Development and evaluation of an artificial intelligence for bacterial growth monitoring in clinical bacteriology.

In clinical bacteriology laboratories, reading and processing of sterile plates remain a significant part of the routine workload (30%-40% of the plates). Here, an algorithm was developed for bacterial growth detection starting with any type of specimens and using the most common media in bacteriology. The growth prediction performance of the algorithm for automatic processing of sterile plates was evaluated not only at 18-24 h and 48 h but also at earlier timepoints toward the development of an early growth monitoring system. A total of 3,844 plates inoculated with representative clinical specimens were used. The plates were imaged 15 times, and two different microbiologists read the images randomly and independently, creating 99,944 human ground truths. The algorithm was able, at 48 h, to discriminate growth from no growth with a sensitivity of 99.80% (five false-negative [FN] plates out of 3,844) and a specificity of 91.97%. At 24 h, sensitivity and specificity reached 99.08% and 93.37%, respectively. Interestingly, during human truth reading, growth was reported as early as 4 h, while at 6 h, half of the positive plates were already showing some growth. In this context, automated early growth monitoring in case of normally sterile samples is envisioned to provide added value to the microbiologists, enabling them to prioritize reading and to communicate early detection of bacterial growth to the clinicians.

Predictors of mortality of Pseudomonas aeruginosa bacteraemia and the role of infectious diseases consultation and source control; a retrospective cohort study.

PURPOSE: To determine predictors of mortality among patients with Pseudomonas aeruginosa bacteraemia. METHODS: Retrospective study. SETTING: This study conducted at the Lausanne University Hospital, Switzerland included adult patients with P. aeruginosa bacteraemia from 2015 to 2021. RESULTS: During the study period, 278 episodes of P. aeruginosa bacteraemia were included. Twenty (7%) isolates were multidrug-resistant. The most common type of infection was low respiratory tract infection (58 episodes; 21%). Sepsis was present in the majority of episodes (152; 55%). Infectious diseases consultation within 48 h of bacteraemia onset was performed in 203 (73%) episodes. Appropriate antimicrobial treatment was administered within 48 h in 257 (92%) episodes. For most episodes (145; 52%), source control was considered necessary, with 93 (64%) of them undergoing such interventions within 48 h. The 14-day mortality was 15% (42 episodes). The Cox multivariable regression model showed that 14-day mortality was associated with sepsis (P 0.002; aHR 6.58, CI 1.95-22.16), and lower respiratory tract infection (P < 0.001; aHR 4.63, CI 1.78-12.06). Conversely, interventions performed within 48 h of bacteraemia onset, such as infectious diseases consultation (P 0.036; HR 0.51, CI 0.27-0.96), and source control (P 0.009; aHR 0.17, CI 0.47-0.64) were associated with improved outcome. CONCLUSION: Our findings underscore the pivotal role of early infectious diseases consultation in recommending source control interventions and guiding antimicrobial treatment for patients with P. aeruginosa bacteraemia.

Clinical significance of concomitant bacteriuria in patients with Staphylococcus aureus bacteraemia.

This retrospective study, conducted at Lausanne University Hospital (2015-2021), compared Staphylococcus aureus bacteraemia (SABA) patients with or without concomitant bacteriuria (SABU). Among 448 included bacteraemic patients, 62 (13.8%) had S. aureus concurrently isolated from urine. In multivariate analysis, there was a significant difference in the odds of community-onset bacteraemia (P 0.030), malignancy (P 0.002), > 1 pair of positive blood cultures (P 0.037), and persistent bacteraemia for at least 48 h (P 0.045) in patients with concurrent SABU. No difference concerning mortality was found. On the other hand, SABU was associated with higher rates of SABA recurrence after antibiotic cessation.

How to Manage Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is a pathogen frequently encountered in healthcare-associated infections and immunocompromised patients. In bacteremia, this pathogen is associated with higher mortality than other Gram-negative pathogens. This increase in mortality was also found globally for multi-resistant compared to susceptible strains. Several factors have been associated with the development of resistance: previous ICU stay, use of carbapenems, and comorbidities were identified in multivariate analysis. In the therapeutic choice, previous antibiotic treatment remains the strongest driver suggesting a potential resistant strain. These risk factors will decide whether multi-resistant strains must be considered in the empiric coverage. For susceptible strains, a single agent can be used, ß-lactams are usually the first choice. Associations do not provide any advantage on mortality. Optimization of pharmacokinetic/pharmacodynamic parameters, such as prolonged infusion (for time-dependent antibiotics), increased dosage (for concentration-dependent antibiotics), and therapeutic drug monitoring, also influences the outcome. The increasing number of resistant strains led the clinician to use either recently approved new molecules but also associations. For multi-resistant strains, new molecules such as ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol have shown an adequate activity against P. aeruginosa. Older molecules like colistin and fosfomycin are also used in this indication. The complexity of the resistance and consequences on a larger scale of antibiotic prescription will probably lead to more individualized prescriptions.

C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence.

C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase-deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.

Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii.

Toxoplasma gondii encodes three protein kinase A catalytic (PKAc1-3) and one regulatory (PKAr) subunits to integrate cAMP-dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 blocks parasite division. Conversely, down-regulation of PKAc1 or stabilisation of a dominant-negative PKAr isoform that does not bind cAMP triggers premature parasite egress from infected cells followed by serial invasion attempts leading to host cell lysis. This untimely egress depends on host cell acidification. A phosphoproteome analysis suggested the interplay between cAMP and cGMP signalling as PKAc1 inactivation changes the phosphorylation profile of a putative cGMP-phosphodiesterase. Concordantly, inhibition of the cGMP-dependent protein kinase G (PKG) blocks egress induced by PKAc1 inactivation or environmental acidification, while a cGMP-phosphodiesterase inhibitor circumvents egress repression by PKAc1 or pH neutralisation. This indicates that pH and PKAc1 act as balancing regulators of cGMP metabolism to control egress. These results reveal a crosstalk between PKA and PKG pathways to govern egress in T. gondii.

Assessment of SARS-CoV-2 Genome Sequencing: Quality Criteria and Low-Frequency Variants.

Although many laboratories worldwide have developed their sequencing capacities in response to the need for SARS-CoV-2 genome-based surveillance of variants, only a few reported some quality criteria to ensure sequence quality before lineage assignment and submission to public databases. Hence, we aimed here to provide simple quality control criteria for SARS-CoV-2 sequencing to prevent erroneous interpretation of low-quality or contaminated data. We retrospectively investigated 647 SARS-CoV-2 genomes obtained over 10 tiled amplicons sequencing runs. We extracted 26 potentially relevant metrics covering the entire workflow from sample selection to bioinformatics analysis. Based on data distribution, critical values were established for 11 selected metrics to prompt further quality investigations for problematic samples, in particular those with a low viral RNA quantity. Low-frequency variants (<70% of supporting reads) can result from PCR amplification errors, sample cross contaminations, or presence of distinct SARS-CoV2 genomes in the sample sequenced. The number and the prevalence of low-frequency variants can be used as a robust quality criterion to identify possible sequencing errors or contaminations. Overall, we propose 11 metrics with fixed cutoff values as a simple tool to evaluate the quality of SARS-CoV-2 genomes, among which are cycle thresholds, mean depth, proportion of genome covered at least 10×, and the number of low-frequency variants combined with mutation prevalence data.

The lectin-specific activity of Toxoplasma gondii microneme proteins 1 and 4 binds Toll-like receptor 2 and 4 N-glycans to regulate innate immune priming.

Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex secreted on the parasite surface that functions to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-γ in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis.

The Conoid Associated Motor MyoH Is Indispensable for Toxoplasma gondii Entry and Exit from Host Cells.

Many members of the phylum of Apicomplexa have adopted an obligate intracellular life style and critically depend on active invasion and egress from the infected cells to complete their lytic cycle. Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa, and as such, the invasive tachyzoite contains an organelle termed the conoid at its extreme apex. This motile organelle consists of a unique polymer of tubulin fibres and protrudes in both gliding and invading parasites. The class XIV myosin A, which is conserved across the Apicomplexa phylum, is known to critically contribute to motility, invasion and egress from infected cells. The MyoA-glideosome is anchored to the inner membrane complex (IMC) and is assumed to translocate the components of the circular junction secreted by the micronemes and rhoptries, to the rear of the parasite. Here we comprehensively characterise the class XIV myosin H (MyoH) and its associated light chains. We show that the 3 alpha-tubulin suppressor domains, located in MyoH tail, are necessary to anchor this motor to the conoid. Despite the presence of an intact MyoA-glideosome, conditional disruption of TgMyoH severely compromises parasite motility, invasion and egress from infected cells. We demonstrate that MyoH is necessary for the translocation of the circular junction from the tip of the parasite, where secretory organelles exocytosis occurs, to the apical position where the IMC starts. This study attributes for the first time a direct function of the conoid in motility and invasion, and establishes the indispensable role of MyoH in initiating the first step of motility along this unique organelle, which is subsequently relayed by MyoA to enact effective gliding and invasion.

Toxoplasma gondii myosin F, an essential motor for centrosomes positioning and apicoplast inheritance.

Members of the Apicomplexa phylum possess an organelle surrounded by four membranes, originating from the secondary endosymbiosis of a red alga. This so-called apicoplast hosts essential metabolic pathways. We report here that apicoplast inheritance is an actin-based process. Concordantly, parasites depleted in either profilin or actin depolymerizing factor, or parasites overexpressing the FH2 domain of formin 2, result in loss of the apicoplast. The class XXII myosin F (MyoF) is conserved across the phylum and localizes in the vicinity of the Toxoplasma gondii apicoplast during division. Conditional knockdown of TgMyoF severely affects apicoplast turnover, leading to parasite death. This recapitulates the phenotype observed upon perturbation of actin dynamics that led to the accumulation of the apicoplast and secretory organelles in enlarged residual bodies. To further dissect the mode of action of this motor, we conditionally stabilized the tail of MyoF, which forms an inactive heterodimer with endogenous TgMyoF. This dominant negative mutant reveals a central role of this motor in the positioning of the two centrosomes prior to daughter cell formation and in apicoplast segregation.

Fundamental Roles of the Golgi-Associated Toxoplasma Aspartyl Protease, ASP5, at the Host-Parasite Interface.

Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite's ability to modulate host signalling pathways and immune responses.

Plasticity between MyoC- and MyoA-glideosomes: an example of functional compensation in Toxoplasma gondii invasion.

The glideosome is an actomyosin-based machinery that powers motility in Apicomplexa and participates in host cell invasion and egress from infected cells. The central component of the glideosome, myosin A (MyoA), is a motor recruited at the pellicle by the acylated gliding-associated protein GAP45. In Toxoplasma gondii, GAP45 also contributes to the cohesion of the pellicle, composed of the inner membrane complex (IMC) and the plasma membrane, during motor traction. GAP70 was previously identified as a paralog of GAP45 that is tailored to recruit MyoA at the apical cap in the coccidian subgroup of the Apicomplexa. A third member of this family, GAP80, is demonstrated here to assemble a new glideosome, which recruits the class XIV myosin C (MyoC) at the basal polar ring. MyoC shares the same myosin light chains as MyoA and also interacts with the integral IMC proteins GAP50 and GAP40. Moreover, a central component of this complex, the IMC-associated protein 1 (IAP1), acts as the key determinant for the restricted localization of MyoC to the posterior pole. Deletion of specific components of the MyoC-glideosome underscores the installation of compensatory mechanisms with components of the MyoA-glideosome. Conversely, removal of MyoA leads to the relocalization of MyoC along the pellicle and at the apical cap that accounts for residual invasion. The two glideosomes exhibit a considerable level of plasticity to ensure parasite survival.

Assessment of phosphorylation in Toxoplasma glideosome assembly and function.

Members of the phylum Apicomplexa possess a highly conserved molecular motor complex anchored in the parasite pellicle and associated with gliding motility, invasion and egress from infected cells. This machinery, called the glideosome, is structured around the acylated gliding-associated protein GAP45 that recruits the motor complex composed of myosin A and two associated myosin light chains (TgMLC1 and TgELC1). This motor is presumably firmly anchored to the inner membrane complex underneath the plasma membrane via an interaction with two integral membrane proteins, GAP50 and GAP40. To determine if the previously mapped phosphorylation sites on TgGAP45 and TgMLC1 have a direct significance for glideosome assembly and function, a series of phospho-mimetic and phospho-null mutants were generated. Neither the overexpression nor the allelic replacement of TgMLC1 with phospho-mutants impacted on glideosome assembly and parasite motility. TgGAP45 phosphorylation mutants were functionally investigated using a complementation strategy in a TgGAP45 inducible knockout background. The loss of interaction with TgGAP50 by one previously reported GAP45-mutant appeared to depend only on the presence of a remaining competing wild type copy of TgGAP45. Accordingly, this mutant displayed no phenotype in complementation experiments. Unexpectedly, GAP45 lacking the region encompassing the cluster of twelve phosphorylation sites did not impact on its dual function in motor recruitment and pellicle integrity. Despite the extensive phosphorylation of TgMLC1 and TgGAP45, this post-translational modification does not appear to be critical for the assembly and function of the glideosome.

The Impact of Laboratory Automation on the Time to Urine Microbiological Results: A Five-Year Retrospective Study.

Total laboratory automation (TLA) is a valuable component of microbiology laboratories and a growing number of publications suggest the potential impact of automation in terms of analysis standardization, streaking quality, and the turnaround time (TAT). The aim of this project was to perform a detailed investigation of the impact of TLA on the workflow of commonly treated specimens such as urine. This is a retrospective observational study comparing two time periods (pre TLA versus post TLA) for urine specimen culture processing. A total of 35,864 urine specimens were plated during the pre-TLA period and 47,283 were plated during the post-TLA period. The median time from streaking to identification decreased from 22.3 h pre TLA to 21.4 h post TLA (p < 0.001), and the median time from streaking to final validation of the report decreased from 24.3 h pre TLA to 23 h post TLA (p < 0.001). Further analysis revealed that the observed differences in TAT were mainly driven by the contaminated and positive samples. Our findings demonstrate that TLA has the potential to decrease turnaround times of samples in a laboratory. Nevertheless, changes in laboratory workflow (such as extended opening hours for plate reading and antibiotic susceptibility testing or decreased incubation times) might further maximize the efficiency of TLA and optimize TATs.

Environmental and geographical factors influencing the spread of SARS-CoV-2 over 2 years: a fine-scale spatiotemporal analysis.

Introduction: Since its emergence in late 2019, the SARS-CoV-2 virus has led to a global health crisis, affecting millions and reshaping societies and economies worldwide. Investigating the determinants of SARS-CoV-2 diffusion and their spatiotemporal dynamics at high spatial resolution is critical for public health and policymaking. Methods: This study analyses 194,682 georeferenced SARS-CoV-2 RT-PCR tests from March 2020 and April 2022 in the canton of Vaud, Switzerland. We characterized five distinct pandemic periods using metrics of spatial and temporal clustering like inverse Shannon entropy, the Hoover index, Lloyd's index of mean crowding, and the modified space-time DBSCAN algorithm. We assessed the demographic, socioeconomic, and environmental factors contributing to cluster persistence during each period using eXtreme Gradient Boosting (XGBoost) and SHapley Additive exPlanations (SHAP), to consider non-linear and spatial effects. Results: Our findings reveal important variations in the spatial and temporal clustering of cases. Notably, areas with flatter epidemics had higher total attack rate. Air pollution emerged as a factor showing a consistent positive association with higher cluster persistence, substantiated by both immission models and, to a lesser extent, tropospheric NO2 estimations. Factors including population density, testing rates, and geographical coordinates, also showed important positive associations with higher cluster persistence. The socioeconomic index showed no significant contribution to cluster persistence, suggesting its limited role in the observed dynamics, which warrants further research. Discussion: Overall, the determinants of cluster persistence remained across the study periods. These findings highlight the need for effective air quality management strategies to mitigate air pollution's adverse impacts on public health, particularly in the context of respiratory viral diseases like COVID-19.

Impact of mutations in the mtrR, rpdlVD and rrl genes on azithromycin resistance in Neisseria gonorrhoeae.

INTRODUCTION: Bacterial sexually transmitted infections (STIs) pose a major public health problem. The emergence of antibiotic-resistant strains of Neisseria gonorrhoeae represents a serious threat to successful treatment and epidemiological control. The first extensively drug-resistant (XDR) strains (ceftriaxone-resistant and high-level azithromycin-resistant [HLR AZY]) have been reported. AIMS: To identify molecular mechanisms implicated in azithromycin resistance in strains isolated from patients over a three-year period in a university hospital in Switzerland. MATERIAL AND METHODS: From January 2020 to December 2022, 34 isolates (one per patient) were recovered from samples analyzed at the University Hospital of Lausanne. Eight genes involved in azithromycin resistance were sequenced: mtrR repressor (mtrCDE operon repressor) and his promotor mtrR-pr, rplD gene (L4 ribosomal protein), rplV gene (L22 ribosomal protein) and the four alleles of the rrl gene (23S rRNA). RESULTS: With a cutoff value of 1 mg/L, 15 isolates were considered as being resistant to azithromycin, whereas the remaining 19 were susceptible. The C2597T mutation in 3 or 4 of the rrl allele confer a medium-level resistance to azithromycin (MIC = 16 mg/L, N = 2). The following mutations were significantly associated with MIC values ≥1 mg/L: the three mutations V125A, A147G, R157Q in the rplD gene (N = 10) and a substitution A->C in the mtrR promotor (N = 9). Specific mutations in the mtrR repressor and its promotor were observed in both susceptible and resistant isolates. CONCLUSIONS: Resistance to azithromycin was explained by the presence of mutations in many different copies of 23S RNA ribosomal genes and their regulatory genes. Other mutations, previously reported to be associated with azithromycin resistance, were documented in both susceptible and resistant isolates, suggesting they play little role, if any, in azithromycin resistance.

SARS-CoV-2 neutralizing antibody response in vaccinated and non-vaccinated hospital healthcare workers with or without history of infection.

Between March 2021 and February 2022, SARS-CoV-2 neutralizing antibodies dynamics was investigated in a prospective observational study in 903 healthcare workers of a hospital in Switzerland. A surrogate neutralization assay measuring the competitive inhibition of the angiotensin converting enzyme 2 (ACE2) binding to the spike protein (S) of the SARS-CoV-2 wild type virus and to five variants of concern (Alpha, Beta, Gamma, Delta, Omicron) was used. We observed a broad distribution of neutralization activity among participants and substantial differences in neutralizing titers against variants. Participants were grouped based on combinations of vaccination status (1, 2 or 3 doses) and/or prior or subsequent SARS-CoV-2 infection/reinfection. Triple vaccination resulted in the highest neutralization response, as did double vaccination with prior or subsequent infection. Double vaccination without infection showed an intermediate neutralization response while SARS-CoV-2 infection in non-vaccinated participants resulted in poor neutralization response. After triple vaccination or double vaccination plus infection, additional vaccination and/or reinfection had no impact on neutralizing antibody titers over the observed period. These results strongly support the booster dose strategy, while additional booster doses within short time intervals might not improve immunization. However, dynamics of neutralizing antibodies titers needs to be monitored individually, over time and include newly emerging variants.

Swiss public health measures associated with reduced SARS-CoV-2 transmission using genome data.

Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Switzerland in 2020-the sixth largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrasted these estimates with simple null models representing the absence of certain public health measures. We show that Switzerland's border closures decoupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86 to 98% reduction in introductions during Switzerland's strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Last, we quantified local transmission dynamics once introductions into Switzerland occurred using a phylodynamic model. We found that transmission slowed 35 to 63% upon outbreak detection in summer 2020 but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics.

Does protein phosphorylation govern host cell entry and egress by the Apicomplexa?

Members of the phylum Apicomplexa are responsible for a wide range of diseases in humans and animals. The absence of an effective vaccine or safe curing drugs and the continuous emergence of resistant parasites to available treatments impose a high demand on the identification of novel targets for intervention against the apicomplexans. Protein kinases are considered attractive potential therapeutic targets not only against cancers but also to combat infectious diseases. The scope and aim of this review is to report on the recent progress in dissecting the impact of protein phosphorylation in regulating motility and invasion.

SARS-CoV-2 seroprevalence in hospital healthcare workers in Western Switzerland at the end of the second pandemic wave.

Introduction. In early January 2020, the pandemic of COVID-19 (coronavirus disease 2019) rapidly spread from China and caused a worldwide pandemic.Hypothesis. Healthcare workers represent a high-risk group for acquiring COVID-19 and for nosocomial transmission of severe acute respiratory coronavirus 2 (SARS-CoV-2).Aim. We aimed to investigate over a 1 year period, across two pandemic waves, the SARS-CoV-2 seroprevalence in employees at a Western Switzerland public hospital.Methodology. A prospective observational SARS-CoV-2 seroprevalence study was proposed to all hospital employees who enrolled on a voluntary basis.Results. Out of 594 participants recruited on a voluntary basis, 269 volunteers (45.3 %) had anti-SARS-CoV-2 antibodies: this seroprevalence was twice higher than that reported in the local community. Healthcare workers with prolonged exposure to patients with COVID-19 showed a significantly higher odds ratio (OR) of having a positive SARS-CoV-2 serology [OR 3.19, 95 % confidence interval (CI) 2.16-4.74]. Symptoms showing the highest association with a positive serology were anosmia (OR 11.9, 95 % CI 5.58-30.9) and ageusia (OR 10.3, 95 % CI 4.8-26.3). A total of 17.1 % (95 % CI 12.2-21.1 %) of SARS-CoV-2 seropositive volunteers did not report a suspicion of COVID-19 in their personal history.Conclusion. Overall, we observed that the impact of the second SARS-CoV-2 pandemic wave was considerable and significantly affected healthcare workers with prolonged exposure to patients with COVID-19.