Sabina Rossi

Evaluation of a real-time polymerase chain reaction assay for the laboratory diagnosis of giardiasis.

A real-time polymerase chain reaction (PCR) assay was evaluated in comparison with the combination of conventional methods (microscopic examination and antigen detection assay) during the period 2006 to 2008 on 771 fecal samples belonging to 386 patients to assess its usefulness for an accurate laboratory diagnosis of giardiasis. The real-time PCR assay detected Giardia intestinalis DNA in 195 samples (106 patients), including 26 samples (21 patients) negative by the conventional assays. Among the 21 patients, in 8 cases, giardiasis was previously diagnosed also by conventional methods in additional samples of the same patients, whereas in 13, it would have been undiagnosed if real-time PCR assay was not used. The real-time PCR assay demonstrated a detection limit of 2 cysts per reaction and 100% specificity and sensitivity compared to conventional methods. A genotype analysis targeting the beta-giardin gene allowed to identify 53 samples (23 patients) containing genotype A and 59 samples (45 patients) containing genotype B.

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.

Accurate identification of the six human Plasmodium spp. causing imported malaria, including Plasmodium ovale wallikeri and Plasmodium knowlesi

BackgroundAccurate identification of Plasmodium infections in non-endemic countries is of critical importance with regard to the administration of a targeted therapy having a positive impact on patient health and management and allowing the prevention of the risk of re-introduction of endemic malaria in such countries. Malaria is no longer endemic in Italy where it is the most commonly imported disease, with one of the highest rates of imported malaria among European non-endemic countries including France, the UK and Germany, and with a prevalence of 24.3% at the University Hospital of Parma. Molecular methods showed high sensitivity and specificity and changed the epidemiology of imported malaria in several non-endemic countries, highlighted a higher prevalence of Plasmodium ovale, Plasmodium vivax and Plasmodium malariae underestimated by microscopy and, not least, brought to light both the existence of two species of P. ovale (Plasmodium ovale curtisi and Plasmodium ovale wallikeri) and the infection in humans by Plasmodium knowlesi, otherwise not detectable by microscopy.MethodsIn this retrospective study an evaluation of two real-time PCR assays able to identify P. ovale wallikeri, distinguishing it from P. ovale curtisi, and to detect P. knowlesi, respectively, was performed applying them on a subset of 398 blood samples belonging to patients with the clinical suspicion of malaria.ResultsThese assays revealed an excellent analytical sensitivity and no cross-reactivity versus other Plasmodium spp. infecting humans, suggesting their usefulness for an accurate and complete diagnosis of imported malaria. Among the 128 patients with malaria, eight P. ovale curtisi and four P. ovale wallikeri infections were detected, while no cases of P. knowlesi infection were observed.Discussion and conclusionsReal-time PCR assays specific for P. ovale wallikeri and P. knowlesi were included in the panel currently used in the University Hospital of Parma for the diagnosis of imported malaria, accomplishing the goal of adhering to the recommendations of the World Health Organization to countries that are malaria-free to include the improvement of the early diagnosis of all cases of imported malaria.

Evaluation of a real-time polymerase chain reaction assay for the detection of Dientamoeba fragilis ☆

The diagnostic value of a real-time polymerase chain reaction (PCR) assay targeting the 5.8S rDNA of Dientamoeba fragilis was investigated as compared with conventional parasitologic methods including cultivation testing 959 fecal samples from 491 patients attending a tertiary-care hospital and suspected of having an intestinal parasitosis. The real-time PCR assay revealed 117 additional D. fragilis-positive samples as compared with conventional methods, showing 100% sensitivity and specificity in our experience. On the whole, D. fragilis infection was detected in 186 samples from 105 patients (21.4%, third in frequency among the diagnosed intestinal parasitoses). The evaluated real-time PCR assay represents an effective tool to obtain both an accurate diagnosis and a reliable epidemiologic picture of dientamoebiasis.

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.

A New Real-Time PCR for the Detection of Plasmodium ovale wallikeri

It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasites: P. ovale curtisi and P. ovale wallikeri. We have extended and optimized a Real-time PCR assay targeting the parasite’s small subunit ribosomal RNA (ssrRNA) gene to detect both these species. When the assay was applied to 31 archival blood samples from patients diagnosed with P. ovale, it was found that the infection in 20 was due to P. ovale curtisi and in the remaining 11 to P. ovale wallikeri. Thus, this assay provides a useful tool that can be applied to epidemiological investigations of the two newly recognized distinct P. ovale species, that might reveal if these species also differ in their clinical manifestation, drugs susceptibility and relapse periodicity. The results presented confirm that P. ovale wallikeri is not confined to Southeast Asia, since the majority of the patients analyzed in this study had acquired their P. ovale infection in African countries, mostly situated in West Africa.

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.

Intestinal parasitoses in a tertiary-care hospital located in a non-endemic setting during 2006–2010

BackgroundThe aim of this study was to assess the epidemiology of intestinal parasitoses during a 5-year period in patients attending a tertiary-care hospital in a non-endemic setting.MethodsIn the period 2006–2010, 15,752 samples from 8,886 patients with clinically suspected parasitosis were subjected to macroscopic and microscopic examination, to parasitic antigen detection assays, and to cultures for protozoa and nematodes. Real-time PCR assays for the differentiation of Entamoeba histolytica and E. dispar and for the detection of Dientamoeba fragilis were also used.A statistical analysis evaluating the demographic data of the patients with intestinal parasitic infections was performed.ResultsIntestinal parasitic infections were diagnosed in 1,477 patients (16.6% prevalence), mainly adults and immigrants from endemic areas for faecal-oral infections; protozoa were detected in 93.4% and helminths in 6.6% of the cases, the latter especially in immigrants. Blastocystis hominis was the most common intestinal protozoan, and G. intestinalis was the most frequently detected among pathogenic protozoa, prevalent in immigrants, males, and pediatric patients. Both single (77.9%) and mixed (22.1%) parasitic infections were observed, the latter prevalent in immigrants.ConclusionsDespite the importance of the knowledge about the epidemiology of intestinal parasitoses in order to adopt appropriate control measures and adequate patient care all over the world, data regarding industrialized countries are rarely reported in the literature. The data presented in this study indicate that intestinal parasitic infections are frequently diagnosed in our laboratory and could make a contribution to stimulate the attention by physicians working in non-endemic areas on the importance of suspecting intestinal parasitoses.

A 22-Year Survey of Leishmaniasis Cases in a Tertiary-Care Hospital in an Endemic Setting

The northward spread of leishmaniasis from Mediterranean to Continental Europe affects our area where it is typically associated with Leishmania infantum infection. In this study a 22-year survey was performed in patients (including both patients with and without history of travel through endemic areas other than Italy) attending the University Hospital of Parma, Northern Italy, in order to make a contribution to describe the cases of the visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) diagnosed in this area. One hundred fifty-six samples from 134 patients with clinical suspicion of leishmaniasis (96 suspected of having VL, 37 CL and one both VL and CL) were analyzed in our laboratory during 1992–2013 by microscopy, culture and, from 2005, also by real-time PCR. Leishmania spp. were detected in 23 samples of 15 patients (seven with VL and eight with CL), representing an infection rate of 11.2%. The figure of the cases of leishmaniasis herein reported, even if not comparable to that described for Italian areas other than Parma, underlines that suitable tools are mandatory for correct diagnosis. Moreover, the severity of this disease, particularly VL with its documented northward spread, requires physicians of continental Europe to increase their attention about the possibility of suspecting leishmaniasis in patients reporting related signs and symptoms and/or risk factors.