First dengue co-infection in a Belgian traveler returning from Thailand, July 2013.

We report a dengue virus (DENV) co-infection in a Belgian traveler after a three-weeks holiday to Thailand. The patient recovered well without any complication. The infection was diagnosed by NS1 antigen testing and the concurrent presence of serotype DENV1 and DENV2 was demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) in acute phase serum sampled three days after symptoms onset. The predominant DENV1 serotype was identified as genotype I, lineage Asia-3 by sequencing. To our knowledge, this is the first time that a dengue co-infection is reported in a European traveler. The co-infection accounts for 1.0% of the total number of RT-PCR-positive samples (n=105) diagnosed in the reference laboratory of Belgium between 2008 and 2013. We expect that the number of reports on acute co-infections will increase in the coming years considering the increasing number of regions that are progressively becoming hyperendemic, especially in Southeast Asia.

Fourth Belgian multicentre survey of antibiotic susceptibility of anaerobic bacteria.

OBJECTIVES: To collect recent data on the susceptibility of anaerobes to antimicrobial agents with known activity against anaerobes, and to compare them with results from previous Belgian multicentre studies. METHODS: Four hundred and three strict anaerobic clinical isolates were prospectively collected from February 2011 to April 2012 in eight Belgian university hospitals. MICs were determined by one central laboratory for 11 antimicrobial agents using Etest methodology. RESULTS: According to EUCAST breakpoints, >90% of isolates were susceptible to amoxicillin/clavulanate (94%), piperacillin/tazobactam (91%), meropenem (96%), metronidazole (92%) and chloramphenicol (98%), but only 70% and 40% to clindamycin and penicillin, respectively. At CLSI recommended breakpoints, only 71% were susceptible to moxifloxacin and 79% to cefoxitin. MIC50/MIC90 values for linezolid and for tigecycline were 1/4 and 0.5/4 mg/L, respectively. When compared with survey data from 2004, no major differences in susceptibility profiles were noticed. However, the susceptibility of Prevotella spp. and other Gram-negative bacilli to clindamycin decreased from 91% in 1993-94 and 82% in 2004 to 69% in this survey. Furthermore, the susceptibility of clostridia to moxifloxacin decreased from 88% in 2004 to 66% in 2011-12 and that of fusobacteria from 90% to 71%. CONCLUSIONS: Compared with previous surveys, little evolution was seen in susceptibility, except a decline in activity of clindamycin against Prevotella spp. and other Gram-negative bacteria, and of moxifloxacin against clostridia. Since resistance was detected to all antibiotics, susceptibility testing of anaerobic isolates is indicated in severe infections to confirm appropriateness of antimicrobial therapy.

Enzymatic pyruvate measurement by Cobas 6000 open channel assay.

BACKGROUND: Blood pyruvate measurement in conjunction with lactic acid is useful for differentiating pyruvate dehydrogenase deficiencies from primary or secondary disorders of mitochondrial electron transport. METHODS: We evaluated the analytical performance of pyruvate measurement by an enzymatic open channel assay on a Roche Cobas 6000. RESULTS: The assay was linear from 0.07 to 0.50 mmol/L pyruvate. Total imprecision ranged from 15.7% to 7.1% at pyruvate levels of 0.08 to 0.31 mmol/L, respectively. Functional sensitivity was 0.07 mmol/L. The assay showed no interference by lipids or bilirubin, whereas haemolysis influenced pyruvate concentrations in a hemoglobin concentration-independent manner. Method comparison with patient samples (n = 41) showed that the Cobas 6000 enzymatic method correlated well (r2 = 0.930) with a similar enzymatic assay on a Cobas Mira platform and showed better accuracy in external control schemes. CONCLUSIONS: Enzymatic pyruvate measurement by a Cobas 6000 open channel shows satisfactory analytical performance. The assay can be integrated in the automated laboratory workflow and is always ready for use thanks to its on-board reagents.

Development of a multipurpose time-resolved fluorescence immunoassay for rat insulin.

We present a time-resolved fluorescence immunoassay (TR-FIA) for the measurement of rat insulin in cell extracts and culture media. This assay is based on the binding of two monoclonal antibodies to different parts of the insulin molecule in a 96-well microtiter plate. For the detection, europium-labeled streptavidin that interacts with the second biotinylated antibody is used. Samples of 25 microl could be analyzed in less than 2 days with a measuring range between 5 and 1250 pg (0.2-50 microg/L or 34.4-8600 pM). The inter- and intraassay percentage coefficients of variation were less than 8.3 and 5.1, respectively. Recoveries of 0.48 to 40 microg/L rat insulin, added to culture medium, ranged between 94 and 107%. Results were significantly correlated with those of an in-house radioimmunoassay (RIA) for rodent insulin (P<0.0001, r(2)=0.99). The TR-FIA method had a similar detection limit (0.16 microg/L), but its working range was at least 5-fold larger. Additional advantages include the lower cost, the applicability to measurements in tissue and serum, and the quantification of insulin from other species.

Simultaneous measurement of plasma concentrations of proinsulin and C-peptide and their ratio with a trefoil-type time-resolved fluorescence immunoassay.

BACKGROUND: When the concentrations of 2 or more substances are measured separately, their molar ratios are subject to the additive imprecisions of the different assays. We hypothesized that the cumulative error for concentration ratios of peptides containing a common sequence might be minimized by measuring the peptides simultaneously with a "trefoil-type" immunoassay. METHODS: As a model of this approach, we developed a dual-label time-resolved fluorescence immunoassay (TRFIA) to simultaneously measure proinsulin, C-peptide, and the proinsulin-C-peptide ratio (PI/C). A monoclonal antibody captures all C-peptide-containing molecules, and 2 differently labeled antibodies distinguish between proinsulin-like molecules and true C-peptide. RESULTS: The trefoil-type TRFIA was capable of measuring plasma C-peptide and proinsulin simultaneously without mutual interference at limits of quantification of 48 and 8125 pmol/L, and 2.1 and 197 pmol/L, respectively. Within-laboratory imprecision values for the trefoil-type TRFIA ranged between 8.4% and 12% for the hormone concentrations. Unlike the hormone results obtained with separate assays, imprecision did not increase when PI/C was calculated from trefoil assay results (P < 0.05). Peptide concentrations were highly correlated with results obtained in individual comparison assays (r(2) > or = 0.965; P < 0.0001). The total error for PI/C obtained with the trefoil-type TRFIA remained < or = 25% over a broader C-peptide range than with separate hormone assays (79-7200 pmol/L vs 590-4300 pmol/L C-peptide). Preliminary data indicate little or no interference by heterophile antibodies. CONCLUSIONS: The developed trefoil-type TRFIA is a reliable method for simultaneous measurement of proinsulin, C-peptide, and PI/C and provides proof of principle for the development of other trefoil-type multiple-label immunoassays.