Wastewater monitoring for detection of public health markers during the COVID-19 pandemic: Near-source monitoring of schools in England over an academic year.

BACKGROUND: Schools are high-risk settings for infectious disease transmission. Wastewater monitoring for infectious diseases has been used to identify and mitigate outbreaks in many near-source settings during the COVID-19 pandemic, including universities and hospitals but less is known about the technology when applied for school health protection. This study aimed to implement a wastewater surveillance system to detect SARS-CoV-2 and other public health markers from wastewater in schools in England. METHODS: A total of 855 wastewater samples were collected from 16 schools (10 primary, 5 secondary and 1 post-16 and further education) over 10 months of school term time. Wastewater was analysed for SARS-CoV-2 genomic copies of N1 and E genes by RT-qPCR. A subset of wastewater samples was sent for genomic sequencing, enabling determination of the presence of SARS-CoV-2 and emergence of variant(s) contributing to COVID-19 infections within schools. In total, >280 microbial pathogens and >1200 AMR genes were screened using RT-qPCR and metagenomics to consider the utility of these additional targets to further inform on health threats within the schools. RESULTS: We report on wastewater-based surveillance for COVID-19 within English primary, secondary and further education schools over a full academic year (October 2020 to July 2021). The highest positivity rate (80.4%) was observed in the week commencing 30th November 2020 during the emergence of the Alpha variant, indicating most schools contained people who were shedding the virus. There was high SARS-CoV-2 amplicon concentration (up to 9.2x106 GC/L) detected over the summer term (8th June - 6th July 2021) during Delta variant prevalence. The summer increase of SARS-CoV-2 in school wastewater was reflected in age-specific clinical COVID-19 cases. Alpha variant and Delta variant were identified in the wastewater by sequencing of samples collected from December to March and June to July, respectively. Lead/lag analysis between SARS-CoV-2 concentrations in school and WWTP data sets show a maximum correlation between the two-time series when school data are lagged by two weeks. Furthermore, wastewater sample enrichment coupled with metagenomic sequencing and rapid informatics enabled the detection of other clinically relevant viral and bacterial pathogens and AMR. CONCLUSIONS: Passive wastewater monitoring surveillance in schools can identify cases of COVID-19. Samples can be sequenced to monitor for emerging and current variants of concern at the resolution of school catchments. Wastewater based monitoring for SARS-CoV-2 is a useful tool for SARS-CoV-2 passive surveillance and could be applied for case identification and containment, and mitigation in schools and other congregate settings with high risks of transmission. Wastewater monitoring enables public health authorities to develop targeted prevention and education programmes for hygiene measures within undertested communities across a broad range of use cases.

Working toward sustainability: Transitioning HIV programs from a USA-based organization to a local partner in Zimbabwe.

BACKGROUND: Despite the history of United States of America (USA)-based partners implementing global health programs in low- and middle-income countries (LMIC), future models for sustainable healthcare rely on local country ownership and leadership. Transition is the process of shifting programs towards country ownership, where local stakeholders plan, manage, and deliver health programs. Transition is not a singular event but a process which may include a phase where health programs are led and managed by local entities but still reliant on awards from international partners. This phase is scarcely described yet can impact long-term program sustainability if navigated poorly. This qualitative study examines the transition of Zimbabwe's voluntary medical male circumcision and HIV care and treatment services from management by a USA-based organization, the International Training and Education Center for Health (I-TECH), to management under a new Zimbabwean organization, the Zimbabwe Technical Assistance, Training and Education Centre for Health (Zim-TTECH). The primary objective of this paper is to explore challenges, successes, and lessons learned during this transition to inform other non-governmental organizations. METHODS: We conducted sixteen virtual, key informant interviews using purposeful sampling, identifying potential participants based on their role in the transition team (leadership, administrative, financial, or human resources) and willingness to consent to the study. We aimed for equal representation from USA-based, I-TECH headquarters staff and Zimbabwe-based, Zim-TTECH staff involved in the transition team. Data were analyzed in Atlas.Ti using deductive and inductive methods, followed by a thematic analysis guided by several frameworks for program transition and organizational change. RESULTS: Findings suggest five themes to guide transition: 1) Develop a vision and empower leadership for change by delegating clear roles and supporting local ownership; 2) Plan and strategize for transition in a manner that accounts for historical context; 3) Communicate with and inform stakeholders to understand transition perceptions, understand barriers to transition, and enable open communications related to risks and benefits; 4) Engage and mobilize staff by constructing necessary infrastructure and providing technical assistance as needed; and 5) Define short-term and long-term success. CONCLUSION: Transition processes were challenged by the local country context, compressed transition timelines, and all-or-nothing measures of transition success. Facilitators included strong staff capacity and a synergistic partnership model between Zim-TTECH and I-TECH. Global funders and international organizations should support local LMIC partners in their pathway to independence by removing restrictions on funding awards, including transitioning ownership mid-stream, and positioning leadership of international awards for in-country entities.

Monitoring occurrence of SARS-CoV-2 in school populations: A wastewater-based approach.

Clinical testing of children in schools is challenging, with economic implications limiting its frequent use as a monitoring tool of the risks assumed by children and staff during the COVID-19 pandemic. Here, a wastewater-based epidemiology approach has been used to monitor 16 schools (10 primary, 5 secondary and 1 post-16 and further education) in England. A total of 296 samples over 9 weeks have been analysed for N1 and E genes using qPCR methods. Of the samples returned, 47.3% were positive for one or both genes with a detection frequency in line with the respective local community. WBE offers a low cost, non-invasive approach for supplementing clinical testing and can provide longitudinal insights that are impractical with traditional clinical testing.

Betulinic Acid-Doxorubicin-Drug Combination Induced Apoptotic Death via ROS Stimulation in a Relapsed AML MOLM-13 Cell Model.

In this study, cell death regulation and induction in AML cell line from a relapsed MLL-rearranged cell model (MOLM-13) was investigated with doxorubin (Dox) and betulinic acid (BetA), singly and in combination. CyQUANT Direct® and Annexin V/propidium iodide double staining were used to measure the cytotoxic and cell death induction effects of the compounds, respectively. Reactive oxygen species (ROS) generation was measured using 2',7'-dichlorofluorescin diacetate staining. Expressions of proteins and genes were examined by Western blot and reverse transcription polymerase chain reaction analysis, respectively. BetA (20 µM) and Dox (1 µM) indicated a synergistic growth inhibitory effect on MOLM-13 cells. The combined drug caused more cells to reside in irreversible late apoptotic stage compared to the single treatments (p < 0.05). Elevation in ROS may be the synergistic mechanism involved in MOLM-13 cell death since ROS can directly disrupt mitochondrial activity. In contrast, in leukaemic U-937 cells, the combination treatments attenuated Dox-induced cell death. Dox and the drug combination selectively reduced (p < 0.05) a recently reported anti-apoptotic Bcl-2 protein isoform p15-20-Bcl-2 in MOLM-13 by our group, without affecting the usually reported p26-Bcl-2-α. Further studies using known inhibitors of apoptosis are required to confirm the potential of Dox-BetA combination to modulate these pathways.

Doxorubicin selectively induces apoptosis through the inhibition of a novel isoform of Bcl­2 in acute myeloid leukaemia MOLM­13 cells with reduced Beclin 1 expression.

The overexpression of anti­apoptotic Bcl­2 in acute myeloid leukaemia (AML) may contribute to difficulties in eradicating these cells during chemotherapy. In the present study, doxorubicin (Dox) was evaluated for its potential to induce selective apoptotic cell death in AML MOLM­13 cells and to modulate autophagy through Bcl­2 and Beclin 1 protein expression. Annexin V/propidium iodide and 5(6)­carboxyfluorescein diacetate succinimidyl ester (CFSE) flow cytometric analyses were conducted to determine the effects of Dox on cell death and cell proliferation, respectively, following 48 h of co­incubation with AML MOLM­13 or U­937 monocytic cells. The protein expression levels of Bcl­2 and Beclin 1 in untreated and treated cells were quantified by western blot analysis. Dox reduced the viability of MOLM­13 cells partly by inhibiting cell division and inducing cell apoptosis. Dox demonstrated a level of selectivity in its cytotoxicity against MOLM­13 compared to U­937 cells (P<0.05). Dox induced a significant decrease in Beclin 1 protein levels in MOLM­13 cells without significantly affecting the protein levels in U­937 monocytes. A novel Bcl­2 15­20 kDa (p15­20­Bcl­2) isoform was found to be selectively expressed in AML MOLM­13 cells (but absent in the leukaemic cell lines tested, OCI­AML2, CML K562 and U­937). Dox induced a highly significant inhibition of p15­20­Bcl­2 at concentrations of 0.5, 0.75 and 1 µM (P<0.01). However, the usual 26 kDa Bcl­2 (p26­Bcl­2­α) isoform protein expression was not affected by the drug in either the MOLM­13 or U­937 cells. It was thus postulated that Dox exhibited some selectivity by targeting the p15­20­Bcl­2 isoform in MOLM­13 cells and activating Beclin 1 to induce cell death.

Antiinflammatory and Hepatoprotective Medicinal Herbs as Potential Substitutes for Bear Bile.

Practitioners of traditional Chinese medicine (TCM) commonly prescribe medicinal formulations relying on the purported synergism of a combination of plant species, sometimes incorporating animal parts and minerals. Bear bile, obtained from either wild or farmed bears, is a commonly used constituent of traditional medicine formulations. With several bear species now listed under Convention on International Trade in Endangered Species of Wild Fauna and Flora as threatened with extinction and with bear farming being actively campaigned against on ethical grounds, it is important to seek and promote alternatives to the use of bear bile as medicine. This chapter describes and evaluates the scientific data relating to the efficacy of bear bile and potential alternatives to its use, including the use of bile from other animal species, the use of synthetic chemical alternatives, and the use of herbal substitutes. Scientific studies have confirmed the efficacy of bear bile as an antiinflammatory and a hepatoprotective agent. Ursodeoxycholic acid (UDCA), the active component of bear bile is used in a synthetic form in Western medicine and can serve as an alternative to bear bile in the treatment and management of certain cholestatic liver conditions. In TCM practice, bile from domesticated animal species (such as cattle, chicken, and pig) has been used as a substitute for bear bile. Following evaluation of TCM literature and pharmacological/clinical data, the authors propose six plant species, either as single herbs or in combination, Gardenia jasminoides (zhi zi; ), Scutellaria baicalensis (huáng qín; ), Coptis chinensis (huáng lián, ), Phellodendron amurense (huáng bai; ), Andrographis paniculata (chuan xin lian; ), and Rheum palmatum (dà huang; ), two medicinal Kampo formulations, Orengedokuto, Dia-Orengedokuto (which originated from traditional Chinese herbal formula Huanglian Jiedu Tang, ), and two individual phytochemicals (berberine and andrographolide) as alternatives to bear bile. The proposed herbal alternatives are frequently found listed in traditional formulations also containing bear bile, usually with different therapeutic roles ascribed to them. The existing evidence base for the effectiveness of herbal alternatives is sufficiently strong for TCM practitioners and consumers to consider using these without the addition of bear bile. This consideration is driven by the imperative to protect populations of bears from overexploitation in the wild and when farmed. However, for the identified alternatives to be accepted by users, it is essential that researchers and TCM practitioners collaborate effectively to initiate consumer behavior change.

Evaluation of Ventilation Tube Placement and Long-term Audiologic Outcome in Children With Cleft Palate.

OBJECTIVE: The purpose of this study was to assess the effect of ventilation tube (VT) placement on long-term hearing outcomes in children with cleft palate. STUDY DESIGN: Case series with chart review. SETTING: Genetic and dysmorphology database at Rady Children's Hospital-San Diego (RCHSD). PATIENTS: Children with cleft palate diagnosis who underwent surgery at RCHSD between 1995 and 2002. MAIN OUTCOME MEASURE: The primary outcome studied was hearing acuity at 10 years of age. Independent variables studied included gender, age at palate repair and first VT placement, total number of VTs, number of complications, and presence of tympanic membrane perforation. RESULTS: An increased number of tubes was associated with a greater incidence of hearing loss at age 10, even after adjusting for total number of otologic complications. The timing of initial tube placement did not have a significant effect on long-term hearing outcome in this study. CONCLUSIONS: While children with worse middle ear disease are more likely to receive more tubes and have long-term conductive hearing loss as a result of ear disease, the results of this study suggest that multiple tube placements may not contribute to improved long-term hearing outcomes. Further research focusing on long-term outcomes is needed to establish patient-centered criteria guiding decision making for ventilation tube placement in children with cleft palate.