Gunnsteinn Haraldsson

Molecular epidemiology of candidemia: evidence of clusters of smoldering nosocomial infections

BACKGROUND Invasive fungal infections pose a serious threat to hospitalized patients worldwide. In particular, the prevalence of clusters of nosocomial infection among patients with candidemia remains unknown. The aim of this study was to investigate the molecular epidemiology of candidemia in a nationwide setting in Iceland during a 16-year period. METHODS The genotypes of all available fungal bloodstream isolates during 1991-2006 (n = 219) were determined by polymerase chain reaction fingerprinting with use of 4 separate primers. Clusters were defined as isolation of > or =2 strains with genotypes that had > or =90% relatedness in the same hospital within a period of 90 days. RESULTS Candida albicans represented 61.6% of isolates, followed by Candida glabrata (13.7%), Candida tropicalis (9.1%), and Candida parapsilosis (8.7%). Polymerase chain reaction fingerprinting revealed 35 clones of C. albicans, 10 clones of C. glabrata, 7 clones of C. tropicalis, 4 clones of C. parapsilosis, and 5 clones of Candida dubliniensis. Overall, 18.7%-39.9% of all infections were part of nosocomial clusters, most commonly caused by C. albicans, C. parapsilosis, and C. tropicalis. Most clusters involved 2 cases and disproportionately affected patients in adult and neonatal intensive care units (P = .045). The 7-day (16%) and 30-day (32%) case-fatality rates among cluster-associated cases did not differ statistically significantly from those for sporadic nosocomial infections. None of the clusters were identified by the hospital surveillance team. CONCLUSIONS In an unselected patient population, as many as one-third of all cases of candidemia may be attributable to nosocomial clusters. The risk is dependent on hospital wards and patient populations; it is highest in intensive care units. Small clusters are not identified by routine hospital surveillance.

Prevalence and Genetic Relatedness of Antimicrobial-Resistant Escherichia coli Isolated From Animals, Foods and Humans in Iceland

The prevalence of resistant bacteria in food products in Iceland is unknown, and little is known of the prevalence in production animals. The aim of this study was to investigate the prevalence and genetic relatedness of antimicrobial‐resistant Escherichia coli from healthy pigs and broiler chicken, pork, broiler meat, slaughterhouse personnel and outpatients in Iceland. A total of 419 E. coli isolates were tested for antimicrobial susceptibility using a microbroth dilution method (VetMIC), and resistant strains were compared using pulsed‐field gel electrophoresis (PFGE). All samples were screened for enrofloxacin‐resistant strains with selective agar plates. The resistance rates among E. coli isolates were moderate to high from caecal and meat samples of pigs (54.1% and 28%), broilers (33.6% and 52%) and slaughterhouse personnel (39.1%), whereas isolates from outpatients showed moderate resistance rates (23.1%). Of notice was resistance to quinolones (minimum inhibitory concentrations: nalidixic acid ≥ 32, ciprofloxacin ≥ 0.12 and enrofloxacin ≥ 0.5), particularly among broiler and broiler meat isolates (18.2% and 36%), as there is no known antimicrobial selection pressure in the broiler production in Iceland. The majority (78.6%) of the resistant E. coli isolates was genotypically different, based on PFGE fingerprint analyses and clustering was limited. However, the same resistance pattern and pulsotype were found among isolates from broiler meat and a slaughterhouse worker, indicating spread of antimicrobial‐resistant E. coli from animals to humans. Diverse resistance patterns and pulsotypes suggest the presence of a large population of resistant E. coli in production animals in Iceland. This study gives baseline information on the prevalence of antimicrobial‐resistant E. coli from production animals, and their food products in Iceland and the moderate to high resistance rates emphasize the need for continuing surveillance. Further studies on the origin of the resistant strains and the genetic relatedness of strains of different origin are needed.

Changing Epidemiology of Methicillin-Resistant Staphylococcus aureus in Iceland from 2000 to 2008: a Challenge to Current Guidelines

ABSTRACT The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is continuously changing. Iceland has a low incidence of MRSA. A “search and destroy” policy (screening patients with defined risk factors and attempting eradication in carriers) has been implemented since 1991. Clinical and microbiological data of all MRSA patients from the years 2000 to 2008 were collected prospectively. Isolates were characterized by pulsed-field gel electrophoresis (PFGE), sequencing of the repeat region of the Staphylococcus protein A gene (spa typing), staphylococcal cassette chromosome mec (SCCmec) typing, and screening for the Panton-Valentine leukocidin (PVL) gene. Two hundred twenty-six infected (60%) or colonized (40%) individuals were detected (annual incidence 2.5 to 16/100,000). From 2000 to 2003, two health care-associated outbreaks dominated (spa types t037 and t2802), which were successfully controlled with extensive infection control measures. After 2004, an increasing number of community-associated (CA) cases without relation to the health care system occurred. A great variety of clones (40 PFGE types and 49 spa types) were found, reflecting an influx of MRSA from abroad. The USA300 and Southwest Pacific (SWP) clones were common. SCCmec type IV was most common (72%), and 38% of the isolates were PVL positive. The incidence of MRSA in Iceland has increased since 1999 but remains low and has been stable in the last years. The search and destroy policy was effective to control MRSA in the health care setting. However, MRSA in Iceland is now shifting into the community, challenging the current Icelandic guidelines, which are tailored to the health care system.

Broiler Chickens as Source of Human Fluoroquinolone- Resistant Escherichia coli, Iceland

To investigate feed as a source for fluoroquinolone-resistant Escherichia coli in broiler chickens, we compared antimicrobial drug–resistant E. coli from broiler feed and broilers with ciprofloxacin-resistant human clinical isolates by using pulsed-field gel electrophoresis. Feed was implicated as a source for ciprofloxacin-resistant broiler-derived E. coli and broilers as a source for ciprofloxacin-resistant human-derived E. coli.

Genomics Reveals the Worldwide Distribution of Multidrug-Resistant Serotype 6E Pneumococci

ABSTRACT The pneumococcus is a leading pathogen infecting children and adults. Safe, effective vaccines exist, and they work by inducing antibodies to the polysaccharide capsule (unique for each serotype) that surrounds the cell; however, current vaccines are limited by the fact that only a few of the nearly 100 antigenically distinct serotypes are included in the formulations. Within the serotypes, serogroup 6 pneumococci are a frequent cause of serious disease and common colonizers of the nasopharynx in children. Serotype 6E was first reported in 2004 but was thought to be rare; however, we and others have detected serotype 6E among recent pneumococcal collections. Therefore, we analyzed a diverse data set of ∼1,000 serogroup 6 genomes, assessed the prevalence and distribution of serotype 6E, analyzed the genetic diversity among serogroup 6 pneumococci, and investigated whether pneumococcal conjugate vaccine-induced serotype 6A and 6B antibodies mediate the killing of serotype 6E pneumococci. We found that 43% of all genomes were of serotype 6E, and they were recovered worldwide from healthy children and patients of all ages with pneumococcal disease. Four genetic lineages, three of which were multidrug resistant, described ∼90% of the serotype 6E pneumococci. Serological assays demonstrated that vaccine-induced serotype 6B antibodies were able to elicit killing of serotype 6E pneumococci. We also revealed three major genetic clusters of serotype 6A capsular sequences, discovered a new hybrid 6C/6E serotype, and identified 44 examples of serotype switching. Therefore, while vaccines appear to offer protection against serotype 6E, genetic variants may reduce vaccine efficacy in the longer term because of the emergence of serotypes that can evade vaccine-induced immunity.

Severity of Influenza A 2009 (H1N1) Pneumonia Is Underestimated by Routine Prediction Rules. Results from a Prospective, Population-Based Study

Background Characteristics of patients with community-acquired pneumonia (CAP) due to pandemic influenza A 2009 (H1N1) have been inadequately compared to CAP caused by other respiratory pathogens. The performance of prediction rules for CAP during an epidemic with a new infectious agent are unknown. Methods Prospective, population-based study from November 2008–November 2009, in centers representing 70% of hospital beds in Iceland. Patients admitted with CAP underwent evaluation and etiologic testing, including polymerase chain reaction (PCR) for influenza. Data on influenza-like illness in the community and overall hospital admissions were collected. Clinical and laboratory data, including pneumonia severity index (PSI) and CURB-65 of patients with CAP due to H1N1 were compared to those caused by other agents. Results Of 338 consecutive and eligible patients 313 (93%) were enrolled. During the pandemic peak, influenza A 2009 (H1N1) patients constituted 38% of admissions due to CAP. These patients were younger, more dyspnoeic and more frequently reported hemoptysis. They had significantly lower severity scores than other patients with CAP (1.23 vs. 1.61, P = .02 for CURB-65, 2.05 vs. 2.87 for PSI, P<.001) and were more likely to require intensive care admission (41% vs. 5%, P<.001) and receive mechanical ventilation (14% vs. 2%, P = .01). Bacterial co-infection was detected in 23% of influenza A 2009 (H1N1) patients with CAP. Conclusions Clinical characteristics of CAP caused by influenza A 2009 (H1N1) differ markedly from CAP caused by other etiologic agents. Commonly used CAP prediction rules often failed to predict admissions to intensive care or need for assisted ventilation in CAP caused by the influenza A 2009 (H1N1) virus, underscoring the importance of clinical acumen under these circumstances.

Putatively novel serotypes and the potential for reduced vaccine effectiveness: capsular locus diversity revealed among 5405 pneumococcal genomes.

The pneumococcus is a leading global pathogen and a key virulence factor possessed by the majority of pneumococci is an antigenic polysaccharide capsule (‘serotype’), which is encoded by the capsular (cps) locus. Approximately 100 different serotypes are known, but the extent of sequence diversity within the cps loci of individual serotypes is not well understood. Investigating serotype-specific sequence variation is crucial to the design of sequence-based serotyping methodology, understanding pneumococcal conjugate vaccine (PCV) effectiveness and the design of future PCVs. The availability of large genome datasets makes it possible to assess population-level variation among pneumococcal serotypes and in this study 5405 pneumococcal genomes were used to investigate cps locus diversity among 49 different serotypes. Pneumococci had been recovered between 1916 and 2014 from people of all ages living in 51 countries. Serotypes were deduced bioinformatically, cps locus sequences were extracted and variation was assessed within the cps locus, in the context of pneumococcal genetic lineages. Overall, cps locus sequence diversity varied markedly: low to moderate diversity was revealed among serogroups/types 1, 3, 7, 9, 11 and 22; whereas serogroups/types 6, 19, 23, 14, 15, 18, 33 and 35 displayed high diversity. Putative novel and/or hybrid cps loci were identified among all serogroups/types apart from 1, 3 and 9. This study demonstrated that cps locus sequence diversity varied widely between serogroups/types. Investigation of the biochemical structure of the polysaccharide capsule of major variants, particularly PCV-related serotypes and those that appear to be novel or hybrids, is warranted.

Prevalence of pilus genes in pneumococci isolated from healthy preschool children in Iceland: association with vaccine serotypes and antibiotic resistance

OBJECTIVES The objective of this study was to investigate the prevalence of pilus islets [pilus islet 1 (PI-1) and pilus islet 2 (PI-2)] in pneumococcal isolates from healthy Icelandic preschool children attending day care centres, prior to the introduction of conjugated pneumococcal vaccine, and the association of the pilus islets with vaccine serotypes and antibiotic resistance. METHODS Nasopharyngeal swabs were collected from 516 healthy children attending day care centres in Reykjavik in March and April 2009. Infant vaccination was started in 2011, thus the great majority of the children were unvaccinated. Pneumococci were cultured selectively, tested for antimicrobial susceptibility and serotyped. The presence of PI-1 and PI-2 was detected using PCR. RESULTS A total of 398 viable isolates were obtained of which 134 (33.7%) showed the presence of PI-1. PI-1-positive isolates were most often seen in serotype 19F [30/31 (96.8%)] and were of clade I, and in 6B [48/58 (82.8%)] of clade II. PI-2-positive isolates were most common in serotype 19F [27/31 (87.1%)]; all of them were also PI-1 positive. Of the PI-1-positive and PI-2-positive isolates, 118 (88.1%) and 31 (81.6%), respectively, were of vaccine serotypes. Both PI-1 and PI-2 were more often present in penicillin-non-susceptible pneumococci (PNSP) than in penicillin-susceptible pneumococci [PI-1 in 41/58 (70.7%) and 93/340 (27.4%), respectively, and PI-2 in 28/58 (48.3%) and 10/340 (2.9%), respectively]. CONCLUSIONS Genes for PI-1 and/or PI-2 in pneumococci isolated from healthy Icelandic children are mainly found in isolates of vaccine serotypes and in PNSP isolates belonging to multiresistant international clones that have been endemic in the country.

Cocolonization of Pneumococcal Serotypes in Healthy Children Attending Day Care Centers: Molecular Versus Conventional Methods.

Objectives: Pneumococci are common colonizer, especially of children, and cocolonization of different serotypes is an important factor for intraspecies genetic exchange. The aim of this study was to analyze pneumococcal carriage and serotype distribution in unvaccinated healthy children in Iceland and compare conventional culture methods and molecular methods using DNA extracted directly from the samples. Methods: Nasopharyngeal swabs were obtained from 514 children aged 2–6 year attending day care centers in Reykjavik in 2009. The swabs were selectively cultured for pneumococci and the isolates serotyped using latex agglutination. DNA was also extracted directly from the swabs and serotyped using a multiplex PCR panel designed to detect vaccine serotypes and the most commonly carried non-vaccine serotypes. Result: Pneumococcal carriage was detected in 391 (76.1%) of the children using polymerase chain reaction (PCR) and in 371 (72.2%) using conventional methods. Cocolonization was detected in 92 (23.5%) of the carriers when PCR method was used and in 30 (8.1%) when conventional methods were used, detecting 500 and 401 strains, respectively (P < 0.0001). The most common serotypes were 23F, 19A, 6B, 6A and 19F, rates 13–8%. The number of isolates of serotypes included in the 10-valent and 13-valent vaccines and detected by PCR were 234 (58.4%) and 363 (90.5%), respectively and by conventional methods 186 (46.4%) and 293 (73.1%), respectively. Conclusion: Cocolonization was detected in a fourth of the children carrying pneumococci using DNA extracted directly from nasopharyngeal swabs. The rate of carriage was very high, but no serotype dominated, and the children were commonly colonized by vaccine serotypes, especially cocolonized children.

Heterogeneity Among Estimates Of The Core Genome And Pan-Genome In Different Pneumococcal Populations

Background Understanding the structure of a bacterial population is essential in order to understand bacterial evolution, or which genetic lineages cause disease, or the consequences of perturbations to the bacterial population. Estimating the core genome, the genes common to all or nearly all strains of a species, is an essential component of such analyses. The size and composition of the core genome varies by dataset, but our hypothesis was that variation between different collections of the same bacterial species should be minimal. To test this, the genome sequences of 3,121 pneumococci recovered from healthy individuals in Reykjavik (Iceland), Southampton (United Kingdom), Boston (USA) and Maela (Thailand) were analysed. Results The analyses revealed a ‘supercore’ genome (genes shared by all 3,121 pneumococci) of only 303 genes, although 461 additional core genes were shared by pneumococci from Reykjavik, Southampton and Boston. Overall, the size and composition of the core genomes and pan-genomes among pneumococci recovered in Reykjavik, Southampton and Boston were very similar, but pneumococci from Maela were distinctly different. Inspection of the pan-genome of Maela pneumococci revealed several >25 Kb sequence regions that were homologous to genomic regions found in other bacterial species. Conclusions Some subsets of the global pneumococcal population are highly heterogeneous and thus our hypothesis was rejected. This is an essential point of consideration before generalising the findings from a single dataset to the wider pneumococcal population.