Validation of Fourier Transform Infrared Spectroscopy for Serotyping of Streptococcus pneumoniae.

Fourier transform infrared (FT-IR) spectroscopy (IR Biotyper; Bruker) allows highly discriminatory fingerprinting of closely related bacterial strains. In this study, FT-IR spectroscopy-based capsular typing of Streptococcus pneumoniae was validated as a rapid, cost-effective, and medium-throughput alternative to the classical phenotypic techniques. A training set of 233 strains was defined, comprising 34 different serotypes and including all 24 vaccine types (VTs) and 10 non-vaccine types (NVTs). The acquired spectra were used to (i) create a dendrogram where strains clustered together according to their serotypes and (ii) train an artificial neural network (ANN) model to predict unknown pneumococcal serotypes. During validation using 153 additional strains, we reached 98.0% accuracy for determining serotypes represented in the training set. Next, the performance of the IR Biotyper was assessed using 124 strains representing 59 non-training set serotypes. In this setting, 42 of 59 serotypes (71.1%) could be accurately categorized as being non-training set serotypes. Furthermore, it was observed that comparability of spectra was affected by the source of the Columbia medium used to grow the pneumococci and that this complicated the robustness and standardization potential of FT-IR spectroscopy. A rigorous laboratory workflow in combination with specific ANN models that account for environmental noise parameters can be applied to overcome this issue in the near future. The IR Biotyper has the potential to be used as a fast, cost-effective, and accurate phenotypic serotyping tool for S. pneumoniae.

Diagnostic performance of seven rapid IgG/IgM antibody tests and the Euroimmun IgA/IgG ELISA in COVID-19 patients.

OBJECTIVES: To evaluate the diagnostic performance of seven rapid IgG/IgM tests and the Euroimmun IgA/IgG ELISA for antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in COVID-19 patients. METHODS: Specificity was evaluated in 103 samples collected before January 2020. Sensitivity and time to seropositivity was evaluated in 167 samples from 94 patients with COVID-19 confirmed with RT-PCR on nasopharyngeal swab. RESULTS: Specificity (confidence interval) of lateral flow assays (LFAs) was ≥91.3% (84.0-95.5) for IgM, ≥90.3% (82.9-94.8) for IgG, and ≥85.4% (77.2-91.1) for the combination IgM OR IgG. Specificity of the ELISA was 96.1% (90.1-98.8) for IgG and only 73.8% (64.5-81.4) for IgA. Sensitivity 14-25 days after the onset of symptoms was between ≥92.1% (78.5-98.0) and 100% (95.7-100) for IgG LFA compared to 89.5% (75.3-96.4) for IgG ELISA. Positivity of IgM OR IgG for LFA resulted in a decrease in specificity compared to IgG alone without a gain in diagnostic performance, except for VivaDiag. The results for IgM varied significantly between the LFAs with an average overall agreement of only 70% compared to 89% for IgG. The average dynamic trend to seropositivity for IgM was not shorter than for IgG. At the time of hospital admission the sensitivity of LFA was <60%. CONCLUSIONS: Sensitivity for the detection of IgG antibodies 14-25 days after the onset of symptoms was ≥92.1% for all seven LFAs compared to 89.5% for the IgG ELISA. The results for IgM varied significantly, and including IgM antibodies in addition to IgG for the interpretation of LFAs did not improve the diagnostic performance.

How small modifications in laboratory workflow of blood cultures can have a significant impact on time to results.

Rapid identification and antimicrobial susceptibility testing of micro-organisms causing bloodstream infections is crucial in the management of septic patients. In this study, we compared a period of twice-daily and a period of thrice-daily reading of subculture agar plates. In 2016, 10,644 positive blood cultures bottles (bioMérieux) from 2608 patients were analyzed at UZ Leuven. Identification and antimicrobial susceptibility testing were performed by MALDI-TOF MS (Bruker Daltonics) and Vitek 2 (bioMérieux) respectively. In period 1 (January to June), subculture plates were read at 8:30 a.m. and 2:00 p.m. during the weekdays. In period 2 (August until December), reading was performed at 8:30 a.m., 2:00 p.m., and 5:00 p.m. Time to identification and time to AST results after positivity were compared. In period 1, median time to identification of all organisms was 22.8 h compared to 20.2 h in period 2 (p < 0.01). Moreover, micro-organisms were identified before 12 h in 9% of samples in period 2, a significant increase compared to 2% in period 1 (p < 0.01). In period 2, AST results were known within 36 h in 39% of samples, compared to 31% in period 1 (p < 0.01). Optimization of the reading frequency of subcultures of blood cultures significantly decreases time to results. Further optimization can be done by introducing lab automation. We will use the data of this study as a baseline to analyze the impact of introducing WASPLab (Copan Diagnostics) automation on time to results.

Compound A influences gene regulation of the Dexamethasone-activated glucocorticoid receptor by alternative cofactor recruitment.

The glucocorticoid receptor (GR) is a transcription factor of which the underlying gene regulatory mechanisms are complex and incompletely understood. The non-steroidal anti-inflammatory Compound A (CpdA), a selective GR modulating compound in various cell models, has been shown to favour GR-mediated gene repression but not GR-mediated gene activation. Shifting balances towards only a particular subset of GR gene regulatory events may be of benefit in the treatment of inflammatory diseases. We present evidence to support that the combination of CpdA with Dexamethasone (DEX), a classic steroidal GR ligand, can shape GR function towards a unique gene regulatory profile in a cell type-dependent manner. The molecular basis hereof is a changed GR phosphorylation status concomitant with a change in the GR cofactor recruitment profile. We subsequently identified and confirmed the orphan nuclear receptor SHP as a coregulator that is specifically enriched at GR when CpdA and DEX are combined. Combining CpdA with DEX not only leads to stronger suppression of pro-inflammatory gene expression, but also enhanced anti-inflammatory GR target gene expression in epithelial cells, making ligand combination strategies in future a potentially attractive alternative manner of skewing and fine-tuning GR effects towards an improved therapeutic benefit.

Added diagnostic value and impact on antimicrobial therapy of 16S rRNA PCR and amplicon sequencing on resected heart valves in infective endocarditis: a prospective cohort study.

OBJECTIVES: For adequate management and therapy of infective endocarditis (IE), identification of the causative pathogen is crucial but molecular testing results are not currently included in diagnostic criteria. The added diagnostic value and impact on antimicrobial therapy of 16S rRNA PCR and amplicon sequencing (16S rRNA PCR) performed on excised heart valves from patients with IE was evaluated alongside the effect of pre-operative antibiotics on the performance of blood culture (BC), valve culture (VC) and 16S rRNA PCR. METHODS: All patients undergoing valve surgery for definite or possible IE, according to modified Duke Criteria, were prospectively included from July 2013 up to and including June 2016. RESULTS: In all, 127 patients were included. Sensitivity for detecting the causative micro-organism in 120 post-operative definite IE patients was 26% for VC and 87% for BC and 16S rRNA PCR. 16S rRNA PCR, VC and BC were equally sensitive for different valve types and causative pathogens. In 27 (21%) definite IE patients, 16S rRNA PCR clarified discrepant culture results or was the only method identifying the causative pathogen. In 12 (10%) post-operative definite IE cases, molecular testing results influenced antimicrobial therapy. CONCLUSIONS: The very good performance characteristics, added diagnostic value and impact on antimicrobial therapy of molecular testing of heart valves should support the incorporation of molecular testing in diagnostic criteria and guidelines for IE.

Performance of three generations of Xpert MRSA in routine practice: approaching the aim?

The aim of this study was to evaluate retrospectively the performance of the Xpert MRSA assay in routine practice and its current use in the intensive care unit (ICU) setting of our hospital, since a pre-emptive isolation strategy has been applied. A total of 6473 patients were routinely screened with ESwab for methicillin-resistant Staphylococcus aureus (MRSA) using three generations of rapid real-time polymerase chain reaction (PCR) (Cepheid GeneXpert) over three consecutive periods of time. Performance was evaluated using broth enrichment culture as the reference method. Our results show that the last generation of Xpert MRSA (NxG) assay is more specific (99.2% vs. 97.9%) but not more sensitive (77.8% vs. 86.9%) than the third generation. Considering the low prevalence of MRSA in our hospital, we obtained an overall low positive predictive value. In conclusion, it remains difficult to abandon the reference method in routine practice considering the possible implications of an erroneous MRSA result in the ICU.

Broad-Range PCR Coupled with Electrospray Ionization Time of Flight Mass Spectrometry for Detection of Bacteremia and Fungemia in Patients with Neutropenic Fever.

Infection is an important complication in patients with hematologic malignancies or solid tumors undergoing intensive cytotoxic chemotherapy. In only 20 to 30% of the febrile neutropenic episodes, an infectious agent is detected by conventional cultures. In this prospective study, the performance of broad-range PCR coupled with electrospray ionization time of flight mass spectrometry (PCR/ESI-MS) technology was compared to conventional blood cultures (BC) in a consecutive series of samples from high-risk hematology patients. In 74 patients, BC and a whole-blood sample for PCR/ESI-MS (Iridica BAC BSI; Abbott, Carlsbad, CA, USA) were collected at the start of each febrile neutropenic episode and, in case of persistent fever, also at day 5. During 100 different febrile episodes, 105 blood samples were collected and analyzed by PCR/ESI-MS. There was evidence of a bloodstream infection (BSI) in 36/105 cases (34%), based on 14 cases with both PCR/ESI-MS and BC positivity, 17 cases with BC positivity only, and 5 cases with PCR/ESI-MS positivity only. The sensitivity of PCR/ESI-MS was 45%, specificity was 93%, and the negative predictive value was 80% compared to blood culture. PCR/ESI-MS detected definite pathogens (Fusobacterium nucleatum and Streptococcus pneumoniae) missed by BC, whereas it missed both Gram-negative and Gram-positive organisms detected by BC. PCR/ESI-MS testing detected additional microorganisms but showed a low sensitivity (45%) compared to BC in neutropenic patients. Our results indicate a lower concordance between BC and PCR/ESI-MS in the neutropenic population than what has been previously reported in other patient groups with normal white blood cell distribution, and a lower sensitivity than other PCR-based methods.

Development of a national EUCAST challenge panel for antimicrobial susceptibility testing.

A challenge panel of bacterial strains useful for clinical laboratories to validate their European Committee on Antimicrobial Susceptibility Testing (EUCAST) antimicrobial susceptibility test (AST) system was established. A total of 117 strains, obtained from Belgian Reference Centres (n = 57) and from routine clinical samples (n = 60) was selected based on resistance pattern. These strains were analysed in seven different laboratories by three different automated AST systems (Vitek (n = 2), Phoenix (n = 2) and Microscan (n = 2)) and by disc diffusion from five different manufacturers (Rosco (n = 2), Becton-Dickinson (n = 2), Biomérieux (n = 1), Bio-rad (n = 1) and i2a (n = 1)). To select the challenge panel, selection criteria were set for categorical agreement between the different systems and the number of very major errors, major errors and minor errors. Very major and major errors for at least two antibiotics were observed in 43% of all strains, leading to the exclusion of these strains from the selected panel. In only 10% of all tested strains was there 100% categorical agreement for all antibiotics. Finally, 28 strains (14 Gram-positive and 14 Gram-negative) covering a wide spectrum of resistance mechanisms were selected. Pilot-testing of this challenge panel in 20 laboratories mainly confirmed the results of the validation study. Only six strains withheld for the pilot study could not be used as challenge strain due to an overall (very) major error rate of >5% for a particular antibiotic (n = 5) or for two antibiotics (n = 1). To conclude, this challenge panel should facilitate the implementation and use of EUCAST breakpoints in laboratories.

Clinical and molecular abnormalities in lipoid proteinosis.

Lipoid proteinosis (hyalinosis cutis et mucosae) is a rare, autosomal recessive disease. The main clinicopathological features comprise skin and mucous membrane infiltration and scarring with deposition of hyaline material. In this report, we describe a 6-year-old boy in whom a diagnosis of lipoid proteinosis was first suspected when he presented with blisters and erosions at 4 years, a history of life-long dysphonia and a previous epileptic convulsion. The diagnosis was confirmed by histology and identification of a homozygous frameshift mutation, 501insC, in exon 6 of the gene encoding extracellular matrix protein 1, ECM1. Lipoid proteinosis may show protean clinical features and be difficult to diagnose on clinical grounds alone. This case report illustrates that lipoid proteinosis may show protean clinical features and yet remain undiagnosed for many years. Although the gold standard for definite diagnosis remains histology, molecular characterisation of the gene mutation will add to our understanding of genotype-phenotype correlation and perhaps to the development of a rationale for future therapeutics.

Expression of the inducible isoform of nitric oxide synthase in the retinas of human subjects with diabetes mellitus.

PURPOSE: Inducible nitric oxide synthase has been implicated in the pathogenesis of cerebral ischemic damage, in the angiogenic process and in diabetic vascular damage. This study was undertaken to determine whether inducible nitric oxide synthase is present in the retinas from human subjects with diabetes mellitus. METHODS: This was an experimental immunohistochemical prospective study. Ten postmortem eyes from five subjects with diabetes mellitus, 10 eyes from five subjects without diabetes and without known ocular disease, and two eyes from one subject with unilateral ocular ischemic syndrome secondary to severe carotid artery obstruction were examined. We used immunohistochemical techniques and antibodies directed against inducible nitric oxide synthase, glial fibrillary acidic protein, and vimentin. The main outcome measure was immunoreactivity for these antibodies. RESULTS: Immunoreactivity for inducible nitric oxide synthase was not observed in retinas from all subjects without diabetes and without ocular disease. Six retinas from three subjects with diabetes and nonproliferative retinopathy, and the retina from the eye with ocular ischemic syndrome showed immunoreactivity for inducible nitric oxide synthase in cells with elongated processes. Based on morphology and on glial fibrillary acidic protein and vimentin immunoreactivity, this inducible nitric oxide synthase immunoreactivity appeared to localize to retinal Müller glial cells. CONCLUSIONS: These observations suggest that Müller cells may be involved in the microvascular remodeling of the diseased retina and that high concentrations of nitric oxide produced by inducible nitric oxide synthase could contribute to neurotoxicity and angiogenesis that occur in diabetic retinopathy.