Serological diagnosis of respiratory viral infections. A five-year study of hospitalised patients.

The results of a five-year study of paired sera from 410 hospitalised patients-mainly children-with respiratory illness are reported. Samples were divided into groups based on clinical diagnosis. The data of each group were analysed in relation to patient age (under or over 1 year of age). The percentage of positive serological diagnoses ranged from 29.4% in the respiratory viral illness group to 46.2% in the bronchiolitis group. Each group showed a prevalent serological diagnosis. Respiratory viral illness patients over 1 year were diagnosed mainly with Influenza virus infection (73.8% positive diagnosis), pharyngotonsillitis patients with Adenovirus infection (72.2%), laryngitis patients with Parainfluenza virus infection (100%), pneumonia patients with Mycoplasma pneumoniae infection (56.7%), and bronchiolitis patients with Respiratory Syncytial virus infection (100%). The serological diagnosis patterns of each group or subgroup were statistically significant with respect to the other groups (chi 2 or Fisher exact tests). Unlike previous reports, none of the patients under 1 year in our study was diagnosed with Influenza virus infection or Parainfluenza virus type 3. Conversely, Respiratory Syncytial virus infection data were in line with previous reports, being the most frequently diagnosed infection in the bronchiolitis group and in the subgroups of patients under 1 year of age. The present report provides new information on patterns of respiratory infections.

M genotyping and DNA fingerprinting of Streptococcus pyogenes isolates from an area of central Italy.

M protein gene typing was used to analyse Streptococcus pyogenes clinical isolates collected between 1983 and 1995 in an area of central Italy from patients presenting different types of infections; the same isolates were also characterized by means of DNA fingerprinting. M type 1 was the most common (50% of study strains), followed by M types 4, 12 and 6. The proportion of M type 12 decreased with time, whereas M type 1 increased, in agreement with data obtained in many different areas. Most invasive strains belonged to types M1 (30%) and M12 (30%); on the other hand, the M1 type did frequently occur also among non-invasive isolates. DNA fingerprinting showed a correlation between M types and DNA patterns. This report provides epidemiological information from a geographic area not sampled recently, and further shows the usefulness of the M genotyping technique, which offers potential advantages over conventional serological typing methods.

Unusual species of campylobacters isolated in the Siena Tuscany area, Italy.

From January 1989 to December 1990, stool samples from 288 children with enteritis were examined for the presence of unusual campylobacters which represented about 20% of all campylobacteria isolated when the filtration technique was used. The isolation percentage was the following: C. jejuni ss. jejuni 6.9%; C. coli 2%; C. jejuni ss. doylei, C. upsaliensis and C. concisus each 0.7%. The atypical Campylobacter isolates were examined for their virulence characteristics. Toxin profiles based on cytotonic, cytotoxic and cytolethal distending factors were determined after analysis responses in Vero, CHO and HeLa cells. Adhesivity and invasivity tests were performed on Intestine 407 cells. No strain was cytotoxic. C. jejuni ss. doylei and C. concisus induced an elongation of CHO cells (a cytotonic-like effect). C. upsaliensis strains provoked a cytolethal distending effect. No strain adhered to cells in vitro. Our results suggest that the filtration technique is excellent for the isolation of atypical campylobacters and indicate that the unusual Campylobacter isolates could be potentially virulent.

Species, biotype and serogroup of Campylobacter spp. isolated from children with diarrhoea over a ten-year period.

From 1981 to 1990, stool samples from 6403 gastroenteritis cases were examined for the presence of campylobacters as well as Salmonella, Shigella, Aeromonas species and Yersinia enterocolitica. The percentages of isolation were the following: campylobacters 10.8 (86.1% of isolates were C. jejuni and 13.9% were C. coli), Salmonella spp. 8.4, Aeromonas spp 1.4, Yersinia enterocolitica 0.3. Shigella spp. were isolated only occasionally. Predominant biotypes of campylobacters were C. jejuni I (69.5%), C. jejuni II (29.5%) and C. coli I (92.7%). The six most common LIO serogroups-36; 4; 1; 28.53; 11; 2-accounted for 50% ca. of typable strains. Campylobacters are the most common etiological agent of bacterial enteritis in children living in this area of Tuscany. The species and serogroup determination can be useful from an epidemiological point of view.

Two-dimensional electrophoretic patterns of acute-phase human serum proteins in the course of bacterial and viral diseases.

Acute-phase serum proteins were analyzed by two-dimensional electrophoresis with isoelectric focusing in 3-10 immobilized pH gradients. Most spots were identified by reference to the plasma map in the SWISS-2DPAGE database. Serum amyloid A protein spots were identified by immunoblotting with specific antiserum and by matching determined with predicted values of electrophoretic parameters. Changes in the concentrations of alpha 1-antitrypsin, leucine-rich glycoprotein, haptoglobin, serum retinol-binding protein and transthyretin were quantitated by densitometry of silver-stained gels. Electrophoretic patterns from 18 patients with bacterial diseases and 16 patients with viral diseases were compared. The incidence of serum amyloid A protein spots was 18/18 in bacterial diseases and 6/16 in viral diseases. As the the other reactants studied, variations were simultaneous in bacterial disease and tended to be staggered in viral diseases.

Two-dimensional electrophoresis of human serum proteins modified by ampicillin during therapeutic treatment.

Two-dimensional electrophoretograms of serum proteins from ampicillin-treated patients were analyzed-by immunoblotting with an antiserum specific for penicilloyl groups. As expected, human serum albumin (HSA) was the main ampicilloylated serum component. Transferrin main form II was found to be the second most important component as regards immunoblotting intensity. Immunoreactive spots were present on the acidic side of the transferrin isoelectric series, suggesting a modification mechanism similar to that observed in HSA, i.e., acylation of basic amino acid residues. Several additional ampicilloylated spots were detected but could not be assigned. Their electrophoretic parameters were determined using internal standards. This is the first description of serum proteins other than HSA being modified by ampicillin in the course of routine therapeutic treatment.

Two-dimensional gel electrophoresis and immunoblotting of human serum albumin modified by reaction with penicillins.

A two-dimensional gel electrophoresis and immunoblotting procedure has been developed to assess the level of modification by penicillins in human serum albumin. The procedure can be used in in vitro experiments and in clinical studies with sera from patients treated with penicillins.

Retrospective survey on the epidemiology of H. influenzae type B meningitis in Italy (1987-91).

A retrospective survey on the epidemiology of H. Influenzae type b (Hib) meningitis was carried out in seven Italian hospitals. During the period 1987-1991, 95 (16.3%) Hib meningitis cases out of 581 bacterial meningitis cases were observed. The proportion of Hib meningitis was lowest in 1987 (11.3%); elevated in 1988 (17.9%); thereafter it did not change. A male preponderance was observed (Sex ratio 1.6). The age distribution showed that 93.7% of cases occurred in subjects < 5 years, 53.7% of cases in those < 1 year. Although Hib meningitis accounts for a small proportion of all bacterial meningitis, it is also a major problem in Italy in early childhood, because nearly all cases occur in children < 5 years.

Antibiotic susceptibility of 206 Haemophilus influenzae isolates collected from children in central Italy.

Susceptibility of 206 H. influenzae isolates was evaluated by disk diffusion method for 11 antimicrobial agents. No isolates were found to be resistant to third-generation cephalosporins, amoxicillin+clavulanic acid, gentamicin and ciprofloxacin. Four untypable isolates (1.9%) were beta-lactamase producing ampicillin-resistant; one of these was also resistant to chloramphenicol. The rate of resistance against rifampin was 0.5 percent.

Two-dimensional electrophoresis analysis of human serum proteins during the acute-phase response.

The serum of patients with meningitis, due to infection by Haemophilus influenzae type b, was analyzed. Several known acute-phase proteins were separated by two-dimensional electrophoresis and estimated quantitatively. In addition, hitherto undescribed reactants were recognized. Gels were calibrated and relevant spots related to master spot numbers in the human serum protein database.

Molecular epidemiology of nasopharyngeal corynebacteria in healthy adults from an area where diphtheria vaccination has been extensively practiced.

In addition to conventional biochemical tests, a DNA probe specific for Corynebacterium diphtheriae was used to characterize 53 cystinase-positive and urease-negative corynebacteria strains isolated from pharyngeal and nasal swabs obtained from 515 healthy adults living in an urban area of central Italy. No Corynebacterium diphtheriae strain was found. Six "atypical" strains were isolated, which could not be classified in any of the species so far defined in the Corynebacterium genus. These strains appeared to be biochemically close to Corynebacterium pseudodiphtheriticum and genetically close to Corynebacterium diphtheriae, since their DNAs strongly hybridized, under relatively low stringency conditions, with a Corynebacterium diphtheriae-specific probe and since insertion sequences which are usually found in Corynebacterium diphtheriae genomes were also found to be present in their genomes. No one of these six strains was either toxigenic or susceptible to lysogenization by beta-corynephage carrying the tox gene. Therefore, they do not seem to have any epidemiological relevance as possible hosts for beta-phages.

The usefulness of the acridine-orange stain in identifying Helicobacter pylori in gastric biopsies.

The efficacy of the Acridine-orange stain (AOS) in identifying Helicobacter pylori (HP)-like organisms in biopsy smears from adults with gastroduodenal disease was studied. The results obtained indicate that AOS can replace Gram Stain in HP organism identification in gastroduodenal mucosa specimen.

Immunity to diphtheria, six to 15 years after a basic three-dose immunization schedule.

The results of a study of the immunity to diphtheria of 283 girls (9-18 years of age) vaccinated at the age of two years with three doses of vaccine, are reported. The rabbit skin test was used to determine the titre of serum diphtheria antitoxin. 55.8% of the subjects were found to be protected (titre greater than or equal to 0.1 IU/ml), 38.9% were only relatively immune (titre greater than or equal to 0.01- less than 0.01 IU/ml), and 5.3% were unprotected (titre less than 0.01 IU/ml). The antitoxin titres showed a tendency to decrease with time. Even so, 6-15 years after vaccination, the percentages of protected and partially protected subjects were still high (95%).

Cytotoxin production by Campylobacter pylori strains isolated from patients with peptic ulcers and from patients with chronic gastritis only.

A total of 66.6% of Campylobacter pylori strains isolated from patients with peptic ulcers produced a cytotoxin active against mammalian cells in vitro, versus 30.1% of strains isolated from patients with chronic gastritis of various degrees of severity only. This difference was statistically significant and suggests that the toxic substance could be involved in the development of peptic ulcers.

[Evaluation of the specific immune response after anti-rubella vaccination]. / Valutazione della risposta immunitaria specifica dopo vaccinazione anti-rosolia.

A selective rubella immunization program was carried out in the Siena (Italy) area since 1984. 912 schoolgirls aged 10 to 18 were screened for rubella antibodies. Seropositivity rates, increasing with age, appeared quite heterogenous among subjects living in different villages. Seronegative girls were partly voluntarily immunized with RA 27/3 live attenuated rubella vaccine. Serological and clinical controls performed on 238 subjects one month after immunization showed a 99.6% vaccine-induced seroconversion rate and mild post-vaccinal reactions in 26% of vaccinees. Serological control performed, on 172 subjects, at 8-12 days after vaccination revealed that in most cases vaccine induced haemagglutination-inhibiting antibodies are not detectable at the time. The results are discussed in relation to a forthcoming introduction of compulsory rubella vaccination in our Country.

Specific antibody patterns over a two-year period after rubella immunization with RA 27/3 live attenuated vaccine.

Screening for rubella antibodies was carried out on 1557 schoolgirls aged 9-20. Of seronegative subjects 70% (442/631) were immunized with RA 27/3 rubella vaccine and some of the vaccinees underwent a serological and clinical follow-up over a two year period. Adverse reactions occurred in 27% of vaccinees, usually 1-2 weeks after immunization; late reactions were never observed. The vaccine-induced seroconversion rate evaluated at 4-5 weeks after immunization was 99.7%. Both one and two years after immunization the seropositivity rate of vaccinees was 100%. The maximum geometric mean antibody titre (GMT) was observed at 4-5 weeks after vaccination and a significant GMT decrease was evident on both the following annual controls. Specific antibody patterns in vaccinees were highly variable and in a small number of subjects a remarkable antibody titre decrease was noticed.

[Comparison of methods for the identification of Campylobacter pylori in gastric biopsies of patients with dyspepsia]. / Confronto di metodiche per l'identificazione di "Campylobacter pylori" in biopsie gastriche di pazienti con dispepsia.

Campylobacter pylori has been associated with gastro-duodenal inflammatory disease. Ninety-five adults with dyspepsia were examined for the presence of C. pylori in the gastric antrum and near gastric or duodenal ulcers (when present) by means of culture, Gram and acridine orange stains, and urease activity of biopsies. C. pylori was identified from 51 out of 67 patients with chronic gastritis, from 9 out of 9 patients with duodenal ulcer, and from 8 out of 10 patients with gastric ulcer. Acridine orange stain revealed the highest number of positive cases, followed by culture, Gram stain and urease test. The latter showed a 100% specificity when carried out with a selective urea broth containing colistin, trimethoprim, vancomycin and amphotericin B. It has to be considered a further diagnostic tool which enables clinicians and microbiologists to diagnose the etiology of a dyspeptic syndrome even at the patient's bedside.

Viral childhood gastroenteritis.

Viruses were detected in 150 hospitalized subjects with gastroenteritis during the years 1983-1984. The samples were collected on admission in the day care unit and after three and seven days. The research was carried out using electron microscopy (E.M.) and isolation in cell cultures in vitro. Faecal samples were also collected from 70 control subjects. Observed and/or isolated viruses were identified by structural features at E.M. and by typical cytopathic effects in cultures. Rotavirus and not cultivable adenovirus proved the most widespread agents in the aetiology of acute viral gastroenteritis in infants.

Rubella in teenagers: epidemiology and prophylaxis in Siena, Italy.

Six hundred and fifty-three teenagers (aged 11-13 year) living in Siena and its surroundings (Tuscany, Italy) were the sample for serological screening intended to ascertain immunity to rubella. It was found that 324 of the teenagers (49.62%) lacked antibodies and, hence, were unprotected against the infection. Out of the 324 girls, 196 (around 3/5) were vaccinated using live vaccine. Post-vaccinal complications, with clinical signs of rubella infection, were recorded in almost one third of the vaccinees. Virus isolation from the blood was, in every case, not possible after either 10 or 30 days from vaccination. The serological findings, expressed in hemagglutination inhibition antibodies, could be summarized in the following way: (i) antibodies at low titre were found in only eight out of 184 girls (4.35%) ten days after vaccination; (ii) serological conversion was recorded in 187 out of 188 girls (99.47%) 30 days after vaccination; (iii) the titres were moderately high but much lower than those recorded for the natural infection. The results are discussed in the context of their implications for the strategies of rubella vaccination as far as the safety and the effectiveness of the vaccine are concerned, with emphasis on the duration of the protective immunity.