Multicenter Evaluation of the Clinical Performance and the Neutralizing Antibody Activity Prediction Properties of 10 High-Throughput Serological Assays Used in Clinical Laboratories.

As the coronavirus disease 2019 (COVID-19) pandemic second wave is emerging, it is of the upmost importance to screen the population immunity in order to keep track of infected individuals. Consequently, immunoassays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and positive predictive values are needed to obtain an accurate epidemiological picture. As more data accumulate about the immune responses and the kinetics of neutralizing-antibody (nAb) production in SARS-CoV-2-infected individuals, new applications are forecast for serological assays such as nAb activity prediction in convalescent-phase plasma from recovered patients. This multicenter study, involving six hospital centers, determined the baseline clinical performances, reproducibility, and nAb level correlations of 10 commercially available immunoassays. In addition, three lateral-flow chromatography assays were evaluated, as these devices can be used in logistically challenged areas. All assays were evaluated using the same patient panels in duplicate, thus enabling accurate comparison of the tests. Seven immunoassays examined in this study were shown to have excellent specificity (98 to 100%) and good to excellent positive predictive values (82 to 100%) when used in a low (5%)-seroprevalence setting. We observed sensitivities as low as 74% and as high as 95% at ≥15 days after symptom onset. The determination of optimized cutoff values through receiver operating characteristic (ROC) curve analyses had a significant impact on the diagnostic resolution of several enzyme immunoassays by increasing the sensitivity significantly without a large trade-off in specificity. We found that spike-based immunoassays seem to be better correlates of nAb activity. Finally, the results reported here will add to the general knowledge of the interlaboratory reproducibility of clinical performance parameters of immunoassays and provide new evidence about nAb activity prediction.

Cathepsins X and B display distinct activity profiles that can be exploited for inhibitor design.

The carboxypeptidase and endopeptidase activities of cathepsins X and B, as well as their inhibition by E-64 derivatives, have been investigated in detail and compared. The results clearly demonstrate that cathepsins X and B do not share similar activity profiles against substrates and inhibitors. Using quenched fluorogenic substrates, we show that cathepsin X preferentially cleaves substrates through a monopeptidyl carboxypeptidase pathway, while cathepsin B displays a preference for the dipeptidyl pathway. The preference for one or the other pathway is about the same for both enzymes, i. e. approximately 2 orders of magnitude. Cleavage of a C-terminal dipeptide of a substrate by cathepsin X can be observed under conditions that preclude efficient monopeptidyl carboxypeptidase activity. In addition, an inhibitor designed to exploit the unique structural features responsible for the carboxypeptidase activity of cathepsin X has been synthesized and tested against cathepsins X, B and L. Although of moderate potency, this E-64 derivative is the first reported example of a cathepsin X-specific inhibitor. By comparison, CA074 was found to inactivate cathepsin B at least 34000-fold more efficiently than cathepsin X.

Cathepsins X and B can be differentiated through their respective mono- and dipeptidyl carboxypeptidase activities.

Several new cysteine proteases of the papain family have been discovered in the past few years. To help in the assignment of physiological roles and in the design of specific inhibitors, a clear picture of the specificities of these enzymes is needed. One of these novel enzymes, cathepsin X, displays a unique specificity, cleaving single amino acid residues at the C-terminus of substrates very efficiently. In this study, the carboxypeptidase activities and substrate specificity of cathepsins X and B have been investigated in detail and compared. Using quenched fluorogenic substrates and HPLC measurements, it was shown that cathepsin X preferentially cleaves substrates through a monopeptidyl carboxypeptidase pathway, while cathepsin B displays a preference for the dipeptidyl pathway. The preference for one or the other pathway is about the same for both enzymes, i.e., approximately 2 orders of magnitude, a result supported by molecular modeling of enzyme-substrate complexes. Cleavage of a C-terminal dipeptide of a substrate by cathepsin X can become more important under conditions that preclude efficient monopeptidyl carboxypeptidase activity, e.g., nonoptimal interactions in subsites S(2)-S(1). These results confirm that cathepsin X is designed to function as a monopeptidyl carboxypeptidase. Contrary to a recent report [Klemencic, I., et al. (2000) Eur. J. Biochem. 267, 5404-5412], it is shown that cathepsins X and B do not share similar activity profiles, and that reagents are available to clearly distinguish the two enzymes. In particular, CA074 was found to inactivate cathepsin B at least 34000-fold more efficiently than cathepsin X. The insights obtained from this and previous studies have been used to produce an inhibitor designed to exploit the unique structural features responsible for the carboxypeptidase activity of cathepsin X. Although of moderate potency, this E-64 derivative is the first reported example of a cathepsin X-specific inhibitor.

Molecular basis of antibiotic resistance and beta-lactamase inhibition by mechanism-based inactivators: perspectives and future directions.

Antibacterial chemotherapy is particularly striking in the family of penicillins and cephalosporins. Over 40 structurally different beta-lactam molecules are available in 73 formulations and the majority of them are currently prescribed for medical use in hospitals. beta-Lactams are well tolerated by humans with few side effects. They interact very specifically with their bacterial target, the D-alanyl-D-alanine carboxypeptidase-transpeptidase usually referred to as DD-peptidase. The outstanding number of beta-lactamases produced by bacteria represent a serious threat to the clinical utility of beta-lactams. The discovery of beta-lactamase inhibitors was thought to solve, in part, the problem of resistance. Unfortunately, bacteria have evolved new mechanisms of resistance to overcome the inhibitory effects of beta-lactamase inactivators. Here, we summarize the diversified mechanistic features of class A beta-lactamases interactions with mechanism-based inhibitors using available microbiological, kinetic and structural data for the prototype TEM beta-lactamases. A brief historical overview of the strategies developed to counteract beta-lactamases will be presented followed by a short description of the chemical events which lead to the inactivation of TEM beta-lactamase by inhibitors from different classes. Finally, an update on the clinical prevalence of natural and inhibitor-resistant enzyme mutants, the total chemical synthesis to design and synthesize a new structure and produced a broad spectrum beta-lactamase inhibitor that mimics the beta-lactam ring, but does not contain it is discussed.

Evaluation of inhibition of the carbenicillin-hydrolyzing beta-lactamase PSE-4 by the clinically used mechanism-based inhibitors.

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing beta-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k(cat)/k(inact)) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 beta-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.

Beta-lactamases and outer membrane investigations in beta-lactam-resistant Comamonas acidovorans strains.

Imipenem-induced beta-lactamase (level of expression, specific activity and kinetic parameters (Vmax and Km) in response to nitrocefin) and outer membrane proteins (OMPs) (hydrophobicity, permeability and electrophoretic pattern) were characterized in, one beta-lactam sensitive (PAC-9), one resistant (PAC-1) and two resistant laboratory mutants (PAC-9M, PAC-9M2) of Comamonas acidovorans strains. Beta-lactamases from both mutant strains showed different Vmax values compared to the parental strains. Beta-lactam resistance was found to be associated in PAC-1 with inducible beta-lactamase production and OMP alteration by the appearance of a 102-KDa protein. Moreover, PAC-1 was less permeable to nitrocefin than PAC-9. These data indicate that C. acidovorans resistance to beta-lactam resulted from synergy between beta-lactamase and OMP alterations.

Roles of amino acids 161 to 179 in the PSE-4 omega loop in substrate specificity and in resistance to ceftazidime.

The PSE-4 enzyme is a prototype carbenicillin-hydrolyzing enzyme exhibiting high activity against penicillins and early cephalosporins. To understand the mechanism that modulates substrate profiles and to verify the ability of PSE-4 to extend its substrate specificity toward expanded-spectrum cephalosporins, we used random replacement mutagenesis to generate six random libraries from amino acids 162 to 179 in the Omega loop. This region is known from studies with TEM-1 to be implicated in substrate specificity. It was found that the mechanism modulating ceftazidime hydrolysis in PSE-4 was different from that in TEM-1. The specificity of class 2c carbenicillin-hydrolyzing enzymes could not be assigned to the Omega loop of PSE-4. Analysis of the percentage of functional enzymes revealed that the hydrolysis of ampicillin was more affected than hydrolysis of carbenicillin by amino acid substitutions at positions 162 to 164 and 165 to 167.

Comparative in vitro activity of nine antistaphylococcal agents against 275 recent isolates of Gram-positive cocci.

The aim of this study was to compare the in vitro activities of 9 antistaphylococcal agents including teicoplanin (TEI) against 275 non-repetitive clinical strains representing 15 species of staphylococci and 27 strains of Enterococcus (E.) faecalis, isolated from various specimens between 1991-1992 at a Canadian teaching hospital. The NCCLS agar dilution method was used (10(4) colonyforming units/spot). In terms of MIC(90), TEI and vancomycin (VAN) appeared to be the most potent antibiotics against all staphylococci tested (TEI: 2.0-4.0 mug/ml; VAN: 1.0-2.0 mug/ml; ciprofloxacin (CPF): 0.25-32 mug/ml; cefazolin (CEF): 8.0-256 mug/ml; methicillin (MET): 2.0->256 mug/ml; imipenem (IMP): 1.0-32 mug/ml; erythromycin (ERT): 16->256 mug/ml; ampicillin (AMP): 16-128 mug/ml; fusidic acid (FSA): 0.5-16 mug/ml). Multiple resistant strains, including MET-resistant Staphylococcus (Staph.) aureus and Staph. epidermidis, were susceptible to TEI and VAN with respective MICs of 2-4 mug/ml and 1-2 mug/ml regardless of specimen type. Moreover, TEI was highly active against E. faecalis (MIC(90) for TEI and VAN: 0.5 and 4.0 mug/ml, respectively).

Properties of separable covariance matrices and their associated gaussian random processes.

A number of properties of separable covariance matrices are summarized. Expressions for the divergence of the corresponding two-dimensional Gaussian random processes are given in terms of row and column covariance matrices, and in terms of linear prediction parameters and maximum likelihood spectral estimates. Such time and frequency domain expressions are not widely known, even for one-dimensional random processes.

Isolation of haploid clones bearing a mating allele from a diploid apomictic isolate of Didymium iridis.

Clones of myxamoebae which fail to undergo a direct morphogenetic change into plasmodia have been isolated from cultures of the Philippine-1 isolates of Didymium iridis. These clones have been maintained continuously for a period of more than 4 years without producing plasmodia. It has been demonstrated by Feulgen-DNA cytophotometry that the mean nuclear DNA content of such clones is equivalent to the haploid replicated (2C) value. Conversely, myxamoebae which produce plasmodia directly have a mean nuclear DNA content which is equivalent to the diploid replicated (4C) value. The haploid Philippine-1 clones have been demonstrated to be cross compatible with isolates bearing the mating alleles A1, and A3 through A11, and incompatible with isolates bearing the mating allele A2. Furthermore, plasmodia which result from the crosses Ph-1n (A2) Hon 1-2 (A1), (A1), Ph-1n (A2) x Pan 2-4 (A7) and Ph-1n (A2) x Pan 2-7 (A8) have mean nuclear DNA values which are equivalent to the replicated diploid value, and produce spores which bear the mating alleles in the predicted ratios. Such data demonstrate that the Ph-1n clones are sexually compatible with haploid myxamoebae of heterothallic isolates. The significance of the isolation of these haploid clones with regard to a recent hypothesis related to the evolution of developmental systems in the Myxomycetes is discussed.