Development of a real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primers for rapid detection of rubella virus.

Rubella virus infection during early pregnancy sometimes causes severe birth defects termed congenital rubella syndrome. Although there are safe and effective live-attenuated vaccines, rubella has only been certified as eliminated in the Americas within the six World Health Organization regions. Rubella remains an endemic disease in many regions, and outbreaks occur wherever population immunity is insufficient. There are two main methods for diagnosis of rubella: detection of anti-rubella IgM antibodies by enzyme immunoassay and detection of the viral genome by real-time RT-PCR. Both of these methods require substantial time and effort. In the present study, a rapid rubella detection assay using real-time fluorescent reverse transcription loop-mediated isothermal amplification with quenching primers was developed. The time required for the new assay was one-half that required for a real-time RT-PCR assay. The assay had 93.6% positive percent agreement and 100% negative percent agreement for clinical specimens compared with the real-time RT-PCR assay. The new assay is considered useful for diagnosis of rubella in areas where rubella is endemic.

Age-specific prevalence of IgG against measles/rubella and the impact of routine and supplementary immunization activities: A multistage random cluster sampling study with mathematical modelling.

BACKGROUND: Vietnam continues to have measles and rubella outbreaks following supplementary immunization activities (SIA) and routine immunization despite both having high reported coverage. To evaluate immunization activities, age-specific immunity against measles and rubella, and the number of averted Congenital Rubella Syndrome (CRS) cases, must be estimated. METHODS: Dried blood spots were collected from 2091 randomly selected individuals aged 1-39 years. Measles and rubella virus-specific immunoglobulin G (IgG) were measured by enzyme immunoassay. Results were considered positive at ≥120 mIU/mL for measles and ≥10 IU/mL for rubella. The number of CRS cases averted by immunization since 2014 were estimated using mathematical modelling. RESULTS: Overall IgG seroprevalence was 99.7% (95%CI: 99.2-99.9) for measles and 83.6% (95%CI: 79.3-87.1) for rubella. Rubella IgG seroprevalence was higher among age groups targeted in the SIA than in non-targeted young adults (95.4% [95%CI: 92.9-97.0] vs 72.4% [95%CI: 63.1-80.1]; P < 0.001). The estimated number of CRS cases averted in 2019 by immunization activities since 2014 ranged from 126 (95%CI: 0-460) to 883 (95%CI: 0-2271) depending on the assumed postvaccination reduction in the force of infection. CONCLUSIONS: The results suggest the SIA was effective, while young adults born before 1998 who remain unprotected for rubella require further vaccination.

Evaluation and validation of a commercial ELISA versus the in vitro toxin neutralization assay for determination of diphtheria anti-toxin in human serum.

Introduction. Diphtheria is a potentially life-threatening infection and remains endemic in many low- and middle-income countries (LMICs). A reliable, low-cost method for serosurveys in LMICs is warranted to estimate the accurate population immunity to control diphtheria.Hypothesis/Gap Statement. The correlation between the ELISA results against diphtheria toxoid and the gold standard diphtheria toxin neutralization test (TNT) values is poor when ELISA values are <0.1 IU ml-1, which results in inaccurate estimates of susceptibility in populations when ELISA is used for measuring antibody levels.Aim. To explore methods to accurately predict population immunity and TNT-derived anti-toxin titres from ELISA anti-toxoid results.Methodology. A total of 96 paired serum and dried blood spot (DBS) samples collected in Vietnam were used for comparison of TNT and ELISA. The diagnostic accuracy of ELISA measurement with reference to TNT was assessed by area under the receiver operating characteristic (ROC) curve (AUC) and other parameters. Optimal ELISA cut-off values corresponding to TNT cut-off values of 0.01 and 0.1 IU ml-1 were identified by ROC analysis. A method based on the multiple imputation approach was also applied to estimate TNT measurements in a dataset that only included ELISA results. These two approaches were then applied to ELISA results previously generated from 510 subjects in a serosurvey in Vietnam.Results. The ELISA results on DBS samples showed a good diagnostic performance compared to TNT. The cut-off values for ELISA measurement corresponding to the TNT cut-off values of 0.01 IU ml-1 were 0.060 IU ml-1 in serum samples, and 0.044 IU ml-1 in DBS samples. When a cut-off value of 0.06 IU ml-1 was applied to the 510 subject serosurvey data, 54 % of the population were considered susceptible (<0.01 IU ml-1). The multiple imputation-based approach estimated that 35 % of the population were susceptible. These proportions were much larger than the susceptible proportion estimated by the original ELISA measurements.Conclusion. Testing a subset of sera by TNT combined with ROC analysis or a multiple imputation approach helps to adjust ELISA thresholds or values to assess population susceptibility more accurately. DBS is an effective low-cost alternative to serum for future serological studies for diphtheria.

[Successful Staged Repair of Double Rupture after Acute Myocardial Infarction:Report of a Case].

Double rupture is a very rare, and life-threatening complication after acute myocardial infection (AMI), which defined as the coexistence of any two of the three types of rupture include left ventricular free wall repture (LVFWR), ventricular septal perforation (VSP) and papillary muscule repture (PMR). We report here a case of successful staged repair of double rupture combined LVFWR and VSP. A 77-year-old woman with diagnosis of AMI in the anteroseptal area fell into cardiogenic shock suddenly just before starting coronary angiography. Echocardiography showed left ventricular free wall rupture, then an emergent operation was performed under intraaortic balloon pumping (IABP) and percutaneous cardiopulmonary support (PCPS) assistance using bovine pericardial patch and felt sandwich technique. Intraoperative transesophageal echocardiography revealed ventricular septal perforation on the apical anterior wall. Her hemodynamic condition was stable, therefore we selected a staged VSP repair to avoid surgery on freshly infarcted myocardium. Twenty-eight days after the initial operation, VSP repair was performed using the extended sandwich patch technique via right ventricle incision. Postoperative echocardiography revealed no residual shunt.

Comparison of population-based measles-rubella immunoglobulin G antibody prevalence between 2014 and 2019 in Lao People's Democratic Republic: Impacts of the national immunization program.

OBJECTIVES: We evaluated the effectiveness of the Lao People's Democratic Republic's measles-rubella immunization program using the seroprevalence from two cross-sectional surveys. METHODS: The nationwide surveys occurred in 2014 and 2019 using a multistage cluster sampling, both requiring samples from 2184 individuals from 52 randomly selected villages. Immunoglobulin G titers, measured using enzyme-linked immunosorbent assay, were considered positive at ≥120 mIU/ml (measles) and ≥10 IU/ml (rubella). We calculated the vaccination-related reduction in the force of rubella infection and the number of congenital rubella syndrome cases averted in 2019. RESULTS: We collected 2135 (women: 55.2%, mean age: 23.2 years) and 2001 (52.7%, 23.1 years) samples in 2014 and 2019, respectively. During 2014-2019, immunoglobulin G prevalence increased from 83.9% (95% confidence interval [CI]: 83.8-84.0) to 98.3% (97.7-98.8) for measles and from 75.4% (75.3-75.5) to 87.8% (86.4-89.2) for rubella. The most plausible reduction in the average force of rubella infection was 100% (95% CI: 28-100) since vaccination started, averting 78 (95% CI: 42-128) congenital rubella syndrome cases in 2019. CONCLUSION: This is the first population-based study for measles and rubella at two different time points in developing countries. Measles and rubella seroprevalence increased significantly during 2014-2019, greatly exceeding the immunity thresholds for their elimination.

Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry.

The lipid composition of the host cell membrane is one of the key determinants of the entry of enveloped viruses into cells. To elucidate the detailed mechanisms behind the cell entry of rubella virus (RuV), one of the enveloped viruses, we searched for host factors involved in such entry by using CRISPR/Cas9 genome-wide knockout screening, and we found sphingomyelin synthase 1 (SMS1), encoded by the SGMS1 gene, as a candidate. RuV growth was strictly suppressed in SGMS1-knockout cells and was completely recovered by the overexpression of enzymatically active SMS1 and partially recovered by that of SMS2, another member of the SMS family, but not by that of enzymatically inactive SMS1. An entry assay using pseudotyped vesicular stomatitis virus possessing RuV envelope proteins revealed that sphingomyelin generated by SMSs is crucial for at least RuV entry. In SGMS1-knockout cells, lipid mixing between the RuV envelope membrane and the membrane of host cells occurred, but entry of the RuV genome from the viral particles into the cytoplasm was strongly inhibited. This indicates that sphingomyelin produced by SMSs is essential for the formation of membrane pores after hemifusion occurs during RuV entry. IMPORTANCE Infection with rubella virus during pregnancy causes congenital rubella syndrome in infants. Despite its importance in public health, the detailed mechanisms of rubella virus cell entry have only recently become somewhat clearer. The E1 protein of rubella virus is classified as a class II fusion protein based on its structural similarity, but it has the unique feature that its activity is dependent on calcium ion binding in the fusion loops. In this study, we found another unique feature, as cellular sphingomyelin plays a critical role in the penetration of the nucleocapsid into the cytoplasm after hemifusion by rubella virus. This provides important insight into the entry mechanism of rubella virus. This study also presents a model of hemifusion arrest during cell entry by an intact virus, providing a useful tool for analyzing membrane fusion, a biologically important phenomenon.

Epidemiology of Congenital Rubella Syndrome Related to the 2012-2013 Rubella Epidemic in Japan.

This study assessed the epidemiological characteristics of 45 congenital rubella syndrome cases in Japan following the 2012-2013 rubella epidemic. Rubella still poses significant health burdens and the uptake of rubella-containing vaccines among women of childbearing age should be improved.

Effectiveness of immunization activities on measles and rubella immunity among individuals in East Sepik, Papua New Guinea: A cross-sectional study.

Objectives: This study aimed to assess measles and rubella immunity by measuring virus-specific immunoglobulin G (IgG) prevalence among individuals and evaluate the effectiveness of recent supplementary immunization activities (SIAs) by comparing the antibody positivity rates of the SIA target age groups in 2015 with those in 2019 as measles and rubella are endemic in Papua New Guinea. Methods: A cross-sectional study. The measles- and rubella-specific IgG levels of patients aged ≥1 year at two clinics in East Sepik province, Papua New Guinea were assessed with commercially available virus-specific IgG EIA kits. Results: In total, 297 people participated in the study and 278 samples with sufficient volume, relevant information, and age inclusion criteria were analyzed. The overall IgG prevalence rates were 62.6% for measles and 82.0% for rubella. The age groups targeted in the 2019 SIAs had a higher IgG prevalence than those targeted in the 2015 SIAs for both the infectious diseases. Moreover, the IgG prevalence for rubella was higher than measles in these groups. Conclusions: The anti-measles and anti-rubella IgG prevalence in the target groups were lower than those required for herd immunity. The immunization program should be emphasized to eliminate measles and rubella. Further population-based studies are warranted.

[Stanford Type A Acute Aortic Dissection after Coronary Artery Bypass Grafting using Automated Proximal Anastomotic Device].

Stanford type A acute aortic dissection after off-pump coronary artery bypass grafting( OPCAB) is a rare but potentially fatal complication. A 61-year-old man with subacute Stanford type B aortic dissection underwent a triple OPCAB using an automated proximal anastomotic device. On postoperative day 4, he had a sudden syncope. An enhanced computed tomography (CT) scan revealed Stanford type A acute aortic dissection. He underwent emergent total aortic arch replacement along with an open stent graft deployment. The entry of the dissection was located at the proximal anastomosis site of the vein graft. This case demonstrates that this device should be used carefully in patients with a history of Stanford type B aortic dissection.

[Stanford Type A Acute Aortic Dissection with Persistent Left Superior Vena Cava in a Turner Syndrome Patient:Report of a Case].

A 48-year-old woman who was diagnosed with Turner syndrome in her childhood presented with sudden onset of low back pain and respiratory discomfort. Contrast enhanced computed tomography scan revealed Stanford type A acute aortic dissection with persistent left superior vena cava (PLSVC). Emergency ascending aortic replacement was performed. After cardiopulmonary bypass was established through cannulating right femoral artery and right superior vena cava, inferior vena cava, another venous cannula was directly placed into the left superior vena cava. After core cooling, the right atrium was incised for retrograde cardioplegia. At a tympanic temperature of 25 ℃, circulatory arrest was started and retrograde cerebral perfusion was performed through right and left superior vena cava. Her postoperative course was uneventful.

Vaccine temperature management in Lao People's Democratic Republic: A nationwide cross-sectional study.

OBJECTIVE: The objective of the study was to evaluate the duration and frequency of vaccine exposure to suboptimal temperatures during transit from the central vaccine storage in the capital to health centers in Lao PDR. METHODS: Temperature data loggers traveled from the capital to the health centre storages (146) with the vaccines to monitor the vaccine temperature nationwide. One health centre per district was selected using a simple random sampling method for the first round of temperature monitoring. One health centre was selected from every forty-nine high risk districts monitor the trend of vaccine temperature at the health centre storage and during outreach sessions in several districts. Vaccines and temperature data loggers were transported using the normal vaccination transportation. FINDINGS: Overall, the vaccines were exposed to temperatures >8 °C for an average of 1648 min, equivalent to 9.0% of the observational period, and to temperatures <0 °C for an average of 184 min, equivalent to 1.35% of the study period. The proportion of exposure to temperatures >8 °C was the highest during the transit from the capital to the province. The proportion of exposure to temperatures <0 °C was the highest during storage at district level. Examined by region, vaccines in the northern provinces had higher risk of exposure to temperatures >8 °C; however, the risk of exposure to temperatures <0 °C was scattered nationwide. Moreover, some health centers showed fluctuations in storage temperature. CONCLUSIONS: Challenges associated with cold chain management, and the resulting deterioration of vaccines, might account for outbreaks of vaccine-preventable diseases. The government should examine and invest in suitable technologies and approaches to ensure consistency in cold chain management.

[Non-anastomotic Pseudoaneurysm of a Prosthetic Graft with Sternal Erosion after Total Aortic Arch Replacement].

An 82-year-old man underwent total aortic arch replacement with a 24 mm Triplex four-branched graft for aortic arch aneurysm. After two years, he was diagnosed with pseudoaneurysms due to bleeding from a non-anastomotic site of the branch graft to the left common carotid artery and minor leakage from a distal anastomotic site of the main graft. A self-expandable Fluency covered stent and cTAG thoracic endograft were used for the aneurysm. After four years, he was referred to our hospital with a complaint of pulsatile swelling of the anterior chest wall. Contrast enhanced computed tomography (CT) revealed a pseudoaneurysm arising from a non-anastomotic site of the branch graft to the left common carotid artery, which extended into the anterior chest wall and the skin through the sternum. He underwent emergency endovascular repair using a Niti-S ComVi covered stent. The postoperative course was uneventful. Postoperative CT showed shrinkage of the pseudoaneurysm. The patient was discharged and required no reintervention during the follow-up.

Rubella outbreak among workers in three small- and medium-size business establishments associated with imported genotype 1E rubella virus-Shizuoka, Japan, 2015.

On 12 February 2015, a local health department (LHD) in Shizuoka prefecture identified two reported rubella cases in its jurisdiction as employees of the same company. As other employees at the company resided both inside and outside of the health department's jurisdiction, it began collaborating with two additional LHDs and the National Institute of Infectious Diseases to investigate and respond to the outbreak, which subsequently identified cases in two additional companies. We obtained epidemiological, clinical, and outbreak response information from the national epidemiological surveillance of infectious disease system's database, the local health departments, and the associated companies. One specimen for genetic sequencing was collected from each of the three companies. The outbreak included a total of twenty-five cases, with seventeen confirmed and eight probable cases from three companies. Among them, 24 (96%) were male, 22 (88%) were employees of one company (Company X), and none had rubella vaccination history. The median age was 45 years (interquartile range: 40-51). Epidemiological information did not reveal the source of infection nor transmission route. All rubella viruses sequenced from the three specimens were classified into genotype 1E. The nucleotide sequences in the 739 bp-window region were completely identical in two specimens, with only one nucleotide difference in the third specimen. According to phylogenetic analysis, these strains were closely related to the Southeast and East Asian lineage. This rubella outbreak at three companies, ranging in size from small- to medium-size, in Japan occurred among unvaccinated employees aged at least 30 years, most of whom were male. Virologic analyses suggest all cases were infected with the same viral strain imported from Southeast Asia. Similar to these companies, most employees at small- and medium-size businesses in Japan are males with no vaccination history for rubella, which poses a serious risk for associated cases of congenital rubella syndrome (CRS).

Summary of the special symposium 'Towards Elimination of Rubella'.

A special symposium titled 'Towards Elimination of Rubella' was held at the 23rd Annual Meeting of the Japanese Society for Vaccinology in Tokyo, Japan on November 30, 2019. Representatives from the Japanese national government, a local government, and a private company presented their efforts to control rubella, and the group discussed the challenges of eliminating rubella in Japan.

Development of Genetic Diagnostic Methods for Detection for Novel Coronavirus 2019(nCoV-2019) in Japan.

During the emergence of novel coronavirus 2019 (nCoV) outbreak in Wuhan city, China at the end of 2019, there was movement of many airline travelers between Wuhan and Japan, suggesting that the Japanese population was at high risk of infection by the virus. Hence, we urgently developed diagnostic systems for detection of 2019 nCoV. Two nested RT-PCR and two real-time RT-PCR assays were adapted for use in Japan. As of February 8, 2020, these assays have successfully detected 25 positive cases of infection in Japan.

Comparison of virological and serological methods for laboratory confirmation of rubella.

BACKGROUND: Work toward rubella elimination has accelerated globally. A reliable laboratory confirmation of rubella-suspected cases is required for effective surveillance in the rubella-elimination phase. The use of adequate specimens is a key to improving the quality of this surveillance. STUDY DESIGN: We conducted rubella virus (RUBV) isolation and RUBV genome or anti-RUBV IgM detection on 1023 specimens from 372 rubella- or measles-suspected cases collected through the national surveillance program in Sakai city of Osaka prefecture, Japan between 2011 and 2013. The resulting data were analyzed by specimen type, collection date, and immunological status. RESULTS: Among the three specimen types (throat swab, serum or plasma, and urine) collected through 10 days post-rash onset, the highest success rates for RUBV genome detection and RUBV isolation were obtained using throat swabs. In agreement with previous work, RUBV-specific IgM were undetectable in 50% of the rubella-confirmed cases until 3 days after rash onset. The success rates of RUBV genome detection and RUBV isolation declined in association with the appearance of RUBV-specific antibodies in blood, especially in serum, plasma, or urine samples. CONCLUSION: Throat swabs are the most optimal specimen types for both RUBV genome detection and RUBV isolation; serum/plasma samples may be suboptimal, especially for RUBV isolation. The findings from this study will provide useful information for improving laboratory surveillance for rubella in the elimination phase.

Endovascular Repair for Abdominal Aortic Rupture Caused by Periaortic Mantle Cell Lymphoma.

A 72-year-old man was referred to our hospital for the suspicion of ruptured abdominal aortic aneurysm. Before admission, he was suspected of having a malignant lymphoma and underwent excisional biopsy in his right groin. A contrast enhanced computed tomography scan revealed a massive retroperitoneal hematoma with an extravasation arising from the infrarenal abdominal aorta coexisting with an extensive retroperitoneal mass surrounding the aorta. An emergency endovascular aneurysm repair was performed and the postoperative course was uneventful. After the treatment, histological examination of the previous biopsy confirmed the diagnosis of mantle cell lymphoma.

Heat Shock Protein 90 Ensures the Integrity of Rubella Virus p150 Protein and Supports Viral Replication.

Two viral nonstructural proteins, p150 and p90, are expressed in rubella virus (RUBV)-infected cells and mediate viral genome replication, presumably using various host machineries. Molecular chaperones are critical host factors for the maintenance of cellular proteostasis, and certain viral proteins use this chaperone system. The RUBV p150 and p90 proteins are generated from a precursor polyprotein, p200, via processing by the protease activity of its p150 region. This processing is essential for RUBV genome replication. Here we show that heat shock protein 90 (HSP90), a molecular chaperone, is an important host factor for RUBV genome replication. The treatment of RUBV-infected cells with the HSP90 inhibitors 17-allylamino-17-desmethoxygeldanamycin (17-AAG) and ganetespib suppressed RUBV genome replication. HSP90α physically interacted with p150, but not p90. Further analyses into the mechanism of action of the HSP90 inhibitors revealed that HSP90 activity contributes to p150 functional integrity and promotes p200 processing. Collectively, our data demonstrate that RUBV p150 is a client of the HSP90 molecular chaperone and that HSP90 functions as a key host factor for RUBV replication.IMPORTANCE Accumulating evidence indicates that RNA viruses use numerous host factors during replication of their genomes. However, the host factors involved in rubella virus (RUBV) genome replication are largely unknown. In this study, we demonstrate that the HSP90 molecular chaperone is needed for the efficient replication of the RUBV genome. Further, we reveal that HSP90 interacts with RUBV nonstructural protein p150 and its precursor polyprotein, p200. HSP90 contributes to the stability of p150 and the processing of p200 via its protease domain in the p150 region. We conclude that the cellular molecular chaperone HSP90 is a key host factor for functional maturation of nonstructural proteins for RUBV genome replication. These findings provide novel insight into this host-virus interaction.

Estimating the immunogenicity of measles-rubella vaccination administered during a mass campaign in Lao People's Democratic Republic using multi-valent seroprevalence data.

Measles and rubella are important causes of morbidity and mortality globally. Despite high coverage reported for measles vaccination, outbreaks continue to occur in some countries. The reasons for these outbreaks are poorly understood. We apply Bayesian methods to multi-valent seroprevalence data for measles and rubella, collected 2 years and 3 months after a mass measles-rubella vaccination campaign in Lao PDR to estimate the immunogenicity and vaccination coverage. When the vaccination coverage was constrained to exceed 95% or 90%, consistent with officially-reported values, the immunogenicity of the measles vaccine component was unexpectedly low (75% (95% CR: 63-82%) and 79% (CR: 70-87%) respectively. The estimated immunogenicity increased after relaxing constraints on the vaccination coverage, with best-fitting values of 83% (95% CR: 73-91%) and 97% (95% CR: 90-100%) for the measles and rubella components respectively, with an estimated coverage of 83% (95% CR: 80-88%). The findings suggest that, if the vaccine coverage was as high as that reported, continuing measles outbreaks in Lao PDR, and potentially elsewhere, may be attributable to suboptimal immunogenicity attained in mass campaigns. Vaccine management in countries with high reported levels of coverage and ongoing measles outbreaks needs to be reviewed if measles elimination targets are to be achieved.