Reduced diversity of faecal microbiota in Crohn's disease revealed by a metagenomic approach.

BACKGROUND AND AIM: A role for the intestinal microbial community (microbiota) in the onset and chronicity of Crohn's disease (CD) is strongly suspected. However, investigation of such a complex ecosystem is difficult, even with culture independent molecular approaches. METHODS: We used, for the first time, a comprehensive metagenomic approach to investigate the full range of intestinal microbial diversity. We used a fosmid vector to construct two libraries of genomic DNA isolated directly from faecal samples of six healthy donors and six patients with CD. Bacterial diversity was analysed by screening the two DNA libraries, each composed of 25,000 clones, for the 16S rRNA gene by DNA hybridisation. RESULTS: Among 1190 selected clones, we identified 125 non-redundant ribotypes mainly represented by the phyla Bacteroidetes and Firmicutes. Among the Firmicutes, 43 distinct ribotypes were identified in the healthy microbiota, compared with only 13 in CD (p<0.025). Fluorescent in situ hybridisation directly targeting 16S rRNA in faecal samples analysed individually (n=12) confirmed the significant reduction in the proportion of bacteria belonging to this phylum in CD patients (p<0.02). CONCLUSION: The metagenomic approach allowed us to detect a reduced complexity of the bacterial phylum Firmicutes as a signature of the faecal microbiota in patients with CD. It also indicated the presence of new bacterial species.

CandidaDB: a genome database for Candida albicans pathogenomics.

CandidaDB is a database dedicated to the genome of the most prevalent systemic fungal pathogen of humans, Candida albicans. CandidaDB is based on an annotation of the Stanford Genome Technology Center C.albicans genome sequence data by the European Galar Fungail Consortium. CandidaDB Release 2.0 (June 2004) contains information pertaining to Assembly 19 of the genome of C.albicans strain SC5314. The current release contains 6244 annotated entries corresponding to 130 tRNA genes and 5917 protein-coding genes. For these, it provides tentative functional assignments along with numerous pre-run analyses that can assist the researcher in the evaluation of gene function for the purpose of specific or large-scale analysis. CandidaDB is based on GenoList, a generic relational data schema and a World Wide Web interface that has been adapted to the handling of eukaryotic genomes. The interface allows users to browse easily through genome data and retrieve information. CandidaDB also provides more elaborate tools, such as pattern searching, that are tightly connected to the overall browsing system. As the C.albicans genome is diploid and still incompletely assembled, CandidaDB provides tools to browse the genome by individual supercontigs and to examine information about allelic sequences obtained from complementary contigs. CandidaDB is accessible at http://genolist.pasteur.fr/CandidaDB.

CAAT-Box, Contigs-Assembly and Annotation Tool-Box for genome sequencing projects.

MOTIVATION: Contigs-Assembly and Annotation Tool-Box (CAAT-Box) is a software package developed for the computational part of a genome project where the sequence is obtained by a shotgun strategy. CAAT-Box contains new tools to predict links between contigs by using similarity searches with other whole genome sequences. Most importantly, it allows annotation of a genome to commence during the finishing phase using a gene-oriented strategy. For this purpose, CAAT-Box creates an Individual Protein file (IPF) for each ORF of an assembly. The nucleotide sequence reported in an IPF corresponds to the sequence of the ORF with 500 additional bases before the ORF and 200 bases after. For annotation, additional information like Blast results can be added or linked to the IPFs as well as automatic and/or manual annotations. When a new assembly is performed, CAAT-Box creates new IPFs according to the old IPF panel. CAAT-Box recognizes the modified IPFs which are the only ones used for a new automatic analysis after each assembly. Using this strategy, the user works with a group of IPFs independently of the closure phase progression. The IPFs are accessible by a web server and can therefore be modified and commented by different groups. RESULT: CAAT-Box was used to obtain and to annotate several complete genomes like Listeria monocytogenes or Streptococcus agalactiae. AVAILABILITY: The program may be obtained from the authors and is freely available to non-profit organisations.

Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Lessons from a multicentre study of the detectability of viral genomes based on a two-round quality control of GB virus C (GBV-C)/hepatitis G virus (HGV) polymerase chain reaction assay.

The aim of this study was to determine whether multicentre quality controls for the detectability of viral genomes could contribute to the improvement of diagnostic performance in the participating laboratories. The study was carried out during two successive rounds, during which 18 laboratories specialized in nucleic acid testing analyzed, through a polymerase chain reaction (PCR) assay, a common panel of GB virus C (GBV-C)/hepatitis G virus (HGV) RNA-positive and -negative samples. During the first round, the laboratories used either an 'in-house' PCR procedure or a partly standardized commercial test. After decoding the results of the first round, the procedures of the participating laboratories were compared in order to establish a consensus procedure deduced from those of the laboratories which provided the best results. During the second round, each participating laboratory could use the resulting consensus procedure, or its own procedure, or both. The results of this quality control study indicated that, whatever method used, even specialized and trained laboratories may give false-negative or false-positive results. The commercial assay did not guarantee a systematic high quality level of results. The striking heterogeneity of results observed among laboratories using the same commercial assay confirm that molecular biology methods need skilled technicians. The results of this quality control study suggest that full standardization of viral genome detection, including all steps of the procedure, is necessary and that the laboratories performing PCR should participate in repeated quality control studies, whatever technique is being used.

The 102-kilobase pgm locus of Yersinia pestis: sequence analysis and comparison of selected regions among different Yersinia pestis and Yersinia pseudotuberculosis strains.

We report the complete 119,443-bp sequence of the pgm locus from Yersinia pestis and its flanking regions. Sequence analysis confirms that the 102-kb unstable pgm locus is composed of two distinct parts: the pigmentation segment and a high-pathogenicity island (HPI) which carries virulence genes involved in iron acquisition (yersiniabactin biosynthetic gene cluster). Within the HPI, three genes coding for proteins related to phage proteins were uncovered. They are located at both extremities indicating that the entire HPI was acquired en bloc by phage-mediated horizontal transfer. We identified, within the pigmentation segment, two novel loci that may be involved in virulence: a fimbriae gene cluster and a locus probably encoding a two component regulatory system similar to the BvgAS regulatory system of Bordetella pertussis. Three genes containing frameshift mutations and two genes interrupted by insertion element insertion were found within this region. To investigate diversity among different Y. pestis and Yersinia pseudotuberculosis strains, the sequence of selected regions of the pgm locus and flanking regions were compared from 20 different Y. pestis and 10 Y. pseudotuberculosis strains. The results showed that the genes interrupted in Y. pestis are intact in Y. pseudotuberculosis. However, one of these mutations, in the bvgS homologue, is only present in Y. pestis strains of biovar Orientalis and not in those of the biovars Antiqua and Medievalis. The results obtained by analysis of variable positions in the sequence are in accordance with historical records, confirming that biovar Orientalis is the most recent lineage. Furthermore, sequence comparisons among 29 Yersinia strains suggest that Y. pestis is a recently emerged pathogen that is probably entering the initial phase of reductive evolution.

Response to interferon alpha treatment and disappearance of cryoglobulinaemia in patients infected by hepatitis C virus.

BACKGROUND: Mixed cryoglobulinaemia is closely associated with hepatitis C virus (HCV) infection. AIM: To assess in a prospective open study the efficiency of interferon alpha treatment of cryoglobulinaemia, as reflected by the disappearance of cryoglobulins and clinical manifestations of the disease, and to analyse the factors predictive of a response to interferon. METHOD: Eighty seven consecutive patients with chronic hepatitis C treated for the first time with interferon at a dose of 3 x 10(6) international units three times a week for six months were studied. Forty three patients had cryoglobulins, which were responsible for clinical manifestations in 12. RESULTS: At the end of interferon treatment, cryoglobulins had disappeared in 39% of the patients. A clinical improvement (except for neuropathies) was observed in all patients. Six months after interferon treatment was stopped, the same rate of response (normal alanine aminotransferase values and undectable HCV RNA) was observed in patients with or without cryoglobulins. Only 14% of patients still had undetectable cryoglobulins, and all of them also had undetectable serum HCV RNA. The disappearance of cryoglobulins was found less frequently in patients with clinical symptoms than in asymptomatic ones, but the difference was not significant. Sustained responders were more often men, infected by genotype 2 or 3, with a lower pretreatment viral load. CONCLUSION: The presence of cryoglobulins does not seem to affect the response to interferon in HCV infected patients. The improvement in cryoglobulinaemia is strongly associated with a virological response, reinforcing the hypothesis of a direct role for HCV in the pathogenesis of this disease.

Comparative evaluation of hepatitis C virus RNA quantitation by branched DNA, NASBA, and monitor assays.

Several studies have shown a relationship between pretreatment hepatitis C virus (HCV) viral load and the response to interferon (IFN) therapy, creating a need for quantitative HCV-RNA assays. Here, we compared three commercial methods: nucleic acid sequence-based amplification NASBA (Organon), branched DNA 2.0 (bDNA) (Chiron), and Monitor (Roche), with reverse-transcription polymerase chain reaction (RT-PCR) as the reference. We assessed sensitivity and reproducibility on a well-characterized panel of sera (EUROHEP), a Chimp Rodney plasma pool, and samples from IFN-treated and -untreated patients with chronic hepatitis C caused by different HCV genotypes. The reproducibility of the NASBA and bDNA methods was slightly better than that of Monitor, especially for genotypes 2 and 4. NASBA had the highest sensitivity (99% vs. 94% and 88% with Monitor and bDNA, respectively), especially for the follow-up of patients on IFN. NASBA gave the highest HCV-RNA concentrations, which were approximately 10-fold more than with the bDNA assay and 100-fold more than with the Monitor kit. The linearity, tested on the chimp Rodney plasma pool, was better with bDNA for high viral load than with NASBA and Monitor, although for low concentration of HCV RNA, bDNA was negative. Pretreatment viral load was lower in patients who had a sustained virological response to IFN, although the bDNA method was not sensitive enough to quantify all pretreatment samples. This study indicates that gene amplification methods (NASBA or Monitor) have better sensitivity than bDNA assays for quantification of HCV RNA in patients with chronic HCV infection, although the bDNA and NASBA methods are more likely to quantify all genotypes. Prospective studies are needed to demonstrate the usefulness of quantitative assays for the follow-up of patients with chronic hepatitis C.

Transfusion-associated or nosocomial hepatitis G virus infection in patients undergoing surgery.

BACKGROUND: Despite blood donor screening, there are still cases of transfusion-associated hepatitis. From 1988 to 1992, a prospective study was conducted on the incidence of non-A, non-B posttransfusion hepatitis (PTH). STUDY DESIGN: The present investigation was designed to determine if transfusion recipients with PTH who are negative for hepatitis C virus (HCV) were positive for hepatitis G virus (HGV). Patients admitted for surgery who had normal liver tests and no transfusions during the previous 6 months were enrolled. Alanine amino transferase levels were determined monthly for 6 months after surgery and for 1 year in the case of PTH (defined as alanine aminotranferase twice the upper limit of normal in two consecutive assays). HGV RNA and E2 antibodies were tested for in samples from transfusion recipients with or without PTH and from nontransfused patients. RESULTS: Of the 308 blood recipients who were enrolled in the study, 21 (6.8%) had PTH. HGV RNA was detected at the onset of hepatitis in 3 patients with PTH (14%), 2 of whom were also anti-HCV and HCV RNA positive. One patient developed E2 antibodies without detectable HGV RNA. Three (10.7%) of 28 recipients of an allogeneic transfusion without PTH developed HGV infection. HGV RNA was also found in two nontransfused patients, which suggests nosocomial transmission of HGV. CONCLUSION: Some cases of PTH are associated with HGV; most cases of postoperative HGV infection are not associated with liver abnormalities; and most PTH cases are not associated with known hepatotropic viruses.

Mutations in NS5A region of hepatitis C virus genome correlate with presence of NS5A antibodies and response to interferon therapy for most common European hepatitis C virus genotypes.

A part of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) amino acid sequence, designated as an interferon (IFN)-sensitive determining region (ISDR), has been shown to be correlated with a response to IFN in Japanese patients. We have shown previously that the presence of NS5A antibodies (Abs) detected by the INNOLIA test (IL-NS5A Ab) is also correlated with a response to IFN. The aim of this study was to investigate, in a wide range of patients, the possible relationship within the NS5A protein between the sequence of ISDR and that used in the INNOLIA test designated as IL3R. Serum samples from 52 patients infected by HCV genotypes 1, 2, and 3 were analyzed before and after treatment. The patients were classified as nonresponders (NRs), responder-relapsers (RRs), or long-term responders (LTRs). We amplified the NS5A region for 42 patients using polymerase chain reaction (PCR), and these amplicons were sequenced directly. The 10 remaining patients were analyzed using PCR with mutation-specific primers. No correlation was found between the IL3R sequence of the HCV strains and the presence of the IL-NS5A Ab for all genotypes. However, for the subtype 1b, only 2 of 11 NR patients tested had an arginin in position 2218 within the ISDR versus 3 of 3 LTR and 10 of 13 RR patients. All patients with R-2218 had IL-NS5A Ab. For the genotype 1a, 2 of 2 LTR and 1 of 3 RR were mutated in position 2216-2218 in comparison to three NR sequences. For the genotype 3, no mutations were found in the region homologous to 1b-ISDR, but 4 of 5 LTR and RR patients had a mutation T-2161 to A or V versus 0 of 3 NR patients. A close correlation was found between arginin in position 2218 in ISDR, the presence of IL-NS5A Ab, and the response to IFN therapy for genotype 1b, but this association did not predict a long-term response. For genotype 3, a potential ISD mutation could be located at the codon 2161.

Pattern of HCV antibodies with special reference to NS5A reactivity in HCV-infected patients: relation to viral genotype, cryoglobulinemia and response to interferon.

BACKGROUND/AIMS: We aimed to compare the anti-hepatitis C virus reactivity in confirmatory assays (RIBA 3.0 Ortho Diagnostic and INNO-LIA HCV Ab III Innogenetics) among patients infected with different hepatitis C virus genotypes, with or without cryoglobulinemia, and in patients treated with interferon. METHODS: One hundred and three patients followed in our hepatogastroenterology unit were included in the study and compared to 320 consecutive patients tested using RIBA 3.0. Seventy-nine of the 103 patients were treated with interferon. Long-term responders to interferon were defined as having normal alanine aminotransferase levels and being HCV RNA negative 6 months after the end of treatment. Initial responders were defined as having normal alanine aminotransferase levels at the end of interferon therapy but abnormal alanine aminotransferase levels and/or detectable HCV RNA during the following 6 months. Non-responders were defined as still having elevated alanine aminotransferase during and after interferon. Serological tests (RIBA and INNO-LIA) were performed according to the manufacturers' instructions. HCV RNA was detected by nested polymerase chain reaction. Hepatitis C virus genotype was determined by using a Line Probe Assay (Innogenetics). RESULTS: There was no significant difference in the pattern of hepatitis C virus reactivity according to the hepatitis C virus genotype or presence of cryoglobulinemia. Twenty-three patients were classified as non-responders, 35 as initial responders, 21 as long-term responders. NS5 reactivity was significantly different (p<0.01) between these three groups: 34% of non-responders (8/23) had RIBA 3.0 NS5 reactivity and 13% (3/23) were reactive in the INNO-LIA III. Almost all long-term responders (95%) had NS5 reactivity by both RIBA 3.0 and INNO-LIA III. CONCLUSION: We conclude that patients who respond to interferon have stronger reactivity against NS5 antigens than non-responders. Molecular changes in the NS5A region may be responsible for such differences, as recently suggested.

GEMHEP multicenter quality control study of PCR detection of GB virus C/hepatitis G virus RNA in serum.

PCR is, to date, the only available tool for the detection of GB virus C (GBV-C) and hepatitis G virus (HGV) RNAs. Twenty-two French laboratories participated in a quality control study to assess the sensitivity and specificity of their procedures. The panel included 13 positive controls and 7 negative controls. The laboratories used either in-house PCR techniques adapted from the literature or partly standardized commercial tests. Three laboratories performed faultlessly with the entire panel. Most laboratories had excellent specificity (100% in 20 of 22 laboratories). Sensitivity was acceptable (85 to 100%) in 15 centers and insufficient (38 to 77%) in 7. As with nonstandardized in-house PCR, the commercial assays gave discrepant performances in different laboratories. These results suggest that laboratories willing to use PCR for detection of GBV-C/HGV RNA for research or diagnostic purposes should participate in multicenter quality control trials.

Variations of serum IgG subclass levels in hepatitis C virus infection during interferon-alpha therapy.

Serum IgG1 levels are selectively increased in patients with chronic hepatitis C virus (HCV) infection. In 15 patients who received interferon (IFN)-alpha therapy, serum levels of immunoglobulin classes and IgG subclasses were measured during treatment and after it was discontinued. In spite of important individual variations, mean IgG, IgG1, IgA and IgM levels decreased during therapy and tended to return to pre-treatment levels afterwards, with no detectable correlation with clinical and biological parameters. These results suggest an effect of IFN-alpha on in vivo immunoglobulin production, in HCV carriers.

Hepatitis C virus genotypes and subtypes in patients with hepatitis C, with and without cryoglobulinemia.

BACKGROUND/AIMS: Recent reports have shown a high frequency of anti-hepatitis C virus antibodies in patients with cryoglobulinemia. The factors involved in the production of cryoglobulins in hepatitis C virus-infected patients are unknown. To assess the role of hepatitis C virus genotypes in the pathogenesis of mixed cryoglobulinemia, we analyzed their prevalence in a group of 118 hepatitis C virus-infected patients according to the presence or absence of cryoglobulins. METHODS: The hepatitis C virus genome was typed using the Line Probe Assay (LiPA, Innogenetics), for the most common genotypes (1a, 1b, 2a, 2b, 3, 4 or 5). RESULTS: Cryoglobulinemia was diagnosed in 60 (51%) patients, 33 (55%) of whom had type II and 27 (45%) type III cryoglobulins. Forty-four (37%) patients had no cryoglobulinemia and 14 (12%) patients had transient cryoglobulins. Cryoglobulins were significantly less prevalent in patients infected by genotype 1a. We found no statistical link between the hepatitis C virus genotype and the presence of symptomatic cryoglobulinemia, or the hepatitis C virus genotype and the type (II or III) of cryoglobulin. Interestingly, all six patients infected by hepatitis C virus genotype 4 or 5 had cryoglobulins. CONCLUSIONS: In patients with hepatitis C virus infection, cryoglobulinemia is not strongly associated with a particular HCV genotype or subtype. The mechanism by which cryoglobulins are produced remains to be elucidated.

Antibody responses to hepatitis C envelope proteins in patients with acute or chronic hepatitis C.

Antibody responses to the hepatitis C virus (HCV) envelope proteins E1 and E2 were analyzed using two original assays in sera from 86 patients in different stages of disease. A Western blot assay and an immunofluorescence assay (IFA) were developed using envelope proteins produced, respectively, in Escherichia coli and in CV1 cells infected with a recombinant SV40. As a third method, the INNO-LIA HCV Ab III assay including E2 synthetic peptides was used. Of 38 chronically infected patients positive for anti-E2 antibodies by IFA, 26 were positive in the Western blot assay (68%) and 25 in the INNO-LIA test (66%). Thus, the detection of anti-envelope antibodies is highly dependent on the antigen formulation, and a native glycosylated form of the proteins is probably needed for their efficient detection. This study shows that the antibody response to HCV envelope proteins depends on the phase of infection. A few acutely infected patients displayed a response to E1 or E2 (36% by Western blot, 7% by IFA), and these antibodies seem to develop in patients evolving toward chronicity. The high prevalence in chronically infected subjects (62% to E2 by Western blot, 90% by IFA), particularly in subjects with essential mixed cryoglobulinemia (68% and 100%), confirms that the resolution of infection involves more than these antibodies. The antienvelope response in patients treated with interferon was investigated, but no significant relationship was found between antibody level prior to treatment and the evolution of hepatitis. The detection of anti-envelope antibodies, therefore, is not predictive of the response to antiviral therapy.

Essential mixed cryoglobulinemia. A comparative study of dermatologic manifestations in patients infected or noninfected with hepatitis C virus.

BACKGROUND AND DESIGN: An association between essential mixed cryoglobulinemia and hepatitis C virus (HCV) infection has been reported. Dermatologic manifestations are a classic presenting complaint in essential mixed cryoglobulinemia. The aim of this study was to compare the frequency and the nature of dermatologic manifestations in essential mixed cryoglobulinemia according to the presence of anti-HCV antibodies. Sixty-two consecutive patients with essential mixed cryoglobulinemia were tested for anti-HCV antibodies. Dermatologic manifestations were systematically assessed. RESULTS: Anti-HCV antibodies were detected in 35 patients. Palpable purpura corresponding histologically to leukocytoclastic vasculitis was the more frequently observed dermatologic manifestation and occurred mainly in HCV-antibody-positive patients. The patients with purpura had significantly higher serum cryoglobulin levels than patients without purpura. CONCLUSIONS: The frequency of palpable purpura is higher in HCV-antibody-positive patients and is related to the serum cryoglobulin levels.

Cryoglobulinemia in chronic liver diseases: role of hepatitis C virus and liver damage.

BACKGROUND/AIMS: Mixed cryoglobulinemia is frequently associated with liver diseases. The respective role of hepatitis C virus (HCV) and liver damage in the pathogenesis of cryoglobulinemia is investigated in this study. METHODS: The prevalence of cryoglobulinemia in 226 consecutive patients with chronic liver diseases (hepatitis C, 127; hepatitis B, 40; other diseases, 59) was studied, and the epidemiological, biological, histological, and virological features in these three groups were analyzed. Anti-HCV antibodies, HCV proteins, and HCV RNA were searched in the cryoprecipitates. RESULTS: The prevalence of mixed cryoglobulinemia was high (41.5%) in patients with liver diseases and higher in patients with hepatitis C (54.3%) than in patients with hepatitis B (15%) or other causes of liver disease (32%). Patients with cryoglobulinemia had cirrhosis more frequently and had a longer history of hepatitis. In patients with hepatitis C, HCV RNA sequences and HCV proteins were detected in the cryoprecipitate. Cryoglobulins became undetectable in 21 of 43 patients treated with interferon. CONCLUSIONS: These findings suggest that HCV is a major cause of cryoglobulinemia. Besides viral infection itself, multiple factors appear to be responsible for the production of cryoglobulins, including cirrhosis and duration of liver disease.