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.

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.

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.

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.

Prolonged carriage of (livestock-associated) MRSA in individuals without professional livestock contact.

OBJECTIVES: To investigate prolonged carriage of MRSA in adults from the general population living in a livestock-dense area, using WGS. METHODS: A cross-sectional study during 2014-15 among 2492 adults without professional livestock contact identified 14 (0.6%) nasal MRSA carriers, 10 of which carried livestock-associated (LA)-MRSA of multiple-locus variable-number tandem repeat analysis (MLVA) complex (MC) 398. Two years later, 12 MRSA-positive and 88 MRSA-negative participants provided a second nasal swab and filled in a short questionnaire. Isolates from persons who were MRSA positive at both timepoints were compared using MLVA and isolates with the same MLVA type were sequenced. The WGS data were used for core-genome MLST (cgMLST) and resistome analysis, including sequenced isolates from the national MRSA surveillance. RESULTS: All MRSA-negative persons tested negative again, while 6 of the 12 initially MRSA-positive persons tested positive again. MLVA revealed that isolate pairs from five individuals had the same MLVA type, of which three were LA-MRSA. cgMLST showed that the distance between these isolate pairs ranged between 3 and 13 genes, while the minimum distance to unrelated isolates from the national MRSA surveillance was 38 genes. Moreover, the resistome present in the five isolate pairs was identical within each pair. None of the prolonged carriers was hospitalized during the 3 months before the sampling moment and none of them with LA-MRSA had contact with livestock in this period. CONCLUSIONS: Prolonged carriage of MRSA, including LA-MRSA, can be demonstrated after more than 30 months in persons without professional livestock contact.

Do vegetarians less frequently carry ESBL/pAmpC-producing Escherichia coli/Klebsiella pneumoniae compared with non-vegetarians?

BACKGROUND: ESBL and plasmid-mediated AmpC (pAmpC)-producing Enterobacteriaceae are frequently found on meat products in Dutch retail, especially on poultry. OBJECTIVES: We investigated whether vegetarians are at lower risk of carrying ESBL/pAmpC-producing Escherichia coli/Klebsiella pneumoniae (ESBL-E/K) compared with persons who consume meat. METHODS: Vegetarians, pescatarians (vegetarians who eat fish) and non-vegetarians (persons who eat meat at least three times per week) were asked to send in a faecal sample and a questionnaire. ESBL-E/K were cultured and MLSTs were determined. ESBL/pAmpC genes were analysed using PCR and sequencing. The risk of ESBL-E/K carriage in the three study groups was analysed using multivariable logistic regression. RESULTS: Prevalence of ESBL-E/K carriage was 8.0% in vegetarians (63/785; 95% CI 6.3-10.1), 6.9% in pescatarians (27/392; 95% CI 4.8-9.8) and 3.8% in non-vegetarians (14/365; 95% CI 2.3-6.3). Multivariable analysis showed an OR for ESBL-E/K carriage of 2.2 for vegetarians (95% CI 1.2-4.0) and 1.6 for pescatarians (95% CI 0.8-3.2) compared with non-vegetarians. The predominant MLST was E. coli ST131 and the most common ESBL genes were blaCTX-M-15, blaCTX-M-27, blaCTX-M-14 and blaCTX-M-1 in all diet groups. Independent risk factors for ESBL-E/K carriage were travel to Africa/Latin America/Asia (OR 4.6; 95% CI 2.8-7.7) in the past 6 months and rarely/never washing hands before food preparation (OR 2.5; 95% CI 1.2-5.0). CONCLUSIONS: Vegetarians and pescatarians did not have a lower risk of ESBL-E/K carriage compared with non-vegetarians, indicating that eating meat is not an important risk factor for ESBL-E/K carriage.

Long-term Carriage of Extended-Spectrum ß-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae in the General Population in The Netherlands.

Background: This longitudinal study aimed to investigate (risk factors for) persistence of carriage and molecular characteristics of extended-spectrum and plasmid-encoded AmpC ß-lactamase-producing (ESBL/pAmpC) Escherichia coli and Klebsiella pneumoniae (ESBL-E/K) in adults in the Dutch community. Methods: Following a cross-sectional study (ESBL-E/K prevalence, 4.5%), a subset of ESBL-E/K-positive (n = 76) and -negative (n = 249) individuals volunteered to provide 5 monthly fecal samples and questionnaires. ESBL-E/K was cultured using selective enrichment/culture, and multilocus sequence types (MLSTs) were determined. ESBL/pAmpC-genes were analyzed using polymerase chain reaction (PCR) and sequencing. Plasmids were characterized and subtyped by plasmid MLST. Risk factors for persistent carriage were analyzed using logistic regression. Results: Of the initially ESBL-E/K-positive participants, 25 of 76 (32.9%) remained positive in all subsequent samples; 51 of 76 persons (67.1%) tested ESBL-E/K negative at some time point during follow-up, of which 31 (40.8%) stayed negative throughout the longitudinal study. Carriers often carried the same ESBL gene and plasmid, but sometimes in different ESBL-E/K strains, indicative for horizontal transfer of plasmids. Of the 249 initially ESBL-E/K-negative participants, the majority (n = 218 [87.6%]) tested negative during 8 months of follow-up, whereas 31 of 249 (12.4%) participants acquired an ESBL-E/K. Escherichia coli phylogenetic group B2 and D and travel to ESBL high-prevalence countries were associated with prolonged carriage. Conclusions: ESBL-E/K carriage persisted for >8 months in 32.9% of the initially ESBL-positive individuals, while 12.4% of initially negative individuals acquired ESBL-E/K during the study. A single positive test result provides no accurate prediction for prolonged carriage. Acquisition/loss of ESBL-E/K does not seem to be a random process, but differs between bacterial genotypes.

MRSA in persons not living or working on a farm in a livestock-dense area: prevalence and risk factors.

Objectives: MRSA emerged in livestock and persons in contact with livestock is referred to as livestock-associated MRSA (LA-MRSA). We assessed the prevalence and risk factors for MRSA carriage in persons not living or working on a farm. Methods: A cross-sectional study was performed among 2492 adults living in close proximity of livestock farms. Persons working and/or living on farms were excluded. Nasal swabs were cultured using selective media. Participants completed questionnaires and the distance from the residential address to the nearest farm was calculated. The Mann-Whitney U -test was used to compare median distances. Risk factors were explored with logistic regression. Results: Fourteen persons carried MRSA (0.56%; 95% CI 0.32%-0.92%), 10 of which carried LA-MRSA of multiple-locus variable-number tandem repeat analysis complex (MC) 398 (0.40%; 95% CI 0.20%-0.71%). MRSA MC 398 carriers lived significantly closer to the nearest farm than non-carriers (median: 184 versus 402 m; P < 0.01). In bivariate analyses correcting for contact with livestock, this difference remained significant. Conclusions: Although the prevalence was low, living near farms increased the risk of MRSA MC 398 carriage for persons not living or working on a farm. Further research is necessary to identify the transmission routes.

Ten years later: still a high prevalence of MRSA in slaughter pigs despite a significant reduction in antimicrobial usage in pigs the Netherlands.

OBJECTIVES: In 2005, 39% of pigs and 81% of the slaughter batches at Dutch slaughterhouses were MRSA positive. The objective of the present study was to investigate whether the 50% reduction of antimicrobial usage in finishing pigs in 2014 compared with 2009 in the Netherlands has led to a lower MRSA prevalence among Dutch slaughter pigs. METHODS: Nasal swabs from eight slaughter batches of on average 10 animals at seven slaughterhouses were taken and cultured using method 1, which was used in 2005, and method 2, using high-salt pre-enrichment. Suspected isolates were confirmed by PCR for two Staphylococcus aureus-specific DNA fragments and the mecA gene. A subset of MRSA isolates were further investigated using spa typing, multiple-locus variable number of tandem repeat analysis (MLVA) and antimicrobial susceptibility testing. RESULTS: Using methods 1 and 2, we found 461 of 558 (83%) and 552 of 558 (99%) of the pigs to carry MRSA in their nares, respectively. All 56 slaughter batches were MRSA positive. All MRSA isolates belonged to the livestock-associated MLVA complex 398, had a non-WT phenotype for tetracycline and spa type t011 predominated. CONCLUSIONS: A very high prevalence of nasal MRSA carriage was found in Dutch slaughter pigs and therefore the reduction in antimicrobial usage at the national level has not yet had an effect on the MRSA carriage rate of pigs entering the slaughterhouse. Therefore, there is still an increased risk of MRSA carriage for personnel working at pig slaughterhouses, particularly those having contact with living animals. Method 2, using high salt pre-enrichment, detected more MRSA-positive pigs and is currently the preferred method for screening of MRSA in livestock in the Netherlands.

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.

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.

Resistance phenotypes and genotypes of methicillin-resistant Staphylococcus aureus isolates from broiler chickens at slaughter and abattoir workers.

OBJECTIVES: To comparatively investigate the resistance phenotypes and genotypes of various methicillin-resistant Staphylococcus aureus (MRSA) isolates from broilers at slaughter and workers at the respective poultry slaughterhouses. METHODS: Forty-six MRSA isolates (28 from broilers and 18 from humans) obtained at four different slaughterhouses were included. In addition to previously determined sequence types (STs) and spa types, the isolates were characterized by dru typing, SCCmec typing and PFGE. Resistance phenotypes were determined by broth microdilution. Resistance genes and clonal complexes (CCs) were detected by DNA microarray or specific PCR assays. RESULTS: MRSA of CC398, spa type t011 and varying dru types represented 23/28 broiler isolates and 12/18 human isolates. Three ST9/t1430/dt10a isolates were each seen among the isolates from the abattoir workers and the broilers. In addition, two human CC398/ST1453/t4652/dt3c isolates, a single human CC398/t034/dt6j isolate and two chicken CC398/t108/dt11a isolates were detected. All CC398 isolates (including ST1453) and some of the ST9 isolates from chickens and humans showed resistance to four to nine classes of antimicrobial agents and carried a wide range of resistance genes. While the resistance phenotypes and genotypes of the chicken isolates of the same flock were closely related, they usually differed from the resistance phenotypes and genotypes of the isolates from the workers at the respective slaughterhouse. CONCLUSIONS: The apparent homogeneity of MRSA isolates from the same flock suggests exchange of isolates between the respective animals. The apparent heterogeneity of MRSA isolates from abattoir workers might reflect their occupational contact with animals from numerous chicken flocks.

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.

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.

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.

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.

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.