Surveillance network for herpes simplex virus resistance to antiviral drugs: 3-year follow-up.

Herpes simplex virus (HSV) infections are very common in the general population and among immunocompromised patients. Acyclovir (ACV) is an effective treatment which is widely used. We deemed it essential to conduct a wide and coordinated survey of the emergence of ACV-resistant HSV strains. We have formed a network of 15 virology laboratories which have isolated and identified, between May 1999 and April 2002, HSV type 1 (HSV-1) and HSV-2 strains among hospitalized subjects. The sensitivity of each isolate to ACV was evaluated by a colorimetric test (C. Danve, F. Morfin, D. Thouvenot, and M. Aymard, J. Virol. Methods 105:207-217, 2002). During this study, 3900 isolated strains among 3357 patients were collected; 55% of the patients were immunocompetent. Only six immunocompetent patients excreted ACV-resistant HSV strains (0.32%), including one female patient not treated with ACV who was infected primary by an ACV-resistant strain. Among the 54 immunocompromised patients from whom ACV-resistant HSV strains were isolated (3.5%), the bone marrow transplantation patients showed the highest prevalence of resistance (10.9%), whereas among patients infected by human immunodeficiency virus, the prevalence was 4.2%. In 38% of the cases, the patients who excreted the ACV-resistant strains were treated with foscarnet (PFA), and 61% of them developed resistance to PFA. The collection of a large number of isolates enabled an evaluation of the prevalence of resistance of HSV strains to antiviral drugs to be made. This prevalence has remained stable over the last 10 years, as much among immunocompetent patients as among immunocompromised patients.

Reduced efficiency of influenza vaccine in prevention of influenza-like illness in working adults: a 7 month prospective survey in EDF Gaz de France employees, in Rhône-Alpes, 1996-1997.

The efficiency of influenza vaccine was evaluated in the working population by comparing the percentage of people presenting with an influenza-like illness (ILI) according to their influenza immunization status, drug expenses and workdays lost. A self-completed questionnaire about the vaccination was sent to 5785 people randomly chosen among 18 249 workers. When any sick leave was incurred amongst the respondents (63.3%), of whom 301 were vaccinated and 3362 unvaccinated, a clinical form was completed by the private physician and the medical adviser of the firm (Electricité de France and Gaz de France). A final self-completed questionnaire was sent to people whose sick leave was not documented by a physician's reported diagnosis. In total, we obtained complete data for 90.9% of the sampling. The vaccine coverage rate of 8.2% [95% confidence interval (95% CI) = 7.4-9.0%] was higher in men than in women, increasing with age and professional category. Among the 775 subjects with a medical diagnosis, the vaccine effectiveness was not significant: 27.3% (95% CI = -13.8 to 53.5%). In the unvaccinated group, 9.6% had days absent from work, versus 7.0% in the vaccinated group; the two populations were comparable in terms of clinical symptoms, smoking habits, exposure to respiratory risk factors and chronic pathology. The average duration of sick leave for ILI was not significantly different between vaccinated (0.5 days) and unvaccinated workers (0.6 days). Despite the large size of the population and the occurrence of an epidemic due to a virus closely related to the vaccine strain (A/Wuhan/359/95), the vaccine did not effectively protect the small vaccine group nor result in an economic benefit, whatever the professional group.

Resistant herpes simplex virus type 1 infection: an emerging concern after allogeneic stem cell transplantation.

Fourteen cases of severe acyclovir-resistant herpes simplex virus type 1 (HSV-1) infection, 7 of which showed resistance to foscarnet, were diagnosed among 196 allogeneic stem cell transplant recipients within a 29-month period. Recipients of unrelated stem cell transplants were at higher risk. All patients received foscarnet; 8 subsequently received cidofovir. Strains were initially foscarnet-resistant in 3 patients and secondarily so in 4 patients. In vitro resistance to acyclovir or foscarnet was associated with clinical failure of these drugs; however, in vitro susceptibility to foscarnet was associated with complete response in only 5 of 7 patients. No strain from any of the 7 patients was resistant in vitro to cidofovir; however, only 3 of 7 patients achieved complete response. Therefore, acyclovir- and/or foscarnet-resistant HSV-1 infections after allogeneic stem cell transplantation have become a concern; current strategies need to be reassessed and new strategies must be evaluated in this setting.

Reactivation of acyclovir-resistant thymidine kinase-deficient herpes simplex virus harbouring single base insertion within a 7 Gs homopolymer repeat of the thymidine kinase gene.

HSV infections are treated efficiently and prevented by acyclovir, although resistant strains have been reported. Resistance to acyclovir involves mainly mutations in the viral gene encoding thymidine kinase; mutations may lead to an altered or, more frequently, deficient TK. These acyclovir-resistant TK deficient strains are not able to reactivate from a latent infection in an experimental model, compared to TK positive strains. A case is reported of a bone marrow transplant child who developed HSV infection at 11 days post-transplantation. Acyclovir-resistant HSV 1 was isolated on day 19 post-transplantation. The patient was cured of his infection. A resistant virus was detected 20 months later that harboured the same TK gene mutation as the first resistant virus. This mutation is an insertion of one guanine in a homopolymer repeat of seven guanines located at codon 146 of TK. It has previously been reported and associated with the expression of a deficient TK activity and the ability to reactivate in mice. These results corroborate the clinical relevance of this mutation, which is associated with acyclovir-resistant recurrent infections in humans.

Genetic characterization of thymidine kinase from acyclovir-resistant and -susceptible herpes simplex virus type 1 isolated from bone marrow transplant recipients.

Emergence of acyclovir (Acy)-resistant herpes simplex virus (HSV) is a major concern in bone marrow transplant recipients. Phenotypic and genetic characterization of thymidine kinase (TK) was done for 7 Acy-susceptible and 11 Acy-resistant HSV-1 isolated from 11 patients. In total, 19 amino acid substitutions were detected that were not related to Acy resistance but to TK gene polymorphism, including 5 mutations that have not been previously reported. The Acy-resistant strain from 1 patient presented no TK gene mutation related to resistance. Five patients (45%) had isolates that harbored point mutations leading to amino acid substitutions that could be associated with Acy resistance. Of the 5 substitutions detected, 3 have not been previously reported (codons 51, 83, and 175). A nucleotide insertion or deletion was detected in resistant isolates from 5 patients (45%); these mutations are located in homopolymer repeats at codon 92 (1 subject) and at codon 146 (4 subjects).

Estudio epidemiológico de virus respiratorios en niños en el Hospital Pediátrico Dr. Elías Toro IVSS, Caracas / Epidemiology study of respiratory viruses in children in Hospital Pediátrico Dr. Elías Toro IVSS, Caracas

Con el objeto de determinar el tipo de virus respiratorios causantes de síndromes tipo influenza en niños, demostrar la circulación de virus influenza y evaluar la compatibilidad con la cepa de las vacunas recomendadas para 1995 y 1996, se realizó un estudio epidemiológico en el Hospital Pediátrico "Elías Toro", (HPET) de Caracas, entre junio de 1995 y diciembre de 1996. Usando hisopos tipo VIROCULT, se recolectaron 70 muestras de secreción nasal en 1995 y 56 en 1996, provenientes de niños entre los 2 meses y los 13 años de edad, llevados al HPET con síndrome tipo influenza. Las muestras se analizaron en Francia, en el Centro Nacional de Referencia de la OMS en Lyon. Los resultados fueron: En 1995 se detectaron 22/70 virus respiratorios (31,4 por ciento), de los cuales el 21 por ciento (15) correspondieron a VSR, 7 por ciento (5) a influenza A, 1,4 por ciento (1) a parainfluenza y 1,4 por ciento (1) a ADV tipo 2. En 1996 se detectaron 11/56 (19,6 por ciento), de los cuales 10,7 por ciento (6) correspondieron a influenza A, 3,5 por ciento (2) a VSR y 1,7 por ciento (1) tanto para ADV tipo 1, ECHO tipo 1 y Coxsackie respectivamente. Los virus influenza aislados estuvieron relacionados con las cepas de las vacunas correspondientes al año de aislamiento. Se confirma la circulación de virus influenza en 1995 y 1996 en Venezuela. Las cepas de la vacuna para 1995 y 1996 se adaptaron a la epidemiología local. Se propone mejorar la toma de muestras, reactivar la vigilancia de virus respiratorios a nivel local, promover la importancia y necesidad de la vigilancia de virus causantes de infecciones respiratorias agudas y continuar chequeando la compatibilidad entre los virus influeza circulantes y las cepas contenidas anualmente en la vacuna

Giant cell arteritis, polymyalgia rheumatica, and viral hypotheses: a multicenter, prospective case-control study. Groupe de Recherche sur l'Artérite à Cellules Géantes.

OBJECTIVE: Although suspected, a viral etiology has never been proven in giant cell arteritis (GCA). We tested for viruses known to induce multinucleated giant cells in human pathology, which include the parainfluenza viruses (HPIV), respiratory syncytial virus, measles virus, herpesviruses type 1 and 2, and the Epstein-Barr virus. METHODS: A multicenter case-control study on incident cases of temporal arteritis (TA) and polymyalgia rheumatica (PMR). Population based age and sex matched controls were randomly selected. Serological tests for IgG and IgM directed against the viruses listed above were performed, on blood samples taken at the time of clinical diagnosis. RESULTS: Three hundred five new patients were included over a 5 year period, of whom 159 presented with positive biopsy TA, 70 with negative biopsy TA, and 76 with negative biopsy PMR. Thirty-eight percent of cases versus 20.9% of controls were positive for IgM directed against HPIV (p = 0.00005). The association was stronger in the positive TA subgroup [positivity rate 43.31%; odds ratio with controls 2.89 (95% CI 1.82-4.60, p = 0.000006)] than in the PMR or negative biopsy TA subgroups. Only HPIV type 1 was associated with the disease, regardless of the season or the geographical origin of the cases. Positivity rates for HPIV types 2 and 3 and for the other viruses tested were similar in cases and controls. CONCLUSION: Our findings suggest that reinfection with HPIV type 1 is associated with the onset of GCA in a subset of patients, particularly in cases with positive TA biopsy.

The mechanism of chronic coxsackievirus B hepatitis in rabbits.

The pathophysiology of chronic hepatitis in rabbits infected with coxsackievirus B5 (CVB5), (strain Mitchell) was investigated. Three-week-old male New Zealand White rabbits were inoculated intraperitoneally with 1 x 10(5) plaque forming units of virus. Every 3 months for 15 months postinoculation (p.i.) groups of animals were sacrificed for the following tests: interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-beta cytokine levels by enzyme-linked immunosorbent assay (ELISA); splenic natural killer (NK) cell function; sequence of a 154-bp section of the 5' noncoding region; antihepatocyte autoantibodies; histologic examination; in situ polymerase chain reaction (ISPCR) of the liver; neutralizing antibody response to CVB5; and viral cultures of liver, spleen, blood, brain, heart, skeletal muscle, and pancreas samples. Histologic evidence of hepatocyte necrosis was evident at each time point, although few inflammatory cells were seen. Liver samples were positive at each time by ISPCR, with viral nucleic acid localized to hepatocyte cytoplasm. Other cells in the liver did not stain. No hepatocyte autoantibodies were detected, and there was no elevation of intrahepatic cytokine levels compared to uninfected controls. There were no mutations in the virus over time. A vigorous neutralizing antibody response to CVB5, Mitchell was generated, but splenic natural killer (NK) function and numbers of splenic NK cells were significantly decreased. Virus culture was positive at 3 months, but negative at further time points. Cultures were negative at 3 months for the other tissues tested. Thus, CVB5, Mitchell causes a chronic hepatitis in rabbits, with virus limited to hepatocyte cytoplasm and no evidence of autoimmunity.

Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: a phase I clinical trial.

Ad CFTR, a replication-deficient adenovirus expressing the human cystic fibrosis transmembrane conductance regulator (CFTR), was administered by aerosolization in a single escalating dose to three pairs (cohorts) of cystic fibrosis (CF) patients. Buffer only was administered to the nose and lungs 9-14 days before nasal instillation of virus followed the day after by aerosolization of Ad CFTR to the lung. Nasal doses (defined in terms of viral plaque forming units, pfu) were 10(5), 10(7), and 4 x 10(8), whereas aerosolized doses were 10(7), 10(8), 5.4 x 10(8) for each cohort, respectively. No acute toxic effects were observed in the first 4 weeks after virus treatment. Shedding of infectious Ad CFTR was never detected, whereas detection of vector DNA sequences and CFTR expression demonstrated DNA transfer to the nose and airways of patients. No significant deviations in immunological and inflammatory parameters were observed in serum and in bronchoalveolar lavage (BAL). Importantly, for all patients, the serum anti-adenovirus antibody levels did not change significantly from baseline and no antibodies against adenovirus were found in BAL.

Surveillance of community-acquired viral infections due to respiratory viruses in Rhone-Alpes (France) during winter 1994 to 1995.

Nasal swab from patients with acute flu-like illness were evaluated for the presence of respiratory viruses in the Rhone-Alpes region of France from 1 October 1994 through 2 May 1995. The relative frequencies and seasonal distributions of the specific viruses were assessed. In addition, virus type was correlated with specific clinical signs and symptoms. During the study, 962 samples were collected by 75 medical practitioners participating in the Groupe Regional d'Observation de la Grippe surveillance network. One or more viruses were detected from 348 samples (36.1%), including 108 respiratory syncytial virus (RSV), 64 influenza virus A type H3N2, 47 influenza virus B, 64 coronavirus, 35 rhinovirus, 22 adenovirus, 5 enterovirus, and 3 parainfluenza-fluenza strains. There were 16 mixed infections. RSV infections peaked in the early winter, and influenza viruses A and B infections peaked during the late winter and early spring. There were two peaks of coronavirus infections (late fall and late winter). Other viruses were detected at lower levels throughout the study period. Patients from whom adenovirus was isolated were significantly more likely to have a fever of > 39.5 degrees C than were patients with other detectable viruses (P < 0.001). Furthermore, there was a significant correlation between influenza and cough (P < 0.01) and RSV and bronchiolitis (P < .001). Thus, the current study defined the overall and relative frequencies of respiratory virus detection from nasal swab specimens in patients with an acute flu-like illness in the Rhone-Alpes region of France during a 7-month period. Correlation with clinical signs and symptoms and provisional conclusions regarding seasonality were also determined.

Evaluation and comparison of PCR and hybridization methods for rapid detection of cytomegalovirus in clinical samples.

Rapid diagnosis of cytomegalovirus (CMV) infection may be obtained by molecular techniques, such as the polymerase chain reaction (PCR) and hybridization assays. The optimal technique to detect CMV in clinical samples was assessed. Two different PCR assays were used, targeting either the major immediate early 1 (MIE 1) or the HXLF 4 gene. The PCR products were detected by gel electrophoresis, dot blotting and an easy to use, rapid, solid phase hybridization assay, DNA enzyme immunoassay (DEIA). Standard tissue culture was also used. Cerebrospinal fluids (18), liver biopsies (9) from hepatic transplant recipients, amniotic fluids (7) from mothers with suspected peripartum infection, and samples (6) of miscellaneous origin (brain and fundus biopsy, pericardial and pleural fluid) were tested. Among the 40 samples, CMV was detected in 19 cases. Three were positive by both molecular techniques and tissue culture, 14 by molecular methods and 2 by culture. 16/19 or 9/19 CMV-positive samples were detected by PCR amplification of the HXLF 4 or MIE 1 gene, respectively and 14/16 HXLF 4-positive samples were detected using either dot-blot or DEIA, compared to 9/16 using gel electrophoresis. Thus, the most sensitive assays for the detection of CMV in clinical samples using the methods compared in the current study were PCR amplification of the HXLF 4 gene followed by dot-blot or DEIA hybridization.

[Aerosol administration of a replication defective recombinant adenovirus expressing normal human cDNA-CFTR in the respiratory tractus in patients with cystic fibrosis]. / Administration par aérosol d'un adénovirus recombinant déficient pour la réplication contenant cADN-CFTR humain normal dans le tractus respiratoire de patients atteints de mucoviscidose.

At present it is conceivable to think that gene therapy represents a way to treat or even prevent the respiratory manifestations of cystic fibrosis. Consistent to such a concept, there is sufficient evidence that Ad-CFTR, a recombinant replication-deficient adenovirus expressing the human cystic fibrosis transmembrane conductance regulator cDNA, can vectorize the expression of a functional CFTR (cystic fibrosis transmembrane conductance regulator) to the nasal and airway epithelia. The clinical protocol was designed to assess the safety of single escalating doses of a replication defective adenovirus expressing the cystic fibrosis transmembrane conductance regulator gene (Ad-CFTR) when administered to the tracheobronchial portion of the airways and whether biological efficacy of CFTR delivery could be demonstrated. Six cystic fibrosis patients received nasal instillation and subsequent aerosol (Optineb, Air Liquide, Paris, France) administration of Ad-CFTR the following day. Doses (pfu) applied to the nose were 10(5) (patients SG and PB), 10(7) (patients FP and EP) and 4 x 10(8) (patients DS and FG), while aerosolised doses were 10(7) (patients SG and PB), 10(8) (patients FP and EP) and 5.4 x 10(8) (patients DS and FG), respectively. No acute toxic effects, no increase in the titer of anti-adenovirus antibodies and no spreading or shedding of Ad-CFTR were detected. In one patient Ad-CFTR DNA was found in the urine and blood two days after aerosolisation. Ad-CFTR DNA was detected in nasal and bronchial brush samples, in BAL, in saliva and tonsils 21, 8, 14 and 4 days post virus administration, respectively. Ad-CFTR mRNA (RT-PCR on bronchial cells) and CFTR protein (immunochemistry on nasal and bronchial cells) were detected up to 14 days following Ad-CFTR administration. These results show that the nebulisation of Ad-CFTR is a possible approach for treating the respiratory manifestation of cystic fibrosis.

Epidemiology of viral infections and evaluation of the potential benefit of OM-85 BV on the virologic status of children attending day-care centers.

Viral investigations were performed during 4 winter seasons (88/89, 89/90, 92/93, 93/94) in children attending day-care centers (DCCs) in the Rhône Département in eastern France. Over the total observation period of 4 winter seasons, 780 children were screened with a nasal swab for the presence of viruses. Of those, 230 (29.5%) had a positive viral culture. The viruses identified were respiratory syncytial virus (RSV), influenza A and B virus, parainfluenza virus, coronavirus, rhinovirus, adenovirus and enterovirus. During that time, 83 epidemic events in 47 DCC were recorded. A particular virus was judged to be causally related to an epidemic if the identical virus was isolated in > or = 3 children during the same outbreak of respiratory diseases. Thus, in 51 cases (61.4%) of all epidemics, the following viruses were responsible for an epidemic: RSV (n = 23), coronavirus (n = 10) (only during the season of 1993-1994), influenza A virus (n = 6), rhinovirus (n = 4), enterovirus (n = 4), adenovirus (n = 3) and parainfluenza virus (n = 1). Except for the somewhat surprising accumulation of coronavirus epidemics during the winter of 1993-1994, there were only minor seasonal variations from one year to another. As expected, RSV accounted for about one third of all respiratory tract infections in children attending DCCs and was therefore the most important single causative agent. These results are compared with data from children who did not attend a DCC and were cared for in a private practice.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid diagnosis of influenza A. Comparison with ELISA immunocapture and culture.

The Directigen Flu-A is an enzyme immunoassay for detecting in 15 min the influenza A nucleoproteinic antigen directly from specimens after passive adsorption on a cellulose membrane. The test was assessed using 160 frozen (-20 degrees C) specimens collected during the 1988-1989 A/H1N1 influenza epidemic and the 1989-1990 A/H3N2 epidemic. Compared to the ELISA immunocapture test, the sensitivity of the commercial test was 87.8% and the specificity was 97.6%. When compared to isolation of viruses on LLCMK2 cells and/or chicken embryo, the sensitivity was 84%. No cross-reaction was found with other respiratory disease viruses. The feasibility, practicability and rapidity of the test make it a test of choice for rapid diagnosis of influenza A.

Comparison between three rapid methods for direct diagnosis of influenza and the conventional isolation procedure.

Besides the rapid diagnostic tests based on influenza A and B antigens nucleoproteins detection, which are routinely used, the isolation of influenza strains is still required to obtain recent variant isolates for full antigenic characterization, in order to up-date the influenza vaccine composition. To increase the rapidity and the efficacy of the virus growth, we implemented a culture test in 24-well plates by centrifugation of samples on to LLCMK2 cells in the presence of trypsin. This test was routinely applied to 331 nasopharyngeal swabs collected during the influenza A outbreak in the winters 1988-1989 and to 962 in 1989-1990. The centrifugation culture assay has been compared with the direct detection of NP antigens in the clinical samples by immunofluorescence and capture ELISA tests and with the conventional virus isolation by inoculation of the samples to embryonated eggs and to LLCMK2 cell cultures. Compared with the NP antigen detection tests, the centrifugation culture assay closely correlated (r = 0.95) and the sensitivity and specificity were also excellent, 93.4% and 99.6%, respectively. Compared with the conventional culture assays, the centrifugation culture markedly increased the performance (five times) and rapidity (2 days) of influenza virus isolation and identification.

Heterogeneity of capsid proteins of echovirus type 25 wild-type strain and prototype strain, studied by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were used to compare the capsid proteins of 19 antigenic variants of echovirus type 25 wild-type strains isolated in France between 1976 and 1987 with those of the prototype JV-4 reference strain isolated in 1957. Immunoblots were developed by using polyclonal sera from rabbits and mice immunized with the reference strain. Immunoblotting patterns revealed reactivity only against viral protein VP1 for sera from both animals. Comparative immunoblotting patterns showed differences in the electrophoretic mobilities of viral protein VP1, especially for the Montpellier 76.1262 wild-type strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of [35S]methioinine-labeled viral polypeptides revealed that the two variant strains, Montpellier 76.1262 and Thionville 86.222, exhibited significant and reproducible shifts in the relative mobilities of VP1 and VP3 and, to a lesser extent, in those of VP0 and VP2. The relative mobility of VP4 seemed very similar for the JV-4 reference strain and the two variants. Interestingly, the structural differences in VP1 and VP3 of Montpellier 76.1262 were not correlated with the pattern of neutralization by monoclonal antibodies, unlike in our previous study, in which this strain differed from the prototype strain in only two epitopes. We concluded that, in addition to the heterogeneity of their biological and antigenic properties that we observed previously, echovirus type 25 wild-type strains may exhibit differences in their structural proteins.