Matthijs Oyaert

Comparison of two immunoassays for measurement of faecal calprotectin in detection of inflammatory bowel disease: (pre)-analytical and diagnostic performance characteristics

Abstract Background: Symptoms of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) can overlap. Faecal calprotectin has recently been established to be a non-invasive marker for neutrophilic intestinal inflammation. We compared two devices for extraction of faecal calprotectin. Based on these results, two immunoassays for measurement of faecal calprotectin were evaluated. Methods: Samples were extracted using the Thermo Fisher extraction device (Thermo Fisher Scientific) and Smart Pep extraction device (Roche Diagnostics) and measured with the EliA Calprotectin immunoassay (Thermo Fisher Scientific) on ImmunoCAP 250. The performance of both assays was investigated by enrolling 183 consecutive patients (79 males, 104 females; median age 32 years) with clinical suspicion of IBD. Faecal calprotectin was measured using a recently launched immunoassay, EliA Calprotectin in comparison with an established immunochomatographic point-of-care-test (POCT, Quantum Blue Calprotectin; Bühlmann). Results were compared with endoscopic and histological findings. Results: The use of the Thermo Fisher extraction device resulted in an underestimation of faecal calprotectin concentrations, especially in liquid stool samples. IBD was diagnosed in 51/183 patients (27.9%) [Crohn’s disease (CD, n=37), ulcerative colitis (UC, n=14)]. After adjusting the optimal cut-off for detection of IBD using receiver operating curve analysis, a sensitivity of 94.1% and 90.2% and specificity of 87.9% and 90.9% for the EliA and POCT assay, respectively, were obtained. Conclusions: The Thermo Fisher device is not reliable for extraction of faecal calprotectin. The performance characteristics of the EliA Calprotectin assay are statistically equivalent to the Bühlmann POCT.

Atopobium deltae sp. nov., isolated from the blood of a patient with Fournier's gangrene

A Gram-stain-positive, obligately anaerobic, short rod, designated strain HHRM1715(T), was isolated from the blood of a patient with Fourniers gangrene, complicated by sepsis. On the basis of 16S rRNA gene sequence analysis, strain HHRM1715(T) was shown to belong to the genus Atopobium and was most closely related to Atopobium minutum (95 % similarity). The results of 16S rRNA-gene-based phylogenetic analysis, cellular fatty acid analysis and differential biochemical tests, showed that strain HHRM1715(T) represented a novel species of the genus Atopobium. We therefore describe Atopobium deltae sp. nov. with HHRM1715(T) ( = LMG 27987(T) = CCUG 65171(T)) as the type strain and propose an emended description of the genus Atopobium with regard to the DNA G+C content.

First Case of Pseudoclavibacter bifida Bacteremia in an Immunocompromised Host with Chronic Obstructive Pulmonary Disease (COPD)

ABSTRACT Pseudoclavibacter spp. are Gram-positive, aerobic, catalase-positive, coryneform bacteria belonging to the family of Microbacteriaceae. Identification of these species with conventional biochemical assays is difficult. This case report of a Pseudoclavibacter bifida bacteremia occurring in an immunocompromised host diagnosed with an acute exacerbation of chronic obstructive pulmonary disease, with a lethal outcome, confirms that this organism may be a human pathogen.

Sepsis with an Atopobium-Like Species in a Patient with Fournier's Gangrene

ABSTRACT Atopobium species are Gram-positive, anaerobic, catalase-negative, fastidious bacteria belonging to the family Coriobacteriaceae. We report the isolation of an Atopobium-like species in a patient with Fourniers gangrene and highlight the role of 16S rRNA gene sequencing in the identification of fastidious organisms in the clinical laboratory.

Analytical performance and diagnostic accuracy of six different faecal calprotectin assays in inflammatory bowel disease

Abstract Background: We evaluated the analytical performance of six different faecal calprotectin immunoassays together with their diagnostic accuracy in the discrimination between functional and organic bowel disorders. Methods: The faecal samples were obtained from inflammatory bowel disease patients (n=27) at the time of diagnosis [Crohn’s disease (n=15), colitis ulcerosa (n=12)], gastroenterologic disease control patients (n=52) and rheumatologic disease control patients (n=26). All individuals included in the study underwent a concurrent ileocolonoscopy. Analytical performance (imprecision, accuracy, carry-over, correlation and agreement) and diagnostic accuracy (sensitivity, specificity, likelihood ratios) of the different assays were evaluated. Results: All methods demonstrated good analytical performance, but within-run and total imprecision varied depending on the assay methodology used. Using Passing Bablok and Bland-Altman analyses, low quantitative agreement was observed between the assays. All assays showed excellent diagnostic accuracy, with areas under the receiver operating characteristic curves (ROC) ranging from 0.974 to 0.998. The AUCs were not significantly different between assays (p>0.05). Diagnostic sensitivity at the cut-off at a fixed specificity of 75% ranged from 95.2% to 100%. Introduction of multiple result intervals increased the clinical interpretation of all the assays. Conclusions: Analytical and diagnostic performance of the evaluated faecal calprotectin assays is good, but numerical values differ substantially between the assays necessitating the use of different clinical cut-offs. Introduction of multiple result intervals aids in clinical decision-making.

Combining antibody tests and taking into account antibody levels improves serologic diagnosis of celiac disease

Abstract Background: The European Society for Pediatric Gastroenterology and Nutrition states that if IgA anti-tissue transglutaminase (tTG) exceeds 10 times the upper limit of normal (ULN), there is the possibility to diagnose celiac disease (CD) without duodenal biopsy, if supported by anti-endomysium testing and human leukocyte antigen (HLA) typing. We aimed to evaluate whether combining IgA tTG and IgG anti-deamidated gliadin peptide (DGP) antibody testing and taking into account the antibody levels improves clinical interpretation. Methods: We calculated likelihood ratios for various test result combinations using data obtained from newly diagnosed CD patients (n=156) [13 children <2 years, 45 children between 2 and 16 years, and 98 adults (>16 years)] and 974 disease controls. All patients and controls underwent duodenal biopsy. IgA anti-tTG and IgG anti-DGP assays were from Thermo Fisher and Inova. Results: Likelihood ratios for CD markedly increased with double positivity and increasing antibody levels of IgA anti-tTG and IgG anti-DGP. Patients with double positivity and high antibody levels (>3 times, >10 times ULN) had a high probability for having CD (likelihood ratio ≥649 for >3 times ULN and ∞ for >10 times ULN). The fraction of CD patients with double positivity and high antibody levels was 59%–67% (depending on the assay) for >3 ULN and 33%–36% (depending on the assay) for >10 ULN, respectively. This fraction was significantly higher in children with CD than in adults. Conclusions: Combining IgG anti-DGP with IgA anti-tTG and defining thresholds for antibody levels improves the serologic diagnosis of CD.

Laboratory diagnosis of urinary tract infections: Towards a BILULU consensus guideline

Urinary tract infections (UTI) are very common throughout life and account for the majority of the workload in the clinical microbiology laboratory. Clear instructions for the interpretation of urine cultures by the laboratory technicians are indispensable to obtain standardized, reliable, and clinically useful results. In literature, there is often a lack of evidence-based practice in processing urinary samples in the laboratory. In this consensus document, the BILULU Study Group presents a practical approach for the implementation of existing guidelines for the culture of urine in the microbiology laboratory and offers answers for issues where no clear solution is available in the guidelines.

Quantitative urine test strip reading for leukocyte esterase and hemoglobin peroxidase

Abstract Background: Recently, urine test strip readers have become available for automated test strip analysis. We explored the possibilities of the Sysmex UC-3500 automated urine chemistry analyzer based on complementary metal oxide semiconductor (CMOS) sensor technology with regard to accuracy of leukocyte esterase and hemoglobin peroxidase results. We studied the influence of possible confounders on these measurements. Methods: Reflectance data of leukocyte esterase and hemoglobin peroxidase were measured using CMOS technology on the Sysmex UC-3500 automated urine chemistry analyzer. Analytical performance (imprecision, LOQ) as well as the correlation with white blood cell (WBC) and red blood cell (RBC) counts (Sysmex UF-5000) were studied. Furthermore, the influence of urinary dilution, haptoglobin, pH and ascorbic acid as confounders was determined. Results: Within- and between-run imprecision (reflectance signal) ranged from 1.1% to 3.6% and 0.9% to 4.2% for peroxidase and 0.4% to 2.5% and 0.4% to 3.3% for leukocyte esterase. Good agreement was obtained between the UF-5000 for RBCs and peroxidase reflectance (r=0.843) and for WBCs and leukocyte esterase (r=0.821). Specific esterase activity decreased for WBC counts exceeding 100 cells/μL. Haptoglobin influenced the peroxidase activity, whereas leukocyte esterase and peroxidase activities showed a pH optimum between 5.0 and 6.5. A sigmoidal correlation was observed between urinary osmolality and peroxidase activity. Conclusions: CMOS technology allows to obtain high quality test strip results for assessing WBC and RBC in urine. Quantitative peroxidase and leukocyte esterase are complementary with flow cytometry and have an added value in urinalysis, which may form a basis for expert system development.

Analytical and pre-analytical performance characteristics of a novel cartridge-type blood gas analyzer for point-of-care and laboratory testing

BACKGROUND Point-of-care blood gas test results may benefit therapeutic decision making by their immediate impact on patient care. We evaluated the (pre-)analytical performance of a novel cartridge-type blood gas analyzer, the GEM Premier 5000 (Werfen), for the determination of pH, partial carbon dioxide pressure (pCO2), partial oxygen pressure (pO2), sodium (Na+), potassium (K+), chloride (Cl-), ionized calcium (iCa2+), glucose, lactate, and total hemoglobin (tHb). METHODS Total imprecision was estimated according to the CLSI EP5-A2 protocol. The estimated total error was calculated based on the mean of the range claimed by the manufacturer. Based on the CLSI EP9-A2 evaluation protocol, a method comparison with the Siemens RapidPoint 500 and Abbott i-STAT CG8+ was performed. Obtained data were compared against preset quality specifications. Interference of potential pre-analytical confounders on co-oximetry and electrolyte concentrations were studied. RESULTS The analytical performance was acceptable for all parameters tested. Method comparison demonstrated good agreement to the RapidPoint 500 and i-STAT CG8+, except for some parameters (RapidPoint 500: pCO2, K+, lactate and tHb; i-STAT CG8+: pO2, Na+, iCa2+ and tHb) for which significant differences between analyzers were recorded. No interference of lipemia or methylene blue on CO-oximetry results was found. On the contrary, significant interference for benzalkonium and hemolysis on electrolyte measurements were found, for which the user is notified by an interferent specific flag. CONCLUSION Identification of sample errors from pre-analytical sources, such as interferences and automatic corrective actions, along with the analytical performance, ease of use and low maintenance time of the instrument, makes the evaluated instrument a suitable blood gas analyzer for both POCT and laboratory use.

Factors impacting unbound vancomycin concentrations in neonates and young infants

Vancomycin pharmacokinetic (PK) and pharmacodynamic (PD) data in neonates are based on total concentrations. However, only unbound vancomycin is pharmacologically active. The objective was to determine vancomycin protein binding and the covariates impacting unbound vancomycin concentration in neonates and young infants. In neonates and young infants to whom vancomycin was administered intermittently for medical indications, total and unbound vancomycin plasma concentrations were determined using LC-MS/MS. Sampling occurred randomly during vancomycin exposure, covering a broad range of concentrations. Impact of covariates on unbound vancomycin concentration was determined using linear regression. Significant results of the univariate regressions were entered in a stepwise multiple regression. Passing-Bablok regression and Bland-Altman were used to assess the difference between measured and calculated unbound vancomycin concentration. Thirty-seven samples in 33 patients (median (interquartile range) gestational age 35 (29–39) weeks) were collected. Median total and unbound vancomycin concentrations were 14.2 (7.4–20.6) and 13.6 (7.2–22.5) mg/L, respectively. Median unbound fraction was 0.90 (0.77–0.98). Multiple regression revealed total vancomycin concentration (β = 0.884, p < 0.001) and albumin (β = − 0.323, p = 0.007) as most important covariates of unbound vancomycin concentrations, with an R2 adjusted of 0.953 (p < 0.0001). Mean absolute difference between calculated and measured unbound vancomycin was − 0.008 (95% CI − 0.92–0.91) mg/L. The unbound vancomycin fraction in neonates is higher compared to that in children and adults, and total vancomycin concentration and albumin were the most important covariates of unbound vancomycin concentration. Integration of protein binding in future PK/PD analyses is appropriate to optimize vancomycin dosing and to determine population-specific vancomycin PD targets for neonates.