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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.

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., Competing Interests: Competing interests: XB has received lecture fees from Thermo Fisher, Inova and Menarini and has been a consultant for Inova., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

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.

Objectives: Free light chain (FLC) measurement gained a lot of interest for diagnostic workup of monoclonal gammopathy., Methods: We evaluated the performance of turbidimetric polyclonal Freelite (The Binding Site, Birmingham, UK) assays on Cobas 6000 (Roche Diagnostics, Rotkreuz, Switzerland) and nephelometric monoclonal N Latex (Siemens Healthcare Diagnostics, Marburg, Germany) assays on BN ProSpec (Dade Behring, Deerfield, IL) vs established nephelometric Freelite assays on BN ProSpec., Results: Analytical performance was acceptable. Method comparison (n = 118) showed significant proportional FLC differences for N Latex assays. However, good correlation and clinical concordance were shown. Recovery study in the low concentration range demonstrated consistent over- and underrecovery for Freelite reagents, hampering future research on prognostic value of suppressed noninvolved FLC. Antigen excess detection was successful for κ FLC in three-fourths of cases with Freelite reagents and in all cases with N Latex reagents. However, the latter resulted in underestimated κ FLC concentrations., Conclusions: FLC analysis requires continuous awareness of analytical limitations. Monitoring of disease response requires FLC analysis on the same platform using the same reagents., (© American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Background . Our study aimed to investigate whether the introduction of automated anti-nuclear antibody (ANA) indirect immunofluorescence (IIF) analysis decreases the interlaboratory variability of ANA titer results. Method . Three serum samples were sent to 10 laboratories using the QUANTA-Lyser® in combination with the NOVA View®. Each laboratory performed the ANA IIF analysis 10x in 1 run and 1x in 10 different runs and determined the endpoint titer by dilution. One of the three samples had been sent in 2012, before the era of ANA IIF automation, by the Belgian National External Quality Assessment (EQA) Scheme. Harmonization was evaluated in terms of variability in fluorescence intensity (LIU) and ANA IIF titer. Results . The evaluation of the intra- and interrun LIU variability revealed a larger variability for 2 laboratories, due to preanalytical and analytical problems. Reanalysis of the EQA sample resulted in a lower titer variability. Diluted endpoint titers were similar to the estimated single well titer and the overall median titer as reported by the EQA in 2012. Conclusion. The introduction of automated microscopic analysis allows more harmonized ANA IIF reporting, provided that this totally automated process is controlled by a thorough quality assurance program, covering the total ANA IIF process., Competing Interests: The authors declare that they have no competing interests.

We report the case of a 56-year-old Caucasian woman in whom hemoglobinopathy screening was triggered following an aberrant Hb A1c analysis. Preliminary diagnosis of the hemoglobin (Hb) variant was obtained through cation exchange high performance liquid chromatography (HPLC) and gel electrophoresis. DNA analysis confirmed the presence of Hb J-Amiens [β17(A14)Lys→Asn; HBB: c.[54G > C or 54G > T)]. However, an unbalanced ratio between wild type and mutant signal after direct sequencing and a lower than expected percentage of this Hb variant led to the suggestion of a mosaic expression. Furthermore, different methods [capillary zone electrophoresis (CZE), cation exchange HPLC and boronate affinity] were tested to study the possible interference of this variant with Hb A1c measurements. These investigations showed a clinically relevant difference between the methods tested. Hb A1c analysis may lead to the discovery of new Hb variants or mosaicism for previously described Hb variants. This may have genetic consequences for the offspring of carriers and brings about the question of partner testing.

Background: The immediate impact of blood gas test results on patient care favors the use of blood gas analyzers as point-of-care-testing (POCT) devices. We performed an analytical performance evaluation of four cartridge-type blood gas analyzers for the determination of pH, partial carbon dioxide pressure (pCO2), partial oxygen pressure (pO2), ionized calcium (iCa2+), potassium (K+), glucose, lactate and total hemoglobin (tHb), in comparison with a traditional blood gas analyzer., Methods: The analyzers included in the study are: RP405, GEM Premier 4000, ABL90 FLEX and Cobas b 123. The ABL700 served as comparator. For each instrument the imprecision was estimated according to the CLSI EP5-A2. Based on the CLSI EP9-A2 evaluation protocol, a method comparison was performed using patient samples. Obtained data were compared against preset quality specifications, based on ABL700 performance and biological variation., Results: The precision of the RP405 and ABL90 FLEX was remarkably better than the preset criteria based on ABL700 performance. The GEM appears to have the worst precision. The RP405 yielded the best overall performance, with exception for tHb and iCa2+. Noteworthy is the very good performance of the glucose determination on RP405. The ABL90 FLEX showed the best performance for pH, K+ and iCa2+ measurements. For tHb determination the Cobas b123 revealed the best results. Regarding practicability, all instruments were found to be user friendly., Conclusions: Globally, all four cartridge-type blood gas analyzers produced clinically acceptable results. The analytical performance, together with the ease of use and low maintenance time of the instruments, demonstrate that these analyzers are perfectly suitable for both POCT and laboratory use.

The present study aimed to determine the diagnostic performance of different testing strategies to diagnose malignant B-cell disorder or monoclonal gammopathy of unknown significance (MGUS). Sensitivity and specificity were determined in 833 consecutive patients investigated for a monoclonal gammopathy. Serum protein electrophoresis (PE), serum kappa/lambda free light chain (FLC) ratio, and serum and urine immunofixation electrophoresis (IFE) were performed in all patients. Twenty-eight patients were diagnosed with a malignant plasma cell disorder, 25 with B-cell non-Hodgkin lymphoma and 156 with MGUS. Serum PE (with follow-up IFE) plus FLC had a sensitivity of 82.3% and a specificity of 96.8% and missed one plasmacytoma and 23 patients with MGUS. Serum IFE plus urine IFE had a sensitivity of 92.3% and a specificity of 100% and missed two MGUS patients. Serum IFE plus FLC had a sensitivity of 93.8% and a specificity of 96.8% and missed one MGUS patient. Serum PE plus FLC had a significantly lower sensitivity than serum IFE plus FLC or serum IFE plus urine IFE for the diagnosis of MGUS. The sensitivity of serum IFE plus FLC was comparable to the sensitivity of serum IFE plus urine IFE. The specificity of serum IFE plus FLC, however, was lower than the specificity of serum IFE plus urine IFE.

Background: Anti-neutrophil cytoplasmic antibodies (ANCA) are associated with primary small vessel vasculitis (SVV). Proteinase-3 (PR3)-ANCA are primarily associated with Wegener granulomatosis, whereas myeloperoxidase (MPO)-ANCA are primarily associated with microscopic polyangiitis (MPA) and vasculitic Churg-Strauss syndrome. We evaluated whether a strategy that is based on screening with ELISA or fluoroenzymeimmunoassay (FEIA) is an accurate alternative to screening with indirect immunofluorescence (IIF)., Methods: C-ANCA and P-ANCA were determined by IIF and PR3-ANCA and MPO-ANCA were determined by ELISA (Inova) or FEIA (Phadia) on 326 patients (38 with newly diagnosed SVV and 288 diseased controls)., Results: Specificity and positive likelihood ratios were higher for ELISA and FEIA than for IIF. Post-test probability for SVV of a positive test result was higher for ELISA and FEIA than for IIF. Decision tree analysis in which several testing strategies were compared revealed that a testing strategy that is based on screening with ELISA or FEIA had an expected clinical utility that was comparable to screening with IIF and confirming with ELISA or FEIA. The highest expected clinical utility was found when both IIF and ELISA or FEIA were performed on all samples., Conclusions: A strategy based on screening for ANCA with ELISA or FEIA (without prior IIF) is a valuable alternative to screening with IIF and confirming with ELISA or FEIA.

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.

Background: Group A rotavirus genotypes G1, G2, G3 and G4 are the main etiological agents of infantile diarrhea. The G9 rotavirus has recently emerged as a fifth important genotype all over the world., Objective: To characterize the VP7 gene of group A rotaviruses from gastroenteritis patients admitted to the Gasthuisberg University Hospital, Leuven, Belgium, during 1999-2003., Study Design: Rotavirus antigen was detected in stool specimens using an enzyme immunoassay. G-typing was performed by reverse transcriptase polymerase chain reaction (RT-PCR) amplification and sequencing of the complete VP7 gene., Results: The genotype distribution varied markedly over the four rotavirus years in Belgium. In the 1999-2000 rotavirus year, G1 was the predominating type (72%), and G9 was present in 5% of the rotavirus-positive patients. In the 2000-2001 and 2002-2003 years, G9 appeared as the dominating strain (45% and 53%, respectively). In the 2001-2002 year, between two G9 epidemic years, G1 was dominating (66%) but G9 was still present in 24%. All the G9 isolates were combined with P[8] and shared a high gene sequence similarity (<3% sequence divergence on the nucleotide and amino acid level). Phylogenetic analysis of the VP7 genes revealed that our Belgian G9 strains clustered together with recent G9 strains from all over the world, distinct from the prototype G9 strains isolated in the 1980s., Conclusion: Our study indicates that although the first introduction of G9 isolates in the Belgian population was recorded in 1997, G9 strains were able to establish themselves quickly as the predominant genotype. The emergence of G9 as an important pathogen in both developing as industrialized countries necessitates the urgent consideration of the G9 moiety in rotavirus vaccines.

Group A rotaviruses are the main cause of severe diarrhoea in humans and animals throughout the world. We report the first description of a clinically apparent infection with a P[14], G3 rotavirus (strain B4106) in a hospitalized 6-year-old child. The VP7 gene of the B4106 strain had the closest sequence similarity (94% and 97% on the nucleotide and amino acid level, respectively) with strain 30/96 (P[14], G3), a lapine rotavirus isolated in an Italian rabbit in 1996 while the VP4 gene had the closest similarity with strain 30/96 on the nucleotide level (96%), and with lapine strains C-11 (P[14], G3) and Alabama (P[14], G3), isolated in the United States in the 1980s on the amino acid level (99%). The host restriction determinant gene NSP4 of B4106 was also most similar to lapine strain Alabama (95% nt identity and 97% aa identity). Phylogenetic analysis showed that the VP4, VP7, and NSP4 genes of the B4106 strain share a common evolutionary lineage with those of lapine rotavirus strains. We therefore hypothesize that a lapine rotavirus was able to cross the host species barrier and caused disease in a new host. The increasing detection of strains in humans that were previously believed to be restricted to animals raises questions whether interspecies transmission of rotaviruses is a common event in nature.

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.

Rotavirus infections are a major cause of severe diarrhea in children younger than 2 years. In Belgium they cause many hospitalizations because of dehydration. A study of the laboratory diagnosis of rotavirus infections in 28.251 stool samples at a university teaching hospital in Belgium during a twenty-year period (1981-2002) showed a marked seasonality. The virus was most often diagnosed during the winter months: 54% of the rotavirus isolates were found in the first three months of the year, with 21% of the positive samples occurring in February. Recently, rotaviruses can be genotyped based on differences in the viral outer capsid protein VP7. Vaccines are currently being developed against the four most prevalent genotypes G1, G2, G3 and G4. During the last three epidemic seasons (1999-2002) in Belgium, G1 was the most prevalent genotype and accounted for 62% of the rotavirus isolates recovered. G2, G3 and G4 were also isolated, and other emerging types need to be carefully monitored too, since G9 (45%) was co-dominant with G1 (42%) in the 2000-2001 rotavirus season in Belgium. The future development of an efficient rotavirus vaccine will need to take the diversity of the rotavirus genotypes into account.