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Background: Acute respiratory viral infections are generally self-limited in healthy subjects but can lead to severe complications in immunocompromised hosts. We report the clinical impact of acute lower respiratory tract [...]

AbstractBackground:The dynamics of coronavirus disease 2019 (COVID-19) seroconversion of hospital employees are understudied. We measured the proportion of seroconverted employees and evaluated risk factors for seroconversion during the first pandemic wave.Methods:In this prospective cohort study, we recruited Geneva University Hospitals employees and sampled them 3 times, every 3 weeks from March 30 to June 12, 2020. We measured the proportion of seroconverted employees and determined prevalence ratios of risk factors for seroconversion using multivariate mixed-effects Poisson regression models.Results:Overall, 3,421 participants (29% of all employees) were included, with 92% follow-up. The proportion of seroconverted employees increased from 4.4% (95% confidence interval [CI], 3.7%–5.1%) at baseline to 8.5% [(95% CI, 7.6%–9.5%) at the last visit. The proportions of seroconverted employees working in COVID-19 geriatrics and rehabilitation (G&R) wards (32.3%) and non–COVID-19 G&R wards (12.3%) were higher compared to office workers (4.9%) at the last visit. Only nursing assistants had a significantly higher risk of seroconversion compared to office workers (11.7% vs 4.9%; P= .006). Significant risk factors for seroconversion included the use of public transportation (adjusted prevalence ratio, 1.59; 95% CI, 1.25–2.03), known community exposure to severe acute respiratory coronavirus virus 2 (2.80; 95% CI, 2.22–3.54), working in a ward with a nosocomial COVID outbreak (2.93; 95% CI, 2.27–3.79), and working in a COVID-19 G&R ward (3.47; 95% CI, 2.45–4.91) or a non–COVID-19 G&R ward (1.96; 95% CI, 1.46–2.63). We observed an association between reported use of respirators and lower risk of seroconversion (0.73; 95% CI, 0.55–0.96).Conclusion:Additional preventive measures should be implemented to protect employees in G&R wards. Randomized trials on the protective effect of respirators are urgently needed.

ObjectivesThis cohort study including essential workers, assessed the risk and incidence of SARS-CoV-2 infection during the second surge of COVID-19 according to baseline serostatus and occupational sector.MethodsEssential workers were selected from a seroprevalence survey cohort in Geneva, Switzerland and were linked to a state centralised registry compiling SARS-CoV-2 infections. Primary outcome was the incidence of virologically confirmed infections from serological assessment (between May and September 2020) to 25 January 2021, according to baseline antibody status and stratified by three predefined occupational groups (occupations requiring sustained physical proximity, involving brief regular contact or others).Results10 457 essential workers were included (occupations requiring sustained physical proximity accounted for 3057 individuals, those involving regular brief contact, 3645 and 3755 workers were classified under ‘Other essential occupations’). After a follow-up period of over 27 weeks, 5 (0.6%) seropositive and 830 (8.5%) seronegative individuals had a positive SARS-CoV-2 test, with an incidence rate of 0.2 (95% CI 0.1 to 0.6) and 3.2 (95% CI 2.9 to 3.4) cases per person-week, respectively. Incidences were similar across occupational groups. Seropositive essential workers had a 93% reduction in the hazard (HR of 0.07, 95% CI 0.03 to 0.17) of having a positive test during the follow-up with no significant between-occupational group difference.ConclusionsA 10-fold reduction in the hazard of being virologically tested positive was observed among anti-SARS-CoV-2 seropositive essential workers regardless of their sector of occupation, confirming the seroprotective effect of a previous SARS-CoV2 exposure at least 6 months after infection.

Infectious viral load (VL) expelled as droplets and aerosols by infected individuals partly determines transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RNA VL measured by qRT–PCR is only a weak proxy for infectiousness. Studies on the kinetics of infectious VL are important to understand the mechanisms behind the different transmissibility of SARS-CoV-2 variants and the effect of vaccination on transmission, which allows guidance of public health measures. In this study, we quantified infectious VL in individuals infected with SARS-CoV-2 during the first five symptomatic days by in vitro culturability assay in unvaccinated or vaccinated individuals infected with pre-variant of concern (pre-VOC) SARS-CoV-2, Delta or Omicron BA.1. Unvaccinated individuals infected with pre-VOC SARS-CoV-2 had lower infectious VL than Delta-infected unvaccinated individuals. Full vaccination (defined as >2 weeks after receipt of the second dose during the primary vaccination series) significantly reduced infectious VL for Delta breakthrough cases compared to unvaccinated individuals. For Omicron BA.1 breakthrough cases, reduced infectious VL was observed only in boosted but not in fully vaccinated individuals compared to unvaccinated individuals. In addition, infectious VL was lower in fully vaccinated Omicron BA.1 -infected individuals compared to fully vaccinated Delta-infected individuals, suggesting that mechanisms other than increased infectious VL contribute to the high infectiousness of SARS-CoV-2 Omicron BA.1 . Our findings indicate that vaccines may lower transmission risk and, therefore, have a public health benefit beyond the individual protection from severe disease.

Recently, cases of multisystem inflammatory syndrome in children (MIS-C) associated with coronavirus disease 2019 (COVID-19) have been reported worldwide. Negative polymerase chain reaction (RT-PCR) testing associated with positive serology in most of the cases suggests a postinfectious syndrome. Because the pathophysiology of this syndrome is still poorly understood, extensive virological and immunological investigations are needed.

Solid organ transplant (SOT) recipients are exposed to respiratory viral infection (RVI) during seasonal epidemics; however, the associated burden of disease has not been fully characterized. We describe the epidemiology and outcomes of RVI in a cohort enrolling 3294 consecutive patients undergoing SOT from May 2008 to December 2015 in Switzerland. Patient and allograft outcomes, and RVI diagnosed during routine clinical practice were prospectively collected. Median follow-up was 3.4 years (interquartile range 1.61–5.56). Six hundred ninety-six RVIs were diagnosed in 151/334 (45%) lung and 265/2960 (9%) non-lung transplant recipients. Cumulative incidence was 60% (95% confidence interval [CI] 53%-69%) in lung and 12% (95% CI 11%-14%) in non-lung transplant recipients. RVI led to 17.9 (95% CI 15.7–20.5) hospital admissions per 1000 patient-years. Intensive care unit admission was required in 4% (27/691) of cases. Thirty-day all-cause case fatality rate was 0.9% (6/696). Using proportional hazard models we found that RVI (adjusted hazard ratio [aHR] 2.45; 95% CI 1.62–3.73), lower respiratory tract RVI (aHR 3.45; 95% CI 2.15–5.52), and influenza (aHR 3.57; 95% CI 1.75–7.26) were associated with graft failure or death. In this cohort of SOT recipients, RVI caused important morbidity and may affect long-term outcomes, underlying the need for improved preventive strategies.

Assessing the burden of COVID-19 on the basis of medically attended case numbers is suboptimal given its reliance on testing strategy, changing case definitions, and disease presentation. Population-based serosurveys measuring anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies provide one method for estimating infection rates and monitoring the progression of the epidemic. Here, we estimate weekly seroprevalence of anti-SARS-CoV-2 antibodies in the population of Geneva, Switzerland, during the epidemic.

ObjectivesTo evaluate the potential associations between fever without a source (FWS) in children and detection of human enterovirus (HEV), human parechovirus (HPeV), adenovirus (AdV) and human herpesvirus type 6 (HHV-6) in the plasma; and to assess whether the detection of viruses in the plasma is associated with a reduced risk of serious bacterial infection (SBI) and antibiotic use.Design and settingBetween November 2015 and December 2017, this prospective, single-centre, diagnostic study tested the plasma of children <3 years old with FWS. Real-time (reverse-transcription) PCR for HEV, HPeV, AdV and HHV-6 was used in addition to the standardised institutional work-up. A control cohort was also tested for the presence of viruses in their blood.ResultsHEV, HPeV, AdV and HHV-6 were tested for in the plasma of 135 patients of median age 2.4 months old. At least one virus was detected in 47 of 135 (34.8%): HEV in 14.1%, HHV-6 in 11.1%, HPeV in 5.9% and AdV in 5.2%. There was no difference in antibiotic use between patients with or without virus detected, despite a relative risk of 0.2 for an SBI among patients with viraemia. Controls were less frequently viraemic than children with FWS (6.0% vs 34.8%; p<0.001).ConclusionsHEV, HPeV, AdV and HHV-6 are frequently detected in the plasma of children with FWS. Antibiotic use was similar between viraemic and non-viraemic patients despite a lower risk of SBI among patients with viraemia. Point-of-care viral PCR testing of plasma might reduce antibiotic use and possibly investigations and admission rates in patients with FWS.Trial registration numberNCT03224026.

Background Neuraminidase (NA) inhibitors (NAIs), including oseltamivir and zanamivir, play an important therapeutic role against influenza infections in immunocompromised patients. In such settings, however, NAI therapy may lead to the emergence of resistance involving mutations within the influenza surface genes. The aim of this study was to investigate the evolution of NA and haemagglutinin (HA) genes of influenza A(H1N1) pdm09 virus in an immunocompromised patient receiving oseltamivir then zanamivir therapies.Methods Nasopharyngeal swab (NPS) samples were collected between 27 January 2018 and 11 April 2018 from a haematopoietic stem cell transplant recipient. These include 10 samples collected either pre-therapy, during oseltamivir and zanamivir treatment as well as after therapy. The A(H1N1)pdm09 HA/NA genes were sequenced. The H275Y NA substitution was quantified by droplet digital RT-PCR assay. A(H1N1)pdm09 recombinant viruses containing HA mutations were tested by HA elution experiments to investigate in vitrobinding properties.Results Oseltamivir rapidly induced the H275Y NA mutation which constituted 98.33% of the viral population after 15 days of oseltamivir treatment. The related HA gene contained S135A and P183S substitutions within the receptor-binding site. After a switch to zanamivir, 275H/Y and 119E/G/D mixed populations were detected. In the last samples, the double H275Y-E119G NA variant dominated with S135A and P183S HA substitutions.Conclusions This report confirms that oseltamivir can rapidly induce the emergence of the H275Y substitution in A(H1N1)pdm09 viruses and subsequent switch to zanamivir can lead to additional substitutions at codon E119 resulting in multi-drug resistance. Such data additionally suggest a potential compensatory role for HA substitutions near the receptor binding site.

Live‐attenuated vaccines are currently contraindicated in solid‐organ transplant recipients. However, the risk of vaccine‐preventable infections is lifelong, and can be particularly severe after transplantation. In this prospective interventional national cohort study, 44 pediatric liver transplant recipients with measles IgG antibodies <150 IU/L (below seroprotection threshold) received measles‐mumps‐rubella vaccine (MMR) at a median of 6.3 years posttransplantation (interquartile range, 4.0 to 10.9). A maximum of two additional doses were administered in nonresponders or when seroprotection was lost. Vaccine responses occurred in 98% (95% confidence interval [CI], 88‐100) of patients. Seroprotection at 1‐, 2‐, and 3‐year follow‐up reached 62% (95% CI, 45‐78), 86% (95% CI, 70‐95), and 89% (95% CI, 67‐99), respectively. All patients responded appropriately to the booster dose(s). Vaccinations were well tolerated and no serious adverse event attributable to vaccination was identified during the 8‐week follow‐up period (or later), using a multimodal approach including standardized telephone interviews, diarized side effect reporting, and monitoring of vaccinal virus shedding. We conclude that live attenuated MMR vaccine can be administered in liver transplant recipients fulfilling specific eligibility criteria (>1 year posttransplantation, low immunosuppression, lymphocyte count ≥0.75 G/L), inducing seroprotection in most subjects. (Clinicaltrials.gov number NCT01770119). This prospective interventional study demonstrates the safety and immunogenicity of the live‐attenuated measles‐mumps‐rubella vaccine in immunocompromised children not seroprotected against measles after liver transplantation.

Children and adolescents are highly vulnerable to the impact of sustained stressors during developmentally sensitive times. We investigated how demographic characteristics intersect with socioeconomic dimensions to shape the social patterning of quality of life and mental health in children and adolescents, two years into the COVID-19 pandemic.

Background: Influenza infections commonly lead to respiratory tract complications that result in antibiotic treatment. Objectives: To determine frequency of respiratory events leading to antibiotic use following influenza illness in adolescents and adults, and to assess whether treatment with topical zanamivir prevents these complications. Methods: Meta-analysis of 7 randomized, double-blind, placebo-controlled trials; 3815 mainly healthy adolescents and adults (mean age, 34 years) with an influenza-like illness of less than 2 days' duration were randomly assigned to receive combined inhaled and intranasal zanamivir, inhaled zanamivir, or corresponding placebos. Twelve percent of enrolled subjects were high-risk patients. The main outcome was the incidence of respiratory events leading to antibiotic prescriptions in patients with proven influenza. Results: Influenza infections were laboratory confirmed in 2499 (66%) of 3815 patients (influenza A in 88% and B in 12%). Placebo recipients developed a respiratory event leading to antibiotic use in 17% of cases, mainly for acute bronchitis or acute sinusitis. Among zanamivir-treated patients (n = 1494) the incidence of respiratory events leading to the use of antimicrobials was 11% (relative risk [RR] compared with placebo, 0.69; 95% confidence interval [CI], 0.57-0.84). Intranasal and inhaled zanamivir seemed to reduce the number of upper (RR, 0.59; 95% CI, 0.36-0.97) and lower respiratory tract events (RR, 0.64; 95% CI, 0.38-1.08). Inhaled zanamivir reduced the number of lower respiratory tract events (RR, 0.60; 95% CI, 0.42-0.85), but the reduction in the number of upper respiratory tract events was not statistically significant (RR, 0.90; 95% CI, 0.63-1.27). Conclusions: Respiratory complications or worsening of symptoms leading to antibiotic use occurred in about 17% of adolescents or adults with influenza infection. Early treatment of influenza illness with zanamivir reduced the number of these antibiotic prescriptions.

Background: Persons infected with human immunodeficiency virus (HIV) are at increased risk for diarrhea and enteric infections. We studied (1) the epidemiology of enteric pathogens associated with diarrhea, (2) the diagnostic yield of stool examination and endoscopic evaluation, (3) risks to develop diarrhea, and (4) the impact of diarrhea on patients' survival. Methods: A total of 1933 participants in the Swiss HIV Cohort Study were prospectively followed up for a median of 25.5 months. A total of 560 diarrheal episodes were evaluated by standardized stool examination. Endoscopic evaluation was performed in 25% of patients with chronic diarrhea. Results: The incidence of diarrhea was 14.2 per 100 person-years (95% confidence interval, 13.0-15.4). Among patients with CD4 cell counts below 0.05 X [10.sup.9]/L, the probability to develop diarrhea within 1, 2, and 3 years was 48.5%, 74.3%, and 95.6%, respectively. The risk to develop diarrhea was increased among patients with severe immunodeficiency, homosexual men, and patients taking antiretroviral therapy. Pneumocystis carinii chemoprophylaxis did not reduce the risk of diarrhea. Diarrhea was an independent negative predictor of survival. Enteric pathogens were detected in 16.5% of 212 acute diarrheal episodes and in 46% of 348 chronic diarrheal episodes. The sensitivity of histological and stool examination was similar except for the diagnosis of intestinal cytomegalovirus infection and leishmaniasis, which required invasive evaluation. Conclusions: Intestinal infections were diagnosed in less than 50% of chronic diarrheal episodes. The prevalence of enteric pathogens tended to decrease during the observation period, possibly because of improved antiretroviral therapy. Endoscopic evaluation did not improve the diagnostic yield compared with stool examination except for the diagnosis of cytomegalovirus enteritis and leishmaniasis. Arch Intern Med. 1999;159:1473-1480

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Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus–cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (∼190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism. These particles show no cytotoxicity, and in vitronanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivoin human cervicovaginal histocultures infected by HSV-2 and in vivoin mice infected with RSV.

Gardnerella vaginalisextra-vaginal infections are seldom reported. A complex clinical case of pyelonephritis imputed to G. vaginalis, with renal abscess and bacteremia in a diabetic non-pregnant woman with polycystic kidney disease is presented. An exhaustive review of the published literature on this topic is presented.Gardnerella vaginalisis infrequently reported as a cause of extragenital infections. Although the pathogenesis of G. vaginalisis not completely understood, available clinical data suggest that it is an opportunistic pathogen that can cause invasive infections.We report a case of pyelonephritis imputed to G. vaginalis, with renal abscess and bacteremia in a diabetic nonpregnant woman with polycystic kidney disease. Gardnerella vaginaliswas isolated from blood, the abscess, and urine obtained by direct placement of a pigtail into the kidney. Identification was made with molecular methods.We present a review of the literature concerning G. vaginalisextragenital infections, with special focus on abscesses and bacteremias.We searched PubMed for articles in English, French, German, and Spanish languages. Only 15 cases of abscesses from diverse body sites have been reported. We found 28 articles describing a total of 117 cases of bacteremia. Most of them occurred in women in the peripartum periods and in newborns. Cases in men have been described with lower frequency. The importance of this agent might be underestimated because microbiological diagnosis is difficult. Most cases in adults were nonfatal and without sequelae. There is no consensus on the antibiotic regimen and duration of treatment for these infections. New microbiological diagnosis tools may lead to prompt identification of this microorganism and help to elucidate its participation in localized and systemic infections.

The recent large outbreak of Ebola virus disease (EVD) in Western Africa resulted in greatly increased accumulation of human genotypic, phenotypic and clinical data, and improved our understanding of the spectrum of clinical manifestations. As a result, the WHO disease classification of EVD underwent major revision.

ABSTRACTConventional tube culture systems have long been the mainstay in clinical virology for the growth and identification of viruses from clinical specimens. Innovations such as centrifugation-enhanced shell vial and multiwell plate cultures and the use of genetically engineered and mixed cell lines, coupled with faster detection of viral replication, have allowed for reasonable turnaround times for even some of the most slowly growing clinically important human viruses. However, molecular methods, in particular, the PCR, have usurped the role of viral culture in many laboratories, limiting the use of this traditional method of virus detection or replacing it altogether. Advances and improvements in molecular technology over time have also resulted in newer generations of more rapid and accurate molecular assays for the detection, quantification, and genetic characterization of viruses. For this point-counterpoint, we have asked two individuals, Richard L. Hodinka of the Children's Hospital of Philadelphia, a clinical virologist whose laboratory has completely eliminated viral culture in favor of molecular methods, and Laurent Kaiser, head of the Virology Laboratory at the University of Geneva Hospital, who continues to be a strong advocate of viral culture, to discuss the relevance of viral culture in the molecular age.

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Serological assays for the presence of anti-SARS-CoV-2 antibodies are crucially needed for research and monitoring of the SARS-CoV-2 pandemic. Antibodies are reliability detected in capillary blood, a minimally invasive and cost-effective alternative to venous blood testing. However, there is a limited knowledge on feasibility of capillary blood self-sampling. This study compared the feasibility of capillary blood self-testing in people aged less than 65 vs.people aged 65 or more. A secondary aim was to investigate the performance of the Hem-Col® (no additive) device compared to venous blood testing.

ABSTRACTHigh-throughput sequencing (HTS) provides the means to analyze clinical specimens in unprecedented molecular detail. While this technology has been successfully applied to virus discovery and other related areas of research, HTS methodology has yet to be exploited for use in a clinical setting for routine diagnostics. Here, a bioinformatics pipeline (ezVIR) was designed to process HTS data from any of the standard platforms and to evaluate the entire spectrum of known human viruses at once, providing results that are easy to interpret and customizable. The pipeline works by identifying the most likely viruses present in the specimen given the sequencing data. Additionally, ezVIR can generate optional reports for strain typing, can create genome coverage histograms, and can perform cross-contamination analysis for specimens prepared in series. In this pilot study, the pipeline was challenged using HTS data from 20 clinical specimens representative of those most often collected and analyzed in daily practice. The specimens (5 cerebrospinal fluid, 7 bronchoalveolar lavage fluid, 5 plasma, 2 serum, and 1 nasopharyngeal aspirate) were originally found to be positive for a diverse range of DNA or RNA viruses by routine molecular diagnostics. The ezVIR pipeline correctly identified 14 of 14 specimens containing viruses with genomes of <40,000 bp, and 4 of 6 specimens positive for large-genome viruses. Although further validation is needed to evaluate sensitivity and to define detection cutoffs, results obtained in this pilot study indicate that the overall detection success rate, coupled with the ease of interpreting the analysis reports, makes it worth considering using HTS for clinical diagnostics.

Cytomegalovirus (CMV) retinitis is a major cause of visual impairment and blindness among patients with uncontrolled HIV infections. Whereas polymorphisms in interferon-lambda 3 (IFNL3, previously named IL28B) strongly influence the clinical course of hepatitis C, few studies examined the role of such polymorphisms in infections due to viruses other than hepatitis C virus.

Rhinovirus (RV)-induced pulmonary exacerbations are common in cystic fibrosis (CF) and have been associated with impaired virus clearance by the CF airway epithelium in vitro. Here, we assess in vivo the association of RV prevalence and load with antiviral defense mechanisms, airway inflammation, and lung function parameters in children with CF compared with a control group and children with other chronic respiratory diseases.

ABSTRACTRhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5'-untranslated regions (5'UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5'UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ±10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible.

Background Solid organ transplant (SOT) recipients are a priority group for influenza vaccination and strategies enhancing immunogenicity are needed.Methods We determined adverse reactions, changes in biomarkers of graft function and immune responses to two doses of the AS03-adjuvanted influenza A/09/H1N1 vaccine in 216 SOT recipients and in 138 controls after one dose. Antibody responses were measured by haemag-glutination inhibition and confirmed by microneutralization. We calculated geometric mean titres (GMT) and seroprotection rates (GMT=40).Results Adverse reactions were fewer than in controls and graft function remained unaffected. Seroprotection was achieved by only 70.3% of SOT recipients, with significant differences between groups (lung 43.6%, heart 72.0%, kidney 83.3%, liver 83.3% and pancreas 85%), compared to 87% of controls (P<0.001). The weakest responses (seroprotection 43.5%) were elicited in lung transplant recipients. GMT remained threefold lower (115 versus 340) in SOT recipients than controls. Multivariate analyses identified increasing age, type of transplant and increasing blood levels of mycophenolate as independently associated to weaker responses. In contrast, even high blood levels of calcineurin inhibitors remained without significant influence on vaccine responses.Conclusions The squalene-based adjuvanted A/09/H1N1 vaccine was safe in SOT recipients. However, even two doses of this adjuvanted influenza vaccine did not provide adequate protection for lung transplant recipients and those with high mycophenolate blood levels. Additional prophylactic measures should, therefore, be considered for these high-risk groups.

Herpes simplex virus (HSV) hepatitis is an uncommon cause of acute liver failure (ALF), primarily affecting immunocompromised patients. So far, 148 cases have been published, of which 9 underwent liver transplantation (LT). The reported post-transplant survival is poor, with over 60% dying in the first year. Dosing and duration of antiviral therapy after LT are not established. Concerns include both the risk of hepatic recurrence after LT and emergence of viral resistance during prolonged therapy. HSV DNA plasma levels might be helpful to monitor therapeutic response and guide duration of therapy. We present a case of ALF complicating a primary HSV-1 infection in an immunocompetent host, who required emergency LT. We further discuss the value of measuring serial HSV DNA plasma loads to monitor antiviral therapy.

ABSTRACTHuman rhinoviruses (HRV), and to a lesser extent human enteroviruses (HEV), are important respiratory pathogens. Like other RNA viruses, these picornaviruses have an intrinsic propensity to variability. This results in a large number of different serotypes as well as the incessant discovery of new genotypes. This large and growing diversity not only complicates the design of real-time PCR assays but also renders immunofluorescence unfeasible for broad HRV and HEV detection or quantification in cells. In this study, we used the 5' untranslated region, the most conserved part of the genome, as a target for the development of both a real-time PCR assay (Panenterhino/Ge/08) and a peptide nucleic acid-based hybridization oligoprobe (Panenterhino/Ge/08 PNA probe) designed to detect all HRV and HEV species members according to publicly available sequences. The reverse transcription-PCR assay has been validated, using not only plasmid and viral stocks but also quantified RNA transcripts and around 1,000 clinical specimens. These new generic detection PCR assays overcame the variability of circulating strains and lowered the risk of missing emerging and divergent HRV and HEV. An additional real-time PCR assay (Entero/Ge/08) was also designed specifically to provide sensitive and targeted detection of HEV in cerebrospinal fluid. In addition to the generic probe, we developed specific probes for the detection of HRV-A and HRV-B in cells. This investigation provides a comprehensive toolbox for accurate molecular identification of the different HEV and HRV circulating in humans.

Replication of picornaviruses is dependent on VPg uridylylation, which is linked to the presence of the internal cis-acting replication element (cre). Cre are located within the sequence encoding polyprotein, yet at distinct positions as demonstrated for poliovirus and coxsackievirus-B3, cardiovirus, and human rhinovirus (HRV-A and HRV-B), overlapping proteins 2C, VP2, 2A, and VP1, respectively. Here we report a novel distinct cre element located in the VP2 region of the recently reported HRV-A2 species and provide evolutionary evidence of its functionality. We also experimentally interrogated functionality of recently identified HRV-B cre in the 2C region that is orthologous to the human enterovirus (HEV) cre and show that it is dispensable for replication and appears to be a nonfunctional evolutionary relic. In addition, our mutational analysis highlights two amino acids in the 2C protein that are crucial for replication. Remarkably, we conclude that each genetic clade of HRV and HEV is characterized by a unique functional cre element, where evolutionary success of a new genetic lineage seems to be associated with an invention of a novel cre motif and decay of the ancestral one. Therefore, we propose that cre element could be considered as an additional criterion for human rhinovirus and enterovirus classification.

Acute respiratory infections (ARI) are a major cause of morbidity in infancy worldwide, with cough and wheeze being alarming symptoms to parents. We aimed to analyze in detail the viral aetiology of ARI with such symptoms in otherwise healthy infants, including rhinoviruses and recently discovered viruses such as human metapneumovirus (HMPV), coronavirus NL63 and HKU1, and human bocavirus (HBoV).

Adults more than 40 years old are not necessarily immune to measles. A measles outbreak that involved healthcare workers occurred after contact with a 44-year-old patient. Results of a hospitalwide program of mass screening revealed that 117 (4.5%) of 2,600 individuals tested seronegative for measles; 31 (26.1%) of these 117 individuals were more than 40 years old.

ABSTRACTA nonfluorescent low-cost, low-density oligonucleotide array was designed for detecting the whole coronavirus genus after reverse transcription (RT)-PCR. The limit of detection was 15.7 copies/reaction. The clinical detection limit in patients with severe acute respiratory syndrome was 100 copies/sample. In 39 children suffering from coronavirus 229E, NL63, OC43, or HKU1, the sensitivity was equal to that of individual real-time RT-PCRs.

We have studied cytomegalovirus (CMV) immunity in 17 CMV-positive recipients of T-cell-depleted or T-cell-replete grafts. In recipients of T-cell-replete grafts, the patient's CMV-specific T-cell response was completely ablated. Because primary anti-CMV responses were rare during the first year, immunity depended essentially on the transfer of donor CMV-specific T cells and, therefore, on the CMV positivity of the donor. In the recipients of T-cell-depleted grafts, CMV-specific cytotoxic T cells were of recipient origin in 2 patients who underwent transplantation with CMV-negative donors and in 3 of 8 patients who underwent transplantation with CMV-positive donors, and they were of mixed or donor origin in the other 5 patients studied. Recipient CMV-specific T cells responded vigorously to antigen ex vivo and persisted for several years without replenishment by donor cells. Furthermore, they appeared to have a protective effect because CMV-related complications were absent in the patients with CMV-specific T cells of recipient origin. Clinical outcomes of a cohort of 91 patients corroborated the experimental results. Patients with recipient T cells in their blood were protected regardless of the donor immune status. Hence, when a T-cell depletion protocol is used that favors the survival of recipient T cells, the patient's pretransplantation CMV-specific immunity protects against posttransplantation CMV-related complications.

ABSTRACTImproved knowledge of the genotypic characteristics of human rhinovirus (HRV) is required, as are nucleic detection assays with the capacity to overcome both the similarities between members of the family Picornaviridaeand the wide diversity of different HRV serotypes. The goal of the present study was to investigate the variability and the genotypic diversity of clinical strains circulating in the community. Since most reverse transcription (RT)-PCR assays available cannot differentiate HRV from other members of the family Picornaviridae, we also validated an assay specific for HRV detection. The 5' noncoding regions of 87 different HRV serotypes and clinical isolates were sequenced. On the basis of sequence analysis and phylogenetic determination, we first confirmed that all clinical isolates were HRV. We then validated a real-time RT-PCR assay that was able not only to detect all HRV serotypes and all clinical isolates tested but also to accurately discriminate between rhinovirus and other viruses from the family Picornaviridae. This assay was negative with isolates of coxsackievirus (types A and B), echovirus, enterovirus, parechovirus, and poliovirus, as well as nonpicornaviruses. Among a series of bronchoalveolar lavage specimens, 4% (7 of 161) were positive by culture, whereas 13% (21 of 161) were positive by RT-PCR. In the present study we showed that to specifically identify HRV in clinical specimens, diagnostic assays need to overcome both the diversities and the similarities of picornaviruses. By sequencing the 5' noncoding regions of rhinoviruses recovered from clinical specimens, we designed probes that could specifically detect rhinovirus.

Transmission of drug-resistant variants is influenced by several factors, including the prevalence of drug resistance in the population of HIV-1-infected patients, HIV-1 RNA levels and transmission by recently infected patients. In order to evaluate the impact of these factors on the transmission of drug-resistant variants, we have defined the population of potential transmitters and compared their resistance profiles to those of newly infected patients. Sequencing of polgene was performed in 220 recently infected patients and in 373 chronically infected patients with HIV-1 RNA >1000 copies/ml. Minimal and maximal drug-resistance profiles of potential transmitters were estimated by weighting resistance profiles of chronically infected patients with estimates of the Swiss HIV-1-infected population, the prevalence of exposure to antiviral drugs and the proportion of infections attributed to primary HIV infections. The drug-resistance prevalence in recently infected patients was 10.5% (one class drug resistance: 9.1%; two classes: 1.4%; three classes: 0%). Phylogenetic analysis revealed significant clustering for 30% of recent infections. The drug-resistance prevalence in chronically infected patients was 72.4% (one class: 29%; two classes: 27.6%; three classes: 15.8%). After adjustment, the risk of transmission relative to wild-type was reduced both for one class drug resistance (minimal and maximal estimates: odds ratio: 0.39, P<0.001; and odds ratio: 0.55, P=0.011, respectively), and for two to three class drug resistance (odds ratios: 0.05 and 0.07, respectively, P<0.001). Neither sexual behaviour nor HIV-1 RNA levels explained the low transmission of drug-resistant variants.These data suggest that drug-resistant variants and in particular multidrug-resistant variants have a substantially reduced transmission capacity.

ABSTRACTEarly and accurate monitoring of cytomegalovirus (CMV) infection in solid-organ transplant recipients is of major importance. We have assessed the potential benefit of an ultrasensitive plasma-based PCR assay for renal transplant recipients. The pp65 CMV antigen (pp65 Ag) assay using leukocytes was employed as a routine test for the monitoring of CMV in 23 transplant recipients. We compared the pp65 antigenemia with the CMV load quantified by an ultrasensitive PCR (US-PCR) with a limit of detection of 20 CMV DNA copies/ml of plasma. CMV infection was detected in 215 (67%) of 321 plasma samples by the US-PCR compared with 124 (39%) of 321 samples by the pp65 Ag assay. The US-PCR assay permitted the detection of CMV infection episodes following transplantation a median of 12 days earlier than the pp65 Ag assay. Moreover, during CMV infection episodes, DNA detection by the US-PCR was consistently positive, whereas false negative results were frequently observed with the pp65 Ag assay. We found a good correlation between the two assays, and the peak viral loads were significantly higher in patients with CMV-related complications (median, 5,000 DNA copies/ml) than in those without symptoms (1,160 DNA copies/ml) (P= 0.048). In addition, patients that did not require preemptive therapy based on the results of the pp65 assay had CMV loads significantly lower (median, 36 DNA copies/ml) than those that needed treatment (median, 4,703 DNA copies/ml) (P< 0.001). These observations provided cutoff levels that could be applied in clinical practice. The ultrasensitive plasma-based PCR detected CMV infection episodes earlier and provided more consistent results than the pp65 Ag assay. This test could improve the monitoring of CMV infection or reactivation in renal transplant recipients.

ABSTRACTCytomegalovirus (CMV) DNA amplification assays in plasma have shown limited sensitivity compared to the detection of pp65 antigen in leukocytes. Our goal was to increase the sensitivity of a commercial CMV DNA PCR quantitative assay. After modification, the new assay was able to reproducibly detect 20 CMV DNA copies/ml of plasma. We compared this new ultrasensitive PCR assay with the standard PCR and the pp65 test for CMV detection and quantification in 22 consecutive allogeneic hematopoietic stem cell recipients. CMV infection or reactivation was detected in 84 of 319 (26%) samples by the ultrasensitive PCR assay compared to 38 of 319 (12%) samples by the pp65 assay (P< 0.01). All samples positive by the pp65 assay were positive by the ultrasensitive PCR, and CMV episodes were detected on average 4 days earlier and 7 days later than the first and the last pp65-positive test, respectively. In addition, during CMV episodes, the ultrasensitive assay identified positive samples that were inconsistently detected by the pp65 assay. The ultrasensitive assay was also much more sensitive than the standard PCR, with 26 versus 12% of CMV DNA-positive samples (P< 0.01). This assay improved the monitoring of CMV infection or reactivation in hematopoietic allogeneic stem cell recipients.

CD4 T-cell–specific memory antiviral responses to human immunodeficiency virus type 1 (HIV-1) and cytomegalovirus (CMV) were investigated in 16 patients with documented primary HIV-1 infection (4 of the 16 subjects also had primary CMV infection) and compared with those observed in patients with chronic HIV-1 and CMV coinfection. Virus-specific memory CD4 T cells were characterized on the basis of the expression of the chemokine receptor CCR7. HIV-1– and CMV-specific interferon-γ–secreting CD4 T cells were detected in patients with primary and chronic HIV-1 and CMV coinfection and were mostly contained in the cell population lacking expression of CCR7. The magnitude of the primary CMV-specific CD4 T-cell response was significantly greater than that of chronic CMV infection, whereas there were no differences between primary and chronic HIV-1–specific CD4 T-cell responses. A substantial proportion of CD4+CCR7− T cells were infected with HIV-1. These results advance the characterization of antiviral memory CD4 T-cell response and the delineation of the potential mechanisms that likely prevent the generation of a robust CD4 T-cell immune response during primary infection.

In experimental human influenza infection initiated by nasal inoculation, the magnitude of viral replication, fever, and symptoms correlate with nasopharyngeal lavage fluid levels of various cytokines. Our aim was to assess these relationships in patients with naturally occurring acute influenza. Patients with culture‐positive influenza illness of less than 36 hr of duration were studied. Nasopharyngeal washing were collected at enrollment and on Day 2, 4, 6 and 8 for quantitative virus isolation and IL‐6, TNF‐α, INF‐α, INF‐γ and IL‐10 determinations. Blood samples collected at entry and on Day 2 and 6 were processed to assess plasma cytokines and circulating influenza RNA. Patients received either oseltamivir or placebo for 5 days. We assessed the correlation between nasopharyngeal lavage fluid or blood levels of cytokines before treatment and viral titers, symptom severity and fever. Sixteen adult subjects (median age of 22 years) were studied. In this small group of patients no significant differences between placebo and oseltamivir patients were found in viral replication or measures of cytokines. Thus the data for all 16 subjects were pooled for analysis. At entry, influenza A viruses were cultured from nasopharyngeal washes at a median titer of 4.8 log10TCID50/ml of wash. Viral titers correlated positively with symptom score (P= 0.006) and temperature values (P< 0.001). Viral titers, fever and symptoms were highest at enrollment and fell in parallel during the subsequent days. RT‐PCR assays failed to detect influenza RNA in the white blood cells from any patient. We observed a significant release, in both nasopharyngeal lavage fluid and in plasma, of IL‐6, TNF‐α, INF‐α, INF‐γ and IL‐10. At entry high IL‐6 levels were detected in the nasopharyngeal lavage fluid (median 10.3 pg/ml) and plasma (median 5.1 pg/ml) of all patients. We found a positive correlation between plasma IL‐6 levels and both symptom scores and temperature values (P< 0.05), as well as a positive correlation between nasopharyngeal lavage fluid levels of IL‐6 and TNF‐α and temperature (P< 0.05). We did not find significant associations between symptoms, fever and levels of INF‐α, INF‐γ or IL‐10. The magnitude of early decrease in viral titers correlated with initial levels of INF‐γ in nasopharyngeal lavage fluid (P< 0.05). Significant production of IL‐6, TNF‐α, INF‐α, INF‐γ and IL‐10 occurs in response to community acquired influenza A illness. As in experimental influenza, symptoms and fever in natural acute influenza correlate with the release of IL‐6. J. Med. Virol. 64:262–268, 2001. © 2001 Wiley‐Liss, Inc.

In a development process, specification and realisation of a real time distributed system can be done by different actors. The test methods are used to ensure the interoperability between the different components . A lot of research have been done on the test generation (Beziers, 1990; Bochmann et al., 1977; Fernandez et al., 1997) but there is only few works which take into account the temporal behaviour of the tested system (Koné, 1995; Laurençot, 1999). Some formal methods have been develop in order to verify that an implementation conforms to its specification (Chow, 1978; Luo et al., 1994; Sabnani and Dahbura, 1988; Sidhu and Leung, 1989). In relation to these works, a test generation approach, based on the on the fly strategy with integrating temporal properties, is proposed.