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

BACKGROUND: 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. AIM: 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. METHODS: 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. FINDINGS: 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. CONCLUSIONS: 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.

Investigation of intra-hospital SARS-CoV-2 transmission using nanopore whole-genome sequencing.

BACKGROUND: During the SARS-CoV-2 pandemic, healthcare workers (HCWs) are being exposed to infection both at work and in their communities. Determining where HCWs might have been infected is challenging based on epidemiological data alone. At Akershus University Hospital, Norway, several clusters of possible intra-hospital SARS-CoV-2 transmission were identified based on routine contact tracing. AIM: To determine whether clusters of suspected intra-hospital SARS-CoV-2 transmission could be resolved by combining whole genome sequencing (WGS) of SARS-CoV-2 with contact tracing data. METHODS: Epidemiological data were collected during routine contact tracing of polymerase chain reaction-confirmed SARS-CoV-2-positive HCWs. Possible outbreaks were identified as wards with two or more infected HCWs defined as close contacts who tested positive for SARS-CoV-2 less than three weeks apart. Viral RNA from naso-/oropharyngeal samples underwent nanopore sequencing in direct compliance to the ARTIC Network protocol. FINDINGS: Five outbreaks were suspected from contact tracing. Viral consensus sequences from 24 HCWs, two patients, and seven anonymous samples were analysed. Two outbreaks were confirmed, one refuted, and two remained undetermined. One new potential outbreak was discovered. CONCLUSION: Combined with epidemiological data, nanopore WGS was a useful tool for investigating intra-hospital SARS-CoV-2 transmission. WGS helped to resolve questions about possible outbreaks and to guide local infection prevention and control measures.

Staphylococcus aureus colonization during military service: a prospective cohort study.

OBJECTIVES: Staphylococcus aureus colonization leading to skin and soft-tissue infections (SSTI) are known challenges in crowded settings such as the military. The aim of the study was to establish and compare the prevalence of S. aureus colonization in recruits at enrolment and discharge after the first year of military service, and to investigate the prevalence of S. aureus SSTI. METHODS: All recruits entering first year of military service in January 2013 to be stationed at three garrisons in the northern part of Norway were invited to join this prospective cohort study. Swabs were taken from nose, throat and perineum. Staphylococcus aureus was identified using standard culturing methods. Methicillin resistance was determined by a cefoxitin disc diffusion test. RESULTS: Of the 923 eligible recruits, 512 were included at enrolment; 265/512 (52%) were also screened at discharge. Staphylococcus aureus colonization was high, and increased significantly during military service (166/265 versus 224/265, p < 0.001) mainly caused by increase in throat colonization alone or in combination with nasal colonization. All S. aureus isolates were susceptible to methicillin. SSTI was self-reported in 7/265 (3%) recruits, of which only one was confirmed by a military physician. CONCLUSION: Staphylococcus aureus colonization increased during military service, but there were few confirmed reports of SSTI. Inclusion of throat swab provides important information as ∼20% of the recruits were only positive in their throat. Further analyses need to be performed to investigate if the increase in colonization is caused by specific S. aureus stains.

Emerging multidrug-resistant Bengal Bay clone ST772-MRSA-V in Norway: molecular epidemiology 2004-2014.

A multidrug-resistant, methicillin-resistant Staphylococcus aureus (MRSA) clone, PVL-positive ST772-MRSA-V, named the Bengal Bay clone, is emerging worldwide. In Norway, where MRSA prevalence is low, a sudden increase in ST772-MRSA-V initiated a nationwide molecular epidemiological study. Clinical data were obtained from the Norwegian Surveillance System for Communicable Diseases (MSIS). S. aureus isolates were characterised by antibiotic susceptibility profiles and comprehensive genotyping (spa typing, MLVA, DNA microarray). ST772-MRSA was detected in 145 individuals during 2004-2014, with 60% of cases occurring in 2013-2014. Median age was 31 years and male/female ratio 1.16. The majority had a family background from the Indian subcontinent (70%). MRSA acquisition was mainly reported as unknown (39%) or abroad (42%), the latter associated with a home-country visit (59%), tourism (16%), and immigration (13%). Clinical infection was present in 75%, predominantly by SSTI (83%), 18% were admitted to hospital and 42% were linked to small-scale outbreaks (n = 25). All isolates were multidrug-resistant. Most isolates were resistant to erythromycin, gentamicin and norfloxacin. Genotyping revealed a conserved clone predominated by spa type t657 (83%), MLVA-type 432 (67%) and the genes lukF/S, sea, sec/sel, egc, scn, cna, ccrAA/ccrC, agrII and cap5. A few untypical ccr gene combinations were detected. Bengal Bay isolates have likely been imported on several occasions and revision of infection control guidelines may prevent further spread.

Bengal Bay clone ST772-MRSA-V outbreak: conserved clone causes investigation challenges.

BACKGROUND: The Bengal Bay clone, ST772-MRSA-V, associated with multi-drug resistance, Panton-Valentine leukocidin (PVL) and skin and soft tissue infections, is emerging worldwide. In Norway, a country with low prevalence of meticillin-resistant Staphylococcus aureus (MRSA), increased occurrence of ST772-MRSA-V has also caused hospital outbreaks. The conserved nature of this clone challenged the outbreak investigations. AIM: To evaluate the usefulness of S. aureus protein A (spa) typing, multiple-locus variable number tandem repeat fingerprinting/analysis (MLVF/MLVA) and pulsed-field gel electrophoresis (PFGE) when investigating outbreaks with a conserved MRSA clone. METHODS: A panel of 25 MRSA isolates collected in 2004-2014, consisting of six hospital outbreak isolates and 19 sporadic isolates, were analysed using spa typing, polymerase chain reaction detection of genes encoding PVL, MLVF/MLVA and PFGE. FINDINGS: All isolates were ST772-MRSA-V-t657 and resistant to erythromycin, gentamicin and norfloxacin, and 88% were PVL positive. PFGE could not discriminate between the isolates (≥85% similarity). MLVF resolved five types [Simpson's index of diversity (SID)=0.56], MLVA resolved six types (SID=0.66), and both methods separated the hospital isolates into two defined outbreaks. CONCLUSION: MLVF/MLVA could not discriminate all epidemiologically unlinked cases and identical genotypes originated from a timespan of 10 years. MLVA was regarded as most suitable due to its higher discriminatory power and ability to provide unambiguous profiles. However, the Bengal Bay clone may require higher resolution methods for exact demarcation of outbreaks due to low diversity among isolates.

DNA microarray analysis of Staphylococcus aureus causing bloodstream infection: bacterial genes associated with mortality?

Providing evidence for microbial genetic determinants' impact on outcome in Staphylococcus aureus bloodstream infections (SABSI) is challenging due to the complex and dynamic microbe-host interaction. Our recent population-based prospective study reported an association between the S. aureus clonal complex (CC) 30 genotype and mortality in SABSI patients. This follow-up investigation aimed to examine the genetic profiles of the SABSI isolates and test the hypothesis that specific genetic characteristics in S. aureus are associated with mortality. SABSI isolates (n = 305) and S. aureus CC30 isolates from asymptomatic nasal carriers (n = 38) were characterised by DNA microarray analysis and spa typing. Fisher's exact test, least absolute shrinkage and selection operator (LASSO) and elastic net regressions were performed to discern within four groups defined by patient outcome and characteristics. No specific S. aureus genetic determinants were found to be associated with mortality in SABSI patients. By applying LASSO and elastic net regressions, we found evidence suggesting that agrIII and cna were positively and setC (=selX) and seh were negatively associated with S. aureus CC30 versus non-CC30 isolates. The genes chp and sak, encoding immune evasion molecules, were found in higher frequencies in CC30 SABSI isolates compared to CC30 carrier isolates, indicating a higher virulence potential. In conclusion, no specific S. aureus genes were found to be associated with mortality by DNA microarray analysis and state-of-the-art statistical analyses. The next natural step is to test the hypothesis in larger samples with higher resolution methods, like whole genome sequencing.

Population-based epidemiology of Staphylococcus aureus bloodstream infection: clonal complex 30 genotype is associated with mortality.

Staphylococcus aureus bloodstream infections (SABSI) are associated with a high burden of morbidity and mortality. The impact of specific S. aureus genotypes on outcome is unclear. The aim of this study was to evaluate the epidemiology and outcome of SABSI, with a special emphasis on the impact of bacterial clonal lineage on mortality. We conducted a 3-year population-based prospective study between 2011 and 2014, including 303 consecutive adult patients. Clinical data were obtained from interviews and medical records. S. aureus isolates were genotyped using DNA microarrays. The incidence rate of SABSI was 27.6 per 100,000 inhabitants [95 % confidence interval (CI) 24.6-31.0]. The median age of the patients was 71 years (interquartile range 56-81 years) and 61.4 % were male. Most SABSI (70.6 %) occurred in hospitals or associated to healthcare, and 34.1 % of these were associated with intravascular catheters. Only five (1.6 %) SABSI were caused by methicillin-resistant S. aureus (MRSA). The 30-day case fatality rate was 20.8 % (95 % CI 16.6-25.7). S. aureus clonal complex 30 [hazard ratio (HR) 3.9; 95 % CI 1.8-8.5, p = 0.001], unknown focus of infection (HR 4.5; 95 % CI 1.9-10.8, p = 0.001) and respiratory tract infection (HR 12.7; 95 % CI 4.6-34.6, p < 0.001) were independent predictors of mortality in a Cox regression analysis after adjusting for age, sex and underlying conditions. A high proportion of potential preventable SABSI calls for effective infection control measures. S. aureus clonal complex 30 genotype was associated with mortality in patients with bloodstream infections. The genetic basis underlying this association remains to be demonstrated.

No change in the distribution of types and antibiotic resistance in clinical Staphylococcus aureus isolates from orthopaedic patients during a period of 12 years.

Staphylococcus aureus (S. aureus) is the most common cause of bone and joint infections. However, limited information is available on the distribution of S. aureus geno- and phenotypes causing orthopaedic infections. The aim of this study was to identify the dominating types causing infections in orthopaedic patients, investigate if the characteristics of these types changed over time and examine if different types were more often associated with surgical site infection (SSI) than primary infection (non-SSI). All clinical S. aureus isolates collected from orthopaedic patients from 2000 through 2011 at Akershus University Hospital, Norway, were characterised by S. aureus protein A (spa) typing and tested for antibiotic resistance. A total of 548 patients with orthopaedic S. aureus infections were included, of which 326 (59 %) had SSI and 222 (41 %) had non-SSI. The median age was 62 years [range 2-97 years] and 54 % were male. Among the 242 unique spa types, t084 was the most common (7 %). Penicillin resistance was identified in 75 % of the isolates, whereas the resistances to the other antibiotics tested were <5 %. Three isolates (0.5 %) were resistant to methicillin. There was no significant difference in the distribution of geno- and phenotypes over time and there was no difference in types between SSI and non-SSI. In this large collection of S. aureus from orthopaedic patients, the S. aureus infections, regardless of origin, were heterogeneous, mainly resistant to penicillin, stable over time and consisted of similar types as previously found in both carrier and other patient populations.

Molecular characterisation of methicillin-sensitive Staphylococcus aureus from deep surgical site infections in orthopaedic patients.

Staphylococcus aureus is a leading cause of surgical site infections (SSIs). The association between S. aureus genotypes and the severity of illness is, however, incompletely understood. The aim of the study was to genotype S. aureus isolates from deep SSI in orthopaedic patients to identify molecular markers associated with invasive S. aureus infections. DNA microarray analysis was performed on S. aureus isolates collected from 60 patients with deep SSI following major orthopaedic surgery, while 57 isolates from nasal carriers served as controls. Genes associated with antibiotic resistance, adhesion, immune evasion, tissue invasion and toxin production were detected. The bone sialoprotein-binding protein gene (bbp) was more frequent in isolates from SSI patients compared to nasal carriers (95.0% vs. 82.5%), suggesting a role in invasive disease. No major differences in other molecular virulence markers could be distinguished among isolates from the two clinical groups, suggesting that any S. aureus strain may cause invasive infection. Our study reveals important genotypic information on isolates obtained from deep SSI following orthopaedic procedures.

A marginal zone phenotype in follicular lymphoma with t(14;18) is associated with secondary cytogenetic aberrations typical of marginal zone lymphoma.

Marginal zone differentiation of follicular lymphomas (FL), sometimes referred to as monocytoid B-cell differentiation, is a relatively uncommon phenomenon. Recently, this type of differentiation was also linked to secondary cytogenetic aberrations of chromosome 3 in a small number of patients. We have analysed 131 primary nodal FL with t(14;18)(q32;q21) for secondary cytogenetic aberrations previously described as recurrent in marginal zone lymphomas (MZL) to identify their frequency and possible association with morphological evidence of marginal zone differentiation. We searched for trisomy of chromosomes 3, 12, and 18, gains of chromosome arm 3q, deletions of chromosome arm 7p, structural anomalies with break-points in 1q21 and 1p34, as well as the t(1;2)(p22;p12), t(1;14)(p22;q32), t(3;14)(q27;q32), t(6;14)(p21;q32), and t(11;18)(q21;q21) translocations. At least focal morphological evidence of marginal zone differentiation occurred in 35/131 (27%) FL with t(14;18)(q32;q21) as the primary chromosomal abnormality. None of the recurrent balanced translocations characteristic of extranodal MZL were seen secondarily in the nodal FLs with t(14;18)(q32;q21). However, 43/131 (33%) cases had at least one of the above secondary cytogenetic aberrations previously reported as recurrent aberrations in MZL and, when combined, these were significantly more frequent in FL with morphological evidence of marginal zone differentiation (p<0.0001, two-sided Fisher's exact test). Aberrations of chromosome 3 and, in particular, trisomy 3 occurred frequently in FL with marginal zone differentiation (p=0.002 and p<0.0001, respectively, two-sided Fisher's exact test), while chromosome 21, 22, and X chromosome aberrations, which have not been described previously as recurrent in MZL, were also significantly associated with marginal zone differentiation in FL (p=0.002, p=0.037, p=0.039, respectively, two-sided Fisher's exact test).