A SARS-CoV-2 and influenza rapid antigen test-based hospital isolation policy awaiting RT-PCR, a prospective observational study.

OBJECTIVES: This study aimed to evaluate the clinical performance of a combined SARS-CoV-2/influenza rapid antigen test (SIRAT) and to evaluate a SIRAT-based hospital isolation policy awaiting RT-PCR results for patients presenting at the emergency department (ED). METHODS: We performed a prospective observational study including all adult patients presenting with influenza-like symptoms at the ED of two hospitals from 31 October 2022 to 31 March 2023. A SIRAT and SARS-CoV-2 and influenza RT-PCR were performed on upper respiratory samples. SIRAT results were compared with RT-PCR. Droplet and contact isolation measures (DCIM) were imposed based on SIRAT results awaiting RT-PCR. We monitored symptomatic nosocomial SARS-CoV-2 and influenza infections potentially caused by delayed isolation of patients with false negative SIRAT and the hours of unnecessary DCIM saved. RESULTS: We included 1740 patients of whom 1296 were hospitalized. SARS-CoV-2 and influenza A/B prevalence were 12.7% (221/1740) and 9.9% (171/1740). Sensitivity and specificity of the SIRAT were 67.7% (95% CI 61.1-73.9%) (149/220) and 99.4% (95% CI 99.0-99.8%) (1510/1518) for SARS-CoV-2 and 52.7% (95% CI 44.9-60.4%) (89/169) and 99.1% (95% CI 98.5-99.5%) (1530/1544) for influenza A/B. We found a 0% nosocomial transmission risk for SARS-CoV-2 (95% CI 0-8.8%) and influenza (95% CI 0-10%). In all, 8712 hours in total or a median up to 6 hours 59 minutes (IQR (interquartile range) 11h03) per patient of unnecessary DCIM were saved. DISCUSSION: A SIRAT-guided hospital isolation policy awaiting RT-PCR results for patients who present at the ED can save unnecessary isolation hours without having to lead to significant symptomatic nosocomial transmission of SARS-CoV-2 or influenza viruses.

A pseudo-outbreak of MRSA due to laboratory contamination related to MRSA carriage of a laboratory staff member.

BACKGROUND: Methicillin resistant Staphylococcus aureus (MRSA) is a major burden for hospitals globally. However, in the Netherlands, the MRSA prevalence is relatively low due to the 'search and destroy' policy. Routine multiple-locus variable-number of tandem repeat analysis (MLVA) of MRSA isolates supports outbreak detection. However, whole genome multiple locus sequence typing (wgMLST) is superior to MLVA in identifying (pseudo-)outbreaks with MRSA. The present study describes a pseudo-outbreak of MRSA at the bacteriology laboratory of a large Dutch teaching hospital. METHODS: All staff members of the bacteriology laboratory of the Elisabeth-TweeSteden hospital were screened for MRSA carriage, after a laboratory contamination with MRSA was suspected. Clonal relatedness between the index isolate and the MRSA isolates from laboratory staff members and all previous MRSA isolates from the Elisabeth-TweeSteden hospital with the same MLVA-type as the index case was examined based on wgMLST using whole genome sequencing. RESULTS: One of the staff members was identified as the probable source of the laboratory contamination, because of carriage of a MRSA possessing the same MLVA-type as the index case. Eleven other isolates with the same molecular characteristics were found in the database, of which seven were retrospectively suspected of contamination. Clonal relatedness was found between ten isolates, including the isolate found in the staff member and the MRSA found in the index patient with a maximum of eleven alleles difference. All isolates were epidemiologically linked through the laboratory staff member, who had worked on all these cultures. CONCLUSIONS: The present study describes a MRSA pseudo-outbreak over a 2.5-year period due to laboratory contamination caused by a MRSA carrying laboratory staff member involving nine patients. In case of unexpected bacteriological findings, the possibility of a laboratory contamination should be considered.

Head-to-head comparison of the accuracy of saliva and nasal rapid antigen SARS-CoV-2 self-testing: cross-sectional study.

BACKGROUND: The diagnostic accuracy of unsupervised self-testing with rapid antigen diagnostic tests (Ag-RDTs) is mostly unknown. We studied the diagnostic accuracy of a self-performed SARS-CoV-2 saliva and nasal Ag-RDT in the general population. METHODS: This large cross-sectional study consecutively included unselected individuals aged ≥ 16 years presenting for SARS-CoV-2 testing at three public health service test sites. Participants underwent molecular test sampling and received two self-tests (the Hangzhou AllTest Biotech saliva self-test and the SD Biosensor nasal self-test by Roche Diagnostics) to perform themselves at home. Diagnostic accuracy of both self-tests was assessed with molecular testing as reference. RESULTS: Out of 2819 participants, 6.5% had a positive molecular test. Overall sensitivities were 46.7% (39.3-54.2%) for the saliva Ag-RDT and 68.9% (61.6-75.6%) for the nasal Ag-RDT. With a viral load cut-off (≥ 5.2 log10 SARS-CoV-2 E-gene copies/mL) as a proxy of infectiousness, these sensitivities increased to 54.9% (46.4-63.3%) and 83.9% (76.9-89.5%), respectively. For the nasal Ag-RDT, sensitivities were 78.5% (71.1-84.8%) and 22.6% (9.6-41.1%) in those symptomatic and asymptomatic at the time of sampling, which increased to 90.4% (83.8-94.9%) and 38.9% (17.3-64.3%) after applying the viral load cut-off. In those with and without prior SARS-CoV-2 infection, sensitivities were 36.8% (16.3-61.6%) and 72.7% (65.1-79.4%). Specificities were > 99% and > 99%, positive predictive values > 70% and > 90%, and negative predictive values > 95% and > 95%, for the saliva and nasal Ag-RDT, respectively, in most analyses. Most participants considered the self-performing and result interpretation (very) easy for both self-tests. CONCLUSIONS: The Hangzhou AllTest Biotech saliva self Ag-RDT is not reliable for SARS-CoV-2 detection, overall, and in all studied subgroups. The SD Biosensor nasal self Ag-RDT had high sensitivity in individuals with symptoms and in those without prior SARS-CoV-2 infection but low sensitivity in asymptomatic individuals and those with a prior SARS-CoV-2 infection which warrants further investigation.

SARS-CoV-2 Alpha-Variant Outbreak Amongst a Partially Vaccinated Long-Term Care Facility Population in The Netherlands-Phylogenetic Analysis and Infection Control Observations.

Despite extensive vaccination and booster programs, SARS-CoV-2 outbreaks in long-term care facilities (LTCF) continue to occur. We retrospectively describe a SARS-CoV-2 outbreak amongst a partially vaccinated LTCF population in The Netherlands which occurred in March 2021. The facility comprised three floors functioning as separate wards. Nasopharyngeal swabs for SARS-CoV-2 qRT-PCR were obtained from residents and staff presenting with COVID-19-like symptoms and from all residents and staff during two point prevalence screenings (PPS). Samples meeting technical criteria were included for phylogenetic analysis. Positive SARS-CoV-2 qRT-PCR were obtained from 11 (18%) of 61 residents and 8 (7%) of 110 staff members between March 8 and March 25. Seven (37%) cases and five (63%) vaccinated cases were diagnosed through PPS. Cases were found on all wards. Phylogenetic analysis (n = 11) showed a maximum difference of four nucleotides between sequences on the outer branches of the tree, but identified two identical sequences on the root differing maximum two nucleotides from all other sequences, suggesting all did belong to the same cluster. Our results imply that PPS is useful in containing SARS-CoV-2 outbreaks amongst (vaccinated) LTCF populations, as an entire LTCF might behave as a single epidemiological unit and it is preferable to maximize the number of samples included for phylogenetic analysis.

Self-testing for the detection of SARS-CoV-2 infection with rapid antigen tests for people with suspected COVID-19 in the community.

OBJECTIVES: To evaluate the performance of nasal mid-turbinate self-testing using rapid antigen detection tests (RDT) for persons with suspected coronavirus disease 2019 (COVID-19) in the community. Self-testing for COVID-19 infection with lateral flow assay severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RDT, provides rapid results and could enable frequent and extensive testing in the community, thereby improving the control of SARS-CoV-2. METHODS: Participants visiting a municipal SARS-CoV-2 testing centre, received self-testing kits containing either the BD Veritor System (BD-RDT) or Roche SARS-CoV-2 antigen detection test (Roche-RDT). Oro-nasopharyngeal swabs were collected from the participants for quantitative RT-PCR (qRT-PCR) testing. As a proxy for contagiousness, viral culture was performed on a selection of qRT-PCR positive samples to determine the Ct-value at which the chance of a positive culture dropped below 0.5 (Ct-value cut-off). Sensitivity and specificity of self-testing were compared to qRT-PCR with a Ct-value below the Ct value cut-off. Determinants independently associated with a false-negative self-test result were determined. RESULTS: A total of 3201 participants were included (BD-RDT n = 1595; Roche-RDT n = 1606). Sensitivity and specificity of self-testing compared with the qRT-PCR results with a Ct-value below the Ct-value cut-off were 78.4% (95% CI 73.2%-83.5%) and 99.4% (95% CI 99.1%-99.7%), respectively. A higher age was independently associated with a false-negative self-testing result with an odds ratio of 1.024 (95% CI 1.003-1.044). CONCLUSIONS: Self-testing using currently available RDT has a high specificity and relatively high sensitivity to identify individuals with a high probability of contagiousness.

Evaluation of the test accuracy of a SARS-CoV-2 rapid antigen test in symptomatic community dwelling individuals in the Netherlands.

BACKGROUND: SARS-CoV-2 real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is well suited for the diagnosis of clinically ill patients requiring treatment. Application for community testing of symptomatic individuals for disease control purposes however raises challenges. SARS-CoV-2 rapid antigen tests might offer an alternative, but quality evidence on their performance is limited. METHODS: We conducted an evaluation of the test accuracy of the 'BD Veritor System for Rapid Detection of SARS-CoV-2' (VRD) compared to qRT-PCR on combined nose/throat swabs obtained from symptomatic individuals at Municipal Health Service (MHS) COVID-19 test centers in the Netherlands. In part one of the study, with the primary objective to evaluate test sensitivity and specificity, all adults presenting at one MHS test center were eligible for inclusion. In part two, with the objective to evaluate test sensitivity stratified by Ct (cycle threshold)-value and time since symptom onset, adults who had a positive qRT-PCR obtained at a MHS test center were eligible. FINDINGS: In part one (n = 352) SARS-CoV-2 prevalence was 4.8%, overall specificity 100% (95%CI: 98·9%-100%) and sensitivity 94·1% (95%CI: 71·1%-100%). In part two (n = 123) the sensitivity was 78·9% (95%CI: 70·6%-85·7%) overall, 89·4% (95% CI: 79·4%-95·6%) for specimen obtained within seven days after symptom onset and 93% (95% CI: 86%-97.1%) for specimen with a Ct-value below 30. INTERPRETATION: The VRD is a promising diagnostic for COVID-19 testing of symptomatic community-dwelling individuals within seven days after symptom onset in context of disease control. Further research on practical applicability and the optimal position within the testing landscape is needed.

Distinguishing blaKPC Gene-Containing IncF Plasmids from Epidemiologically Related and Unrelated Enterobacteriaceae Based on Short- and Long-Read Sequence Data.

Limited information is available on whether blaKPC-containing plasmids from isolates in a hospital outbreak can be differentiated from epidemiologically unrelated blaKPC-containing plasmids based on sequence data. This study aimed to evaluate the performance of three approaches to distinguish epidemiologically related from unrelated blaKPC-containing pKpQiL-like IncFII(k2)-IncFIB(pQiL) plasmids. Epidemiologically related isolates were subjected to short- and long-read whole-genome sequencing. A hybrid assembly was performed, and plasmid sequences were extracted from the assembly graph. Epidemiologically unrelated plasmid sequences were extracted from GenBank. Pairwise comparisons of epidemiologically related and unrelated plasmids based on SNPs using snippy and of phylogenetic distance using Roary and using a similarity index that penalizes size differences between plasmids (Stoesser index) were performed. The percentage of pairwise comparisons misclassified as genetically related or as clonally unrelated was determined using different genetic thresholds for genetic relatedness. The ranges of number of SNPs, Roary phylogenetic distance, and Stoesser index overlapped between the epidemiologically related and unrelated plasmids. When a genetic similarity threshold that classified 100% of epidemiologically related plasmid pairs as genetically related was used, the percentages of plasmids misclassified as epidemiologically related ranged from 6.7% (Roary) to 20.8% (Stoesser index). Although epidemiologically related plasmids can be distinguished from unrelated plasmids based on genetic differences, blaKPC-containing pKpQiL-like IncFII(k2)-IncFIB(pQiL) plasmids show a high degree of sequence similarity. The phylogenetic distance as determined using Roary showed the highest degree of discriminatory power between the epidemiologically related and unrelated plasmids.

Limited Genetic Diversity of blaCMY-2-Containing IncI1-pST12 Plasmids from Enterobacteriaceae of Human and Broiler Chicken Origin in The Netherlands.

Distinguishing epidemiologically related and unrelated plasmids is essential to confirm plasmid transmission. We compared IncI1-pST12 plasmids from both human and livestock origin and explored the degree of sequence similarity between plasmids from Enterobacteriaceae with different epidemiological links. Short-read sequence data of Enterobacteriaceae cultured from humans and broilers were screened for the presence of both a blaCMY-2 gene and an IncI1-pST12 replicon. Isolates were long-read sequenced on a MinION sequencer (OxfordNanopore Technologies). After plasmid reconstruction using hybrid assembly, pairwise single nucleotide polymorphisms (SNPs) were determined. The plasmids were annotated, and a pan-genome was constructed to compare genes variably present between the different plasmids. Nine Escherichia coli sequences of broiler origin, four Escherichia coli sequences, and one Salmonella enterica sequence of human origin were selected for the current analysis. A circular contig with the IncI1-pST12 replicon and blaCMY-2 gene was extracted from the assembly graph of all fourteen isolates. Analysis of the IncI1-pST12 plasmids revealed a low number of SNP differences (range of 0-9 SNPs). The range of SNP differences overlapped in isolates with different epidemiological links. One-hundred and twelve from a total of 113 genes of the pan-genome were present in all plasmid constructs. Next generation sequencing analysis of blaCMY-2-containing IncI1-pST12 plasmids isolated from Enterobacteriaceae with different epidemiological links show a high degree of sequence similarity in terms of SNP differences and the number of shared genes. Therefore, statements on the horizontal transfer of these plasmids based on genetic identity should be made with caution.

Clinical performance and sample freeze-thaw stability of the cobas®6800 SARS-CoV-2 assay for the detection of SARS-CoV-2 in oro-/nasopharyngeal swabs and lower respiratory specimens.

INTRODUCTION: Studies describing the performance characteristics of the cobas®6800 system for SARS-CoV-2 detection in deep respiratory specimens and freeze-thaw stability are limited. The current study compares the clinical performance of the automated SARS-CoV-2 assay on the cobas®6800 system to a lab-developed assay (LDA) and the cobas impact of freeze-thawing combined with lysis buffer. METHODS: Both retrospective and prospectively selected deep respiratory samples and oro- and nasopharyngeal samples in either E-swab® or GLY- were tested using the SARS-CoV-2 assay on the cobas®6800 System and compared to a lab developed assay. Additonally, SARS-CoV-2 RNA stability was assessed after one freeze-thaw cycle with or without lysis buffer. RESULTS: In total, 221 (58.3 %) oro- and nasopharyngeal swabs, 131 (34.6 %) deep respiratory specimens, and n = 25 (6.6 %) swabs of unknown origin were included to study clinical performance. Only 4 samples gave discrepant results, all being positive in the LDA and not the cobas®6800 system. For stability testing, 66 samples without and 110 with lysis buffer were included. No clinically significant difference was found in test results after one freeze-thaw cycle and addition of lysis buffer. CONCLUSION: Based on our findings, the cobas®6800 SARS-CoV-2 RNA assay yielded similar results as the LDA in oro-/nasopharyngeal swabs and deep respiratory specimens. Moreover, the cobas®6800 SARS-CoV-2 RNA assay yielded similar results before and after a freeze-thaw cycle, with better preservation of low viral loads in lysis buffer.

Detection of extended-spectrum beta-lactamase (ESBL) genes and plasmid replicons in Enterobacteriaceae using PlasmidSPAdes assembly of short-read sequence data.

Knowledge of the epidemiology of plasmids is essential for understanding the evolution and spread of antimicrobial resistance. PlasmidSPAdes attempts to reconstruct plasmids using short-read sequence data. Accurate detection of extended-spectrum beta-lactamase (ESBL) genes and plasmid replicon genes is a prerequisite for the use of plasmid assembly tools to investigate the role of plasmids in the spread and evolution of ESBL production in Enterobacteriaceae. This study evaluated the performance of PlasmidSPAdes plasmid assembly for Enterobacteriaceae in terms of detection of ESBL-encoding genes, plasmid replicons and chromosomal wgMLST genes, and assessed the effect of k-mer size. Short-read sequence data for 59 ESBL-producing Enterobacteriaceae were assembled with PlasmidSPAdes using different k-mer sizes (21, 33, 55, 77, 99 and 127). For every k-mer size, the presence of ESBL genes, plasmid replicons and a selection of chromosomal wgMLST genes in the plasmid assembly was determined. Out of 241 plasmid replicons and 66 ESBL genes detected by whole-genome assembly, 213 plasmid replicons [88 %; 95 % confidence interval (CI): 83.9-91.9] and 43 ESBL genes (65 %; 95 % CI: 53.1-75.6) were detected in the plasmid assemblies obtained by PlasmidSPAdes. For most ESBL genes (83.3 %) and plasmid replicons (72.0 %), detection results did not differ between the k-mer sizes used in the plasmid assembly. No optimal k-mer size could be defined for the number of ESBL genes and plasmid replicons detected. For most isolates, the number of chromosomal wgMLST genes detected in the plasmid assemblies decreased with increasing k-mer size. Based on our findings, PlasmidSPAdes is not a suitable plasmid assembly tool for short-read sequence data for ESBL-encoding plasmids of Enterobacteriaceae.

Within-patient plasmid dynamics in Klebsiella pneumoniae during an outbreak of a carbapenemase-producing Klebsiella pneumoniae.

INTRODUCTION: Knowledge of within-patient dynamics of resistance plasmids during outbreaks is important for understanding the persistence and transmission of plasmid-mediated antimicrobial resistance. During an outbreak of a Klebsiella pneumoniae carbapenemase-producing (KPC) K. pneumoniae, the plasmid and chromosomal dynamics of K. pneumoniae within-patients were investigated. METHODS: During the outbreak, all K. pneumoniae isolates of colonized or infected patients were collected, regardless of their susceptibility pattern. A selection of isolates was short-read and long-read sequenced. A hybrid assembly of the short-and long-read sequence data was performed. Plasmid contigs were extracted from the hybrid assembly, annotated, and within patient plasmid comparisons were performed. RESULTS: Fifteen K. pneumoniae isolates of six patients were short-read whole-genome sequenced. Whole-genome multi-locus sequence typing revealed a maximum of 4 allele differences between the sequenced isolates. Within patients 1 and 2 the resistance gene- and plasmid replicon-content did differ between the isolates sequenced. Long-read sequencing and hybrid assembly of 4 isolates revealed loss of the entire KPC-gene containing plasmid in the isolates of patient 2 and a recombination event between the plasmids in the isolates of patient 1. This resulted in two different KPC-gene containing plasmids being simultaneously present during the outbreak. CONCLUSION: During a hospital outbreak of a KPC-producing K. pneumoniae isolate, plasmid loss of the KPC-gene carrying plasmid and plasmid recombination was detected within the isolates from two patients. When investigating outbreaks, one should be aware that plasmid transmission can occur and the possibility of within- and between-patient plasmid variation needs to be considered.

Development of amoxicillin resistance in Escherichia coli after exposure to remnants of a non-related phagemid-containing E. coli: an exploratory study.

OBJECTIVE: To determine the effect of exposure to remnants of a phagemid-containing E. coli, killed by treatment with a propanol-based hand rub, on antimicrobial resistance in E. coli isolates. METHODS: An in vitro model was developed in which a clinical E. coli isolate (EUR1) was exposed to remnants of an E. coli K-12 strain containing a phagemid (pBS-E12) strain treated with Sterillium®. A series of 200 experiments was performed using this in vitro model. As a control, a series of 400 experiments was performed where the EUR1 was exposed either to the remnants of an E. coli K-12 strain (not containing a phagemid) (E12) treated with Sterillium® (n = 200) or to dried Sterillium® only (n = 200). The number of experiments that showed growth of an amoxicillin-resistant EUR1 isolate was evaluated in all three groups. An additional 48 experiments were performed in which a different clinical E. coli isolate (EUR2) was exposed to remnants of the pBS-E12 treated with Sterillium®. Whole-genome sequencing and phenotypic testing for AmpC beta-lactamase production was performed to investigate the mechanism behind this resistance development. RESULTS: In 22 (11.0%) of 200 experiments in which the EUR1 isolate was exposed to remnants of a pBS-E12 an amoxicillin-resistant mutant isolate was obtained, as opposed to only 2 (1.0%) of 200 experiments involving the exposure of the EUR1 to Sterillium® only (risk difference: 10.0%; 95% CI 5.4-14.6%)) and 1 (0.5%) of 200 experiments involving the exposure of the EUR1 isolate to the remnants of the phagemid-free E12 (risk difference: 10.5%; 95% CI 6.1-14.9%). In 1 (2.1%) of the 48 experiments in which the EUR2 isolate was exposed to remnants of a pBS-E12 an amoxicillin-resistant mutant isolate was obtained. The development of resistance in all experiments was due to mutations in the promoter/attenuator region of the chromosomal AmpC beta-lactamase (cAmpC) gene leading to cAmpC hyperproduction. CONCLUSION: Exposure of an E. coli isolate to another phagemid-containing E. coli that was treated with propanol-based hand rub increased the development of amoxicillin resistance. Although phagemids are cloning vectors that are not present in clinical isolates, this finding may have implications for hand disinfection practices in healthcare facilities.