Evaluation of the ELITe InGenius and Bordetella ELITe MGB Kit for the detection and identification of B. pertussis, B. parapertussis and B. holmesii.

Bordetella pertussis is the causative pathogen of whooping cough or pertussis, a contagious respiratory disease. Aside from serodiagnosis, laboratory confirmation of pertussis is done through PCR, as B. pertussis is difficult to culture. The ELITe InGenius instrument (ELITechGroup, France) with accompanying Bordetella ELITe MGB Kit was evaluated against a laboratory-developed assay. Both assays combine two screening (IS481, IS1001) and two confirmation targets (recA, ptxA-Pr or IS1002) for optimal sensitivity and specificity. The company's stated claims on sensitivity and reproducibility were confirmed. Accuracy testing showed full concordance between both assays for the screening targets. Minor discrepancies were seen for the B. pertussis confirmation target. Some cross-reactivity with other Bordetella species was observed for the IS481-target, however, none of these were confirmed in the ptxA-Pr target. These results show the suitability of the Bordetella ELITe MGB Kit for the detection and differentiation of B. pertussis, B. parapertussis and B. holmesii.

Novel Ralstonia species from human infections: improved matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based identification and analysis of antimicrobial resistance patterns.

A collection of 161 Ralstonia isolates, including 90 isolates from persons with cystic fibrosis, 27 isolates from other human clinical samples, 8 isolates from the hospital environment, 7 isolates from industrial samples, and 19 environmental isolates, was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification and yielded confident species level identification scores for only 62 (39%) of the isolates, including four that proved misidentified subsequently. Whole-genome sequence analysis of 32 representative isolates for which no confident MALDI-TOF MS species level identification was obtained revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively, and provided the basis for updating an in-house MALDI-TOF MS database. A reanalysis of all mass spectra with the updated MALDI-TOF MS database increased the percentage of isolates with confident species level identification up to 77%. The antimicrobial susceptibility of 30 isolates mainly representing novel human clinical and environmental Ralstonia species was tested toward 17 antimicrobial agents and demonstrated that the novel Ralstonia species were generally multi-resistant, yet susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline. An analysis of genomic antimicrobial resistance genes in 32 novel and publicly available genome sequences revealed broadly distributed beta-lactam resistance determinants.IMPORTANCEThe present study demonstrated that a commercial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification database can be tailored to improve the identification of Ralstonia species. It also revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively. An analysis of minimum inhibitory concentration values demonstrated that the novel Ralstonia species were generally multi-resistant but susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline.

Evaluation of two automated real-time PCR-based quantification methods for whole blood Epstein-Barr viral load.

Quantification of EBV DNA is important in transplantation settings for the diagnosis of post-transplantation. We evaluated the performance of the AltoStar® EBV PCR Kit 1.5 on whole blood specimens: limit of detection, linearity, accuracy, and precision were determined using the WHO NIBSC 09/260 international standard. Results of 69 clinical samples were compared between the AltoStar® EBV PCR Kit 1.5 (altona Diagnostics) and the RealTime EBV assay (Abbott). The LoD of the AltoStar® Kit was 148 IU/mL and linearity was between 375 and 500000. A high concordance was found between nominal value of the NIBSC dilutions and the AltoStar EBV result. The total variation ranged from 2.2% to 9.6%. Out of 69 clinical samples tested, there was a high concordance between the 22 paired results within the overlapping linear ranges of both tests. The AltoStar® EBV assay is reliable and accurate for EBV viral load determination on whole blood samples.

Aztreonam-avibactam synergy, a validation and comparison of diagnostic tools.

Introduction: Antimicrobial resistance is a growing problem that necessitates the development of new therapeutic options. Cefiderocol and aztreonam (AT) are often the last active ß-lactams for treating metallo-ß-lactamases (MBL)-producing Gram-negative bacilli. In these difficult-to-treat bacterial strains, AT resistance is frequently attributed to the co-occurrence of other resistance mechanisms. In the case of ß-lactamases they can often be inhibited by avibactam. In the present study, we evaluated the use of the double-disc synergy test (DDST) as a screening tool for the detection of synergy between AT-avibactam (ATA). We validated both the Gradient Diffusion Strips (GDSs) superposition method and the commercially available Liofilchem's ATA GDS. Materials and methods: We tested AT susceptibility in combination with ceftazidime-avibactam for 65 strains, including 18 Serine-ß-Lactamase (SBL)- and 24 MBL-producing Enterobacterales, 12 MBL-producing P. aeruginosa, and 11 S. maltophilia isolates. Interpretation was done with EUCAST breakpoints (version 13.0), AT breakpoints being used for ATA. The accuracy and validity of the GDSs superposition method and ATA GDS were evaluated using an AT GDS applied on Mueller Hinton Agar plates supplemented with avibactam (MH-AV). A DDST was performed to screen for synergy between antibiotic combinations. Results: Using MH-AV, all SBL- and MBL-positive Enterobacterales were susceptible or susceptible at increased exposure to the combination AT-avibactam. In contrast, only 2 out of the 12 (17%) P. aeruginosa strains and 9/11 (82%) of the S. maltophilia strains were susceptible- or susceptible at increased exposure for the combination of AT-avibactam. The DDST detected all synergies, demonstrating a 100% sensitivity and 100% negative predictive value for all bacterial strains. Conclusion: The DDST is a sensitive tool for screening for antibiotic synergy. Unlike S. maltophilia and SBL- and MBL-positive Enterobacterales, most MBL-positive P. aeruginosa strains remain resistant to AT-avibactam. ATA GDS should be preferred for MIC determination of the AT-avibactam combination, while the GDSs superposition method can be used as an alternative to the commercial test.

Culturomics in Unraveling the Upper Female Reproductive Tract Microbiota.

In recent years, the study of the human microbiome has surged, shedding light on potential connections between microbiome composition and various diseases. One specific area of intense interest within this research is the female reproductive tract, as it holds the potential to influence the process of embryo implantation. Advanced sequencing technologies have delivered unprecedented insights into the microbial communities, also known as microbiota, residing in the female reproductive tract. However, their efficacy encounters significant challenges when analyzing low-biomass microbiota, such as those present in the endometrium. These molecular techniques are susceptible to contamination from laboratory reagents and extraction kits, leading to sequencing bias that can significantly alter the perceived taxonomy of a sample. Consequently, investigating the microbiota of the upper female reproductive tract necessitates the exploration of alternative methods. In this context, the current review delves into the application of culturomics in unraveling the upper female reproductive tract microbiota. While culturomics holds value in research, its transition to routine clinical practice appears remote, at least in the foreseeable future.

Antimicrobial Activities of Aztreonam-Avibactam and Comparator Agents against Enterobacterales Analyzed by ICU and Non-ICU Wards, Infection Sources, and Geographic Regions: ATLAS Program 2016-2020.

Increasing antimicrobial resistance among multidrug-resistant (MDR), extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing Enterobacterales (CPE), in particular metallo-ß-lactamase (MBL)-positive strains, has led to limited treatment options in these isolates. This study evaluated the activity of aztreonam-avibactam (ATM-AVI) and comparator antimicrobials against Enterobacterales isolates and key resistance phenotypes stratified by wards, infection sources and geographic regions as part of the ATLAS program between 2016 and 2020. Minimum inhibitory concentrations (MICs) were determined per Clinical and Laboratory Standards Institute (CLSI) guidelines. The susceptibility of antimicrobials were interpreted using CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. A tentative pharmacokinetic/pharmacodynamic breakpoint of 8 µg/mL was considered for ATM-AVI activity. ATM-AVI inhibited ≥99.2% of Enterobacterales isolates across wards and ≥99.7% isolates across infection sources globally and in all regions at ≤8 µg/mL. For resistance phenotypes, ATM-AVI demonstrated sustained activity across wards and infection sources by inhibiting ≥98.5% and ≥99.1% of multidrug-resistant (MDR) isolates, ≥98.6% and ≥99.1% of ESBL-positive isolates, ≥96.8% and ≥90.9% of carbapenem-resistant (CR) isolates, and ≥96.8% and ≥97.4% of MBL-positive isolates, respectively, at ≤8 µg/mL globally and across regions. Overall, our study demonstrated that ATM-AVI represents an important therapeutic option for infections caused by Enterobacterales, including key resistance phenotypes across different wards and infection sources.

Outbreak of Corynebacterium diphtheriae among asylum seekers in Belgium in 2022: operational challenges and lessons learnt.

Since 2022, European countries have been facing an outbreak of mainly cutaneous diphtheria caused by toxigenic Corynebacterium diphtheriae among asylum seekers. In Belgium, between 1 March and 31 December 2022, 25 cases of toxigenic C. diphtheriae infection were confirmed among asylum seekers, mostly among young males from Afghanistan. Multi-locus sequence typing showed that most isolates belonged to sequence types 574 or 377, similar to the majority of cases in other European countries. The investigation and management of the outbreak, with many asylum seekers without shelter, required adjustments to case finding, contact tracing and treatment procedures. A test-and-treat centre was organised by non-governmental organisations, the duration of the antimicrobial treatment was shortened to increase compliance, and isolation and contact tracing of cases was not possible. A vaccination centre was opened, and mobile vaccination campaigns were organised to vaccinate a maximum of asylum seekers. No more cases were detected between end December 2022 and May 2023. Unfortunately, though, three cases of respiratory diphtheria, including one death, were reported at the end of June 2023. To prevent future outbreaks, specific attention and sufficient resources should be allocated to this vulnerable population, in Belgium and at international level.

Transforming Shiga toxin-producing Escherichia coli surveillance through whole genome sequencing in food safety practices.

Introduction: Shiga toxin-producing Escherichia coli (STEC) is a gastrointestinal pathogen causing foodborne outbreaks. Whole Genome Sequencing (WGS) in STEC surveillance holds promise in outbreak prevention and confinement, in broadening STEC epidemiology and in contributing to risk assessment and source attribution. However, despite international recommendations, WGS is often restricted to assist outbreak investigation and is not yet fully implemented in food safety surveillance across all European countries, in contrast to for example in the United States. Methods: In this study, WGS was retrospectively applied to isolates collected within the context of Belgian food safety surveillance and combined with data from clinical isolates to evaluate its benefits. A cross-sector WGS-based collection of 754 strains from 1998 to 2020 was analyzed. Results: We confirmed that WGS in food safety surveillance allows accurate detection of genomic relationships between human cases and strains isolated from food samples, including those dispersed over time and geographical locations. Identifying these links can reveal new insights into outbreaks and direct epidemiological investigations to facilitate outbreak management. Complete WGS-based isolate characterization enabled expanding epidemiological insights related to circulating serotypes, virulence genes and antimicrobial resistance across different reservoirs. Moreover, associations between virulence genes and severe disease were determined by incorporating human metadata into the data analysis. Gaps in the surveillance system were identified and suggestions for optimization related to sample centralization, harmonizing isolation methods, and expanding sampling strategies were formulated. Discussion: This study contributes to developing a representative WGS-based collection of circulating STEC strains and by illustrating its benefits, it aims to incite policymakers to support WGS uptake in food safety surveillance.

In Vitro Susceptibility of Achromobacter Species Isolated from Cystic Fibrosis Patients: a 6-Year Survey.

We conducted in vitro antimicrobial susceptibility testing of 267 Achromobacter isolates for 16 antibiotics from 2017 to 2022. The highest susceptibility was found for piperacillin-tazobactam (70%) and ceftazidime-avibactam (62%). Between 30% and 49% of strains were susceptible to tigecycline, ceftazidime, and meropenem. We applied species-specific Achromobacter xylosoxidans breakpoints for piperacillin-tazobactam, meropenem, and trimethoprim-sulfamethoxazole and EUCAST pharmacokinetic/pharmacodynamic (PK/PD) breakpoints for the others. A. xylosoxidans was the most frequently isolated species, followed by Achromobacter insuavis and Achromobacter ruhlandii.

Epidemiology of Resistance Determinants Identified in Meropenem-Nonsusceptible Enterobacterales Collected as Part of a Global Surveillance Study, 2018 to 2019.

The objective of this study was to describe the frequency of resistance determinants in meropenem-nonsusceptible (MEM-NS) Enterobacterales isolates collected in 2018 and 2019 as a part of the ATLAS global surveillance program. Among a total of 39,368 Enterobacterales isolates collected in 2018 and 2019, 5.7% were MEM-NS (MIC ≥2 µg/mL). Among the different regions, the proportion of MEM-NS isolates ranged from 1.9% (North America) to 8.4% (Asia/Pacific). The majority of MEM-NS isolates collected were of the species Klebsiella pneumoniae (71.5%). Among the MEM-NS Enterobacterales isolates collected, metallo-ß-lactamases (MBL) were identified in 36.7%, KPC in 25.5%, and OXA-48-like in 24.1%. The predominance of resistance mechanisms among MEM-NS isolates varied by region: MBLs were dominant in isolates collected in Africa and Middle East (AfME, 49%) and Asia/Pacific (59.4%), OXA-48-like carbapenemases were predominant in Europe (30%), and KPC in Latin America (51.9%) and North America (53.6%). NDM ß-lactamases accounted for the majority of MBLs identified (88.4%). Of the 38 carbapenemase variants identified, NDM-1 (68.7%), KPC-2 (54.6%), OXA-48 (54.3%), and VIM-1 (76.1%) were the common variants within their respective families. Among the MEM-NS isolates, 7.9% co-carried two carbapenemases. Notably, the proportion of MEM-NS Enterobacterales increased from 4.9% in 2018 to 6.4% in 2019. The results of this study show a continuation of the trend of increasing carbapenem-resistance within clinical Enterobacterales with mechanisms of resistance varying across different regions. The existential threat to public health posed by the continued spread of nearly untreatable pathogens requires a multifaceted approach to prevent the collapse of modern medicine.

Comparing Vaginal and Endometrial Microbiota Using Culturomics: Proof of Concept.

It is generally accepted that microorganisms can colonize a non-pathological endometrium. However, in a clinical setting, endometrial samples are always collected by passing through the vaginal-cervical route. As such, the vaginal and cervical microbiomes can easily cross-contaminate endometrial samples, resulting in a biased representation of the endometrial microbiome. This makes it difficult to demonstrate that the endometrial microbiome is not merely a reflection of contamination originating from sampling. Therefore, we investigated to what extent the endometrial microbiome corresponds to that of the vagina, applying culturomics on paired vaginal and endometrial samples. Culturomics could give novel insights into the microbiome of the female genital tract, as it overcomes sequencing-related bias. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy were included. An additional vaginal swab was taken from each participant right before hysteroscopy. Both endometrial biopsies and vaginal swabs were analyzed using our previously described WASPLab-assisted culturomics protocol. In total, 101 bacterial and two fungal species were identified among these 10 patients. Fifty-six species were found in endometrial biopsies and 90 were found in vaginal swabs. On average, 28 % of species were found in both the endometrial biopsy and vaginal swab of a given patient. Of the 56 species found in the endometrial biopsies, 13 were not found in the vaginal swabs. Of the 90 species found in vaginal swabs, 47 were not found in the endometrium. Our culturomics-based approach sheds a different light on the current understanding of the endometrial microbiome. The data suggest the potential existence of a unique endometrial microbiome that is not merely a presentation of cross-contamination derived from sampling. However, we cannot exclude cross-contamination completely. In addition, we observe that the microbiome of the vagina is richer in species than that of the endometrium, which contradicts the current sequence-based literature.

Global population structure, genomic diversity and carbohydrate fermentation characteristics of clonal complex 119 (CC119), an understudied Shiga toxin-producing E. coli (STEC) lineage including O165:H25 and O172:H25.

Among Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of various serotypes, O157:H7 and five major non-O157 STEC (O26:H11, O111:H8, O103:H2, O121:H19 and O145:H28) can be selectively isolated by using tellurite-containing media. While human infections by O165:H25 STEC strains have been reported worldwide, their detection and isolation are not easy, as they are not resistant to tellurite. Systematic whole-genome sequencing (WGS) analyses have not yet been conducted. Here, we defined O165:H25 strains and their close relatives, including O172:H25 strains, as clonal complex 119 (CC119) and performed a global WGS analysis of the major lineage of CC119, called CC119 sensu stricto (CC119ss), by using 202 CC119ss strains, including 90 strains sequenced in this study. Detailed comparisons of 13 closed genomes, including 7 obtained in this study, and systematic analyses of Stx phage genomes in 50 strains covering the entire CC119ss lineage, were also conducted. These analyses revealed that the Stx2a phage, the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS), many prophages encoding T3SS effectors, and the virulence plasmid were acquired by the common ancestor of CC119ss and have been stably maintained in this lineage, while unusual exchanges of Stx1a and Stx2c phages were found at a single integration site. Although the genome sequences of Stx2a phages were highly conserved, CC119ss strains exhibited notable variation in Stx2 production levels. Further analyses revealed the lack of SpLE1-like elements carrying the tellurite resistance genes in CC119ss and defects in rhamnose, sucrose, salicin and dulcitol fermentation. The genetic backgrounds underlying these defects were also clarified.

Nursery outbreak caused by enteroaggregative Escherichia coli serogroup O111:H21.

This study describes, for the first time, the occurrence of an epidemic of enteroaggregative Escherichia coli (EAEC) O111:H21 in a Belgian nursery and describes a practical approach concerning its management. Few data exist in the literature on this type of outbreak. Clinical and microbiological investigations were needed to find a link between the cases and identify the causative agent. The microbiological procedure followed was first based on conventional analyses: isolation using selective cultures, identification by MALDI-TOF MS, antibiogram, determination of the serogroup by agglutination, then whole genome sequencing. A total of 7/21 children were infected with this pathogen. Four cases could be confirmed by a molecular technique, wgMLST, as belonging to the same bacterial clone. The action plan put in place focused on symptomatic case eviction, strict general hygiene precaution as well as specific cleaning and disinfection measures. The epidemic did last only a few days. It appears important, in the context of an epidemic, that clinical laboratories standardize their practice by equipping themselves with molecular techniques such as a multiplex which does not focus only on the serotype O157:H7 and which make it possible to distinguish the different pathotypes of E. coli by targeting several virulence genes (stx, aggR…). However, cost/effectiveness studies are awaited to confirm the interest of a systematic search by molecular method for the pathogen involved in a suspected outbreak occurring in a nursery.

Beyond Guidelines and Reports on Bacterial Co-/Superinfections in the Context of COVID-19: Why Uniformity Matters.

BACKGROUND: In the period following the declaration of the COVID-19 pandemic, more evidence became available on the epidemiology of bacterial co-/superinfections (bCSs) in hospitalized COVID-19 patients. Various European therapeutic guidelines were published, including guidance on rational antibiotic use. METHODS: In this letter to the editor, we provide an overview of the largest meta-analyses or prospective studies reporting on bCS rates in COVID-19 patients and discuss why the reader should interpret the results of those reports with care. Moreover, we compare different national and international COVID-19 therapeutic guidelines from countries of the European Union. Specific attention is paid to guidance dedicated to rational antibiotic use. RESULTS: We found a significant heterogeneity in studies reporting on the epidemiology of bCSs in COVID-19 patients. Moreover, European national and international guidelines differ strongly from each other, especially with regard to the content and extent of antibiotic guidance in hospitalized COVID-19 patients. CONCLUSION: A standardized way of reporting on bCSs and uniform European guidelines on rational antibiotic use in COVID-19 patients are crucial for antimicrobial stewardship teams to halt unnecessary antibiotic use in the COVID-19 setting.

Healthcare-Associated COVID-19 across Five Pandemic Waves: Prediction Models and Genomic Analyses.

BACKGROUND: Healthcare-associated SARS-CoV-2 infections need to be explored further. Our study is an analysis of hospital-acquired infections (HAIs) and ambulatory healthcare workers (aHCWs) with SARS-CoV-2 across the pandemic in a Belgian university hospital. METHODS: We compared HAIs with community-associated infections (CAIs) to identify the factors associated with having an HAI. We then performed a genomic cluster analysis of HAIs and aHCWs. We used this alongside the European Centre for Disease Control (ECDC) case source classifications of an HAI. RESULTS: Between March 2020 and March 2022, 269 patients had an HAI. A lower BMI, a worse frailty index, lower C-reactive protein (CRP), and a higher thrombocyte count as well as death and length of stay were significantly associated with having an HAI. Using those variables to predict HAIs versus CAIs, we obtained a positive predictive value (PPV) of 83.6% and a negative predictive value (NPV) of 82.2%; the area under the ROC was 0.89. Genomic cluster analyses and representations on epicurves and minimal spanning trees delivered further insights into HAI dynamics across different pandemic waves. The genomic data were also compared with the clinical ECDC definitions for HAIs; we found that 90.0% of the 'definite', 87.8% of the 'probable', and 70.3% of the 'indeterminate' HAIs belonged to one of the twenty-two COVID-19 genomic clusters we identified. CONCLUSIONS: We propose a novel prediction model for HAIs. In addition, we show that the management of nosocomial outbreaks will benefit from genome sequencing analyses.

Culturomics to Investigate the Endometrial Microbiome: Proof-of-Concept.

The microbiome of the reproductive tract has been associated with (sub)fertility and it has been suggested that dysbiosis reduces success rates and pregnancy outcomes. The endometrial microbiome is of particular interest given the potential impact on the embryo implantation. To date, all endometrial microbiome studies have applied a metagenomics approach. A sequencing-based technique, however, has its limitations, more specifically in adequately exploring low-biomass settings, such as intra-uterine/endometrial samples. In this proof-of-concept study, we demonstrate the applicability of culturomics, a high-throughput culturing approach, to investigate the endometrial microbiome. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy, as part of their routine work-up at Brussels IVF, were included after their informed consent. Biopsies were used to culture microbiota for up to 30 days in multiple aerobic and anaerobic conditions. Subsequent WASPLab®-assisted culturomics enabled a standardized methodology. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) or 16S rRNA sequencing was applied to identify all of bacterial and fungal isolates. Eighty-three bacterial and two fungal species were identified. The detected species were in concordance with previously published metagenomics-based endometrial microbiota analyses as 77 (91%) of them belonged to previously described genera. Nevertheless, highlighting the added value of culturomics to identify most isolates at the species level, 53 (62.4%) of the identified species were described in the endometrial microbiota for the first time. This study shows the applicability and added value of WASPLab®-assisted culturomics to investigate the low biomass endometrial microbiome at a species level.

Identification of Five Serotypes of Enteropathogenic Escherichia coli from Diarrheic Calves and Healthy Cattle in Belgium and Comparative Genomics with Shigatoxigenic E. coli.

Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (AE) lesions and cause non-bloody diarrhea in mammals. A minority of bovine EPEC belong to one of the ten classical serotypes of human and bovine AE-STEC. The purpose of this study was to identify five non-classical O serotypes (O123/186, O156, O177, O182, and O183) among bovine EPEC and to characterize their virulence repertoires by whole genome sequencing. Around 40% of the 307 EPEC from 307 diarrheic calves, 368 EPEC from 47 healthy cattle, and 131 EPEC from 36 healthy calves in dairy farms were analyzed. Serotype O177 was the most frequent among EPEC from diarrheic and healthy calves, while the O156 was the most frequent in healthy cattle. The genomic analysis identified different H serotypes, MLSTypes, and/or eae gene subtypes among the O156 and O177 EPEC, while the O182 was homogeneous. The virulence gene profiles of bovine EPEC were closely related to each other and to the profiles of ten bovine and human AE-STEC. These results emphasize the need for additional studies to identify more O:H serotypes of bovine EPEC and to elucidate their origin and evolution of EPEC with regard to AE-STEC belonging to the same O:H serotypes.

Healthcare-Associated SARS-CoV-2 Reinfection after 3 Months with a Phylogenetically Distinct Omicron Variant: A Case Report.

This case report describes a 60-year-old female patient suffering from systemic sclerosis, for which she received immunomodulatory drugs. Her first SARS-CoV-2-positive nasopharyngeal sample was obtained in the emergency department, on 31 January 2022. Whole genome sequencing confirmed infection with Omicron BA.1.1. Her hospital stay was long and punctuated by many complications, including admission to the intensive care unit. At the beginning of April 2022, she started complaining of increased coughing, for which another SARS-CoV-2 RT-qPCR test was performed. The latter nasopharyngeal swab showed a strongly positive result. To support the theory of healthcare-associated reinfection, whole genome sequencing was performed and confirmed reinfection with Omicron BA.2. Since this patient was one of ten positive cases in this particular ward, a hospital outbreak investigation was performed. Whole genome sequencing data were available for five of these ten patients and showed a cluster of four patients with ≤2 small nucleotide polymorphisms difference.

Validation of the Colibrí Instrument for Automated Preparation of MALDI-TOF MS Targets for Yeast Identification.

Recently, Copan (Italy) introduced the Colibrí instrument for automated colony picking and preparation of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) target plates. Our study aimed to validate this system for yeasts as such testing has not been performed yet and is a missing link needed to implement the system for routine use. Fifty-five Candida strains were selected to evaluate the accuracy of Colibrí. For each strain, a sheep blood agar plate supplemented with X and V factors (HEM) and a Sabouraud agar plate (SAB) were inoculated and incubated using the WASPlab specimen processing system (Copan). After 18 h and 36 h of incubation, the isolates were spotted in parallel using Colibrí and manually onto MALDI-TOF target plates with the addition of formic acid and identified using MALDI-TOF mass spectrometry. The reproducibility was evaluated using ATCC reference and clinical isolate-derived strains. The cumulative percentage of acceptable identification scores (IDs) after 36 h was 91% for strains cultured on HEM plates using both Colibrí and the manual method. The SAB plates showed inferior results for both Colibrí (76%) and the manual method (78%). We observed an overall agreement of 92% at 18 h for identification of the strains on the HEM plates between Colibrí and the manual method and 94% after 36 h. For the SAB plates, the agreement was 78% after 18 h and 84% after 36 h. Apart from Candida dubliniensis and Candida tropicalis, all Candida species were identified with 100% accuracy using Colibrí on HEM plates. We observed good agreement between Colibrí and the manual reference method. These results demonstrate that Colibrí is a reliable system for MALDI-TOF target preparation for yeast identification, allowing increased standardization and less hands-on time.