BackgroundMALDI-TOF MS recently emerged as a valuable identification tool for bacteria and yeasts and revolutionized the daily clinical laboratory routine. But it has not been established for routine mould identification. This study aimed to validate a standardized procedure for MALDI-TOF MS-based mould identification in clinical laboratory.Materials and MethodsFirst, pre-extraction and extraction procedures were optimized. With this standardized procedure, a 143 mould strains reference spectra library was built. Then, the mould isolates cultured from sequential clinical samples were prospectively subjected to this MALDI-TOF MS based-identification assay. MALDI-TOF MS-based identification was considered correct if it was concordant with the phenotypic identification; otherwise, the gold standard was DNA sequence comparison-based identification.ResultsThe optimized procedure comprised a culture on sabouraud-gentamicin-chloramphenicol agar followed by a chemical extraction of the fungal colonies with formic acid and acetonitril. The identification was done using a reference database built with references from at least four culture replicates. For five months, 197 clinical isolates were analyzed; 20 were excluded because they were not identified at the species level. MALDI-TOF MS-based approach correctly identified 87% (154/177) of the isolates analyzed in a routine clinical laboratory activity. It failed in 12% (21/177), whose species were not represented in the reference library. MALDI-TOF MS-based identification was correct in 154 out of the remaining 156 isolates. One Beauveria bassiana was not identified and one Rhizopus oryzae was misidentified as Mucor circinelloides.ConclusionsThis work's seminal finding is that a standardized procedure can also be used for MALDI-TOF MS-based identification of a wide array of clinically relevant mould species. It thus makes it possible to identify moulds in the routine clinical laboratory setting and opens new avenues for the development of an integrated MALDI-TOF MS-based solution for the identification of any clinically relevant microorganism.

Acanthamoeba polyphaga mimivirus is the largest known virus in both particle size and genome complexity. Its 1.2-Mb genome encodes 911 proteins, among which only 298 have predicted functions. The composition of purified isolated virions was analyzed by using a combined electrophoresis/mass spectrometry approach allowing the identification of 114 proteins. Besides the expected major structural components, the viral particle packages 12 proteins unambiguously associated with transcriptional machinery, 3 proteins associated with DNA repair, and 2 topoisomerases. Other main functional categories represented in the virion include oxidative pathways and protein modification. More than half of the identified virion-associated proteins correspond to anonymous genes of unknown function, including 45 "ORFans." As demonstrated by both Western blotting and immunogold staining, some of these "ORFans," which lack any convincing similarity in the sequence databases, are endowed with antigenic properties. Thus, anonymous and unique genes constituting the majority of the mimivirus gene complement encode bona fide proteins that are likely to participate in well-integrated processes.Acanthamoeba polyphaga mimivirus (mimivirus) is the largest virus isolated so far (23). Based on its highly specific characteristics, this double-stranded-DNA icosahedral virus (47) is the first member of the new Mimiviridae family (33, 43). Computational annotation of its 1.2-Mb genome (33) revealed many atypical features, including the presence of key translation enzymes, a full complement of DNA repair pathway components, and the unique presence of three different topoisomerases (of types IA, IB, and II) (2, 33). Another unique characteristic of mimivirus is the presence of nearly identical promoter sequence motifs upstream of half of its 911 proteinencoding genes (42), which are presumably associated with proteins expressed during the early or late-early phase. Only 23% of the predicted coding genes exhibit convincing homology to proteins of known function, and 39% of them do not exhibit a clear (E values, Ͻ10 Ϫ5 ) sequence database match (33). Such coding regions without sequence similarity to other genes in databases are considered orphan open reading frames (ORFs) and termed "ORFans" (12). The origin and function of ORFan genes are still a matter of controversy, with opinions ranging from considering them pieces of junk DNA (1,8,40,44) to seeing them as quickly evolving sequences encoding normally expressed functional proteins (38, 39). Recent clinical evidence raised the possibility that mimivirus might be a human pathogen causing pneumonia (4, 24, 34), as suspected when it was first isolated from a cooling tower following an outbreak of pneumonia (23).Mass spectrometry-based analysis has recently emerged as a technique of choice to identify more comprehensively the set of viral proteins associated with viral particles (19,29,49). We now present the application of this technique to the largest known, and presumably most complex, viral particle,...

“Nanobacteria” are nanometer-scale spherical and ovoid particles which have spurred one of the biggest controversies in modern microbiology. Their biological nature has been severely challenged by both geologists and microbiologists, with opinions ranging from considering them crystal structures to new life forms. Although the nature of these autonomously replicating particles is still under debate, their role in several calcification-related diseases has been reported. In order to gain better insights on this calciferous agent, we performed a large-scale project, including the analysis of “nanobacteria” susceptibility to physical and chemical compounds as well as the comprehensive nucleotide, biochemical, proteomic, and antigenic analysis of these particles. Our results definitively ruled out the existence of “nanobacteria” as living organisms and pointed out the paradoxical role of fetuin (an anti-mineralization protein) in the formation of these self-propagating mineral complexes which we propose to call “nanons.” The presence of fetuin within renal calculi was also evidenced, suggesting its role as a hydroxyapatite nucleating factor.