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.

Infective colitis associated with human intestinal spirochetosis.

AIM: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Human intestinal spirochaetosis in Parma: a focus on a selected population during 2002-2005.

BACKGROUND AND AIM OF THE WORK: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Colonic amoebiasis and spirochetosis: morphological, ultrastructural and microbiological evaluation.

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Entamoeba histolytica and Entamoeba dispar: comparison of two PCR assays for diagnosis in a non-endemic setting.

Detection of Entamoeba histolytica, the causative agent of amoebiasis, is an important goal of the clinical parasitology laboratory. The identification of Entamoeba dispar as a morphologically identical but non-pathogenic species has highlighted the need for non-microscopic detection methods able to differentiate between the two organisms. In this study we evaluated the utility of conventional PCR and real-time PCR as methods for identification and differentiation of E. histolytica and E. dispar. The second aim of this study was to determine the relative proportions of infections caused by E. histolytica and the non-pathogenic E. dispar, allowing a picture of the epidemiological situation in a non-endemic setting to be obtained. One hundred and sixty-six clinical samples (faecal and liver abscess samples and one intestinal biopsy) belonging to 108 patients were analysed. More patients with E. dispar infection (8.3%) than patients with E. histolytica infection (5.6%) were found by both PCR assays. It is concluded that routine diagnosis of invasive amoebiasis performed by a combination of microscopy, culture and serology should be complemented with a PCR assay such as real-time PCR that offers a practical and clinically acceptable alternative for rapid and accurate diagnosis of amoebic infection in patients presenting with symptoms indicative of this disease.

Prevalence of intestinal parasites in the area of Parma during the year 2005.

BACKGROUND AND AIM OF THE WORK: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Rapid detection and identification of Brachyspira aalborgi from rectal biopsies and faeces of a patient.

This study reports for the first time the detection of Brachyspira aalborgi in faeces and rectal biopsies of a female suffering for 3-4 months of abdominal pain with long-standing mucosal diarrhoea, rectal bleeding and suspected carcinoma of the rectum. After pre-treatment of samples (faeces and biopsies) with a liquid medium (trypticase soy broth-TSB) containing foetal calf serum (FCS, 10%) and spectinomycin and rifampicin (TSB-SR) the first detection of B. aalborgi isolate HBS1 was observed after 48 h in the primary plates of selective blood agar modified medium (BAM) containing spectinomycin and rifampicin (BAM-SR), where growth zones were signalled by a small weakly beta-haemolytic halo. Attempts to subculture spirochaetes in agar media failed. The new HBS1 isolate was only propagated in TSB broth and at electron microscopy it showed 4 endoflagella inserted at each tapered end. The phenotypic characterization of HBS1 demonstrated absence of hippurate hydrolysis, indole production, alpha-galactosidase, alpha- and beta-glucosidase activities in accordance with the B. aalborgi type strain. Rapid identification of B. aalborgi isolate HBS1 was performed directly from faeces and rectal biopsies and subsequently from pure cultures by a genetic method based on 16S DNA restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR). The sequence of 16S DNA amplicon of the isolate HBS1 was found 99.2% corresponding to that of the B. aalborgi type strain. Our results encourage further investigations for the development of a suitable selective agar medium for the isolating and cultivating B. aalborgi from human specimens.