Analytical and Clinical Analysis of Two Automated Anti-SARS-CoV-2 Immunoassays in Pre-Pandemic and Pandemic Patient Populations.

BACKGROUND: In the absence of a safe, effective vaccine, the worldwide spread of COVID-19 (SARS-CoV-2) infection will continue. Laboratory tests with ideal precision, sensitivity, and specificity should be used in public health and clinical settings to gauge the extent of virus exposure. Toward this end, we evaluated the analytical and clinical performance of the Abbott SARS-CoV-2 IgG and the Roche Anti-SARS-CoV-2 immunoassays. METHODS: Quality control, pooled COVID-19, and non-COVID-19 patient specimens were used for the imprecision study. Two hundred and forty-six specimens from 70 patients with COVID-19 diagnosis were tested to study the sensitivity. Seventy-three non-COVID-19 control specimens were measured to study the specificity. All specimens were analyzed by both assays. RESULTS: Total analytic variability (CV) of the negative and positive controls were 5.5% and 3.6% for the Abbott assay and 4.5% and 1.9% for the Roche assay. Both assays demonstrated 100% qualitative reproducibility of negative and positive controls. The clinical specificities of the Abbott and the Roche assays were 100% (95% CI: 94%-100%) and 97% (95% CI: 90%-100%), respectively. The clinical sensitivities of the Abbott assay were 49% (95% CI: 41%-56%), 86% (95% CI: 74%-93%), and 100% (95% CI: 76%-100%) for samples collected at 0-6 days, 7-13 days, and ≥14 days after the first RT-PCR, while the sensitivities of the Roche assay were 55% (95% CI: 47%-62%), 86% (95% CI: 74%-93%), and 100% (95% CI: 76%-100%). CONCLUSIONS: This study demonstrates similar analytical and clinical performance of the Abbott and the Roche SARS-CoV-2 antibody assays, but the Roche assay may be slightly more sensitive for patients tested within 0-6 days after first positive RT-PCR of SARS-CoV-2.COVID-19 is a respiratory infectious disease caused by SARS-CoV-2. Laboratory tests with ideal precision, sensitivity, and specificity should be used in public health and clinical settings. We analyzed analytical and clinical performance of the Roche and Abbott SARS-CoV-2 antibody assays in pre-pandemic and pandemic patient populations. Additionally, we analyzed the sensitivity of both assays in patients at different stages of the disease. The 2 assays showed similar analytical and clinical performance, but the Roche assay may be slightly more sensitive for patients tested within 0-6 days after first positive RT-PCR of SARS-CoV-2. Our findings help other clinical labs select appropriate assays for SARS-CoV-2 antibody testing.

Characterization of methicillin-resistant Staphylococcus aureus displaying increased MICs of ceftaroline.

OBJECTIVES: To characterize the mechanisms responsible for elevated MICs of ceftaroline for methicillin-resistant Staphylococcus aureus (MRSA). METHODS: During the 2008 Assessing Worldwide Antimicrobial Resistance Evaluation ('AWARE') surveillance programme, four S. aureus collected from separate patients in Athens, Greece, demonstrated ceftaroline MICs of 4 mg/L. These isolates were clonally related and one strain (13101) was selected for further characterization. Two strains (4981 and 4977) displaying ceftaroline MICs of 1 and 2 mg/L, respectively, were included for comparison. All strains originated from the same hospital. Penicillin-binding protein (PBP) affinities for ceftaroline and comparators were determined. Strains were typed by single-locus typing (i.e. spa typing), multilocus sequence typing ('MLST') and by multiple-locus variable-number tandem repeat fingerprinting (MLVF). The presence of Pantone-Valentine leucocidin and the staphylococcal cassette chromosome mec types was assessed. We also performed nucleotide sequencing of the mecA (encoding PBP2a) promoter and ribosomal binding site (rbs) regions and mecR1. RESULTS: Ceftaroline demonstrated the highest PBP2a affinity with strain 4981 (ST5-MRSA-II) (IC(50) 0.06 mg/L; MIC 1 mg/L). Strains 4977 and 13101 (both ST239-MRSA-III) showed indistinguishable MLVF profiles. Ceftaroline PBP2a binding affinity in strains 4977 (IC(50) 0.25 mg/L; MIC 2 mg/L) and 13101 (IC(50) 1 mg/L; MIC 4 mg/L) was 4- and 16-fold lower than 4981, respectively. Strain 4981 contains a wild-type PBP2a, while strains 4977 and 13101 have N(146)K and E(150)K alterations in the non-penicillin-binding domain. Additionally, 13101 has one substitution (H(351)N) in the transpeptidase domain. Alterations in the mecR1, mecA promoter or rbs regions were not observed. CONCLUSIONS: Increased ceftaroline MICs were associated with decreased PBP2a binding affinity and reflected alterations in PBP2a.

In vitro activity of fusidic acid (CEM-102, sodium fusidate) against Staphylococcus aureus isolates from cystic fibrosis patients and its effect on the activities of tobramycin and amikacin against Pseudomonas aeruginosa and Burkholderia cepacia.

We tested the MICs of fusidic acid (CEM-102) plus other agents against 40 methicillin-resistant Staphylococcus aureus (MRSA) isolates from cystic fibrosis patients and the activities of fusidic acid with or without tobramycin or amikacin against Pseudomonas aeruginosa, MRSA, and Burkholderia cepacia isolates from cystic fibrosis patients in a 24-h time-kill study. Fusidic acid was potent (MICs, 0.125 to 0.5 µg/ml; a single 500-mg dose of fusidic acid at 8 h averaged 8 to 12. 5 µg/ml with 91 to 97% protein binding) against all MRSA strains. No antagonism was observed; synergy occurred for one MRSA strain treated with fusidic acid plus tobramycin.

Multistep resistance development studies of ceftaroline in gram-positive and -negative bacteria.

Ceftaroline, the active component of the prodrug ceftaroline fosamil, is a novel broad-spectrum cephalosporin with bactericidal activity against Gram-positive and -negative isolates. This study evaluated the potential for ceftaroline and comparator antibiotics to select for clones of Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, Moraxella catarrhalis, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis with elevated MICs. S. pneumoniae and S. pyogenes isolates in the present study were highly susceptible to ceftaroline (MIC range, 0.004 to 0.25 µg/ml). No streptococcal strains yielded ceftaroline clones with increased MICs (defined as an increase in MIC of >4-fold) after 50 daily passages. Ceftaroline MICs for H. influenzae and M. catarrhalis were 0.06 to 2 µg/ml for four strains and 8 µg/ml for a ß-lactamase-positive, efflux-positive H. influenzae with a mutation in L22. One H. influenzae clone with an increased ceftaroline MIC (quinolone-resistant, ß-lactamase-positive) was recovered after 20 days. The ceftaroline MIC for this isolate increased 16-fold, from 0.06 to 1 µg/ml. MICs for S. aureus ranged from 0.25 to 1 µg/ml. No S. aureus isolates tested with ceftaroline had clones with increased MIC (>4-fold) after 50 passages. Two E. faecalis isolates tested had ceftaroline MICs increased from 1 to 8 µg/ml after 38 days and from 4 to 32 µg/ml after 41 days, respectively. The parental ceftaroline MIC for the one K. pneumoniae extended-spectrum ß-lactamase-negative isolate tested was 0.5 µg/ml and did not change after 50 daily passages.

Molecular and epidemiologic characteristics of linezolid-resistant coagulase-negative staphylococci at a tertiary care hospital.

We investigated emergence of linezolid resistance among coagulase-negative staphylococci at our tertiary care center in 2007. All 17 cases were healthcare associated, and prior administration of linezolid was documented

In vitro activity of CEM-101 against Streptococcus pneumoniae and Streptococcus pyogenes with defined macrolide resistance mechanisms.

CEM-101 had MIC ranges of 0.002 to 0.016 microg/ml against macrolide-susceptible pneumococci and 0.004 to 1 microg/ml against macrolide-resistant phenotypes. Only 3 strains with erm(B), with or without mef(A), had CEM-101 MICs of 1 microg/ml, and 218/221 strains had CEM-101 MICs of 64 microg/ml, while 17/19 strains had telithromycin MICs of 4 to 16 microg/ml; CEM-101 MICs were 0.015 to 1 microg/ml. By comparison, erm(A) and mef(A) strains had CEM-101 MICs of 0.015 to 0.5 microg/ml, clindamycin and telithromycin MICs of 64 microg/ml. Pneumococcal multistep resistance studies showed that although CEM-101 yielded clones with higher MICs for all eight strains tested, seven of eight strains had clones with CEM-101 MICs that rose from 0.004 to 0.03 microg/ml (parental strains) to 0.06 to 0.5 microg/ml (resistant clones); for only one erm(B) mef(A) strain with a parental MIC of 1 microg/ml was there a resistant clone with a MIC of 32 microg/ml, with no detectable mutations in the L4, L22, or 23S rRNA sequence. Among two of five S. pyogenes strains tested, CEM-101 MICs rose from 0.03 to 0.25 microg/ml, and only for the one strain with erm(B) did CEM-101 MICs rise from 1 to 8 microg/ml, with no changes occurring in any macrolide resistance determinant. CEM-101 had low MICs as well as low potential for the selection of resistant mutants, independent of bacterial species or resistance phenotypes in pneumococci and S. pyogenes.

Comparative study of the mutant prevention concentrations of moxifloxacin, levofloxacin, and gemifloxacin against pneumococci.

We tested the propensity of three quinolones to select for resistant Streptococcus pneumoniae mutants by determining the mutant prevention concentration (MPC) against 100 clinical strains, some of which harbored mutations in type II topoisomerases. Compared with levofloxacin and gemifloxacin, moxifloxacin had the lowest number of strains with MPCs above the susceptibility breakpoint (P<0.001), thus representing a lower selective pressure for proliferation of resistant mutants. Only moxifloxacin gave a 50% MPC (MPC50) value (1 microg/ml) within the susceptible range.

Activity of telavancin against staphylococci and enterococci determined by MIC and resistance selection studies.

This study used CLSI broth microdilution to test the activity of telavancin and comparator antimicrobial agents against 67 methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates. Twenty-six vancomycin-intermediate S. aureus (VISA) strains were among the isolates tested; all strains were susceptible to telavancin at < or = 1 microg/ml, whereas 12/26 (46%) of these isolates were nonsusceptible to daptomycin at the same concentration. All strains were susceptible to quinupristin-dalfopristin, while resistance was found to all other drugs tested. Telavancin demonstrated potent activity against all vancomycin-susceptible isolates as well as against heterogeneously VISA and VISA resistance phenotypes. In multistep resistance selection studies, telavancin yielded one stable mutant after 43 days in one MRSA strain out of the 10 MRSA strains tested with the MIC rising eightfold from 0.25 microg/ml (parent) to 2 microg/ml. MICs for this clone did not increase further when passages were continued for the maximum 50 days. In contrast, daptomycin selected stable resistant clones (MIC increase of >4x) after 14 to 35 days in 4 of 10 MRSA strains with MICs increasing from 1 to 2 microg/ml (parents) to 4 to 8 microg/ml (resistant clones). Sequencing analysis of daptomycin resistance determinants revealed point mutations in the mprF genes of all four stable daptomycin-resistant clones. Teicoplanin gave rise to resistant clones after 14 to 21 days in 2 of 10 MRSA strains with MICs rising from 1 to 2 microg/ml (parents) to 4 to 16 microg/ml (stable resistant clones). Linezolid selected stable resistant clones after 22 to 48 days in 2 of 10 MRSA strains with MICs rising from 2 to 4 microg/ml (parents) to 32 microg/ml (resistant clones). Vancomycin yielded no resistant clones in 10 MRSA strains tested; however, MICs increased two- to fourfold from 1 to 8 microg/ml to 2 to 16 microg/ml after 50 days. No cross-resistance was found with any clone/antimicrobial combination. The two enterococci developed resistance to daptomycin, and one developed resistance to linezolid. Single-step mutation frequencies for telavancin (<4.0 x 10(-11) to <2.9 x 10(-10) at 2x MIC) were lower than the spontaneous mutation frequencies obtained with the comparators.

Resistance selection studies comparing the activity of razupenem (PTZ601) to vancomycin and linezolid against eight methicillin-resistant and two methicillin-susceptible Staphylococcus aureus strains.

Multistep and single-step resistance selection studies were performed with razupenem, linezolid, and vancomycin against 10 methicillin (meticillin)-resistant and -susceptible Staphylococcus aureus strains. After 20 daily subcultures, razupenem yielded only clones with MICs of >4 microg/ml in one strain (8 microg/ml) whose parent's MIC was already 4 microg/ml. After 18 to 49 passages in 6/10 strains, razupenem MICs rose from 0.016 to 2 microg/ml (parents) to 0.125 to 8 microg/ml (with clones stable after 10 drug-free subcultures). Single-step mutant selection frequencies were similarly low for razupenem and comparators.

Comparative antipneumococcal activities of sulopenem and other drugs.

For 297 penicillin-susceptible, -intermediate, and -resistant pneumococcal strains, the sulopenem MIC(50)s were 0.008, 0.06, and 0.25, respectively, and the sulopenem MIC(90)s were 0.016, 0.25, and 0.5 microg/ml, respectively. The MIC(50)s of amoxicillin for the corresponding strains were 0.03, 0.25, and 2.0 microg/ml, respectively, and the MIC(90)s were 0.03, 1.0, and 8.0 microg/ml, respectively. The combination of amoxicillin and clavulanate gave MICs similar to those obtained with amoxicillin alone. The sulopenem MICs were similar to those of imipenem and meropenem. The MICs of ss-lactams increased with those of penicillin G, and among the quinolones tested, moxifloxacin had the lowest MICs. Additionally, 45 strains of drug-resistant type 19A pneumococci were tested by agar dilution and gave sulopenem MIC(50)s and MIC(90)s of 1.0 and 2.0 microg/ml, respectively. Both sulopenem and amoxicillin (with and without clavulanate) were bactericidal against all 12 strains tested at 2x MIC after 24 h. Thirty-one strains from 10 countries with various penicillin, amoxicillin, and carbapenems MICs, including those with the highest sulopenem MICs, were selected for sequencing analysis of the pbp1a, pbp2x, and pbp2b regions encoding the transpeptidase active site and MurM. We did not find any correlations between specific penicillin-binding protein-MurM patterns and changes in the MICs.

Antistaphylococcal activity of dihydrophthalazine antifolates, a family of novel antibacterial drugs.

For a panel of 153 Staphylococcus aureus clinical isolates (including 13 vancomycin-intermediate or heterogeneous vancomycin-intermediate and 4 vancomycin-resistant strains), MIC(50)s and MIC(90)s of three novel dihydrophthalazine antifolates, BAL0030543, BAL0030544, and BAL0030545, were 0.03 and 0.25 microg/ml, respectively, for methicillin-susceptible strains and 0.03 and 128 microg/ml), although rates of endogenous resistance development were much lower for the dihydrophthalazines than for trimethoprim. Single-step platings of naïve staphylococci onto media containing dihydrophthalazine antifolates indicated considerable variability among strains with respect to preexistent subpopulations nonsusceptible to dihydrophthalazine antifolates.

Binding of faropenem and other beta-lactam agents to penicillin-binding proteins of pneumococci with various beta-lactam susceptibilities.

Faropenem demonstrated low MICs (< or = 1 microg/ml) for all penicillin-susceptible and nonsusceptible pneumococci and exhibited very strong abilities to bind to Streptococcus pneumoniae penicillin-binding proteins (PBPs), except for PBP2X. The lower faropenem affinity for PBP2X did not affect MICs for any strains tested, and only imipenem had lower MICs, with much lower binding affinities for all PBPs tested, than faropenem.

In vitro activity of the quinolone WCK 771 against recent U.S. hospital and community-acquired Staphylococcus aureus pathogens with various resistance types.

WCK 771 demonstrated MIC(50) and MIC(90)s of 0.03 and 1 microg/ml, respectively, against 297 recent U.S. community-acquired and hospital strains of Staphylococcus aureus, irrespective of quinolone or glycopeptide resistance. Against quinolone-resistant strains, MIC(90)s of WCK 771 and moxifloxacin were 1 and 16 microg/ml, respectively.

Incidence and characteristics of vancomycin nonsusceptible strains of methicillin-resistant Staphylococcus aureus at Hershey Medical Center.

All 982 methicillin-resistant Staphylococcus aureus strains collected from August 2006 to December 2007 were tested for vancomycin susceptibility by using 3-microg/ml vancomycin brain heart infusion screening plates, a vancomycin Etest, and a vancomycin/teicoplanin macro Etest. Three vancomycin-intermediate Staphylococcus aureus (VISA) (0.3%) and two heterogeneous VISA (0.2%) isolates were identified. The screening method yielded 895 cases of < or =1 colony and 87 positive results (with growth of >1 colony after 48 h); further Etests showed 82/87 isolates with growth on screening plates to be false positive. Repeat testing showed a false-positivity rate of only 15 of the original 87 isolates by plate screening.

In vitro activity of DC-159a, a new broad-spectrum fluoroquinolone, compared with that of other agents against drug-susceptible and -resistant pneumococci.

DC-159a yielded MICs of or=4 microg/ml). Although the MICs for DC-159a against quinolone-susceptible pneumococci were a few dilutions higher than those of gemifloxacin, the MICs of these two compounds against 28 quinolone-resistant pneumococci were identical. The DC-159a MICs against quinolone-resistant strains did not appear to depend on the number or the type of mutations in the quinolone resistance-determining region. DC-159a, as well as the other quinolones tested, was bactericidal after 24 h at 2x MIC against 11 of 12 strains tested. Two of the strains were additionally tested at 1 and 2 h, and DC-159a at 4x MIC showed significant killing as early as 2 h. Multistep resistance selection studies showed that even after 50 consecutive subcultures of 10 strains in the presence of sub-MICs, DC-159a produced only two mutants with maximum MICs of 1 microg/ml.

Antistaphylococcal activity of CG400549, a new experimental FabI inhibitor, compared with that of other agents.

Among 203 strains of Staphylococcus aureus, the MICs of CG400549 were 0.06 to 1.0 microg/ml, with MIC(50) and MIC(90) values of 0.25 microg/ml each. All strains were susceptible to linezolid and quinupristin-dalfopristin (MICs, 0.25 to 2.0 microg/ml). The daptomycin MICs were 0.25 to 2.0 microg/ml for methicillin-susceptible and 0.25 to 4.0 microg/ml against methicillin-resistant strains (including vancomycin-intermediate strains). Single-passage selection testing showed low resistance frequencies with CG400549, but multistep analysis showed that CG400549 yielded resistant mutants after 14 to 17 days in all strains tested.

Multistep resistance selection and postantibiotic-effect studies of the antipneumococcal activity of LBM415 compared to other agents.

LBM415 is a peptide deformylase inhibitor active against gram-positive bacterial species and some gram-negative species. In multiselection studies, LBM415 had low MICs against all Streptococcus pneumoniae strains tested, regardless of their genotype, and selected resistant clones after 14 to 50 days. MIC increases correlated with changes mostly in the 70GXGXAAXQ77 motif in peptide deformylase. The postantibiotic effect of LBM415 ranged from 0.3 to 1.4 h.

Activity of LBM415 compared to those of 11 other agents against Haemophilus species.

When tested against 254 Haemophilus influenzae strains, LBM415, a peptide deformylase inhibitor, gave MIC50 and MIC90 values of 2.0 microg/ml and 8.0 microg/ml, respectively. The MICs were independent of beta-lactam or quinolone susceptibility and the presence or absence of macrolide efflux or ribosomal protein mutations. The MICs of LBM415 against 23 H. parainfluenzae strains were similar to those against H. influenzae. In contrast, erythromycin, azithromycin, and clarithromycin gave unimodal MIC distributions, and apart from beta-lactamase-negative, ampicillin-resistant strains, all strains were susceptible to the beta-lactams tested. Apart from selected quinolone-resistant strains, all strains were susceptible to ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, and gemifloxacin. Resistance to trimethoprim-sulfamethoxazole was common. The potencies of all drugs against 23 H. parainfluenzae strains were similar to those against H. influenzae. Time-kill studies with 10 Haemophilus strains showed LBM415 to be bactericidal at 2 x the MIC against 8 of 10 strains after 24 h. For comparison, the macrolides and beta-lactams were bactericidal against 8 to 10 strains each at 2 x the MIC after 24 h. Quinolones were bactericidal against all 10 strains tested at 2 x the MIC after 24 h. Against six H. influenzae strains, postantibiotic effects for LBM415 lasted between 0.8 and 2.2 h. In multistep resistance selection studies, LBM415 produced resistant clones in 7 of the 10 strains tested, with MICs ranging from 4 to 64 microg/ml. No mutations in deformylase (def) and formyltransferase (fmt) genes were detected in any of the LBM415-resistant mutants.

Antipneumococcal activity of DW-224a, a new quinolone, compared to those of eight other agents.

DW-224a is a new broad-spectrum quinolone with excellent antipneumococcal activity. Agar dilution MIC was used to test the activity of DW-224a compared to those of penicillin, ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, gemifloxacin, amoxicillin-clavulanate, cefuroxime, and azithromycin against 353 quinolone-susceptible pneumococci. The MICs of 29 quinolone-resistant pneumococci with defined quinolone resistance mechanisms against seven quinolones and an efflux mechanism were also tested. DW-224a was the most potent quinolone against quinolone-susceptible pneumococci (MIC(50), 0.016 microg/ml; MIC(90), 0.03 microg/ml), followed by gemifloxacin, moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin. beta-Lactam MICs rose with those of penicillin G, and azithromycin resistance was seen mainly in strains with raised penicillin G MICs. Against the 29 quinolone-resistant strains, DW-224a had the lowest MICs (0.06 to 1 microg/ml) compared to those of gemifloxacin, clinafloxacin, moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin. DW-224a at 2x MIC was bactericidal after 24 h against eight of nine strains tested. Other quinolones gave similar kill kinetics relative to higher MICs. Serial passages of nine strains in the presence of sub-MIC concentrations of DW-224a, moxifloxacin, levofloxacin, ciprofloxacin, gatifloxacin, gemifloxacin, amoxicillin-clavulanate, cefuroxime, and azithromycin were performed. DW-224a yielded resistant clones similar to moxifloxacin and gemifloxacin but also yielded lower MICs. Azithromycin selected resistant clones in three of the five parents tested. Amoxicillin-clavulanate and cefuroxime did not yield resistant clones after 50 days.

Single- and multistep resistance selection studies on the activity of retapamulin compared to other agents against Staphylococcus aureus and Streptococcus pyogenes.

Retapamulin had the lowest rate of spontaneous mutations by single-step passaging and the lowest parent and selected mutant MICs by multistep passaging among all drugs tested for all Staphylococcus aureus strains and three Streptococcus pyogenes strains which yielded resistant clones. Retapamulin has a low potential for resistance selection in S. pyogenes, with a slow and gradual propensity for resistance development in S. aureus.