Reconstructing household transmission dynamics to estimate the infectiousness of asymptomatic influenza virus infections.

There has long been controversy over the potential for asymptomatic cases of the influenza virus to have the capacity for onward transmission, but recognition of asymptomatic transmission of COVID-19 stimulates further research into this topic. Here, we develop a Bayesian methodology to analyze detailed data from a large cohort of 727 households and 2515 individuals in the 2009 pandemic influenza A(H1N1) outbreak in Hong Kong to characterize household transmission dynamics and to estimate the relative infectiousness of asymptomatic versus symptomatic influenza cases. The posterior probability that asymptomatic cases [36% of cases; 95% credible interval (CrI): 32%, 40%] are less infectious than symptomatic cases is 0.82, with estimated relative infectiousness 0.57 (95% CrI: 0.11, 1.54). More data are required to strengthen our understanding of the contribution of asymptomatic cases to the spread of influenza.

Assessing the longitudinal effects of the continuation and discontinuation of the school-located influenza vaccination programme on parental vaccine hesitancy in Hong Kong.

BACKGROUND: School-located influenza vaccination programme (SIVP) can effectively promote childhood seasonal influenza vaccination (SIV). However, the longitudinal effects of continuation and discontinuation of the SIVP on parents' vaccine hesitancy remained unknown. METHODS: A two-wave longitudinal study recruited adult parents who had at least one child attending a kindergarten or primary school using random-digital-dialled telephone interviews. Generalized estimating equation and structural equation modelling were used to examine the impact of changes in schools' SIVP participation status on parents' vaccine-related attitudes, and childhood SIV acceptance over 2 years in Hong Kong. RESULTS: Children's SIV uptake varied by the schools' SIVP participation status. The highest SIV uptake was found in schools that consistently participated in SIVP (Consistent participation group) (2018/2019: 85.0%; 2019/2020: 83.0%) but lowest in the Consistent non-Participation group (2018/2019: 45.0%; 2019/2020: 39.0%). SIV uptake increased in the Late Initiation group but declined in the Discontinuation group. An increasing trend of parental vaccine-hesitant attitudes was observed in the Consistent non-Participation group. CONCLUSIONS: Initiation and continuation of the SIVP can reduce parental vaccine hesitancy to achieve a high childhood SIV uptake. Conversely, discontinuation of the SIVP or persistent resistance to the implementation of SIVP can increase parental vaccine hesitancy and reduce childhood SIV uptake.

Effectiveness of BNT162b2 and CoronaVac COVID-19 vaccination against asymptomatic and symptomatic infection of SARS-CoV-2 omicron BA.2 in Hong Kong: a prospective cohort study.

BACKGROUND: COVID-19 vaccines provide protection against symptomatic infection that might require medical attention and against severe outcomes; however, there is a paucity of evidence regarding the effectiveness of the BNT162b2 and CoronaVac vaccines and their booster regimens against asymptomatic or mild omicron infections in the community. We aimed to measure the effectiveness of BNT162b2 and CoronaVac vaccines against asymptomatic and symptomatic SARS-CoV-2 omicron infections, during a period of omicron BA.2 predominance in Hong Kong. METHODS: In this prospective cohort study in a population that was generally infection-naive before the large omicron BA.2 wave between January and late May, 2022, we established a public health surveillance platform to monitor the evolving activity of SARS-CoV-2 infections in the community. We recruited a cohort of individuals aged 5 years and older between March 1 and March 7, 2022, from the general population. Individuals were enrolled from all 18 districts of Hong Kong, according to a predefined age-stratified quota, primarily by random digit dialing (generating suitable eight-digit local telephone numbers by randomly picking sets of the first four digits from a sampling frame, and randomly generating the last four digits), and supplemented by our existing cohorts (which included cohorts for studying influenza vaccination from school-based vaccination programmes and cohorts for SARS-CoV-2 seroprevalence from the community), to ensure representativeness of the population in Hong Kong. Participants did weekly rapid antigen testing with a self-collected pooled nasal and throat swab, regardless of symptom and exposure status, from March 1 to April 15, 2022. Individuals reporting a history of SARS-CoV-2 infection confirmed by laboratory PCR testing before enrolment were excluded from the vaccine effectiveness analysis to avoid potential bias due to infection-induced immunity. The primary outcomes of the study were the incidence of SARS-CoV-2 infection, including asymptomatic and symptomatic infections, and the vaccine effectiveness of BNT162b2 and CoronaVac vaccines. The effectiveness of one, two, and three doses of vaccination was estimated with a Cox proportional hazards regression model with time-dependent covariates, allowing for changes in vaccination status over time, after adjustment for demographic factors and pre-existing medical conditions. FINDINGS: Of the 8636 individuals included in the analysis, 7233 (84%) received at least two doses of vaccine, 3993 (46%) received booster doses, and 903 (10%) reported SARS-CoV-2 infection. Among these infections 589 (65·2%) were symptomatic and 314 (34·8%) were asymptomatic at the time of testing. Statistically significant protection against asymptomatic and symptomatic SARS-CoV-2 omicron infection was found only for those who received a BNT162b2 or CoronaVac booster dose, with a vaccine effectiveness of 41·4% (23·2 to 55·2; p=0·0001) and 32·4% (9·0 to 49·8; p=0·0098), respectively. The vaccine effectiveness of BNT162b2 and CoronaVac boosters was further increased to 50·9% (95% CI 31·0-65·0; p<0·0001) and 41·6% (15·0-59·8; p=0·0049), respectively, for symptomatic omicron infections. A similar pattern of vaccine effectiveness (55·8%, 22·9-74·6; p=0·0040) was also conferred after receipt of a BNT162b2 booster by individuals who received a CoronaVac primary vaccination series. INTERPRETATION: Two doses of either vaccine did not provide significant protection against COVID-19 infection. However, receipt of a BNT162b2 booster or CoronaVac booster was associated with a significantly lower risk of omicron BA.2 infection and symptomatic infection. Our findings confirm the effectiveness of booster doses to protect against mild and asymptomatic infection. FUNDING: Henry Fok Foundation and Hong Kong Health Bureau.

MicroRNAs in extracellular vesicles released from epicardial adipose tissue promote arrhythmogenic conduction slowing.

Background: Patients with excess epicardial adipose tissue (EAT) are at increased risk of developing cardiac arrhythmias. EAT promotes arrhythmias by depolarizing the resting membrane of cardiomyocytes, which slows down conduction and facilitates re-entrant arrhythmias. We hypothesized that EAT slows conduction by secreting extracellular vesicles (EVs) and their microRNA (miRNA) cargo. Objective: We aimed to determine the role of EAT-derived EVs and their miRNA cargo in conduction slowing. Methods: EAT and subcutaneous adipose tissue (SAT) were collected from patients with atrial fibrillation. Adipose tissue explants were incubated in culture medium and secretome was collected. The numbers of EVs in the EAT and SAT secretome were measured by calibrated flow cytometry. EVs in the EAT secretome were isolated by size exclusion chromatography and miRNAs were sequenced. Pathway analysis was performed to predict candidates involved in cardiac electrophysiology. The candidates were validated in the EAT and SAT by quantitative real-time polymerase chain reaction. Finally, miRNA candidates were overexpressed in neonatal rat ventricular myocytes. Results: The EV concentration was higher in the EAT secretome than in the SAT and control secretomes. miRNA sequencing of EAT-derived EVs detected a total of 824 miRNAs. Pathway analysis led to the identification of 7 miRNAs potentially involved in regulation of cardiac resting membrane potential. Validation of those miRNA candidates showed that they were all expressed in EAT, and that miR-1-3p and miR-133a-3p were upregulated in EAT in comparison with SAT. Overexpression of miR-1-3p and miR-133a-3p in neonatal rat ventricular myocytes led to conduction slowing and reduced Kcnj2 and Kcnj12 expression. Conclusion: miR-1-3p and miR-133a-3p are potential mediators of EAT arrhythmogenicity.

Indirect Protection from Vaccinating Children against Influenza A Virus Infection in Households.

Influenza vaccination is an important intervention to prevent influenza virus infection. Our previous analysis suggested that indirect protection is limited in an influenza B epidemic in Hong Kong. We further analyzed six influenza A epidemics to determine such potential. We applied a statistical model to estimate household transmission dynamics in the 3 influenza A(H3N2) and 3 pandemic influenza A(H1N1) epidemics. Then, we estimated the reduction in infection risk among unvaccinated household members when all children in households are vaccinated, with different assumptions on vaccine efficacy (VE). In the optimal scenario that VE was 70%, the reduction to the total probability of infection was only marginal, with relative probabilities ranged from 0.91-0.94 when all children in households were vaccinated because community was by far the main source of infection during the six epidemics in our study. The proportion of cases attributed to household transmission was 10% (95% CrI: 7%, 13%). Individual influenza vaccination is important even when other household members are vaccinated, given the degree of indirect protection is small.

Performance of saline and water gargling for SARS-CoV-2 reverse transcriptase PCR testing: a systematic review and meta-analysis.

The performance of gargling for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcriptase (RT)-PCR testing has not been previously reviewed. This review systematically assessed the performance of saline and water gargling for SARS-CoV-2 RT-PCR testing in the settings of diagnosing and monitoring viral shedding.We included original studies comparing the performance of gargling and (oropharyngeal-)nasopharyngeal swabs for SARS-CoV-2 RT-PCR testing. Studies conducted in either suspected individuals or confirmed cases were included and analysed separately. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were examined using random-effects models.Gargles achieved a high overall sensitivity (91%), specificity (97%), PPV (95%) and NPV (91%) for SARS-CoV-2 RT-PCR testing. Studies using saline gargle and water gargle have an overall sensitivity of 97% and 86%, respectively. The sensitivity values were largely maintained for saline and water gargling on stratified analysis, for both diagnosis (96% and 92%) and viral shedding monitoring (98% and 78%). A higher sensitivity was also reported by studies using sterile saline (100%), a smaller amount of gargling solution (92% versus 87%) and a longer gargling duration (95% versus 86%).Our results supported the use of gargling as a sampling approach for SARS-CoV-2 RT-PCR testing, which achieved a high sensitivity for both diagnosis and viral shedding monitoring purposes. Further investigation on the comparative performance of different gargling mediums is needed to draw a definitive conclusion.

Extracellular vesicles from amniotic fluid, milk, saliva, and urine expose complexes of tissue factor and activated factor VII.

BACKGROUND: Tissue factor (TF) is expressed in the adventitia of the vessel wall and on extracellular vesicles (EVs) in body fluids. TF and activated coagulation factor (F) VII(a) together form the so-called extrinsic tenase complex, which initiates coagulation. AIM: We investigated whether EVs in amniotic fluid, milk, saliva, and urine expose functional extrinsic tenase complexes that can trigger coagulation. METHODS: Milk, saliva, and urine were collected from healthy breastfeeding women (n = 6), and amniotic fluid was collected from healthy women undergoing routine amniocentesis (n = 7). EVs were isolated from body fluids by size exclusion chromatography (SEC) and clotting experiments were performed in the presence and absence of antibodies against TF and FVIIa in normal plasma and in FVII-deficient plasma. The ability of body fluids to generate FXa also was determined. RESULTS: Amniotic fluid, milk, saliva, and urine triggered clotting of normal plasma and of FVII-deficient plasma, which was almost completely inhibited by an anti-FVII antibody and to a lesser extent by an anti-TF antibody. Fractionation of body fluids by SEC showed that only the fractions containing EVs triggered clotting in normal plasma and FVII-deficient plasma and generated FXa, which again was almost completely inhibited by an anti-FVII antibody and partially by an anti-TF antibody. CONCLUSION: Here we show that EVs from amniotic fluid, milk, saliva, and urine expose complexes of TF and FVIIa (i.e., extrinsic tenase complexes) that directly activate FX. Based on our present findings we propose that these EVs from normal body fluids provide hemostatic protection.

Protocol for Measuring Concentrations of Extracellular Vesicles in Human Blood Plasma with Flow Cytometry.

Extracellular vesicles (EVs) are lipid membrane enclosed particles that are released from cells into body fluids, such as blood. EVs offer potential new biomarkers of diseases, because the cellular origin, composition, concentration, and function of EVs change in health and disease. The concentration of EVs from specific cell types in blood can be determined with flow cytometry. A flow cytometer measures fluorescence and light scattering signals from single EVs, but only if these signals are sufficiently bright to be detected. Measured concentrations of EVs are therefore only reproducible and comparable if the detection ranges are known and reported in standard units, such as molecules of equivalent soluble fluorophore (MESF) for fluorescence signals and the diameter in nm for scatter signals. The goal of this protocol is to discuss all steps needed to derive the concentration of cell-type specific EVs within a known diameter range and fluorescence range. More specifically, this protocol describes how to determine the concentration of CD61+ (Integrin beta-3, platelet marker), CD235a+ (Glycophorin A, erythrocyte marker), and CD45+ (leukocyte common antigen) EVs in human blood plasma with an Apogee A60-Micro flow cytometer using scatter-based triggering. The principles behind this protocol could lay a firm basis for the design of a protocol suitable for other flow cytometers and body fluids.

Reconstructing antibody dynamics to estimate the risk of influenza virus infection.

For >70 years, a 4-fold or greater rise in antibody titer has been used to confirm influenza virus infections in paired sera, despite recognition that this heuristic can lack sensitivity. Here we analyze with a novel Bayesian model a large cohort of 2353 individuals followed for up to 5 years in Hong Kong to characterize influenza antibody dynamics and develop an algorithm to improve the identification of influenza virus infections. After infection, we estimate that hemagglutination-inhibiting (HAI) titers were boosted by 16-fold on average and subsequently decrease by 14% per year. In six epidemics, the infection risks for adults were 3%-19% while the infection risks for children were 1.6-4.4 times higher than that of younger adults. Every two-fold increase in pre-epidemic HAI titer was associated with 19%-58% protection against infection. Our inferential framework clarifies the contributions of age and pre-epidemic HAI titers to characterize individual infection risk.

A Mixed-Methods Study to Evaluate Elementary School Staff's Acceptability, Delivery Challenges, and Communication Regarding the Implementation of School-Located Influenza Vaccination Program in Hong Kong.

This was a mixed-methods study comprising a questionnaire-based survey, a qualitative study, and analysis of school newsletters to evaluate elementary school staff's acceptability, delivery challenges and communication about school-located influenza vaccination program (SIVP) in Hong Kong. We found that school staff with lower intention to implement SIVP perceived greater logistical difficulties in arranging SIVP. Challenges regarding program delivery included schools' limited infrastructure, the burden of paperwork, the fear of being overwhelmed by multiple school-based vaccination schedules, lacking confidence in communicating with parents about influenza vaccines, and the difficulties in managing vaccination-related anxiety among children with intellectual disability. School staff were generally passive in communicating with parents and students about influenza vaccines. We also found that schools may use the school newsletters as a substitute of the formal informed consent forms. Good partnerships among government, service providers and schools should be established to minimize the burden of paperwork for school staff, facilitate early planning of SIVP, and support schools with limited infrastructure and the vaccination of children with intellectual disabilities. Training is needed to enhance school staff's confidence in communicating with parents and students about influenza vaccines and improve information delivery to support parents' informed decisions for children's vaccination.

Diagnostic performance of different sampling approaches for SARS-CoV-2 RT-PCR testing: a systematic review and meta-analysis.

BACKGROUND: The comparative performance of different clinical sampling methods for diagnosis of SARS-CoV-2 infection by RT-PCR among populations with suspected infection remains unclear. This meta-analysis aims to systematically compare the diagnostic performance of different clinical specimen collection methods. METHODS: In this systematic review and meta-analysis, we systematically searched PubMed, Embase, MEDLINE, Web of Science, medRxiv, bioRxiv, SSRN, and Research Square from Jan 1, 2000, to Nov 16, 2020. We included original clinical studies that examined the performance of nasopharyngeal swabs and any additional respiratory specimens for the diagnosis of SARS-CoV-2 infection among individuals presenting in ambulatory care. Studies without data on paired samples, or those that only examined different samples from confirmed SARS-CoV-2 cases were not useful for examining diagnostic performance of a test and were excluded. Diagnostic performance, including sensitivity, specificity, positive predictive value, and negative predictive value, was examined using random effects models and double arcsine transformation. FINDINGS: Of the 5577 studies identified in our search, 23 studies including 7973 participants with 16 762 respiratory samples were included. Respiratory specimens examined in these studies included 7973 nasopharyngeal swabs, 1622 nasal swabs, 6110 saliva samples, 338 throat swabs, and 719 pooled nasal and throat swabs. Using nasopharyngeal swabs as the gold standard, pooled nasal and throat swabs gave the highest sensitivity of 97% (95% CI 93-100), whereas lower sensitivities were achieved by saliva (85%, 75-93) and nasal swabs (86%, 77-93) and a much lower sensitivity by throat swabs (68%, 35-94). A comparably high positive predictive value was obtained by pooled nasal and throat (97%, 90-100) and nasal swabs (96%, 87-100) and a slightly lower positive predictive value by saliva (93%, 88-97). Throat swabs have the lowest positive predictive value of 75% (95% CI 45-96). Comparably high specificities (range 97-99%) and negative predictive value (range 95-99%) were observed among different clinical specimens. Comparison between health-care-worker collection and self-collection for pooled nasal and throat swabs and nasal swabs showed comparable diagnostic performance. No significant heterogeneity was observed in the analysis of pooled nasal and throat swabs and throat swabs, whereas moderate to substantial heterogeneity (I2 ≥30%) was observed in studies on saliva and nasal swabs. INTERPRETATION: Our review suggests that, compared with the gold standard of nasopharyngeal swabs, pooled nasal and throat swabs offered the best diagnostic performance of the alternative sampling approaches for diagnosis of SARS-CoV-2 infection in ambulatory care. Saliva and nasal swabs gave comparable and very good diagnostic performance and are clinically acceptable alternative specimen collection methods. Throat swabs gave a much lower sensitivity and positive predictive value and should not be recommended. Self-collection for pooled nasal and throat swabs and nasal swabs was not associated with any significant impairment of diagnostic accuracy. Our results also provide a useful reference framework for the proper interpretation of SARS-CoV-2 testing results using different clinical specimens. FUNDING: Hong Kong Research Grants Council.

Cellular origin and microRNA profiles of circulating extracellular vesicles in different stages of diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is a major complication of diabetes and the main cause of end-stage renal disease. Extracellular vesicles (EVs) are small cell-derived vesicles that can alter disease progression by microRNA (miRNA) transfer. METHODS: In this study, we aimed to characterize the cellular origin and miRNA content of EVs in plasma samples of type 2 diabetes patients at various stages of DN. Type 2 diabetes patients were classified in three groups: normoalbuminuria, microalbuminuria and macroalbuminuria. The concentration and cellular origin of plasma EVs were measured by flow cytometry. A total of 752 EV miRNAs were profiled in 18 subjects and differentially expressed miRNAs were validated. RESULTS: Diabetic patients with microalbuminuria and/or macroalbuminuria showed elevated concentrations of total EVs and EVs from endothelial cells, platelets, leucocytes and erythrocytes compared with diabetic controls. miR-99a-5p was upregulated in macroalbuminuric patients compared with normoalbuminuric and microalbuminuric patients. Transfection of miR-99a-5p in cultured human podocytes downregulated mammalian target of rapamycin (mTOR) protein expression and downregulated the podocyte injury marker vimentin. CONCLUSIONS: Type 2 diabetes patients with microalbuminuria and macroalbuminuria display differential EV profiles. miR-99a-5p expression is elevated in EVs from macroalbuminuria and mTOR is its validated mRNA target.

Platelet removal by single-step centrifugation.

The study of extracellular vesicles (EVs) in plasma requires removal of cells including platelets. At present, a two-step centrifugation protocol is recommended and commonly used. A simpler protocol that is less operator dependent is likely to improve the quality of plasma samples collected for EV research. The objective of this study is to develop an easy, fast and clinically applicable centrifugation protocol to produce essentially platelet-free plasma with a high yield for EV research. We compared the two-step centrifugation protocol to a single-step protocol at 5,000 g for 20 minutes. The removal of platelets was computationally predicted and experimentally validated. Flow cytometry was used to detect residual platelets and platelet-derived (CD61+) EVs. The single-step protocol at 5,000 g (i) is less laborious and approximately ten minutes faster, (ii) removes platelets as effective as the two-step centrifugation protocol, and (iii) has a ~ 10% higher plasma yield, whereas (iv) the recovery of platelet-derived EVs is comparable. For future research on plasma EVs we recommend the newly developed, easy and fast single-step protocol for preparation of platelet-free plasma for research on plasma biomarkers including EVs.

Human milk triggers coagulation via tissue factor-exposing extracellular vesicles.

Almost a century ago, it was discovered that human milk activates the coagulation system, but the milk component that triggers coagulation had until now been unidentified. In the present study, we identify this component and demonstrate that extracellular vesicles (EVs) present in normal human milk expose coagulant tissue factor (TF). This coagulant activity withstands digestive conditions, mimicking those of breastfed infants, but is sensitive to pasteurization of pooled donor milk, which is routinely used in neonatal intensive care units. In contrast to human milk, bovine milk, the basis of most infant formulas, lacks coagulant activity. Currently, the physiological function of TF-exposing vesicles in human milk is unknown, but we speculate that these vesicles may be protective for infants. Another explanation could be nipple skin damage, which occurs in most breastfeeding women. Milk-derived TF-exposing EVs may seal the wound and thereby reduce bleeding and breast inflammation.

Rate zonal centrifugation can partially separate platelets from platelet-derived vesicles.

BACKGROUND: Centrifugation is commonly used as a first step to enrich biomarkers from blood. Biomarkers are separated on the basis of density and/or diameter. However, the centrifugation protocol affects the yield and purity of biomarkers, for example, isolation of platelets results in co-isolation with extracellular vesicles (EVs). OBJECTIVE: To assess the ability of rate zonal centrifugation (RZC) to separate platelets from co-isolated EVs. METHODS: Using a linear Optiprep gradient, RZC was able to separate a mixture of beads with different diameters but similar density. Next, RZC was applied to samples containing both platelets and platelet-derived EVs (n = 3). After RZC, all fractions were collected and stained with anti-CD61-Alexa 488 to measure the concentrations of platelets and platelet-derived EVs by flow cytometry. RESULTS: We confirm that RZC separates polystyrene beads with diameters of 140 nm, 380 nm and 1,000 nm. Next, we show that the majority of platelets occur in fractions 8-19, whereas the majority of platelet-derived EVs are detectable in fractions 1-7. Furthermore, each fraction contains a different diameter range of platelets, which suggests that separation is indeed diameter based. CONCLUSION: RZC can partially separate platelets from EVs.

Comparative Reactogenicity of Enhanced Influenza Vaccines in Older Adults.

BACKGROUND: We analyzed data from a randomized controlled trial on the reactogenicity of 3 enhanced influenza vaccines compared with standard-dose (SD) inactivated influenza vaccine. METHODS: We enrolled community-dwelling older adults in Hong Kong, and we randomly allocated them to receive 2017-2018 northern hemisphere formulations of SD vaccine (FluQuadri; Sanofi Pasteur), MF59-adjuvanted vaccine (FLUAD; Seqirus), high-dose (HD) vaccine (Fluzone High-Dose; Sanofi Pasteur), or recombinant hemagglutinin vaccine (Flublok; Sanofi Pasteur). Local and systemic reactions were evaluated at days 1, 3, 7, and 14 after vaccination. RESULTS: Reported reactions were generally mild and short-lived. Systemic reactions occurred in similar proportions of participants by vaccine. Some local reactions were slightly more frequently reported among recipients of the MF59-adjuvanted and HD vaccines than among SD vaccine recipients. Participants reporting feverishness 1 day after vaccination had mean fold rises in postvaccination hemagglutination inhibition titers that were 1.85-fold higher (95% confidence interval, 1.01-3.38) for A(H1N1) than in those who did not report feverishness. CONCLUSIONS: Some acute local reactions were more frequent after vaccination with MF59-adjuvanted and HD influenza vaccines, compared with SD inactivated influenza vaccine, whereas systemic symptoms occurred at similar frequencies in all groups. The association between feverishness and immunogenicity should be further investigated in a larger population. CLINICAL TRIALS REGISTRATION: NCT03330132.