Detection of antimicrobial resistance in <5 h in Neisseria gonorrhoeae isolates using flow cytometry-proof of concept for seven clinically relevant antimicrobials.

INTRODUCTION: Antimicrobial resistance in Neisseria gonorrhoeae compromises gonorrhoea treatment and rapid antimicrobial susceptibility testing (AST) would be valuable. We have developed a rapid and accurate flow cytometry method (FCM) for AST of gonococci. METHODS: The 2016 WHO gonococcal reference strains, and WHO Q, R and S (n = 17) were tested against seven clinically relevant antibiotics (ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline and gentamicin). After 4.5 h incubation of inoculated broth, the fluorescent dye Syto™ 9 was added, followed by FCM analysis. After gating, the relative remaining population of gonococci, compared with unexposed growth control samples, was plotted against antimicrobial concentration, followed by non-linear curve regression analysis. Furthermore, the response at one single concentration/tested antibiotic was evaluated with the intention to use as a screening test for detection of resistant gonococci. RESULTS: A dose-dependent response was seen in susceptible isolates for all tested antimicrobials. There was a clear separation between susceptible/WT and resistant/non-WT isolates for ceftriaxone, cefixime, spectinomycin, ciprofloxacin and tetracycline. In contrast, for azithromycin, only high-level-resistant isolates were distinguished, while resistant isolates with MICs of 4 mg/L were indistinguishable from WT (MIC ≤ 1 mg/L) isolates. For gentamicin, all tested 17 isolates were WT and FCM analysis resulted in uniform dose-response curves. Using a single antibiotic concentration and a 50% remaining cell population cut-off, the overall sensitivity and specificity for resistance detection were 93% and 99%, respectively. CONCLUSIONS: By providing results in <5 h for gonococcal isolates, FCM-based AST can become a rapid screening method for antimicrobial resistance or antimicrobial susceptibility in gonococci.

Rapid high-resolution detection of colistin resistance in Gram-negative bacteria using flow cytometry: a comparison with broth microdilution, a commercial screening test and WGS.

BACKGROUND: Even though both EUCAST and CLSI consider broth microdilution (BMD) as the reference method for antimicrobial susceptibility testing (AST) of colistin, the method exhibits potential flaws related to properties of the colistin molecule. OBJECTIVES: To develop a flow cytometry method (FCM) for colistin AST and to validate it against BMD, a commercial screening test and WGS. METHODS: Colistin-mediated loss of membrane integrity in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. was detected with the fluorescent probe YoPro-1 by FCM. An international collection of 65 resistant and 109 susceptible isolates were analysed and the colistin concentration required to reach the EC50 was compared with the BMD MIC and the presence of genotypic resistance markers. RESULTS: The overall FCM sensitivity and specificity for colistin resistance was 89% and 94%, with E. coli > K. pneumoniae > P. aeruginosa, whereas the performance for Acinetobacter spp. was poor. All tested E. coli were correctly categorized. Three K. pneumoniae isolates with genotypic findings consistent with colistin resistance were detected by FCM but not BMD. Compared with BMD, FCM delivered AST results with a 75% reduction of time. CONCLUSIONS: Here, we present a rapid FCM-based AST assay for qualitative and quantitative testing of colistin resistance in E. coli and K. pneumoniae. The assay revealed probable chromosomal colistin resistance in K. pneumoniae that was not detected by BMD. If confirmed, these results question the reliability of BMD for colistin testing.

High-throughput immunoassays for SARS-CoV-2 - considerable differences in performance when comparing three methods.

BACKGROUND: The recently launched high-throughput assays for detecting antibodies against SARS-CoV-2 has contributed to the managing strategies for the COVID-19 pandemic. This study aimed to investigate the performance of three high-throughput assays and one rapid lateral flow test relative to regulatory authorities' recommended criteria. METHODS: A total of 315 samples, including 150 pre-pandemic samples, 152 samples from SARS-CoV-2 RT-PCR positive individuals and 13 potentially cross-reactive samples were analysed with SARS-CoV-2 IgG (Abbott, Abbott Park, IL), Elecsys Anti-SARS-CoV-2 (Roche, Solna, Sweden), LIAISON SARS-CoV-2 S1/S2 IgG (DiaSorin, Saluggia, Italy) and 2019-nCOV IgG/IgM Rapid Test (Dynamiker Biotechnology Co., Tianjin, China). RESULTS: All assays performed with a high level of specificity ranging from 96.7% to 99.3%. Sensitivity differed more between the assays, Roche exhibiting the highest sensitivity of 98.7%. The corresponding figures for Abbott, DiaSorin and Dynamiker Biotechnology were 80.9%, 89.0% and 72.4%, respectively. CONCLUSIONS: The results of the evaluated SARS-CoV-2 assays vary considerably, as well as their ability to fulfil the performance criteria proposed by regulatory authorities. Introduction into clinical use in low-prevalent settings, should, therefore, be made with caution.

Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction.

Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50-60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LG-domains.

Erythrocyte-derived microparticles supporting activated protein C-mediated regulation of blood coagulation.

Elevated levels of erythrocyte-derived microparticles are present in the circulation in medical conditions affecting the red blood cells. Erythrocyte-derived microparticles expose phosphatidylserine thus providing a suitable surface for procoagulant reactions leading to thrombin formation via the tenase and prothrombinase complexes. Patients with elevated levels of circulating erythrocyte-derived microparticles have increased thrombin generation in vivo. The aim of the present study was to investigate whether erythrocyte-derived microparticles are able to support the anticoagulant reactions of the protein C system. Erythrocyte-derived microparticles were isolated using ultracentrifugation after incubation of freshly prepared erythrocytes with the ionophore A23187 or from outdated erythrocyte concentrates, the different microparticles preparations yielding similar results. According to flow cytometry analysis, the microparticles exposed phoshatidylserine and bound lactadherin, annexin V, and protein S, which is a cofactor to activated protein C. The microparticles were able to assemble the tenase and prothrombinase complexes and to stimulate the formation of thrombin in plasma-based thrombin generation assay both in presence and absence of added tissue factor. The addition of activated protein C in the thrombin generation assay inhibited thrombin generation in a dose-dependent fashion. The anticoagulant effect of activated protein C in the thrombin generation assay was inhibited by a monoclonal antibody that prevents binding of protein S to microparticles and also attenuated by anti-TFPI antibodies. In the presence of erythrocyte-derived microparticles, activated protein C inhibited tenase and prothrombinase by degrading the cofactors FVIIIa and FVa, respectively. Protein S stimulated the Arg306-cleavage in FVa, whereas efficient inhibition of FVIIIa depended on the synergistic cofactor activity of protein S and FV. In summary, the erythrocyte-derived microparticle surface is suitable for the anticoagulant reactions of the protein C system, which may be important to balance the initiation and propagation of coagulation in vivo.

Protein S and factor V in regulation of coagulation on platelet microparticles by activated protein C.

INTRODUCTION: Platelets are the main source of microparticles in plasma and the concentration of microparticles is increased in many diseases. As microparticles expose negatively charged phospholipids, they can bind and assemble the procoagulant enzyme-cofactor complexes. Our aim was to elucidate possible regulation of these complexes on microparticles by the anticoagulant protein C system. MATERIALS AND METHODS: Platelets were activated with thrombin ± collagen or the calcium ionophore A23187 ± thrombin to generate microparticles. The microparticles were analyzed using flow cytometry and functional coagulation assays to characterize parameters with importance for the activated protein C system. RESULTS: Activation with A23187+thrombin was most efficient, fully converting the platelets to microparticle-like vesicles, characterized by high lactadherin and protein S binding capacity. Suppression of thrombin generation by activated protein C in plasma spiked with these microparticles was dependent on the presence of plasma protein S. Experiments with purified components showed that activated protein C inhibited both factor Va and factor VIIIa on the microparticle surface. Inhibition of factor Va was stimulated by, but not fully dependent on, the presence of protein S. In the factor VIIIa-degradation, activated protein C was dependent on the addition of protein S, and exogenous factor V further increased the efficiency. CONCLUSIONS: Protein S is crucial for activated protein C-mediated inhibition of thrombin generation on platelet-derived microparticles in plasma. Moreover, protein S and factor V are synergistic cofactors in the inhibition of factor VIIIa. The results demonstrate that the activated protein C system has the capacity to counterbalance the procoagulant ability of microparticles.