Functional Evaluation and Genetic Landscape of Children and Young Adults Referred for Assessment of Bronchiectasis.

Bronchiectasis is the abnormal dilation of the airway which may be caused by various etiologies in children. Beyond the more recognized cause of bacterial and viral infections and primary immunodeficiencies, other genetic conditions such as cystic fibrosis and primary ciliary dyskinesia (PCD) can also contribute to the disease. Currently, there is still debate on whether genome sequencing (GS) or exome sequencing reanalysis (rES) would be beneficial if the initial targeted testing results returned negative. This study aims to provide a back-to-back comparison between rES and GS to explore the best integrated approach for the functional and genetics evaluation for patients referred for assessment of bronchiectasis. In phase 1, an initial 60 patients were analyzed by exome sequencing (ES) with one additional individual recruited later as an affected sibling for ES. Functional evaluation of the nasal nitric oxide test, transmission electron microscopy, and high-speed video microscopy were also conducted when possible. In phase 2, GS was performed on 30 selected cases with trio samples available. To provide a back-to-back comparison, two teams of genome analysts were alternatively allocated to GS or rES and were blinded to each other's analysis. The time for bioinformatics, analysis, and diagnostic utility was recorded for evaluation. ES revealed five positive diagnoses (5/60, 8.3%) in phase 1, and four additional diagnoses were made by rES and GS (4/30, 13%) during phase 2. Subsequently, one additional positive diagnosis was identified in a sibling by ES and an overall diagnostic yield of 10/61 (16.4%) was reached. Among those patients with a clinical suspicion of PCD (n = 31/61), the diagnostic yield was 26% (n = 8/31). While GS did not increase the diagnostic yield, we showed that a variant of uncertain significance could only be detected by GS due to improved coverage over ES and hence is a potential benefit for GS in the future. We show that genetic testing is an essential component for the diagnosis of early-onset bronchiectasis and is most effective when used in combination with functional tools such as TEM or HSVM. Our comparison of rES vs. GS suggests that rES and GS are comparable in clinical diagnosis.

Assessment of SARS-CoV-2 Immunity in Convalescent Children and Adolescents.

Persistence of protective immunity for SARS-CoV-2 is important against reinfection. Knowledge on SARS-CoV-2 immunity in pediatric patients is currently lacking. We opted to assess the SARS-CoV-2 adaptive immunity in recovered children and adolescents, addressing the pediatrics specific immunity towards COVID-19. Two independent assays were performed to investigate humoral and cellular immunological memory in pediatric convalescent COVID-19 patients. Specifically, RBD IgG, CD4+, and CD8+ T cell responses were identified and quantified in recovered children and adolescents. SARS-CoV-2-specific RBD IgG detected in recovered patients had a half-life of 121.6 days and estimated duration of 7.9 months compared with baseline levels in controls. The specific T cell response was shown to be independent of days after diagnosis. Both CD4+ and CD8+ T cells showed robust responses not only to spike (S) peptides (a main target of vaccine platforms) but were also similarly activated when stimulated by membrane (M) and nuclear (N) peptides. Importantly, we found the differences in the adaptive responses were correlated with the age of the recovered patients. The CD4+ T cell response to SARS-CoV-2 S peptide in children aged <12 years correlated with higher SARS-CoV-2 RBD IgG levels, suggesting the importance of a T cell-dependent humoral response in younger children under 12 years. Both cellular and humoral immunity against SARS-CoV-2 infections can be induced in pediatric patients. Our important findings provide fundamental knowledge on the immune memory responses to SARS-CoV-2 in recovered pediatric patients.

Current Perspectives and Unmet Needs of Primary Immunodeficiency Care in Asia Pacific.

Background: The Asia Pacific Society for Immunodeficiencies (APSID) conducted nine primary immunodeficiency (PID) Schools in 5 years since inauguration to provide PID care training for early career physicians in Asia Pacific, a region with divergent needs in PID resources and training. Objective: To identify differences in PID patient care resource and training needs across Asia Pacific and propose a corresponding action plan. Methods: The Human Development Index (HDI) indicates the degree of socio-economic development in each country/region. Information related to investigations and learning issues were extracted from the abstracts and personal statements from all Schools and mapped onto resource and training needs. Correlations between HDI and country/region-specific parameters were tested by two-tailed Pearson correlation. Results: A total of 427 abstracts were received in nine Schools between 2015 and 2020, predominantly on immunodeficiencies affecting cellular and humoral immunity. Genetic confirmation was described in 61.8% of abstracts, and its absence negatively correlated with HDI (r = -0.696, p = 0.004). Essential immunologic and genetic tests were not available in 25.4 and 29.5% of abstracts, respectively, and their absence negatively correlated with HDI (r = -0.788, p < 0.001; r = -0.739, p = 0.002). HDI positively correlated with average testing level (r = 0.742, p = 0.002). Cases from medium-HDI countries/regions focused on learning how to investigate a patient for PIDs in cases of severe or atypical infections, whereas those from very-high-HDI countries/regions, from which most faculty members originated, listed hematopoietic stem cell transplantation and gene therapy, newborn screening, and research as learning issues more frequently. Conclusion: There are unique HDI-related PID resource and training needs in each country/region. APSID proposes HDI group-specific strategies to improve PID care and education in her member countries/regions. Further quantitative analysis of needs in PID care in Asia Pacific is needed for lobbying governments to increase their support for PID care and research.

Rapid whole-exome sequencing facilitates precision medicine in paediatric rare disease patients and reduces healthcare costs.

BACKGROUND: Rapid whole-exome sequencing (rWES) offers the potential for early diagnosis-predicated precision medicine. Previous evidence focused predominantly on infants from the intensive care unit (ICU). This study sought to examine the diagnostic and clinical utility, and the economic impact on clinical management of rWES in patients beyond infancy and ICU setting. METHODS: rWES was performed on a prospective cohort of patients with suspected monogenic disorder referred from territory-wide paediatric ICUs and non-ICUs in Hong Kong urging for rapid genetic diagnosis. All eligible families were invited. We aimed to achieve a rapid turnaround time (TAT) of 14 days. Clinical utility and costs associated with clinical management were assessed in diagnosed cases. Actual quantitative changes in healthcare utilisation were compared with a counterfactual diagnostic trajectory and/or with matched historical control whenever possible. FINDINGS: rWES were offered to 102 families and 32/102 (31%) patients received a molecular diagnosis, with a median TAT of 11 days. Clinical management changed in 28 of 32 diagnosed patients (88%), including but not limited to modifications in treatment, avoidance of surgeries, and informing decisions on redirection of care. Cost analysis was performed in eight patients. rWES was estimated to reduce hospital length of stay by 566 days and decrease healthcare costs by HKD$8,044,250 (GBP£796,460) for these eight patients. The net cost-savings after inclusion of rWES costs were estimated to be HKD$5,325,187 (GBP£527,246). INTERPRETATION: This study replicates the diagnostic capacity and rapid TAT of rWES in predominantly Chinese patients, and demonstrates diagnosis-predicated precision medicine and net healthcare savings. Findings were corroborated by evidence from multinational cohorts, combined as part of a meta-analysis. rWES merits consideration as a first-tier diagnostic tool for patients with urgent needs in the clinical setting. FUNDING: Health and Medical Research Fund, HKU Seed Fund for Basic Research, The Society for the Relief of Disabled Children, and Edward and Yolanda Wong Fund.

HPV vaccination in Hong Kong: uptake and reasons for non-vaccination amongst Chinese adolescent girls.

OBJECTIVES: The study aims to determine HPV vaccine uptake (≥ 1 dose) amongst adolescent girls in Hong Kong and to explore the reasons for non-acceptance of the vaccine. STUDY DESIGN: A total of 1832 secondary school girls (15.5 ± 2.0 years) were randomly surveyed. Their HPV vaccine uptake was estimated, and their reasons for non-vaccination summarised. RESULTS: A total of 131 (7.2%, 95% CI: 6.0-8.4%) adolescent girls had received the HPV vaccine (≥ 1 dose). Vaccine uptake was positively associated with a higher maternal education level and locally born status. Amongst the non-vaccinated girls, 20.6% had never heard of or knew little about the vaccine, 20.2% 'did not know where to receive', and 17.8% were concerned about the cost. CONCLUSIONS: The HPV vaccine uptake amongst adolescent girls in Hong Kong is very low. A school-based education and service programme is needed to improve uptake and prevent disparities in the Chinese population.

The infection attack rate and severity of 2009 pandemic H1N1 influenza in Hong Kong.

BACKGROUND: Serial cross-sectional data on antibody levels to the 2009 pandemic H1N1 influenza A virus from a population can be used to estimate the infection attack rates and immunity against future infection in the community. METHODS: From April through December 2009, we obtained 12,217 serum specimens from blood donors (aged 16-59 years), 2520 specimens from hospital outpatients (aged 5-59 years), and 917 specimens from subjects involved in a community pediatric cohort study (aged 5-14 years). We estimated infection attack rates by comparing the proportions of specimens with antibody titers ≥ 1:40 by viral microneutralization before and after the first wave of the pandemic. Estimates were validated using paired serum samples from 324 individuals that spanned the first wave. Combining these estimates with epidemiologic surveillance data, we calculated the proportion of infections that led to hospitalization, admission to the intensive care unit (ICU), and death. RESULTS: We found that 3.3% and 14% of persons aged 5-59 years had antibody titers ≥ 1:40 before and after the first wave, respectively. The overall attack rate was 10.7%, with age stratification as follows: 43.4% in persons aged 5-14 years, 15.8% in persons aged 15-19 years, 11.8% in persons aged 20-29 years, and 4%-4.6% in persons aged 30-59 years. Case-hospitalization rates were 0.47%-0.87% among persons aged 5-59 years. Case-ICU rates were 7.9 cases per 100,000 infections in persons aged 5-14 years and 75 cases per 100,000 infections in persons aged 50-59 years, respectively. Case-fatality rates were 0.4 cases per 100,000 infections in persons aged 5-14 years and 26.5 cases per 100,000 infections in persons aged 50-59 years, respectively. CONCLUSIONS: Almost half of all school-aged children in Hong Kong were infected during the first wave. Compared with school children aged 5-14 years, older adults aged 50-59 years had 9.5 and 66 times higher risks of ICU admission and death if infected, respectively.

In-depth cDNA library sequencing provides quantitative gene expression profiling in cancer biomarker discovery.

Quantitative gene expression analysis plays an important role in identifying differentially expressed genes in various pathological states, gene expression regulation and co-regulation, shedding light on gene functions. Although microarray is widely used as a powerful tool in this regard, it is suboptimal quantitatively and unable to detect unknown gene variants. Here we demonstrated effective detection of differential expression and co-regulation of certain genes by expressed sequence tag analysis using a selected subset of cDNA libraries. We discussed the issues of sequencing depth and library preparation, and propose that increased sequencing depth and improved preparation procedures may allow detection of many expression features for less abundant gene variants. With the reduction of sequencing cost and the emerging of new generation sequencing technology, in-depth sequencing of cDNA pools or libraries may represent a better and powerful tool in gene expression profiling and cancer biomarker detection. We also propose using sequence-specific subtraction to remove hundreds of the most abundant housekeeping genes to increase sequencing depth without affecting relative expression ratio of other genes, as transcripts from as few as 300 most abundantly expressed genes constitute about 20% of the total transcriptome. In-depth sequencing also represents a unique advantage of detecting unknown forms of transcripts, such as alternative splicing variants, fusion genes, and regulatory RNAs, as well as detecting mutations and polymorphisms that may play important roles in disease pathogenesis.

Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an important role in the induction and maintenance of immune tolerance. Although adoptive transfer of bulk populations of Treg can prevent or treat T cell-mediated inflammatory diseases and transplant allograft rejection in animal models, optimal Treg immunotherapy in humans would ideally use antigen-specific rather than polyclonal Treg for greater specificity of regulation and avoidance of general suppression. However, no robust approaches have been reported for the generation of human antigen-specific Treg at a practical scale for clinical use. Here, we report a simple and cost-effective novel method to rapidly induce and expand large numbers of functional human alloantigen-specific Treg from antigenically naive precursors in vitro using allogeneic nontransformed B cells as stimulators. By this approach naive CD4(+)CD25(-) T cells could be expanded 8-fold into alloantigen-specific Treg after 3 weeks of culture without any exogenous cytokines. The induced alloantigen-specific Treg were CD45RO(+)CCR7(-) memory cells, and had a CD4(high), CD25(+), Foxp3(+), and CD62L (L-selectin)(+) phenotype. Although these CD4(high)CD25(+)Foxp3(+) alloantigen-specific Treg had no cytotoxic capacity, their suppressive function was cell-cell contact dependent and partially relied on cytotoxic T lymphocyte antigen-4 expression. This approach may accelerate the clinical application of Treg-based immunotherapy in transplantation and autoimmune diseases.