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Bone and joint infection (BJI) epidemiology and outcomes in solid organ transplant recipients (SOTr) remain largely unknown. We aim to describe BJI in a multi-center cohort of SOTr (Swiss Transplant Cohort Study). All consecutive SOTr with BJI (01.05.2008-31.12.2019) were included. A nested case-control study to identify risk factors for BJI was performed. Among 4482 patients, 61 SOTr with 82 BJI were included, at an incidence of 1.4% (95% CI 1.1-1.7), higher in heart and kidney-pancreas SOTr (Gray's test p .01). Although BJI were predominately late events (median of 18.5 months post-SOT), most infections occurred during the first year post-transplant in thoracic SOTr. Diabetic foot osteomyelitis was the most frequent infection (38/82, 46.3%), followed by non-vertebral osteomyelitis (26/82, 31.7%). Pathogens included Gram-positive cocci (70/131, 53.4%), Gram-negative bacilli (34/131, 26.0%), and fungi (9/131, 6.9%). BJI predictors included male gender (OR 2.94, 95% CI 1.26-6.89) and diabetes (OR 2.97, 95% CI 1.34-6.56). Treatment failure was observed in 25.9% (21/81) patients and 1-year mortality post-BJI diagnosis was 14.8% (9/61). BJI remain a rare event in SOTr, associated with subtle clinical presentations, high morbidity and relapses, requiring additional studies in the future.

BACKGROUND SARS-CoV-2 infection triggers different auto-antibodies, including anti-apolipoprotein A-1 IgGs (AAA1), which could be of concern as mediators of persistent symptoms. We determined the kinetics of AAA1 response over after COVID-19, and the impact of AAA1 on the inflammatory response and symptoms persistence. METHODS All serologies were assessed at one, three, six, and twelve months in 193 hospital employees with COVID-19. ROC curve analyses and logistic regression models (LRM) were used to determine the prognostic accuracy of AAA1 and their association with patient-reported COVID-19 symptoms persistence at 12 months. Interferon (IFN)-α and-γ production by AAA1-stimulated human monocyte-derived macrophages (HMDM) was assessed in vitro. RESULTS AAA1 seropositivity was 93% at one month and declined to 15% at 12 months after COVID-19. Persistent symptoms at 12 months were observed in 45.1% of participants, with a predominance of neurological (28.5%), followed by general (15%) and respiratory symptoms (9.3%). Over time, strength of correlations between AAA1 and anti-SARS-COV2 serologies decreased, but remained significant. From the 3rd month on, AAA1 levels predicted persistent respiratory symptoms (area under the curves 0.72-0.74; p

Heteroresistance refers to subpopulation-mediated differential antimicrobial susceptibility within a clonal bacterial population. Usually, it designates a resistant subpopulation identified within an isolate considered susceptible by classical antimicrobial susceptibility testing. Heteroresistance lacks a uniform microbiological definition for diagnostic laboratories, and its clinical impact remains unclear for most bacterial species.This narrative review aims to provide a practical overview on the latest developments in the field of heteroresistance for both clinical microbiologists and physicians, with a particular focus on ESKAPE pathogens.A literature search was performed on Pubmed and Google with the key words heteroresistance (heterogeneity OR heterogeneous) AND antibiotic resistance. Among the 836 publications selected based on their abstracts, the most relevant for the detection, epidemiology and clinical impact of heteroresistance in ESKAPE pathogens are discussed here.Heteroresistance is only clearly defined for heterogeneous vancomycin intermediate Staphylococcus aureus. We compiled a larger microbiological definition to be applicable to other bacterial species and antibiotics in the clinical context. We highlighted the key technical points of population analysis profile, which is the reference standard for detecting heteroresistance. Heteroresistance to polymyxins, β-lactams (carbapenems, cefiderocol), fosfomycin, tigecycline and aminoglycosides is frequently reported in multidrug-resistant gram-negative pathogens. Treatment failure due to heteroresistance has been described in case reports or retrospective studies, so far confirmed by meta-analyses in the case of heterogeneous vancomycin intermediate S. aureus only. Finally, to treat pandrug-resistant bacterial infections, the option of targeting susceptible subpopulations of resistant isolates using tailored antibiotic combinations is also discussed.Systematic heteroresistance screening by clinical laboratories is not currently recommended. Nevertheless, we should be aware of this phenomenon, and in specific cases, such as treatment failure, heteroresistance should be tested by reference laboratories. Additional studies using standardized methods are needed to improve our understanding of heteroresistance and further assess its clinical impact.

In 208 children seeking medical care, the seropositivity rate of anti–SARS-CoV-2 IgG antibodies was 8.7%, suggesting an infection rate similar to that observed in adults but >100-fold the incidence of RT-PCR–confirmed pediatric cases. Compared with the gold-standard combined ELISA + immunofluorescence, the MEDsan IgG rapid diagnostic test performed accurately.

Patients treated with anti-CD20 therapy are particularly at risk of developing severe COVID-19, however little is known regarding COVID-19 vaccine effectiveness in this population. This study assesses humoral and T-cell responses to mRNA-based COVID-19 vaccines in patients treated with rituximab for rheumatic diseases or ocrelizumab for multiple sclerosis (n=37), compared to immunocompetent individuals (n=22). SARS-CoV-2-specific antibodies were detectable in only 69.4% of patients and at levels that were significantly lower compared to controls who all seroconverted. In contrast to antibodies, Spike (S)-specific CD4+ T cells were equally detected in immunocompetent and anti-CD20 treated patients (85-90%) and mostly of a Th1 phenotype. Response rates of S-specific CD8+T cells were higher in ocrelizumab (96.2%) and rituximab-treated patients (81.8%) as compared to controls (66.7%). Vaccine-specific CD4+and CD8+T cells were polyfunctional but expressed more IL-2 in patients than in controls. In summary, our study suggests that patients on anti-CD20 treatment are able to mount potent T-cell responses to mRNA COVID-19 vaccines, despite impaired humoral responses. This could play an important role in the prevention of severe COVID-19.

Background Carbapenemase-producing Klebsiella pneumoniae has become a global priority, not least in low- and middle-income countries. Here, we report the emergence and clinical impact of a novel Klebsiella pneumoniae carbapenemase–producing K. pneumoniae (KPC-KP) sequence type (ST) 16 clone in a clonal complex (CC) 258–endemic setting. Methods In a teaching Brazilian hospital, a retrospective cohort of adult KPC-KP bloodstream infection (BSI) cases (January 2014 to December 2016) was established to study the molecular epidemiology and its impact on outcome (30-day all-cause mortality). KPC-KP isolates underwent multilocus sequence typing. Survival analysis between ST/CC groups and risk factors for fatal outcome (logistic regression) were evaluated. Representative isolates underwent whole-genome sequencing and had their virulence tested in a Galleria larvae model. Results One hundred sixty-five unique KPC-KP BSI cases were identified. CC258 was predominant (66%), followed by ST16 (12%). The overall 30-day mortality rate was 60%; in contrast, 95% of ST16 cases were fatal. Patients’ severity scores were high and baseline clinical variables were not statistically different across STs. In multivariate analysis, ST16 (odds ratio [OR], 21.4; 95% confidence interval [CI], 2.3–202.8; P = .008) and septic shock (OR, 11.9; 95% CI, 4.2–34.1; P < .001) were independent risk factors for fatal outcome. The ST16 clone carried up to 14 resistance genes, including blaKPC-2 in an IncFIBpQIL plasmid, KL51 capsule, and yersiniabactin virulence determinants. The ST16 clone was highly pathogenic in the larvae model. Conclusions Mortality rates were high in this KPC-KP BSI cohort, where CC258 is endemic. An emerging ST16 clone was associated with high mortality. Our results suggest that even in endemic settings, highly virulent clones can rapidly emerge demanding constant monitoring., An emerging clone, Klebsiella pneumoniae carbapenemase 2–producing K. pneumoniae ST16, associated with high mortality rates in a CC258 endemic setting, was identified. This clone carried the KL51 capsule and displayed higher virulence in a Galleria larvae pathogenicity model than CC258 clones.

Until recently, the role of insects, and particularly flies, in disseminating antimicrobial resistance (AMR) has been poorly studied. In this study, we screened blowflies (Chrysomya spp.) from different areas near the city of Phitsanulok, Northern Thailand, for the presence of AMR genes and in particular, mcr-1, using whole genome sequencing (WGS). In total, 48 mcr-1-positive isolates were recovered, consisting of 17 mcr-1-positive Klebsiella pneumoniae (MCRPKP) and 31 mcr-1-positive Escherichia coli (MCRPEC) strains. The 17 MCRPKP were shown to be clonal (ST43) with few single poly nucleomorphs (SNPs) by WGS analysis. In in-vitro models, the MCRPKP were shown to be highly virulent. In contrast, 31 recovered MCRPEC isolates are varied, belonging to 12 different sequence types shared with those causing human infections. The majority of mcr-1 gene are located on IncX4 plasmids (29/48, 60.42%), sharing an identical plasmid backbone. These findings highlight the contribution of flies to the AMR contagion picture in low- and middle-income countries and the challenges of tackling global AMR. Keywords: Blow flies, mcr-1 gene, Klebsiella pneumoniae, IncX4 plasmid, Multidrug resistance

To evaluate longitudinally the persistence of humoral immunity for up to 6 months in a cohort of hospital employees with mild coronavirus disease 2019 (COVID-19).We measured anti-RBD (receptor binding domain of viral spike protein), anti-N (viral nucleoprotein) and neutralizing antibodies at 1, 3 and 6 months after mostly mild COVID-19 in 200 hospital workers using commercial ELISAs and a surrogate virus neutralization assay.Antibodies specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persisted in all participants for up to 6 months. Anti-RBD geometric mean concentrations (GMCs) progressively increased between months 1 (74.2 U/mL, 95%CI: 62.7-87.8), 3 (103.2 U/mL, 95%CI: 87.9-121.2; p 0.001), and 6 (123.3 U/mL, 95%CI: 103.4-147.0; p 0.001) in the whole cohort. Anti-N antibodies were detectable in97% at all times. Neutralizing antibodies were detectable in 99.5% of participants (195/196) at 6 months post infection. Their GMC progressively decreased between months 1 (20.1 AU/mL, 95%CI: 16.9-24.0), 3 (15.2 AU/mL, 95%CI: 13.2-17.6; p 0.001) and 6 (9.4 AU/mL, 95%CI: 7.7-11.4; p 0.001). RBD-ACE2-inhibiting antibody titres and anti-RBD antibody concentrations strongly correlated at each timepoint (all r 0.86, p 0.001). Disease severity was associated with higher initial anti-RBD and RBD-ACE2-inhibiting antibody titres, but not with their kinetics.Neutralizing antibodies persisted at 6 months in almost all participants, indicating more durability than initially feared. Anti-RBD antibodies persisted better and even increased over time, possibly related to the preferential detection of progressively higher-affinity antibodies.

Significance COVID-19, caused by SARS-CoV-2, led to an unprecedented global health crisis. As the majority of people infected with SARS-CoV-2 have no or only mild symptoms, many cases aren’t captured by direct testing. However, it is important to establish the true spread of the virus by identifying how many people have been exposed. Detection of anti–SARS-CoV-2–specific antibodies in blood samples can help us understand how the pandemic is evolving over time. We developed a sensitive and specific assay that performs 1,024 measurements in parallel. To enable decentralized blood sample collection, the method can detect antibodies in a small drop of blood obtainable by finger pricking, and the blood can be collected and shipped with a simple, low-cost blood glucose test strip., Novel technologies are needed to facilitate large-scale detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific antibodies in human blood samples. Such technologies are essential to support seroprevalence studies and vaccine clinical trials, and to monitor quality and duration of immunity. We developed a microfluidic nanoimmunoassay (NIA) for the detection of anti–SARS-CoV-2 IgG antibodies in 1,024 samples per device. The method achieved a specificity of 100% and a sensitivity of 98% based on the analysis of 289 human serum samples. To eliminate the need for venipuncture, we developed low-cost, ultralow-volume whole blood sampling methods based on two commercial devices and repurposed a blood glucose test strip. The glucose test strip permits the collection, shipment, and analysis of 0.6 μL of whole blood easily obtainable from a simple finger prick. The NIA platform achieves high throughput, high sensitivity, and specificity based on the analysis of 289 human serum samples, and negligible reagent consumption. We furthermore demonstrate the possibility to combine NIA with decentralized and simple approaches to blood sample collection. We expect this technology to be applicable to current and future SARS-CoV-2 related serological studies and to protein biomarker analysis in general.

Objectives: Carbapenem resistance in Klebsiella pneumoniae is a major clinical challenge. Aminoglycosides remain an important asset in the current therapeutic arsenal to treat these infections. We examined aminoglycoside resistance phenotypes and genomics in a collection of 100 invasive KPC-producing K. pneumoniae isolates sequentially collected in a Brazilian tertiary hospital between 2014 and 2016.Methods: Aminoglycoside susceptibility testing was performed. We used a combined long-read (MinION) and short-read (Illumina) whole-genome sequencing strategy to provide a genomic picture of aminoglycoside resistance genes, with particular emphasis on 16S rRNA methyltransferases and related plasmids.Results: 68% of the strains were resistant to gentamicin and 42% to amikacin, with 35% resistant to both of these commonly used aminoglycosides. We identified the 16S rRNA methyltransferase gene rmtB in 30% of these isolates: 97% (29/30) belonged to sequence type 258 (ST258) and a single isolate to the emergent ST16 clone. In ST258 and ST16 the rmtB gene was located on large IncC plasmids of 177 kb and 174 kb, respectively, highly similar to a plasmid previously identified in Proteus mirabilis in the same hospital. Moreover, 99% of the isolates remained susceptible to the veterinary-approved drug apramycin, currently under clinical development for human medicine.Conclusion: Such findings in geographically and temporally related isolates suggest a combination of vertical clonal spread as well as horizontal interspecies and intraspecies plasmid transfer. This broad rmtB dissemination in an endemic setting for KPC-producing clones is worrisome since it provides resistance to most clinically available aminoglycosides, including the novel aminoglycoside-modifying enzyme-resistant plazomicin.

Polymyxin resistance is a public health concern – present in humans, animals and the environment – caused by chromosomal-encoding or plasmid-encoding mechanisms. Chromosomal alterations in MgrB are frequently detected in Klebsiella spp., but not yet reported and characterised in Klebsiella variicola (K. variicola). This study performed microbiological and genomic characterisation of three polymyxin-resistant K. variicola isolates (M14, M15 and M50) recovered from the microbiota of migratory birds in Brazil. The isolates were submitted to SpeI-PFGE, broth microdilution and whole genome sequencing using Illumina MiSeq for analysis of genetic relatedness, sequence typing and detection of antimicrobial-resistance genes. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance only for polymyxins. Sequences of chromosomal two-component systems (PmrAB, PhoPQ, RstAB, CrrAB) and MgrB were evaluated by blastN and blastP against a polymyxin-susceptible K. variicola (A58243), and mutations with biological effect were checked by the PROVEAN tool. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance for polymyxins. In M14 and M15, phoQ deleterious mutations (D90N, I122S and G385S) were identified, while an mgrB variant containing a single deletion (C deletion on position 93) leading to the production of a non-functional protein was detected in M50. mgrB complementation studies showed restoration of polymyxin susceptibility (64 to ≤ 0.25 mg/L) as a wild-type mgrB was inserted into the mgrB-deficient M50. This study confirmed the role of a non-functional mgrB variant in conferring polymyxin resistance, stressing the role of this regulator in K. variicola and drawing attention to novel polymyxin resistance mechanisms emerging in wildlife.

Serologic studies have been critical in tracking the evolution of the COVID-19 pandemic. The reliability of serologic studies for quantifying the proportion of the population that have been infected depends on the extent of antibody decay as well as on assay performance in detecting both recent and older infections. Data on anti-SARS-CoV-2 antibodies persistence remain sparse, especially from infected individuals with few to no symptoms. In a cohort of mostly mild/asymptomatic SARS-CoV-2-infected individuals tested with three widely-used immunoassays, antibodies persisted for at least 8 months after infection, although detection depended on immunoassay choice, with one of them missing up to 40% of past infections. Simulations reveal that without appropriate adjustment for time-varying assay sensitivity, seroprevalence surveys may underestimate infection rates. As the immune landscape becomes more complex with naturally-infected and vaccinated individuals, assay choice and appropriate assay-performance-adjustment will become even more important for the interpretation of serologic studies.

Purpose: To assess the diagnostic performances of five automated anti-SARS-CoV-2 immunoassays, Epitope (N), Diasorin (S1/S2), Euroimmun (S1), Roche N (N), and Roche S (S-RBD), and to provide a testing strategy based on pre-test probability. Methods: We assessed the receiver operating characteristic (ROC) areas under the curve (AUC) values, along with the sensitivity, specificity, positive predictive values (PPVs), and negative predictive values (NPVs), of each assay using a validation sample set of 172 COVID-19 sera and 185 negative controls against a validated S1-immunofluorescence as a reference method. The three assays displaying the highest AUCs were selected for further serodetection of 2033 sera of a large population-based cohort. Results: In the validation analysis (pre-test probability: 48.1%), Roche N, Roche S and Euroimmun showed the highest discriminant accuracy (AUCs: 0.99, 0.98, and 0.98) with PPVs and NPVs above 96% and 94%, respectively. In the population-based cohort (pre-test probability: 6.2%) these three assays displayed AUCs above 0.97 and PPVs and NPVs above 90.5% and 99.4%, respectively. A sequential strategy using an anti-S assay as screening test and an anti-N as confirmatory assays resulted in a 96.7% PPV and 99.5% NPV, respectively. Conclusions: Euroimmun and both Roche assays performed equally well in high pre-test probability settings. At a lower prevalence, sequentially combining anti-S and anti-N assays resulted in the optimal trade-off between diagnostic performances and operational considerations.

Objectives To report a case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection 6 months after the first infection in a young healthy female physician. Both episodes led to mild coronavirus disease 2019 (COVID-19). Methods SARS-CoV-2 infections were detected by real-time reverse transcriptase PCR (RT-PCR) on nasopharyngeal specimens. Reinfection was confirmed by whole-genome sequencing. Kinetics of total anti-S receptor binding domain immunoglobulins (Ig anti–S RBD), anti-nucleoprotein (anti-N) and neutralizing antibodies were determined in serial serum samples retrieved during both infection episodes. Memory B-cell responses were assessed at day 12 after reinfection. Results Whole-genome sequencing identified two different SARS-CoV-2 genomes both belonging to clade 20A, with only one nonsynonymous mutation in the spike protein and clustered with viruses circulating in Geneva (Switzerland) at the time of each of the corresponding episodes. Seroconversion was documented with low levels of total Ig anti–S RBD and anti-N antibodies at 1 month after the first infection, whereas neutralizing antibodies quickly declined after the first episode and then were boosted by the reinfection, with high titres detectable 4 days after symptom onset. A strong memory B-cell response was detected at day 12 after onset of symptoms during reinfection, indicating that the first episode elicited cellular memory responses. Conclusions Rapid decline of neutralizing antibodies may put medical personnel at risk of reinfection, as shown in this case. However, reinfection leads to a significant boosting of previous immune responses. Larger cohorts of reinfected subjects with detailed descriptions of their immune responses are needed to define correlates of protection and their duration after infection.

Background: Since the beginning of the COVID-19 pandemic, no direct antiviral treatment is effective as post-exposure prophylaxis (PEP). Lopinavir/ritonavir (LPV/r) was repurposed as a potential PEP agent against COVID-19. Methods: We conducted a pragmatic open-label, parallel, cluster-randomized superiority trial in four sites in Switzerland and Brazil. Clusters were randomized to receive LPV/r PEP (400/100 mg) twice daily for 5 days or no PEP (surveillance). The primary outcome is the occurrence of COVID-19 within 21 days post-enrollment. Findings: Of 318 participants, 157 (49.4%) were women, median age was 39 (interquartile range, 28-50) years. A total of 209 (179 clusters) participants were randomized to LPV/r PEP and 109 (95 clusters) to surveillance. Baseline characteristics were similar, with the exception of baseline SARS-CoV-2 PCR positivity, which was 3-fold more frequent in the LPV/r arm (34/209 [16.3%] vs 6/109 [5.5%], respectively). During 21-day follow-up, 48/318 (15.1%) participants developed COVID-19: 35/209 (16.7%) in the LPV/r group and 13/109 (11.9%) in the surveillance group (unadjusted hazard ratio 1.44; 95% CI, 0.76 to 2.73). In the primary endpoint analysis adjusted for propensity score to receive LPV/r, the hazard ratio for developing COVID-19 in the LPV/r group vs surveillance was 0.53 (95% CI, 0.23 to 1.23, respectively; P =.14). Interpretation: LPV/r role as PEP for COVID-19 remains unanswered. In this trial, LPV/r over 5 days did not significantly reduce incidence of COVID-19 in exposed individuals. We observed a change in directionality of the effect in favor of LPV/r after adjusting for baseline SARS-CoV-2 PCR results, indicating a potential role of antivirals in COVID-19 prevention. Clinical Trial Registration Details: ClinicalTrials.gov (Identifier: NCT04364022); Swiss National Clinical Trial Portal: SNCTP 000003732. Funding Information: Fondation privee des HUG and Swiss National Fund (project number: 33IC30_166819). Declaration of Interests: None reported. Ethics Approval Statement: The protocol and amendments were approved by Swissmedic and local ethics committees in Switzerland and Brazil. Participants provided written informed consent before study entry.

To validate the diagnostic accuracy of the Augurix SARS-CoV-2 IgM/IgG rapid immunoassay diagnostic test (RDT) for COVID-19.In this unmatched 1:1 case-control study, blood samples from 46 real-time RT-PCR-confirmed SARS-CoV-2 hospitalized cases and 45 healthy donors (negative controls) were studied. Diagnostic accuracy of the IgG RDT was assessed against both an in-house recombinant spike-expressing immunofluorescence assay (rIFA), as an established reference method (primary endpoint), and the Euroimmun SARS-CoV-2 IgG enzyme-linked immunosorbent assays (ELISA) (secondary endpoint).COVID-19 patients were more likely to be male (61% vs 20%; P = .0001) and older (median 66 vs 47 years old; P .001) than controls. Whole blood IgG-RDT results showed 86% and 93% overall Kendall concordance with rIFA and IgG ELISA, respectively. IgG RDT performances were similar between plasma and whole blood. Overall, RDT sensitivity was 88% (95% confidence interval [95%CI]: 70-96), specificity 98% (95%CI: 90-100), PPV 97% (95%CI: 80-100) and NPV 94% (95%CI: 84-98). The IgG-RDT carried out from 0 to 6 days, 7 to 14 days and 14 days after the SARS-CoV-2 RT-PCR test displayed 30%, 73% and 100% positivity rates in the COVID-19 group, respectively. When considering samples taken14 days after RT-PCR diagnosis, NPV was 100% (95%CI:90-100), and PPV was 100% (95%CI:72-100).The Augurix IgG-RDT done in whole blood displays a high diagnostic accuracy for SARS-CoV-2 IgG in high COVID-19 prevalence settings, where its use could be considered in the absence of routine diagnostic serology facilities.

Background: Comparative data of SARS-CoV-2 IgM/IgG serology rapid diagnostic tests (RDTs) is scarce. We thus performed a head-to-head comparison of three RDTs. Methods: In this unmatched case-control study, blood samples from 41 RT-PCR-confirmed COVID-19 cases and 50 negative controls were studied. The diagnostic accuracy of three commercially available COVID-19 RDTs: NTBIO (RDT-A), Orient-Gene (RDT-B), and MEDsan (RDT-C), against both a recombinant spike-expressing immunofluorescence assay (rIFA) and Euroimmun IgG ELISA, was assessed. RDT results concordant with the reference methods, and between whole blood and plasma, were established by the Kendall coefficient. Results: COVID-19 cases&rsquo, median time from RT-PCR to serology was 22 days (interquartile range (IQR) 13&ndash, 31 days). Whole-blood IgG detection with RDT-A, -B, and -C showed 0.93, 0.83, and 0.98 concordance with rIFA. Against rIFA, RDT-A sensitivity (SN) was 92% (95% CI: 78&ndash, 98) and specificity (SP) 100% (95% CI: 91&ndash, 100), RDT-B showed 87% SN (95% CI: 72&ndash, 95) and 98% SP (95% CI: 88&ndash, 100), and RDT-C 100% SN (95% CI: 88&ndash, 100) and 98% SP (95% CI: 88&ndash, 100). Against ELISA, SN and SP were above 90% for all three RDTs. Conclusions: RDT-A and RDT-C displayed IgG detection SN and SP above 90% in whole blood. These RDTs could be considered in the absence of routine diagnostic serology facilities.

The emergence ofmobilecolistinresistance (mcr) threatens to undermine the clinical efficacy of the last antibiotic that can be used to treat serious infections caused by Gram-negative pathogens. Here we measure the fitness cost of a newly discovered MCR-3 using in vitro growth and competition assays.mcr-3expression confers a lower fitness cost thanmcr-1, as determined by competitive ability and cell viability. Consistent with these findings, plasmids carryingmcr-3have higher stability thanmcr-1plasmids across a range ofEscherichia colistrains. Crucially,mcr-3plasmids can stably persist, even in the absence of colistin. Recent compensatory evolution has helped to offset the cost ofmcr-3expression, as demonstrated by the high fitness ofmcr-3.5as opposed tomcr-3.1. Reconstructing all of the possible evolutionary trajectories frommcr-3.1tomcr-3.5reveals a complex fitness landscape shaped by negative epistasis between compensatory and neutral mutations. Our findings highlight the importance of fitness costs and compensatory evolution in driving the dynamics and stability of mobile colistin resistance in bacterial populations, and they highlight the need to understand how processes (other than colistin use) impactmcrdynamics.

Background: Little is known about the duration and breadth of humoral immunity against SARS-CoV-2 to better inform appropriate pandemic control measures. Methods: We measured anti-RBD, -N and neutralizing antibodies at one, three and six months after mostly mild COVID-19 in 200 hospital workers using commercial ELISAs and a surrogate virus neutralization assay. Findings: Anti-RBD antibodies were detected in all participants at each timepoint. Geometric mean concentrations (GMCs) progressively increased between month one (74 . 2 U/ml [95%CI 62 . 7-87 . 8), three (103 . 2 U/ml [95%CI 87 . 9-121 . 2]; p 97% at all times. Neutralizing antibodies were detectable in 92 . 5% (184/199), 94 . 4% (185/196) and 76 . 5% (150/196) participants at one, three and six months, respectively. Their GMC progressively decreased between months one (20 . 1 AU/ml [95%CI 16 . 9-24 . 0]), three (15 . 2 AU/ml [95%CI 13 . 2-17 . 6]; p 0 . 86, p

Objectives Serological studies have been critical in tracking the evolution of the COVID-19 pandemic. Data on anti-SARS-CoV-2 antibodies persistence remain sparse, especially from infected individuals with few to no symptoms. The objective of the study was to quantify the sensitivity for detecting historic SARS-CoV-2 infections as a function of time since infection for three commercially available SARS-CoV-2 immunoassays and to explore the implications of decaying immunoassay sensitivity in estimating seroprevalence. Methods We followed a cohort of mostly mild/asymptomatic SARS-CoV-2-infected individuals (n = 354) at least 8 months after their presumed infection date and tested their serum for anti-SARS-CoV-2 antibodies with three commercially available assays: Roche-N, Roche-RBD and EuroImmun-S1. We developed a latent class statistical model to infer the specificity and time-varying sensitivity of each assay and show through simulations how inappropriately accounting for test performance can lead to biased serosurvey estimates. Results Antibodies were detected at follow-up in 74–100% of participants, depending on immunoassays. Both Roche assays maintain high sensitivity, with the EuroImmun assay missing 40% of infections after 9 months. Simulations reveal that without appropriate adjustment for time-varying assay sensitivity, seroprevalence surveys may underestimate infection rates. Discussion Antibodies persist for at least 8 months after infection in a cohort of mildly infected individuals with detection depending on assay choice. Appropriate assay performance adjustment is important for the interpretation of serological studies in the case of diminishing sensitivity after infection.

We report a clonal outbreak of multidrug-resistant (MDR) Klebsiella variicola (sequence type [ST] 771) in a Bangladeshi neonatal unit from October 2016 to January 2017, associated with high mortality (54.5%). During the outbreak, K. variicola ST771 acquired an MDR plasmid harboring blaNDM-1, linked to high exposure to ceftriaxone and amikacin.

The microbiological safety of islet preparations is paramount. Preservation medium contamination is frequent, and its impact on islet yield and function remains unclear. Microbiological samples collected during islet isolations from 2006 to 2016 were analyzed and correlated to isolation and allo- and autotransplantation outcomes. Microbial contamination of preservation medium was found in 64.4% of processed donor pancreases (291/452). We identified 464 microorganisms including Staphylococcus (253/464, 54.5%), Streptococcus (31/464, 6.7%), and Candida species (25/464, 5.4%). Microbial contamination was associated with longer warm and cold ischemia times and lower numbers of postpurification islet equivalents, purity, transplant rate, and stimulation index (all P < 0.05). Six percent of the preparations accepted for transplantation showed microbial contamination after isolation (12/200); 9 of 12 were Candida species. Six patients were transplanted with a sample with late microbial growth discovered after the infusion. Insulin independence rate was not affected. This risk of transplanting a contaminated islets preparation was reduced by half following the implementation of an additional sampling after 24 h of islet culture. Pancreas preservation fluid microbial contamination is associated with lower transplant rate and poorer in vitro function, but not with changes in graft survival. Culture medium testing 1 day after isolation reduces the risk of incidental transplantation with contaminated islets.

Ceftaroline is a broad-spectrum antibiotic with activity against methicillin-resistant Staphylococcus aureus (MRSA) strains. Ceftaroline susceptibility of an MRSA set archived between 1994 and 2003 in the Geneva University Hospitals detected a high percentage (66 %) of ceftaroline resistance in clonotypes ST228 and ST247 and correlated with mutations in PBP2a. The ceftaroline mechanism of action is based on the inhibition of PBP2a; thus, the identification of PBP2a mutations of recently circulating clonotypes in our institution was investigated. We analyzed ceftaroline susceptibility in MRSA isolates (2013 and 2014) and established that resistant strains correlated with PBP2a mutations and specific clonotypes. Ninety-six MRSA strains were analyzed from independent patients and were isolated from blood cultures (23 %), deep infections (38.5 %), and superficial (skin or wound) infections (38.5 %). This sample showed a ceftaroline minimum inhibitory concentration (MIC) range between 0.25 and 2 μg/ml and disk diameters ranging from 10 to 30 mm, with a majority of strains showing diameters ≥20 mm. Based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, 76 % (73/96) of isolates showed susceptibility to ceftaroline. Nevertheless, we still observed 24 % (23/96) of resistant isolates (MIC = 2 μg/ml). All resistant isolates were assigned to clonotype ST228 and carried the N146K mutation in PBP2a. Only two ST228 isolates showed ceftaroline susceptibility. The decreasing percentage of ceftaroline-resistant isolates in our hospital can be explained by the decline of ST228 clonotype circulating in our hospital since 2008. We present evidence that ceftaroline is active against recent MRSA strains from our hospital; however, the presence of PBP2a variants in particular clonotypes may affect ceftaroline efficacy.

Mucormycosis (zygomycosis) is an emerging fungal disease in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients. A 30-year-old woman diagnosed with acute myelomonocytic leukemia and needing allo-HSCT presented pulmonary and cerebral infection due to Rhizomucor pusillus. This fungal infection was treated with surgical treatment and posaconazole delayed-release tablets. This strategy allowed reaching high drug levels that could not be obtained with the posaconazole solution.

Although there have been many publications regarding the risk factors for implant-associated orthopaedic infections, none have investigated how clinical presentations and epidemiology may differ between infections with and without osteosynthetic material. We pooled clinical data from several databases of adult patients with orthopaedic infections hospitalized at Geneva University Hospitals from January 2004 to December 2014. Among 2740 episodes of orthopaedic infections, 76% were implant-free osteoarticular or soft tissue infections. Among the 665 (24% of the total episodes) infections that involved osteosynthetic material, 319 (49%) were total joint arthroplasties, 143 single plates, and 50 single nails. The remainders were mixed implant infections, pins, wires, screws, cerclages or spondylodeses. The implant-associated, compared to the implant-free, infections were significantly more frequently associated with female sex, older age, bacteraemia and skin commensal infections, e.g. coagulase-negative staphylococci, corynebacteria or propionibacteria. In contrast, implant-associated infections were significantly less frequently associated with immune suppression, abscess formation, infections due to Staphylococcus aureus or streptococci, polymicrobial pathogens and foot infections. The serum CRP levels at admission were similar (median 82 vs. 75 mg/L). Compared to implant-free infections, implant-associated orthopaedic infections are more likely monomicrobial, bacteraemic and due to skin commensals. They involve more often female and older patients, but are less often associated with immune suppression, abscess formation and foot infections.

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 Δ spx 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 Δ spx 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.

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.

The agr locus of Staphylococcus aureus is composed of two divergent transcripts (RNAII and RNAIII) driven by the P2 and P3 promoters. The P2-P3 intergenic region comprises the SarA/SarR binding sites and the four AgrA boxes to which AgrA binds. We reported here the role of AgrA, SarA, and SarR on agr P2 and P3 transcription. Using real-time reverse transcription (RT)-PCR and promoter fusion studies with selected single, double, triple, and complemented mutants, we showed that AgrA is indispensable to agr P2 and P3 transcription, whereas SarA activates and SarR represses P2 transcription. In vitro runoff transcription assays revealed that AgrA alone promoted transcription from the agr P2 promoter, with SarA enhancing it and SarR inhibiting agr P2 transcription in the presence of AgrA or with SarA and AgrA. Electrophoretic mobility shift assay (EMSA) analysis disclosed that SarR binds more avidly to the agr promoter than SarA and displaces SarA from the agr promoter. Additionally, SarA and AgrA bend the agr P2 promoter, whereas SarR does not. Collectively, these data indicated that AgrA activates agr P2 and P3 promoters while SarA activates the P2 promoter, presumably via bending of promoter DNA to bring together AgrA dimers to facilitate engagement of RNA polymerase (RNAP) to initiate transcription.

The Staphylococcus aureus SarA global regulator controls the expression of numerous virulence genes, often in conjunction with the agr quorum-sensing system and its effector RNA, RNAIII. In the present study, we have examined the role of both SarA and RNAIII on the regulation of the promoter of tst , encoding staphylococcal superantigen toxic shock syndrome toxin 1 (TSST-1). In vitro DNA-protein interaction studies with purified SarA using gel shift and DNase I protection assays revealed one strong SarA binding site and evidence for a weaker site nearby within the minimal 400-bp promoter region upstream of tst. In vivo analysis of tst promoter activation using a p tst - luxAB reporter inserted in the chromosome revealed partial but not complete loss of tst expression in a Δ hld - RNAIII strain. In contrast, disruption of sarA abrogated tst expression. No significant tst expression was found for the double Δ hld-RNAIII- Δ sarA mutant. Introduction of a plasmid containing cloned hld-RNAIII driven by a non- agr -dependent promoter, p HU , into isogenic parental wild-type or Δ sarA strains showed comparable levels of RNAIII detected by quantitative reverse transcription-PCR (qRT-PCR) but a two-log 10 reduction in p tst -luxAB reporter expression in the Δ sarA strain, arguing that RNAIII levels alone are not strictly determinant for tst expression. Collectively, our results indicate that SarA binds directly to the tst promoter and that SarA plays a significant and direct role in the expression of tst .

International audience; Diagnosis of nonmenstrual staphylococcal toxic shock syndrome (TSS) is often challenging. A female medical colleague had a rare entity, a staphylococcal pharyngitis complicated by TSS. The diagnosis was confirmed by isolation of tst-positive Staphylococcus aureus in throat culture and by identification of a specific Vβ2 expansion pattern of her T lymphocytes. Recent improvements in microbiology can be of great help for the diagnosis of nonmenstrual TSS.

Staphylococcus aureus is an important pathogen manifesting virulence through diverse disease forms, ranging from acute skin infections to life-threatening bacteremia or systemic toxic shock syndromes. In the latter case, the prototypical superantigen is TSST-1 (Toxic Shock Syndrome Toxin 1), encoded by tst(H), and carried on a mobile genetic element that is not present in all S. aureus strains. Transcriptional regulation of tst is only partially understood. In this study, we dissected the role of sarA, sarS (sarH1), RNAIII, rot, and the alternative stress sigma factor sigB (σB). By examining tst promoter regulation predominantly in the context of its native sequence within the SaPI1 pathogenicity island of strain RN4282, we discovered that σB emerged as a particularly important tst regulator. We did not detect a consensus σB site within the tst promoter, and thus the effect of σB is likely indirect. We found that σB strongly repressed the expression of the toxin via at least two distinct regulatory pathways dependent upon sarA and agr. Furthermore rot, a member of SarA family, was shown to repress tst expression when overexpressed, although its deletion had no consistent measurable effect. We could not find any detectable effect of sarS, either by deletion or overexpression, suggesting that this regulator plays a minimal role in TSST-1 expression except when combined with disruption of sarA. Collectively, our results extend our understanding of complex multifactorial regulation of tst, revealing several layers of negative regulation. In addition to environmental stimuli thought to impact TSST-1 production, these findings support a model whereby sporadic mutation in a few key negative regulators can profoundly affect and enhance TSST-1 expression.