Maternal SARS-CoV-2 infection in pregnancy disrupts gene expression in Hofbauer cells with limited impact on cytotrophoblasts.

BACKGROUND: Hofbauer cells (HBCs) and cytotrophoblasts (CTBs) are major cell populations in placenta. The indirect impact of maternal SARS-CoV-2 disease on these cells that are not directly infected has not been extensively studied. Herein, we profiled gene expression in HBCs and CTBs isolated from placentae of recovered pregnant subjects infected with SARS-CoV-2 during all trimesters of pregnancy, placentae from subjects with active infection, SARS-CoV-2 vaccinated subjects, and those who were unexposed to the virus. METHODS: Placentae were collected within 4 h post-delivery and membrane-free tissues were enzymatically digested for the isolation of HBCs and CTBs. RNA extracted from HBCs and CTBs were sequenced using 150bp paired-end reads. Differentially expressed genes (DEGs) were identified by DESeq2 package in R and enriched in GO Biological Processes, KEGG Pathway, Reactome Gene Sets, Hallmark Gene Sets, and Canonical Pathways. Protein-protein interactions among the DEGs were modelled using STRING and BioGrid. RESULTS: Pregnant subjects (n = 30) were recruited and categorized into six groups: infected with SARS-CoV-2 in i) the first (1T, n = 4), ii) second (2T, n = 5), iii) third (3T, n = 5) trimester, iv) tested positive at delivery (Delivery, n = 5), v) never infected (Control, n = 6), and vi) fully mRNA-vaccinated by delivery (Vaccinated, n = 5). Compared to the Control group, gene expression analysis showed that HBCs from infected subjects had significantly altered gene expression profiles, with the 2T group having the highest number of DEGs (1,696), followed by 3T and 1T groups (1,656 and 958 DEGs, respectively). These DEGs were enriched for pathways involved in immune regulation for host defense, including production of cytokines, chemokines, antimicrobial proteins, ribosomal assembly, neutrophil degranulation inflammation, morphogenesis, and cell migration/adhesion. Protein-protein interaction analysis mapped these DEGs with oxidative phosphorylation, translation, extracellular matrix organization, and type I interferon signaling. Only 95, 23, and 8 DEGs were identified in CTBs of 1T, 2T, and 3T groups, respectively. Similarly, 11 and 3 DEGs were identified in CTBs and HBCs of vaccinated subjects, respectively. Reassuringly, mRNA vaccination did not induce an inflammatory response in placental cells. CONCLUSIONS: Our studies demonstrate a significant impact of indirect SARS-CoV-2 infection on gene expression of inner mesenchymal HBCs, with limited effect on lining CTB cells isolated from pregnant subjects infected and recovered from SARS-CoV-2. The pathways associated with these DEGs identify potential targets for therapeutic intervention.

16S rRNA Gene PCR/Sequencing of Heart Valves for Diagnosis of Infective Endocarditis in Routine Clinical Practice.

Sequencing is increasingly used for infective endocarditis (IE) diagnosis. Here, the performance of 16S rRNA gene PCR/sequencing of heart valves utilized in routine clinical practice was compared with conventional IE diagnostics. Subjects whose heart valves were sent to the clinical microbiology laboratory for 16S rRNA gene PCR/sequencing from August 2020 through February 2022 were studied. A PCR assay targeting V1 to V3 regions of the 16S rRNA gene was performed, followed by Sanger and/or next-generation sequencing (NGS) (using an Illumina MiSeq), or reported as negative, depending on an algorithm that included the PCR cycle threshold value. Fifty-four subjects, including 40 with IE, three with cured IE, and 11 with noninfective valvular disease, were studied. Thirty-one positive results, 11 from NGS and 20 from Sanger sequencing, were generated from analysis of 16S rRNA gene sequence(s). Positivity rates of blood cultures and 16S rRNA gene PCR/sequencing of valves were 55% and 75%, respectively (P = 0.06). In those with prior antibiotic exposure, positivity rates of blood cultures and 16S rRNA gene PCR/sequencing of valves were 11% and 76%, respectively (P < 0.001). Overall, 61% of blood culture-negative IE subjects had positive valve 16S rRNA gene PCR/sequencing results. 16S rRNA gene-based PCR/sequencing of heart valves is a useful diagnostic tool for pathogen identification in patients with blood culture-negative IE undergoing valve surgery in routine clinical practice.

Fetal surgery is not associated with increased inflammatory placental pathology.

OBJECTIVE: Fetal surgery has improved neonatal outcomes; however, it is unknown if the intervention contributes to the developmental of inflammatory pathologies in the placenta. Here, an association between fetal surgery and placental pathology was examined. METHOD: This case-control study compared pregnancies with fetal surgery (n = 22), pregnancies with an indication for fetal surgery but without an intervention being done (n = 13), and gestational-age and fetus-number matched controls (n = 36). Data on maternal, infant, and placental outcomes were abstracted. Additionally, immunohistochemistry identified expression of lymphoid and myeloid cells in the placenta on a subset of cases. Comparisons were performed using Kruskal-Wallis or Pearson's chi-squared tests. RESULTS: Maternal characteristics were comparable between groups. Most fetal interventions were for diaphragmatic hernia, spina bifida, or twin-to-twin transfusion syndrome. Fetuses who were operated on before birth were more likely to be born preterm (p = 0.02). There was no increase in the rate of observed placental pathologies or immune cell infiltration in fetal surgery cases compared to controls. CONCLUSION: The data suggest that fetal surgery is not associated with increased inflammatory or morphologic pathology in the placenta. This observation supports the growing field of fetal surgery.

Maternal Factors and Placental Pathologies Associated with a Diagnosis of Chronic Villitis.

OBJECTIVE: To evaluate maternal risk factors associated with chronic villitis of unknown etiology (VUE) and to describe cooccurring placental pathologies. STUDY DESIGN: A retrospective case-control study was conducted using placental pathology records from deliveries ≥ 20 weeks between 2010 and 2018. Cases were placentas with documented chronic villitis without infectious cause, hereafter called VUE. Controls were placentas without this diagnosis, matched to the cases 2:1. Maternal and neonatal demographic and clinical data were collected. Descriptive statistics are reported with Fisher's exact test or a chi-squared test, as appropriate, and multivariable conditional logistic regression was conducted. RESULTS: Our study included 352 cases with VUE and 657 controls. A diagnosis of gestational diabetes (p = 0.03) and gestational hypertension (p = 0.06) was 1.5 times more likely to occur in those with a VUE diagnosis. A trend was also seen for chronic hypertension (odds ratio [OR] = 1.7, p = 0.07) and preeclampsia (OR = 1.5, p = 0.09) compared with controls. Placentas with VUE, specifically high-grade VUE, were more likely to be small for gestational age (p = 0.01), and to be diagnosed with other placental findings including lymphoplasmacytic or chronic deciduitis (p < 0.01), maternal (p < 0.01) and fetal vascular malperfusion (p = 0.02), and chorionitis (acute or chronic; p < 0.01). CONCLUSION: Gestational diabetes and hypertension were associated with a diagnosis of VUE, and overall, VUE placentas have more abnormal placental findings compared with control. Understanding VUE risk factors may facilitate prenatal care strategies and counseling to achieve the best outcomes for pregnant patients and their neonates. KEY POINTS: · VUE is a common inflammatory lesion of the placenta.. · Gestational diabetes and hypertension are associated with a VUE diagnosis.. · Findings of other placental pathologies increase in VUE..

In from the cold: M-protein light chain glycosylation is positively associated with cold agglutinin titer levels.

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.

Error simulation modeling to assess the effects of bias and precision on bilirubin measurements used to screen for neonatal hyperbilirubinemia.

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 <75th percentile TBS). 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 <75th percentile bilirubin values was observed when positive systematic bias was 17.1 µmol/L (1 mg/dL) or less at CV ≤ 10%. Critical errors did not occur with a frequency >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.

Retrospective Review of Clinical Utility of Shotgun Metagenomic Sequencing Testing of Cerebrospinal Fluid from a U.S. Tertiary Care Medical Center.

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.

Development and performance characteristics of Platelet Virtual Crossmatch (PLT VXM), a software application for the evaluation and management of platelet transfusion-refractory patients.

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.

Parasitic Infections of the Skin and Subcutaneous Tissues.

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.

The use of cytapheresis in the treatment of infectious diseases.

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.

How do I manage the platelet transfusion-refractory patient?

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.

Carbapenem- and Colistin-Resistant Enterobacter cloacae from Delta, Colorado, in 2015.

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 blaIMI-1 and blaAmpC, as well as numerous efflux pump genes.

An Immunocompromised Child with Bloodstream Infection Caused by Two Escherichia coli Strains, One Harboring NDM-5 and the Other Harboring OXA-48-Like Carbapenemase.

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 blaNDM positive, the other demonstrated meropenem susceptibility yet harbored blaOXA181 (which encodes a blaOXA48-like enzyme). This report highlights the challenge of laboratory detection of blaOXA48-like enzymes and the clinical implications of genotypic resistance detection in carbapenemase-producing Enterobacteriaceae.

Vps4 stimulatory element of the cofactor Vta1 contacts the ATPase Vps4 α7 and α9 to stimulate ATP hydrolysis.

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 ß-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.

Relief of autoinhibition enhances Vta1 activation of Vps4 via the Vps4 stimulatory element.

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.

Performance of externally validated machine learning models based on histopathology images for the diagnosis, classification, prognosis, or treatment outcome prediction in female breast cancer: A systematic review.

Numerous machine learning (ML) models have been developed for breast cancer using various types of data. Successful external validation (EV) of ML models is important evidence of their generalizability. The aim of this systematic review was to assess the performance of externally validated ML models based on histopathology images for diagnosis, classification, prognosis, or treatment outcome prediction in female breast cancer. A systematic search of MEDLINE, EMBASE, CINAHL, IEEE, MICCAI, and SPIE conferences was performed for studies published between January 2010 and February 2022. The Prediction Model Risk of Bias Assessment Tool (PROBAST) was employed, and the results were narratively described. Of the 2011 non-duplicated citations, 8 journal articles and 2 conference proceedings met inclusion criteria. Three studies externally validated ML models for diagnosis, 4 for classification, 2 for prognosis, and 1 for both classification and prognosis. Most studies used Convolutional Neural Networks and one used logistic regression algorithms. For diagnostic/classification models, the most common performance metrics reported in the EV were accuracy and area under the curve, which were greater than 87% and 90%, respectively, using pathologists' annotations/diagnoses as ground truth. The hazard ratios in the EV of prognostic ML models were between 1.7 (95% CI, 1.2-2.6) and 1.8 (95% CI, 1.3-2.7) to predict distant disease-free survival; 1.91 (95% CI, 1.11-3.29) for recurrence, and between 0.09 (95% CI, 0.01-0.70) and 0.65 (95% CI, 0.43-0.98) for overall survival, using clinical data as ground truth. Despite EV being an important step before the clinical application of a ML model, it hasn't been performed routinely. The large variability in the training/validation datasets, methods, performance metrics, and reported information limited the comparison of the models and the analysis of their results. Increasing the availability of validation datasets and implementing standardized methods and reporting protocols may facilitate future analyses.

Spatial proteomics reveals phenotypic and functional differences in T cell and macrophage subsets during villitis of unknown etiology.

Villitis of unknown etiology (VUE) is a prevalent inflammatory pathology of the placenta characterized by infiltration of maternal T cells and accumulation of fetal macrophages into chorionic villi. VUE is associated with a variety of adverse clinical outcomes, including fetal growth restriction and fetal demise. Evaluation of the phenotypic and functional differences between two immune cell types associated with this pathology, namely T cells and macrophages, was completed to gain a deeper understanding of the immuno-pathogenesis of VUE. GeoMx Digital Spatial Profiling was performed on placental tissue from 4 high grade VUE cases and 4 controls with no underlying pathology. Placental tissues were fluorescently labeled with CD3 and CD68 antibodies and oligo-conjugated antibodies against 48 protein targets. Overall, T cells in VUE exhibited upregulated markers of activation, memory, and antigen experience compared to controls and were altered based on placental location (villi vs. decidua). Additionally, villous macrophages in VUE upregulated costimulatory and major histocompatibility complex class I and II molecules compared to controls and macrophage subtypes in the decidua. Data herein provides new mechanistic insights into T cell and macrophage biology in VUE which contribute to this abnormal immune response to pregnancy.