Mirko Buttrini

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry applied to virus identification

Virus detection and/or identification traditionally rely on methods based on cell culture, electron microscopy and antigen or nucleic acid detection. These techniques are good, but often expensive and/or time-consuming; furthermore, they not always lead to virus identification at the species and/or type level. In this study, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was tested as an innovative tool to identify human polioviruses and to identify specific viral protein biomarkers in infected cells. The results revealed MALDI-TOF MS to be an effective and inexpensive tool for the identification of the three poliovirus serotypes. The method was firstly applied to Sabin reference strains, and then to isolates from different clinical samples, highlighting its value as a time-saving, sensitive and specific technique when compared to the gold standard neutralization assay and casting new light on its possible application to virus detection and/or identification.

MALDI-TOF MS analysis of human and animal Brachyspira species and benefits of database extension.

Spirochaetes belonging to the genus Brachyspira are anaerobic bacteria that colonize the large intestine of humans and animals, mainly pigs. The main species are namely, B. hyodysenteriae, the etiological agent of swine dysentery, B. pilosicoli, a zoonotic agent causing colonic spirochaetosis both in humans and different animal species, B. aalborgi, exclusively infecting humans causing colonic spirochaetosis, B. intermedia, a potential animal pathogen, B. innocens and B. murdochii, generally commensal of pigs, and B. alvinipulli, found in egg laying hens with diarrhea. In this study, for the first time, MALDI-TOF MS was applied on Brachyspira strains of human and animal origins, supplementing the existing database, limited to the species B. murdochii only, with spirochaetal protein profiles and demonstrating its usefulness in the rapid, cheap and reliable identification of Brachyspira strains at the species level, overcoming the problems previously encountered in the identification of these spirochaetes when using biochemical and genetic-based methods. Moreover, a dendrogram based on protein profiles of the different spirochaetal species was generated reflecting their host spectrum, showing in the same branch the only two species able to infect humans (B. aalborgi and B. pilosicoli) and in the other branch the spirochaetes infecting exclusively animals.

Identification of Borrelia species after creation of an in-house MALDI-TOF MS database.

Lyme borreliosis (LB) is a multisystemic disease caused by Borrelia burgdorferi sensu lato (sl) complex transmitted to humans by Ixodes ticks. B. burgdorferi sl complex, currently comprising at least 19 genospecies, includes the main pathogenic species responsible for human disease in Europe: B. burgdorferi sensu stricto (ss), B. afzelii, and B. garinii. In this study, for the first time, MALDI-TOF MS was applied to Borrelia spp., supplementing the existing database, limited to the species B. burgdorferi ss, B . spielmanii and B. garinii, with the species B. afzelii, in order to enable the identification of all the species potentially implicated in LB in Europe. Moreover, we supplemented the database also with B. hermsii, which is the primary cause of tick-borne relapsing fever in western North America, B. japonica, circulating in Asia, and another reference strain of B. burgdorferi ss (B31 strain). The dendrogram obtained by analyzing the protein profiles of the different Borrelia species reflected Borrelia taxonomy, showing that all the species included in the Borrelia sl complex clustered in a unique branch, while Borrelia hermsii clustered separately. In conclusion, in this study MALDI-TOF MS proved a useful tool suitable for identification of Borrelia spp. both for diagnostic purpose and epidemiological surveillance.

Leptospira species and serovars identified by MALDI-TOF mass spectrometry after database implementation

BackgroundLeptospirosis, a spirochaetal zoonotic disease of worldwide distribution, endemic in Europe, has been recognized as an important emerging infectious disease, though yet it is mostly a neglected disease which imparts its greatest burden on impoverished populations from developing countries. Leptospirosis is caused by the infection with any of the more than 230 serovars of pathogenic Leptospira sp. In this study we aimed to implement the MALDI-TOF mass spectrometry (MS) database currently available in our laboratory with Leptospira reference pathogenic (L. interrogans, L. borgpetersenii, L. kirschneri, L. noguchii), intermediate (L. fainei) and saprophytic (L. biflexa) strains of our collection in order to evaluate its possible application to the diagnosis of leptospirosis and to the typing of strains. This was done with the goal of understanding whether this methodology could be used as a tool for the identification of Leptospira strains, not only at species level for diagnostic purposes, but also at serovar level for epidemiological purposes, overcoming the limits of serological and molecular conventional methods. Twenty Leptospira reference strains were analysed by MALDI-TOF MS. Statistical analysis of the protein spectra was performed by ClinProTools software.ResultsThe spectra obtained by the analysis of the reference strains tested were grouped into 6 main classes corresponding to the species analysed, highlighting species-specific protein profiles. Moreover, the statistical analysis of the spectra identified discriminatory peaks to recognize Leptospira strains also at serovar level extending previously published data.ConclusionsIn conclusion, we confirmed that MALDI-TOF MS could be a powerful tool for research and diagnostic in the field of leptospirosis with broad applications ranging from the detection and identification of pathogenic leptospires for diagnostic purposes to the typing of pathogenic and non-pathogenic leptospires for epidemiological purposes in order to enrich our knowledge about the epidemiology of the infection in different areas and generate control strategies.

MALDI-TOF mass spectrometry for the detection and differentiation of Entamoeba histolytica and Entamoeba dispar.

Detection of Entamoeba histolytica and its differentiation from Entamoeba dispar is an important goal of the clinical parasitology laboratory. The aim of this study was the identification and differentiation of E. histolytica and E. dispar by MALDI-TOF MS, in order to evaluate the application of this technique in routine diagnostic practice. MALDI-TOF MS was applied to 3 amebic reference strains and to 14 strains isolated from feces that had been differentiated by molecular methods in our laboratory. Protein extracts from cultures of these strains (axenic cultures for the 3 reference strains and monoxenic cultures for the 14 field isolates) were analyzed by MALDI-TOF MS and the spectra obtained were analyzed by statistical software. Five peaks discriminating between E. histolytica and E. dispar reference strains were found by protein profile analysis: 2 peaks (8,246 and 8,303 Da) specific for E. histolytica and 3 (4,714; 5,541; 8,207 Da) for E. dispar. All clinical isolates except one showed the discriminating peaks expected for the appropriate species. For 2 fecal samples from which 2 strains (1 E. histolytica and 1 E. dispar) out of the 14 included in this study were isolated, the same discriminating peaks found in the corresponding isolated amebic strains were detected after only 12h (E. histolytica) and 24h (E. dispar) of incubation of the fecal samples in Robinson’s medium without serum. Our study shows that MALDI-TOF MS can be used to discriminate between E. histolytica and E. dispar using in vitro xenic cultures and it also could have potential for the detection of these species in clinical samples.

Identification of Dermatophyte Species after Implementation of the In-House MALDI-TOF MS Database

Despite that matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool in the clinical microbiology setting, few studies have till now focused on MALDI-TOF MS-based identification of dermatophytes. In this study, we analyze dermatophytes strains isolated from clinical samples by MALDI-TOF MS to supplement the reference database available in our laboratory. Twenty four dermatophytes (13 reference strains and 11 field isolated strains), identified by both conventional and molecular standard procedures, were analyzed by MALDI-TOF MS, and the spectra obtained were used to supplement the available database, limited to a few species. To verify the robustness of the implemented database, 64 clinical isolates other than those used for the implementation were identified by MALDI-TOF MS. The implementation allowed the identification of the species not included in the original database, reinforced the identification of the species already present and correctly identified those within the Trichophyton mentagrophytes complex previously classified as Trichophyton. tonsurans by MALDI-TOF MS. The dendrogram obtained by analyzing the proteic profiles of the different species of dermatophytes reflected their taxonomy, showing moreover, in some cases, a different clusterization between the spectra already present in the database and those newly added. In this study, MALDI-TOF MS proved to be a useful tool suitable for the identification of dermatophytes for diagnostic purpose.

Preliminary molecular analysis of Clostridium difficile isolates from healthy horses in northern Italy

Clostridium difficile, associated with a wide spectrum of diseases in humans, as well as in several animal species, is an important cause of colitis in adult horses and foals. The aim of this study was to investigate by toxin gene profile and PCR-ribotyping the molecular characteristics of 14 C. difficile strains isolated from 42 faeces of healthy horses. Both toxin genes, tcdA and tcdB, were present in only 1 isolate (7.1%). Six isolates (42.9%) demonstrated tcdA-/tcdB+ genotype, and seven isolates (50.0%) were tcdA-/tcdB-. All strains were binary toxin genes negative (cdtA-/cdtB-). The PCR-positive strains, except for the tcdA+/tcdB+ isolate, tested negative for, in vitro, A and/or B toxins production by EIA. Eleven distinct ribotypes were observed. In conclusion, C. difficile can be present in the normal intestinal flora of healthy adult horses, in addition to foals. These animals could therefore play an important role as potential reservoirs of toxigenic strains.

Evaluation of a modified meropenem hydrolysis assay on a large cohort of KPC and VIM carbapenemase-producing Enterobacteriaceae

Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. The introduction of rapid and sensitive methods for the detection of carbapenemase-producing bacteria is of increasing importance. The carbapenemase production can be detected using non-molecular methods (such as the modified Hodge test, the synergy test, the Carba NP test and the antibiotic hydrolysis assays) and DNA-based methods. In this study, we propose a modified version of a previously described meropenem hydrolysis assay (MHA) by MALDI-TOF MS for the phenotypic detection in 2h of carbapenemase-producing Enterobacteriaceae. The MHA was successfully applied to detect carbapenemase activity in 981 well-characterized Enterobacteriaceae strains producing KPC or VIM carbapenemases, and in 146 carbapenem fully susceptible strains. This assay, applied also to NDM and OXA-48-producing strains and to CRE with resistance mechanisms other than carbapenemase production, has proved to be able to distinguish between carbapenemase-producing and -nonproducing Enterobacteriaceae. As already stated and as observed in our hands, MHA by MALDI-TOF MS analysis is independent from the type of carbapenemases involved, it is faster and easier to perform/interpret than culture-based methods. On the other hand, it cannot detect other carbapenem resistance mechanisms, such as porin alterations and efflux mechanisms.

Identification of different respiratory viruses, after a cell culture step, by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)

In this study matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), a reliable identification method for the diagnosis of bacterial and fungal infections, is presented as an innovative tool to investigate the protein profile of cell cultures infected by the most common viruses causing respiratory tract infections in humans. MALDI-TOF MS was applied to the identification of influenza A and B viruses, adenovirus C species, parainfluenza virus types 1, 2 and 3, respiratory syncytial virus, echovirus, cytomegalovirus and metapneumovirus. In this study MALDI-TOF MS was proposed as a model to be applied to the identification of cultivable respiratory viruses using cell culture as a viral proteins enrichment method to the proteome profiling of virus infected and uninfected cell cultures. The reference virus strains and 58 viruses identified from respiratory samples of subjects with respiratory diseases positive for one of the above mentioned viral agents by cell culture were used for the in vitro infection of suitable cell cultures. The isolated viral particles, concentrated by ultracentrifugation, were used for subsequent protein extraction and their spectra profiles were generated by MALDI-TOF MS analysis. The newly created library allowed us to discriminate between uninfected and respiratory virus infected cell cultures.

MALDI-TOF MS as a new tool for the identification of Dientamoeba fragilis

BackgroundIn this study for the first time, a Dientamoeba fragilis protein profile by MALDI-TOF MS was created in order to identify specific markers for the application of this technology in the laboratory diagnosis of dientamoebiasis. In particular, one D. fragilis reference strain was used to create a reference spectrum and 14 clinical isolates to verify the reliability of the obtained results.ResultsWhile 15 peaks were found to be discriminating between the reference strain and the culture medium used, six peaks, observed in all the 14 strains tested, were considered as markers able to identify D. fragilis.ConclusionsIn our hands, MALDI-TOF MS technology was demonstrated as a useful tool to be used in association with or in replacement of the real-time PCR assay for the identification of D. fragilis used in our laboratory on xenic cultures, due to its accuracy, rapidity and low cost.