Direct antimicrobial susceptibility testing (AST) from positive blood cultures using Microscan system for early detection of bacterial resistance phenotypes.

Antimicrobial Susceptibility Testing is mandatory for Bloodstream Infections management in order to establish appropriate antimicrobial therapy. Herein we evaluated new approach based on AST results directly from positive blood cultures, using Microscan WA to carry out rapid phenotypical profile of antibiotic resistance. Our investigations allow to reduce time versus traditional results.

Epidemiological features and specificities of HCV infection: a hospital-based cohort study in a university medical center of Calabria region.

The epidemiological status of HCV in Europe, and in particular in Mediterranean countries, is continuously evolving. The genotype distribution is related to improvement of healthcare conditions, expansion of intravenous drug use and immigration. We review and characterize the epidemiology of the distribution of HCV genotypes within Calabria, an area of Southern Italy. We focus on the pattern of distinct HCV genotype changes over the last 16 years; particularly subtype 1b and genotype 4. We collected data by evaluating a hospital-based cohort of chronic hepatitis C patients; in addition, we report an update including new patients enrolled during last eight months.

Future research and collaboration: the "SINERGIE" project on HCV (South Italian Network for Rational Guidelines and International Epidemiology).

The SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology) project is intended to set up a collaborative network comprising virologists, clinicians and public health officials dealing with patients affected by HCV disease in the Calabria Region. A prospective observational data-base of HCV infection will be developed and used for studies on HCV natural history, response to treatment, pharmaco-economics, disease complications, and HCV epidemiology (including phylogenetic analysis). With this approach, we aim at improving the identification and care of patients, focusing on upcoming research questions. The final objective is to assist in improving care delivery and inform Public Health Authorities on how to optimize resource allocation in this area.

Long-term immunogenicity of hepatitis B vaccination in children and adolescents in a southern Italian town.

PURPOSE: Universal anti-hepatitis B vaccination of infants and of 12-year-old children became mandatory in Italy in 1991. The purpose of this study was to evaluate the persistence of anti-hepatitis B surface (HBs) antibodies several years after a primary course of vaccination. METHODS: In 2010, anti-HBs titers were measured in all subjects aged between 5 and 25 years residing in a southern Italian town. Individuals with an anti-hepatitis B antibody concentration of 10 IU/ml or more were considered to be protected. RESULTS: Of the 671 subjects evaluated, 149 (30%) lacked protective antibodies. Fifty-three (29.4%) of the subjects had been vaccinated ≤10 years earlier and 96 (30.3%) more than 10 years earlier (P = not significant). Subjects vaccinated in infancy were more likely to lack protective anti-HBs antibodies than subjects vaccinated at 12 years of age, regardless of the years elapsed since immunization. CONCLUSIONS: Most subjects maintained protective antibodies for a considerable number of years after vaccination. Vaccination in adolescence results in more prolonged immunogenicity than vaccination in infancy.

Effects of budesonide on P38 MAPK activation, apoptosis and IL-8 secretion, induced by TNF-alpha and Haemophilus influenzae in human bronchial epithelial cells.

Non-typeable Haemophilus influenzae (NTHi) is one of the most frequently involved pathogens in bacterial exacerbations of chronic obstructive pulmonary disease (COPD). In the airways, the main tissue target of NTHi is bronchial epithelium, where this pathogen can further amplify the inflammatory and structural changes induced by proinflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha). Therefore, the aim of this study is to investigate, in primary cultures of human bronchial epithelial cells, the effects of NTHi on signal transduction pathways, apoptotic events and chemokine production activated by TNF-alpha. Moreover, we also evaluated the effects exerted on such cellular and molecular phenomena by a corticosteroid drug. p38 mitogen-activated protein kinase (MAPK) phosphorylation was analyzed by Western blotting, using an anti-phospho-p38 MAPK monoclonal antibody. Apoptosis was assayed by active caspase-3 expression. Interleukin-8 (IL-8/CXCL8) was detected in cell-free culture supernatants by ELISA. TNF-alpha induced a significant increase in p38 MAPK phosphorylation. NTHi was able to potentiate the stimulatory actions of TNF-alpha on caspase-3 expression and, to a lesser extent, on IL-8 secretion. These effects were significantly (P less than 0.01) inhibited by a pharmacological pre-treatment with budesonide. These results suggest that TNF-alpha is able to stimulate, via activation of p38 MAPK signalling pathway, IL-8 release and airway epithelial cell apoptosis; the latter effect can be markedly potentiated by NTHi. Furthermore, budesonide can be very effective in preventing, through inhibition of p38 MAPK phosphorylation, both structural and proinflammatory changes elicited in bronchial epithelium by TNF-alpha and NTHi.

Applications of LightCycler Staphylococcus M-GRADE assay to detect Staphylococcus aureus and coagulase-negative staphylococci in clinical blood samples and in blood culture bottles.

We evaluated the applicability of the LightCycler Staphylococcus M(GRADE0 assay on artificially infected blood samples from healthy donors and on clinical specimens of 31 hospitalized patients. The sensitivity and specificity of the assay for detecting Staphylococcus aureus was 100% in blood samples, and 100% in blood culture bottles, when data from the BACTEC 9120 blood culture system were taken as gold standard. The same specificity and sensitivity was found during the search for CoNS (Coagulase Negative Staphylococci) in blood culture bottles, whereas a 93.33% sensitivity and 100% specificity was observed for detecting CoNS directly in blood clinical specimens.

Fourteen-year experience with imported malaria.

Geographical position, an increasing flow of immigrants and refugees coming from regions where malaria is endemic might further increase those cases of malaria imported to Calabria due to travel on military missions, visiting relatives, business and leisure. However, few reports have been published regarding malaria imported into the southern Italian region of Calabria. Based on data from our laboratory, official reports received from the Italian Ministry of Health and Regional Health Offices, an epidemiological analysis of malaria cases registered in Calabria from January 1988 to December 2001 is reported. The epidemiological and clinical features concerning the cases are discussed. A total of 34 slide-confirmed malaria cases were observed in Calabria during the period in question. Infections were mostly acquired in Africa (84.8%), while remaining infections came from Asia (9.1%) and South America and Europe (6.0%). Length of stay in the endemic area did not increase the infection risk. Etiological diagnosis indicated Plasmodium falciparum as the species most often involved (60.6%), followed by Plasmodium vivax (36.3%) and P. vivax/Plasmodium malariae mixed infection (3.0%). The mortality rate was about 3.0%. The number of cases during the second seven-year period of this study was almost double that of the first seven-year period. Correct chemoprophylaxis was performed by only 27.3% of our studied subjects. Delay of malaria diagnosis ranged between 4 days and 1 month. In conclusion, increases in malaria cases, mostly due to P. falciparum, delay in diagnosis and reporting to the Regional Health Office, as well as the increasing arrival of refugees from endemic areas, are epidemiological concerns in Calabria, the southernmost region of continental Italy.

Pathogenic mechanisms of Bartonella quintana.

Bartonella quintana is an epi- and intracellular gram-negative rod responsible for both acute and chronic clinical manifestations. We review the literature about pathogenic mechanisms of B. quintana and discuss our data. Our efforts to clarify Bartonella quintana pathogenesis run on two parallel tracks. The first one concerns interactions between Bartonella quintana and endothelial cells by evaluation and modulation of apoptosis, signal transduction pathways and inflammation. The second one concerns some biological activities of Bartonella quintana endotoxin on human whole blood and endothelium. The elucidation of the mechanisms regulating the inflammatory/proliferative pattern of chronic clinical manifestations of Bartonella quintana infections may offer a contribution for addressing the pathogenesis of intracellular bacterial persistence.

Early detection of Leishmania promastigotes in dog bone marrow cultures by acridine orange stain.

An acridine orange staining technique was evaluated in comparison with other well-known methods for the laboratory diagnosis of leishmaniasis. A higher number of promastigotes was found in Novy-MacNeal-Nicolle (NNN) cultures inoculated with canine bone marrow, when culture samples were stained with acridine orange vital stain, compared with those detected using either Giemsa staining or unstained wet mount examination. Based on our data the acridine orange stain is a useful and timely technique in reflecting the true numbers of microorganisms present in a culture and also enhances the visualization of the parasites. The present results warrant further studies with human samples from suspected leishmaniasis patients.

Evidence favouring the gastro-oral route in the transmission of Helicobacter pylori infection in children.

OBJECTIVE: Several studies support the view that Helicobacter pylori is acquired in early life and within families. However, the exact route of transmission remains unknown. Given that H. pylori colonizes only the human gastric mucosa, the hypothesis that history of vomiting in siblings may be a relevant risk factor was tested in a paediatric setting. METHODS: One hundred urban children (age range 0.8-16.6 years, median 9), 44% with evidence of active H. pylori infection, were recruited. A structured questionnaire dealing with socio-economic issues was completed. Vomiting siblings and siblings of vomiting index children were screened for H. pylori by means of (13)C-urea breath test. Serum samples from index children were assayed for immunoglobulin G to hepatitis A (HAV) and Epstein-Barr virus (EBV) in order to check for faecal-oral and oral-oral exposure, respectively. RESULTS: Vomiting siblings of H. pylori-infected index children and siblings of H. pylori-infected vomiting index children had a high rate of active H. pylori infection (60 and 67%, respectively). History of vomiting in siblings was positively associated with active H. pylori infection in the index children (multivariate odds ratio 2.4, 95% confidence interval 1.3-4.3). Seropositivity for HAV and EBV was found in 1 and 68 index children, respectively. The agreement between active H. pylori infection and EBV seropositivity was not significant (kappa = 0.26). CONCLUSIONS: History of vomiting in siblings is an independent risk factor for H. pylori. Nowadays, transmission of H. pylori in urban children may involve the gastro-oral route more than the faecal-oral or oral-oral pathways.

Altered cytokine production after human herpes virus type 6 infection.

Several strategies allow viruses to elude the surveillance of the immune system and to establish persistent infection in the host. One of such mechanisms is the immunosuppression caused by the direct infection and functional impairment of immune cells. Human Herpes virus type 6 (HHV-6) is a typical immunosuppressive agent, as suggested by its tropism for both CD4+ and CD8+ T cells, B cells, monocytes/macrophages, megakaryocytes and NK cells. In this study the production of IL-10 and IL-12 by peripheral blood mononuclear cells (PBMC) was evaluated during HHV-6 infection "in vitro". Our results demonstrate that HHV-6 up-regulates IL-10 production by PBMC. Furthermore, our data suggest that rhIFN gamma addition counteracts the effect of HHV-6 in promoting IL-10 release. To gain more insight into the role of IFN gamma, anti-IFN gamma monoclonal antibodies were added to PBMC stimulated with LPS. Neutralization of endogenous IFN gamma upregulated IL-10 release. Furthermore, HHV-6 infection inhibited IFN gamma release induced by LPS in PBMC. No basal production of IL-12 was found in PBMC. Moreover, HHV-6 infection did not induce IL-12 release by PBMC. On the contrary, IL-12 was detected in the supernatants of PBMC treated with LPS with or without rhIFN gamma. In these experimental conditions the further addition of HHV-6 markedly impaired IL-12 production. Moreover, the neutralization of IL-10 resulted in a significant up-regulation of IL-12. Finally our data suggest that the immunodysregulation induced by HHV-6 could be accounted for by a shift from a Th-1 to a Th-2 type cytokine profile.

[Extraction and characterization of the lipopolysaccharide of Bartonella quintana] / Estrazione e caratterizzazione del lipopolisaccaride di Bartonella quintana.

Bartonella quintana has been reported as the cause of trench fever, persistent endocarditis, bacteriaemia and has been isolated with an increasing incidence in clinical specimens from AIDS patients. One of the main pathogenic factors of gram-negative bacteria, including B. quintana, is the lipopolysaccharide (LPS). However, very little information is available on the features of Bartonella LPS. The aim of the present study was to extract, purify and characterise B. quintana LPS. The effect of the LPS under scrutiny was also evaluated on TNFa release by means of the "in vitro" human whole blood model of sepsis. The Oklahoma strain of B. quintana was grown on sheep blood agar, at 37 C, in a moist atmosphere containing 5% carbon dioxide. Cells were harvested and washed in sterile and apyrogenic saline solution and LPS extracted following the procedure of Westphal e Jann (1965), modified by Minnick (1994). The LPS of B. quintana showed the migration pattern of a deep rough chemotype, and the chromogenic limulus amoebocyte lysate test (LAL test) revealed strong reactivity at low concentrations (6.2 pg/ml). Samples of human whole blood stimulated by 1000 ng/ml of B. quintana LPS released 1707 378 pg/ml of TNFa.

Kinetics of IL-8, MIP-1 alpha, TNF alpha, IL-1 beta, IL-1ra and IL-10 in human whole blood samples triggered by smooth and rough LPS.

An in vitro model for the study of sepsis mediators was used to investigate the effects of two different lipopolysaccharides (LPS), a smooth (LPS-S) and a rough (LPS-R) type, on the release of chemokines (IL-8 and MIP-1 alpha) and cytokines (TNF alpha, IL-1 beta, IL-1ra and IL-10) from human whole blood samples. TNF alpha level increased significantly vs. control, at 4 h and 8 h after the challenge with smooth and rough type of LPS respectively. Concentrations of the two chemokines studied, IL-8 and MIP-1 alpha, were significantly elevated following stimulation by both LPS, and reached concentrations significantly different from controls at 4, 8 and 24 h. After 24 h of incubation both LPS produced a significant IL-10 increase, although such change was more substantial with the rough type. Present data suggest an early and maintained release of chemokines regardless of the type of LPS used and often in absence of a significant increase in primary pro-inflammatory cytokines.

Productive HHV-6 infection in differentiated U937 cells: role of TNF alpha in regulation of HHV-6.

This study characterizes the effect of differentiation on the resistance of the human monocytic cell line U937 to human herpes virus type 6 (HHV-6). The use of monocytic cell line has the advantage of avoiding genetic variations among different donors. The HHV-6 infection was compared in undifferentiated U937 cells and U937 cells differentiated with a combination of vitamin D3 and retinoic acid. Undifferentiated U937 cells were highly resistant to HHV-6 infection. Differentiation of U937 cells was accompanied by an increase in permissiveness for HHV-6 demonstrated in terms of extracellular virus production and viral antigen positive immunofluorescent cells. Tumor necrosis factor alpha (TNF alpha) appears to be an essential mediator during the first line defences of the host against viruses, even though its role during viral infection remains controversial. For this reason we examined the behaviour of TNF alpha in differentiated U937 upon HHV-6 infection. No basal production of TNF alpha was found in culture supernatants, while HHV-6 infection up-regulated TNF alpha release. The addition of human recombinant-TNF alpha to HHV-6 infected cells induced a marked cytotoxic effect accompanied by an increased release of extracellular virus, whereas it did not affect viral replication, as shown by the unmodified percentage of antigen positive cells. In conclusion, TNF alpha acts as a soluble mediator of cytotoxicity against HHV-6 infected U937 cells, but it fails to induce an antiviral state.

Impairment of immunological functions in genetically epilepsy-prone rats.

1. In genetically epilepsy-prone rats (GEPR-9s), which represent a natural genetic model of epilepsy, we observed that the number of peritoneal macrophages was significantly lower with respect to normal rats, and that some functional parameters (i.e. phagocytosis and intracellular killing) of these macrophages were impaired. 2. The count of lymphocyte populations showed a predominance of T-helper over T-cytotoxic/suppressor both in the spleen and lymph nodes. Moreover, an increased T-cell/B-cell ratio was observed in GEPR-9s. Flow cytometry revealed that GEPR-9s spleens possessed a large percentage of T-helper cells in comparison to normal rats. 3. By using concanavalin A-induced proliferation of GEPR-9s cultured lymphocytes, we have shown increased functional activation. 4. We suggest that the alterations in T-cell functions in GEPR-9s could be due to the involvement of the neuroendocrine system in the modulation of immunity, in the shift between Th1 and Th2, and in the activation of stress response.

Teicoplanin reduces in-vitro reactivity and murine lethality of Salmonella minnesota R595 lipopolysaccharide.

Three different tests were performed to investigate the effect of teicoplanin on lipopolysaccharide (LPS). After incubation for 3 h with teicoplanin, LPS from Salmonella minnesota R595 showed reduced reactivity in the metachromatic dimethyl-methylene blue assay and the limulus amoebocyte lysate test. In addition, galactosamine-sensitized mice had an increased survival rate, from 29% to 72%, when teicoplanin was pre-incubated for 3 h with the LPS to be injected intraperitoneally. The results suggest that teicoplanin may have a neutralizing effect on LPS.

Aminoglycosides modify the in vitro metachromatic reaction and murine generalized Shwartzman phenomenon induced by Salmonella minnesota R595 lipopolysaccharide.

Endotoxin-neutralizing activity may be an important property for antibiotics to be used in severe sepsis. Several antibiotics, belonging to different classes, were evaluated as to their endotoxin-neutralizing ability, using the inhibition of an in vitro metachromatic assay for lipopolysaccharides and a murine generalized Shwartzman reaction model. Gentamicin, amikacin, and sisomicin have been found to share significant in vitro antiendotoxin activity at an antibiotic/endotoxin ratio as low as 1.0/5 (by weight) and to reduce the murine generalized Shwartzman reaction at an antibiotic/endotoxin ratio of 3.3/5.