How to prevent rubella epidemics and congenital rubella syndrome: lessons from 42 years of longitudinal epidemiology in Osaka Prefecture, Japan (1982-2023).

BACKGROUND: Despite the introduction of rubella-containing vaccine into routine immunization in 1977, rubella has not been eliminated in Japan. This study aimed to validate the immunization strategy and to highlight the crucial elements of elimination program. METHODS: We scrutinized cases of rubella and congenital rubella syndrome (CRS). Additionally, we analyzed the national vaccination coverage, seroprevalence, and number of maternal rubella-related spontaneous or artificial fetal deaths. RESULTS: The shift from selective to universal immunization significantly reduced rubella cases coupled with increased seroprevalence in children. However, rubella resurged in 2012-2013 and 2018-2019, which was virologically and serologically confirmed to be associated with imported rubella virus (RuV) and susceptible males. Although the disease burden of CRS may have been suppressed in the past by the large number of spontaneous or artificial fetal deaths, the incidence rate of CRS was comparable to that of the 1960s to 1980s. Cases of breakthrough infection and CRS were identified in females who were considered to have a history of single-dose vaccination. CONCLUSIONS: Even with universal immunization, future epidemics and severe outcomes cannot be prevented unless immunization gaps are closed. Furthermore, CRS and breakthrough infection are not completely prevented by single-dose vaccination, indicating the need for second-dose vaccination.

The Role of Human Mobility Flow in the Region-to-Region Spread of Respiratory Syncytial Virus Infection among Infants: An Infographic Analysis.

The region-to-region spread of human infectious diseases is considered to be dependent on the human mobility flow (HMF). However, it has been hard to obtain the evidence for this. Since the onset of the coronavirus disease 2019 (COVID-19) pandemic in Japan 2020, the government has enforced countermeasures against COVID-19 nationwide, namely the restriction of personal travelling, universal masking, and hand hygiene. As a result, the spread of acute respiratory infections had been effectively controlled. However, COVID-19 as well as pediatric respiratory syncytial virus (RSV) infections were not well-controlled. The region-to-region spread of pediatric RSV infections in 2020-2021 was recognizable unlike those in 2018 and 2019. In this study, we investigated the correlation between the trend of regional reports of the pediatric RSV infections and the HMF based on cellular phone signal data. Upon closer examination of both epidemiological trend and HMF data, the spread of pediatric RSV infection from one region to another was logically explained by HMF, which would serve as the evidence of the dependence of regional transmission on HMF. This is the first solid evidence where this correlation has been clearly observed for the common respiratory infections. While social implementation of infection control measures has successfully suppressed the droplet-mediated respiratory infections, such as influenza, but not the airborne infections, it was suggested that the aerosol transmission and adult asymptomatic carrier were involved in the transmission of RSV akin to COVID-19.

Sphingomyelin synthase 1 supports two steps of rubella virus life cycle.

Our knowledge of the regulatory mechanisms that govern the replication of the rubella virus (RV) in human cells is limited. To gain insight into the host-pathogen interaction, we conducted a loss-of-function screening using the CRISPR-Cas9 system in the human placenta-derived JAR cells. We identified sphingomyelin synthase 1 (SGMS1 or SMS1) as a susceptibility factor for RV infection. Genetic knockout of SGMS1 rendered JAR cells resistant to infection by RV. The re-introduction of SGMS1 restored cellular susceptibility to RV infection. The restricted step of RV infection was post-endocytosis processes associated with the endosomal acidification. In the late phase of the RV replication cycle, the maintenance of viral persistence was disrupted, partly due to the attenuated viral gene expression. Our results shed light on the unique regulation of RV replication by a host factor during the early and late phases of viral life cycle.

Development and Crosslinking Properties of Psoralen-Conjugated Triplex-Forming Oligonucleotides as Antigene Tools Targeting Genome DNA.

Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been utilized for genome editing and anti-gene experiments for over thirty years. However, the research on Ps-TFOs employing artificial nucleotides is still limited, and their photo-crosslinking properties have not been thoroughly investigated in relation to biological activities. In this study, we extensively examined the photo-crosslinking properties of Ps-TFOs to provide fundamental insights for future Ps-TFO design. We developed novel Ps-TFOs containing 2'-O,4'-C-methylene-bridged nucleic acids (Ps-LNA-mixmer) and investigated their photo-crosslinking properties using stable cell lines that express firefly luciferase constitutively to evaluate the anti-gene activities of Ps-LNA-mixmer. As a result, Ps-LNA-mixmer successfully demonstrated suppression activity, and we presented the first-ever correlation between photo-crosslinking properties and their activities. Our findings also indicate that the photo-crosslinking process is insufficient under cell irradiation conditions (365 nm, 2 mW/cm2 , 60 min). Therefore, our results highlight the need to develop new psoralen derivatives that are more reactive under cell irradiation conditions.

Impact of COVID-19 Countermeasures on Pediatric Infections.

(1) Background: General infection control measures have been implemented at the societal level against COVID-19 since the middle of 2020, namely, hand hygiene, universal masking, and social distancing. The suppressive effect of the social implementation of general infection control measures on pediatric infections has not been systematically assessed. (2) Methods: We addressed this issue based on publicly available data on 11 pediatric infections reported weekly by sentinel sites in Osaka and Iwate prefectures in Japan since 2010. We obtained the 5-year average for 2015-2019 and compared it to the weekly report for 2020-2021. (3) Results: The rate of 6 of the 11 pediatric infections decreased significantly during 2020-2021, regardless of the magnitude of the prevalence of COVID-19 in both areas. However, only RSV infection, one of the six infections, was endemic in 2021. Exanthem subitum was not as affected by COVID-19 countermeasures as other diseases. (4) Conclusions: The social implementation of infectious disease control measures was effective in controling certain infectious diseases in younger age groups, where compliance with the countermeasures should not be as high as that of adults.

Clinical perspectives of Treponema pallidum subsp. Endemicum infection in adults, particularly men who have sex with men in the Kansai area, Japan: A case series.

Bejel, caused by Treponema pallidum subsp. Endemicum (TEN), is a locally transmitted disease among children and juveniles in hot and dry regions. The number of adult cases of TEN infection outside of endemic areas has recently increased. We clinically examined five cases of TEN infection among adult cases previously reported in Japan. TEN infection mainly developed among young to middle-aged men who have sex with men (MSM). The clinical features of cases of TEN infection were similar to those of primary- and secondary-stage T. pallidum subsp. pallidum (TPA) infection. Genital lesions were common as the primary lesion. The clinical features and laboratory parameters of cases of TEN infection were similar to those of TPA infection. Most of the isolated strains had the A2058G mutation in 23S rDNA, which is responsible for resistance to macrolides. We also performed the systemic literature review of the TEN cases outside the endemic countries. The recent reported cases diagnosed with molecular methods shared the clinical features, occurred in young-to middle-aged sexually active persons in urban areas of developed countries and often accompanied with genital lesions, which were distinct from the classic description of bejel. This case series and the literature review provides important clinical insights and will contribute to the clinical detection of this rarely identified disease in developed countries. The surveillance of treponematoses, including TEN infection, using molecular diagnostic techniques is also warranted in developed countries, for the purpose of grasping the epidemic situation and control the local transmission.

Iron-Utilization System in Vibrio vulnificus M2799.

Vibrio vulnificus is a Gram-negative pathogenic bacterium that causes serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In the ferric-utilization system in V. vulnificus M2799, an isochorismate synthase (ICS) and an outer membrane receptor, VuuA, are required under low-iron conditions, but alternative proteins FatB and VuuB can function as a periplasmic-binding protein and a ferric-chelate reductase, respectively. The vulnibactin-export system is assembled from TolCV1 and several RND proteins, including VV1_1681. In heme acquisition, HupA and HvtA serve as specific outer membrane receptors and HupB is a sole periplasmic-binding protein, unlike FatB in the ferric-vulnibactin utilization system. We propose that ferric-siderophore periplasmic-binding proteins and ferric-chelate reductases are potential targets for drug discovery in infectious diseases.

Development of 7SK snRNA Mimics That Inhibit HIV Transcription.

The 332-nucleotide small nuclear RNA (snRNA) 7SK is a highly conserved non-coding RNA that regulates transcriptional elongation. By binding with positive transcriptional elongation factor b (P-TEFb) via HEXIM1, 7SK snRNA decreases the kinase activity of P-TEFb and inhibits transcriptional elongation. Additionally, it is reported that 7SK inhibition results in the stimulation of human immunodeficiency virus (HIV)-specific transcription. These reports suggest that 7SK is a naturally occurring functional molecule as negative regulator of P-TEFb and HIV transcription. In this study, we developed functional oligonucleotides that mimic the function of 7SK (7SK mimics) as novel inhibitors of HIV replication. We defined the essential region of 7SK regarding its suppressive effects on transcriptional downregulation using an antisense strategy. Based on the results, we designed 7SK mimics containing the defined region. The inhibitory effects of 7SK mimics on HIV-1 long terminal repeat promoter specific transcription was drastic compared with those of the control mimic molecule. Notably, these effects were found to be more enhanced by co-transfection with Tat-expressing plasmids. From these results, it is indicated that 7SK mimics may have great therapeutic potential for HIV/AIDS treatment.

Relationship between biochemical markers and measles viral load in patients with immunologically naive cases and secondary vaccine failure: LDH is one of the potential auxiliary indicators for secondary vaccine failure.

This study investigated the correlation between biochemical markers and viral load among 38 measles cases, including 15 immunologically naive patients and 23 patients with secondary vaccine failure (SVF). We examined four biochemical markers, namely, aspartate aminotransferase, alanine aminotransferase, C-reactive protein, and lactate dehydrogenase (LDH) and their relationship between virus genome copy numbers in peripheral blood mononuclear cells (PBMCs) and throat swabs as well as the concentration of measles-specific IgG. Although viral genome copies in both clinical specimens showed a significant correlation with specific IgG concentration, they had a higher correlation in PBMCs (Pearson's product-moment correlation coefficient, -0.662; p < .0001) than in throat swabs (Spearman's rank correlation coefficient, -0.443; p = .0078). The viral load in PBMCs also significantly correlated with LDH values (correlation coefficient, 0.360; p = .036). Thus, the serum LDH level might be a potential auxiliary indicator to distinguish immunologically naive patients with measles from those with SVF.

VuuB and IutB reduce ferric-vulnibactin in Vibrio vulnificus M2799.

Vibrio vulnificus, a pathogenic bacterium that causes serious infections in humans, requires iron for growth. Clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, namely, vulnibactin, to capture iron (III) from the environment. Growth experiments using a deletion mutant indicated that VuuB, a member of the FAD-containing siderophore-interacting protein family, plays a crucial role in Fe3+-vulnibactin reduction. IutB, a member of the ferric-siderophore reductase family, stands a substitute for VuuB in its absence. It remained unclear why V. vulnificus M2799 has two proteins with relevant functions. Here we biochemically characterized VuuB and IutB using purified recombinant proteins. Purified VuuB, a flavoprotein, catalyzed the reduction of Fe3+-nitrilotriacetic acid as its electron acceptor, in the presence of NADH as its electron donor and FAD as its cofactor. IutB catalyzed the reduction of Fe3+-nitrilotriacetic acid, in the presence of NADH, NADPH, or reduced glutathione as its electron donor. The optimal pH values and temperatures of VuuB and IutB were 7.0 and 37 °C, and 8.5 and 45 °C, respectively. On analyzing their ferric-chelate reductase activities, both VuuB and IutB were found to catalyze the reduction of Fe3+-aerobactin, Fe3+-vibriobactin, and Fe3+-vulnibactin. When the biologically relevant substrate, Fe3+-vulnibactin, was used, the levels of ferric-chelate reductase activities were similar between VuuB and IutB. Finally, the mRNA levels were quantified by qRT-PCR in M2799 cells cultivated under low-iron conditions. The number of vuuB mRNA was 8.5 times greater than that of iutB. The expression ratio correlated with the growth of their mutants in the presence of vulnibactin.

Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping.

Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.

First isolation and characterization of vancomycin-resistant Enterococcus faecium harboring vanD5 gene cluster recovered from a 79-year-old female inpatient in Japan.

This study reports the first isolation and characterization of a vanD5 genotype vancomycin-resistant Enterococcus faecium strain (E. faecium IPHb306) recovered from a 79-year-old Japanese female inpatient. Species identification was determined by biochemical testing, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and species-specific PCR. Susceptibility tests indicated that E. faecium IPHb306 was resistant to vancomycin but susceptible to teicoplanin. Southern hybridization analyses indicated that E. faecium IPHb306 harbored a vanD5 gene cluster on chromosomal DNA. Growth curve analyses showed that a vancomycin resistance phenotype could be inducible. Sequencing analyses of the vanD5 gene cluster and the ddlE. faecium gene demonstrated several point mutations were present. Because this strain belongs to ST203, a major hospital-adapted lineage, spread of the vanD5 genotype E. faecium ST203 is considered a clinical threat in Japan.

Bejel, a Nonvenereal Treponematosis, among Men Who Have Sex with Men, Japan.

Bejel, an endemic treponematosis caused by infection with Treponema pallidum subspecies endemicum, has not been reported in eastern Asia and the Pacific region. We report local spread of bejel among men who have sex with men in Japan. Spread was complicated by venereal syphilis.

Clinical value of enzyme immunoassay that detects rubella-specific immunoglobulin M immediately after disease onset.

A total of 300 patients with nucleic acid test-confirmed rubella, mostly adults, were investigated to determine the clinical value of a rubella-specific IgM test using an EIA kit. IgM titers increased after rash onset, the median IgM titer being significantly higher 3 days post-onset than on previous days (P < 0.0001). Similarly, the IgM-positive rate at 3 days post-onset (61.5%) was significantly higher than on previous days (P < 0.0001). This IgM test against rubella at 3 days or more post-disease onset provides the clinically relevant information.

Circulation of Distinct Treponema pallidum Strains in Individuals with Heterosexual Orientation and Men Who Have Sex with Men.

Human treponematosis is caused by various pathogenic Treponema pallidum subspecies, including T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, T. pallidum subsp. endemicum, and Treponema carateum The global prevalence of syphilis has been increasing since the 2000s. Men account for more than 90% of the cases, with the majority being men who have sex with men (MSM). In Japan, the increase in the number of syphilis patients began in 2011, a 10-year delay from the global trend. In 2017, a total of 5,829 syphilis cases (3,934 men and 1,895 women) were reported, with an outstanding increase in cases among young adult women; the number reported for women age 15 to 20 years was 1,100. Hence, a molecular epidemiological study was conducted on circulating T. pallidum strains using two strain typing methods, the enhanced CDC method and sequencing-based molecular typing. Clinical specimens from 95 adults suspected of syphilis were collected from September 2013 to August 2017 in Osaka, Japan. T. pallidum DNA was detected in specimens from 25 males and 11 females, including seven MSM. The majority of the heterosexual patients (66.7% and 90.9% of males and females, respectively) were positive for 14d/f-SSR8. In contrast, the genotypes identified in the MSM group were significantly divergent. T. pallidum subsp. endemicum was notably identified in two MSM patients. Macrolide-sensitive or Nichols-like strains were significantly associated with the MSM group. These data suggest that distinct T. pallidum strains were circulating in the heterosexual and MSM groups. Our findings imply that independent factors may contribute to the increased syphilis prevalence in heterosexual and MSM populations.

Foodborne Outbreaks Caused by Human Norovirus GII.P17-GII.17-Contaminated Nori, Japan, 2017.

Seven foodborne norovirus outbreaks attributable to the GII.P17-GII.17 strain were reported across Japan in 2017, causing illness in a total of 2,094 persons. Nori (dried shredded seaweed) was implicated in all outbreaks and tested positive for norovirus. Our data highlight the stability of norovirus in dehydrated food products.

High transmissibility of norovirus among infants and school children during the 2016/17 season in Osaka, Japan.

The number of person-to-person transmitted norovirus cases (n = 4,712) in school children in Osaka, Japan, during 2016/17 was the largest since 2012/13. Norovirus outbreaks were reported by 101 schools including 53 nursery schools (1,927 cases), 18 kindergartens (1,086 cases) and 30 elementary schools (1,699 cases). The dominant genotype among outbreaks was GII.P16-GII.2 (57.4%; 58/101), followed by GII.P2-GII.2 (8.9%; 9/101) and GII.P7-GII.6 (5.9%; 6/101). GII.4 was not detected despite dominance in previous years.

Current prevention and control of health care-associated infections in long-term care facilities for the elderly in Japan.

Residents of long-term care facilities for the elderly are vulnerable to health care-associated infections. However, compared to medical institutions, long-term care facilities for the elderly lag behind in health care-associated infection control and prevention. We conducted a epidemiologic study to clarify the current status of infection control in long-term care facilities for the elderly in Japan. A questionnaire survey on the aspects of infection prevention and control was developed according to SHEA/APIC guidelines and was distributed to 617 long-term care facilities for the elderly in the province of Osaka during November 2016 and January 2017. The response rate was 16.9%. The incidence rates of health care-associated infection outbreaks and residents with health care-associated infections were 23.4 per 100 facility-years and 0.18 per 1,000 resident-days, respectively. Influenza and acute gastroenteritis were reported most frequently. Active surveillance to identify the carrier of multiple drug-resistant organisms was not common. The overall compliance with 21 items selected from the SHEA/APIC guidelines was approximately 79.2%. All facilities had infection control manuals and an assigned infection control professional. The economic burdens of infection control were approximately US$ 182.6 per resident-year during fiscal year 2015. Importantly, these data implied that physicians and nurses were actively contributed to higher SHEA/APIC guideline compliance rates and the advancement of infection control measures in long-term care facilities for the elderly. Key factors are discussed to further improve the infection control in long-term care facilities for the elderly, particularly from economic and social structural standpoints.

A novel cell-based high throughput assay to determine neutralizing antibody titers against circulating strains of rubella virus.

A large rubella outbreak occurred in Japan 2013, and 14,344 rubella and 45 congenital rubella syndrome (CRS) cases were reported. At that time, the populational immunity was above the protective threshold assessed by hemmaglutination inhibition (HI) titer. The genotype 2B rubella virus (RV) strains were responsible for the outbreak, which are non-indigenous in Japan. In this work, a cell-based high throughput assay was established to measure the neutralizing antibody (NA) titer against circulating RV isolates. RV infection poorly induces cytopathic effects in tissue culture, preventing the casual measurement of NA titer. Our assay system has overcome this hurdle. Using this assay, we re-evaluated the antibody prevalence rate against circulating viral isolates using human sera collected before the outbreak. Individuals with protective IgG titer (≥10 IU/ml) represented 88.1% of the population. Consistently, 85.2% of the population had protective neutralizing antibody titers (≥1:8) against the vaccine strain. In contrast, 50.5% of the population had protective neutralizing antibody titers against circulating genotype 2B RV strains. These data suggest that the herd immunity assessed by HI titer should have been appreciated deliberately.