Alfredo Chiarini

European Sero-Epidemiology Network: standardisation of the assay results for pertussis

A standardisation process was developed in order to compare and harmonize serological results of pertussis toxin (PT) antibody measurements performed by laboratories using different technical procedures for detection. This involved the development of a common panel, of sera by a designed reference centre, the distribution of the panel to each participating laboratory for testing with their routine methods, the comparison of the obtained results to those of the reference centre, and the calculation of standardisation equations by regressing the quantitative results against those of the reference centre. As a cut-off indicative of protection against pertussis has not yet been defined, a particular emphasis was laid upon achieving standardisation of high titre results that would allow epidemiological evaluations based on the estimation of the incidence of recent infections rather than on the traditional approach of determining the population immunity profile. A generally good agreement was achieved between the participating laboratories, all using ELISA procedures very similar in many crucial aspects, and standardisation equations were produced useful to enable inter-country comparison during the next stages of the European Sero-Epidemiology Network (ESEN) project concerning the serological surveillance of immunity to pertussis in Europe.

Detection of Bacterial and Yeast Species with the Bactec 9120 Automated System with Routine Use of Aerobic, Anaerobic, and Fungal Media

ABSTRACT During the period 2006 and 2007, all blood cultures required by four units at high infective risk and most of those required by other units of the University Hospital of Palermo, Palermo, Italy were performed using a Bactec 9120 automated blood culture system with a complete set of Plus Aerobic/F, Plus Anaerobic/F, and Mycosis IC/F bottles. The aim of the study was to enable the authors to gain firsthand experience of the culture potentialities of the three different media, to obtain information regarding the overall and specific recovery of bacteria and yeasts from blood cultures in the hospital, and to reach a decision as to whether and when to utilize anaerobic and fungal bottles. Although very few bloodstream infections (1.8%) were associated with obligate anaerobes, the traditional routine use of anaerobic bottles was confirmed because of their usefulness, not only in the detection of anaerobes, but also in that of gram-positive cocci and fermentative gram-negative bacilli. In this study, Mycosis IC/F bottles detected 77.4% of all the yeast isolates, 87.0% of yeasts belonging to the species Candida albicans, and 45.7% of nonfermentative gram-negative bacilli resistant to chloramphenicol and tobramycin. In order to improve the diagnosis of fungemia in high-risk patients, the additional routine use of fungal bottles was suggested when, as occurred in the intensive-care unit and in the hematology unit of the University Hospital of Palermo, high percentages of bloodstream infections are associated with yeasts, and/or antibiotic-resistant bacteria and/or multiple bacterial isolates capable of inhibiting yeast growth in aerobic bottles.

Prevalence of virulence-associated genotypes of Helicobacter pylori and correlation with severity of gastric pathology in patients from western Sicily, Italy

In a bacterium like Helicobacter pylori, which is characterized by a recombinant population structure, the associated presence of genes encoding virulence factors might be considered an expression of a selective advantage conferred to strains with certain genotypes and, therefore, a potentially useful tool for predicting the clinical outcome of infections. However, differences in the geographical and ethnic prevalence of the H. pylori virulence-associated genotypes can affect their clinical predictive value and need to be considered in advance. In this study we carried out such an evaluation in a group of patients living in Sicily, the largest and most populous island in the Mediterranean Sea. cagA, vacA, babA2, hopQ, oipA, sabA, and hopZ were the H. pylori virulence-associated genes assayed; their presence, expression status or allelic homologs were detected in H. pylori DNA samples and/or isolated strains, obtained by gastric biopsy from 90 Sicilian patients with chronic gastritis, inactive (n = 37), active (n = 26), or active with peptic ulcer (n = 27). Genotypes cagA+, vacAs1, vacAm1, babA2+, and hopQ I, I/II were identified in 51.8, 80.4, 35.2, 47.3, and 67.7% of the different samples respectively. Only these genotypes were associated with each other and with the active form of chronic gastritis, irrespective of the presence of a peptic ulcer. In our isolates their prevalence was more similar to values observed in the north of Italy and France than to those observed in Spain or other Mediterranean countries that are closer and climatically more similar to western Sicily.

Analogous IgG subclass response to pertussis toxin in vaccinated children, healthy or affected by whooping cough.

The study of antigen specific IgG subclass distribution during disease, or during any other natural or artificial immunisation, can provide useful information on the kind of the immune response and the expected levels of protection. This is particularly true for diseases, such as pertussis in which the mechanisms underlying specific defence are still not completely understood. An investigation was therefore performed to evaluate the IgG subclass response to pertussis toxin (PT) in sera from 89 healthy vaccinated children and 131 vaccinated or unvaccinated children convalescent after a confirmed B. pertussis symptomatic infection. Antibody titres were expressed in arbitrary ELISA units/ml, and statistical analyses were performed. In unvaccinated convalescent children IgG1 and IgG3 were prevalent whereas in children immunised with two different acellular pertussis (aP) vaccines, both healthy and convalescent, IgG1, IgG2 and IgG4 antibodies were mainly produced. Maintenance of the same anti-PT antibody response pattern in healthy acellular pertussis vaccine recipients and in vaccinated children who later acquire the disease is an interesting result indicative of the priming effect induced by these vaccines in the direction of a relatively higher Th2 cell-polarisation of the immune response.

Changing patterns of hepatitis A virus infection in children in Palermo, Italy.

In 1988 in Palermo, Italy the prevalence of antibodies to hepatitis A virus (anti-HAV) in a sample of 490 children 6–13 years old was 10.6%; it increased from 6.3% among children 6–10 years old to 14.7% in children 11–13 years old (P < 0.01).Compared with findings from a survey conducted in 1978 in the same area, the results of the present study show a significant (P < 0.01) reduction in the anti-HAV prevalence in both age groups.Anti-HAV prevalence was inversely related to the fathers years of education and positively related to the family size. Children of fathers with less than 6 years of schooling had a 3.2-fold risk (C.I. 95% = 1.3–8.1), and children with five or more members in their households had a 2.7-fold risk (C.I. 95% = 1.1–6.4) of previous exposure to hepatitis A virus (HAV) infection.Our findings indicate that exposure of children in Palermo to HAV is decreasing significantly, probably because of improvements in socio-economic conditions during recent years; however socio-demographic factors appear to be important determinants of infection.

European Sero-Epidemiology Network 2: standardisation of immunoassay results for pertussis requires homogeneity in the antigenic preparations.

A standardisation process, already developed during the earlier European Sero-Epidemiology Network (ESEN) project, was employed with a more robust algorithm to harmonise results of pertussis serological assays performed in 12 European and non-European countries. Initially, results from each countrys own assay were compared with those obtained at the reference laboratory by means of an in-house pertussis toxin (PT)-based ELISA: seven countries used in-house or commercial PT-ELISAs; the other countries used assays based on Bordetella pertussis whole cell extracts (WCE) (three countries) or on combined PT-FHA (filamentous haemagglutinin) antigenic preparations (two countries). The WCE assays, although admitted for diagnostic purposes, confirmed their low correlation with the PT-ELISAs and their results could not be used for standardisation; the PT-FHA ELISAs gave results that were suitable for standardisation in one country but unsatisfactory in the other; the use of purified PT in serological assays confirmed its better reliability than other preparations and all PT-ELISAs results could be calibrated against those of the reference centre. In the standardisation process two high-titre cut-offs indicative of likelihood of recent infection (from within 4 weeks of disease onset up to 1 year after) were included for evaluations as they are suggested to be more useful, for the sero-epidemiological assays of immunity to pertussis, than the cut-off of protection, commonly employed, but still not defined for pertussis. Providing PT-ELISAs are used, standardisation of pertussis assay results is always possible and, when standardisation is performed, evaluation and comparison of the impact of different interventions can be also allowed, by measuring at the distribution of high antibody titres in the populations.

Seroepidemiology of pertussis infection in an urban childhood population in Cameroon

In 1989, the prevalence of IgG antibodies to pertussis toxin (PT) in a sample of 367 unvaccinated apparently healthy children 5–14 years old was estimated by ELISA in Kumba City (Cameroon). Children were recruited using a systematic random sampling from six primary schools located in different districts of the city. The sample was representative of the various socio-economic classes.The overall prevalence was 75%; it increased from 62% in 5 year old children to 81% in children 12–14 years old (P < 0.01). IgG antibody prevalence was positively related to the family size. Children belonging to households of nine or more members had a 2.2-fold risk (C.I. 95 per cent =1.1–4.6) of previous exposure to B. pertussis infection. No association was found with the fathers occupation (O.R. = 1). These findings demonstrate a great impact of pertussis infection in Cameroon, with a nearly total exposure by late childhood.

Prevalence of pertussis IgG antibodies in children in Palermo, Italy

SummaryThe prevalence of IgG antibodies toBordetella pertussis in a sample of 615 1–12-year-old unvaccinated children in Palermo was estimated by ELISA. The overall prevalence was 56%; it increased from 24% in one to three-year-old children to 67% in 11–12-year-old children (p<0.01). IgG antibody prevalence was not associated with the fathers years of schooling (OR 1), nor with the family size (OR 1.3; C.I. 95%=0.8−2.2). For children aged one the three years, serological results showed that the history of pertussis reported by parents in questionnaires gave high specificity (93.2%) and negative predictive value (85.4%). Our seroepidemiological study evidences a great exposure of children toB. pertussis in Palermo, with a high proportion of infections occurring after three years of age.ZusammenfassungBei 615 ein bis zwölf Jahre alten Kindern, die in Palermo leben und nicht gegen Pertussis geimpft wurden, wurde die Prävalenz von IgG-Antikörpern gegenBordetella pertussis mittels ELISA bestimmt. Die Gesamtprävalenz betrug 65%; sie nahm von 24% bei ein bis drei Jahre alten Kindern auf 67% bei 11–12jährigen zu (p < 0,01). Es bestand keine Beziehung zwischen der IgG-Antikörperprävalenz und der Schulbildung des Vaters (OR 1) oder der Größe der Familie (OR 1,3; C.I. 95%=0,8−2.2). Bei ein- bis dreijährigen Kindern zeigten die serologischen Ergebnisse, daß die auf den Fragebogen von den Eltern angegebene Pertussisanamnese hochspezifisch war (93,2%) und einen hohen negativen prädiktiven Wert hatte (85,4%). Unsere seroepidemiologische Studie belegt, daß Kinder in Palermo dem KeuchhustenerregerB. pertussis stark ausgesetzt sind und ein großer Teil der Infektionen nach dem dritten Lebensjahr auftritt.

Ganglioside Stimulation of Neuronal Maturation in vitro

Neurons from fetal rat brain hemispheres cultured in synthetic medium were stimulated to aggregate into clusters and to send out neurites by the early addition of gangliosides. This stimulatory effect was limited at the beginning of the culture. In fact the ganglioside addition did not affect either the axonal sprouting or the survival time of the neurons.

A combined molecular typing approach does not discriminate Legionella pneumophila serogroup 1 strains of a predominant sequence-based type in Palermo, Italy.

The sequence-based type 1,4,3,1,1,1 of Legionella pneumophila sg.1 is predominant in the Palermo city environment since several years. In this study, extended sequence-based typing and pulsed field gel electrophoresis were used in a combined approach in the aim to enhance discriminatory power of the molecular typing procedures. However, probably due to a common environmental reservoir and genetic stability, most of the strains circulating in the geographic area under study belong to the same clone and are, consequently, indistinguishable by molecular typing. Investigations of clinical cases and tracing to their environmental source require caution and support from sound epidemiological data.