Biennial Upsurge and Molecular Epidemiology of Enterovirus D68 Infection in New York, USA, 2014 to 2018.

Enterovirus D68 (EV-D68) infection has been associated with outbreaks of severe respiratory illness and increased cases of nonpolio acute flaccid myelitis. The patterns of EV-D68 circulation and molecular epidemiology are not fully understood. In this study, nasopharyngeal (NP) specimens collected from patients in the Lower Hudson Valley, New York, from 2014 to 2018 were examined for rhinovirus/enterovirus (RhV/EV) by the FilmArray respiratory panel. Selected RhV/EV-positive NP specimens were analyzed using two EV-D68-specific real-time RT-PCR assays, Sanger sequencing and metatranscriptomic next-generation sequencing. A total of 2,398 NP specimens were examined. EV-D68 was detected in 348 patients with NP specimens collected in 2014 (n = 94), 2015 (n = 0), 2016 (n = 160), 2017 (n = 5), and 2018 (n = 89), demonstrating a biennial upsurge of EV-D68 infection in the study area. Ninety-one complete or nearly complete EV-D68 genome sequences were obtained. Genomic analysis of these EV-D68 strains revealed dynamics and evolution of circulating EV-D68 strains since 2014. The dominant EV-D68 strains causing the 2014 outbreak belonged to subclade B1, with a few belonging to subclade B2. New EV-D68 subclade B3 strains emerged in 2016 and continued in circulation in 2018. Clade D strains that are rarely detected in the United States also arose and spread in 2018. The establishment of distinct viral strains and their variable circulation patterns provide essential information for future surveillance, diagnosis, vaccine development, and prediction of EV-D68-associated disease prevalence and potential outbreaks.

Integrated Genome-Wide Analysis of an Isogenic Pair of Pseudomonas aeruginosa Clinical Isolates with Differential Antimicrobial Resistance to Ceftolozane/Tazobactam, Ceftazidime/Avibactam, and Piperacillin/Tazobactam.

Multidrug-resistant (MDR) Pseudomonas aeruginosa is one of the main causes of morbidity and mortality in hospitalized patients and the leading cause of nosocomial infections. We investigated, here, two MDR P. aeruginosa clinical isolates from a hospitalized patient with differential antimicrobial resistance to ceftazidime/avibactam (CZA), ceftolozane/tazobactam (C/T), and piperacillin/tazobactam (P/T). Their assembled complete genomes revealed they belonged to ST235, a widespread MDR clone; and were isogenic with only a single nucleotide variant, causing G183D mutation in AmpC ß-lactamase, responsible for a phenotypic change from susceptible to resistant to CZA and C/T. Further epigenomic profiling uncovered two conserved DNA methylation motifs targeted by two distinct putative methyltransferase-containing restriction-modification systems, respectively; more intriguingly, there was a significant difference between the paired isolates in the pattern of genomic DNA methylation and modifications. Moreover, genome-wide gene expression profiling demonstrated the inheritable genomic methylation and modification induced 14 genes being differentially regulated, of which only toxR (downregulated), a regulatory transcription factor, had its promoter region differentially methylate and modified. Since highly expressed opdQ encodes an OprD porin family protein, therefore, we proposed an epigenetic regulation of opdQ expression pertinent to the phenotypic change of P. aeruginosa from resistant to susceptible to P/T. The disclosed epigenetic mechanism controlling phenotypic antimicrobial resistance deserves further experimental investigation.

Emergence and Evolution of Multidrug-Resistant Klebsiella pneumoniae with both blaKPC and blaCTX-M Integrated in the Chromosome.

The extended-spectrum-ß-lactamase (ESBL)- and Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae represent serious and urgent threats to public health. In a retrospective study of multidrug-resistant K. pneumoniae, we identified three clinical isolates, CN1, CR14, and NY9, carrying both blaCTX-M and blaKPC genes. The complete genomes of these three K. pneumoniae isolates were de novo assembled by using both short- and long-read whole-genome sequencing. In CR14 and NY9, blaCTX-M and blaKPC were carried on two different plasmids. In contrast, CN1 had one copy of blaKPC-2 and three copies of blaCTX-M-15 integrated in the chromosome, for which the blaCTX-M-15 genes were linked to an insertion sequence, ISEcp1, whereas the blaKPC-2 gene was in the context of a Tn4401a transposition unit conjugated with a PsP3-like prophage. Intriguingly, downstream of the Tn4401a-blaKPC-2-prophage genomic island, CN1 also carried a clustered regularly interspaced short palindromic repeat (CRISPR)-cas array with four spacers targeting a variety of K. pneumoniae plasmids harboring antimicrobial resistance genes. Comparative genomic analysis revealed that there were two subtypes of type I-E CRISPR-cas in K. pneumoniae strains and suggested that the evolving CRISPR-cas, with its acquired novel spacer, induced the mobilization of antimicrobial resistance genes from plasmids into the chromosome. The integration and dissemination of multiple copies of blaCTX-M and blaKPC from plasmids to chromosome depicts the complex pandemic scenario of multidrug-resistant K. pneumoniae Additionally, the implications from this study also raise concerns for the application of a CRISPR-cas strategy against antimicrobial resistance.

Single-cell RNA sequencing reveals an altered gene expression pattern as a result of CRISPR/cas9-mediated deletion of Gene 33/Mig6 and chronic exposure to hexavalent chromium in human lung epithelial cells.

Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently reported that depletion of this protein promotes lung epithelial cell transformation induced by hexavalent chromium [Cr(VI)]. However, the early molecular events that mediate this process are not clear. In the present study, we used single-cell RNA sequencing to compare gene expression profiles between BEAS-2B lung epithelial cells chronically exposed to a sublethal dose of Cr(VI) with or without CRISPR/cas9-mediated deletion of Gene 33. Our data reveal 83 differentially expressed genes. The most notable changes are genes associated with cell adhesion, oxidative stresses, protein ubiquitination, epithelial-mesenchymal transition/metastasis, and WNT signaling. Up-regulation of some neuro-specific genes is also evident, particularly ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a deubiquitinase and potential biomarker for lung cancer. Gene 33 deletion and/or Cr(VI) exposure did not cause discernable changes in cell morphology. However, Gene 33 deletion led to a modest but significant reduction of cells in the G2/M phase of the cell cycle regardless of Cr(VI) exposure. Gene 33 deletion also significantly reduced cell proliferation. Interestingly, Cr(VI) exposure eliminated the difference in cell proliferation between the two genotypes. Gene 33 deletion also significantly elevated cell migration. Our data indicate that combined Gene 33 deletion and chronic Cr(VI) exposure produces a gene expression pattern and a phenotype resemble those of the transformed lung epithelial cells. Given the known association of UCHL1 with lung cancer, we propose that UCHL1 is an important player in the early stage of lung epithelial cell transformation and tumorigenesis.

Recurrent TERT promoter mutations in urothelial carcinoma and potential clinical applications.

Increased telomerase activity is associated with almost all types of advanced human cancers with unknown molecular mechanism(s). Two recurrent point mutations in the promoter region of telomerase reverse transcriptase (TERT)--the key subunit of telomerase--have recently been identified in melanoma as well as a small sample of bladder cancer cell lines. However, the incidence and clinical-pathological significance of these mutations in urothelial carcinoma have not been well established yet. We collected 86 specimens of urothelial carcinoma including upper and lower urinary tract: high grade and low grade, invasive and noninvasive, and primary and metastatic. We also included some matched benign urothelium and common benign bladder lesions: cystitis, nephrogenic adenoma, and inverted papilloma. In addition, we collected urine samples for urothelial carcinoma workup; blood samples from patients underwent cystectomy with extensive lymphovascular invasion. All specimens were subject to polymerase chain reaction amplification and bidirectional Sanger sequencing for the TERT promoter mutations: C228T and C250T. We found that 64 (74%) of 86 carcinoma samples harbored 1 of the 2 TERT promoter mutations (C228T, n = 54; C250T, n = 10); the incidences were roughly equal regardless of site of origin, histologic grade, and invasive status. All matched benign and benign lesion samples showed wild-type sequence. These TERT promoter mutations are the most common genetic alterations in urothelial carcinoma and are not associated with tumor locations, grade, or invasiveness. Importantly, the feasibility of detecting these mutations in urine samples may provide a novel method to detect urothelial carcinoma in urine.

Identification of a novel PARP14-TFE3 gene fusion from 10-year-old FFPE tissue by RNA-seq.

Xp11 (TFE3) translocation renal cell carcinoma (RCC) is officially recognized as a distinct subtype of RCC in the 2004 WHO classification. This neoplasm is characterized by several chromosomal translocations between the TFE3-involving Xp11.2 breakpoint and various fusion partners. To date, five partner genes have been identified, that is, PRCC in 1q21, PSF in 1q34, ASPL in 17q25, CLTC in 17q23, and NONO in Xq12; and three additional translocations have been reported with no partner gene being defined: t(X;3)(p11;q23), t(X;10)(p11;q23), and t(X;19)(p11;q13). Here, we report the identification of a novel TFE3 fusion partner, PARP14 in chromosome band3q21. We used RNA-seq on a 10-year-old FFPE (formalin-fixed, paraffin-embedded) tissue sample, which carried t(X;3)(p11;q23) as detected in the original cytogenetic study. The fusion transcript connected the 5'-end of the first two exons of PARP14 to the 3'-end of five exons of TFE3, which was verified by reverse transcription PCR (RT-PCR) and Sanger sequencing. Similar to other TFE3 fusions previously reported, the predicted PARP14-TFE3 product retains the nuclear localization and DNA-binding domains of TFE3. This finding expands the list of TFE3 translocation partner genes and re-emphasizes the essential oncogenic role of TFE3 fusion proteins in this tumor. Our result also clearly demonstrated the feasibility of identifying chromosomal translocation by RNA-seq in clinical FFPE, which are easily accessible and associated with valuable clinical information. © 2015 Wiley Periodicals, Inc.

Evaluation of a Real-Time Reverse Transcription-PCR Assay for Detection of Enterovirus D68 in Clinical Samples from an Outbreak in New York State in 2014.

An outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68) infection was reported in mid-August 2014 in the United States. In this study, we evaluated the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (rRT-PCR) that was recently developed by the Centers for Disease Control and Prevention in clinical samples. Nasopharyngeal (NP) swab specimens from patients in a recent outbreak of respiratory illness in the lower Hudson Valley, New York State, were collected and examined for the presence of human rhinovirus or enterovirus using the FilmArray Respiratory Panel (RP) assay. Samples positive by RP were assessed using EV-D68 rRT-PCR, and the data were compared to results from sequencing analysis of partial VP1 and 5' untranslated region (5'-UTR) sequences of the EV genome. A total of 285 RP-positive NP specimens (260 from the 2014 outbreak and 25 from 2013) were analyzed by rRT-PCR; EV-D68 was detected in 74 of 285 (26.0%) specimens examined. Data for comparisons between rRT-PCR and sequencing analysis were obtained from 194 NP specimens. EV-D68 detection was confirmed by sequencing analysis in 71 of 74 positive and in 1 of 120 randomly selected negative specimens by rRT-PCR. The EV-D68 rRT-PCR showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. Our data suggest that the EV-D68 rRT-PCR is a reliable assay for detection of EV-D68 in clinical samples and has a potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories.

Telomerase reverse transcriptase promoter mutations in glandular lesions of the urinary bladder.

Glandular lesions of the urinary bladder include a broad spectrum of entities ranging from completely benign to primary and secondary malignancies. The accurate diagnosis of these lesions is both important and challenging. Recently, studies suggest that telomerase reverse transcriptase (TERT) promoter mutations could be a biomarker for urothelial carcinoma (UC). We hypothesized that these mutations can distinguish UC with glandular differentiation from nephrogenic adenoma, primary adenocarcinoma of the urinary bladder (PAUB), or secondary malignancies. Twenty-five cases of benign glandular lesions (including nephrogenic adenoma); 29 cases of UC with glandular differentiation; 10 cases of PAUB; and 10 cases each of metastatic colon cancer, prostatic carcinoma, and carcinoma from Mullerian origin were collected. Slides were reviewed and selected to make sure the lesion was at least 10% to 20% of all tissue. Macrodissection was performed in some of cases, and genomic DNA was extracted from the tissue. Telomerase reverse transcriptase promoter mutations were determined by standard polymerase chain reaction sequencing. Twenty-one cases (72%) of UC with glandular differentiation were positive for TERT promoter mutations. However, none of the remaining cases (total 65 cases of benign lesions, PAUB, and metastatic carcinomas) was positive for TERT promoter mutation. Telomerase reverse transcriptase promoter mutations were highly associated with UC including UC with glandular differentiation but not other glandular lesions of bladder. Therefore, in conjunction with morphologic features, Immunohistochemistry stain profile, and clinical information, TERT promoter mutations could distinguish UC with glandular differentiation from other bladder glandular lesions. In addition, lack of TERT promoter mutations in primary adenocarcinoma of bladder suggests that this entity may have different origin or carcinogenesis from those of UC.

Heterogeneity of TERT promoter mutations status in squamous cell carcinomas of different anatomical sites.

Squamous cell carcinoma (SCC) can arise from different anatomical sites including the skin, head and neck, lung, esophagus, genital area, and so on. Despite the same histopathologic features and immunohistochemistry profile, the SCCs of different body sites can show tremendous differences in their presenting symptoms, risk factor associations, natural history, prognosis, and response to treatment. This may reflect the fact that SCCs are heterogenous and likely have unique molecular characteristics at different anatomical sites. Recurrent somatic mutations in the TERT promoter region were first reported in human melanomas. Subsequently, other tumors including cutaneous SCC were found to demonstrate high frequencies of the same mutations. However, the incidences of TERT promoter mutation in noncutaneous SCCs have not been systemically studied. We investigated the TERT promoter mutation status among SCCs from different sites. We collected 84 cases of SCC from the skin (27), head and neck (12), lung (25), and cervix (10), as well as 10 cases of urothelial carcinoma with squamous differentiation (UC-SqD). We found that the frequencies of TERT promoter mutation among SCC of different sits are quite heterogenous: ~70% in skin SCC and UC-SqD, 16.67% in head and neck SCC, and 0% in lung and cervix SCC. These results may support the hypothesis of different carcinogenesis mechanisms of SCC in different sites. It also indicates that TERT promoter mutation could be a biomarker for distinguishing skin SCC or UC-SqD vs pulmonary SCC.

Identification of a novel clone, ST736, among Enterococcus faecium clinical isolates and its association with daptomycin nonsusceptibility.

Resistance to daptomycin in enterococcal clinical isolates remains rare but is being increasingly reported in the United States and worldwide. There are limited data on the genetic relatedness and microbiological and clinical characteristics of daptomycin-nonsusceptible enterococcal clinical isolates. In this study, we assessed the population genetics of daptomycin-nonsusceptible Enterococcus faecium (DNSE) clinical isolates by multilocus sequence typing (MLST) and whole-genome sequencing analysis. Forty-two nonduplicate DNSE isolates and 43 randomly selected daptomycin-susceptible E. faecium isolates were included in the analysis. All E. faecium isolates were recovered from patients at a tertiary care medical center in suburban New York City from May 2009 through December 2013. The daptomycin MICs of the DNSE isolates ranged from 6 to >256 µg/ml. Three major clones of E. faecium (ST18, ST412, and ST736) were identified among these clinical isolates by MLST and whole-genome sequence-based analysis. A newly recognized clone, ST736, was seen in 32 of 42 (76.2%) DNSE isolates and in only 14 of 43 (32.6%) daptomycin-susceptible E. faecium isolates (P < 0.0001). This report provides evidence of the association between E. faecium clone ST736 and daptomycin nonsusceptibility. The identification and potential spread of this novel E. faecium clone and its association with daptomycin nonsusceptibility constitute a challenge for patient management and infection control at our medical center.

Clostridioides difficile Infection: Diagnosis and Treatment Challenges.

Clostridioides difficile is the most important cause of healthcare-associated diarrhea in the United States. The high incidence and recurrence rates of C. difficile infection (CDI), associated with high morbidity and mortality, pose a public health challenge. Although antibiotics targeting C. difficile bacteria are the first treatment choice, antibiotics also disrupt the indigenous gut flora and, therefore, create an environment that is favorable for recurrent CDI. The challenge of treating CDI is further exacerbated by the rise of antibiotic-resistant strains of C. difficile, placing it among the top five most urgent antibiotic resistance threats in the USA. The evolution of antibiotic resistance in C. difficile involves the acquisition of new resistance mechanisms, which can be shared among various bacterial species and different C. difficile strains within clinical and community settings. This review provides a summary of commonly used diagnostic tests and antibiotic treatment strategies for CDI. In addition, it discusses antibiotic treatment and its resistance mechanisms. This review aims to enhance our current understanding and pinpoint knowledge gaps in antimicrobial resistance mechanisms in C. difficile, with an emphasis on CDI therapies.

Advances in Development of Novel Therapeutic Strategies against Multi-Drug Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) with multi-drug resistance (MDR) is a major cause of serious healthcare-associated infections, leading to high morbidity and mortality. This opportunistic pathogen is responsible for various infectious diseases, such as those seen in cystic fibrosis, ventilator-associated pneumonia, urinary tract infection, otitis externa, and burn and wound injuries. Due to its relatively large genome, P. aeruginosa has great diversity and can use various molecular mechanisms for antimicrobial resistance. For example, outer membrane permeability can contribute to antimicrobial resistance and is determined by lipopolysaccharide (LPS) and porin proteins. Recent findings on the regulatory interaction between peptidoglycan and LPS synthesis provide additional clues against pathogenic P. aeruginosa. This review focuses on recent advances in antimicrobial agents and inhibitors targeting LPS and porin proteins. In addition, we explore current and emerging treatment strategies for MDR P. aeruginosa, including phages, vaccines, nanoparticles, and their combinatorial therapies. Novel strategies and their corresponding therapeutic agents are urgently needed for combating MDR pathogens.

Differential Deep RNA Sequencing for Diagnostic Detection of Microbial Infections in Inflammatory Cardiomyopathy.

BACKGROUND: Inflammatory heart disease can be triggered by a variety of causes, both infectious and noninfectious in nature. We hypothesized that inflammatory cardiomyopathy is potentially related to microbial infection. METHODS: In this retrospective study, we used deep RNA sequencing on formalin-fixed paraffin-embedded heart tissue specimens to detect pathogenic agents. We first investigated 4 single-sample cases to test the feasibility of this diagnostic protocol and further 3 control-sample paired cases to improve the protocol with differential metatranscriptomics next-generation sequencing (mtNGS) analysis. RESULTS: We demonstrate that differential mtNGS allows identification of various microbials as potentially pathogenic, for example, Cutibacterium acnes, Corynebacterium aurimucosum, and Pseudomonas denitrificans, which are usually commensal in healthy individuals. Differential mtNGS also allows characterization of human host response in each individual by profiling alterations of gene expression, networked pathways, and inferred immune cell compositions, information of which is beneficial for us to understand different etiologies and immunity roles in each case. Additionally, differential mtNGS allows the identification of genetic variants in patients that may contribute to their susceptibility to particular microbial infections. CONCLUSIONS: The demonstrated power of differential mtNGS in simultaneous capture of both the infectious microbial(s) and the status of human host immune response could help us better understand the pathogenesis of complex inflammatory cardiomyopathy, if conducted on a larger scale of the population. The developed differential mtNGS method could also shed light on its translation and adoption of such a laboratory test in clinic practice, allowing for a more effective diagnosis to guide therapeutic treatment of the disease.

Wastewater based epidemiology as a surveillance tool during the current COVID-19 pandemic on a college campus (East Carolina University) and its accuracy in predicting SARS-CoV-2 outbreaks in dormitories.

The COVID-19 outbreak led governmental officials to close many businesses and schools, including colleges and universities. Thus, the ability to resume normal campus operation required adoption of safety measures to monitor and respond to COVID-19. The objective of this study was to determine the efficacy of wastewater-based epidemiology as a surveillance method in monitoring COVID-19 on a college campus. The use of wastewater monitoring as part of a surveillance program to control COVID-19 outbreaks at East Carolina University was evaluated. During the Spring and Fall 2021 semesters, wastewater samples (N = 830) were collected every Monday, Wednesday, and Friday from the sewer pipes exiting the dormitories on campus. Samples were analyzed for SARS-CoV-2 and viral quantification was determined using qRT-PCR. During the Spring 2021 semester, there was a significant difference in SARS-CoV-2 virus copies in wastewater when comparing dorms with the highest number student cases of COVID-19 and those with the lowest number of student cases, (p = 0.002). Additionally, during the Fall 2021 semester it was observed that when weekly virus concentrations exceeded 20 copies per ml, there were new confirmed COVID-19 cases 85% of the time during the following week. Increases in wastewater viral concentration spurred COVID-19 swab testing for students residing in dormitories, aiding university officials in effectively applying COVID testing policies. This study showed wastewater-based epidemiology can be a cost-effective surveillance tool to guide other surveilling methods (e.g., contact tracing, nasal/salvia testing, etc.) to identify and isolate afflicted individuals to reduce the spread of pathogens and potential outbreaks within a community.

Recombinational exchange of M-fibril and T-pilus genes generates extensive cell surface diversity in the global group A Streptococcus population.

Among genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as "pilin types." Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools (pubmlst.org) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies.IMPORTANCEPrecision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.

Host Immune Responses to Clostridioides difficile Infection and Potential Novel Therapeutic Approaches.

Clostridioides difficile infection (CDI) is a leading nosocomial infection, posing a substantial public health challenge within the United States and globally. CDI typically occurs in hospitalized elderly patients who have been administered antibiotics; however, there has been a rise in the occurrence of CDI in the community among young adults who have not been exposed to antibiotics. C. difficile releases toxins, which damage large intestinal epithelium, leading to toxic megacolon, sepsis, and even death. Unfortunately, existing antibiotic therapies do not always prevent these consequences, with up to one-third of treated patients experiencing a recurrence of the infection. Host factors play a crucial role in the pathogenesis of CDI, and accumulating evidence shows that modulation of host immune responses may potentially alter the disease outcome. In this review, we provide an overview of our current knowledge regarding the role of innate and adaptive immune responses on CDI outcomes. Moreover, we present a summary of non-antibiotic microbiome-based therapies that can effectively influence host immune responses, along with immunization strategies that are intended to tackle both the treatment and prevention of CDI.

Commentary on why implementing and standardizing histologic diagnosis of myocarditis is crucial for the clinical setting and patient care.

Histological examination of endomyocardial biopsy or myocardium at autopsy is key to the diagnosis of myocarditis. Among pathologists there is currently extensive variability in routine practice and criteria used to define, diagnose, and report myocarditis as well as to achieve consensus on cases. Two manuscripts emphasizing the need to standardize and implement histopathological diagnostic criteria for myocarditis are reviewed.

Dynamic Evolution of SARS-CoV-2 in a Patient on Chemotherapy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved significantly during the pandemic and resulted in daunting numbers of genomic sequences. Tracking SARS-CoV-2 evolution during persistent cases could provide insight into the origins and dynamics of new variants. We report here a case of B-cell acute lymphocytic leukemia on chemotherapy with infection of SARS-CoV-2 for more than two months. Genomic surveillance of his serial SARS-CoV-2-positive specimens revealed two unprecedented large deletions, Δ15-26 and Δ138-145, in the viral spike protein N-terminal domain (NTD) and demonstrated their dynamic shifts in generating these new variants. Located at antigenic supersites, these large deletions are anticipated to dramatically change the spike protein NTD in three-dimensional protein structure prediction, which may lead to immune escape but reduce their viral transmissibility. In summary, we present here a new viral evolutionary trajectory in a patient on chemotherapy.

Myocarditis and endomyocardial biopsy: achieving consensus diagnosis on 100 cases.

The two histopathology benchmarks used to diagnose myocarditis are the Dallas Criteria, developed in 1984 and the European Society of Cardiology criteria, developed in 2013, which added immunohistochemistry for the detection of CD3+ T cells (lymphocytes) and CD68+ macrophages. Despite their near universal acceptance, the extent to which pathologists use these criteria or their own criteria to consistently render the diagnosis of myocarditis on endomyocardial biopsy (EMB) is unknown. We digitally scanned slides from 100 heart biopsies, including a trichrome stain and immunostaining, that were chosen as representative of myocarditis, non-myocarditis, and borderline myocarditis, as diagnosed per one institution's use of the Dallas Criteria. Eight blinded international cardiovascular experts were asked to render diagnoses and offer a confidence score on each case. No clinical histories were shared. There was full initial agreement across all experts on 37 cases (16 myocarditis and 21 non-myocarditis) and moderate consensus on 35 cases. After individual inquiries and group discussion, consensus was reached on 90 cases. Diagnostic confidence was highest among the myocarditis diagnoses, lowest for borderline cases, and significantly different between the three diagnostic categories (myocarditis, borderline myocarditis, non-myocarditis; P-value=8.49 × 10-57; ANOVA). Diagnosing myocarditis, particularly in cases with limited inflammation and injury, remains a challenge even for experts in the field. Intermediate cases, termed "borderline" in the Dallas Criteria, represent those for which consensus is particularly hard to achieve. To increase consistency for the histopathologic diagnosis of myocarditis, we will need more specifically defined criteria, more granular descriptions of positive and negative features, clarity on how to incorporate immunohistochemistry findings, and improved nomenclature.

Sudden cardiac death in the young: A consensus statement on recommended practices for cardiac examination by pathologists from the Society for Cardiovascular Pathology.

Sudden cardiac death is, by definition, an unexpected, untimely death caused by a cardiac condition in a person with known or unknown heart disease. This major international public health problem accounts for approximately 15-20% of all deaths. Typically more common in older adults with acquired heart disease, SCD also can occur in the young where the cause is more likely to be a genetically transmitted process. As these inherited disease processes can affect multiple family members, it is critical that these deaths are appropriately and thoroughly investigated. Across the United States, SCD cases in those less than 40 years of age will often fall under medical examiner/coroner jurisdiction resulting in scene investigation, review of available medical records and a complete autopsy including toxicological and histological studies. To date, there have not been consistent or uniform guidelines for cardiac examination in these cases. In addition, many medical examiner/coroner offices are understaffed and/or underfunded, both of which may hamper specialized examinations or studies (e.g., molecular testing). Use of such guidelines by pathologists in cases of SCD in decedents aged 1-39 years of age could result in life-saving medical intervention for other family members. These recommendations also may provide support for underfunded offices to argue for the significance of this specialized testing. As cardiac examinations in the setting of SCD in the young fall under ME/C jurisdiction, this consensus paper has been developed with members of the Society of Cardiovascular Pathology working with cardiovascular pathology-trained, practicing forensic pathologists.