Use Of Whole-genome Sequencing Research Articles

Utility of SARS-CoV-2 Genomic Sequencing for Understanding Transmission and School Outbreaks.

An understanding of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) transmission in schools is important. It is often difficult, using epidemiological information alone, to determine whether cases associated with schools represent multiple introductions from the community or transmission within the school. We describe the use of whole genome sequencing (WGS) in multiple schools to investigate outbreaks of SARS-CoV-2 in the pre-Omicron period. School outbreaks were identified for sequencing by local public health units based on multiple cases without known epidemiological links. Cases of SARS-CoV-2 from students and staff from 4 school outbreaks in Ontario underwent WGS and phylogenetic analysis. The epidemiological clinical cohort data and genomic cluster data are described to help further characterize these outbreaks. A total of 132 positive SARS-CoV-2 cases among students and staff from 4 school outbreaks were identified with 65 (49%) of cases able to be sequenced with high-quality genomic data. The 4 school outbreaks consisted of 53, 37, 21 and 21 positive cases; within each outbreak there were between 8 and 28 different clinical cohorts identified. Among the sequenced cases, between 3 and 7 genetic clusters, defined as different strains, were identified in each outbreak. We found genetically different viruses within several clinical cohorts. WGS, together with public health investigation, is a useful tool to investigate SARS-CoV-2 transmission within schools. Its early use has the potential to better understand when transmission may have occurred, can aid in evaluating how well mitigation interventions are working and has the potential to reduce unnecessary school closures when multiple genetic clusters are identified.

Point-Counterpoint: What's in a Name? Clinical Microbiology Laboratories Should Use Nomenclature Based on Current Taxonomy.

INTRODUCTION The mnemonic SPICE (Serratia, Pseudomonas, indole-positive Proteus, Citrobacter, and Enterobacter) has served as a reminder to consider when a Gram-negative organism may carry a chromosomal copy of blaampC, with the associated risk of developing resistance to first-, second-, and third-generation cephalosporins. However, in 2017, there was a well-founded proposal to rename Enterobacter aerogenes to Klebsiella aerogenes, based on whole-genome sequencing (WGS), and the SPICE mnemonic lost its relevance. With the increased use of WGS for taxonomy, it seems like bacteria and fungi are undergoing constant name changes. These changes create unique challenges for clinical microbiology laboratories, who would like to issue reports that are readily understood and that help clinicians determine empirical antibiotic therapy, interpret antimicrobial resistance, and understand clinical significance. In this Point-Counterpoint, Drs. Karen Carroll and Erik Munson discuss the pros of updating bacterial taxonomy and why clinical labs must continue to update reporting, while Drs. Susan Butler-Wu and Sheila Patrick argue for caution in adopting new names for microorganisms.

Whole-Genome and Plasmid Comparative Analysis of Campylobacter jejuni from Human Patients in Toyama, Japan, from 2015 to 2019.

Campylobacter jejuni is a major causative agent of food poisoning, and increasing antimicrobial resistance is a concern. This study investigated 116 clinical isolates of C. jejuni from Toyama, Japan, which were isolated from 2015 to 2019. Antimicrobial susceptibility testing and whole-genome sequencing were used for phenotypic and genotypic characterization to compare antimicrobial resistance (AMR) profiles and phylogenic linkage. The multilocus sequence typing approach identified 37 sequence types (STs) and 15 clonal complexes (CCs), including 7 novel STs, and the high frequency CCs were CC21 (27.7%), CC48 (10.9%), and CC354 (9.9%). The AMR profiles and related resistant factors were as follows: fluoroquinolones (51.7%), mutation in quinolone resistance-determining region (QRDRs) (GyrA T86I); tetracyclines (27.6%), acquisition of tet(O); ampicillin (7.8%), harboring blaOXA184 or a promoter mutation in blaOXA193; aminoglycosides (1.7%), acquisition of ant(6)-Ia and aph(3')-III; chloramphenicol (0.9%), acquisition of cat. The acquired resistance genes tet(O), ant(6)-Ia, aph(3')-III, and cat were located on pTet family plasmids. Furthermore, three pTet family plasmids formed larger plasmids that incorporated additional genes such as the type IV secretion system. Sequence type 4526 (ST4526; 10.9%), which is reported only in Japan, was the most predominant, suggesting continued prevalence. This study reveals the sequences of the pTet family plasmids harbored by C. jejuni in Japan, which had been unclear, and the acquisition of the insertion sequences in a part of the pTet family plasmids. Because pTet family plasmids can be horizontally transmitted and are a major factor in acquired resistance in Campylobacter, the risk of spreading pTet that has acquired further resistance should be considered. IMPORTANCE Campylobacter jejuni is among the major causes of enteritis and diarrhea in humans in many countries. Drug-resistant Campylobacter is increasing in both developing and developed countries, and in particular, fluoroquinolone-resistant Campylobacter was one of the species included on the priority list of antibiotic-resistant bacteria. Campylobacter drug resistance surveillance is important and has been conducted worldwide. In this study, we performed whole-genome analysis of Campylobacter jejuni isolated from diarrhea patients at a hospital in Toyama, Japan. This revealed the continued prevalence of Campylobacter jejuni ST4526, which has been reported to be prevalent in Japan, and the acquisition of resistance and virulence factors in the pTet family plasmids. The diversity of pTet family plasmids, the major resistance transmission factor, is expected to potentially increase the risk of Campylobacter. The usefulness of whole-genome sequencing in Campylobacter surveillance was also demonstrated.

Use of Whole Genome Sequencing as Routine Typing Method — Improvements in the Investigation of Foodborne Outbreaks of Listeria monocytogenes

Use of Whole Genome Sequencing as Routine Typing Method — Improvements in the Investigation of Foodborne Outbreaks of <i>Listeria monocytogenes</i>

How can DAFM best make use of whole genome sequencing to improve the effectiveness of the TB eradication programme?

How can DAFM best make use of whole genome sequencing to improve the effectiveness of the TB eradication programme?

Reader Response: Use of Whole-Genome Sequencing for Mitochondrial Disease Diagnosis.

We were interested to read this study, in which the authors applied a “genomics first” approach in 242 adult patients.1 They identified 3 patients from 2 families with an adult-onset peroxisomal disorder, alpha-methylacyl-CoA racemase deficiency. This condition was identified in 3 families with suspected mitochondrial disorders in the 100,000 Genomes Project and 1 patient in the whole-exome sequencing study by Theunissen et al.2,3

Author Response: Use of Whole-Genome Sequencing for Mitochondrial Disease Diagnosis.

We strongly agree with Dr. Schon and Prof. Chinnery that alpha-methylacyl-CoA racemase (AMACR) deficiency should be considered in the differential diagnosis of patients presenting with combinations of stroke-like episodes, seizures, encephalopathy, and retinal pigmentary changes.1 As described in our study,1 causative AMACR variants in patients with clinical features suggestive of mitochondrial disease highlight a broader issue of mitochondrial disease phenocopies that require consideration by a comprehensive sequencing approach for suspected mitochondrial disease cases. Examples of this include 3 individuals from 2 families in our study,1 3 individuals in the 100,000 Genomes Project,2 an individual in the study by Theunissen et al,3 and a recent case of an individual with late-onset disease associated with stroke-like episodes and seizures.4

Antimicrobial Resistance and Whole Genome Sequencing Analysis of Neisseria gonorrhoeae Isolates in Bulgaria, 2019

Neisseria gonorrhoeae is evolving into a superbug with resistance to previously and currently recommended antimicrobials for treatment of gonorrhoea, which creates a significant public health concern globally. Antimicrobial resistance (AMR) poses a great threat; it causes longer duration of illness and treatment, and increases health care costs and the social, psychological and financial burden to families and societies. In response to this worrying situation laboratory research should be strengthened with implementation of newer molecular methods for monitoring and detecting AMR, and evaluation of the correlation of obtained genetic data with corresponding phenotypes.&#x0D; This study aimed to implement whole genome sequencing analysis for the first time of N. gonorrhoeae isolates from Bulgaria and to acquire molecular epidemiological and AMR information linked with phenotypic resistance data.&#x0D; Although the tested strains were susceptible to current recommended dual therapy, genetic analysis showed the presence of several mutations responsible for reducing susceptibility to third generation cephalosporins, namely the presence of genotype G1407 strains in the Bulgarian population. Regarding previously recommended antimicrobials, penicillin resistance and intermediate sensitivity to tetracyclines was demonstrated in all strains, and fluoroquinolone resistance in two strains.&#x0D; The obtained results showed that the use of whole genome sequencing was feasible, could describe current circulating gonococcal strains and predict and infer transmission of antimicrobial resistance. Therefore, gonococcal infections prevention and control programmes will be aided to target interventions where needed and to revise treatment guidelines that will help improve patient care.

A Practical Bioinformatics Workflow for Routine Analysis of Bacterial WGS Data.

The use of whole-genome sequencing (WGS) for bacterial characterisation has increased substantially in the last decade. Its high throughput and decreasing cost have led to significant changes in outbreak investigations and surveillance of a wide variety of microbial pathogens. Despite the innumerable advantages of WGS, several drawbacks concerning data analysis and management, as well as a general lack of standardisation, hinder its integration in routine use. In this work, a bioinformatics workflow for (Illumina) WGS data is presented for bacterial characterisation including genome annotation, species identification, serotype prediction, antimicrobial resistance prediction, virulence-related genes and plasmid replicon detection, core-genome-based or single nucleotide polymorphism (SNP)-based phylogenetic clustering and sequence typing. Workflow was tested using a collection of 22 in-house sequences of Salmonella enterica isolates belonging to a local outbreak, coupled with a collection of 182 Salmonella genomes publicly available. No errors were reported during the execution period, and all genomes were analysed. The bioinformatics workflow can be tailored to other pathogens of interest and is freely available for academic and non-profit use as an uploadable file to the Galaxy platform.

Use of Whole-Genome Sequencing to Estimate the Contribution of Immune Evasion and Waning Immunity on Decreasing COVID-19 Vaccine Effectiveness.

The impact variant-specific immune evasion and waning protection have on declining coronavirus disease 2019 (COVID-19) vaccine effectiveness (VE) remains unclear. Using whole-genome sequencing (WGS), we examined the contribution these factors had on the decline that followed the introduction of the Delta variant. Furthermore, we evaluated calendar-period-based classification as a WGS alternative. We conducted a test-negative case-control study among people tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) between 1 April and 24 August 2021. Variants were classified using WGS and calendar period. We included 2029 cases (positive, sequenced samples) and 343 727 controls (negative tests). VE 14-89 days after second dose was significantly higher against Alpha (84.4%; 95% confidence interval [CI], 75.6%-90.0%) than Delta infection (68.9%; 95% CI, 58.0%-77.1%). The odds of Delta infection were significantly higher 90-149 than 14-89 days after second dose (P value = .003). Calendar-period-classified VE estimates approximated WGS-classified estimates; however, calendar-period-based classification was subject to misclassification (35% Alpha, 4% Delta). Both waning protection and variant-specific immune evasion contributed to the lower effectiveness. While calendar-period-classified VE estimates mirrored WGS-classified estimates, our analysis highlights the need for WGS when variants are cocirculating and misclassification is likely.

Evaluation of molecular typing for national surveillance of invasive clinical Haemophilus influenzae isolates from Denmark.

Haemophilus influenzae is a gram-negative coccobacillus known to cause respiratory and invasive infections. It can possess a polysaccharide capsule that can be categorized into six different serotypes (i.e., Hia, Hib, Hic, Hid, Hie, and Hif) and non-encapsulated strains that are defined as non-typeable. Furthermore, H. influenzae can be characterized into eight biotypes (I-VIII). Traditionally, isolates have been serotyped and biotyped using phenotypic methods; however, these methods are not always reliable. In this study, we evaluate the use of whole-genome sequencing (WGS) for national surveillance and characterization of clinical Danish H. influenzae isolates. In Denmark, all clinical invasive isolates between 2014 and 2021 have been serotyped using a traditional phenotypic latex agglutination test as well as in silico serotyped using the in silico programs "hinfluenzae_capsule_characterization" and "hicap" to compare the subsequent serotypes. Moreover, isolates were also biotyped using a phenotypic enzyme test and the genomic data for the detection of the genes encoding ornithine, tryptophan, and urease. The results showed a 99-100% concordance between the two genotypic approaches and the phenotypic serotyping, respectively. The biotyping showed a 95% concordance between genotyping and phenotyping. In conclusion, our results show that in a clinical surveillance setting, in silico serotyping and WGS-based biotyping are a robust and reliable approach for typing clinical H. influenzae isolates.

Diagnostic Utility of Whole Genome Sequencing in Adults with B-Other Acute Lymphoblastic Leukaemia

Background B-other ALL represents a working definition for patients with B cell precursor (BCP) ALL without a known primary chromosomal abnormality at diagnosis. In this study we use whole genome sequencing (WGS) to characterize the molecular pathogenesis of adult B-other cases (age ≥ 25yrs) from the UKALL14 trial (NCT01085617). Methods Of 652 patients aged 25-65yrs enrolled onto UKALL14, 333 (51%) had B-other ALL. Sufficient material was available to screen 156/333 B-other cases for recurrent Ph-like fusion events (CRLF2, JAK2, ABL1, ABL2 & PDGFRB) using FISH and MLPA (kit P335). This identified 28 (18%) "Ph-like" fusion events (21 CRLF2, 5 ABL-class and 2 JAK2). Of the remaining 128 B-other cases 57 had available samples for tumor normal paired WGS (read depth 60x and 30x respectively). Diagnostic RNA was available for 33 of the 57 cases Bioinformatic analysis was performed to determine small somatic mutations; single nucleotide variants (SNVs) and insertion/deletions (INDELs) as well as copy number aberrations (CNA) and structural variants (SV). We developed WGS approaches to identify IGH enhancer hijack and DUX4 rearrangements, utilizing gGnome and GRIDSS respectively. For 33 cases RNA-seq data was analyzed to validate fusion genes and genetic classification into one of 18 ALL subtypes using the ALLSorts v2 classifier. Results 52/57 cases passed QC. In 5 (10%) cases an established WHO2016 genetic subtype was identified that was missed by standard of care including three high-risk (2 low hypodiploid and a near haploid) and 2 standard risk (high hyperdiploid and TCF3::PBX1) cases. Cytogenetic analysis for these 5 cases either failed (n=2) or produced a normal karyotype (n=3). The WGS inferred tumor purity for these 5 cases was variable with a median of 46% and a range of 33% to 96% compared to 89% (range 13%-99%) for the remaining cases (n=47) in the dataset. Among the remaining 47 B-other cases, WGS analysis identified abnormalities enabling 41 (87%) cases to be classified into a genetic ALL subtype: DUX4 rearrangements (DUX4r, n=8), PAX5alt (n=7), PAX5 P80R (n=4), ZNF384 rearrangements (ZNF384r, n=5), ZEB2/CEBP (n=4), MEF2D rearrangements (MEF2Dr, n=3), UBTF::ATXN7L3 fusions (n=2), IDH1/2 mutations (n=2), JAK-STAT abnormalities (n=2), along with single cases of IGK::BCL2, IGH::CEBPA, IGH::ID4 and IGH::MIR125B1. WGS resolves 8 cases with complex karyotype into one of 6 different genomic subtypes: 2xDUX4r, 2xMEF2Dr, ZEB2/CEBP, UBTF::ATXN7L3, IGK::BCL2 & IGH::ID4. The MEF2Dr subtype (n=3), is enriched in chromothripsis (2/3), and complex SVs targeting the MEF2D gene. Lastly, in all cases with PAX5 mutations (PAX5 P80R (n=4) and PAX5alt (n=4)) we observe bi-allelic targeting of locus by mutations, deletions or LOH. The remaining 6 (13%) B-other cases could not be classified into a recognized B-other subtype. A MYO18A::FGFR1 fusion (t(8;17)(p11;q23)), which most likely maps to the WHO2016 "Lymphoid neoplasms with FGFR1 rearrangement" classification. Two cases had clonal frameshifts in genes associated with T-ALL (PTPN2 p.A108fs*5 and WT1 p.V371fs*14 respectively). A case with a somatic ETV6 p.R399C previously reported in germline risk with a ZNF292::KDM6A fusion. Lastly a case with a clonal mutation typically associated with myeloid disease ASXL1 p.R417*. Among the 52 cases studied, WGS only failed to identify a driver or putative driver in one case, which had the lowest burden of SNVs (861) and an estimated tumor purity, 28%. For the 31 cases with both DNA and RNA, WGS and RNA analysis were concordant for 100% of cases, with RNA identifying an additional KDM6A::CGA fusion. In 3 cases WGS provides evidence that would have challenged diagnosis and/or treatment; IGK::BCL2 with BCL2 and MYC overexpression, IGH::DUX4 with hypermutation (MSH6 loss) and IGH::ID4 with homozygous loss of CD58 and concurrent LOH of HLA-B. Conclusions WGS assigned 88% (46/52) of cases called B-other to an established genetic subtype of ALL. This includes 5 cases with WHO2016 defined subtypes, 3 were high-risk ploidy subtypes that would have changed their UKALL14 risk group and post-induction treatment in the absence of other risk factors. Among the true B-other cases, 87% (41/47) were assigned to one of the newly described genetic subtypes of ALL. Taken together our findings demonstrate the clinical utility of WGS as a diagnostic assay to inform and improve the management of adult B-Other ALL patients in the future.

Genomic Characterization of Colistin-Resistant Isolates from the King Fahad Medical City, Kingdom of Saudi Arabia.

Whole-genome sequencing is one of the best ways to investigate resistance mechanisms of clinical isolates as well as to detect and identify circulating multi-drug-resistant (MDR) clones or sub-clones in a given hospital setting. Here, we sequenced 37 isolates of Acinetobacter baumannii, 10 Klebsiella pneumoniae, and 5 Pseudomonas aeruginosa collected from the biobank of the hospital setting of the King Fahad Medical City. Complete phenotypic analyses were performed, including MALDI-TOF identification and antibiotic susceptibility testing. After the genome assembly of raw data, exhaustive genomic analysis was conducted including full resistome determination, genomic SNP (gSNP) analysis, and comparative genomics. Almost all isolates were highly resistant to all tested antibiotics, including carbapenems and colistin. Resistome analysis revealed many antibiotic resistance genes, including those with resistance to β-lactams, aminoglycosides, macrolides, tetracyclines, sulfamids, quinolones, and phenicols. In A. baumannii isolates, the endemic carbapenemase blaOXA-23 gene was detected in 36 of the 37 isolates. Non-synonymous mutations in pmrB were detected in almost all of the isolates and likely mediated colistin resistance. Interestingly, while classical analyses, such as MLST, revealed the predominance of an ST2 clone in A. baumannii isolates, the genomic analysis revealed the presence of five circulating sub-clones and identified several isolate transmissions between patients. In the 10 K. pneumoniae isolates, several resistance genes were identified, and the observed carbapenem resistance was likely mediated by overexpression of the detected extended-spectrum-β-lactamase (ESBL) genes associated with low membrane permeability as few carbapenemase genes were detected with just blaOXA-48 in three isolates. Colistin resistance was mediated either by non-synonymous mutations in the MgrB regulator, PmrA, PmrB, and PhoQ proteins or the presence of the MCR-1 protein. Here, gSNP analysis also revealed the existence of bacterial clones and cases of isolate transmissions between patients. The five analyzed P. aeruginosa isolates were highly resistant to all tested antibiotics, including carbapenems mediated by loss or truncated OprD porin, and colistin resistance was associated with mutations in the genes encoding the PmrA, PmrB, or PhoQ proteins. We demonstrate here the usefulness of whole-genome sequencing to exhaustively investigate the dissemination of MDR isolates at the sub-clone level. Thus, we suggest implementing such an approach to monitor the emergence and spread of new clones or sub-clones, which classical molecular analyses cannot detect. Moreover, we recommend increasing the surveillance of the endemic and problematic colistin resistance mcr-1 gene to avoid extensive dissemination.

Phylogenetic analyses of Salmonella detected along the broiler production chain in Trinidad and Tobago

This study was conducted to determine the phylogenies of Salmonella strains isolated from cross-sectional studies conducted at hatcheries, broiler farms, processing plants, and retail outlets (broiler production chain) in Trinidad and Tobago over 4 yr (2016-2019). Whole-genome sequencing (WGS) was used to characterize Salmonella isolates. Core genome phylogenies of 8 serovars of public health significance were analyzed for similarities in origin and relatedness. In addition, Salmonella strains isolated from human salmonellosis cases in Trinidad were analyzed for their relatedness to the isolates detected along the broiler production chain. The common source of these isolates of diverse serovars within farms, within processing plants, between processing plants and retail outlets, and among farm-processing plant-retail outlet continuum was well-supported (bootstrap value >70%) by the core genome phylogenies for the respective serovars. Also, genome analyses revealed clustering of Salmonella serovars of regional (intra-Caribbean) and international (extra-Caribbean) origin. Similarly, strains of S. Enteritidis and S. Infantis isolated from human clinical salmonellosis in 2019 from Trinidad and Tobago clustered with our processing plant isolates recovered in 2018. This study is the first phylogenetic analysis of Salmonella isolates using WGS from the broiler industry in the Caribbean region. The use of WGS confirmed the genetic relatedness and transmission of Salmonella serovars contaminating chickens in broiler processing, and retailing in the country, with zoonotic and food safety implications for humans.

The Development of an Infrastructure to Facilitate the Use of Whole Genome Sequencing for Population Health.

The clinical use of genomic analysis has expanded rapidly resulting in an increased availability and utility of genomic information in clinical care. We have developed an infrastructure utilizing informatics tools and clinical processes to facilitate the use of whole genome sequencing data for population health management across the healthcare system. Our resulting framework scaled well to multiple clinical domains in both pediatric and adult care, although there were domain specific challenges that arose. Our infrastructure was complementary to existing clinical processes and well-received by care providers and patients. Informatics solutions were critical to the successful deployment and scaling of this program. Implementation of genomics at the scale of population health utilizes complicated technologies and processes that for many health systems are not supported by current information systems or in existing clinical workflows. To scale such a system requires a substantial clinical framework backed by informatics tools to facilitate the flow and management of data. Our work represents an early model that has been successful in scaling to 29 different genes with associated genetic conditions in four clinical domains. Work is ongoing to optimize informatics tools; and to identify best practices for translation to smaller healthcare systems.

Lessons learned: use of WGS in real-time investigation of suspected intrahospital SARS-CoV-2 outbreaks

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been a continuing source of hospital-acquired infection and outbreaks. At Akershus University Hospital in Norway, traditional contact tracing has been combined with whole-genome sequencing (WGS) surveillance in real-time to investigate potential hospital outbreaks. To describe the advantages and challenges encountered when using WGS as a real-time tool in hospital outbreak investigation and surveillance during the SARS-CoV-2 pandemic. Routine contact tracing in the hospital was performed for all healthcare workers (HCWs) who tested positive for SARS-CoV-2. Viral RNA from all positive patient and HCW samples was sequenced in real-time using nanopore sequencing and the ARTIC Network protocol. Suspected outbreaks involving five or more individuals with viral sequences were described. Nine outbreaks were suspected based on contact tracing, and one outbreak was suspected based on WGS results. Five outbreaks were confirmed; of these, two outbreaks were supported but could not be confirmed by WGS with high confidence, one outbreak was found to consist of two different lineages, and two outbreaks were refuted. WGS is a valuable tool in hospital outbreak investigations when combined with traditional contact tracing. Inclusion of WGS data improved outbreak demarcation, identified unknown transmission chains, and highlighted weaknesses in existing infection control measures.

WIN Symposium 2022 - Abstract Poster Presentations.

WIN Symposium 2022 - Abstract Poster Presentations.

Whole-Genome Sequencing-Based Characterization of Listeria Isolates from Produce Packinghouses and Fresh-Cut Facilities Suggests Both Persistence and Reintroduction of Fully Virulent L. monocytogenes.

The contamination of ready-to-eat produce with Listeria monocytogenes (LM) can often be traced back to environmental sources in processing facilities and packinghouses. To provide an improved understanding of Listeria sources and transmission in produce operations, we performed whole-genome sequencing (WGS) of LM (n = 169) and other Listeria spp. (n = 107) obtained from 13 produce packinghouses and three fresh-cut produce facilities. Overall, a low proportion of LM isolates (9/169) had inlA premature stop codons, and a large proportion (83/169) had either or both of the LIPI-3 or LIPI-4 operons, which have been associated with hypervirulence. The further analysis of the WGS data by operation showed a reisolation (at least 2 months apart) of highly related isolates (<10 hqSNP differences) in 7/16 operations. Two operations had highly related strains reisolated from samples that were collected at least 1 year apart. The identification of isolates collected during preproduction (i.e., following sanitation but before the start of production) that were highly related to isolates collected during production (i.e., after people or products have entered and begun moving through the operation) provided evidence that some strains were able to survive standard sanitation practices. The identification of closely related isolates (<20 hqSNPs differences) in different operations suggests that cross-contamination between facilities or introductions from common suppliers may also contribute to Listeria transmission. Overall, our data suggest that the majority of LM isolates collected from produce operations are fully virulent and that both persistence and reintroduction may lead to the repeat isolation of closely related Listeria in produce operations. IMPORTANCE Listeria monocytogenes is of particular concern to the produce industry due to its frequent presence in natural environments as well as its ability to survive in packinghouses and fresh-cut processing facilities over time. The use of whole-genome sequencing, which provides high discriminatory power for the characterization of Listeria isolates, along with detailed source data (isolation date and sample location) shows that the presence of Listeria in produce operations appears to be due to random and continued reintroduction as well as to the persistence of highly related strains in both packinghouses and fresh-cut facilities. These findings indicate the importance of using high-resolution characterization approaches for root cause analyses of Listeria contamination issues. In cases of repeat isolation of closely related Listeria in a given facility, both persistence and reintroduction need to be considered as possible root causes.

A two-ward acute care hospital outbreak of SARS-CoV-2 delta variant including a point-source outbreak associated with the use of a mobile vital signs cart and sub-optimal doffing of personal protective equipment

BackgroundWe conducted a detailed epidemiological investigation of a linked 2-ward COVID-19 Delta variant outbreak to elucidate its source, risk factors, and control measures.MethodsInvestigations included epidemiologic analysis, detailed case review serial SARS-CoV-2 RT-PCR testing of patients and healthcare workers (HCWs), viral culture, environmental swabbing, HCW-unaware personal protective equipment (PPE) audits, ventilation assessments, and the use of whole genome sequencing (WGS).ResultsThis linked 2-ward outbreak resulted in 17 patient and 12 HCW cases, despite an 83% vaccination rate. In this setting suboptimal adherence and compliance to PPE protocols, suboptimal hand hygiene, multi-bedded rooms, and a contaminated vital signs cart (VSC) with potential fomite or spread via the hands of HCWs were identified as significant risk factors for nosocomial COVID-19 infection.Sudden onset of symptoms, within 72h, was observed in 79% of all Ward 2 patients and 93% of all cases (patients and HCWs) on Ward 2 occurred within one incubation period, consistent with a point source outbreak. RT-PCR assays showed low Cycle threshold (Ct) values, indicating high viral load from environmental swabs including the VSC. WGS results with ≤ 3 SNP differences between specimens were observed.ConclusionsOutbreaks on both wards settled rapidly, within 3 weeks, using a ‘back-to-basics’ approach without extraordinary measures or changes to standard PPE requirements. Strict adherence to recommended PPE, hand hygiene, education, assistance from infected cases (interviews and testing), and additional measures such as limiting movement of patients and staff temporarily were all deemed to have contributed to prompt resolution of the outbreak.

Multidrug-resistant tuberculosis in Finland: treatment outcome and the role of whole-genome sequencing.

Treatment of multidrug-resistant tuberculosis (MDR-TB) is a global challenge requiring long treatment with costly drugs. We assessed treatment combinations, outcome and the utility of whole-genome sequencing (WGS) in MDR-TB cases. Clinical, demographic and microbiological data were obtained of all patients with MDR-TB who started treatment in Finland in 2007-2016. Definitions of MDR, pre-extensively drug-resistant (pre-XDR) and XDR tuberculosis were those applicable at the study period. Treatment outcome was defined according to World Health Organization (WHO) guidelines. Mycobacterium tuberculosis isolates were analysed by WGS in addition to routinely performed phenotypic drug susceptibility testing and genotyping. Among the 47 cases, 35 (74%) had a successful treatment outcome. Risk factors for non-successful outcome were Finnish origin and XDR. Almost 90% of our cases had an adverse event for at least one drug. Phenotypic and WGS drug resistance results were fully concordant for isoniazid, fluoroquinolones and amikacin, and >90% concordant for rifampicin, pyrazinamide, kanamycin and capreomycin. >60% of phenotypically ethambutol-susceptible isolates were genotypically resistant. The results of the rifampicin and isoniazid nucleic acid amplification tests (NAATs) performed for the isolates were identical to the WGS results except for three isolates having uncommon resistance mutations not included in the NAATs. WGS did not reveal unexpected clustering. More training is needed for physicians treating MDR-TB, and especially XDR-TB, to improve treatment outcome. Phenotypic drug susceptibility testing was shown to be unreliable for ethambutol. WGS could aid in the selection of optimal treatment regimen in the future.