Clinical severity and molecular characteristics of circulating and emerging rotaviruses in young children attending hospital emergency departments in France.

Group A rotavirus (RVA) is the leading cause of acute gastroenteritis in young children worldwide. A prospective surveillance network has been set up to investigate the virological and clinical features of RVA infections and to detect the emergence of potentially epidemic strains in France. From 2009 to 2014, RVA-positive stool samples were collected from 4800 children <5 years old attending the paediatric emergency units of 16 large hospitals. Rotaviruses were then genotyped by RT-PCR with regard to their outer capsid proteins VP4 and VP7. Genotyping of 4708 RVA showed that G1P[8] strains (62.2%) were predominant. The incidence of G9P[8] (11.5%), G3P[8] (10.4%) and G2P[4] (6.6%) strains varied considerably, whereas G4P[8] (2.7%) strains were circulating mostly locally. Of note, G12P[8] (1.6%) strains emerged during the seasons 2011-12 and 2012-13 with 4.1% and 3.0% prevalence, respectively. Overall, 40 possible zoonotic reassortants, such as G6 (33.3%) and G8 (15.4%) strains, were detected, and were mostly associated with P[6] (67.5%). Analysis of clinical records of 624 hospitalized children and severity scores from 282 of them showed no difference in clinical manifestations or severity in relation to the genotype. The relative stability of RVA genotypes currently co-circulating and the large predominance of P[8] type strains may ensure vaccine effectiveness in France. The surveillance will continue to monitor the emergence of new reassortants that might not respond to current vaccines, all the more so as all genotypes can cause severe infections in infants.

Polymerase chain reaction detection of cytomegalovirus DNA in peripheral blood leukocytes as a predictor of cytomegalovirus disease in HIV-infected patients.

OBJECTIVE: To describe and evaluate a polymerase chain reaction (PCR) method for early diagnosis and prompt management of cytomegalovirus (CMV) retinitis in HIV-infected patients. METHODS: A total of 110 HIV-infected patients (Centers for Disease Control and Prevention stages II to IV) were sampled sequentially for isolation of CMV from peripheral blood leukocytes (PBL; n = 560) and for amplification of CMV DNA in PBL. Semiquantitative analysis of the PCR product was performed and each PCR-positive specimen was assigned a score between 1+ and 4+ (corresponding to four points on a standard curve of dilutions: 80, 800, 8000 and 80,000 CMV genome copies). RESULTS: Levels of CMV DNA in blood increased with HIV infection stage. We focused on eight patients who developed one or more episodes of retinitis during longitudinal follow-up, in whom we found a strong correlation between viraemia, high PCR signal (3+ or 4+) (P < 0.0001) and clinical symptoms. Relapse was preceded by an increase in CMV DNA and resolution correlated with clearance of CMV DNA from blood. CONCLUSIONS: Persistent high PCR levels always preceded virus isolation and may be the first indication of organ involvement and thus early treatment. PCR scores were consistently useful as indicators of drug efficacy and for monitoring of treatment.

Polymerase chain reaction detection of human cytomegalovirus in over 2000 blood specimens correlated with virus isolation and related to urinary virus excretion.

The polymerase chain reaction (PCR) as applied to human cytomegalovirus (HCMV) detection should provide a valuable tool for rapid, reliable diagnosis of infection, thereby allowing prompt treatment. However, to date the high sensitivity of this technique and the lack of semi-quantitative interpretation have hindered establishing its validity for diagnosing systemic infection. We describe a rapid, simple, semi-quantitative PCR technique for HCMV detection. The validity of the technique was tested objectively by analyzing over 2000 leukocytes specimens by PCR and comparing the results with virus isolation from urine and blood in concomitant samples in the absence of any clinical data. It could thus be established that this technique had a sensitivity and specificity of 97%. When the PCR signal corresponded to > or = 8000 genome equivalents for 10(4) leukocytes, the predictive value for viremia was 86%. This semi-quantitative PCR technique should allow rapid diagnosis of systemic infection and provide a reliable means of monitoring clearance of CMV from blood during drug therapy.

New types of primers (stair primers) for PCR amplification of the variable V3 region of the human immunodeficiency virus.

The aim of the study was to develop a reliable PCR method for the detection of viral genomes with frequent mutations like HIV and hepatitis C virus. A system of 'stair' primers is suggested which allows amplification of a genomic sequence despite the presence of mutations in the region of the primers. In this system, classical primers are replaced with primers composed of a mixture of equimolar oligonucleotides in which the 5' end remains constant (single sized fragment) and the 3' end is displaced base by base. By PCR, 'stair' primers (HIV set) were compared to single-sequence primers of 20 and 25 nucleotides chosen in the same hypervariable region of the HIV gp120 (on both sides of V3 region), as well as to classical primers chosen in the conserved pol (polV2) and gag (SK38-39) regions of the genome. Of 17 HIV isolates obtained by co-culture of lymphocytes from HIV-seropositive patients, 17/17 (100%) were amplified using stair primers, 14/17 (82%) with 25-nucleotide primers, and 12/17 (70%) with 20-nucleotide primers. Amplification occurred in 17/17 instances with polV2 primers and in 16/17 instances with SK38-39. In addition, 55 other isolates were tested comparatively using stair, polV2 and SK38-39 primers. All isolates were amplified using stair and SK38-39 primers and 54/55 isolates with polV2 primers. When applied to 22 extracts of patients' lymphocytes DNA, stair primers amplified all 22 extracts to the same degree as polV2 and SK38-39, whereas the 20 and 25 nucleotide primers chosen in the variable region were not as reliable. This new primer system allows reliable detection of variable genomic regions of the HIV genome and amplification of such regions directly in patient leukocytes. In addition, the contribution of this system to microbiology and human genetics in general may be important.

Antibiotic-lock technique is an effective treatment of bacterial catheter-related sepsis during parenteral nutrition.

Catheter-related sepsis is one of the most frequent and troublesome complications of parenteral nutrition. In a 2-year survey of 19 home parenteral nutrition patients, with a total of 25.2 years of cyclic nocturnal parenteral nutrition, the annual incidence of catheter-related sepsis was 1.27, of which 84% were due to bacterial catheter infection without any cutaneous focus. These 27 episodes were treated by a daily, 2 ml injection of antibiotic-saline solution, mainly amikacin, locked for 12 h per day within the infected catheter for 15 (7-20) days. On admission the parenteral nutrition was halted for 2 days and the catheter hub was changed. In 7 cases, an average of 3 days (2-5) of systemic antibiotic therapy was given in addition to the 2-week antibiotic-lock. Control of catheter-sepsis was achieved in 93% of the 27 episodes and parenteral nutrition was resumed using the same catheter with only one episode of recurrent sepsis. The present data confirm our preliminary report of the efficacy of the antibiotic-lock technique for the control of bacterial catheter-related sepsis. This treatment offers the advantage over current therapies of avoiding repeated catheter change and 2-6 weeks of systemic antibiotic therapy.

[Epidemiology of hepatitis C virus infection in 1,304 HCV positive patients: variations according to the origin of transmission and year of diagnosis]. / Epidémiologie de l'infection virale C chez 1,304 sujets VHC positif. Variations en fonction du mode de contamination et de l'année du diagnostic.

UNLABELLED: The evolution of epidemiological data on hepatitis C virus infection is poorly documented and thus the impact of screening is difficult to evaluate. AIM: To study epidemiological variations based on the origin of transmission and the year of diagnosis of hepatitis C virus infection. METHODS: The files of all 1304 patients seen in the hepatology unit of the Rennes University Hospital were analyzed (retrospectively before and prospectively after October 1995) in relation to epidemiological features. RESULTS: Despite widespread screening which is the source of 60% of the diagnoses, the total number of new cases of hepatitis C infection per year has not increased. Compared to patients diagnosed in the first years following the discovery of the virus, patients recently identified were younger (42 +/- 14 years) and frequently drug addicts (40%). Aminotransaminases were normal in 20% of cases. The frequency of cirrhosis has declined (17%). There has been a decrease in the proportion of patients who undergo liver biopsy (50%) and treatment with interferon (one third of patients). CONCLUSIONS: The impact of screening on the number of newly treated patients seems to be lower than previously predicted.

[Diagnostic value of the study of anti-Chlamidia antibodies in salpingitis. 379 cases]. / Intérêt diagnostique de la recherche des anticorps anti-chlamydiens au cours des salpingites. 379 observations.

Antibodies to Chlamydia were assayed by complement fixation (CF) and inclusion indirect immunofluorescence (IFI) in sera collected from 379 patients with salpingitis: 30.3% of the patients had total and IgM antibodies at IFI and CF antibodies (profile I); 26.6% of the patients had total and IgM antibodies at IFI without CF antibodies (profile II); 31.6% of the patients had only total antibodies at IFI without specific IgM and without CF antibodies (profile III); 11.3% of the patients were Chlamydia antibody negative (profile IV). In the control group of 50 pregnant women apparently non infected, the profile distribution was 2% profile I, 8% profile II, 38% profile III, and 54% profile IV. Detection of IgM antibodies to Chlamydia trachomatis in 57% of patients with salpingitis, taking only one specimen, suggested recent or active chlamydial infection. CF antibodies indicated diffuse infection. Total antibodies correlated well with IgG antibodies detected by ELISA. Their finding was by no means diagnosis for Chlamydia being the cause of tubal infection, although titers observed in salpingitis patients were higher than in controls.

[Non-gonococcal urethritis and cervicitis. Isolation of Chlamydia trachomatis and detection of anti-Chlamydia antibodies by indirect immunofluorescence]. / Urétrites et cervicites non gonococciques. Isolement de Chlamydia trachomatis et détection des anticorps anti-chlamydiens par immunofluorescence indirecte.

The responsibility of Chlamydia trachomatis in non-gonococcal urethritis and cervicitis was investigated in 267 patients of both sexes. It was confirmed in 36.3% of patients with urethritis and 20.9% of patients with cervicitis by isolating C. trachomatis on Hela 229 cells in the presence of cytochalasin B. No clinical feature specific of C. trachomatis infection could be elicited. The patients were tested for total IgM-type serum anti-chlamydia antibodies by indirect immunofluorescence (IF), using as antigen the inclusions formed in Hela 229 cells by an L2 serotype of C. trachomatis. The serological study was also performed in 86 blood-donors used as controls. The diagnostic value of IF serology is limited in lower genito-urinary infections; the presence of specific IgM's correlates well with the isolation of C. trachomatis, but these IgM's are not detected in protracted urethritis or cervicitis. In such cases, the aetiological diagnosis can only be made by isolation of C. trachomatis from the focus of infection.

[Direct diagnostic methods for the demonstration of cytomegaloviruses in the blood]. / Méthodes de diagnostic direct appliquées à la mise en évidence du cytomégalovirus dans le sang.

CMV can be detected in blood with two techniques: isolation of CMV from the buffy-coat on human embryonic fibroblasts; CMV genome detection by DNA hybridization technique. Isolation of CMV from blood plated on embryonic fibroblasts necessitates a delay of 10-30 days. Using monoclonal antibodies we have developed a 96 hours test to diagnose viremia. Fibroblasts grown on coverslip in tubes are inoculated with buffy-coat. Indirect immunofluorescence test is performed using the monoclonal antibodies E 13, 48 and 96 hours post-infection. DNA-DNA hybridization technique necessitates purification of CMV DNA. CMV DNA is then labelled with 32 P by nick-translation. This labelled DNA is used as probe for detection of CMV genome in DNA extracted from leukocytes. The hybridization is virus-specific. Cytomegaloviremia correlates with active infection and is not indicate of the carrier state of blood donors. CMV genome research in blood can potentially identify infective donors. Further studies are needed to correlate CMV genome detection in blood with presence of CMV antibodies.

[A method for the preparation of a Chlamydiae group-specific antigen on hela-229 cells infected with a strain of "Chlamydia trachomatis" for use in the complement fixation test (author's transl)]. / Préparation sur cellules HeLa-229 infectées par une souche de Chlamydia trachomatis d'un antigène de groupe des Chlamydiae fixant le complément.

A simple method for the preparation of a potent group-specific antigen on HeLa-229 cells infected with MRC-1 (LB) (TRIC/GB/MRC-1 Gf) strain of Chlamydia trachomatis is outlined. HeLa-229 cells are infected (MRC-1 strain, 102 inclusion-forming units per cell) with centrifugation at 4,000 g for 1 h in flat-bottomed vials. The cells are removed by brief trypsinization with 0.25% trypsin and put into 75 cm2 culture flasks (12 x 106 cells by flask) in BHK-21 medium supplemented with foetal bovine serum. The flasks are incubated for 5 days at 37 degrees C. The destroyed cell monolayer and the supernatant are centrifuged at 100,000 g for 1 h. The pellet is collected and resuspended in PBS and subjected to ultrasonic vibration for 30 min. This antigen may be used for detection of complement-fixing antibodies in lymphogranuloma venereum and ornithosis.

Conservation and map location of human cytomegalovirus strain Ad-169 transforming sequences in the DNA of clinical isolates.

All DNAs from human cytomegalovirus studied (57 fresh isolates, 3 reference strains) showed hybridization under stringent conditions to a plasmid containing strain Ad-169 transforming sequences. Analysis of hybridization patterns obtained with DNAs digested by three different enzymes allowed construction of a global map for this highly conserved area of the genome.

Demonstration of the colinearity of human cytomegalovirus genomes and construction of restriction maps of unknown isolates using cloned subgenomic fragments.

In this study, we have established the colinearity of human cytomegalovirus (HCMV) genomes using stringent conditions of DNA-DNA filter hybridization of HCMV HindIII fragments and cosmid-cloned AD169 strain HCMV DNA fragments. Large cosmid-cloned fragments of AD169 DNA were used for the preparation of radioactive probes by nick translation. These probes were hybridized to HindIII digests of DNA from three fresh isolates of HCMV and to that of the Davis strain. Using published HindIII restriction maps for the AD169 strain as a reference, the results obtained by hybridization allowed us to construct HindIII restriction maps for the genomes of the three fresh isolates. Confirmation of our methodology was found in the correspondence between the HindIII map we constructed for the Davis strain and that published previously. Furthermore, it is shown that variation in the restriction profiles of the unique regions of the genome are due to the absence or gain of restriction sites, and not to a rearrangement of fragments. This technique allows rapid construction of physical maps of the DNA of any fresh isolate for a given restriction enzyme provided the corresponding restriction map of a strain to be used as reference is available.

Comparison of six methods for detecting human rotavirus in stools.

The following six methods for detecting rotavirus in human faecal samples were compared: electron microscopy, immune electron microscopy, immunofluorescence in cell culture, two enzyme immunoassays (Rotazyme, Enzygnost ) and a latex agglutination test ( Rotalex ). Specimens were collected from 112 children with diarrhoea. The relative sensitivities of the different assays for human rotavirus were as follows: electron microscopy, 84%; immunofluorescence, 86%; Rotalex , 88%; Rotazyme, 89%; immune electron microscopy, 93%; Enzygnost , 98%. According to our findings Enzygnost is the most sensitive method, but Rotalex is more valuable for screening a small number of faecal samples. No false-positive results were observed in the two enzyme immunoassays or in Rotalex .

Detection of cytomegaloviremia using monoclonal antibodies.

Isolation of cytomegalovirus (CMV) from blood plated on embryonic fibroblasts necessitates a delay of 10-30 days. Using monoclonal antibodies (McAb) we have developed a 96 hour test to diagnose viremia. We produced monoclonal antibodies directed against CMV (strain AD 169). Clone E 13 McAb detected a CMV early antigen in nuclei using indirect immunofluorescence. These monoclonal antibodies recognized reference strains (Davis and Towne) and 20 field isolates of CMV. E 13 McAb were therefore selected for the diagnostic tests. MRC 5 cells grown on coverslips in tubes were inoculated with buffy-coat from 51 blood samples from bone marrow allograft recipients and Guillain Barre polyneuritis, and centrifuged on hour at 4000 g at 36 degrees C. The indirect immunofluorescence test (IIF) was performed using E 13 McAb, 48 and 96 hours post infection. Three viremia were detected within 48 hours and five others within 96 hours. Plasma and buffy-coat were also inoculated onto MRC 5 cells grown in flasks. Flasks were observed daily for CMV cytopathic effect (CPE). CPE appeared within an average of 14 days. When CMV was detected by CPE, the IF tests were always positive within 96 hours after drawing the blood.