Emergence and spread of Mycobacterium ulcerans at different geographic scales.

The classical lineage of Mycobacterium ulcerans is the most prevalent clonal group associated with Buruli ulcer in humans. Its reservoir is strongly associated with the environment. We analyzed together 1,045 isolates collected from 13 countries on two continents to define the evolutionary history and population dynamics of this lineage. We confirm that this lineage spread over 7,000 years from Australia to Africa with the emergence of outbreaks in distinct waves in the 18th and 19th centuries. In sharp contrast with its global spread over the last century, transmission chains are now mostly local, with little or no dissemination between endemic areas. This study provides new insights into the phylogeography and population dynamics of M. ulcerans, highlighting the importance of comparative genomic analyses to improve our understanding of pathogen transmission. IMPORTANCE: Mycobacterium ulcerans is an environmental mycobacterial pathogen that can cause Buruli ulcer, a severe cutaneous infection, mostly spread in Africa and Australia. We conducted a large genomic study of M. ulcerans, combining genomic and evolutionary approaches to decipher its evolutionary history and pattern of spread at different geographic scales. At the scale of villages in an endemic area of Benin, the circulating genotypes have been introduced in recent decades and are not randomly distributed along the river. On a global scale, M. ulcerans has been spreading for much longer, resulting in distinct and compartmentalized endemic foci across Africa and Australia.

All Roads Lead to Rome: YAP/TAZ Activity Influences Efficacy of KRASG12C Inhibitors.

The development of direct inhibitors of KRASG12C represents a monumental step forward in the field of oncology. Nevertheless, there is considerable opportunity to enhance response rates to KRASG12C inhibitors. In this issue of Cancer Research, three investigative teams explore the modulation of KRASG12C inhibitor activity in lung, colorectal, and pancreatic cancers using CRISPR-based knockout screens. While each group identified and validated a variety of genes and pathways conferring resistance to KRASG12C inhibition, all three groups converged upon activation of YAP/TAZ as a common means of resistance. While coinhibition of KRASG12C and YAP/TAZ did not cause complete tumor regression in xenograft models, combining YAP/TAZ inhibition was capable of significantly extending the response of tumors to KRASG12C inhibition. See related articles by Mukhopadhyay et al., p. 4095, Edwards et al., p. 4112, and Prahallad et al., p. 4130.

Allosteric site variants affect GTP hydrolysis on Ras.

RAS GTPases are proto-oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP-bound and GTP-bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4, and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF-RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP-bound state of RAS.

Allosteric Regulation of Switch-II Domain Controls KRAS Oncogenicity.

RAS proteins are GTPases that regulate a wide range of cellular processes. RAS activity is dependent on its nucleotide-binding status, which is modulated by guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAP). KRAS can be acetylated at lysine 104 (K104), and an acetylation-mimetic mutation of K104 to glutamine (K104Q) attenuates the in vitro-transforming capacity of oncogenic KRAS by interrupting GEF-induced nucleotide exchange. To assess the effect of this mutation in vivo, we used CRISPR-Cas9 to generate mouse models carrying the K104Q point mutation in wild-type and conditional KrasLSL-G12D alleles. Homozygous animals for K104Q were viable, fertile, and arose at the expected Mendelian frequency, indicating that K104Q is not a complete loss-of-function mutation. Consistent with our previous findings from in vitro studies, however, the oncogenic activity of KRASG12D was significantly attenuated by mutation at K104. Biochemical and structural analysis indicated that the G12D and K104Q mutations cooperate to suppress GEF-mediated nucleotide exchange, explaining the preferential effect of K104Q on oncogenic KRAS. Furthermore, K104 functioned in an allosteric network with M72, R73, and G75 on the α2 helix of the switch-II region. Intriguingly, point mutation of glycine 75 to alanine (G75A) also showed a strong negative regulatory effect on KRASG12D. These data demonstrate that lysine at position 104 is critical for the full oncogenic activity of mutant KRAS and suggest that modulating the sites in its allosteric network may provide a unique therapeutic approach in cancers expressing mutant KRAS. SIGNIFICANCE: An allosteric network formed by interaction between lysine 104 and residues in the switch-II domain is required for KRAS oncogenicity, which could be exploited for developing inhibitors of the activated oncoprotein.

Changes in Inflammatory Markers in Patients Treated for Buruli Ulcer and Their Ability to Predict Paradoxical Reactions.

Mycobacterium ulcerans causes Buruli ulcer, the third most frequent mycobacterial disease after tuberculosis and leprosy. Transient clinical deteriorations, known as paradoxical reactions (PRs), occur in some patients during or after antibiotic treatment. We investigated the clinical and biological features of PRs in a prospective cohort of 41 patients with Buruli ulcer from Benin. Neutrophil counts decreased from baseline to day 90, and interleukin 6 (IL-6), granulocyte colony-stimulating factor, and vascular endothelial growth factor were the cytokines displaying a significant monthly decrease relative to baseline. PRs occurred in 10 (24%) patients. The baseline biological and clinical characteristics of the patients presenting with PRs did not differ significantly from those of the other patients. However, the patients with PRs had significantly higher IL-6 and tumor necrosis factor alpha (TNF-α) concentrations on days 30, 60, and 90 after the start of antibiotic treatment. The absence of a decrease in IL-6 and TNF-α levels during treatment should alert clinicians to the possibility of PR onset.

States' Methods for Capturing and Reporting Local Responses to Suspected Nonfatal Drug Overdoses.

This study examines the available data used to surveil state-level drug overdoses and characterizes them based on their timeliness, drug involvement, and specification of nonfatal outcomes.

Integrating third-party apps with electronic health records to support COVID-19 response.

OBJECTIVES: To (1) track the integration of telehealth- and COVID-19-related apps with electronic health records (EHRs) over time, (2) identify the primary functionality of apps designed to support the COVID-19 response, and (3) examine whether apps available prior to the pandemic added new telehealth- or COVID-19-related functionalities during the pandemic. STUDY DESIGN: Data were collected from public EHR app galleries on a monthly basis from December 31, 2019, through June 1, 2021. METHODS: Apps were identified as relating to COVID-19 or telehealth using text analysis of the app marketing materials. Descriptive analyses were conducted to characterize telehealth- and COVID-19-related apps discovered through the app galleries, identify their primary functionality, and examine whether any apps added new telehealth- or COVID-19-related functionalities during the pandemic. RESULTS: The number of COVID-19-related apps increased from 0 in March 2020 to 19 a month later and continued to grow to 62 as of June 2021. The number of telehealth-related apps more than doubled from prepandemic levels (n = 41) to a total of 87 apps by June 2021. These apps were 2 times more likely to contain specialized capabilities used to support COVID-19 response efforts, such as secure messaging, vaccine administration, and laboratory testing, compared with all apps listed in the EHR app galleries. CONCLUSIONS: These findings demonstrate the potential of integrating third-party apps into EHRs to expand the range of tools that health care providers can use to diagnose, treat, and communicate with patients.

Disparities in patient portal access and the role of providers in encouraging access and use.

OBJECTIVE: The aim of this study was to identify racial and ethnic disparities in patient portal offers, access, and use and to examine the role of providers in facilitating access to electronic health information (EHI) by offering patient portals and encouraging their use. MATERIALS AND METHODS: Using nationally representative survey data from 2019 and 2020 (N = 8028), we examined disparities in patients being offered access to a portal by their provider and differences in subsequent access and use. Using multivariable models, we estimated the effect of race and ethnicity on the likelihood of being offered, accessing or using a portal. Among those offered, we examined the relationship between provider encouragement and portal access; and for those who did not access their portal, we explored reasons for nonuse. RESULTS: Black and Hispanic individuals were offered and accessed patient portals at significantly lower rates than White individuals. Compared to Whites, Black and Hispanic individuals were 5.2 percentage-points less likely to be offered a portal (P < .05) and, among those offered, 7.9 percentage-points less likely to access their portal (P < .05). Black and Hispanic individuals who were offered and accessed a portal were 12 percentage-points more likely than Whites to use it to download or transmit information (P < .01). Individuals who were offered a portal and encouraged to use it were 21 percentage-points more likely to access it. DISCUSSION: Differences in patient portal access and use are likely driven by disparities in which groups of patients reported being offered a portal. CONCLUSIONS: Providers play an important role in increasing access to EHI by facilitating access to patient portals.

New EHR certification requirements and their association with duplicate tests and images.

BACKGROUND: Certified electronic health record (EHR) technology has been adopted by most hospitals and health care providers. In 2015, the Office of the National Coordinator for Health Information Technology (ONC) published new EHR certification requirements, known as the 2015 Edition. To date, no research has examined the impact of hospitals' adoption of the 2015 Edition on health care delivery. METHODS: We analyzed aggregated, longitudinal data drawn from a repository of deidentified health insurance claims collected by FAIR Health, the repository was estimated to represent about 75% of the privately insured in the United States. These data were linked with the American Hospital Association (AHA) Information Technology Supplement Survey to obtain hospitals' health information technology characteristics. A fixed effects specification was used to assess the incidence of duplicate testing and imaging in both inpatient and outpatient settings before and after the hospitals' adoption of the 2015 Edition. RESULTS: Hospitals with the 2015 Edition were less likely to perform duplicate imaging for inpatients by 5 percentage points (or 50% from baseline). Hospitals that adopted the 2015 Edition and actively engaged in interoperable data exchange were even less likely to perform duplicate lab tests. CONCLUSIONS: Adoption of the 2015 Edition certified EHR was negatively associated with the incidence of lab and imaging test duplication in both the outpatient and inpatient settings. However, the results were not robust across specifications. Given that multiple factors influence care delivery decisions, improvements in certification standards alone are unlikely to eliminate unneeded duplicate lab and imaging tests.

Classification of KRAS-Activating Mutations and the Implications for Therapeutic Intervention.

Members of the family of RAS proto-oncogenes, discovered just over 40 years ago, were among the first cancer-initiating genes to be discovered. Of the three RAS family members, KRAS is the most frequently mutated in human cancers. Despite intensive biological and biochemical study of RAS proteins over the past four decades, we are only now starting to devise therapeutic strategies to target their oncogenic properties. Here, we highlight the distinct biochemical properties of common and rare KRAS alleles, enabling their classification into functional subtypes. We also discuss the implications of this functional classification for potential therapeutic avenues targeting mutant subtypes. SIGNIFICANCE: Efforts in the recent past to inhibit KRAS oncogenicity have focused on kinases that function in downstream signal transduction cascades, although preclinical successes have not translated to patients with KRAS-mutant cancer. Recently, clinically effective covalent inhibitors of KRASG12C have been developed, establishing two principles that form a foundation for future efforts. First, KRAS is druggable. Second, each mutant form of KRAS is likely to have properties that make it uniquely druggable.

The Impact of the COVID-19 Pandemic on Drug Use Behaviors, Fentanyl Exposure, and Harm Reduction Service Support among People Who Use Drugs in Rural Settings.

BACKGROUND: The COVID-19 pandemic has worsened the opioid overdose crisis in the US. Rural communities have been disproportionately affected by opioid use and people who use drugs in these settings may be acutely vulnerable to pandemic-related disruptions due to high rates of poverty, social isolation, and pervasive resource limitations. METHODS: We performed a mixed-methods study to assess the impact of the pandemic in a convenience sample of people who use drugs in rural Illinois. We conducted 50 surveys capturing demographics, drug availability, drug use, sharing practices, and mental health symptoms. In total, 19 qualitative interviews were performed to further explore COVID-19 knowledge, impact on personal and community life, drug acquisition and use, overdose, and protective substance use adaptations. RESULTS: Drug use increased during the pandemic, including the use of fentanyl products such as gel encapsulated "beans" and "buttons". Disruptions in supply, including the decreased availability of heroin, increased methamphetamine costs and a concomitant rise in local methamphetamine production, and possible fentanyl contamination of methamphetamine was reported. Participants reported increased drug use alone, experience and/or witness of overdose, depression, anxiety, and loneliness. Consistent access to harm reduction services, including naloxone and fentanyl test strips, was highlighted as a source of hope and community resiliency. CONCLUSIONS: The COVID-19 pandemic period was characterized by changing drug availability, increased overdose risk, and other drug-related harms faced by people who use drugs in rural areas. Our findings emphasize the importance of ensuring access to harm reduction services, including overdose prevention and drug checking for this vulnerable population.

Regulation of GTPase function by autophosphorylation.

A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts.

A combined field study of Buruli ulcer disease in southeast Benin proposing preventive strategies based on epidemiological, geographic, behavioural and environmental analyses.

Buruli ulcer is a neglected tropical disease caused by M. ulcerans, an environmental mycobacterium. This cutaneous infectious disease affects populations with poor access to sanitation, safe water and healthcare living in rural areas of West and Central Africa. Stagnant open bodies of surface water and slow-running streams are the only risk factor identified in Africa, and there is no human-to-human transmission. Appropriate and effective prevention strategies are required for populations living in endemic areas. Based on a multidisciplinary approach in an area in which Buruli ulcer is endemic in South Benin, we investigated the link between all human-environment interactions relating to unprotected water and behaviors associated with Buruli ulcer risk likely to affect incidence rates. We characterised the sources of water as well as water bodies and streams used by communities, by conducting a prospective case-control study directly coupled with geographic field observations, spatial analysis, and the detection of M. ulcerans in the environment. A full list of the free surface waters used for domestic activities was generated for a set of 34 villages, and several types of human behaviour associated with a higher risk of transmission were identified: (i) prolonged walking in water to reach cultivated fields, (ii) collecting water, (iii) and swimming. Combining the results of the different analyses identified the risk factor most strongly associated with Buruli ulcer was the frequency of contact with unprotected and natural water, particularly in regularly flooded or irrigated lowlands. We confirm that the use of clean water from drilled wells confers protection against Buruli ulcer. These specific and refined results provide a broader scope for the design of an appropriate preventive strategy including certain practices or infrastructures observed during our field investigations. This strategy could be improved by the addition of knowledge about irrigation practices and agricultural work in low-lying areas.

Fear of falling avoidance behavior affects the inter-relationship between vision impairment and diminished mobility in community-dwelling older adults.

BACKGROUND: Age-related decline in vision may contribute to the development of fear of falling (FOF) behavior and reduced mobility, which are related to increased fall risk in older adults. PURPOSE: To investigate the inter-relationship between vision impairment, physical mobility performance, and FOF behavior in community-dwelling older adults. METHODS: A total of 400 participants from community centers (267 females; age = 74.8 (6.4), range = 65-97 years) participated in this cross-sectional study. Presence of age-related eye diseases (e.g. macular degeneration, cataracts, glaucoma, and retinopathy) and visual acuity (VA) was assessed. Physical mobility and FOF avoidance behavior were assessed using the Timed Up-and-Go (TUG) test and the Fear of Falling Avoidance Behavior Questionnaire (FFABQ). The inter-relationships between parameters were analyzed using mediation model analysis. RESULTS: Significant decreases in mobility performance were observed in those with eye disease (eye disease = 9.56 [5.2] sec, no eye disease = 8.54 [2.75] sec; p = .037) and FOF avoidance behavior (avoiders = 12.87 [6.04] sec, non-avoiders = 8.51 [3.56] sec; p < .001). Furthermore, FOF behavior was found to significantly influence the inter-relationship between presence of eye disease and TUG performance (p = .004). VA alone had no significant effect on mobility (p = .69). CONCLUSION: The presence of eye disease and the associated FOF behavior was related to decreased mobility and potentially increased fall risk. We recommend clinicians to inquire about the presence of eye disease and FOF behavior to identify risk factors related to falls in older adults.

Molecular and epidemiological characterization of recurrent Mycobacterium ulcerans infections in Benin.

BACKGROUND: Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans, an environmental mycobacterium. Although transmission of M. ulcerans remains poorly understood, the main identified risk factor for acquiring Buruli ulcer is living in proximity of potentially contaminated water sources. Knowledge about the clinical features of Buruli ulcer and its physiopathology is increasing, but little is known about recurrence due to reinfection. METHODOLOGY/PRINCIPAL FINDINGS: We describe two patients with Buruli ulcer recurrence due to reinfection with M. ulcerans, as demonstrated by comparisons of DNA from the strains isolated at the time of the first diagnosis and at recurrence. Based on the spatial distribution of M. ulcerans genotypes in this region and a detailed study of the behavior of these two patients with respect to sources of water as well as water bodies and streams, we formulated hypotheses concerning the sites at which they may have been contaminated. CONCLUSIONS/SIGNIFICANCE: Second episodes of Buruli ulcer may occur through reinfection, relapse or a paradoxical reaction. We formally demonstrated that the recurrence in these two patients was due to reinfection. Based on the sites at which the patients reported engaging in activities relating to water, we were able to identify possible sites of contamination. Our findings indicate that the non-random distribution of M. ulcerans genotypes in this region may provide useful information about activities at risk.

Challenges and Prospects of Development of Herbal Biomaterial Based Ethical Wound Care Products-A Scoping Review.

Total wound care is an unmet therapeutic need considering the morbidity and mortality associated with the rising prevalence of nonhealing/chronic wounds. Current wound management fails to address all aspects/types of wounds despite the availability of scores of traditional and modern, investigational products. Traditional medicine drugs of wound healing repute validated to target multiple biological pathways and key events in the mammalian wound healing cascade, reportedly affecting wound healing phases. Advances in the development of biocomposite matrices and their analytical characterization warrant a relook at consolidating time-tested wound healing properties of herbal bioactives for prospective development as ethical wound care products. Aside from the bottlenecks of their multiconstituent profiling and clinical trial data generation, regulatory hurdles also cloister any systematic attempts at their re-engineering into clinical deliverables. In the context of national policy changes to bring in totally indigenous solutions, countries with a huge knowledge/material resource on wound healing bioactives need to essentially facilitate the same.

The ecosystem of apps and software integrated with certified health information technology.

OBJECTIVE: This study: 1) characterized the app market by EHR app gallery and type of app; 2) tracked changes in the EHR app galleries from the end of 2019 through 2020; and 3) examined how apps connect to EHR data systems, and if the apps support the HL7 FHIR standard. MATERIALS AND METHODS: We developed a program that gathered data from the public app galleries hosted by Allscripts, athenahealth, Cerner Corporation, Epic Systems Corporation, and SMART. Data collection for this study began in December 2019 and ended December 2020. The program was run 11 times during this period, and the data collected were used to generate the findings and trends observed in this study. RESULTS: The total number of unique apps increased from 600 to 734 during the study period. The most common types of apps marketed were intended for administrative (42%) and clinical use (38%). About 1 in 5 apps (22%) described support for the FHIR standard. Support for FHIR varied by intended functionality and gallery. DISCUSSION: This study provides early insights into the number of third-party apps that are connecting to EHRs, what services they provide, and if these connections use standards-based application programming interfaces (APIs). CONCLUSION: It is a federal government priority to improve the access and use of electronic health information, including third-party apps that can introduce competition as well as best-of-breed functions and user experiences. This study shows that there is room for growth, and variation exists among some of the largest EHR developers.

Measuring time clinicians spend using EHRs in the inpatient setting: a national, mixed-methods study.

OBJECTIVE: To understand hospitals' use of EHR audit-log-based measures to address burden associated with inpatient EHR use. MATERIALS AND METHODS: Using mixed methods, we analyzed 2018 American Hospital Association Information Technology Supplement Survey data (n = 2864 hospitals; 64% response rate) to characterize measures used and provided by EHR vendors to track clinician time spent documenting. We interviewed staff from the top 3 EHR vendors that provided these measures. Multivariable analyses identified variation in use of the measures among hospitals with these 3 vendors. RESULTS: 53% of hospitals reported using EHR data to track clinician time documenting, compared to 68% of the hospitals using the EHR from the top 3 vendors. Among hospitals with EHRs from these vendors, usage was significantly lower among rural hospitals and independent hospitals (P < .05). Two of these vendors provided measures of time spent doing specific tasks while the third measured an aggregate of auditable activities. Vendors varied in the underlying data used to create measures, measure specification, and data displays. DISCUSSION: Tools to track clinicians' documentation time are becoming more available. The measures provided differ across vendors and disparities in use exist across hospitals. Increasing the specificity of standards underlying the data would support a common set of core measures making these measures more widely available. CONCLUSION: Although half of US hospitals use measures of time spent in the EHR derived from EHR generated data, work remains to make such measures and analyses more broadly available to all hospitals and to increase its utility for national burden measurement.

Barriers to accessing online medical records in the United States.

OBJECTIVES: Patients' access to and use of online medical records (OMRs) can facilitate better management of their health and health care. However, health care disparities continue to exist. This study aimed to (1) determine the prevalence and predictors of individuals being offered access to OMRs, (2) identify predictors of individuals accessing their OMR, and (3) describe barriers to accessing one's OMR. STUDY DESIGN: Secondary analyses of cross-sectional data from Health Information National Trends Survey 5, cycles 1 and 2 (n = 6670). METHODS: Multivariable logistic regression analyses were used to examine the association between sociodemographic and health care-related factors on being offered access to OMRs, accessing OMRs, and cited reasons for not accessing OMRs. RESULTS: In 2017-2018, 54% of US adults reported having been offered access to OMRs, and among those offered, 57% reported accessing their records. The groups who were less likely to be offered OMRs included men, middle-aged adults, members of racial/ethnic minority groups, individuals with lower education and household incomes, those who do not use the internet, and those living in rural areas. Respondents who were less likely to access their OMRs despite being offered included individuals with lower education and household incomes and rural residents. Among the 43% who did not access their records, the primary reason for not accessing was their preference to speak to their provider directly. CONCLUSIONS: Sociodemographic and health care-related factors are associated with variation in use of OMRs. To realize the intended value of OMR use for patients, it is important to address barriers to OMR access and integrate OMRs into patient-provider communication and clinical care.

Manufacturing of convalescent plasma of COVID-19 patients: Aspects of quality.

The ongoing coronavirus disease 2019 (COVID-19) pandemic emerged in December 2019. Convalescent plasma represents a promising COVID-19 treatment. Here, we report on the manufacturing of a plasma-based product containing antibodies specific to SARS-CoV-2 obtained from recently recovered COVID-19 patients. Convalescent plasma donors were screened as follows: 1) previously confirmed SARS-CoV-2 infection (by real-time PCR (RT-PCR)); 2) a subsequent negative PCR test followed by a 2-week waiting period; 3) an additional negative PCR test prior to plasmapheresis; and 4) confirmation of the presence of SARS-CoV-2 specific antibodies. Convalescent plasma was stored fresh (2-6°C) for up to 5 days or frozen (-30°C) for long-term storage. Donor peripheral blood and final plasma product were assayed for binding antibodies targeting the SARS-CoV-2 S-protein receptor-binding domain (RBD) and their titers measured by an enzyme-linked immunosorbent assay (ELISA). We performed 72 plasmaphereses resulting in 248 final products. Convalescent plasma contained an RBD-specific antibody titer (IgG) ranging from 1:100 to 1:3200 (median 1:800). The titer was congruent to the titer of the blood (n = 34) before collection (1:100-1:6400, median 1:800). Levels of IL-8 and LBP of donors were slightly increased. Therapeutic products derived from a human origin must undergo rigorous testing to ensure uniform quality and patient safety. Whilst previous publications recommended RBD-specific binding antibody titers of ≥ 1:320, we selected a minimum titer of 1:800 in order to maximize antibody delivery. Production of highly standardized convalescent plasma was safe, feasible and was readily implemented in the treatment of severely ill COVID-19 patients.