Jean-Sébastien Casalegno

Rhinoviruses delayed the circulation of the pandemic influenza A (H1N1) 2009 virus in France

In contrast to the experience in other European countries, the onset of the A(H1N1)2009 influenza virus epidemic was unexpectedly slow in France during the first part of autumn 2009. Our objective was to test the hypothesis that intense circulation of rhinoviruses might have reduced the probability of infection by A(H1N1)2009 virus at the beginning of autumn 2009. Systematic analysis for the detection of A(H1N1)2009 (H1N1) and human rhinovirus (HRV) was performed by RT-PCR from week 36 to week 48 on respiratory samples sent to the diagnostic laboratory by the paediatric hospital (n = 2121). Retrospective analysis of the obtained data, using 2 x 2 contingency tables with Fishers exact test, revealed evidence of an inverse relationship between HRV and H1N1 detection. Between weeks 36 and 48 of 2009, both HRV and H1N1 were detected but in different time frames. HRV dispersed widely during early September, peaking at the end of the month, whereas the H1N1 epidemic began during mid-October and was still active at the end of this survey. During the co-circulation period of these two respiratory viruses (weeks 43-46), HRV detection appeared to reduce the likelihood of H1N1 detection in the same sample (OR = 0.08-0.24 p <0.0001). These results support the hypothesis that HRV infections can reduce the probability of A(H1N1) infection. This viral interference between respiratory viruses could have affected the spread of the H1N1 viruses and delayed the influenza pandemic at the beginning of autumn in France.

Pandemic A(H1N1)2009 influenza virus detection by real time RT-PCR : is viral quantification useful?

The emergence of the influenza A(H1N1) 2009 virus prompted the development of sensitive RT-PCR detection methods. Most are real time RT-PCRs which can provide viral quantification. In this manuscript, we describe a universal influenza A RT-PCR targeting the matrix (M) gene, combined with an RNaseP RT-PCR. These PCRs allow the detection of all influenza A virus subtypes, including A(H1N1)2009, together with a real-time assessment of the quality of the specimens tested. These PCR procedures were evaluated on 209 samples collected from paediatric patients. Viral loads determined through Ct values were corrected according to the RNaseP Ct value. The mean viral load in the collected samples was estimated to be 6.84 log RNA copies/mL. For poor quality samples (RNaseP Ct > 27), corrections resulted in +3 to +8 Ct values for the M gene RT-PCR. Corrected influenza Ct values were lower in late samples. No correlation was established between viral loads and clinical severity or duration of disease.This study shows that real time RT-PCR targeting the matrix gene is a reliable tool for quantification of type A influenza virus but emphasises the need for sample quality control assessment through cellular gene quantification for reliable estimation of the viral load. This method would be useful for disease management when repeated specimens are collected from an infected individual.

Resistance of herpes simplex viruses to acyclovir: an update from a ten-year survey in France.

The widespread use of acyclovir (ACV) and the increasing number of immunocompromised patients have raised concern about an increase in ACV-resistant herpes simplex virus (HSV). ACV resistance has traditionally been a major concern for immunocompromised patients with a frequency reported between 2.5% and 10%. The aim of this study was to reassess the status of HSV resistance to ACV in immunocompetent and immunocompromised patients over a ten year period, between 2002 and 2011. This was done by retrospectively following 1425 patients. In immunocompetent patients, prevalence of resistance did not exceed 0.5% during the study period; whereas in immunocompromised patients, a significant increase was observed, rising from 3.8% between 2002 and 2006 (7/182 patients) to 15.7% between 2007 and 2011 (28/178) (p=0.0001). This sharp rise in resistance may largely be represented by allogeneic hematopoietic stem cell transplant patients, in which the prevalence of ACV resistance rose similarly from 14.3% (4/28) between 2002 and 2006 to 46.5% (26/56) between 2007 and 2011 (p=0.005). No increase in ACV resistance was detected in association with other types of immune deficiencies. Genotypic characterization of HSV UL23 thymidine kinase and UL30 DNA polymerase genes revealed 11 and 7 previously unreported substitutions, respectively. These substitutions may be related to potential polymorphisms, drug resistance, or other mutations of unclear significance.

High Risk HPV Contamination of Endocavity Vaginal Ultrasound Probes: An Underestimated Route of Nosocomial Infection?

Background Endocavity ultrasound is seen as a harmless procedure and has become a common gynaecological procedure. However without correct disinfection, it may result in nosocomial transmission of genito-urinary pathogens, such as high-risk Human Papillomavirus (HR-HPV). We aimed to evaluate the currently recommended disinfection procedure for covered endocavity ultrasound probes, which consists of “Low Level Disinfection” (LLD) with “quaternary ammonium compounds” containing wipes. Methods From May to October 2011 swabs were taken from endovaginal ultrasound probes at the Gynecology Department of the Lyon University Hospital. During the first phase (May–June 2011) samples were taken after the ultrasound examination and after the LLD procedure. In a second phase (July–October 2011) swab samples were collected just before the probe was used. All samples were tested for the presence of human DNA (as a marker for a possible transmission of infectious pathogens from the genital tract) and HPV DNA with the Genomica DNA microarray (35 different HPV genotypes). Results We collected 217 samples before and 200 samples after the ultrasound examination. The PCR was inhibited in two cases. Human DNA was detected in 36 (18%) post-examination samples and 61 (28%) pre-examination samples. After the ultrasound LLD procedure, 6 (3.0%) samples contained HR-HPV types (16, 31, 2×53 and 58). Similarly, HPV was detected in 6 pre-examination samples (2.7%). Amongst these 4 (1.9%) contained HR-HPV (types 53 and 70). Conclusion Our study reveals that a considerable number of ultrasound probes are contaminated with human and HR-HPV DNA, despite LLD disinfection and probe cover. In all hospitals, where LLD is performed, the endovaginal ultrasound procedure must therefore be considered a source for nosocomial HR-HPV infections. We recommend the stringent use of high-level disinfectants, such as glutaraldehyde or hydrogen peroxide solutions.

Combining High-Resolution Contact Data with Virological Data to Investigate Influenza Transmission in a Tertiary Care Hospital

OBJECTIVE Contact patterns and microbiological data contribute to a detailed understanding of infectious disease transmission. We explored the automated collection of high-resolution contact data by wearable sensors combined with virological data to investigate influenza transmission among patients and healthcare workers in a geriatric unit. DESIGN Proof-of-concept observational study. Detailed information on contact patterns were collected by wearable sensors over 12 days. Systematic nasopharyngeal swabs were taken, analyzed for influenza A and B viruses by real-time polymerase chain reaction, and cultured for phylogenetic analysis. SETTING An acute-care geriatric unit in a tertiary care hospital. PARTICIPANTS Patients, nurses, and medical doctors. RESULTS A total of 18,765 contacts were recorded among 37 patients, 32 nurses, and 15 medical doctors. Most contacts occurred between nurses or between a nurse and a patient. Fifteen individuals had influenza A (H3N2). Among these, 11 study participants were positive at the beginning of the study or at admission, and 3 patients and 1 nurse acquired laboratory-confirmed influenza during the study. Infectious medical doctors and nurses were identified as potential sources of hospital-acquired influenza (HA-Flu) for patients, and infectious patients were identified as likely sources for nurses. Only 1 potential transmission between nurses was observed. CONCLUSIONS Combining high-resolution contact data and virological data allowed us to identify a potential transmission route in each possible case of HA-Flu. This promising method should be applied for longer periods in larger populations, with more complete use of phylogenetic analyses, for a better understanding of influenza transmission dynamics in a hospital setting.

Functional Balance between the Hemagglutinin and Neuraminidase of Influenza A(H1N1)pdm09 HA D222 Variants

D222G/N substitutions in A(H1N1)pdm09 hemagglutinin may be associated with increased binding of viruses causing low respiratory tract infections and human pathogenesis. We assessed the impact of such substitutions on the balance between hemagglutinin binding and neuraminidase cleavage, viral growth and in vivo virulence.Seven viruses with differing polymorphisms at codon 222 (2 with D, 3 G, 1 N and 1 E) were isolated from patients and characterized with regards hemagglutinin binding affinity (Kd) to α-2,6 sialic acid (SAα-2,6) and SAα-2,3 and neuraminidase enzymatic properties (Km, Ki and Vmax). The hemagglutination assay was used to quantitatively assess the balance between hemagglutinin binding and neuraminidase cleavage. Viral growth properties were compared in vitro in MDCK-SIAT1 cells and in vivo in BALB/c mice. Compared with D222 variants, the binding affinity of G222 variants was greater for SAα-2,3 and lower for SAα-2,6, whereas that of both E222 and N222 variants was greater for both SAα-2,3 and SAα-2,6. Mean neuraminidase activity of D222 variants (16.0 nmol/h/106) was higher than that of G222 (1.7 nmol/h/106 viruses) and E/N222 variants (4.4 nmol/h/106 viruses). The hemagglutination assay demonstrated a deviation from functional balance by E222 and N222 variants that displayed strong hemagglutinin binding but weak neuraminidase activity. This deviation impaired viral growth in MDCK-SIAT1 cells but not infectivity in mice. All strains but one exhibited low infectious dose in mice (MID50) and replicated to high titers in the lung; this D222 strain exhibited a ten-fold higher MID50 and replicated to low titers. Hemagglutinin-neuraminidase balance status had a greater impact on viral replication than hemagglutinin affinity strength, at least in vitro, thus emphasizing the importance of an optimal balance for influenza virus fitness. The mouse model is effective in assessing binding to SAα-2,3 but cannot differentiate SAα-2,3- from SAα-2,6- preference, nor estimate the hemagglutinin-neuraminidase balance in A(H1N1)pdm09 strains.

Fatal Influenza A(H1N1)pdm09 Encephalopathy in Immunocompetent Man

We report an immunocompetent patient who had fatal encephalopathy after mild influenza. He rapidly died after unusual symptoms related to intracerebral thrombosis and hemorrhage. A brain biopsy specimen was positive for influenza A(H1N1)pdm09 virus RNA, but a lung biopsy specimen and cerebrospinal spinal fluid samples were negative.

Novel influenza A(H1N1) 2009 in vitro reassortant viruses with oseltamivir resistance.

BACKGROUND With the recent emergence of the novel A(H1N1) virus in 2009, the efficacy of available drugs, such as neuraminidase (NA) inhibitors, is of great concern for good patient care. Influenza viruses are known to be able to acquire resistance. In 2007, A(H1N1) viruses related to A/Brisbane/59/2007 (H1N1) (A[H1N1] Brisbane-like virus), which are naturally resistant to oseltamivir, emerged. Resistance to oseltamivir can be acquired either by spontaneous mutation in the NA (H275Y in N1), or by reassortment with a mutated NA. It is therefore crucial to determine the risk of pandemic A(H1N1) 2009 virus acquiring resistance against oseltamivir by reassortment. METHODS We estimated the capacity of reassortment between the A(H1N1) 2009 virus and an oseltamivir-resistant A(H1N1) Brisbane-like virus by in vitro coinfections of influenza-permissive cells. The screening and the analysis of reassortant viruses was performed by specific reverse transcriptase PCRs and by sequencing. RESULTS Out of 50 analysed reassortant viruses, two harboured the haemagglutinin (HA) segment from the pandemic A(H1N1) 2009 virus and the mutated NA originated from the A(H1N1) Brisbane-like virus. The replicating capacities of these viruses were measured, showing no difference as compared to the two parental strains, suggesting that acquisition of the mutated NA segment did not impair viral fitness in vitro. CONCLUSIONS Our results suggest that the novel A(H1N1) 2009 virus can acquire by in vitro genetic reassortment the H275Y mutated NA segment conferring resistance to oseltamivir.

Knowledge and risk perception of measles and factors associated with vaccination decisions in subjects consulting university affiliated public hospitals in Lyon, France, after measles infection

In 2011, a large number of European countries faced measles outbreaks, France accounting for more than half of the reported cases. The Rhône-Alpes region, located in south-east France, was one of the most affected provinces, with an incidence rate of 97.9 cases per 100 000 inhabitants. We conducted a retrospective survey of adults and parents of children consulting university affiliated public hospitals because of measles infections between January 1, 2010 and September 2012 in Lyon, France. Our main objectives were to evaluate (1) the level of study population knowledge of measles, (2) vaccination practices, and (3) changes in opinion with regard to measles vaccination after disease onset. Overall, 73.64% of patients were not vaccinated or partially vaccinated. The main reason for non-vaccination in children was inappropriate age while among non-vaccinated adults, 29.3% could not give any reason. In total, 29.1% of the responding parents and 24.2% of adult cases were opposed to vaccination “in principle.” A large number of patients did not recognize measles as a serious illness and were unaware of its complications. Among parents of infected children, knowledge of transmission mode (odds ratio [OR] = 5.9; 95% confidence interval [95% CI]: 1.64–21.26), perceived severity of measles (OR = 1.5; 95% CI: 1.06–2.13), and absence of hepatitis B vaccination (OR = 0.17; 95% CI: 0.04–0.65) were independently associated with a more positive opinion about measles vaccination after disease onset. In adult patients, low education level (OR = 3.39; 95% CI: 1.03–11.11) and lack of knowledge of sequelae (OR = 10.19; 95% CI: 1.14–91.31) were linked with a more positive opinion. Individuals affected by vaccine-preventable diseases are interesting populations to study disease impact on vaccine perception.

The clinical interest of HSV1 semi-quantification in bronchoalveolar lavage

BACKGROUND Detecting high herpes simplex (HSV) viral load in lower respiratory tract samples is reported to be significantly associated with the severity of the illness in critical patients, particularly in patients on mechanical ventilation. It may therefore be of interest to quantify HSV in bronchoalveolar lavage (BAL). Quantitative PCR for HSV is not commonly available in clinical routine. Real-time PCR tests are, however, used commonly and provide semi-quantitative information based on the cycle threshold (Ct). OBJECTIVES Our objectives were to determine the clinically significant threshold and to study the impact of viral load normalisation in relation to cell quantity in samples using real-time PCR. STUDY DESIGN During the period 2011-2012, 59 HSV1 positive BAL were included. HSV viral load was determined by a quantitative real-time PCR (R-gene, Argène BioMérieux, France) and compared to a semi-quantitative real-time PCR (SmartCycler®HerpesSimplex, Cepheid, USA). Viral load normalisation was determined using a real-time PCR targeting a cellular gene (Cc r-gene kit, Argène BioMérieux, France). The significant threshold was determined versus clinical features by statistical analysis (Epiinfo Software v3.5.1 CDC). RESULTS A viral load of 10(4) copies/ml of BAL was significantly associated with admission to the intensive care unit (p<0.001), mechanical ventilation (p<0.01) and death (p<0.01), with no influence of viral load normalisation in relation to cell quantity in the sample. This viral load was equivalent to a Ct value of 31 in the semi-quantitative technique. CONCLUSIONS As semi-quantitative techniques are currently used in many labs, determining this Ct value could be useful for interpreting the clinical advantages of detecting HSV in BAL.