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.

Amplification of the six major human herpesviruses from cerebrospinal fluid by a single PCR.

We used a novel type of primer system, a system that uses stair primers, in which the primer sequences are based on consensus sequences in the DNA polymerase gene of herpesvirus to detect herpesviruses by PCR. A single PCR in a single tube detected the six major herpesviruses that infect the central nervous system: herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and human herpesvirus 6 (HHV-6). We used the technique to analyze 142 cerebrospinal fluid (CSF) samples that had been stored at -80 degrees C and compared the results with those obtained previously for the same samples by standard, targeted PCR. Four hundred one targeted PCR tests had been run with the 142 samples to detect HSV-1, HSV-2, CMV, and VZV; screening for EBV and HHV-6 was not prescribed when the samples were initially taken. Eighteen CSF samples tested positive by classic targeted PCR. The herpesvirus consensus PCR detected herpesviruses in 37 samples, including 3 samples with coinfections and 17 viral isolates which were not targeted. Two samples identified as infected by the targeted PCR tested negative by the consensus PCR, and eight samples that tested positive by the consensus PCR were negative by the targeted PCR. One hundred three samples scored negative by both the targeted and the consensus PCRs. This preliminary study demonstrates the value of testing for six different herpesviruses simultaneously by a sensitive and straightforward technique rather than screening only for those viruses that are causing infections as suggested by clinical signs.

[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.

Spontaneous recovery from severe parvovirus B19 pure red cell aplasia, in a heart transplant recipient, as demonstrated by marrow culture.

A 61-year-old heart transplant recipient with parvovirus B19 infection, presented as a severe pure red cell aplasia (PRCA) with hemoglobin level of 5 g/dl. Both blood and bone marrow cells were positive for parvovirus B19 DNA, whereas specific immunoglobulins IgG and IgM were not informative. Bone marrow smears revealed erythroid hypoplasia without giant pronormoblasts. Autologous and allogenic bone-marrow cultures revealed a high inhibition by patient's serum on BFU-E growth whereas the number of CFU-GM were normal. Spontaneous remission of the anemia was observed despite the persistence of severe immunodeficiency as demonstrated by development of a monoclonal EBV lymphoproliferative disorder two months later. The "recovery" serum reversed the initial serum BFU-E inhibiting property. This case pinpointed the usefulness of blood or marrow cultures in parvovirus B19 infection of immunocompromised patients without normal Ig responses, as in other PRCA. Further, it argues that the usual immunoglobulin therapy may not be necessary in order to obtain a viral clearance.

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.

Automated determination of amplified PCR products: application to HCV viremia detection and quantification.

A simple and reliable automated method was developed for the detection of amplified products after PCR, which provides an alternative to the time-consuming Southern blotting and hybridization procedure. For the determination and quantification of hepatitis C viremia, the digoxigenin labelling process was applied during the PCR of the amplicons, followed by an inverse hybridization assay performed with biotinylated probes. The detection of the PCR amplified products could be processed as simply as an ELISA, or by means of an automated analyser.

The prognostic value of plasma viremia in HIV-infected patients under AZT treatment: a two-year follow-up study.

To determine the prognostic value of plasma viremia in long-term zidovudine (AZT)-treated HIV-infected patients, HIV-1 plasma viremia (PV) was quantified in 28 HIV-infected patients before and during AZT long-term treatment; the follow-up also included p24 antigenemia and CD4 cell counts. The variations of these markers during the follow-up period, the correlation with the clinical outcome (progressors versus nonprogressors), and the discrepancies between PV and surrogate markers were then analyzed. A significant and stable decrease in PV titer was observed in only nonprogressors (Friedman test, p < 0.005). At the end of follow-up, 11 (73%) of the 15 non-progressors were PV responders (patients who remained or became PV- long-term), whereas all the 13 progressors were PV nonresponders (patients who remained or became PV+). These results indicated a strong correlation between PV and clinical outcome (Fischer's exact test, p < 0.0001). The persistence, increase, or reappearance of viral replication appeared to be an important predictor of poor clinical outcome in HIV-infected patients under AZT treatment. This finding could provide a rational basis to help the clinician's decision in the clinical treatment of HIV-infected patients.

Monitoring levels of human cytomegalovirus DNA in blood after liver transplantation.

We evaluated a semiquantitative PCR assay prospectively in 40 liver transplant recipients as an aid in making a prompt diagnosis of cytomegalovirus (CMV) infection. For 2 months after transplantation, clinical specimens from patients were tested weekly by PCR, virus isolation from peripheral blood and urine, and CMV serology. The incidence of active CMV infection was 70%. The levels of CMV DNA determined by hybridization of PCR samples and densitometric scanning of blots were assigned a score of 1 to 4 by comparison with four external standards amplified in parallel and corresponding to a range of 80 to 80,000 genomes. The first detection of CMV in blood by PCR occurred at a mean of 15 days, and high-level PCR scores of 3 or 4 were obtained 21 days after transplantation, whereas viremia occurred 33 days after transplantation. Significantly higher levels of CMV DNA were seen in patients with CMV disease (P < 0.05) than in asymptomatic patients. The prevalence of symptomatic CMV infection was 30%. The positive predictive value of PCR was 48%, while the negative predictive value was 100%. After treatment, the clearance of CMV DNA was always observed and the disappearance of symptoms occurred concomitantly with undetectable PCR signals.

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.

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.

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.

A human cytomegalovirus-neutralizing monoclonal antibody recognizes a normal cell protein.

A monoclonal antibody (MAb), N2, neutralized human cytomegalovirus (HCMV) infectivity in the absence of complement and recognized a normal cell protein. By immunofluorescence, MAb N2 detected antigens in uninfected human cells, but not in cells of non-human origin. Antigens were present at the membrane and were dispersed diffusely within the cytoplasm. MAb N2 immunoprecipitated a glycoprotein with an Mr of 94K from uninfected and infected cells. In infected cells only, it also recognized a protein of Mr 34K which was not linked to the 94K Mr glycoprotein by disulphide bonds. N2 neutralized both laboratory and field isolates of HCMV. A study of the distribution of the N2 neutralization epitope recognized among fresh isolates of HCMV showed that only 67% of the isolates could be neutralized by this antibody. There was no correlation between the number of in vitro passages and the level of neutralization. The 34K Mr polypeptide was present in cells infected by all isolates. It thus appears that, during virus assembly, HCMV acquires a normal cell protein that bears a neutralization epitope.

[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.