The variation in the amount of parvovirus B19 DNA and different classes of RNA in permissive and non-permissive infected cells was analysed by means of quantitative real-time PCR and RT-PCR assays. In the permissive bone marrow mononuclear cells, UT7/Epo and KU812Ep6 cells, viral DNA usually increased within 48 hpi, rarely exceeding 2 Logs with respect to input DNA. Viral RNA was always present within 2-6 hpi, its increase paralleled that of viral DNA up to 36-48 hpi, and all the different classes of viral RNA were constantly represented in stable relative amounts throughout the infection cycle. In the non-permissive TF-1 cells, viral DNA did not increase and only one most represented single class of viral RNA was detected. Our data do not support the current model for B19 virus replication and transcription, consisting in different early and late expression patterns, but suggest an alternative model, indicating that the B19 virus genome should be considered a single, two-state replicative and transcriptional unit.

tion rates than those with moderate impairment (P Ͻ0.05, Mann-Whitney rank-sum test). Furthermore, in patients with severe renal impairment, there was a linear relationship between the urinary free cortisol excretion rate and CrCl (r 2 ϭ 0.83, linear regression; Fig. 1B). On the other hand, we observed no linear relationship between urinary cortisol excretion rates and random total serum cortisol or the degree of proteinuria in the studied patients (data not shown). In contrast to the strong relationship between CrCl and urinary cortisol excretion rate, there was only a weak correlation between the volume of the 24-h urine collection and urinary free cortisol (r 2 ϭ 0.16, Spearman correlation). Therefore, it is unlikely that the observed reduction in urinary free cortisol excretion was attributable to the incomplete collection of 24-h urine.Because the estimation of CrCl involves collection of urine over 24 h, we further investigated whether the reduced urinary cortisol excretion could be predicted by use of the serum creatinine concentration alone. We performed a ROC curve analysis to determine the cutoff value for serum creatinine concentrations for the prediction of CrCl of Ͻ60 mL/min and found that the cutoff values for males and females were 126 and 94 mol/L, respectively. The distributions of urinary free cortisol excretion rates and CrCl in patients with normal and increased serum creatinine concentrations are shown in Fig. 1A. The mean urinary free cortisol excretion rates in individuals with normal and increased serum creatinine concentrations were 168 and 61 nmol/day, respectively (P Ͻ0.001, Mann-Whitney rank-sum test).Our study has shown that CrCl is a major determinant for urinary cortisol excretion rate in patients with severe renal impairment. Because there is a theoretical possibility that the low urinary cortisol excretion rate may reflect the low blood cortisol concentrations in these patients, we compared the total random morning serum cortisol concentrations for patients with different degrees of renal impairment and found that serum total cortisol was significantly higher in patients with CrCl Ͻ20 mL/min (P ϭ 0.002, Kruskal-Wallis test). This finding is consistent with previous reports that the serum cortisol concentrations of patients with end stage renal failure were higher than those of healthy individuals (9 ). Therefore, it is unlikely that the observed decrease in urinary free cortisol excretion in patients with moderate to severe renal impairment was attributable to hypoadrenalism.In summary, the urinary free cortisol excretion rate is significantly reduced in patients with moderate to severe renal impairment (CrCl Ͻ60 mL/min) and consequently has diminished sensitivity in the diagnosis of Cushing syndrome in such patients. Moreover, this diminished urinary free cortisol excretion rate could be accurately predicted by the increased serum creatinine concentrations. Therefore, it would be more appropriate to use late-night serum salivary cortisol or other blood-based diagnostic tests for...

Serum samples from 446 Italian blood donors between 18 and 65 years of age were analysed for the presence of IgG against parvovirus B19 capsid proteins VP1 and VP2 including conformational and linear epitopes. The overall prevalence of IgG against parvovirus B19 capsid proteins VP1 and VP2 against at least one antigen type was 79.1 %. No significant difference was found between men and women. In the 18-27 years age group, 77.0 % of the population had experienced infection with the virus, reaching 88.5 % in the 48-57 years age group. The overall prevalence of IgG was 78.0 % against conformational VP1 + VP2 antigens, 74.9 % against conformational VP2, 70.9 % against linear VP1 and 23.3 % against linear VP2 in the analysis of the IgG response against different conformational and linear epitopes of VP1 and VP2. Although IgG against conformational VP1+VP2, conformational VP2 and linear VP1 was present in more than 60 % of subjects in all age groups, IgG against VP2 linear antigens was present in only 32% of subjects in the 18-27 years age group and then decreased to 20.5 % in the 28-37 years age group. A different trend was noted when IgG positivity against linear and conformational epitopes was analysed separately in men and women. A significant increase was found in seroprevalence of IgG against VP2 conformational antigens with increasing age in males and a significant decrease in seroprevalence of IgG against VP2 linear antigens in women with increasing age.

B19 virus can be transmitted by contaminated blood or blood products. Recent observations, in healthy volunteers, suggest that active B19 infection can follow the administration of plasma pools with a concentration > or =10(7) genome equivalents/ml (geq/ml) of B19 DNA. However, patients receiving batches with levels of virus DNA lower than 10(4) geq/ml do not show any evidence of transmission of the virus. The aim of the study was to show, by in vitro assays, a threshold of viral load in B19 contaminated plasma pools over which the infection can be transmitted. Twenty plasma pools, each containing 960 single donations, were tested to correlate the viral load and the level of antibodies anti-B19 with the in vitro infectivity and expression of B19 virus. All the plasma pools, titrated for B19 viral load by competitive PCR, were inoculated into KU812Ep6 erythroid human cell line. Five of the nine contaminated plasma pools, with a B19 DNA concentration > or =3.60 x 10(6) geq/ml, were able to infect KU812Ep6 cells. In vitro infectivity was shown in KU812Ep6 cells at 24 h post-infection by in situ hybridisation and amplification assays for viral DNA and RNAs. Plasma pools with a viral load in the range of 6.00 x 10(3)-8.96 x 10(4) geq/ml did not show infectivity when inoculated into KU812Ep6 cells. Medium-high titres of IgG antibodies anti-B19 were detectable in all the plasma pools and the neutralising activity associated with specific IgG anti-B19 may explain the lack of infectivity of plasma pools contaminated with a low viral load. In conclusion, in situ hybridisation and amplification assays for viral DNA and RNAs in KU812Ep6 cells inoculated with plasma pools can be valid assays to test for the presence of infectious virus in the production of B19-safe material.

Obiettivo : verificare l'utilità del genotipo virale nella scelta e nella modulazione della terapia. Metodi : popolazione di 30 pazienti, età media 44.9, range 21-72 anni di cui 6 con cirrosi ( 20%) trattati inizialmente con interferone . Determinazione su tutti i pazienti della carica virale con AMPLICOR HCV Monitor e del genotipo con saggio di ibridazione inversa su striscia. (Genotype HCV III Nuclear Laser Medicine). Scopo del presente lavoro è la descrizione di un caso d'infezione acuta da HCV dopo incidente ospedaliero. Una colluttazione avvenuta fra un operatore sanitario ed un degente ha provocato escoriazioni in entrambi i soggetti con contatto reciproco. Il paziente, in seguito ha firmato la dimissione volontaria e si è allontanato dal Presidio Ospedaliero: non è stato quindi possibile effettuare accertamenti sierologici. L'operatore sanitario è stato monitorato, secondo i protocolli, e,dopo 3 mesi, anticorpi anti-HCV presenti, RIBA III positivo per core C22, NS3 C33, NS4 C100, PCR quantitativa 941 UI-mL, genotipo 3°. Seguiva un decorso clinico, nei successivi 6 mesi, apparentemente favorevole con negativizzazione della PCR quantitativa: si decideva di non eseguire alcuna terapia. Il valore dell'ALT presentava andamento sinusoide. Il controllo dopo 30 giorni rivelava un movimento antigenico (176 U.I.-mL) che aveva un'impennata al controllo del mese successivo (28.000 U.I.-mL): si decideva d'intervenire con RIBAVIRINA 1000-mg/die. I controlli successivi dimostrano ALT normali e PCR quantitativa negativa. La metodica PCR monitor Ampliprep-Amplicor correlata col quadro emato-chimico può essere impiegata per prevenire la cronicizzazione di un'infezione da HCV, anticipando l'intervento terapeutico. Mod. trasmissione/Gen P162 STUDIO DELL'ESPRESSIONE DI PARVOVIRUS B19 IN SISTEMI PERMISSIVI E NON MEDIANTE REAL-TIME PCRBonvicini F., Gallinella G., Manaresi E., Filippone C., Delbarba S., Gentilomi G., Musiani M., Zerbini M. Dip. di Medicina Clinica, Specialistica e Sperimentale, Sez. di Microbiologia, Università di Bologna, Via Massarenti 9, 40138 BolognaScopo studiare la replicazione e l'espressione del parvovirus B19 in sistemi sperimentali in vitro, come modello per l'interpretazione delle interazioni virus-cellula in vivo. Metodologie cellule permissive e non alla replicazione virale sono state infettate in vitro con parvovirus B19. Gli acidi nucleici e le proteine virali sono stati analizzati con metodi qualitativi e quantitativi a diversi tempi dopo l'infezione. In particolare, gli acidi nucleici virali, estratti dalle cellule infettate, sono stati analizzati mediante Real-Time PCR (sistema Light-Cycler Roche) utilizzando coppie di primer specifiche per il DNA e per le diverse classi di messaggeri virali. Risultati le coppie di primer disegnate per la determinazione quantitativa del genoma virale e, in maniera selettiva, delle diverse classi di messaggeri virali, hanno consentito di identificare diversi livelli di replicazione e pattern di espressione del genoma virale. Sono state analizzate sia colture pri...