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.

[Incidence of rotavirus gastroenteritis among children under 5 years consulting a paediatrician or a general practitioner in France]. / Incidence des gastroentérites à rotavirus chez les enfants de moins de cinq ans consultant un pédiatre ou un médecin généraliste en France.

BACKGROUND: Rotavirus (RV) is the main infectious agent of severe acute gastroenteritis (AGE) in infants and children under 5 years. Given the recent availability of new vaccines, it is important to accurately assess the incidence of rotavirus gastroenteritis (GERV) and their medical and epidemiological consequences. METHODS: This work is the French part of study program called SPRIK, a multicenter, prospective, observational study conducted from October 2005 to May 2007 to estimate the annual incidence of GERV within children under 5 years visiting a general practitioner or pediatrician. It presents data collected by 41 general practitioners and 36 paediatricians located throughout the French metropolitan territory. A stool sample was taken for every child. Rotavirus presence was sought by the physician using a rapid immunochromatographic test. French results are presented in this article. RESULTS: A total of 1648 GEA episodes corresponding to 1463 eligible patients were included in the study mainly from December to May (peak in February-March). The incidence rate of GERV leading to consultations in general practice was 1357 cases per 100,000 patient-years (PY) (1.36%), with a 95% confidence interval of [1345-1368]. The peak incidence occurs before 2 years. GERV accounted for 21% of all GEA cases seen by paediatricians and general practitioners. Patients with GERV were younger (14.1 ± 10.8 versus 18.4 ± 13.9 months for other GEA, P<0.0001) and had more severe clinical symptoms: presence of fever (32.6% versus 20.0%, P<0.0001), behavioural symptoms (45.6% versus 20.8%, P<0.0001) and dehydration (48.7% versus 21.2%, P<0.0001). GERV episodes were considered severe in 79.7% of cases, using the Vesikari scale. More than 86% patients received oral rehydration during the episode and 13 patients (5.8%) were hospitalized. Nearly 80% GERV episodes were considered severe using the Vesikari scale. Main genotypes were G1P[8] rotavirus (44%) and G9P[8] rotavirus (35%) types. CONCLUSION: The incidence rate reported in this study is close to results of previous studies done in Europe. The frequency and severity relative to GERV support vaccination in very young children to reduce the burden associated with this pathology.

[From structural and antigenic features of the rotavirus to the development of the vaccines]. / Des caractéristiques structurales et antigéniques des rotavirus au développement des vaccins.

Rotaviruses are the main etiologic agent of severe acute diarrhoea in children under the age of 5, world-wide. Given that the currently available preventive measures to fight against the transmission of RV disease are not sufficiently effective, vaccination likely represents the only efficacious adapted response to the massive impact of this infection. Two RV vaccines (RotaTeq and Rotarix) have recently been developed and licensed in the United States and in Europe. The development of these two vaccines has followed two different strategies. Despite their differences, these vaccines are both safe and efficient in protecting young infants against severe rotavirus acute gastroenteritis.

Lack of association between B19 or V9 erythrovirus infection and ANCA-positive vasculitides: a case-control study.

OBJECTIVES: To examine the potential association of human B19 or V9 erythrovirus infection and onset of ANCA-positive vasculitides. METHODS: We tested the sera of 13 adults with newly diagnosed ANCA-positive vasculitides. Each was age- and sex-matched to three sera obtained from healthy controls. All samples were tested for B19- and V9-specific immunoglobulin (Ig) G and IgM antibodies (Ab) (third-generation ELISA), and B19 or V9 DNA was sought with the polymerase chain reaction. Statistical analysis was performed by conditional logistic regression. RESULTS: Patient diagnoses comprised six cases of Wegener's granulomatosis, six of microscopic polyangiitis and one of Churg-Strauss syndrome. IgG Ab to B19 were detected equally in patient and control sera (77 and 79% respectively) (odds ratio=0.84, P=0.84). All 13 cases and 39 controls were negative for IgM Ab and viral DNA. CONCLUSION: These results suggest that neither acute nor chronic B19 or V9 infection is an aetiological factor in ANCA-associated vasculitides.

Real-time PCR quantification of human cytomegalovirus DNA in amniotic fluid samples from mothers with primary infection.

A real-time PCR assay was developed to quantify human cytomegalovirus (HCMV) DNA in amniotic fluid (AF) samples collected from 30 pregnant women with primary HCMV infection as detected either from HCMV-immunoglobulin G (IgG) seroconversion or by the presence of HCMV-specific IgG and IgM associated with a low IgG avidity. Clinical information available for each case included ultrasonographic examination and fetal or newborn outcome. HCMV infection of fetuses or newborns was confirmed for the 30 studied cases. AF samples were subdivided into three groups. In group A (n = 13), fetuses presented major ultrasound abnormalities, and pregnancy was terminated. In group B (n = 13), fetuses had normal ultrasound findings, the pregnancy went to term, and the newborns were asymptomatic at birth. In group C (n = 4), fetuses had no or minor ultrasonographic signs, and pregnancy was terminated. The HCMV DNA load values in AF samples were significantly higher in group A (median, 2.8 x 10(5) genome equivalents [GE]/ml) than in group B (median, 8 x 10(3) GE/ml) (P = 0.014). Our findings suggest that HCMV load level in AF samples correlates with fetal clinical outcome but might also be dependent on other factors, such as the gestational age at the time of AF sampling and the time elapsed since maternal infection.

Quantification of Epstein-Barr virus load in peripheral blood of human immunodeficiency virus-infected patients using real-time PCR.

Epstein-Barr virus (EBV) reactivation is more likely to occur in immunocompromised patients with subsequent higher susceptibility to EBV-associated lymphoproliferations. In contrast to transplant recipients, limited data are available concerning the EBV load in HIV-infected patients, with or without AIDS-related non-Hodgkin's lymphomas. We developed a TaqMan real-time PCR assay, allowing both the EBV genome and a cellular gene to be quantified in order to obtain a reliable normalized measurement of the EBV load in peripheral blood mononuclear cells (PBMCs). With a wide 6-log(10) quantification range and inter-assay variations of less than 24%, this quantitative PCR was sufficiently accurate and reproducible for routine follow-up. The EBV load was determined in PBMCs from 113 HIV-infected patients, 11 patients with primary HIV infection and 24 HIV-seronegative healthy controls. The rates of EBV detection were similar in the three groups. However, EBV loads were higher in the HIV-infected group (P < 0.00001) except for the patients with primary HIV infection. Unexpectedly, EBV loads were not correlated with the clinical stages of HIV infection or HIV replication, and did not depend on the degree of immunodepression, as judged by CD4+ counts. This study contributes towards the definition of the baseline EBV load during HIV infection and stresses the broad inter-individual variability of the EBV load in HIV-infected patients. Real-time PCR provides a useful tool that can be used in further longitudinal studies to assess the relevance of the EBV load to identify HIV-infected patients with a high risk of EBV-associated lymphoproliferations.

A human rotavirus with rearranged genes 7 and 11 encodes a modified NSP3 protein and suggests an additional mechanism for gene rearrangement.

A human rotavirus (isolate M) with an atypical electropherotype with 14 apparent bands of double-stranded RNA was isolated from a chronically infected immunodeficient child. MA-104 cell culture adaptation showed that the M isolate was a mixture of viruses containing standard genes (M0) or rearranged genes: M1 (containing a rearranged gene 7) and M2 (containing rearranged genes 7 and 11). The rearranged gene 7 of virus M1 (gene 7R) was very unusual because it contained two complete open reading frames (ORF). Moreover, serial propagation of virus M1 in cell culture indicated that gene 7R rapidly evolved, leading to a virus with a deleted gene 7R (gene 7RDelta). Gene 7RDelta coded for a modified NSP3 protein (NSP3m) of 599 amino acids (aa) containing a repetition of aa 8 to 296. The virus M3 (containing gene 7RDelta) was not defective in cell culture and actually produced NSP3m. The rearranged gene 11 (gene 11R) had a more usual pattern, with a partial duplication leading to a normal ORF followed by a long 3' untranslated region. The rearrangement in gene 11R was almost identical to some of those previously described, suggesting that there is a hot spot for gene rearrangements at a specific location on the sequence. It has been suggested that in some cases the existence of short direct repeats could favor the occurrence of rearrangement at a specific site. The computer modeling of gene 7 and 11 mRNAs led us to propose a new mechanism for gene rearrangements in which secondary structures, besides short direct repeats, might facilitate and direct the transfer of the RNA polymerase from the 5' to the 3' end of the plus-strand RNA template during the replication step.

Quantification of human cytomegalovirus DNA by real-time PCR.

A quantitative real-time PCR assay was developed to measure human cytomegalovirus (HCMV) DNA load in peripheral blood leukocytes (PBLs). The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin). The results of the real-time PCR assay correlated with those of the HCMV pp65-antigenemia assay (P < 0.0001).

Ralstonia paucula (Formerly CDC group IV c-2): unsuccessful strain differentiation with PCR-based methods, study of the 16S-23S spacer of the rRNA operon, and comparison with other Ralstonia species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum).

Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNA(Ile) and tRNA(Ala) genes, which are identical to genes described for R. pickettii and R. solanacearum.

[Acute diarrhea and rotavirus infection in the child: assessment of data from emergency care and and the microbiology laboratory of the Armand-Trousseau (Paris) Hospital between 1988 and 2001]. / La diarrhée aiguë et l'infection à rotavirus chez l'enfant: confrontation des données d'activité des urgences médicales et du laboratoire de microbiologie de l'hôpital Armand-Trousseau (Paris) entre 1988 et 2001.

MATERIAL AND METHODS: Between October 1, 1988 and March 31, 2001, a longitudinal survey was conducted at the French pediatric hospital Armand-Trousseau in Paris. Following data were simultaneously collected: consultations and hospitalizations for acute diarrhea at the emergency room, and identifications of rotavirus from diarrheic stools at the laboratory. RESULTS: Acute diarrhea represented 9.3% to 11.1% of all consultations. The activity was continuous through the year with several epidemic peaks, the largest occurring during the winter months. The hospitalization rate was high and stable since 1989 (16.5% to 21%), reaching 22-26% at the winter peak. Overall, rotaviruses were identified in 22.4% of stool samples but the detection rate increased from 10% in 1989 to 31% in 1997. Rotaviruses were isolated mainly in winter, reaching the rate of 50-70% at the peak. DISCUSSION: Despite numerous biases of methodology and the fact that data were extracted from two different sources, acute diarrhea appeared as a major epidemic phenomenon in Paris, and rotaviruses were the main pathogens identified in diarrheic infants in winter. The extent of the winter epidemic increased each year since ten years, in parallel with the increase of the global activity of the emergency room. Despite attempts to develop ambulatory care, admission rates remained high in patients with acute diarrhea and searching for care at the emergency room of our hospital, especially in winter. CONCLUSION: These preliminary data were restricted to a single pediatric hospital in Paris. They need to be extended to a national level before considering a strategy for prevention using vaccination.

Novel human erythrovirus associated with transient aplastic anemia.

Erythrovirus (formerly parvovirus) B19 causes a wide range of diseases in humans, including anemia due to aplastic crisis. Diagnosis of B19 infection relies on serology and the detection of viral DNA by PCR. These techniques are usually thought to detect all erythrovirus field isolates, since the B19 genome is known to undergo few genetic variations. We have detected an erythrovirus (V9) markedly different from B19 in the serum and bone marrow of a child with transient aplastic anemia. The B19 PCR assay yielded a product that hybridized only very weakly to the B19-specific probe and whose sequence diverged more from those of 24 B19 viruses (11 to 14%) than the divergence found within the B19 group (

Distribution of human rotavirus G types circulating in Paris, France, during the 1997-1998 epidemic: high prevalence of type G4.

Group A human rotavirus G genotypes were determined by means of reverse transcription-PCR in 170 stool specimens from children with acute diarrhea admitted to a Paris children's hospital during a 1-year survey (1997 to 1998). The isolates all belonged to types G1 to G4, with type G4 predominating (60%).

CDC group IV c-2: a new Ralstonia species close to Ralstonia eutropha.

CDC group IV c-2, an environmental gram-negative bacillus recently proposed for inclusion in the genus Ralstonia, has been isolated in several human infections. Biochemical characterization and 16S ribosomal DNA (rDNA) sequencing with phylogenetic analysis were used to characterize eight clinical isolates and four type strains. Other typing tools, such as pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis, were also used. PFGE typing of clinical isolates was unsuccessful because the DNA was degraded, and RAPD analysis was poorly discriminatory. In contrast, the type strains were clearly distinguished with both PFGE and RAPD analysis. All of the 16S rDNA sequences were identical. Comparison of the 16S rDNA sequences to the GenBank sequences showed that they were consistent with CDC group IV c-2 belonging to the genus Ralstonia. The closest matches were obtained with Ralstonia eutropha. However, four differences in 32 biochemical tests separated R. eutropha from CDC group IV c-2, which suggests that CDC group IV c-2 is a new species of the genus Ralstonia.

Comparison of randomly amplified polymorphic DNA analysis and pulsed-field gel electrophoresis for typing of Moraxella catarrhalis strains.

Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) for the analysis of 13 Moraxella catarrhalis isolates, 11 successive strains isolated from sputa of five children and 2 isolates obtained the same day from twins, were compared. RAPD and PFGE both yielded nine types from the 13 isolates, showing a chronic colonization with one strain in three patients and a successive colonization with different strains in two patients. The promising results obtained with RAPD should be confirmed with a larger number of strains, but RAPD seems as suitable as PFGE for the typing of M. catarrhalis.

Investigation of an outbreak of wound infections due to Alcaligenes xylosoxidans transmitted by chlorhexidine in a burns unit.

Alcaligenes xylosoxidans, an environmental gram-negative bacillus, was isolated within a 1-month period from six patients in a pediatric burns unit. Twelve isolates were studied, one from each of the six patients (five from wound cultures and one from a blood culture) and one from each of six contaminated atomizers containing chlorhexidine diluted to 600 mg/l. The biochemical and susceptibility patterns of all the isolates were similar, and their DNA enzyme restriction patterns were identical. The epidemic strain of Alcaligenes xylosoxidans was probably introduced into the atomizers during handling of the diluted solution, which failed to eliminate it.

Parvovirus B19 infection, hepatitis C virus infection, and mixed cryoglobulinaemia.

BACKGROUND: Infection with human parvovirus B19 (B19) has been reported in a few patients with various vasculitis syndromes. Mixed cryoglobulinaemia (MC), a model of small vessel size vasculitis, may result from numerous infectious diseases, particularly hepatitis C virus (HCV) infection. AIM: To assess the prevalence of seric B19 infection markers in a large series of patients with MC, with or without HCV infection. PATIENTS AND METHODS: Sixty-four patients were studied: essential MC (EMC, n = 19), MC associated with non-infectious diseases (non-essential MC, n = 9), and patients with HCV infection with (HCV-MC, n = 18) or without MC (HCV-no-MC, n = 18). Patients were considered to have MC if two successive determinations of their serum cryoglobulin concentration were above 0.05 g/l. Serum samples were analysed for specific IgG and IgM antibodies to B19 by enzyme immunoassay. B19 DNA detection was performed by polymerase chain reaction using a set of primers located in the VP1 gene, separately in serum and in cryoprecipitates to investigate a possible capture of B19 DNA in cryoprecipitate. The study also looked for a possible enrichment for of IgG antibodies to B19 in MC. RESULTS: The presence of specific IgG antibodies to B19 was found in 68% EMC, 56% non-essential MC, 78% HCV-MC, and 78% HCV-no-MC. No patient of either group had specific IgM antibodies to B19, or B19 DNA in serum or in cryoprecipitate. Overall, IgG antibodies to B19 were found in 46 of 64 (72%) serum samples, a prevalence quite similar to the prevalence in general adult population (> 60%). A specific enrichment of IgG antibodies to B19 in the MC was not found. CONCLUSION: These results suggest that B19 infection is neither an aetiological factor of EMC, nor a cofactor that may lead to MC production in patients with chronic HCV infection.

[Cytomegalovirus, pregnancy and occupational risk]. / Cytomégalovirus, grossesse et risque professionnel.

BACKGROUND: Female health professionals are not more likely to contract cytomegalovirus (CMV) infection than the general population. CASE REPORT: Generalized congenital infection was diagnosed in a neonate. His mother was a nurse working the 2 first trimesters of her pregnancy in close-contact with AIDS patients chronically infected with CMV. CONCLUSION: Preventive measures to avoid CMV transmission among health-care professionals are controversial. The only guideline actually receiving universal agreement consists of standard hospital measures of hygiene.