Variations in the recognition of echovirus type 11 strains by a group-specific anti-VP1 monoclonal antibody.

BACKGROUND: Mab 5-D8/1 is a monoclonal antibody (Mab) that was shown to be directed towards a conserved epitope of the capsid protein VP1 among the genus enterovirus. The use of this Mab for the routine detection of enteroviruses in clinical specimens led to the observation that several strains of echovirus type 11 (EV-11) could not be detected on spontaneously detached cells from 26-h cultures using a two-step immunofluorescence (IF) assay. Conversely, these strains were detected positive with the same Mab when tested on adherent or trypsinizated cells. OBJECTIVES: The aim of this study was to understand the misrecognition of some strains of EV-11 by this Mab. STUDY DESIGN AND RESULTS: IF tests at different times of the viral cycle brought evidence that the detection of a variant strain of EV-11 decreased rapidly with time, becoming undetectable 26 h post-infection, since the reference strain remained positive up to 46 h post-infection. The infective titres of the variant strains were shown to be high in comparison with those of well-recognised strains. Sequencing the Mab binding epitope confirmed that the variant strains exhibited no antigenic shift. CONCLUSION: These results suggest that the poor recognition of some strains of EV-11 by Mab 5-D8/1 is due to a rapid decrease of the expression of the binding epitope in the cell, maybe in relation with the high lytic power of these strains. From a practical point of view, our data indicate that a negative result when Mab 5-D8/1 is used for enterovirus typing must be interpreted cautiously with highly replicative strains and that detached cells should not be used for enterovirus identification under these circumstances.

Characterization of nosocomial strains of Enterobacter aerogenes by arbitrarily primed-PCR analysis and ribotyping.

OBJECTIVE: To study the spread of strains of Enterobacter aerogenes in our hospital in 1992 and 1993 by using two genotypic markers, and to evaluate these methods for the epidemiological investigation of this species. DESIGN: Ribotyping (using two endonucleases) and arbitrarily primed (AP)-PCR (using two different 10-mer primers) were applied to the epidemiological typing of clinical strains of E aerogenes isolated from hospitalized patients. SETTING AND PATIENTS: The intensive care unit (ICU; 5 patients, 13 isolates), nephrology units (3 patients, 5 isolates), and surgery units (2 patients, 2 isolates) of the university hospital of Saint-Etienne (France). RESULTS: Eight epidemiologically unrelated isolates, chosen as controls, exhibited distinct profiles, both by AP-PCR and ribotyping. Two clones of E aerogenes circulated in the ICU; both were isolated successively from samples of a single patient who stayed in the unit for almost 1 year. A third clone was recovered from patients of surgery units. A fourth clone was shown to have infected patients of nephrology units. CONCLUSIONS: Ribotyping and AP-PCR appear to be reliable methods for typing E aerogenes strains implicated in nosocomial infection. The spread of independent clones of E aerogenes in different units of our hospital in 1992 and 1993 was demonstrated by both methods. This study emphasizes the need to choose the endonucleases or primers with care to obtain high discriminatory results in genotypic investigations.

Differentiation of Pseudomonas aeruginosa strains by ribotyping: high discriminatory power by using a single restriction endonuclease.

The genotypic diversity of 40 presumably epidemiologically unrelated strains of Pseudomonas aeruginosa belonging to nine different O-serotypes was analysed according to ribosomal DNA fingerprints. Ribotyping was performed with a digoxigenin-labelled DNA probe and four restriction endonucleases. Characteristic banding patterns of three to 12 bands were obtained with the different endonucleases. Among the 40 strains, eight, nine, 10 and 29 different ribotypes were differentiated with EcoRI, the combination EcoRI+HindIII, BamHI and PvuII, respectively. Poor correlations were noted between the results of serotyping and those of ribotyping. With the latter method, indices of discrimination were calculated for each enzyme from the data of the 40 unrelated strains: the values ranged from 0.678 for EcoRI to 0.979 for PvuII. Epidemiologically related samples were also tested; this enabled assessment of whether the method was able to cluster strains from a common origin with each of the enzymes tested. Ribotyping with PvuII endonuclease is proposed for screening large numbers of P. aeruginosa strains in epidemiological studies. Additional enzymes could be used to further increase the discrimination between isolates found to be indistinguishable with PvuII enzyme.

Immunoblotting patterns with Mycoplasma pneumoniae of serum specimens from infected and non-infected subjects.

Two hundred and ninety-four serum specimens from 248 subjects, whose complement fixation (CF) titres to Mycoplasma pneumoniae were known, were further investigated by IgG immunoblotting. After analysis of M. pneumoniae proteins by SDS-PAGE, nine polypeptides (p) with mol. wts of 180-43 Kda were selected for immunoblotting studies. Antibodies to M. pneumoniae measured by immunoblotting appeared progressively with age; most subjects more than 19 years old gave positive results. For most of the polypeptides, there was an increase in the frequency of band detection when the CF titres were higher. Furthermore, paired serum specimens from 10 patients with M. pneumoniae infection, as demonstrated by a rise in CF antibody titre, were tested for IgG blotting patterns. Generally, p180 (the P1 adhesin of M. pneumoniae), p172 and p84 were shown to be the dominant targets of the immune response to this organism and may have diagnostic value.

Competition binding studies with biotinylated echovirus 11 in cytofluorimetry analysis.

Competition binding studies between viruses are usually performed with radiolabelled probes. In this report, a cytofluorimetric method using biotinylated echovirus (EV) 11 is described for the study of competition of enteroviruses for a common cell receptor site. An N-hydroxysuccinimide ester biotin spacer arm was used for biotinylation of CsSO4-purified EV 11. Biotinylation did not change the infectivity of the virus (attachment to and replication in susceptible cells). With the exception of EV 22 and EV 23, all the echovirus serotypes and also coxsackievirus A9 (CA 9) were able to inhibit the absorption of biotinylated EV 11 onto cells. The taxonomic implications of these findings are discussed.

Detection of immunoglobulin G, M, and A antibodies to enterovirus structural proteins by immunoblot technique in echovirus type 4-infected patients.

Paired serum specimens from 24 patients with echovirus (EV) type 4 infection by virus isolation were tested by the immunoblot technique for the presence of IgG, IgM, and IgA antibodies to EV4 structural proteins. Single sera from 20 patients without neutralizing enterovirus IgM were used as controls. All the sera from EV4-infected patients had IgG antibodies to VP1 of EV4 but also 13 out of the 20 controls. 23 out of 24 EV4-infected patients elicited IgM and IgA specific antibodies to VP1, a pattern highly significant as compared with controls (3/20 for IgM and 8/20 for IgA). In 16 out of the 24 EV4-infected patients, the IgM antibodies were also directed against VP2 (versus 2 out of 20 in the control group). Anti-VP2 IgA were detected in 4 out of the 24 EV4 patients (versus 0 in controls). The 24 paired sera from EV4-infected subjects were also tested by immunoblot technique against three other enteroviruses: EV21, coxsackievirus A9 and poliovirus 1. Cross-reactivities were observed to a large extent against VP1 and VP2 proteins with the three classes of antibodies. These results confirm the data of previous studies on the reactivity of IgM antibodies to various structural proteins that IgG antibodies react exclusively to VP1. Furthermore, this study demonstrates the occurrence of circulating IgA antibodies directed to VP1 and sometimes VP2 in the course of enterovirus infection. The potential interest of this latter finding for diagnosis requires further investigation.

Ventilator temperature sensors: an unusual source of Pseudomonas cepacia in nosocomial infection.

A prospective study was undertaken to determine the source of Pseudomonas cepacia colonization and infection that had affected ventilated patients in an Intensive Care Unit (ICU) for three years. Thirty-eight patients undergoing mechanical ventilation were enrolled during a six-week period. Samples were taken from patients, ventilator circuits and the environment for culture. P. cepacia was isolated from the condensate formed in the ventilator circuit and the source of the contamination was shown to be the temperature sensor. Ribotyping of the representative strains of P. cepacia performed with two endonucleases, EcoRI and PvuII, confirmed the homogeneity of the isolates from patients and ventilator circuits. A modification of the procedure for disinfection of the temperature sensors resulted in the eradication of P. cepacia from the ICU.

A gel filtration method to test the efficiency of virucidal disinfectants.

We have described a gel filtration technique for assay of the virucidal potency of disinfectants. It allows a complete separation of disinfectant from the virus after contact, thus preventing chemical cytotoxicity. Very low residual infectious activity can be measured. There is no need for a high viral titer antigen.

Live poliovirus vaccine in patients with chronic glomerulonephritis: effects on renal function and specific antibody response.

Twenty-one patients with chronic glomerulonephritis (GN) (5 with renal failure) received three doses of live trivalent poliovirus vaccine administered orally. The effect of the polio vaccination on the renal function and the titers of antibodies to poliovirus were studied. No significant consequence was observed in renal disease. Before vaccination, titers of poliovirus type 1 and 3 antibodies were significantly decreased as compared to healthy adult subjects. After vaccination, the patients exhibited a significant rise in poliovirus antibody titers for the three serotypes, although some of them failed to develop a fourfold or greater antibody rise to at least one of the three serotypes, especially in the group of patients with renal failure. These results indicate that live poliovirus vaccination is not deleterious in patients with GN and can provide a good protection.

[The concept of risk in poliomyelitis (author's transl)]. / La notion de risque dans la poliomyélite

In poliomyelitis the risk is of neurological accidents resulting either from the circulation of wild viruses (spontaneous risk) or from the introduction of virus-vaccine (vaccine risk). The spontaneous risk varies both according to the virus type and according to various factors which determine host resistance. Well known among these are: the intrinsic characteristics of the population at a given period (genetic factors and previous experiences of this population with poliomyelitis viruses); environmental factors (water supply, living conditions, rapid urbanization...) which are extremely variable among geographic zones, and cultural factors just as variable among populations. The vaccine risk involves two aspects: the risk of neurological accidents due to use of live vaccine, which has changed from the beginning of vaccination and risk of inefficacy due either to the vaccine itself, whether live or inactived or to temporary ill-understood unfitness of a vaccine to provoke an immunitary response. At the present time, spontaneous and vaccine risks are balanced only in the so called developed countries in which the vaccine risk is accepted as necessary to maintain the spontaneous risk at the lowest level.

[Viral flora (coliphages and human enteroviruses) found in river water after an urban district (Saint-Etienne). I. Comparative study of four simple and inexpensive methods for concentration and isolation of river-water viruses (author's transl)]. / Inventaire virologique (coliphages et entérovirus humains) dans une eau de rivière en aval d'une zone urbaine (Saint-Etienne). I. Etude comparée de quatre méthodes simples et économiques de concentration et d'isolement des virus

Four methods for the concentration of human enteroviruses in the waters of the river Furan were surveyed: the original method described by WALLIS, of adsorption of viruses on PE-60 polyelectrolyte; a modified version of the Wallis method; a method using a different polyelectrolyte available in France, the POE; the two-phase system described by SHUVAL. The best results were obtained when using the method of adsorption on polyelectrolytes, and the second method was finally chosen for field surveys.

[Polio immunity in North African children hospitalized for paralytic poliomyelitis due to type 1: relative deficiency in humoral immunity compared with European children (author's transl)]. / Immunité antipoliomyélitique acquise par les enfants d'Afrique du Nord après poliomyélite paralytique à type 1. Déficience relative de l'immunité humorale comparée à celle des enfants européens.

Three points may be emphasized: 1. The antibody titers in North African children are lower than previously reported for European children during the period 1950-1960. 2. In both groups of children, there is a positive correlation between the antibody titers and the age of infection (paralysis or vaccination). 3. The non polio enterovirus present in the intestine of paralyzed children at the same time as the poliovirus seem to suppress the polio immunity. This last point should be confirmed on a larger scale.

[Viral flora (coliphages and human enteroviruses) found in river water after an urban district (Saint-Etienne). II. A virological and epidemiological survey (author's transl)]. / Inventaire virologique (coliphages et entérovirus humains) dans une eau de rivière en aval d'une zone urbaine (Saint-Etienne). II. Etude virologique et épidémiologique

A survey was carried out from March 1972 to February 1973 to identify viral flora found in the river Furan ater St. Etienne. The 54 samples examined revealed the following data: 1) more accurate results are obtained when viral concentration values are expressed in terms of m3/sec., taking into account the flow of the river and eliminating the seasonal dilution factor; 2) rates of enteroviruses remain constant throughout the year, in spite of a relatively rapid spontaneous inactivation of the viruses; 3) rates of coliphages vary considerably according to seasons, with a notable increase in summer; 4) the two previous data are unrelated; 5) 147 enterovirus strains were isolated, of which 44% were polioviruses; 6) virulent and attenuated types 2 and 3 polioviruses were found simultaneously at certain periods; 7) only virulent strains of type 1 poliovirus were isolated; 8) this type of survey may be useful in controlling the endemic residual poliomyelitis in the region of St. Etienne.

Evaluation of five commercial tests: complement fixation, microparticle agglutination, indirect immunofluorescence, enzyme-linked immunosorbent assay and latex agglutination, in comparison to immunoblotting for Mycoplasma pneumoniae serology.

A panel of 68 serum specimens from 41 subjects exhibiting various immunological patterns to Mycoplasma pneumoniae as determined by detection of a 180 kDa protein in immunoblotting was used to compare five commercially available tests based on different methods: complement fixation test (CFT), microparticle agglutination (MAG), indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (Elisa), and latex agglutination (LA). The tests were performed according to the manufacturers' instructions. For the determination of immunity to M pneumoniae, the five tests were in good accordance with immunoblotting: sensitivity was 100% for all the five assays, specificity ranged from 95.6% (MAG) to 82.6% (Elisa) and overall agreement ranged from 98.2% (MAG) to 92.8% (Elisa). The comparisons of antibody rates obtained by the four quantitative tests (CFT, MAG, IFA, Elisa) showed correlation coefficients ranging from 0.87 (CFT-IFA) to 0.67 (CFT-Elisa). Six significant antibody rises demonstrated by immunoblotting patterns were detected by all the tests but Elisa in one case. As a whole, the commercial assays gave satisfactory results for routine determination of immune status to M pneumoniae: CFT was the cheapest test and MAG and LA were the easiest to perform.

[Enterovirus infections and systemic clinical manifestations with prolonged inflammatory syndrome: association with a persistence of specific IGM antibodies]. / Entérovirose et manifestations cliniques systémiques avec syndrome inflammatoire prolongé: association avec une persistance des anticorps IGM spécifiques.

We report 9 cases of enteroviral infection associated with systemic inflammatory disease (including 4 cases of vasculitis, 1 case of periarteritis nodosa, 1 case of Sharp's syndrome). We then reviewed 36 cases of enteroviral infection with persistent IgM antibodies diagnosed in the virology laboratory in a 6-year period: among them 11 cases were found to present with subacute or chronic inflammatory disease. We conclude that enteroviruses might be important triggers of systemic inflammatory disease.

The respective advantages of complement fixation and ELISA for routine diagnosis of cytomegalovirus infection.

The results of CF and ELISA tests for cytomegalovirus performed on 270 sera of hospitalized patients show a positive correlation. As a general rule, ELISA is more sensitive than CF, except for a few sera collected from patients with immunological disorders. When two sequential sera are available, the CF remains a reliable and inexpensive method. But when only one serum can be obtained, the probability of an active CMV infection can be estimated on the IgM/IgG ratio. In 26% of the patients, this ratio was greater than or equal to 1. The ELISA is twice as expensive as the CF test. To reduce its cost, a simple method for preparing ELISA antigen from commercially-obtained CF antigens is described.

Rapid diagnosis of echovirus 33 infection by neutralizing specific IgM antibody.

In 1982, we isolated 20 strains of echovirus type 33 (EV33) from 18 patients. We studied the humoral response to EV33 of 2,437 subjects from whom at least one serum was available during the same year. In 388 subjects with the neutralizing antibody level at 64 or more, we assayed the EV33 IgM antibodies by seroneutralization test after fractionation of sera by ion exchange chromatography. One hundred ninety-five subjects (8.0%) had a high titre (greater than or equal to 32) of EV33 IgM antibodies, which was considered as evidence of recent infection. The EV33-positive IgM fractions were assayed against five other enteroviruses. Sixty percent of the IgM fractions did not cross-react with any of the five serotypes, 8.7% cross-reacted with at least one serotype but with predominant EV33 IgM response, and 31.3% had an equivalent or greater amount of non-EV33 IgM antibodies; the type specificity of the assay was directly related to the age of the subjects. These findings suggest that determination of neutralizing specific IgM antibody is a sensitive and rapid test for the diagnosis of enterovirus infections, especially in young people.