Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Identification of Clinically Relevant Bacteria

Background: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows rapid and reliable identification of microorganisms, particularly clinically important pathogens. Methodology/Principal Findings: We compared the identification efficiency of MALDI-TOF MS with that of PhoenixH, APIH and 16S ribosomal DNA sequence analysis on 1,019 strains obtained from routine diagnostics. Further, we determined the agreement of MALDI-TOF MS identifications as compared to 16S gene sequencing for additional 545 strains belonging to species of Enterococcus, Gardnerella, Staphylococcus, and Streptococcus. For 94.7 % of the isolates MALDI-TOF MS results were identical with those obtained with conventional systems. 16S sequencing confirmed MALDI-TOF MS identification in 63 % of the discordant results. Agreement of identification of Gardnerella, Enterococcus, Streptococcus and Staphylococcus species between MALDI-TOF MS and traditional method was high (Crohn’s kappa values: 0.9 to 0.93). Conclusions/Significance: MALDI-TOF MS represents a rapid, reliable and cost-effective identification technique for clinically relevant bacteria

Rapid Species Diagnosis for Invasive Candidiasis Using Mass Spectrometry

BACKGROUND: Matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF-MS) allows the identification of most bacteria and an increasing number of fungi. The potential for the highest clinical benefit of such methods would be in severe acute infections that require prompt treatment adapted to the infecting species. Our objective was to determine whether yeasts could be identified directly from a positive blood culture, avoiding the 1-3 days subculture step currently required before any therapeutic adjustments can be made. METHODOLOGY/PRINCIPAL FINDINGS: Using human blood spiked with Candida albicans to simulate blood cultures, we optimized protocols to obtain MALDI TOF-MS fingerprints where signals from blood proteins are reduced. Simulated cultures elaborated using a set of 12 strains belonging to 6 different species were then tested. Quantifiable spectral differences in the 5000-7400 Da mass range allowed to discriminate between these species and to build a reference database. The validation of the method and the statistical approach to spectral analysis were conducted using individual simulated blood cultures of 36 additional strains (six for each species). Correct identification of the species of these strains was obtained. CONCLUSIONS/SIGNIFICANCE: Direct MALDI TOF-MS analysis of aliquots from positive blood cultures allowed rapid and accurate identification of the main Candida species, thus obviating the need for sub-culturing on specific media. Subsequent to this proof-of-principle demonstration, the method can be extended to other clinically relevant yeast species, and applied to an adequate number of clinical samples in order to establish its potential to improve antimicrobial management of patients with fungemia

Filamentous fungal characterizations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ⁄ ionization time-of-flight intact cell mass spectrometry (MALDI-TOF ICMS) is coming of age for the identification and characterization of fungi. The procedure has been used extensively with bacteria. UV-absorbing matrices function as energy mediators that transfer the absorbed photoenergy from an irradiation source to the surrounding sample molecules, resulting in minimum fragmentation. A surprisingly high number of fungal groups have been studied: (i) the terverticillate penicillia, (ii) aflatoxigenic, black and other aspergilli, (iii) Fusarium, (iv) Trichoderma, (iv) wood rotting fungi (e.g. Serpula lacrymans) and (v) dermatophytes. The technique has been suggested for optimizing quality control of fungal Chinese medicines (e.g. Cordyceps). MALDI-TOF ICMS offers advantages over PCR. The method is now used in taxonomic assessments (e.g. Trichoderma) as distinct from only strain characterization. Low and high molecular mass natural products (e.g. peptaibols) can be analysed. The procedure is rapid and requires minimal pretreatment. However, issues of reproducibility need to be addressed further in terms of strains of species tested and between run variability. More studies into the capabilities of MALDI-TOF ICMS to identify fungi are required.R.R.M. Paterson is grateful for FCT-Portugal Grant (SFRH/BPD/34879/2007) and IOI Professorial Chair, Malaysia where some of this review was first revised. C. Santos acknowledges FACEPE/CNPq-Brazil financial support. N. Lima appreciates the stay in the Biochemistry Department of UFPE-Brazil as Visiting Full Professor where this review was drafted. C. Santos and N. Lima acknowledge Laboratorio de Imunopatologia Keizo Asami (LIKA/UFPE) for the consent and support given to use their MALDI-TOF MS equipment

New insights for diagnosis of Pineapple Fusariosis by MALDI-TOF MS technique

Fusarium is one of the most economically important fungal genus, since it includes many pathogenic species which cause a wide range of plant diseases. Morphological or molecular biology identification of Fusarium species is a limiting step in the fast diagnosis and treatment of plant disease caused by these fungi. Mass spectrometry by matrix-assisted laser/desorption ionisation-time-of-flight (MALDI-TOF)-based fingerprinting approach was applied to the fungal growth monitoring and direct detection of strain Fusarium guttiforme E-480 inoculated in both pineapple cultivars Pérola and Imperial side shoots, that are susceptible and resistant, respectively, to this fungal strain. MALDI-TOF MS technique was capable to detect fungal molecular mass peaks in the susceptible pineapple stem side shoot tissue. It is assumed that these molecular masses are mainly constituted by ribosomal proteins. MALDI-TOF-based fingerprinting approach has herein been demonstrated to be sensitive and accurate for the direct detection of F. guttiforme E-480 molecular masses on both susceptible and resistant pineapple side stem free of any pre-treatment. According to the results obtained, the changing on molecular mass peaks of infected susceptible pineapple tissue together with the possibility of fungal molecular masses analysis into this pineapple tissue can be a good indication for an early diagnosis by MALDI-TOF MS of pineapple fusariosis

Mould Routine Identification in the Clinical Laboratory by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry

BACKGROUND: MALDI-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 METHODS: First, 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. RESULTS: The 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. CONCLUSIONS: This 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

Temperature Shift and Host Cell Contact Up-Regulate Sporozoite Expression of Plasmodium falciparum Genes Involved in Hepatocyte Infection

Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria

Couplage orthogonal entre un piège ionique et un analyseur temps de vol IT/O-reTOF

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Preparing for Transmission: Gene Regulation in Plasmodium Sporozoites

International audiencePlasmodium sporozoites are transmitted to mammals by anopheline mosquitoes and first infect the liver, where they transform into replicative exoerythrocytic forms, which subsequently release thousands of merozoites that invade erythrocytes and initiate the malaria disease. In some species, sporozoites can transform into dormant hypnozoites in the liver, which cause malaria relapses upon reactivation. Transmission from the insect vector to a mammalian host is a critical step of the parasite life cycle, and requires tightly regulated gene expression. Sporozoites are formed inside oocysts in the mosquito midgut and become fully infectious after colonization of the insect salivary glands, where they remain quiescent until transmission. Parasite maturation into infectious sporozoites is associated with reprogramming of the sporozoite transcriptome and proteome, which depends on multiple layers of transcriptional and post-transcriptional regulatory mechanisms. An emerging scheme is that gene expression in Plasmodium sporozoites is controlled by alternating waves of transcription activity and translational repression, which shape the parasite RNA and protein repertoires for successful transition from the mosquito vector to the mammalian host