Clostridioides difficile in calves, cattle and humans from Dutch dairy farms: Predominance of PCR ribotype 695 (clade 5, sequence type 11) in cattle.

Background: Clostridioides difficile is a leading cause of infectious diarrhea in both humans and livestock. In particular, C. difficile strains belonging to sequence type (ST) 11 are common enteropathogens. The aim of this study was to determine the presence and genetic relatedness of C. difficile types in dairy cattle and calves. Method: Dutch dairy farms were visited between February and December 2021. Feces was collected from adult dairy cattle and calves of two age categories (<4 weeks and 4 weeks-4 months). Fecal samples were also requested from dairy farmers, family members and employees. Fecal samples were cultured in an enrichment medium for 10-15 days and subcultured on solid media for capillary PCR ribotyping and whole genome sequencing. Results: C. difficile was detected on 31 out of 157 (19.8%) dairy farms. The highest prevalence was found in calves <4 weeks (17.5%). None of the 99 human samples collected were positive. Thirty-seven cultured isolates belonged to 11 different PCR ribotypes (RT) of which RT695 (56.8%) and RT078/126 (16.2%) were most abundant. In the database of the Netherlands National Expertise Centre for C. difficile infections (CDI, >10.000 patient isolates), RT695 was found in only two patients with hospital-onset CDI, diagnosed in 2020 and 2021. Sequence analysis of 21C. difficile RT695 from cattle revealed that all isolates belonged to clade 5, ST11 and contained genes encoding toxin A, toxin B and binary toxin. RT695 strains carried antimicrobial resistance genes typically found in clade 5C. difficile. Groups of genetically related RT695 isolates were found between dairy farms, whereas identical strains were only present in individual farms. Conclusions: C. difficile was found in ∼20% of dairy farms with a predominance of the relatively unknown RT695. Isolates of RT695 belonged to the same clade and sequence type as RT078/126, which is recognized as an important zoonotic type.

B cell receptor signaling proteins as biomarkers for progression of CLL requiring first-line therapy.

The molecular landscape of chronic lymphocytic leukemia (CLL) has been extensively characterized, and various potent prognostic biomarkers were discovered. The genetic composition of the B-cell receptor (BCR) immunoglobulin (IG) was shown to be especially powerful for discerning indolent from aggressive disease at diagnosis. Classification based on the IG heavy chain variable gene (IGHV) somatic hypermutation status is routinely applied. Additionally, BCR IGH stereotypy has been implicated to improve risk stratification, through characterization of subsets with consistent clinical profiles. Despite these advances, it remains challenging to predict when CLL progresses to requiring first-line therapy, thus emphasizing the need for further refinement of prognostic indicators. Signaling pathways downstream of the BCR are essential in CLL pathogenesis, and dysregulated components within these pathways impact disease progression. Considering not only genomics but the entirety of factors shaping BCR signaling activity, this review offers insights in the disease for better prognostic assessment of CLL.

High-throughput Proteomics Identifies THEMIS2 as Independent Biomarker of Treatment-free Survival in Untreated CLL.

It remains challenging in chronic lymphocytic leukemia (CLL) to distinguish between patients with favorable and unfavorable time-to-first treatment (TTFT). Additionally, the downstream protein correlates of well-known molecular features of CLL are not always clear. To address this, we selected 40 CLL patients with TTFT ≤24 months and compared their B cell intracellular protein expression with 40 age- and sex-matched CLL patients with TTFT >24 months using mass spectrometry. In total, 3268 proteins were quantified in the cohort. Immunoglobulin heavy-chain variable (IGHV) mutational status and trisomy 12 were most impactful on the CLL proteome. Comparing cases to controls, 5 proteins were significantly upregulated, whereas 3 proteins were significantly downregulated. Of these, only THEMIS2, a signaling protein acting downstream of the B cell receptor, was significantly associated with TTFT, independently of IGHV and TP53 mutational status (hazard ratio, 2.49 [95% confidence interval, 1.62-3.84]; P < 0.001). This association was validated on the mRNA and protein level by quantitative polymerase chain reaction and ELISA, respectively. Analysis of 2 independently generated RNA sequencing and mass spectrometry datasets confirmed the association between THEMIS2 expression and clinical outcome. In conclusion, we present a comprehensive characterization of the proteome of untreated CLL and identify THEMIS2 expression as a putative biomarker of TTFT.

Genome-scale functional genomics identify genes preferentially essential for multiple myeloma cells compared to other neoplasias.

Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

Genomic comparison of mecC-carrying methicillin-resistant Staphylococcus aureus from hedgehogs and humans in the Netherlands.

OBJECTIVES: MRSA carrying the mecC gene (mecC-MRSA) have been found in humans and animals worldwide. A high carriage rate of mecC-MRSA has been described among hedgehogs in different countries. We performed genomic comparison of mecC-MRSA from hedgehogs and humans using next-generation sequencing (NGS) to investigate possible zoonotic transmission in the Netherlands. METHODS: Nasal swabs from hedgehogs (n = 105) were cultured using pre-enrichment and selective plates. Isolates were sequenced using Illumina NGS platforms. These data were compared with sequence data of mecC-MRSA (n = 62) from the Dutch national MRSA surveillance in humans. RESULTS: Fifty hedgehogs were found to be MRSA positive, of which 48 carried mecC. A total of 60 mecC-MRSA isolates derived from 50 hedgehogs were compared with the human isolates. Fifty-nine mecC-MRSA from hedgehogs and all but one isolate from humans belonged to clonal complexes CC130 and CC1943. The mecC gene was located within the SCCmec XI element. Most mecC-MRSA did not carry other resistance genes besides mecC and blaZ. Two human isolates carried erm(C). Isolates differed in the presence of various virulence genes, which were linked to distinct STs and clonal complexes. Some isolates had up to 17 virulence genes, which underlines their pathogenic potential. No genetic clusters of hedgehog and human isolates were found. CONCLUSIONS: mecC-MRSA from hedgehogs and humans mainly belonged to the same two clonal complexes, indicating a common source. No firm evidence for recent zoonotic transmission was found. Further studies are needed to investigate the role of hedgehogs in the occurrence of mecC-MRSA in humans.

Detecting measurable residual disease beyond 10-4 by an IGHV leader-based NGS approach improves prognostic stratification in CLL.

The sensitivity of conventional techniques for reliable quantification of minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) is limited to MRD 10-4. Measuring MRD <10-4 could help to further distinguish between patients with CLL with durable remission and those at risk of early relapse. We herein present an academically developed immunoglobulin heavy-chain variable (IGHV) leader-based next-generation sequencing (NGS) assay for the quantification of MRD in CLL. We demonstrate, based on measurements in contrived MRD samples, that the linear range of detection and quantification of our assay reaches beyond MRD 10-5. If provided with sufficient DNA input, MRD can be detected down to MRD 10-6. There was high interassay concordance between measurements of the IGHV leader-based NGS assay and allele-specific oligonucleotide quantitative polymerase chain reaction (PCR) (r = 0.92 [95% confidence interval {CI}, 0.86-0.96]) and droplet digital PCR (r = 0.93 [95% CI, 0.88-0.96]) on contrived MRD samples. In a cohort of 67 patients from the CLL11 trial, using MRD 10-5 as a cutoff, undetectable MRD was associated with superior progression-free survival (PFS) and time to next treatment. More important, deeper MRD measurement allowed for additional stratification of patients with MRD <10-4 but ≥10-5. PFS of patients in this MRD range was significantly shorter, compared with patients with MRD <10-5 (hazard ratio [HR], 4.0 [95% CI, 1.6-10.3]; P = .004), but significantly longer, compared with patients with MRD ≥10-4 (HR, 0.44 [95% CI, 0.23-0.87]; P = .018). These results support the clinical utility of the IGHV leader-based NGS assay.

cfr and fexA genes in methicillin-resistant Staphylococcus aureus from humans and livestock in the Netherlands.

Background: Although the Netherlands is a country with a low endemic level of methicillin-resistant Staphylococcus aureus (MRSA), a national MRSA surveillance has been in place since 1989. In 2003 livestock emerged as a major reservoir of MRSA and currently livestock-associated MRSA (clonal complex CC398) make up 25% of all surveillance isolates. To assess possible transfer of resistant strains or resistance genes, MRSA obtained from humans and animals were characterized in detail. Methods: The sequenced genomes of 6327 MRSA surveillance isolates from humans and from 332 CC398 isolates from livestock-related samples were analyzed and resistance genes were identified. Several isolates were subjected to long-read sequencing to reconstruct chromosomes and plasmids. Results: Here we show the presence of the multi-resistance gene cfr in seven CC398 isolates obtained from humans and in one CC398 isolate from a pig-farm dust sample. Cfr induces resistance against five antibiotic classes, which is true for all but two isolates. The isolates are genetically unrelated, and in seven of the isolates cfr are located on distinct plasmids. The fexA gene is found in 3.9% surveillance isolates and in 7.5% of the samples from livestock. There is considerable sequence variation of fexA and geographic origin of the fexA alleles. Conclusions: The rare cfr and fexA resistance genes are found in MRSA from humans and animals in the Netherlands, but there is no evidence for spread of resistant strains or resistance plasmids. The proportion of cfr-positive MRSA is low, but its presence is worrying and should be closely monitored.

Zoonotic Pathogens in Eurasian Beavers (Castor fiber) in the Netherlands.

Successful repopulation programs of Eurasian beavers (Castor fiber) have resulted in an increase in beaver populations throughout Europe. This may be of public health relevance because beavers can host multiple zoonotic pathogens. From March 2018 to March 2020, opportunistic testing of dead beavers was performed for hepatitis E virus, orthohantavirus, Anaplasma phagocytophilum, Bartonella spp., extended-spectrum-betalactamase or AmpC (ESBL/AmpC-)-producing Enterobacteriaceae, Francisella tularensis, Leptospira spp., Neoehrlichia mikurensis, Babesia spp., Echinococcus multilocularis, Toxoplasma gondii, and Trichinella spp. From the 24 beavers collected, three zoonotic pathogens were detected. One beaver was positive for T. gondii, one was positive for ESBL/AmpC-producing Enterobacteriaceae, and one was positive for N. mikurensis. The latter finding indicates that beavers can be bitten by Ixodes ricinus and be exposed to tick-borne pathogens. The detected ESBL/AmpC-gene was blaCMY-2 in an Escherichia coli ST6599. The findings suggest that the role of beavers in the spread of zoonotic diseases in the Netherlands is currently limited.

Faecal carriage of Clostridioides difficile is low among veterinary healthcare workers in the Netherlands.

Veterinary healthcare workers are in close contact with many different animals and might be at an increased risk of acquiring Clostridioides difficile. In this cross-sectional study, we assessed the prevalence and risk factors of C. difficile carriage in Dutch veterinary healthcare workers. Participants provided a faecal sample and filled out a questionnaire covering potential risk factors for C. difficile carriage. C. difficile culture positive isolates were polymerase chain reaction (PCR) ribotyped and the presence of toxin genes tcdA, tcdB and cdtA/cdtB was determined. Eleven of 482 [2.3%; 95% confidence interval (CI) 1.3-4.0] veterinary healthcare workers were carriers of C. difficile. Three persons carried C. difficile ribotype 078 (0.6%; 95% CI 0.2-1.8). Risk factors for carriage were health/medication and hygiene related, including poor hand hygiene after patient (animal) contact, and did not include occupational contact with certain animal species. In conclusion, the prevalence of C. difficile carriage in veterinary healthcare workers was low and no indications were found that working in veterinary care is a risk for C. difficile carriage.

High-risk subtypes of chronic lymphocytic leukemia are detectable as early as 16 years prior to diagnosis.

Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B-cell lymphocytosis (MBL), a CLL precursor state with a prevalence of up to 12% in aged individuals; however, the duration of MBL and the mechanisms of its evolution to CLL remain largely unknown. In this study, we sequenced the B-cell receptor (BcR) immunoglobulin heavy chain (IGH) gene repertoire of 124 patients with CLL and 118 matched controls in blood samples taken up to 22 years prior to diagnosis. Significant skewing in the BcR IGH gene repertoire was detected in the majority of patients, even before the occurrence of lymphocytosis and irrespective of the clonotypic IGH variable gene somatic hypermutation status. Furthermore, we identified dominant clonotypes belonging to major stereotyped subsets associated with poor prognosis up to 16 years before diagnosis in 14 patients with CLL. In 22 patients with longitudinal samples, the skewing of the BcR IGH gene repertoire increased significantly over time to diagnosis or remained stable at high levels. For 14 of 16 patients with available samples at diagnosis, the CLL clonotype was already present in the prediagnostic samples. Overall, our data indicate that the preclinical phase of CLL could be longer than previously thought, even in adverse-prognostic cases.

Treatment Approaches to Chronic Lymphocytic Leukemia With High-Risk Molecular Features.

The clinical course of chronic lymphocytic leukemia (CLL) is highly variable. Over the past decades, several cytogenetic, immunogenetic and molecular features have emerged that identify patients suffering from CLL with high-risk molecular features. These biomarkers can clearly aid prognostication, but may also be capable of predicting the efficacy of various treatment strategies in subgroups of patients. In this narrative review, we discuss treatment approaches to CLL with high-risk molecular features. Specifically, we review and provide a comprehensive overview of clinical trials evaluating the efficacy of chemotherapy, chemoimmunotherapy and novel agent-based treatments in CLL patients with TP53 aberrations, deletion of the long arm of chromosome 11, complex karyotype, unmutated IGHV, B cell receptor stereotypy, and mutations in NOTCH1 or BIRC3. Furthermore, we discuss future pharmaceutical and immunotherapeutic perspectives for CLL with high-risk molecular features, focusing on agents currently under investigation in clinical trials.

ESBL/pAmpC-producing Escherichia coli and Klebsiella pneumoniae carriage among veterinary healthcare workers in the Netherlands.

BACKGROUND: Animals are a reservoir for ESBL/pAmpC-producing Escherichia coli/Klebsiella pneumoniae (ESBL-E/K). We investigated the association between occupational contact with different types of animals and the prevalence of ESBL-E/K carriage among veterinary healthcare workers, assessed molecular characteristics of ESBL-E/K, and followed-up on the ESBL-E/K carriage status of participants and their household members. METHODS: Participants completed a questionnaire about their contact with animals at work and at home, health status, travel behaviour and hygiene, and sent in a faecal sample which was tested for the presence of ESBL-E/K. Resistance genes were typed using PCR and sequencing. ESBL-E/K positive participants and their household members were followed up after 6 months. Risk factors were analysed using multivariable logistic regression methods. RESULTS: The prevalence of ESBL-E/K carriage was 9.8% (47/482; 95%CI 7.4-12.7). The most frequently occurring ESBL genes were blaCTX-M-15, blaCTX-M-14 and blaDHA-1. The predominant sequence type was ST131. None of the occupation related factors, such as contact with specific animal species, were significantly associated with ESBL-E/K carriage, whereas travel to Africa, Asia or Latin America in the past 6 months (OR 4.4), and stomach/bowel complaints in the past 4 weeks (OR 2.2) were. Sixteen of 33 initially ESBL-E/K positive participants (48.5%) tested positive again 6 months later, in 14 persons the same ESBL gene and E. coli ST was found. Four of 23 (17.4%) household members carried ESBL-E/K, in three persons this was the same ESBL gene and E. coli ST as in the veterinary healthcare worker. CONCLUSIONS: Despite the absence of specific occupation related risk factors, ESBL-E/K carriage in veterinary healthcare workers was high compared to the prevalence in the general Dutch population (5%). This indicates that occupational contact with animals is a potential source of ESBL-E/K for the population at large.

Consistent B Cell Receptor Immunoglobulin Features Between Siblings in Familial Chronic Lymphocytic Leukemia.

Key processes in the onset and evolution of chronic lymphocytic leukemia (CLL) are thought to include chronic (antigenic) activation of mature B cells through the B cell receptor (BcR), signals from the microenvironment, and acquisition of genetic alterations. Here we describe three families in which two or more siblings were affected by CLL. We investigated whether there are immunogenetic similarities in the leukemia-specific immunoglobulin heavy (IGH) and light (IGL/IGK) chain gene rearrangements of the siblings in each family. Furthermore, we performed array analysis to study if similarities in CLL-associated chromosomal aberrations are present within each family and screened for somatic mutations using paired tumor/normal whole-genome sequencing (WGS). In two families a consistent IGHV gene mutational status (one IGHV-unmutated, one IGHV-mutated) was observed. Intriguingly, the third family with four affected siblings was characterized by usage of the lambda IGLV3-21 gene, with the hallmark R110 mutation of the recently described clinically aggressive IGLV3-21R110 subset. In this family, the CLL-specific rearrangements in two siblings could be assigned to either stereotyped subset #2 or the immunogenetically related subset #169, both of which belong to the broader IGLV3-21R110 subgroup. Consistent patterns of cytogenetic aberrations were encountered in all three families. Furthermore, the CLL clones carried somatic mutations previously associated with IGHV mutational status, cytogenetic aberrations and stereotyped subsets, respectively. From these findings, we conclude that similarities in immunogenetic characteristics in familial CLL, in combination with genetic aberrations acquired, point towards shared underlying mechanisms behind CLL development within each family.

Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

Reading the B-cell receptor immunome in chronic lymphocytic leukemia: revelations and applications.

B-Cell receptor (BCR) sequencing has been the force driving many recent advances in chronic lymphocytic leukemia (CLL) research. Here, we discuss the general principles, revelations, and applications of reading the BCR immunome in the context of CLL. First, IGHV mutational status, obtained by measuring the mutational imprint on the IGHV gene of the CLL clonotype, is the cornerstone of CLL risk stratification. Furthermore, the discovery of "BCR-stereotyped" groups of unrelated patients that share not only a highly similar BCR on their leukemic clone, but also certain clinical characteristics has provided insights key to understanding disease ontogeny. Additionally, whereas the BCR repertoire of most CLL patients is characterized by a single dominant rearrangement, next-generation sequencing (NGS) has revealed a rich subclonal landscape in a larger than previously expected proportion of CLL patients. We review the mechanisms underlying these "multiple dominant" cases, including V(D)J-recombination errors, failure of allelic exclusion, intraclonal diversification, and "true" bi- or oligoclonality, and their implications, in detail. Finally, BCR repertoire sequencing can be used for sensitive quantification of minimal residual disease to potentially unprecedented depth. To surmount pitfalls inherent to this approach and develop internationally harmonized protocols, the EuroClonality-NGS Working Group has been established.

Disseminated cryptococcal disease during treatment with idelalisib and corticosteroids for follicular lymphoma.

A patient on a regimen of idelalisib and corticosteroids for a relapse of follicular lymphoma presented to our emergency ward with a fever of unknown origin. Despite the initiation of broad-spectrum antibiotics and fluids, the patient's clinical condition deteriorated. Eventually, a diagnosis of disseminated cryptococcosis was established and immunophenotyping revealed complete absence of circulating B and CD4+-T lymphocytes, and a markedly diminished CD8+-T lymphocyte count. In this case, treatment with idelalisib and corticosteroids likely resulted in profound lymphopenia and the first reported instance of disseminated cryptococcosis under this regimen. After the withdrawal of idelalisib and steroids and initiation of antifungal therapy, lymphocyte counts partially recovered. After clinical improvement, the patient could be discharged from the hospital. This case highlights that the combination of idelalisib and corticosteroids can cause significant immunocompromise and opportunistic infections. Additionally, we illustrate the rate of lymphocyte reconstitution after withdrawal from idelalisib and corticosteroids.