Lieve Van Hoovels

First Case of Staphylococcus pseudintermedius Infection in a Human

ABSTRACT We present the first clinical report of a Staphylococcus pseudintermedius infection in a human. Biochemically, S. pseudintermedius can be easily misidentified as S. aureus. Therefore, the final microbiological identification requires the combination of phenotypic and genotypic tests.

Genetic Characterization of a Novel, Naturally Occurring Recombinant Human G6P[6] Rotavirus

ABSTRACT A binary classification system has been established for group A rotaviruses, with the viral capsid protein VP7 defining G types and VP4 defining P types. At least 15 G types and 21 P types have been isolated globally with various G and P combinations. Most of the currently circulating human rotaviruses belong to G1P[8], G2P[4], G3P[8], and G4P[8]. We report a human rotavirus strain (B1711) with a novel genotypic VP7/VP4 combination of G6P[6]. This unique rotavirus was isolated from a 13-month-old human immunodeficiency virus (HIV)- negative child of an HIV-seropositive Malian mother that was hospitalized with severe diarrhea in Belgium after returning from a trip to Mali. The VP7 and VP4 genes of the rotavirus strain were sequenced, and phylogenetic trees were constructed. Nucleotide and amino acid sequence comparisons with 15 known G genotypes indicated that the VP7 sequence of strain B1711 was most closely related to an American (Se584) and an Italian (PA151) human G6 strain (95 to 96% nucleotide and 98% amino acid identity). Comparison of the VP4 sequence with 21 P types showed the closest similarity to P[6] genotypes, with greatest similarity to a G8P[6] Malawi strain (mw131) (97% nucleotide and 98% amino acid identity). The B1711 strain is the first reported rotavirus isolate with a G6P[6] genotypic combination. The discovery and surveillance of novel human and nonhuman rotavirus G or P types or of novel G/P combinations is essential for the design of future rotavirus vaccines and for our understanding of rotavirus diversity and evolution.

The importance of detecting anti-DFS70 in routine clinical practice: comparison of different care settings

Abstract Background: Screening for antinuclear antibodies by indirect immunofluorescence (ANA-IIF) is essential in the diagnostic workup of ANA-associated autoimmune rheumatic diseases (AARDs). However, also healthy individuals may test positive, making the interpretation challenging. Recent reports suggest that dense fine speckled 70 antibodies (anti-DFS70) may facilitate this challenge. Here, we investigate their clinical importance based on data from four Belgian laboratories (one primary, two secondary and one tertiary care). Methods: At least one specific DFS70 assay (DFS70 IgG ELISA or lineblot [Euroimmun, full length antigen] and/or DFS70 IgG CLIA [Inova Diagnostics, truncated antigen]) was performed on four consecutive cohorts of homogeneous-like ANA-IIF samples (n=697). Co-occurrence with AARD-specific ANA and clinical information were documented in the anti-DFS70-positive samples. Results: Using a combination of solid phase techniques, we found between 7.6% and 26% anti-DFS70 in the different cohorts. Focusing on anti-DFS70 CLIA-positive samples without co-occurrence of AARD-specific ANA, we observed a trend towards lower frequency in tertiary (8% [p=0.0786]) and secondary care (12% [p=0.1275] and 6% [p<0.001]) compared to primary care (21%). Moreover, in this specific subpopulation, AARD was less frequent (0%–50% compared to 6%–77% in the total anti-DFS70-positive group). Conclusions: Anti-DFS70 prevalence depends on the applied assay and care setting. Our data suggest that, for an ANA-IIF-positive patient, it is rather the absence of AARD-associated ANA and clinical symptoms that contribute to the exclusion of AARD than the presence of anti-DFS70. Nevertheless, isolated anti-DFS70 helps to clarify positive ANA-IIF results, especially if pretest probability for AARD is low.

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.

Performance characteristics of rheumatoid factor and anti-cyclic citrullinated peptide antibody assays may impact ACR/EULAR classification of rheumatoid arthritis

Objectives Rheumatoid factor (RF) and anti-cyclic citrullinated protein/peptide antibodies (ACPA) are integrated in the 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria for rheumatoid arthritis (RA). The objectives of this study were to evaluate the technical and diagnostic performance of different RF and ACPA assays and to evaluate whether differences in performance impact RA classification. Methods Samples from 594 consecutive patients who for the first time consulted a rheumatologist (44 of whom were diagnosed with RA) and 26 extra newly diagnosed patients with RA were analysed with six different RF assays (Menarini, Thermo Fisher, Inova, Roche, Abbott, Euroimmun) and seven different ACPA assays (Menarini, Thermo Fisher, Inova, Roche, Abbott, Euro Diagnostica, Euroimmun). Results We found differences in analytical performance between assays. There was poor numerical agreement between the different RF and ACPA assays. For all assays, the likelihood ratio for RA increased with increasing antibody levels. The areas under the curve of receiver operating characteristic analysis of the RF (range 0.676–0.709) and ACPA assays (range 0.672–0.769) only differed between some ACPA assays. Nevertheless, using the cut-off proposed by the manufacturer, there was a large variation in sensitivity and specificity between assays (mainly for RF). Consequently, depending on the assay used, a subgroup of patients (13% for RF, 1% for ACPA and 9% for RF/ACPA) might or might not be classified as RA according to the 2010 ACR/EULAR criteria. Conclusion Due to poor harmonisation of RF and ACPA assays and of test result interpretation, RA classification according to 2010 ACR/EULAR criteria may vary when different assays are used.

Analytical performance of the single well titer function of NOVA View®: good enough to omit ANA IIF titer analysis?

Antinuclear antibodies (ANAs) are a diagnostic marker for ANA-associated rheumatic diseases [1]. The gold standard method for ANA detection is considered indirect immunofluorescence (IIF) on human epithelial (HEp-2) cells because of its high sensitivity [2]. Most recent ANA IIF guidelines recommend not only to report the presence of ANA as positive or negative but also to give a quantitative result [3, 4]. Nowadays, automated digital reading systems for ANAs by IIF testing have been integrated in routine immunodiagnostic laboratory practice [5, 6]. Several studies already showed a good correlation between fluorescence intensity by automated reading and end point titer (ET) obtained by manual reading [7, 8]. Furthermore, recent studies objectified that the likelihood for a systemic rheumatic disease increases with increasing ANA IIF fluorescence intensity [8, 9]. This illustrates that estimation of fluorescence intensity by automated IIF systems (without serial dilution) has clinical utility. NOVA View® (Inova, San Diego, CA, USA) is a digital IIF microscope that can be used for automated ANA detection. The system is able to assign five basic fluorescent ANA patterns (homogeneous, speckled, centromere, nucleolar and nuclear dots) and reports the measured average nuclear fluorescence intensity in nominal units, called Light-intensity units (LIUs). For positive ANA IIF samples, the instrument allows estimation of a “single well titer” (SWT) based on the LIUs measured at the 1/80 screening dilution and pattern-specific dilution curves [5, 7]. We evaluated the analytical performance (total imprecision and accuracy) of the SWT function on the NOVA View® for different ANA IIF nuclear patterns. Total imprecision of LIU measurement and of SWT function was evaluated by analyzing samples with a high and low level of a homogeneous, speckled, centromere and nucleolar pattern in 10 different ANA IIF runs. The target ET of the samples was determined by manual serial dilution. The total imprecision values of LIU measurement for the high-level and low-level samples were, respectively, 8% and 34% for a homogeneous pattern, 26% and 45% for a speckled pattern, 35% and 47% for a centromere pattern, and 38% and 39% for a nucleolar pattern ( Supplemental Data, Table 1). When results were expressed as SWT, the estimated SWT deviated from the target ET by maximum 1 SWT for all patterns except for the centromere pattern where the deviation was 2 titers in 30% of the sample with a low antibody level and in 10% of the sample with a high antibody level. Of note, for the sample with a high level of nucleolar antibodies, 60% of the determinations deviated by 1 titer from the target value. To evaluate the accuracy of the SWT function, samples with an isolated homogeneous (n = 389), speckled (n = 458), centromere (n = 101) and nucleolar (n = 69) ANA IIF pattern were analyzed in four hospitals: University Hospital Leuven, Gasthuiszusters Hospital Antwerp, OLV *Corresponding author: Lieve Van Hoovels, Department of Laboratory Medicine, OLV Hospital Aalst, Moorselbaan 164, 9300 Aalst, Belgium, Phone: +32 (0)53/72 42 91, Fax: +32 (0)53/72 45 88, E-mail: lieve.van.hoovels@olvz-aalst.be Sofie Schouwers and Laura Bogaert: Department of Laboratory Medicine, GZA Hospitals, Antwerp, Belgium Stefanie Van den Bremt: Department of Laboratory Medicine, OLV Hospital, Aalst, Belgium Nathalie Vandeputte: Department of Immunodiagnostics, Werfen N.V./S.A., Breda, Netherlands Martine Vercammen: Department of Laboratory Medicine, AZ SintJan Hospital Brugge, Brugge, Belgium Xavier Bossuyt: Department of Laboratory Medicine, University Hospital Leuven, Leuven, Belgium

Revised 2017 international consensus on ANCA testing in small vessel vasculitis: support from an external quality assessment

We read with great interest the multicentre study of Damoiseaux et al 1–3 on the detection of antineutrophil cytoplasmic antibodies (ANCA). ANCAs are important laboratory markers to support the diagnosis of ANCA-associated small vessel vasculitis (AAV), including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). Traditionally, laboratories screen for ANCA by indirect immunofluorescence (IIF) and IIF positive samples are further evaluated for antibodies to proteinase 3 (PR3) or myeloperoxidase (MPO) by specific immunoassays. Such diagnostic algorithm is based on an international consensus statement on ANCA testing issued in 1999.4 Over the last two decades, the diagnostic performance of immunoassays has significantly improved. The recent multicentre study showed a …