Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis.

Leptospira biflexa is a free-living saprophytic spirochete present in aquatic environments. We determined the genome sequence of L. biflexa, making it the first saprophytic Leptospira to be sequenced. The L. biflexa genome has 3,590 protein-coding genes distributed across three circular replicons: the major 3,604 chromosome, a smaller 278-kb replicon that also carries essential genes, and a third 74-kb replicon. Comparative sequence analysis provides evidence that L. biflexa is an excellent model for the study of Leptospira evolution; we conclude that 2052 genes (61%) represent a progenitor genome that existed before divergence of pathogenic and saprophytic Leptospira species. Comparisons of the L. biflexa genome with two pathogenic Leptospira species reveal several major findings. Nearly one-third of the L. biflexa genes are absent in pathogenic Leptospira. We suggest that once incorporated into the L. biflexa genome, laterally transferred DNA undergoes minimal rearrangement due to physical restrictions imposed by high gene density and limited presence of transposable elements. In contrast, the genomes of pathogenic Leptospira species undergo frequent rearrangements, often involving recombination between insertion sequences. Identification of genes common to the two pathogenic species, L. borgpetersenii and L. interrogans, but absent in L. biflexa, is consistent with a role for these genes in pathogenesis. Differences in environmental sensing capacities of L. biflexa, L. borgpetersenii, and L. interrogans suggest a model which postulates that loss of signal transduction functions in L. borgpetersenii has impaired its survival outside a mammalian host, whereas L. interrogans has retained environmental sensory functions that facilitate disease transmission through water.

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.

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.

Comparison between two real-time PCR assays and a nested-PCR for the detection of Toxoplasma gondii.

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

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.

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.