Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile

A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A−B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A−B−, and A−B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A−B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A−B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections.     

Strain-Dependent Migration of CD4 and CD8 Lymphocyte Subsets to Lymph Nodes in NOD (Nonobese Diabetic) and Control Mice

Subpopulations of lymphoid cells were compared with respect to their ability to migrate into peripheral lymphoid organs of nonobese diabetic (NOD) mice and various strains of control mice. In short-term, in vivo homing studies, no major differences in the pattern of homing of B and T cells were observed among all mouse strains studied. On the other hand, CD4 cells localized consistently more efficiently than CD8 cells in both PP and LN of adult NOD and BALB/c mice, whereas both populations migrated roughly equivalently in LN of adult DBA/2, CBA, and C57BL/6 mice. No age-dependent differences in the homing of CD4 and CD8 cells were observed in BALB/c mice. On the contrary, in 2-week-old NOD mice, CD4 and CD8 cells migrated equally well. The preferential entry of CD4 cells in adult NOD and BALB/c did not result from increased blood transit time of CD8 cells. On the other hand, the preferential migration of CD8 cells was observed in the liver, whereas the two T-cell subsets migrated equally well in the lungs. The differences in the homing characteristics of CD4 and CD8 cells among NOD, BALB/c, and C57BL/6 mice were not related to modifications in the level of expression of adhesion molecules such as MEL-14, LFA-1, and Pgp-1.     

OH treatment of tetanus toxin reduces its susceptibility to limited proteolysis with more efficient presentation to specific T cells

At inflammatory sites, before their processing, antigens are exposed to oxygen free radicals released by activated cells. The effect of hydroxyl radicals (OH·) on the structure of a protein antigen, tetanus toxin (TT) was investigated, as well as the consequences on processing and presentation. A chemical system composed of Fe-EDTA, ascorbate and H₂O₂ was used to produce physiological amounts of OH^• radicals. TT exposed to OH· radicals presented a marked decrease of its intrinsic fluorescence with a concomitant increase of the content of bityrosine, but no fragmentation of the protein was detected by SDS-PAGE. Processing of the modified TT was analysed, by incubating TT at acidic pH with fractions enriched in plasma membranes and lysosomes obtained from a lymphoblastoid cell line (LCL). Proteolysis of OH·-treated TT was less important than proteolysis of native TT, especially upon prolonged incubations. Oxidized TT presented by LCL cells induced a greater proliferation of three different TT specific T cell clones, compared to native TT. When proteolytic digests of TT were presented by fixed LCL cells to a homologous T cell line, the proliferative response obtained in the presence of digests of OH·-treated TT was sustained, even in the case of prolonged proteolysis, whereas the response to digests of native TT fell rapidly. The relative resistance of OH·-treated TT to proteolysis appears thus responsible for its greater presentation to specific T cells, probably by protecting epitopes.     

Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay

We describe a rapid and reproducible PCR assay for quantitation of the Candida albicans ribosomal DNA (rDNA) in clinical blood samples based on the TaqMan principle (Applied Biosystems), in which a signal is generated by cleavage of a template-specific probe during amplification. We used two fluorogenic probes based on universal, fungus-specific primers, one for the detection of C. albicans species DNA and one for the detection of all Candida genus DNA. C. albicans blastoconidia mixed with whole blood in a titration experiment yielded a linear PCR signal over a range of 3 orders of magnitude. The TaqMan-based PCR assay for C. albicans exhibited a low limit of detection (5 CFU/ml of blood) and an excellent reproducibility (96 to 99%). While the C. albicans species-specific probe had 100% specificity for C. albicans, all Candida genus-specific probes cross-reacted with other organisms likely to coinfect patients with C. albicans infections. On the basis of these data, we determined the C. albicans loads with a species-specific probe from 122 blood samples from 61 hematology or oncology patients with clinically proven or suspected systemic Candida infections. Eleven positive samples exhibited a wide range of C. albicans loads, extending from 5 to 100,475 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97%, respectively, compared with the results of blood culture. These data indicate that the TaqMan-based PCR assay for quantitation of C. albicans with a species-specific probe provides an attractive alternative for the identification and quantitation of C. albicans rDNA in pure cultures and blood samples.     

Evaluation of three commercially available hepatitis C virus antibody detection assays under the conditions of a clinical virology laboratory.

BACKGROUND: Most studies evaluating antibody detection assays are conducted on samples from healthy blood donors but not on samples of hospitalized patients which can show non-specific reactions. OBJECTIVES: To compare the performance of three commercial automated assays for the detection of hepatitis C virus (HCV) antibodies, Monolisa anti-HCV Plus version 2, Axsym anti-HCV 3.0 and Vitros anti-HCV, on a population of hospitalized patients. STUDY DESIGN: The specificity of the assays was prospectively evaluated in 2020 routine serum samples. In order to assign the serostatus of each sample, those giving positive or discordant results were further tested by three immunoblots and by RT-PCR (Roche). Moreover, the sensitivity was evaluated on eight commercial HCV seroconversion panels. RESULTS: The Monolisa, Axsym and Vitros assays showed specificities of 99.64%, 99.12% and 99.33%, respectively. Concerning the sensitivity, among 49 samples, the number of positive results was 21, 24 and 24 for the Monolisa, Axsym and Vitros kits, respectively. The differences were not statistically significant at an alpha risk of 5%. CONCLUSIONS: All assays appeared to be reliable for routine screening, but there were a surprising number of indeterminate samples that could not be resolved by confirmatory tests.     

Clinical evidence of the nonpathogenic nature of the M34T variant in the connexin 26 gene

Mutations in GJB2 are the most common cause of congenital nonsyndromic hearing loss. The controversial allele variant M34T has been hypothesized to cause autosomal dominant or recessive nonsyndromic hearing impairment and some in vitro data has been consistent with this hypothesis. In this report, we present the clinical and genotypic study of 11 families (seven familial forms of nonsyndromic sensorineural hearing loss (NSSNHL) and four sporadic cases) in which the M34T GJB2 variant has been identified. The M34T mutation did not segregate with the deafness in six of the seven familial forms of NSSNH. Eight persons with normal audiogram presented a heterozygous M34T variation and five normal hearing individuals were composite heterozygous for M34T and another GJB2 mutation. Four normal hearing individuals with a documented audiogram were M34T/35delG and one was M34T/(GJB6-D13S1830)del. Screening a French control population of 116 subjects we have found an M34T allele frequency of 1.72%. This percentage was not significatively different from the prevalence of the M34T allele in the deaf population, which was 2.12%. All these data suggest that the M34T variant is not clinically significant in human and is a frequent polymorphism in France.     

Glucocorticoids down-regulate dendritic cell function in vitro and in vivo

Exogenous glucocorticoid hormones are widely used as therapeutical agents, whereas endogenous glucocorticoids may act as physiological immunosuppressants involved in the control of immune and inflammatory responses. The optimal activation of T lymphocytes requires two distinct signals: the major histocompatibility complex-restricted presentation of the antigen and an additional co-stimulatory signal provided by the antigen-presenting cells. There is ample evidence that, among the cells able to present the antigen, the dendritic cells (DC) have the unique property to activate antigen-specific, naive T cells in vitro and in vivo, and are therefore required for the induction of primary immune responses. In this work, we tested whether glucocorticoids affected the capacity of DC to sensitize naive T cells. Our data show that, in vitro, the steroid hormone analog dexamethasone (Dex) affects the viability of DC, selectively downregulates the expression of co-stimulatory molecules on viable DC, and strongly reduces their immunostimulatory properties. In vivo, a single injection of Dex results in impaired antigen presenting function, a finding which correlates with reduced numbers of splenic DC. These results show that glucocorticoids regulate DC maturation and immune function in vitro and in vivo and suggest that this mechanism may play a role in preventing overstimulation of the immune system.     

Efficacy and tolerability of prolonged linezolid therapy in the treatment of orthopedic implant infections

The aim of the study presented here was to assess the efficacy and tolerability of linezolid in the treatment of orthopedic implant infections (OII). Eighty-five patients with an OII treated with linezolid were prospectively followed up for a minimum of 12 months from the end of antibiotic therapy. Outcome was evaluated in relation to the duration and type of symptoms (acute or chronic) and the retention or removal of the implant. For acute and chronic infections, the respective success rates were 100 and 92.3% when the implant was removed and 72.2 and 42.8% when it was not. The median length of linezolid treatment in acute and chronic infections was 47 and 60 days, respectively. Thrombocytopenia was observed in four (4.7%) patients and anemia in five (5.8%). The results suggest oral linezolid is an effective and well-tolerated alternative for treating OII.     

Cloning,sequencing and analysis of the yeastS. uvarum ERG10 gene encoding acetoacetyl CoA thiolase

Summary TheERG10 gene specific toS. uvarum, a brewing yeast, has been cloned by complementation of anS. cerevisiae erg10 mutant.S. uvarum contains two differentERG10 genes. One of these is similar to theS. cerevisiae ERG10 gene; they are structurally different, but functionally homologous. The clonedERG10 gene has been located on chromosome XVI, and we have shown that it is allelic to the previously isolatedtsm0115 mutants. Northern blot and sequence analysis indicate that theERG10 gene is highly expressed, and biochemical and genetic evidence show that it encodes the cytoplasmic acetoacetyl CoA thiolase.