Abstract: Blood platelets link the processes of haemostasis and inflammation. This study examined the immunomodulatory factors released by platelets after Toll-Like Receptor 4 (TLR4) engagement on their surfaces. Monoclonal anti-human FcgammaRII Ab (IV.3)-treated human platelets were cultured with TLR4 ligands in the presence or absence of blocking monoclonal antibody to human TLR4. The release of sCD62p, epidermal growth factor (EGF), transforming growth factor beta (TGFbeta), interleukin (IL)-8, platelet activating factor 4 (PAF4), platelet-derived growth factor, alpha, beta polypeptide (PDGF-AB), Angiogenin, RANTES (regulated upon activation, normal T-cell expressed, and presumably secreted) and sCD40L were measured by specific enzyme-linked immunosorbent assay. TLR4 ligand [Escherichia coli lipopolysaccharide (LPS)] bound platelet TLR4, which differentially modulates the release of cytokines by platelets. It was noted that (i) sCD62p, IL-8, EGF and TGFbeta release were each independent of platelet activation after TLR4 engagement; (ii) RANTES, Angiogenin and PDGF-AB concentration were weaker in platelet supernatant after TLR4 engagement; (iii) sCD40L and PAF4 are present in large concentration in the releaseate of platelets stimulated by TLR4 ligand. The effects of LPS from E. coli on the modulation of secretory factors were attenuated by preincubation of platelets with an anti-TLR4 monoclonal antibody, consistent with the immunomodulation being specifically mediated by the TLR4 receptor. We propose that platelets adapt the subsequent responses, with polarized cytokine secretion, after TLR4 involvement.

Abstract: We calculated the incidence of nosocomial infection in 2 intensive care units (ICUs) on the basis of prevalence data recorded from 1997 through 2002 and compared these estimates to cumulative incidences measured in the 2 ICUs during the same period to investigate the feasibility and the reliability of converting prevalence data to incidence estimates. Decreases in the calculated and measured incidences over time in the ICUs were found to be statistically significantly related.

Abstract: We report 32 cases of acute encephalitis consecutively hospitalized in one hospital, from January 1991 to December 2002. The causative agent was identified in 26 cases (81%). The main associated viruses were varicella-zoster (10 children; 31%), Herpes simplex (6 children; 19%), and enteroviruses (4 children; 13%). At the acute phase, the most relevant biological findings were electroencephalogram results and CSF analysis. The initial encephalic imaging was primarily helpful to exclude other acute neurological diseases whereas long-term imaging was a prognostic factor for necrotizing encephalitis. The microbiological diagnosis required several days or weeks to be determined. It did not influence the initial management. In addition to the 6 cases of herpetic encephalitis, 19 children (78% altogether) were then treated by acyclovir before a definitive diagnosis was made. Twenty-two children (69%) had a favorable outcome, 2 (6%) had moderate sequels, 2 (6%) had important ones, and 5 (16%) had major ones. One (3%) child died. The outcome was highly dependant on the causative agent and the mechanism of encephalitis. This series gives information on the epidemiology of encephalitis in children in our region over a period of 12 years.

Abstract: Recombination between two strains is a known phenomenon for enteroviruses replicating within a single cell. We describe a recombinant strain recovered from human stools, typed as coxsackievirus B4 (CV-B4) and CV-B3 after partial sequencing of the VP1 and VP2 coding regions, respectively. The strain was neutralized by a polyclonal CV-B3-specific antiserum but not by a CV-B4-specific antiserum. The nucleotide sequence analysis of the whole structural genomic region showed the occurrence of a recombination event at position 1950 within the VP3 capsid gene, in a region coding for the 2b antigenic site previously described for CV-B3. This observation evidences for the first time the occurrence of an interserotypic recombination within the VP2-VP3-VP1 capsid region between two nonpoliovirus enterovirus strains. The neutralization pattern suggests that the major antigenic site is located within the VP2 protein.

Abstract: BACKGROUND: Various pathogens have been suspected to play a role in the initiation or amplification of the atherosclerotic lesions. Both experimental and epidemiological arguments plead for a possible role of enterovirus in this process. OBJECTIVE: To determine the prevalence of enterovirus genome in atherosclerotic plaques, in comparison with Chlamydia pneumoniae, human cytomegalovirus (hCMV) and herpes simplex virus. STUDY DESIGN: Pilot study on 18 patients who underwent artery resection. Five artery samples were tested for each patient and each pathogen by using PCR techniques whose sensitivity was evaluated for this kind of specimen. The quality of the extraction step was assessed by amplification of a fragment of the human aldolase A gene. RESULTS: The genome of at least one infectious agent was detected in artery samples from 7 of the 18 patients (38.9%). In all cases, only one of the five aliquots was found positive; a confirmation was done by sequencing the PCR product. With regards to enterovirus, four patients (22.2%) were detected positive (one of them being also positive for hCMV). CONCLUSIONS: These results suggest that small amounts of enterovirus genome are commonly found in lesions of patients with advanced arteriosclerosis. Further studies are needed to evaluate the clinical significance of this association.

Abstract: OBJECTIVES: In this study, we evaluated the potential direct role of enterovirus (EV) cardiac infections in the pathogenesis of myocardial infarction (MI). BACKGROUND: Enteroviruses (Picornaviridae) have been suspected to play a role in the development of acute MI. METHODS: The presence of EV ribonucleic acid (RNA) sequences and capsid viral protein 1 (VP1) and the virus-mediated focal disruption of dystrophin were retrospectively investigated by reverse transcriptase-polymerase chain reaction and immunohistochemistry assays in endomyocardial tissues of patients who died suddenly of acute MI by comparison with similar samples of control patients matched for gender, residence area, and year of death. RESULTS: Enterovirus infection markers were detected in 20 (40%) of 50 patients who died suddenly of MI, 2 (4%) of 50 matched subjects without cardiac disease (p < 0.001), and 4 (8%) of 50 matched patients exhibiting a noncoronary chronic cardiopathy (p < 0.001). All of the EV RNA-positive patients exhibited VP1, which provided evidence of viral protein synthesis activity. The VP1 gene sequences amplified after cloning from myocardial or coronary samples of 8 of the MI patients and showed a strong homology with sequences of coxsackievirus B2 and B3 serotypes. Moreover, in the endomyocardial tissue of these 8 patients, immunohistochemical analyses demonstrated that there was disruption of the sarcolemmal localization of dystrophin in the same tissue areas that were infected by coxsackieviruses. CONCLUSIONS: Our findings demonstrate a significantly higher proportion of active coxsackievirus B cardiovascular infections in patients who suddenly died of MI compared with matched control subjects, suggesting that these EVs may significantly contribute to the pathogenesis of acute MI by a focal disruption of the dystrophin-glycoprotein complex.

Abstract: The sequencing of the VP1 hypervariable region of the human enterovirus (HEV) genome has become the reference test for typing field isolates. This study describes a new strategy for typing HEV at the serotype level that uses a reverse transcription-PCR assay targeting the central part of the VP2 capsid protein. Two pairs of primers were used to amplify a fragment of 584 bp (with reference to the PV-1 sequence) or a part of it (368 bp) for typing. For a few strains not amplified by the first PCR, seminested primers enhanced the sensitivity (which was found to be approximately 10(-1) and 10(-4) 50% tissue culture infective dose per reaction tube for the first and seminested assay, respectively). The typing method was then applied to 116 clinical and environmental strains of HEV. Sixty-one typeable isolates were correctly identified at the serotype level by comparison to seroneutralization. Forty-eight of 55 "untypeable" strains (87.3%) exhibited the same serotype using VP1 and VP2 sequencing methods. For six strains (four identified as EV-71, one as E-9, and one as E-30 by the VP2 method), no amplification was obtained by the VP1 method. The last strain, typed as CV-B4 by VP1 and CV-B3 by VP2 and monovalent antiserum, could exhibit recombination within the capsid region. Although the VP2 method was tested on only 36 of the 68 HEV serotypes, it appears to be a promising strategy for typing HEV strains isolated on a routine basis. The good sensitivity of the seminested technique could avoid cell culture and allow HEV typing directly from PCR products.

Abstract: This study, involving 20 laboratories and using currently available assays for hepatitis C virus RNA quantification, demonstrated that differences in viral load values are due not to interlaboratory variations but rather to the nature of the assay itself. This underlines the importance of using the same assay in multicenter studies or when monitoring antiviral therapy.

Abstract: Enterovirus is a genus of the Picornaviridae family including more than 80 serotypes belonging to four species designed Human enterovirus A to D. The antigens of the structural proteins support the subdivision of enteroviruses into multiple serotypes. Comparative phylogeny based on molecular typing methods has been of great help to classify former and new types of enterovirus, and to investigate the diversity of enteroviruses and the evolutionary mechanisms involved in their diversity. By now, molecular typing methods of enterovirus rely mainly on the sequencing of an amplicon targeting a variable part of the region coding for the capsid proteins (VP1 and, alternatively, VP2 or VP4), either from a strain recovered by cell culture or, more recently, by direct amplification of a clinical or environmental specimen. In the future, microarrays are thought to play a major role in enterovirus typing and in the analysis of the determinants of virulence that support the puzzling diversity of the pathological conditions associated with human infection by these viruses.

Abstract:

Abstract: This study evaluated a multidisciplinary strategy to decrease the rate of invasive pulmonary aspergillosis (IPA) among adult patients hospitalised in two haematology wards in a single 560-bed building at the University Hospital of Saint-Etienne. Upgrading of the air filtration system and construction of an air-lock chamber at the entrance to the unit were completed during 1994. In 1995, specific hygienic measures were introduced during hospital building work, including the use of plastic barriers, watering during demolition work, reduction of pedestrian traffic in construction areas, and the wearing of high-efficiency filtration masks by immunosuppressed patients when outside the protected unit. This strategy was evaluated by a prospective survey of IPA cases between 1993 and 2001, coupled with environmental surveillance. The number and risk-level of hospital renovation projects increased between 1995 and 2001 (p < 0.01). In parallel, the rate of IPA decreased globally in the haematology unit from 0.85% (1.19/1,000 patients) in 1993 to 0.28% (0.21/1,000 patients) in 2001. The incidence of IPA decreased significantly between 1993-1996 and 1997-2001 (p 0.02, Mann-Whitney test). These results show that a multidisciplinary approach involving engineers, infection control practitioners, mycologists and clinicians enables IPA rates among patients hospitalised in haematology wards to be significantly decreased.

Abstract: This study focuses on the interest of the hypervariable 23S-5S ribosomal intergenic spacer region (ISR) of the genus Legionella to analyze the phylogenic diversity of Legionella at the species and subspecies levels and to identify isolates directly from clinical specimens. The method, using a real-time PCR assay with a single primer pair followed by sequencing, was able to identify correctly 49 reference strains of Legionella belonging to 37 different species, including those implicated in human infections, and to clearly differentiate the three subspecies of L. pneumophila. Based on sequence similarities, the 23S-5S ISR sequences were much more variable than the rpoB and mip sequences (P<0.0001 by the Wilcoxon signed rank test). The 23S-5S ISR method was able to cluster Legionella species in accordance with phenotypic traits, such as autofluorescence or fatty acid membrane composition. Using maximum parsimony methods, the rpoB and 23S-5S ISR data sets were shown to be incongruent (P<0.001). In contrast, the 23S-5S ISR and the mip data sets were found to be congruent (P=0.313), suggesting the interest of combining these two regions to demonstrate phylogenetic links between Legionella species. This molecular assay was shown able to both detect Legionella DNA directly in respiratory specimens from patients exhibiting a Legionella infection and provide accurate identification of the bacterium at the species level in the tested specimens. These properties open a wide range of applications to the 23S-5S ISR sequencing method, from taxonomic analyses to clinical and epidemiological investigations.

Abstract: BACKGROUND: Cytokines present in the sperm could influence the heterosexual transmission of HIV by modulating viral titers and influencing the early immune response in the vaginal mucosa. OBJECTIVES: To assess the relation between cytokine concentrations and HIV status in the seminal plasma. STUDY DESIGN: Twenty-five HIV positive subjects were tested for cytokine content and HIV-1 load in seminal and blood plasma through a cross-sectional study. RESULTS: HIV positive subjects exhibited a significantly higher amount of seminal IL-1beta as compared to a group of 33 HIV negative controls. In HIV positive subjects, amounts of IL-1beta and HIV-1 RNA in semen were significantly correlated and a trend for correlation was found between seminal IL-1beta and blood HIV-1 RNA. Amount of seminal IL-1beta was significantly lower in patients under HAART, according to the decrease of their viral loads in blood and semen. CONCLUSIONS: Considering that IL-1beta is known to enhance viral replication and to promote immune response, its dosage in semen could represent an interesting marker for identifying patients at high risk for HIV heterosexual transmission.

Abstract: SUMMARY:: The proportion of non-B HIV-1 variants is increasing in Western Europe. The impact of the high polymorphism in the protease and reverse transcriptase genes, as recently described for CRF02-AG isolates of African origin, on antiretroviral resistance is still disputed. We first examined the polymorphism of these genes in CRF02-AG strains recovered from drug-naive patients followed at the University Hospital of Saint-Etienne in France, most of these of French origin and harboring a clonal strain as elicited by phylogenic analysis. The first plasma sample detected positive from 31 CRF02-AG and 23 B strains was used to compare sequences with their respective subtype consensus strain. The overall number of mutations was dramatically higher for CRF02-AG strains than for B strains in both protease and reverse transcriptase genes (P < 0.0001 and 0.009, respectively). In addition, no statistically significant difference in the number of therapeutic failures, mean CD4 cell count, and viral load was observed between 22 and 45 patients infected with CRF02-AG or B strains, respectively, during a mean treatment period of 25.5 months. Even if no striking antiretroviral failure linked to this polymorphism was observed during short-term follow-up, its impact on long-term therapy will have to be extensively evaluated in patients infected by non-B HIV-1 variants.

Abstract: BACKGROUND: After Bacillus cereus recovery in opened boxes of disposable gloves, the bacteriological contamination of disposable nonsterile gloves kept stored in native packages was investigated prospectively. METHODS: Thirty-six commercially available nonsterile nonpowdered disposable gloves made of latex, vinyl, or nitrile were cultured. RESULTS: A large variety of spore-forming and non-spore-forming bacteria was recovered, including Bacillus cereus and Clostridium perfringens. CONCLUSION: This finding must be taken into consideration for care involving gloves in very immunocompromised patients.

Abstract: A multicenter study of NS5b hepatitis C virus (HCV) genotype determination involving 12 laboratories demonstrates that any laboratory with expertise in sequencing techniques would be able to provide a reliable HCV genotype for clinical and epidemiological purposes as long as they are provided a consensus reference sequence database.

Abstract: A national evaluation study was performed in 14 specialized laboratories with the objective of assessing their capacities to provide (i) hepatitis B virus (HBV) viral loads (VL), (ii) HBV genotypes, and(iii) identification of precore/core mutants. The panel consisted of 12 HBV DNA-positive samples with VLs from 2.8 to 9.1 log(10) copies/ml, different HBV genotypes (A to F), and 3 mutant and 9 wild-type samples at nucleotide 1896. The coefficients of variation of the mean VLs ranged from 2.4% to 10.4% with the Cobas HBV Monitor assay, from 1.8% to 5.5% with the Cobas TaqMan 48, from 1.5 to 26.2% with RealArt HBV PCR, and from 0 to 7% with branched DNA (bDNA). The Cobas Monitor assay underestimated the VLs of genotype F samples, with differences ranging from 1.4 to 2.4 log(10) copies/ml. The accuracies of genotype determinations ranged from 33% to 100%, and those of precore mutant determinations ranged from 25 to 100%. This study showed some drawbacks of two widely used assays: (i) Cobas Monitor has a narrow dynamic range and underestimates genotype F sample VLs and (ii) bDNA shows poor sensitivity and may fail to identify patients with low VLs. With higher performance in terms of analytical sensitivity combined with a larger dynamic range and an ability to quantify the main genotypes equally, real-time PCR methods appear more appropriate for accurate monitoring of HBV DNA quantification. Furthermore, the clinical implications of HBV genotyping and the determination of precore/core mutants need to be clearly stated to justify the standardization of these methods.

Abstract: BACKGROUND: Heterosexual human immunodeficiency virus (HIV) transmission implies the crossing of the vaginal mucosa by virions present in the semen, potentially using Langerhans cells as transporters. The recruitment of these cells in the mucosa is mediated by the chemokine macrophage inflammatory protein 3alpha (CCL20). The aim of this study was to evaluate the capacity of the semen to induce Langerhans cell recruitment via the production of CCL20 by vaginal epithelial cells. METHODS AND RESULTS: Using a vaginal epithelium model based on the SiHa cell line and human seminal plasma, we demonstrated that semen enhanced the production of CCL20. This secretion was regulated by the nuclear factor-kappaB intracellular signalling pathway. Fractionation of the seminal plasma indicated that the secretion of CCL20 was stimulated by high molecular weight compounds present in semen. Migration assays demonstrated that secreted CCL20 was able to promote the recruitment of Langerhans cell precursors (LCps), which remain permissive to X4 and R5 HIV infection. CONCLUSIONS: Our results demonstrate that epithelial cells respond to factors present in semen by secreting CCL20, leading to the enhancement of LCp recruitment. These data argue in favour of the implication of epithelial cells in the heterosexual transmission of HIV.

Abstract: In aged-care facilities, gastroenteritis outbreaks are responsible for big trouble in the management of cares to the elderly. In November 2002, a gastroenteritis outbreak was observed in 5 of the 6 wards of the geriatric hospital La Charité, University Hospital of Saint-Etienne, France, with an attack rate of 38.5% in the elderly (70 infected from 182 patients) and of 26.0% in the nursing staff (40 infected from 154 agents). The outbreak lasted 30 days with a peak corresponding to 79.8% of the cases between the 11(th) and the 20(th) of November. The first cases were observed in the two short-term-care wards; then, the outbreak spread rapidly to 3 of the 4 long-term care units. Health care workers were contaminated later than the elderly (P < 0.001 by Kruskal-Wallis test). A self-administered questionnaire was documented by most of the nursing staff; the most frequently observed clinical symptoms in this population were nausea (82.5%), abdominal pain (80.0%), diarrhoea (70.5%), asthenia (67.5%) and vomiting (62.5%). Thirty-five percent of the health care workers ceased their work. The causative agent of the gastroenteritis was identified by RT-PCR in the stools of 5 aged persons as a norovirus close to the Lordsdale strain (genogroup II). These findings illustrate the respective role of elderly and health care workers in the spread of the gastroenteritis outbreak inside the geriatric hospital.

Abstract: Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.

Abstract: The aim of the study was to evaluate the performance of the combined antigen and antibody HIV screening assay VIDAS HIV DUO Ultra (BioMérieux, Marcy l'Etoile, France) in comparison with two other combined tests: the former version of the same test (VIDAS HIV DUO, BioMérieux) and the AxSYM HIV Ag/Ab Combo assay (Abbott Laboratories, Rungis, France). A prospective study was performed on serum specimens received on a routine basis for HIV testing: 1443 blood samples were tested with the three assays. Sensitivity was 100% for the three tests. Specificity assessed on repeated false-positive samples was 99.86, 99.03 and 99.65% for VIDAS HIV DUO Ultra, VIDAS HIV DUO and AxSYM HIV Ag/Ab Combo, respectively. In addition, 14 seroconversion panels were tested with the VIDAS DUO Ultra and AxSYM HIV Ag/Ab Combo assays. For four of these panels, a positive signal was detected one blood sampling point earlier with the VIDAS DUO Ultra assay, corresponding to a higher sensitivity of the HIV antigen test. These results indicate that the VIDAS HIV DUO Ultra exhibits an improved specificity with comparison to the former version of this assay and an excellent sensitivity for early detection of HIV seroconversion.

Abstract: OBJECTIVE: The authors had for aim to study the distribution of HIV-1 subtypes in a cohort of HIV-1 positive patients in the University hospital of Saint-Etienne, France, and to describe the epidemiological characteristics of patients infected with a non-B subtype strain. DESIGN: An epidemiological study was made on 271 HIV-1 positive patients followed up in the Infectious Diseases Department over 20 years. All patients sample were subtyped by serotyping and some samples were also tested by genotyping. RESULTS: Two hundred and sixty-four patients (191 men and 73 women) were found infected by an HIV-1 strain belonging to the M group. After combining serotyping and genotyping results, 195 patients were found infected by a B subtype and 69 by a non-B subtype. Most of the latter strains belonged to an A subtype or related ones. The following factors were shown to be linked to an infection by a non-B strain: being born abroad, having contracted the infection though heterosexual practice, and being a woman. The incidence of non-B strains increased regularly over time (to reach more than 40% in 2003). This progression was especially noted for men born in France with risky sexual behaviour. CONCLUSION: These results indicate that more than 40% of HIV-1 new cases detected in the Saint-Etienne area are related to non-B strains and that strains of A and related subtypes are common in the local population with risky sexual behaviour.

Abstract: A national evaluation study was performed in 11 specialized laboratories with the objective of assessing their capacities to genotype hepatitis C virus (HCV) and define the applicability of a given genotyping method. The panel consisted of 14 samples positive for HCV RNA of different genotypes (including 3 samples with two different artificially mixed genotypes) and 1 HCV-negative sample. Seventeen sets of data were gathered from the 11 participating laboratories. The sensitivities ranged from 64.3 to 100% and from 42.7 to 85.7% for the methods that used sequencing of the NS5b region and the 5' noncoding (5' NC) region, respectively. When the data for the artificially mixed samples were excluded, NS5b genotyping gave correct results for 80% of the samples, 1.7% of the samples were misclassified, and 18.3% of the samples had false-negative results. By 5' NC-region genotyping methods, 58.3% of the results were correct, 29.7% were incomplete, 8.3% were misclassifications, 1.2% were false positive, and 2.4% were false negative. Only two procedures based on NS5b sequencing correctly identified one of the three samples with mixtures of genotypes; the other methods identified the genotype corresponding to the strain with the highest viral load in the sample. Our results suggest that HCV 5' NC-region genotyping methods give sufficient information for clinical purposes, in which the determination of the subtype is not essential, and that NS5b genotyping methods are more reliable for subtype determination, which is required in epidemiological studies.

Abstract: Enteroviruses and other cardiotropic viruses have been associated with the development of late severe adverse cardiac events in infants receiving heart transplants. However, the source and the chronology of cardiac allograft infection by an enterovirus in patients receiving heart transplants remain unknown. Using RT-PCR and immunohistochemistry assays, endomyocardial tissue samples of 30 adult patients were tested to detect the presence of specific enterovirus 5' non-coding (5'NC) sequences and of VP1 capsid protein, and this at the time of cardiac transplantation and at the 12-month biopsy for graft rejection control. Moreover, the endomyocardial detection of genomic sequences of enteroviruses, Epstein-Barr virus, herpes simplex virus, cytomegalovirus (CMV), varicella-zoster virus, adenoviruses, and parvovirus B19 was carried out by RT-PCR and polymerase chain reaction (PCR) assays at the time of late severe cardiac events. Enterovirus RNA and VP1 antigen were both detected in 4 (13%) of 30 patients at the time of the 12-month biopsy for graft rejection control, whereas no enterovirus component was detected in the explanted and implanted heart tissues taken from these 4 patients at the time of transplantation. At the time when severe cardiac events were developed, within 3 months after the positive enterovirus cardiac detection, these four patients demonstrated the presence of endomyocardial enterovirus RNA sequences whereas they were tested negative for the endomyocardial detection of genomic sequences from DNA viruses (except for CMV in two cases), and for a significant level of pp65 CMV antigenemia. Taken together, these findings indicate that enteroviruses could be acquired as a new endomyocardial infection within 12 months after transplantation in adults receiving heart transplants, and suggest that this infection might be an etiological cause for unexplained late severe adverse cardiac events in the heart-transplantated adults.

Abstract: This 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 Legionella species, as well as PCR inhibitors, by using a multiplex PCR and microplate hybridization. The sensitivities of Chlamylege were 1 x 10(-3) IFU, 5 x 10(-2) color-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. pneumoniae infection, 9 samples from patients with M. pneumoniae infection, 19 samples from patients with Legionella species 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. pneumoniae and six were found to be positive for M. pneumoniae by using both strategies. The Chlamylege kit detected two additional samples positive for M. pneumoniae and one additional sample positive for a Legionella species 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.

Abstract: OBJECTIVE: Most of coxsackieviruses A (CV-A) are difficult to isolate in cell culture and are responsible for flask paralysis in suckling mice. The aim of the present work was to analyze the ability of immune and RT-PCR techniques to detect viral components of three different serotypes, CV-A6, CV-A13, and CV-A14, in skeletal muscles of experimentally infected suckling mice. MATERIAL AND METHODS: The antigen detection was done by immunofluorescence technique on trypsinized muscular cells and by immunoperoxidase assay on frozen sections of skeletal muscle, using a monoclonal antibody directed towards a conserved epitope of the VP1 capsid protein among enteroviruses. The nested RT-PCR technique used primers located in the 5' non coding region of viral RNA. RESULTS: The group antigen was present in muscle cells of suckling mice infected by the three serotypes of CV-A which were assayed. Similarly, the muscle specimens were positive by nested RT-PCR. A kinetic study performed with CV-A13 and CV-A14 showed that the RT-PCR assay was positive as soon as 24 h after infection whereas the detection of VP1 antigen and symptoms of flask paralysis were observed only 48 and 72 h after infection, respectively. CONCLUSION: These results show that the tested serotypes of CV-A can be easily detected in muscle specimens of suckling mice by using antigenic and molecular techniques currently available for the diagnosis of enterovirus infections.

Abstract: 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.

Abstract: Prevalence of hepatitis G virus among Tunisian blood donors Hepatitis G virus (GBV-C/HGV) is a recently identified virus which occurs worldwide. The prevalence of GBV-C/HGV in Tunisia has not been previously studied. We aimed to assess the prevalence of GBV-C/HGV infection in Tunisian blood donors. A total of 912 blood donors were tested for anti-E2 antibodies of GBV-C/HGV by enzyme-linked immunosorbent assay and 600 were tested by reverse transcriptase polymerase chain reaction. GBV-C/HGV RNA was found in 5.3% of the sample and HGV antibodies occurred in 4.9%. A correlation was noticed between GBV-C/HGV infection and hepatitis C virus (P = 0.006). The prevalence of GBV-C/HGV is similar to that reported worldwide.

Abstract: X4 and R5 HIV strains are present in the semen of men infected with HIV but R5 isolates are transmitted preferentially. The role of human epithelial cells in this selection is addressed. Three human cervical cell lines-CaSki, SiHa, and HEC1A-and normal human vaginal cells from HIV-negative donors were characterized for HIV receptor expression and incubated with X4 and R5 laboratory-adapted strains or primary isolates. The infection was assessed by detection of intracellular HIV DNA. The three cell lines were shown to express on their surface the CXCR4 and GalCer molecules, but not the CD4 and CCR5 ones. The three cell lines and normal human vaginal cells were found to be selectively permissive to X4 HIV entry; the preincubation of the cell lines with rhSDF-1 inhibited this infection. The detection of the intracellular proviral DNA in the cell lines and in normal human vaginal cells demonstrated a selective integration of X4 strains. Additional experiments showed that no extracellular RNA was detected in the supernatants of HEC1A cells infected by X4 isolates either after 18 days of culture or after incubation with PHA-stimulated PBMCs and that no transmission occurred after co-culture between infected HEC1A cells and PHA-stimulated PBMCs. These results suggest specific sequestration of X4 strains by genital epithelial cells, which could explain, at least in part, the HIV tropism selection process during sexual intercourse.

Abstract: Non-fermentative Gram negative rods are opportunistic pathogens responsible for nosocomial infections. Using phenotypic markers (serotypes for Pseudomonas aeruginosa and antibiotic susceptibility) allows a preliminary screening of epidemiologically-related strains. However, genotypic markers are necessary to better characterize nosocomial strains for the investigation of outbreaks or cross-transmissions in the hospital setting. Infections due to P. aeruginosa, Burkholderia. cepacia or Stenotrophomonas. maltophilia are usually hospital-acquired and responsible for a high mortality rate as illustrated by the lethality of nosocomial pneumonia due to P. aeruginosa. The severity of these infections is due to the virulence factors of the bacteria and to their occurrence in debilitated patients in whom invasives devices are used. The hospital environment can act as a reservoir with a rate of exogeneous transmission of these bacteria as high as 50% in some studies. To better prevent nosocomial infections related to Gram negative non fermentative rods, the control of the aqueous hospital environment, the strict application of hand disinfection and the investigation of potential cross-transmission in the hospital setting are needed.

Abstract: OBJECTIVE: To evaluate the presence of IgA directed to the CD4-binding domain of gp120 and to a conserved region of gp41 (the Kennedy epitope) in serum and parotid saliva of HIV-1-seropositive patients. METHODS: IgA were separated from IgG by anion-exchange chromatography and protein G treatment. The reactivity of IgA was tested against peptides and fusion proteins of the maltose-binding protein (MBP) and the CD4-binding site (MBP24) and MBP and the Kennedy epitope (MBP42). The capacity of serum and saliva IgA to interfere with the gp120-soluble CD4 (sCD4) interaction was examined. IgA were also purified by affinity chromatography using the MBP proteins adsorbed to a resin. RESULTS: Peptides representing the CD4-binding domain and the Kennedy epitope were recognized by serum and saliva IgA of HIV-1-seropositive patients. Of the sera and saliva samples tested, 6/26 serum IgA and 5/25 saliva IgA inhibited the gp120-sCD4 interaction by approximately 50%. The gp120-sCD4 interaction was inhibited by MBP24 affinity-purified IgA but not by MBP42 affinity-purified IgA. CONCLUSION: Immunogens capable of eliciting IgA antibodies that inhibit gp120-CD4 binding might be efficiently used in vaccine to prevent mucosal transmission of HIV-1.

Abstract: An audit was carried out in paediatric wards to study the compliance of healthcare workers (HCWs) to the procedures recommended for the control of hospital-acquired diarrhoea. Thirty-two paediatric wards in the southeast of France participated on a voluntary basis in this prospective observational study after completing a self-administered questionnaire recording measures of hygiene. All the observations were made by the same investigator and focused on preventive procedures: use of single room, handwashing, hand disinfection, overclothing, single-use gloves and masks. Two hundred and seventy patient-HCW contacts were observed, including mainly diapering, temperature measurement, collection of blood sample and catheter care. The isolation of patients in a single room and use of gowns by HCWs were significantly associated with diarrhoea. Whereas handwashing before care was performed by HCWs in more than 95% of all the procedures, the compliance in the use of disposable gloves by HCWs was only of 39.4% for technical procedures (including those with potential exposure to blood) and 20.3% for diapering or temperature measurement. A substantial agreement between reported and observed measures of hygiene was observed for handwashing before contact and hand disinfection with antimicrobial soap before contact. In contrast, this agreement was moderate for use of single room, handwashing after contact, overclothing and wearing disposable gloves after a diaper change. Despite the excellent compliance of HCWs to handwashing, clearer recommendations for the indication and use of disinfectants and disposable gloves are urgently needed.

Abstract: The aim of this study was to evaluate the risk of hospital-acquired diarrhoea during an epidemic period through a prospective multicentre observational study. A systemic investigation of the hospital-acquired diarrhoea (occurring at least 48 h after hospital admission) was conducted through a standardised questionnaire from January to March 1999 in patients of 5 years old or less hospitalised in 28 wards (620 beds) belonging to 20 hospitals located in the south-east part of France. Overall, 241 cases of hospital-acquired diarrhoea were collected, corresponding to a prevalence of 3.3% (3.6% after exclusion of patients admitted for diarrhoea) and a density of incidence of 0.81 per 100 days of hospitalisation. The mean stay duration of hospital-infected patients was greater than 10 days, versus 3.9 days for the other children (P < 0.001). A readmission was required in 27% of the infected children. Rotavirus was involved in 97.8% of microbiologically documented cases (88%). In 50% of the cases, the hospital-acquired diarrhoea was seen in patients with bronchiolitis. Contact isolation measures were prescribed in 88.4% of the cases. These results stress that hospital-acquired diarrhoea represent an important medical and economic load for paediatric units and could be used as reference data to evaluate the impact of preventive measures, especially to reduce readmission and mean stay duration.

Abstract: Herpes simplex virus (HSV) infections are very common in the general population and among immunocompromised patients. Acyclovir (ACV) is an effective treatment which is widely used. We deemed it essential to conduct a wide and coordinated survey of the emergence of ACV-resistant HSV strains. We have formed a network of 15 virology laboratories which have isolated and identified, between May 1999 and April 2002, HSV type 1 (HSV-1) and HSV-2 strains among hospitalized subjects. The sensitivity of each isolate to ACV was evaluated by a colorimetric test (C. Danve, F. Morfin, D. Thouvenot, and M. Aymard, J. Virol. Methods 105:207-217, 2002). During this study, 3900 isolated strains among 3357 patients were collected; 55% of the patients were immunocompetent. Only six immunocompetent patients excreted ACV-resistant HSV strains (0.32%), including one female patient not treated with ACV who was infected primary by an ACV-resistant strain. Among the 54 immunocompromised patients from whom ACV-resistant HSV strains were isolated (3.5%), the bone marrow transplantation patients showed the highest prevalence of resistance (10.9%), whereas among patients infected by human immunodeficiency virus, the prevalence was 4.2%. In 38% of the cases, the patients who excreted the ACV-resistant strains were treated with foscarnet (PFA), and 61% of them developed resistance to PFA. The collection of a large number of isolates enabled an evaluation of the prevalence of resistance of HSV strains to antiviral drugs to be made. This prevalence has remained stable over the last 10 years, as much among immunocompetent patients as among immunocompromised patients.

Abstract: Hepatitis C virus (HCV) strains isolated from 68 haemodialysis Tunisian patients exhibiting chronic infection were genotyped targeting the NS5b region of the HCV genome using a prototype assay developed by Bayer HealthCare-Diagnostics (TRUGENE NS5b HCV). The overall results were compared to those obtained with another assay of the same company based on sequencing of the 5' non-coding region (TRUGENE HCV 5'NC genotyping kit). All strains could be typed by the 5'NC typing kit, but only 62 (91; 2%) by the NS5b prototype assay. All the 62 strains typed by both methods exhibited the same pattern at the type level: 57 were type 1, 3 were type 2, and 2 were type 4. At the subtype level, eight strains that gave undetermined results by the 5'NC kit were successfully typed by the NS5b kit; eight additional strains exhibited discrepant results. The overall agreement between the two assays was 74.2% at the subtype level. In conclusion, the NS5b region appears to be much more accurate than the 5'NC region to subtype HCV strains, especially in those isolated from patients attending haemodialysis centres where the subtype distribution suggests frequent nosocomial transmissions.

Abstract: Many mouse models of human enterovirus disease have been pro- posed, concerning both acute and persistent infection. However, rather paradoxically since the usual way of contamination is fecal-oral, most of them used a systemic route of infection. The aim of the present work was to follow the development of an experimental enterovirus infection and to study the viral persistence at the organ level. Twenty-eight female 3-week old BALB/c mice were infected with 5 x 10(4) TCID(50) of coxsackievirus B3 (CV-B3), Nancy strain, by oral route using a rigid cannula introduced into the stomach. The kinetics of infection was studied by sacrificing 2 animals at different times post infection (from 1 hour to 90 days). The presence of the virus in various organs (small intestine, heart, pancreas, lung, spleen, kidney, liver) was studied by cell culture and RT-PCR. As soon as one hour post infection, the virus was detected in the small intestine. In the heart, the virus was present at 24 and 48 hours post infection by RT-PCR and culture, respectively. At 5 days post infection, all the organs but the liver were found infected. The virus was detected up to 15 days in kidney, 21 days in pancreas, 30 days in lung and spleen, and 45 days in intestine, by both culture and PCR. The heart was still found infected 90 days post infection by both techniques. These results show the dramatic cardiotropism of CV-B3 inoculated by oral route, with a detection of the virus very soon in the course of infection (24 hours) and a persistence of the virus for more than 3 months. The intestine, the initial target of enterovirus infection, can also be considered as a site of viral persistence.

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Abstract: Although the transmission of coxsackievirus B3 occurs mainly via the oral route, little is known about the primary replication and persistence of this agent in the intestine. To address this question, BALB/c mice were inoculated by gavage with coxsackievirus B3, Nancy strain. The mice were killed from 1 hr to 90 days after infection. The viral markers were detected in the small intestine using RT-PCR, cell culture and detection of VP1 protein. Coxsackievirus B3 was detected positive by the three methods from hr 2 to day 45 after infection. By using monoclonal antibodies directed towards VP1, CD40 and CD26, the virus was shown to be present in the lymphocytes of the mucosa as soon as 2 hr after infection; in contrast, no virus was detected in the epithelial cells lining the intestinal lumen. Further experiments were performed to evaluate the capacity of coxsackievirus B3 to establish a persistent infection in two intestinal cell lines. In contrast to HT29 cells, the CaCo-2 cells were shown to develop a persistent infection for up to 20 passages, as demonstrated by the detection of viral RNA and VP1 protein. This study provides further evidence that, after infection by the oral route, the viral particles are concentrated in the lymphocytes of the mucosal layer. In addition, the results suggest that coxsackievirus B3 is capable of establishing a persistent infection in the small intestine that may act as a reservoir of viral particles for the delayed spread of the virus to other target organs.

Abstract: BACKGROUND: In France, assisted reproductive technologies involving a hepatitis C virus (HCV)-infected man requires the cryopreservation of potentially infected semen (in order to establish the presence of HCV), hence the need for a safe and secure storage system. We evaluated the safety of high-security straws for the conservation of semen containing HCV RNA under routine conditions. METHODS: Ionomeric resin (IR) straws were filled with seminal plasma spiked with different concentrations of HCV RNA and sealed using a thermo-solder. After a 4% sodium hypochlorite treatment and/or cryopreservation for 7 days in liquid nitrogen, the outside ends of each straw were rinsed with RNAse-free water. RESULTS: No HCV RNA could be detected in any of the water samples. Additional samples included the rinsing water from straws sealed by thermo-solder and from the heating wire used to cut the end of straws containing HCV-positive semen. The latter samples were found positive for both HCV RNA and the protamine-2 gene expressed by spermatozoa. CONCLUSIONS: These results demonstrate the safety of IR straws, the filling system and the thermo-solder for cryopreservation of semen containing HCV in liquid nitrogen. Decontamination of the straw after sealing and the use of disposable scissors to open the straws are strongly recommended.

Abstract: Hemodialysed patients are recognised as a group at increased risk of infection with hepatitis C virus (HCV). The aim of this study was to determine the prevalence and incidence of HCV infection among dialysis patients of the east-centre part of Tunisia. Two hundred and seventy-six patients dialysed until 2001 were recruited within seven hemodialysis units located in the cities of Sousse, Monastir and Mahdia. The serum markers of HCV infection were tested over the period of March 2000-December 2002, by a 3rd generation ELISA test for antibodies and by qualitative RT-PCR technique for viral RNA. The prevalence of anti-HCV antibodies and of HCV RNA was 32.6% (90 patients) and 25.7% (71 patients), respectively. Between 1998 and 2002, 20 new infections were documented in five of the seven dialysis units corresponding to an incidence of 2.34% per year, with an average time of contamination after the beginning of dialysis of 4.6 years. If all the infections are assessed to have occurred during dialysis, the density of incidence of HCV contamination was 4.4% per year of dialysis. A high correlation was noticed between the presence of HCV markers in serum and the duration of dialysis (F = 34.15, P < 0.0001). In the absence of other risk factors (transfusion, drug-addiction), these results plead for the nosocomial transmission of the observed HCV infections. A phylogenetic analysis of the E2 hypervariable region of the viral genome is in progress to confirm this assumption.

Abstract: Prior to sperm cryopreservation, French guidelines only recommend viral screening for serological status towards human immunodeficiency virus, hepatitis B and C viruses and Treponema palidum. The probability of semen infection by other bacterial pathogens is not taken into consideration by the current recommendations. The objective of the present study was to evaluate this risk and a strategy to reduce it prospectively. Ninety-six patients consulting for sperm cryopreservation underwent a semen culture simultaneously to cryopreservation. The patients were classified into three groups following semen culture results: negative culture (group 1, 77/96, 80.2%), positive culture with saprophytic agents (group 2, 9/96, 9.4%) and positive culture with pathogen agents (group 3, 10/96, 10.4%). For six patients of the latter group showing a genital infection with Ureaplasma urealyticum, a discontinuous gradient selection performed on the cryopreserved sample was efficient to discard bacteria. These data emphasize the usefulness to cultivate semen simultaneously to cryopreservation and demonstrate the ability to remove some microbial agents from semen before its use in assisted reproductive techniques.

Abstract: BACKGROUND AND OBJECTIVES: Currently, the bacterial contamination of blood constitutes one of the major infectious risks of transfusion. The aim of this study was to evaluate the bactericidal effect of blood on various bacterial species and to determine the influence of prestorage conditions and white blood cell (WBC) filtration on the reduction of the bacterial load in isolated red blood cells (RBCs). MATERIALS AND METHODS: The growth kinetics of eight different species of bacteria were studied at 20 degrees C in deliberately contaminated RBC units. Further experiments evaluated the effect of prestorage conditions and WBC filtration on the viability of two model bacteria (Klebsiella oxytoca and Staphylococcus epidermidis) in comparison to previous results obtained with Yersinia enterocolitica. RESULTS: For bacteria susceptible to the bactericidal effect of blood (mainly Gram-negative rods), a reduction of the bacterial load was obtained within 2 h of prestorage at 20 degrees C. When the prestorage period was prolonged beyond 3 h at 20 degrees C, rapid growth was observed with some Enterobacteriaceae. Whereas WBC filtration reduced dramatically the viability of Y. enterocolitica, it had only a minimal effect on the viability of S. epidermidis and K. oxytoca. However, the two latter species of bacteria did not survive prolonged storage at 4 degrees C. CONCLUSIONS: Experiments conducted under realistic conditions are needed to determine whether it would be worthwhile recommending the rapid storage of RBCs at 4 degrees C after WBC reduction of the blood product.

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Abstract: BACKGROUND: The effect of age on declining immunity is well established, but its influence on humoral responses to influenza vaccines is still debated. OBJECTIVE: To compare prospectively the immunogenicity of an influenza vaccine in elderly and healthy control subjects and to search for correlations between this specific humoral protection and clinical and virological parameters. METHODS: A prospective study of the humoral response to influenza vaccine was conducted over 9 consecutive years in the long-term care units of the Saint-Etienne University Hospital, France. Antibodies directed against the vaccinal strains in an inactivated trivalent vaccine were tested by radial haemolysis before and 1 month after 477 and 242 cumulative annual vaccinations in elderly people and in healthy controls, respectively. During the last 6 years of the study, clinical samples from patients with fever > or =38 degrees C were tested systematically for the diagnosis of an influenza infection. RESULTS: A significant rise in anti-A/H3N2 antibody titres was observed in 49.3% of the elderly subjects and in 48.3% of the controls (not significant). The rises for the A/H1N1 and B strains were 31.4 and 39.2% (p < 0.001) and 30.6 and 40.5 (p < 0.001), respectively. When control subjects under 31 years old were excluded, no significant difference was recorded for any strain. Only 3.9% of the vaccinated elderly experienced any clinical influenza infection as defined by fever >/=38 degrees C and virological proof. CONCLUSIONS: The humoral response to influenza immunization is not impaired in the elderly compared with middle-aged controls, and the incidence of febrile episodes due to influenza is low in this vaccinated elderly population. These findings provide further evidence supporting the recommendation for annual influenza vaccination in the elderly.

Abstract: This outbreak of colonization of neonates in a 10-bed pediatric intensive care unit illustrates the probable role of a healthcare worker (HCW) in the transmission of methicillin-resistant Staphylococcus aureus, despite good hygienic practices. It raises the issue of preventive exclusion of HCWs affected by chronic skin disease from high-risk units.

Abstract: The discrepant results available in the literature about the presence of hepatitis C virus (HCV) RNA in seminal plasma of men chronically infected by this agent are related, at least in part, to the molecular techniques used and particularly to the wide range of protocols dedicated to RNA extraction. In order to evaluate these protocols and to standardize the method of detection of HCV RNA in this fluid, a panel of coded specimens was tested blindly in 12 French laboratories; it included 14 seminal plasma specimens and four water controls spiked with HCV RNA ranging from 10 to 20000 IU/ml and two HCV-negative seminal plasma specimens. The extraction step was performed according to methods using either silica beads (NucliSens [Organon Teknika S.A., Fresnes, France]; RNA viral kit [Qiagen, Courtaboeuf, France]) or guanidinium thiocyanate (Amplicor HCV assay; Roche Diagnostics, Meylan, France), preceded or not by a centrifugation of the seminal plasma. For the amplification step, all the laboratories performed the same reverse transcription-PCR technique (Amplicor HCV Cobas assay). The percentage of correct results ranged from 53.3 to 100, the poorest results being obtained when no centrifugation step preceded the Amplicor extraction protocol. The rate of correct results was significantly higher in laboratories using a preliminary centrifugation of the specimen (P = 0.034 by chi-square test). By contrast, the overall number of correct results was not correlated to the initial volume of sample used for the test. These results allowed us to validate standardized techniques adapted to the performance of this test on a routine basis, especially in men infected with HCV and involved in programs of medically assisted reproduction.

Abstract: OBJECTIVES: French guidelines recommend screening all patients for virus infection prior to cryopreservation of their semen. In case of viral risk, the use of specific high secure CBS straws is recommended. The objective of this work was to evaluate the microbiological risk by testing all semen samples before cryopreservation. PATIENTS AND METHODS: Fifty one patients underwent a semen culture before cryopreservation. RESULTS: The fifty one patients were classed into 3 groups following semen culture results: group I: negative culture (39/51, 76.47%), group II: positive culture with microbiological contamination (7/51, 13.73%) and group III: positive culture with pathogen (5/51, 9.8%). For 3 patients of the latter group, we tested a three-layer density gradient to eliminate bacteria before Assisted Reproductive Techniques. DISCUSSION AND CONCLUSIONS: This paper discusses the risks related to microbiological contamination and the options available in this case.

Abstract: We describe a new PCR test (Penter RT-PCR) that recognizes all 64 prototypes of enterovirus. Sixty clinical samples were analyzed in parallel with this Penter RT-PCR and previously described PCR tests: 34 and 32 samples tested positive, respectively. This assay is suitable for use in clinical diagnosis, and its ability to amplify all known serotypes makes it more useful than other consensus PCR tests.

Abstract: The type III secretion system (TTSS) of Pseudomonas aeruginosa was characterized genetically and phenotypically in 92 epidemiologically unrelated bacteremic strains. Four groups of strains (TTSS types) were defined according to the level of type III protein secretion and kinetics of cytotoxicity. Type 1 strains (n=26) were highly and rapidly cytotoxic and secreted ExoU, type 2 strains (n=48) exhibited slower cytotoxic rates and expressed ExoS but not ExoU, type 3 strains (n=14) were poorly cytotoxic, and type 4 strains (n=4) were not cytotoxic. Type 3 and 4 strains did not have detectable secretion phenotype; however, some type 4 strains were able to reach a level of cytotoxicity similar to that of type 1 and type 2 strains when complemented in trans by a functional exsA gene. A statistically significant association (P<.001) was found between TTSS types and detection of the mutually exclusive exoU and exoS genes. In addition, 24 of 25 serotype O:1, O:10, and O:11 strains contained exoU, whereas 54 of 55 serotype O:3, O:4, O:6, O:12, and O:16 strains contained exoS (P<.001). Our results demonstrate correlations among exoU or exoS genotype, TTSS phenotype, and O serotype in bacteremic P. aeruginosa isolates.

Abstract: We report an outbreak of infections due to methicillin-resistant Staphylococcus aureus (MRSA) in a medical unit and the possible implication of student nurses in the dissemination of the epidemic strain. A retrospective epidemiological study looking for hospitalised patients colonised or infected with MRSA from the 1st of June to the 30th of September 2001 in the unit was conducted. An audit of delivered cares and a nasal screening of health care workers (HCW) was performed. Six patients were colonised or infected with a MRSA strain, four of them exhibiting a bacteremia. Six HCW had a nasal carriage of MRSA. Typing of the MRSA strains by pulsed field gel electrophoresis demonstrated an epidemic clone isolated from five of six patients, two student nurses and one HCW not implicated in nursing cares. This report illustrates the risk of nosocomial outbreak linked to cares delivered by student nurses.

Abstract: OBJECTIVE: To investigate the relationship between hygienic measures reported for the prevention of hospital-acquired diarrhea and incidence rates of hospital-acquired diarrhea. DESIGN: A survey of hospital-acquired diarrhea was conducted between January 1 and March 31, 1999. Multivariate analysis of reported measures of hygiene according to the observed incidence rates of hospital-acquired diarrhea was performed. SETTING: Thirty-one pediatric or neonatal wards located in hospitals in the southeast of France, selected as a convenience sample of wards volunteering to participate. PATIENTS: A total of 6,726 children younger than 5 years. RESULTS: The overall incidence rate of hospital-acquired diarrhea was 3.6%. Rotavirus was responsible for 69% of the cases of hospital-acquired diarrhea. Among the hygienic measures reported by the wards for preventing hospital-acquired diarrhea were using a single room or cohorting (77.4%), washing hands (83.9%), wearing gowns (80.6%), and wearing disposable single-use gloves for diapering a patient (51.6%). By multivariate analysis, the variables statistically associated with a lower incidence of hospital-acquired diarrhea were restricting the patient's mobility outside his or her room, keeping the patient's door closed, and having fewer than 20 beds in the ward, with adjusted odds ratios of 0.34 (95% confidence interval [CI95], 0.18 to 0.65), 0.33 (CI95, 0.23 to 0.47), and 0.42 (CI95 0.30 to 0.60), respectively. CONCLUSION: Simple preventive measures can decrease the rate of hospital-acquired diarrhea in pediatric wards.

Abstract: This study describes two epidemic outbreaks involving Staphylococcus aureus with reduced sensitivity to glycopeptides, one in 2000 involving eight patients and the other in 2001-2002 involving 16 patients. These strains were detected rapidly, thanks to routine screening for the offending organisms in the bacteriology laboratory of our hospital. The clonal character of these strains was confirmed by pulsed field electrophoresis. The management of these epidemic outbreaks confirmed (i) the need for systematic adoption of standard precautions, (ii) the importance of circulating information in combating multi-resistant bacteria, as well as the difficulties in transferring colonised patients to different hospital wards, and (iii) the intermittent nature of S. aureus carriage, resulting in a need for prolonged surveillance of colonised and/or infected patients. In addition, our study underlines the value of a multi-disciplinary approach to the management of diffusion of multi-resistant bacteria.

Abstract: Enterovirus RNA has been found previously in specimens of muscle biopsy from patients with idiopathic dilated cardiomyopathy, chronic inflammatory muscle diseases, and fibromyalgia or chronic fatigue syndrome (fibromyalgia/chronic fatigue syndrome). These results suggest that skeletal muscle may host enteroviral persistent infection. To test this hypothesis, we investigated by reverse transcription-polymerase chain reaction (RT-PCR) assay the presence of enterovirus in skeletal muscle of patients with chronic inflammatory muscle diseases or fibromyalgia/chronic fatigue syndrome, and also of healthy subjects. Three of 15 (20%) patients with chronic inflammatory muscle diseases, 4 of 30 (13%) patients with fibromyalgia/chronic fatigue syndrome, and none of 29 healthy subjects was found positive. The presence of VP-1 enteroviral capsid protein was assessed by an immunostaining technique using the 5-D8/1 monoclonal antibody; no biopsy muscle from any patient or healthy subject was found positive. The presence of viral RNA in some muscle biopsies from patients exhibiting muscle disease, together with the absence of VP-1 protein, is in favor of a persistent infection involving defective viral replication.

Abstract: The Authors comment on the difficulty of diagnosing and treating duodenal tumours. The most appropriate indications and extent of resection of these neoplasms are discussed. The Authors report 4 cases of primitive adenocarcinoma of the duodenum treated by pancreaticoduodenectomy (2 cases), segmental resection (1 case) and palliative surgery (1 case) for the presence of omental and lymph-node metastases. Survival was 18 and 14 months in the patients who underwent pancreaticoduodenectomy and 9 months for the patient receiving palliative treatment; the patient who underwent segmental resection is still alive and healthy after 12 months. The Authors point out that adenocarcinoma of the duodenum is an uncommon neoplasm and stress the difficulty encountered in establishing an accurate diagnosis and appropriate surgical management. Better results can be obtained only with an early diagnosis. Chemotherapy and radiotherapy do not significantly improve survival.

Abstract: BACKGROUND: Yersinia enterocolitica is known to cause severe infections in patients who receive transfusions. STUDY DESIGN AND METHODS: The aim of the study was to define the best strategy for reducing the bacterial load in blood that was deliberately contaminated with Y. enterocolitica by combining prestorage temperature and WBC filtration with conditions of blood processing close to those applied in blood banks. RESULTS: The effects of three prestorage temperatures (4 degrees C, 20 degrees C, 37 degrees C) were evaluated at various times after infection. The best reduction of bacterial load was achieved after 3 hours at 20 degrees C. In further experiments, conducted according to the former specifications, filtration of whole blood from eight and six donors with an inoculum of 100 and 500 to 1000 CFUs per mL, respectively, resulted in a total inhibition of bacterial growth up to 42 days after infection. After fractionation of blood components, in contrast to plasma and RBCs, filtration was shown to reduce dramatically the bacterial growth in buffy coats, demonstrating that the antibacterial effect of filtration was supported by the removal of infected WBCs from blood samples. CONCLUSION: These results provide support for the systematic use of blood filtration in the preparation of blood components to prevent Y. enterocolitica infection of patients receiving transfusions.

Abstract: OBJECTIVES: Report of epidemiological, clinical and virological data collected from the prospective surveillance of febrile episodes observed in aged residents of a long-stay care unit of 33 beds, at the University Hospital of Saint-Etienne, during the 1997-1998 winter season. METHODS: Systematic collection of clinical and biological data from febrile patients (> or = 38 degrees C) on a form, including virological findings obtained from a nasal swab and paired serum specimens. RESULTS: From 38 patients (37 of them having been vaccinated against influenza in October 1997), 18 febrile episodes were recorded in 16 subjects, including 3 respiratory syncytial virus infections and a late-occurring outbreak (March 1998) of influenza due to a A/H3N2 strain (15 cases, 14 of them virologically confirmed). No death was noted after the influenza outbreak. In 8 of the 9 tested patients with influenza, "protective" titres of antibodies directed towards the hemagglutinin of the vaccinal strain were present by radial hemolysis test three months before the beginning of the outbreak. During the influenza outbreak, the attack rate of symptomatic infection was 45.5% in elderly and 47.5% in healthcare workers (mainly unvaccinated). The occurrence of the first cases in the latter suggests their possible role in the transmission of the virus to the aged. CONCLUSION: This study underlines the epidemic circulation of multiple respiratory viruses during the same winter season in long-stay care facilities, the occurrence of clinical influenza infections in vaccinated patients exhibiting protective antibody titres and the role of unvaccinated healthcare workers in the propagation of influenza in institutionalised aged.

Abstract: In France, assisted reproductive technology (ART) for hepatitis C virus (HCV)-infected patients is now subject to strict control after the publication of recent guidelines. Infertile serodiscordant couples (HCV-viraemic men and their seronegative female partners) require special care to carried out in designated 'viral risk' laboratories. Twelve sequential semen samples taken from an HCV chronically infected patient were analysed within 22 months. HCV RNA was detected in all the seminal plasma sampled before antiviral treatment with relatively high viral loads, and in two of the corresponding fractions of motile sperm obtained after a gradient selection, suggesting that a contamination risk by HCV through ART cannot be excluded. When the selection of sperm on a discontinuous gradient was followed by an additional swim-up step, HCV RNA was never detected in the motile sperm suspension that was frozen in highly secure straws. IVF was performed using cryopreserved sperm that tested negative for HCV RNA, resulting in a pregnancy. One month after embryo transfer, testing for HCV RNA and antibodies in the woman gave negative results.

Abstract: OBJECTIVE: To investigate the presence of the genome of GB virus C/hepatitis G virus (GBV-C/HGV) in semen and saliva from HIV-1-infected men. METHODS: Samples of blood from 33 men seropositive for HIV-1 were tested for the presence of GBV-C/HGV markers of infection, RNA by RT-PCR, and anti-E2 antibodies by ELISA, respectively. The cell-free fractions of seminal fluid and saliva samples of the patients with positive blood samples for GBV-C/HGV RNA or anti-E2 antibodies were then analyzed for the presence of the RNA of this virus. In addition, six semen samples and 11 saliva samples from GBV-C/HGV-negative men were tested. RESULTS: The GBV-C/HGV RNA tested by RT-PCR was recovered from blood in 11 patients of 33 (33.3%), and the antibodies to E2 envelope protein were detected in six patients (18.2%). Since no patient was positive for both markers, the overall prevalence of GBV-C/HGV infection was 51.5% in the studied population. Four-all belonging to the homosexual risk group-of the 17 men with markers to GBV-C/HGV in blood were found to be positive for GBV-C/HGV RNA in mucosal samples: two of them exhibited genomic RNA in both semen and saliva, and two others were positive for semen only. The absence of inhibitors of the PCR technique was confirmed in all mucosal fractions found negative for GBV-C/HGV RNA, except for one saliva sample and one seminal fluid sample. CONCLUSION: These results confirm the high prevalence of GBV-C/HGV infection in patients infected with HIV-1 by sexual exposure and the presence of GBV-C/HGV RNA in seminal fluid and saliva of men with markers of this virus in the blood, suggesting that mucosal fluids could be a potential source for the spread of the GBV-C/HGV infection.

Abstract: To investigate the risk of transmission of hepatitis C virus (HCV) via semen in assisted reproduction techniques, semen samples from 32 men chronically infected with HCV attending a center for assisted procreation were tested for HCV RNA by a reverse transcription-PCR protocol by using a modified version of the Cobas AMPLICOR HCV assay (version 2.0; Roche Diagnostics). The sensitivity of the test was 40 copies/ml. Four of 32 seminal plasma samples (12.5%) were found to be positive for the presence of HCV RNA. The median HCV load in blood was significantly higher in patients who were found to be positive for the presence of HCV RNA in semen than in those who tested negative (P = 0.02). In one man, seven consecutive seminal plasma samples tested positive for HCV RNA, as did two consecutive motile spermatozoon fractions; the corresponding fractions obtained after migration of the spermatozoa remained negative. Despite the absence of the proven infectivity of virus in semen samples that test positive for HCV RNA, these findings highlight the fact that seminal fluid may exhibit prolonged HCV RNA excretion. The usefulness of HCV RNA detection in both seminal plasma and spermatozoon fractions before the start of a program of medically assisted reproduction in couples in whom the male partner is chronically infected with HCV would need to be evaluated prospectively with a larger population of subjects exhibiting HCV RNA in their semen.

Abstract: OBJECTIVE: To investigate the respective contribution of endogenous and exogenous transmission of Pseudomonas aeruginosa in the colonization of lungs in the mechanically ventilated patient, to estimate the role of P. aeruginosa colonization in the occurrence of severe infections, and to extrapolate appropriate control measures for the prevention of P. aeruginosa ventilator-associated pneumonia. DESIGN: Prospective study of the presence of P. aeruginosa (in stomach fluid, throat specimens, stool, and sputum) on admission, twice a week throughout the patient's stay, and in their environment. O-serotyping, pulsed-field gel electrophoresis, and arbitrarily-primed polymerase chain reaction were used to characterize the strains. SETTING: The two intensive care units (ICUs 1 and 2) of a university hospital. PATIENTS: During a 6-month period, 59 patients were included (21 in ICU 1 and 38 in ICU 2). RESULTS: P. aeruginosa was isolated in 26 patients, including ten pneumonia cases and seven colonizations on admission. The incidence of acquired colonization was statistically different between the two ICUs: 5.5 and 20.5 per 1000 days of mechanical ventilation, in ICUs 1 and 2, respectively. Endogenous acquisition was the main origin of P. aeruginosa colonization (21 of 26 patients) and the upper respiratory tract was the main bacterial reservoir in broncho-pulmonary colonization and infection. However, during the 6-month period of the study, a multidrug-resistant strain of P. aeruginosa O:11, isolated in the sink of the room of 12 patients, was found responsible for two colonizations (1 digestive, 1 throat/lungs) and one pneumonia. As a whole, from 26 cases of colonization/infection with P. aeruginosa, 5 were related to an exogenous contamination (environmental reservoir in 4 patients and cross-contamination in one patient). CONCLUSIONS: These results emphasize the need for applying various infection control measures to prevent colonization of patients with P. aeruginosa, including strategies to limit the potential of sinks from acting as a source or reservoir for this bacterium.

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Abstract: OBJECTIVE: To investigate the persistence of colonization of premature babies by Klebsiella oxytoca, with special emphasis on the mode of transmission of the bacterium and evaluation of Standard Precautions to stop the epidemic. DESIGN: Retrospective analysis of cases and prospective study of systematic bacteriological samples (stools and throat) from babies, healthcare workers (HCWs), and environment, with genotyping of strains by arbitrarily primed polymerase chain reaction. SETTING: A premature baby unit (PBU) and a neonatal intensive care unit in the university hospital of Saint-Etienne, France. RESULTS: An outbreak of K oxytoca was suspected in two pediatric wards after the occurrence of a fatal bacteremia in a newborn hospitalized in the PBU and the colonization of other babies 2 months later. Retrospective analysis showed that 24 babies' digestive tract had been colonized. No environmental reservoir was recovered in the units nor in enteral feeding. No K oxytoca was isolated from HCW samples. Genotyping confirmed the presence of epidemic strains, although independent clones were responsible for infections or colonizations in each of the two units. The chronology and the site of babies' colonization (isolation of K oxytoca in stools before throat) were determined during a prospective study and suggested that enteral feeding procedures could be the source of contamination. Therefore, use of gloves during this practice by HCWs was recommended and, after readjustment of Standard Precautions, stopped the outbreak. CONCLUSION: To prevent cross-contamination among high-risk babies, careful attention must be paid to Standard Precautions. Bacteriological surveillance of the digestive tract of neonates could help to check compliance with these guidelines

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Abstract: PURPOSE: In the geriatric units of the University Hospital of Saint-Etienne, 129 cases of fever (38 degrees C or more) were recorded prospectively during the 1995-1996 winter period in a population of 503 hospitalised patients (25.6%), and were investigated for the detection of a viral aetiology. METHODS: In febrile patients, a standard form was used to record clinical and biological parameters, including the results of investigations for respiratory viruses from a nasal swab and dual serum specimens. RESULTS: A clinical or radiological respiratory infection was found in 69 cases (53.5% of all cases of fever), including 14 respiratory syncytial virus (RSV) infections. In comparison to nonviral respiratory infections, the RSV infections were characterised by the prevalence of anorexia (57% vs 20%, P < 0.05) and rhinorrhea (64% vs 5%, P < 0.01). No influenza infection was recorded despite the concomitant circulation of influenza virus in the community. A nosocomial outbreak of RSV infection (nine cases, attack rate of 18.7%) was identified in a long-stay care unit. CONCLUSION: This study illustrates the high prevalence (10.9%) of RSV infections in elderly patients with fever during this season and the importance of hygienic measures to control the spread of nosocomial outbreaks.

Abstract: BACKGROUND: There are still discrepancies in the association of enterovirus and myocardial disease, partially due to lack of data on the detection of virus antigens in tissues. It is desirable to localize enteroviral antigens so as to establish a link between the two and to study mechanisms of virus persistence. METHODS AND RESULTS: Nineteen fixed explanted or postmortem myocardial samples were obtained from patients with myocarditis or dilated cardiomyopathy (DCM). Control samples were collected from 11 subjects who had died accidentally or of noncardiovascular disease. Viral antigen was detected by an improved immunohistochemical technique using an enterovirus group-specific antibody to viral capsid protein VP1. Nine of 11 myocarditis cases (81.8%) and 6 of 8 DCM cases (75%) were positive. Signals were localized in the cytoplasm of myocytes. Intense immunostaining was observed in acute myocarditis, whereas VP1 was detected in scattered myocytes in chronic myocarditis or DCM. Enteroviral RNA was detected in 6 of 11 myocarditis samples (54.5%) and 3 of 8 DCM samples (37.5%) by the reverse transcription-nested polymerase chain reaction, correlating with antigen detection (kappa=0.6+/-0.21). Neither viral antigen nor RNA was detected in any controls. CONCLUSIONS: Our findings demonstrate a direct link between enterovirus infection and some myocarditis or DCM cases. The pattern of VP1 detection may correlate with disease stage and severity. The data suggest that viral protein synthesis may be involved in persistent enterovirus infection in the pathogenesis of DCM.

Abstract: Infection with human immunodeficiency virus type 1 (HIV-1) has been shown to elicit a serum antibody response with neutralizing activity against T cell line-adapted HIV strains and primary HIV-1 isolates. Mucosal surfaces are the primary route of HIV-1 infection. Evidence is presented here for the presence of HIV-neutralizing antibodies in secretions. Infection of mucosal cells with HIV stimulates systemic and mucosal immune responses and results in the generation of neutralizing antibodies. Serum IgG and IgA neutralize HIV-1MN infection of susceptible T cell lines; serum IgG inhibits more effectively. Mucosal IgA purified from parotid saliva of HIV-1-seropositive individuals could neutralize both a T cell line-adapted strain and a primary isolate. The neutralizing activity of IgA was not directed against the anti-third-variable-loop or the anti-ELDKWA epitope. Thus, the specificity of mucosal IgA for HIV-1 neutralization epitopes remains to be determined and may provide insight into development of a mucosal vaccine.

Abstract: OBJECTIVE: Study the influence of hepatitis C virus (HCV) serology on the course of HIV disease in AIDS patients. PATIENTS AND METHODS: A prospective study of survival prognosis in HIV infected patients who had reached the AIDS stage was conducted in the Saint-Etienne, Clermond-Ferrand and Lyons infectious disease centers to compare patients with positive and negative HCV serology. Data were collected using the clinico-epidemiological software DMI II. The effect of HCV ìco-infectionî defined by RIBA II or III confirmed seropositivity, was studied using Kaplan-Meier survival plots. RESULTS: Among the 1,005 HIV-infected subjects included in the study, 219 had AIDS and 43 of them (19.6%) were HCV positive. Survival curves in HIV/HCV positive patients with AIDS were not significantly different from those of HCV-negative AIDS patients (median 17.8 versus 18.6 months respectively, p = 0.93). This result was confirmed by univariate Kaplan-Meier analysis. Only 2 patients were treated with interferon and no deaths were attributed to liver disease. CONCLUSION: HCV positivity in AIDS patients does not appear to influence survival. The longer survival obtained with the new anti-retroviral treatments may have an effect on the HIV-HCV interaction.

Abstract: OBJECTIVES: To investigate an outbreak of Serratia marcescens in a maternity hospital (November 1994 to May 1995). DESIGN: Retrospective analysis of epidemiological data and prospective study of systematic bacteriological samples from patients and environment, with genotyping of strains by arbitrarily primed polymerase chain reaction. SETTING: A private maternity hospital, Saint-Etienne, France. RESULTS: In the neonatal unit, 1 newborn developed a bacteremia, and 36 were colonized in stools with S marcescens. As the colonization of some newborns was shown to occur only a few hours after delivery, the inquiry was extended to other maternity wards, where 8 babies and 4 mothers were found to be colonized. Environmental sampling led to the isolation of S marcescens from a bottle of enteral feed additive in the neonatal unit and from the transducers of two internal tocographs in the delivery rooms. The genotyping of 27 strains showed two different profiles: a major epidemic profile shared by 22 strains (18 from babies of the neonatal unit, 2 from babies of other units, and 2 from breast milk) and another profile shared by 5 strains (2 from transducers of internal tocographs, 2 from babies, and 1 from a mother). The strain isolated from lipid enteral feeding was not available for typing. Although this source of contamination was removed soon from the neonatal unit, the outbreak stopped only when infection control measures were reinforced in the delivery rooms, including the nonreuse of internal tocographs. CONCLUSIONS: In delivery rooms, the quality of hygiene needs to be as high as in surgery rooms to prevent nosocomial colonization or infection of neonates at birth.

Abstract: GB virus C (GBV-C) or hepatitis G virus (HGV) is transmitted by the parenteral route but the importance of sexual transmission needs to be ascertained. GBV-C/HGV infections were investigated using RNA and E2-antibody detection methods in 80 subjects infected by the human immunodeficiency virus type 1 (HIV-1) divided into 4 groups of 20 individuals each according to their main risk factor for HIV-1 infection: blood product recipients (group 1), intravenous drug users (group 2), homosexuals (group 3), or heterosexual exposure (group 4). The overall prevalence of GBV-C/HGV infection was 66.3%. No significant difference was observed in GBV-C/ HGV prevalence among the four groups: 75, 75, 55, and 60% in groups 1, 2, 3, and 4, respectively. Hepatitis C virus (HCV) antibodies, used as a control for parenteral exposure, were found in 70% and 90% of the subjects in groups 1 and 2 versus only 15% and 20% of the subjects in groups 3 and 4, respectively (P< .001). Similarly, coinfections with GBV-C/HGV and HCV were significantly associated with the parenteral route (P <.001). These data emphasized the usefulness of combining the detection of RNA and the E2 antibody to determine the actual prevalence of GBV-C/HGV infection. The high prevalence of the GBV-C/HGV markers among the HIV-1-infected subjects, especially those with sexual exposure, provides additional evidence that this route of transmission plays a key role in the epidemiology of GBV-C/HGV. The potential influence of GBV-C/HGV infection on the course of HIV-1 disease needs further evaluation.

Abstract: Enterovirus infections in childhood (echoviruses, coxsackie viruses A and B, unclassified enteroviruses) are varied, nonspecific and benign (except for poliovirus infections). However, in infants, they may lead to severe neurological or cardiac lesions. In neonates, enterovirus infections are difficult to distinguish from late-onset bacterial infection. Maternal-fetal or postnatal transmission can induce early spontaneous abortions or severe neonatal infections. Diagnosis is usually based on viral cultures and, in recent years, on PCR techniques. At the present time, no potential effective drug is available: intravenous immunoglobulins, immunization or anti-proteases may be of interest.

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Abstract: Two very unusual cases of aplastic anemia complicating mycoplasma infection are described. Each patient had preexisting hematologic abnormalities at the time of the infection: reactive hemophagocytic syndrome in one, and autoimmune hemolytic anemia associated with cold autoantibodies in the other. This adds another entity to the protean manifestations of Mycoplasma pneumoniae infection.

Abstract: BACKGROUND: Mab 5-D8/1 is a monoclonal antibody (Mab) that was shown to be directed towards a conserved epitope of the capsid protein VP1 among the genus enterovirus. The use of this Mab for the routine detection of enteroviruses in clinical specimens led to the observation that several strains of echovirus type 11 (EV-11) could not be detected on spontaneously detached cells from 26-h cultures using a two-step immunofluorescence (IF) assay. Conversely, these strains were detected positive with the same Mab when tested on adherent or trypsinizated cells. OBJECTIVES: The aim of this study was to understand the misrecognition of some strains of EV-11 by this Mab. STUDY DESIGN AND RESULTS: IF tests at different times of the viral cycle brought evidence that the detection of a variant strain of EV-11 decreased rapidly with time, becoming undetectable 26 h post-infection, since the reference strain remained positive up to 46 h post-infection. The infective titres of the variant strains were shown to be high in comparison with those of well-recognised strains. Sequencing the Mab binding epitope confirmed that the variant strains exhibited no antigenic shift. CONCLUSION: These results suggest that the poor recognition of some strains of EV-11 by Mab 5-D8/1 is due to a rapid decrease of the expression of the binding epitope in the cell, maybe in relation with the high lytic power of these strains. From a practical point of view, our data indicate that a negative result when Mab 5-D8/1 is used for enterovirus typing must be interpreted cautiously with highly replicative strains and that detached cells should not be used for enterovirus identification under these circumstances.

Abstract: It was shown in children that serum procalcitonin was the best marker to use to differentiate bacterial from viral meningitis. To evaluate procalcitonin in the diagnosis of acute bacterial and viral meningitis, we conducted a prospective study including adult patients who were suspected of having meningitis and who were admitted to an emergency department. Cerebrospinal fluid (CSF) and serum levels of procalcitonin were measured in 105 consecutive patients. The diagnosis of meningitis was based on clinical findings, gram staining, culture, and chemical analysis of CSF. Twenty-three patients had bacterial meningitis, 57 had viral meningitis, and 25 did not have meningitis. Bacteriologic and chemical analysis of CSF did not allow correct differentiation of viral from bacterial meningitis. On the other hand, a serum procalcitonin level >0.2 ng/mL had a sensitivity and specificity of up to 100% in the diagnosis of bacterial meningitis. Serum procalcitonin levels seem to be the best marker in differentiating between bacterial and viral meningitis in adults.

Abstract: OBJECTIVE: To investigate the epidemiologic relatedness of nosocomial infections due to Legionella pneumophila serogroup 1 diagnosed between 1992 and 1994 in six immunocompromised patients of the same hospital and to describe the measures which were developed to control the outbreak. METHODS: Legionella strains isolated from patients and from potable hot water were compared using three typing methods: monoclonal antibody analysis, arbitrarily primed PCR and ribotyping. RESULTS: Environmental investigations revealed the presence of high levels of L. pneumophila serogroup 1 in hot water. The typing methods gave concordant results for demonstrating (1) the persistence of an epidemic strain of L. pneumophila serogroup 1 in the major water distribution circuit of the hospital over a 3-year period, and (2) the identity between patients' and environmental strains. Five of the six patients were probably infected via aerosols of hot tap water following inappropriate therapeutic procedures. Repetitive heat flushings associated with regular bacteriologic surveillance allowed correct disinfection of the water distribution systems. Specific recommendations concerning aerosol delivery and oxygen therapy were implemented in order to prevent further nosocomial legionellosis. CONCLUSIONS: The same strain of L. pneumophila had been able to colonize the main water circuit of the hospital for at least 3 years; the relatedness between clinical and environmental strains was easily confirmed by the use of molecular markers.

Abstract: BACKGROUND: Coxsackievirus B3 (CVB3) causes myocarditis in the SWR (H2q) mouse model and persistence of CVB3 in myocardium disposes to the development of dilated cardiomyopathy. An attenuated strain of CVB3 has been isolated, sequenced and several candidate mutations for attenuation identified. Derivation of a revertant to cardiovirulence allows the significance of these mutations to be assessed. OBJECTIVES: To ascertain which candidate mutation(s) determine(s) the attenuated phenotype. STUDY DESIGN: A revertant to cardiovirulence was isolated following passage through severe combined immunodeficient disease (SCID) mouse heart. The 5'-non-translated region (NTR) and region coding for capsid proteins were sequenced and compared to the wildtype and attenuant. RESULTS: There are five candidates for attenuation: (1) A-G at base 580 in the 5'-NTR; (2) A-T at base 690 in the 5'-NTR; (3) CG-GC at bases 1401/2 (Thr to Ser at amino acid 151 in VP2); (4) AA-GT at bases 2691/2 (Lys to Ser at amino acid 80 in VP1); (5) A-G at base 2916 (Asp to Gly at amino acid 155 in VP1). It was shown previously that mutations at 580, 690 and 2691/2 are not important in attenuation. Additionally, there are three novel mutations in the coding region of the revertant and one in the 5'-NTR which are unlikely to be relevant for attenuation as they are not present in the attenuant. Of nucleotide changes seen at 1401/2 and 2916 in the attenuant, only 2916 reverts to the wildtype sequence and so is a strong candidate for a determinant of attenuation.

Abstract: Between december 1996 and february 1998, rhinopharyngeal and tracheal aspirates from 165 children exhibiting symptoms compatible with M. pneumoniae infection were tested by a PCR method using in the same tube primers specific for M. pneumoniae P1 adhesin gene and for a human control gene. The positive cases were controlled using culture and/or serology. PCR was positive in 22 out of 165 samples (13.3%); an evaluation of the clinical and biological data was possible in 20 of these infected children. From 17 PCR positive respiratory samples tested by culture, 13 (76.5%) grew M. pneumoniae. From 14 serum specimens tested by ELISA, 12 exhibited specific IgM (3 of them with low titers); cold agglutinins were detected in all 7 tested sera. Only one case was not confirmed by any of the 3 former markers. The mean age of patients was 8.1 years. The main clinical symptoms included fever > 38 degrees C, cough, clinical and radiological pneumonia in 90, 95, 50 and 85% of cases, respectively. Neurological symptoms were the main clinical manifestation in 3 patients; another child exhibited a pneumonia associated to an hemophagocytic syndrome and a bone marrow failure which needed a graft. These results emphasize the value of PCR for the rapid diagnosis of M. pneumoniae infection in children.

Abstract: In order to shorten the time required for the detection of enteroviruses in stool specimens, an 18-h immunoperoxidase test combining low-speed centrifugation and the use of a group specific anti-VP1 monoclonal antibody (5-D8/1, Dako) was developed. This rapid culture assay (RCA) was compared blindly to a conventional culture assay (CCA) on a panel of 180 children's stool specimens received for routine diagnosis of enterovirus infection. The same cell lines (human embryonic fibroblasts and KB continuous cell line) were used in both tests. Discrepancies in results were analysed by a PCR technique with primers located in a conserved part of the 5' non-coding region of the enterovirus genome. Fourteen specimens were positive and 158 were negative with both tests. Four samples were positive with the RCA yet negative with the CCA and 3 others showed the opposite pattern; an additional sample positive by RCA was uninterpretable by CCA due to bacterial contamination. Subsequent PCR testing of these 8 samples showed no discrepancies; all were positive. Using CCA as the reference, the sensitivity and specificity of RCA were 77.8 and 98% respectively. Kinetic studies using enterovirus isolates demonstrated that RCA was much more sensitive than CCA during the first three days of culture. These results further suggested that RCA sensitivity could be improved by a factor of at least 10 times by prolonging the incubation period by 24 hr. With this change, the RCA assay described below is suggested as a rapid alternative to CCA for the routine diagnosis of enterovirus infection in stool specimens. When an identification at the serotype level is required, samples found positive using RCA could then be subjected to CCA.

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Abstract: Enteroviruses are small RNA viruses belonging to the Picornaviridae family. At least 65 serotypes have been described, including polioviruses, coxsackieviruses A and B, echoviruses and unclassified enteroviruses. Because of the absence of envelope they are relatively resistant to physical and chemical agents. They are mainly transmitted by the oral-fecal mode, but respiratory and mucosal transmissions are also possible. In humans, enteroviruses have been involved in miscellaneous acute infections and more recently in persistent infections (chronic meningoencephalitis in agammaglobulinemic patients, post-polio syndrome, chronic myocarditis and dilated cardiomyopathy, insulin-dependent diabetes mellitus...). Hypotheses in the relation between enterovirus persistence and chronic infections are formulated. The virological diagnosis of enterovirus infections is discussed, with a special focus on genomic application techniques (PCR) that are renewing the interest for this family of viruses in clinical pathology. If the role of enteroviruses in chronic pathologies is confirmed, the development of new therapeutic approaches (including vaccines and antiviral agents) will be needed.

Abstract: Paired sera and parotid saliva from 75 HIV-1-infected patients, divided in three equal groups with CD4+ cell counts > 500, 200-500 and < 200/mm3, respectively, were analysed for IgG, IgA and secretory IgA (sIgA) concentrations and for IgG and IgA antibody directed to HIV-1. Twenty-nine age-matched HIV-subjects were used as controls. In serum the concentrations of immunoglobulins were significantly increased in HIV-infected subjects compared with controls, and a progressive increase of IgA and sIgA was noticed while the CD4+ cell count decreased. In contrast, concentrations of IgA and sIgA were not different in parotid saliva between the four subject groups. By an ELISA test directed towards HIV-1 proteins, 73 of the 75 serum specimens from the HIV-infected subjects (97%) and 43 of the corresponding saliva (57%) were found positive for specific IgA antibodies to HIV-1, with an even distribution among the three groups of patients. By Western blotting multiple specificities of IgA to HIV-1 proteins were not frequently found in patients. By contrast, in spite of an IgG concentration in saliva about 100 times lower than that of IgA, reactivities were significantly higher for IgG than for IgA antibodies, especially to env and to pol HIV-1 products. Altogether, these data suggest that the regulation of IgA production in HIV-infected subjects is independent in serum and in parotid saliva. This imbalance of IgA/IgG antibodies to HIV-1 at the mucosal level appears to be a specific feature of HIV-1 infection, and may raise important issues in terms of local protection after immunization.

Abstract: 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 x 10(5) 50% tissue culture infective doses (TCID50) 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 TCID50/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.

Abstract: In order to study the humoral immune defences in the respiratory tract during HIV-1 infection, we measured the levels, local productions and anti-HIV and antibacterial activities of IgG and IgA in the bronchoalveolar lavage fluid (BALF) and serum of 61 adult patients with severe HIV infection and of 56 HIV- controls. Albumin was used as the serum transudation factor. The increase of immunoglobulin levels in the serum of HIV-infected patients was confirmed. The IgG level was also increased in epithelial lining fluid (ELF), whereas the total IgA level was unchanged and secretory IgA (SIgA) level was decreased. The ELF/serum immunoglobulin ratios suggested that the IgG present in ELF resulted mainly from transudation, in contrast to SIgA, which was synthesized locally in controls but greatly diminished in HIV-infected patients. IgG to HIV-1 could be detected in BALF of all the patients, but IgA to HIV-1 only in 30% of patients. BAL IgG reacted more consistently and with a broader array of HIV-1 antigens than did IgA. BAL IgA, when present in samples, reacted primarily with viral envelope antigens. Because IgA specificities to some HIV-1 antigens were detected more intensively by BAL than by serum immunoglobulins, we conclude that the mucosal immune response is distinct from that in serum. IgG antibody activity to Streptococcus pneumoniae was decreased in HIV-infected patients' sera, and IgA antibody activities to S. pneumoniae and to Pseudomonas aeruginosa were decreased in ELF in HIV-infected patients.

Abstract: Hepatitis C virus (HCV) is an enveloped, single-stranded RNA virus that has been classified in the Flaviviridae family. The genome of 9400 nucleotides comprises two non-coding regions in 5' and 3' flanking a large reading frame which codes for a polyprotein of 3000 amino acids; this polyprotein is further cleaved into structural (C, E1, E2) and non-structural (NS1, NS2, NS3, NS4, NS5) proteins. The positive RNA acts as a cap-independent messenger; the transcription is mediated by the NS5 RNA polymerase. After the maturation step, the virion is liberated by budding through the cytoplasmic membrane. As for many other RNA viruses, the HCV genome exhibits a high degree of variability, especially in the E2/NS1, E1, NS3 and NS5b regions. Conversely the 5' non-coding region is highly conserved, at least in part, and can be used for diagnostic purposes by PCR technique. Six genotypes of HCV have already been reported, numbered from 1 to 6 in Simmonds' classification. The same genotype can be divided into subtypes (for instance, genotype 1 comprises three subtypes: 1a, 1b and 1c). Various minor variants of the same strain, called quasispecies, are commonly present in the blood of the same patient. Strains of genotype 1b--which is the most widespread worldwide--are correlated with more severe clinical manifestations, greater viral loads and lower response to interferon treatment. The high variability of the HCV genome contributes greatly to the difficulty of designing potent vaccines.

Abstract: The prevalence of HCV infection is high in renal transplantation (RT) patients: 29% in our cohort of 399 RT recipients. The consequences of that infection on the liver have to be carefully assessed. Clinical chronic hepatitis was detected from ALT concentrations (> x 1.5 N) in only 26 patients (22%) with constant (15%) or fluctuating (85%) ALT elevation. Only three of 117 cases developed cirrhosis (3%). No liver cancer was noted. Liver biopsy was performed (mean interval = 60.2 months) in 62 patients with HCV infection alone. We found 26 cases (42%) of chronic active hepatitis (CAH) with a mean Knodell score as low as 6.1 (range: 3-12), a mean activity grade of 4.9, and a fibrosis stage of 1.3. Twelve patients (19%) presented with normal liver pathology and met the criteria of healthy HCV carriers (positive viraemia, normal ALT and normal liver). The rest presented with portal lesions, either inflammation or fibrosis. In addition, patient and graft survival rates did not differ in HCV+ recipients. To conclude, HCV infection did not appear too deleterious for the liver in this cohort of patients. There is therefore no contraindication for HCV-positive recipients to undergo renal transplantation.

Abstract: 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.

Abstract: Thirteen laboratories participated in blind tests of a panel of 20 coded cerebrospinal fluid specimens (7 uninfected samples, 3 samples infected with 1 50% tissue culture infective dose [TCID50]/0.1 ml [nonenterovirus strains], and 10 samples infected with 10, 1, or 0.1 TCID50/0.1 ml [three different enterovirus serotypes]) on the Amplicor enterovirus PCR assay (Roche Diagnostic Systems). The panel was also evaluated by in-house PCR (two nested-PCR and three one-step PCR assay) or tissue culture (eight laboratories). The viral load was shown to influence greatly the sensitivity of the assay. The average sensitivity of the Amplicor test ranged from 67 to 98% for viral titers of 1 to 10 TCID50/0.1 ml, respectively; titers of 0.1 TCID50/0.1 ml resulted in a sensitivity of only 16%. The overall specificity of the Amplicor test was 98%. The Amplicor assay compared favorably to the five in-house PCR tests (no significant difference in either sensitivity or specificity) and was much more sensitive than tissue culture (P < 0.001), even for high viral loads. It was easy to perform, rapid (about 6 h), well-standardized, and appeared to be suitable for the diagnosis of enterovirus meningitis on a routine basis in laboratories trained in molecular biology techniques.

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Abstract: OBJECTIVES: In industrialized countries with a high level of sanitation, immunity against hepatitis A (HVA) is not acquired during childhood, and infection typically occurs in adults, mainly in travelers returning from developing countries where infection is endemic. However, the introduction of hepatitis A virus (HAV) among certain population groups, such as intravenous drug users (IVDU) or homosexual men, leads to a significant increase in the disease. We conducted a retrospective analysis of seroprevalence of anti-HAV antibodies. METHODS: The study group included 296 patients (174 homosexual men and 122 IVDU) for comparison with 76 control subjects (nurses in pediatric wards and workers in hospital kitchen). RESULTS: We found a significantly higher anti-HAV seroprevalence among less than 35-year old IVDU, HIV positive or negative, in comparison with control subjects but not among homosexual men, whatever their HIV status. CONCLUSION: Our experience illustrates that HVA is a health risk for IVDU in industrialized nations, and given its morbidity among adults population, IVDU should receive HVA vaccine.

Abstract: OBJECTIVE: To study the spread of strains of Enterobacter aerogenes in our hospital in 1992 and 1993 by using two genotypic markers, and to evaluate these methods for the epidemiological investigation of this species. DESIGN: Ribotyping (using two endonucleases) and arbitrarily primed (AP)-PCR (using two different 10-mer primers) were applied to the epidemiological typing of clinical strains of E aerogenes isolated from hospitalized patients. SETTING AND PATIENTS: The intensive care unit (ICU; 5 patients, 13 isolates), nephrology units (3 patients, 5 isolates), and surgery units (2 patients, 2 isolates) of the university hospital of Saint-Etienne (France). RESULTS: Eight epidemiologically unrelated isolates, chosen as controls, exhibited distinct profiles, both by AP-PCR and ribotyping. Two clones of E aerogenes circulated in the ICU; both were isolated successively from samples of a single patient who stayed in the unit for almost 1 year. A third clone was recovered from patients of surgery units. A fourth clone was shown to have infected patients of nephrology units. CONCLUSIONS: Ribotyping and AP-PCR appear to be reliable methods for typing E aerogenes strains implicated in nosocomial infection. The spread of independent clones of E aerogenes in different units of our hospital in 1992 and 1993 was demonstrated by both methods. This study emphasizes the need to choose the endonucleases or primers with care to obtain high discriminatory results in genotypic investigations.

Abstract: Seventy-nine serum specimens from pregnant women and 29 from immunocompromised patients (12 from graft recipients and 17 from patients with acquired immunodeficiency syndrome) were classified into three groups according to their serologic status to Toxoplasma gondii as determined by immunofluorescence and an enzyme-linked immunosorbent assay (ELISA): no antibodies (group 1), acute acquired infection (group 2), and reactivation (group 3). These samples were tested for the presence of circulating antigens (CAg) of T. gondii by capture ELISA and immunoblotting. The presence of CAg was detected by at least one of the two techniques in six of 31 subjects in group 1, 51 of 68 subjects in group 2, and seven of nine subjects in group 3. Of a total of 108 serum specimens, 28 were found to be T. gondii-positive by capture ELISA, 57 by immunoblotting, and 21 by both techniques. Among the nine polypeptides detected by immunoblotting, 38 recognized p14, 17 recognized p8, and 16 recognized p8, and 16 recognized p30. These results demonstrate that the detection of CAg can aid in the diagnosis of infection by T. gondii in humans, especially in immunocompromised patients whose serologic response can be impaired.

Abstract: Fourteen serotypes are currently recognized in the Ureaplasma urealyticum species. 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 14 U. urealyticum serotypes. 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 of U. urealyticum recovered 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.

Abstract: The rates of colonization by Ureaplasma urealyticum and Mycoplasma hominis were evaluated in 208 women at delivery and in their neonates. Mycoplasmas were isolated from the cervicovaginal specimens of 100 mothers (48.1%) and from the gastric secretions of 40 neonates (19.2%). The prevalences of U. urealyticum and M. hominis were 47.6% and 11.0% in women and 19.2% and 1.0% in neonates, respectively. Premature rupture of membranes was significantly associated with colonization of women by U. urealyticum (P = 0.031), and colonization of their neonates by U. urealyticum (P = 0.002) and/or M. hominis (P = 0.023). Forty-four selected strains of mycoplasmas were further characterized by arbitrarily primed polymerase chain reaction. All strains of U. urealyticum belonged to the parvo biovar of the species. Arbitrarily primed polymerase chain reaction demonstrated the similarity of strains isolated from mother-neonate pairs, confirming the importance of vertical transmission of mycoplasmas at delivery.

Abstract: 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.

Abstract: The genotypic diversity of 40 presumably epidemiologically unrelated strains of Pseudomonas aeruginosa belonging to nine different O-serotypes was analysed according to ribosomal DNA fingerprints. Ribotyping was performed with a digoxigenin-labelled DNA probe and four restriction endonucleases. Characteristic banding patterns of three to 12 bands were obtained with the different endonucleases. Among the 40 strains, eight, nine, 10 and 29 different ribotypes were differentiated with EcoRI, the combination EcoRI+HindIII, BamHI and PvuII, respectively. Poor correlations were noted between the results of serotyping and those of ribotyping. With the latter method, indices of discrimination were calculated for each enzyme from the data of the 40 unrelated strains: the values ranged from 0.678 for EcoRI to 0.979 for PvuII. Epidemiologically related samples were also tested; this enabled assessment of whether the method was able to cluster strains from a common origin with each of the enzymes tested. Ribotyping with PvuII endonuclease is proposed for screening large numbers of P. aeruginosa strains in epidemiological studies. Additional enzymes could be used to further increase the discrimination between isolates found to be indistinguishable with PvuII enzyme.

Abstract: 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.

Abstract: Except for Ig E, serum immunoglobulin abnormalities in persons with human immunodeficiency virus (HIV) infection have been well described. Serum IgE levels have been shown to rise with progressive disease. The authors evaluated IgE in 148 HIV-seropositive individuals with or without acquired immunodeficiency syndrome (AIDS). Mean serum IgE levels were compared between groups based on absolute CD4 lymphocyte counts or clinical status (CDC) and with a seronegative control group. Higher serum IgE levels were observed in seropositive-patients. A rise in IgE serum is common in patients with HIV infection; it could be link with an earlier dysregulation in the IgE synthesis. No correlation was found between IgE level and CD4 counts.

Abstract: The tolerance of influenza vaccination (Vaxigrip, Pasteur-Mérieux) was evaluated during four consecutive years (1989-1992) in the geriatric hospital of Saint-Etienne from questionnaires concerning 327 vaccinations in the aged (group 1) and 88 vaccinations in members of the nursing staff (group 2). Minor local symptoms were the more common incidents, respectively for each group: blotch (9.8 and 21.6%), pain (9.2 and 47.7%), nodule (2.4 and 13.6%). Fatigue (4.0 vs 12.5%) and fever (6.1 vs 4.5%) were the more frequent among general symptoms, respectively in each group. No major vaccinal accident was recorded. These results underline that influenza vaccination is well tolerated, much more in aged people than in members of the nursing staff.

Abstract: This study was performed in 77 HIV1 seropositive adult patients to characterise the IgA hyperglobulinaemia seen in the serum during the course of HIV infection. It was shown that both IgA1 and IgA2 subclass concentrations were simultaneously increased but the IgA1 increase was predominant. Secretory IgA (SIgA) concentration was significantly increased and IgA activity to gliadin, bovine serum albumin, and casein could be detected and was correlated with SIgA concentration. In contrast, IgA activity to cytomegalovirus and to tetanus toxoid did not correlate with total IgA concentration. These data suggest the presence of IgA from gut mucosal origin in the serum of these patients. Hyper IgA was inversely correlated with the CD4+ cell number. The increase of all parameters studied varied according to the total IgA concentration in the serum but was also directly related to the stage of immune deficiency in patients with hyper IgA.

Abstract: We report 9 cases of enteroviral infection associated with systemic inflammatory disease (including 4 cases of vasculitis, 1 case of periarteritis nodosa, 1 case of Sharp's syndrome). We then reviewed 36 cases of enteroviral infection with persistent IgM antibodies diagnosed in the virology laboratory in a 6-year period: among them 11 cases were found to present with subacute or chronic inflammatory disease. We conclude that enteroviruses might be important triggers of systemic inflammatory disease.

Abstract: The spread in Europe of a single multiresistant strain of Pseudomonas 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 these Pseudomonas aeruginosa O12 strains, despite a relative increase in their antibiotic resistance.

Abstract: During a winter epidemic of A/H1N1 influenza virus, we evaluated the protection conferred by vaccination of 285 residents of a nursing home. Fifteen of 204 members of the nursing staff were also vaccinated. Serological determinations were performed before and after vaccination using radial hemolysis (RH) and neuraminidase inhibition (NI) tests. In the outbreak period, only one influenza case was noted in the vaccinated elderly and none among the vaccinated nursing staff. On the other hand, 38 cases (20%) occurred in the unvaccinated hospital personnel. Twenty-one percent of the elderly people exhibited seroconversion to the vaccinal strain by RH and NI while 27 and 20% of the vaccinated nursing staff seroconverted by the same tests, respectively. Thus, the clinical protection conferred by influenza vaccination was excellent and much greater than expected from serological results.

Abstract: 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.

Abstract: This study was designed to explore the relationship between malnutrition, inflammation, and the specific antibody response after influenza vaccination in the elderly. Eighty-two aged subjects, immunized annually against influenza with a trivalent inactivated vaccine, were evaluated for 9 protein markers (albumin, thyroxin-binding prealbumin, transferrin, immunoglobulins (Ig) G, M, and A, orosomucoid, haptoglobin, and C reactive protein) and for their antibody response to influenza viruses in comparison to 29 younger adults who received the same vaccine and 21 unvaccinated adults. IgM and nutritional markers were significantly reduced in the aged as compared to controls, while the opposite pattern was seen for IgA and inflammatory markers. No difference was observed between the elderly and the controls with regard to the antibody response to influenza virus after vaccination. Reciprocally, influenza immunization had no influence on the levels of the protein variables. These results suggest that the protein status does not play an important role in the antibody response to influenza vaccination in the elderly, a fact which could be related to the slight involvement of cellular immunity in the defense against influenza reinfection.

Abstract: Serum specimens from 66 HIV-1-infected subjects were tested by ELISA for the presence of IgA antibodies to HIV-1: 44 samples were found positive and 37 were confirmed by immunoblot. In these subjects, the presence of anti-HIV IgA antibodies was studied in relation to the total count of circulating CD4+ lymphocytes and to the level of serum IgA. A significative correlation (P < 0.03) was found between the absence of IgA to the subunit p68 of the reverse transcriptase and a count of CD4+ cell < 400/mm3 or total IgA level over 4.25 g/l. The same pattern was observed for the IgA antibodies to the p52 subunit but the association was just not significant (P < 0.07). No significant decrease was noted for the IgA directed towards the other proteins of HIV-1, especially the products of the gag gene.

Abstract: A prospective study was undertaken to determine the source of Pseudomonas cepacia colonization and infection that had affected ventilated patients in an Intensive Care Unit (ICU) for three years. Thirty-eight patients undergoing mechanical ventilation were enrolled during a six-week period. Samples were taken from patients, ventilator circuits and the environment for culture. P. cepacia was isolated from the condensate formed in the ventilator circuit and the source of the contamination was shown to be the temperature sensor. Ribotyping of the representative strains of P. cepacia performed with two endonucleases, EcoRI and PvuII, confirmed the homogeneity of the isolates from patients and ventilator circuits. A modification of the procedure for disinfection of the temperature sensors resulted in the eradication of P. cepacia from the ICU.

Abstract: We report a case of transient erythroblastopenia in a three-year-old girl presenting with echovirus 11 infection. Viral infection was demonstrated by isolation of echovirus 11 in stool cultures and the presence of echovirus 11-specific IgM antibody in serum. We suggest that echovirus may have played a role in the pathogenesis of transient erythroblastopenia of childhood in this patient.

Abstract: Cell lines of primate origin carry membrane receptors which are specific for echoviruses (EV). The present report describes isolation and characterization of a monoclonal antibody (Mab 143) reacting with the membrane of KB cells. The Mab was selected for its protection of different cell lines from primate origin against the CPE of EV-11. This protection was found to extend to most EV serotypes and to coxsackievirus A9, while the replication of several other picornaviruses was not affected. The fluorecein isothiocyanate labelled Mab did not react with cell lines from bovine, canine, or rabbit origin, but bound specifically to the cell lines from human or simian origin. These results suggest that a unique receptor site is used by most EV serotypes for binding onto and penetration into susceptible cells.

Abstract: Interaction between herpesviruses and human immunodeficiency virus (HIV)1 is postulated in the progression of HIV disease. In order to evaluate the specific antibody responses directed to Epstein-Barr virus (EBV) and cytomegalovirus (CMV) and to provide serological evidence suggesting reactivation of these viruses able to accelerate the immunodeficiency, we studied IgA and IgG titres to EBV and CMV in the serum of HIV positive patients in relation to the CD4 cell number. The titres of IgG antibodies to EBV and the prevalence of IgG to CMV were significantly higher in HIV positive patients compared to control high risk HIV negative subjects. In HIV infected patients, anti-VCA IgG antibodies increased and anti-EBNA IgG antibodies decreased progressively in relation to the decline of CD4 cell number whereas anti-CMV IgG antibodies did not varied significantly at the same time. Anti-VCA IgA and anti-EA IgG antibodies were found uncommonly and with low titres. IgA antibodies to EA and CMV were not detected in any patient. The variations in EBV antibody response that we describe in HIV infection were previously reported in other immunodeficiency states and could be distinctive of these diseases.

Abstract: Emergence of resistance to fluoroquinolones was observed in two clinical isolates of Pseudomonas aeruginosa after ciprofloxacin or norfloxacin monotherapy. In the first case, the resistant variants exhibited quinolone-imipenem cross-resistance (MIC of norfloxacin and ciprofloxacin: 16 mg/L; MIC of imipenem: 8 mg/L), although the patient had never received imipenem treatment, while the strain from the second case remained imipenem-susceptible (MIC of norfloxacin or ciprofloxacin: 8 mg/L; MIC of imipenem: 2 mg/I). The frequency of in-vitro emergence of variants resistant to imipenem and fluoroquinolones was studied for the two strains, with imipenem or fluoroquinolones as selecting agents. Ciprofloxacin and three other quinolones (norfloxacin, temafloxacin and tosufloxacin) selected imipenem-resistant variants in a similar way to imipenem for the first strain, but not for the other. In contrast, imipenem did not select quinolone-resistant variants from either strain. For both strains, killing curves demonstrated that a bactericidal effect could be obtained with a drug combination (2 x MIC of ciprofloxacin and 2 x MIC of imipenem) without any selection of resistant mutants after 24 h, thereby suggesting the possible use of this combined regimen for treating severe P. aeruginosa infection.

Abstract: A panel of 68 serum specimens from 41 subjects exhibiting various immunological patterns to Mycoplasma pneumoniae as determined by detection of a 180 kDa protein in immunoblotting was used to compare five commercially available tests based on different methods: complement fixation test (CFT), microparticle agglutination (MAG), indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (Elisa), and latex agglutination (LA). The tests were performed according to the manufacturers' instructions. For the determination of immunity to M pneumoniae, the five tests were in good accordance with immunoblotting: sensitivity was 100% for all the five assays, specificity ranged from 95.6% (MAG) to 82.6% (Elisa) and overall agreement ranged from 98.2% (MAG) to 92.8% (Elisa). The comparisons of antibody rates obtained by the four quantitative tests (CFT, MAG, IFA, Elisa) showed correlation coefficients ranging from 0.87 (CFT-IFA) to 0.67 (CFT-Elisa). Six significant antibody rises demonstrated by immunoblotting patterns were detected by all the tests but Elisa in one case. As a whole, the commercial assays gave satisfactory results for routine determination of immune status to M pneumoniae: CFT was the cheapest test and MAG and LA were the easiest to perform.

Abstract: Cellular receptors play an important role in viral pathogenesis. Until now little was known on echovirus (EV) receptor. Using detergent-treated KB cell extracts as immunogen, a mouse monoclonal antibody (Mab 143) was produced that selectively blocks the attachment of EV-11 to KB and other susceptible cells. By immunoblotting, Mab 143 detected a 44,000 protein on susceptible cell lines but not on cell lines from nonprimate origin. The receptor protein complex, purified from KB cell membranes by immunoaffinity using Mab 143 as ligand, was shown to contain a single glycoprotein with apparent molecular weight of 44,000 (gp44). The role of gp44 in the attachment of EV-11 onto KB cells was demonstrated by the ability (i) of affinity-purified gp44 to reduce the infectivity of EV-11 and (ii) of rabbit polyclonal antisera raised against gp44 to protect cells from the replication of various EV, as did Mab 143.

Abstract: We previously reported that infection of KB cells by echoviruses (EV) was inhibited by a KB-derived EV receptor murine monoclonal antibody (mAb 143). This antibody enabled the identification of a cellular receptor common to all echoviruses (with the exception of EV-22 and -23) and coxsackievirus (CV) A9, but different from the receptor of other picornaviruses. We now present results of cell protection assays conducted with human and simian cell lines different from the KB cell line used for production of mAb 143. When human embryonic lung fibroblasts were pretreated with 150 micrograms/ml of mAb 143, EV-11 and CV-A9 were completely inhibited (more than a 2-log difference compared to untreated cells). When the cell protection experiments were performed with Vero cells, the same results were observed with EV-33, but not with EV-22. The protection afforded human fibroblast cells by mAb 143 persisted for at least 5 days after 2-h exposure to 100 TCID50 of EV-11. These results suggest that EV receptors can be effectively blocked for prolonged periods in susceptible cells.

Abstract: Toxoplasma gondii trophozoites (RH strain) were cultured in embryonic fibroblasts in order to study the kinetics of production of excretory/secretory antigens, and the results were compared to the production of circulating antigens in an in vivo mouse model. By capture-ELISA, excretory/secretory antigens were first detected on the fourth day of culture whereas circulating antigens were first detected 1 day after infection. Similar concentrations of antigens were detected in both models as evidenced by comparable absorbance values. By immunoblotting, the excretory/secretory antigens were also detected later compared to circulating antigens (day 4 vs day 1). Seven major polypeptides were detected in both antigen preparations, six of them having the same molecular mass (110, 75, 48, 30, 24 and 22 kDa).

Abstract: Two hundred and ninety-four serum specimens from 248 subjects, whose complement fixation (CF) titres to Mycoplasma pneumoniae were known, were further investigated by IgG immunoblotting. After analysis of M. pneumoniae proteins by SDS-PAGE, nine polypeptides (p) with mol. wts of 180-43 Kda were selected for immunoblotting studies. Antibodies to M. pneumoniae measured by immunoblotting appeared progressively with age; most subjects more than 19 years old gave positive results. For most of the polypeptides, there was an increase in the frequency of band detection when the CF titres were higher. Furthermore, paired serum specimens from 10 patients with M. pneumoniae infection, as demonstrated by a rise in CF antibody titre, were tested for IgG blotting patterns. Generally, p180 (the P1 adhesin of M. pneumoniae), p172 and p84 were shown to be the dominant targets of the immune response to this organism and may have diagnostic value.

Abstract: Little is known about the cellular immune response during the acute phase of enterovirus infection. This was studied in patients with echovirus meningitis by analysing changes in the lymphocyte subset distribution both in blood and in cerebrospinal fluid (CSF) and their stage of activation. A significant increase of whole T-cell and CD4+ T-cell counts was observed in CSF in parallel with a slight decrease of T-cell percentage in blood. Activation of the cellular immune response is supported by the observation of elevated neopterin levels in serum and CSF. However, the phenotypic markers of T-cell activation, IL-2 receptor (CD25), and HLA-DR antigens were not detected in blood or in CSF.

Abstract: 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.

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Abstract: The authors report on a pediatric case of transient neutropenia with erythroblastopenia secondary to human parvovirus infection occurring in a child without underlying hemolytic disease. The heterogeneity of hematologic manifestations in such infections is discussed.

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Abstract: Tosufloxacin tosylate (TOSU) and temafloxacin hydrochloride (TEMA) were tested in vitro against 248 clinical isolates of various species of streptococci recovered in a hospital microbiology laboratory (Bellevue Regional Teaching Hospital). Species included S. pneumoniae (n = 20), group A streptococci (n = 22), group B streptococci (n = 30), group G streptococci (n = 17), group D S. bovis (n = 19), Enterococcus faecium (n = 45), Enterococcus faecalis (n = 28), S. sanguis (n = 21), S. milleri (n = 29) and S. mitis (n = 17). Activities of each of the two study drugs were evaluated comparatively with two other fluoroquinolones, i.e., ciprofloxacin (CIP) and pefloxacin (PEF). Activities of each of these four antibiotics, expressed as the MIC 90%, varied as follows according to the species of streptococci: TOSU, 0.25 to 1 mg/l, TEMA 0.5 to 2 mg/l, CIP, 1 to 4 mg/l and PEF 8 to 32 mgl. Overall, TOSU and TEMA exhibited the greatest activity of the various species. The size of the inoculum had no significant effect on MIC values.

Abstract: Combinations of one of the new fluoroquinolones (ciprofloxacin, temafloxacin or tosufloxacin) with a betalactam (amoxicillin, piperacillin, or imipenem) were tested in vitro using the checkboard method against 28 strains of Streptococcus faecalis recovered in 1990 from a variety of specimens. No instance of antagonism (FIC greater than 2) was recorded. Effects of the two agents were usually additive (0.5 less than FIC less than or equal to 1). An indifferent effect (1 less than FIC less than or equal to 2) was seen in 32.1% of cases (9 strains) with the amoxicillin-tosufloxacin combination. Synergy (FIC less than or equal to 0.5) was uncommon with combinations including imipenem: effects were synergistic for only three strains with imipenem and either ciprofloxacin or tosufloxacin and for six strains with the imipenem-temafloxacin combination. Synergy was more common with combinations including ciprofloxacin and either piperacillin (10 strains, 35.7%) or amoxicillin (13 strains, 46.4%).

Abstract: Competition binding studies between viruses are usually performed with radiolabelled probes. In this report, a cytofluorimetric method using biotinylated echovirus (EV) 11 is described for the study of competition of enteroviruses for a common cell receptor site. An N-hydroxysuccinimide ester biotin spacer arm was used for biotinylation of CsSO4-purified EV 11. Biotinylation did not change the infectivity of the virus (attachment to and replication in susceptible cells). With the exception of EV 22 and EV 23, all the echovirus serotypes and also coxsackievirus A9 (CA 9) were able to inhibit the absorption of biotinylated EV 11 onto cells. The taxonomic implications of these findings are discussed.

Abstract: A case of pneumonia related to 2 serogroups (1 and 8) of Legionella pneumophila (Lp) in a 10-day-old boy is described together with the epidemiological survey in the maternity ward which made it possible to establish its nosocomial origin. Rodshaped bacteria reacting with an Lp genus-specific monoclonal antibody and serogroup 1 and 8 polyclonal sera were detected in bronchoalveolar lavages (BAL) collected on day 13. Serogroups 1 and 8 were recovered from cultures of BAL collected on days 12 and 13. Fourfold or more antibody rises to serogroups 1, 5, 8 and 10 of Lp were observed in sequential serum specimens. Water samples collected from the tank and mixer of the maternity ward grew serogroups 1 and 8 of Lp. Serogroup 1 was detected in large amounts in water samples taken at several points of the hot water supply system and from the oxygen nebulizers and the feeding-bottle heater. Analysis of the Lp serogroup 1 strains isolated from the water by subgroup-specific monoclonal antibodies revealed the presence of 4 different subgroups, one of which was identical to the Lp 1 subgroup isolated from the neonate's BAL. This latter subgroup, reactive with McKinney monoclonal antibody Mab 2, has been described as highly virulent. No other case of legionellosis was recorded in the maternity ward.

Abstract: Paired serum specimens from 24 patients with echovirus (EV) type 4 infection by virus isolation were tested by the immunoblot technique for the presence of IgG, IgM, and IgA antibodies to EV4 structural proteins. Single sera from 20 patients without neutralizing enterovirus IgM were used as controls. All the sera from EV4-infected patients had IgG antibodies to VP1 of EV4 but also 13 out of the 20 controls. 23 out of 24 EV4-infected patients elicited IgM and IgA specific antibodies to VP1, a pattern highly significant as compared with controls (3/20 for IgM and 8/20 for IgA). In 16 out of the 24 EV4-infected patients, the IgM antibodies were also directed against VP2 (versus 2 out of 20 in the control group). Anti-VP2 IgA were detected in 4 out of the 24 EV4 patients (versus 0 in controls). The 24 paired sera from EV4-infected subjects were also tested by immunoblot technique against three other enteroviruses: EV21, coxsackievirus A9 and poliovirus 1. Cross-reactivities were observed to a large extent against VP1 and VP2 proteins with the three classes of antibodies. These results confirm the data of previous studies on the reactivity of IgM antibodies to various structural proteins that IgG antibodies react exclusively to VP1. Furthermore, this study demonstrates the occurrence of circulating IgA antibodies directed to VP1 and sometimes VP2 in the course of enterovirus infection. The potential interest of this latter finding for diagnosis requires further investigation.

Abstract: We used ganciclovir to treat 11 renal transplant recipients with symptomatic cytomegalovirus infection (seven primary), including one severe, five mild and five moderate cases. Two patients exhibited a non-mechanically ventilated pneumonitis and two others a gastrointestinal involvement. Ganciclovir was used intravenously according to a schedule which took into account renal function, for a median time of 14 days. All patients survived. Cytomegalovirus infection was cured in all patients but two: in the first an early clinical relapse required a second successful ganciclovir course; in the other graftectomy was needed to control infection. Graft was lost in an additional cured patient. Ganciclovir was well tolerated, especially with regard to haematological status. At the current follow-up of at least one month after the end of ganciclovir therapy, no further clinical relapse was observed; however, in one clinically cured patient cytomegalovirus was isolated from blood one week after ganciclovir cessation. These encouraging preliminary data suggest that ganciclovir therapy should be started as soon as cytomegalovirus infection is suspected, especially in cytomegalovirus seronegative recipients receiving a seropositive graft.

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Abstract: A total of 314 patients exhibiting symptoms consistent with a viral disease provided, during the early stage of hospitalization, at least one specimen from a peripheral site (throat or stools or both) and a serum specimen in order to evaluate the neutralizing immunoglobulin M (IgM) antibody response in acute-phase serum in comparison with virus isolation for the rapid diagnosis of enterovirus (EV) infection. IgM antibodies were fractionated by ion-exchange chromatography and tested by seroneutralization against the various types of EV that have been recently circulating. A total of 189 patients (60%) were negative, and 21 (7%) were positive by both methods; in 51 patients (16%), a virus was isolated without IgM antibody response; 53 patients (17%) showed the opposite pattern. In all age groups except for children under 6 months, the frequency of positive results was higher with IgM serology than with virus isolation (27 and 22%, respectively). Apart from meningitis, for which isolation was more efficient, the other clinical conditions were associated with similar percentages of positivity by both methods. Regarding the 21 cases with positive results by the two techniques, the same serotype was detected in 9 cases and different serotypes were detected in 12, suggesting crossreactivities. Thus, IgM neutralizing antibody response on acute-phase serum appears to be of limited value in the rapid diagnosis of acute EV infection but may prove useful for the investigation of the wide range of chronic diseases associated with EV.

Abstract: Data are presented of IgM detection by a neutralization test used routinely in 1,062 patients. Antigens isolated during the period of investigation were EV4, EV7, EV11, EV18, EV21, EV24, EV33, CA9, CB2, CB4, and CB5. No difference was observed in the distribution of IgM-positive sera according to age and sex. Total antibodies are at higher titres when IgM antibodies are present. Polytypic IgM responses are not frequent (less than 10%). The frequency of the IgM-positive sera for a given serotype correlated with the frequency of isolates for the serotype except for CA9. Other than for babies under age 6 months, IgM detection is more frequent than is isolation. The susceptibility of the elderly and the frequency of IgM-positive sera among adults over age 40 years suggests possible underestimation of enterovirus infections in adults. The duration of IgM remains a major question.

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Abstract: Zinc chloride (0.9 mM)--an inhibitor of the processing of the initial polypeptides of Picornaviridae--was used to accumulate large precursors of echovirus 33 (EV33) and notably two proteins of 147- and 97-kDa. Polyclonal hyperimmune sera were raised in mice against these polypeptides and assayed by immunoblotting against EV33-infected cells blocked or not with ZnCl2, showing that protein 147--2ABC3ABCD according to the L434 convention--can be considered to be the precursor of the two non-structural proteins P2 and P3. Sixty-three serum specimens from subjects exhibiting varying antibody status against EV33 by seroneutralization were investigated by immunoblotting against an EV33 ZnCl2-blocked antigen. Some subjects infected with EV33 were shown to elicit antibodies which recognize the non-structural precursor polypeptides, a fact whose clinical significance needs further evaluation.

Abstract: The incidence and severity of cytomegalovirus (CMV) infection was examined in groups of consecutive renal transplant patients: group A (50 patients) transplanted from August 1984 to October 1985 received cyclosporin (from the day of transplantation) and steroid therapy; group B (50 patients) transplanted between June and July 1984 received conventional therapy (azathioprine and steroids, and antilymphocyte globulin for 14 days). In groups A and B there were respectively 5 (10%) and 14 (28%) seronegative patients prior to transplantation (CMV antibody titre less than 128 by ELISA); the overall incidence of CMV was 38% vs 74% (P less than 0.001); the incidence of primary infection was 0% (0 of 5) vs 36% (5 of 14) (NS); the incidence of secondary infection was 42% (19 of 45) vs 89% (32 of 36) (P less than 0.00001); and the total incidence of symptomatic infection was 10% vs 26% (P = 0.04). Thus we conclude that initial cyclosporin therapy leads to a reduction in CMV infection.

Abstract: Antibodies against initial polypeptides have been detected in sera of patients infected with an Echo virus 33. The technique is an IF test using infected cells in which the virus multiplication cycle is blocked at 2 hrs p.i.

Abstract: A commercially available direct immunofluorescence (IF) test using a reagent consisting of a pool of two monoclonal antibodies against selected surface and internal proteins of respiratory syncytial virus (RSV) was evaluated in comparison with the indirect IF technique using a commercial bovine anti-RSV hyperimmune serum for the rapid detection of RSV in nasopharyngeal aspirates from 228 hospitalized children. Overall agreement between the two IF methods was 95%. The direct IF test was quicker to perform and easier to interpret than the indirect IF test.

Abstract: After transplantation, the frequency of reactivation (or superinfection) by CMV is high. Less frequent, but more severe, is the primary form of the disease. The virological procedure to follow up the graft recipients includes: 1) The detection of the infectious virus and/or its antigens in urine, blood, bronchial secretions or brushing, bronchiolar and alveolar washings, ... The laboratory will perform a rapid culture for detection of early CMV antigen (24-72 h) associated with the more classical long-term culture, a reliable but slow method (2-3 weeks). 2) The detection of antibodies, primarily IgM, or even IgA. The usual ELISA for IgG and ELISA modified by addition of an immunocapture on a solid phase are the more reliable techniques. As a rule, the pattern IgM+/IgG- is common for the primary infection. In the case of reactivation (or superinfection), the pattern may be either IgM+/IgG+ or IgM-/IgG+. In the latter case, the increase of the IgG during the next weeks will bring evidence of a reactivation process.

Abstract: We report four cases of fulminant hepatitis in children (4 to 15 years) who developed an hepatic encephalopathy grade III to IV, 4 to 13 days after the onset of their illness. Three patients recovered without sequelae. The complications were neurological: one child showed elevation of the intracranial pressure, successfully treated after monitoring of extra-dural pressure; one suffered from cerebral death. Hepatitis A was diagnosed by the presence in serum of the IgM component of hepatitis A antibody, but another etiologic factor was present in two cases: an halothane anesthesia and an Epstein Barr virus infection which could explain the severity of the hepatitis.

Abstract: Twenty-one patients with chronic glomerulonephritis (GN) (5 with renal failure) received three doses of live trivalent poliovirus vaccine administered orally. The effect of the polio vaccination on the renal function and the titers of antibodies to poliovirus were studied. No significant consequence was observed in renal disease. Before vaccination, titers of poliovirus type 1 and 3 antibodies were significantly decreased as compared to healthy adult subjects. After vaccination, the patients exhibited a significant rise in poliovirus antibody titers for the three serotypes, although some of them failed to develop a fourfold or greater antibody rise to at least one of the three serotypes, especially in the group of patients with renal failure. These results indicate that live poliovirus vaccination is not deleterious in patients with GN and can provide a good protection.

Abstract:

Abstract: In 1982, we isolated 20 strains of echovirus type 33 (EV33) from 18 patients. We studied the humoral response to EV33 of 2,437 subjects from whom at least one serum was available during the same year. In 388 subjects with the neutralizing antibody level at 64 or more, we assayed the EV33 IgM antibodies by seroneutralization test after fractionation of sera by ion exchange chromatography. One hundred ninety-five subjects (8.0%) had a high titre (greater than or equal to 32) of EV33 IgM antibodies, which was considered as evidence of recent infection. The EV33-positive IgM fractions were assayed against five other enteroviruses. Sixty percent of the IgM fractions did not cross-react with any of the five serotypes, 8.7% cross-reacted with at least one serotype but with predominant EV33 IgM response, and 31.3% had an equivalent or greater amount of non-EV33 IgM antibodies; the type specificity of the assay was directly related to the age of the subjects. These findings suggest that determination of neutralizing specific IgM antibody is a sensitive and rapid test for the diagnosis of enterovirus infections, especially in young people.

Abstract: We have summarized 14 human cases with antibodies to interferon (anti-IFN) reported in the literature. In 4 cases, the patients developed auto-antibodies without any injection of exogenous interferon (IFN). In most cases, antibodies were directed against alpha IFN (13/14) and especially against recombinant alpha 2 IFN (8/14). When tested, antibodies to IFN were always IgG. In a few subjects, the IFN system has been studied more completely with the following results: the competent cells are able, after induction, to produce IFN; the endogenous IFN is neutralized by the antibodies to IFN; the antibodies to IFN are able to inhibit the interaction of IFN with its specific cellular receptors. Clinically, the raising of an anti-IFN immunization does not seem to impair the antiviral or antitumoral effects of IFN. These data can be related to the relative low neutralizing titres of antibodies to IFN with regard to levels of IFN produced at the site of the viral infection. We discuss the putative benefit of antibodies on the availability of circulating IFN and the possible role of antibodies to IFN in autoimmune diseases.

Abstract: During an outbreak with Mycoplasma pneumoniae, a serological survey was performed with 3,165 sera from 1,900 hospitalized patients over a 33 months period. Four hundred and eleven patients exhibit serological pattern suggestive for a recent infection. The main points are the following: the infection was more frequently (21.6%) detected in females than males, in the patients 5 to 19 years than in the other age groups; the incidence of the infection is the same in the group of patients hospitalized for extra-respiratory syndromes compared to respiratory infections; children and teen-agers exhibit the higher antibody titers; no modification of the type of infection was found in relation with age groups; the antibody titers are higher when patients are hospitalized with respiratory diseases, whatever their age group may be; there is no relation between sex and the type of infection, pulmonary or extra-pulmonary; a surprisingly high incidence of infections was detected in patients hospitalized with renal failure.

Abstract: Adult female Swiss mice showed a greater resistance to intraperitoneal (i.p.) infection with encephalomyocarditis virus (EMCV) than male mice. This difference was not observed in weanling mice, in castrated adult mice or in adult mice injected intracerebrally. Administration of antibody to mouse interferon alpha/beta enhanced the virulence of EMCV for both sexes and no difference was then observed in susceptibility between male and female mice. Six h after EMCV infection, serum interferon titres were higher in adult female mice than in male mice. There was a close correlation between the early serum interferon titre (at 6 h) and survival of EMCV-infected mice. No differences in serum interferon titres were observed between male or female weanling mice or castrated adult mice. Potent preparations of exogenous interferon provided the same degree of protection against EMCV infection in male and female mice. We conclude that the more marked early interferon response of female mice to i.p. EMCV infection is one of the important factors underlying the differential susceptibility to EMCV. It is possible that the interferon system is also involved in the reported greater prevalence of picornavirus infections of men compared with women.

Abstract: We analysed HLA-A, B, DR antigens and Gm allotypes in 45 Caucasian patients treated by alpha methyl dopa. 30 had a positive antiglobulin direct Coombs test of the pure IgG type and 15 showed no erythrocyte antibody. We found no difference in the HLA and Gm gene frequencies between the 2 patient groups and the normal control group (nb 104). Furthermore we appreciated the T cell subsets in 11 Coombs positive patients. They showed an increase of the helper/suppressor T cell ratio, due to a significant decrease of the suppressor T cell subset. These data are compatible with Kirtland findings which demonstrated a methyl dopa inhibition of suppressor lymphocyte function. However they do not support the hypothesis that genetic factors may have a role in the susceptibility of patients to methyl dopa induced suppressor-cell disfunction.

Abstract: A patient with varicella-zoster disease was found to have antibodies to human interferon. These antibodies included all IgG subclasses, showed a high combined affinity (average dissociation constant, 10(-11)M), and were present in serum at a concentration of 10(-9)M. The antibodies neutralized the activity of a series of human alpha-interferons prepared from recombinant DNA as well as that of naturally occurring mixtures of alpha-type interferon prepared by viral stimulation of human cells. Examination of the patient's lymphoid cells revealed normal production of interferon and normal expression of interferon receptors. Interferon from the patient's cells was neutralized by her serum. The neutralizing capacity of this serum was analyzed with regard to levels of interferon previously detected in patients with varicella-zoster infections.

Abstract: The results of CF and ELISA tests for cytomegalovirus performed on 270 sera of hospitalized patients show a positive correlation. As a general rule, ELISA is more sensitive than CF, except for a few sera collected from patients with immunological disorders. When two sequential sera are available, the CF remains a reliable and inexpensive method. But when only one serum can be obtained, the probability of an active CMV infection can be estimated on the IgM/IgG ratio. In 26% of the patients, this ratio was greater than or equal to 1. The ELISA is twice as expensive as the CF test. To reduce its cost, a simple method for preparing ELISA antigen from commercially-obtained CF antigens is described.

Abstract: In a prospective work, we have studied the non-polio enterovirus (NPEV) excretion in spot urine and stools by four appropriate cell cultures, in four different groups: 20 exposed controls (GI), 88 patients with renal biopsy 'proven GN' (GII), 38 cases with 'proven nonGN' (GIII), and 9 with 'probable nonGN' (GIV). The positive excretion in stools and/or urine is respectively 0 and 5% in Group I, 14 and 33% in Group II, 6 and 19% in Group III, and 13 and 22% in Group IV. Viruria, the consequence of a viraemia, is therefore associated with 'proven GN' (p less than 0.05). In the majority of patients with positive NPEV excretion, we have made an additional but similar study, 3-12 months later. Persistent excretion was confirmed in 22/30 cases in Group II (73%) versus 0/8 in Group III-IV (p less than 0.001). These data concerned patients with membranoproliferative GN [5], membranous GN [6], mesangial IgA GN [6], endocapillary GN [2] or minimal lesions [3]. Thus we have demonstrated a significant relation between persistent NPEV urine/stools excretion and the occurrence of active GN in humans. Such persistent viral infections may represent the cause of some GN, probably mediated by an immune complex mechanism with viral antigens.