Hospital Outbreak Investigation Research Articles

Identifying Candida auris transmission in a hospital outbreak investigation using whole-genome sequencing and SNP phylogenetic analysis.

Candida auris poses a global public health challenge, causing multiple outbreaks within healthcare facilities. Despite advancements in strain typing for various infectious diseases, a consensus on the genetic relatedness threshold for identifying C. auris transmission in local hospital outbreaks remains elusive. We investigated genetic variations within our local isolate collection using whole-genome-based single nucleotide polymorphism (SNP) phylogenetic analysis. A total of 74 C. auris isolates were subjected to whole-genome sequencing (WGS) and SNP phylogenetic analysis via the QIAGEN CLC Genomics Workbench. Isolates included known related strains from the same patient, strains from different hospitals, strains from our hospital patients with no epidemiological link, and 19 patient isolates from a recent C. auris outbreak. All but three isolates were identified to be Clade IV. By examining the genetic diversities of C. auris within patients and between patients, we identified a SNP variation range of 0-13 for identifying related isolates. During an outbreak investigation, utilizing this range, maximum likelihood phylogenetic analysis revealed two distinct clusters that aligned with the epidemiological links. Determining a SNP variation range to delineate genetic relatedness among isolates is crucial for the application of WGS and SNP phylogenetic analysis in identifying C. auris transmission during hospital outbreak investigations. The use of WGS SNP phylogenetic analysis via the CLC Genomics Workbench has emerged as a valuable method for typing C. auris in clinical microbiology laboratories.

Antimicrobial Resistance Surveillance: Data Harmonisation and Data Selection within Secondary Data Use.

Resistance to last-resort antibiotics is a global threat to public health. Therefore, surveillance and monitoring systems for antimicrobial resistance should be established on a national and international scale. For the development of a One Health surveillance system, we collected exemplary data on carbapenem and colistin-resistant bacterial isolates from human, animal, food, and environmental sources. We pooled secondary data from routine screenings, hospital outbreak investigations, and studies on antimicrobial resistance. For a joint One Health evaluation, this study incorporates epidemiological metadata with phenotypic resistance information and molecular data on the isolate level. To harmonise the heterogeneous original information for the intended use, we developed a generic strategy. By defining and categorising variables, followed by plausibility checks, we created a catalogue for prospective data collections and applied it to our dataset, enabling us to perform preliminary descriptive statistical analyses. This study shows the complexity of data management using heterogeneous secondary data pools and gives an insight into the early stages of the development of an AMR surveillance programme using secondary data.

Investigation of a Donor-derived Carbapenamase-producing Carbapenem-resistant Enterobacterales Hospital Outbreak

Carbapenamase-producing carbapenem-resistant Enterobacterales (CP-CRE) is an urgent public health threat for healthcare facilities. Solid organ transplant (SOT) recipients carry an increased risk for CRE infection and colonization due to prolonged exposures to antimicrobials, healthcare facilities and immunosuppression. CRE infection in SOT patients is associated with an increase in morbidity and mortality. Here, we describe a hospital outbreak investigation of three cases of New Delhi metallo-beta-lactamase (NDM) - CRE that led to novel findings with implications for further interdisciplinary investigations. An NDM-CRE infection in a critically-ill patient was identified during passive surveillance and prompted an investigation. Previous CP-CRE passive surveillance cases were reviewed. Rectal screening was performed for potentially exposed patients. 403 rectal swabs were tested for carbapenemase genes in active surveillance. Patients identified to have a new NDM-CRE isolate on active or passive surveillance were considered cases and underwent in-depth chart review including possible patient-to-patient exposures, hospital locations, procedures, devices, and consultations. NDM-CRE isolates were sent to the Minnesota Department of Health (MDH) for whole genome sequencing (WGS) to assess relatedness. Five NDM-CRE cases were identified, with all isolates harboring blaNDM including three NDM-Klebsiella pneumoniae (NDM-KP) cases (Figure 1). The first NDM-KP case, patient 1, developed mediastinal infection following lung transplantation. Review of United Network for Organ Sharing revealed that respiratory specimens from patient 1’s donor grew NDM-KP and a bronchial wash at the time of transplant yielded NDM-KP. The second NDM-KP case (patient 3) developed ventilator-associated pneumonia and was found to have used sequentially the same ventilator as patient 1. The third NDM-KP case (patient 4) was detected via rectal swab in active surveillance and shared wound care personnel in common with patients 1 and 3 (Figure 2). WGS demonstrated two single nucleotide polymorphisms (SNP) among all three isolates, strongly suggesting relatedness (Figure 3). Best practices for infection prevention were reviewed with wound care personnel. To date, no further NDM-KP isolates have been identified. Investigation was facilitated by in-depth chart review and WGS via the Central Region Antimicrobial Resistance Laboratory Network at MDH. Detection of the NDM-KP from a lung donor specimen appears genetically linked to clinical isolates in other patients, raising the possibility of a donor-derived hospital outbreak. This investigation is the first to describe a donor-derived NDM outbreak in a healthcare facility. Communication between organ procurement agencies, transplant centers, and infection prevention must be optimized to prevent CRE-associated morbidity in SOT receipts and CRE hospital outbreaks.

VirulenceFinder for Enterococcus faecium and Enterococcus lactis: an enhanced database for detection of putative virulence markers by using whole-genome sequencing data.

Enterococcus faecium (Efm) is a leading cause of hospital-associated (HA) infections, often enriched in putative virulence markers (PVMs). Recently, the Efm clade B was assigned as Enterococcus lactis (Elts), which usually lack HA-Efm infection markers. Available databases for extracting PVM are incomplete and/or present an intermix of genes from Efm and Enterococcus faecalis, with distinct virulence profiles. In this study, we constructed a new database containing 27 PVMs [acm, scm, sgrA, ecbA, fnm, sagA, hylEfm, ptsD, orf1481, fms15, fms21-fms20 (pili gene cluster 1, PGC-1), fms14-fms17-fms13 (PGC-2), empA-empB-empC (PGC-3), fms11-fms19-fms16 (PGC-4), ccpA, bepA, gls20-glsB1, and gls33-glsB] from nine reference genomes (seven Efm + two Elts). The database was validated against these reference genomes and further evaluated using a collection of well-characterized Efm (n = 43) and Elts (n = 7) control strains, by assessing PVM presence/absence and its variants together with a genomic phylogeny constructed as single-nucleotide polymorphisms. We found a high concordance between the phylogeny and in silico findings of the PVM, with Elts clustering separately and mostly carrying Elts-specific PVM gene variants. Based on our validation results, we recommend using the database with raw reads instead of assemblies to avoid missing gene variants. This newly constructed database of 27 PVMs will enable a more comprehensive characterization of Efm and Elts based on WGS data. The developed database exhibits scalability and boasts a range of applications in public health, including diagnostics, outbreak investigations, and epidemiological studies. It can be further used in risk assessment for distinguishing between safe and unsafe enterococci.IMPORTANCEThe newly constructed database, consisting of 27 putative virulence markers, is highly scalable and serves as a valuable resource for the comprehensive characterization of these closely related species using WGS data. It holds significant potential for various public health applications, including hospital outbreak investigations, surveillance, and risk assessment for probiotics and feed additives.

2476. Universal detection of healthcare-related bacterial transmissions using de novo assembly with short-read whole genome sequencing

Abstract Background Whole genome sequencing (WGS) is the gold standard for molecular evidence of pathogen transmission. To date, most WGS investigations within healthcare settings have used low cost short-read technologies (e.g., Illumina), aligning reads to a high quality reference genome. However, this relies on either public repositories having closely-related high quality references, which is not guaranteed for arbitrary clinical isolates, or additional long-read sequencing. De novo assembly avoids reliance on references, but the utility of fragmented assemblies of short reads is not well-established in hospital outbreak investigations. Methods 24 clusters of clinical isolates were identified by automated and routine methods at 17 U.S. hospitals and underwent paired-end WGS on Illumina platforms, followed by two parallel analyses. For a reference-free analysis, de novo assembly was performed with Shovill, assemblies were clustered with Mash, core-genome alignments were created with Harvest, and finally single nucleotide polymorphism (SNP) distances were visualized using the PathoSPOT phylogenomics toolkit to highlight likely transmission events. For a reference-guided analysis, references were selected for each isolate using StrainGST, and SNPs called by read alignment were used to define phylogenomic distances. Finally, conclusions from both analyses were compared. Results 92 bacterial isolates underwent WGS, of which 86 (93%) completed de novo assembly and passed quality control, comprising 10 isolates of Acinetobacter baumanii, 4 of Klebsiella oxytoca, 46 of Klebsiella pneumoniae, and 26 of Staphylococcus aureus. Reference-free analysis with PathoSPOT identified close relatedness consistent with transmission in 13 of 24 clusters (54%), while the reference-guided analysis identified the same in 12 clusters (50%). Overall, 21 clusters (88%) showed concordance between reference-free and reference-guided methods. Cohen’s kappa indicated substantial agreement (0.75; 95% CI, 0.49–1.0). Conclusion De novo assembly of short reads is tenable for WGS investigation of hospital outbreaks, elides the need for reference genomes or long-read sequencing, and compares favorably with SNP calling via read alignment for detecting likely healthcare-related transmissions. Disclosures Laura E. McLean, M.Ed., HCA Healthcare: Stocks/Bonds Susan S. Huang, MD MPH, Medline Industries, Inc: Conducted studies whereby participating nursing homes and hospital patients received cleaning & antiseptic products|Xttrium Laboratories: Conducted studies where participating nursing homes and hospital patients received antiseptic products Yonatan H. Grad, MD, PhD, Day Zero Diagnostics: Board Member|GSK: Advisor/Consultant

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.

NGSocomial Infections: High-Resolution Views of Hospital-Acquired Infections Through Genomic Epidemiology.

Hospital outbreak investigations are high-stakes epidemiology. Contacts between staff and patients are numerous; environmental and community exposures are plentiful; and patients are highly vulnerable. Having the best data is paramount to understanding an outbreak in order to stop ongoing transmission and prevent future outbreaks. In the past 5 years, the high-resolution view of transmission offered by analyzing pathogen whole-genome sequencing (WGS) is increasingly part of hospital outbreak investigations. Concerns over speed and actionability, assay validation, liability, cost, and payment models lead to further opportunities for work in this area. Now accelerated by funding for COVID-19, the use of genomics in hospital outbreak investigations has firmly moved from the academic literature to more quotidian operations, with associated concerns involving regulatory affairs, data integration, and clinical interpretation. This review details past uses of WGS data in hospital-acquired infection outbreaks as well as future opportunities to increase its utility and growth in hospital infection prevention.

Mucormycosis in pediatric oncology patients: a hospital outbreak investigation report

Mucormycosis in pediatric oncology patients: a hospital outbreak investigation report

A two-step approach for the investigation of a Clostridium difficile outbreak by molecular methods

Abstract The recent review “How to: molecular investigation of a hospital outbreak” written by Nutman and Marchaim published in Clinical Microbiology and Infection journal summarized data on typing methods used for a hospital outbreak investigation. Regarding Clostridium difficile infection (CDI), a whole genome sequencing (WGS) was recommended as the initial and a single molecular test for characterisation of C. difficile isolates in a suspected outbreak situation. According to results from our survey on typing capacity for C. difficile in Europe, whole genome sequencing was available only in five National reference laboratories for C. difficile in 2017 in Europe. Because of limited availability of WGS, we suggest a two-step approach that includes the typing of C. difficile isolates with a high discriminatory method, capillary electrophoresis ribotyping and the subtyping of C. difficile isolates by Multilocus Variable-number Tandem-repeats Analysis (MLVA) and/or WGS when a common cluster of CDIs is confirmed. This two-step approach would provide an epidemiological relevant timeframe for confirmation of a suspected CDI outbreak as well as being cost-effective by selecting highly related C. difficile isolates for further investigation.

Methodology for Whole-Genome Sequencing of Methicillin-Resistant Staphylococcus aureus Isolates in a Routine Hospital Microbiology Laboratory.

There is growing evidence for the value of bacterial whole-genome sequencing in hospital outbreak investigations. Our aim was to develop methods that support efficient and accurate low-throughput clinical sequencing of methicillin-resistant Staphylococcus aureus (MRSA) isolates. Using a test panel of 25 MRSA isolates previously associated with outbreak investigations, we devised modifications to library preparation that reduced the processing time by 1 hour. We determined the maximum number of isolates that could be sequenced per run using an Illumina MiniSeq platform and a 13-hour (overnight) run time, which equated to 21 MRSA isolates and 3 controls (no template, positive, and negative). Repeatability and reproducibility assays based on this sequencing methodology demonstrated 100% accuracy in assigning species and sequence type (ST) and in detecting mecA Established genetic relatedness between isolates was recapitulated. Quality control (QC) metrics were evaluated over nine sequencing runs. Of the test panel MRSA genomes, 168/173 (97%) passed QC metrics based on the correct species assigned, detection of mecA and ST, and depth/coverage metrics. An evaluation of contamination in these 9 runs showed that positive and negative controls and test MRSA sequence files contained <0.14% and <0.48% of fragments that matched another species, respectively. Deliberate contamination experiments confirmed that this was insufficient to impact data interpretation. These methods support reliable and reproducible clinical MRSA sequencing with a turnaround time (from DNA extraction to availability of data files) of 24 hours.

Next-Generation-Sequencing-Based Hospital Outbreak Investigation Yields Insight into Klebsiella aerogenes Population Structure and Determinants of Carbapenem Resistance and Pathogenicity.

Klebsiella aerogenes is a nosocomial pathogen associated with drug resistance and outbreaks in intensive care units. In a 5-month period in 2017, we experienced an increased incidence of cultures for carbapenem-resistant K. aerogenes (CR-KA) from an adult cardiothoracic intensive care unit (CICU) involving 15 patients. Phylogenomic analysis following whole-genome sequencing (WGS) identified the outbreak CR-KA isolates to group together as a tight monoclonal cluster (with no more than six single nucleotide polymorphisms [SNPs]), suggestive of a protracted intraward transmission event. No clonal relationships were identified between the CICU CR-KA strains and additional hospital CR-KA patient isolates from different wards and/or previous years. Carbapenemase-encoding genes and drug-resistant plasmids were absent in the outbreak strains, and carbapenem resistance was attributed to mutations impacting AmpD activity and membrane permeability. The CICU outbreak strains harbored an integrative conjugative element (ICE) which has been associated with pathogenic Klebsiella pneumoniae lineages (ICEKp10). Comparative genomics with global K. aerogenes genomes showed our outbreak strains to group closely with global sequence type 4 (ST4) strains, which, along with ST93, likely represent dominant K. aerogenes lineages associated with human infections. For poorly characterized pathogens, scaling analyses to include sequenced genomes from public databases offer the opportunity to identify emerging trends and dominant clones associated with specific attributes, syndromes, and geographical locations.

Prospective study of the feasibility of point-of-care testing strategy for carbapenem-resistant organism detection.

Background/aims In an investigator-initiated, prospective study, we evaluated the feasibility of a five-gene sequence point-of-care (POC) testing strategy (Xpert CARBA-R Assay, Cepheid Inc., Sunnyvale, CA, USA), compared to reference laboratory PCR (48 – 72 hours turnaround time, two gene sequences), in patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) and in a hospital outbreak investigation. Methods After informed consent, patients undergoing ERCP (September 2015 – April 2016, n = 191) at Mayo Clinic and potential hospital contacts (n = 9) of an index carbapenem-resistant organism (CRO)-positive inpatient were included. Two rectal swabs, one each for reference and POC assays were obtained. The Xpert CARBA-R Assay enables qualitative rapid detection of five beta-lactamase gene sequences associated with carbapenem-non-susceptibility in Gram-negative bacteria. Feasibility parameters (specimen processing and assay run time, ease of use) and percent agreement between the tests were calculated using JMP Pro11 (SAS Corp, Cary, NC, USA). Results Mean age was 62 ± 15 years; 108 (54 %) were male. Both tests were successfully performed in all patients. The POC test was rated by endoscopy nurses as easy/very easy to conduct in 193 patients (97 %); median assay run time and median time for specimen collection and processing were 55 minutes (interquartile range IQR: 53 – 55 minutes) and 3 minutes (IQR: 3 – 6 minutes), respectively. In 200/201 (99.5 %) tests, there was agreement between the POC and reference PCR. Conclusions The more comprehensive POC CRO testing of patients in the endoscopy suite is feasible and results are available in < 1 hour. This strategy may enable rapid risk stratification of duodenoscope exposure to CRO and potentially improve operational efficiency and decrease costs.

A Novel Peptidomic Approach to Strain Typing of Clinical Acinetobacter baumannii Isolates Using Mass Spectrometry.

Acinetobacter baumannii is a common nosocomial pathogen and strain-typing methods play an important role in hospital outbreak investigations and epidemiologic surveillance. We describe a method for identifying strain-specific peptide markers based on LC-MS/MS profiling of digested peptides. This method classified a test set of A. baumannii isolates collected from a hospital outbreak with discriminatory performance exceeding that of MALDI-TOF mass spectrometry. Following the construction of a species "pan-peptidome" by in silico translation and digestion of whole genome sequences, a hypothetical set of genome-specific peptides for an isolate was constructed from the disjoint set of the pan-peptidome and the isolate's calculated peptidome. The genome-specific peptidome guided selection of highly expressed genome-specific peptides from LC-MS/MS experimental profiles as potential peptide markers. The species specificity of each experimentally identified genome-specific peptide was confirmed through a Unipept lowest common ancestor analysis. Fifteen A. baumannii isolates were analyzed to derive a set of genome- and species-specific peptides that could be used as peptide markers. Identified peptides were cross-checked with protein BLAST against a set of 22 A. baumannii whole genome sequences. A subset of these peptide markers was confirmed to be present in the actual peptide profiles generated by multiple reaction monitoring and targeted LC-MS/MS. The experimentally identified peptides separated these isolates into 6 strains that agreed with multilocus sequence typing analysis performed on the same isolates. This approach may be generalizable to other bacterial species, and the peptides may be useful for rapid MS strain tracking of isolates with broad application to infectious disease diagnosis.

Sequence analysis of the capsid gene during a genotype II.4 dominated norovirus season in one university hospital: identification of possible transmission routes.

Norovirus (NoV) is a leading cause of gastroenteritis and genotype II.4 (GII.4) is responsible for the majority of nosocomial NoV infections. Our objective was to examine whether sequencing of the capsid gene might be a useful tool for the hospital outbreak investigation to define possible transmission routes. All NoV positive samples submitted from one university hospital during the 2007/8 season were selected. Genotyping of selected samples by partial polymerase gene sequencing had shown that the majority belonged to the GII.4 variant Den Haag 2006b and had identical polymerase sequences. Sequences of the capsid gene (1412 nucleotides) were obtained from the first available sample from 55 patients. From six immunocompromised patients with persistent infections a second sample was also included. As a control for a point-source outbreak, five samples from a foodborne outbreak caused by the same GII.4 variant were analyzed. Forty-seven of the inpatients (85%) were infected with the GII.4 variant Den Haag 2006b. Phylogenetic analysis of the Den Haag 2006b sequences identified four distinct outbreaks in different departments and a fifth outbreak with possible inter-department spread. In addition, a more heterogeneous cluster with evidence of repeated introductions from the community, but also possible inter-department spread was observed. In all six patients with paired sequences, evidence for in vivo evolution of the virus was found. Capsid gene sequencing showed substantial sequence variation among NoV GII.4 variant Den Haag 2006b strains from one single institution during a nine months’ period. This method proved useful to understand the local epidemiology and, when used promptly, has the potential to make infection control measures more targeted.

Mucormycosis Outbreak Associated With Hospital Linens

Mucormycosis is an invasive fungal infection with a high fatality rate. We investigated an outbreak of mucormycosis in a pediatric hospital to determine routes of pathogen transmission from the environment and prevent additional infections. A case was defined as a hospital-onset illness consistent with mucormycosis, confirmed by culture or histopathology. Case-patient medical records were reviewed for clinical course and exposure to items and locations within the hospital. Environmental samples were collected from air and surfaces. Fungal isolates collected from case-patients and the environmental samples were identified using DNA sequencing. Five case-patients had hospital-associated cutaneous mucormycosis over an 11-month period; all subsequently died. Three case-patients had conditions known to be associated with susceptibility to mucormycosis, while 2 had cardiac conditions with persistent acidosis. The cases occurred on several different wards throughout the hospital, and hospital linens were the only exposure identified as common to the case-patients. Rhizopus species were recovered from 26 (42%) of 62 environmental samples from clean linens and associated areas and from 1 (4%) of 25 samples from nonlinen-related items. Case-patients were infected with Rhizopus delemar, which was also isolated from cultures of clean linens and clean linen delivery bins from the off-site laundry facility. Hospital linens were identified as a vehicle that carried R. delemar into contact with susceptible patients. Fungal species identification using DNA-based methods is useful for corroborating epidemiologic links in hospital outbreak investigations. Hospital linens should be laundered, packaged, shipped and stored in a manner that minimizes exposure to environmental contaminants.

Genome-wide single nucleotide polymorphism-based assay for high-resolution epidemiological analysis of the methicillin-resistant Staphylococcus aureus hospital clone EMRSA-15

The EMRSA-15 clone is a major cause of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections in the UK and elsewhere but existing typing methodologies have limited capacity to discriminate closely related strains, and are often poorly reproducible between laboratories. Here, we report the design, development and validation of a genome-wide single nucleotide polymorphism (SNP) typing method and compare it to established methods for typing of EMRSA-15. In order to identify discriminatory SNPs, the genomes of 17 EMRSA-15 strains, selected to represent the breadth of genotypic and phenotypic diversity of EMRSA-15 isolates in Scotland, were determined and phylogenetic reconstruction was carried out. In addition to 17 phylogenetically informative SNPs, five binary markers were included to form the basis of an EMRSA-15 genotyping assay. The SNP-based typing assay was as discriminatory as pulsed-field gel electrophoresis, and significantly more discriminatory than staphylococcal protein A (spa) typing for typing of a representative panel of diverse EMRSA-15 strains, isolates from two EMRSA-15 hospital outbreak investigations, and a panel of bacteraemia isolates obtained in healthcare facilities in the east of Scotland during a 12-month period. The assay is a rapid, and reproducible approach for epidemiological analysis of EMRSA-15 clinical isolates in Scotland. Unlike established methods the DNA sequence-based method is ideally suited for inter-laboratory comparison of identified genotypes, and its flexibility lends itself to supplementation with additional SNPs or markers for the identification of novel S. aureus strains in other regions of the world.

A “pre-seasonal” hospital outbreak of influenza pneumonia caused by the drift variant A/Victoria/361/2011-like H3N2 viruses, Hong Kong, 2011

BackgroundBeginning from late 2011 and early 2012, increasing circulation of antigenically drifted influenza A/Victoria/361/2011-like H3N2 viruses within genotype 3 of the A/Victoria/208/2009 clade have been reported in multiple European countries and elsewhere. Whether these emerging viruses are associated with increased disease severity is unclear. ObjectivesTo report the clinical and virological findings of a moderately severe hospital outbreak of A/Victoria/361/2011-like viruses that occurred in November 2011 in Hong Kong. Study designClinical and virological hospital outbreak investigation. ResultsThe outbreak occurred in an adult psychiatric ward in November 2011, a time well before the usual local seasonal influenza winter peak. Altogether, 7 patients and 1 healthcare-worker were affected (mean age, 47 [range, 34–61] years). The attack rates among patients and healthcare-workers were 33% (7/21) and 7% (1/15), respectively. Pneumonia developed in 38% (3/8) of cases; none had underlying immunocompromised conditions. High nasopharyngeal viral loads were detected. All cases responded to antiviral treatment. Multiple amino acid mutations with reference to earlier A(H3N2) vaccine strains were mapped to key antigenic sites on hemagglutinin; however, no critical mutations on receptor binding sites were detected. Viral sequence variations jeopardized the performance of molecular diagnostic assays. ConclusionsSevere disease and pneumonia occurred in a substantial proportion of non-immunocompromised adults in a hospital outbreak attributed to the emerging antigenically drifted A/Victoria/361/2011-like H3N2 viruses. Close monitoring of the transmission of this drift variant is required. Further studies are also necessary to determine virus virulence.

Comparison of Two Multilocus Variable-Number Tandem-Repeat Methods and Pulsed-Field Gel Electrophoresis for Differentiating Highly Clonal Methicillin-Resistant Staphylococcus aureus Isolates

In the United Kingdom, EMRSA-15 and EMRSA-16 account for the majority (∼90%) of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections. Currently, the standard typing technique, pulsed-field gel electrophoresis (PFGE), is laborious and insufficient for discriminating between closely related subtypes of EMRSA-15 and -16. The objective of the present study was to compare the usefulness of multilocus variable-number tandem-repeat fingerprinting (MLVF) and multilocus variable-number tandem-repeat analysis (MLVA) with PFGE for subtyping these highly clonal MRSA lineages. A panel of 85 MRSA isolates (41 EMRSA-15, 20 EMRSA-16, and 24 MRSA isolates with diverse PFGE patterns) was investigated. In addition, a further 29 EMRSA-15s with identical PFGE patterns from two geographically linked but epidemiologically distinct outbreaks and several sporadic cases were analyzed. PFGE, MLVF, and MLVA resolved 66 (Simpson's index of diversity [SID] = 0.984), 51 (SID = 0.95), and 42 (SID = 0.881) types, respectively, among the 85 MRSA isolates. MLVF was more discriminatory than MLVA for EMRSA-15 and -16 strains, but both methods had comparable discriminatory powers for distinguishing isolates in the group containing diverse PFGE types. MLVF was comparable to PFGE for resolving the EMRSA-15s but had a lower discriminatory power for the EMRSA-16s. MLVF and MLVA resolved the 29 isolates with identical PFGE patterns into seven and six subtypes, respectively. Importantly, both assays indicated that the two geographically related outbreaks were caused by distinct subtypes of EMRSA-15. Taken together, the data suggest that both methods are suitable for identifying and tracking specific subtypes of otherwise-indistinguishable MRSA. However, due to its greater discriminatory power, MLVF would be the most suitable alternative to PFGE for hospital outbreak investigations.