Virological investigation on aerosol from waste depuration plants.

Aerosol from activated mud decontamination plants used for the treatment of urban sewage can represent a vehicle for bacteria, virus and fungi. As a result, they become an infective hazard for plant personnel, the general population residing in the surrounding area and the occasional visitor. The present investigation focuses on the identification of enteric-type viruses in this kind of aerosol. The following methods were employed on 214 samples collected in the 1999-2000 period: cell culture (BGM, RD, Hep-2), electron microscopy, and polymerase chain reaction (PCR). Cytopathic effect was mild in 180 samples, and severe in 14, upon their first passage in culture. Virus identification was based on positivity to both electron microscopy (EM) and PCR. Thus, one positive sample was recognized to be of enteric-type virus and two positive samples were recognized as reovirus-type. All samples were negative for Norwalk-type virus or HAV. There was considerable discrepancy between electron microscopy and PCR concerning the number of enteric-type viruses recognized. A possible explanation is contamination with animal-type enterovirus.

[Research on Pisa University student's life style and health]. / Indagine sugli stili di vita e la salute degli studenti universitari dell'ateneo pisano.

Surveys on life and health conditions of university students besides providing useful epidemiologic data partly related to the young population. They are potentially useful for the intervention planning aimed to remove possible environmental or behavioural risk factors. Following these purpose a survey sample about 1200 student attending the University of Pisa was carried out through an anonymous questionnaire about 41 questions focused on behaviours and lifestyles of young people, possible health problems, self-perception of the health state, the use of public health services, and the more commonly used (or preferred) sources of information on health topics. The results suggest that although the self perception of the state of health is broadly positive for the majority of students, there is also a significant frequency of individuals declaring significant at risk behaviours (persistence of sexual practices at risk, drug use, alcohol and smoke abuse). The consumption, also sporadic, of psicoattive substances has been declared gives beyond 40% of the students, in particular 37% asserts to have tried drugs. The habit to smoke appeared diffused much between the students; the percentage of smokers turns out equal to 41% in total (40% females, 42% males). 76% of the interviewed ones has declared to be sexually active, the percentage of those who have declared multiple relations or occasional is elevated (12%), and 47% of the students asserts to use the condom with fixed partner and 77% with occasional partner. Moreover there seems to be a significant association between the changes in the diet and other lifestyles that are due to the "university lifestyle" (as a consequence of leaving the family or increased commuting) and the insurgence of several types of sickness, e.g. gastroenteric disturbances and fever attacks. Finally, the present investigation suggests that few interventions on services offered to students (such as accommodation, teaching structures and especially the creation of a "health office" where students can acquire information on health and other topics related to their condition) could have a significantly beneficial impact on the general state of health of the university population.

Identification of cytopathogenic isolates from environmental matrices: application of analytical protocols.

This work was aimed at studying cytopathogenic agents isolated from the environment by adopting, in parallel, various techniques currently used in clinical virology. The purpose of this research was not only to identify the viral species isolated but also to verify the most reliable protocols for environmental applications.

Virological control of groundwater quality using biomolecular tests.

Deep groundwater, even if generally protected, could be contaminated by surface or rain water infiltration through soil fractures, septic tanks, cesspits, land irrigation, disposal of wastewater and disposal of muds from depuration systems. The sanitary importance of such possible contamination is related to the different uses of the water and it is at the maximum level when it is intended for human use. Routine microbiological analyses do not consider viruses, only bacterial parameters, as contamination indicators. However, it is known that enteric viruses can survive a long time in deep aquifers and that they may not always be associated with bacterial indicators. The virological analysis of waters intended for drinking use is provided only as an occasional control exercised at the discretion of the sanitary authority. Technological difficulties with obtaining data about groundwater viral contamination led to a study to devise rapid and efficient methods for their detection and the application of these methods to samples from different sources. Four acid nucleic extraction techniques have been tested (classic proteinase K- phenol/chloroform, QIAamp Viral RNA Kit (Qiagen), SV Total RNA Isolation System (Promega) and NucleoSpin Virus L (Macherey-Nagel). Sensitivity and specificity of RT-PCR protocols for entero- (EV), hepatitis A (HAV) and small round structured (SRSV) viruses have been verified. Deep groundwater samples (100 L) were concentrated (2-step tangential flow ultrafiltration) and the concentrate contaminated with serial 10-fold dilutions of a known titre of poliovirus type 3. Extracted RNA was concentrated (microcon-100) and analysed by RT-PCR using specific EV primers and visualising amplification products by agarose gel electrophoresis. In addition, two different methods of RT-PCR for non-cultivable viruses have been tested: (a) RT-PCR and nested RT-PCR for HAV and (b) RT-PCR with generic primers and RT-PCR with specific primers for SRSV. Different specificity tests have been carried out in the presence of some of the commoner microorganisms. The most efficient, sensitive and specific protocols were used to test 35 x 100L deep groundwater samples. Sample concentrates were split with one part treated with chloroform and analysed by cell culture (BGM and Frp/3, derived from FrHK/4, cells) and the other tested by RT-PCR for HAV, EV and SRSV. Results demonstrated the high efficiency of the classic and QIAamp methods. Microcon-100 did not increase the sensitivity of the technique used. The highest sensitivity was observed for RT-PCR with specific primers for SRSV and for nested RT-PCR for HAV. One sample showed a cytopathic effect, not confirmed at the third subculture, while the RT-PCR allowed the detection of echovirus 7. Cell culture did not allow detection of the majority of the enteric viruses while PCR gave sensitive, specific and rapid detection of a range of agents in the same samples. Even if it was impossible to fix a virological quality standard, it would be necessary to find a viral indicator in order to achieve a complete preventive check which would be particularly useful in some cases (e.g. water never used before, after pollution accidents, for seasonal checking).

Indication of hysteroscopy in tamoxifen treated breast cancer patients.

The aim of this study was to indicate the patients treated with tamoxifen for breast cancer in which hysteroscopy with biopsy should be considered mandatory. 414 breast cancer patients who underwent hysteroscopy with bioptic evaluation were enrolled in the study. 334 subjects were treated with 20 mg of tamoxifen daily as adjuvant therapy for six up to a hundred months. Of the remaining 80 control patients, which had not received tamoxifen, 30 were in premenopause (Group IA) and 50, in postmenopause (Group IIA). The tamoxifen-treated patients were subdivided in premenopausal (Group IB = 72 patients) and in postmenopausal (Group IIB = 262 patients) groups. All patients were further classified in asymptomatic or symptomatic groups considering whether uterine bleeding was absent or present. The evaluation of the endometrial mucosa was performed by office hysteroscopy. In group IIB patients presenting uterine bleeding, malignant lesions were found in 7.8% of the cases. The incidence of premalignant and malignant lesions in IIB patients treated for longer than 3 years (11.7%) was higher than that observed in IIB patients treated for less than 3 years (1.3%). There was a significant difference in terms of endometrial pathology between Group IIB (32.8%) and Group IIA (8%) (p < 0.001); and between Group IIB (32.8%) and Group IB (13.9%) women (p = 0.003). Among IA and IIA patients there were no cases of endometrial hyperplasia or cancer; on the contrary, in IB and IIB women, 2 and 22 cases of atypical hyperplasia were observed, respectively. All cases of endometrial cancer were observed in Group IIB and had a diagnosis of poor prognosis. In conclusion the hysteroscopy with biopsy should be considered the first diagnostic procedure to perform in tamoxifen-treated postmenopausal patients presenting uterine bleeding and in postmenopausal women treated for longer than 3 years. In premenopause, hysteroscopy should be proposed to women with ultrasonographic abnormalities and/or with uterine bleeding to patients at high risk for endometrial cancer.

Detection and potential indicators of the presence of hepatitis C virus on surfaces in hospital settings.

AIMS: The risk of hepatitis C virus infection in hospital environments can be assessed not only by studying epidemiological data and work practices, but also by the detection of these viruses (or indicators thereof) in health-care settings, on instruments etc. METHODS: Since standardized techniques specific to this end do not exist, this study was undertaken to apply methods currently used on clinical samples to the assessment of environmental HCV risk, either through direct detection of the virus (RT-PCR), or by probing for haemoglobin as a potential indicator of blood contamination. The tested techniques were applied in a trial environmental monitoring programme undertaken in various hospital laboratories and clinics, during which total bacterial count determinations were performed in parallel with haemoglobin and hepatitis C virus detection. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that the applied methods are of value in detecting low levels of contamination in a hospital environment.

Interference between enterovirus and reovirus as a limiting factor in environmental virus detection.

AIMS: Faecal material from raw sewage or other sources lacking effective treatment sometimes contaminates water for human consumption. The relevant Italian regulations therefore call for testing drinking and recreational water for the presence of enterovirus. METHODS AND RESULTS: Traditional methods of analysis are based on revealing the typical cytopathic effects of enterovirus on cell cultures. However, the presence in environmental samples of different types of virus may cause interference phenomena that mask such cytopathic effects. The paper reports on an experimental test of this interference hypothesis. Buffalo Green Monkey cell cultures were co-infected via mixed suspensions of the polio type 3 virus and reovirus type 1. Cytopathic effects were then sought and the presence of enterovirus tested for via RT-PCR. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained indicate that the normally high sensitivity of tests for the detection of enterovirus in samples is considerably decreased by the simultaneous presence of reovirus.

Expression and prognostic significance of urokinase and plasminogen activator inhibitor type-1 in endometrial hyperplasia and cancer.

Proteolytic enzymes, like urokinase (uPA) and plasminogen activator inhibitor type-1 (PAI-1), are involved in remodelling tissues during invasion and metastasis of tumor cells. The purpose of the study is to evaluate the expression and the prognostic significance of these enzymes in endometrial hyperplasia and cancer. We used immunohistochemical staining to localize uPA and PAI-1 antigens and evaluate their expression, and the enzyme-linked immunosorbent assay (ELISA) to measure their levels during the progression of endometrial carcinoma. The results show that the levels of uPA and PAI-1 detection are systematically weak in simplex hyperplasia and are moderate in complex hyperplasia. In the endometrial carcinoma a very strong reaction was observed in the most aggressive variant of epithelial tumors. A positive signal for uPA was found only in the cytoplasm of normal and hyperplastic cells while, in tumors, uPA was present also in the cellular areas surrounding the neoplastic glands and at the apex of the malignant cells. The PAI-1 immunoreactivity was weak to moderate in 95.4% of carcinomas, with a diffuse signal mostly distributed in the cytoplasm of neoplastic cells and tumor stroma. UPA antigen concentrations were significantly higher in endometrial carcinoma than in endometrial hyperplasia (p<0.05) and in normal endometrium (p<0.001). PAI-1 antigen concentrations in carcinoma samples were significantly higher than in normal endometrium (p=0.002), but the difference was not statistically significant with respect to that in endometrial hyperplasia. We did not find any correlation between uPA and PAI-1 concentrations and the standard prognostic parameters for evaluating endometrial carcinoma. In conclusion, this study demonstrates that in hyperplastic endometria and in endometrial carcinoma there is a progressive increase in expression of uPA and PAI-1 than in normal endometrial tissue. In carcinoma tissues, the high expression of uPA is unregulated in the surrounding stroma tissue, particularly in the most aggressive histopathologic variants. UPA and PAI-1 may be factors associated with invasive behavior in endometrial carcinoma independent of other clinicopathological parameters.

Nitroxides and malignant human tissues: electron spin resonance in colorectal neoplastic and healthy tissues.

Healthy and neoplastic colorectal human tissues of as many as 12 patients have been studied, immediately after surgery, by electron spin resonance (ESR) of stable nitroxides at physiological temperature. Cells were maintained in a living state using the McCoy's 5A culture medium. The very low concentration changes of hydrophilic and lipophilic nitroxides allowed us to establish that the response to the oxidative stress induced by the occurrence of nitroxides in healthy and tumor cells was very weak, thus suggesting these compounds are good candidates for contrast enhancement agents in magnetic resonance imaging of colorectal tumor. The analysis of the computed ESR line shape of lipophilic nitroxides in both healthy and malignant cells of the same patient agreed for an unmodified physical status of the membranes where they were mainly localized. The results reported here proved that the comparison between ESR results must be made in tissues from the same patient and that the physical status of the membranes depended more on the patient history than on changes in the colorectal cell membrane fluidity induced by the neoplastic process.

Comparison of three assays for HIV antibodies detection in urine to be applied to epidemiological setting.

Three immune-enzymatic tests for urinary HIV antibodies were examined in order to assess their sensibility, specificity and delta value. The highest sensibility was noticed for the Seradyn test (98.8%), followed by the Wellcozyme test (98.1%) and finally the SUDS rapid test (56.8%). The resultant specificity was 98.5% for the Seradyn test, 91.3% for the Wellcozyme test and 97.3% for the SUDS test. The measurement of delta value showed a higher capability of discrimination for Seradyn test, that could be considered the most reliable for epidemiological purposes.

Cord plasma corticotropin-releasing factor-binding protein (CRF-BP) in term and preterm labour.

Corticotropin-releasing factor-binding protein (CRF-BP) in pregnant women is measurable in maternal and fetal plasma as well as in amniotic fluid. The concentration of CRF-BP in maternal plasma and amniotic fluid changes significantly at the time of parturition. The aim of the present study was to evaluate fetal plasma CRF-BP levels in women delivering at term or with preterm labour. CRF-BP levels were measured the in umbilical cord plasma of women subdivided into two groups: (1) healthy pregnant women throughout the last 5 weeks of pregnancy either (a) out of labour (n = 21) or (b) at delivery after spontaneous labour (n = 64); and (2) patients with preterm labour (a) gone to delivery (n = 12) or (b) responding to tocolysis (n = 10). In the group of healthy women at term, CRF-BP levels were also measured in maternal plasma. CRF-BP was measurable in all specimens of umbilical cord plasma. Mean values +/- SEM at 40 weeks (5.85 +/- 0.65 nmol/l) were significantly lower than those obtained at 37 (6.48 +/- 0.47 nmol/l) or 38 (6.95 +/- 1.16 nmol/l) weeks of pregnancy. Similarly, mean +/- SEM maternal plasma CRF-BP levels in women at term out of labour were lowest at 40 weeks (3.57 +/- 0.22 nmol/l). In these women, mean +/- SEM CRF-BP levels in cord plasma (37 weeks: 6.47 +/- 0.47; 38 weeks: 6.95 +/- 1.16; 40 weeks: 5.85 +/- 0.65 nmol/l) were significantly higher than in maternal plasma at the same gestational age (37 weeks: 4.29 +/- 0.2; 38 weeks: 4.35 +/- 0.205; 40 weeks: 3.57 +/- 0.22 nmol/l). Mean +/- SEM levels of cord blood collected at delivery at term (4.93 +/- 0.14 nmol/l) showed lower CRF-BP levels than in women out of labour (6.18 +/- 0.55 nmol/l). Patients with preterm labour, with delivery within 48 h, showed significantly lower levels of cord plasma CRF-BP (4.21 +/- 0.29 nmol/l) than women at term out of labour (6.18 +/- 0.55 nmol/l) and than those at term with labour (4.93 +/- 0.14 nmol/l). Cord plasma CRF-BP levels decreased in the last 5 weeks of pregnancy, similar to maternal plasma CRF-BP levels, the lowest values resulting in women at labour or with preterm labour, thus suggesting that changes of CRF-BP in cord plasma are associated with the events of parturition.