F. Morfin

Genotypic detection of acyclovir-resistant HSV-1: Characterization of 67 ACV-sensitive and 14 ACV-resistant viruses

Infections due to herpes simplex virus (HSV) resistant to acyclovir (ACV) represent an important clinical concern in immunocompromised patients. In order to switch promptly to an appropriate treatment, rapid viral susceptibility assays are required. We developed herein a genotyping analysis focusing on thymidine kinase gene (TK) mutations in order to detect acyclovir-resistant HSV in clinical specimens. A total of 85 HSV-1 positive specimens collected from 69 patients were analyzed. TK gene could be sequenced directly for 81 clinical specimens (95%) and 68 HSV-1 specimens could be characterized as sensitive or resistant by genotyping (84%). Genetic characterization of 67 susceptible HSV-1 specimens revealed 10 polymorphisms never previously described. Genetic characterization of 14 resistant HSV-1 revealed 12 HSV-1 with either TK gene additions/deletions (8 strains) or substitutions (4 strains) and 2 HSV-1 with no mutation in the TK gene. DNA polymerase gene was afterwards explored. With this rapid PCR-based assay, ACV-resistant HSV could be detected directly in clinical specimens within 24 h.

Human parechovirus infections, Lyon, France, 2008-10: evidence for severe cases.

BACKGROUNDnAlthough data documenting the frequency and severity of human parechovirus type 3 (HPeV-3) infection in infants have been published in Canada, the USA, the UK and the Netherlands, no data from France are available.nnnOBJECTIVESnTo determine the detection frequency of HPeV in cerebrospinal fluid (CSF) samples collected from children aged <5 years hospitalized between 2008 and 2010 in the University Hospital of Lyon and to describe the clinical, virological and biological characteristics associated with HPeV infection.nnnSTUDY DESIGNnA total of 1128 CSF samples were retrospectively tested using the Parechovirus-Rgene™ real-time RT-PCR assay. Positive samples were typed by sequencing using the CDC method. Retrospective analysis of the medical charts was performed.nnnRESULTSnOver a 3-year period, 33/1128 (2.9%) CSF samples were found to be HPeV-positive. In 2010, 9.3% of the children aged <3 months (32% in June) were detected HPeV-positive. The median age at diagnosis was 26 days (8-131 days). Most patients (86%) presented with fever or a sepsis-like syndrome. Three patients (2 with septic shock syndrome, 1 with severe respiratory distress) required hospitalization in an intensive care unit. An HPeV-3 acute infection was identified in an 11-day-old girl who died from sudden infant death syndrome. Of 29 patients genotyped, 28 were infected with HPeV-3 and one with HPeV-4.nnnCONCLUSIONSnHPeV is a significant cause of sepsis and severe sepsis in children <3 months. Routine screening for HPeV in CSF and blood should thus be performed more extensively and could improve clinical management.

The clinical interest of HSV1 semi-quantification in bronchoalveolar lavage

BACKGROUNDnDetecting 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).nnnOBJECTIVESnOur 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.nnnSTUDY DESIGNnDuring 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).nnnRESULTSnA 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.nnnCONCLUSIONSnAs 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.

Intérêts des inhibiteurs de la neuraminidase dans la prise en charge des infections dues aux virus influenza

Oseltamivir and zanamivir are two neuraminidase inhibitors (NAIs) active on A and B influenza viruses. These analogues have been developed from the structure of sialic acid, the neuraminidase (NA) substrate. Resistance to NAIs have been detected. They are mainly associated to mutations located on the NA gene. The use of these antiviral drugs remains low in the context of seasonal flu, even the duration of symptoms can be reduced of one day if an antiviral treatment is started within 48 hours after disease onset. NAIs also present a significant effect when used in postexposition prophylaxis. Resistance, mainly to oseltamivir, have been detected but remained rare until the spontaneous emergence in 2007-2008 winter of a seasonal A(H1N1) variant resistant to this drug. NAIs are also interesting for the treatment of severe flu infections, specially those associated to A(H5N1). Finally, because of the pandemic A(H1N1)2009 virus, NAIs use has largely increased for prophylactic and therapeutic treatment of severe and non severe infections. This large use may be associated to an increased risk of selection of resistant viruses. Up to now, this phenomenon remains fortunately limited but has to be closely monitored.