Identification of the flotillin-1/2 heterocomplex as a target of autoantibodies in bona fide multiple sclerosis.

BACKGROUND: Autoantibodies, in particular those against aquaporin-4 and myelin-oligodendrocyte glycoprotein (MOG), aid as biomarkers in the differential diagnosis of demyelination. Here, we report on discovery of autoantibodies against flotillin in patients with multiple sclerosis (MS). METHODS: The target antigen was identified by histo-immunoprecipitation using the patients' sera and cryosections of rat or pig cerebellum combined with mass spectrometrical analysis. Correct identification was ascertained by indirect immunofluorescence and neutralization tests using the target antigens recombinantly expressed in HEK293 cells. RESULTS: Serum and CSF of the index patient produced a fine-granular IgG indirect immunofluorescence staining of the hippocampal and cerebellar molecular layers. Flotillin-1 and flotillin-2 were identified as target autoantigens. They also reacted with recombinant human flotillin-1/2 co-expressed in HEK293 cells, but not with the individual flotillins in fixed- and live-cell assays. Moreover, neutralization using flotillin-1/2, but not the single flotillins, abolished the tissue reactivity of patient serum. Screening of 521 patients, for whom anti-aquaporin-4 testing was requested and negative, revealed 8 additional patients with anti-flotillin-1/2 autoantibodies. All eight were negative for anti-MOG. Six patients ex post fulfilled the revised McDonald criteria for MS. Vice versa, screening of 538 MS sera revealed anti-flotillin-1/2 autoantibodies in eight patients. The autoantibodies were not found in a cohort of 67 patients with other neural autoantibody-associated syndromes and in 444 healthy blood donors. CONCLUSIONS: Autoantibodies against the flotillin-1/2 heterocomplex, a peripheral membrane protein that is involved in axon outgrowth and regeneration of the optic nerve, are present in 1-2% of patients with bona fide MS.

Evaluation of species-specific score cutoff values of routinely isolated clinically relevant bacteria using a direct smear preparation for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based bacterial identification.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was introduced a few years ago as a new method for bacterial identification. A variety of studies have been published concerning MALDI-TOF MS-based identification, most of them using culture collections for the validation of the respective databases in a retrospective manner in favor of a parallel investigation. The score cutoff value is of special importance for reliable species identification in the Biotyper database. The score cutoff values suggested by the manufacturer have been validated using a previously published formic acid extraction protocol. In most of the previously published studies investigating the Biotyper database, only little information was given concerning species-specific score values. In addition, the mass spectrometer instruments, the number of replicates, the number of spectra used to calculate a sum-spectrum by the supplied software, and the score cutoff values which have been applied varied within these studies. In this study, we compared a straightforward direct smear preparation and measurement without replicate testing to defined biochemical identifications in a parallel manner. In addition, we described new species-specific score cutoff values for the identification of certain bacteria.

False-negative test results in the Slidex Staph Plus (bioMérieux) agglutination test are mainly caused by spa-type t001 and t001-related strains.

The relative sensitivity of commercial agglutination kits for fast identification of S. aureus is usually given to be about 98%. This reported sensitivity has sometimes been questioned. In this study, three collections of molecularly defined, single-copy strains of S. aureus were used to compare the sensitivities of agglutination-based identification and the MALDI-TOF mass spectrometry-based identification using the Biotyper 2.0 database to a molecularly defined reference method. Clinical isolates (n = 363) of methicillin-susceptible S. aureus (MSSA) and 240 clinical isolates of methicillin-resistant S. aureus (MRSA) were included. In order to rule out a predominance of local MRSA-strains, a collection of 104 pulsed-field-gel electrophoresis divergent MRSA strains were also tested. MALDI-TOF MS using Biotyper database (Bruker) identified all isolates, whereas the Slidex Staph Plus (bioMérieux) detected only 98.0% of the MSSA, 94.5% of the MRSA and only 70.1% of the MRSA of the molecularly divergent strain collection. Interestingly, strains with a false-negative test result in the agglutination methods were mostly spa-type t001 and t001 related. The MALDI-TOF MS based identification can thus be used as an alternative identification method for suspected false-negative results from the agglutination tests, especially if the local prevalence of t001 and t001 related strains is high.

Vancomycin MIC creep in MRSA blood culture isolates from Germany: a regional problem?

The aim of this study was to assess the vancomycin MIC distribution for MRSA blood culture isolates over a period of six years in Germany. The study examined 287 MRSA isolates from blood cultures collected at several hospitals in two German cities between 2004 and 2009. The vancomycin MIC was determined by Etest. Genotypic features of the MRSA strains with vancomycin MIC ≥ 1 mg/L were determined by semiautomated repetitive-sequence-based polymerase chain reaction. The range of vancomycin MIC as determined by Etest was 0.25 to 2.0 mg/L. The geometric mean MIC increased by 1.34-fold in city A over the study period (p < 0.05), but there was no meaningful change in city B (a 1.09-fold increase, p > 0.05). Furthermore, in city A a shift in vancomycin MICs occurred as an increase in the percentage of isolates with MIC ≥ 1 mg/L from period one (2004-2006) to period two (2007-2009) (p < 0.0001). Typing results showed that in city A a single clone was predominant (55% of the creep isolates). In this study, the creep phenomenon seems to be a regional problem. We suggest that all hospitals should monitor their local status of elevated vancomycin MICs in invasive MRSA isolates.

The sensitivity of direct identification from positive BacT/ALERT™ (bioMérieux) blood culture bottles by matrix-assisted laser desorption ionization time-of-flight mass spectrometry is low.

Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been presented as a novel method for the direct identification of bacteria from positive blood culture bottles. The rate of the MALDI TOF MS-based identification in the present study from positive BacT/ALERT (bioMérieux, Marcy l'Etoile, France) blood culture bottles was 30%, which is far below the previously reported sensitivities using the BACTEC (Becton Dickinson, Franklin Lakes, NJ, USA) system. We also found evidence that the Biotyper algorithm did not identify a second pathogen in cases of positive BacT/ALERT blood culture bottles containing two different species.

Comparison of phenotypic methods for penicillinase detection in Staphylococcus aureus.

Penicillinase testing is required for Staphylococcus aureus isolates with a penicillin MIC of