Use Of Whole-genome Sequencing Research Articles

Listeria monocytogenes Strains Persisting in a Meat Processing Plant in Central Italy: Use of Whole Genome Sequencing and In Vitro Adhesion and Invasion Assays to Decipher Their Virulence Potential.

In this study, we used both a WGS and an in vitro approach to study the virulence potential of nine Listeria monocytogenes (Lm) strains belonging to genetic clusters persisting in a meat processing plant in Central Italy. The studied clusters belonged to CC1-ST1, CC9-ST9, and CC218-ST2801. All the CC1 and CC218 strains presented the same accessory virulence genes (LIPI-3, gltA, gltB, and aut_IVb). CC1 and CC9 strains presented a gene profile similarity of 22.6% as well as CC9 and CC218 isolates. CC1 and CC218 showed a similarity of 45.2% of the same virulence profile. The hypervirulent strains of lineage I (CC1 and CC218) presented a greater ability to adhere and invade Caco-2 cells than hypovirulent ones (CC9). CC1 strains were significantly more adhesive and invasive compared with CC9 and CC218 strains, although these last CCs presented the same accessory virulence genes. No statistically significant difference was found comparing CC218 with CC9 strains. This study provided for the first time data on the in vitro adhesiveness and invasiveness of CC218-ST2801 and added more data on the virulence characteristics of CC1 and CC9. What we observed confirmed that the ability of Lm to adhere to and invade human cells in vitro is not always decipherable from its virulence gene profile.

Investigating the Genetic Diversity of H5 Avian Influenza Viruses in the United Kingdom from 2020-2022.

Since 2020, the United Kingdom and Europe have experienced annual epizootics of high-pathogenicity avian influenza virus (HPAIV). The first epizootic, during the autumn/winter of 2020-2021, involved six H5Nx subtypes, although H5N8 HPAIV dominated in the United Kingdom. While genetic assessments of the H5N8 HPAIVs within the United Kingdom demonstrated relative homogeneity, there was a background of other genotypes circulating at a lower degree with different neuraminidase and internal genes. Following a small number of detections of H5N1 in wild birds over the summer of 2021, the autumn/winter of 2021-2022 saw another European H5 HPAIV epizootic that dwarfed the prior epizootic. This second epizootic was dominated almost exclusively by H5N1 HPAIV, although six distinct genotypes were defined. We have used genetic analysis to evaluate the emergence of different genotypes and proposed reassortment events that have been observed. The existing data suggest that the H5N1 viruses circulating in Europe during late 2020 continued to circulate in wild birds throughout 2021, with minimal adaptation, but then went on to reassort with AIVs in the wild bird population. We have undertaken an in-depth genetic assessment of H5 HPAIVs detected in the United Kingdom over two winter seasons and demonstrate the utility of in-depth genetic analyses in defining the diversity of H5 HPAIVs circulating in avian species, the potential for zoonotic risk, and whether incidents of lateral spread can be defined over independent incursions of infections from wild birds. This provides key supporting data for mitigation activities. IMPORTANCE High-pathogenicity avian influenza virus (HPAIV) outbreaks devastate avian species across all sectors, having both economic and ecological impacts through mortalities in poultry and wild birds, respectively. These viruses can also represent a significant zoonotic risk. Since 2020, the United Kingdom has experienced two successive outbreaks of H5 HPAIV. While H5N8 HPAIV was predominant during the 2020-2021 outbreak, other H5 subtypes were also detected. The following year, there was a shift in the subtype dominance to H5N1 HPAIV, but multiple H5N1 genotypes were detected. Through the thorough utilization of whole-genome sequencing, it was possible to track and characterize the genetic evolution of these H5 HPAIVs in United Kingdom poultry and wild birds. This enabled us to assess the risk posed by these viruses at the poultry-wild bird and the avian-human interfaces and to investigate the potential lateral spread between infected premises, a key factor in understanding the threat to the commercial sector.

Salmonella Typhi whole genome sequencing in Rwanda shows a diverse historical population with recent introduction of haplotype H58.

Salmonella enterica serovar Typhi (S. Typhi) is the cause of typhoid fever, presenting high rates of morbidity and mortality in low- and middle-income countries. The H58 haplotype shows high levels of antimicrobial resistance (AMR) and is the dominant S. Typhi haplotype in endemic areas of Asia and East sub-Saharan Africa. The situation in Rwanda is currently unknown and therefore to reveal the genetic diversity and AMR of S. Typhi in Rwanda, 25 historical (1984-1985) and 26 recent (2010-2018) isolates from Rwanda were analysed using whole genome sequencing (WGS). WGS was locally implemented using Illumina MiniSeq and web-based analysis tools, thereafter complemented with bioinformatic approaches for more in-depth analyses. Whereas historical S. Typhi isolates were found to be fully susceptible to antimicrobials and show a diversity of genotypes, i.e 2.2.2, 2.5, 3.3.1 and 4.1; the recent isolates showed high AMR rates and were predominantly associated with genotype 4.3.1.2 (H58, 22/26; 84,6%), possibly resulting from a single introduction in Rwanda from South Asia before 2010. We identified practical challenges for the use of WGS in endemic regions, including a high cost for shipment of molecular reagents and lack of high-end computational infrastructure for the analyses, but also identified WGS to be feasible in the studied setting and giving opportunity for synergy with other programs.

Clinical Accuracy and Utility of Plasma Microbial Cell-Free DNA Whole-Genome Sequencing for the Diagnosis of Invasive Aspergillosis in Patients with Hematologic Malignancies or Coronavirus Disease 2019.

We evaluated the clinical accuracy and utility of whole-genome sequencing (WGS) of plasma microbial cell-free DNA (cfDNA) as a novel non-invasive method in diagnosing invasive aspergillosis (IA) in patients with hematologic malignancy (HM) or coronavirus disease 2019 (COVID-19). Adults with HM or COVID-19 and suspected IA were recruited. IA cases were retrospectively diagnosed according to EORTC/MSG definitions and ECMM/ISHAM criteria for HM and COVID-19 patients, respectively. The results of cfDNA WGS were compared with the conventional diagnosis. Microbial cfDNA WGS was performed 53 times from 41 participants (19 from HM, 16 from COVID-19, and 7 from the control group). In participants with HM, Aspergillus cfDNA was detected in 100% of proven IA and 91.7% of probable IA cases. In participants with COVID-19, 50.0% of probable IA were positive for Aspergillus in cfDNA WGS. Concordance between Aspergillus cfDNA detection and proven/probable IA conventional diagnosis was significantly higher in participants with HM than in those with COVID-19. IA diagnosed using EORTC/MGS definitions showed significantly high concordance between Aspergillus cfDNA detection and proven/probable IA. Aspergillus cfDNA detection strongly correlated with proven/probable IA diagnosed using EORTC/MSG definitions and could be used as an additional diagnostic tool for IA.

Use of whole genome sequencing to determine the genetic basis of visceral myopathies including Prune Belly syndrome

Objectives/aimsThe visceral myopathies (VM) are a group of disorders characterised by poorly contractile or acontractile smooth muscle. They manifest in both the GI and GU tracts, ranging from megacystis to Prune Belly syndrome. We aimed to apply a bespoke virtual genetic panel and describe novel variants associated with this condition using whole genome sequencing data within the Genomics England 100,000 Genomes Project.MethodsWe screened the Genomics England 100,000 Genomes Project rare diseases database for patients with VM-related phenotypes. These patients were screened for sequence variants and copy number variants (CNV) in ACTG2, ACTA2, MYH11, MYLK, LMOD1, CHRM3, MYL9, FLNA and KNCMA1 by analysing whole genome sequencing data. The identified variants were analysed using variant effect predictor online tool, and any possible segregation in other family members and novel missense mutations was modelled using in silico tools. The VM cohort was also used to perform a genome-wide variant burden test in order to identify confirm gene associations in this cohort.ResultsWe identified 76 patients with phenotypes consistent with a diagnosis of VM. The range of presentations included megacystis/microcolon hypoperistalsis syndrome, Prune Belly syndrome and chronic intestinal pseudo-obstruction. Of the patients in whom we identified heterozygous ACTG2 variants, 7 had likely pathogenic variants including 1 novel likely pathogenic allele. There were 4 patients in whom we identified a heterozygous MYH11 variant of uncertain significance which leads to a frameshift and a predicted protein elongation. We identified one family in whom we found a heterozygous variant of uncertain significance in KCNMA1 which in silico models predicted to be disease causing and may explain the VM phenotype seen. We did not find any CNV changes in known genes leading to VM-related disease phenotypes. In this phenotype selected cohort, ACTG2 is the largest monogenic cause of VM-related disease accounting for 9% of the cohort, supported by a variant burden test approach, which identified ACTG2 variants as the largest contributor to VM-related phenotypes.ConclusionsVM are a group of disorders that are not easily classified and may be given different diagnostic labels depending on their phenotype. Molecular genetic analysis of these patients is valuable as it allows precise diagnosis and aids understanding of the underlying disease manifestations. We identified ACTG2 as the most frequent genetic cause of VM. We recommend a nomenclature change to ‘autosomal dominant ACTG2 visceral myopathy’ for patients with pathogenic variants in ACTG2 and associated VM phenotypes.

Abstract PR005: Multi-level functional genomics reveals molecular and cellular oncogenicity of patient-based 3’ untranslated region mutations

Abstract It is clear from the high mortality rate in advanced cancers that we need a more complete understanding of the many sources of oncogenic dysregulation in tumors. The importance of the non-coding genome in disease has recently begun to be revealed through expanded use of whole genome sequencing. In particular, 3’ untranslated region (3’UTR) somatic mutations represent an important but largely unexplored avenue of alternative oncogenic gene dysregulation. Individual instances of 3’UTR-mediated oncogenicity are known, including oncogenic RNA binding proteins and microRNAs. However, a comprehensive, high-throughput study of patient-based 3’UTR mutations and their effect on post-transcriptional gene regulation in cancer has yet to be undertaken. To determine the significance of 3’UTR mutations in advanced disease, we identify 3’UTR somatic variants across 185 metastatic castration-resistant prostate tumors, discovering 14,497 single-nucleotide mutations, which are enriched in oncogenic pathways and 3’UTR regulatory elements. We develop two complementary massively parallel reporter assays (MPRAs) to measure how thousands of these patient-based mutations affect two distinct levels of post-transcriptional gene regulation: mRNA translation and stability. These MPRAs identify hundreds of functional variants that allow us to define three determinants of mutation significance: sequence conservation, known regulatory elements, and RNA structure. Furthermore, we demonstrate the clinical relevance of these mutations, observing that CRISPR-Cas9 base editing of distinct patient 3’UTR mutations into endogenous cellular loci increases oncogenic mRNA translation and cellular stress resistance. Finally, we go back to patients, illustrating that those harboring oncogenic 3’UTR mutations discovered by our methods display particularly poor prognosis. This work represents an unprecedented view of the extent to which disease-relevant 3’UTR mutations affect mRNA stability, translation, and cancer progression, uncovering principles of regulatory functionality and potential therapeutic targets in previously unexplored regulatory regions. Citation Format: Samantha L. Schuster, Sonali Arora, Cynthia L. Wladyka, Lukas Corey, Bethany L. Stackhouse, Lori Kollath, Eva Corey, Lawrence D. True, Dave Young, Patrick J. Paddison, Andrew C. Hsieh. Multi-level functional genomics reveals molecular and cellular oncogenicity of patient-based 3’ untranslated region mutations [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR005.

Surveillance of healthcare-onset clinical cultures using whole-genome sequencing reveals hidden nosocomial transmission

Background: Traditional hospital outbreak-detection methods are typically limited to select multidrug-resistant pathogens in a single unit, which can miss transmission of many medically important healthcare-transmissible pathogens. Whole-genome sequencing (WGS) enables comprehensive genomic resolution for accurate identification of clonal transmission. Previously, lack of scalability limited the use of WGS for hospital surveillance. Methods: We conducted prospective surveillance of select bacteria from all inpatient clinical cultures plus all bacteria from clinical cultures from ICUs and oncology units at the University of California Irvine (UCI) Clinical Microbiology Laboratory from September 2021 to February 2022. Due to pandemic stressors, this pilot test was a prelude to a real-time demonstration project. Its goal was to demonstrate the efficiency and scalability of the WGS platform when receiving samples monthly and analyzing results quarterly without the intent for real-time response. Bacterial isolates slated for discard were collected weekly and sent monthly to Day Zero Diagnostics for sequencing. In total, 1,036 samples from 926 patients were analyzed for genomic relatedness, a scalable and automated analysis pipeline already in use for rapid (days) characterization of genomic-relatedness in small and large sets of isolates. Mapping and SNP calling was performed against high-quality, best-match reference genomes. Sets of samples with pairwise distance of 2 persons with genomically related isolates and were denoted as “clusters.” Separately, we also investigated within-patient diversity by quantifying the genomic relatedness of isolates collected from individual patients. Results: Isolates represented 28 distinct species. We identified 10 Escherichia coli clusters (range, 2–4 patients; median, 2 patients), 2 Klebsiella pneumoniae clusters (range, 2–4 patients), and 1 Enterococcus faecium cluster (3 patients). All but 1 involved genomically matched isolates from multiple hospital locations. There were 4 Escherichia coli ST131 clusters spanning 4 months, including 1 with 4 patients across 3 different hospital locations. At a species level, there were distinct differences between the observed SNP distances between samples isolated from the same versus different patients (Fig. 1). All identified clusters had not been flagged by routine outbreak detection methods used by the UCI infection prevention program. Conclusions: Comprehensive WGS-based surveillance of hospital clinical isolates identified multiple potential transmission events between patients not in the same unit at the time cultures were taken. Combining WGS detection and real-time epidemiologic investigation may identify new avenues of transmission risk and could provide early warnings of clonal transmission to prevent larger outbreaks. High-volume surveillance of hospital isolates can also provide species- and context-specific clonality.Financial support: This study was funded by Day Zero Diagnostics.Disclosures: None

Organizational Aspects of the Implementation and Use of Whole Genome Sequencing and Whole Exome Sequencing in the Pediatric Population in Italy: Results of a Survey.

This study explores the organizational aspects of whole genome sequencing (WGS) implementation for pediatric patients with suspected genetic disorders in Italy, comparing it with whole exome sequencing (WES). Health professionals' opinions were collected through an internet-based survey and analyzed using a qualitative summative content analysis methodology. Among the 16 respondents, most were clinical geneticists performing only WES, while 5 also used WGS. The key differences identified include higher needs for analyzing genome rearrangements following WES, greater data storage and security requirements for WGS, and WGS only being performed in specific research studies. No difference was detected in centralization and decentralization issues. The main cost factors included genetic consultations, library preparation and sequencing, bioinformatic analysis, interpretation and confirmation, data storage, and complementary diagnostic investigations. Both WES and WGS decreased the need for additional diagnostic analyses when not used as last-resort tests. Organizational aspects were similar for WGS and WES, but economic evidence gaps may exist for WGS in clinical settings. As sequencing costs decline, WGS will likely replace WES and traditional genetic testing. Tailored genomic policies and cost-effectiveness analyses are needed for WGS implementation in health systems. WGS shows promise for enhancing genetics knowledge and expediting diagnoses for pediatric patients with genetic disorders.

Whole genome sequencing of Mycobacterium bovis directly from clinical tissue samples without culture.

Advancement in next generation sequencing offers the possibility of routine use of whole genome sequencing (WGS) for Mycobacterium bovis (M. bovis) genomes in clinical reference laboratories. To date, the M. bovis genome could only be sequenced if the mycobacteria were cultured from tissue. This requirement for culture has been due to the overwhelmingly large amount of host DNA present when DNA is prepared directly from a granuloma. To overcome this formidable hurdle, we evaluated the usefulness of an RNA-based targeted enrichment method to sequence M. bovis DNA directly from tissue samples without culture. Initial spiking experiments for method development were established by spiking DNA extracted from tissue samples with serially diluted M. bovis BCG DNA at the following concentration range: 0.1 ng/μl to 0.1 pg/μl (10-1 to 10-4). Library preparation, hybridization and enrichment was performed using SureSelect custom capture library RNA baits and the SureSelect XT HS2 target enrichment system for Illumina paired-end sequencing. The method validation was then assessed using direct WGS of M. bovis DNA extracted from tissue samples from naturally (n = 6) and experimentally (n = 6) infected animals with variable Ct values. Direct WGS of spiked DNA samples achieved 99.1% mean genome coverage (mean depth of coverage: 108×) and 98.8% mean genome coverage (mean depth of coverage: 26.4×) for tissue samples spiked with BCG DNA at 10-1 (mean Ct value: 20.3) and 10-2 (mean Ct value: 23.4), respectively. The M. bovis genome from the experimentally and naturally infected tissue samples was successfully sequenced with a mean genome coverage of 99.56% and depth of genome coverage ranging from 9.2× to 72.1×. The spoligoyping and M. bovis group assignment derived from sequencing DNA directly from the infected tissue samples matched that of the cultured isolates from the same sample. Our results show that direct sequencing of M. bovis DNA from tissue samples has the potential to provide accurate sequencing of M. bovis genomes significantly faster than WGS from cultures in research and diagnostic settings.

New Horizons in the Diagnosis of Tuberculosis of the Spine: The Role of Whole Genome Sequencing.

Prospective study. To evaluate the utility of whole genome sequencing (WGS) in drug resistance testing, lineage of the organisms, and organism- related factors responsible for bacilli settling in the spine. The workstream for the diagnosis of tuberculosis (TB) involves isolation and culture of the organism and drug resistance testing using phenotypic methods. Xpert MTB/RIF Ultra is a genetic-based method that detects for Mycobacterium tuberculosis DNA in the rpoB gene. Meanwhile, WGS is a newer genetic-based method that assesses the whole genome of the bacterium. Very few studies have reported the use of WGS for extrapulmonary TB. Herein, we used WGS to diagnose spinal TB. Tissues from 61 patients undergoing surgery for spinal TB underwent histologic examination, Xpert MTB/RIF Ultra, and culture and sensitivity testing. DNA from the cultured bacteria was sent for WGS. The test bacterial genome was compared to a reference strain of pulmonary TB. Acid-fast bacilli were observed in 9/58 specimens. Meanwhile, histology confirmed TB in all the patients. Bacilli were cultured in 28 patients (48.3%), and the average time to culture was 18.7 days. Xpert MTB/RIF Ultra was positive in 47 patients (85%). WGS was performed in 23 specimens. Overall, 45% of the strains belonged to lineage 2 (East Asian). There was one case of multidrug- resistant TB and two cases of non-tuberculous mycobacteria on WGS. We could not confirm any genomic difference between pulmonary and spinal TB strains. Xpert MTB/RIF Ultra of tissues or pus is the investigation of choice when diagnosing spinal TB. Meanwhile, WGS can diagnose multidrug-resistant TB and non-tuberculous mycobacteria more accurately. No mutations were identified in spinal and pulmonary TB bacteria.

A Genome-Wide Association Study of the Chest Circumference Trait in Xinjiang Donkeys Based on Whole-Genome Sequencing Technology.

Animal genotyping by means of genome-wide association studies is important for connecting phenotypes of interest with their underlying genetics in livestock. However, the use of whole genome sequencing to investigate chest circumference (CC) in donkeys has rarely been reported. We aimed to use the genome-wide association study approach to detect significant single nucleotide polymorphisms (SNPs) and key genes associated with chest circumference traits in Xinjiang donkeys. We assessed 112 Xinjiang donkeys in this study. The chest circumference of each was measured 2 h before milking. We re-sequenced blood samples from the Xinjiang donkeys, and genome-wide association study analyses were performed using a mixed model with the PLINK, GEMMA, and REGENIE programs. We tested 38 donkeys for candidate SNPs for genome-wide association study using three software programs. Additionally, 18 SNP markers reached genome-wide significance (p < 1.61 × 10-9). On the basis of these, 41 genes were identified. Previously proposed candidate genes for CC traits were supported by this study, including NFATC2 (Nuclear Factor of Activated T Cells 2), PROP1 (PROP Paired-Like Homeobox 1), UBB (Ubiquitin B), and HAND2 (Heart and Neural Crest Derivatives Expressed 2). These promising candidates provide a valuable resource for validating potential meat production genes and will facilitate the development of high-yielding Xinjiang donkey breeds through marker-assisted selection or gene editing.

Microbiological sampling and analyses in the food business operators’ HACCP-based self-control programmes

A questionnaire was developed within the OH-HARMONY-CAP project providing an overview of current procedures for microbiological sampling and analyses in food business operators’ HACCP-based self-control programmes in EU/EEA countries. It focused on six bacterial species: Salmonella spp., Listeria monocytogenes, Campylobacter spp., Shiga toxin-producing Escherichia coli, Shigella spp. and Yersinia spp.; and five parasites: Trichinella spp., Cryptosporidium spp., Echinococcus granulosus (Sensu lato), Echinococcus multilocularis and Toxoplasma gondii. Participating EU/EEA countries distributed the questionnaire to food business operators’ laboratories within their countries and responses were received from nine countries. Feedback from 35 laboratories among 554 were considered for data analysis. Results showed that dairy products were analysed most frequently and the majority of laboratories analysed both ready-to-eat and non-ready-to-eat products. Laboratories analysing Salmonella spp. and Listeria monocytogenes processed the majority of samples. Accreditation for ISO-standards or an alternative method was in place in a considerable proportion of laboratories, but did not cover all pathogens investigated. Sending isolates for further confirmation to external laboratories was not common. In contrast, storing isolates was more frequently established. Around 60% of laboratories used more than one typing or characterisation method, predominantly MALDI-TOF, antimicrobial resistance typing and PCR, while 40% did not use any of these methods. Variability was observed as regards use of Whole Genome Sequencing; and participation in External Quality Assessment programmes. The study gathered insight into current practices of microbiological sampling and analyses performed in food business operators’ HACCP-based self-control programmes, and showed that further efforts are needed for harmonisation of analytical protocols and characterisation of foodborne pathogens.

Genomic Surveillance of Salmonella from the Comunitat Valenciana (Spain).

Salmonella enterica subspecies enterica is one of the most important foodborne pathogens and the causative agent of salmonellosis, which affects both humans and animals producing numerous infections every year. The study and understanding of its epidemiology are key to monitoring and controlling these bacteria. With the development of whole-genome sequencing (WGS) technologies, surveillance based on traditional serotyping and phenotypic tests of resistance is being replaced by genomic surveillance. To introduce WGS as a routine methodology for the surveillance of food-borne Salmonella in the region, we applied this technology to analyze a set of 141 S. enterica isolates obtained from various food sources between 2010 and 2017 in the Comunitat Valenciana (Spain). For this, we performed an evaluation of the most relevant Salmonella typing methods, serotyping and sequence typing, using both traditional and in silico approaches. We extended the use of WGS to detect antimicrobial resistance determinants and predicted minimum inhibitory concentrations (MICs). Finally, to understand possible contaminant sources in this region and their relationship to antimicrobial resistance (AMR), we performed cluster detection combining single-nucleotide polymorphism (SNP) pairwise distances and phylogenetic and epidemiological data. The results of in silico serotyping with WGS data were highly congruent with those of serological analyses (98.5% concordance). Multi-locus sequence typing (MLST) profiles obtained with WGS information were also highly congruent with the sequence type (ST) assignment based on Sanger sequencing (91.9% coincidence). In silico identification of antimicrobial resistance determinants and minimum inhibitory concentrations revealed a high number of resistance genes and possible resistant isolates. A combined phylogenetic and epidemiological analysis with complete genome sequences revealed relationships among isolates indicative of possible common sources for isolates with separate sampling in time and space that had not been detected from epidemiological information. As a result, we demonstrate the usefulness of WGS and in silico methods to obtain an improved characterization of S. enterica enterica isolates, allowing better surveillance of the pathogen in food products and in potential environmental and clinical samples of related interest.

Epidemiological transmission patterns and use of whole genome sequencing in investigating campus based covid-19 outbreaks during the second and third waves of SARS-Cov-2 infection

Epidemiological transmission patterns and use of whole genome sequencing in investigating campus based covid-19 outbreaks during the second and third waves of SARS-Cov-2 infection

An Overview of Foodborne Sample-Initiated Retrospective Outbreak Investigations and Interagency Collaboration in the United States

Foodborne outbreak investigations have traditionally included the detection of a cluster of illnesses first, followed by an epidemiologic investigation to identify a food of interest. The increasing use of whole genome sequencing (WGS) subtyping technology for clinical, environmental, and food isolates of foodborne pathogens, and the ability to share and compare the data on public platforms, present new opportunities to identify earlier links between illnesses and their potential sources. We describe a process called sample-initiated retrospective outbreak investigations (SIROIs) used by federal public health and regulatory partners in the United States. SIROIs begin with an evaluation of the genomic similarity between bacterial isolates recovered from food or environmental samples and clusters of clinical isolates while subsequent and parallel epidemiologic and traceback investigations are initiated to corroborate their connection. SIROIs allow for earlier hypothesis generation, followed by targeted collection of information about food exposures and the foods and manufacturer of interest, to confirm a link between the illnesses and their source. This often leads to earlier action that could reduce the breadth and burden of foodborne illness outbreaks. We describe two case studies of recent SIROIs and present the benefits and challenges. Benefits include insight into foodborne illness attribution, international collaboration, and opportunities for enhanced food safety efforts in the food industry. Challenges include resource intensiveness, variability of epidemiologic and traceback data, and an increasingly complex food supply chain. SIROIs are valuable in identifying connections among small numbers of illnesses that may span significant time periods; detecting early signals for larger outbreaks or food safety issues associated with manufacturers; improving our understanding of the scope of contamination of foods; and identifying novel pathogen/commodity pairs.

Green Day: An Interview with NHGRI Director Eric Green

Green Day: An Interview with NHGRI Director Eric Green

A One Health approach revealed the long-term role of Mycobacterium caprae as the hidden cause of human tuberculosis in a region of Spain, 2003 to 2022.

IntroductionMycobacterium caprae is a member of the Mycobacterium tuberculosis complex (MTBC) not routinely identified to species level. It lacks specific clinical features of presentation and may therefore not be identified as the causative agent of tuberculosis. Use of whole genome sequencing (WGS) in the investigation of a family microepidemic of tuberculosis in Almería, Spain, unexpectedly identified the involvement of M. caprae.AimWe aimed to evaluate the presence of additional unidentified M. caprae cases and to determine the magnitude of this occurrence.MethodsFirst-line characterisation of the MTBC isolates was done by MIRU-VNTR, followed by WGS. Human and animal M. caprae isolates were integrated in the analysis.ResultsA comprehensive One Health strategy allowed us to (i) detect other 11 M. caprae infections in humans in a period of 18 years, (ii) systematically analyse M. caprae infections on an epidemiologically related goat farm and (iii) geographically expand the study by including 16 M. caprae isolates from other provinces. Integrative genomic analysis of 41 human and animal M. caprae isolates showed a high diversity of strains. The animal isolates' diversity was compatible with long-term infection, and close genomic relationships existed between isolates from goats on the farm and recent cases of M. caprae infection in humans.DiscussionZoonotic circulation of M. caprae strains had gone unnoticed for 18 years. Systematic characterisation of MTBC at species level and/or extended investigation of the possible sources of exposure in all tuberculosis cases would minimise the risk of overlooking similar zoonotic events.

A pseudo-outbreak of Cyberlindnera fabianii funguria: Implication from whole genome sequencing assay.

Although the yeast Cyberlindnera fabianii (C. fabianii) has been rarely reported in human infections, nosocomial outbreaks caused by this organism have been documented. Here we report a pseudo-outbreak of C. fabianii in a urology department of a Chinese hospital over a two-week period. Three patients were admitted to the urology department of a tertiary teaching hospital in Beijing, China, from Nov to Dec 2018, for different medical intervention demands. During the period Nov 28 to Dec 5, funguria occurred in these three patients, and two of them had positive urine cultures multiple times. Sequencing of rDNA internal transcribed spacer (ITS) region and MALDI-TOF MS were applied for strain identification. Further, sequencing of rDNA non-transcribed spacer (NTS) region and whole genome sequencing approaches were used for outbreak investigation purpose. All the cultured yeast strains were identified as C. fabianii by sequencing of ITS region, and were 100% identical to the C. fabianii type strain CBS 5640T. However, the MALDI-TOF MS system failed to correctly identify this yeast pathogen. Moreover, isolates from these three clustered cases shared 99.91%-100% identical NTS region sequences, which could not rule out the possibility of an outbreak. However, whole genome sequencing results revealed that only two of the C. fabianii cases were genetically-related with a pairwise SNP of 192 nt, whilst the third case had over 26,000 SNPs on its genome, suggesting a different origin. Furthermore, the genomes of the first three case strains were phylogenetically even more diverged when compared to a C. fabianii strain identified from another patient, who was admitted to a general surgical department of the same hospital 7 months later. One of the first three patients eventually passed away due to poor general conditions, one was asymptomatic, and other clinically improved. In conclusion, nosocomial outbreaks caused by emerging and uncommon fungal species are increasingly being reported, hence awareness must be raised. Genotyping with commonly used universal gene targets may have limited discriminatory power in tracing the sources of infection for these organisms, requiring use of whole genome sequencing to confirm outbreak events.

Genome-wide association studies for additive and dominance effects for body composition traits in commercial crossbred Piétrain pigs.

Fat depth (FD) and muscle depth (MD) are economically important traits and used to estimate carcass lean content (LMP), which is one of the main breeding objectives in pig breeding programmes. We assessed the genetic architectures of body composition traits for additive and dominance effects in commercial crossbred Piétrain pigs using both 50 K array and sequence genotypes. We first performed a genome-wide association study (GWAS) using single-marker association analysis with a false discovery rate of 0.1. Then, we estimated the additive and dominance effects of the most significant variant in the quantitative trait loci (QTL) regions. It was investigated whether the use of whole-genome sequence (WGS) will improve the QTL detection (both additive and dominance) with a higher power compared with lower density SNP arrays. Our results showed that more QTL regions were detected by WGS compared with 50 K array (n= 54 vs. n= 17). Of the novel associated regions associated with FD and LMP and detected by WGS, the most pronounced peak was on SSC13, situated at ~116-118, 121-127 and 129-134 Mbp. Additionally, we found that only additive effects contributed to the genetic architecture of the analysed traits and no significant dominance effects were found for the tested SNPs at QTL regions, regardless of panel density. The associated SNPs are located in or near several relevant candidate genes. Of these genes, GABRR2, GALR1, RNGTT, CDH20 and MC4R have been previously reported as being associated with fat deposition traits. However, the genes on SSC1 (ZNF292, ORC3, CNR1, SRSF12, MDN1, TSHZ1, RELCH and RNF152) and SSC18 (TTC26 and KIAA1549) have not been reported previously to our best knowledge. Our current findings provide insights into the genomic regions influencing composition traits in Piétrain pigs.

Drug-Resistant Tuberculosis on the Balkan Peninsula: Determination of Drug Resistance Mechanisms with Xpert MTB/XDR and Whole-Genome Sequencing Analysis.

The new molecular assay Xpert MTB/XDR (Cepheid, Sunnyvale, CA, USA) was launched in 2021 to detect Mycobacterium tuberculosis (MT) complex with mutations conferring resistance to isoniazid (INH), ethionamide (ETH), fluoroquinolone (FQ), and second-line injectable drugs (SLIDs). The aim of our study was to evaluate the performance of the Xpert MTB/XDR rapid molecular assay on rifampicin-resistant, multidrug-resistant, and pre-extensively resistant tuberculosis (TB) isolates in a clinical laboratory in the Balkan Peninsula compared to a phenotypic drug susceptibility test (pDST). Xpert MTB/XDR was used to test positive Bactec MGIT 960 (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) cultures or DNA isolates. In the case of discrepant results between Xpert MTB/XDR and pDST, the usefulness of whole-genome sequencing (WGS) was emphasized. In our study, 80 MT isolates from different Balkan countries were selectively chosen from the National Mycobacterial Strain Collection in Golnik, Slovenia. Isolates were tested with the Xpert MTB/XDR assay, conventional pDST, and WGS. Xpert MTB/XDR showed high sensitivities of 91.9%, 100%, and 100% for detecting INH, FQ, and SLID resistance, respectively, compared to pDST. In contrast, low sensitivity (51.9%) for ETH resistance was achieved because isolates harbored widespread mutations across the ethA gene. The specificity of Xpert MTB/XDR was 100% for all drugs except for INH (66.7%). Further investigation with WGS revealed -57c→t mutations in the oxyR-ahpC region marked with uncertain significance, which caused the low specificity for detecting INH resistance with the new assay. Xpert MTB/XDR can be used in clinical laboratories for the rapid detection of INH, FQ, and SLID resistance. Moreover, it can be used to rule in resistance to ETH. Additional use of WGS is recommended in cases of discrepant results between pDST and Xpert MTB/XDR. Future improvements of Xpert MTB/XDR with the inclusion of additional genes may increase the usefulness of the assay. IMPORTANCE The Xpert MTB/XDR was tested on drug-resistant Mycobacterium tuberculosis complex isolates from the Balkan Peninsula. Positive Bactec MGIT 960 cultures or DNA isolates were tested as starting material. According to the results of our study with Xpert MTB/XDR, sensitivities for the detection of SLID, FQ, and INH resistance were sufficient (>90%) for the assay to be implemented into diagnostic algorithms. In our study, WGS revealed lesser-known mutations in genes conferring INH and ETH resistance, and their impact on resistance is still unknown. Mutations in the ethA gene causing resistance to ETH were scattered along structural gene without high-confidence markers for resistance. Therefore, resistance to ETH should be reported based on a combination of methods. Because the Xpert MTB/XDR assay was found to have good performance, we propose that it should be the method of choice for confirming resistance to INH, FQ, and SLID and conditionally for resistance to ETH.