In-Host Emergence of Linezolid Resistance in a Complex Pattern of Toxic Shock Syndrome Toxin-1-Positive Methicillin-Resistant Staphylococcus aureus Colonization in Siblings with Cystic Fibrosis

Methicillin-resistant Staphylococcus aureus (MRSA) can cause chronic lung infections in patients with Cystic Fibrosis (CF). One option for managing them is the use of linezolid. We hereby report the in-host emergence of linezolid resistance (LR) in MRSA in CF siblings via a population analysis. A collection of 171 MRSA strains from 68 samples were characterized by determining their linezolid Minimal Inhibitory Concentrations (MICs), analyzing the locus of staphylococcal protein A (spa) and whole genome sequencing. Courses of linezolid were retraced. Strains belonged to three spa types (t002, t045, t127) and two sequence types (ST1, ST5). Emergence of LR occurred under treatment, one year apart in both siblings, in the CC5-MRSA-I Geraldine clone harboring the toxic shock syndrome toxin-1-encoding gene. Resistance was related to a G2576T substitution present in a variable number of 23S rRNA gene copies. Susceptible and resistant strains were co-isolated within samples. Single Nucleotide Polymorphism-based analysis revealed complex colonizations by highly diversified, clonally related populations. LR remains rare in MRSA and there are very few longitudinal analyses documenting its emergence. Analyzing a large MRSA collection revealed new aspects of LR emergence: it emerges in specific subclonal lineages resulting from adaptive diversification of MRSA in the CF lung and this heterogeneity of intra-sample resistance may contribute to compromising antibiotic management

Limitation of Screening of Different Variants of SARS-CoV-2 by RT-PCR

Since January 2021, the diffusion of the most propagated SARS-CoV-2 variants in France (UK variant 20I/501Y.V1 (lineage B.1.1.7), 20H/H501Y.V2 (lineage B.1.351) and 20J/H501Y.V3 (lineage P.1)) were urgently screened, needing a surveillance with an RT-PCR screening assay. In this study, we evaluated one RT-PCR kit for this screening (ID SARS-CoV-2/UK/SA Variant Triplex®, ID Solutions, Grabels, France) on 2207 nasopharyngeal samples that were positive for SARS-CoV-2. Using ID Solutions kit, 4.1% (92/2207) of samples were suspected to belonged to B.1.351 or P.1 variants. Next-generation sequencing that was performed on 67.4% (62/92) of these samples confirmed the presence of a B.1.351 variant in only 75.8% of the samples (47/62). Thirteen samples belonged to the UK variant (B.1.1.7), and two to A.27 with N501Y mutation. The thirteen with the UK variant presented one mutation in the S-gene, near the ΔH69/ΔV70 deletion (S71F or A67S), which impacted the detection of ΔH69/ΔV70 deletion. Using another screening kit (PKampVariantDetect SARS-CoV-2 RT-PCR combination 1 and 3® PerkinElmer, Waltham, MA, USA) on the misidentified samples, we observed that the two mutations, S71F or A67S, did not impact the detection of the UK variant. In conclusion, this study highlights the limitations of the screening strategy based on the detection of few mutations/deletions as well as it not being able to follow the virus evolution

Intégration chromosomique de HHV6, un piège diagnostique potentiel

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Evaluation of Two Methods for the Detection of Third Generation Cephalosporins Resistant Enterobacterales Directly From Positive Blood Cultures

International audienceDue to the importance of a rapid determination of patients infected by multidrug resistant bacteria, we evaluated two rapid diagnostic tests for the detection of third-generation cephalosporins (3GC)-resistant Enterobacterales directly from positive blood cultures within 1 h: BL-REDTM (electrochemical method) and β-LACTATM test (chromogenic method). A panel of 150 clinical strains characterized for their resistance profiles (e.g., penicillinases, extended-spectrum beta-lactamases (ESBLs), overproduction of cephalosporinase, carbapenemases, impermeability) was tested. Approximately 100 CFU of each isolate was spiked into sterile blood culture bottles and incubated in a BD BACTECTM FX automated system (Becton Dickinson, USA). Positive blood cultures were examined to parallel testing using the BL-REDTM and β-LACTATM tests and conventional susceptibility method (disc diffusion following EUCAST recommendations). For all phenotypes combined, the sensitivity, specificity, positive predictive value, and negative predictive value in the detection of 3GC resistance were, respectively (i) with BL-REDTM: 45.7, 100, 100, and 54.2% and (ii) with β-LACTATM test: 52.2, 100, 100, and 56.9%. The positivity of tests allows to adapt antibiotic treatment whereas the negative result requires other tests. Moreover, these tests detect most Ambler class A-producing Enterobacterales (KPC, ESBL, extended-spectrum OXY) with sensitivities and specificities of 87.5 and 99% for BL-REDTM, respectively and both 100% for β-LACTATM test (47/47 isolates). These two rapid tests failed to detect AmpC overexpressed (sensitivities of 2.7% for BL-REDTM and 0% for β-LACTATM test) and Ambler class B-producing Enterobacterales (sensitivities of 40% for both tests) notably strains without ESBLs associated (sensitivities of 0% for both tests). BL-REDTM and β-LACTATM tests are easy-to-use and mainly attractive when a positive result is obtained notably to detect most of the Ambler class A-producing Enterobacterales in <1 h after the positivity of the blood culture, allowing a rapid adaptation of the antibiotic therapy in patients

Limitation of Screening of Different Variants of SARS-CoV-2 by RT-PCR

International audienceSince January 2021, the diffusion of the most propagated SARS-CoV-2 variants in France (UK variant 20I/501Y.V1 (lineage B.1.1.7), 20H/H501Y.V2 (lineage B.1.351) and 20J/H501Y.V3 (lineage P.1)) were urgently screened, needing a surveillance with an RT-PCR screening assay. In this study, we evaluated one RT-PCR kit for this screening (ID SARS-CoV-2/UK/SA Variant Triplex®, ID Solutions, Grabels, France) on 2207 nasopharyngeal samples that were positive for SARS-CoV-2. Using ID Solutions kit, 4.1% (92/2207) of samples were suspected to belonged to B.1.351 or P.1 variants. Next-generation sequencing that was performed on 67.4% (62/92) of these samples confirmed the presence of a B.1.351 variant in only 75.8% of the samples (47/62). Thirteen samples belonged to the UK variant (B.1.1.7), and two to A.27 with N501Y mutation. The thirteen with the UK variant presented one mutation in the S-gene, near the ΔH69/ΔV70 deletion (S71F or A67S), which impacted the detection of ΔH69/ΔV70 deletion. Using another screening kit (PKampVariantDetect SARS-CoV-2 RT-PCR combination 1 and 3® PerkinElmer, Waltham, MA, USA) on the misidentified samples, we observed that the two mutations, S71F or A67S, did not impact the detection of the UK variant. In conclusion, this study highlights the limitations of the screening strategy based on the detection of few mutations/deletions as well as it not being able to follow the virus evolution

In-Host Emergence of Linezolid Resistance in a Complex Pattern of Toxic Shock Syndrome Toxin-1-Positive Methicillin-Resistant Staphylococcus aureus Colonization in Siblings with Cystic Fibrosis

International audienceMethicillin-resistant Staphylococcus aureus (MRSA) can cause chronic lung infections in patients with Cystic Fibrosis (CF). One option for managing them is the use of linezolid. We hereby report the in-host emergence of linezolid resistance (LR) in MRSA in CF siblings via a population analysis. A collection of 171 MRSA strains from 68 samples were characterized by determining their linezolid Minimal Inhibitory Concentrations (MICs), analyzing the locus of staphylococcal protein A (spa) and whole genome sequencing. Courses of linezolid were retraced. Strains belonged to three spa types (t002, t045, t127) and two sequence types (ST1, ST5). Emergence of LR occurred under treatment, one year apart in both siblings, in the CC5-MRSA-I Geraldine clone harboring the toxic shock syndrome toxin-1-encoding gene. Resistance was related to a G2576T substitution present in a variable number of 23S rRNA gene copies. Susceptible and resistant strains were co-isolated within samples. Single Nucleotide Polymorphism-based analysis revealed complex colonizations by highly diversified, clonally related populations. LR remains rare in MRSA and there are very few longitudinal analyses documenting its emergence. Analyzing a large MRSA collection revealed new aspects of LR emergence: it emerges in specific subclonal lineages resulting from adaptive diversification of MRSA in the CF lung and this heterogeneity of intra-sample resistance may contribute to compromising antibiotic management

Use of Deep Learning to Detect the Maternal Heart Rate and False Signals on Fetal Heart Rate Recordings

We have developed deep learning models for automatic identification of the maternal heart rate (MHR) and, more generally, false signals (FSs) on fetal heart rate (FHR) recordings. The models can be used to preprocess FHR data prior to automated analysis or as a clinical alert system to assist the practitioner. Three models were developed and used to detect (i) FSs on the MHR channel (the FSMHR model), (ii) the MHR and FSs on the Doppler FHR sensor (the FSDop model), and (iii) FSs on the scalp ECG channel (the FSScalp model). The FSDop model was the most useful because FSs are far more frequent on the Doppler FHR channel. All three models were based on a multilayer, symmetric, GRU, and were trained on data recorded during the first and second stages of delivery. The FSMHR and FSDop models were also trained on antepartum recordings. The training dataset contained 1030 expert-annotated periods (mean duration: 36 min) from 635 recordings. In an initial evaluation of routine clinical practice, 30 fully annotated recordings for each sensor type (mean duration: 5 h for MHR and Doppler sensors, and 3 h for the scalp ECG sensor) were analyzed. The sensitivity, positive predictive value (PPV) and accuracy were respectively 62.20%, 87.1% and 99.90% for the FSMHR model, 93.1%, 95.6% and 99.68% for the FSDop model, and 44.6%, 87.2% and 99.93% for the FSScalp model. We built a second test dataset with a more solid ground truth by selecting 45 periods (lasting 20 min, on average) on which the Doppler FHR and scalp ECG signals were recorded simultaneously. Using scalp ECG data, the experts estimated the true FHR value more reliably and thus annotated the Doppler FHR channel more precisely. The models achieved a sensitivity of 53.3%, a PPV of 62.4%, and an accuracy of 97.29%. In comparison, two experts (blinded to the scalp ECG data) respectively achieved a sensitivity of 15.7%, a PPV of 74.3%, and an accuracy of 96.91% and a sensitivity of 60.7%, a PPV of 83.5% and an accuracy of 98.24%. Hence, the models performed at expert level (better than one expert and worse than the other), although a well-trained expert with good knowledge of FSs could probably do better in some cases. The models and datasets have been included in the Fetal Heart Rate Morphological Analysis open-source MATLAB toolbox and can be used freely for research purposes

Environmental detection of SARS-CoV-2 in hospital rooms in different wards of a university hospital

International audienceBackground: transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can occur through direct, indirect, or close contact with infected people. However, the extent of environmental contamination is unknown. The nature of the relation between patients' symptoms and SARS-CoV-2 environmental shedding remains unclear. The aim of this study was to assess the relationship between patient coronavirus disease 2019 (COVID-19) status and environmental contamination.Methods: between May and November 2020, environmental swabs were taken before and after room disinfection at day 7 after symptom onset in a cohort of patients clinically or biologically diagnosed with COVID-19. Twelve surfaces per room were collected in 13 rooms. Sample analysis was performed by reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 detection [SARS-CoV-2 R-Gene (biomérieux, Marcy l'Etoile, France)]. Clinical data (day of illness, symptoms, RT-PCR results) was collected from the clinical software.Results: five medical units were included in the study. Of 156 samples collected in 13 rooms, five rooms (38.5%) presented 11 SARS-CoV-2-positive samples. These positive samples were detected on eight different surfaces. There was no association between detection of SARS-CoV-2 and patient age (P=1) or patient symptoms (P=0.3).Conclusion: viral shedding during COVID-19 appears to be unrelated to the presence of symptoms, patient age, and low-value cycle threshold of patient's test. This study supports the evidence for the environmental shedding of SARS-CoV-2 until at least 7 days after symptom onset. It emphasizes the need for strict compliance with contact precautions, hand hygiene, the correct use of personal protective equipment and room disinfection for the routine care of patients with COVID-19

Fetal heart rate baseline computation with a weighted median filter

International audienceBackgroundAutomated fetal heart rate (FHR) analysis removes inter- and intra-expert variability, and is a promising solution for reducing the occurrence of fetal acidosis and the implementation of unnecessary medical procedures. The first steps in automated FHR analysis are determination of the baseline, and detection of accelerations and decelerations (A/D). We describe a new method in which a weighted median filter baseline (WMFB) is computed and A/Ds are then detected.MethodThe filter weightings are based on the prior probability that the sampled FHR is in the baseline state or in an A/D state. This probability is computed by estimating the signal’s stability at low frequencies and by progressively trimming the signal. Using a competition dataset of 90 previously annotated FHR recordings, we evaluated the WMFB method and 11 recently published literature methods against the ground truth of an expert consensus. The level of agreement between the WMFB method and the expert consensus was estimated by calculating several indices (primarily the morphological analysis discordance index, MADI). The agreement indices were then compared with the values for eleven other methods. We also compared the level of method-expert agreement with the level of interrater agreement.ResultsFor the WMFB method, the MADI indicated a disagreement of 4.02% vs. the consensus; this value is significantly lower () than that calculated for the best of the 11 literature methods (7.27%, for Lu and Wei’s empirical mode decomposition method). The level of inter-expert agreement (according to the MADI) and the level of WMFB-expert agreement did not differ significantly (p=0.22).ConclusionThe WMFB method reproduced the expert consensus analysis better than 11 other methods. No differences in performance between the WMFB method and individual experts were observed. The method Matlab source code is available under General Public Licence at http://utsb.univ-catholille.fr/fhr-wmfb

Biofilm Formation by <i>Staphylococcus aureus</i> in the Specific Context of Cystic Fibrosis

Staphylococcus aureus is a major human pathogen whose characteristics support its success in various clinical settings including Cystic Fibrosis (CF). In CF, S. aureus is indeed the most commonly identified opportunistic pathogen in children and the overall population. S. aureus colonization/infection, either by methicillin-susceptible or methicillin-resistant strains, will become chronic in about one third of CF patients. The persistence of S. aureus in CF patients’ lungs, despite various eradication strategies, is favored by several traits in both host and pathogen. Among the latter, living in biofilm is a highly protective way to survive despite deleterious environmental conditions, and is a common characteristic shared by the main pathogens identified in CF. This is why CF has earned the status of a biofilm-associated disease for several years now. Biofilm formation by S. aureus, and the molecular mechanisms governing and regulating it, have been extensively studied but have received less attention in the specific context of CF lungs. Here, we review the current knowledge on S. aureus biofilm in this very context, i.e., the importance, study methods, molecular data published on mono- and multi-species biofilm and anti-biofilm strategies. This focus on studies including clinical isolates from CF patients shows that they are still under-represented in the literature compared with studies based on reference strains, and underlines the need for such studies. Indeed, CF clinical strains display specific characteristics that may not be extrapolated from results obtained on laboratory strains