[Bronchiolitis viruses]. / Les virus des bronchiolites aiguës.

In Normandy (France), human respiratory syncytial virus (hRSV) was detected in 64.1% of acute bronchiolitis in hospitalized children, rhinovirus in 26.8%, human metapneumovirus (hMPV) in 7.6%, and parainfluenza virus (PIV) in 3.4%. The viruses causing acute bronchiolitis in the community were hRSV (42%), rhinovirus (19.5%), coronavirus (8%), PIV (3.5%), and hMPV (2.5%). In 53.7% of the cases, hRSV infected infants (86.9%), 53.7% being less than 6 months of age. Of the hRSV cases, 48.2% were detected in November and December and 44.5% in January and February. The hRSV epidemic started the 1st or 2nd week of October but it varied from one year to another and from one region to another. hRSV acute bronchiolitis increased from 261 cases in epidemics from 1999-2003 to 341 cases from 2004-2009. Rhinoviruses gave acute bronchiolitis in 38.4% of cases. A rate of 54.6% of viruses was detected in September and October and 38.5% in March and April. A total of 34.2% of infected infants were under 6 months of age, 37.8% between 6 months and 2 years, and 19.5% were between 2 and 5 years old. hMPV epidemics coincided with hRSV epidemics, but they accounted for one-sixth the number of cases. HMPV infected infants (74%) who were older than those infected with hRSV, and the diagnosis was bronchiolitis (59%) and pneumonia (17%). PIV infections (about 100 cases per year) included PIV3 (62.7%), PIV1 (25.3%), and PIV2 (7.3%). PIV1 infections occurred every 2 years in the fall. PIV3 infections were observed every year during the fall and winter, with peaks of infections in the spring in the years without PIV1. There were acute cases of bronchiolitis in 29.8% of PIV3 infections and 18.3% in PIV1 infections.

[Seasonal flu]. / La grippe saisonnière.

Seasonal flu is caused by influenza viruses A and B. These enveloped viruses have a genome made up of seven or eight RNA fragments. The different subtypes are determined by the nature of the two surface glycoproteins HA and NA. Seasonal flu is an epidemic wintertime illness occurring in temperate climate zones. Its epidemiology is linked to the great variability of the virus in time, necessitating an alert system that detects dominating circulating variants each year and that determines the vaccination composition. Clinical flu symptoms are not sufficiently specific to allow for diagnosis with virological tests. This is especially true during non-epidemic periods as well as in subjects older than 65 and younger than five. Children are especially vulnerable to influenza virus infections. Hospitalization occurs more frequently, the younger the child. In children younger than two years, the infection can be pauci-symptomatic and is sometimes detected from non-respiratory symptoms such as lethargy, convulsions, and dizziness. In all cases of respiratory syndrome compatible with influenza virus infection in hospitalized subjects, virological flu diagnosis is of utmost interest. Several tools are available to allow for direct viral detection in respiratory specimens: cell culture isolation, antigenic detection, RNA molecular detection. Choice of method is based on the characteristics of the test: sensibility, specificity, speed and ease of realization, and cost.

Prevalence and risk factors of the whole spectrum of sexually transmitted diseases in male incoming prisoners in France.

Sexually transmitted diseases (STD) are a public health issue in prison. As inmates are eventually released, it is also a community concern. There are very few data on the entire spectrum of STDs, particularly condyloma among prisoners. To determine the prevalence of all STDs: infection with human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus (HSV), Chlamydia trachomatis, Neisseria gonorrhoea, syphilis, and condyloma among entering inmates. A cross-sectional study was conducted in France from November 2000 to June 2003. Male adults entering a prison remand center in Caen had a medical consultation and physical examination including external genital organs and perianal area for condyloma and herpes infection, a urethral swab for Chlamydia trachomatis and Neisseria gonorrhoea detection, and a blood sample for HBV, HCV, HIV, and syphilis serology. Five hundred and ninety-seven inmates agreed to participate in the study. Sixteen percent had at least one STD: 4.0% had condyloma, 4.0% chlamydia infection, and 4.9% were positive for HCV antibodies. Two had early syphilis and 1 had acute HBV, but no HIV infection, neither genital herpes nor gonorrhea. The analysis of the STD risk behaviors did not show any difference between the infected and uninfected participants, except that HCV-positive participants were more likely to be intravenous drug users. Results suggest that a systematic screening of all STDs should be at least proposed to every entering inmate since no demographic or sexual characteristics are consistently associated with STDs.

Study of influenza C virus infection in France.

From November 2004 to April 2007, specimens were obtained from 2,281 patients with acute respiratory tract illness in Normandy, France. Eighteen strains of influenza C virus were detected in these samples using a combined tissue culture/RT-PCR diagnostic method. Most patients with influenza C virus infection (13/18) were infants or young children (<2 years of age). The most frequent symptoms were fever and cough, and the clinical presentation of influenza C virus infection was similar to that of other respiratory viruses. Thirteen of the 18 infected patients were hospitalized; 3 presented with a severe lower respiratory infection. The hemagglutinin-esterase (HE) gene of 10 isolates was sequenced to determine the lineages of the circulating influenza C viruses. Phylogenetic analysis revealed that most of the isolated strains had an HE gene belonging to the C/Yamagata/26/81-related lineage. These results show that influenza C virus regularly circulates in Normandy and generally causes a mild upper respiratory infection. Because the differential clinical diagnosis of influenza C virus infection is not always easy, it is important to identify viral strains for both patient management and epidemiological purposes.

Molecular diversity of the aminoterminal region of the G protein gene of human respiratory syncytial virus subgroup B.

Two major antigenic subgroups (designated A and B) have been described for human respiratory syncytial virus (HRSV). Between and within the two main subgroups, there is antigenic variation in the attachment protein G. The variability of the G protein is known to be located in two hypervariable regions of the ectodomain. Most investigators have studied the gene segment coding the C-terminal end of the protein, and little is known about the N-terminal variable region. In the present study, the genetic variability of HRSV subgroup B was evaluated by nucleotide sequencing of the N-terminal region of the G gene of 52 Tunisian isolates. Tunisian subgroup B isolates clustered into two main lineages designated arbitrarily as Tu-GB1 and Tu-GB2. Three distinct subtypes were identified within genotype Tu-GB2. The inter- and intragenotype nucleotide variability ranged from 4 to 8% and from 0 to 4%, respectively. Overall divergence values of the G sequences were inferior or equal to 15% at the aminoacid level. Comparison of sequences among Tunisian HRSV strains and viruses isolated in other geographical areas during different epidemics demonstrated close similarity to strains from Kenya, Belgium, the UK, Qatar, Canada and South Korea.

Multicentric evaluation of a new commercial cytomegalovirus real-time PCR quantitation assay.

Automated real-time PCR systems have become the most common method in the quantitation of viral load during cytomegalovirus (CMV) infection in immuno-compromised patients. In order to evaluate a new commercially available CMV real-time PCR assay (CMV R-gene, Argene, France), a pp65 antigenemia assay and four different "in-house" real-time PCR assays were compared to the CMV R-gene for the detection and the quantitation of CMV load in 506 specimens of whole blood from transplant patients in four French hospital laboratories. The CMV R-gene was more sensitive than the pp65 antigenemia: there were 18% antigenemia-negative versus CMV R-gene-positive samples. A significant correlation was found between DNA quantitation by CMV R-gene and the number of positive cells detected by the pp65 antigenemia test (Spearman's rank test, r=0.63, p<0.0001). A CMV DNA load equivalent to 50 pp65-positive cells/200000 polymorphonuclear leukocytes was 5.26log(10)copies/mL of whole blood. When the CMV R-gene kit was compared to the four other "in-house" real-time PCR assays, there were few discordant results (6.7% total for the four laboratories), all detected with a weak positive CMV DNA viral load. Spearman's coefficients showed a good (r=0.82 for laboratory 1, r=0.66 for laboratory 3) to excellent (r=0.99 for laboratory 2, r=0.94 for laboratory 4) correlation between CMV R-gene and the four real-time "in-house" PCR assays. However, the results of CMV DNA viral load generated by CMV R-gene test were constantly higher than those generated by three out of four "in-house" PCR assays. This mean variation in CMV DNA viral load measured by CMV R-gene and "in-house" PCRs was of 0.77log(10), 0.04log(10), 0.77log(10) and 0.97log(10), for laboratories 1, 2, 3 and 4, respectively. We concluded that there was variability between results of different real-time PCR assays for CMV DNA quantitation. This observation emphasized the need of a standardised commercial assay to allow an "inter-laboratory" comparison of results. Our study showed that CMV R-gene is an accurate, efficient, reliable and versatile tool for rapid diagnosis and monitoring of CMV disease in transplantation recipients.

[Current techniques used for the diagnosis of respiratory virus infectious in intensive care units]. / Techniques actuelles de diagnostic des infections virales respiratoires en réanimation.

Hundred viruses can be isolated in patients suffering from respiratory virus infections and hospitalised in intensive care unit (ICU): influenza virus, respiratory syncytial virus, para-influenza virus, adenovirus, coronavirus, rhinovirus, enterovirus, human metapneumovirus, bocavirus… Nasal or tracheobronchial specimens, which contain many epithelial cells will be used to isolate these common viruses. In immunocompromised patients a bronchoalveolar lavage has to be added to these specimens in order to detect cytomegalovirus and some adenovirus. The immunofluorescence or immunoenzymatic assays, which detect viral antigens in the infected cells are the easiest and fastest diagnostic methods, theoretically. As with other techniques, specimen quality is a major determinant of their performance. Unfortunately, the sensitivity of the antigen detection assays is low in respiratory infections in adults. Then the virus recovery by cell culture, which is usually more sensitive than the antigen detection assays, can be helpful. Many studies have reported more respiratory virus detections using nucleic acid testing such as PCR. They detect viruses, which are missed by conventional methods and increase the detection of common respiratory virus. Multiplex PCR assays have been developed, and these can simultaneously detect several viruses directly in clinical specimens. Nucleic acid testing can subtype viruses using subtype-specific primers, and analyse strain variation through genetic. It can be used also to quantify the viral load in clinical specimens. More recently real-time RT-PCR assays have been developed to get more rapidly the results of the nucleic acids assays. Specimen quality, timing and transportation conditions may be less critical for nucleic acid testing than for culture or antigen detection, as viable virus and intact infected cells need not to be preserved. Moreover, viral nucleic acids are detectable for several days longer into the clinical course than is cultivable virus, potentially allowing a diagnosis to be made in late-presenting patients. However, in a clinical virology laboratory, where the speed, low cost, and high sensitivity of the methods are required, the sequential use of antigen detection tests and multiplex PCR could be the best choice, particularly in the clinical setting of respiratory virus infections in adults hospitalised in ICU. In the future, the development of real-time multiplex PCR is likely to be top-priority.

Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens.

BACKGROUND: Detection of enteroviral nucleic acid in cerebrospinal fluid (CSF) specimens has been demonstrated to improve the management of patients with aseptic meningitis. OBJECTIVE: To develop on the LightCycler (LC) instrument a real-time RT-PCR assay based on TaqMan technology for the detection of enteroviruses (EV) in cerebrospinal fluid (CSF) specimens. STUDY DESIGN: After evaluation of the analytical performances, seventy-four CSF samples collected prospectively from patients who have been suspected for a clinical diagnosis of meningitis were evaluated by two LC real-time RT-PCR assays and one conventional RT-PCR assay. RESULTS: Our assay detected all 30 different EV species tested, whereas no reactivity was observed with other neurotropic viruses. The analytical sensitivity of both LC RT-PCR real-time assays was 1 TCID50 for LC one-step and two-step RT-PCR assays. Results for LC one-step and LC two-step RT-PCR were compared to results of the conventional RT-PCR: of the 74 CSF specimens tested, 11 were positive and 56 were negative by all methods. Four other specimens were positive for EV by at least two of the methods (including the LC two-step RT-PCR and the conventional RT-PCR), two other CSF specimens were positive by the LC two-step RT-PCR assay only, and another one CSF specimen was positive by the LC one-step RT-PCR assay only. No CSF specimens were negative by the LC two-step RT-PCR assay and positive by the conventional RT-PCR assay. The sensitivity, specificity, positive and negative predictive values of both LC RT-PCR assays by using conventional RT-PCR as the "gold standard" were, respectively, 73.3, 98.3, 91.7, 93.5% for the LC one-step RT-PCR and 100, 96.6, 88.2, 100% for the LC two-step RT-PCR. There was substantial agreement between the three assays (k=0.80). CONCLUSIONS: The LC two-step RT-PCR assay is a rapid, sensitive and reliable method which can be routinely performed with CSF samples for diagnosis of EV infection and is an important improvement for optimal patient management.

[Epidemiology and diagnosis of respiratory syncitial virus in adults]. / Epidémiologie et diagnostic des infections à virus respiratoire syncytial de l'adulte.

INTRODUCTION: Respiratory syncytial virus (RSV) is rarely searched for in respiratory infections in adults. This study assessed its frequency and diagnosis. METHODS: Three separate studies were conducted in adults presenting with (1) a flu-like illness, (2) a lower respiratory tract infection in the community, and (3) a severe pneumonia requiring hospitalisation. The diagnosis of RSV infection was sought by PCR in all cases, and compared to antigen detection and culture in two studies. RESULTS: RSV was identified in 20 (11.7%) of 170 influenza-vaccinated adults suffering from flu-like symptoms. In the 270 cases of non-severe lower respiratory tract illnesses in the community, viruses were identified in 86 (31.8%) cases, with RSV accounting for 13 (4.8%). In the 164 cases of acute bronchitis, a virus was detected in 64 (36.7%) of which 11 (6.3%) were RSV, 37 (21.3%) rhinovirus, 5 influenza viruses A and B, and 12 other viruses. In the 60 cases of infective exacerbations of chronic bronchitis, rhinovirus was detected in 9 (15%) and para-influenza 3 virus in 2 cases. In the 21 acute pneumonia's, 1 RSV, 1 influenza virus A and 2 rhinovirus cases were detected as well as 1 RSV, 1 parainfluenza 3 viruses and 4 rhinovirus cases in the 11 lower respiratory tract illnesses in patients with pre-existing lung disease. There were overall 19 viral and bacterial associated infections. Finally, in the 51 acute pneumonias hospitalised with respiratory distress syndrome, a virus was identified in 17 (33.3%) cases, including 3 (5.5%) RSV, 6 influenza A, 3 rhinovirus, 2 adenovirus, 2 herpes simplex virus and 1 cytomegalovirus. There were 6 bacterial-associated infections, and 4 were hospital-acquired. All RSV-infected patients were old people and had chronic pulmonary or cardiac disease. CONCLUSIONS: In adults, RSV is a frequent cause of flu-like symptoms. It can sometimes cause lower respiratory tract illness, which can be severe, and should be considered in the differential diagnosis in such cases. The PCR method is a particularly effective diagnostic test, but as yet is not routinely available.

Quantitative analysis of HCMV DNA load in whole blood of renal transplant patients using real-time PCR assay.

BACKGROUND: Preemptive antiviral treatment of Human Cytomegalovirus (HCMV) disease is a major goal in the management of organ transplant patients. It requires sensitive diagnostic methods. Automated real-time PCR systems have been recently proposed to monitor HCMV infection in such patients. OBJECTIVE: Objectives of this study was to compare a real-time quantitative PCR on whole blood with the HCMV pp65 antigenemia assay in renal transplant recipients, and also to evaluate two different DNA extraction methods. STUDY DESIGN: A total of 248 specimens from 21 patients were tested by quantitative pp65 antigenemia and quantitative real-time PCR. DNA was extracted from whole blood samples using two different methods: a conventional column manual assay and an automated system. RESULTS: Quantification of HCMV DNA using the two extraction methods showed highly similar results (Spearman rank test, r=0.863). We found a significant correlation between DNA quantification by real-time PCR in whole blood and pp65 antigenemia test (Spearman rank test, r=0.767). This correlation was not modified when the HCMV DNA results were normalized by quantification of the albumin cellular gene. In eight patients, HCMV infection was detected earlier with quantitative PCR than with the antigenemia test (mean delay of 11.25 days). HCMV DNA load equivalent of 50 pp65 positive cells/200,000 polymorphonuclear leukocytes (PMNLs) is log4.095 copies per ml of blood. CONCLUSIONS: Real-time PCR in whole blood is a sensitive method for estimating the HCMV genome load in renal transplant patients, and is more rapid and practicable than using PMNLs for pp65 antigenemia tests.

Quantitation of respiratory syncytial virus RNA in nasal aspirates of children by real-time RT-PCR assay.

A method was developed for the quantitation of respiratory syncytial virus (RSV) based on real-time RT-PCR using a LightCycler instrument. A control real-time RT-PCR was undertaken on GAPDH mRNA (a human housekeeping gene) was carried out to standardise the non-homogeneous respiratory samples. The real-time RT-PCR method was one log more sensitive for the detection of RSV according to the endpoint dilution technique than the culture method or a conventional qualitative RT-PCR-hybridization-EIA. No cross-reactivity was observed with any of the viruses that could be found in the respiratory tract. RSV and GAPDH were quantified in nasal aspirates from 75 children hospitalised for acute respiratory tract disease: 31 (41.3%) were positive according to the immunofluorescence assay (IFA), 34 (45.3%) were culture-positive and 42 (56%) were positive according to our real-time RT-PCR method. The sensitivity, specificity, positive and negative predictive values of the real-time RT-PCR were 100, 90, 92, 100%, respectively. The samples found to be positive for RSV were classified according to the severity of the disease. The mean number of RSV RNA copies was higher in the severe disease group than in the non-severe group 4.05 x 10(7) vs 9.1 x 10(6) (P=0.055). However, the mean ratio of RSV RNA copies to GAPDH mRNA copies was 42.8 in the severe group, and 22.2 in non-severe group (P=NS).

[Evaluation of four commercial immunoglobulin G (IgG)- and IgM- specific enzyme immunoassays for diagnosis of Mycoplasma pneumoniae infections]. / Evaluation de quatre tests immunoenzymatiques (ELISA IgG et IgM) pour le diagnostic des infections à Mycoplasma pneumoniae.

The four following commercially available enzyme immunoassays (EIAs) were assessed and compared for their performance in detecting Mycoplasma pneumoniae specific IgG and IgM antibodies: EIA-Platelia, EIA-Bmd, EIA-Sorin and EIA-Biotest. Three groups of patients were investigated: 39 patients (27 children and 12 adults) with respiratory infections and a M. pneumoniae PCR-positive in respiratory specimens (group I; 52 sera), 61 healthy children and adults (group II; 61 sera) and 20 patients with rheumatoid factor, antinuclear antibodies or positive antiviral IgM (group III; 20 sera). In group III, the IgM specificity for the EIA-Platelia, EIA-Bmd, EIA-Biotest and EIA-Sorin was 100%, 90%, 65% and 25%, respectively. In the children from group I, the four EIAs had similar IgM sensitivity (89 to 92%) but a striking difference in IgM sensitivity was observed in adult patients: 16% EIA-Platelia and EIA-Bmd, 50% EIA-Biotest, 58% EIA-Sorin. The sensitivity for IgG was greater with EIA-Bmd and EIA-Biotest, especially in detection of IgG in acute-phase serum : 61% EIA-Bmd and EIA-Biotest, 15% EIA-Platelia and 31% EIA-Sorin. Discrepant and unexpected results were observed in IgM detection from control healthy patients using EIA-Sorin and EIA-Biotest, confirming the lack of specificity of these two EIA-tests and making them inaccurate for routine diagnosis. A high IgG seroprevalence were found in healthy adults by the four EIAs (43-70%). In healthy children, EIA-Bmd and EIA-Biotest gave a higher IgG seroprevalence than EIA-Sorin and EIA-Platelia (45% each for the former as compared to 17% and 20%, respectively, for the latter).These results confirm that the IgM EIA serology test is a valuable tool for the early diagnosis of M. pneumoniae infections in children, as long as the EIA test used is specific. In adults, the difficult interpretation of EIA tests suggests that paired sera, combined with PCR detection on respiratory tract specimens collected on admission of patient, should be required for accurate diagnosis.

[Role of viral infections and Chlamydia pneumoniae and Mycoplasma pneumoniae infections in asthma in infants and young children. Epidemiologic study of 118 children]. / Rôle des infections virales et des infections à Chlamydia pneumoniae et à Mycoplasma pneumoniae au cours de l'asthme du nourrisson et du jeune enfant. A propos d'une étude épidémiologique chez 118 enfants.

Wheezing associated with upper respiratory tract infections is common in children. Using conventional techniques (viral culture and immunofluorescence) and molecular techniques (PCR), we studied the prevalence of viral, Chlamydia pneumoniae (CP) and Mycoplasma pneumoniae (MP) infections in 118 children hospitalised for acute asthma exacerbation. A virus was identified by conventional techniques in 40 of the 118 nasal swabs (34%), while PCR allowed identification of virus CP and MP in 80 samples (68%). Combination of both techniques allowed identification of an infectious agent in 91 cases (77%). More than one agent was isolated in 15 cases (23%). Rhinovirus (RV) (45%) were prevalent, followed by respiratory syncytial virus (RSV) (28%) and enterovirus (8.5%). RV and RSV have a similar prevalence (42% and 36% respectively) before two years of age, as compared with 66% and 27% respectively in older children. CP and MP were identified by PCR in only 6 cases. Molecular techniques of identification demonstrated a clear advantage in sensitivity compared with conventional techniques. The high prevalence of RV and RSV infections is remarkable, while CP and MP do not seem particularly involved in children acute asthma exacerbation.

Evaluation of four commercial immunoglobulin G (IgG)- and IgM-specific enzyme immunoassays for diagnosis of Mycoplasma pneumoniae infections.

The four following commercially available enzyme immunoassays (EIAs) were assessed and compared for their performance in detecting Mycoplasma pneumoniae immunoglobulin G (IgG)- and IgM-specific antibodies Platelia EIA, ImmunoWELL M. pneumoniae ELISA IgG and IgM, ETI-MP-IgG and IgM EIAs and Biotest anti-M. pneumoniae IgG and IgM ELISA (referred to herein as EIA-Platelia, EIA-BMD, EIA-Sorin, and EIA-Biotest). Three groups of patients were investigated: 39 patients (27 children and 12 adults) with respiratory infections who tested positive by PCR for M. pneumoniae in respiratory specimens (group I; 52 serum samples), 61 healthy children and adults (group II; 61 serum samples), and 20 patients with rheumatoid factor or antinuclear antibodies, or who tested positive for antiviral IgM (group III; 20 serum samples). In group III, the IgM specificity for EIA-Platelia, EIA-BMD, EIA-Biotest, and EIA-Sorin was 100, 90, 65, and 25%, respectively. In the children from group I, the four EIAs had similar IgM sensitivities (89 to 92%); the sensitivity for IgG was greater with EIA-BMD and EIA-Biotest than with EIA-Platelia and EIA-Sorin (66 and 78% versus 55 and 52%, respectively). In adult patients from group I, 9 to 10 serum samples were positive for IgG with a concordant sensitivity of 75 to 83% between the four EIAs but a striking difference in IgM sensitivity: 16% by EIA-Platelia and EIA-BMD, 50% by EIA-Biotest, and 58% by EIA-Sorin. Discrepant and unexpected results were observed in IgM detection from control healthy patients using EIA-Sorin and EIA-Biotest, confirming the lack of specificity of these two EIAs and making them inaccurate for routine diagnosis. A good concordance of IgG seroprevalence in healthy adults was found between the four EIAs (66 to 70%), though this concordance was lower with EIA-Platelia (43%). In healthy children, EIA-BMD and EIA-Biotest gave a higher IgG seroprevalence than EIA-Sorin and EIA-Platelia (45% each for the former compared to 17 and 20%, respectively, for the latter). These results confirm that the IgM EIA serology test is a valuable tool for the early diagnosis of M. pneumoniae infections in children, as long as the EIA used is specific. In adults, the difficult interpretation of EIAs suggests that paired sera, combined with PCR detection on respiratory tract specimens collected on admission of patient, should be required for accurate diagnosis.

Direct diagnosis of human respiratory coronaviruses 229E and OC43 by the polymerase chain reaction.

An RT-PCR-hybridization was developed that amplified genetic material from the M protein gene of HCoV-229E and HCoV-OC43. The analytic sensitivity of these original primers were compared with primers defined in the N gene and described previously. The results show that 0.05 TCID50 of HCoV-229E and 0.01 TCID50 of HCoV-OC43 can be detected by this molecular method using the original method. Detection of HCoV-229E and HCoV-OC43 in clinical specimens is possible using this method: 348 respiratory specimens (202 sputum and 146 nasal aspirates) were tested with this RT-PCR-hybridization and 12 human coronavirus are detected (3%). The method could provide a useful tool for demonstrating the role of human coronavirus in infections of the respiratory tract.

[Epidemiology of respiratory virus infections]. / Epidémiologie des infections virales respiratoires.

Respiratory viral infections are very common in young children. They sometimes occur as primary infections (and sometimes re-infections) by influenza and parainfluenza virus, respiratory syncytial virus (VRS), adenovirus, rhinovirus and coronavirus. The clinical pictures are very varied and without strict clinico-virological correlation. In adults the role of the site (frail lung, aged persons) and the type of virus play an important part. Many viral infections develop in an epidemiological way (influenza, VRS bronchiolitis, rhinovirus infections...) and several epidemics by different viruses overlap from September-October to March-April making it very difficult to decide the precise cause. Epidemics are followed thanks to networks of medical practitioners (GROG, SENTINELLE...) and by data from hospitalised patients, but precise identification of epidemic viruses is only possible and validated by virological analysis of samples taken from patients.

[Viral identification, Chlamydia pneumoniae and Mycoplasma pneumoniae during asthma exacerbation: comparative epidemiology between infants and children.] / Exacerbations de l'asthme et identification virale associée à celle du Chlamydiae pneumoniae et du Mycoplasma pneumoniae : épidémiologie comparative entre nourrissons et enfants âgés de plus de deux ans.

Purpose.- Molecular processes can have a different impact on epidemiological data. Patients and methods. - The study covers 118 nasal aspirate samples taken on children hospitalized for acute asthma exacerbation for 2 years. Conventional techniques associated viral culture and immunofluorescence while molecular techniques used polymerase chain reaction (PCR). Results. - Virus presence was revealed with conventional techniques in 34% of the respiratory samples (40/118), while PCR study of viruses and genomes of Chlamydia pneumoniae and Mycoplasma pneumoniae allowed positive identification in 68% of the samples (80/118). The combination of both techniques allowed identification of an infectious agent in 77% of cases (91/118). More than one pathogenic agent was isolated in 23% of positive samples. Epidemiological study shows prevalence of rhinovirus (45%), then respiratory syncytial virus (28%) and enterovirus (8.5%). In children under 2 years of age, rhinovirus and respiratory syncytial virus have a close prevalence (respectively 42 and 36%), which is not the same result as in older children (respectively 66 and 27%). Moreover, PCR techniques allowed the identification of just a few Chlamydia pneumoniae and Mycoplasma pneumoniae (6/118). Conclusion. - In this study, molecular techniques of identification demonstrate a clear advantage in sensitivity compared to performances of viral cultures or immunofluorescence. The importance of rhinovirus and respiratory syncytial virus is remarkable while Chlamydia pneumoniae an Mycoplasma pneumoniae do not seem to be particularly involved.

Comparison of three non-nested RT-PCR for the detection of influenza A viruses.

BACKGROUND: The viral isolation technique (VIT) is largely used as a gold standard for the detection of influenza A and B viruses in respiratory samples. Some recent studies have pointed out that the polymerase chain reaction (PCR) assays allow sensitive and rapid detection of influenza viruses, also providing excellent correlation with traditional methods. OBJECTIVES AND DESIGN STUDY: The aim of this study was to evaluate the efficiency of three non-nested PCR, two PCR-hybridization assays using primers defined in M and NS genes, and one PCR which uses primers defined in NP, NS and HA genes and combines the detection of H3N2 and H1N1 hemagglutinin genes using defined primers in NP, NS and HA genes (PCR3), in comparison with an IF assay (IFA) and viral isolation technique (VIT). The study was carried out on 244 nasal samples collected mainly by practitioners of the GROG surveillance network during winter 1998-1999 for the detection of influenza A virus. RESULTS: Overall influenza viruses were detected more frequently by PCR techniques in 157 (64.3%), 147 (60.2%), 110 (45%) cases for PCR1, PCR2, PCR3, respectively, than by VIT or IFA, in 100 (40.9%) and 74 (30.3%) cases, respectively. Taking the positive culture samples as a reference, 100 (41.8%) samples were found to be positive for influenza A, and the sensitivity of IFA, PCR 1, PCR 2 and PCR3 techniques were 70, 100, 99, and 90%, respectively as compared with viral isolation cultures. On the other hand, as 86.5% of positive samples were positive with at least two different techniques, the sensitivity, specificity, VPP and VPN of each technique were recalculated taking into account a further criterion defining a positive sample: positivity with two techniques. We observe that techniques PCR 2 and particularly PCR 1 have very good sensitivity, respectively 98.6 and 100%, far better than the traditional techniques, IFA and culture, whilst maintaining acceptable specificity: 94.1 and 86.1%, respectively. In both cases they enable 141 (57.7%) A-positive influenza samples to be detected instead of the 100 (40.9%) obtained when culture is the reference test. IFA, culture and PCR 3 are highly specific (VPP=100%), but in comparison with PCR 1 and 2 their sensitivity, respectively 51.7, 69. 9, 77.6%, and negative predictive value are unsatisfactory. PCR 1 and 2 are superior to the other techniques to a statistically highly significant degree in terms of sensitivity, but the difference between the two is not significant.

Detection of viral, Chlamydia pneumoniae and Mycoplasma pneumoniae infections in exacerbations of asthma in children.

BACKGROUND: A high frequency of virus infections has been recently pointed out in the exacerbations of asthma in children. OBJECTIVES: To confirm this, using conventional and molecular detection methods, and expanding the study to younger children. STUDY DESIGN: One hundred and thirty-two nasal aspirates from 75 children hospitalized for a severe attack of asthma were studied (32 infants, mean age 9.1 months; and 43 children, mean age 5.6 years). According to the virus, a viral isolation technique, immunofluorescence assays (IFA) or both were used for the detection of rhinovirus, enterovirus, respiratory syncytial (RS) virus, adenovirus, coronavirus 229E, influenza and parainfluenza virus. Polymerase chain reaction (PCR) assays were used for the detection of rhinovirus, enterovirus, RS virus, adenovirus, coronavirus 229E and OC43, Chlamydia pneumoniae and Mycoplasma pneumoniae. RESULTS: Using IFA and viral isolation techniques, viruses were detected in 33.3% of cases, and by PCR techniques, nucleic acid sequences of virus, Chlamydia pneumoniae and Mycoplasma pneumoniae were obtained in 71.9% of cases. The combination of conventional and molecular techniques detects 81.8% of positive samples. Two organisms were identified in the same nasal sample in 20.4% of the cases. The percentage of detections was higher (85.9%) in the younger group than in the other (77%). The most frequently detected agents were rhinovirus (46.9%) and RS virus (21.2%). Using PCR rather than conventional techniques, the detection rates were increased 5.8- and 1.6-fold in rhinovirus and RS virus infections, respectively. The detection levels of the other organisms are as follows: 9.8, 5.1, 4.5, 4.5, 4.5, 3.7, and 2.2% for enterovirus, influenza virus, Chlamydia pneumoniae, adenovirus, coronavirus, parainfluenza virus, and Mycoplasma pneumoniae, respectively. CONCLUSION: These results confirm the previously reported high frequency of rhinovirus detection in asthmatic exacerbations in children. They also point out the frequency of RS virus detection, and emphasize the fact that PCR assays may be necessary to diagnose respiratory infections in asthma.