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SARS-CoV-2 has had an unprecedented impact on human health and highlights the need for genomic epidemiology studies to increase our understanding of the evolution and spread of pathogens and to inform policy decisions. Most efforts have focused on international or country-wide transmission, which are unable to highlight state-wide trends. We sequenced virus genomes from over 22,000 patients tested at Mayo Clinic Laboratories between 2020-2022 and leveraged detailed patient metadata to describe county-to-county spread in Minnesota. Our findings indicate that spread in the state was mostly dominated by viruses from Hennepin County, which contains the largest metropolis. For many counties, we found that state government restrictions eventually led to a decrease in the diversity of circulating viruses from other counties and that their complete removal in May of 2021 saw a drastic revert to levels at or greater than those observed during the months before. We also linked over 14,000 genomes with patient risk characteristics and infection-related phenotypes from the Mayo Clinic electronic health record. We found that the genetic relationship of Omicron viruses was structured by clinical outcomes when stratifying by patient risk factor and variant of concern. However, we were unable to identify nucleotide variants that drove this association.

SARS-CoV-2 has had an unprecedented impact on human health and highlights the need for genomic epidemiology studies to increase our understanding of virus evolution and spread, and to inform policy decisions. We sequenced viral genomes from over 22,000 patient samples tested at Mayo Clinic Laboratories between 2020-2022 and use Bayesian phylodynamics to describe county and regional spread in Minnesota.The earliest introduction into Minnesota was to Hennepin County from a domestic source around January 22, 2020; six weeks before the first confirmed case in the state. This led to the virus spreading to Northern Minnesota, and eventually, the rest of the state. International introductions were most abundant in Hennepin (home to the Minneapolis/St. Paul International (MSP) airport) totaling 45 (out of 107) over the two-year period. Southern Minnesota counties were most common for domestic introductions with 19 (out of 64), potentially driven by bordering states such as Iowa and Wisconsin as well as Illinois which is nearby. Hennepin also was, by far, the most dominant source of in-state transmissions to other Minnesota locations (n=772) over the two-year period.We also analyzed the diversity of the location source of SARS-CoV-2 viruses in each county and noted the timing of state-wide policies as well as trends in clinical cases. Neither the number of clinical cases or major policy decisions, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, appeared to have impact on virus diversity across each individual county.ImportanceWe analyzed over 22,000 SARS-CoV-2 genomes of patient samples tested at Mayo Clinic Laboratories during a two-year period in the COVID-19 pandemic that included Alpha, Delta, and Omicron VoCs to examine the roles and relationships of Minnesota virus transmission.We found that Hennepin County, the most populous county, drove the transmission of SARS-CoV-2 viruses in the state after including the formation of earlier clades including 20A, 20C, and 20G, as well as variants of concern Alpha and Delta. We also found that Hennepin County was the source for most of the county-to-county introductions after its initial introduction with the virus in early 2020 from an international source, while other counties acted as transmission “sinks”. In addition, major policies such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, did not appear to have an impact on virus diversity across individual counties.

Objectives Error simulation models have been used to understand the relationship between analytical performance and clinical outcomes. We developed an error simulation model to understand the effects of method bias and precision on misclassification rate for neonatal hyperbilirubinemia using an age-adjusted risk assessment tool. Methods For each of 176 measured total bilirubin (TSBM) values, 10,000 simulated total bilirubin (TBS) values were generated at each combination of bias and precision conditions for coefficient of variation (CV) between 1 and 15%, and for biases between −51.3 μmol/L and 51.3 μmol/L (−3 and 3 mg/dL) fixed bias. TBS values were analyzed to determine if they were in the same risk zone as the TSBM value. We then calculated sensitivity and specificity for prediction of ≥75th percentile for postnatal age values as a function of assay bias and precision, and determined the rate of critical errors (≥95th percentile for age TSBM with S). Results A sensitivity >95% for predicting ≥75th percentile bilirubin values was observed when there is a positive fixed bias of greater than 17.1 μmol/L (1.0 mg/dL) and CV is maintained ≤10%. A specificity >70% for predicting 0.2% until negative bias was −17.1 μmol/L (−1 mg/dL) or lower. Conclusions A positive systematic bias of 17.1 μmol/L (1 mg/dL) may be optimal for balancing sensitivity and specificity for predicting ≥75th percentile TSB values. Negative systematic bias should be avoided to allow detection of high risk infants and avoid critical classification errors.

The emergence of highly transmissible SARS-CoV-2 variants and vaccine breakthrough infections globally mandated the characterization of the immuno-evasive features of SARS-CoV-2. Here, we systematically analyzed 2.13 million SARS-CoV-2 genomes from 188 countries/territories (up to June 2021) and performed whole-genome viral sequencing from 102 COVID-19 patients, including 43 vaccine breakthrough infections. We identified 92 Spike protein mutations that increased in prevalence during at least one surge in SARS-CoV-2 test positivity in any country over a 3-month window. Deletions in the Spike protein N-terminal domain were highly enriched for these 'surge-associated mutations' (Odds Ratio = 14.19, 95% CI 6.15-32.75, p value = 3.41 × 10

Variants of SARS-CoV-2 are evolving under a combination of immune selective pressure in infected hosts and natural genetic drift, raising a global alarm regarding the durability of COVID-19 vaccines. Here, we conducted longitudinal analysis over 1.8 million SARS-CoV-2 genomes from 183 countries or territories to capture vaccination-associated viral evolutionary patterns. To augment this macroscale analysis, we performed viral genome sequencing in 23 vaccine breakthrough COVID-19 patients and 30 unvaccinated COVID-19 patients for whom we also conducted machine-augmented curation of the electronic health records (EHRs). Strikingly, we find the diversity of the SARS-CoV-2 lineages is declining at the country-level with increased rate of mass vaccination (n = 25 countries, mean correlation coefficient = −0.72, S.D. = 0.20). Given that the COVID-19 vaccines leverage B-cell and T-cell epitopes, analysis of mutation rates shows neutralizing B-cell epitopes to be particularly more mutated than comparable amino acid clusters (4.3-fold, p < 0.001). Prospective validation of these macroscale evolutionary patterns using clinically annotated SARS-CoV-2 whole genome sequences confirms that vaccine breakthrough patients indeed harbor viruses with significantly lower diversity in known B cell epitopes compared to unvaccinated COVID-19 patients (2.3-fold, 95% C.I. 1.4-3.7). Incidentally, in these study cohorts, vaccinated breakthrough patients also displayed fewer COVID-associated complications and pre-existing conditions relative to unvaccinated COVID-19 patients. This study presents the first known evidence that COVID-19 vaccines are fundamentally restricting the evolutionary and antigenic escape pathways accessible to SARS-CoV-2. The societal benefit of mass vaccination may consequently go far beyond the widely reported mitigation of SARS-CoV-2 infection risk and amelioration of community transmission, to include stemming of rampant viral evolution.

The raging COVID-19 pandemic in India and reports of “vaccine breakthrough infections” globally have raised alarm mandating the characterization of the immuno-evasive features of SARS-CoV-2. Here, we systematically analyzed 1.57 million SARS-CoV-2 genomes from 187 countries/territories and performed whole-genome viral sequencing from 53 COVID-19 patients, including 20 vaccine breakthrough infections. We identified 89 Spike protein mutations that increased in prevalence during at least one surge in SARS-CoV-2 test positivity in any country over a three-month window. Deletions in the Spike protein N-terminal domain (NTD) are highly enriched for these ‘surge-associated mutations’ (Odds Ratio = 41.8, 95% CI: 6.36-1758, p-value = 7.7e-05). In the recent COVID-19 surge in India, an NTD deletion (ΔF157/R158) increased over 10-fold in prevalence from February 2021 (1.1%) to April 2021 (15%). During the recent surge in Chile, an NTD deletion (Δ246-253) increased rapidly over 30-fold in prevalence from January 2021 (0.86%) to April 2021 (33%). Strikingly, these simultaneously emerging deletions associated with surges in different parts of the world both occur at an antigenic supersite that is targeted by neutralizing antibodies. Finally, we generated clinically annotated SARS-CoV-2 whole genome sequences and identified deletions within this NTD antigenic supersite in a patient with vaccine breakthrough infection (Δ156-164) and other deletions from unvaccinated severe COVID-19 patients that could represent emerging deletion-prone regions. Overall, the expanding repertoire of Spike protein deletions throughout the pandemic and their association with case surges and vaccine breakthrough infections point to antigenic minimalism as an emerging evolutionary strategy for SARS-CoV-2 to evade immune responses. This study highlights the urgent need to sequence SARS-CoV-2 genomes at a larger scale globally and to mandate a public health policy for transparent reporting of relevant clinical annotations (e.g. vaccination status) in order to aid the development of comprehensive therapeutic strategies.

Objectives Pathogenic free-living amebae (FLAs) cause skin, ocular, and central nervous system (CNS) infections with significant morbidity and mortality. Diagnosis of FLA infections by pathologic examination of tissue sections can be aided using molecular assays. This study investigated the performance characteristics of a multiplex real-time polymerase chain reaction (PCR) assay (FLA-PCR) for detection and differentiation of FLAs in clinical specimens. Methods FLA-PCR was performed on 39 human specimens comprising one cutaneous, 14 corneal, and 24 CNS formalin-fixed, paraffin-embedded (FFPE) tissues with a histopathologic diagnosis of FLA infection and four CNS FFPE tissues with inflammation but no evidence of FLAs. In addition, clinical specificity and assay limit of detection were determined. Results FLA detection sensitivities ranged from 79% to 84% in FFPE tissues. No cross-reactivity was observed. Conclusions While sensitivity is limited, FLA-PCR assay may serve as a useful adjunct for detection or confirmation of FLA infections in FFPE tissues.

Context.—Preanalytic errors, including specimen labeling errors and specimen loss, occur frequently during specimen collection, transit, and accessioning. Radio-frequency identification tags can decrease specimen identification and tracking errors through continuous and automated tracking of specimens.Objective.—To implement a specimen tracking infrastructure to reduce preanalytic errors (specimen mislabeling or loss) between specimen collection and laboratory accessioning. Specific goals were to decrease preanalytic errors by at least 70% and to simultaneously decrease employee effort dedicated to resolving preanalytic errors or investigating lost specimens.Design.—A radio-frequency identification specimen-tracking system was developed. Major features included integral radio-frequency identification labels (radio-frequency identification tags and traditional bar codes in a single printed label) printed by point-of-care printers in collection suites; dispersed radio-frequency identification readers at major transit points; and systems integration of the electronic health record, laboratory information system, and radio-frequency identification tracking system to allow for computerized physician order entry driven label generation, specimen transit time tracking, interval-based alarms, and automated accessioning.Results.—In the 6-month postimplementation period, 6 mislabeling events occurred in collection areas using the radio-frequency identification system, compared with 24 events in the 6-month preimplementation period (75% decrease; P = .001). In addition, the system led to the timely recovery of 3 lost specimens. Labeling expenses were decreased substantially in the transition from high-frequency to ultrahigh frequency radio-frequency identification tags.Conclusions.—Radio-frequency identification specimen tracking prevented several potential specimen-loss events, decreased specimen recovery time, and decreased specimen labeling errors. Increases in labeling/tracking expenses for the system were more than offset by time savings and loss avoidance through error mitigation.

Introduction Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are common laboratory assays used as markers of inflammation. ESR suffers from higher false positive and false negative rates than CRP. To that end, the American Board of Internal Medicine's (ABIM's) Choosing Wisely campaign has recommended against ESR testing for those with undiagnosed conditions in favor of CRP testing. This study describes the impact of a computerized provider order entry (CPOE) decision support rule against ESR/CRP co-ordering within a community health system that predates the ABIM's Choosing Wisely national guidance. To demonstrate the potential impact of such a CPOE rule within other healthcare settings, ESR/CRP ordering data from a multi-site tertiary care practice and from the commercially insured population in the OptumLabs® Data Warehouse (OLDW) were analyzed and the relative reduction in ESR/CRP co-ordering achieved within the community health system was projected onto these populations. Materials and methods ESR and/or CRP orders from a community health system were assessed from 2012 to 2016. Co-ordering and test concordance rates between ESR and CRP were compared before and after CPOE decision support rule launch. Similarly, ESR/CRP co-ordering across three tertiary care sites from 2015 to 2016 and the OLDW from 2009 to 2013 were assessed and the co-ordering rate reduction achieved in the community health system was mathematically projected onto these populations. Estimated payer savings from the rule's effect were calculated within each population using Medicare reimbursement rates. Results The CPOE decision support rule realized an unadjusted 42% relative rate reduction in ESR/CRP co-ordering within the community health system yielding an annual payer savings of $15,000 with a modest increase in ESR/CRP concordance rates. Projecting a 40% relative reduction in ESR/CRP co-ordering rates from a similarly effective CPOE rule, annual payer cost reductions exceeding $100,000 within a multi-site tertiary care setting and $1,000,000 within the OLDW would be expected. Conclusion ESR/CRP co-ordering represents an opportunity to eliminate testing waste and reduce payer costs. A CPOE decision support rule stably reduces ESR/CRP co-ordering rates. Similar results may occur as one component of new commercially available decision support platforms.

Newborn screening for congenital hypothyroidism remains challenging decades after broad implementation worldwide. Testing protocols are not uniform in terms of targets (TSH and/or T4) and protocols (parallel vs. sequential testing, one or two specimen collection times), and specificity (with or without collection of a second specimen) is overall poor. The purpose of this retrospective study is to investigate the potential impact of multivariate pattern recognition software (CLIR) to improve the post-analytical interpretation of screening results. Seven programs contributed reference data (N = 1,970,536) and two sets of true (TP, N = 1369 combined) and false (FP, N = 15,201) positive cases for validation and verification purposes, respectively. Data were adjusted for age at collection, birth weight, and location using polynomial regression models of the fifth degree to create three-dimensional regression surfaces. Customized Single Condition Tools and Dual Scatter Plots were created using CLIR to optimize the differential diagnosis between TP and FP cases in the validation set. Verification testing correctly identified 446/454 (98%) of the TP cases, and could have prevented 1931/5447 (35%) of the FP cases, with variable impact among locations (range 4% to 50%). CLIR tools either as made here or preferably standardized to the recommended uniform screening panel could improve performance of newborn screening for congenital hypothyroidism.

Context.—Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in formalin-fixed, paraffin-embedded tissue using reverse transcription–polymerase chain reaction, immunohistochemistry (IHC), and/or RNA in situ hybridization (RNAish).Objective.—To compare droplet digital polymerase chain reaction, IHC, and RNAish to detect SARS-CoV-2 in formalin-fixed, paraffin-embedded tissue in a large series of lung specimens from coronavirus disease 2019 (COVID-19) patients.Design.—Droplet digital polymerase chain reaction and RNAish used commercially available probes; IHC used clone 1A9. Twenty-six autopsies of COVID-19 patients with formalin-fixed, paraffin-embedded tissue blocks of 62 lung specimens, 22 heart specimens, 2 brain specimens, and 1 liver, and 1 umbilical cord were included. Control cases included 9 autopsy lungs from patients with other infections/inflammation and virus-infected tissue or cell lines.Results.—Droplet digital polymerase chain reaction had the highest sensitivity for SARS-CoV-2 (96%) when compared with IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between droplet digital polymerase chain reaction and IHC or RNAish was fair (κ = 0.23 and κ = 0.35, respectively). Agreement between IHC and in situ hybridization was substantial (κ = 0.75). Interobserver reliability was almost perfect for IHC (κ = 0.91) and fair to moderate for RNAish (κ = 0.38–0.59). Lung tissues from patients who died earlier after onset of symptoms revealed higher copy numbers by droplet digital polymerase chain reaction (P = .03, Pearson correlation = −0.65) and were more likely to be positive by RNAish (P = .02) than lungs from patients who died later. We identified SARS-CoV-2 in hyaline membranes, in pneumocytes, and rarely in respiratory epithelium. Droplet digital polymerase chain reaction showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative.Conclusions.—Droplet digital polymerase chain reaction was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients.

Background Primary cold agglutinin disease (CAD) is a monoclonal antibody (M-protein) and complement-mediated chronic hemolytic disease process. Antibody glycosylation can play a role in both antibody half-life and complement fixation. Recently, M-protein light chain (LC) glycosylation has been shown to be associated with AL amyloidosis. We hypothesized that M-protein LC glycosylation is also associated with cold agglutinin (CA) titers and CA-mediated hemolysis. Study design and methods A cross-sectional study of patients undergoing CA titer evaluation underwent mass spectrometric analysis for M-proteins and M-protein LC glycosylation. A subset of serum samples also underwent evaluation for the ability to trigger cold hemolysis in vitro. M-protein and M-protein LC glycosylation rates were compared across CA titer groups, clinical diagnosis, direct antiglobulin testing (DAT) results, and cold in vitro hemolysis rates. Results Both M-protein and M-protein LC glycosylation rates significantly differed across CA titer groups with the highest rates in those with elevated CA titers. M-protein LC glycosylation occurred almost exclusively on IgM kappa M-proteins and was significantly associated with positive DAT results and a clinical diagnosis of CAD. Cold in vitro hemolysis was demonstrated in two patients who both had a CA titer of more than 512 but there was no significant association with CA titer group or M-protein LC glycosylation status. Conclusion M-protein LC glycosylation is significantly associated with higher CA titer levels. Given the role that antibody glycosylation can play in antibody half-life and complement fixation, further studies are needed to clarify the effects of LC glycosylation within the context of CAD.

Shotgun metagenomic sequencing can detect nucleic acids from bacteria, fungi, viruses, and/or parasites in clinical specimens; however, little data exist to guide its optimal application to clinical practice. We retrospectively reviewed results of shotgun metagenomic sequencing testing requested on cerebrospinal fluid samples submitted to an outside reference laboratory from December 2017 through December 2019. Of the 53 samples from Mayo Clinic patients, 47 were requested by neurologists, with infectious diseases consultation in 23 cases. The majority of patients presented with difficult-to-diagnose subacute or chronic conditions. Positive results were reported for 9 (17%) Mayo Clinic patient samples, with 6 interpreted as likely contamination. Potential pathogens reported included bunyavirus, human herpesvirus 7, and enterovirus D-68, ultimately impacting care in two cases. Twenty-seven additional samples were submitted from Mayo Clinic Laboratories reference clients, with positive results reported for three (11%): two with potential pathogens (West Nile virus and Toxoplasma gondii) and one with Streptococcus species with other bacteria below the reporting threshold (considered to represent contamination). Of 68 negative results, 10 included comments on decreased sensitivity due to high DNA background (n = 5), high RNA background (n = 1), insufficient RNA read depth (n = 3), or quality control (QC) failure with an external RNA control (n = 1). The overall positive-result rate was 15% (12/80), with 58% (7/12) of these interpreted as being inconsistent with the patient’s clinical presentation. Overall, potential pathogens were found in a low percentage of cases, and positive results were often of unclear clinical significance. Testing was commonly employed in cases of diagnostic uncertainty and when immunotherapy was being considered.

Background Platelet (PLT) transfusion refractoriness increases bleeding complications, hospital stays, and PLT inventory usage. Immune-mediated refractoriness can be evaluated for using a physical PLT crossmatch with ABO-compatible inventory and, if positive, managed with HLA-compatible PLT inventory and donors. Manual completion of these complex tasks can be time-consuming and potentially error-prone. This study was conducted to determine if a Web-based software application could improve process efficiency and accuracy. Study design and methods Workflow analysis was performed to identify process, data, and analytic requirements for a software application for three PLT transfusion-refractoriness associated tasks: (a) physical PLT crossmatch inventory selection, (b) HLA-compatible inventory selection, and (c) HLA-compatible donor selection. After software application development, a comparison study was performed over 10 consecutive days, with each task performed manually and with the software application (Platelet Virtual Crossmatch [PLT VXM]) for a different unique immune-mediated PLT transfusion-refractory recipient. Task completion time, number of incompatible units/donors presented, and number of documentation errors were compared. Results PLT VXM is a Web-based software application developed using R and the Shiny Web application framework. PLT VXM significantly reduced median task completion times by 4.5 (49%), 11.2 (79%), and 59.1 minutes (94%), respectively. PLT VXM did not present any incompatible PLT units or donors for user consideration. PLT VXM also had a lower number of documentation errors than the manual process, and none of these documentation errors were software generated. Conclusion Computer-aided evaluation and management of immune-mediated PLT transfusion-refractory recipients can significantly improve workflow and reduce manual errors in this complex process.

BACKGROUND Platelet transfusion–refractoriness is a challenging and expensive clinical scenario seen most often in patients with hematologic malignancies. Although the majority of platelet transfusion–refractory cases are due to nonimmune causes, a significant minority are caused by alloimmunization against Class I human leukocyte antigens (HLAs) or human platelet antigens (HPAs). Such platelet transfusion–refractory patients can be effectively managed with appropriate antigen-negative products. STUDY DESIGN AND METHODS Our institution has developed a diagnostic and management algorithm for the platelet transfusion–refractory patient with an early focus on identifying those cases caused by immune-mediated factors. Using physical platelet cross-matches to initially classify platelet transfusion–refractory patients as immune-mediated or not, cross-match–compatible inventory is then provided to immune-mediated patients, whereas subsequent HLA (with or without HPA) testing is performed. RESULTS Our blood donor program performs Class I HLA typing of all repeat platelet donors to facilitate the identification of antigen-negative platelet units (virtual cross-matching) as well as the recruitment of HLA-matched donors. The platelet transfusion–refractoriness algorithm realizes an initial net cost savings once two apheresis platelets are saved from use for each newly identified, immune-mediated platelet transfusion–refractory patient. CONCLUSION An algorithm utilizing physical platelet cross-matches, Class I HLA and HPA antibody testing, and upfront Class I HLA typing of platelet donors leads to overall resource savings and improved clinical management for platelet transfusion–refractory patients.

Introduction/Objective Advances in whole slide imaging have enabled the application of machine learning algorithms to anatomic pathology. In the current state, the development of accurate algorithms requires robust training data with correctly assigned diagnostic and classification labels. Increasingly, institutions have looked to their archival slides as a source of “ground truth” for algorithm development. However, the curation and use of archival data poses several challenges. Here, we share lessons learned from reviewing head and neck pathology consult cases spanning a 10- year period at Mayo Clinic Rochester. Methods Archived surgical pathology slides from 2,590 consult cases were reviewed. Clinical and demographic information was recorded for each case, including surgical date, surgical procedure, anatomic site, age, gender and diagnosis. Cases were excluded from the curated archive if there was insufficient volume or quality of tissue to render a specific diagnosis (141 cases, 5.6%). Slides with a range of tissue size and quality, from numerable laboratories were included in the curated archive. Selected cases were collated by anatomic site: ear, gnathic, larynx, nasopharynx, neck, oral cavity, oropharynx, salivary gland and sinonasal tract. Results Common diagnostic reconciliations (115 cases, 4.4%) fell within the following categories: (1) novel entities (59 cases, 2.3%), including biphenotypic sinonasal sarcoma and clear cell carcinoma; (2) novel classifications (21 cases, 0.8%), as seen in HPV-related oropharyngeal squamous cell carcinoma and polymorphous adenocarcinoma; and (3) novel grading schema (35 cases, 1.4%), as seen in keratinizing dysplasia and oropharyngeal malignancies. Conclusion Several nuances emerged in the process of reviewing slides, highlighting the need for continual amendment of any machine learning dataset over time. Curating anatomic pathology cases for machine learning algorithm development requires the recognition of emerging entities, with re-classification and re-grading as needed.

Resistance to carbapenems in Enterobacteriaceae is a clinical problem of growing significance. Difficulty in treating multidrug-resistant Gram-negative organisms with conventional antibiotics has led to a renewed and increasing use of polymyxin compounds, such as colistin. Here, we report the isolation of carbapenem- and colistin-resistant Enterobacter cloacae from a polymicrobial lower extremity wound in an ambulatory patient. Whole-genome sequencing demonstrated the presence of chromosomal bla IMI-1 and bla AmpC , as well as numerous efflux pump genes.

Whole-slide images (WSIs) are a rich new source of biomedical imaging data. The use of automated systems to classify and segment WSIs has recently come to forefront of the pathology research community. While digital slides have obvious educational and clinical uses, their most exciting potential lies in the application of quantitative computational tools to automate search tasks, assist in classic diagnostic classification tasks, and improve prognosis and theranostics. An essential step in enabling these advancements is to apply advances in machine learning and artificial intelligence from other fields to previously inaccessible pathology datasets, thereby enabling the application of new technologies to solve persistent diagnostic challenges in pathology. Here, we applied convolutional neural networks to differentiate between two forms of melanocytic lesions (Spitz and conventional). Classification accuracy at the patch level was 99.0%–2% when applied to WSI. Importantly, when the model was trained without careful image curation by a pathologist, the training took significantly longer and had lower overall performance. These results highlight the utility of augmented human intelligence in digital pathology applications, and the critical role pathologists will play in the evolution of computational pathology algorithms.

Objectives To examine the performance of a commercial off-the-shelf (COTS) monitor vs a medical-grade (MG) monitor for small object enumeration in standardized digital pathology images. Methods Pathologists reviewed 35 melanoma or 35 gastric biopsy images using the MG and COTS displays, with a 2-week washout period. Mitotic figure or Helicobacter pylori burden enumerations were compared with reference values reported by an expert subspecialist pathologist using a light microscope. Subjective evaluations of image color, brightness, and overall quality were also obtained. Results There was substantial agreement between the mitotic counts obtained by the evaluating pathologists between monitors and the reference mitotic figure or H pylori burden assessments. Six of the nine evaluating pathologists subjectively evaluated the monitors as substantially similar. Conclusions These findings are consistent with previous studies demonstrating that color calibration has limited impact on diagnostic accuracy and suggest that noncalibrated displays could be considered for fine assessment tasks.

A variety of arthropods, protozoa, and helminths infect the skin and subcutaneous tissues and may be identified by anatomic pathologists in standard cytology and histology preparations. The specific organisms seen vary greatly with the patient's exposure history, including travel to or residence in endemic countries. Arthropods are the most commonly encountered parasites in the skin and subcutaneous tissues and include Sarcoptes scabei, Demodex species, Tunga penetrans, and myiasis-causing fly larvae. Protozoal parasites such as Leishmania may also be common in some settings. Helminths are less often seen, and include round worms (eg, Dirofilaria spp.), tapeworms (eg, Taenia solium, Spirometra spp.), and flukes (eg, Schistosoma spp.). This review covers the epidemiologic and histopathologic features of common parasitic infections of the skin and subcutaneous tissues.

Cytapheresis (removal of cellular blood components) has been employed for treatment of infectious diseases since the 1960s. Techniques have included thrombocytapheresis (buffy coat apheresis) for loiasis, erythrocytapheresis for malaria and babesiosis, and leukocytapheresis for pertussis-associated lymphocytosis. Published data on these applications is largely limited to case level data and small observational studies; as such, recommendations for or against the use of cytapheresis in the treatment of infections have been extrapolated from these limited (and at times flawed) data sets. Consequently, utilization of cytapheresis in many instances is not uniform between institutions, and typically occurs at the discretion of treating medical teams. This review revisits the existing literature on the use of cytapheresis in the treatment of four infections (loasis, malaria, babesiosis, and pertussis) and examines the rationale underlying current treatment recommendations concerning its use.

The endosomal sorting complexes required for transport (ESCRTs) function in a variety of membrane remodeling processes including multivesicular body sorting, abscission during cytokinesis, budding of enveloped viruses, and repair of the plasma membrane. Vps4 ATPase activity modulates ESCRT function and is itself modulated by its cofactor Vta1 and its substrate ESCRT-III. The carboxyl-terminal Vta1/SBP-1/Lip5 (VSL) domain of Vta1 binds to the Vps4 β-domain to promote Vps4 oligomerization-dependent ATP hydrolysis. Additionally, the Vps4 stimulatory element (VSE) of Vta1 contributes to enhancing Vps4 oligomer ATP hydrolysis. The VSE is also required for Vta1-dependent stimulation of Vps4 by ESCRT-III subunits. However, the manner by which the Vta1 VSE contributes to Vps4 activation is unknown. Existing structural data were used to generate a model of the Vta1 VSE in complex with Vps4. This model implicated residues within the small ATPase associated with various activities (AAA) domain, specifically α-helices 7 and 9, as relevant contact sites. Rational generation of Vps4 mutants defective for VSE-mediated stimulation, as well as intergenic compensatory mutations, support the validity of this model. These findings have uncovered the Vps4 surface responsible for coordinating ESCRT-III-stimulated Vta1 input during ESCRT function and identified a novel mechanism of Vps4 stimulation.

Platelet transfusion-refractoriness is a challenging and expensive clinical scenario seen most often in patients with hematologic malignancies. Although the majority of platelet transfusion-refractory cases are due to nonimmune causes, a significant minority are caused by alloimmunization against Class I human leukocyte antigens (HLAs) or human platelet antigens (HPAs). Such platelet transfusion-refractory patients can be effectively managed with appropriate antigen-negative products.Our institution has developed a diagnostic and management algorithm for the platelet transfusion-refractory patient with an early focus on identifying those cases caused by immune-mediated factors. Using physical platelet cross-matches to initially classify platelet transfusion-refractory patients as immune-mediated or not, cross-match-compatible inventory is then provided to immune-mediated patients, whereas subsequent HLA (with or without HPA) testing is performed.Our blood donor program performs Class I HLA typing of all repeat platelet donors to facilitate the identification of antigen-negative platelet units (virtual cross-matching) as well as the recruitment of HLA-matched donors. The platelet transfusion-refractoriness algorithm realizes an initial net cost savings once two apheresis platelets are saved from use for each newly identified, immune-mediated platelet transfusion-refractory patient.An algorithm utilizing physical platelet cross-matches, Class I HLA and HPA antibody testing, and upfront Class I HLA typing of platelet donors leads to overall resource savings and improved clinical management for platelet transfusion-refractory patients.

Adrenal insufficiency when associated with giant cell arteritis (GCA) is typically the consequence of treatment with exogenous glucocorticoids. To our knowledge, this is the first reported case of ...

We describe a 16-year-old neutropenic patient from the Middle East with bloodstream infection caused by two carbapenemase-producing Escherichia coli isolates that we characterized by whole-genome sequencing. While one displayed meropenem resistance and was bla NDM positive, the other demonstrated meropenem susceptibility yet harbored bla OXA181 (which encodes a bla OXA48 -like enzyme). This report highlights the challenge of laboratory detection of bla OXA48 -like enzymes and the clinical implications of genotypic resistance detection in carbapenemase-producing Enterobacteriaceae .

Background Adjunctive stenting has increasingly become an acceptable option for the endovascular treatment of unruptured aneurysms. The Nationwide Inpatient Sample (NIS) was used to compare US in-hospital outcomes related to coiling with and without adjunctive stenting for unruptured aneurysms. Methods Hospitalizations for coiling of unruptured cerebral aneurysms from 2004 to 2008 were identified in the NIS by extracting ICD-9-CM codes for the diagnosis of unruptured aneurysm (437.3) and intracranial stenting (00.65) with coiling (39.52, 39.79 or 39.72) of cerebral aneurysms. All patients with a diagnosis of subarachnoid hemorrhage (430) and/or intracerebral hemorrhage (431) were excluded. Mortality and discharge to a long-term facility were compared between stent and non-stent patient groups using multivariate regression analysis. Results Patients treated with stent-assisted coiling had an in-hospital mortality rate of 0.08–0.8% compared with a death rate of 0.5% (95% CI 0.3% to 0.7%) for patients who did not receive a stent during coiling (p=0.36). Patients in the stent group had a 3% rate of discharge to a care facility (95% CI 1.5% to 5.8%) compared with 5% (95% CI 4.5% to 5.6%) for those in the non-stent group (p=0.14). Patients treated with a stent had a similar likelihood of in-hospital mortality (adjusted OR, 2.12 (95% CI 0.32 to 7.11), p=0.34) and a lower likelihood of discharge to a long-term care facility (adjusted OR 0.59 (95% CI 0.24 to 1.16), p=0.16) compared with the non-stent group. Conclusions Adjunctive stenting adds little in-hospital risk to the endovascular treatment of cerebral aneurysms. However, the need for dual antiplatelet therapy may predispose to delayed hemorrhagic complications and discontinuation of dual antiplatelet therapy may lead to delayed thromboembolic complications.

Ubiquitin modification of endosomal membrane proteins is a signal for active inclusion into the Multivesicular Body (MVB) pathway, resulting in lysosomal degradation. However, the endosome represents a dynamic site of protein sorting with a majority of proteins destined for recycling, rather than MVB targeting. Substrate recognition by ubiquitin ligases is therefore highly regulated. We have investigated substrate recognition by the Nedd4 ortholog Rsp5 as a model for understanding ligase-substrate interactions. Rsp5 interacts directly with its substrate Cps1 via a novel interaction mode. Perturbation of this mode of interaction revealed a compensatory role for the Rsp5 adaptor Bsd2. These results highlight the ability of Rsp5 to interact with substrates via multiple modalities, suggesting additional mechanisms of regulating this interaction and relevant outcomes.

Foodborne disease leads to about 325,000 hospitalizations and 5000 deaths each year in the United States. The irradiation of food could sharply reduce the incidence of foodborne disease, but currently it is rarely performed. This article examines the reasons for the slow growth in the use of food irradiation. Like pasteurization, irradiation is an effective strategy to improve food safety.

The endosomal sorting complexes required for transport (ESCRTs) impact multiple cellular processes including multivesicular body sorting, abscission, and viral budding. The AAA-ATPase Vps4 is required for ESCRT function, and its full activity is dependent upon the co-factor Vta1. The Vta1 carboxyl-terminal Vta1 SBP1 Lip5 (VSL) domain stimulates Vps4 function by facilitating oligomerization of Vps4 into its active state. Here we report the identification of the Vps4 stimulatory element (VSE) within Vta1 that is required for additional stimulation of Vps4 activity in vitro and in vivo. VSE activity is autoinhibited in a manner dependent upon the unstructured linker region joining the amino-terminal microtubule interacting and trafficking domains and the carboxyl-terminal VSL domain. The VSE is also required for Vta1-mediated Vps4 stimulation by ESCRT-III subunits Vps60 and Did2. These results suggest that ESCRT-III binding to the Vta1 microtubule interacting and trafficking domains relieves linker region autoinhibition of the VSE to produce maximal activation of Vps4 during ESCRT function.

Background The gold standard for malaria diagnosis is the examination of thick and thin blood films. Thick films contain 10 to 20 times more blood than thin films, correspondingly providing increased sensitivity for malaria screening. A potential complication of thick film preparations is sloughing of the blood droplet from the slide during staining or rinsing, resulting in the loss of sample. In this work, two methods for improving thick film slide adherence (‘scratch’ (SCM) and ‘acetone dip’ (ADM) methods) were compared to the ‘standard method’ (SM) of thick film preparation. Methods Standardized blood droplets from 26 previously examined EDTA whole blood specimens (22 positive and four negative) were concurrently spread on glass slides using the SM, ADM, and SCM. For the SM and ADM prepared slides, the droplet was gently spread to an approximate 22 millimeters in diameter spot on the slide using the edge of a second glass slide. For the SCM, the droplet was spread by carefully grinding (or scratching) it into the slide with the point of a second glass slide. Slides were dried for one hour in a laminar flow hood. For the ADM, slides were dipped once in an acetone filled Coplin jar and allowed to air dry. All slides were then Giemsa-stained and examined in a blinded manner. Adherence was assessed by blinded reviewers. Results No significant or severe defects were observed for slides prepared with the SCM. In contrast, 8 slides prepared by the ADM and 3 prepared using the SM displayed significant or severe defects. Thick films prepared by the three methods were microscopically indistinguishable and concordant results (positive or negative) were obtained for the three methods. Estimated parasitaemia of the blood samples ranged from 25 to 429,169 parasites/μL of blood. Conclusions The SCM is an inexpensive, rapid, and simple method that improves the adherence of thick blood films to standard glass slides without altering general slide preparation, microscopic appearance or interpretability. Using the SCM, thick films can be reliably examined less than two hours after sample receipt. This represents a significant diagnostic improvement over protocols requiring extended drying periods.