A Case of Mpox Reinfection.

A healthy young man first diagnosed with mpox in May 2022 presented again in November 2022 with anal proctitis and a positive polymerase chain reaction on a rectal swab for Monkeypox virus after a recent trip to Brazil, where he engaged in condomless sexual intercourse with multiple male partners.

MazF toxin causes alterations in Staphylococcus aureus transcriptome, translatome and proteome that underlie bacterial dormancy.

Antibiotic resistance is a serious problem which may be caused by bacterial dormancy. It has been suggested that bacterial toxin-antitoxin systems induce dormancy. We analyzed the genome-wide role of Staphylococcus aureus endoribonuclease toxin MazF using RNA-Seq, Ribo-Seq and quantitative proteomics. We characterized changes in transcriptome, translatome and proteome caused by MazF, and proposed that MazF decreases translation directly by cleaving mRNAs, and indirectly, by decreasing translation factors and by promoting ribosome hibernation. Important pathways affected during the early stage of MazF induction were identified: MazF increases cell wall thickness and decreases cell division; MazF activates SsrA-system which rescues stalled ribosomes, appearing as a result of MazF mRNA cleavage. These pathways may be promising targets for new antibacterial drugs that prevent bacteria dormancy. Finally, we described the overall impact of MazF on S. aureus cell physiology, and propose one of the mechanisms by which MazF might regulate cellular changes leading to dormancy.

Insights into the global effect on Staphylococcus aureus growth arrest by induction of the endoribonuclease MazF toxin.

A crucial bacterial strategy to avoid killing by antibiotics is to enter a growth arrested state, yet the molecular mechanisms behind this process remain elusive. The conditional overexpression of mazF, the endoribonuclease toxin of the MazEF toxin-antitoxin system in Staphylococcus aureus, is one approach to induce bacterial growth arrest, but its targets remain largely unknown. We used overexpression of mazF and high-throughput sequence analysis following the exact mapping of non-phosphorylated transcriptome ends (nEMOTE) technique to reveal in vivo toxin cleavage sites on a global scale. We obtained a catalogue of MazF cleavage sites and unearthed an extended MazF cleavage specificity that goes beyond the previously reported one. We correlated transcript cleavage and abundance in a global transcriptomic profiling during mazF overexpression. We observed that MazF affects RNA molecules involved in ribosome biogenesis, cell wall synthesis, cell division and RNA turnover and thus deliver a plausible explanation for how mazF overexpression induces stasis. We hypothesize that autoregulation of MazF occurs by directly modulating the MazEF operon, such as the rsbUVW genes that regulate the sigma factor SigB, including an observed cleavage site on the MazF mRNA that would ultimately play a role in entry and exit from bacterial stasis.

Sensitivity of nasopharyngeal, oropharyngeal, and nasal wash specimens for SARS-CoV-2 detection in the setting of sampling device shortage.

In the context of an unprecedented shortage of nasopharyngeal swabs (NPS) or sample transport media during the coronavirus disease 2019 (COVID-19) crisis, alternative methods for sample collection are needed. To address this need, we validated a cell culture medium as a viral transport medium, and compared the analytical sensitivity of SARS-CoV-2 RT-PCR in nasal wash (NW), oropharyngeal swab (OPS), and NPS specimens. Both the clinical and analytical sensitivity were comparable in these three sample types. OPS and NW specimens may therefore represent suitable alternatives to NPS for SARS-CoV-2 detection.

Sub-Inhibitory Doses of Individual Constituents of Essential Oils Can Select for Staphylococcus aureus Resistant Mutants.

Increased bacterial resistance to food preservation technologies represents a risk for food safety and shelf-life. The use of natural antimicrobials, such as essential oils (EOs) and their individual constituents (ICs), has been proposed to avoid the generation of antimicrobial resistance. However, prolonged application of ICs might conceivably lead to the emergence of resistant strains. Hence, this study was aimed toward applying sub-inhibitory doses of the ICs carvacrol, citral, and (+)-limonene oxide to Staphylococcus aureus USA300, in order to evaluate the emergence of resistant strains and to identify the genetic modifications responsible for their increased resistance. Three stable-resistant strains, CAR (from cultures with carvacrol), CIT (from cultures with citral), and OXLIM (from cultures with (+)-limonene oxide) were isolated, showing an increased resistance against the ICs and a higher tolerance to lethal treatments by ICs or heat. Whole-genome sequencing revealed in CAR a large deletion in a region that contained genes encoding transcriptional regulators and metabolic enzymes. CIT showed a single missense mutation in aroC (N187K), which encodes for chorismate synthase; and in OXLIM a missense mutation was detected in rpoB (A862V), which encodes for RNA polymerase subunit beta. This study provides a first detailed insight into the mechanisms of action and S. aureus resistance arising from exposure to carvacrol, citral, and (+)-limonene oxide.

Whole-Genome Sequencing and Genetic Analysis Reveal Novel Stress Responses to Individual Constituents of Essential Oils in Escherichia coli.

Food preservation by the use of essential oils (EOs) is being extensively studied because of the antimicrobial properties of their individual constituents (ICs). Three resistant mutants (termed CAR, CIT, and LIM) of Escherichia coli MG1655 were selected by subculturing with the ICs carvacrol, citral, and (+)-limonene oxide, respectively. These derivative strains showed increased MIC values of ICs and concomitantly enhanced resistance to various antibiotics (ampicillin, trimethoprim, chloramphenicol, tetracycline, kanamycin, novobiocin, norfloxacin, cephalexin, and nalidixic acid) compared to those for the parental strain (wild type [WT]). Whole-genome sequencing (WGS) of these hyperresistant strains permitted the identification of single nucleotide polymorphisms (SNPs) and deletions in comparison to the WT. In order to analyze the contribution of these mutations to the increased antimicrobial resistance detected in hyperresistant strains, derivative strains were constructed by allelic reversion. A role of the SoxR D137Y missense mutation in CAR was confirmed by growth in the presence of some ICs and antibiotics and by its tolerance to ICs but not to lethal heat treatments. In CIT, increased resistance relied on contributions by several detected SNPs, resulting in a frameshift in MarR and an in-frame GyrB ΔG157 mutation. Finally, both the insertion resulting in an AcrR frameshift and large chromosomal deletions found in LIM were correlated with the hyperresistant phenotype of this strain. The nature of the obtained mutants suggests intriguing links to cellular defense mechanisms previously implicated in antibiotic resistance.IMPORTANCE The antimicrobial efficacy of ICs has been proven over the years, together with their potential to improve traditional heat treatments by reducing treatment intensity and, consequently, adverse effects on food quality. However, the mechanisms of bacterial inactivation by ICs are still not well understood, in contrast to antibiotics. We performed WGS of three E. coli strains that are hyperresistant to ICs. The information provided detailed insight into the mechanisms of bacterial resistance arising from exposure to carvacrol, citral, and (+)-limonene oxide. Future experiments will undoubtedly yield additional insights into genes and pathways contributing to the acquisition of endogenous resistance to ICs.

Molecular Bases Determining Daptomycin Resistance-Mediated Resensitization to ß-Lactams (Seesaw Effect) in Methicillin-Resistant Staphylococcus aureus.

Antimicrobial resistance is recognized as one of the principal threats to public health worldwide, yet the problem is increasing. Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains are among the most difficult to treat in clinical settings due to the resistance of MRSA to nearly all available antibiotics. The cyclic anionic lipopeptide antibiotic daptomycin (DAP) is the clinical mainstay of anti-MRSA therapy. The decreased susceptibility to DAP (DAP resistance [DAPr]) reported in MRSA is frequently accompanied by a paradoxical decrease in ß-lactam resistance, a process known as the "seesaw effect." Despite the observed discordance in resistance phenotypes, the combination of DAP and ß-lactams has been proven to be clinically effective for the prevention and treatment of infections due to DAPr MRSA strains. However, the mechanisms underlying the interactions between DAP and ß-lactams are largely unknown. In the study described here, we studied the role of mprF with DAP-induced mutations in ß-lactam sensitization and its involvement in the effective killing by the DAP-oxacillin (OXA) combination. DAP-OXA-mediated effects resulted in cell wall perturbations, including changes in peptidoglycan insertion, penicillin-binding protein 2 (PBP 2) delocalization, and reduced membrane amounts of PBP 2a, despite the increased transcription of mecA through mec regulatory elements. We have found that the VraSR sensor-regulator is a key component of DAP resistance, triggering mutated mprF-mediated cell membrane (CM) modifications that result in impairment of PrsA location and chaperone functions, both of which are essential for PBP 2a maturation, the key determinant of ß-lactam resistance. These observations provide for the first time evidence that synergistic effects between DAP and ß-lactams involve PrsA posttranscriptional regulation of CM-associated PBP 2a.

Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus.

UNLABELLED: Staphylococcus aureus is capable of causing a remarkable spectrum of disease, ranging from mild skin eruptions to life-threatening infections. The survival and pathogenic potential of S. aureus depend partly on its ability to sense and respond to changes in its environment. Spx is a thiol/oxidative stress sensor that interacts with the C-terminal domain of the RNA polymerase RpoA subunit, leading to changes in gene expression that help sustain viability under various conditions. Using genetic and deep-sequencing methods, we show that spx is essential in S. aureus and that a previously reported Δspx strain harbored suppressor mutations that allowed it to grow without spx One of these mutations is a single missense mutation in rpoB (a P-to-L change at position 519 encoded by rpoB [rpoB-P519L]) that conferred high-level resistance to rifampin. This mutation alone was found to be sufficient to bypass the requirement for spx The generation of rifampin resistance libraries led to the discovery of an additional rpoB mutation, R484H, which supported strains with the spx disruption. Other rifampin resistance mutations either failed to support the Δspx mutant or were recovered at unexpectedly low frequencies in genetic transduction experiments. The amino acid residues encoded by rpoB-P519L and -R484H map in close spatial proximity and comprise a highly conserved region of RpoB. We also discovered that multicopy expression of either trxA (encoding thioredoxin) or trxB (encoding thioredoxin reductase) supports strains with the deletion of spx Our results reveal intriguing properties, especially of RNA polymerase, that compensate for the loss of an essential gene that is a key mediator of diverse processes in S. aureus, including redox and thiol homeostasis, antibiotic resistance, growth, and metabolism. IMPORTANCE: The survival and pathogenicity of S. aureus depend on complex genetic programs. An objective for combating this insidious organism entails dissecting genetic regulatory circuits and discovering promising new targets for therapeutic intervention. In this study, we discovered that Spx, an RNA polymerase-interacting stress regulator implicated in many stress responses in S. aureus, including responses to oxidative and cell wall antibiotics, is essential. We describe two mechanisms that suppress the lethality of spx disruption. One mechanism highlights how only certain rifampin resistance-encoding alleles of RpoB confer new properties on RNA polymerase, with important mechanistic implications. We describe additional stress conditions where the loss of spx is deleterious, thereby highlighting Spx as a multifaceted regulator and attractive drug discovery target.

Missense mutations in PBP2A Affecting ceftaroline susceptibility detected in epidemic hospital-acquired methicillin-resistant Staphylococcus aureus clonotypes ST228 and ST247 in Western Switzerland archived since 1998.

The development and maintenance of an arsenal of antibiotics is a major health care challenge. Ceftaroline is a new cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA); however, no reports concerning MRSA ceftaroline susceptibility have been reported in Switzerland. We tested the in vitro activity of ceftaroline against an archived set of 60 MRSA strains from the University Hospital of Geneva collected from 1994 to 2003. Our results surprisingly revealed ceftaroline-resistant strains (MIC, >1 µg/ml in 40/60 strains; EUCAST breakpoints, susceptible [S], ≤1 µg/ml; resistant [R], >1 µg/ml) were present from 1998 to 2003. The detected resistant strains predominantly belonged to sequence type 228 (ST228) (South German clonotype) but also to ST247 (Iberian clonotype). A sequence analysis of these strains revealed missense mutations in the penicillin-binding protein 2A (PBP2A) allosteric domain (N146K or E239K and N146K-E150K-G246E). The majority of our ST228 PBP2A mutations (N146K or E150K) were distinct from ST228 PBP2A allosteric domain mutations (primarily E239K) recently described for MRSA strains collected in Thailand and Spain during the 2010 Assessing Worldwide Antimicrobial Resistance Evaluation (AWARE) global surveillance program. We also found that similar allosteric domain PBP2A mutations (N146K) correlated with ceftaroline resistance in an independent external ST228 MRSA set obtained from the nearby University Hospital of Lausanne, Lausanne, Switzerland, collected from 2003 to 2008. Thus, ceftaroline resistance was observed in our archived strains (including two examples of an MIC of 4 µg/ml for the Iberian ST247 clonotype with the triple mutation N146K/E150K/G246E), at least as far back as 1998, considerably predating the commercial introduction of ceftaroline. Our results reinforce the notion that unknown parameters can potentially exert selective pressure on PBP2A that can subsequently modulate ceftaroline resistance.

The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A.

Expression of the methicillin-resistant S. aureus (MRSA) phenotype results from the expression of the extra penicillin-binding protein 2A (PBP2A), which is encoded by mecA and acquired horizontally on part of the SCCmec cassette. PBP2A can catalyze dd-transpeptidation of peptidoglycan (PG) because of its low affinity for ß-lactam antibiotics and can functionally cooperate with the PBP2 transglycosylase in the biosynthesis of PG. Here, we focus upon the role of the membrane-bound PrsA foldase protein as a regulator of ß-lactam resistance expression. Deletion of prsA altered oxacillin resistance in three different SCCmec backgrounds and, more importantly, caused a decrease in PBP2A membrane amounts without affecting mecA mRNA levels. The N- and C-terminal domains of PrsA were found to be critical features for PBP2A protein membrane levels and oxacillin resistance. We propose that PrsA has a role in posttranscriptional maturation of PBP2A, possibly in the export and/or folding of newly synthesized PBP2A. This additional level of control in the expression of the mecA-dependent MRSA phenotype constitutes an opportunity to expand the strategies to design anti-infective agents.

Exposure of Staphylococcus aureus to subinhibitory concentrations of ß-lactam antibiotics induces heterogeneous vancomycin-intermediate Staphylococcus aureus.

Glycopeptides are known to select for heterogeneous vancomycin-intermediate Staphylococcus aureus (h-VISA) from susceptible strains. In certain clinical situations, h-VISA strains have been isolated from patients without previous exposure to glycopeptides, such as cystic fibrosis patients, who frequently receive repeated treatments with beta-lactam antibiotics. Our objective was to determine whether prolonged exposure to beta-lactam antibiotics can induce h-VISA. We exposed 3 clinical vancomycin-susceptible methicillin-resistant Staphylococcus aureus (MRSA) strains to ceftazidime, ceftriaxone, imipenem, and vancomycin (as a control) at subinhibitory concentrations for 18 days in vitro. Population analyses showed progressive increases in vancomycin resistance; seven of the 12 derived strains obtained after induction were classified as h-VISA according to the following criteria: area under the curve (AUC) on day 18/AUC of Mu3 of ≥90% and/or growth on brain heart infusion (BHI) agar with 4 mg/liter vancomycin. The derived isolates had thickened cell walls proportional to the level of glycopeptide resistance. Genes known to be associated with glycopeptide resistance (vraSR, yvqF, SA1703, graRS, walKR, and rpoB) were PCR sequenced; no de novo mutations were observed upon beta-lactam exposure. To determine whether trfA, a gene encoding a glycopeptide resistance factor, was essential in the selection of h-VISA upon beta-lactam pressure, a trfA-knockout strain was generated by allelic replacement. Indeed, beta-lactam exposure of this mutated strain showed no capacity to induce vancomycin resistance. In conclusion, these results showed that beta-lactam antibiotics at subinhibitory concentrations can induce intermediate vancomycin resistance in vitro. This induction required an intact trfA locus. Our results suggest that prior use of beta-lactam antibiotics can compromise vancomycin efficacy in the treatment of MRSA infections.

The Staphylococcus aureus thiol/oxidative stress global regulator Spx controls trfA, a gene implicated in cell wall antibiotic resistance.

S. aureus combats cell wall antibiotic stress by altered gene expression mediated by various environmental signal sensors. In this study, we examined the transcriptional regulation of trfA, a gene related to mecA of Bacillus subtilis encoding an adaptor protein implicated in multiple roles, notably, proteolysis and genetic competence. Despite strong sequence similarity to B. subtilis mecA, the function of S. aureus trfA remains largely unexplored; however, its deletion leads to almost complete loss of resistance to oxacillin and glycopeptide antibiotics in glycopeptide-intermediate S. aureus (GISA) derivatives of methicillin-susceptible or methicillin-resistant S. aureus (MRSA) clinical or laboratory isolates. Northern blot analysis and 5' rapid amplification of cDNA ends (RACE) mapping revealed that trfA was expressed monocistronically by three promoters. Cell wall-active antibiotic exposure led to both increased trfA transcription and enhanced steady-state TrfA levels. trfA promoter regulation was not dependent upon the cell wall stress sentinel VraSR and other sensory stress systems, such as GraRS, WalkRK, Stk1/Stp1, and SigB. Notably, we discovered that the global oxidative-stress regulator Spx controlled trfA transcription. This finding was also confirmed using a strain with enhanced Spx levels resulting from a defect in yjbH, encoding a Spx-interacting protein governing Spx proteolytic degradation. A cohort of clinical GISA strains revealed significant steady-state upregulation of trfA compared to corresponding susceptible parental strains, further supporting a role for trfA in antibiotic resistance. These data provide strong evidence for a link between cell wall antibiotic stress and evoked responses mediated by an oxidative-stress sensor.

The posttranslocational chaperone lipoprotein PrsA is involved in both glycopeptide and oxacillin resistance in Staphylococcus aureus.

Understanding in detail the factors which permit Staphylococcus aureus to counteract cell wall-active antibiotics is a prerequisite to elaborating effective strategies to prolong the usefulness of these drugs and define new targets for pharmacological intervention. Methicillin-resistant S. aureus (MRSA) strains are major pathogens of hospital-acquired and community-acquired infections and are most often treated with glycopeptides (vancomycin and teicoplanin) because of their resistance to most penicillins and a limited arsenal of clinically proven alternatives. In this study, we examined PrsA, a lipid-anchored protein of the parvulin PPIase family (peptidyl-prolyl cis/trans isomerase) found ubiquitously in all Gram-positive species, in which it assists posttranslocational folding at the outer surface of the cytoplasmic membrane. We show by both genetic and biochemical assays that prsA is directly regulated by the VraRS two-component sentinel system of cell wall stress. Disruption of prsA is tolerated by S. aureus, and its loss results in no detectable overt macroscopic changes in cell wall architecture or growth rate under nonstressed growth conditions. Disruption of prsA leads, however, to notable alterations in the sensitivity to glycopeptides and dramatically decreases the resistance of COL (MRSA) to oxacillin. Quantitative transcriptional analysis reveals that prsA and vraR are coordinately upregulated in a panel of stable laboratory and clinical glycopeptide-intermediate S. aureus (GISA) strains compared to their susceptible parents. Collectively, our results point to a role for prsA as a facultative facilitator of protein secretion or extracellular folding and provide a framework for understanding why prsA is a key element of the VraRS-mediated cell wall stress response.

High prevalence of isolates with reduced glycopeptide susceptibility in persistent or recurrent bloodstream infections due to methicillin-resistant Staphylococcus aureus.

Reduced susceptibility to glycopeptides in methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates is considered a risk factor for failure of glycopeptide therapy. We compared the prevalences of MRSA isolates with reduced glycopeptide susceptibility in patients with versus without persistent or recurrent MRSA bloodstream infections. A retrospective cohort study at the University Hospital of Geneva identified 27 patients with persistent or recurrent clonally related MRSA bacteremic episodes over an 8-year period, which included 208 consecutive nosocomial MRSA bacteremic episodes. Vancomycin and teicoplanin MICs were determined by a modified macrodilution assay allowing improved detection of glycopeptide-intermediate MRSA isolates (GISA), characterized by elevated teicoplanin or/and vancomycin MICs (≥ 4 µg/ml). For 16 patients (59%), their pretherapy and/or posttherapy MRSA isolates showed elevated teicoplanin MICs, among which 10 (37%) concomitantly displayed elevated vancomycin MICs. In contrast, 11 other patients (41%) were persistently or recurrently infected with non-GISA isolates. In comparison, only 39 (22%) of 181 single isolates from patients with no microbiological evidence of persistent or recurrent infections showed elevated teicoplanin MICs, among which 14 (8%) concomitantly displayed elevated vancomycin MICs. Clinical, microbiological, and pharmacokinetic variables for patients persistently or recurrently infected with GISA or non-GISA isolates were similar. Bacteremic patients with a poor response to glycopeptide therapy had a 2.8-fold- and 4.8-fold-higher rates of MRSA isolates displaying elevated teicoplanin and vancomycin MICs, respectively, than patients with single isolates (P < 0.0001). Detection of elevated teicoplanin MICs may help to predict a poor response to glycopeptide therapy in MRSA bacteremic patients.

Site-specific mutation of Staphylococcus aureus VraS reveals a crucial role for the VraR-VraS sensor in the emergence of glycopeptide resistance.

An initial response of Staphylococcus aureus to encounter with cell wall-active antibiotics occurs by transmembrane signaling systems that orchestrate changes in gene expression to promote survival. Histidine kinase two-component sensor-response regulators such as VraRS contribute to this response. In this study, we examined VraS membrane sensor phosphotransfer signal transduction and explored the genetic consequences of disrupting signaling by engineering a site-specific vraS chromosomal mutation. We have used in vitro autophosphorylation assay with purified VraS[64-347] lacking its transmembrane anchor region and tested site-specific kinase domain histidine mutants. We identified VraS H156 as the probable site of autophosphorylation and show phosphotransfer in vitro using purified VraR. Genetic studies show that the vraS(H156A) mutation in three strain backgrounds (ISP794, Newman, and COL) fails to generate detectable first-step reduced susceptibility teicoplanin mutants and severely reduces first-step vancomycin mutants. The emergence of low-level glycopeptide resistance in strain ISP794, derived from strain 8325 (ΔrsbU), did not require a functional σ(B), but rsbU restoration could enhance the emergence frequency supporting a role for this alternative sigma factor in promoting glycopeptide resistance. Transcriptional analysis of vraS(H156A) strains revealed a pronounced reduction but not complete abrogation of the vraRS operon after exposure to cell wall-active antibiotics, suggesting that additional factors independent of VraS-driven phosphotransfer, or σ(B), exist for this promoter. Collectively, our results reveal important details of the VraRS signaling system and predict that pharmacologic blockade of the VraS sensor kinase will have profound effects on blocking emergence of cell wall-active antibiotic resistance in S. aureus.

The Role of ArlRS and VraSR in Regulating Ceftaroline Hypersusceptibility in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus infections are a global health problem. New control strategies, including fifth-generation cephalosporins such as ceftaroline, have been developed, however rare sporadic resistance has been reported. Our study aimed to determine whether disruption of two-component environmental signal systems detectably led to enhanced susceptibility to ceftaroline in S. aureus CA-MRSA strain MW2 at sub-MIC concentrations where cells normally continue to grow. A collection of sequential mutants in all fifteen S. aureus non-essential two-component systems (TCS) was first screened for ceftaroline sub-MIC susceptibility, using the spot population analysis profile method. We discovered a role for both ArlRS and VraSR TCS as determinants responsible for MW2 survival in the presence of sub-MIC ceftaroline. Subsequent analysis showed that dual disruption of both arlRS and vraSR resulted in a very strong ceftaroline hypersensitivity phenotype. Genetic complementation analysis confirmed these results and further revealed that arlRS and vraSR likely regulate some common pathway(s) yet to be determined. Our study shows that S. aureus uses particular TCS environmental sensing systems for this type of defense and illustrates the proof of principle that if these TCS were inhibited, the efficacy of certain antibiotics might be considerably enhanced.

Analytical Evaluation of Visby Medical RT-PCR Portable Device for Rapid Detection of SARS-CoV-2.

Extended community testing constitutes one of the main strategic pillars in controlling the COVID-19 pandemic. Reverse transcription PCR (RT-PCR) targeting the SARS-CoV-2 genome on nasopharyngeal swab samples is currently the reference test. While displaying excellent analytical sensitivity and specificity, this test is costly, often requires a substantial turnaround time, and, more importantly, is subject to reagent and other material shortages. To complement this technology, rapid antigen tests have been developed and made available worldwide, allowing cheap, quick, and decentralized SARS-CoV-2 testing. The main drawback of these tests is the reduced sensitivity when RT-PCR is the gold standard. In this study, we evaluate Visby an innovative, portable, easy-to-use RT-PCR point-of-care (POC) diagnostic device. Our retrospective analysis shows that overall, compared to the Cobas 6800 RT-qPCR assay (Roche), this RT-PCR POC technology detects SARS-CoV-2 RNA with 95% sensitivity (95%CI = 86.3-99%) and 100% specificity (95% CI = 80.5-100%). For samples with cycle-threshold values below 31, we observed 100% sensitivity (95% CI = 66.4-100%). While showing an analytical sensitivity slightly below that of a standard RT-qPCR system, the evaluated Visby RT-PCR POC device may prove to be an interesting diagnostic alternative in the COVID-19 pandemic, potentially combining the practical advantages of rapid antigen tests and the robust analytical performances of nucleic acid detection systems.

Hydrogen Peroxide Affects Growth of S. aureus Through Downregulation of Genes Involved in Pyrimidine Biosynthesis.

Reactive oxygen species (ROS) play a crucial role in the cellular defense against S. aureus, as evidenced by the importance of this pathogen in patients lacking the ROS-generating phagocyte NADPH oxidase NOX2. ROS concentrations required to kill S. aureus in vitro are much higher than those found in the phagosome. We therefore hypothesized that sublethal ROS concentrations may play a role in S. aureus gene dysregulation and investigated the in vitro transcriptomic response of S. aureus to sublethal concentrations of hydrogen peroxide (H2O2). A striking observation of these experiments was a coordinated and massive downregulation of genes involved in pyrimidine metabolism. Using transposon insertion mutants, we demonstrated that deletion of carA, a gene involved in pyrimidine synthesis, led to a significant growth defect and to an increased sensitivity of S. aureus to added H2O2. The phenotype of the carA mutant could be reversed through supplementation with the pyrimidine precursor uracil, or with a multicopy vector encoding carA. As opposed to the impact of ROS on extracellular survival, carA deletion did not affect the intracellular survival in neutrophils. Our results raise the possibility that ROS-dependent downregulation of pyrimidine metabolism might be a survival strategy of S. aureus, allowing colonization through intracellular survival, while decreasing the risk of killing the host through dampened extracellular growth.

Diagnostic accuracy of Panbio rapid antigen tests on oropharyngeal swabs for detection of SARS-CoV-2.

BACKGROUND: Antigen-detecting rapid diagnostic tests (Ag-RDTs) for the detection of SARS-CoV-2 offer new opportunities for testing in the context of the COVID-19 pandemic. Nasopharyngeal swabs (NPS) are the reference sample type, but oropharyngeal swabs (OPS) may be a more acceptable sample type in some patients. METHODS: We conducted a prospective study in a single screening center to assess the diagnostic performance of the Panbio™ COVID-19 Ag Rapid Test (Abbott) on OPS compared with reverse-transcription quantitative PCR (RT-qPCR) using NPS during the second pandemic wave in Switzerland. RESULTS: 402 outpatients were enrolled in a COVID-19 screening center, of whom 168 (41.8%) had a positive RT-qPCR test. The oropharyngeal Ag-RDT clinical sensitivity compared to nasopharyngeal RT-qPCR was 81% (95%CI: 74.2-86.6). Two false positives were noted out of the 234 RT-qPCR negative individuals, which resulted in a clinical specificity of 99.1% (95%CI: 96.9-99.9) for the Ag-RDT. For cycle threshold values ≤ 26.7 (≥ 1E6 SARS-CoV-2 genomes copies/mL, a presumed cut-off for infectious virus), 96.3% sensitivity (95%CI: 90.7-99.0%) was obtained with the Ag-RDT using OPS. INTERPRETATION: Based on our findings, the diagnostic performance of the Panbio™ Covid-19 RDT with OPS samples, if taken by a trained person and high requirements regarding quality of the specimen, meet the criteria required by the WHO for Ag-RDTs (sensitivity ≥80% and specificity ≥97%) in a high incidence setting in symptomatic individuals.

Diagnostic accuracy of two commercial SARS-CoV-2 antigen-detecting rapid tests at the point of care in community-based testing centers.

OBJECTIVES: Determine the diagnostic accuracy of two antigen-detecting rapid diagnostic tests (Ag-RDT) for SARS-CoV-2 at the point of care and define individuals' characteristics providing best performance. METHODS: We performed a prospective, single-center, point of care validation of two Ag-RDT in comparison to RT-PCR on nasopharyngeal swabs. RESULTS: Between October 9th and 23rd, 2020, 1064 participants were enrolled. The PanbioTM Covid-19 Ag Rapid Test device (Abbott) was validated in 535 participants, with 106 positive Ag-RDT results out of 124 positive RT-PCR individuals, yielding a sensitivity of 85.5% (95% CI: 78.0-91.2). Specificity was 100.0% (95% CI: 99.1-100) in 411 RT-PCR negative individuals. The Standard Q Ag-RDT (SD Biosensor, Roche) was validated in 529 participants, with 170 positive Ag-RDT results out of 191 positive RT-PCR individuals, yielding a sensitivity of 89.0% (95%CI: 83.7-93.1). One false positive result was obtained in 338 RT-PCR negative individuals, yielding a specificity of 99.7% (95%CI: 98.4-100). For individuals presenting with fever 1-5 days post symptom onset, combined Ag-RDT sensitivity was above 95%. Lower sensitivity of 88.2% was seen on the same day of symptom development (day 0). CONCLUSIONS: We provide an independent validation of two widely available commercial Ag-RDTs, both meeting WHO criteria of ≥80% sensitivity and ≥97% specificity. Although less sensitive than RT-PCR, these assays could be beneficial due to their rapid results, ease of use, and independence from existing laboratory structures. Testing criteria focusing on patients with typical symptoms in their early symptomatic period onset could further increase diagnostic value.