Validation of a high performance liquid chromatographic method for alpha amanitin determination in urine.

The objective of the present study was to develop and validate a liquid chromatographic method with electrochemical detection to measure alpha amanitin concentrations in urine after sample pretreatment with double mechanism (reversed phase/cation exchange) solid-phase extraction cartridges. The urine samples (10 ml) were purified and concentrated to 1 ml with elimination of matrix contaminants. The extracts were then separated by isocratic reversed-phase chromatography using a C18 column (4.6 mm x 25 cm) with a mobile phase composed of 0.005 M phosphate buffer (pH 7.2) and acetonitrile (90:10). Coulometric detection was performed by applying an oxidation potential of +500 mV to a porous graphite electrode in a low-volume analytical cell. The limit of quantitation was 10 ng/ml with a signal-to-noise ratio = 25. The linearity studied on spiked urine was satisfactory (r = 0.9966) from 10 ng/ml to 200 ng/ml. The average extraction recovery of alpha amanitin was 78%, determined using spiked urine samples ranging from 10-300 ng/ml. The intra-assay precision was checked at 10, 50 and 100 ng/ml levels (n = 10) in spiked urine samples, with resulting coefficients of variation of 3.6%, 2% and 1.5%, respectively.

Human leptospirosis in Italy, 1986-1993.

In the eight-year period 1986-1993, the Italian National Center for Leptospirosis and the Regional Leptospira Laboratories confirmed 312 cases of clinical leptospirosis by using the microscopic agglutination (MA) assay. The majority of cases was observed in Northern regions of the Country. Cases were reported in all age groups, but were most common in the working-age population. Of 312 cases, 291 (93.3%) occurred among males. The largest number of infections was ascribed to occupational activities (45.8%). The typical leptospiral seasonal course, with a peak during the summer, was observed. Involvement of the liver was the most frequent manifestation. Influenza-like symptoms were the only signs of illness in 11.1% of cases. Anti-leptospira antibodies, cross-reacting with two or more serovars, were found in 28.2% of sera. The most frequent serovar-specific antibodies were those against poi, icterohaemorrhagiae, bratislava, copenhageni and sejroe.

Antibiotic sensitivity of Salmonella strains from food-handlers in the period 1980-1988 in Italy.

Throughout the past nine years, between 1980 and 1988, we tested 1,205,257 food-handlers as Salmonella carriers in Milan. We isolated 20,286 Salmonella strains with annual average prevalence of 1.68%, fairly costant in recent years. The serotype isolated confirm the prevalence of minor Salmonella, such as S. thypimurium, S. panama, S. anatum, S. london, S. derby, S. infantis, S. heidelberg, S. bredeney, S. braenderup. The frequency of new strains decreased from 15 in 1980 to 1 in 1988 and these were infrequent serogroups. Salmonella strains isolated were generally sensitive to aminoglycoside antibiotics (tobramycin, netilmicin), while they were often resistant to rifampicin, aztreonam, and sometimes to cefuroxime and tetracycline with multiple-resistance.

Serological determination of rabies antibodies in vaccinated subjects.

This study reports the immunological response in subjects vaccinated against rabies. Four serological methods were compared: the immunoenzymatic test with HDCV virus vaccine (EIA-V) and with purified viral glycoprotein (EIA-P), the neutralizing in vitro rapid fluorescent focus inhibition test (RFFIT) and the indirect immunofluorescent test (IFI). A concordance of 93.3% was found with RFFIT and EIA-V, 86.8% with RFFIT and EIA-P and 80.5% with RFFIT and IFI. The RFFIT is the most sensitive and specific method for rabies antibodies but more expensive and difficult. IFI recognizes late nucleocapsid antibodies but reports more false positive and negative results. The EIA-V and EIA-P methods are efficient, sensitive, specific and inexpensive for routine use to detect protective titres of antibodies above 0.5 IU/ml fifteen days after the inoculation of HDCV vaccine.

[Comparison of methods for the demonstration of Treponema-specific IgM]. / Metodologie a confronto per la ricerca di IgM Treponema-specifiche.

In this research 113 sera have been analysed with three methods for IgM treponema-specific determination: IgM-SPHA, IgM-EIA and 19S IgM FTA-ABS. Among these sera, 33 samples related to non-treated patients at different stages of infection, and 80 samples to treated patients. The results point out a light sensibility of the IgM-SPHA in primary lues. The IgM-EIA test has performed a good specificity, but displayed a sensibility lower to the 19S IgM FTA-ABS, which proved the best of tests. About the cases of treated lues at different stages, in 23 samples with VDRL negative has been found no positivity at three tests used, while in 49 samples with VDRL positive 8 are resulted positive at 19S IgM FTA-ABS.

[Seroepidemiologic study of human leptospirosis in Lombardy]. / Indagine sieroepidemiologica sulla leptospirosi umana in Lombardia.

In 1987 a seroepidemiological research was performed to verify the prevalence of leptospira agglutinins on the urban and rural population of Milan, Bergamo and Mantova Provinces in Lombardia Region. Were controlled 150 subjects (75 males and 15 females) in Milan town, 58 (49 males and 9 females) in municipalities of Bergamo Province, and 68 (55 males and 13 females) of Mantova Province. La prevalence of leptospira agglutinins in exposed subjects was 14.3% and in unexposed subjects 8.6%. In the females was noted a significative higher prevalence of agglutinins (14.4%) than in males (9.5%), especially in the females exposed (36.3%) than in females unexposed (9.6%). The leptospira serovars most interested were L. sejroe (15.9%), L. poi (13.6%), L. bratislava, L. canicola and L. Zanoni (9.1%). The sera tested proved reactivity of 56.8% also against two or three different serovars of pathogenic leptospira and prevalence of 13.6% of coagglutinins against nonpathogenic L. patoc serovar.

[Multicenter study of antirabies antibodies in vaccinated subjects: comparison of serologic methods]. / Ricerca multicentrica di anticorpi antirabbici in soggetti vaccinati: confronto tra metodiche sierologiche.

Polycentric research is performed in three laboratories to value the immunological response in vaccinated persons against rabies. The serological methods were: the immunoenzymatic test with virus of HDCV vaccine (EIA-V) and viral glycoprotein purified (EIA-P), the neutralizing in vitro test (RFFIT) and the immunofluorescent indirect test (IFI). The sera of 119 subjects were examined by three laboratories, every performed a different test. It is observed a concordance of 98.3% between RFFIT and EIA-V, of 86.8% between RFFIT and EIA-P, of 80.5% between RFFIT and IFI. The RFFIT is the method more sensible and specific for rabies antibody, but more expensive and difficult. The IFI showed late nucleocapsid antibodies and more false positive and negative cases. The EIA-V and EIA-P are efficient, sensible, specific, and not expensive routine methods to point out protective titres of antibodies, above 0.5 UI/ml after 15 day the inoculation of vaccine HDCV.

[Seroepidemiologic case finding in HTLV III infection, lues and HBV in a prison]. / Indagine sieroepidemiologica per infezione da HTLV III, lue e HBV in una collettività carceraria.

We report serological screening of 1217 prisoners for HTLV III antibodies, HBV markers and lues tests. The prevalence for HTLV III Abs on the whole of prisoners controlled is 30.8%, among the drug-users is 60.7%, with a relative risk (RR) of 1.18 between males and females in this group. It is been observed a significant association between aspecific positivity to VDRL and HTLV III Abs in the serum.

[IgM in the diagnosis of syphilis: results on the use of the IgM solid-phase hemadsorption test]. / Ricerca delle IgM nella diagnostica della sifilide: risultati sull'impiego del test IgM-SPHA (IgM solid-phase hemadsorption).

IgM-SPHA test was improved for IgM treponema-specific determination. The test can find routinary application because it does not require for its execution special equipments, and is very easy. With this test 160 sera of luetic subjects were examined 103 were treated and 57 were affected by lues at different stages and untreated. The test is rather sensitive in the secondary and latent lues and in the reinfections, while the results are paradoxically less satisfactory in the primary syphilis. The test can be a precious diagnostic tool since, beside allowing to decide the recovery from the disease from an immunological point, finds further applications in the connatal and neurological lues.

[Diagnostic research on leptospirosis in the Milanese territory]. / Indagini diagnostiche sulle leptospirosi nel territorio Milanese.

168 serum specimens have been tested for Leptospirosis in the years from 1980 to 1983. The identification procedure was a macroscopic plate agglutination screening test with formalin fied antigens and a confirmatory microscopic agglutination-lysis test of live antigens of actively growing cultures of different pathogenic serotypes propagated in a fluid medium. 34 serum specimens (23.8%) relevant to 18 cases were positive. L. icterohaemorrhagiae was positive in 12 cases (63.1%), L. pomona in 2 cases (10.5%), L. canicola in 1 case (5.3%), 1 case was positive for both L. grippotyphosa and L. bataviae and 1 case was positive for L. poi and L. sejroe. During the summer-fall of 1981 40 specimens of surface water from 4 pre-alpin lakes of the Provincia of Como were tested for Leptospiras with the cultural and biological examinations, following the detection of several leptospirosis cases on the relevant area. All water specimens gave negative results.

[Experiences with the control of antitetanus immunity at National Health Service units]. / Esperienze di controllo della immunità antitetanica nell'ambito del Servizio Sanitario Nazionale.

The results obtained in the PMIP of Milan for tetanus antitoxin titration in the serum of 772 subjects vaccinated permits a discussion on the problems posed by the antitetanus prophylaxis in the population and on the suitable methods for its control.

Resistance to chemioantibiotics of Salmonella strains isolated from people employed in food production and distribution in 1982.

From healthy carriers, working in the alimentation field in the milanese area 2144 serotypes of Salmonella have been isolated in 1982, on which sensitivity towards 19 chemioantibiotics has been tested selecting them among those mostly employed in therapy. A remarkable resistance of isolated strains has been observed towards streptomycin (91.77%) and rifampicin (82.00%) and a low resistance towards sisomycin (1.21%) and cefotaxime (1.30%). 402 resistance patterns with various degree have been noticed towards up to 11 chemioantibiotics, particularly among Salmonella strains belonging to those of long lasting circulation of B, C and D serological groups. The importance from the epidemiological point of view of the presence of Salmonella strains with multiple resistance among staff working in the alimentation field is stressed, because they may be considered as incubation sites for the appearance of alimentary salmonellosis.

[Organizational and methodologic problems in a human rabies prevention service]. / Problemi organizzativi e metodologici di un servizio per la profilassi antirabbica nell'uomo.

On the basis of attained experiences by Antirabies Centre of Milano are described the organization and structure of the antirabies services, the modality of correct performance of the immune prophylactic treatment, the estimation of the antibodies titre considered protective.

[Radioimmunological survey of serological markers of viral hepatitis]. / Rilevamento radioimmunologico dei markers sierologici dell'epatite virale.

230 serum samples of HBsAg carriers have been investigated for the presence of the combination HBeAg/anti-HBe as an index infectivity: an infectivity titre of 5,65% has been found for HBeAg and a positivity titre of 83,04% for anti-HBe antibody. In the institutionalized patients of an isolated community the positive titre of HBeAg resulted 28,57% whilst in the relevant health care staff the positivity titre was the same as detected for the carriers mentioned above. In a episode of viral hepatitis A in a child community a serological survey for IgM anti-HAV revealed 8 asymptomatic positive individuals referable to exposure to A type virus: it proves the utility of the individuation of IgM anti-HAV for an early diagnosis of the viral infection.

[Findings on the spread of anthrax spores in the provincial territory of Milan with tanneries]. / Indagini sulla diffusione di spore carbonchiose in territorio provinciale milanese con concerie.

This paper presents the results of a research, started since 1976, on the environmental spreading of the Anthrax spores in the county of Milan, where there is an high concentration of leather manufactures preparing and tanning skins mostly imported from Africa. B. anthracis spores were found in the raw skins, in the sewage water from the tanning units and in the fresh and dry sludges from the tannery water treatment plants. We propose hereby few operational procedures for the sanitizing of the imported skins and for the treatment of the tannery sludges in order to avoid the spreading of the Anthrax spores on the territory and the consequent threat to the health of the population and of the live-stock.