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.

CXCL13 in laboratory diagnosis of Lyme neuroborreliosis-the performance of the recomBead and ReaScan CXCL13 assays in human cerebrospinal fluid samples.

The chemokine CXCL13 is used as complement to serology in the diagnostics of Lyme neuroborreliosis (LNB). We evaluated and compared the semi-quantitative, cassette-based ReaScan CXCL13 assay with the quantitative recomBead CXCL13 assay using a collection of 209 cerebrospinal fluid samples. The categorical agreement between results interpreted as negative, grey zone, and positive by the two methods was 87%. The diagnostic sensitivity was higher using the recomBead assay, whereas specificity was higher using ReaScan. Few manual steps, and a short turn-around time with no batching of samples makes the ReaScan CXCL13 assay an attractive complement to serology in the diagnostics of LNB.

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.

Reconciling the Potentially Irreconcilable? Genotypic and Phenotypic Amoxicillin-Clavulanate Resistance in Escherichia coli.

Resistance to amoxicillin-clavulanate, a widely used beta-lactam/beta-lactamase inhibitor combination antibiotic, is rising globally, and yet susceptibility testing remains challenging. To test whether whole-genome sequencing (WGS) could provide a more reliable assessment of susceptibility than traditional methods, we predicted resistance from WGS for 976 Escherichia coli bloodstream infection isolates from Oxfordshire, United Kingdom, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines). A total of 339/976 (35%) isolates were amoxicillin-clavulanate resistant. Predictions based solely on beta-lactamase presence/absence performed poorly (sensitivity, 23% [78/339]) but improved when genetic features associated with penicillinase hyperproduction (e.g., promoter mutations and copy number estimates) were considered (sensitivity, 82% [277/339]; P < 0.0001). Most discrepancies occurred in isolates with MICs within ±1 doubling dilution of the breakpoint. We investigated two potential causes: the phenotypic reference and the binary resistant/susceptible classification. We performed reference standard, replicated phenotyping in a random stratified subsample of 261/976 (27%) isolates using agar dilution, following both EUCAST and CLSI guidelines, which use different clavulanate concentrations. As well as disagreeing with each other, neither agar dilution phenotype aligned perfectly with genetic features. A random-effects model investigating associations between genetic features and MICs showed that some genetic features had small, variable and additive effects, resulting in variable resistance classification. Using model fixed-effects to predict MICs for the non-agar dilution isolates, predicted MICs were in essential agreement (±1 doubling dilution) with observed (BD Phoenix) MICs for 691/715 (97%) isolates. This suggests amoxicillin-clavulanate resistance in E. coli is quantitative, rather than qualitative, explaining the poorly reproducible binary (resistant/susceptible) phenotypes and suboptimal concordance between different phenotypic methods and with WGS-based predictions.

Rapid antimicrobial susceptibility tests for sepsis; the road ahead.

Current methods for antimicrobial susceptibility testing (AST) are too slow to affect initial treatment decisions in the early stages of sepsis, when the prescriber is most concerned to select effective therapy immediately, rather than finding out what will not work 1 or 2 days later. There is a clear need for much faster differentiation between viral and bacterial infection, and AST, linked to earlier aetiological diagnosis, without sacrificing either the accuracy of quantitative AST or the low cost of qualitative AST. Truly rapid AST methods are eagerly awaited, and there are several candidate technologies that aim to improve the targeting of our limited stock of effective antimicrobial agents. However, none of these technologies are approaching the point of care and nor can they be described as truly culture-independent diagnostic tests. Rapid chemical and genomic methods of resistance detection are not yet reliable predictors of antimicrobial susceptibility and often rely on prior bacterial isolation. In order to resolve the trade-off between diagnostic confidence and therapeutic efficacy in increasingly antimicrobial-resistant sepsis, we propose a series of three linked decision milestones: initial clinical assessment (e.g. qSOFA score) within 10 min, initial laboratory tests and presumptive antimicrobial therapy within 1 h, and definitive AST with corresponding antimicrobial amendment within an 8 h window (i.e. the same working day). Truly rapid AST methods therefore must be integrated into the clinical laboratory workflow to ensure maximum impact on clinical outcomes of sepsis, and diagnostic and antimicrobial stewardship. The requisite series of development stages come with a substantial regulatory burden that hinders the translation of innovation into practice. The regulatory hurdles for the adoption of rapid AST technology emphasize technical accuracy, but progress will also rely on the effect rapid AST has on prescribing behaviour by physicians managing the care of patients with sepsis. Early adopters in well-equipped teaching centres in close proximity to large clinical laboratories are likely to be early beneficiaries of rapid AST, while simplified and lower-cost technology is needed to support poorly resourced hospitals in developing countries, with their higher burden of AMR. If we really want the clinical laboratory to deliver a specific, same-day diagnosis underpinned by definitive AST results, we are going to have to advocate more effectively for the clinical benefits of bacterial detection and susceptibility testing at critical decision points in the sepsis management pathway.