Evolutionary implications of the frequent horizontal transfer of mismatch repair genes.

Mutation and subsequent recombination events create genetic diversity, which is subjected to natural selection. Bacterial mismatch repair (MMR) deficient mutants, exhibiting high mutation and homologous recombination rates, are frequently found in natural populations. Therefore, we have explored the possibility that MMR deficiency emerging in nature has left some "imprint" in the sequence of bacterial genomes. Comparative molecular phylogeny of MMR genes from natural Escherichia coli isolates shows that, compared to housekeeping genes, individual functional MMR genes exhibit high sequence mosaicism derived from diverse phylogenetic lineages. This apparent horizontal gene transfer correlates with hyperrecombination phenotype of MMR-deficient mutators. The sequence mosaicism of MMR genes may be a hallmark of a mechanism of adaptive evolution that involves modulation of mutation and recombination rates by recurrent losses and reacquisitions of MMR gene functions.

A novel HLA-B null allele (B*4022N) generated by a nonsense codon in the alpha1 domain.

We describe in this work a novel HLA-B null allele designated B*4022N. This new variant was found in a Caucasian individual who was serologically typed for one HLA-B allele as a B-blank, Bw-blank. Retrospective DNA typing by polymerase chain reaction using sequence-specific primers (PCR-SSP) has established the correspondence of this blank allele with the classical HLA-B*4001 allele. Nucleotide sequence analysis of exon 2 and 3 has revealed the presence of two adjacent point mutations at position 170 and 171 of exon 2 (GG to TT). While the first difference is silent, the second leads to the creation of a nonsense codon at position 58 of the alpha1 domain, providing the most likely mechanism underlying the observed null phenotype.

Prospective virological follow-up of hepatitis C infection in a haemodialysis unit.

Hepatitis C virus (HCV) is of major concern in the management of patients on maintenance haemodialysis. Many studies have reported a high prevalence of HCV infection in dialysis centres. The objective of our study was first, to perform a prospective follow-up of the evolution of HCV infection in a haemodialysis centre, and second, to assess the rate of viral clearance in patients on dialysis. For this, genotypes, HCV antibodies (anti-HCV) and HCV RNA were evaluated initially and 9 months later. HCV RNA quantification was also performed. Of 136 patients, 62 (45.6%) were anti-HCV positive by third-generation enzyme immunoassay (EIA 3) in the first survey and 64 of 136 (47.1%) were anti-HCV positive by EIA 3 in the second survey. The rate of new HCV infection, estimated from the two seroconversions between the surveys, was 1.9% per year. One of the two patients was initially HCV RNA positive, with a titre of 0.6 x 10(6) eq ml-1. The viral load measured in the dialysis patients was low and does not seem to be influenced by dialysis. No significant difference was observed in viral load between the two periods nor were there any gender-related differences in viral load. In conclusion, detection of antibodies to HCV, together with HCV RNA, seems to be relevant in haemodialysis patients, but this strategy is not suitable for use in all haemodialysis centres because of its high cost.

Characterization of avian Chlamydia psittaci strains using omp1 restriction mapping and serovar-specific monoclonal antibodies.

In the present study, 60 avian Chlamydia psittaci isolates were characterized using restriction fragment length polymorphism as well as serovar-specific monoclonal antibodies, enabling a comparison between the two characterization methods. Sixty avian C. psittaci isolates were characterized by Alul restriction mapping of the major outer membrane protein gene omp1 obtained after amplification by the polymerase chain reaction. The 60 avian C. psittaci strains were also characterized using serovar-specific monoclonal antibodies in a microimmunofluorescence test. Digestion of 60 avian C. psittaci omp1 amplicons by Alul generated 5 of the 6 known distinct restriction patterns (A, B, D, E and F). Restriction pattern C was not observed. Serotyping revealed 4 avian C. psittaci serovars (A, B, C and D). None of the 60 isolates was typed as serovar E. AluI restriction patterns A, B, D and E corresponded in 98% of the cases to serovars A, B, C and D, respectively. One isolate, classified as serovar A, generated restriction pattern F instead of A. Genotyping enabled a more precise differentiation of avian C. psittaci serovar A strains. Serovar A strains were divided into two groups according to their Alul restriction pattern (A or F). For epidemiological studies, genotyping can thus be a highly valuable alternative to serotyping, especially when applied directly to the clinical samples.

[Clinical significance of detection and quantification of hepatitis C virus RNA in hemodialysis: proposal for a rational diagnostic strategy]. / Intérêt clinique de la recherche et de la quantification de l'ARN du virus de l'hépatite C chez les hémodialysés: proposition d'une stratégie diagnostique rationnelle.

Viral infections due to hepatitis C virus in hemodialysis patients are frequent and have a potential risk of progression towards chronicity. Biochemical and viral markers of infection, are transaminases level, anti-HCV serology and the detection of HCV RNA, respectively. A rational strategy based on routine use of these three diagnostic tools is proposed in order to avoid unnecessary assays and to increase in the case of health cost control, the cost/efficacy ratio. The latter is set up, in the sero-negative hemodialysed, on the early detection of infection by the hepatitis C virus in order to consider of a therapeutic which is able to cure patients and to avoid the ineluctable passage towards chronicity; in hemodialysed with positive HCV serology, the detection of HCV RNA allows to establish the infectiosity status of these hemodialysis patients. It is therefore very important to evaluate prospectively this diagnosis approach in hemodialysis patients.

Kinetics of HCV viremia in kidney transplant recipients during and after alpha-interferon therapy.

Fifteen kidney transplant recipients with chronic hepatitis C were given 3 million units recombinant alpha2b-interferon for 142+/-35 days. There were significant decreases in hepatitis C virus (HCV) RNA 1 month after the initiation of treatment (p < 0.01), and at the end of treatment (p < 0.05). HCV RNA was undetectable by PCR analysis during treatment in 5 patients. But HCV RNA reappeared in all patients 1 month after the cessation of therapy, and the level of viremia returned to baseline. While all patients had normalized alanine aminotransferase (ALT) activities at the end of therapy, 11 experienced a relapse during the follow-up period (1 year). There was a correlation between the amount of HCV RNA at the end of treatment and the time of relapse. Serum IgM against core protein of HCV were detected in 7/15 patients. Anti-core IgM remained detectable during treatment and afterwards. There was no correlation between IgM status and other virological parameters, or ALT activity.

A pilot study of a combination of three reverse transcriptase inhibitors in HIV-1 infection.

The objective of this study was to evaluate the efficacy and tolerability of a combination of three reverse transcriptase inhibitors in patients with human immunodeficiency virus type 1 (HIV-1) infection. The investigation was an open pilot study of 48 weeks duration. Forty-five patients with CD4 cell counts between 50 and 500 cells/mm3 received a combination of oral zidovudine (200 mg three times daily) plus didanosine (200 mg twice daily) and lamivudine (150 mg twice daily). Plasma HIV-1 RNA and CD4 cell levels were measured weekly during the first month, at weeks 6, 8 and monthly thereafter. HIV-1 RNA levels were also measured sequentially in the lymph nodes of five patients after the initiation of therapy, and after several months of undetectable plasma RNA in 10 additional cases. Sequencing was performed on virus from the peripheral blood mononuclear cells of a subset of 14 patients after a mean period of 11+/-1 months on therapy. The mean (+/-SE) plasma viral load was 5.04+/-0.09 log10 copies/ml and the mean CD4 cell count was 339+/-14 cells/mm3 at baseline. Plasma HIV-1 RNA levels decreased exponentially in each case and became undetectable in 36 out of 42 cases who continued therapy for 24 weeks. HIV-1 RNA levels were < 20 copies/ml in 73% of these cases with undetectable HIV RNA. HIV-1 RNA decreased exponentially in lymph nodes after the initiation of therapy. The mean residual lymph node HIV-1 RNA level was 3.06+/-0.58 log10 copies/10(6) cells in 10 patients evaluated after several months of having undetectable plasma HIV RNA levels. A mean gain of 212 and 237 CD4 cells/mm3 was observed at 24 and 48 weeks, respectively. Proviral DNA sequencing showed that the main resistance codon mutations were absent in each case. Only one patient presented with a mutation resulting in the K219Q substitution, and one other with a T200I substitution. We conclude that this combination can achieve a significant decrease in HIV-1 replication in both plasma and lymph nodes in most cases. It is safe, able to delay the selection of resistant mutants, and keeps open the option for the use of protease inhibitors in case of therapeutic failure.

HIV-1 replication in the plasma and cerebrospinal fluid.

The HIV-1 RNA in plasma and CSF samples from 40 HIV-1 infected patients was measured by a polymerase chain reaction (PCR) technique. The possible implication of cytokines in HIV-1 replication was investigated by measuring the concentrations of tumor necrosis factor alpha (TNF-alpha), macrophage colony stimulation factor (M-CSF) and interleukin-6 (IL-6) in these fluids. HIV-1 RNA was quantified in all plasma samples and in 87.5% of the CSF samples. CSF HIV-1 RNA titers did not correlate with the stage of disease or the CD4+ T cell counts, unlike the plasma HIV-1 RNA titers. These results were confirmed when patients with a blood brain barrier damage, as assessed by the CSF/ plasma albumin ratio, were excluded from the analysis. TNF-alpha levels were statistically correlated with the HIV-1 RNA in plasma and CSF. These data demonstrate that HIV-1 replication in the CSF at each clinical stage can be accurately measured with PCR and, although the titers of HIV-1 RNA copies in the CSF are correlated with those in the plasma, the magnitude of HIV-1 replication in CSF is not directly linked to the stage of disease, or to the CD4+ T cell count. The significance of early high levels of HIV-1 RNA in CSF is now being studied prospectively.

Assessment of a standardized reverse-transcriptase PCR assay for quantifying HIV-1 RNA in plasma and serum.

The analytical variability of the new commercially available Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay, Amplicor HIV-1 Monitor, has been assessed to establish criteria for assessing the significance of HIV-1 RNA level measurements. Estimations of the standard deviations (SD) of log-copies in inter-assay (mean 0.09 log) and in inter-laboratory (mean 0.14 log) reproducibility experiments demonstrated that the assay can discriminate with 95% confidence between 3-fold (inter-assay) and 5-fold differences (inter-laboratory). The inter-lot reproducibility (mean 0.10 log) was similar to the inter-assay reproducibility. The HIV-1 RNA concentrations measured in plasma collected in potassium EDTA anticoagulant were slightly higher than those measured in plasma collected in sodium citrate. The HIV-1 RNA concentrations measured in sera were about 50% of the HIV-1 RNA concentrations measured in paired plasma samples. However, there was a strong correlation between these two measurements (P < 0.0001). The assay was used to measure viral RNA in the plasma of 50 HIV-1 positive individuals at different stages of infection. All the individuals had detectable HIV-1 RNA (300-957000 copies/ml). There was no correlation between HIV-1 RNA and Immune Complex Dissociated (ICD) p24 antigen, but HIV-1 RNA was correlated with CD4+ cell counts (P < 0.0001) and the clinical stage (P = 0.0042), with higher HIV-1 RNA concentrations in patients with a more advanced stage of the disease. The significant association of HIV-1 RNA with major markers of HIV infection and the reliability of this sensitive, easy-to-use RT-PCR assay indicate its suitability for use in clinical trials and suggest that this assay is appropriate for routine clinical applications.

Significance of indeterminate third-generation hepatitis C virus recombinant immunoblot assay.

Indeterminate hepatitis C virus (HCV) third-generation recombinant immunoblot assay (RIBA3.0; Ortho Diagnostic Systems) patterns were arbitrarily defined by the manufacturer as the detection of only one antibody out of the four that were sought, namely, c100 (NS4 encoded), c22 (core encoded), c33c (NS3 encoded), and NS5 (NS5 encoded). The aims of the present study were (i) to determine the prevalence of indeterminate RIBA3.0 patterns in patients consecutively tested for anti-HCV antibodies in a university hospital; (ii) to evaluate the significance of these patterns in terms of viral replication, liver disease, and risk factors for HCV; and (iii) to get an insight into the mechanism underlying this peculiar immune response. Among 3,074 serum samples consecutively tested for anti-HCV antibodies, 588 were found to be positive by screening assays. Fifty-nine of them (10%) were RIBA3.0 indeterminate and were compared with 59 RIBA3.0-positive ones. Thirty-one RIBA3.0-indeterminate and 53 RIBA3.0-positive serum samples were HCV RNA positive by PCR (53 versus 90%; P < 10(-6). RIBA3.0-indeterminate and RIBA-3.0-positive patients with positive PCR results were not significantly different for the prevalence of risk factors for HCV infection and elevated serum alanine aminotransferase activities. Immunosuppression, attributable to coexisting human immunodeficiency virus infection, organ transplantation, or the administration of immunosuppressive drugs, was significantly more frequent in PCR-positive, RIBA3.0-indeterminate patients than in PCR-negative, RIBA3.0 indeterminate patients (P < 0.001) and PCR-positive patients with a positive RIBA3.0 result (P < 0.01). The distribution of HCV genotypes did not differ significantly between HCV RNA-positive patients with indeterminate or positive RIBA3.0 results. In conclusion, the prevalence of indeterminate RIBA3.0 patterns in virology laboratories is about 10%; in about half of these patients HCV replication is detected by PCR; the main factor responsible for indeterminate RIBA3.0 patterns could be immunosuppression, whereas HCV genotypes do not seem to play major role.

Heterogeneity within the first constant segment of the major outer membrane protein gene in Chlamydia trachomatis serovar D/Da distinguishes 2 lineages.

Restriction fragment length polymorphism of the major outer membrane protein gene (ompl) was studied in 73 epidemiologically unrelated Chlamydia trachomatis serovar D (n = 64) and Da (n = 9) strains isolated between 1983 and 1991 in various european countries from patients suffering from sexually transmitted diseases, as well as 3 reference strains D/IC-Cal-8 (USA), D/UW3 (UK) and Da/TW-448 (Taiwan). Among these strains, 5 different genotypic groups were distinguished: 3 for the serovar D strains and 2 for the serovar Da strains. Comparisons of the complete ompl nucleotide sequence of 1 member of each group revealed 2 distinct lineages, independently of the D/Da classification, presumably as the result of an intragenic DNA recombination event within the first constant segment. The observed ompl genetic diversity of serovar D/Da strains in regions involved in T- and B-cell responses might add another level of complexity to the design of a subunit vaccine.

Denaturing gradient gel electrophoresis analysis for the detection of point mutations in the Chlamydia trachomatis major outer-membrane protein gene.

Fifty clinical strains of Chlamydia trachomatis were studied by denaturing gradient gel electrophoresis (DGGE) of bacterial DNA amplified by the polymerase chain reaction (PCR). The strains belonged to the three most commonly encountered serovars in developed countries--D, E and F. Six reference strains, including the serovar Da strain, were also studied. The DNA sequences explored encompassed the four variable domains (VDs) of omp1, the gene encoding the major outer-membrane protein (MOMP). The corresponding regions in the MOMP contain the species-, subspecies- and serovar-specific epitopes. The four distinct serovars were clearly differentiated by specific migration pattern. No sequence variations were observed among strains of serovar F. However, variant strains within serovars D and E were found, which exhibited migration patterns different from those of the reference strains and these were sequenced directly. According to the observed sequence variations, serovar D strains could be divided into three stable representative groups (D, D1 and D2). Two variants were identified among serovar E strains. No biological differences were observed for the variant strains in terms of growth properties, ecology or pathogenicity. All the nucleotide substitutions detected in the VDs were non-synonymous at the protein level and, for the serovar D strains, could account for differences identified by specific monoclonal antibodies. These substitutions could be involved in antigenic drift, driven by the immune pressure of the host, leading to the emergence of serovariants. The data may explain, in part, chlamydial infection recurrences and could have critical implications for developing rational vaccine strategies.

Improvement of cervical Chlamydia detection in asymptomatic family planning attendees by using Amplicor Chlamydia trachomatis assay.

We evaluated the Amplicor C. trachomatis PCR assay (Roche Molecular Systems, N.J.) for the diagnosis of cervical infection in asymptomatic women attending a family planning clinic, aged between 18 and 25 years. Culture onto McCoy cells with fluorescent monoclonal staining was the reference system. Cervical specimens from 485 women were tested. The prevalence of C. trachomatis was 10.5% by culture and 11% by Amplicor. No specimen was positive by culture and negative by PCR. Three PCR-positive, culture-negative specimens were positive by MOMP-PCR and a second plasmid-based PCR. The resolved sensitivity of PCR and culture were 100% and 94.5%, respectively. Specificities for both were 100%, positive and negative predictive values for culture were 100% and 99.3%. Total test efficiency was 99.4%. The Amplicor C. trachomatis assay gave very clear results, quite above or below the cut-off value, and showed high sensitivity and specificity, improved ease of handling and represented a good alternative to culture for large scale diagnosis of asymptomatic C. trachomatis infection.

Usefulness of omp1 restriction mapping for avian Chlamydia psittaci isolate differentiation.

Sixty-five avian Chlamydia psittaci isolates collected worldwide, including 27 previously characterized reference strains, were analysed by restriction mapping of the major outer membrane protein gene (omp1) obtained after DNA amplification by PCR. They were compared to 2 ruminant isolates, a feline pneumonitis and a guinea pig inclusion conjunctivitis (GPIC) isolate. According to their omp1 restriction patterns, avian strains were heterogeneous in that they exhibited 6 and 4 distinct patterns using AluI and MboII restriction enzymes, respectively, thus defining 7 groups. However, 84% of the studied strains belonged to groups 1 to 4, which share a specific fragment triplet of 411, 282 and 102 base pairs in their AluI digestion patterns. Comparisons with serological classifications showed a strict correlation and allowed further intraserovar differentiation. Furthermore, this classification based upon a single gene (omp1) roughly correlated with the data obtained by RFLP of native DNA and DNA/DNA hybridization studies. There was no host or geographic specificity in the pattern exhibited by these strains. The ruminant, feline pneumonitis and GPIC C. psittaci isolates were clearly distinguished from each other and the avian strains. Moreover, this method was clearly able to identify dubiously designated strains as well as mixtures of isolates within a single sample. In conclusion, this PCR approach based upon omp1 restriction mapping enables the differentiation of avian C. psittaci isolates and can be proposed as a taxonomic and epidemiologic tool.

A simple procedure to differentiate ailA and ailNA gene variants among human pathogenic Yersinia enterocolitica strains.

The identification and differentiation of the two variants of the ail gene, ailA from the more virulent American serotypes (08, 013a, 13b, 018, 020, 021) and ailNA from the less virulent non-American serotypes (03, 04, 05, 06, 09, 027 and 07, 8) was studied in a panel of 32 Yersinia enterocolitica human pathogenic isolates. A 444 bp fragment corresponding to the ail gene was amplified using a PCR procedure in all tested strains. Subsequent digestion of the PCR product by Rsal and by HaeIII endonucleases, provide electrophoretic patterns that clearly discriminate ailA and ailNA variants. This non-radioactive and reliable procedure allows large clinical and epidemiological studies, and could be proposed to survey the spread of virulent clones.

Genomic fingerprinting of Yersinia enterocolitica species by degenerate oligonucleotide-primed polymerase chain reaction.

A novel version of the polymerase chain reaction (PCR) termed degenerate oligonucleotide-primed PCR (DOP-PCR) was used to fingerprint the bacterial genome of Yersinia enterocolitica species. Eight well-characterized reference strains of Y. enterocolitica (ribotype, serotype, biotype and zymotype) were examined. Optimal experimental conditions for reproducibility, sensitivity and specificity were obtained using a single DOP-primer. All the strains gave distinct DOP-PCR profiles composed of 12 to 19 fragments with sizes from 200 to 1.500 bp. Thus, the DOP-PCR, which has so far been used to fingerprint human, mouse, fruitfly and plant DNA, is also well-suited to bacterial DNA.

[Alpha-thalassemia]. / Les alpha-thalassémies.

alpha-Thalassaemias are probably the most common genetic disorder worldwide. alpha-Thalassaemias are haemolytic anaemias resulting from inherited deficient synthesis of alpha-globin chains. In this paper, the classification and nomenclature of alpha-thalassaemias are developed. Procedures to ensure the laboratory diagnosis are explained. The heterozygous carrier states for these disorders are, in most cases, not associated with any easily discernible change in the haemoglobin pattern, except, sometimes, a reduced MCV in the blood picture. Heterozygotes for alpha-thalassaemia deletions are now detectable by the accurate PCR method. Because of the high prevalence of these disorders in large segments of the world population, alpha-thalassemia and haemoglobinopathies often occur in the same individual. The laboratory features of these interactions and, particularly, the role of alpha-thalassaemia as a potential modulator of sickling haemoglobinopathies are discussed.