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ABSTRACT: The increased incidence and severity of Clostridium difficile infection, particularly in North America and Europe, have brought renewed focus on the most appropriate method to detect C. difficile and/or its toxins in stools. This prospective study evaluated the usefulness of the Illumigene TM C. difficile assay in diagnostic practice for the detection of toxigenic C. difficile DNA in clinical samples. A total of 88 out of 306 stool samples analysed were positive both by Illumigene and the combination of toxigenic C. difficile culture (TC) and immunochromatographic assay (IC) with a concordance of 100%. Of the 218 samples negative by the combination of TC and IC, 204 were negative also by Illumigene with a concordance of 93.57%. In our experience, compared to conventional assays Illumigene assay proved to be easy to perform, accurate and prompt giving results within 1 hour at a cost of 28 euro per sample.
The new microbiologica: official journal of the Italian Society for Medical, Odontoiatric, and Clinical Microbiology (SIMMOC) 01/2013; 36(1):57-63. · 1.00 Impact Factor
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ABSTRACT: 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.
Journal of proteomics 10/2012; · 5.07 Impact Factor
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ABSTRACT: Intestinal spirochaetosis is a human colonic infection due to Brachyspira aalborgi and Brachyspira pilosicoli causing various abdominal complaints. Although the presence of healthy epithelial cells was hypothesized to be essential for the adhesion of spirochaetes to the colonic mucosa, their adhesion to hyperplastic and adenomatous colonic polyps has been observed recently. We report a case of a woman with long-standing abdominal symptoms, in whom spirochaetes were found on the colonic mucosa surrounding an adenocarcinoma in the biopsies collected during eight years of follow-up. Spirochaetes were found attached to normal mucosa, to hyperplastic and to adenomatous polyps, but not to the epithelium of the carcinoma. The rectal biopsy collected during the last follow-up colonoscopy was subjected to histopathology and to a specific examination for brachyspires, demonstrating the presence of B. pilosicoli DNA. This report could stimulate microbiological investigations during the follow-up of colonic polyps in order to explain whether the persistence of abdominal symptoms in such patients could be caused by a colonic spirochaetosis susceptible to eradication by a targeted therapy.
Pathology - Research and Practice 01/2012; 208(3):177-80. · 1.21 Impact Factor
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ABSTRACT: 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.
PLoS ONE 01/2012; 7(10):e48033. · 4.09 Impact Factor
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ABSTRACT: The diagnostic value of a real-time PCR assay for the detection of Cryptosporidium spp. in fecal samples was assessed as compared to the combination of the immunocromatographic assay (IC) and immunofluorescence assay (IF) currently performed in our laboratory for the diagnosis of cryptosporidiosis. On a total of 1040 samples collected from 2006 to 2010 and belonging to 533 patients suspected of having an intestinal parasitosis, Cryptosporidium spp. was detected in 31 samples (belonging to 12 patients) by IC and IF; the real-time PCR assay revealed Cryptosporidium spp. DNA in 5 additional samples for a total of 36 samples (13 patients). The real-time PCR assay exhibited higher sensitivity than IC and IF; however, its application to the diagnosis of cryptosporidiosis should be evaluated by every single laboratory, depending on the availability of trained personnel, financial resources, and the cost/effectiveness related to the prevalence of cryptosporidiosis.
Diagnostic microbiology and infectious disease 05/2011; 70(1):72-7. · 2.45 Impact Factor
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ABSTRACT: Lyme borreliosis (LB) is a multisystemic disease caused by Borrelia burgdorferi sensu lato complex transmitted to humans by Ixodes ticks and whose epidemiology is poorly investigated in Europe. In this study an epidemiologic survey on the prevalence of the causative agent of such infectious disease was performed in the area of Parma (Northern Italy) during 2002-2008. Serum samples belonging to 2336 patients and cerebrospinal fluid samples (belonging to 42 of the same patients) were analyzed for serologic diagnosis of LB. Direct laboratory assays [polymerase chain reaction (PCR) and cultivation] were performed on samples belonging to patients with the clinical suspicion of LB. The seroprevalence was 0.55% considering the subjects with both anti-B. burgdorferi IgG and IgM. The samples tested by culture and PCR were all negative except for a tick removed from the skin of a healthy man. The results suggest that infection by B. burgdorferi in this area quite rarely occurs.
Diagnostic microbiology and infectious disease 03/2011; 70(4):455-60. · 2.45 Impact Factor
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ABSTRACT: 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.
Diagnostic microbiology and infectious disease 07/2010; 67(3):239-45. · 2.45 Impact Factor
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ABSTRACT: 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.
Diagnostic microbiology and infectious disease 11/2009; 66(3):261-7. · 2.45 Impact Factor
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ABSTRACT: Our study aimed to describe the occurrence of imported malaria in a nonendemic area (Parma, Italy) during the period 2000 to 2007, comparing the data obtained by microscopy and molecular assays targeting plasmodial 18S subunit rRNA gene. The prevalence of imported malaria in Parma was 21.8% by microscopy and 22.7% by polymerase chain reaction (PCR). Plasmodium falciparum accounted for 81.1% of the cases, followed by Plasmodium ovale (8.8%), Plasmodium vivax (3.8%), and Plasmodium malariae (1.9%). Mixed infections accounted for 4.4% of the cases. In this study, PCRs proved to be more sensitive and specific than microscopy and changed the picture of malaria epidemiology in Parma, detecting additional cases of malaria undiagnosed by microscopy and allowing speciation of plasmodia in cases misidentified by microscopy. Generally, imported malaria cases reflect the number of immigrants who visit their native countries, in particular, West Africa, explaining the increased prevalence of P. ovale cases among non-P. falciparum infections in Parma.
Diagnostic Microbiology and Infectious Disease 09/2008; 61(4):434-9. · 2.53 Impact Factor
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ABSTRACT: The new Vidia system is a fully automated system based on chemiluminescence and antigen bound to magnetic microparticles, which allows a fast measurement of Toxoplasma gondii-specific immunoglobulin G (IgG) and IgM levels. The analytical performances of the Vidia Toxo IgG and IgM assays were compared with those of the automated Vidas, AxSYM, and Liaison Toxo IgG and IgM assays. The comparative evaluation was performed utilizing 204 frozen sera belonging to 166 subjects and 201 fresh sera collected from 198 subjects. For the Vidia Toxo IgG system, the sensitivities were 100% in both the retrospective and prospective studies, and specificities were 98.39% in the retrospective study and 100% in the prospective study, respectively. The sensitivities of the other three Toxo IgG assays were 100%, and the specificities ranged from 96.77% to 100%. For the Vidia Toxo IgM assay, the sensitivity and specificity were 100% in both the retrospective and prospective studies. The overall sensitivities and specificities of the other three Toxo IgM assays ranged from 80% to 100% and from 99.44% to 100%, respectively. In our study, the Vidia system revealed excellent sensitivity (100% for both IgG and IgM assays) and good specificity (99.25% for IgG and 100% for IgM assays).
Clinical and vaccine immunology: CVI 08/2008; 15(7):1076-9. · 2.37 Impact Factor
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ABSTRACT: This article describes a case of myiasis by Dermatobia hominis diagnosed in a young Italian man returning from a vacation through Brazil. Considering the increasing number of travels to tropical and subtropical areas, clinicians in nonendemic areas must think about the possibility of imported unusual infestations during their daily practice.
Diagnostic Microbiology and Infectious Disease 05/2008; 60(4):417-8. · 2.53 Impact Factor
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ABSTRACT: Entamoeba histolytica infection may have various clinical manifestations. Nine out of ten E. histolytica infections remain asymptomatic, while the remainder become invasive and cause disease. The most common form of invasive infection is amebic diarrhea and colitis, whereas the most common extra-intestinal disease is amebic liver abscess. The underlying reasons for the different outcomes are unclear, but a recent study has shown that the parasite genotype is a contributor. To investigate this link further we have examined the genotypes of E. histolytica in stool- and liver abscess-derived samples from the same patients. Analysis of all 18 paired samples (16 from Bangladesh, one from the United States of America, and one from Italy) revealed that the intestinal and liver abscess amebae are genetically distinct. The results suggest either that E. histolytica subpopulations in the same infection show varying organ tropism, or that a DNA reorganization event takes place prior to or during metastasis from intestine to liver.
PLoS Neglected Tropical Diseases 02/2008; 2(4):e219. · 4.69 Impact Factor
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PLoS Neglected Tropical Diseases 02/2008; 2(6). · 4.69 Impact Factor
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ABSTRACT: Malaria is a protozoan infection caused by parasites of the genus Plasmodium (P. falciparum, P. ovale, P. vivax, P. malariae) that is transmitted from one human to another by female Anopheles mosquitoes. It can be considered a reemerging imported disease in our area because of increasing of movements from endemic countries, and nowadays it is the most common imported infection in Italy. This study describes the occurrence of imported malaria in our area between January 2005 and May 2006.
During 17 months we analysed 170 blood samples belonging to 139 patients (95 foreigners and 44 Italians) with the clinical suspect of malaria. Samples were used to prepare orange acridine and Giemsa stained thin blood films for microscopic observation and to perform an immunochromatographic assay for the detection of specific plasmodia antigens. Molecular assays (nested-PCR and Real-time PCR) were also performed in order to confirm the diagnosis.
Thirty-six cases of malaria were diagnosed: 35 in foreigners coming from Africa and only one in an Italian who lived in Chad. Thirty-three patients were infected by P. falciparum, 1 by P. ovale, 1 by P. vivax, and a mixed infection by P. falciparum, P. ovale and P. malariae was also found.
Malaria is usually associated with travels within areas where the infection is endemic and our data demonstrated that imported malaria in our area has a prevalence of 25.89%.
Acta bio-medica: Atenei Parmensis 01/2008; 78(3):170-5.
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ABSTRACT: Our study reports the detection and identification of intestinal spirochetosis in patients with colonic diseases in a tertiary-care hospital over a 12-year period, and includes a description of all cases we diagnosed.
Our patients (8323) underwent colonoscopy and histopathological examinations including transmission electron microscopy (TEM) and light microscopy. Specimens from patients suspected of intestinal spirochetosis at histopathology (17 patients) underwent microbiological investigation performed by culture and molecular methods (16S restriction fragment length polymorphism-polymerase chain reaction [RFLP-PCR], nox RFLP-PCR assays).
Seventeen cases were diagnosed: seven patients were infected by B. aalborgi, one by B. pilosicoli, two by both species and four by Brachyspira spp. diagnosed both histopathology and microbiology (culture and molecular methods: 16S RFLP-PCR and nox RFLP-PCR assays). Three cases were referred to as Brachyspira spp. infections using only histopathology, including TEM.
Our results demonstrated that intestinal spirochetosis, although rarely occurring, might play a role in chronic diarrhea and suggested a pathogenetic mechanism of intestinal spirochetosis based on the destruction of colonic microvilli and colitis histologically documented, providing additional clinical and pathological information on this entity. This study suggests that metronidazole seems to be the drug of choice for the eradication of intestinal spirochetosis.
Journal of Gastroenterology and Hepatology 12/2007; 22(11):1772-9. · 2.87 Impact Factor
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ABSTRACT: The aim of this study was to investigate the occurrence of human intestinal spirochetosis (IS) by a 16S rRNA restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) in a selected group (234) of patients with gastrointestinal complaints and/or potential risk factors for IS in comparison with the occurrence of infections by other enteropathogenic agents. By using 16S rRNA RFLP-PCR, 16 patients (6.8%) with IS were found (11 infected by Brachyspira aalborgi, 3 by Brachyspira pilosicoli, and 2 by both species); moreover, 10 patients with gastroenteric viruses (4.2%), 13 with enteropathogenic bacteria other than intestinal spirochetes (5.5%), and 24 with intestinal parasites (10.2%) were found. This study provides an enhancement of the knowledge about the distribution of IS, suggesting that it may be more frequent than suspected and that clinicians should consider IS when patients present with long-standing diarrhea. Moreover, 16S rRNA RFLP-PCR might be a powerful tool not only for diagnostic purpose but also to investigate the occurrence of IS just on fecal samples, not requiring invasive diagnostic techniques.
Diagnostic Microbiology and Infectious Disease 11/2007; 59(2):157-63. · 2.53 Impact Factor
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ABSTRACT: Human intestinal spirochaetosis (HIS) is a large bowel infection characterised by the colonization of the intestinal mucosa by spirochaetes belonging to the genus Brachyspira. The causative agents of HIS are Brachyspira aalborgi and Brachyspirapilosicoli. Symptoms of the infection, even if not specific, are long standing diarrhoea, abdominal pain, meteorism and rectal bleeding and sometimes they can suggest the clinical suspect of inflammatory bowel diseases or rectal carcinoma. Since poor data were available on the prevalence of this infection, the aim of our study was to describe the occurrence of this infection in our area in the period 2002-2005.
During a period of 4 years we analysed 297 faecal samples from 99 patients selected by potential risk factors and symptomatology suspected for HIS. The diagnosis of HIS was performed by isolation and a molecular assay based on 16S rDNA restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR).
From 2002 to 2005 we detected 12 cases of intestinal spirochaetosis, 7 caused by Brachyspira aalborgi, 4 by Brachyspirapilosicoli and one by both spirochaetes, which represented the first case of a mixed infection by 2 intestinal spirochaetes in our area.
Despite the fact that HIS seems to be a low prevalence infection in our area, in a strongly selected population we found 12 cases of this infection (12.12%). These results stimulate us to extend the research of intestinal spirochaetosis in the general population, when long standing gastrointestinal disorders and potential risk factors are present.
Acta bio-medica: Atenei Parmensis 09/2007; 78(2):128-32.
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ABSTRACT: The present study reports on a prompt diagnosis of colonic amoebiasis with colonic spirochetosis by Brachyspira aalborgi and B. pilosicoli; such diagnosis allowed exclusion of other diseases and resolution of the case after specific treatment.
A 37-year-old Italian man with a history of several months' mucosal diarrhea travelled to Greece, Romania and Tunisia. After his last trip he presented with an increase of up to 3-5 discharges daily, associated with bloody diarrhea, supporting the clinical suspect of inflammatory bowel disease. Colonoscopy revealed erosions from the cecum to the rectum, and ulcers both in the descending and sigmoid colon. Structures resembling amoebic trophozoites and sinusoidal microorganisms were observed in the colonic biopsies at histopathology and electron microscopy. Entamoeba histolytica DNA was detected by small-subunit rDNA polymerase chain reaction (PCR) from feces, rectal biopsies and isolated trophozoites. Spirochetes were identified from feces, colonic biopsies and cultures using a 16S rDNA restriction fragment length polymorphism-PCR specific for the detection of B. aalborgi and B. pilosicoli. After therapy, the patient was restored to health.
The rapid identification of E. histolytica, B. aalborgi and B. pilosicoli using traditional and specific and sensitive molecular methods permitted an accurate diagnosis and a specific therapy. It is suggested that mixed infection by parasites and spirochetes might occur more frequently than expected: it would be of extreme interest and importance to intensify clinical findings, and one infection should not prompt the pathologist/clinician to stop looking.
Journal of Gastroenterology and Hepatology 02/2007; 22(1):64-7. · 2.87 Impact Factor
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ABSTRACT: Intestinal parasitosis represent a relevant clinical problem, especially in developing countries, where they are responsible for morbidity and mortality in adults and children and many epidemiological data are available for these areas. The actual situation of intestinal parasitosis in Europe is not yet well investigated since they are usually not notified. We describe the occurrence of intestinal parasitosis in our laboratory from January to December 2005.
We considered all patients (1117) whose stool samples were sent to our laboratory with the suspect of intestinal parasitosis during the year 2005. Each specimen was subjected to macroscopic and microscopic examination to demonstrate the presence of worm eggs, larvae, protozoan trophozoites or cysts and to an immunochromatographic assay to detect Giardia intestinalis and Cryptosporidium spp. specific antigens. Cultures for protozoa and helminths were carried out and a PCR specific for Entamoeba histolytica/Entamoeba dispar was also performed.
Our results indicated that 148 patients (13.24%) were affected by intestinal parasitosis. Among the 951 Italians, 96 (10%) were infected, while out of a total of 166 foreigners 52 had intestinal parasitosis (31%). Moreover, we found that 113 infections were caused by only one parasite while 35 were mixed infections.
Intestinal parasitosis represent a remarkable cause of gastrointestinal disease and our study demonstrates that these infections are quite common in our area, affecting both Italians and non European citizens from developing countries.
Acta bio-medica: Atenei Parmensis 01/2007; 77(3):147-51.
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ABSTRACT: In recent years, the diagnosis of toxoplasmosis has been improved by Real-time PCR assays. In this study we compared the performances of two Real-time PCRs (FRET and TaqMan protocols) already described in the literature, and one nested-PCR, currently used in our laboratory for the molecular diagnosis of toxoplasmosis.
We evaluated the sensitivity and the specificity of a FRET- and a TaqMan-based Real-time PCRs targeting a 529 bp repeat region and the 18S RNA gene, respectively, and a nested-PCR, targeting the B1-gene of Toxoplasma gondii. We also tested, through nested-PCR, 46 biological samples obtained during a period of 29 months from pregnant women or immunocompromised patients with suspected T. gondii infection.
The analytical sensitivity of nested and TaqMan PCRs was approximately 10(3) tachyzoites/ml. FRET assay showed a sensitivity of 102 tachyzoites/ml. Three out of 46 biological samples were nested-PCR-positive and these results were also confirmed by both Real-time PCRs.
Nested- and real-time PCRs evaluated in this study resulted very sensitive and specific; in particular FRET PCR resulted more sensitive than the other assays, probably because of the greater copy number of the target sequence. Real-time PCR assays are easy-to-use, producing results faster than conventional PCR systems and reducing contamination risks.
Acta bio-medica: Atenei Parmensis 09/2006; 77(2):75-80.
