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Hemodialysis patients are recognized as a group at high risk of infection with hepatitis C virus (HCV). Therefore, such a population should be screened routinely for the presence of HCV viremia. Since nucleic acid techniques remain expensive and largely unavailable in many laboratories in the developing world, the present study assesses the clinical usefulness of the HCV core antigen enzyme immunoassay for the diagnosis of HCV infection in dialysis patients. One hundred seventy‐five dialysis patients were screened for the presence of anti‐HCV antibodies and HCV RNA in the serum. One hundred twenty‐eight serum samples were collected from the 76 patients who were anti‐HCV antibody‐ and/or HCV RNA‐positive. These were evaluated for total HCV core antigen. Of these samples, 55 had sufficient volume to be further tested to quantify HCV RNA by reverse transcription polymerase chain reaction (RT‐PCR). Genotyping of the HCV strains showed that the majority belonged to genotype 1b (77%). The HCV core antigen assay showed a sensitivity and specificity of 84% and 89%, respectively. The use of core antigen assay has enabled the early detection of three patients who developed an acute hepatitis C infection during the period of study. A correlation study was undertaken between the quantitative values of viral load, expressed as pg/ml of HCV core antigen in serum, and viral RNA in UI/ml. A significant correlation was observed (Pearson's correlation coefficient: 0.552; P< 0.001). In conclusion, detection of HCV core antigen in serum is an inexpensive, reliable, and highly specific assay that can be useful in most laboratory settings to diagnose HCV infection, and especially in laboratories where nucleic acid technologies are not yet available. J. Med. Virol.77:502–508, 2005. © 2005 Wiley‐Liss, inc.

ABSTRACTThis study describes the development and evaluation of a new commercial test, Chlamylege (Argene Inc.), which allows the simultaneous detection in respiratory samples of Chlamydophila pneumoniae, Mycoplasma pneumoniae, and most Legionellaspecies, as well as PCR inhibitors, by using a multiplex PCR and microplate hybridization. The sensitivities of Chlamylege were 1 × 10-3IFU, 5 × 10-2color-changing units, and 1 CFU per reaction tube for C. pneumoniae, M. pneumoniae, and Legionella pneumophila, respectively. A cohort of 154 clinical samples from patients with documented respiratory infections was analyzed by the kit, including 2 samples from patients with C. pneumoniaeinfection, 9 samples from patients with M. pneumoniaeinfection, 19 samples from patients with Legionellaspecies infection, and 114 samples that tested negative for the three pathogens. All the positive specimens were correctly detected and identified by the Chlamylege kit, and no false-positive result was observed with the negative samples. The kit was then evaluated in a pediatric prospective study that included 220 endotracheal aspirates, and the results were compared with those obtained by three single in-house PCR assays. Four specimens were found to be positive for C. pneumoniaeand six were found to be positive for M. pneumoniaeby using both strategies. The Chlamylege kit detected two additional samples positive for M. pneumoniaeand one additional sample positive for a Legionellaspecies other than L. pneumophila; these three samples were shown to be true positive by other techniques. These overall results demonstrate that the Chlamylege assay is sensitive, specific, and convenient for the rapid detection and identification of atypical pathogens in clinical samples from patients with respiratory infections.

Fourteen serotypes are currently recognized in theUreaplasma urealyticumspecies. These serotypes have been divided into two genomic clusters or biovars by a large number of typing methods. The parvo-biovar includes strains of serotypes 1, 3, 6 and 14 and the T960-biovar, strains belonging to the ten other serotypes. In this study, arbitrarily primed polymerase chain reaction (AP-PCR) has been applied to the analysis of reference strains of the 14U. urealyticumserotypes. By using two different sets of 10-mer oligonucleotide primers, the method allowed the clear differentiation between the two known biovars of the species. However, further differentiation within a same biovar was only achieved for a few standard strains of the T960-biovar analysed by using a pairwise combination of primers. The reproducibility of AP-PCR profiles was shown on strains tested after repeated subcultures and with different thermal cyclers. Additional experiments were performed on forty isolates ofU. urealyticumrecovered from subjects of various origins. They confirmed that AP-PCR was able to identify the strains at the biovar level. With reference to the other typing methods, AP-PCR is easy to perform and can be applied to large numbers of strains for epidemiological purposes.

The spread in Europe of a single multiresistant strain ofPseudomonas aeruginosa serotype O12 has been suggested. This bacterium was responsible for a nosocomial outbreak in our hospital in 1988–1989. Three different epidemiological methods were used to analyze 30 strains isolated during five consecutive years. Protein profile analysis and chromosomal DNA fingerprinting with four different enzymes revealed closely related patterns. rRNA gene restriction fragment length analysis performed with a digoxigenin-labelled probe showed identical hybridization patterns with four to six bands according to the endonuclease used. Combination of the three typing methods showed genotypic homogeneity of thesePseudomonas aeruginosa O12 strains, despite a relative increase in their antibiotic resistance.

Arbitrarily primed PCR with two different primers was compared with ribotyping and monoclonal antibody analysis for typing Legionella strains. Applied to 11 epidemiologically unrelated strains, arbitrarily primed PCR resulted in an index of discrimination of 100% with both primers. It was found able to identify an epidemic clone of Legionella pneumophila serogroup 1 that was isolated from both patients and a hot water circuit of the same hospital.

We evaluated the usefulness of a commercially available monoclonal antibody (MAb) directed against a group-specific epitope of the capsid protein VP1 of enteroviruses for the rapid identification of these viruses in cell culture. The MAb was assayed in an indirect immunofluorescence test with cultured cells infected by various serotypes of enterovirus; all 39 serotypes tested, including echoviruses 22 and 23, which are considered atypical enteroviruses, were reactive. The MAb was also tested with 61 strains recovered from clinical specimens inoculated into cell cultures in comparison with seroneutralization with intersecting pools of hyperimmune sera and PCR with primers from the 5' untranslated region of enteroviruses. There was total agreement between the results obtained with the MAb and those obtained by PCR, even for those strains of enteroviruses which were found to be untypeable with polyclonal antisera. These data demonstrate the usefulness of the MAb for rapid identification of enteroviruses in cell culture.

The molecular characteristics of 31 unrelated strains of Staphylococcus schleiferi isolated from 13 hospitals between 1973 and 1991 were determined by ribosomal DNA fingerprinting by using a digoxigenin-labeled DNA probe, genomic DNA restriction patterns, and plasmid profiles. Only six strains harbored one or two plasmids. DNA restriction analysis, which was carried out with five endonucleases (EcoRI, HindIII, PstI, PvuII, and ClaI), did not allow us to discriminate between isolates. Ribotyping with HindIII, ClaI, or EcoRI enzymes generated six, seven, and nine distinct patterns, respectively. With the combination ClaI-EcoRI, 13 ribotypes were obtained among the 31 strains, suggesting a relative heterogeneity within the species. Moreover, all strains shared two or three common bands, according to the endonuclease used, which were relatively specific for S. schleiferi in comparison with the ribosomal banding patterns described for other coagulase-negative staphylococci. These results illustrate that ribotyping can be used for the epidemiological investigation of S. schleiferi isolates and possibly for taxonomic analysis in this species.

Background: Previous studies have reported the role of enteroviruses in chronic diseases, using in-house RT-PCR protocols. A well-standardized PCR assay (Amplicor enterovirus, Produits Roche) designed for the diagnosis of enterovirus meningitis in cerebrospinal fluids (CSF) was recently described. Objectives: To evaluate this commercially-available PCR assay for the detection of enterovirus in intestinal biopsies. Study design: In order to obtain large quantities of infected material, eight mice were inoculated orally with 2×10 50% tissue culture infective doses (TCID 50 ) of coxsackievirus B3 (CBV3); two mice were sacrificed every day from day 1 to day 4 post-infection. Stool specimens and small bowel fragments were taken from infected animals and controls. Four protocols of RNA extraction from intestinal tissue were compared. Extracted RNA was then tested by the Amplicor assay and by a seminested in-house PCR. Results: The best results were obtained with a commercial reagent using a combination of guanidium thiocyanate and phenol (TRI Reagent, Sigma). This procedure allowed the detection of enteroviral RNA in intestinal samples of 7/8 and 8/8 infected mice by Amplicor assay and seminested PCR, respectively, whereas only five samples were tested positive by conventional cell culture. When tested on serial dilutions of CBV3 mixed with intestinal tissue, a sensitivity of 0.2 TCID 50 /mg was achieved with both PCR assays. Conclusions: The data demonstrate that the Amplicor enterovirus assay, which is designed to avoid false-positive amplifications, can be used, with a slight modification of the RNA extraction step, for the detection of enterovirus in specimens different from CSF such as intestinal tissue.

In December 1992, Enterobacter cloacae was isolated from the oropharynx and respiratory tract of six ventilated neonates hospitalized in the intensive care unit (ICU) of our hospital. To establish the spread of the outbreak, 41 strains of E. cloacae were analyzed for genotypic markers by three methods: plasmid profile analysis, ribotyping with EcoRI or PvuII endonuclease, and arbitrarily primed (AP) PCR. The tested strains included 12 isolates from the 6 epidemic cases, 4 isolates from the respiratory tract of 4 children hospitalized in other wards during the same period, 13 isolates from 12 children hospitalized in pediatric units before or after the outbreak, and 12 epidemiologically unrelated isolates. Ribotyping and AP PCR demonstrated that each of the last 12 strains exhibited distinct genomic patterns, as did each of the strains isolated from neonates hospitalized before or after the epidemic peak. Conversely, two clones of strains were found among the isolates recovered in December, with concordant results being obtained by the three typing methods: the first clone included seven strains from five ventilated children in the ICU and two children from another ward; another clone was shared by one neonate in the ICU and an infant from another ward. These results indicate that ribotyping and AP PCR-the latter applied, to our knowledge, for the first time to the genotypic analysis of E. cloacae--represent very discriminatory tools for the investigation of nosocomial outbreaks caused by this species.

Four methods for rapid detection of adenovirus were evaluated by testing retrospectively 28 frozen clinical specimens from which an adenovirus strain had been isolated. After thawing all specimens were retested for the presence of adenovirus by conventional culture on KB cells and found to be positive. The four tests used for rapid detection of adenovirus were a 48-hour culture technique, and an immunoassay, a latex agglutination test and an immunofluorescence assay for direct detection of viral antigen using commercially available reagents. Of the 28 specimens all were positive in the 48-hour culture, 25 (89 %) positive in the immunoassay and 10 (36 %) positive in the latex agglutination test. Six of eight nasopharyngeal aspirate specimens were positive in the immunofluorescence assay. Twenty-five clinical specimens negative for adenovirus on conventional culture were also negative in the 48-hour culture technique. Overall, the rapid (48-hour) culture technique was 100 % sensitive and 100 % specific compared to conventional culture. The direct detection of viral antigen by immunoassay was less sensitive, however results were available within a few hours. Prospective comparative studies are warranted to determine whether these rapid techniques could replace conventional culture in the routine diagnosis of adenovirus infection.

Les Staphylococcus aureusrésistants à la méticilline (SAMR) sont un enjeu majeur de santé publique. Nous avons souhaité savoir si des données biométriques ou biologiques pouvaient caractériser les patients atteints de SAMR hospitalisés dans notre service, afin de procéder à une identification de la population à risque pour réaliser un isolement préventif et limiter sa diffusion dans le service.