Validation of an automated, end-to-end metagenomic sequencing assay for agnostic detection of respiratory viruses.

BACKGROUND: Current molecular diagnostics are limited in the number and type of detectable pathogens. Metagenomic next generation sequencing (mNGS) is an emerging, and increasingly feasible, pathogen-agnostic diagnostic approach. Translational barriers prohibit the widespread adoption of this technology in clinical laboratories. We validate an end-to-end mNGS assay for detection of respiratory viruses. Our assay is optimized to reduce turnaround time, lower cost-per-sample, increase throughput, and deploy secure and actionable bioinformatic results. METHODS: We validated our assay using residual nasopharyngeal swab specimens from Vancouver General Hospital (n = 359), RT-PCR-positive, or negative for Influenza, SARS-CoV-2, and RSV. We quantified sample stability, assay precision, the effect of background nucleic acid levels, and analytical limits of detection. Diagnostic performance metrics were estimated. RESULTS: We report that our mNGS assay is highly precise, semi-quantitative, with analytical limits of detection ranging from 103-104 copies/mL. Our assay is highly specific (100%) and sensitive (61.9% Overall: 86.8%; RT-PCR Ct < 30). Multiplexing capabilities enable processing of up to 55-specimens simultaneously on an Oxford Nanopore GridION device, with results reported within 12-hours. CONCLUSIONS: This study outlines the diagnostic performance and feasibility of mNGS for respiratory viral diagnostics, infection control, and public health surveillance. We addressed translational barriers to widespread mNGS adoption.

Successful haploidentical hematopoietic stem cell transplantation (HSCT) and durable engraftment by repeated donor lymphocyte infusions for a Chinese patient with transfusion-dependent hemoglobin (Hb) Hammersmith and massive splenomegaly.

BACKGROUND: Hemoglobin (Hb) Hammersmith is a rare form of unstable ß-chain hemoglobinopathy causing hemolytic anemia. This rare event led to a more serious transfusion-dependent phenotype in a patient. It was successfully cured by haploidentical hematopoietic stem cell transplantation (HSCT). METHODS AND RESULTS: A 9-year-old mainland Chinese male with a history of neonatal unconjugated hyperbilirubinemia was diagnosed to have hemoglobin (Hb) Hammersmith. He required regular blood transfusion but was unable to be transfused to desired parameters for 8 years prior to transplant due to social and geographical reasons. He subsequently developed marrow hyperplasia and progressive splenomegaly (down to umbilicus level), suggestive of extramedullary hematopoiesis. Eventually, the family came to Hong Kong and complied to a more intensive transfusion regimen and preconditioning chemotherapy 3 months prior to transplant. He underwent haploidentical HSCT using paternal TCRαß/CD45RA-depleted graft but suffered from graft rejection, despite splenic irradiation for massive splenomegaly. It was successfully salvaged with second HSCT with unmanipulated graft from the same donor with additional serotherapy and donor lymphocyte infusions. CONCLUSION: Allogenic haploidentical HSCT for hemoglobin Hammersmith is feasible but adequate immunosuppression during conditioning is crucial. Precise adoptive cell therapy can promote durable engraftment.

Epidemiology and outcomes of pediatric transplant-associated thrombotic microangiopathy in Hong Kong.

BACKGROUND: Transplant-associated thrombotic microangiopathy (TA-TMA) is an under-recognized yet potentially devastating complication of hematopoietic stem cell transplantation (HSCT) which had increased awareness in recent years. This report summarizes the demographics and outcomes of pediatric TA-TMA in Hong Kong. METHODS: All patients aged below 18 years who underwent HSCT in the Hong Kong Children's Hospital and were diagnosed to have TA-TMA during the 2-year period from April 1, 2019 to March 31, 2021 were included. RESULTS: A total of 73 transplants (51 allogeneic and 22 autologous) in 63 patients had been performed. Six patients (four males and two females) developed TA-TMA at a median duration of 2.5 months post-HSCT. The incidence rate was 9.52%. Of the six TA-TMA patients, five underwent allogenic one underwent autologous HSCT, respectively. Three of them were histologically proven. All four patients with cyclosporine had stopped the drug once TA-TMA was suspected. Median six doses of eculizumab were administered to five out of six patients. Three patients died (two due to fungal infection and one due to acute-on-chronic renal failure) within 3 months upon diagnosis of TA-TMA. Among three survivors, two stabilized with mild stage 2 chronic kidney disease (CKD) while the other suffered from stage 5 end-stage CKD requiring lifelong dialysis. CONCLUSION: In conclusion, recognition and diagnosis of TA-TMA are challenging. Early recognition and prompt administration of complement blockage with eculizumab may be beneficial in selected cases. Further prospective research studies are recommended to improve the management and outcomes of TA-TMA.

Minimally Invasive Management of Second Trimester Placenta Percreta.

STUDY OBJECTIVE: To describe the diagnostic and surgical challenges in the management of second trimester placenta percreta. DESIGN: Stepwise demonstration of the surgical technique with the use of an educational video. SETTING: Second trimester placenta percreta is a rare entity, with very few case reports in the literature. Our video demonstrates the challenges of a minimally invasive approach toward definitive surgical management with hysterectomy. A 39-year-old G7P3 (3 previous cesarean deliveries) female at 17 weeks and 2 days gestation presented with acute abdominal pain to a community hospital. This was a spontaneously conceived pregnancy. Her hemoglobin level on admission was 92 g/L. An ultrasound showed a normal uterus, and the appendix was not visualized. One unit of packed red blood cells was transfused, and she underwent exploratory laparoscopy for a possible retrocecal hematoma/mass seen on computerized tomography. In the operating room, acute hemoperitoneum was visualized with placenta-like tissue invading through the anterior lower uterine segment (Figures 2 & 3). A hemostatic agent (Floseal, Baxter) was placed over the bleeding, and she was then transferred to a tertiary academic center for further management. INTERVENTIONS: Magnetic resonance imaging was performed on the following day after transfer to our facility, which confirmed placenta percreta at the level of the bladder (Figure 1). Following counseling with a multidisciplinary team and given that there was ongoing bleeding from the invading placental tissue, pregnancy continuation and uterine conservation were not possible. The patient was offered preprocedure termination of pregnancy with intra-cardiac injection of potassium chloride and 350 cc of amniotic fluid was drained at that time. This was done to facilitate visualization for a minimally invasive approach. We describe 5 main challenges of minimally invasive hysterectomy for placental percreta and provide a stepwise approach to mitigating them: visibility, vascular control, bladder dissection, colpotomy, and specimen retrieval. We adapted the previously described laparotomy techniques of progressive uterine devascularization and approach to bladder dissection and colpotomy to laparoscopy [1,2]. In addition, we performed dilatation and evacuation to allow for vaginal specimen removal. The patient's postoperative course was uncomplicated, and she was discharged home in a stable condition. CONCLUSION: Midtrimester placenta percreta poses significant challenges in diagnosis and surgical management. Total laparoscopic hysterectomy for this condition poses unique challenges but is feasible and safe.

A Purse-String Approach to Laparoscopic Cornuotomy for Interstitial Ectopic Pregnancy.

An interstitial ectopic refers to the implantation of a pregnancy in the proximal fallopian tube where it passes through the myometrium. This type of ectopic pregnancy presents a distinct surgical challenge, as it often presents with rupture and carries a significant risk of hemorrhage at resection. This video demonstrates a four-step approach to the resection of an interstitial ectopic pregnancy with laparoscopic cornuotomy. This approach includes (1) isolating the pregnancy by performing a salpingectomy and identifying the utero-ovarian ligament; (2) ensuring hemostasis with the injection of vasopressin, followed by application of the purse string suture around the pregnancy at its equatorial line; (3) performing the resection using a linear incision; and (4) repairing the uterine defect with layered closure. The purse-string suture is shown to be a useful tool in minimizing bleeding, and this sequential approach allows for interstitial ectopic pregnancies to be excised with a minimally invasive cornuotomy, even in cases of significant anatomical distortion.

Repeated CD45RA-depleted DLI successfully increases donor chimerism in a patient with beta-thalassemia major after haploidentical stem cell transplant.

Allogeneic hematopoietic stem cell transplantation is curative for transfusion-dependent thalassemia, but mixed chimerism (MC) may herald graft rejection. We report a child who failed bone marrow transplant (BMT) from matched unrelated donor (MUD) successfully salvaged with haploidentical peripheral blood stem cell transplant (PBSCT), but had MC in T-lymphocyte compartment despite near-complete donor chimerism in myeloid compartment. MC was successfully improved by repeated CD45RA-depleted donor lymphocyte infusion (DLI). A 2-year-old Chinese girl with beta-thalassemia major underwent 12/12-MUD BMT with HU/AZA/Cy/Flu/Bu/TT conditioning resulted in graft rejection. As donor refused second donation, rescue haploidentical PBSCT was performed with alemtuzumab/fludarabine/treosulfan conditioning. Harvest product was CD3/CD45RA depleted with extra products cryopreserved. Split cell chimerism performed 1-month after haplo-transplant showed 97% mother, 3% MUD, and 0% host for granulocytes but 38% mother, 62% MUD, and 0% host for CD3 + T cells. In view of low haploidentical donor chimerism in T-lymphocyte compartment, CD45RA-depleted DLI using cryopreserved product was performed on day + 38, after thymoglobulin 3 mg/kg given as T-cell depletion 3 days beforehand. T-cell chimerism improved to 51% mother and 49% MUD post-DLI. Second cryopreserved CD45RA-depleted DLI was given 17 days after the first DLI (day + 55), and 100% full chimerism of mother's T cells was gradually established without significant graft-versus-host disease (GVHD) or viral reactivation. To conclude, split lineage chimerism determination is beneficial to guide management strategy. For MC in T-cell compartment, CD45RA-depleted DLI is a potential alternative to unselected T cells as it carries lower risk of GVHD and infection.

Outcomes of allogeneic transplantation for hemoglobin Bart's hydrops fetalis syndrome in Hong Kong.

BACKGROUND: Hemoglobin Bart's hydrops fetalis syndrome (BHFS) was once considered a fatal condition universally. Medical advances over the past three decades have resulted in increasing numbers of BHFS survivors. This retrospective review summarized local territory-wide experience and outcomes of BHFS patients who received allogeneic hematopoietic stem cell transplantation (HSCT) in Hong Kong. METHODS: All BHFS patients who underwent allogeneic HSCT in Hong Kong, either in one of the two former pediatric transplant centers (Queen Mary Hospital and Prince of Wales Hospital) on or before 2019 or in the single territory-wide pediatric transplant center (Hong Kong Children's Hospital) since 2019, from January 1, 1996, till December 31, 2020, were included. Basic demographic data, perinatal history, transplant details, long-term outcomes, and morbidities were reviewed. RESULTS: Total five allogeneic HSCT were performed in two males and three females at a median age of 22 months, which include one 8/8 matched-sibling bone marrow transplant, one 5/6 matched-sibling cord blood transplant with HLA-DR antigenic mismatch, two 12/12 matched-unrelated peripheral blood stem cell transplant (PBSCT), and one haploidentical PBSCT with TCRαß/CD45RA depletion from maternal donor. Neutrophil and platelet engrafted (>20 × 109 /L) at a median of 15 and 22 days, respectively. All achieved near full donor chimerism at 1 month. All patients survived and remained transfusion-independent without significant morbidities with median follow-up duration of 10 years. CONCLUSION: To conclude, local data demonstrated favorable outcome of allogeneic HSCT for BHFS patients, but sample number is small. Non-directive approach in counseling and international collaboration is recommended.

Opioid Prescribing Practices for Women Undergoing Elective Gynecologic Surgery.

STUDY OBJECTIVE: To describe the opioid prescribing practices in opioid-naive women undergoing elective gynecologic surgery for benign indications and identify risk factors associated with increased perioperative opioid use. We also explored factors associated with new persistent opioid use in women with perioperative opioid use. DESIGN: Retrospective, population-based cohort study. SETTING: We used linked administrative data from a government-administered single-payer provincial healthcare system in Canada. This study was undertaken at ICES, a not-for-profit research institute in Ontario, Canada. PATIENTS: We followed opioid-naive adult women who underwent benign elective gynecologic surgery between 2013 and 2018. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was perioperative opioid use defined as ≥1 opioid prescription from 30 days before to 14 days after surgery. New persistent opioid use after gynecologic surgery was defined as having filled 1 or more opioid prescriptions between 91 days and 180 days postoperatively. Multivariable log-linear regression analyses were employed to adjust for clinical and demographic data. Of the 132 506 patients included in our cohort, most (74.3%) underwent minor gynecologic procedures. Perioperative opioid use was documented in 27 763 (21.0%) patients, and there was a significant decreasing trend (p <.001) in the proportion of patients with perioperative opioid use from 21.8% in 2013 to 18.5% in 2018. Factors associated with increased perioperative opioid use included younger age; higher income quintile; urban dwellers; and diagnosis of infertility, endometriosis, or adnexal mass. Perioperative opioid use was an independent risk factor for persistent use (adjusted relative risk 1.40; 95% confidence interval, 1.13-1.72) and for every 65 patients prescribed opioids associated with gynecologic surgery, one developed new persistent opioid use. The highest risk factor for developing persistent use was filling a high-dose opioid prescription (adjusted relative risk5th quintileOME 2.33; 95% confidence interval, 1.83-2.96). CONCLUSION: One in 5 women who undergo a gynecologic procedure has a new exposure to opioids. For every 65 patients who fill an opioid prescription after their gynecologic surgery, one will experience prolonged opioid use.

Retrograde Bladder Filling after Laparoscopic Gynecologic Surgery: A Double-blind Randomized Controlled Trial.

STUDY OBJECTIVE: To evaluate whether retrofilling the bladder on completion of elective laparoscopic gynecologic surgery for benign indications has an effect on the timing of the first postoperative void and the timing of discharge from the hospital. DESIGN: Double-blind randomized controlled trial. SETTING: Single academic surgical day hospital. PATIENTS: Patients undergoing outpatient laparoscopic gynecologic surgery, excluding hysterectomy or pelvic reconstructive surgery. INTERVENTIONS: On completion of surgery, patients were randomized to either retrograde filling of the bladder with 200 mL of saline before catheter removal or standard care (immediate catheter removal). Patients and postanesthesia care unit nurses (outcome assessors) were both blinded. MEASUREMENTS AND MAIN RESULTS: The primary outcome was the time to first void. The secondary outcomes were time to hospital discharge, postoperative urinary tract infection, and patient satisfaction. Over a 3-month period, 47 patients were approached on the day of surgery, 42 consented and were randomized (21 to intervention and 21 to control). There were no significant differences in baseline demographics between the groups. The median time to first void was significantly shorter for patients in the intervention arm than controls (104 ± 75 minutes vs 162 ± 76 minutes, p <.001). Patients who had retrofilled bladders were discharged faster from post-anesthesia care unit compared to controls (155.0 ± 74 minutes vs 227 ± 58 minutes, p = .001). There were no urinary tract infections in either group, and the proportion of satisfied or very satisfied patients was high (93.8% vs 88.2%, p = .512). CONCLUSION: Retrograde filling of the bladder after outpatient laparoscopic gynecologic surgery is a safe, effective method that significantly reduces the length of hospital stay.

Risk and resilience of vulnerable families in Hong Kong under the impact of COVID-19: an ecological resilience perspective.

PURPOSE: Hong Kong has experienced four waves of COVID-19 since the first case was confirmed in January 2020. Several studies have highlighted the psychological impacts of the outbreak in Hong Kong but have largely ignored the protective factors that contribute to resilience among vulnerable families. This study adopted an ecological resilience framework to explore the impact of this epidemic on members of families with youth with a delinquent tendency/mental health concerns and the ecological protective factors for these vulnerable families. METHODS: Random sampling based on a sampling frame provided by one of the largest local social service organizations in Hong Kong led to the recruitment of 407 respondents who were interviewed using a battery of standardized questionnaires. RESULTS: The results showed that 30.6% and 11.5% of respondents reported a moderate and a severe level of psychological distress, respectively, almost double the percentages reported in a previous study conducted in Hong Kong before the COVID-19 outbreak. Around 36.6% of respondents indicated they had encountered financial problems and almost 40% indicated aggravated financial circumstances since the outbreak. Hierarchical regression analysis revealed that financial stress was the strongest predictor of psychological distress. Structural equation modeling indicated that family support, indoor leisure activities and community resources significantly mediated the negative influence of COVID-19-related stressors on psychological distress of family members. CONCLUSION: Family leisure activities, family support, community spirit and mutual help within the context of social-distancing restrictions may need to be promoted to benefit vulnerable families in Hong Kong under the COVID-19 epidemic.

Regulation and Role of Transcription Factors in Osteogenesis.

Bone is a dynamic tissue constantly responding to environmental changes such as nutritional and mechanical stress. Bone homeostasis in adult life is maintained through bone remodeling, a controlled and balanced process between bone-resorbing osteoclasts and bone-forming osteoblasts. Osteoblasts secrete matrix, with some being buried within the newly formed bone, and differentiate to osteocytes. During embryogenesis, bones are formed through intramembraneous or endochondral ossification. The former involves a direct differentiation of mesenchymal progenitor to osteoblasts, and the latter is through a cartilage template that is subsequently converted to bone. Advances in lineage tracing, cell sorting, and single-cell transcriptome studies have enabled new discoveries of gene regulation, and new populations of skeletal stem cells in multiple niches, including the cartilage growth plate, chondro-osseous junction, bone, and bone marrow, in embryonic development and postnatal life. Osteoblast differentiation is regulated by a master transcription factor RUNX2 and other factors such as OSX/SP7 and ATF4. Developmental and environmental cues affect the transcriptional activities of osteoblasts from lineage commitment to differentiation at multiple levels, fine-tuned with the involvement of co-factors, microRNAs, epigenetics, systemic factors, circadian rhythm, and the microenvironments. In this review, we will discuss these topics in relation to transcriptional controls in osteogenesis.

Activating the unfolded protein response in osteocytes causes hyperostosis consistent with craniodiaphyseal dysplasia.

Bone remodeling is a balanced process between bone synthesis and degradation, maintaining homeostasis and a constant bone mass in adult life. Imbalance will lead to conditions such as osteoporosis or hyperostosis. Osteoblasts build bone, becoming embedded in bone matrix as mature osteocytes. Osteocytes have a role in sensing and translating mechanical loads into biochemical signals, regulating the differentiation and activity of osteoblasts residing at the bone surface through the secretion of Sclerostin (SOST), an inhibitor of WNT signaling. Excessive mechanical load can lead to activation of cellular stress responses altering cell behavior and differentiation. The unfolded protein response (UPR) is a shared pathway utilized by cells to cope with stress stimuli. We showed that in a transgenic mouse model, activation of the UPR in early differentiating osteocytes delays maturation, maintaining active bone synthesis. In addition, expression of SOST is delayed or suppressed; resulting in active WNT signaling and enhanced periosteal bone formation, and the combined outcome is generalized hyperostosis. A clear relationship between the activation of the unfolded protein response was established and the onset of hyperostosis that can be suppressed with a chemical chaperone, sodium 4-phenobutyrate (4-PBA). As the phenotype is highly consistent with craniodiaphyseal dysplasia (CDD; OMIM 122860), we propose activation of the UPR could be part of the disease mechanism for CDD patients as these patients are heterozygous for SOST mutations that impair protein folding and secretion. Thus, therapeutic agents ameliorating protein folding or the UPR can be considered as a potential therapeutic treatment.

Characterization of Histone Deacetylase Expression Within In Vitro and In Vivo Bladder Cancer Model Systems.

Epigenetic aberrations are prominent in bladder cancer (BC) and contribute to disease pathogenesis. We characterized histone deacetylase (HDAC) expression, a family of deacetylation enzymes, in both in vitro and in vivo BC model systems and analyzed expression data from The Cancer Genome Atlas (TCGA). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analysis was used to determine the expression status of Class I and II HDACs in ten human BC cell lines, while qRT-PCR was used to determine HDAC expression in 24 human tumor specimens. The TCGA cohort consists of 408 muscle invasive BC (MIBC) clinical samples and analysis of this data set identified expression of HDAC4 and -9 as being associated with basal-squamous disease. These findings agree with qRT-PCR results identifying increased expression of HDAC4, -7, and -9 in basal BC cell lines (p < 0.05; Kruskal-Wallis test) and in clinical specimens with invasive bladder cancer (not statistically significant). We also observed increased expression in Hdac4, -7, and -9 in commonly used BC mouse models. Here, we identify suitable preclinical model systems for the study of HDACs, and show increased expression of Class IIa HDACs, specifically HDAC4 and HDAC9, in basal BC cell lines and in invasive clinical specimens. These results suggest this class of HDACs may be best suited for targeted inhibition in patients with basal BC.

Obstetrical provider knowledge and attitudes towards cell-free DNA screening: results of a cross-sectional national survey.

BACKGROUND: Cell-free DNA (cfDNA) screening has recently acquired tremendous attention, promising patients and healthcare providers a more accurate prenatal screen for aneuploidy than other current screening modalities. It is unclear how much knowledge regarding cfDNA screening obstetrical providers possess which has important implications for the quality and content of the informed consent patients receive. METHODS: A survey was designed to assess obstetrical provider knowledge and attitudes towards cfDNA screening and distributed online through the Society of Obstetricians & Gynecologists of Canada (SOGC). Chi-squared tests were used to detect differences in knowledge and attitudes between groups. RESULTS: 207 respondents completed the survey, composed of 60.6% Obstetricians/Gynecologists (OB/GYN), 15.4% Maternal Fetal Medicine (MFM) specialists, 16.5% General Practitioners (GP), and 7.5% Midwives (MW). MFM demonstrated a significant trend of being most knowledgeable about cfDNA screening followed by OB/GYN, GP, and lastly MW in almost all aspects of cfDNA screening. All groups demonstrated an overall positive attitude towards cfDNA screening; however, OB/GYN and MFM demonstrated a significantly more positive attitude than GP and MW. Despite not yet being a diagnostic test, 19.4% of GP would offer termination of pregnancy immediately following a positive cfDNA screen result compared to none of the MFM and only few OB/GYN or MW. CONCLUSIONS: We have demonstrated that different types of obstetrical providers possess varying amounts of knowledge regarding cfDNA screening with MFM currently having greater knowledge to all other groups. All obstetrical providers must have adequate prenatal screening understanding so that we can embrace the benefits of this novel and promising technology while protecting the integrity of the informed consent process.

Sociodemographic correlates of urine culture test utilization in Calgary, Alberta.

BACKGROUND: Many clinical practice guidelines encourage diagnosis and empiric treatment of lower urinary tract infection without laboratory investigation; however, urine culture testing remains one of the largest volume tests in the clinical microbiology laboratory. In this study, we sought to determine if there were specific patient groups to which increased testing was directed. To do so, we combined laboratory data on testing rates with Census Canada sociodemographic data. METHODS: Urine culture testing data was obtained from the Calgary Laboratory Services information system for 2011. We examined all census dissemination areas within the city of Calgary and, for each area, testing rates were determined for age and gender cohorts. We then compared these testing rates to sociodemographic factors obtained from Census Canada and used Poisson regression and generalized estimating equations to test associations between testing rates and sociodemographic variables. RESULTS: Per capita urine culture testing is increasing in Calgary. For 2011, 100,901 individuals (9.2% of all people) received urine cultures and were included in this analysis. The majority of cultures were received from the community (67.9%). Substantial differences in rate of testing were observed across the city. Most notably, urine culture testing was drastically lower in areas of high (≥ $100000) household income (RR = 0.07, p < 0.0001) and higher employment rate (RR = 0.36, p < 0.0001). Aboriginal - First Nations status (RR = 0.29, p = 0.0008) and Chinese visible minority (RR = 0.67, p = 0.0005) were also associated with decreased testing. Recent immigration and visible minority status of South Asian, Filipino or Black were not significant predictors of urine culture testing. Females were more likely to be tested than males (RR = 2.58, p < 0.0001) and individuals aged 15-39 were the most likely to be tested (RR = 1.69, p < 0.0001). CONCLUSIONS: Considerable differences exist in urine culture testing across Calgary and these are associated with a number of sociodemographic factors. In particular, areas of lower socioeconomic standing had significantly increased rates of testing. These observations highlight specific groups that should be targeted to improve healthcare delivery and, in turn, enhance laboratory utilization.