Antimicrobial activity of mucosal-associated invariant T cells.

Mucosal-associated invariant T lymphocytes (MAIT lymphocytes) are characterized by two evolutionarily conserved features: an invariant T cell antigen receptor (TCR) alpha-chain and restriction by the major histocompatibility complex (MHC)-related protein MR1. Here we show that MAIT cells were activated by cells infected with various strains of bacteria and yeast, but not cells infected with virus, in both humans and mice. This activation required cognate interaction between the invariant TCR and MR1, which can present a bacteria-derived ligand. In humans, we observed considerably fewer MAIT cells in blood from patients with bacterial infections such as tuberculosis. In the mouse, MAIT cells protected against infection by Mycobacterium abscessus or Escherichia coli. Thus, MAIT cells are evolutionarily conserved innate-like lymphocytes that sense and help fight off microbial infection.

Labeled TEMPO-Oxidized Mannan Differentiates Binding Profiles within the Collectin Families.

Establishing the rapid and accurate diagnosis of sepsis is a key component to the improvement of clinical outcomes. The ability of analytical platforms to rapidly detect pathogen-associated molecular patterns (PAMP) in blood could provide a powerful host-independent biomarker of sepsis. A novel concept was investigated based on the idea that a pre-bound and fluorescent ligand could be released from lectins in contact with high-affinity ligands (such as PAMPs). To create fluorescent ligands with precise avidity, the kinetically followed TEMPO oxidation of yeast mannan and carbodiimide coupling were used. The chemical modifications led to decreases in avidity between mannan and human collectins, such as the mannan-binding lectin (MBL) and human surfactant protein D (SP-D), but not in porcine SP-D. Despite this effect, these fluorescent derivatives were captured by human lectins using highly concentrated solutions. The resulting fluorescent beads were exposed to different solutions, and the results showed that displacements occur in contact with higher affinity ligands, proving that two-stage competition processes can occur in collectin carbohydrate recognition mechanisms. Moreover, the fluorescence loss depends on the discrepancy between the respective avidities of the recognized ligand and the fluorescent mannan. Chemically modulated fluorescent ligands associated with a diversity of collectins may lead to the creation of diagnostic tools suitable for multiplex array assays and the identification of high-avidity ligands.

Four-Hour Immunochromatographic Detection of Intestinal Carriage of Carbapenemase-Producing Enterobacteriaceae: a Validation Study.

The increasing incidence of carbapenemase-producing Gram-negative bacilli (C-PGNB) represents a major public health challenge. Rapid detection of digestive colonization with C-PGNB is fundamental to control their spread. We performed the validation of a rapid protocol for C-PGNB detection directly on rectal swabs. We developed a protocol combining enrichment by a rapid selective subculture of the rectal swab medium and realization of a Resist-4 O.K.N.V. K-SeT test on the bacterial pellet obtained. The limit of detection and performances of this protocol were validated in vitro on 52 C-PGNB strains spiked on a calibrated sample suspension and confirmed in clinical settings on 144 rectal swabs sampled from patients with C-PGNB digestive colonization (n = 48) and controls (patients with extended-spectrum beta-lactamase [ESBL] colonization [n = 48] and without carbapenemase/ESBL [n = 48]). The protocol detected, with 100% sensitivity, the presence of the 15 OXA-48-, 14 KPC-, 13 NDM-, and 10 VIM-producing GNB from 103 CFU/ml. The limit of detection was 2 × 102 CFU/ml. Among the 48 C-PGNB-containing rectal swabs of the validation cohort, 46 were accurately detected. False negative were observed for 1 NDM-producing Acinetobacter baumannii strain and 1 OXA-48-producing Escherichia coli strain. The 96 control swabs were negative. Sensitivity and specificity for C-PGNB detection were 97.7% (95% confidence interval [CI], 87.7 to 100) and 100% (95% CI, 96.2 to 100). The negative likelihood ratio was 0.04 (95% CI, 0.01 to 0.16). Considering a C-PGNB digestive colonization prevalence between 0.01% and 0.1%, positive and negative predictive values were 100%. Our protocol is a rapid and low-cost method detecting accurately the digestive colonization with carbapenemase-producing Enterobacteriaceae in 4 h without any requirement for specific equipment.

A mobile DNA laboratory for forensic science adapted to coronavirus SARS-CoV-2 diagnosis.

The Forensic Science Institute of the French "Gendarmerie Nationale" (IRCGN™) developed in 2015 an ISO 17025 certified mobile DNA laboratory for genetic analyses. This Mobil'DNA laboratory is a fully autonomous and adaptable mobile laboratory to perform genetic analyses in the context of crime scenes, terrorism attacks or disasters. To support the hospital task force in Paris during the peak of the COVID-19 epidemic, we adapted this mobile genetic laboratory to perform high-throughput molecular screening for coronavirus SARS-CoV-2 by real-time PCR. We describe the adaptation of this Mobil'DNA lab to assist in Coronavirus SARS-CoV-2 diagnosis.

Cutibacterium acnes clonal complexes display various growth rates in blood culture vials used for diagnosing orthopedic device-related infections.

OBJECTIVES: Blood culture bottles (BCBs) are commonly used for the diagnosis of infections associated with orthopedic devices. Although Cutibacterium acnes is an important pathogen in orthopedics, relatively little is known about its growth characteristics in BCBs. This prompted us to analyze the influence of bacterial genotype and clinical significance on time-to-detection (TTD) in BCBs. METHODS: We reviewed 59 cases of orthopedic device-related infections in which at least one intraoperative specimen yielded a pure C. acnes culture from anaerobic BCBs (BD Bactec Lytic/10 Anaerobic/F; Lytic-Ana) and/or solid media. A strain was considered infectant if the same genotype was present in two or more intraoperative samples. From these cases, we isolated a total of 72 unique C. acnes strains belonging to four multilocus sequence type clonal complexes (CCs): CC18, CC28, CC36 and CC53. Growth rate and TTD in Lytic-Ana BCB were studied under experimental conditions (inoculation of standard inoculum) and in clinical samples (inoculation of periprosthetic tissue samples). RESULTS: Median TTD values were shorter for CC53 compared to other CCs under experimental conditions (69 vs. 103 h; p < 0.001) and from clinical specimens (70 vs. 200 h; p = 0.02). Infectant strains had a shorter median TTD compared to contaminant strains in a clinical situation, while the difference was not observed under experimental conditions. CONCLUSIONS: The detection dynamics of C. acnes in Lytic-Ana BCBs were associated with genotype. Thus, TTD not only reflects the bacterial load in clinical samples, but may also reflect the intrinsic properties of the clonal complex of C. acnes.

Predictive factors for positive disco-vertebral biopsy culture in pyogenic vertebral osteomyelitis, and impact of fluoroscopic versus scanographic guidance.

BACKGROUND: The aims of this study were to identify the predictive factors for microbiological diagnosis through disco-vertebral biopsy (DVB) in patients with pyogenic vertebral osteomyelitis (PVO) and negative blood cultures, and compare the performance of DVB under fluoroscopic versus scanographic guidance. METHODS: We performed a cohort study comparing positive and negative DVB among patients with PVO. All cases of PVO undergoing a DVB for microbiological diagnosis in our center were retrospectively reviewed. Infections due to Mycobacterium tuberculosis, infections on foreign device, and non-septic diseases were excluded. Anamnestic, clinical, biological, microbiological, as well as radiological data were collected from medical charts thanks to a standardized data set. RESULTS: A total of 111 patients were screened; 88 patients were included. Microbiological cultures were positive in 53/88 (60.2%) patients. A thickening of the paravertebral tissue ≥10 mm on magnetic resonance imaging (MRI) in axial MR scans was a predictive factor of DVB microbiological positivity (52.4% vs. 13.3%; p = 0.006; OR = 5.4). Overall, 51 DVB were performed under fluoroscopic guidance and 37 under scanographic guidance. Considering lumbar DVB, 25/36 (69.4%) of cases yielded positive results under fluoroscopic guidance versus 5/15 (33.3%) under scanographic guidance (p = 0.02; OR = 4.4). No adverse event linked to DVB was notified. CONCLUSION: Every patient with PVO and negative blood cultures should undergo a DVB. A thickening of the paravertebral tissue ≥10 mm on MRI is associated with a higher rate of positive DVB culture. A lumbar DVB under fluoroscopic guidance is more sensitive than under scanographic guidance to identify the micro-organism involved.

Measurement of Serum Anti-staphylococcal Antibodies Increases Positive Predictive Value of Preoperative Aspiration for Hip Prosthetic Joint Infection.

BACKGROUND: Preoperative synovial fluid culture is pivotal in the early diagnosis of prosthetic joint infection (PJI) but may yield false-positive and false-negative results. We evaluated the predictive value of synovial fluid culture results combined with the measurement of serum anti-staphylococcal antibodies (SASA). QUESTIONS/PURPOSES: (1) For hip and knee PJI, does combining positive SASA results with preoperative synovial culture results improve the positive predictive value (PPV) of preoperative synovial fluid culture alone? (2) Does combining preoperative synovial fluid culture results with a positive cell count and differential result increase the PPV of preoperative synovial fluid culture alone? (3) What proportion of isolated organisms exhibit concordance in antibiotic susceptibility: preoperative aspiration versus intraoperative isolates? METHODS: A prospective study was conducted at two French reference centers that manage bone and joint infections and included 481 adult patients who had a revision or resection arthroplasty between June 25, 2012 and June 23, 2014. Exclusion criteria including no serum sample available for immunoassay, the lack of microbiological documentation, and the absence of preoperative aspiration reduced the patient number to 353. Seven patients with an undetermined SASA result were excluded from the analysis. We also excluded patients with PJI involving more than one Staphylococcus species (polystaphylococcal infection) and those in whom more than one Staphylococcus species was recovered from the preoperative synovial fluid culture (polystaphylococcal synovial fluid culture). In total, 340 patients were included in the analysis (no infection, 67% [226 of 340]; staphylococcal infection, 21% [71 of 340]; other infection, 13% [43 of 340]). The preoperative synovial fluid analysis included a cell count and differential and bacterial culture. SASAs were measured using a multiplex immunoassay. The diagnosis of PJI was determined using the Infectious Diseases Society of America (IDSA) criteria [] and intraoperative tissue culture at the time of revision surgery was used as the gold standard (at least one positive intraoperative sample for a "virulent" organism (such as S. aureus) or two positive samples for a "non-virulent" (for example S. epidermidis). RESULTS: SASA increased the PPV compared with synovial fluid culture alone (92% [95% CI 82 to 97] versus 79% [95% CI 68 to 87]; p = 0.04); when stratified by site, an increase in PPV was seen in hip infections (100% [95% CI 89 to 100] versus 77% [95% CI 63 to 88]; p = 0.01) but not in knee infections (84% [95% CI 66 to 95] versus 80% [95% CI 64 to 91]; p = 0.75). A positive cell count and differential result increased the PPV of staphylococcal synovial fluid cultures compared with synovial fluid culture alone (86% [95% CI 70 to 95] versus 79% [95% CI 68 to 87]; p = 0.36); when stratified by site, no difference in hip and knee infections was observed (86% [95% CI 67 to 96] versus 77% [95% CI 63 to 88]; p = 0.42) and 86% [95% CI 70 to 95] versus 80% [95% CI 64 to 91]; p = 0.74). CONCLUSION: SASA measurement improves the predictive value of synovial fluid cultures of the hip for all staphylococcal organisms, including coagulase-negative staphylococci, but the PPV of SASA plus synovial fluid culture it is not superior to the PPV of synovial fluid cell count/differential plus synovial culture for the knee. LEVEL OF EVIDENCE: Level III, diagnostic study.

Growth detection of Cutibacterium acnes from orthopaedic implant-associated infections in anaerobic bottles from BACTEC and BacT/ALERT blood culture systems and comparison with conventional culture media.

Cutibacterium acnes is a major etiologic agent of orthopaedic implant-associated infections (IAIs) and requires up to 14 days of incubation in an anaerobic atmosphere for growth detection. As blood culture (BC) systems are increasingly being used to monitor the growth of IAI specimens, we compared different BC media for growth detection of C. acnes. Non-duplicate C. acnes isolates (n = 99) obtained from sonicate-fluid cultures of orthopaedic IAIs from Slovenia (n = 54), conventional tissue samples of monomicrobial orthopaedic IAIs from France (n = 43) and two reference strains were inoculated to anaerobic BC bottles of two major BC systems and 3 conventional culture media types (thioglycolate broth, Schaedler and chocolate agar). Growth and time-to-detection (TTD) were recorded. Only Lytic (BACTEC) and SN (BacT/ALERT) bottles consistently detected growth of C. acnes within 14 days with 94% (n = 93) and 92% (n = 91) detection rates, respectively (p = 0.79). Lytic was superior to Plus BACTEC medium (p < 0.001), while SN was superior to all other BacT/ALERT media (p < 0.001). Mean TTD was 128 ± 43 h (61-336 h) for Lytic and 158 ± 65 h (77-336 h) for SN medium. Among the conventional media, 99% (n = 98) of the isolates grew on Schaedler agar, 96% (n = 95) in thioglycolate broth and 74% (n = 73) on chocolate agar. Inconsistent growth of C. acnes in different BC media can critically influence the detection of this major IAI pathogen. Only Lytic (BACTEC) and SN (BacT/ALERT) BC media types were consistently able to detect C. acnes within 14 days of incubation. However, visible growth was observed faster in thioglycolate broth and Schaedler agar media.

Fluorescein Derivatives as Fluorescent Probes for pH Monitoring along Recent Biological Applications.

Potential of hydrogen (pH) is one of the most relevant parameters characterizing aqueous solutions. In biology, pH is intrinsically linked to cellular life since all metabolic pathways are implicated into ionic flows. In that way, determination of local pH offers a unique and major opportunity to increase our understanding of biological systems. Whereas the most common technique to obtain these data in analytical chemistry is to directly measure potential between two electrodes, in biological systems, this information has to be recovered in-situ without any physical interaction. Based on their non-invasive optical properties, fluorescent pH-sensitive probe are pertinent tools to develop. One of the most notorious pH-sensitive probes is fluorescein. In addition to excellent photophysical properties, this fluorophore presents a pH-sensitivity around neutral and physiologic domains. This review intends to shed new light on the recent use of fluorescein as pH-sensitive probes for biological applications, including targeted probes for specific imaging, flexible monitoring of bacterial growth, and biomedical applications.

Molecular Typing of Multiple Isolates Is Essential to Diagnose Cutibacterium acnes Orthopedic Device-related Infection.

Cutibacterium acnes orthopedic device-related infections (ODRIs) range from obvious infections to solely culture-based diagnoses. Multilocus sequence typing of multiple isolates from the same procedure revealed that most cases with normal C-reactive protein levels that were classified as C. acnes ODRI would be considered contaminations when accounting for genotypic data.

Monomicrobial bone and joint infection due to Corynebacterium striatum: literature review and amoxicillin-rifampin combination as treatment perspective.

Corynebacterium striatum is a ubiquitous colonizer of human skin and mucous membranes. It is increasingly involved in infections, especially with prosthetic devices or in immunocompromised individuals. Microbiological diagnosis is challenging and bacterial resistance is a major concern. We performed a retrospective study of monomicrobial bone and joint infections (BJI) due to C. striatum in two referral centers from April 2012 to July 2017. We collected the patients' clinical and microbiological characteristics and outcomes. We also performed a literature review of BJI due to C. striatum. We identified 12 cases (nine prosthetic joint infections, one osteosynthetic device infection, one non-union, and one arthritis) in 11 patients, five of which were immunocompromised. Microbiological diagnosis was performed with prolonged culture media. Ten out of 12 strains were susceptible to aminopenicillin, a drug class not recommended for testing by the EUCAST/CASFM guidelines, and 8/12 patients were treated with amoxicillin-rifampicin. The cure rate was 8/12, after a median follow-up period of 487.5 days (IQR 140.3-1348.5). Twelve cases of BJI due to C. striatum were previously reported. Among them, 5/12 patients were immunocompromised, 3/12 cases were acute BJI, and 2/12 were device-related infections. The diagnosis was performed by PCR in one case, and 10/12 patients were treated with glycolipopeptides, with a cure rate of 11/12. We report the largest cohort of monomicrobial BJI with C. striatum. Determination of aminopenicillin susceptibility is essential since it is frequently active in our experience, even in BJI. The cure rate of this infection seems high.

In-Host Adaptation of Salmonella enterica Serotype Dublin during Prosthetic Hip Joint Infection.

Genome degradation has been central to the adaptation of Salmonella enterica serotypes to their hosts throughout evolution. We witnessed the patho-adaptation of a strain of Salmonella Dublin (a cattle-adapted serotype) to a human host during the course of a recurrent prosthetic hip joint infection evolving over several years.

Case of femoral pseudarthrosis due to Scedosporium apiospermum in an immunocompetent patient with successful conservative treatment and review of literature.

Scedosporium apiospermum is a ubiquitous filamentous fungus, commonly found in soil, sewage and polluted waters. It is rarely pathogenic but can cause a broad spectrum of clinical diseases, which can be localised or disseminate to distant organs. The disseminated form of the disease is mostly seen among immunocompromised patients. However, some rare cases of disseminated disease have been reported in immunocompetent individuals. Treatment of these infections is challenging because of their natural resistance to many antifungal agents. Here, we report the case of a 57-year-old immunocompetent patient diagnosed with femoral pseudarthrosis due to S. apiospermum, despite having no obvious clinical sign of infection. Previously, the patient had undergone four iterative femoral surgeries following a road traffic accident which occurred 20 years before. During its last surgery for pseudarthrosis, no clinical or biological signs of infection were present. Per operative samples tested positive for S. apiospermum. The patient was successfully treated with oral voriconazole during 6 months with an excellent tolerance. We also provide a review of literature on bone and joint infections due to Scedosporium spp. (S. apiospermum, Scedosporium boydii and Scedosporium aurantiacum), discussing the evolution of their management and outcome which seems to improve since the use of voriconazole.

Emergence of Plasmid-Mediated Fosfomycin-Resistance Genes among Escherichia coli Isolates, France.

FosA, a glutathione S-transferase that inactivates fosfomycin, has been reported as the cause of enzymatic resistance to fosfomycin. We show that multiple lineages of FosA-producing extended spectrum ß-lactamase Escherichia coli have circulated in France since 2012, potentially reducing the efficacy of fosfomycin in treating infections with antimicrobial drug-resistant gram-negative bacilli.

Multiplex Antibody Detection for Noninvasive Genus-Level Diagnosis of Prosthetic Joint Infection.

We developed and evaluated a multiplex antibody detection-based immunoassay for the diagnosis of prosthetic joint infections (PJIs). Sixteen protein antigens from three Staphylococcusspecies (Staphylococcus aureus,Staphylococcus epidermidis, and Staphylococcus lugdunensis) (8 antigens),Streptococcus agalactiae(4 antigens), and Propionibacterium acnes(4 antigens) were selected by comparative immune proteomics using serum samples from PJI cases versus controls. A bead-based multiplex immunoassay that measured serum IgG against purified, recombinant forms of each of the 16 antigens was developed. We conducted a prospective study to evaluate the performance of the assay. A PJI was defined by the presence of a sinus tract and/or positive intraoperative sample cultures (at least one sample yielding a virulent organism or at least two samples yielding the same organism). A total of 455 consecutive patients undergoing revision or resection arthroplasty (hip, 66.3%; knee, 29.7%; shoulder, 4%) at two French reference centers for the management of PJI were included: 176 patients (38.7%) were infected and 279 (61.3%) were not. About 60% of the infections involved at least one of the species targeted by the assay. The sensitivity/specificity values were 72.3%/80.7% for targeted staphylococci, 75%/92.6% forS. agalactiae, and 38.5%/84.8% forP. acnes The assay was more sensitive for infections occurring >3 months after arthroplasty and for patients with an elevated C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR). However, it detected 64.3% and 58.3% of targeted staphylococcal infections associated with normal CRP and ESR values, respectively. This new multiplex immunoassay approach is a novel noninvasive tool to evaluate patients suspected of having PJIs and provides information complementary to that from inflammatory marker values.

Mycobacterium abscessus phospholipase C expression is induced during coculture within amoebae and enhances M. abscessus virulence in mice.

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium involved in pulmonary and cutaneo-mucous infections worldwide, to which cystic fibrosis patients are exquisitely susceptible. The analysis of the genome sequence of M. abscessus showed that this bacterium is endowed with the metabolic pathways typically found in environmental microorganisms that come into contact with soil, plants, and aquatic environments, where free-living amoebae are frequently present. M. abscessus also contains several genes that are characteristically found only in pathogenic bacteria. One of them is MAB_0555, encoding a putative phospholipase C (PLC) that is absent from most other rapidly growing mycobacteria, including Mycobacterium chelonae and Mycobacterium smegmatis. Here, we report that purified recombinant M. abscessus PLC is highly cytotoxic to mouse macrophages, presumably due to hydrolysis of membrane phospholipids. We further showed by constructing and using an M. abscessus PLC knockout mutant that loss of PLC activity is deleterious to M. abscessus intracellular survival in amoebae. The importance of PLC is further supported by the fact that M. abscessus PLC was found to be expressed only in amoebae. Aerosol challenge of mice with M. abscessus strains that were precultured in amoebae enhanced M. abscessus lung infectivity relative to M. abscessus grown in broth culture. Our study underlines the importance of PLC for the virulence of M. abscessus. Despite the difficulties of isolating M. abscessus from environmental sources, our findings suggest that M. abscessus has evolved in close contact with environmental protozoa, which supports the argument that amoebae may contribute to the virulence of opportunistic mycobacteria.

Comparative Evaluation of Two PCR-Based Methods for Detection of Methicillin-Resistant Staphylococcus aureus (MRSA): Xpert MRSA Gen 3 and BD-Max MRSA XT.

We compared two walk-away molecular diagnostic assays, the GeneXpert MRSA Gen 3 assay and the BD-Max MRSA XT assay. A total of 119 prospective swabs and 36 culture-positive samples were tested. Xpert MRSA Gen 3 had sensitivity of 95.7% and specificity of 100% versus 87.5% and 97.1% for BD-Max. The difference in agreement with the enriched culture results was significantly in favor of the Xpert assay (P < 0.02, McNemar nonparametric text).

Ruminococcus gnavus total hip arthroplasty infection in a 62-year-old man with ulcerative colitis.

We report the case of a total hip arthroplasty infection caused by Ruminococcus gnavus in a 62-year-old man with ulcerative colitis. The bacterium was perfectly identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Combined coagulation and inflammation markers as predictors of venous thrombo-embolism and death in COVID-19.

Background: The COVID-19 pandemic related to SARS-CoV-2 virus was responsible for global pandemic. The severe form of the disease was linked to excessive activation of immune pathways together with a systemic cytokine storm response and thrombotic venous or arterial complications. Factors predicting severe outcomes including venous and/or pulmonary thrombosis (VT) and death were identified, but the prognostic role of their combination was not addressed extensively. Objectives: We investigated the role of prognostic factors from the coagulation or inflammatory pathways to better understand the outcome of the disease. Methods: For this, we prospectively studied 167 SARS-CoV-2-positive patients from admission in intensive care units (ICU) or emergency departments from four academic hospitals over a 14-month period. Besides standard biology, we assessed serum concentrations of inflammatory markers, coagulation factors and peripheral blood cells immunophenotyping. Results: Thirty-nine patients (23.3%) developed VT and 30 patients (18%) died. By univariate analysis, C-reactive protein (CRP) level > 150 mg/L, interleukin-6 (IL-6) ≥ 20 pg/mL, D-dimers > 1,500 µg/L, ADAMTS13 activity ≤ 50%, Von. Conclusion: A combination of coagulation and inflammatory markers can refine the prognostication of severe outcome in COVID-19, and could be useful for the initial evaluation of other types of viral infection.

Genetic analysis of glycopeptide-resistant Staphylococcus epidermidis strains from bone and joint infections.

Glycopeptide-resistant Staphylococcus epidermidis (GRSE) strains are of increasing concern in bone and joint infections (BJIs). Using multilocus sequence typing and multilocus variable-number tandem repeat analysis, we show that BJI-associated GRSE strains are genetically diverse but arise from related, multiresistant hospital sequence types (STs), mostly ST2, ST5, and ST23.