[Enteroviruses responsible for acute hemorrhagic conjunctivitis]. / Les Enterovirus responsables de conjonctivite aiguë hémorragique.

Acute hemorrhagic conjunctivitis (AHC) is an epidemic form of highly contagious conjunctivitis, characterized by conjunctival hemorrhages. The first AHC outbreak was described in 1969 in Ghana, West Africa, and was called Apollo disease, from the Apollo landing on the moon. This outbreak was caused by Enterovirus 70 (EV70) together with a Coxsackievirus A24 (CVA24v) variant, which are the major etiological agents involved in AHC outbreaks worldwide. AHC is known to be directly transmitted by close person-to-person contact or indirectly through soiled ophthalmological materials or unsafe recreational water. Recently, a possible airborne virus spread was suggested which could explain the high transmission rate of the disease. In the absence of a specific antiviral therapy, a rapid diagnosis of the causative agent is required to distinguish AHC due to enteroviruses from other ocular infectious diseases, for there are active drugs, or to quickly implement proper public health measures to limit the extension of the outbreak. However, virus identification remains difficult and time-consuming. Moreover, virological diagnosis is difficult to implement in developing countries where AHC has recently become a major problem for public health.

[Characterisation of viral agents with potential to cause diarrhea in Djibouti]. / Caractérisation des virus agents potentiels des diarrhées chez l'adulte à Djibouti.

Due to limited laboratory facilities in the tropics, the exact role of enteric viruses in causing diarrhea among adults in the tropics is unknown. The purpose of this report is to describe a multicenter study undertaken in Djibouti to determine the prevalence of a large panel of enteric viruses using immunochromatography; antigenic detection by ELISA, RT-PCR cellular inoculation, sequence analysis; and indirect serology. Study samples were collected from 108 patients presenting acute and sporadic diarrhea. Although they are well known causes of diarrhea in children, rotavirus and adenovirus were identified in only 2 and 5% of adults respectively. In contrast human caliciviruses (HuCVs) and enterovirus were identified in 25 and 42% of adult cases respectively. Uncommon genotypes of HuCVs and recombinant forms (junction pol/l cap) as well as a significant number of sapovirus (30%) were identified. Further study is needed to clarify the role of enterovirus (echovirus) in the etiology of acute diarrhea in adults. No polivirus was identified. These new data from the Horn of Africa increase our knowledge about the epidemiology of acute infectious diarrhea that is a major public health problem and potential danger for travelers.

Surveillance of enteroviruses in France, 2000-2004.

In the context of poliomyelitis eradication, a reinforced sentinel laboratory network for surveillance of enteroviruses (RSE) was implemented in France in January 2000, and the purpose of this report is to describe the results of the five first years of surveillance. From 2000 to 2004, the RSE laboratory network performed detailed surveillance of the circulating enteroviruses. No wild-type poliovirus was isolated from humans during the 5 years of surveillance, although two imported vaccine polioviruses were detected. During the same period, Sabin-like polioviruses were identified on five occasions in the sludge from sewage treatment plants, but no wild-type poliovirus was found. Over the 5 years of surveillance, information was collected from 192,598 clinical samples, including 39,276 cerebrospinal fluid specimens, of which 14.7% were positive for enteroviruses, 45,889 stool samples (4.3% positive for enteroviruses), 70,330 throat swabs (2.2% positive) and 14,243 sera (1.4% positive). The ten main nonpolio enteroviruses typed were as follows, in decreasing order of frequency: E-30, E-13, E-6, CV-B5, E-11, CV-B4, E-9, E-7, CV-B1, and CV-B2. During the year 2000, an outbreak of aseptic meningitis due to three main enteroviruses (echoviruses type 30, 13, and 6) was monitored. Continued surveillance of enteroviruses is important to alert physicians and public health officials to changes in disease trends. Although the geographical coverage of the RSE network as well as the percentage of enteroviruses identified must be improved, the large number of samples tested for enteroviruses shows the ability of virology laboratories to detect the circulation of enteroviruses and to report the possible identification of poliovirus (wild-type, vaccine-derived, or Sabin-like).

Two outbreaks of acute hemorrhagic conjunctivitis in Africa due to genotype III coxsackievirus A24 variant.

Reported here are two outbreaks of acute hemorrhagic conjunctivitis that occurred in the Democratic Republic of the Congo and in Morocco in the summers of 2003 and 2004, respectively, with a large impact on public health. Virus was isolated from the conjunctival swabs of 30 Congolese and 20 Moroccan patients. Enterovirus-specific cytopathic effect was observed in all samples. None of the strains could be typed using a conventional neutralization assay with the Melnick intersecting pools; however, by sequencing the VP1 region, the viruses could be identified as coxsackie A24 variants. Phylogenetic analysis of the 3C protease region revealed that these strains were closely related to each other as well as to genotype III isolates detected in Korea in 2002, thus proving their worldwide spread. This is the first report of an epidemic of acute hemorrhagic conjunctivitis due to a coxsackievirus A24 variant in Africa since 1987 and the first ever from Morocco.

Caracterisation des virus agents potentiels des diarrhees chez l'adulte a Djibouti

Chez l'adulte; en zone tropicale; la part exacte des virus enteriques dans les diarrhees infectieuses aigues; demeure inconnue faute de moyens diagnostiques. Une etude multicentrique a permis de preciser la prevalence virale; chez des patients presentant des diarrhees aigues sporadiques a Djibouti. A partir d'un echantillon de 108 sujets; un large panel de virus enteriques a ete recherche par immunochromatographie; detection antigenique en ELISA immunocapture; RT-PCR; inoculation sur cellules permissives; analyse de sequences; et methodes indirectes en serologie. Les rotavirus et adenovirus agents principaux des gastroenterites chez l'enfant representent respectivement 2 et 5des cas. Les astrovirus; 5des cas. En revanche; les calicivirus humains (HuCVs) et enterovirus sont retrouves dans 25et 42des cas respectivement. Des genotypes inhabituels de HuCVs et des formes recombinantes (jonction pol / cap) ont ete mis en evidence; ainsi qu'un nombre relatif eleve de sapovirus (30). La participation des enterovirus (echovirus) dans l'etiologie des diarrhees sporadiques de l'adultemerite d'etre approfondie.Aucune souche de poliovirus n'a etemise en evidence. Ces nouvelles donnees concernant la Corne de l'Afrique renforcent nos connaissances epidemiologiques sur les diarrhees infectieuses aigues; probleme majeur de sante publique; et danger potentiel pour les voyageurs

Caracterisation des virus agents potentiels des diarrhees chez l'adulte a Djibouti

Chez l'adulte; en zone tropicale; la part exacte des virus enteriques dans les diarrhees infectieuses aigues; demeure inconnue faute de moyens diagnostiques. Une etude multicentrique a permis de preciser la prevalence virale; chez des patients presentant des diarrhees aigues sporadiques a Djibouti. A partir d'un echantillon de 108 sujets; un large panel de virus enteriques a ete recherche par immunochromatographie; detection antigenique en ELISA immunocapture; RT-PCR; inoculation sur cellules permissives; analyse de sequences; et methodes indirectes en serologie. Les rotavirus et adenovirus agents principaux des gastroenterites chez l'enfant representent respectivement 2 et 5des cas. Les astrovirus; 5des cas. En revanche; les calicivirus humains (HuCVs) et enterovirus sont retrouves dans 25 et 42des cas respectivement. Des genotypes inhabituels de HuCVs et des formes recombinantes (jonction pol / cap) ont ete mis en evidence; ainsi qu'un nombre relatif eleve de sapovirus (30). La participation des enterovirus (echovirus) dans l'etiologie des diarrhees sporadiques de l'adulte merite d'etre approfondie.Aucune souche de poliovirus n'a ete mise en evidence. Ces nouvelles donnees concernant la Corne de l'Afrique renforcent nos connaissances epidemiologiques sur les diarrhees infectieuses aigues; probleme majeur de sante publique; et danger potentiel pour les voyageurs

Three ECHOvirus serotypes responsible for outbreak of aseptic meningitis in Rhône-Alpes region, France.

During the year 2000 in the Rhône-Alpes region of France, 559 cases of aseptic meningitis due to enterovirus infection were recorded (approximate incidence, 10 cases per 100,000 population compared with a mean of 0.3 cases during endemic years in this region; P<0.001). Cerebrospinal fluid samples were collected from all 559 patients and processed for enterovirus RNA detection using a reverse transcriptase polymerase chain reaction assay only. In addition to the cerebrospinal fluid samples, 40 stool and 76 throat samples were collected from 116 patients (20.7% of cases); the following three enterovirus serotypes were isolated from the stool and throat samples only: ECHOvirus 13 (48/116; 41.3%), ECHOvirus 30 (44/116;37.9%) and ECHOvirus 6 (24/116, 20.7%). This is the first report that demonstrates the involvement of three different ECHOvirus serotypes, particularly ECHOvirus 13, in an outbreak of aseptic meningitis, in the French Rhône-Alpes region.

Application of a real-time polymerase chain reaction with internal positive control for detection and quantification of enterovirus in cerebrospinal fluid.

A quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method based on TaqMan technology was developed to determine the presence and amount of enterovirus RNA. In order to prevent false-negative results, a one-step multiplex RT-PCR was optimized. It contains two dual-labelled fluorogenic probes to quantify the 5' noncoding region of enterovirus and detect an internal positive control. In the present study, 104 cerebrospinal fluid samples collected during an outbreak of enteroviral meningitis were analyzed using this method. Amplification of the internal positive control was effective in all but two specimens, confirming the absence of PCR inhibitors and allowing the results of amplification to be validated. The sensitivity of the RT-PCR was 96.8%, while that of cell culture was 34.9%. Genomic viral loads found ranged between 3.3 and 5.9 log(10) copies per milliliter of cerebrospinal fluid (mean, 4.8 log(10) copies/ml). This fluorogenic enterovirus RT-PCR allows large numbers of samples to be screened rapidly. Moreover, its sensitivity and reproducibility make it highly reliable. With these characteristics, the enterovirus RT-PCR can be a useful tool that may offer considerable benefit in the clinical management of patients with enteroviral infections.

Paralytic poliomyelitis associated with the Sabin 3 revertant strain of poliovirus in Bahrain.

We report a case of vaccine-associated paralytic poliomyelitis (VAPP) in Bahrain. The case occurred in an 8-week-old infant who had received a dose of oral polio vaccine (OPV) 7 days after birth. She was in contact with two vaccinees who had received OPV during the national immunisation campaign conducted 10 days before her birth. Specimens from the infant were sent to the WHO Collaborating Centre for Virus Reference and Research Laboratory for serological testing and virus detection, including genomic sequencing. Clinical and virological features are presented of a case of VAPP caused by the Sabin 3 strain of poliovirus that had reverted towards neurovirulence. The case represents one in 51,879 first doses of OPV distributed between 1995 and 1998. In order to reduce further the risk of VAPP, the dose of OPV at birth has been discontinued and a sequential schedule of inactivated polio vaccine (IPV) followed by OPV will be recommended.

Role of cytomegalovirus infection in the incidence of viral acute respiratory infections in children attending day-care centers.

In a prospective study carried out in Lyon, France, the association between the excretion of cytomegalovirus (CMV) and the increasing frequency and severity of viral respiratory infections in children attending day-care centers was evaluated. Urine samples were collected in November 1992 (S1) and 4 months later in February 1993 (S4). A total of 246 children aged 6-12 months attending 29 day-care centers from 1 November to 28 February were screened for the excretion of CMV in urine. The diagnosis of viral acute respiratory infection was performed in the case of outbreaks only. Forty-eight (19.5%) children were both S1 and S4 positive for CMV, 30 (12.4%) became CMV positive (S1-/S4+), 4 (1.6%) became negative (S1+/S4-) and 164 (66.7%) remained negative. The percentage of children becoming CMV positive was significantly (P<0.001) higher in day-care centers where more than 40 children were enrolled. Nine outbreaks due to respiratory syncytial virus, rhinovirus and enterovirus were recorded in 8 of 29 (27.6%) day-care centers. Viral acute respiratory infections were significantly (P<0.05) more frequently recorded in day-care centers in which CMV and respiratory viruses cocirculated and were significantly (P<0.001) more frequently reported in CMV-infected children. These findings suggest that viral acute respiratory infections are significantly more likely to occur in CMV-infected children.

Rapid direct diagnosis of mumps meningitis by ELISA capture technique.

ELISA capture technique (ELISAc) was carried out using a rabbit hyperimmune serum attached to a solid phase for capturing mumps antigens in cerebrospinal fluid (CSF) in patients with meningitis and/or in supernatants of infected Vero cells. A biotin-labelled rabbit serum prepared from the previous serum was added and the reaction was read by an enzymatic (avidine-peroxidase) reaction by automated reading. The cut-off was calculated in 100 CSFs negative for viruses by conventional diagnosis. The specificity was evaluated in Vero cells infected with 22 CSFs collected from vaccinated children (URABE AM9 attenuated vaccine) who developed meningitis. A guinea pig hyperimmune serum confirmed the specificity. Results in culture correlated with the ELISA capture technique (ELISAc). No cross-reactivity was observed with parainfluenza 1, 2, 3 human reference strains. At least 2.5 ngs of purified mumps proteins were detected corresponding to 10(1.5) infectious particles per ml. ELISAc applied directly to 14 CSFs collected from unvaccinated children with meningitis diagnosed five positive cases, whereas in four cases conventional diagnosis had to be undertaken twice. ELISAc permitted the diagnosis of one additional patient. The test can be carried out in 3 h.

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)

Stimulation of nonspecific immunity to reduce the risk of recurrent infections in children attending day-care centers. The Epicrèche Research Group.

A randomized, double blind, placebo-controlled clinical trial was performed in 423 children attending day-care centers to assess whether stimulating nonspecific immunity would reduce the incidence of recurrent infections. The drug used for the trial (Imocur) is an extract obtained from eight different species of bacteria. At the end of the total follow-up period (3 months with treatment and 4.5 months without), the risk for > or = 4 episodes of upper respiratory infections was not significantly lower in the treated group than in the placebo group (26.7% vs. 33.8%, relative risk, 0.79; 95% confidence interval, 0.59 to 1.06). In an exploratory analysis limited to the 3-month treatment period, however, we observed a 48% reduction in the risk of presenting > or = 3 episodes of upper respiratory infections: 9.5% vs. 18.3%, respectively, in the treatment group and the placebo group (relative risk, 0.52; 95% confidence interval, 0.31 to 0.86). Similar results were found for the risk of > or = 1 episode of gastroenteritis. We also observed a strong correlation between the drug efficacy and age; this observation is coherent with the underlying pathophysiologic model in which the immune system matures with age.

A nylon membrane enzyme immunoassay for rapid diagnosis of influenza A infection.

A new membrane-enzyme immunofiltration assay (MIFA) was developed for rapid diagnosis of influenza A infection. The pretreated specimens were dispensed into a 1.2 micron Biodyne B nylon membrane-bottomed microplate and vacuum filtration was applied. Blocking solution, peroxidase-conjugated anti-influenza A nucleoprotein monoclonal antibody, washing buffer and substrate were added in that order. The assay was completed within 30 min. Out of 103 nasopharyngeal swabs collected in transport medium, 31 isolates of influenza A virus were obtained and 22 specimens were detected directly by the MIFA technique. The 9 isolation-positive MIFA-negative specimens required 6 days or more for viral detection in cell culture, and probably contained a very low quantity of virus. The 72 cell culture negative specimens were also negative by MIFA. Comparison with a classical immunocapture assay (ICA) gave a better sensitivity for MIFA, as only 15/103 specimens were positive by ICA. MIFA is a rapid test with 71% sensitivity and 100% specificity. It was also very useful to test the cell culture supernatants, as a sensitivity of 100% was obtained with MIFA when the immunofluorescence technique was positive. The same technique could be readily carried out on the same plate for other respiratory viruses since capture antibody is not used.

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.