Usutu virus infections in humans: a retrospective analysis in the municipality of Modena, Italy.

OBJECTIVE: To monitor the spread and to evaluate the role for public health of Usutu virus (USUV) in an endemic area of Italy. METHODS: The survey was retrospectively conducted by detecting USUV RNA and USUV antibodies in cerebrospinal fluid and serum samples collected between 2008 and 2011 from 915 patients with or without neurologic impairments in the area of the municipality of Modena, Italy. Organs of birds and pools of mosquitoes were also tested for USUV RNA. Positive samples were partially sequenced and used for phylogenetic analysis. RESULTS: The presence of USUV RNA (1.1%; 95% confidence interval (CI) 0.6-2.0) was significantly (p <0.05) higher than that of West Nile virus (0%; 95% CI 0-0.33). USUV antibody level was 6.57% (95% CI 4.87-8.82), and it was significantly higher (p <0.05) compared to that of West Nile virus (p 2.96, 95% CI 1.89-4.62). Partial genome sequencing of USUV strains detected in humans, birds and mosquitoes revealed high nucleotide sequence identity within them and with the USUV strains isolated in Central Europe. CONCLUSIONS: USUV infection in humans is not a sporadic event in the studied area, and USUV neuroinvasiveness has been confirmed.

Hospital-acquired Legionella infections: an update on the procedures for controlling environmental contamination.

The waterborne healthcare-associated infections are mainly sustained by Legionella and Pseudomonas spp. Various water factors and plumbing characteristics, and the interaction with other water microorganisms are considered to be predictive of Legionella contamination. It is therefore mandatory to organize plans of surveillance, prevention and control in order to avoid disease appearance in immunosuppressed patients, with higher risk of death. Guidelines for the prevention of Legionnaires' disease have been published, benefiting those who face this problem, but definitive standardized solutions do not exist yet. Here we describe fifteen years of activity, during which our study group gathered interesting data on the control of Legionella contamination. Water disinfection is not generally sufficient to control the risk of infection, but a complex water safety plan should be developed, including system maintenance, training of staff and implementation of a clinical surveillance system aimed at early detection of cases. Concerning the control measures, we evaluated the effectiveness of different treatments suggested to reduce Legionella spp contamination, comparing our results with the current literature data. The performance ranking was highest for the filter, followed by boilers at high temperature, monochloramine and, at a lower level, chlorine dioxide; the effectiveness of hyperchlorination was limited, and thermal shock was even more ineffective.

Characteristics and outcome predictors of patients involved in an outbreak of Burkholderia cepacia complex.

A Burkholderia cepacia complex outbreak occurred among ventilated non-cystic fibrosis patients in an intensive care unit (ICU) in Italy: 33 colonized and 13 infected patients were included in a retrospective study aimed at investigating factors related to clinical infection and mortality. Demographic/clinical conditions and mortality did not vary significantly between colonized and infected patients, both groups showing high mortality rates compared with the overall ICU population and similar to that observed in patients with other infections. In multivariate regression analysis, disease severity (defined by the Simplified Acute Physiology Score II) and age were the only independent predictors of early mortality (odds ratio: 1.12; 95% confidence interval: 1.02-1.26; and 1.07; 1.01-1.15, respectively).

Effectiveness of different methods to control legionella in the water supply: ten-year experience in an Italian university hospital.

We report our ten-year experience of hyperchlorination, thermal shock, chlorine dioxide, monochloramine, boilers and point-of-use filters for controlling legionella contamination in a hospital hot water distribution system. Shock disinfections were associated with a return to pre-treatment contamination levels within one or two months. We found that chlorine dioxide successfully maintained levels at <100 cfu/L, whilst preliminary experiments gave satisfactory results with monochloramine. No contamination was observed applying point-of-use filters and electric boilers at temperatures of >58°C and no cases of nosocomial legionellosis were detected in the ten-year observation period. Our performance ranking in reducing legionella contamination was filter, boiler, chlorine dioxide, hyperchlorination and thermal shock. Chlorine dioxide was the least expensive procedure followed by thermal shock, hyperchlorination, boiler and filter. We suggest adopting chlorine dioxide and electric boilers in parallel.

[Active clinical surveillance for detection of Legionnaires' disease: implications for health care structures]. / Sorveglianza clinica attiva per la ricerca di polmoniti da Legionella: risultati e considerazioni per le strutture sanitarie.

In an university hospital of about 900 beds, a clinical surveillance was activated to detect cases of Legionnaires' disease in patients affected by community and/or nosocomial-acquired pneumonia. In the hospital Legionella spp was detected in the hot water distribution system and various disinfecting and control procedures were adopted to reduce contamination. Contemporary, the clinical surveillance began with the systematic detection of Legionella urinary antigen among recovered pneumonia, seroconversion as confirmation test and the collection of respiratory secretions or other biological materials to isolate the microorganism in patients positive to the urinary antigen. From September 2003 to May 2005, 486 pneumonia were followed, 98 of which considered of nosocomial origin. In total, 15 cases of community-acquired Legionnaires' disease were detected by the urinary test, whereas no cases of nosocomial origin were found. The characteristics of the detected cases are described in comparison with the other pneumonia and the surveillance cost was evaluated. The systematic clinical surveillance for Legionella infections is feasible with limit costs, allows to detect community-acquired cases otherwise unknown and to ascertain the absence/presence of nosocomial-acquired pneumonia, irrespective of the environment contamination.

A simultaneous outbreak of Serratia marcescens and Klebsiella pneumoniae in a neonatal intensive care unit.

We describe two concurrent outbreaks of Serratia marcescens and Klebsiella pneumoniae in a neonatal intensive care unit (NICU). Over a 16-month period, a total of 27 infants were either colonized (N=14) or infected (N=13). There were 15 cases of S. marcescens and 11 cases of K. pneumoniae. Both micro-organisms were involved in one fatal case. Seven preterm babies developed septicaemia, two had bacteraemia, three had respiratory infections and one had purulent conjunctivitis. The S. marcescens and K. pneumoniae isolates were investigated by three molecular methods: enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR), arbitrary primed PCR with M13 primer, and random amplification of polymorphic DNA. Different patterns were found in the 16 S. marcescens epidemic isolates from 16 newborn infants. The major epidemic-involved genotype was linked to the first nine cases and this was subsequently replaced by different patterns. Eight different typing profiles were also determined for the 13 K. pneumoniae isolates from 12 newborn infants. Four K. pneumoniae bacteraemic strains proved to be identical. In conclusion, the typing results revealed that two different micro-organisms (S. marcescens and K. pneumoniae) were simultaneously involved in invasive nosocomial infections in preterm newborns. Two simultaneous clusters of cases were documented. Heterogeneous genotypes among both species were also demonstrated to be present in the NICU at the same time. A focal source for both micro-organisms was not identified but cross-transmission through handling was probably an important route in this outbreak. Strict adherence to handwashing policies, cohorting, isolation of colonized and infected patients, and rigorous environmental hygiene were crucial measures in the containment of the epidemic.

"Side" effects of HAART: decreasing and changing occupational exposure to HIV-infected patients.

To investigate percutaneous exposures to HIV in the highly active antiretroviral therapy (HAART) era, we performed an analysis of all percutaneous exposures reported from January 1994 to December 1998 in 18 Italian acute-care hospitals. Frequency and rate per 100 prevalent AIDS cases of HIV exposures decreased by 40% (from 4.3% to 2.6%, and from 1.0% to 0.6%, respectively; p<0.001), which were mainly those related to the insertion/manipulation of peripheral vascular access devices (from 7.2% to 4.8%; p=0.05). We conclude that the benefits of HAART have changed the complexity of care required and therefore, the number and type of procedures performed on HIV patients that place the HCW at risk of injury.

Evaluation of a western blot test as a potential screening tool for occupational exposure to Mycobacterium tuberculosis in health care workers.

Health care workers (HCWs) have a higher than average risk for contracting Mycobacterium tuberculosis (MTB) infection and tuberculosis (TB). No markers of MTB-exposure are available, and TB risk assessment is performed by tuberculin screening, identifying individuals with acquired MTB infection. This study evaluated a western blot (WB) anti-M. bovis A60 complex antibody as a MTB-exposure marker. WB reactivity was evaluated on 127 exposed and 28 non-exposed HCWs from four divisions of the Policlinico Hospital of Modena, and 140 non-exposed bacille Calmette-Guérin-vaccinated controls. Excess of occupational TB risk according to the Occupational Safety and Health Administration (OSHA) was calculated in each division. WB-positivity (%) was: (1) significantly higher in exposed HCWs compared with non-exposed (72% vs 25%, P < 0.00001), (2) highly related (r = 0.99) to OSHA risk excess in all divisions, (3) higher than non-exposed in HCWs with short (< 5 years) MTB-exposure (purified protein derivative [PPD], P > 0.18; WB, P < 0.04). PPD-positivity (%) was higher than controls only in HCWs with longer (> 5 years) MTB-exposure. The study suggests that the WB antibody might represent a more sensitive biological marker of MTB contact among exposed HCWs, related to the level of TB risk and detectable earlier than the PPD skin test, thus providing new tools for TB risk assessment in health care facilities.

Transport of sulfobromophthalein and taurocholate in the HepG2 cell line in relation to the expression of membrane carrier proteins.

The transport of two different classes of organic anions (cholephilic dyes; the sulfobromophthalein, BSP, and bile acids; taurocholate, TC) was investigated in the HepG2 cell line. At 37 degrees C, BSP uptake was found to be biphasic with an apparent saturative curve in the concentration range between 0-6 microM followed by a linear component up to 18 microM. Kinetic constant determination showed an apparent Km of 26.6 +/- 3.1 microM and a Vmax of 5.64 +/- 0.82 nmol BSP.min-1.mg prot-1. At 4 degrees C, uptake was linear. By subtracting this latter component from the total uptake, a saturable, carrier mediated uptake was found with an apparent Km of 3.6 +/- 1.0 microM BSP and a Vmax of 0.37 +/- 0.04 nmol BSP.min-1.mg prot-1 (m +/- SEM, n = 6). These values were fully comparable with those found in freshly isolated male hepatocyte. Immunoblot analysis of HepG2 cell plasma membrane revealed the presence of bilitranslocase when tested against a monospecific antibody against this carrier molecule. On the contrary, TC uptake was linear up to concentration of 100 microM TC. No difference was observed in the presence or absence of Na+. Immunoprecipitation analysis showed the absence of the putative carrier of TC. These data indicate that the HepG2 cell line expresses a functioning bilitranslocase-mediated system. Conversely, carrier mediated bile acid uptake is absent in line with the lack of expression of the carrier protein.

Immunogenicity and safety of a recombinant hepatitis B vaccine produced in mammalian cells and containing the S and the preS2.

A group of 273 health care workers, at risk of HBV infection, underwent vaccination with recombinant HBsAg produced in mammalian cells and containing protein sequences coded by both the S and pre-S2 regions (Genhevac B). Preliminary results show that a very early pre-S2 response occurred which may be useful in post-exposure prophylaxis. This observation, in addition to reduced influence by the vaccination protocol, provides grounds for optimism in spite of the fact that the efficiency spectrum of this vaccine was not superior to that of recombinant vaccines produced in yeast.

A simple score for the identification of patients at high risk of organic diseases of the colon in the family doctor consulting room. The Local IBS Study Group.

In order to develop a scoring system for selecting patients at high risk of organic diseases of the colon, who would need a colonoscopy or a barium enema, we conducted a study with 14 GPs in the local health care district of Modena. Over one year, 254 consecutive patients who consulted their GP for chronic abdominal pain were asked to answer a guided questionnaire. A checklist of simple parameters suggestive of the presence of organic diseases of the colon was also registered by the GP. For the final diagnosis, the patients underwent either a colonoscopy or a barium enema. Data collected were analysed by means of a stepwise logistic regression analysis to obtain a weighted score for the diagnosis of either irritable bowel syndrome (score less than 0) or organic disease (score greater than 0). Out of the 25 parameters explored, six were significantly more common among patients with organic disease and weighted as positive score (namely ESR greater than 17 mm, first hour, history of blood in the stool, leukocytosis greater than 10,000 cm3, age greater than 45 years, slight fever and presence of neoplastic colonic diseases in first-degree relatives). On the contrary, five parameters were more frequent among patients with irritable bowel syndrome and weighted as negative score (namely visible distension of the abdomen, feeling of distension, presence of irritable bowel syndrome in first degree relatives, flatulence and irregularities of bowel movement). Our scoring system correctly classified 83.5% of the cases, and it was very sensitive (82.4%) for the diagnosis of organic disease.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of calcium precipitation in the sulfoglycolithocholate-induced cholestasis of the bile fistula hamster.

Sulfate glycolithocholic acid (SGLC) has been shown to be highly cholestatic in the rat. This study was performed in order to gain understanding of the mechanisms of SGLC-induced cholestasis and the aim of the investigation was to explore the hypothesis that SGLC could cause a precipitation of calcium in bile. We studied the effects of intravenously administrated SGLC on bile flow, biliary lipids secretion and calcium excretion in the female bile fistula hamster. We also performed in-vitro studies with a Ca2(+)-selective electrode in order to measure the calcium binding capacity of SGLC. The results showed that after 1 h of infusion of 8 mumol/100 g body weight [14C]SGLC bile flow dropped to zero. During the infusion period a fine white sludge was visible in the test tube used for bile collection. TLC and HPLC analysis of both the supernatant and the precipitate showed that unchanged SGLC was excreted into bile. Up to 20% of biliary SGLC and more than 50% of the total Ca2+ present in bile was precipitated. The SGLC/Ca2+ molar ratio in the precipitate was 1.12 +/- 0.3 (mean +/- S.D. of four experiments). Light and electron microscopy of the liver did not show any specific abnormalities. The Ca2+ binding activity of SGLC in vitro, was highest among the bile acids tested at a concentration of 0.1 mM, when almost 100% of bile acids are in the monomeric (non-micellar) form. This suggests that among the bile acids, SGLC exerts the strongest binding activity on free calcium ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex steroid modulation of the hepatic uptake of organic anions in rat.

To investigate the role of sex steroids in the sex-related difference in the hepatic uptake of organic anions, sulphobromophthalein (bromsulphalein, BSP) transport was measured in hepatocytes isolated from rats either deprived of hormonal influence by castration at prepubertal age or after hormonal substitution. In control animals, the kinetics of BSP uptake showed the presence of two components: one saturable (0-3 microM), with high affinity and low capacity, and the other linear (9-30 microM), probably related to the non-specific component of BSP uptake. Sex difference was detected only in the saturable portion of the uptake process as the apparent Km was significantly lower in females than in males (3.8 +/- 0.7 vs. 6.1 +/- 1.8 microM, mean +/- S.D. of six animals, P less than 0.01). In contrast, no difference was observed in Vmax (2.3 +/- 0.3 vs. 2.2 +/- 0.7 nmol BSP.(mg protein)-1.min-1). Castration was associated with the disappearance of the saturable uptake site and abolished the sex difference. Progesterone treatment of castrated males failed to restore the saturable kinetics of BSP uptake. In contrast, administration of oestradiol to castrated males or testosterone to castrated females did restore the saturable kinetics of the high-affinity BSP uptake. Km and Vmax were comparable to those of adult females and males, respectively, with the exception of testosterone which induced a Vmax value higher than that observed in the other groups of animals. These data suggest that the influence of oestrogen and testosterone is necessary for the expression of the high-affinity, low-capacity carrier-mediated process of hepatic BSP uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Taurine increases bile acid pool size and reduces bile saturation index in the hamster.

There is evidence that increased availability of taurine enhances the proportion of taurine-conjugated bile acids in bile. To explore the possibility that taurine treatment could also influence hepatic cholesterol and bile acid metabolism, we fed female hamsters for 1 week and measured both the biliary lipid content and the microsomal level of the rate-limiting enzymes of cholesterol and bile acid synthesis. In these animals the cholesterol 7 alpha-hydroxylase activity was significantly greater in respect to controls (P less than 0.05). The total HMG-CoA reductase activity, as well as that of the active form, was similarly increased. The stimulation of 7 alpha-hydroxycholesterol synthesis was associated with an expansion of the bile acid pool size in taurine-fed animals. Taurine feeding was observed to induce an increase in bile flow as well as in the rate of excretion of bile acids, whereas the secretion rate of cholesterol in bile was decreased. As a consequence, the saturation index was significantly lower in taurine-fed animals (P less than 0.05). The possible mechanisms through which taurine exhibits the modification of the enzyme activities and of the biliary lipid composition are discussed.

Bile acid inhibition of taurocholate uptake by rat hepatocytes: role of OH groups.

To define further the structural specificity of the taurocholate uptake site, we studied the ability of a variety of taurine-conjugated bile acids with differing hydroxyl substituents on the sterol moiety to inhibit [14C]taurocholate uptake. Rat hepatocytes isolated by collagenase perfusion were incubated in a tris(hydroxymethyl)aminomethane-phosphate buffer containing [14C]taurocholate (2.5-100 microM) in the presence or absence of inhibitor bile acid. Stronger inhibitors were studied at a fixed concentration of 5 microM, weaker ones at 25 microM. Initial uptake velocity was measured by sedimenting an aliquot of cells through silicone oil into 3 N KOH every 15 s for 1 min. Uptake velocity (nmol X mg protein-1 X min-1) could then be related to taurocholate concentration and a Vmax and Km could be determined by applying a nonlinear least squares fit to the data obtained with or without inhibitor. The kinetic parameters allowed the determination of the type of inhibition and of inhibition constants (Ki) of the various test bile acids. The data indicate that bile acids containing a 6- or 7-OH group exhibit competitive inhibition, whereas bile acids with no 6- or 7-OH group exhibit noncompetitive inhibition. Of the compounds exhibiting competitive inhibition, Ki varied with the number of hydroxyl groups on the sterol moiety. We conclude that the presence or absence of a 6- or 7-OH group dictates the mechanism of inhibition; the number of hydroxyl substituents determines the potency of competitive inhibition.