Trends in COVID-19 admissions and deaths among people living with HIV in South Africa: analysis of national surveillance data.

BACKGROUND: In 2021, the HIV prevalence among South African adults was 18% and more than 2 million people had uncontrolled HIV and, therefore, had increased risk of poor outcomes with SARS-CoV-2 infection. We investigated trends in COVID-19 admissions and factors associated with in-hospital COVID-19 mortality among people living with HIV and people without HIV. METHODS: In this analysis of national surveillance data, we linked and analysed data collected between March 5, 2020, and May 28, 2022, from the DATCOV South African national COVID-19 hospital surveillance system, the SARS-CoV-2 case line list, and the Electronic Vaccination Data System. All analyses included patients hospitalised with SARS-CoV-2 with known in-hospital outcomes (ie, who were discharged alive or had died) at the time of data extraction. We used descriptive statistics for admissions and mortality trends. Using post-imputation random-effect multivariable logistic regression models, we compared characteristics and the case fatality ratio of people with HIV and people without HIV. Using modified Poisson regression models, we compared factors associated with mortality among all people with COVID-19 admitted to hospital and factors associated with mortality among people with HIV. FINDINGS: Among 397 082 people with COVID-19 admitted to hospital, 301 407 (75·9%) were discharged alive, 89 565 (22·6%) died, and 6110 (1·5%) had no recorded outcome. 270 737 (68·2%) people with COVID-19 had documented HIV status (22 858 with HIV and 247 879 without). Comparing characteristics of people without HIV and people with HIV in each COVID-19 wave, people with HIV had increased odds of mortality in the D614G (adjusted odds ratio 1·19, 95% CI 1·09-1·29), beta (1·08, 1·01-1·16), delta (1·10, 1·03-1·18), omicron BA.1 and BA.2 (1·71, 1·54-1·90), and omicron BA.4 and BA.5 (1·81, 1·41-2·33) waves. Among all COVID-19 admissions, mortality was lower among people with previous SARS-CoV-2 infection (adjusted incident rate ratio 0·32, 95% CI 0·29-0·34) and with partial (0·93, 0·90-0·96), full (0·70, 0·67-0·73), or boosted (0·50, 0·41-0·62) COVID-19 vaccination. Compared with people without HIV who were unvaccinated, people without HIV who were vaccinated had lower risk of mortality (0·68, 0·65-0·71) but people with HIV who were vaccinated did not have any difference in mortality risk (1·08, 0·96-1·23). In-hospital mortality was higher for people with HIV with CD4 counts less than 200 cells per µL, irrespective of viral load and vaccination status. INTERPRETATION: HIV and immunosuppression might be important risk factors for mortality as COVID-19 becomes endemic. FUNDING: South African National Institute for Communicable Diseases, the South African National Government, and the United States Agency for International Development.

Using generalized structured additive regression models to determine factors associated with and clusters for COVID-19 hospital deaths in South Africa.

BACKGROUND: The first case of COVID-19 in South Africa was reported in March 2020 and the country has since recorded over 3.6 million laboratory-confirmed cases and 100 000 deaths as of March 2022. Transmission and infection of SARS-CoV-2 virus and deaths in general due to COVID-19 have been shown to be spatially associated but spatial patterns in in-hospital deaths have not fully been investigated in South Africa. This study uses national COVID-19 hospitalization data to investigate the spatial effects on hospital deaths after adjusting for known mortality risk factors. METHODS: COVID-19 hospitalization data and deaths were obtained from the National Institute for Communicable Diseases (NICD). Generalized structured additive logistic regression model was used to assess spatial effects on COVID-19 in-hospital deaths adjusting for demographic and clinical covariates. Continuous covariates were modelled by assuming second-order random walk priors, while spatial autocorrelation was specified with Markov random field prior and fixed effects with vague priors respectively. The inference was fully Bayesian. RESULTS: The risk of COVID-19 in-hospital mortality increased with patient age, with admission to intensive care unit (ICU) (aOR = 4.16; 95% Credible Interval: 4.05-4.27), being on oxygen (aOR = 1.49; 95% Credible Interval: 1.46-1.51) and on invasive mechanical ventilation (aOR = 3.74; 95% Credible Interval: 3.61-3.87). Being admitted in a public hospital (aOR = 3.16; 95% Credible Interval: 3.10-3.21) was also significantly associated with mortality. Risk of in-hospital deaths increased in months following a surge in infections and dropped after months of successive low infections highlighting crest and troughs lagging the epidemic curve. After controlling for these factors, districts such as Vhembe, Capricorn and Mopani in Limpopo province, and Buffalo City, O.R. Tambo, Joe Gqabi and Chris Hani in Eastern Cape province remained with significantly higher odds of COVID-19 hospital deaths suggesting possible health systems challenges in those districts. CONCLUSION: The results show substantial COVID-19 in-hospital mortality variation across the 52 districts. Our analysis provides information that can be important for strengthening health policies and the public health system for the benefit of the whole South African population. Understanding differences in in-hospital COVID-19 mortality across space could guide interventions to achieve better health outcomes in affected districts.

Genome Engineering of Human Urine-Derived Stem Cells to Express Lactoferrin and Deoxyribonuclease.

Urine-derived stem cells (USCs) are adult kidney cells that have been isolated from a urine sample and propagated in tissue culture on gelatin-coated plates. Urine is a practical and completely painless source of cells for gene and cell therapy applications. We have isolated, expanded, and optimized transfection of USCs to develop regenerative therapies based on piggyBac transposon modification. USCs from a healthy donor sample were isolated according to established protocols. Within 2 months, 10 clones had been expanded, analyzed, and frozen. Fluorescence-activated cell sorting analysis of individual clones revealed that all 10 clones expressed characteristic USC markers (97-99% positive for CD44, CD73, CD90, and CD146; negative for CD31, CD34, and CD45). The isolated USCs were successfully differentiated along the osteogenic, adipogenic, and chondrogenic lineages, suggesting multipotent differentiation capacity. Additionally, the USCs were differentiated into podocytes positive for NEPHRIN (NPHS1), podocalyxin, and Wilms tumor 1 (WT1). Transfection of USCs with a strongly expressing Green fluorescent protein plasmid was optimized to achieve 61% efficiency in live cells using several commercially available lipophilic reagents. Transgene promoters were compared in five luciferase-expressing piggyBac transposons by live animal imaging. The CMV promoter produced the highest luciferase signal, followed by EF1-α. Finally, HEK-293 and USCs were transfected with piggyBac transposons expressing lactoferrin and DNase1 for treatment of acute kidney injury associated with rhabdomyolysis. We found that both proteins were expressed in USCs and that lactoferrin was successfully secreted into the cell culture media. In conclusion, USCs represent a clinically relevant cell type that can express nonviral transgenes. Impact statement Acute kidney injury (AKI) affects over 13 million people worldwide each year, with hospitalization rates on the rise. There are no therapies that directly regenerate the kidney after AKI. Each human kidney contains approximately one million nephrons that process ∼100 L of urinary filtrate each day. Thousands of kidney cells become detached and are excreted in the urine. A small percentage of these cells can be clonally derived into urine-derived stem cells. We have optimized methods for genome engineering of adult human urine-derived stem cells for future applications in regenerative approaches to treat kidney injury.

Sustained Low Incidence of Severe and Fatal COVID-19 Following Widespread Infection Induced Immunity after the Omicron (BA.1) Dominant in Gauteng, South Africa: An Observational Study.

We conducted an epidemiologic survey to determine the seroprevalence of SARS-CoV-2 anti-nucleocapsid (anti-N) and anti-spike (anti-S) protein IgG from 1 March to 11 April 2022 after the BA.1-dominant wave had subsided in South Africa and prior to another wave dominated by the BA.4 and BA.5 (BA.4/BA.5) sub-lineages. We also analysed epidemiologic trends in Gauteng Province for cases, hospitalizations, recorded deaths, and excess deaths were evaluated from the inception of the pandemic through 17 November 2022. Despite only 26.7% (1995/7470) of individuals having received a COVID-19 vaccine, the overall seropositivity for SARS-CoV-2 was 90.9% (95% confidence interval (CI), 90.2 to 91.5) at the end of the BA.1 wave, and 64% (95% CI, 61.8 to 65.9) of individuals were infected during the BA.1-dominant wave. The SARS-CoV-2 infection fatality risk was 16.5-22.3 times lower in the BA.1-dominant wave compared with the pre-BA.1 waves for recorded deaths (0.02% vs. 0.33%) and estimated excess mortality (0.03% vs. 0.67%). Although there are ongoing cases of COVID-19 infections, hospitalization and death, there has not been any meaningful resurgence of COVID-19 since the BA.1-dominant wave despite only 37.8% coverage by at least a single dose of COVID-19 vaccine in Gauteng, South Africa.

A cohort study of post-COVID-19 condition across the Beta, Delta, and Omicron waves in South Africa: 6-month follow-up of hospitalized and nonhospitalized participants.

OBJECTIVES: The study aimed to describe the prevalence of and risk factors for post-COVID-19 condition (PCC). METHODS: This was a prospective, longitudinal observational cohort study. Hospitalized and nonhospitalized adults were randomly selected to undergo telephone assessment at 1, 3, and 6 months. Participants were assessed using a standardized questionnaire for the evaluation of symptoms and health-related quality of life. We used negative binomial regression models to determine factors associated with the presence of ≥1 symptoms at 6 months. RESULTS: A total of 46.7% of hospitalized and 18.5% of nonhospitalized participants experienced ≥1 symptoms at 6 months (P ≤0.001). Among hospitalized people living with HIV, 40.4% had persistent symptoms compared with 47.1% among participants without HIV (P = 0.108). The risk factors for PCC included older age, female sex, non-Black race, presence of a comorbidity, greater number of acute COVID-19 symptoms, hospitalization/COVID-19 severity, and wave period (lower risk of persistent symptoms for the Omicron compared with the Beta wave). There were no associations between self-reported vaccination status with persistent symptoms. CONCLUSION: The study revealed a high prevalence of persistent symptoms among South African participants at 6 months but decreased risk for PCC among participants infected during the Omicron BA.1 wave. These findings have serious implications for countries with resource-constrained health care systems.

Trends in Cases, Hospitalizations, and Mortality Related to the Omicron BA.4/BA.5 Subvariants in South Africa.

BACKGROUND: In this study, we compared admission incidence risk and the risk of mortality in the Omicron BA.4/BA.5 wave to previous waves. METHODS: Data from South Africa's SARS-CoV-2 case linelist, national COVID-19 hospital surveillance system, and Electronic Vaccine Data System were linked and analyzed. Wave periods were defined when the country passed a weekly incidence of 30 cases/100 000 population. In-hospital case fatality ratios (CFRs) during the Delta, Omicron BA.1/BA.2, and Omicron BA.4/BA.5 waves were compared using post-imputation random effect multivariable logistic regression models. RESULTS: The CFR was 25.9% (N = 37 538 of 144 778), 10.9% (N = 6123 of 56 384), and 8.2% (N = 1212 of 14 879) in the Delta, Omicron BA.1/BA.2, and Omicron BA.4/BA.5 waves, respectively. After adjusting for age, sex, race, comorbidities, health sector, and province, compared with the Omicron BA.4/BA.5 wave, patients had higher risk of mortality in the Omicron BA.1/BA.2 wave (adjusted odds ratio [aOR], 1.3; 95% confidence interval [CI]: 1.2-1.4) and Delta wave (aOR, 3.0; 95% CI: 2.8-3.2). Being partially vaccinated (aOR, 0.9; 95% CI: .9-.9), fully vaccinated (aOR, 0.6; 95% CI: .6-.7), and boosted (aOR, 0.4; 95% CI: .4-.5) and having prior laboratory-confirmed infection (aOR, 0.4; 95% CI: .3-.4) were associated with reduced risks of mortality. CONCLUSIONS: Overall, admission incidence risk and in-hospital mortality, which had increased progressively in South Africa's first 3 waves, decreased in the fourth Omicron BA.1/BA.2 wave and declined even further in the fifth Omicron BA.4/BA.5 wave. Mortality risk was lower in those with natural infection and vaccination, declining further as the number of vaccine doses increased.

Clinical severity of SARS-CoV-2 Omicron BA.4 and BA.5 lineages compared to BA.1 and Delta in South Africa.

Omicron lineages BA.4 and BA.5 drove a fifth wave of COVID-19 cases in South Africa. Here, we use the presence/absence of the S-gene target as a proxy for SARS-CoV-2 variant/lineage for infections diagnosed using the TaqPath PCR assay between 1 October 2021 and 26 April 2022. We link national COVID-19 individual-level data including case, laboratory test and hospitalisation data. We assess severity using multivariable logistic regression comparing the risk of hospitalisation and risk of severe disease, once hospitalised, for Delta, BA.1, BA.2 and BA.4/BA.5 infections. After controlling for factors associated with hospitalisation and severe outcome respectively, BA.4/BA.5-infected individuals had a similar odds of hospitalisation (aOR 1.24, 95% CI 0.98-1.55) and severe outcome (aOR 0.72, 95% CI 0.41-1.26) compared to BA.1-infected individuals. Newly emerged Omicron lineages BA.4/BA.5 showed similar severity to the BA.1 lineage and continued to show reduced clinical severity compared to the Delta variant.

Methods for early characterisation of the severity and dynamics of SARS-CoV-2 variants: a population-based time series analysis in South Africa.

BACKGROUND: Assessment of disease severity associated with a novel pathogen or variant provides crucial information needed by public health agencies and governments to develop appropriate responses. The SARS-CoV-2 omicron variant of concern (VOC) spread rapidly through populations worldwide before robust epidemiological and laboratory data were available to investigate its relative severity. Here we develop a set of methods that make use of non-linked, aggregate data to promptly estimate the severity of a novel variant, compare its characteristics with those of previous VOCs, and inform data-driven public health responses. METHODS: Using daily population-level surveillance data from the National Institute for Communicable Diseases in South Africa (March 2, 2020, to Jan 28, 2022), we determined lag intervals most consistent with time from case ascertainment to hospital admission and within-hospital death through optimisation of the distance correlation coefficient in a time series analysis. We then used these intervals to estimate and compare age-stratified case-hospitalisation and case-fatality ratios across the four epidemic waves that South Africa has faced, each dominated by a different variant. FINDINGS: A total of 3 569 621 cases, 494 186 hospitalisations, and 99 954 deaths attributable to COVID-19 were included in the analyses. We found that lag intervals and disease severity were dependent on age and variant. At an aggregate level, fluctuations in cases were generally followed by a similar trend in hospitalisations within 7 days and deaths within 15 days. We noted a marked reduction in disease severity throughout the omicron period relative to previous waves (age-standardised case-fatality ratios were consistently reduced by >50%), most substantial for age strata with individuals 50 years or older. INTERPRETATION: This population-level time series analysis method, which calculates an optimal lag interval that is then used to inform the numerator of severity metrics including the case-hospitalisation and case-fatality ratio, provides useful and timely estimates of the relative effects of novel SARS-CoV-2 VOCs, especially for application in settings where resources are limited. FUNDING: National Institute for Communicable Diseases of South Africa, South African National Government.

Podocytes derived from human induced pluripotent stem cells: characterization, comparison, and modeling of diabetic kidney disease.

BACKGROUND: In diabetic kidney disease, high glucose damages specialized cells called podocytes that filter blood in the glomerulus. In vitro culture of podocytes is crucial for modeling of diabetic nephropathy and genetic podocytopathies and to complement animal studies. Recently, several methods have been published to derive podocytes from human-induced pluripotent stem cells (iPSCs) by directed differentiation. However, these methods have major variations in media composition and have not been compared. METHODS: We characterized our accelerated protocol by guiding the cells through differentiation with four different medias into MIXL1+ primitive streak cells with Activin A and CHIR for Wnt activation, intermediate mesoderm PAX8+ cells via increasing the CHIR concentration, nephron progenitors with FGF9 and Heparin for stabilization, and finally into differentiated podocytes with Activin A, BMP-7, VEGF, reduced CHIR, and retinoic acid. The podocyte morphology was characterized by scanning and transmission electron microscopy and by flow cytometry analysis for podocyte markers. To confirm cellular identity and niche localization, we performed cell recombination assays combining iPSC-podocytes with dissociated mouse embryonic kidney cells. Finally, to test iPSC-derived podocytes for the modeling of diabetic kidney disease, human podocytes were exposed to high glucose. RESULTS: Podocyte markers were expressed at similar or higher levels for our accelerated protocol as compared to previously published protocols that require longer periods of tissue culture. We confirmed that the human podocytes derived from induced pluripotent stem cells in twelve days integrated into murine glomerular structures formed following seven days of culture of cellular recombinations. We found that the high glucose-treated human podocytes displayed actin rearrangement, increased cytotoxicity, and decreased viability. CONCLUSIONS: We found that our accelerated 12-day method for the differentiation of podocytes from human-induced pluripotent stem cells yields podocytes with comparable marker expression to longer podocytes. We also demonstrated that podocytes created with this protocol have typical morphology by electron microscopy. The podocytes have utility for diabetes modeling as evidenced by lower viability and increased cytotoxicity when treated with high glucose. We found that multiple, diverse methods may be utilized to create iPSC-podocytes, but closely mimicking developmental cues shortened the time frame required for differentiation.

Clinical severity of COVID-19 in patients admitted to hospital during the omicron wave in South Africa: a retrospective observational study.

BACKGROUND: Up to the end of January, 2022, South Africa has had four recognisable COVID-19 pandemic waves, each predominantly dominated by one variant of concern: the ancestral strain with an Asp614Gly mutation during the first wave, the beta variant (B.1.351) during the second wave, the delta variant (B.1.617.2) during the third wave, and lastly, the omicron variant (B.1.1.529) during the fourth wave. We aimed to assess the clinical disease severity of patients admitted to hospital with SARS-CoV-2 infection during the omicron wave and compare the findings with those of the preceding three pandemic waves in South Africa. METHODS: We defined the start and end of each pandemic wave as the crossing of the threshold of weekly incidence of 30 laboratory-confirmed SARS-CoV-2 cases per 100 000 population. Hospital admission data were collected through an active national COVID-19-specific surveillance programme. We compared disease severity across waves by post-imputation random effect multivariable logistic regression models. Severe disease was defined as one or more of the following: acute respiratory distress, receipt of supplemental oxygen or mechanical ventilation, admission to intensive care, or death. FINDINGS: We analysed 335 219 laboratory-confirmed SARS-CoV-2 hospital admissions with a known outcome, constituting 10·4% of 3 216 179 cases recorded during the four waves. During the omicron wave, 52 038 (8·3%) of 629 617 cases were admitted to hospital, compared with 71 411 (12·9%) of 553 530 in the Asp614Gly wave, 91 843 (12·6%) of 726 772 in the beta wave, and 131 083 (10·0%) of 1 306 260 in the delta wave (p<0·0001). During the omicron wave, 15 421 (33·6%) of 45 927 patients admitted to hospital had severe disease, compared with 36 837 (52·3%) of 70 424 in the Asp614Gly wave, 57 247 (63·4%) of 90 310 in the beta wave, and 81 040 (63·0%) of 128 558 in the delta wave (p<0·0001). The in-hospital case-fatality ratio during the omicron wave was 10·7%, compared with 21·5% during the Asp614Gly wave, 28·8% during the beta wave, and 26·4% during the delta wave (p<0·0001). Compared with those admitted to hospital during the omicron wave, patients admitted during the other three waves had more severe clinical presentations (adjusted odds ratio 2·07 [95% CI 2·01-2·13] in the Asp614Gly wave, 3·59 [3·49-3·70] in the beta wave, and 3·47 [3·38-3·57] in the delta wave). INTERPRETATION: The trend of increasing cases and admissions across South Africa's first three waves shifted in the omicron wave, with a higher and quicker peak but fewer patients admitted to hospital, less clinically severe illness, and a lower case-fatality ratio compared with the preceding three waves. Omicron marked a change in the SARS-CoV-2 epidemic curve, clinical profile, and deaths in South Africa. Extrapolations to other populations should factor in differing vaccination and previous infection levels. FUNDING: National Institute for Communicable Diseases.

Population Immunity and Covid-19 Severity with Omicron Variant in South Africa.

BACKGROUND: The B.1.1.529 (omicron) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified on November 25, 2021, in Gauteng province, South Africa. Data regarding the seroprevalence of SARS-CoV-2 IgG in Gauteng before the fourth wave of coronavirus disease 2019 (Covid-19), in which the omicron variant was dominant, are needed. METHODS: We conducted a seroepidemiologic survey from October 22 to December 9, 2021, in Gauteng to determine the seroprevalence of SARS-CoV-2 IgG. Households included in a previous seroepidemiologic survey (conducted from November 2020 to January 2021) were contacted; to account for changes in the survey population, there was a 10% increase in the households contacted, with the use of the same sampling framework. Dried-blood-spot samples were tested for IgG against SARS-CoV-2 spike protein and nucleocapsid protein with the use of quantitative assays. We also evaluated Covid-19 epidemiologic trends in Gauteng, including cases, hospitalizations, recorded deaths, and excess deaths from the start of the pandemic through January 12, 2022. RESULTS: Samples were obtained from 7010 participants, of whom 1319 (18.8%) had received a Covid-19 vaccine. The seroprevalence of SARS-CoV-2 IgG ranged from 56.2% (95% confidence interval [CI], 52.6 to 59.7) among children younger than 12 years of age to 79.7% (95% CI, 77.6 to 81.5) among adults older than 50 years of age. Vaccinated participants were more likely to be seropositive for SARS-CoV-2 than unvaccinated participants (93.1% vs. 68.4%). Epidemiologic data showed that the incidence of SARS-CoV-2 infection increased and subsequently declined more rapidly during the fourth wave than it had during the three previous waves. The incidence of infection was decoupled from the incidences of hospitalization, recorded death, and excess death during the fourth wave, as compared with the proportions seen during previous waves. CONCLUSIONS: Widespread underlying SARS-CoV-2 seropositivity was observed in Gauteng before the omicron-dominant wave of Covid-19. Epidemiologic data showed a decoupling of hospitalizations and deaths from infections while omicron was circulating. (Funded by the Bill and Melinda Gates Foundation.).

Early assessment of the clinical severity of the SARS-CoV-2 omicron variant in South Africa: a data linkage study.

BACKGROUND: The SARS-CoV-2 omicron variant of concern was identified in South Africa in November, 2021, and was associated with an increase in COVID-19 cases. We aimed to assess the clinical severity of infections with the omicron variant using S gene target failure (SGTF) on the Thermo Fisher Scientific TaqPath COVID-19 PCR test as a proxy. METHODS: We did data linkages for national, South African COVID-19 case data, SARS-CoV-2 laboratory test data, SARS-CoV-2 genome data, and COVID-19 hospital admissions data. For individuals diagnosed with COVID-19 via TaqPath PCR tests, infections were designated as either SGTF or non-SGTF. The delta variant was identified by genome sequencing. Using multivariable logistic regression models, we assessed disease severity and hospitalisations by comparing individuals with SGTF versus non-SGTF infections diagnosed between Oct 1 and Nov 30, 2021, and we further assessed disease severity by comparing SGTF-infected individuals diagnosed between Oct 1 and Nov 30, 2021, with delta variant-infected individuals diagnosed between April 1 and Nov 9, 2021. FINDINGS: From Oct 1 (week 39), 2021, to Dec 6 (week 49), 2021, 161 328 cases of COVID-19 were reported in South Africa. 38 282 people were diagnosed via TaqPath PCR tests and 29 721 SGTF infections and 1412 non-SGTF infections were identified. The proportion of SGTF infections increased from two (3·2%) of 63 in week 39 to 21 978 (97·9%) of 22 455 in week 48. After controlling for factors associated with hospitalisation, individuals with SGTF infections had significantly lower odds of admission than did those with non-SGTF infections (256 [2·4%] of 10 547 vs 121 [12·8%] of 948; adjusted odds ratio [aOR] 0·2, 95% CI 0·1-0·3). After controlling for factors associated with disease severity, the odds of severe disease were similar between hospitalised individuals with SGTF versus non-SGTF infections (42 [21%] of 204 vs 45 [40%] of 113; aOR 0·7, 95% CI 0·3-1·4). Compared with individuals with earlier delta variant infections, SGTF-infected individuals had a significantly lower odds of severe disease (496 [62·5%] of 793 vs 57 [23·4%] of 244; aOR 0·3, 95% CI 0·2-0·5), after controlling for factors associated with disease severity. INTERPRETATION: Our early analyses suggest a significantly reduced odds of hospitalisation among individuals with SGTF versus non-SGTF infections diagnosed during the same time period. SGTF-infected individuals had a significantly reduced odds of severe disease compared with individuals infected earlier with the delta variant. Some of this reduced severity is probably a result of previous immunity. FUNDING: The South African Medical Research Council, the South African National Department of Health, US Centers for Disease Control and Prevention, the African Society of Laboratory Medicine, Africa Centers for Disease Control and Prevention, the Bill & Melinda Gates Foundation, the Wellcome Trust, and the Fleming Fund.

Health professionals' views and experiences of discussing weight with children and their families: A systematic review of qualitative research.

BACKGROUND: Healthcare professionals are ideally placed to discuss weight management with children and families to treat and prevent childhood obesity. The aim of this review was to collect and synthesize primary research evidence relating to health professional's views and experiences of discussing weight with children and their families. METHODS: Systematic searches were conducted using the following databases: MEDLINE (OVID), Cumulative Index to Nursing and Allied Health Literature (CINAHL), EMBASE (OVID), PsycINFO (OVID) and Healthcare Management Information Consortium (HMIC). Twenty-six full text qualitative studies published in English Language journals since inception to October 2019 were included. Papers were quality assessed and synthesized using an inductive thematic analysis approach. RESULTS: Data analysis generated five themes: sensitivity of the issue, family-professional relationships, whole systems approach, professional competency and sociocultural context. CONCLUSION: Supporting behaviour change through discussion of healthy weight with children and families is an important part of the health professional's role. Tailored information for professionals, including resources and training, facilitates them to confidently talk to children and families about weight prioritized within interventions. The success of such interventions requires commitment from a range of professionals to ensure healthy weight is tackled through a whole system approach.

Genome Engineering Renal Epithelial Cells for Enhanced Volume Transport Function.

INTRODUCTION: Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na+/H+ exchange and H2O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously. METHODS: We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport. RESULTS: piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport. CONCLUSIONS: Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy.

CRISPR/Cas9 engineering of albino cystinuria Type A mice.

Cystinuria Type A is a relatively common genetic kidney disease occurring in 1 in 7,000 people worldwide that results from mutation of the cystine transporter rBAT encoded by Slc3a1. We used CRISPR/Cas9 technology to engineer cystinuria Type A mice via genome editing of the C57BL/6NHsd background. These mice are an improvement on currently available models as they are on a coisogenic genetic background and have a single defined mutation. In order to use albinism to track Cas9 activity, we co-injected gRNAs targeting Slc3a1 and tyrosinase (Tyr) with Cas9 expressing plasmid DNA into mouse embryos. Two different Slc3a1 mutational alleles were derived, with homozygous mice of both demonstrating elevated urinary cystine levels, cystine crystals, and bladder stones. We used whole genome sequencing to evaluate for potential off-target editing. No off-target indels were observed for the top 10 predicted off-targets for Slc3a1 or Tyr. Therefore, we used CRISPR/Cas9 to generate coisogenic albino cystinuria Type A mice that could be used for in vivo imaging, further study, or developing new treatments of cystinuria.

Metabolic consequences of cystinuria.

BACKGROUND: Cystinuria is an inherited disorder of renal amino acid transport that causes recurrent nephrolithiasis and significant morbidity in humans. It has an incidence of 1 in 7000 worldwide making it one of the most common genetic disorders in man. We phenotypically characterized a mouse model of cystinuria type A resultant from knockout of Slc3a1. METHODS: Knockout of Slc3a1 at RNA and protein levels was evaluated using real-time quantitative PCR and immunofluorescence. Slc3a1 knockout mice were placed on normal or breeder chow diets and evaluated for cystine stone formation over time suing x-ray analysis, and the development of kidney injury by measuring injury biomarkers. Kidney injury was also evaluated via histologic analysis. Amino acid levels were measured in the blood of mice using high performance liquid chromatography. Liver glutathione levels were measured using a luminescent-based assay. RESULTS: We confirmed knockout of Slc3a1 at the RNA level, while Slc7a9 RNA representing the co-transporter was preserved. As expected, we observed bladder stone formation in Slc3a1-/- mice. Male Slc3a1-/- mice exhibited lower weights compared to Slc3a1+/+. Slc3a1-/- mice on a regular diet demonstrated elevated blood urea nitrogen (BUN) without elevation of serum creatinine. However, placing the knockout animals on a breeder chow diet, containing a higher cystine concentration, resulted in the development of elevation of both BUN and creatinine indicative of more severe chronic kidney disease. Histological examination revealed that these dietary effects resulted in worsened kidney tubular obstruction and interstitial inflammation as well as worsened bladder inflammation. Cystine is a precursor for the antioxidant molecule glutathione, so we evaluated glutathione levels in the livers of Slc3a1-/- mice. We found significantly lowered levels of both reduced and total glutathione in the knockout animals. CONCLUSIONS: Our results suggest that that diet can affect the development and progression of chronic kidney disease in an animal model of cystinuria, which may have important implications for patients with this disease. Additionally, reduced glutathione may predispose those with cystinuria to injury caused by oxidative stress. Word count: 327.

Screening tool for malignant bowel obstruction in relapsed, metastatic ovarian cancer.

BACKGROUND: Malignant bowel obstruction (MBO) is a common cause of morbidity and mortality in women diagnosed with ovarian cancer. Earlier detection of MBO may improve patient outcomes. There are currently no screening tools to assist detection. AIM: We report a screening questionnaire that can be used to detect MBO, and how the severity score for key clinical symptoms correlate with radiological evidence of MBO from ovarian cancer. DESIGN: A case-control study in which patients with relapsed, metastatic ovarian cancer were asked to answer 10 questions related to key clinical symptoms associated with intestinal obstruction. The study group included women with CT-confirmed MBO, whereas the control group had no evidence of MBO. Patients scored each question according to severity from 1 (least severe) to 5 (most severe). SETTING/PARTICIPANTS: Between 1 June and 31 December 2016, 37 women completed the screening questionnaire. RESULTS: Patients in the study group (n=17) reported significantly higher (ie, more severe) scores for abdominal pain, nausea, vomiting and constipation. In contrast, differences in severity scores between groups did not differ significantly in response to questions regarding abdominal swelling, borborygmi, diarrhoea or loss of appetite. All patients in the study group more frequently stated that their symptoms had deteriorated within the 2 months prior to completing the questionnaire. CONCLUSION: Here we report the key clinical symptoms associated with radiologically-confirmed MBO in relapsed, metastatic ovarian cancer. We recommend healthcare practitioners focus on these specific symptoms during patient consultations in order to improve risk stratification of MBO.

Dose-dense cisplatin with gemcitabine for relapsed platinum-resistant ovarian cancer.

INTRODUCTION: Standard of care treatment for women who develop relapsed ovarian cancer includes sequential platinum- and/or paclitaxel-based chemotherapy, with reducing disease-free intervals. Once platinum resistance develops, treatment options become limited and dose-dense regimens may be offered. We report the efficacy and safety of dose-dense cisplatin with gemcitabine chemotherapy for relapsed platinum-resistant ovarian cancer. METHODS: A retrospective analysis of all patients with relapsed, platinum-resistant ovarian, primary peritoneal or fallopian tube cancer treated with cisplatin 35 mg/m2 of body surface area by intravenous infusion with gemcitabine 1000 mg/m2 of body surface area by intravenous infusion on days 1 and 8 of every 21-day treatment cycle between 1 January 2009 and 1 June 2017. RESULTS: Ninety-four eligible patients had received a median of three (range one-eight) prior lines of cytotoxic therapy for relapsed ovarian cancer. Sixty patients (64%) had received ≥ 1 prior dose-dense chemotherapy regimen. Dose-dense cisplatin with gemcitabine was associated with a median progression-free survival (PFS) of 4.4 months (95% CI 3.6 to 5.3) and overall survival of 7.6 months (95% CI 5.6 to 9.6). The median PFS for dose-dense cisplatin with gemcitabine as first- (n = 34), second- (n = 42), and third-line or later (n = 18) dose-dense therapy was 4.2 (95% CI 3.2 to 5.2), 5.0 (95% CI 3.5 to 6.5), and 4.2 (95% CI 3.3 to 5.1) months respectively. The RECIST objective response rate for first-, second-, and third-line dose-dense cisplatin with gemcitabine was 23%, 14 %, and 7 % respectively. The most common grade 3 - 4 adverse events were thrombocytopenia (20%), anemia (18%), and neutropenia (14%). DISCUSSION: Dose-dense cisplatin with gemcitabine provides modest efficacy whether it is used as a first- or subsequent line of dose-dense chemotherapy to treat relapsed platinum-resistant ovarian cancer and the toxicity is manageable with supportive measures.

Long QT syndrome KCNH2 mutation with sequential fetal and maternal sudden death.

We report a case of a woman who experienced intrauterine fetal death at full term pregnancy, and then died suddenly soon after learning about the death of her fetus. At autopsy, previously undiagnosed neurofibromatosis and an adrenal gland pheochromocytoma were discovered in the mother. Genetic screening also revealed a novel KCNH2mutation in both fetus and mother indicating type 2 congenital long-QT syndrome (LQTS). A catecholamine surge was suspected as the precipitating event of fetal cardiac arrhythmia and sudden fetal death, while the addition of emotional stress provoked a lethal cardiac event in the mother. This case illustrates the potential for lethal interactions between two occult diseases (pheochromocytoma, LQTS).

Transposon-modified antigen-specific T lymphocytes for sustained therapeutic protein delivery in vivo.

A cell therapy platform permitting long-term delivery of peptide hormones in vivo would be a significant advance for patients with hormonal deficiencies. Here we report the utility of antigen-specific T lymphocytes as a regulatable peptide delivery platform for in vivo therapy. piggyBac transposon modification of murine cells with luciferase allows us to visualize T cells after adoptive transfer. Vaccination stimulates long-term T-cell engraftment, persistence, and transgene expression enabling detection of modified cells up to 300 days after adoptive transfer. We demonstrate adoptive transfer of antigen-specific T cells expressing erythropoietin (EPO) elevating the hematocrit in mice for more than 20 weeks. We extend our observations to human T cells demonstrating inducible EPO production from Epstein-Barr virus (EBV) antigen-specific T lymphocytes. Our results reveal antigen-specific T lymphocytes to be an effective delivery platform for therapeutic molecules such as EPO in vivo, with important implications for other diseases that require peptide therapy.