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

Background: Leptospirosis, 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. Results: The 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. Conclusions: In 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

Periostin improves cell adhesion to implantable biomaterials and osteoblastic differentiation on implant titanium surfaces in a topography-dependent fashion.

Periostin is a matricellular protein highly expressed in periodontal ligament and periostium and has been shown to be required for tissue development and maintenance. We showed that the adhesion of murine osteoblastic MC3T3 cells to thiolated hyaluronic acid/polyethyleneglycol hydrogels was greatly improved by enrichment with periostin. Polished or sand-blasted/acid-etched (SLA) commercially pure titanium surfaces were also coated with this protein and periostin ameliorated cell adhesion and dramatically affected cell morphology on both surfaces, as assessed at fluorescence microscopy, scanning electron microscopy, and chemiluminescence-based viability assay. Moreover, periostin increased the expression of alkaline phosphatase, osteoprotegerin, connective tissue growth factor, collagen 1a1, osteocalcin, Runx2, and osterix transcription factors on smooth surfaces. However, it did not affect, or even decreased, the expression of these genes on SLA discs. Transcript levels for connexin 43 were greatly increased on both surfaces in the presence of periostin. Taken together, these results show that periostin coatings can be a viable approach to improve cell adhesion and differentiation on implantable biomaterials

MALDI-TOF mass spectrometry as a potential tool for Trichomonas vaginalis identification

Background: Trichomonas vaginalis is a flagellated protozoan causing trichomoniasis, a sexually transmitted human infection, with around 276.4 million new cases estimated by World Health Organization. Culture is the gold standard method for the diagnosis of T. vaginalis infection. Recently, immunochromatographic assays as well as PCR assays for the detection of T. vaginalis antigen or DNA, respectively, have been also available. Although the well-known genome sequence of T. vaginalis has made possible the application of proteomic studies, few data are available about the overall proteomic expression profiling of T. vaginalis. The aim of this study was to investigate the potential application of MALDI-TOF MS as a new tool for the identification of T. vaginalis. Methods: Twenty-one isolates were analysed by MALDI-TOF MS after the creation of a Main Spectrum Profile (MSP) from a T. vaginalis reference strain (G3) and its subsequent supplementation in the Bruker Daltonics database, not including any profile of protozoa. This was achieved after the development of a new identification method created by modifying the range setting (6-10 kDa) for the MALDI-TOF MS analysis in order to exclude the overlapping of peaks derived from the culture media used in this study. Results: Two MSP reference spectra were created in 2 different range: 3-15 kDa (standard range setting) and 6-10 kDa (new range setting). Both MSP spectra were deposited in the MALDI BioTyper database for further identification of additional T. vaginalis strains. All the 21 strains analysed in this study were correctly identified by using the new identification method. Conclusions: In this study it was demonstrated that changes in the MALDI-TOF MS standard parameters usually used to identify bacteria and fungi allowed the identification of the protozoan T. vaginalis. This study shows the usefulness of MALDI-TOF MS in the reliable identification of microorganism grown on complex liquid media such as the protozoan T. vaginalis, on the basis of the proteic profile and not on the basis of single markers, by using a "new range setting" different from that developed for bacteria and fungi

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

Sviluppo di un database per l’identificazione di Borrelia spp. mediante spettrometria di massa MALDI-TOF.

Le borrelie sono batteri gram-negativi microaerofili a lenta crescita trasmessi all'uomo mediante il morso di un artropode vettore. Il complesso Borrelia burgdorferi sensu lato comprende gli agenti eziologici di Borreliosi di Lyme (LB), una malattia sistemica trasmessa all'uomo dal morso di una zecca dura. In Europa le specie responsabili della LB sono: B. burgdorferi sensu stricto (ss), B. afzelii e B. garini. In questo studio la spettrometria di massa con tecnologia MALDI-TOF è stata applicata all’identificazione di borrelie per l’implementazione del database dello strumento in uso nel nostro laboratorio, già comprendente le specie Borrelia burgdorferi ss, B. garini e B. spielmanii, con B. afzelii, unica specie mancante delle tre agenti eziologici di LB in Europa. Sono stati inoltre inclusi nello studio 3 ceppi di riferimento appartenenti alle specie B. hermsii, responsabile di febbre ricorrente in Nord America, B. japonica, circolante in Asia, e B. burgdorferi ss (B31) per rendere più accurate future applicazioni volte all’identificazione di borrelie. E' stato inoltre possibile verificare l'efficacia del database creato identificando un ceppo di isolamento clinico (UCSC) precedentemente caratterizzato mediante studi biochimici e molecolari come B. burgdorferi ss. L'analisi mediante MALDI-TOF MS ha prodotto per 3 dei ceppi di riferimento analizzati (B. afzelii, B. hermsii e B. japonica) un profilo proteico specifico non riconducibile a nessuno dei profili presenti nel database dello strumento. Il profilo proteico ottenuto dal ceppo di riferimento B. burgdorferi ss (B31) è stato correttamente associato a quello del ceppo di B. burgdorferi ss già presente nel database, così come per il ceppo di isolamento clinico UCSC. Infine, dagli spettri ottenuti mediante MALDI-TOF è stato possibile creare un dendrogramma che riflette la separazione delle specie appartenenti al complesso Borrelia burgdorferi sensu lato da B. hermsii che si posiziona in un gruppo separato. I risultati riportati in questo studio dimostrano che la tecnologia MALDI-TOF MS è utile sia a fini diagnostici per il rilevamento e l'identificazione di specie di Borrelia sia a scopo epidemiologico per la eventuale realizzazione di strategie di sorveglianza

Diagnostic performances of antigen detection compared to conventional and nucleic acid detection of Entamoeba histolytica in a non-endemic setting

This study evaluated the immunochromatographic (IC) assay "TECHLAB® E. HISTOLYTICA QUIK CHEK™" analysing 36 faecal samples and 7 cultured strains. This assay was compared to the methods performed in our laboratory for the diagnosis of amoebiasis. The IC assay revealed a detection limit of 103 trophozoites/g faeces and no cross-reactivity with other parasites and failed to detect E. histolytica antigen in frozen faeces. In our laboratory located in a non-endemic setting this assay could not replace the methods currently used for the diagnosis of amoebiasis

Anisakidae: i primi due rilevamenti a Parma nel 2013.

L'anisakiasi è un'infestazione sostenuta dai nematodi delle specie Anisakis simplex e Pseudoterranova decipiens; nell’uomo è caratterizzata dalla localizzazione di larve in stadio avanzato di maturazione nella parete dello stomaco e dell'intestino, oppure nella cavità addominale o in altri siti extraintestinali. La trasmissione all'uomo è mediata dall'assunzione di pesci di mare o di cefalopodi crudi o poco cotti contenenti le larve al terzo stadio (L3) del parassita. In Italia i casi segnalati sono rari, riguardanti principalmente soggetti immigrati; il ridotto numero di casi è il risultato di efficaci controlli sanitari nel pesce e dell'ancora limitata abitudine al consumo di pesce crudo nel nostro paese. In questo studio sono riportati i primi due casi di ritrovamento di larve di Anisakidi a Parma a distanza di soli 5 mesi nel corso del 2013. Nel primo caso, un nematode è stato rinvenuto nelle carni di un pesce sottoposto dapprima a congelamento e poi a cottura. Nel secondo caso l'elminta vitale è stato rimosso dal cavo orale del paziente che riportava un pasto recente con pesce crudo. L'elminta del primo caso si presentava di colore rossastro, di dimensioni pari a 3,5cm x 1,5mm. L'elminta del secondo caso si presentava di colore rosa-biancastro, di dimensioni pari a circa 1,8cm x 1,5mm. Per entrambi i reperti erano visibili 3 labbra prominenti all'estremità prossimale e una cuticola esterna dotata di striatura. Le caratteristiche presentate hanno permesso di identificare il primo nematode come una larva L3 di Pseudoterranova decipiens e il secondo come una larva L3 di Anisakis simplex. I dati riportati si rivelano di particolare importanza in quanto il ritrovamento di larve di anisakidi nel nostro paese è un fenomeno relativamente recente ed assume una particolare importanza in considerazione del fatto che l'abitudine al consumo di pesce marino crudo, un tempo diffusa in altri paesi, si sta sempre più estendendo anche in Itali

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

<div><p>Carbapenem-resistant <i>Enterobacteriaceae</i> (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 <i>Enterobacteriaceae</i>. The MHA was successfully applied to detect carbapenemase activity in 981 well-characterized <i>Enterobacteriaceae</i> 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 <i>Enterobacteriaceae</i>.</p><p>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.</p></div

Identification of <i>Borrelia</i> Species after Creation of an In-House MALDI-TOF MS Database

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

Frequency of the carbapenemase activity values of 180 strains analysed with the degraded drug.

<p>In brackets, the percentage of each frequency range calculated on the total of 180 strains are indicated.</p