Chronic active Epstein-Barr virus infection with cutaneous and sinus lymphoproliferation in a white female patient with 25 years' follow-up: an original case report.

Chronic active Epstein-Barr virus infection (CAEBV) is characterized by chronic infectious mononucleosis-like symptoms associated with very high viral load, as assessed by quantitative polymerase chain reaction. We present an unusual case in a French woman who was followed up over 25 years with cutaneous and sinus lymphoproliferation. This white woman presented with a long history of recurrent cutaneous necrotic papules of the skin, which started during childhood and healed spontaneously with depressed scars. The lesions spread to the left maxillary sinus and were associated with hepatomegaly and splenomegaly with no other visceral locations. Pathological examination of the skin and sinus revealed a dermal monoclonal T-cell lymphoproliferative disorder, CD7(+) and CD20(-) , with no epidermotropism. T-cell receptor rearrangement was positive, showing the monoclonality from the first biopsy. This T-cell proliferation was positive for EBV-encoded small RNA and was associated with a high EBV viral load. Since then, the patient has been in good health, despite a permanently high EBV viral load. Hydroa vacciniforme (HV)-like lymphoma and natural killer/T-cell lymphoma were discussed, but none really fit our case. Natural killer cell lymphoma was ruled out because of the indolent course, but sinus lesions do not exist in HV-like lymphoma. A therapeutic approach is difficult because of the coexistence of viral infection and monoclonal T-cell proliferation. Chemotherapy is not efficient and induces immunosuppression, which may worsen the prognosis. Although rituximab may have an immunomodulatory function, it was not effective in our case.

[Molecular diagnosis of respiratory enterovirus infections: Use of PCR and molecular identification for a best approach of the main circulating strains during 2008]. / Diagnostic moléculaire des infections respiratoires à entérovirus : apport de la PCR et du génotypage pour une meilleure approche de la circulation des souches en Basse-Normandie au cours de l'année 2008.

UNLABELLED: The PCR assays are currently used in diagnosis of enterovirus (EV) meningitis. Nevertheless, the use of molecular diagnosis of EV should be investigated in respiratory tract infections (RTI). OBJECTIVES: To perform enterovirus molecular diagnostic tools, PCR and genotyping, in nasal samples for diagnostic and epidemiologic purposes. METHODS: During 2008, 3612 nasal specimen (NS) were studied by IFD and MRC5 culture. Next, we realised successively viral isolation on HuH7 culture (for NS negative by IFD assay) and a duplex PCR enterovirus-rhinovirus for the 816 HuH7 positive supernatants. Furthermore, 327 NS collected from neonates were systematically tested by a real-time RT-PCR. This assay was used in routine for EV diagnosis setting in cerebrospinal fluid. Enterovirus genotyping was then performed for the 68 positive supernatants. RESULTS: Thirty-five NS (0.97%) were positive for EV by culture (MRC5). A combination of both PCR assays, PEVRV and PEV, allowed an additional identification of 41 EV, eight EV-RV and 12 RV, increasing the number of positive to 96 NS (2.6%). Among the neonates, 32 NS (11.3%) were positive for EV by PEV. Of the 98 NS tested by the two PCR assays (PEV and PEVRV), 27 were positive and we detected 10 EV, five EV-RV and 12 RV. From January to December 2008, the circulation of EV showed the usual peak in June-July when a small outbreak of aseptic meningitis occurred and an additional autumnal peak corresponding to respiratory tract infections. Five main serotypes were isolated: 19 EV68 (29.7%), 12 CB3 (18.7%), nine E3 (14,1%), six CA9 (9.4%) and six CB1 (9.4%); the 19 EV68 were isolated in October-November and 17/19 (89.5%) of positive patients were hospitalised for severe respiratory diseases. CONCLUSION: The use of molecular screening techniques (PCR assays and genotyping) on nasal samples collected from patients with respiratory infections allowed a prospective, effective and precise identification of circulating strains.

Epstein-Barr virus involvement in the pathogenesis of hydroa vacciniforme: an assessment of seven adult patients with long-term follow-up.

BACKGROUND: Hydroa vacciniforme (HV) is a chronic papulovesicular photodermatosis of childhood, with some cases persisting through adulthood. In children, the Epstein-Barr virus (EBV) has been detected in typical HV and in HV evolving into natural killer/T-cell lymphoma. No exploration of EBV infection has been performed in adult patients with HV with long-term follow-up. OBJECTIVES: To assess EBV infection systematically in blood and in experimentally photoinduced lesions in adult patients with HV. METHODS: Repeated tests for EBV DNA blood load using real-time polymerase chain reaction (PCR) and serological EBV tests were performed in seven adult patients with long-term follow-up. Skin samples from phototest-induced lesions and surrounding normal skin were studied using PCR, in situ hybridization and electron microscopy. ZEBRA protein was detected using immunostaining. Thirty-five patients with other photosensitive disorders were included as controls. RESULTS: The EBV DNA blood load was strongly positive in the seven patients with HV and negative in 34 of 35 of the patients with other photosensitive disorders (P < 0.001). The levels were higher in photosensitive patients with HV than in patients with HV in clinical remission. Ultrastructurally, viral particles were detected in lymphocytes and also in keratinocytes in three experimentally phototest-induced lesions; they were not found in the surrounding normal skin. ZEBRA protein was also detected in phototest-induced lesions, but not in the surrounding normal skin. CONCLUSION: EBV is involved in HV pathogenesis and persists in adult patients with HV. A positive EBV DNA load, specific to HV in the spectrum of photosensitive disorders, might be a useful biomarker in HV.

[Mycoplasma pneumoniae and Chlamydophila pneumoniae coinfection in severe pneumoniae among a hospitalized child with respiratory distress: what are the best diagnostic tools for an optimal care?]. / Coinfection à Mycoplasma pneumoniae et Chlamydophila pneumoniae chez un enfant hospitalisé pour une pneumonie sévère avec détresse respiratoire: choix des outils diagnostiques pour une prise en charge optimale.

The role for Mycoplasma pneumoniae and Chlamydophila pneumoniae in lower and upper respiratory tract infections in childhood increased by use of specialised diagnostic techniques, more and more performant for the early diagnosis of these infections. However, the prevalence of M. pneumoniae and C. pneumoniae as a cause of severe pneumoniae among hospitalized children has been rarely described. We report a case of M. pneumoniae et C. pneumoniae coinfection in a 10-year-old child hospitalized with a respiratory distress.

[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.

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.

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.

Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses.

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

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.

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.

Congenital HCMV infection: a collaborative and comparative study of virus detection in amniotic fluid by culture and by PCR.

Cytomegalovirus (HCMV) infection is the leading cause of congenital virus infection in developed countries, affecting an estimated 1% of births. This antenatal infection can cause serious sequelae. Strategies for prevention and treatment must, therefore, be agreed upon, entailing a preliminary performance assessment of antenatal virus diagnosis techniques. Between 1992 and 1999, HCMV serology status was established for 19456 pregnant women in four French hospitals. Seronegative patients (55.4%) were given serology screening, and antenatal diagnosis was given to 152 women who had shown seroconversion during their pregnancies (1.4%). The detection of HCMV transmission from mother to fetus was finally established in 95 cases, using polymerase chain reaction (PCR) and viral culture methods for detecting HCMV in the amniotic fluid. These results were compared with viral culture of children's urine after birth, enabling us to distinguish between children really infected in utero (30%) and non-infected children (70%). The results of the virus culture and those of PCR were identical in 94 of the 95 cases, with one discrepancy (culture-/PCR+). The two diagnosis techniques had identical sensitivity (72%), with culture proving slightly more specific than PCR (98.4% as opposed to 96.9%). Positive prediction values for culture and for PCR were, respectively, 95.6 and 91.3%. Antenatal virus diagnosis on amniotic fluid was negative with both techniques in 8 out of 29 cases of children born with HCMV infection (VPN=89%). Over half of these wrongly negative results can be explained by amniocentesis carried out too early in the pregnancy or too early with respect to the mother's primary infection.

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).

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.

[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.

[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.

[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.

[Epidemiology of viral infection and asthma]. / Épidémiologie de l'infection virale et asthme.

The first epidemiological data concerning viruses and asthma were obtained in the 1970s and 1980s by viral isolation and serology. Viral infection can be identified in 24 % to 31.9 % of children, and in 13.3 % of adults. The three most frequent viruses are rhinovirus (RV), respiratory syncytial virus (RSV), and parainfluenza viruses (PIV), detected in 8.8 %, 6.4 % and 6 % of cases, respectively. Due to its amplifying properties, the use of PCR increases the frequency of viral detection, and appears particularly appropriate in asthma where the viral load can be reduced. In a study of bronchiolitis, RSV, PIV3, AdV and RV were identified in 39.3 %, 4.3 %, 1.4 % and 3.9 % of cases, respectively, by IF or culture, and in 62.4 %, 8.3 %, 10.8 % and 12.6 % of cases, respectively, by PCR. Two recent epidemiological surveys used molecular diagnosis in asthma attacks. In a series of 61 adults, 27 (44 %) infections were identified: 16 RV, 4 CV OC43, 3 PIV, 1 RSV, 1 VI, 1 Chlamydia psitacci. In children, viral infection was detected in 226 cases (77 %) : 84 RV, 38 CV, 21 IV, 21 PIV, 12 RSV. We have performed a short retrospective survey for 1997, using molecular biology, on 39 nasal aspirates from children consulting for asthma or wheezing bronchitis. Testing for respiratory viruses by conventional techniques identified 8 (20.5 %) viral infections: 3 RV, 3 RSV, 1 IBV and 1 VPI2. After nucleic acid extraction, PCR-hybridization techniques were applied to these samples to detect RSV, AdV, RV, CV 229E, CV OC43, CP and MP sequences. Twenty six aspirates (54 %) were positive only on molecular biology techniques: 11 RSV, 12 RV, 2 enterovirus, 1 CV OC43. Overall 34 (82 %) viral infections were detected in these children, and a mixed RSV-RV infection was identified in 6 cases. Compared to the studies reported in the literature, we observed the same predominance of RV infections, more RSV infections, probably related to the use of PCR, and a lower incidence of CV infections.