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[1] Abraham J. Gitlitz Memorial Lecture: Development of a BroadSpectrum Antiviral-based Intranasal Spray as a Pandemic Preparedness Strategy. Kenneth E. Palmer, Ph.D, Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases and Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY In early 2020, the public health emergency prompted many researchers to contemplate how they might contribute to control of the COVID-19 pandemic. This presentation will detail the research steps taken to bring a novel broad spectrum antiviral protein into a first in humans clinical trial. We had been developing a broad spectrum antiviral protein, the lectin Q-Griffithsin (Q-GRFT), as a topical microbicide for HIV-1 prevention. We knew that Q-GRFT also inhibited replication of many members of the Coronavirus family of pathogens, so initiated a rapid preclinical and clinical development program of a Q-GRFT intranasal spray as a prophylaxis modality against SARS-CoV-2, the virus that causes COVID-19. We finalized a suitable formulation to deliver Q-GRFT to the nasopharynx, the initial site of replication of SARS-CoV-2. Non-clinical toxicology studies supported first-in-human clinical studies. Efficacy studies in mice and hamsters provided proof of concept that Q-GRFT can protect animals against challenge. We filed an investigational new drug (IND) application, and received study may proceed authorization from the FDA. We conducted a randomized, placebo-controlled single dose safety and pharmacokinetics clinical study in 18 volunteers, all of whom had been vaccinated against SARS-CoV-2. The product was safe, and enhanced the levels of SARS-CoV-2 as well as MERS-CoV inhibitory activity present in the nasal and naso-pharyngeal swabs. The safety and pharmacokinetics profile of our Q-GRFT intranasal spray supports a multi-dose Phase 1b clinical study, and further development as a general pandemic preparedness strategy. Upon completion of this activity, participants should be able to: describe the preclinical and clinical strategies employed to support a first in humans clinical trial of a novel broad-spectrum antiviral protein containing nasal spray; discuss planning, design and execution of a first-in-humans Phase 1a clinical trial for evaluation of safety, and selection of a dosing strategy for a Phase 1b multiple dose safety and pharmacokinetics study. [2] Acetylation pharmacogenomics: paradigm for informed individual risk assessment following environmental carcinogen exposure. David W. Hein, Ph.D, University of Louisville, Louisville, KY Human epidemiological studies associating chemical exposures to cancer risk often are inconsistently validated across studies. Examples include the effect of smoking on cancer etiology other than the lung, such as urinary bladder and breast. Research findings from the laboratory have improved the understanding of arylamine carcinogen metabolism leading to improved design and interpretation of human molecular epidemiology investigations. Laboratory studies that infer and test biological plausibility, including cancer risks modified by differential metabolism of arylamine carcinogens in rapid and slow arylamine N-acetyltransferase (NAT2) acetylators, have been critical for investigating the role of smoking in the etiology of human cancers. This chapter will illustrate these concepts with an example of a cancer in which the role of smoking has largely been validated (urinary bladder cancer) and examples where a consensus has yet to be achieved. Portions of this work were funded by the following NIH grants: R01-CA034627; T32-ES011564; P42-ES023716; P20-GM113226; R25-CA134283; and P30-ES030283. Upon completion of this activity, participants should be able to: 1. Assess the role of genetic polymorphisms in individual risk assessments following exposures to environmental carcinogens. 2. Recognize the importance of laboratory-based data in the biological plausibility of individual risk assessments. 3. Recognize the genetic complexity inherent in human epidemiological studies. [3] Rolling with the punches: A biosafety program at a research university addresses COVID-19. Allen Helm, PhD, The University of Chicago, Chicago, IL This presentation describes the biosafety program at a research university and medical center, demonstrating how the program was modified during the COVID-19 pandemic. Biosafety professionals are tasked with facilitating biomedical research by enacting coordinated efforts to protect research staff and students, the larger research community, and the environment and ecosystem from biological hazards used in the laboratory. These hazards include recombinant organisms ranging from microorganisms to animals, microbial pathogens, human-derived material, and biological toxins. The University of Chicago (UChicago) is an academic research institution affiliated with an urban medical center. There are approximately 300 biomedical principal investigator-led laboratories conducting investigations in basic, translational, and clinical science. The biosafety program at UChicago is part of the Office of Research Safety and consists of a director and four biosafety officers who perform a variety of duties, including: 1) Assisting researchers in planning experiments that utilize biohazards; 2) Developing and delivering biosafety training courses; 3) Performing annual inspections of laboratories; 4) Working with the university's Institutional Biosafety Committee; 5) Ensuring compliance with local, state, and federal agencies. The COVID-19 pandemic affected the biosafety program in several ways, as follows: 1) Altering administrative operations; 2) Developing biosafety standards for investigators working with SARS-CoV-2; 3) Establishing a "COVID Core" to handle influx of said researchers; 4) Enhanced biosafety interactions with clinical laboratories due to a demand for vaccines and treatments. Upon completion of this activity, participants should be able to: 1. Identify the role of biosafety professionals in basic, translational, and clinical research. 2. Recognize how a pandemic can alter the way biosafety services are delivered to research programs. 3. Recognize the need for flexibility in a successful biosafety program. [4] An interdisciplinary Post Operative Personalized Pain Management Clinical Trial. Loralie J. Langman1, Jeremy Gaskins2, Gwendolyn A. McMillin3, Paul J. Jannetto1, Brandi Hartley4, Arthur Malkani4, and Saeed A. Jortani5, department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, 2Departments of Bioinformatics and Biostatistics, University of Louisville School of Medicine, Louisville, KY, 3Department of Pathology, University of Utah, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, 4Departments of Orthopedic Surgery, University of Louisville School of Medicine, Louisville, KY, and 5Departments of Pathology, and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY Various sources of variability in response and toxicity to hydrocodone were investigated. In a cohort of orthopedic surgery patients, we interrogated the associations between genetic, intrinsic and extrinsic patient factors, plasma concentrations of hydrocodone and metabolites, common side effects, and pain score. Data for each patient was collected by the review of the electronic medical record and a patient interview at the time of sample collection. Patients with trauma or undergoing scheduled elective surgery for total knee or total hip replacement at the University of Louisville, Baptist East, and Jewish Hospitals, Louisville, KY. Plasma opiate concentrations and a targeted genotyping panel were performed. We observed statistically significant correlations for daily (p<0.001) and total dose (p=0.002) of hydrocodone. Duration of in-hospital and duration of opioid therapy for patients were also significantly different based on their genotypes. The length of opioid administration was significantly shorter in CYP2D6 EM/UM compared to patients with CYP2D6 PM/IM genotypes (p=0.018). Subjects with the OPRM1 c.118G polymorphism were also on opioids for a longer period of time (p=0.022). Co-administration of medications with CYP2D6 inhibitor activity had a significant effect on the length of opioid therapy (P<0.001). Both the hospital stay period and days of opioid use post hospital discharge were greater in patients with the inhibitor-adjusted CYP2D6 phenotype (p<0.001). This trial showed that patients should be evaluated for the use of inhibitors of CYP2D6. Interaction with these therapeutics while administering hydrocodone therapy can alter the phenotype of the patient (phenocopy) and result in longer opioid therapy duration. Upon completion of this activity, participants should be able to: 1. to discuss various sources of variability to response and toxicity of analgesics. 2. to present the post-operative pain management trial in a cohort of women just undergone Cesarean Section. 3. to present the post-operative pain management trial in a cohort of lower extremity orthopedic patients. [6] Claude P. Brown Memorial Lecture: The Many Roads to Steatohepatitis and its Treatment. Craig J. McClain, MD, University of Louisville School of Medicine,Louisville, KY Hepatic steatosis and steatohepatitis are common histologic findings that can be caused by multiple etiologies. The three most frequent causes for steatosis/ steatohepatitis are alcohol (alcohol-associated steatohepatitis, ASH), obesity/metabolic syndrome (nonalcoholic steatohepatitis, NASH), and environmental toxicants (toxicant-associated steatohepatitis, TASH). Hepatic steatosis is an early occurrence in all three forms of liver disease, and they often share common pathways to disease progression/severity. Disease progression is a result of both direct effects on the liver as well as indirect alterations in other organs/tissues such as intestine, adipose tissue, and the immune system. Although the three liver diseases (ASH, NASH, and TASH) sharemany common pathogenic mechanisms, they also exhibit distinct differences. Both shared and divergent mechanisms can be potential therapeutic targets. I will provide an overview of selected important mechanistic similarities and differences in ASH, NASH, and TASH, and discuss the importance of a multidisciplinary and personalized approach. Upon completion of this activity, participants should be able to: 1. understand the mechanisms including similarities and differences between ASH, NASH and TASH. 2. understand targets to treat ASH, NASH and TASH. 3. recognize tests to help distinguish between ASH, NASH and TASH. [7] Bioactive lipid metabolites: biomarkers and therapeutic targets in alcohol-associated liver disease. Dennis Warner, Josiah Hardesty, Jeff Warner, Craig McClain, Irina Kirpich, University of Louisville, Louisville, KY Alcohol-associated liver disease (ALD) is a spectrum of liver disorders ranging from steatosis to steatohepatitis, fibrosis and cirrhosis. The mechanisms and mediators of ALD progression are not well understood and effective therapeutic options are limited. Various bioactive oxidized lipid mediators (oxylipins) have recently emerged as important factors in ALD pathogenesis. The current study aimed to examine plasma linoleic acid (LA)-derived lipid metabolites in heavy drinking individuals and to evaluate associations between these molecules and markers of liver injury. Analysis of plasma LA-derived metabolites was performed by HPLC/MS on 66 heavy drinking individuals and 29 socially drinking but otherwise healthy volunteers. Based on plasma ALT levels, 15 patients had no liver injury (ALT ≤ 40 U/L), 33 patients had mild liver injury (ALT > 40 U/L), and 18 were diagnosed with moderate Alcohol-associated Hepatitis (mAH). Statistically significant differences (set at p<0.05) were determined by One-way ANOVA. Lipoxygenase-derived LA metabolites, 13-HODE and 13-oxoODE, were markedly elevated only in mAH patients. The CYP450-derived LA epoxides, 9,10-EpOME and 12.13-EpOME were decreased in all patients regardless of the presence or the absence of liver injury. LA-derived diols, 9,10-DiHOME and 12.13-DiHOME, were elevated only in the mAH group. The current study provides evidence that specific changes in LA-metabolites in heavy drinking individuals can distinguish individuals with mAH from those with mild ALD. Upon completion of this activity, participants should be able to: 1. identify effects of alcohol consumption on circulating oxylipins; 2. determine the role of oxylipins in ALD pathogenesis; 3. associate severity of ALD with the specific changes in oxylipins [8] Hepatic protein and phosphoprotein signatures of alcohol-associated hepatitis. Josiah Hardesty, Jeffrey Warner, Dennis Warner, Craig McClain, and Irina Kirpich, University of Louisville, Louisville, KY The objective of the current study was to identify hepatic proteome and phosphoproteome signatures of alcohol-associated hepatitis (AH). AH is a clinical manifestation of ALD characterized by compromised liver function contributing to a 6-month mortality rate as high as 50%. Proteomic and phosphoproteomic analyses were conducted on explant liver tissue from AH patients (n=6) and non-AH controls (n=12). Data were statistically compared by an unpaired Student's t-test and a p < 0.05 was considered statistically significant. Alterations the expression of multiple proteins involved in various biological processes were observed in AH. Among significant findings in AH included elevated expression of pro-fibrotic transcription factors, reduced albumin (ALBU) phosphorylation, and diminished expression of functional mitochondria proteins. One transcription factor involved in fibrogenesis, yes1-associated transcriptional regulator (YAP1) was elevated in AH (p=0.003), along with increased phosphorylation at pS105 (p=0.01). In addition, expression of hepatic ALBU was elevated in AH (p=0.04) concomitant with diminished ALBU phosphorylation (p=0.02), which may prevent ALBU release leading to hypoalbuminemia. Lastly, we found a loss in the expression of mitochondria proteins in AH, including enzymes essential for mitochondria function and biogenesis (e.g., hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha, [ECHA] p=0.04). This study identified hepatic protein and phosphoprotein signatures of AH which may facilitate the development of novel therapeutic strategies. Upon completion of this activity, participants should be able to: 1. understand the hepatic proteomic changes that occur in AH, 2. recognize hepatic protein and phosphoprotein signatures of AH and 3. identify novel mechanisms and pathways implicated in AH. [9] Soluble epoxide hydrolase inhibition in alcohol-associated liver disease: liver-specific drug delivery. Jeffrey Warner, Josiah Hardesty, Ying Song, Philip Bauer, Chirag Soni, Claudio Maldonado, Craig McClain, Dennis Warner, Irina Kirpich, University of Louisville, Louisville, KY Alcohol-associated liver disease (ALD) is a prevalent condition resulting from excessive alcohol consumption. Advanced stages of ALD, such as alcohol-associated hepatitis (AH), cause significant mortality and lack effective therapies. Previous data established that soluble epoxide hydrolase (sEH, an enzyme which degrades beneficial lipid epoxides) is induced in clinical/ experimental ALD, and that sEH inhibition may be an effective treatment for this disease. This study aimed to improve this approach by using liverspecific drug delivery via fusogenic lipid vesicles (FLVs) to increase efficacy and avoid extra-hepatic side effects. We prepared fluorescent-labeled FLVs loaded with the sEH inhibitor t-TUCB (t-TUCB-FLVs) at various doses. t-TUCB-FLV preparations had an appropriate size and charge and were confirmed to target the liver by fluorescent microscopy. Flow cytometry demonstrated that hepatocytes and macrophages were most responsible for t-TUCB-FLV uptake. In a dose response experiment using a chronic-binge ethanol feeding model mimicking AH, mice receiving ethanol+3.0 mg/kg t-TUCB-FLVs had the greatest reduction in liver injury by plasma ALT. This treatment was more efficacious than systemically delivered (non-FLV-encapsulated) t-TUCB at the same dose. t-TUCB-FLVs also decreased liver cell death and ER stress but had no effect on steatosis or neutrophil infiltration. These data demonstrated that liver-specific delivery of t-TUCB was more efficacious than systemic delivery. This drug delivery platform may help increase the efficacy of sEH inhibition in ALD while reducing extra-hepatic side effects, improving translation to humans. Upon completion of this activity, participants should be able to: 1.recognize the pathogenic role of sEH in ALD; 2. Describe basic concepts in liver-specific drug delivery by nanoparticle systems; and 3. Evaluate the beneficial effects of a liver-targeted sEH inhibitor in experimental ALD in mice. [10] COVID-19 and early post-primary TB: commonalities of pathobiology in pneumonitis and therapies. Robert L. Hunter and Robert E.Brown, UTHealth McGovern Medical School Houston, TX Concurrent infection with COVID-19 and M. tuberculosis has been reported to be more severe than either alone, resulting in increased mortality. Our objective was to define the shared pathobiology of COVID-19 and the developmental stage of TB in the lung and explore adjunctive therapies to treat such commonalities. We used similar morphoproteomic analyses to study lung tissues of patients with early postprimary tuberculosis or COVID-19 infection.These studies showed colocalization of the COVID-19 virus and M. tuberculosis antigens with cyclo-oxygenase-2 and fatty acid synthase in the reactive alveolar pneumocytes and with programmed death-ligand 1 expression on the alveolar interstitium and alveolar pneumocytes.This was associated with accumulation of pro-infectious M2 polarized macrophages in the alveolar spaces.The commonalities in these pathways suggest that they might be susceptible to adjunctive therapies with metformin and vitamin D3. This is supported by published studies that metformin and vitamin D3 could reduce the severity of both COVID-19 and early post-primary TB infections. Upon completion of this activity, participants should be able to identify and define the commonalities in the pathobiology of COVID-19 and early post-primary TB pneumonitis and describe the potential targets for therapy with metformin and vitamin D3. [11] Plasma ctDNA for Monitoring Response to Immune Check Point Inhibitors. Mark W. Linder PhD, DABCC, FAACC. Professor Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY Circulating tumor DNA (ctDNA) measurements from a variety of malignancies are aggressively being investigated in the context of a "liquid biopsy" to provide for a minimally invasive means of monitoring tumor status and therapeutic response. This session will discuss what is known about the biologic and physiological characteristics of plasma ctDNA. We will then describe the what is known regarding the relationship between plasma ctDNA and tumor characteristics such as tumor burden, proliferative activity and therapeutic response . Understanding of these relationships is central to developing a fundamental framework for interpretation longitudinal plasma ctDNA measurements in light of other routine measurements such as radiographic assessments and other blood based biomarkers. Upon completion of this activity, participants should be able to: 1. Explain the sources and methods of measurement of ctDNA. 2. Describe the relationships between plasma ctDNA, disease burden and therapeutic response. 3. Discuss how these relationships influence the clinical utility of routine plasma ctDNA testing. [12] Quantitative Methods of Monitoring Circulating Tumor DNA. Evan M. Alexander, PhD, Roland Valdes Jr., PhD, DABCC, Mark Linder, PhD, DABCC, Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY Circulating tumor DNA (ctDNA) is emerging as a new, robust biomarker that can be used for early cancer diagnosis, disease prognosis and even guiding treatment through precision medicine. This non-invasive tumor monitoring tool can be performed on the scale of a single gene mutation to an entire sequenced genome. Quantitation of ctDNA is complicated by two factors: wide ranges and low concentrations (0.003%-95% mutant allele frequency (MAF); 5-1500 ng/mL). Currently, the field of monitoring ctDNA is dominated by next-generation sequencing (NGS) and polymerase chain reaction (PCR) based technologies. Understandably, these different monitoring techniques require specialized analytical instrumentation. Furthermore, the sensitivity and specificity of these methods are not universal. There are many considerations a clinician needs to be mindful of when utilizing these technologies to monitor ctDNA (known vs unknown mutation, absolute quantitation vs MAF). Understanding the strengths and weaknesses of ct DNA quantification methods in a given clinical situation is paramount in utilizing this biomarker to its fullest and most appropriate potential. Upon completion of this activity, participants should be able to: 1. Identify which quantitation technique for ctDNA is most appropriate to use in a given clinical scenario 2. Recognize the difference between quantitation of mutant allele frequency and absolute quantitation of ctDNA 3. Recognize the importance of assay sensitivity when serially monitoring patient cancer progression. [13] The Value of Next Generation Sequencing in Myeloid Neoplasia. Mustafa Al-Kawaaz, University of Louisville, Louisville, KY This presentation will demonstrate some of the most important utilities of next generation sequencing (NGS) in a spectrum of acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and overlap syndromes. Interrogation of nucleic acid (DNA and/or RNA) to look for certain genomic alterations is essential for diagnostic, prognostic, and therapeutic purposes. Classification of myeloid neoplasms continues to evolve by including entities defined by specific genomic alterations. NGS is an essential utility to guide management decisions. Upon completion of this activity, participants should be able to: 1. Recognize changes to classification of myeloid neoplasms and some categories definite by genetic alterations. 2. Identify the utility of different vendors/ platforms offering certain advantages in next generation sequencing. 3. Recommend testing utility by evaluating the targeted genetic alteration as well as clinical scenario. [14] Forecasting clinical behavior and therapeutic response of breast carcinoma using gene expression. James L. Wittliff and Michael W. Daniels, University of Louisville, Louisville, KY Our goal is to associate expression of nuclear and peptide hormone receptor genes with biomarker status of breast carcinoma and risk of recurrence, to advance clinical management. Cellular heterogeneity of tissue specimens is a complicating factor in determining analyte (protein or gene) levels of specific cell types. A unique deidentified database was analyzed that contained microarray results of 22,000 genes derived only from total RNA extracted from breast carcinoma cells procured by laser capture microdissection (Pixcell IIe:Arcturus®/ Thermo Fisher) of 247 de-identified primary tissue biopsies. Relative expression levels of each gene candidate for 49 nuclear receptors as well as 61 peptide/protein hormones and 81 of their cognate receptor proteins were selected for this retrospective investigation. Assessment of a patient's risk of recurrence primarily utilizes estrogen (ER) and progestin receptor proteins (PR), quantified by radio-ligand binding (NEN/DuPont) and/or enzyme immunoassay (Abbott Labs). Parameters and clinical outcomes were analyzed by univariable and multivariable Cox regressions, Fisher's Exact Test, Kaplan Meier plots and with R software v4.0.0. Examples of multivariable Cox regression models of candidates of nuclear receptor genes, fit to predict disease-free survival (DFS) and overall survival (OS), revealed that only NR4A2, PGR, PPARA and THRB were required to predict DFS and NR3C2, PGR and THRB were necessary to predict OS. Of 142 candidate genes for peptide hormones and their cognate receptors, 30 exhibited expression levels that individually predicted DFS and/or OS. When pairs of genes for a peptide hormone and its receptor were evaluated by multivariable Cox Regression with interaction, complexes were identified that predicted DFS and OS (EDN1-ENDRA, GHRL-GHSR, INHBB-ACVR2B, NPY-NPY1R, INHBB-ACVR1B, RLN2-RXFP3 and NPY-NPY6R) based on unadjusted p-value for the interaction term. These investigations also revealed numerous over-expressed genes in carcinomas with poor clinical outcomes suggesting candidates for development of novel therapeutics. Collectively, use of small molecular signatures (gene subsets) with quantified ER/PR protein or ESR1/ PGR expression in a breast carcinoma with clinical outcomes enhanced prediction of risk of recurrence and identification drug development candidates. Upon completion of this activity, participants should be able to: 1. recognize the power of LCM to non-disruptively capture populations of specific cell types for genomics testing, 2. differentiate gene expression patterns based upon protein tumor marker status groups and 3. predict risk of recurrence for patients based upon expression of gene molecular sign. [15] Imaging Mass Spectrometry: Applications in Biomedical Research and Clinical Diagnosis. Yusheng Zhu, PhD; Pennsylvania State University College of Medicine Hershey Medical Center, Hershey, PA Mass spectrometry tissue imaging is a technology used in mass spectrometry to visualize the spatial distribution of molecules in tissues by their molecular masses. Compared to traditional tissue imaging methods such as immunohistochemistry, mass spectrometry imaging does not need any antibodies and tracers; it can detect and map multiple analytes including proteins, peptides, nucleic acids, lipids, carbohydrates, metabolites, drugs, toxins, and even elements simultaneously; researchers do not need prior knowledge of molecules in the samples; the analysis can be qualitative and/or quantitative. Therefore, mass spectrometry imaging has become a powerful technology for biomedical research and biomarker discovery. It is widely used in proteomic, peptidomic, lipidomic, glycomic, metabolomic, pharmacological and toxicological studies. This session will introduce common mass spectrometry imaging techniques including matrix assistant laser desorption ionization-time of flight (MALDI-TOF) imaging, time of flight-secondary ion mass spectrometry (TOF-SIMS) imaging, and desorption electrospray ionization (DESI)-mass spectrometry imaging. In addition, the basic principle, procedure, and application of these methods in biomedical research and clinical diagnosis will be discussed. Upon completion of this activity, participants should be able to: 1. Define tissue imaging mass spectrometry. 2. Explain basic principle of common types of imaging mass spectrometry. 3. Describe application of tissue imaging mass spectrometry in biomedical research and clinical diagnosis. [16] Immune Landscape in Sentinel Lymph Nodes from Melanoma Patients by Single-Cell Mass Cytometry (CyTOF) Analysis. Kavitha Yaddanapudi, University of Louisville, Louisville, KY We describe the development of a multiscale immune profiling strategy to map the immune landscape of sentinel lymph nodes (SLN) in our search for tumor-driven immune changes that can guide the design of novel immunotherapeutic strategies for patients with early-stage melanoma. We used mass cytometry by time-of-flight (CyTOF), flow cytometry, and T cell receptor immunosequencing to conduct simultaneous single-cell analyses of immune cells in the SLNs of melanoma patients. We identified unique tumordriven T, NK, and innate immune cell signatures that are present in stage III melanoma-bearing SLNs, but absent in stage I/II non-melanoma-bearing SLNs. We found increased effector-memory T cells and TCR clonality selectively in the melanoma-bearing SLNs relative to non-melanoma-bearing SLNs, consistent with possible activation of an anti-tumor immune response. However, we also observed a markedly immunotolerant environment in the melanoma-bearing SLNs indicated by reduced and impaired NK cells and increased levels of CD8+CD57+PD-1+ cells which are known to display low melanoma killing capabilities. Other changes observed in melanoma-bearing SLNs when compared to non-melanoma bearing SLNs include reduced CD8+CD69+ T cells/T regulatory cells ratio and high PD-1 expression on CD4+ and CD8+ T cells. Our data suggests that these immunological changes compromise anti-melanoma immunity and contribute to a high relapse rate. Upon completion of this activity, participants should be able to describe mass cytometry analysis of melanoma patient sentinel lymph node samples, identify new immunologic and therapeutic targets for preventing melanoma recurrence and identify unique melanoma-driven immune cell signatures. [17] Application of MassARRAY System in Molecular Diagnostics - Pharmacogenomics and Oncology. Shuko Harada and Alexander C. Mackinnon, University of Alabama at Birmingham, Birmingham, AL In molecular diagnostic laboratories, multiplex genetic analysis, for example Next Generation Sequencing (NGS), is increasingly utilized. NGS is expensive, laborious, and requires complicated bioinformatics analysis. The MassARRAY system provides accurate, low cost, facile, multiplexed analysis of hundreds of clinically relevant mutations with relatively simple analytics. In this study, we evaluated the utility of this system for two multiplex, molecular diagnostic applications: detection of somatic variants found in brain (CNS) tumors and germline variants (SNPs) found in metabolic enzymes as part of a pharmacogenomics (PGDX) assay. The CNS assay utilizes a custom design to identify deletions involving chromosomes 1p and 19q and somatic variants in IDH1, IDH2 and TERT. The PGDX panel targets 68 SNPs and several copy number variants (CNV) in 20 metabolic genes implicated in drug metabolisms. Genetic targets are PCR amplified followed by primer extension using allele-specific, specialized primers. Allotypes are detected by the MassARRAY system and results are generated using the MassArray software. Thirty CNS tumor samples with known 1p19q and IDH1/IDH2 mutation status were analyzed using the CNS panel. The results were >95% concordant to orthogonal test results. 48 samples with known various genotypes were analyzed using the PGDX assay. The results were >99% concordant; intra- and inter-run reproducibility was 100% (n=7). Overall, the MassARRAY system is reliable, cost effective and easy to operate. Performance is consistent and analysis is streamlined. The MassARRAY platform has broad application across multiple indications for molecular diagnostic testing. Upon completion of this activity, participants should be able to understand the utility of MassARRAY system in molecular diagnostics. [18] Alzheimer's disease CSF biomarkers in clinical practice: analytical and clinical considerations. Alicia Algeciras-Schimnich, Susan Ashrafzadeh-Kian, Wentao Li, Michelle R. Campbell, Ronald Petersen, and Joshua Bornhorst, Mayo Clinic, Rochester, MN This presentation will describe analytical and clinical considerations for the appropriate use of the cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers: amyloid beta 1-42 (Aβ4 2), total Tau (t-Tau), and phosphorylated Tau (p-Tau). Changes in these biomarkers reflect key changes in AD pathophysiology and are being incorporated into clinical practice. One of the challenges with the interpretation of CSF biomarkers has been the lack of standardized procedures for sample collection and handling as well as the lack of robust assays to measure these biomarkers. The analytical characteristics of the Roche Elecsys immunoassays for the quantitation of Aβ42, t-Tau, and p-Tau in CSF were established. All assays demonstrated robust analytical performance suitable for clinical laboratory utilization. To evaluate clinical performance, these biomarkers were measured in a cohort of clinically characterized samples (n=161). Using a p-Tau/Aβ42 ratio cutoff of >0.023, 20% of cognitively unimpaired, 41% of mild cognitive impairment, and 100% of AD dementia patients were classified as positive. The use of the p-Tau/Aβ42 ratio showed optimal concordance with amyloid PET exhibiting 100% negative and 86% positive concordance. Finally, the pattern of these biomarkers was evaluated in 535 Mayo Clinic patients that underwent testing as part of a cognitive evaluation over a 1-year period. In 27% of patients all biomarkers were normal and not consistent with AD; in 25% of patients all biomarkers were consistent with AD. A normal p-Tau/ Aβ42 ratio with low/abnormal Aβ42 was observed in 19% of patients and was most often associated with the clinical presentation of normal pressure hydrocephalus (NPH). An abnormal p-Tau/Aβ42 ratio displayed the strongest relationship with AD. Upon completion of this activity, participants should be able to 1. identify the role of Aβ42, t-Tau, and p-Tau in the differential diagnosis of AD and 2 discuss how various biomarker patterns are associated with other non-AD cognitive disorders. [19] Evaluation of SARS-CoV-2viral RNA detection, using Massarray, RT-qPCR and UltraFast RT-PCR assays. Bene Ekene-Afolabi1, John Patrick Alao1, Solomon Rotimi1,2, and John Bolodeoku1,3, 1ZEAB Therapeutic Ltd. Discovery Park, Ramsgate Road, Sandwich, Kent CF13 9FF, United Kingdom, 2Department of Biochemistry, Covenant University, Ota, Nigeria, and 3JB Consulting MDP Ltd, 1 Bell Street, Maidenhead, Berkshire, SL6 1 BU, United Kingdom The Covid-19 pandemic caused by the SARS-CoV-2 virus revealed short comings in the global ability to effectively deal with such crises. In particular, the molecular diagnostics market was flooded with a diverse array of RT-PCR kits. These kits target different regions of the SARS-CoV-2 genome with the United States Centres for Disease Control (CDC) recommended nucleocapsid N1 and N2 genes being the most common. In addition, these kits use various cycling parameters and cut-offs despite being approved for diagnostic testing in various countries. Detailed information on the suitability of these tests for the clinical detection of SARS-CoV-2 and their performance under laboratory conditions remain scarce. In this study, three assays evaluated viral genes in synthetic RNA and RNA extracted from SARS-CoV-2 samples stored in viral transport medium (VTM) (30 panel certified reference materials), UK NEQAS samples and 100 patient samples: 1) Agilent SARS-CoV-2 RT-qPCR kit for detecting the N1 and N2; 2) Agena Bioscience MassArray (MALDI-ToF) SARS-CoV-2 for detecting N1, N2, N3, ORF1 & ORF1ab, variant SARS detection, and 3) Molecular Biology System UltraFast RT-PCR SARS-CoV-2 Kit for detecting N1, N2, and ORF1ab These assays showed good performance, sensitivity and specificity with a limit of detection (LOD) as low as 1 copy/ μL. The specificity and sensitivity are 100% and 98% respectively. These kits thus provide a robust assay for the detection of SARS-CoV-2 in clinical samples. Conclusion: These assays are suitable for routine diagnostic. The UltraFast NextGenPCR is the fastest with average time (30mins), followed by Agilent (2 hrs) and MassArray (6hrs). Upon completion of this activity, participants should be able to examine, measure and compare results from different assays for SARS detection, evaluate and diagnose accurately, as well as being able to plan, organize and recommend a diagnostic procedure for diagnostic laboratory. Key words: SARS-CoV-2, RNA extraction, RT-PCR, limit of detection, quantification cycle, COVID-19, in vitro diagnostic tests, Agilent, Massarray, Ultrafast. [20] From the Microbiology Lab to the Operating Room: Advanced Development of the MasSpec Pen for Broad Clinical Use. Livia Eberlin, PhD, Associate Professor, Department of Surgery, Baylor College of Medicine, Houston, TX Ambient ionization mass spectrometry techniques that enable direct, gentle, and rapid analysis of samples offer exciting opportunities to provide clinicians with rich molecular data to enhance decision-making. Here, an overview of several ongoing clinical projects in the Eberlin laboratory related to the development of a handheld MSbased device, the MasSpec Pen technology, for improving patient care will be discussed. In particular, I will describe results centered around our efforts in employing the MasSpec Pen for intraoperative tissue analysis and cancer detection, identification of infectious microorganisms, and detection of drugs of abuse. Current challenges and opportunities towards incorporating this technology into clinical practice will also be addressed. Upon completion of this activity, participants should be able to 1. describe operating principles of direct mass spectrometry techniques for rapid molecular analysis that are being explored for clinical use; 2. evaluate analytical and diagnostic performance metrics of direct mass spectrometry techniques to address unmet clinical needs. [21] Triggered MRM for Urine Drug Testing: Finding What You Aren't Looking For. Joshua Hayden, Norton Healthcare, Louisville, KY This work aimed to develop a triggered multiple reaction monitoring (MRM) method for urine drug testing that allows simultaneous targeted quantitation of commonly used/abused drugs and qualitative detection of a larger array of uncommonly used drugs. Targeted quantitation is most often accomplished with triple quadruple mass spectrometers but these instruments suffer in their ability to do untargeted analysis. This limitation is especially challenging given the ever increasing number of drugs that are produced and abused-including fentanyl analogues, bath salts, and a broad group of drugs known as spice. Ideally, clinical laboratories would be able to perform both targeted quantitation and some level of qualitative detection of infrequently encountered drugs. Towards this end, we developed a method that allows us to utilize our triple quadruple mass spectrometer to do targeted quantitation of commonly used/ abused drugs and qualitative detection of a larger range of drugs. This qualitative detection is accomplished using a triggered MRM method and a spectral database. To validate this method, deidentified, remnant urine samples were spiked with a variety of uncommonly encountered illicit drugs including various spice derivatives, bath salts, and fentanyl derivatives. The performance of the method was evaluated based on its ability to perform accurate quantitation of targeted compounds and its ability to qualitatively detect the presence of the spiked drugs. The method demonstrated excellence performance. The success of this method suggests such an approach could find widespread use in clinical laboratories; however, questions remain with regards to the regulatory requirements (quality control, proficiency testing, etc). Upon completion of this activity, participants should be able to: 1 .Describe the advantages and limitations of triple quadruple mass spectrometers, 2. Discuss the advantage of screening for a wide array of illicit drugs, 3.Summarize how a triggered MRM method might add value to a urine drug assay. [22] Advances in the assessment of Orexin-A (hypocretin-1) deficiency in the diagnosis of Type 1 Narcolepsy. Joshua Bornhorst, Bethany Larson, and Alicia Algeciras-Schimnich, Mayo Clinic, Rochester, MN This presentation will describe the clinical performance s of an assay for orexin-A (also known as hypocretin-1), which is a neuropeptide involved in the sleep/wake cycle. Impairment of orexin-A production and orexin-A modulated neurotransmission is associated with narcolepsy with cataplexy (episodes of muscle weakness in response to emotional stimuli), and deficiency of orexin-A in cerebrospinal fluid (CSF) is a hallmark of type 1 narcolepsy. The diagnostic criteria for type 1 narcolepsy in the third edition of the International Classification of Sleep Disorders (2014) includes the presence of cataplexy and/ or measured CSF orexin concentrations less than or equal to 110 pg/mL. However, clinical testing for orexin-A in CSF had been unavailable in the United States. A competitive radioimmunoassay for orexin-A quantitation in CSF was characterized for clinical use in the diagnosis of type 1 narcolepsy. This assay demonstrates acceptable analytical precision, accuracy, and stability. To evaluate clinical performance, 100 residual CSF specimens from individuals without suspicion of type 1 narcolepsy all exhibited orexin-A concentrations of >200 pg/mL (mean: 531pg/mL). Additionally, samples from 20 patients with clinical suspicion of type 1 narcolepsy were evaluated. All nine confirmed 9 type 1 narcolepsy patients exhibited orexin concentrations of <50 pg/mL. All 11 patients that were subsequently deemed to have hypersomnias other than type 1 narcolepsy had orexin concentrations of >200pg/mL (range 352-600 pg/mL). Clinical introduction of this test fulfilled a pressing diagnostic need for differentiation of type 1 narcolepsy from other causes of hypersomnolence. Emerging information on how this assay compares to other methods of patient assessment for potential type 1 narcolepsy will also be discussed. Upon completion of this activity, participants should be able to identify the role of orexin deficiency in type 1 narcolepsy as well as recommend appropriate testing for the evaluation of individuals who potential have this disorder. [23] Zika outbreak in Dominican Republic 2016: A review. Frederick L. Kiechle1, Angelica Freddo1, and Henry Quezada2. 1Pompano Beach, FL and 2Santo Domingo, Dominican Republic We mined the data in the Biorepository at Boca Biolistics obtained during the Zika outbreak for symptomatic pregnant (PG) and non-pregnant (NP) women in the Dominican Republic in 2016. Zika virus (ZIKV) is a Flavivirus transmitted in humans by Aedes mosquitoes. There was a biphasic distribution of 305 symptomatic (NP) and 65 (PG) women (266 NAAT positive by Hologic, Aptima ZIKV assay) in April and late May/June 2016. To determine previous exposure to dengue (DENV) infection, anti-DENV IgG was measured in serum or plasma by ELISA (Euroimmun). (Science 2019; 363: 307). In 65 symptomatic PG women the outcome was 4.6 % spontaneous abortion, 2 % fetal demise and 4.6 % other. We evaluated the presence of 6 presenting symptoms and 3 lab tests (Aptima ZIKA assay; EUROIMMUN ELISA IgG; CDC MAC-ELISA ZiKV IgM - performed by FL Dept of Health) over 8 time intervals (12 - 36 repeat PG or NP patients per interval; total PR 131, total NP 171). The prevalence of 4 symptoms were less frequent in PG women vs NP, including fever, severe eye pain, head pain and joint and muscle pain. Conjunctivitis occurred more frequently in PG vs NP and rash had an equal occurrence. ZIKV IgG and MAC-ELISA-IgM were positive with similar frequency in both groups 1 through 8. NAAT ZIKV results were positive for all 8 intervals (87.5% to 72.2%) in PG patients and in NP (52% in interval 1 and 0.0 to 4.0% in intervals 2 to 8). PG patients with rash were 98% positive for DENV IgG. In conclusion, NP and PG women exhibited a biphasic spike of ZIKV positive infections; from 0 to 86 days post symptoms, PG exhibited a lower prevalence of fever, head pain, severe eye pain and joint and muscle pain compared to NP ZIKV symptomatic patients. The results suggest that PG provides protection from clinical symptoms compared to NP during infection cycle in spite of persistent viremia in PG vs NP. Upon completion of this activity, participants should be able to describe the clinical and laboratory findings in Zika infection and differences in PG and NP patient presentation. [24] Rapidly evolving and fatal miliary tuberculosis and COVID-19 infection inan infant.Anindita Ghosh1,Amanda Tchakarov1, Norma Perez1, Nina Tatevian2, Meenakshi Bhattacharjee1. 1University of Texas Health Science Center at Houston, Houston, TX, and 2Women and Infant Hospital of Rhode Island, Providence, RI Background: Tuberculosis (TB) and SARS-CoV-2 (COVID-19) are two important infectious diseases causing morbidity and mortality worldwide. Active TB infection can stimulate host immune responses and together with COVID-19, may lead to cytokine storm and immune dysregulation leading to multi-organ failure. Studies have reported flare up of pulmonary TB after SARS-CoV-2 infection in adults. We present a unique case of both miliary tuberculosis and SARS-CoV-2 coinfection in an infant, indicating that children can acquire both infections concurrently, and have rapid progression with fatal outcome. Case: Our patient was a 6-month-old previously healthy term boy. He had persistent cough and congestion, became severely ill, and was brought to emergency department. Chest X-ray showed diffuse alveolar and interstitial airspace opacities. He was found to be COVID-19 positive by PCR test. Laboratory studies showed pancytopenia with left shift, elevated transaminases (ALT 77, AST 313), low albumin, elevated inflammatory markers (CRP 103, IL-6 19000), and abnormal coagulation profile with coagulopathy and disseminated intravascular coagulation. He developed strokes, severe sepsis and electrolyte abnormalities, and declined rapidly with death within 6 days. Autopsy examination showed hepatosplenomegaly. His lungs, liver, kidneys, and spleen showed multifocal micro-abscesses, which on microscopic examination showed necrotic foci teeming with mycobacteria, and were culture positive for M. tuberculosis, i.e. miliary tuberculosis. Neuropathological examination showed infarction in the right middle and posterior cerebral artery territories. Conclusions: This patient helps illuminate some immunological and pathological aspects of two co-occurring infectious diseases and the susceptibility for development of fatal complications with SARS-CoV-2 infection in the pediatric population. Upon completion this activity the learner will be able to: 1. recognition of COVID-19 (SARS-CoV-2) infection cooccurrence with another infectious disease in children. 2. challenges in treatment of COVID-19 infection with other/associated infectious disease. 3. recognition of the complexity of pathological processes in childhood infectious disease. [25] A Postulated Cross Talk between Insulin Growth Factor Binding Protein 3 (IGFBP-3) and IGFBP-7 May Suggest a Critical Role in Carcinogenesis. Fan Shen1 and Consolato M. Sergi1,2. 1Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada and 2Anatomic Pathology Division, Children's Hospital of Eastern Ontario, University of Ottawa, ON, Canada Objective: The insulin/insulin-like growth factors (IGFs) have crucial tasks in the growth, differentiation, and proliferation of healthy and pernicious cells. They are involved in coordinated complexes, including receptors, ligands, binding proteins, and proteases. However, the systems can become dysregulated in tumorigenesis. Insulinlike growth factor-binding protein 7 (IGFBP7) is a protein belonging to the IGFBP superfamily. We intended to explore the cross-talk between IGFBP-3 and IGFBP-7. Methods: Analysis of text/ data mining tools using the NCBI platform was used. Results: Numerous studies have provided evidence that IGFBP-3 and IGFBP-7 are involved in a variety of cancers, including hepatocellular carcinoma (HCC), breast cancer, gastroesophageal cancer, colon cancer, prostate cancer, among many others. Still, very few suggest an interaction between these two molecules. We found that both proteins share some crucial signaling pathways. P53, and growth inhibitory agents, including retinoic acid (RA), transforming growth factor beta (TGF-β), and anti-estrogens, can give rise to increased expression of IGFBP-3. IGFBP-7 can regulate the growth-suppressing effects of the TGF-β superfamily, and similarly the expression of IGFBP-7 can be upregulated by cellular treatment with TGF-β1 and RA. Conclusions: A comprehensive overview of the relationship between IGFBP-3, IGFBP-7, and cancer highlighted the IGFBP-3 crosstalk with IGFBP-7, which may suggest a promotion other than initiation in carcinogenesis. Upon completion of this learning activity participants should be able to: 1. to define to role of Insulin Growth Factor Binding Protein superfamily in carcinogenesis 2. to compare the respective role of IGFBP-3 and IGFBP7 in cancer 3. to determine the relevance of a cross talk between IGFBP-3 and IGFBP-7 for carcinogenesis [26] Measure Directly, Treat Efficiently. Critical Bleeding Management at the Speed of Sound. Oksana Volod, Cedars Sinai, Los Angeles, CA, USA Viscoelastic Testing (VET) is a category of functional whole blood tests used to assay hemostatic competence and optimize blood product transfusions in patients during or after surgical procedures where acute bleeding may occur. Efficient use of these tests has become an elevated need for a comprehensive patient blood management program. Cup and pin legacy systems and those that display the traditional interpretation "curves", such as with thromboelastography (TEG®) and rotational thromboelastometry (ROTEM®), require varying levels of oversight to maintain compliance and optimal efficient clinical use. Over the last decade, advances have been made with the integration of cartridge-based VET systems to improve upon those legacy systems. During this presentation, the legacy and new generation VETs available in the US are compared based on their unique hemostatic parameters that define contributions of coagulation factors, fibrinogen/fibrin, platelets, and clot lysis as related to the lifespan of a clot. The balance between the needs to act on relevant critical data in near real-time and compliance with the laboratory that must ensure quality is also discussed. The Quantra® system is the newest VET. It is a fully sealed, cartridgebased, automated, four-channel device that provides the fastest hemostasis assessment (12 minutes on average). The Quantra is based on a new ultrasound technology termed sonic estimation of elasticity via resonance (SEER). The important differences between Quantra and other VETs are its ease of interpretation, optimizations for use at the point of patient need, and its ability to directly measure blood viscoelastic properties. Through real case examples, this talk will also focus on Quantra specific hemostasis alterations and optimal management strategies for bleeding patients. Upon completion of this learning activity participants should be able to: 1. Describe Viscoelastic Testing as part of a Patient Blood Management initiative. 2. Categorize various viscoelastic assay methods and classify clinical and operational needs related to implementation and accessibility (i.e., speed, ease of use and location) 3. Interpret and explain the results of Quantra via participation in real-world case example. [27] Managing the challenges of running a transfusion service during the COVID-19 pandemic Claire Meena-Leist, Amanda Riggs and Mohamed Elkady, University of Louisville School of Medicine, Louisville, KY Hospitals have developed systems to adjust to periodic blood product shortages. Apheresis platelets and group O Rh(D) negative red blood cell shortages are common during summer months and holidays and can be managed well by having a strong blood management program and obtaining contracts with secondary blood suppliers. The COVID-19 pandemic, however, caused historic blood product shortages that resulted in red blood cell rationing and requests from our primary blood supplier for hospitals to consider canceling non-urgent surgical procedures. With virtually no lead time, in December 2021, our blood supplier began rationing all group O red blood cells to their customers. For the first time blood was allotted to customers based on blood center supply, not patient need. The rationing quickly expanded to include all group A and B red blood cells, with no information about how long the shortage would last. Hospitals were forced to develop more creative blood management methods, and to develop criteria for surgery cancellations. We discuss our experience with providing patient care in a hospital system that includes the region's only American College of Surgeons (ACS) Verified Level 1 Trauma centers for adults in the Commonwealth of KY. Upon completion of this activity, participants should be able to 1. Describe how one hospital system approached blood management during a historic blood shortage 2. Explain why hospital blood management is not the sole answer to providing patient care during extreme blood shortages 3. Identify ways that blood centers can improve strategic planning for the next pandemic. [28] Cells,wells, & spells: the evolution of HLA testing methods. Tiffany K Bratton, University of Louisville, Louisville, KY Organ transplantation is a life-saving treatment modality for patients with end stage organ failure. However, allograft rejection remains an obstacle for long term outcomes. An evolution in the understanding of transplant immunology over the past fifty years has led to continuous development and improvement of methods for detecting, not only HLA antibodies, but also other markers of the overall immune status of transplant patients. HLA testing began in the 1960's with donor cells; the cytotoxic crossmatch caused a paradigm shift in transplantation with immediate improvements in short term outcomes. In the early 2000's the organ allocation system was radically changed by the development of solid phase technology. This presentation will cover the evolution of HLA testing methods from the very beginning to current state as well as speculation on new technologies that could be developed and used to continue to improve long term outcomes for transplant patients. Upon completion of this learning activity participants should be able to: 1. Describe the different testing methods used for detection of HLA sensitization. 2. Identify the current HLA testing methods. 3. Evaluate new technologies that may be developed for both HLA and overall transplant testing. [29] The CNS-penetrating taxane drug TPI 287 potentiates the antiglioma activity of the AURKA inhibitor alisertib in vivo. Müge Sak, Brian J. Williams, Cory T. Zumbar, Mustafa N. G. Al-Kawaaz, Aastha Kakar, Andrew J. Hey, Leslie M. Scheir, Landon Teer, Joseph Chen and Norman L. Lehman. University of Louisville, Louisville KY Glioblastoma, IDH wildtype (GBM) is the most common malignant primary brain tumor in adults and has a poor prognosis. We previously found cytotoxic synergy between the AURKA inhibitor alisertib and the novel CNS-penetrating taxane TPI 287 against GBM tumor stem-like cells in vitro. Here we used an orthotopic human GBM xenograft mouse model to test the ability of TPI 287 to potentiate alisertib's antitumor activity in vivo. At two weeks, animal tumor volume was significantly decreased by alisertib, TPI 287, and combination treatments compared to controls. At four weeks both alisertib and TPI 287 groups had reduced tumor volume and a statistically significant decrease was again observed in the combination therapy group. Alisertib monotherapy improved animal survival, which was further improved with the addition of TPI 287 (p=0.0058). TPI 287 alone did not significantly improve animal survival. We also investigated the mechanism of apoptotic synergy between alisertib and TPI 287. Alisertib + TPI 287 combination treatment decreased Bcl-2 levels in vivo and in vitro. In contrast, this treatment increased expression of pro-apoptotic proteins Bim and Bak in synchronized GBM cells. Bim knockdown by siRNA inhibited synergistic cytotoxicity caused by alisertib + TPI 287 (p=0.0042). These results suggest that these drugs cause synergistic apoptosis in GBM cells partially through effects on Bcl-2 family proteins. Additionally, both alisertib and TPI 287 significantly reduced GBM cell invasion (p<0.0001). However, this effect was greater with TPI 287, and the drug combination was no more effective at inhibiting invasion than TPI 287 alone. These findings support the potential use of this combination therapy against GBM in clinical trials. Upon completion of this activity, participants should be able to understand the Bcl-2 family protein apoptotic cascade, the mechanism of action of taxanes and AURKA inhibitors and the rationale for combination treatment for GBM. [30] SARS-CoV-2 antibody testing in the post-vaccine era: comparing assays for standardization. Sarrah Lahorewala and Xin Yi, Houston Methodist Hospital, Houston, TX The interpretation of results of various SARS-CoV-2 antibody tests is challenging due to non-standardization among assays. The aims of this study were: to compare three commonly used semi-quantitative/quantitative SARS-CoV-2 antibody tests with our institutional Anti-SARS-CoV-2 Spike ELISA assay; and, to validate the ELISA assay against known National Standard (CRM-NIBSC21/234), thus correlating calculated ELISA concentrations (U/ mL) and the international standard units for binding assay formats, i.e. Binding Antibody Units/mL (BAU/ mL). The Anti-SARS-CoV-2 Spike ELISA assay detects IgG antibodies against the viral anti-S Ectodomain. The three commercial tests compared to the ELISA assay were the Roche Elecsys® Anti-SARS-CoV-2S (U/mL), Siemens Atellica COV2G (Index) and VITROS Anti-SARS-CoV-2 IgG Quantitative (BAU/mL). The Roche and Siemens test comparisons included 95 patient samples, with 51 samples evaluated for the VITROS assay. Statistical analyses were performed using the GraphPad Prism9 software. We mapped the antibody concentrations obtained on the ELISA assay against that of CRM-NIBSC21/234 and validated their linear relationship. The commercial assays evaluated showed good correlation with the Anti-SARS-CoV-2 Spike ELISA, across all ELISA titers. The corresponding assay result ranges (lower and upper 95% CI of mean) for each titer group is as follows: <1:50: Roche: <0.4-<0.4; Siemens: 0.0070.020; Vitros: 1.854-4.606; 1:50: Roche: 26.01-88.39; Siemens: 0.585-0.966; Vitros: 33.47-50.83; 1:150: Roche: 622.9-1095; Siemens: 6.82-214.38; Vitros: 52.35-141.4; 1:450: Roche: 1321-2091; Siemens: 16.74-19.55; Vitros: 112.4-192.2; 1:1350: Roche: 2067->2500; Siemens: >20->20; Vitros: >200->200. The calculated concentration (U/mL) obtained by the Anti-SARS-CoV-2 Spike ELISA and the expected concentration of CRM were strongly correlated and results were found to be linear in the range from 3.25-832 BAU/mL covering titers ranging from <1:50 to >1:1350. The calibration of our ELISA assay to the International Standard, and its correlation with commercial assays, allows for standardization between assays and a better understanding of the various antibody titers in the context of immune protection against SARS-CoV-2. Upon completion of this activity, participants should be able to: 1. Describe the clinical utility of antibody testing in SARS-CoV-2 2. Understand the various kinds of SARS-CoV-2 antibody tests commonly utilized 3. Interpret results of commonly used semi-quantitative/quantitative assays and correlate assay titers with the International Standard Units (BAU/ mL). [31] How to be a better surgical pathology consultant. Neda Zarrin-Khameh, Baylor College of Medicine, Houston, TX Consultation on surgical pathology specimens is part of the daily professional practice of every pathologist. We evaluated the characteristics of a good consultant and the habits that should be avoided. A 1-page questionnaire was prepared to evaluate how pathologists select their consultants. The questionnaire was emailed to 106 pathologists. Fifty-eight pathologists completed the questionnaire (55% response rate). The most important criteria for a consultant were knowledge and expertise. Accessibility, turnaround time, and teaching (providing explanation about the case) were selected next for choosing a consultant. The 2 factors that contributed to avoiding a consultant were expensive workup and changing the diagnosis. Open questions about "definition of best/worst consultant," "when to change the consultant," and "if the criteria for consultant have changed over time" provided additional valuable information. Accessibility, short turnaround time, and teaching are the most important reasons for selecting a consultant. Performing an expensive workup and being in the habit of changing the diagnosis are the factors that make a consultant less favorable. Upon completion of this activity, participants should be able to: 1. recognize three characteristics of a consultant that are important for requesting consultation, 2. identify three characteristics that are not recommended for a consultant and 3. determine when a pathologists may decide to change their consultant. [32] The Diagnostic Role of Fusion-Gene Analysis in Ambiguous Soft Tissue Sarcomas. Haider A. Mejbel, Alexander C. Mackinnon, and Shuko Harada, The University of Alabama at Birmingham, Department of Pathology, Birmingham, The diagnosis of soft tissue sarcoma often requires adequate clinicopathologic correlation as well as the appropriate application of immunohistochemical studies. However, certain ambiguous/undifferentiated soft tissue sarcomas can pose a diagnostic challenge. We retrospectively reviewed the cases submitted for RNA-based fusion panel (Archer FusionPlex assay) to evaluate the diagnostic utility of fusion gene analysis in soft tissue sarcomas. Herein we present the clinicopathologic and molecular alterations of three challenging soft tissue sarcomas, on which, the application of fusion-gene analysis has entirely altered the initial diagnosis. These cases include Ewing sarcoma, Ewing sarcoma with melanocytic differentiation, and MEIS1::NOCA1 sarcoma that were initially diagnosed as carcinoid tumor of the lung, melanoma, and low grade endometrial stromal sarcoma, respectively. In addition to providing the valuable prognostic information, the application of fusion-gene analysis has resulted in the reclassification of these neoplasms and altered the type of therapy. Currently, all patients are alive at the 6-, 26-, and 4-month of their final diagnosis. In conclusion, although adequate histopathologic examination and extensive immunohistochemical study were performed, the final diagnoses and classification of these sarcomas were only rendered after the application of the appropriate molecular testing. Upon completion of this activity, participants should be able to understand the role of molecular fusiongene analysis in further classifying ambiguous soft tissue sarcomas that can help arrive at the accurate diagnosis, alter disease stage, and inform therapy. [33] Distinct lesions of host defense and bacterial offense in tuberculosis. Robert L. Hunter and Robert E. Brown, University of Texas Health Science Center, Houston, TX The continued existence of M. tuberculosis depends on production of two distinct disease processes in humans. Primary tuberculosis (TB), the host's defense, protects against disseminated infection. Post-primary TB, in contrast, is the bacteria's offense. It facilitates transmission of infection to new hosts. Host defense is mediated by granulomas as are widely studied in human TB and animal models. People may succumb to lack of defense with disseminated infection, but this does not benefit the organism since it also dies. Bacterial offense, the lesions that mediate transmission of infection to new hosts, is mediated by post-primary TB. It begins as a subclinical obstructive lobular pneumonia that slowly accumulates materials for a sudden massive necrotizing pneumonia that can be coughed out to produce a cavity from which the organism can escape to infect new people. The early lesions of post-primary TB were well known to investigators in the preantibiotic era when autopsies were cutting edge science, but are unknown to the majority of current investigators who study only animal models. While modern technologies are making unprecedented progress in understanding tuberculous granulomas, they are making very little in understanding post-primary TB that does not exist in most animal models. Fortunately, new multiplex technologies make it possible to study human FFPE slides with great depth and precision. Such studies are necessary to gain a better understanding to the key developmental stage of TB. Upon completion of the activity, participants should be able to recognize and describe the differences between primary and post-primary TB. [34] Agonal-stress vesicles in critically ill children confused with microvesicular steatosis. John Hicks, Children's Hospital, Baylor College of Medicine Houston, TX Background: Acute or prolonged systemic organ failure, perinatal demise, or unexpected death in the neonates and children raises a concern for metabolic, lysosomal storage or mitochondrial diseases and infectious etiologies. A potential pitfall is the presence of cytoplasmic vacuoles particularly within hepatocytes that appear as microvesicular steatosis on routine staining. Microvesicular and macrovesicular steatosis may indicate lipid metabolic or mitochondrial disorders. Ultrastructural examination (EM) is useful in identifying abnormal lipid accumulation, mitochondrial structural abnormalities, and metabolic and lysosomal storage diseases. Particular structures indicative of post-mortem agonal or pre-mortem stress have been termed agonal-stress vesicles. It has not been shown, that these agonal-stress vesicles contain acute phase reactants in response to cellular ischemic or asphyxia. Design: EM laboratory archives were searched for cases with vesicles fitting the ultrastructural features of agonal-stress vesicles. 40 cases (20 males; 20 females) were identified with liver tissue submitted for routine processing (paraffin-embedded formalin fixed) and EM. Study population had variety of medical conditions associated with ischemic and asphyxial stress. Immunohistochemical staining (IHC) with acute phase reactant antibodies was performed (c-reactive protein, fibrinogen, alpha-2-macroglobulin, alpha-1-antitrypsin, alpha-1-chymotrypsin). Results: All routinely stained liver tissue sections shows variably-sized vacuoles within hepatocyte cytoplasm, mimicking microvesicular steatosis. IHC for acute phase reactants highlighted the vacuoles with all antibodies (c-reactive protein, fibrinogen, alpha-2-macroglobulin, alpha-1-antitrypsin, alpha-1-chymotrypsin), with greatest intensity with alpha-1-antitrypsin and alpha-1-chymotrypsin. EM showed frequent vesicles with limiting membranes, containing finely granular, homogeneous content. Conclusion: Agonal-stress vesicles are under recognized structure and may be interpreted as microvesicular steatosis, leading to a workup for metabolic or mitochondrial disease. EM assists in identifying these structures as agonal-stress vesicles. Also, residual formalin-fixed, frozen tissue and tissue recovered from paraffin tissue blocks may be utilized for EM. IHC for acute phase reactants in this study confirms the contents of agonal-stress vacuoles being acute phase reactants. Upon completion of this activity, participants should be able to describe the histopathologic, immunohistochemical and ultrastructural features of agonal-stress vacuoles. [35] Transmission Electron Microscopy (TEM): A Tool often Underused for Pediatric Small Round Blue Cell Tumors opening New Venues for Single-Cell Technologies. Consolato M. Sergi1,2, 1Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, and 2Anatomic Pathology Division, Children's Hospital of Eastern Ontario, University of Ottawa, ON, Canada Aims: Pediatric small round blue cell tumors (PSRBCT) are an intriguing and challenging collection of neoplasms. Light microscopy of small round blue cell tumors identifies small round cells. Pediatric small round blue cell tumors include several entities, such as nephroblastoma, neuroblastoma, rhabdomyosarcoma, Ewing's sarcoma, retinoblastoma, malignant lymphoma, and small cell osteosarcoma among others. The differential diagnosis of these neoplasms may be controversial at a light microscopy level, even using immunohistochemistry. A faint staining or an ambiguous background can deter pathologists from making the proper diagnostic decision. Methods: A review of the personal experience at four centers is considered here. In addition, single cell technologies are added to renew the interest of TEM. Results: Molecular biology may provide an overwhelming amount of data challenging to distinguish them, and some translocations may be seen in more than one category. Thus, TEM can be extremely valuable. In particular, tumor cells associated with tangles of cytoplasmic processes containing neurosecretory granules can diagnose neuroblastoma. Conversely, a marked variation in size, shape and cytoplasmic differentiation with most tumor cells containing prominent dilated cisterns of rough endoplasmic reticulum and bundles of thick and thin filaments with well-formed Z-bands may infer the diagnosis of rhabdomyosarcoma. The presence of an intracytoplasmic deposit of glycogen may suggest Ewing's sarcoma. At the same time, a cellular arrangement in a tubular configuration with a well-formed basal lamina may advocate the diagnosis of nephroblastoma. Single-cell sequencing are booming. Conclusion: Single-cell sequencing technologies are useful to discover the genome, transcriptome, metabolome, and epigenome of single cells. These techniques can show the differences and evolutionary relationships of innumerable cells. Here, we speculate that TEM may have an intriguing role for single-cell sequencing technologies and their applications in oncology, microbiology, reproductive and environmental sciences emphasizing the essential role that single-cell sequencing methods play in these areas. Upon completion of this activity, participants should be able to: 1. To define to general role of TEM in pathology. 2. To identify the role of TEM for PSRBCTs. 3. To determine the relevance in applying new technologies to TEM studies. [36] Chondroid Chordoma Presenting as Oropharyngeal Mass in Pediatrics. John Hicks, Texas Children's Hospital, Baylor College of Medicine, Houston, TX Introduction: Chordoma is a low to intermediate grade malignancy, resembling embryonal notochord. This tumor occurs in sacrococcygeal (60%), craniocervical (25%, clivus most common) and vertebral (15%) sites. Chordoma is a rare tumor (incidence 5/10,000,000; 2% of bone tumors). Peak incidence is in 4th decade. Clinical Presentation and Pathology Findings: 8 year-old girl with asthma history presented with 6-12 months of progressively worsening snoring, muffled voice and saliva pooling, attributed to seasonal allergies. Upon oral examination, a firm, protruding right tonsillar bed (oropharyngeal) mass with intact overlying vascularized mucosa was noted. Limited CT imaging identified an oropharyngeal mass with bony destruction. Biopsy was performed which showed a hyalinized chondroid to cartilaginous mass. Tumor cells immunoreacted with brachyury, EMA, S100 and SOX9, while negative for D2-40, with retained nuclear INI-1. Low-grade chondroid chordoma diagnosis was rendered. Molecular tumor testing identified p53 mutation, and KMT2B and ROS1 mutations of unknown significance. Conventional tumor karyotype was 46,XX. Germline testing was negative for p53 mutation. Additional imaging identified an 8.5 cm clivus origin tumor. The patient underwent additional surgery for tumor debulking, followed by oncologic (radiationtherapy) management. Conclusion: Chondroid chordoma involving oro/nasopharyngeal region is rare (0.2% of oro/nasopharyngeal tumors). Differential diagnosis includes chondrosarcoma, chordoid meningioma, myoepithelioma/myoepithelial carcinoma, extraskeletal myxoid chondrosarcoma, and chordoid meningioma. Treatment is surgical, with complete resection difficult due to anatomic location. Although the tumor tends to be radioresistant, high-dose radiation therapy is usuallyemployed. Overall survival with aggressive surgery is up to 75% at 5yrs and up to 65% at 10yrs. Recurrence is common (up to 90% at 10 years). Chondroid variant has somewhat better prognosis. Sonic hedgehog homolog protein gene (7q33), T (brachyury) gene duplication (6q27), and TSC1 or TSC2 (tuberous sclerosis) gene inactivation are associated with chordoma. Autosomal dominant familial tumors associated with T gene duplication are rare. Upon completion of this activity, participants should be able to describe the clinical, radiologic histopathologic, immunohistochemistry and molecular features in the diagnosis of chordoma. [37] Deciduosis adjacent an ileouterine fistula in the setting of Crohn's disease: Case report and literature review. Lance Truong1, Yigit Baykara1, and Nina Tatevian2. 1Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, RI and 2Women & Infants Hospital, Warren Alpert Medical School of Brown University, Providence, RI Deciduosis is a rare diagnosis which refers to extrauterine decidual tissue, found usually in adjacent gynecological structures including the cervix and ovaries, and more rarely in the peritoneum and in other abdominal organs. Fistulas are a known and frequent complication of Crohn's disease, commonly involving adjacent bowel and rarely involving gynecological structures. Here, we report a case of deciduosis of the distal ileum occurring in the setting of long-standing Crohn's disease in a 34-year-old patient at 31 5/7 weeks of gestation, admitted to our hospital with acute right lower quadrant pain. Imaging was suggestive of complex fistulae and abscess formation. She underwent a cesarean delivery followed by hysterectomy and ileocolectomy with an ileouterine fistula tract leading to a complex subserosal uterine abscess on intraoperative and pathological gross examination. On histological examination, a focus of ectopic decidua adjacent the fistula was incidentally found on H&E section. Review of the current literature on gastrointestinal deciduosis identified nine publications reporting cases primarily involving the appendix and only a single previously reported case involving the ileum. Crohn's disease cases with reported ileouterine fistulas are extremely rare, with only four previously reported cases in the literature. The utility in recognizing this extremely rare presentation is in avoiding misinterpreting this entity as a neoplastic process. Upon completion of this activity, participants should be able to 1. define deciduosis, 2. recognize complications associated with Crohn's disease, 3. compare neoplastic mimickers of ectopic decidual tissue [38] Modern implementation of Digital Pathology with Artificial Intelligence in an Academic Medical Center. Dibson Dibe Gondim, Department of Pathology and Laboratory Medicine at University of Louisville, KY Digital pathology refers to the digital transformation of histopathology - turning histologic tissue sections into multi-gigabyte whole slide images. These images can be evaluated by quantitative and reproducible computational techniques, including artificial intelligence-based algorithms. While the value of this technology has been increasingly recognized, clinical adoption has been limited in most of the world, including in the United States. Our academic center and health care system have embraced the concept of digital pathology and artificial intelligence. My goal is to present the digital pathology implementation proposal and discuss our progress. Overall, digital pathology implementation tends to be a slow process occurring over years. However, we decided that the technology was mature enough for an accelerated adoption plan. From the beginning, we focused on 100% prospective glass slide scanning 100%, in contrast with most institutions which start with limited retrospective scanning. To achieve this, we created a multidisciplinary team with expertise in pathology, histotechnology, project management, software engineering, and data science. We integrated scanning in the histology laboratory using LEAN principles and updated our staining equipment to allow compatibility with the slide scanners. We installed high-throughput slide scanners that require minimal user experience or training. We adopted cloud-based software as the platform product from a vendor with expertise in artificial intelligence. This software platform provides image viewer, worklist, storage, and artificial intelligence-based algorithms. Cloud storage allowed us to scale our operation without major overhead. We started scanning 100% of slides in less than 9 months after the beginning of the project. We deployed the integration interface and the first FDA-approved artificial intelligence-based tool in the testing environment within months. Our experience shows that the current technologies allow for an accelerated adoption of digital pathology. However, advanced understanding of digital pathology technologies and assembling a multidisciplinary task force have been instrumental in determining how to best put together all the pieces and to overcome multiple challenges that emerged during the implementation period. Upon completion of this activity, participants should be able to 1. understand how digital pathology fits in the clinical workflow, 2. describe digital pathology implementation strategies, and 3. explain how integration of multiple hospital and laboratory information systems for a productive user experience. [39] Establishing a Hospital AI Committee: Enhancing Precision Medicine. Andrew A. Borkowski, M.D. James A. Haley Veterans' Hospital James A. Haley Veterans' Hospital AI Committee was established in May 2021. Vision: To improve outcomes and experiences for our Veterans by developing trustworthy Artificial Intelligence capabilities to support the Department of Veterans Affairs mission. Mission: To build robust capacity in Artificial Intelligence to develop and apply innovative AI solutions and transform the VA by facilitating a learning environment that supports the delivery of world-class benefits and services to our veterans. Accomplishments: Establishment of the AI email group, MS Teams AI Group, and SharePoint site. AI Ethics Guidelines. Clinical AI Product Evaluation Guidelines. Education programs with "AI Article of the Week", AI Newsletter, and AI Conference. Collaboration with the National AI Institute. Partnership with the Moffitt Machine Learning League. AI in Healthcare Workshop during the 2021 VISN 8 Improvement and Innovation Forum. 2022 VISN 8 AI Conference Upon completion of this activity, participants should be able to familiarize themselves with the formal process of promoting AI throughout their institutions. [40] Artificial Intelligence in Radiology: Hype, Hope, or Hysteria.Narayan Viswanadhan, Chief of Radiology at the Tampa VA, and is a clinical faculty at USF Health Morsani College of Medicine. Tampa, FL There is particularly increasing application of AI tools within Diagnostic Imaging and Pathology. Clinical applications of AI in imaging will be discussed, with focus on current uses, and future directions. Additional areas covered will be utilization of AI in Neuroimaging, population health, Breast Imaging, and Precision Medicine. Utilization of AI in the face of the pandemic will also be discussed.. Upon completion of this activity, participants should be able to 1. develop an understanding of Artificial Intelligence as it relates to imaging 2.understand current clinical use cases and future directions, and 3. appreciate challenges of AI in Radiology. [41] Data literacy in healthcare: a vital component of precision medicine. Joyce J. Ou, Alpert Medical School, Brown University, Providence, RI The accelerating volume of heterogenous, multimodal data that is generated in modern healthcare highlights an urgent need for data literacy across a broad range of stakeholders. As information sources, analytics, and use cases rapidly evolve, data-powered augmented intelligence is becoming a major driver of precision medicine. The growing influence of machine generated insights on decisionmaking underscores the importance of ensuring that health data producers and consumers have the skills to critically evaluate, interpret, communicate, and act on these insights. Data literacy has, at times, been equated with specific data science tools and processes. However, fluency with data in the current era requires additional knowledge of its value and limitations with respect to data life cycles and governance, along with technical, social, and ethical implications. To achieve this breadth of understanding, a practical framework can be developed to identify and address gaps in data literacy. This framework focuses on identifying core categories of data skills, assessing stakeholder roles and needs relative to these categories, and developing processes for continuous learning. Implementation of such a model can promote systemic innovation by enabling individuals to stay current with the pace of technological changes that impact all stages of precision medicine discovery and delivery. Upon completion of this activity, participants should be able to 1. Identify core competencies needed to achieve healthcare data literacy 2. Evaluate the impact of a data literacy framework on promoting collaborations between data producers and consumers in precision medicine 3. Describe a data education model that supports continuous learning for healthcare stakeholders. [42] Digital health applications and key characteristics related to at-home testing outcomes. Lee B. Springer, Brio Systems, Bellevue, OH Digital health technology applications (DHT) have been minimally utilized to support at-home rapid testing in the past. With the emergence of SARS-CoV-2, their use has increased significantly. One of the primary contributors to this growth is the widespread adoption of at-home testing throughout the progression of the pandemic to increase the publics access to testing. Given the increased need for at-home testing, digital health technology applications have been utilized to aid users throughout all testing processes and efficiently document results. These applications are considered to play a vital role in ensuring optimal accuracy of the test device and facilitate public health reporting. A step-by-step systematic analysis of digital health technology supporting at-home point-of-care testing was performed to identify key areas of task related dependence to predicted outcome in relation to technological support. The aim of the analysis was to assess the quality and features in relation to engagement during testing phases utilizing the mobile application rating scale (MARS). This analysis included 27 digital health applications designed to complement at-home testing for glucose, creatinine, coagulation, hemoglobin A1c, and SARS-CoV-2 with more than 5,000 downloads or registered users. A nonparametric rank sum test was used to determine correlating performance between sections of the mobile application rating scale and aligned testing phases. High scores in pre-analytical and post-analytical sections of the mobile application rating scale correlated to an overall high level of satisfaction and desired outcome. Additional correlations were seen between sections related to overall functionality, and applications viewed to support pre-analytical processes effectively, these were deemed as being able to consistently produce better patient and result outcomes. Upon completion of this activity, participants should be able to identify digital health technology that would optimize accurate results, predict overall patient engagement in clinical application and design clinical practices around digital health technology to optimize patient outcomes. [43] Validation of Artificial Intelligence-Based System for Prostate Cancer Detection and Grading. Tarymé López Díaz and Dibson Dibe Gondim, Department of Pathology and Laboratory Medicine at University of Louisville, KY Paige Prostate Detect (Paige.AI Inc., New York, USA) is the first FDA-approved artificial intelligence (AI)-based system created to assist pathologists to detect and grade prostate cancer. Our goal is to present the results of a validation study of this system in an academic center. 40 prostate biopsy cases were randomly selected from a list of 60 consecutive cases. All HE-stained slides (619) were scanned in a Leica Aperio GT450 (400x magnification). Paige Prostate Detect was applied to each slide and results were compared with the original diagnoses. Diagnostic discrepancies were evaluated by pathologists. AI output was recorded for each whole slide image. On the other hand, pathologists provided results based on the evaluation of two or more glass slides per biopsy location. 36 disagreements were identified (5.8%) in all slides. Agreement on the case level required that all slides of a case had concordant diagnosis between AI-system and pathologist. 16/40 cases had disagreements. The AI-system helped to detect focal cancer in a case with no prior definitive cancer diagnosis. Multiple discrepancies related to atypical small acinar proliferation (ASAP) were identified. Since the system was not trained to make a diagnosis of ASAP, these cases were either diagnosed as carcinoma or benign. Validation of pathology AI-systems is essential before clinical adoption. This system shows potential to locate low-volume cancer in cases with no definitive diagnosis of malignancy. In the case of ASAP, pathologists should use well-defined criteria to render this diagnosis and should not rely on AI impression. [44] Respiratory Pathology of Acute Respiratory distress Syndrome (ARDS). Henry Oh, PhD, FACSc, FRSB, FAPSR, RRT, MT, CSci, Health Occupations Department, Idaho State University, Pocatello, ID The body's immune system response to infection is primarily an inflammation. Cytokines cause inflammation of tissue by making the cell walls of blood vessels become more permeable, thus allowing leakage of blood with immune cells into the surrounding tissue to start the healing process of the damaged tissue. The inflammation is triggered by the release of cytokines from interferons, interleukins, and tumor necrosis factor (TNF). Cells that release cytokines include leucocytes or interleukins, neutrophils, macrophages, monocytes, and many other cells. When the inflammatory response go out of control, it can cause more harm to the lungs in COVID-19 infection where cytokines can destroy normal, healthy alveoli. Cytokine storm occurs when there is an overwhelming inflammatory response due to the increase release of cytokines. The inflammatory cytokines "storms" the lungs which is the leading cause of mortality rates in patients with COVID-19 infection in ICU. Accumulated dead cells and other debris in the lungs can set the stage for the development of acute respiratory distress syndrome (ARDS). The effects of cytokine storm amplify the severity and extent of ARDS. In ARDS, the alveoli become filled with dead cells, debris and leaked fluids from damaged interstitial cells. The lungs become stiff because of very low compliance. Oxygen saturation levels fall below 90% and the partial pressure of oxygen (PaO2) fall below 60 mmHg which can lead to severe hypoxemia. Patients with ARDS develop refractory hypoxemia which is unresponsive to oxygen therapy. Patient needs to be intubated and connected to a ventilator. The positive pressure effects of mechanical ventilation may not be adequate to improve the oxygenation status of the patient. Positive End Expiratory Pressure (PEEP) would need to be added to keep the oxygen saturation at 90%. However, caution must be observed when ventilating patients with high positive pressure since this can cause detrimental effects on the cardiovascular system. It can decrease blood pressure, cardiac output, urine output leading to cardiovascular collapse, multi-organ failure, and cardiopulmonary arrest. Monitoring of fluids and electrolytes, vital signs, lung compliance, blood gases and patient's response to positive pressure ventilation are extremely important in critical care management. Upon completion of this activity, participants should be able to to briefly discuss how cytokines affect the lungs as a result of COVID-19 infection, to identify ventilator strategies and initial management of patients with ARDS, and to identify the common complications when ventilating patients with ARDS.

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(2015) Sponge species composition of north-east Atlantic cold-water coral reefs compared in a bathyal to inshore gradient. Journal of the Marine Biological Association of the United Kingdom, 95 (7), 1461-1474.        https://doi.org/10.1017/S0025315413001410Vanhöffen, E. (1910) Die Hydroiden der Deutschen Südpolar-Expedition 1901-1903. Deutsche Südpolar-Expedition 1901-1903, 11, Zoologie 3, 269-340.Vervoort, W. (1946) Exotic hydroids in the collections of the Rijksmuseum van Natuurlijke Historie and the Zoological Museum at Amsterdam. Zoologische Mededelingen, Leiden, 26 (1-4), 287-351.Vervoort, W. (1946) Exotic hydroids in the collections of the Rijksmuseum van Natuurlijke Historie and the Zoological Museum at Amsterdam. Zoologische Mededelingen, Leiden, 26 (1-4), 287-351.Vervoort, W. (1972) Hydroids from the Theta, Vema and Yelcho cruises of the Lamont-Doherty geological observatory. Rijksmuseum van Natuurlijke Historie, Leiden, Netherlands, 120, 1-247.Vervoort, W. 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Institut für Meereskunde der Universität Hamburg, 136 pp.Wienberg, C., Titschack, J., Freiwald, A., Frank, N., TomasLundälv, T., Taviani, M., Beuck, L., Schröder-Ritzrau, A., Krengel, T. and Hebbeln, D. (2018) The giant Mauritanian cold-water coral mound province: Oxygen control on coral mound formation. Quaternary Science Reviews, 185, 135-152.        https://doi.org/10.1016/j.quascirev.2018.02.012Wright, T.S. (1859) Observations on British zoophytes. Edinburgh new Philosophical Journal, 10, 105-114.

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Description of the condition Malaria, an infectious disease transmitted by the bite of female mosquitoes from several Anopheles species, occurs in 87 countries with ongoing transmission (WHO 2020). The World Health Organization (WHO) estimated that, in 2019, approximately 229 million cases of malaria occurred worldwide, with 94% occurring in the WHO's African region (WHO 2020). Of these malaria cases, an estimated 409,000 deaths occurred globally, with 67% occurring in children under five years of age (WHO 2020). Malaria also negatively impacts the health of women during pregnancy, childbirth, and the postnatal period (WHO 2020). Sulfadoxine/pyrimethamine (SP), an antifolate antimalarial, has been widely used across sub-Saharan Africa as the first-line treatment for uncomplicated malaria since it was first introduced in Malawi in 1993 (Filler 2006). Due to increasing resistance to SP, in 2000 the WHO recommended that one of several artemisinin-based combination therapies (ACTs) be used instead of SP for the treatment of uncomplicated malaria caused by Plasmodium falciparum (Global Partnership to Roll Back Malaria 2001). However, despite these recommendations, SP continues to be advised for intermittent preventive treatment in pregnancy (IPTp) and intermittent preventive treatment in infants (IPTi), whether the person has malaria or not (WHO 2013). Description of the intervention Folate (vitamin B9) includes both naturally occurring folates and folic acid, the fully oxidized monoglutamic form of the vitamin, used in dietary supplements and fortified food. Folate deficiency (e.g. red blood cell (RBC) folate concentrations of less than 305 nanomoles per litre (nmol/L); serum or plasma concentrations of less than 7 nmol/L) is common in many parts of the world and often presents as megaloblastic anaemia, resulting from inadequate intake, increased requirements, reduced absorption, or abnormal metabolism of folate (Bailey 2015; WHO 2015a). Pregnant women have greater folate requirements; inadequate folate intake (evidenced by RBC folate concentrations of less than 400 nanograms per millilitre (ng/mL), or 906 nmol/L) prior to and during the first month of pregnancy increases the risk of neural tube defects, preterm delivery, low birthweight, and fetal growth restriction (Bourassa 2019). The WHO recommends that all women who are trying to conceive consume 400 micrograms (µg) of folic acid daily from the time they begin trying to conceive through to 12 weeks of gestation (WHO 2017). In 2015, the WHO added the dosage of 0.4 mg of folic acid to the essential drug list (WHO 2015c). Alongside daily oral iron (30 mg to 60 mg elemental iron), folic acid supplementation is recommended for pregnant women to prevent neural tube defects, maternal anaemia, puerperal sepsis, low birthweight, and preterm birth in settings where anaemia in pregnant women is a severe public health problem (i.e. where at least 40% of pregnant women have a blood haemoglobin (Hb) concentration of less than 110 g/L). How the intervention might work Potential interactions between folate status and malaria infection The malaria parasite requires folate for survival and growth; this has led to the hypothesis that folate status may influence malaria risk and severity. In rhesus monkeys, folate deficiency has been found to be protective against Plasmodium cynomolgi malaria infection, compared to folate-replete animals (Metz 2007). Alternatively, malaria may induce or exacerbate folate deficiency due to increased folate utilization from haemolysis and fever. Further, folate status measured via RBC folate is not an appropriate biomarker of folate status in malaria-infected individuals since RBC folate values in these individuals are indicative of both the person's stores and the parasite's folate synthesis. A study in Nigeria found that children with malaria infection had significantly higher RBC folate concentrations compared to children without malaria infection, but plasma folate levels were similar (Bradley-Moore 1985). Why it is important to do this review The malaria parasite needs folate for survival and growth in humans. For individuals, adequate folate levels are critical for health and well-being, and for the prevention of anaemia and neural tube defects. Many countries rely on folic acid supplementation to ensure adequate folate status in at-risk populations. Different formulations for folic acid supplements are available in many international settings, with dosages ranging from 400 µg to 5 mg. Evaluating folic acid dosage levels used in supplementation efforts may increase public health understanding of its potential impacts on malaria risk and severity and on treatment failures. Examining folic acid interactions with antifolate antimalarial medications and with malaria disease progression may help countries in malaria-endemic areas determine what are the most appropriate lower dose folic acid formulations for at-risk populations. The WHO has highlighted the limited evidence available and has indicated the need for further research on biomarkers of folate status, particularly interactions between RBC folate concentrations and tuberculosis, human immunodeficiency virus (HIV), and antifolate antimalarial drugs (WHO 2015b). An earlier Cochrane Review assessed the effects and safety of iron supplementation, with or without folic acid, in children living in hyperendemic or holoendemic malaria areas; it demonstrated that iron supplementation did not increase the risk of malaria, as indicated by fever and the presence of parasites in the blood (Neuberger 2016). Further, this review stated that folic acid may interfere with the efficacy of SP; however, the efficacy and safety of folic acid supplementation on these outcomes has not been established. This review will provide evidence on the effectiveness of daily folic acid supplementation in healthy and malaria-infected individuals living in malaria-endemic areas. Additionally, it will contribute to achieving both the WHO Global Technical Strategy for Malaria 2016-2030 (WHO 2015d), and United Nations Sustainable Development Goal 3 (to ensure healthy lives and to promote well-being for all of all ages) (United Nations 2021), and evaluating whether the potential effects of folic acid supplementation, at different doses (e.g. 0.4 mg, 1 mg, 5 mg daily), interferes with the effect of drugs used for prevention or treatment of malaria. To examine the effects of folic acid supplementation, at various doses, on malaria susceptibility (risk of infection) and severity among people living in areas with various degrees of malaria endemicity. We will examine the interaction between folic acid supplements and antifolate antimalarial drugs. Specifically, we will aim to answer the following. Among uninfected people living in malaria endemic areas, who are taking or not taking antifolate antimalarials for malaria prophylaxis, does taking a folic acid-containing supplement increase susceptibility to or severity of malaria infection? Among people with malaria infection who are being treated with antifolate antimalarials, does folic acid supplementation increase the risk of treatment failure? Criteria for considering studies for this review Types of studies Inclusion criteria Randomized controlled trials (RCTs) Quasi-RCTs with randomization at the individual or cluster level conducted in malaria-endemic areas (areas with ongoing, local malaria transmission, including areas approaching elimination, as listed in the World Malaria Report 2020) (WHO 2020) Exclusion criteria Ecological studies Observational studies In vivo/in vitro studies Economic studies Systematic literature reviews and meta-analyses (relevant systematic literature reviews and meta-analyses will be excluded but flagged for grey literature screening) Types of participants Inclusion criteria Individuals of any age or gender, living in a malaria endemic area, who are taking antifolate antimalarial medications (including but not limited to sulfadoxine/pyrimethamine (SP), pyrimethamine-dapsone, pyrimethamine, chloroquine and proguanil, cotrimoxazole) for the prevention or treatment of malaria (studies will be included if more than 70% of the participants live in malaria-endemic regions) Studies assessing participants with or without anaemia and with or without malaria parasitaemia at baseline will be included Exclusion criteria Individuals not taking antifolate antimalarial medications for prevention or treatment of malaria Individuals living in non-malaria endemic areas Types of interventions Inclusion criteria Folic acid supplementation Form: in tablet, capsule, dispersible tablet at any dose, during administration, or periodically Timing: during, before, or after (within a period of four to six weeks) administration of antifolate antimalarials Iron-folic acid supplementation Folic acid supplementation in combination with co-interventions that are identical between the intervention and control groups. Co-interventions include: anthelminthic treatment; multivitamin or multiple micronutrient supplementation; 5-methyltetrahydrofolate supplementation. Exclusion criteria Folate through folate-fortified water Folic acid administered through large-scale fortification of rice, wheat, or maize Comparators Placebo No treatment No folic acid/different doses of folic acid Iron Types of outcome measures Primary outcomes Uncomplicated malaria (defined as a history of fever with parasitological confirmation; acceptable parasitological confirmation will include rapid diagnostic tests (RDTs), malaria smears, or nucleic acid detection (i.e. polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), etc.)) (WHO 2010). This outcome is relevant for patients without malaria, given antifolate antimalarials for malaria prophylaxis. Severe malaria (defined as any case with cerebral malaria or acute P. falciparum malaria, with signs of severity or evidence of vital organ dysfunction, or both) (WHO 2010). This outcome is relevant for patients without malaria, given antifolate antimalarials for malaria prophylaxis. Parasite clearance (any Plasmodium species), defined as the time it takes for a patient who tests positive at enrolment and is treated to become smear-negative or PCR negative. This outcome is relevant for patients with malaria, treated with antifolate antimalarials. Treatment failure (defined as the inability to clear malaria parasitaemia or prevent recrudescence after administration of antimalarial medicine, regardless of whether clinical symptoms are resolved) (WHO 2019). This outcome is relevant for patients with malaria, treated with antifolate antimalarials. Secondary outcomes Duration of parasitaemia Parasite density Haemoglobin (Hb) concentrations (g/L) Anaemia: severe anaemia (defined as Hb less than 70 g/L in pregnant women and children aged six to 59 months; and Hb less than 80 g/L in other populations); moderate anaemia (defined as Hb less than 100 g/L in pregnant women and children aged six to 59 months; and less than 110 g/L in others) Death from any cause Among pregnant women: stillbirth (at less than 28 weeks gestation); low birthweight (less than 2500 g); active placental malaria (defined as Plasmodium detected in placental blood by smear or PCR, or by Plasmodium detected on impression smear or placental histology). Search methods for identification of studies A search will be conducted to identify completed and ongoing studies, without date or language restrictions. Electronic searches A search strategy will be designed to include the appropriate subject headings and text word terms related to each intervention of interest and study design of interest (see Appendix 1). Searches will be broken down by these two criteria (intervention of interest and study design of interest) to allow for ease of prioritization, if necessary. The study design filters recommended by the Scottish Intercollegiate Guidelines Network (SIGN), and those designed by Cochrane for identifying clinical trials for MEDLINE and Embase, will be used (SIGN 2020). There will be no date or language restrictions. Non-English articles identified for inclusion will be translated into English. If translations are not possible, advice will be requested from the Cochrane Infectious Diseases Group and the record will be stored in the "Awaiting assessment" section of the review until a translation is available. The following electronic databases will be searched for primary studies. Cochrane Central Register of Controlled Trials. Cumulative Index to Nursing and Allied Health Literature (CINAHL). Embase. MEDLINE. Scopus. Web of Science (both the Social Science Citation Index and the Science Citation Index). We will conduct manual searches of ClinicalTrials.gov, the International Clinical Trials Registry Platform (ICTRP), and the United Nations Children's Fund (UNICEF) Evaluation and Research Database (ERD), in order to identify relevant ongoing or planned trials, abstracts, and full-text reports of evaluations, studies, and surveys related to programmes on folic acid supplementation in malaria-endemic areas. Additionally, manual searches of grey literature to identify RCTs that have not yet been published but are potentially eligible for inclusion will be conducted in the following sources. Global Index Medicus (GIM). African Index Medicus (AIM). Index Medicus for the Eastern Mediterranean Region (IMEMR). Latin American & Caribbean Health Sciences Literature (LILACS). Pan American Health Organization (PAHO). Western Pacific Region Index Medicus (WPRO). Index Medicus for the South-East Asian Region (IMSEAR). The Spanish Bibliographic Index in Health Sciences (IBECS) (ibecs.isciii.es/). Indian Journal of Medical Research (IJMR) (journals.lww.com/ijmr/pages/default.aspx). Native Health Database (nativehealthdatabase.net/). Scielo (www.scielo.br/). Searching other resources Handsearches of the five journals with the highest number of included studies in the last 12 months will be conducted to capture any relevant articles that may not have been indexed in the databases at the time of the search. We will contact the authors of included studies and will check reference lists of included papers for the identification of additional records. For assistance in identifying ongoing or unpublished studies, we will contact the Division of Nutrition, Physical Activity, and Obesity (DNPAO) and the Division of Parasitic Diseases and Malaria (DPDM) of the CDC, the United Nations World Food Programme (WFP), Nutrition International (NI), Global Alliance for Improved Nutrition (GAIN), and Hellen Keller International (HKI). Data collection and analysis Selection of studies Two review authors will independently screen the titles and abstracts of articles retrieved by each search to assess eligibility, as determined by the inclusion and exclusion criteria. Studies deemed eligible for inclusion by both review authors in the abstract screening phase will advance to the full-text screening phase, and full-text copies of all eligible papers will be retrieved. If full articles cannot be obtained, we will attempt to contact the authors to obtain further details of the studies. If such information is not obtained, we will classify the study as "awaiting assessment" until further information is published or made available to us. The same two review authors will independently assess the eligibility of full-text articles for inclusion in the systematic review. If any discrepancies occur between the studies selected by the two review authors, a third review author will provide arbitration. Each trial will be scrutinized to identify multiple publications from the same data set, and the justification for excluded trials will be documented. A PRISMA flow diagram of the study selection process will be presented to provide information on the number of records identified in the literature searches, the number of studies included and excluded, and the reasons for exclusion (Moher 2009). The list of excluded studies, along with their reasons for exclusion at the full-text screening phase, will also be created. Data extraction and management Two review authors will independently extract data for the final list of included studies using a standardized data specification form. Discrepancies observed between the data extracted by the two authors will be resolved by involving a third review author and reaching a consensus. Information will be extracted on study design components, baseline participant characteristics, intervention characteristics, and outcomes. For individually randomized trials, we will record the number of participants experiencing the event and the number analyzed in each treatment group or the effect estimate reported (e.g. risk ratio (RR)) for dichotomous outcome measures. For count data, we will record the number of events and the number of person-months of follow-up in each group. If the number of person-months is not reported, the product of the duration of follow-up and the number of children evaluated will be used to estimate this figure. We will calculate the rate ratio and standard error (SE) for each study. Zero events will be replaced by 0.5. We will extract both adjusted and unadjusted covariate incidence rate ratios if they are reported in the original studies. For continuous data, we will extract means (arithmetic or geometric) and a measure of variance (standard deviation (SD), SE, or confidence interval (CI)), percentage or mean change from baseline, and the numbers analyzed in each group. SDs will be computed from SEs or 95% CIs, assuming a normal distribution of the values. Haemoglobin values in g/dL will be calculated by multiplying haematocrit or packed cell volume values by 0.34, and studies reporting haemoglobin values in g/dL will be converted to g/L. In cluster-randomized trials, we will record the unit of randomization (e.g. household, compound, sector, or village), the number of clusters in the trial, and the average cluster size. The statistical methods used to analyze the trials will be documented, along with details describing whether these methods adjusted for clustering or other covariates. We plan to extract estimates of the intra-cluster correlation coefficient (ICC) for each outcome. Where results are adjusted for clustering, we will extract the treatment effect estimate and the SD or CI. If the results are not adjusted for clustering, we will extract the data reported. Assessment of risk of bias in included studies Two review authors (KSC, LFY) will independently assess the risk of bias for each included trial using the Cochrane 'Risk of bias 2' tool (RoB 2) for randomized studies (Sterne 2019). Judgements about the risk of bias of included studies will be made according to the recommendations outlined in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2021). Disagreements will be resolved by discussion, or by involving a third review author. The interest of our review will be to assess the effect of assignment to the interventions at baseline. We will evaluate each primary outcome using the RoB2 tool. The five domains of the Cochrane RoB2 tool include the following. Bias arising from the randomization process. Bias due to deviations from intended interventions. Bias due to missing outcome data. Bias in measurement of the outcome. Bias in selection of the reported result. Each domain of the RoB2 tool comprises the following. A series of 'signalling' questions. A judgement about the risk of bias for the domain, facilitated by an algorithm that maps responses to the signalling questions to a proposed judgement. Free-text boxes to justify responses to the signalling questions and 'Risk of bias' judgements. An option to predict (and explain) the likely direction of bias. Responses to signalling questions elicit information relevant to an assessment of the risk of bias. These response options are as follows. Yes (may indicate either low or high risk of bias, depending on the most natural way to ask the question). Probably yes. Probably no. No. No information (may indicate no evidence of that problem or an absence of information leading to concerns about there being a problem). Based on the answer to the signalling question, a 'Risk of bias' judgement is assigned to each domain. These judgements include one of the following. High risk of bias Low risk of bias Some concerns To generate the risk of bias judgement for each domain in the randomized studies, we will use the Excel template, available at www.riskofbias.info/welcome/rob-2-0-tool/current-version-of-rob-2. This file will be stored on a scientific data website, available to readers. Risk of bias in cluster randomized controlled trials For the cluster randomized trials, we will be using the RoB2 tool to analyze the five standard domains listed above along with Domain 1b (bias arising from the timing of identification or recruitment of participants) and its related signalling questions. To generate the risk of bias judgement for each domain in the cluster RCTs, we will use the Excel template available at https://sites.google.com/site/riskofbiastool/welcome/rob-2-0-tool/rob-2-for-cluster-randomized-trials. This file will be stored on a scientific data website, available to readers. Risk of bias in cross-over randomized controlled trials For cross-over randomized trials, we will be using the RoB2 tool to analyze the five standard domains listed above along with Domain 2 (bias due to deviations from intended interventions), and Domain 3 (bias due to missing outcome data), and their respective signalling questions. To generate the risk of bias judgement for each domain in the cross-over RCTs, we will use the Excel template, available at https://sites.google.com/site/riskofbiastool/welcome/rob-2-0-tool/rob-2-for-crossover-trials, for each risk of bias judgement of cross-over randomized studies. This file will be stored on a scientific data website, available to readers. Overall risk of bias The overall 'Risk of bias' judgement for each specific trial being assessed will be based on each domain-level judgement. The overall judgements include the following. Low risk of bias (the trial is judged to be at low risk of bias for all domains). Some concerns (the trial is judged to raise some concerns in at least one domain but is not judged to be at high risk of bias for any domain). High risk of bias (the trial is judged to be at high risk of bias in at least one domain, or is judged to have some concerns for multiple domains in a way that substantially lowers confidence in the result). The 'risk of bias' assessments will inform our GRADE evaluations of the certainty of evidence for our primary outcomes presented in the 'Summary of findings' tables and will also be used to inform the sensitivity analyses; (see Sensitivity analysis). If there is insufficient information in study reports to enable an assessment of the risk of bias, studies will be classified as "awaiting assessment" until further information is published or made available to us. Measures of treatment effect Dichotomous data For dichotomous data, we will present proportions and, for two-group comparisons, results as average RR or odds ratio (OR) with 95% CIs. Ordered categorical data Continuous data We will report results for continuous outcomes as the mean difference (MD) with 95% CIs, if outcomes are measured in the same way between trials. Where some studies have reported endpoint data and others have reported change-from-baseline data (with errors), we will combine these in the meta-analysis, if the outcomes were reported using the same scale. We will use the standardized mean difference (SMD), with 95% CIs, to combine trials that measured the same outcome but used different methods. If we do not find three or more studies for a pooled analysis, we will summarize the results in a narrative form. Unit of analysis issues Cluster-randomized trials We plan to combine results from both cluster-randomized and individually randomized studies, providing there is little heterogeneity between the studies. If the authors of cluster-randomized trials conducted their analyses at a different level from that of allocation, and they have not appropriately accounted for the cluster design in their analyses, we will calculate the trials' effective sample sizes to account for the effect of clustering in data. When one or more cluster-RCT reports RRs adjusted for clustering, we will compute cluster-adjusted SEs for the other trials. When none of the cluster-RCTs provide cluster-adjusted RRs, we will adjust the sample size for clustering. We will divide, by the estimated design effects (DE), the number of events and number evaluated for dichotomous outcomes and the number evaluated for continuous outcomes, where DE = 1 + ((average cluster size 1) * ICC). The derivation of the estimated ICCs and DEs will be reported. We will utilize the intra-cluster correlation coefficient (ICC), derived from the trial (if available), or from another source (e.g., using the ICCs derived from other, similar trials) and then calculate the design effect with the formula provided in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2021). If this approach is used, we will report it and undertake sensitivity analysis to investigate the effect of variations in ICC. Studies with more than two treatment groups If we identify studies with more than two intervention groups (multi-arm studies), where possible we will combine groups to create a single pair-wise comparison or use the methods set out in the Cochrane Handbook to avoid double counting study participants (Higgins 2021). For the subgroup analyses, when the control group was shared by two or more study arms, we will divide the control group (events and total population) over the number of relevant subgroups to avoid double counting the participants. Trials with several study arms can be included more than once for different comparisons. Cross-over trials From cross-over trials, we will consider the first period of measurement only and will analyze the results together with parallel-group studies. Multiple outcome events In several outcomes, a participant might experience more than one outcome event during the trial period. For all outcomes, we will extract the number of participants with at least one event. Dealing with missing data We will contact the trial authors if the available data are unclear, missing, or reported in a format that is different from the format needed. We aim to perform a 'per protocol' or 'as observed' analysis; otherwise, we will perform a complete case analysis. This means that for treatment failure, we will base the analyses on the participants who received treatment and the number of participants for which there was an inability to clear malarial parasitaemia or prevent recrudescence after administration of an antimalarial medicine reported in the studies. Assessment of heterogeneity Heterogeneity in the results of the trials will be assessed by visually examining the forest plot to detect non-overlapping CIs, using the Chi2 test of heterogeneity (where a P value of less than 0.1 indicates statistical significance) and the I2 statistic of inconsistency (with a value of greater than 50% denoting moderate levels of heterogeneity). When statistical heterogeneity is present, we will investigate the reasons for it, using subgroup analysis. Assessment of reporting biases We will construct a funnel plot to assess the effect of small studies for the main outcome (when including more than 10 trials). Data synthesis The primary analysis will include all eligible studies that provide data regardless of the overall risk of bias as assessed by the RoB2 tool. Analyses will be conducted using Review Manager 5.4 (Review Manager 2020). Cluster-RCTs will be included in the main analysis after adjustment for clustering (see the previous section on cluster-RCTs). The meta-analysis will be performed using the Mantel-Haenszel random-effects model or the generic inverse variance method (when adjustment for clustering is performed by adjusting SEs), as appropriate. Subgroup analysis and investigation of heterogeneity The overall risk of bias will not be used as the basis in conducting our subgroup analyses. However, where data are available, we plan to conduct the following subgroup analyses, independent of heterogeneity. Dose of folic acid supplementation: higher doses (4 mg or more, daily) versus lower doses (less than 4 mg, daily). Moderate-severe anaemia at baseline (mean haemoglobin of participants in a trial at baseline below 100 g/L for pregnant women and children aged six to 59 months, and below 110 g/L for other populations) versus normal at baseline (mean haemoglobin above 100 g/L for pregnant women and children aged six to 59 months, and above 110 g/L for other populations). Antimalarial drug resistance to parasite: known resistance versus no resistance versus unknown/mixed/unreported parasite resistance. Folate status at baseline: Deficient (e.g. RBC folate concentration of less than 305 nmol/L, or serum folate concentration of less than 7nmol/L) and Insufficient (e.g. RBC folate concentration from 305 to less than 906 nmol/L, or serum folate concentration from 7 to less than 25 nmol/L) versus Sufficient (e.g. RBC folate concentration above 906 nmol/L, or serum folate concentration above 25 nmol/L). Presence of anaemia at baseline: yes versus no. Mandatory fortification status: yes, versus no (voluntary or none). We will only use the primary outcomes in any subgroup analyses, and we will limit subgroup analyses to those outcomes for which three or more trials contributed data. Comparisons between subgroups will be performed using Review Manager 5.4 (Review Manager 2020). Sensitivity analysis We will perform a sensitivity analysis, using the risk of bias as a variable to explore the robustness of the findings in our primary outcomes. We will verify the behaviour of our estimators by adding and removing studies with a high risk of bias overall from the analysis. That is, studies with a low risk of bias versus studies with a high risk of bias. Summary of findings and assessment of the certainty of the evidence For the assessment across studies, we will use the GRADE approach, as outlined in (Schünemann 2021). We will use the five GRADE considerations (study limitations based on RoB2 judgements, consistency of effect, imprecision, indirectness, and publication bias) to assess the certainty of the body of evidence as it relates to the studies which contribute data to the meta-analyses for the primary outcomes. The GRADEpro Guideline Development Tool (GRADEpro) will be used to import data from Review Manager 5.4 (Review Manager 2020) to create 'Summary of Findings' tables. The primary outcomes for the main comparison will be listed with estimates of relative effects, along with the number of participants and studies contributing data for those outcomes. These tables will provide outcome-specific information concerning the overall certainty of evidence from studies included in the comparison, the magnitude of the effect of the interventions examined, and the sum of available data on the outcomes we considered. We will include only primary outcomes in the summary of findings tables. For each individual outcome, two review authors (KSC, LFY) will independently assess the certainty of the evidence using the GRADE approach (Balshem 2011). For assessments of the overall certainty of evidence for each outcome that includes pooled data from included trials, we will downgrade the evidence from 'high certainty' by one level for serious (or by two for very serious) study limitations (risk of bias, indirectness of evidence, serious inconsistency, imprecision of effect estimates, or potential publication bias).

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Our expanded efforts in genomic sequencing to cover additional skipper butterfly (Lepidoptera: Hesperiidae) species and populations, including primary type specimens, call for taxonomic changes to restore monophyly and correct misidentifications by moving taxa between genera and proposing new names. Reconciliation between phenotypic characters and genomic trees suggests three new tribes, two new subtribes, 23 new genera, 17 new subgenera and 10 new species that are proposed here: Psolosini Grishin, <bnew tribe</b (type genus <iPsolos</i Staudinger, 1889), Ismini Grishin, <bnew tribe</b (type genus <iIsma</i Distant, 1886), Eetionini Grishin, <bnew tribe</b (type genus <iEetion</i de Nicéville, 1895), Orphina Grishin, <bnew subtribe</b (type genus <iOrphe</i Godman, 1901), Carystoidina Grishin, <bnew subtribe</b (type genus <iCarystoides</i Godman, 1901), <iFulvatis</i Grishin, <bnew genus</b (type species <iTelegonus fulvius</i Plötz, 1882), <iAdina</i Grishin, <bnew genus</b (type species <iNascus adrastor</i Mabille and Boullet, 1912), <iOrnilius</i Grishin, <bnew genus</b (type species <iOrnilius rotundus</i Grishin, <bnew species</b), <iTolius</i Grishin, <bnew genus</b (type species <iAntigonus tolimus</i Plötz, 1884), <iLennia</i Grishin, <bnew genus</b (type species <iLeona lena</i Evans, 1937), <iTrida</i Grishin, <bnew genus</b (type species <iCyclopides barberae</i Trimen, 1873), <iNoxys</i Grishin, <bnew genus</b (type species <iOxynthes viricuculla</i Hayward, 1951), <iGracilata</i Grishin, <bnew genus</b (type species <iEnosis quadrinotata</i Mabille, 1889), <iHermio</i Grishin, <bnew genus</b (type species <iFalga</i ? <ihermione</i Schaus, 1913), <iEutus</i Grishin, <bnew genus</b (type species <iCobalus rastaca</i Schaus, 1902), <iGufa</i Grishin, <bnew genus</b (type species <iPhlebodes gulala</i Schaus, 1902), <iGodmia</i Grishin, <bnew genus</b (type species <iEuroto chlorocephala</i Godman, 1900), <iRhomba</i Grishin, <bnew genus</b (type species <iEutychide gertschi</i Bell, 1937), <iRectava</i Grishin, <bnew genus</b (type species <iMegistias ignarus</i Bell, 1932), <iContrastia</i Grishin, <bnew genus</b (type species <iHesperia distigma</i Plötz, 1882), <iMit</i Grishin, <bnew genus</b (type species <iMnasitheus badius</i Bell, 1930), <iPicova</i Grishin, <bnew genus</b (type species <iVorates steinbachi</i Bell, 1930), <iLattus</i Grishin, <bnew genus</b (type species <iEutocus arabupuana</i Bell, 1932), <iGubrus</i Grishin, <bnew genus</b (type species <iVehilius lugubris</i Lindsey, 1925), <iKoria</i Grishin, <bnew genus</b (type species <iHesperia kora</i Hewitson, 1877), <iCorta</i Grishin, <bnew genus</b (type species <iEutychide lycortas</i Godman, 1900), <iCalvetta</i Grishin, <bnew genus</b (type species <iHesperia calvina</i Hewitson, 1866), <iOz</i Grishin, <bnew genus</b (type species <iAstictopterus ozias</i Hewitson, 1878), <iPraxa</i Grishin, <bnew subgenus</b (type species <iNascus prax</i Evans, 1952), <iBron</i Grishin, <bnew subgenus</b (type species <iPapilio broteas</i Cramer, 1780), <iTuris</i Grishin, <bnew subgenus</b (type species <iPyrgus (Scelothrix) veturius</i Plötz, 1884), <iTiges</i Grishin, <bnew subgenus</b (type species <iAntigonus liborius</i Plötz, 1884), <iOcrypta</i Grishin, <bnew subgenus</b (type species <iNotocrypta caerulea</i Evans, 1928), <iTixe</i Grishin, <bnew subgenus</b (type species <iCobalus quadrata</i Herrich-Schäffer, 1869), <iNycea</i Grishin, <bnew subgenus</b (type species <iPamphila hycsos</i Mabille, 1891), <iNausia</i Grishin, <bnew subgenus</b (type species <iOenus</i [sic] <inausiphanes</i Schaus, 1913), <iFlor</i Grishin, <bnew subgenus</b (type species <iStomyles florus</i Godman, 1900), <iGeia</i Grishin, <bnew subgenus</b (type species <iPamphila geisa</i Möschler, 1879), <iRotundia</i Grishin, <bnew subgenus</b (type species <iEnosis schausi</i Mielke and Casagrande, 2002), <iVolus</i Grishin, <bnew subgenus</b (type species <iEutocus volasus</i Godman, 1901), <iPseudopapias</i Grishin, <bnew subgenus</b (type species <iPapias tristissimus</i Schaus, 1902), <iSeptia</i Grishin, <bnew subgenus</b (type species <iJustinia septa</i Evans, 1955), <iBrasta</i Grishin, <bnew subgenus</b (type species <iLychnuchus brasta</i Evans, 1955), <iBina</i Grishin, <bnew subgenus</b (type species <iCobalus gabina</i Godman, 1900), <iBalma</i Grishin, <bnew subgenus</b (type species <iCarystoides balza</i Evans, 1955), <iOrnilius rotundus</i Grishin, <bnew species</b (type locality in Brazil: Santa Catarina), <iSalantoia metallica</i Grishin, <bnew species</b (type locality in Guyana: Acarai Mts.), <iDyscophellus australis</i Grishin, <bnew species</b (type locality in Paraguay: Sapucay), <iDyscophellus basialbus</i Grishin, <bnew species</b (type locality in Brazil: Rondônia), <iTelegonus subflavus</i Grishin, <bnew species</b (type locality in Ecuador: Riobamba), <iDecinea colombiana</i Grishin, <bnew species</b (type locality in Colombia: Bogota), <iLerema lucius</i Grishin, <bnew species</b (type locality in Panama: Colón), <iCynea rope</i Grishin, <bnew species</b (type locality in Nicaragua: Chontales), <iLerodea sonex</i Grishin, <bnew species</b (type locality in Peru: Cuzco), and <iMetiscus goth</i Grishin, <bnew species</b (type locality in Costa Rica). <bLectotypes</b are designated for the following 17 taxa: <iTelegonus gildo</i Mabille, 1888, <iNetrocoryne damias</i Plötz, 1882, <iTelegonus erythras</i Mabille, 1888, <iTelegonus galesus</i Mabille, 1888, <iEudamus cretellus</i Herrich-Schäffer, 1869, <iLeucochitonea chaeremon</i Mabille, 1891, <iAntigonus aura</i Plötz, 1884, <iPamphila voranus</i Mabille, 1891, <iHesperia pupillus</i Plötz, 1882, <iCobalus lumina</i Herrich-Schäffer, 1869, <iCobalus stigmula</i Mabille, 1891, <iMegistias isus</i Godman, 1900, <iCobalopsis latonia</i Schaus, 1913, <iPamphila nubila</i Mabille, 1891, <iMetiscus atheas</i Godman, 1900, <iMnasalcas amatala</i Schaus, 1902, and <iHesperia ina</i Plötz, 1882. The lectotype of <iHesperia infuscata</i Plötz, 1882 is <binvalid</b because it does not agree with the original description and illustration by Plötz, is not from the locality listed in the original description, and therefore is not a syntype. <bNeotypes</b are designated for the following five taxa: <iTelegonus corentinus</i Plötz, 1882, <iHesperia dido</i Plötz, 1882, <iHesperia distigma</i Plötz, 1882, <iHesperia infuscata</i Plötz, 1882, and <iHesperia pruinosa</i Plötz, 1882. As a result, the following five taxa are <bjunior objective synonyms</b: <iTelegonus diophorus</i Möschler, 1883 of <iTelegonus corentinus</i Plötz, 1882, <iPamphila puxillius</i Mabille, 1891 of <iHesperia pupillus</i Plötz, 1882, <iCobalus stigmula</i Mabille, 1891 of <iHesperia distigma</i Plötz, 1882, <iMnasalcas amatala</i Schaus, 1902 of <iHesperia infuscata</i Plötz, 1882, and <iHesperia pruinosa</i Plötz, 1882 of <iHesperia uza</i Hewitson, 1877. <iMorys valerius valda</i Evans, 1955 is fixed as the <btype species</b of <iMorys</i Godman, 1900, and <iPamphila compta</i Butler, 1877 is reaffirmed as the <btype species</b of <iEuroto</i Godman, 1900. Furthermore, the following <btaxonomic changes</b are suggested. <iProsopalpus</i Holland, 1896, <iLepella</i Evans, 1937, and <iCreteus</i de Nicéville, 1895 are placed in Aeromachini Tutt, 1906. <iTriskelionia</i Larsen and Congdon, 2011 is transferred from Celaenorrhinini Swinhoe, 1912 to Tagiadini Mabille, 1878. <iKobelana</i Larsen and Collins, 2013 is transferred from Tagiadini Mabille, 1878 to Celaenorrhinini Swinhoe, 1912. The following nine genus-group names are <bresurrected from synonymy</b and treated as valid genera: <iAbaratha</i Moore, 1881 (not in <iCaprona</i Wallengren, 1857), <iBibla</i Mabille, 1904 (not in <iTaractrocera</i Butler, 1870), <iKerana</i Distant, 1886 and <iTamela</i Swinhoe, 1913 (not in <iAncistroides</i Butler, 1874), <iMetrocles</i Godman, 1900 (not in <iMetron</i Godman, 1900), <iAlerema</i Hayward, 1942 (not in <iTigasis</i Godman, 1900), <iMetiscus</i Godman, 1900 (not in <iEnosis</i Mabille, 1889), <iVistigma</i Hayward, 1939 (not in <iPhlebodes</i Hübner, [1819]), and <iMnasalcas</i Godman, 1900 (not in <iMnasitheus</i Godman, 1900). The genus-group names <iDaimio</i Murray, 1875 and <iPterygospidea</i Wallengren, 1857 are <bresurrected from synonymy</b and treated as valid subgenera of <iTagiades</i Hübner, [1819]. We confirm <iApallaga</i Strand, 1911 as a valid genus. The following 24 genera are placed as subgenera, <bnew status</b: <iPseudonascus</i Austin, 2008 of <iNascus</i Watson, 1893; <iAlbiphasma</i Huang, Chiba, Wang and Fan, 2016 of <iPintara</i Evans, 1932; <iCtenoptilum</i de Nicéville, 1890 of <iTapena</i Moore, [1881]; <iOdontoptilum</i de Nicéville, 1890 of <iAbaratha</i Moore, 1881; <iCaprona</i Wallengren, 1857 of <iAbantis</i Hopffer, 1855; <iTimochreon</i Godman and Salvin, 1896 of <iZopyrion</i Godman and Salvin, 1896; <iPulchroptera</i Hou, Fan and Chiba, 2021 of <iHeteropterus</i Duméril, 1806; <iStimula</i de Nicéville, 1898 of <iKoruthaialos</i Watson, 1893; <iUdaspes</i Moore, [1881] and <iNotocrypta</i de Nicéville, 1889 of <iAncistroides</i Butler, 1874; <iCravera</i de Jong, 1983 of <iXeniades</i Godman, 1900; <iCobaloides</i Hayward, 1939 of <iOligoria</i Scudder, 1872; <iSaniba</i O. Mielke and Casagrande, 2003 of <iPsoralis</i Mabille, 1904; <iQuinta</i Evans, 1955 of <iCynea</i Evans, 1955; <iStyriodes</i Schaus, 1913 and <iRemella</i Hemming, 1939 of <iMnasicles</i Godman, 1901; <iRepens</i Evans, 1955 of <iEprius</i Godman, 1901; <iMorys</i Godman, 1900 of <iLerema</i Scudder, 1872; <iEnosis</i Mabille, 1889 of <iLychnuchus</i Hübner, [1831]; <iPenicula</i Evans, 1955 of <iVistigma</i Hayward, 1939; <iMnasinous</i Godman, 1900 of <iMethionopsis</i Godman, 1901; and <iMoeros</i Evans, 1955, <iArgon</i Evans, 1955, and <iSynale</i Mabille, 1904 of <iCarystus</i Hübner, [1819]. The following 20 genera are treated as junior subjective synonyms: <iLeucochitonea</i Wallengren, 1857 of <iAbantis</i Hopffer, 1855; <iSapaea</i Plötz, 1879 and <iNetrobalane</i Mabille, 1903 of <iCaprona</i Wallengren, 1857; <iParasovia</i Devyatkin, 1996 of <iSebastonyma</i Watson, 1893; <iPemara</i Eliot, 1978 of <iOerane</i Elwes and Edwards, 1897; <iAnkola</i Evans, 1937 of <iPardaleodes</i Butler, 1870; <iArotis</i Mabille, 1904 of <iMnaseas</i Godman, 1901; <iChalcone</i Evans, 1955, <iHansa</i Evans, 1955, and <iPropertius</i Evans, 1955 of <iMetrocles</i Godman, 1900; <iJongiana</i O. Mielke and Casagrande, 2002 of <iCobaloides</i Hayward, 1939; <iPamba</i Evans, 1955 of <iPsoralis</i Mabille, 1904; <iBrownus</i Grishin, 2019 of <iStyriodes</i Schaus, 1913; <iMnasilus</i Godman, 1900 of <iPapias</i Godman, 1900; <iSucova</i Evans, 1955 of <iMnasitheus</i Godman, 1900; <iPyrrhocalles</i Mabille, 1904 and <iAsbolis</i Mabille, 1904 of <iChoranthus</i Scudder, 1872; <iMiltomiges</i Mabille, 1903 of <iMethionopsis</i Godman, 1901; <iSacrator</i Evans, 1955 of <iThracides</i Hübner, [1819]; and <iLychnuchoides</i Godman, 1901 of <iPerichares</i Scudder, 1872. <iArunena</i Swinhoe, 1919 is a <bjunior subjective synonym</b of <iStimula</i de Nicéville, 1898 (not of <iKoruthaialos</i Watson, 1893). The following 27 names are species-level taxa (some in new combinations) <breinstated from synonymy</b: <iSalantoia gildo</i (Mabille, 1888) (not <iSalatis cebrenus</i (Cramer, 1777)), <iBungalotis corentinus</i (Plötz, 1882) (not <iBungalotis midas</i (Cramer, 1775)), <iTelegonus cretellus</i (Herrich-Schäffer, 1869) (not <iTelegonus cassander</i (Fabricius, 1793)), <iSanta palica</i (Mabille, 1888) (not <iChiothion asychis</i (Stoll, 1780)), <iCamptopleura cincta</i Mabille and Boullet, 1917 (not <iCamptopleura auxo</i (Möschler, 1879)), <iCamptopleura orsus</i (Mabille, 1889) (not <iNisoniades mimas</i (Cramer, 1775)), <iMetron voranus</i (Mabille, 1891) and <iMetron fasciata</i (Möschler, 1877) (not <iMetron zimra</i (Hewitson, 1877)), <iLimochores catahorma</i (Dyar, 1916) (not <iLimochores pupillus</i (Plötz, 1882)), <iPares viridiceps</i (Mabille, 1889) (not <iThoon modius</i (Mabille, 1889)), <iTigasis wellingi</i (Freeman, 1969) (not <iTigasis arita</i (Schaus, 1902)), <iRectava sobrinus</i (Schaus, 1902) (not <iPapias phainis</i Godman, 1900), <iNastra subsordida</i (Mabille, 1891) (not <iAdlerodea asema</i (Mabille, 1891), previously in <iEutychide</i Godman, 1900), <iLerema pattenii</i Scudder, 1872 (not <iLerema accius</i (J. E. Smith, 1797)), <iLerema (Morys) ancus</i (Möschler, 1879) (not <iCymaenes tripunctus theogenis</i (Capronnier, 1874)), <iCobalopsis zetus</i (Bell, 1942) (not <iCobalopsis nero</i (Herrich-Schäffer, 1869)), <iLerema (Geia) etelka</i (Schaus, 1902) (not <iLerema (Geia) geisa</i (Möschler, 1879), previously in <iMorys</i Godman, 1900), <iCymaenes isus</i (Godman, 1900) (not <iCymaenes trebius</i (Mabille, 1891)), <iVehilius labdacus</i (Godman, 1900) (not <iVehilius inca</i (Scudder, 1872)), <iPapias amyrna</i (Mabille, 1891) (not <iPapias allubita</i (Butler, 1877), previously in <iMnasilus</i Godman, 1900), <iPapias integra</i (Mabille, 1891) (not <iPapias subcostulata</i (Herrich-Schäffer, 1870)), <iMetiscus atheas</i Godman, 1900 (not <iHesperia achelous</i Plötz, 1882), <iDion agassus</i (Mabille, 1891) (not <iDion uza</i (Hewitson, 1877), previously in <iEnosis</i Mabille, 1889), <iPicova incompta</i (Hayward, 1942) (not <iLerema (Morys) micythus</i (Godman, 1900), previously in <iMorys</i Godman, 1900), <iLucida melitaea</i (Draudt, 1923) (not <iLucida lucia</i (Capronnier, 1874)), <iMethionopsis modestus</i Godman, 1901 (not <iMethionopsis ina</i (Plötz, 1882)), and <iThargella (Volus) volasus</i (Godman, 1901) (not <iEutocus facilis</i (Plötz, 1884)). The following 57 taxa are elevated from subspecies to species, <bnew status</b (some in <bnew combinations</b): <iDyscophellus doriscus</i (Hewitson, 1867) (not <iDyscophellus porcius</i (C. Felder and R. Felder, 1862), <iPhocides vida</i (A. Butler, 1872) (not <iPhocides urania</i (Westwood, 1852)), <iTagiades (Daimio) ceylonica</i Evans, 1932 (not <iTagiades litigiosa</i Möschler, 1878), <iTagiades (Daimio) tubulus</i Fruhstorfer, 1910 (not <iTagiades sambavana</i Elwes and Edwards, 1897), <iTagiades (Daimio) kina</i Evans, 1934, <iTagiades (Daimio) sheba</i Evans, 1934, <iTagiades (Daimio) martinus</i Plötz, 1884, <iTagiades (Daimio) sem</i Mabille, 1883, and <iTagiades (Daimio) neira</i Plötz, 1885 (not <iTagiades trebellius</i (Hopffer, 1874)), <iTagiades (Daimio) korela</i Mabille, 1891 and <iTagiades (Daimio) presbyter</i Butler, 1882 (not <iTagiades nestus</i (C. Felder, 1860)), <iTagiades obscurus</i Mabille, 1876, <iTagiades ravi</i (Moore, [1866]), <iTagiades atticus</i (Fabricius, 1793), <iTagiades titus</i Plötz, 1884, <iTagiades janetta</i Butler, 1870, <iTagiades inconspicua</i Rothschild, 1915, and <iTagiades hovia</i Swinhoe, 1904 (not <iTagiades japetus</i (Stoll, [1781])), <iTagiades silvia</i Evans, 1934 and <iTagiades elegans</i Mabille, 1877 (not <iTagiades gana</i (Moore, [1866])), <iTapena bornea</i Evans, 1941 and <iTapena minuscula</i Elwes and Edwards, 1897 (not <iTapena thwaitesi</i Moore, [1881]), <iDarpa dealbata</i (Distant, 1886) (not <iDarpa pteria</i (Hewitson, 1868)), <iPerus manx</i (Evans, 1953) (not <iPerus minor</i (Schaus, 1902)), <iCanesia pallida</i (Röber, 1925) (not <iCarrhenes canescens</i (R. Felder, 1869)), <iCarrhenes conia</i Evans, 1953 (not <iCarrhenes fuscescens</i (Mabille, 1891)), <iAnisochoria extincta</i Hayward, 1933 and <iAnisochoria polysticta</i Mabille, 1876 (not <iAnisochoria pedaliodina</i (Butler, 1870)), <iAnisochoria verda</i Evans, 1953 (not <iAnisochoria minorella</i Mabille, 1898), <iBralus alco</i (Evans, 1953) (not <iBralus albida</i (Mabille, 1888)), <iEphyriades jamaicensis</i (Möschler, 1879) (not <iEphyriades brunnea</i (Herrich-Schäffer, 1865)), <iKoruthaialos (Stimula) frena</i Evans, 1949 (not <iKoruthaialos focula</i (Plötz, 1882)), <iEuphyes kiowah</i (Reakirt, 1866) (not <iEuphyes vestris</i (Boisduval, 1852)), <iMnaseas inca</i Bell, 1930 (not <iMnaseas bicolor</i (Mabille, 1889)), <iMetron hypochlora</i (Draudt, 1923) (not <iMetrocles schrottkyi</i (Giacomelli, 1911), previously in <iMetron</i Godman, 1900), <iDecinea huasteca</i (H. Freeman, 1969), <iDecinea denta</i Evans, 1955, and <iDecinea antus</i (Mabille, 1895) (not <iDecinea decinea</i (Hewitson, 1876)), <iXeniades pteras</i Godman, 1900 (not <iXeniades chalestra</i (Hewitson, 1866)), <iXeniades difficilis</i Draudt, 1923 (not <iXeniades orchamus</i (Cramer, 1777)), <iXeniades hermoda</i (Hewitson, 1870) (not <iTisias quadrata</i (Herrich-Schäffer, 1869)), <iHermio vina</i (Evans, 1955) (not <iHermio hermione</i (Schaus, 1913), previously in <iLento</i Evans, 1955), <iCymaenes loxa</i Evans, 1955, (not <iCymaenes laureolus</i (Schaus, 1913)), <iNiconiades peri</i (Evans, 1955) (not <iRhinthon bajula</i (Schaus, 1902), previously in <iNeoxeniades</i Hayward, 1938), <iGallio danius</i (Bell, 1941) (not <iVehilius seriatus</i (Mabille, 1891)), <iGallio massarus</i (E. Bell, 1940) (not <iGallio garima</i (Schaus, 1902) previously in <iTigasis</i Godman, 1900), <iCymaenes edata</i (Plötz, 1882), <iCymaenes miqua</i (Dyar, 1913) and <iCymaenes aequatoria</i (Hayward, 1940) (not <iCymaenes odilia</i (Burmeister, 1878)), <iLychnuchus (Enosis) demon</i (Evans, 1955) (not <iLychnuchus (Enosis) immaculata</i (Hewitson, 1868), previously in <iEnosis</i Mabille, 1889), <iNaevolus naevus</i Evans, 1955 (not <iNaevolus orius</i (Mabille, 1883)), <iLucida scopas</i (Mabille, 1891), <iLucida oebasus</i (Godman, 1900), and <iLucida leopardus</i (Weeks, 1901) (not <iLucida lucia</i (Capronnier, 1874)), <iCorticea schwarzi</i (E. Bell, 1941) and <iCorticea sylva</i (Hayward, 1942) (not <iCorticea mendica</i (Mabille, 1898)), and <iChoranthus orientis</i (Skinner, 1920) (not <iChoranthus antiqua</i (Herrich-Schäffer, 1863), previously in <iPyrrhocalles</i Mabille, 1904). <iBorbo impar bipunctata</i (Elwes and J. Edwards, 1897) is a valid subspecies, not a synonym of <iBorbo impar tetragraphus</i (Mabille, 1891), here placed in synonymy with <iLotongus calathus</i (Hewitson, 1876), <bnew synonym</b. We confirm the species status of <iTelegonus cassius</i (Evans, 1952) and <iLerema (Morys) valda</i Evans, 1955. <iEuphyes chamuli</i Freeman, 1969 is placed as a subspecies of <iEuphyes kiowah</i (Reakirt, 1866), <bnew status</b. The following 41 taxa are <bjunior subjective synonyms</b, either newly proposed or transferred from synonymy with other species or subspecies: <iTelegonus mutius</i Plötz, 1882 of <iEuriphellus phraxanor</i (Hewitson, 1876), <iTelegonus erythras</i Mabille, 1888 of <iDyscophellus damias</i (Plötz, 1882), <iAethilla jaira</i Butler, 1870 of <iTelegonus cretellus</i (Herrich-Schäffer, 1869), <iPaches era</i Evans, 1953 of <iSanta palica</i (Mabille, 1888), <iAntigonus alburnea</i Plötz, 1884 of <iTolius tolimus robigus</i (Plötz, 1884) (not of <iEchelatus sempiternus simplicior</i (Möschler, 1877)), <iEchelatus depenicillus</i Strand, 1921 of <iE. sempiternus simplicior</i (not of <iT. tolimus robigus</i), <iAntigonus aura</i Plötz, 1884 of <iTheagenes dichrous</i (Mabille, 1878) (not of <iHelias phalaenoides palpalis</i (Latreille, [1824])), <iAchlyodes impressus</i Mabille, 1889 of <iCamptopleura orsus</i (Mabille, 1889), <iAugiades tania</i Schaus, 1902 of <iMetron voranus</i (Mabille, 1891), <iPamphila verdanta</i Weeks, 1906 of <iMetron fasciata</i (Möschler, 1877), <iNiconiades viridis vista</i Evans, 1955 of <iNiconiades derisor</i (Mabille, 1891), <iPamphila binaria</i Mabille, 1891 of <iConga chydaea</i (A. Butler, 1877) (not of <iCynea cynea</i (Hewitson, 1876)), <iPsoralis concolor</i Nicolay, 1980 of <iRalis immaculatus</i (Hayward, 1940), <iHesperia dido</i Plötz, 1882 of <iCynea (Quinta) cannae</i (Herrich-Schäffer, 1869) (not of <iLerema lochius</i (Plötz, 1882)), <iProteides osembo</i Möschler, 1883 of <iCynea (Cynea) diluta</i (Herrich-Schäffer, 1869) (not of <iCynea (Quinta) cannae</i (Herrich-Schäffer, 1869)), <iCobalopsis brema</i E. Bell, 1959 of <iEutus rastaca</i (Schaus, 1902), <iPsoralis panamensis</i Anderson and Nakamura, 2019 of <iRhomba gertschi</i (Bell, 1937), <iCobalus asella</i Herrich-Schäffer, 1869 of <iAmblyscirtes alternata</i (Grote and Robinson, 1867) (not of <iAmblyscirtes vialis</i (W. H. Edwards, 1862)), <iPapias trimacula</i Nicolay, 1973 of <iNastra subsordida</i (Mabille, 1891), <iPamphila bipunctata</i Mabille, 1889 and <iSarega staurus</i Mabille, 1904 of <iLerema pattenii</i Scudder, 1872 (not of <iCymaenes lumina</i (Herrich-Schäffer, 1869), previously in <iLerema</i Scudder, 1872), <iHesperia aethra</i Plötz, 1886 of <iLerema lineosa</i (Herrich-Schäffer, 1865) (not of <iLerema (Morys) compta</i Butler, 1877), <iMegistias miaba</i Schaus, 1902 of <iCobalopsis valerius</i (Möschler, 1879), <iPhanis sylvia</i Kaye, 1914 of <iLerema etelka</i (Schaus, 1902) (not of <iLerema (Geia) geisa</i (Möschler, 1879), previously in <iMorys</i Godman, 1900), <iCarystus odilia</i Burmeister, 1878, <iPamphila trebius</i Mabille, 1891 and <iMegistias corescene</i Schaus, 1902 of <iCymaenes lumina</i (Herrich-Schäffer, 1869), <iHesperia phocylides</i Plötz, 1882 of <iCymaenes edata</i (Plötz, 1882) (not of <iLerema accius</i (J. E. Smith, 1797)), <iPamphila xenos</i Mabille, 1898 of <iVehilius inca</i (Scudder, 1872), <iMnasilus guianae</i Lindsey, 1925 of <iPapias amyrna</i (Mabille, 1891), <iPamphila nubila</i Mabille, 1891 of <iPapias integra</i (Mabille, 1891) (not of <iCynea corisana</i (Plötz, 1882)), <iEnosis matheri</i H. Freeman, 1969 of <iMetiscus atheas</i Godman, 1900 (previously in <iEnosis</i Mabille, 1889), <iHesperia infuscata</i Plötz, 1882 of <iMnaseas derasa derasa</i (Herrich-Schäffer, 1870) (previously <iArotis</i Mabille, 1904), (not of <iPapias subcostulata</i (Herrich-Schäffer, 1870)), <iPamphila astur</i Mabille, 1891 of <iMetiscus angularis</i (Möschler, 1877) (not of <iCymaenes tripunctus theogenis</i (Capronnier, 1874)), <iAnthoptus macalpinei</i H. Freeman, 1969 of <iAnthoptus inculta</i (Dyar, 1918), <iMethionopsis typhon</i Godman, 1901 of <iMethionopsis ina</i (Plötz, 1882), <iMethionopsis dolor</i Evans, 1955 of <iThargella volasus</i (Godman, 1901), <iHesperia cinica</i Plötz, 1882 of <iDubiella dubius</i (Stoll, 1781), <iCobalus disjuncta</i Herrich-Schäffer, 1869 of <iDubiella dubius</i (Stoll, 1781) (not of <iVettius lafrenaye</i (Latreille, [1824])), and <iSaliana vixen</i Evans, 1955 of <iNeoxeniades parna</i (Evans, 1955). The following are <bnew and revised genus-species combinations</b: <iEuriphellus cebrenus</i (Cramer, 1777) (not <iSalatis</i Evans, 1952), <iGorgopas extensa</i (Mabille, 1891) (not <iPolyctor</i Evans, 1953), <iClytius shola</i (Evans, 1953) (not <iStaphylus</i Godman and Salvin, 1896), <iPerus narycus</i (Mabille, 1889) (not <iOuleus</i Lindsey, 1925), <iPerus parvus</i (Steinhauser and Austin, 1993) (not <iStaphylus</i Godman and Salvin, 1896), <iPholisora litus</i (Dyar, 1912) (not <iBolla</i Mabille, 1903), <iCarrhenes decens</i (A. Butler, 1874) (not <iAntigonus</i Hübner, [1819]), <iSanta palica</i (Mabille, 1888) (not <iChiothion</i Grishin, 2019), <iBralus nadia</i (Nicolay, 1980) (not <iAnisochoria</i Mabille, 1876), <iAcerbas sarala</i (de Nicéville, 1889) (not <iLotongus</i Distant, 1886), <iCaenides sophia</i (Evans, 1937) (not <iHypoleucis</i Mabille, 1891), <iHypoleucis dacena</i (Hewitson, 1876) (not <iCaenides</i Holland, 1896), <iDotta tura</i (Evans, 1951) (not <iAstictopterus</i C. Felder and R. Felder, 1860), <iNervia wallengrenii</i (Trimen, 1883) (not <iKedestes</i Watson, 1893), <iTestia mammaea</i (Hewitson, 1876) (not <iDecinea</i Evans, 1955), <iOxynthes trinka</i (Evans, 1955) (not <iOrthos</i Evans, 1955), <iMetrocles argentea</i (Weeks, 1901) (not <iParatrytone</i Godman, 1900), <iMetrocles scitula</i (Hayward, 1951) (not <iMucia</i Godman, 1900), <iMetrocles schrottkyi</i (Giacomelli, 1911) (not <iMetron</i Godman, 1900), <iNiconiades derisor</i (Mabille, 1891) (not <iDecinea</i Evans, 1955), <iParatrytone samenta</i (Dyar, 1914) (not <iOchlodes</i Scudder, 1872), <iOligoria (Cobaloides) locutia</i (Hewitson, 1876) (not <iQuinta</i Evans, 1955), <iPsoralis (Saniba) laska</i (Evans, 1955) (not <iVidius</i Evans, 1955), <iPsoralis (Saniba) arva</i (Evans, 1955) and <iPsoralis (Saniba) umbrata</i (Erschoff, 1876) (not <iVettius</i Godman, 1901), <iPsoralis (Saniba) calcarea</i (Schaus, 1902) and <iPsoralis (Saniba) visendus</i (E. Bell, 1942) (not <iMolo</i Godman, 1900), <iAlychna gota</i (Evans, 1955) (not <iPsoralis</i Mabille, 1904), <iAdlerodea asema</i (Mabille, 1891) and <iAdlerodea subpunctata</i (Hayward, 1940) (not <iEutychide</i Godman, 1900), <iRalis immaculatus</i (Hayward, 1940) (not <iMucia</i Godman, 1900), <iRhinthon braesia</i (Hewitson, 1867) and <iRhinthon bajula</i (Schaus, 1902) (not <iNeoxeniades</i Hayward, 1938), <iCymaenes lochius</i Plötz, 1882 (not <iLerema</i Scudder, 1872), <iParacarystus ranka</i (Evans, 1955) (not <iThoon</i Godman, 1900), <iTricrista aethus</i (Hayward, 1951), <iTricrista canta</i (Evans, 1955), <iTricrista slopa</i (Evans, 1955), <iTricrista circellata</i (Plötz, 1882), and <iTricrista taxes</i (Godman, 1900) (not <iThoon</i Godman, 1900), <iGallio madius</i (E. Bell, 1941) and <iGallio seriatus</i (Mabille, 1891) (not <iVehilius</i Godman, 1900), <iGallio garima</i (Schaus, 1902) (not <iTigasis</i Godman, 1900), <iTigasis corope</i (Herrich-Schäffer, 1869) (not <iCynea</i Evans, 1955), <iTigasis perloides</i (Plötz, 1882) (not <iCymaenes</i Scudder, 1872), <iAmblyscirtes (Flor) florus</i (Godman, 1900) (not <iRepens</i Evans, 1955), <iVidius fraus</i (Godman, 1900) (not <iCymaenes</i Scudder, 1872), <iNastra celeus</i (Mabille, 1891) (not <iVehilius</i Godman, 1900), <iNastra nappa</i (Evans, 1955) (not <iVidius</i Evans, 1955), <iVehilius warreni</i (Weeks, 1901) and <iVehilius limae</i (Lindsey, 1925) (not <iCymaenes</i Scudder, 1872), <iCymaenes lumina</i (Herrich-Schäffer, 1869) (not <iLerema</i Scudder, 1872), <iCobalopsis valerius</i (Möschler, 1879) (not <iCobalopsis</i Godman, 1900), <iCobalopsis dictys</i (Godman, 1900) (not <iPapias</i Godman, 1900), <iLerema (Morys) venias</i (Bell, 1942) (not <iCobalopsis</i Godman, 1900), <iPapias latonia</i (Schaus, 1913) (not <iCobalopsis</i Godman, 1900), <iDion iccius</i (Evans, 1955) and <iDion uza</i (Hewitson, 1877) (not <iEnosis</i Mabille, 1889), <iVistigma (Vistigma) opus</i (Steinhauser, 2008) (not <iThoon</i Godman, 1900), <iSaturnus fartuga</i (Schaus, 1902) (not <iParphorus</i Godman, 1900), <iPhlebodes fuldai</i (E. Bell, 1930) (not <iVettius</i Godman, 1901), <iMnasitheus padus</i (Evans, 1955) (not <iMoeris</i Godman, 1900), <iNaevolus brunnescens</i (Hayward, 1939) (not <iPsoralis</i Mabille, 1904), <iLamponia ploetzii</i (Capronnier, 1874) (not <iVettius</i Godman, 1901), <iMnestheus silvaticus</i Hayward, 1940 (not <iLudens</i Evans, 1955), <iRigga spangla</i (Evans, 1955) (not <iSodalia</i Evans, 1955), <iCorticea vicinus</i (Plötz, 1884) (not <iLento</i Evans, 1955), <iMnasalcas thymoetes</i (Hayward, 1942) (not <iMnasicles</i Godman, 1901), <iMnasalcas boyaca</i (Nicolay, 1973) (not <iPamba</i Evans, 1955), <iVertica brasta</i (Evans, 1955) (not <iLychnuchus</i Hübner, [1831]), <iCarystina discors</i Plötz, 1882 (not <iCobalus</i Hübner, [1819]), <iZetka irena</i (Evans, 1955) (not <iNeoxeniades</i Hayward, 1938), and <iNeoxeniades parna</i (Evans, 1955) (not <iNiconiades</i Hübner, [1821]). The following are <bnew or revised species-subspecies combinations</b: <iTagiades neira moti</i Evans, 1934, <iTagiades neira canonicus</i Fruhstorfer, 1910, <iTagiades sheba vella</i Evans, 1934, <iTagiades sheba lola</i Evans, 1945, <iTagiades korela biakana</i Evans, 1934, <iTagiades korela mefora</i Evans, 1934, <iTagiades korela suffusus</i Rothschild, 1915, <iTagiades korela brunta</i Evans, 1949, <iTagiades ravi ravina</i Fruhstorfer, 1910, <iTagiades atticus carnica</i Evans, 1934, <iTagiades atticus nankowra</i Evans, 1934, <iTagiades atticus helferi</i C. Felder, 1862, <iTagiades atticus balana</i Fruhstorfer, 1910, <iTagiades inconspicua mathias</i Evans, 1934, <iTagiades hovia kazana</i Evans, 1934, <iTagiades elegans fuscata</i de Jong and Treadaway, 2007, <iTagiades elegans semperi</i Fruhstorfer, 1910, <iMetron hypochlora tomba</i Evans, 1955, <iDecinea denta pruda</i Evans, 1955, and <iChoranthus orientis eleutherae</i (Bates, 1934) (previously in <iPyrrhocalles</i Mabille, 1904). In addition to the abovementioned changes, the following <bnew combinations</b involve newly proposed genus group names: <iFulvatis fulvius</i (Plötz, 1882) and <iFulvatis scyrus</i (E. Bell, 1934) (not <iSalatis</i Evans, 1952); <iAdina adrastor</i (Mabille and Boullet, 1912) (not <iBungalotis</i Watson, 1893); <iNascus (Praxa) prax</i Evans, 1952<i, Nascus (Bron) broteas</i (Cramer, 1780), and <iNascus (Bron) solon</i (Plötz, 1882) (not <iPseudonascus</i Austin, 2008); <iChirgus (Turis) veturius</i (Plötz, 1884); <iPaches (Tiges) liborius</i (Plötz, 1884), and <iPaches (Tiges) mutilatus</i (Hopffer, 1874) (not <iAntigonus</i Hübner, [1819]); <iPaches (Tiges) exosa</i (A. Butler, 1877); <iTolius tolimus</i (Plötz, 1884) and <iTolius luctuosus</i (Godman &amp; Salvin, 1894) (not <iEchelatus</i Godman and Salvin, 1894); <iAncistroides (Ocrypta) caerulea</i (Evans, 1928), <iAncistroides (Ocrypta) renardi</i (Oberthür, 1878), <iAncistroides (Ocrypta) waigensis</i (Plötz, 1882)<i, Ancistroides (Ocrypta) aluensis</i (Swinhoe, 1907)<i, Ancistroides (Ocrypta) flavipes</i (Janson, 1886), and <iAncistroides (Ocrypta) maria</i (Evans, 1949) (not <iNotocrypta</i de Nicéville, 1889); <iLennia lena</i (Evans, 1937)<i, Lennia binoevatus</i (Mabille, 1891)<i, Lennia maracanda</i (Hewitson, 1876), and <iLennia lota</i (Evans, 1937) (not <iLeona</i Evans, 1937); <iTrida barberae</i (Trimen, 1873) and <iTrida sarahae</i (Henning and Henning, 1998) (not <iKedestes</i Watson, 1893); <iNoxys viricuculla</i (Hayward, 1951) (not <iOxynthes</i Godman, 1900); <iXeniades (Tixe) quadrata</i (Herrich-Schäffer, 1869)<i, Xeniades (Tixe) rinda</i (Evans, 1955)<i, Xeniades (Tixe) putumayo</i (Constantino and Salazar, 2013) (not <iTisias</i Godman, 1901); <iGracilata quadrinotata</i (Mabille, 1889) (not <iStyriodes</i Schaus, 1913); <iHermio hermione</i (Schaus, 1913) (not <iLento</i Evans, 1955); <iCynea (Nycea) hycsos</i (Mabille, 1891)<i, Cynea (Nycea) corisana</i (Plötz, 1882)<i, Cynea (Nycea) popla</i Evans, 1955<i, Cynea (Nycea) iquita</i (E. Bell, 1941)<i, Cynea (Nycea) robba</i Evans, 1955<i, Cynea (Nycea) melius</i (Geyer, 1832), and <iCynea (Nycea) irma</i (Möschler, 1879); <iEutus rastaca</i (Schaus, 1902) (not <iEutychide</i Godman, 1900); <iEutus yesta</i (Evans, 1955) (not <iThoon</i Godman, 1900); <iEutus mubevensis</i (E. Bell, 1932) (not <iTigasis</i Godman, 1900); <iGufa gulala</i (Schaus, 1902) (not <iMucia</i Godman, 1900); <iGufa fusca</i (Hayward, 1940) (not <iTigasis</i Godman, 1900); <iGodmia chlorocephala</i (Godman, 1900) (not <iOnophas</i Godman, 1900); <iRhomba gertschi</i (E. Bell, 1937) (not <iJustinia</i Evans, 1955); <iMnasicles (Nausia) nausiphanes</i (Schaus, 1913) (not <iTigasis</i Godman, 1900); <iAmblyscirtes (Flor) florus</i (Godman, 1900) (not <iRepens</i Evans, 1955); <iRectava ignarus</i (E. Bell, 1932) (not <iPapias</i Godman, 1900); <iRectava vorgia</i (Schaus, 1902) (not <iCobalopsis</i Godman, 1900); <iRectava nostra</i (Evans, 1955) (not not <iVidius</i Evans, 1955); <iLerema (Geia) geisa</i (Möschler, 1879) and <iLerema (Geia) lyde</i (Godman, 1900) (not <iMorys</i Godman, 1900); <iContrastia distigma</i (Plötz, 1882) (not <iCymaenes</i Scudder, 1872); <iMit (Mit) badius</i (E. Bell, 1930) (not <iStyriodes</i Schaus, 1913); <iMit (Mit) gemignanii</i (Hayward, 1940), (not <iMnasitheus</i Godman, 1900); <iMit (Rotundia) schausi</i (Mielke and Casagrande, 2002), (not <iEnosis</i Mabille, 1889); <iPicova steinbachi</i (E. Bell, 1930) (not <iSaturnus</i Evans, 1955); <iLattus arabupuana</i (E. Bell, 1932) (not <iEutocus</i Godman, 1901); <iGubrus lugubris</i (Lindsey, 1925) (not <iVehilius</i Godman, 1900); <iThargella (Pseudopapias) tristissimus</i (Schaus, 1902) (not <iPapias</i Godman, 1900); <iKoria kora</i (Hewitson, 1877) (not <iJustinia</i Evans, 1955); <iJustinia (Septia) septa</i Evans, 1955; <iCorta lycortas</i (Godman, 1900) (not <iOrthos</i Evans, 1955); <iVertica (Brasta) brasta</i (Evans, 1955) (not <iLychnuchus</i Hübner, [1831]); <iCalvetta calvina</i (Hewitson, 1866) (not <iCobalus</i Hübner, [1819]); <iNeoxeniades (Bina) gabina</i (Godman, 1900) (not <iOrthos</i Evans, 1955); <iOz ozias</i (Hewitson, 1878) and <iOz sebastiani</i Salazar and Constantino, 2013 (not <iLychnuchoides</i Godman, 1901); and <iCarystoides (Balma) balza</i Evans, 1955 and <iCarystoides (Balma) maroma</i (Möschler, 1877). Finally, unless stated otherwise, all subgenera, species, subspecies and synonyms of mentioned genera and species are transferred together with their parent taxa, and taxa not mentioned in this work remain as previously classified.

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