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Influenza viruses and severe acute respiratory syndrome coronavirus 2 are respiratory viruses with a high potential for evolution. Their circulation must be carefully monitored.. The development of diagnostic tools, as well as the search for universal vaccines and new treatments, are strategic tools in public health that allow us to be ready to face future emergencies.

Les virus grippaux et le severe acute respiratory syndrome coronavirus 2 sont des virus respiratoires dont le potentiel évolutif est important. Leur circulation doit être surveillée attentivement. Le développement des moyens de diagnostic, ainsi que la recherche de vaccins universels et de nouveaux traitements sont des outils stratégiques en santé publique permettant de se tenir prêts pour faire face à de futures émergences.

Background Tenofovir and emtricitabine interfere with the SARS CoV-2 ribonucleic acid (RNA)-dependent RNA polymerase (RdRp). Several cohorts reported that people treated by tenofovir disoproxil fumarate and emtricitabine are less likely to develop SARS CoV-2 infection and related severe COVID-19. Methods We conducted a pilot randomized, open-label, controlled, phase 2 trial at two hospitals in France. Eligible patients were consecutive outpatients (aged ≥18 years) with RT-PCR-confirmed SARS-CoV-2 infection and an interval from symptom onset to enrolment of 7 days or less. Patients were randomly assigned in a 1:1 ratio to receive oral tenofovir disoproxil fumarate and emtricitabine (2 pills on day 1 followed by 1 pill per day on days 2–7) or the standard of care. The primary and secondary endpoints were SARS-CoV-2 viral clearance from baseline assessed by cycle threshold (Ct) RT-PCR on nasopharyngeal swab collected at day 4 and day 7, respectively. A higher Ct corresponds to a lower SARS CoV-2 viral burden. Other endpoints were the time to recovery and the number of adverse events. This trial is registered with ClinicalTrials.gov, NCT04685512. Findings From November, 20th 2020 to March, 19th 2021, 60 patients were enrolled and randomly assigned to a treatment group (30 to tenofovir disoproxil fumarate and emtricitabine and 30 to standard of care). The median number of days from symptom onset to inclusion was 4 days (IQR 3–5) in both groups. Amongst patients who received tenofovir disoproxil fumarate, the difference from standard of care in the increase in Ct RT-PCR from baseline was 2.3 (95% confidence interval [-0.6 to 5.2], p = 0.13) at day 4 and 2.9 (95% CI [0.1 to 5.2], p = 0.044) at day 7. At day 7, 6/30 in the tenofovir disoproxil fumarate and emtricitabine group and 3/30 in the standard of care group reported no COVID-related symptoms. Adverse events included 11 cases of gastrointestinal side effects (grade ≤ 2), three of which leaded to drug discontinuation. Three patients had COVID-19 related hospitalisation, no participant died. Interpretation In this pilot study of outpatients adult with recent non-severe COVID-19, tenofovir disoproxil fumarate plus emtricitabine appeared to accelerate the natural clearance of nasopharyngeal SARS-CoV-2 viral burden. These findings support the conduct of larger trials of tenofovir-based therapies for the prevention and early treatment of COVID-19. Funding No external funding.

Background Successful 2-drug regimens (2DRs) for HIV were made possible by the availability of drugs combining potency and tolerability with a high genetic barrier to resistance. How these deal with resistance development/re-emergence, compared with 3DRs, is thus of paramount importance. Materials and methods A national survey including patients who were either naive or experienced with any 2DR or 3DR but failing integrase strand transfer inhibitor (INSTI)-containing regimens [two consecutive plasma viral load (VL) values >50 copies/mL] was conducted between 2014 and 2019. Genotypic resistance tests were interpreted with the v28 ANRS algorithm. Results Overall, 1104 patients failing any INSTI-containing regimen (2DRs, n = 207; 3DRs, n = 897) were analysed. Five hundred and seventy-seven (52.3%) patients were infected with a B subtype and 527 (47.3%) with non-B subtypes. Overall, 644 (58%) patients showed no known integrase resistance mutations at failure. In multivariate analysis, factors associated with the emergence of at least one integrase mutation were: high VL at failure (OR = 1.24 per 1 log10 copies/mL increase); non-B versus B subtype (OR = 1.75); low genotypic sensitivity score (GSS) (OR = 0.10 for GSS = 2 versus GSS = 0–0.5); and dolutegravir versus raltegravir (OR = 0.46). Although 3DRs versus 2DRs reached statistical significance in univariate analysis (OR = 0.59, P = 0.007), the variable is not retained in the final model. Conclusions This study is one of the largest studies characterizing integrase resistance in patients failing any INSTI-containing 2DR or 3DR in routine clinical care and reveals factors associated with emergence of integrase resistance that should be taken into consideration in clinical management. No difference was evidenced between patients receiving a 2DR or a 3DR.

Background Measles remains endemic worldwide, despite current vaccination recommendations, and is associated with high morbidity and mortality rates. We describe all cases hospitalized in Bordeaux University Hospital (BUH), the starting point of a national significant measles outbreak in 2017–2018. Methods In this retrospective study, we included all patients hospitalized in BUH from September 1, 2017, to May 31, 2018. Inclusion criteria were age >1 year, clinical symptoms, and biological confirmation by measles immunoglobulin M or measles reverse transcription polymerase chain reaction positivity. Results We included 171 patients. Most patients were immunocompetent; only 19% had preexisting medical histories. Most patients had rash and fever (97%), but some cases were atypical and difficult to diagnose. Köplik’s spots were reported in 66 cases (38%). The most frequent biological markers were blood inflammation markers (96%) and lymphopenia (81%). Unexpectedly, we found hyponatremia ( Conclusions During the BUH outbreak, measles was often complicated and sometimes atypical. Vaccination coverage was dramatically insufficient. We also describe vaccination failure cases that must be better investigated.

Prolonged measles virus detection in maternal saliva and blood was evidenced in 6 pregnant women. Maternal-fetal transmission was evidenced in 2 of 4 infants who were asymptomatic at birth, 21–24 weeks after maternal infection. Whereas peripartum congenital measles is severe, asymptomatic measles virus vertical transmission can occur earlier in pregnancy.

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.

International audience; Objectives: To describe integrase strand transfer inhibitor (INSTI) resistance profiles and factors associated with resistance in antiretroviral-naive and -experienced patients failing an INSTI-based regimen in clinical practice.Methods: Data were collected from patients failing an INSTI-containing regimen in a multicentre French study between 2014 and 2017. Failure was defined as two consecutive plasma viral loads (VL) >50 copies/mL. Reverse transcriptase, protease and integrase coding regions were sequenced at baseline and failure. INSTI resistance-associated mutations (RAMs) included in the Agence Nationale de Recherches sur le SIDA genotypic algorithm were investigated.Results: Among the 674 patients, 359 were failing on raltegravir, 154 on elvitegravir and 161 on dolutegravir therapy. Overall, 90% were experienced patients and 389 (58%) patients showed no INSTI RAMs at failure. The strongest factors associated with emergence of at least one INSTI mutation were high VL at failure (OR = 1.2 per 1 log10 copies/mL increase) and low genotypic sensitivity score (GSS) (OR = 0.08 for GSS ≥3 versus GSS = 0-0.5). Patients failing dolutegravir also had significantly fewer INSTI RAMs at failure than patients failing raltegravir (OR = 0.57, P = 0.02) or elvitegravir (OR = 0.45, P = 0.005). Among the 68 patients failing a first-line regimen, 11/41 (27%) patients on raltegravir, 7/18 (39%) on elvitegravir and 0/9 on dolutegravir had viruses with emergent INSTI RAMs at failure.Conclusions: These results confirmed the robustness of dolutegravir regarding resistance selection in integrase in the case of virological failure in routine clinical care.

On 30 October 2017, an outbreak of measles started in the Nouvelle-Aquitaine (NA) region in France among Bordeaux University students before spreading to other regions. Until 1 July 2018, 1,101 cases were reported in NA, including 98 complications and two deaths. Cases were related to clusters (e.g. students, healthcare workers) in 16%; 81% of cases were not vaccinated against measles as recommended. Vaccination coverage above herd immunity threshold remains the main preventative outbreak measure.

International audience; ObjectivesTo assess the phenotypic susceptibility of the E157Q polymorphism in HIV-1 integrase (IN) and the virological outcome of patients infected with E157Q-mutated virus initiating an IN inhibitor (INI)-based regimen.MethodsThis was a multicentre study assessing IN sequences from INI-naive patients among 17 French HIV clinical centres. E157Q site-directed mutants in pNL4.3 and pCRF02_AG contexts were assessed in a recombinant phenotypic assay.ResultsPrevalence of the E157Q polymorphism was 2.7% among 8528 IN sequences from INI-naive patients and its distribution was 1.7%, 5.6% and 2.2% in B, CRF02_AG and various non-B subtypes, respectively. Thirty-nine INI-naive patients with E157Q-mutated virus initiated an INI-based regimen. Among them, 15 had a viral load (VL) 50 copies/mL at the time of INI-based regimen initiation. Among them eight were receiving a first-line regimen and the only two patients who did not reach VL

Objectives There is no consensus about the performances of genotypic rules for predicting HIV-1 non-B subtype tropism. Three genotypic methods were compared for CRF01_AE HIV-1 tropism determination. Methods The V3 env region of 207 HIV-1 CRF01_AE and 178 B subtypes from 17 centers in France and 1 center in Switzerland was sequenced. Tropism was determined by Geno2Pheno algorithm with false positive rate (FPR) 5% or 10%, the 11/25 rule or the combined criteria of the 11/25, net charge rule and NXT/S mutations. Results Overall, 72.5%, 59.4%, 86.0%, 90.8% of the 207 HIV-1 CRF01_AE were R5-tropic viruses determined by Geno2pheno FPR5%, Geno2pheno FPR10%, the combined criteria and the 11/25 rule, respectively. A concordance of 82.6% was observed between Geno2pheno FPR5% and the combined criteria for CRF01_AE. The results were nearly similar for the comparison between Geno2pheno FPR5% and the 11/25 rule. More mismatches were observed when Geno2pheno was used with the FPR10%. Neither HIV viral load, nor current or nadir CD4 was associated with the discordance rate between the different algorithms. Conclusion Geno2pheno predicted more X4-tropic viruses for this set of CRF01_AE sequences than the combined criteria or the 11/25 rule alone. For a conservative approach, Geno2pheno FPR5% seems to be a good compromise to predict CRF01_AE tropism.

International audience; Background: Human respiratory syncytial virus (HRSV) is the main viral cause of severe lower respiratory tract disease in infants and young children. The aim of this study was to describe for the first time the genetic variability of HRSV in Cameroonian patients living in Yaounde for three consecutive epidemic seasons.Methods: HRSV-positive nasopharyngeal samples detected in children less than 15 years in Yaounde were collected from September 2011 to December 2013. Semi-nested RT-PCR, sequencing, and phylogenetic analyses of the second hypervariable region of the G gene were performed.Results: A total of 57 HRSV-positive samples were collected during the study period. Among these, 46 (80.7%) could be amplified in the G gene. HRSV group A (HRSV-A) and group B (HRSV-B) co-circulated in this population at 17.4 and 82.6%, respectively. HRSV-A strains clustered in the NA-1 genotype while HRSV-B strains clustered in the BA-9 genotype. HRSV-A strains accounted for 33.3% (2/6), 4.3% (1/23), and 29.4% (5/17) of the viruses isolated in 2011, 2012, and 2013, respectively.Conclusions: This study reports molecular epidemiology data of HRSV in Cameroon for the first time. Additional studies are required to clarify evolutionary patterns of HRSV throughout sub-Saharan Africa to support antiviral and vaccine development.

International audience; `Objectives: To analyse a measles outbreak in a Roma community. Methods: We describe a community-wide outbreak of genotype D8 measles that took place in southeastern France, between May and July 2017, along with the control measures adopted. Results: We identified a total of eighteen cases, between six months and 24 years old. All cases were unvaccinated or incompletely vaccinated and belonged to a sedentary French Roma community. Most of them (67%) were hospitalised, with three cases (17%) of severe measles including one death of a 16-year-old girl who had previously received oral corticosteroids. The latter was the only lethal case notified in France during theyear 2017. Control measures included intensification of surveillance, isolation of cases, and a large vaccination campaignin this Romacommunity. During the outbreak period, there was no caseof healthcareassociated measles transmission. A broad adherence to vaccination through the mediating role of both the chief of the community and the pastor allowed reaching completed vaccination coverage of 90%. Conclusions: Efforts should be concentrated to enhance access to health services for minorities such as the Roma community characterized by low vaccination coverage. A trustful relationship with leaders of the community is essential to ensure adherence to vaccination. In France, attributable mortality to measles is low and concerns mainly unvaccinated and immunodepressed patients. (c) 2018 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

Background: Viral reservoirs represent an important barrier to HIV cure. Accurate markers of HIV reservoirs are needed to develop multicenter studies. The aim of this multicenter quality control (QC) was to evaluate the inter-laboratory reproducibility of total HIV-1-DNA quantification. Methods: Ten laboratories of the ANRS-AC11 working group participated by quantifying HIV-DNA with a real-time qPCR assay (Biocentric) in 4 samples (QCMD). Results: Good reproducibility was found between laboratories (standard deviation ≤ 0.2 log10 copies/106PBMC) for the three positive QC that were correctly classified by each laboratory (QC1

Measles outbreaks are regularly reported in European countries despite efforts to improve vaccination coverage. In January 2016, an outbreak occurred in a refugee settlement in Calais, France. We report here the complete genome sequence of a wild-type measles virus isolated from a health care worker (MVi/Calais. FRA/01.16) infected during this outbreak.

Viral respiratory infections are common in children, most of which are due to a virus. They can lead to serious infections, and these children may require treatment in a pediatric intensive care unit (PICU). This prospective study evaluated the epidemiology of respiratory viruses and associated illnesses among children hospitalized in a PICU during the three winter months of 2012-2013.All the children admitted to the PICU, University Hospital of Caen, France, were included. Nasal swabs were collected and specimens were tested using a single real-time PCR (polymerase chain reaction).Of the 105 patients admitted to the PICU during the study period, 84 fulfilled the inclusion criteria. The "respiratory group" included 37 patients with respiratory symptoms at admission while the "nonrespiratory group" included 47 patients with no respiratory symptoms. The 84 nasal swabs collected included 54 that were considered positive (64.3%) and 70 viruses were detected. The most commonly detected virus was RSV (n=28; 40.0% positive samples), followed by HRV (n=24; 34.3%). Viruses were more frequently detected in the respiratory (86.5%) than in the nonrespiratory (42.6%) group (P0.001). Statistical analysis by subgroups revealed that RSV infections were significantly more frequent in the respiratory group (54.1%) than in the nonrespiratory group (6.4%) (P0.001). There was no difference for HRV (32.4% and 27.7%) or for the other viruses. No difference in duration of hospitalization or duration of mechanical ventilation was demonstrated depending on the virus detected.The use of the very sensitive multiplex PCR technique increased virus detection rates in both symptomatic and asymptomatic subjects.We have confirmed the frequency of RSV infections in a PICU and found that many patients without respiratory symptoms have respiratory infections caused by viruses. The impact of these infections on patient outcome should now be analyzed in order to demonstrate the role played by respiratory viruses.

The objective of this study is primarily to compare the performance of the VIDAS(®) Measles immunoglobulin (Ig)G assay to that of two other serological assays using an immunoassay technique, Enzygnost(®) Anti-measles Virus/IgG (Siemens) and Measles IgG CAPTURE EIA(®) (Microimmune). The sensitivity and the agreement of the VIDAS(®) Measles IgG assay compared to the Enzygnost(®) Anti-measles Virus/IgG assay and the Measles IgG CAPTURE EIA(®) assay are 100%, 97.2% and 99.0%, 98.4%, respectively. The very low number of negative sera for IgG antibodies does not allow calculation of specificity. As a secondary objective, we have evaluated the ability of the VIDAS(®) Measles IgG assay to measure anti-measles virus IgG antibody avidity with the help of the VIDAS(®) CMV IgG Avidity reagent, using 76 sera from subjects with measles and 238 other sera. Different groups of populations were analyzed. In the primary infection measles group, the mean IgG avidity index was 0.16 (range of 0.07 to 0.93) compared to 0.79 (range of 0.25 to 1) in the serum group positive for IgG antibodies and negative for IgM. These data allow to define a weak anti-measles virus IgG antibody avidity as an avidity index (AI)0.3 and a strong avidity as an AI0.6. The VIDAS(®) Measles IgG assay has a performance equivalent to that of other available products. Its use, individual and quick, is well adapted to testing for anti-measles immunity in exposed subjects.

Background Respiratory viral diagnosis of upper respiratory tract infections has largely developed through multiplex molecular techniques. Although the sensitivity of different types of upper respiratory tract samples seems to be correlated to the number of sampled cells, this link remains largely unexplored. Methods Our study included 800 upper respiratory tract specimens of which 400 negative and 400 positive for viral detection in multiplex PCR. All samples were selected and matched for age in these 2 groups. For the positive group, samples were selected for the detected viral species. Results Among the factors influencing the cellularity were the type of sample (p

Resume Alors que la place de la PCR est bien reconnue pour le diagnostic des infections neuromeningees a enterovirus (EV), l’utilisation des techniques de biologie moleculaire dans les infections respiratoires a EV est encore mal definie. Objectifs Definir les apports des outils moleculaires (PCR et genotypage) dans le diagnostic et l’epidemiologie des infections respiratoires a EV. Methodes Au cours de l’annee 2008, 3612 secretions nasales ont eu une recherche virale par IFD et culture MRC5. Dans un second temps, sont realises une recherche virale sur culture HuH7 (SN negative en IF) et une PCR duplex enterorhinovirus (PEVRV) sur 816 surnageants de culture HuH7 presentant un ECP. De plus, 327 SN de nouveau-nes ont eu une recherche systematique par la PCR EV temps reel « maison » (PEV) utilisee pour le diagnostic des infections meningees. Un genotypage a ete realise chez 68 patients presentant une identification d’EV sur surnageants de cultures MRC5 ou HuH7. Resultats Trente-cinq EV ont ete isoles en culture (0,97 %) ; la combinaison des deux PCR, PEVRV et PEV, a permis d’identifier respectivement, 41 EV, 8 EV-RV et 12 RV supplementaires, portant le nombre de positif a 96 (2,6 %). Chez les NN, 32 (11,3 %) SN sont positives avec la PEV. Parmi les 98 SN testees par PEV et PEVRV, 27 etaient positives et nous avons detecte dix EV, cinq EV-RV et 12 RV. De janvier a decembre 2008, la distribution saisonniere montre le pic estival habituel d’infections meningees et un pic automnal correspondant d’infections respiratoires. Cinq serotypes predominent : 19 EV68 (29,7 %), 12 CB3 (18,7 %), neuf E3 (14,1 %), six CA9 (9,4 %) et six CB1 (9,4 %). Les EV68 sont tous isoles en octobre a novembre et 17/19 (89,5 %) des patients positifs ont ete hospitalises avec des signes d’insuffisance respiratoire aigue. Conclusion Les methodes de diagnostic moleculaires des infections respiratoires a EV (PCR et genotypage) permettent d’identifier precisement les souches circulantes dans un temps relativement court, apportant un benefice epidemiologique, clinique et economique de sante.

Resume En Normandie, le virus respiratoire syncytial (hRSV) est trouve dans 64,1 % des cas de bronchiolites aigues chez l’enfant hospitalise, les rhinovirus dans 26,8 %, le metapneumovirus humain (hMPV) dans 7,6 % et les virus para-influenza (PIV) dans 3,4 %. Dans les bronchiolites aigues communautaires, on a une repartition differente : hRSV : 42 %, rhinovirus : 19,5 %, coronavirus : 8 %, PIV : 3,5 %, hMPV : 2,5 %. Le hRSV infecte les nourrissons (86,9 %), dont 53,7 % ont moins de 6 mois et 48,2 % des hRSV sont detectes en novembre et decembre, et 44,5 % en janvier et fevrier. L’epidemie debute la 1re ou 2e semaine d’octobre, mais elle varie d’une annee a l’autre et d’une region a l’autre. A l’hopital, les bronchiolites a hRSV sont passees de 261 cas par epidemies de 1999 a 2003, a 341 cas de 2004 a 2009. Les rhinovirus sont associes a des bronchiolites aigues dans 38,4 % des cas ; 54,6 % des souches etant identifiees en septembre et octobre, et 38,5 % en mars et avril ; 34,2 % infectent des nourrissons de moins de 6 mois, 37,8 % entre 6 mois et 2 ans, et 19,5 % des enfants entre 2 et 5 ans. Les epidemies a hMPV coincident avec celles du hRSV mais ont 6 fois moins de cas hospitalises. Le hMPV infecte les nourrissons (74 %) mais plus âges que ceux infectes par le hRSV. Il entraine une bronchiolite aigue dans 59 % des cas, une pneumonie dans 17 %. Les infections a PIV (environ 100 cas hospitalises par an) se repartissent en PIV3 : 62,7 %, PIV1 : 25,3 % et PIV2 : 7,3 %. Les PIV1 apparaissent tous les 2 ans, a l’automne. Les infections a PIV3 sont automnales et hivernales, avec des infections au printemps les annees sans PIV1. Il y a des bronchiolites aigues dans 29,8 % des infections a PIV3 et 18,3 % a PIV1.

Resume La grippe saisonniere est due aux virus influenza A et B. Il s’agit de virus enveloppes dont le genome est constitue de sept a huit fragments d’ARN. Les differents sous-types sont determines par la nature des deux glycoproteines de surface HA et NA. La grippe saisonniere est une maladie epidemique et hivernale dans les zones a climat tempere. Son epidemiologie est liee a la grande variabilite du virus au cours du temps, necessitant la mise en place d’un systeme d’alerte detectant chaque annee les variants circulants dominant et determinant la composition vaccinale. Les symptomes cliniques de la grippe ne sont pas suffisamment specifiques pour permettre le diagnostic sans examen virologique. Cela est particulierement vrai en periode non epidemique, chez les sujets de plus de 65 ans et chez les enfants de moins de cinq ans. L’enfant represente une cible privilegiee des infections a virus influenza. Le recours a l’hospitalisation est d’autant plus eleve que l’enfant est jeune. Chez le nourrisson, l’infection peut etre paucisymptomatique et s’accompagner de manifestations non respiratoires (lethargie, convulsions, malaises). Le diagnostic virologique de la grippe est justifie chez tous les sujets hospitalises pour un syndrome respiratoire compatible avec une infection a virus influenza. Il existe plusieurs outils permettant une recherche directe du virus dans les secretions respiratoires : isolement du virus en culture, detection d’antigenes, detection moleculaire de l’ARN. Le choix de la methode se fait selon les caracteristiques du test : sensibilite, specificite, rapidite et simplicite de realisation, cout.

A method was developed for the detection and quantitation of HAdV (human adenovirus) and HBoV (human bocavirus) based on a duplex real-time PCR, the AB PCR, using a Smartcycler instrument. A control real-time PCR was carried out on albumin DNA to standardise the non-homogenous respiratory samples. No cross-reactivity was observed with viruses or bacteria that could be found in the respiratory tract. The diagnosis rate using the AB PCR on clinical samples was 10.7%: 3.4% for HBoV detection, 6.9% for HAdV detection and 0.3% double detection HBoV–HAdV. The clinical and epidemiological characteristics of the HAdV- and HBoV-infected patients were evaluated. In the HAdV-positive group and the HBoV-positive group the samples were classified according to the severity of the disease. The HAdV viral load did not appear to be linked to the severity of the disease. Conversely, the difference between the two HBoV groups, severe and non-severe, was significant statistically when the comparison was based on the viral load (P = 0.006) or after adjustment of the viral load to the number of cells in the samples (P = 0.02).

Resume Les coronavirus forment un grand groupe de virus infectant les mammiferes et les oiseaux ; cinq d’entre eux infectent l’homme : HCoV 229E et OC43, connus depuis les annees 1960, SARS-CoV identifie en mars 2003 lors de l’epidemie de syndrome de detresse respiratoire aigu severe, NL63 et HKU1, identifies respectivement en 2004 et 2005. Ce sont des virus enveloppes dont le genome est une molecule d’ARN de polarite positive de tres grande taille. Leur mode evolutif fait intervenir plusieurs parametres : la generation de nombreux mutants lors de la replication, responsable d’une distribution en quasi-especes de la population virale, la capacite a etablir des infections persistantes, la possibilite de deletions importantes, la grande flexibilite du genome due a un fort taux de recombinaisons homologues et heterologues, la capacite a franchir les barrieres d’especes et a s’adapter au nouvel environnement. Les coronavirus (hors SARS-CoV) sont ubiquitaires et circulent sous forme epidemique. Les differents HCoV co-circulent avec une distribution entre les types qui est variable selon les annees et les regions geographiques. Ce sont essentiellement des virus responsables d’infections respiratoires hautes et basses. Le SARS-CoV est, lui, un virus emergent responsable de l’epidemie de pneumopathies atypiques entre novembre 2002 et juillet 2003. Sa circulation a ete interrompue grâce a la mise en place de mesures sanitaires drastiques. L’implication des HCoV dans des pathologies digestives et neurologiques reste a preciser. La detection des coronavirus est difficile, et fait appel surtout a des techniques moleculaires. Il n’existe actuellement aucun traitement specifique des infections a HCoV.

Aim: This study has two objectives: to study the clinical symptoms associated with the detection of the four human coronaviruses (HCoVs), 229E, OC43, NL63 and HKU1 types, in the respiratory specimens sampled from hospitalised children in France between September 2004 and May 2005; and to develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay allowing for the simultaneous detection of the four HCoVs. Methods: 1002 respiratory specimens were tested for HCoVs. The clinical and epidemiological data were compared on the basis of the type HCoV infection. Results: A hundred coronaviruses, 33 NL63, 2229E, 27 OC43 and 38 HKU1, were detected in 97 (9.8%) of 1002 samples negative in routine tests. The clinical and epidemiological characteristics of the study children were compared in three groups, 24 OC43-, 27 NL63- and 34 HKU1-infected children. HCoVs were identified mainly in children with upper and lower respiratory tract infections (50.5% vs. 29.4%). The significant difference in clinical presentation between the three coronavirus groups was the very low association between lower respiratory tract illness and HKU1 detection. Conclusions: HCoV detection in hospitalised children without any other respiratory virus detection was associated with upper and a significant rate of lower respiratory tract illness. The four types of HCoVs were detected, and new types NL63 and HKU1 represented a substantial portion of detection. The multiplex RT-PCR enabled a sensitive one-time detection and the characterisation of all of the known HCoV types with the exception severe acute respiratory syndrome-coronavirus.

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 (

The nucleocapsid (N) gene of human coronavirus strain OC43 (HCoV-OC43) was amplified by reverse transcriptase-polymerase chain reaction, and cloned in pENTR™/D-TOPO® plasmid. This plasmid containing the N gene was recombined with in a BaculoDirect™ baculovirus DNA designed in order to express N protein in fusion with a C-terminal polyhistidine tag containing V5 epitope. Sf21 cells were transfected with recombinant baculovirus DNA. Recombinant N protein was extracted from infected cells, analysed by SDS-PAGE and Western blot, and purified by Ni2+ affinity procedure. Sera from 100 healthcare workers and five 2–3-year-old children were tested in a Western blot assay using the purified recombinant N protein. All of the sera from adults and two of the sera from children have a positive result.

Resume But de l'etude L'objectif de cette etude est de decrire l'epidemiologie des infections a Mycoplasma pneumoniae (Mpn) en Basse-Normandie par une analyse retrospective des resultats serologiques et PCR, et de valider ces deux outils pour un diagnostic precoce de ces infections. Patients et methodes De 1997 a aout 2005, 6156 serums et 6123 prelevements respiratoires ont ete realises chez des patients hospitalises et analyses pour une recherche d'infection a Mpn, soit par PCR, soit par serologie Elisa, soit par les deux. Pendant la periode epidemique (2004–2005), les resultats de 1489 patients ont ete analyses. Resultats Sur les neuf annees, la seroprevalence moyenne se chiffre a 40,4 % et 525 cas d'infection ont ete diagnostiques par PCR et/ou serologie, se profilant nettement sous l'aspect de deux pics epidemiques espaces de sept ans. Pendant la periode epidemique, la seroprevalence augmente (50,2 %) et l'incidence est de 8,3 %. L'analyse des caracteristiques epidemiologiques des 124 cas diagnostiques montre : une predominance chez l'enfant et l'adulte jeune, une frequence saisonniere estivohivernale, l'existence de co-infection virale. L'analyse des resultats chez 36 patients ayant beneficie des deux tests diagnostiques, PCR et serologie, montre une concordance dans seulement 41,7 % des cas. Conclusion Les infections a Mpn surviennent sur un mode endemo-epidemique, avec une incidence elevee lors des pics epidemiques, particulierement chez l'enfant. Nous disposons actuellement d'outils bacteriologiques performants permettant de realiser un diagnostic precoce et fiable. Chez l'enfant, il convient d'associer une serologie (IgM) et une PCR sur un prelevement nasopharynge ; chez l'adulte il faudra privilegier la PCR.

Coronavirus NL63 was found in hospitalized children with upper and lower respiratory infections., The human coronavirus NL63 (HCoV-NL63) was first identified in the Netherlands, and its circulation in France has not been investigated. We studied HCoV-NL63 infection in hospitalized children diagnosed with respiratory tract infections. From November 2002 to April 2003, we evaluated 300 respiratory specimens for HCoV-NL63. Of the 300 samples, 28 (9.3%) were positive for HCoV-NL63. The highest prevalence was found in February (18%). The main symptoms were fever (61%), rhinitis (39%), bronchiolitis (39%), digestive problems (33%), otitis (28%), pharyngitis (22%), and conjunctivitis (17%). A fragment of the spike protein gene was sequenced to determine the variety of circulating HCoV-NL63. Phylogenetic analysis indicated that strains with different genetic markers cocirculate in France.

International audience; Equine coronavirus (ECoV) is involved mainly in enteric infections. Following the recent description of ECoV in 2000, this study reports for the first time the presence of ECoV in France and, on a wider scale, in Europe. ECoV was molecularly detected from diarrheic and respiratory specimens. Sequencing and phylogenetic analyses demonstrated that European strains are most closely related to the reference North American strain (ECoV-NC99) than the Asian strain (ECoV-Tokachi09).

We compared the coreceptor tropism-predicting performance of a specific genotypic algorithm for HIV-1 subtype D and that of the geno2pheno algorithm with different cutoffs. The D-specific algorithm and geno2pheno with a false-positivity rate cutoff of 2.5% had the same concordance with the phenotypic determination. The geno2pheno algorithm with a false-positivity rate cutoff of 2.5%, more sensitive but slightly less specific, seems to be an appropriate alternative.

The molecular diagnosis of respiratory infection can be performed using different commercial multiplex-based PCR kits whose performances have been previously compared individually to those of conventional techniques. This study compared the practicability and the diagnostic performances of six CE-marked kits available in 2011 on the French market, including 2 detecting viruses and atypical bacteria (from Pathofinder and Seegene companies) and 4 detecting only viruses (from Abbott, Genomica, Qiagen and Seegene companies). The respective sensitivity, specificity, accuracy and agreement of each multiplex technique were calculated by comparison to commercial duplex PCR tests (Argene/bioMerieux) used as gold standard. Eighty-eight respiratory specimens with no pathogen (n = 11), single infections (n = 33) or co-infections (n = 44) were selected to cover 9 viruses or groups of viruses and 3 atypical bacteria. All samples were extracted using the NUCLISENS® easyMAG™ instrument (bioMerieux). The overall sensitivity ranged from 56.25% to 91.67% for viruses and was below 50% with both tests for bacteria. The overall specificity was excellent (>94% for all pathogens). For each tested kit, the overall agreement with the reference test was strong for viruses (kappa test >0.60) and moderate for bacteria. After the extraction step, the hands-on time varied from 50 min to 2h30 and the complete results were available in 2h30 to 9 h. The spectrum of tested agents and the technology used to reveal the PCR products as well as the laboratory organization are determinant for the selection of a kit.

Human metapneumovirus (hMPV) is responsible for respiratory tract disease, particularly in the young and elderly population. An epidemiological and phylogenic study was performed on children admitted to hospital with an acute lower respiratory tract infection (LRI). Data were obtained and analyzed over three consecutive winters, from 2002–2003 to 2004–2005. Each year during the winter period, from November to March, 2,415 nasal swabs were tested by a direct immunofluorescence assay (DFA) for influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses, and adenoviruses. Rhinoviruses, enteroviruses, and coronaviruses OC43 and 229E were detected by RT‐PCR. A RT‐PCR designed for the M gene was performed on negative samples for hMPV detection and phylogenic analyses. For the three consecutive winters, hMPV represented 10%, 22.6%, and 8.8% of virus‐negative samples, respectively. In most cases, clinical symptoms indicated a LRI with a final diagnosis of bronchiolitis. During the winter of 2003–2004, all viral clusters (A1, A2, B1, and B2) that circulated in France shifted progressively from the A group to the B group. This study determined the prevalence of hMPV in Normandy, its clinical impact and permitted the analysis of the molecular evolution during the successive outbreaks. J. Med. Virol. 83:517–524, 2011. © 2011 Wiley‐Liss, Inc.

Human coronavirus OC43 (HCoV-OC43) causes acute, self-limited respiratory infections. A close relationship between bovine coronaviruses (BCoVs) and HCoV-OC43 has recently been demonstrated. This study includes seven clinical, non-cell culture-adapted, contemporary HCoV-OC43 strains detected in France in 2003. By using RT-PCR and clonal sequencing of the S1 gene of HCoV-OC43, the inter-variant heterogeneity of the HCoV-OC43 circulating strains was studied and the intra-variant diversity was assessed by investigation of a quasispecies cloud. This paper brings to the forefront a high genetic diversity of circulating HCoV-OC43 variants. Genetically different groups are defined among the variants described in this study. One of these variants holds characteristics of an outlier and presents a deletion of 12 nt, also found in BCoV strains. Moreover, the presence of HCoV-OC43 as a quasispecies cloud in vivo during an acute respiratory-tract illness was discovered. It has also been revealed that quasispecies-cloud sizes are similar for the two viral populations tested.

The performances of four multiplex PCR (m‐PCR) were compared to direct immunofluorescence assay (DFA) and HuH7 cell culture for the detection of viruses in 263 children admitted to hospital with an acute respiratory illness. One hundred fifty (57.6%) nasal aspirates were found DFA‐positive; 188 (72.3%) were found positive by both DFA and HuH7 cell culture, and 242 (92%) were PCR‐positive. The m‐PCR detected 124 viruses which were not found by conventional methods: 68 rhinovirus, 17 human metapneumovirus, 15 respiratory syncytial virus (RSV), 8 parainfluenza virus (PIV), 5 coronavirus 229E, 3 OC43 and 3 NL63, 4 enterovirus, 2 influenza virus B and C virus. The m‐PCR were more sensitive, had the advantages of a shorter delay in specific diagnosis, and a lower cost than DFA and culture. Using these m‐PCR, the prevalence of each virus was compared between in‐patient and out‐patient groups of children attending the emergency unit of the hospital. Nasal aspirates from 411 (91.5%) children were found positive by the PCRs. RSV, rhinovirus, and influenza virus were the most frequent viruses detected in this population, representing 43.6%, 31.8%, and 8.8% of the virus found, respectively, followed by human metapneumovirus (4.4%), coronavirus (3.4%), parainfluenza virus (3.2%), adenovirus (2.3%), and enterovirus (2.1%). RSVs were detected more significantly in the in‐patient group than in the out‐patient group, and influenza viruses were detected more frequently in the out‐patient group than in the in‐patient group. Moreover, the use of m‐PCR pointed out the frequency of rhinovirus and mixed viral detections in these patients. In conclusion, according to the requirements of speed and low cost of the methods, and to achieve the highest rate of detection of respiratory viruses, the combined use of DFA and m‐PCR is today likely to be the best way to improve diagnosis of respiratory illnesses in children. J. Med. Virol. 78:1498–1504, 2006. © 2006 Wiley‐Liss, Inc.

Background. Human coronavirus HKU1 (HCoV-HKU1), a new group 2 coronavirus, was first characterized in 2005 from 2 adults with pneumonia in Hong Kong, China. To the best of our knowledge, there is no other report to date about the detection of this new virus. We report a molecular method allowing for the detection of HCoV-HKU1 and also report the clinical presentation of 6 infected patients. Methods. We screened 141 specimens (135 nasal samples and 6 stool samples) received in February and March 2005 in our laboratory and obtained from 135 hospitalized patients (61.5% of whom were 20 years old) for HCoV-HKU1. Results. HCoV-HKU1 was detected in 6 (4.4%) of the 135 nasal specimens and in 2 (33.3%) of the 6 stool samples; the positive samples were obtained from 6 patients (5 children and 1 adult). The clinical presentation of these 6 patients was as follows: 3 were admitted to the hospital for acute enteric disease resulting in severe dehydration associated with upper respiratory symptoms; 1 had fever, otitis, and febrile seizure; 1 had a sample obtained to investigate failure to thrive; and 1 had a sample obtained for exploration of X-linked agammaglobulinemia and hyperleucocytosis. Conclusion. HCoV-HKU1 can be detected in respiratory and stool samples from children and adults in a part of the world other than Hong Kong. Our results suggest that HCoV-HKU1 could be associated with respiratory and enteric diseases, and its detection can be related to a persistent asymptomatic infection in patients with poor underlying conditions.

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.

The lsa gene confers intrinsic resistance to lincosamides and streptogramins A in Enterococcus faecalis , probably by active efflux. The lsa -like genes of two clinical isolates of E. faecalis susceptible to lincosamides and dalfopristin contained mutations that produced premature termination codons. Revertant mutants were obtained by selection on agar plates containing clindamycin.