiome and its disturbances in preterm birth although clinical utility of these techniques remains to be determined. We tested whether a broad-range polymerase chain reaction (PCR) technique is useful for detection of culture-negative intra-amniotic infection (IAI). Pregnant women who underwent amniocentesis for the management of preterm birth with or without premature rupture of membranes. Bacterial 16S ribosomal DNA in the amniotic fluid was detected by PCR using primers for a sequence shared by Ureaplasma, Mycoplasma, and other bacteria. Sixty-four women were enrolled, 9 of whom were culture-positive. Of the 55 culture-negative women, 13 were PCR-positive and exhibited significantly higher interleukin 6 and 8 levels and lower glucose levels in the amniotic fluid than the remaining 42 women did, who were PCR- and culture-negative. C-reactive protein concentrations were elevated in cord and neonatal blood in the culture-negative, PCR-positive subgroup, whereas maternal C-reactive protein concentrations, white blood cell counts, and body temperatures were alike. The placental inflammation score (Blanc stage≥2) was significantly higher in the PCR-positive than in PCR-negative subgroup. This PCR-based method could be useful for identifying bacterial-culture-negative subclinical IAI and could help with predicting the severity of IAI.

Phenotypic detection of extended-spectrum β-lactamase (ESBL) is important for public health and infection control; however, plasmid-mediated AmpC β-lactamases (pAmpCs) can interfere with the ESBL phenotyping. We focused on Enterobacteriaceae strains that were susceptible to cefepime but had a mildly elevated minimum inhibitory concentration (MIC) of ceftazidime and studied the effect of pAmpC on the ESBL phenotyping in this population. Genotyping of ESBL and pAmpC was performed on 528 clinical isolates of Escherichia coli, Klebsiella spp., and Proteus spp. with a ceftazidime MIC of ≥2 μg/mL and cefepime MIC≤8 μg/mL; these isolates were collected at Nagasaki University Hospital from January 2005 to March 2011. In this sample, 145 isolates (27.5%) tested positive for pAmpC (pAmpC group). The concordance rates of phenotypic and genotypic detection of ESBLs were 69.2% in the pAmpC group and 88.8% in the non-pAmpC group (P=0.04). pAmpC was more commonly detected in isolates with non-CTX-M genes (5/53, 9.4%) than in isolates with CTX-M genes (8/121, 6.6%). Our data suggest that the presence of pAmpC increases the false negative detection of ESBL. When ESBL phenotyping is used, the underestimation of the prevalence of ESBL producers should be taken into account.

Vaccinations with habu (Protobothrops flavoviridis) venom toxoid were administered to individuals living in Amami Oshima from 1965 to 2002, and its effectiveness was investigated in 1991. The results raised the possibility that normal human serum inherently contains an inhibitor of the hemorrhagic metalloproteinase HR2, considered to be one of the major components of habu venom. In this study, we investigated the interaction between the hemorrhagic metalloproteinases HR1 and HR2 from habu-venom and human alpha 2-macroglobulin (α2M). Hemorrhagic activity of HR2 was completely inhibited by human α2M. However, the hemorrhagic activity of the large molecule HR1a was not inhibited. Size exclusion chromatography revealed that human α2M captured the HR2 molecule and formed a complex with it, thus inhibiting hemorrhagic activity. These results suggest that human α2M plays an important role in the inhibition of hemorrhage induced by HR2 from habu venom.

To understand human parechovirus (HPeV) infections in Taiwanese children, we analyzed data for 112 children (age≤10 years) with HPeV infection diagnosed between July 2007 and June 2016 in a medical center in Kaohsiung, southern Taiwan. The patients were infected with HPeV1 (n=94), HPeV3 (n=3), HPeV4 (n=3), HPeV6 (n=1) and non-typeable HPeV (n=11). We compared the clinical implications for children younger than 3 months (n=56) and 3 months and older (n=31), excluding 25 children with concomitant infections. Fever was noted in almost half of the children younger than 3 months but was more frequent in older than in younger children (83.9% vs 46.4%). As compared with older children, children younger than 3 months had a lower incidence of respiratory symptoms (30.1% vs 83.9%), more frequently required intensive care unit admission (28.6% vs 3.2%), and had longer hospital stays (mean 10.95 vs 5.13 days). Importantly, about one-third of the children were suspected to have hospital-acquired or cluster infections in the environment of medical institutions, with a significantly high proportion of 42.9% (24/56) in younger infants. Hospital-acquired infections might play a key role in the spread of HPeV, especially in children younger than 3 months.

Human parechovirus (HPeV) infections in Yokohama City, Japan, were surveyed from 2000 to 2016. The sequence of the VP1 region of HPeVs was used to construct a phylogenetic tree and to reveal the putative amino acid (aa) sequences. Phylogenetic analysis showed the presence of 3 genotypes in Yokohama City: HPeV1 (25 specimens), HPeV3 (86 specimens), and HPeV4 (2 specimens). HPeV1 was detected nearly every year, with the highest number detected in 2014. HPeV3 was not detected until 2005, but was detected over a 1- or 3-yr period thereafter. HPeV1 was most prevalent from July to November, whereas HPeV3 peaked in July and August each year. HPeV1 was mainly detected in patients with infectious gastroenteritis or respiratory tract infections. In contrast, 87% of HPeV3-positive cases were in patients less than 2 months of age with a viral-induced fever. An analysis of the aa sequence of VP1 revealed a divergence within the same HPeV genotype, which was useful in analyzing the emergence and re-emergence of HPeV infections during the survey period. These findings suggest that molecular analysis of HPeVs may contribute to a better understanding of its epidemiology.

Anaplasma phagocytophilum DNA was detected from a dog with canine granulocytic anaplasmosis (CGA) in Japan. Phylogenetic analysis of the DNA using 16S rRNA, gltA, and groEL sequences revealed that the strain was nearly identical to A. phagocytophilum detected from Apodemus agrarius (black-striped field mouse) in China and Korea. To our knowledge, this is the first report of molecular detection and phylogenetic analysis of A. phagocytophilum from a clinical case of CGA in Japan.

A simulation experiment was conducted to examine hand contamination from wiping the buttocks after the use and non-use of an electric toilet seat with water spray. A model of the buttocks was smeared with artificial diarrheal feces containing Serratia marcescens, and wiped by the participants wearing disposable gloves with 4 sheets of toilet paper after the use and non-use of the water spray of an electric toilet seat. Subsequently, the presence of S. marcescens on the surface of the gloves was quantified. After using the water spray, the mean count±standard deviation of S. marcescens was 0.067±0.249 colony-forming units (cfu)/glove, and it was 4,275±6,069 cfu/glove when water spray was not used. The cfu of S. marcescens was significantly lower when the water spray was used (p<0.00001) prior to wiping the artificial diarrheal feces. This result supports the effectiveness of water spray to prevent defecation-related hand contamination.

Cytomegalovirus (CMV) is the most common cause of congenital infection. Pneumonitis is considered to be a rare manifestation although congenital CMV infection presents with various non-specific findings. Ganciclovir and valganciclovir are beneficial for improving neurodevelopmental sequelae and hearing outcomes of congenital CMV infection; however, treatment response evaluation is not well reported. We report a female case of congenital CMV infection presenting with pneumonitis, meningoencephalitis, and chorioretinitis. She was treated with intravenous ganciclovir for 6 weeks, and clinical features improved. Measurement of the CMV genome load by real-time polymerase chain reaction assay was performed during treatment. After the administration of ganciclovir, the CMV genome was not detected in the blood and levels decreased gradually in the urine. Physicians should consider the possibility of congenital CMV infection in neonates who present with respiratory distress. Furthermore, measurement of the CMV genome load in blood and urine may be useful for evaluating treatment response.

In this study, we investigated the fosfomycin susceptibility rates among different methicillin-resistant Staphylococcus aureus (MRSA) clones. A total of 293 MRSA isolates obtained from Sir Run Run Shaw hospital during 2013–2015 were tested for fosfomycin susceptibility. The overall fosfomycin resistance rate among these MRSA isolates was 53.2%. Although 91.9% of the ST5 MRSA isolates (MIC50>1,024 mg/L) were resistant to fosfomycin, no fosfomycin-resistant isolate was found among the 69 ST59 MRSA isolates (MIC50/90, 0.5/4 mg/L). The fosfomycin resistance rate among the MRSA isolates recovered from skin and soft tissue infections was 19.1%, which was lower than the rates detected among MRSA isolates from other types of invasive infections. The fosfomycin resistance rate in community-onset MRSA was 30.2%, which was lower than that detected in healthcare-associated MRSA of 70.7%. One MRSA isolate had the fosB7 gene, whereas most (127/156) of the fosfomycin-resistant MRSA isolates had deletions in glpT genes. These findings highlight the importance of monitoring the fosfomycin susceptibility in MRSA isolates for epidemiological purposes.