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.

COVID-19 vaccine effectiveness against severe COVID-19 requiring oxygen therapy, invasive mechanical ventilation, and death in Japan: A multicenter case-control study (MOTIVATE study).

INTRODUCTION: Since the SARS-CoV-2 Omicron variant became dominant, assessing COVID-19 vaccine effectiveness (VE) against severe disease using hospitalization as an outcome became more challenging due to incidental infections via admission screening and variable admission criteria, resulting in a wide range of estimates. To address this, the World Health Organization (WHO) guidance recommends the use of outcomes that are more specific to severe pneumonia such as oxygen use and mechanical ventilation. METHODS: A case-control study was conducted in 24 hospitals in Japan for the Delta-dominant period (August-November 2021; "Delta") and early Omicron (BA.1/BA.2)-dominant period (January-June 2022; "Omicron"). Detailed chart review/interviews were conducted in January-May 2023. VE was measured using various outcomes including disease requiring oxygen therapy, disease requiring invasive mechanical ventilation (IMV), death, outcome restricting to "true" severe COVID-19 (where oxygen requirement is due to COVID-19 rather than another condition(s)), and progression from oxygen use to IMV or death among COVID-19 patients. RESULTS: The analysis included 2125 individuals with respiratory failure (1608 cases [75.7%]; 99.2% of vaccinees received mRNA vaccines). During Delta, 2 doses provided high protection for up to 6 months (oxygen requirement: 95.2% [95% CI:88.7-98.0%] [restricted to "true" severe COVID-19: 95.5% {89.3-98.1%}]; IMV: 99.6% [97.3-99.9%]; fatal: 98.6% [92.3-99.7%]). During Omicron, 3 doses provided high protection for up to 6 months (oxygen requirement: 85.5% [68.8-93.3%] ["true" severe COVID-19: 88.1% {73.6-94.7%}]; IMV: 97.9% [85.9-99.7%]; fatal: 99.6% [95.2-99.97]). There was a trend towards higher VE for more severe and specific outcomes. CONCLUSION: Multiple outcomes pointed towards high protection of 2 doses during Delta and 3 doses during Omicron. These results demonstrate the importance of using severe and specific outcomes to accurately measure VE against severe COVID-19, as recommended in WHO guidance in settings of intense transmission as seen during Omicron.

Immune escape and waning immunity of COVID-19 monovalent mRNA vaccines against symptomatic infection with BA.1/BA.2 and BA.5 in Japan.

BACKGROUND: Repeated emergence of variants with immune escape capacity and waning immunity from vaccination are major concerns for COVID-19. We examined whether the surge in Omicron subvariant BA.5 cases was due to immune escape or waning immunity through vaccine effectiveness (VE) evaluation. METHODS: A test-negative case-control study was conducted in 16 clinics/hospitals during the BA.1/BA.2-dominant and BA.5-dominant periods. VE against symptomatic infection was estimated after adjusting for age, sex, comorbidity, occupation, testing frequency, prior infection, close contact history, clinic/hospital, week, and preventive measures. Absolute VE (aVE) was calculated for 2/3/4 doses, compared to the unvaccinated. Relative VE (rVE) was calculated, comparing 3 vs 2 and 4 vs 3 doses. RESULTS: 13,025 individuals were tested during the BA.1/BA.2-dominant and BA.5-dominant periods with similar baseline characteristics. For BA.1/BA.2, aVE was 52 % (95 %CI:34-66) 14 days-3 months post-dose 2, 42 % (29-52) > 6 months post-dose 2, 71 % (64-77) 14 days-3 months post-dose 3, and 68 % (52-79) 3-6 months post-dose 3. rVE was 49 % (38-57) 14 days-3 months post-dose 3 and 45 % (18-63) 3-6 months post-dose 3. For BA.5, aVE was 56 % (27-73) 3-6 months post-dose 2, 32 % (12-47) > 6 months post-dose 2, 70 % (61-78) 14 days-3 months post-dose 3, 59 % (48-68) 3-6 months post-dose 3, 50 % (29-64) > 6 months post-dose 3, and 74 % (61-83) ≥ 14 days post-dose 4. rVE was 56 % (45-65) 14 days-3 months post-dose 3, 39 % (27-48) 3-6 months post-dose 3, 25 % (-2-45) > 6 months post-dose 3, and 30 % (-6-54) ≥ 14 days post-dose 4. CONCLUSIONS: Booster doses initially provided high protection against BA.5 at a level similar to that against BA.1/BA.2. However, the protection seemed shorter-lasting against BA.5, which likely contributed to the surge. Furthermore, rVE post-dose 4 was low even among recent vaccinees. These results support the introduction of variant-containing vaccines and emphasize the need for vaccines with longer duration of protection.

Effectiveness of BA.1- and BA.4/BA. 5-Containing Bivalent COVID-19 mRNA Vaccines Against Symptomatic SARS-CoV-2 Infection During the BA.5-Dominant Period in Japan.

In this multicenter, prospective, test-negative, case-control study in Japan, the effectiveness of both BA.1-containing and BA.4/BA.5-containing bivalent coronavirus disease 2019 mRNA vaccines against symptomatic infection during the BA.5-dominant period was high compared with no vaccination (65% and 76%) and moderate compared with monovalent vaccines administered over half a year earlier (46% combined).

Coronavirus Disease 19 (COVID-19) Vaccine Effectiveness Against Symptomatic Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection During Delta-Dominant and Omicron-Dominant Periods in Japan: A Multicenter Prospective Case-control Study (Factors Associated with SARS-CoV-2 Infection and the Effectiveness of COVID-19 Vaccines Study).

BACKGROUND: Although several coronavirus disease 2019 (COVID-19) vaccines initially showed high efficacy, there have been concerns because of waning immunity and the emergence of variants with immune escape capacity. METHODS: A test-negative design case-control study was conducted in 16 healthcare facilities in Japan during the Delta-dominant period (August-September 2021) and the Omicron-dominant period (January-March 2022). Vaccine effectiveness (VE) against symptomatic severe acute respiratory syndrome coronavirus 2 infection was calculated for 2 doses for the Delta-dominant period and 2 or 3 doses for the Omicron-dominant period compared with unvaccinated individuals. RESULTS: The analysis included 5795 individuals with 2595 (44.8%) cases. Among vaccinees, 2242 (55.8%) received BNT162b2 and 1624 (40.4%) received messenger RNA (mRNA)-1273 at manufacturer-recommended intervals. During the Delta-dominant period, VE was 88% (95% confidence interval [CI], 82-93) 14 days to 3 months after dose 2 and 87% (95% CI, 38-97) 3 to 6 months after dose 2. During the Omicron-dominant period, VE was 56% (95% CI, 37-70) 14 days to 3 months since dose 2, 52% (95% CI, 40-62) 3 to 6 months after dose 2, 49% (95% CI, 34-61) 6+ months after dose 2, and 74% (95% CI, 62-83) 14+ days after dose 3. Restricting to individuals at high risk of severe COVID-19 and additional adjustment for preventive measures (ie, mask wearing/high-risk behaviors) yielded similar estimates, respectively. CONCLUSIONS: In Japan, where most are infection-naïve, and strict prevention measures are maintained regardless of vaccination status, 2-dose mRNA vaccines provided high protection against symptomatic infection during the Delta-dominant period and moderate protection during the Omicron-dominant period. Among individuals who received an mRNA booster dose, VE recovered to a high level.

Cerebral blood flow abnormalities with central sparing on arterial spin labeling in mild encephalopathy associated with excitotoxicity: a case report.

BACKGROUND: Acute encephalopathy with biphasic seizures and late reduced diffusion (AESD) and mild encephalopathy associated with excitotoxicity (MEEX) are the most frequent acute encephalopathies in pediatric patients in Japan. AESD typically presents with biphasic seizures and delayed reduced diffusion in the subcortical area, called bright tree appearance (BTA), on radiological examination. In patients with AESD, arterial spin labeling (ASL) shows decreased cerebral blood flow (CBF) in the hyperacute stage and increased CBF in the acute stage, suggesting the usefulness of ASL for the early diagnosis of AESD. Additionally, proton magnetic resonance spectroscopy (MRS) shows elevated glutamate (Glu) and glutamine (Gln) in AESD. MEEX is a group of mild encephalopathies with transient elevation of Gln on MRS similar to that in AESD; however, MEEX does not include any clinical biphasic course or abnormalities, including BTA on diffusion-weighted imaging. Although the usefulness of ASL for AESD has been reported, there are no reports for patients with MEEX. In this study, we report our experience with a 4-year-old girl diagnosed with MEEX who showed unique findings on ASL. CASE PRESENTATION: The patient was a 4-year-old girl admitted to the emergency room with febrile status epilepticus. Considering the possibility of AESD, vitamin therapy was initiated. ASL-MR imaging (MRI) of the brain performed on the second day showed increased blood flow in the frontal, temporal, and occipital regions with spared central sulcus, which indicated AESD with central sparing. The patient was diagnosed with AESD, and the treatment included pulse steroid therapy and immunoglobulin therapy from day 3. The patient remained mildly unconscious but gradually became conscious by day 7 with no seizures. Brain MRI performed on day 8 did not show any characteristic AESD findings, such as BTA. Furthermore, MRS showed elevated Gln, which, along with the clinical course, led to the diagnosis of MEEX. The patient was discharged on day 16 without obvious sequelae. CONCLUSIONS: ASL may be useful in the early diagnosis of MEEX as well as AESD, facilitating early intervention.

Behavioral factors associated with SARS-CoV-2 infection in Japan.

BACKGROUND: The relative burden of COVID-19 has been less severe in Japan. One reason for this may be the uniquely strict restrictions imposed upon bars/restaurants. To assess if this approach was appropriately targeting high-risk individuals, we examined behavioral factors associated with SARS-CoV-2 infection in the community. METHODS: This multicenter case-control study involved individuals receiving SARS-CoV-2 testing in June-August 2021. Behavioral exposures in the past 2 weeks were collected via questionnaire. SARS-CoV-2 PCR-positive individuals were cases, while PCR-negative individuals were controls. RESULTS: The analysis included 778 individuals (266 [34.2%] positives; median age [interquartile range] 33 [27-43] years). Attending three or more social gatherings was associated with SARS-CoV-2 infection (adjusted odds ratio [aOR] 2.00 [95% CI 1.31-3.05]). Attending gatherings with alcohol (aOR 2.29 [1.53-3.42]), at bars/restaurants (aOR 1.55 [1.04-2.30]), outdoors/at parks (aOR 2.87 [1.01-8.13]), at night (aOR 2.07 [1.40-3.04]), five or more people (aOR 1.81 [1.00-3.30]), 2 hours or longer (aOR 1.76 [1.14-2.71]), not wearing a mask during gatherings (aOR 4.18 [2.29-7.64]), and cloth mask use (aOR 1.77 [1.11-2.83]) were associated with infection. Going to karaoke (aOR 2.53 [1.25-5.09]) and to a gym (aOR 1.87 [1.11-3.16]) were also associated with infection. Factors not associated with infection included visiting a cafe with others, ordering takeout, using food delivery services, eating out by oneself, and work/school/travel-related exposures including teleworking. CONCLUSIONS: We identified multiple behavioral factors associated with SARS-CoV-2 infection, many of which were in line with the policy/risk communication implemented in Japan. Rapid assessment of risk factors can inform decision making.

SARS-CoV-2 Omicron-neutralizing memory B cells are elicited by two doses of BNT162b2 mRNA vaccine.

Multiple SARS-CoV-2 variants have mutations in the spike receptor binding domain (RBD) with potential to evade neutralizing antibody. In particular, the Beta and Omicron variants escape from antibody neutralizing activity in those who received two doses of BNT162b2 mRNA vaccine. Nonetheless, boosting with a third vaccine dose or by breakthrough infection improves the overall breadth of the neutralizing antibodies, but the mechanism remains unclear. Here, we longitudinally profiled the cellular composition of RBD-binding memory B cell subsets and their antibody binding and neutralizing activity against SARS-CoV-2 variants after the second dose of mRNA vaccine. Two doses of the mRNA vaccine elicited plasma neutralizing antibodies with a limited activity against Beta and Omicron but induced an expanded antibody breadth overtime, up to 4.9 months after vaccination. In contrast, more than one-third of RBD-binding IgG+ memory B cells with a resting phenotype initially bound the Beta and Omicron variants and steadily increased the B cell receptor breadth overtime. As a result, a fraction of the resting memory B cell subset secreted Beta and Omicron-neutralizing antibody when stimulated in vitro. The neutralizing breadth of the resting memory B cell subset helps us understand the prominent recall of Omicron-neutralizing antibodies after an additional booster or breakthrough infection in fully vaccinated individuals.

Leucine-rich alpha-2-glycoprotein 1 and angiotensinogen as diagnostic biomarkers for Kawasaki disease.

OBJECTIVE: Kawasaki disease (KD) is a systemic vasculitis in childhood that can lead to coronary artery lesions (CALs). Although early diagnosis and treatment is important for preventing KD patients from development of CALs, diagnosis depends on the clinical features of KD. We studied the usefulness of leucine-rich alpha-2-glycoprotein 1 (LRG1) and angiotensinogen (AGT), previously reported as KD-related proteins, for KD diagnosis and estimation of intravenous immunoglobulin (IVIG) efficacy. METHODS: We undertook a prospective cohort study with patients having two or more KD symptoms in multiple centers in Japan, between July 2017 and February 2019. RESULTS: Two hundred forty-two patients were included. In multivariable analysis, one unit increase in LRG1 was associated with higher odds of KD diagnosis (Odds ratio [OR] 1.02 [95% confidence interval (CI) 1.001-1.03]). Double-positivity for AGT (≥ 26 µg/mL) and LRG1 (≥ 123.5 µg/mL) was an independent biomarker for KD diagnosis in both the total cohort and the subgroup of patients with two to four KD symptoms (OR 5.01 [95% CI 1.86-13.50] and 3.71 [95% CI 1.23-11.16], respectively). There was no association between LRG1/AGT and IVIG efficacy. CONCLUSION: Double-positivity for LRG1 and AGT is an biomarker for KD diagnosis, especially useful in diagnosing incomplete KD from non-KD. Future studies with larger cohorts should seek to determine whether LRG1 and AGT are valuable as definitive data referred at the diagnosis of KD and for estimating the risk of CALs.

Publisher Correction: Clinical evaluation of fully automated molecular diagnostic system "Simprova" for influenza virus, respiratory syncytial virus, and human metapneumovirus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Clinical evaluation of fully automated molecular diagnostic system "Simprova" for influenza virus, respiratory syncytial virus, and human metapneumovirus.

Influenza virus, respiratory syncytial virus, and human metapneumovirus commonly cause acute upper and lower respiratory tract infections, especially in children and the elderly. Although rapid antigen detection tests for detecting these infections have been introduced recently, these are less sensitive than nucleic acid amplification tests. More recently, highly sensitive point-of-care testings (POCTs) have been developed based on nucleic acid amplification tests, which are easy to use in clinical settings. In this study, loop-mediated isothermal amplification (LAMP)-based POCT "Simprova" to detect influenza A and B viruses, respiratory syncytial virus, and human metapneumovirus was developed. Simprova system is fully automated and does not require skilled personnel. In addition, positive results can be achieved faster than with PCR. In this study, the accuracy of the POCT was retrospectively analyzed using 241 frozen stocked specimens. Additionally, the usability of the Simprova at clinical sites was assessed in a prospective clinical study using 380 clinical specimens and compared to those of real-time PCR and rapid antigen detection test. The novel LAMP-based POCT demonstrated high sensitivity and specificity in characterizing clinical specimens from patients with influenza-like illnesses. The Simprova is a powerful tool for early diagnosis of respiratory viral infections in point-of-care settings.

Neurogenic pulmonary edema following febrile status epilepticus in a 22-month-old infant with multiple respiratory virus co-detection: a case report.

BACKGROUND: Neurogenic pulmonary edema is a rare but serious complication of febrile status epilepticus in children. Comprehensive screening for viral pathogens is seldomly performed in the work-up of febrile children. CASE PRESENTATION: A 22-month-old girl presented with her first episode of febrile status epilepticus, after which she developed acute pulmonary edema and respiratory failure. After the termination of seizure activity, the patient was intubated and managed on mechanical ventilation in the emergency room. The resolution of respiratory failure, as well as the neurological recovery, was achieved 9 h after admission, and the patient was discharged 6 days after admission without any complications. Molecular biological diagnostic methods identified the presence of human coronavirus HKU1, influenza C virus, and human parainfluenza virus 2 from the patient's nasopharyngeal specimens. CONCLUSIONS: Neurogenic pulmonary edema following febrile status epilepticus was suspected to be the etiology of our patient's acute pulmonary edema and respiratory failure. Timely seizure termination and rapid airway and respiratory intervention resulted in favorable outcomes of the patient. Molecular biological diagnostic methods identified three respiratory viruses; however, their relevance and association with clinical symptoms remain speculative.

Aseptic meningitis caused by torque teno virus in an infant: a case report.

BACKGROUND: Torque teno virus-induced aseptic meningitis has not been documented, although torque teno virus infections still remain under consideration for etiological agents. This study identified a torque teno virus sequence using next generation sequencing and immunoglobulin M response to the torque teno virus antigen, therefore, that would be a comprehensive diagnosis for torque teno virus infection. CASE PRESENTATION: A 2-month-old Japanese boy was brought to our hospital because he was irritable, drowsy, and lethargic. He was admitted based on his test results which indicated the possibility of septic meningitis. He was started on treatment with high-dose antibiotics and steroids. On the third day of hospitalization, he became afebrile with improvement in his general status and was discharged on the sixth day. He had no developmental problems for up to 1 year after discharge. Metagenomic ribonucleic acid-Seq pathogen detection using next generation sequencing of a sample of his cerebrospinal fluid, which was collected at admission, revealed three short reads homologous to those in torque teno virus out of a total of 1,708,516 reads. This finding indicated that our patient was positive compared to the torque teno virus-negative cerebrospinal fluid samples (controls) from 13 other patients. The torque teno virus has been shown to have a whole genome sequence of 2810 nt by polymerase chain reaction. We prepared a recombinant GP2 antigen from torque teno virus and used it to study our patient's anti-torque teno virus immune response. An anti-GP2 serum immunoglobulin M response was detected, providing further supportive evidence of torque teno virus infection. CONCLUSIONS: This case speculates that torque teno virus-induced aseptic meningitis has a good course. New technologies like next generation sequencing can help in the identification of such cases, and an accumulation of future cases is expected.

Development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primer for influenza virus and respiratory syncytial virus.

Influenza virus and respiratory syncytial virus cause acute upper and lower respiratory tract infections, especially in children and the elderly. Early treatment for these infections is thought to be important, so simple and sensitive detection methods are needed for use at clinical sites. Therefore, in this study, real-time reverse transcription loop-mediated isothermal amplification assays with quenching primer for influenza virus and respiratory syncytial virus were developed. Evaluation of a total of 113 clinical specimens compared to real-time RT-PCR assays showed that the novel assays could distinguish between the types and subtypes of influenza virus and respiratory syncytial virus and had 100% diagnostic specificity. The diagnostic sensitivity of each assay exceeded 85.0% and the assays showed sufficient clinical accuracy. Furthermore, positive results could be obtained in around 15 min using the novel assays in cases with high concentrations of virus. The developed assays should be useful for identifying influenza virus and respiratory syncytial virus cases not only in experimental laboratories but also in hospital and quarantine laboratories.

Propagation of Rhinovirus C in Differentiated Immortalized Human Airway HBEC3-KT Epithelial Cells.

Rhinoviruses (RVs) are classified into three species: RV-A, B, and C. Unlike RV-A and -B, RV-C cannot be propagated using standard cell culture systems. In order to isolate RV-Cs from clinical specimens and gain a better understanding of their biological properties and pathogenesis, we established air⁻liquid-interface (ALI) culture methods using HBEC3-KT and HSAEC1-KT immortalized human airway epithelial cells. HBEC3- and HSAEC1-ALI cultures morphologically resembled pseudostratified epithelia with cilia and goblet cells. Two fully sequenced clinical RV-C isolates, RV-C9 and -C53, were propagated in HBEC3-ALI cultures, and increases in viral RNA ranging from 1.71 log10 to 7.06 log10 copies were observed. However, this propagation did not occur in HSAEC1-ALI cultures. Using the HBEC3-ALI culture system, 11 clinical strains of RV-C were isolated from 23 clinical specimens, and of them, nine were passaged and re-propagated. The 11 clinical isolates were classified as RV-C2, -C6, -C9, -C12, -C18, -C23, -C40, and -C53 types according to their VP1 sequences. Our stable HBEC3-ALI culture system is the first cultivable cell model that supports the growth of multiple RV-C virus types from clinical specimens. Thus, the HBEC3-ALI culture system provides a cheap and easy-to-use alternative to existing cell models for isolating and investigating RV-Cs.

Development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assays for rhinovirus detection.

Human rhinoviruses (RVs) belong to the genus Enterovirus of the family Picornaviridae, and are classified into RV-A, -B, and -C species. Two assays were developed to detect RVs by a real-time fluorescent reverse transcription loop-mediated isothermal amplification method: one was designed based on the 5'-untranslated regions (UTRs) of RV-A and -B, and the other was designed based on the 5'-UTR of RV-C. The competence of both assays for the diagnosis of RV infection was tested using isolated viruses and compared with real-time reverse transcription polymerase chain reaction assays on clinical specimens. Neither assay demonstrated cross-reactivity with other tested enteroviruses, and they detected 19 out of 21 tested RV-As and seven out of eight tested RV-Cs. The specificity of the assays was 100% for the detection of RVs and their sensitivity for RV-A and RV-C was 86.3% and 77.3%, respectively, on clinical specimens by the combined use of both assays. Considering that both developed assays were highly specific and detected the majority of recently circulating RVs, they are helpful for the diagnosis of RV infection. Consequently, the results generated by these assays will enhance the surveillance of respiratory illness and the study of the roles of RVs associated with clinical features and disease severity.

Development and evaluation of a new real-time RT-PCR assay for detecting the latest H9N2 influenza viruses capable of causing human infection.

The H9N2 subtype of avian influenza A viruses (AIV) has spread among domestic poultry and wild birds worldwide. H9N2 AIV is sporadically transmitted to humans from avian species. A total of 42 laboratory-confirmed cases of non-fatal human infection with the Eurasian Y280 and G1 lineages have been reported in China, Hong Kong, Bangladesh and Egypt since 1997. H9N2 AIV infections in poultry have become endemic in Asia and the Middle East and are a major source of viral internal genes for other AIV subtypes, such that continuous monitoring of H9N2 AIV is recommended. In this study, a new, one-step, real-time RT-PCR assay was developed to detect two major Eurasian H9 lineages of AIV capable of causing human infection. The sensitivity of this assay was determined using in vitro-transcribed RNA, and the detection limit was approximately 3 copies/reaction. In this assay, no cross-reactivity was observed against RNA from H1-15 subtypes of influenza A viruses, influenza B viruses and other viral respiratory pathogens. In addition, this assay could detect the H9 hemagglutinin (HA) gene from artificially reconstituted clinical samples spiked with H9N2 virus without any non-specific reactions. Therefore, this assay is highly sensitive and specific for H9 HA detection. The assay is useful both for diagnostic purposes in cases of suspected human infection with influenza H9N2 viruses and for the surveillance of both avian and human influenza viruses.