Diagnosing COVID-19; towards a feasible COVID-19 rule-out protocol.

INTRODUCTION: We present the results of the COVID-19 rule-out protocol at Ghent University Hospital, a step-wise testing approach which included repeat NFS SARS-CoV-2 rRT-PCR, respiratory multiplex RT-PCR, low-dose chest CT and bronchoscopy with BAL to confirm or rule-out SARS-CoV-2 infection in patients admitted with symptoms suggestive of COVID-19. RESULTS: Between 19 March 2020 and 30 April 2020, 455 non-critically ill patients with symptoms suspect for COVID-19 were admitted. The initial NFS for SARS-CoV-2 rRT-PCR yielded 66.9%, the second NFS 25.4% and bronchoscopy with BAL 5.9% of total COVID-19 diagnoses. In the BAL fluid, other respiratory pathogens were detected in 65% (13/20) of the COVID-19 negative patients and only in 1/7 COVID-19 positive patients. Retrospective antibody testing at the time around BAL sampling showed a positive IgA or IgG in 42.9 % of the COVID-19 positive and 10.5% of the COVID-19 negative group. Follow-up serology showed 100% COVID-19 positivity in the COVID-19 positive group and 100% IgG negativity in the COVID-19 negative group. CONCLUSION: In our experience, bronchoscopy with BAL can have an added value to rule-in or rule-out COVID-19 in patients with clinical and radiographical high-likelihood of COVID-19 and repeated negative NFS testing. Furthermore, culture and respiratory multiplex PCR on BAL fluid can aid to identify alternative microbial etiological agents in this group. Retrospective analysis of antibody development in this selected group of patients suggests that the implementation of serological assays in the routine testing protocol will decrease the need for invasive procedures like bronchoscopy.

Impact of the introduction of EUCAST's concept of "area of technical uncertainty".

On the first of January 2019, the European Committee on Antimicrobial Susceptibility Testing, EUCAST, introduced the concept of "area of technical uncertainty" (ATU). The aim was to report on the incidence of ATU test results in a selection of common bacterial species and the subsequent impact on antimicrobial resistance categorization and workload. A retrospective analysis of clinical samples collected from February 2019 until November 2019 was performed. Susceptibility to amoxicillin-clavulanic acid and piperacillin-tazobactam in Enterobacterales (Escherichia spp., Klebsiella spp., Proteus spp.), piperacillin-tazobactam in Pseudomonas aeruginosa, and amoxicillin-clavulanic acid and cefuroxime in Haemophilus influenzae was studied. Disk diffusion antibiotic susceptibility testing was read and interpreted by ADAGIO 93400 automated system (Bio-Rad, France). In case of an inhibition zone in the ATU, strains were retested using gradient minimal inhibitory concentration method (Etest, BioMérieux, France). Overall, 14,164 isolate-antibiotic combinations were tested in 7922 isolates, resulting in 1204 (8.5%) disk zone diameters in the ATU region. Retesting of ATUs with Etest resulted in a category change from S to R for amoxicillin-clavulanic acid in 63/498 (12.7%) of Escherichia spp., 2/58 (3.4%) of Klebsiella spp., 2/37 (5.4%) of Proteus spp., and 6/125 (4.8%) of Haemophilus influenzae. For piperacillin-tazobactam, a category change from S to R was found in 33/92 (35.9%) of Pseudomonas aeruginosa. We conclude that ATU testing has a substantial impact on the correct interpretation of antimicrobial resistance, at the expense of turn-around time and with the cost of additional workload.

Toilet drain water as a potential source of hospital room-to-room transmission of carbapenemase-producing Klebsiella pneumoniae.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) have rapidly emerged in Europe, being responsible for nosocomial outbreaks. AIM: Following an outbreak in the burn unit of Ghent University Hospital, we investigated whether CPE can spread between toilets through drain water and therefrom be transmitted to patients. METHODS: In 2017, the burn centre of our hospital experienced an outbreak of OXA-48-producing Klebsiella pneumoniae that affected five patients staying in three different rooms. Environmental samples were collected from the sink, shower, shower stretcher, hand rail of the bed, nursing carts, toilets, and drain water to explore a common source. Whole-genome sequencing and phylogenetic analysis was performed on K. pneumoniae outbreak isolates and two random K. pneumoniae isolates. FINDINGS: OXA-48-producing K. pneumoniae was detected in toilet water in four out of six rooms and drain water between two rooms. The strain persisted in two out of six rooms after two months of daily disinfection with bleach. All outbreak isolates belonged to sequence type (ST) 15 and showed isogenicity (<15 allele differences). This suggests that the strain may have spread between rooms by drain water. Unexpectedly, one random isolate obtained from a patient who became colonized while residing at the geriatric ward clustered with the outbreak isolates, suggesting the outbreak to be larger than expected. Daily application of bleach tended to be superior to acetic acid to disinfect toilet water; however, disinfection did not completely prevent the presence of carbapenemase-producing K. pneumoniae in toilet water. CONCLUSION: Toilet drain water may be a potential source of hospital room-to-room transmission of carbapenemase-producing K. pneumoniae.

CC398 Staphylococcus aureus subpopulations in Belgian patients.

Studies based on genome-wide single nucleotide polymorphisms (SNPs) supported the existence of two subpopulations in clonal complex (CC) 398 Staphylococcus aureus: an ancestral human-adapted clade (HC) and an animal-associated clade (AC). In this study, we have investigated the occurrence of genetic markers that allow discrimination of these subpopulations among CC398 isolates collected during 2014 to 2016 from human patients in Belgium. A collection of isolates was investigated by means of spa-typing and 16S-mecA-nuc PCR. CC398 isolates were classified as belonging to the human or the animal clade by using a canonical SNPs PCR and further studied by antimicrobial susceptibility and the presence of toxins, immune evasion cluster (IEC), and resistance genes. A total of 124 (7.8%) human isolates belonged to CC398. They were grouped into HC (n = 58) or AC (n = 66). The genes erm(T), pvl, chp, and scn were predominantly found in HC-CC398, while AC-CC398 isolates carried more frequently than the mecA, erm(C), tet(K), tet(M), and tet(L) genes. Different combinations of gene profiles were observed according to the clade. CC398 isolates from Belgian patients belonged to different subpopulations including typical HC and AC-isolates. Few HC-strains with mecA and AC-isolates harboring IEC were found. CC398 isolates from Belgian patients belonged to different subpopulations including typical HC and AC-isolates, as well as new emerging subpopulations that underline the ability of this lineage to acquire resistance and virulence genes. Further research is needed to evaluate the emergence of these subpopulations in the clinical setting.

Characterisation of Staphylococcus aureus isolates from bloodstream infections, Democratic Republic of the Congo.

Staphylococcus aureus is known worldwide as an invasive pathogen, but information on S. aureus from bloodstream infections in Central Africa remains scarce. A collection of S. aureus blood culture isolates recovered from hospitals in four provinces in the Democratic Republic of the Congo (2009-2013) was assessed. A total of 27/108 isolates were methicillin-resistant S. aureus (MRSA), of which >70% were co-resistant to aminoglycosides, tetracyclines, macrolides and lincosamides. For MRSA and methicillin-susceptible S. aureus (MSSA) isolates, resistance to chloramphenicol and trimethoprim-sulphamethoxazole (TMP-SMX) was <10%. However, 66.7% (72/108) of all isolates harboured the trimethoprim resistance gene dfrG. More than three-quarters (84/108, 77.8%) of isolates belonged to CC5, CC8, CC121 or CC152. Genetic diversity was higher among MSSA (31 spa types) compared to MRSA (four spa types). Most MRSA (23/27, 85.2%) belonged to CC8-spa t1476-SCCmec V and 17/23 (73.9%) MRSA ST8 were oxacillin susceptible but cefoxitin resistant. Among MRSA and MSSA combined, 49.1% (53/108) and 19.4% (21/108) contained the genes encoding for Panton-Valentine leucocidin (lukS-lukF PV, PVL) and toxic shock syndrome toxin-1 (tst, TSST-1), respectively. PVL was mainly detected among MSSA (51/53 isolates harbouring PVL were MSSA, 96.2%) and associated with CC121, CC152, CC1 and CC5. TSST-1 was associated with CC8-spa t1476-SCCmec V. The immune evasion cluster (IEC) genes scn, sak and chp were detected in 81.5% of isolates (88/108, equally represented among MSSA and MRSA). The present study confirms the occurrence of MRSA with high levels of multidrug co-resistance and PVL-positive MSSA among invasive S. aureus isolates in Central Africa.

Low occurrence of the new species Staphylococcus argenteus in a Staphylococcus aureus collection of human isolates from Belgium.

Staphylococcus argenteus is a novel Staphylococcus species closely related to Staphylococcus aureus that has been recently described. In this study, we investigated the proportion and the characteristics of S. argenteus recovered from humans in Belgium. S. aureus. human isolates collected in Belgium from 2006 to 2015 (n = 1,903) were retrospectively characterised via the presence of non-pigmented colonies on chocolate agar, spa typing and rpoB sequencing to determine if some of them were in fact S. argenteus. Out of 73 strains non-pigmented on chocolate plates, 3 isolates (0.16 %) showed rpoB sequences, in addition to spa and sequence types (ST2250/t5787, ST2250/t6675, ST3240/t6675), related to S. argenteus. Two of them were methicillin-resistant, harbouring a SCCmec type IV. The three S. argenteus isolates carried genes (sak, scn) of the immune evasion cluster. This first Belgian nationwide analysis showed a low occurrence of S. argenteus. Further studies should be conducted to identify the distribution range and the clinical impact of this new species.

Dynamics of nasal carriage of methicillin-resistant Staphylococcus aureus among healthcare workers in a tertiary-care hospital in Peru.

The study aims were to describe the frequency and dynamics of methicillin-resistant Staphylococcus aureus (MRSA) carriage among healthcare workers (HCWs), and to compare the molecular epidemiology of MRSA isolates from HCWs with those from patients with bacteremia. HCWs were interviewed and three nasal swabs were collected in a hospital in Lima, Peru, during 2009-2010. Consecutive S. aureus blood culture isolates from patients with bacteremia in the same hospital were also collected. SCCmec, multilocus sequence typing (MLST), and spa typing were performed. Persistent carriage was defined if having at least two consecutive cultures grown with S. aureus harboring an identical spa type. Among 172 HCWs included, the proportions of S. aureus and MRSA nasal carriage during first sampling were 22.7 % and 8.7 %, respectively. From 160 HCWs who were sampled three times, 12.5 % (20/160) were persistent S. aureus carriers and 26.9 % (43/160) were intermittent carriers. MRSA carriage among persistent and intermittent S. aureus carriers was 45.0 % (9/20) and 37.2 % (16/43), respectively. Fifty-six S. aureus blood culture isolates were analyzed, and 50 % (n = 28) were MRSA. Multidrug resistant ST5-spa t149-SCCmec I and ST72-spa t148-SCCmec non-typeable were the two most frequent genotypes detected among HCWs (91.7 %, i.e., 22/24 HCW in whom MRSA was isolated in at least one sample) and patients (24/28, 85.7 %). In conclusion, we found high proportions of MRSA among persistent and intermittent S. aureus nasal carriers among HCWs in a hospital in Lima. They belonged to similar genetic lineages as those recovered from patients with bacteremia.

Staphylococcus aureus nasal carriage among healthcare workers in Kisangani, the Democratic Republic of the Congo.

Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, but there are few data from Central Africa. The objective of our study was to characterise S. aureus colonisation isolates from healthcare-exposed professionals in the Democratic Republic of the Congo (DRC). Healthcare workers and medical students (n = 380) in Kisangani, DRC were screened for S. aureus nasal carriage in a single-centre cross-sectional study in the University Hospital of Kisangani. The isolates were identified and characterised using phenotypic and genotypic methods. The nasal carriage rate of S. aureus was 16.6 % and 10 out of 63 isolates (15.9 %) were MRSA. We found 28 different spa types. Most MRSA isolates belonged to ST8-spa t1476-SCCmec V. The majority of MRSA were multidrug-resistant to non-beta-lactam antibiotics. Overall, 28.5 % of S. aureus carried Panton-Valentine leucocidin (PVL)-encoding genes (all methicillin-sensitive) and 17.5 % carried toxic shock syndrome toxin-1 (TSST-1)-encoding genes. The finding of MRSA carriage among healthcare workers in a setting with limited access to diagnostic microbiology and appropriate therapy calls for improved education on infection control practices and supports the introduction of surveillance programmes.

Nonlinear superchiral meta-surfaces: tuning chirality and disentangling non-reciprocity at the nanoscale.

Circularly polarized light is incident on a nanostructured chiral meta-surface. In the nanostructured unit cells whose chirality matches that of light, superchiral light is forming and strong optical second harmonic generation can be observed.

Poly(3-alkylthiophene)s show unexpected second-order nonlinear optical response.

Regioregular poly(3-hexylthiophene)s with chain lengths varying from 5 to 100 monomers are synthesized. Poly(3-hexylthiophene)s show in solution an unexpectedly significant second-order nonlinear optical response. The increase in transition dipole moment upon oligomerisation causes the significant second-order nonlinear optical response.

Previous healthcare exposure is the main antecedent for methicillin-resistant Staphylococcus aureus carriage on hospital admission in Belgium.

This study aimed to estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) carriage upon hospital admission and to study the molecular epidemiology of MRSA in order to assess the proportion of Panton-Valentine leukocidin (PVL)-positive community-associated (CA) and livestock-associated (LA) MRSA strains. Epidemiological data on MRSA carriage upon hospital admission (2006-2009) were collected in a compulsory, continuous, national MRSA surveillance in Belgian acute-care hospitals. Additionally, 328 MRSA strains in 2005 and 314 strains in 2008 were collected in a separate, multicenter microbiological survey. Spa-typing, SCCmec-typing and MLST were performed; toxin genes were detected by PCR. The overall prevalence of MRSA carriage upon hospital admission was 8.9 cases/1,000 admissions between 2006 and 2009. Of MRSA carriers, 37.5% had a known MRSA history, 39.4% had stayed in a care facility, 12.2% reported no contact with healthcare. Over 90% of MRSA belonged to five healthcare-associated clones. Of these, MRSA spa-CC038-ST45-IV was in decline, mainly in favor of spa-CC008-ST8-IV. MRSA spa-CC002-ST5-IV, spa-CC002-ST5-II and spa-CC032-ST22-IV remained relatively stable. The proportion of PVL-positive CA-MRSA and LA-MRSA ST398 was below 2% of all MRSA. The extra-hospital MRSA reservoir in Belgium mainly consists of persons with previous healthcare exposure. PVL-positive CA-MRSA and LA-MRSA strains remained infrequent among hospitalized patients.

The role of chiral local field enhancements below the resolution limit of Second Harmonic Generation microscopy.

While it has been demonstrated that, above its resolution limit, Second Harmonic Generation (SHG) microscopy can map chiral local field enhancements, below that limit, structural defects were found to play a major role. Here we show that, even below the resolution limit, the contributions from chiral local field enhancements to the SHG signal can dominate over those by structural defects. We report highly homogeneous SHG micrographs of star-shaped gold nanostructures, where the SHG circular dichroism effect is clearly visible from virtually every single nanostructure. Most likely, size and geometry determine the dominant contributions to the SHG signal in nanostructured systems.