Usefulness of human papilloma virus testing in the screening of cervical cancer precursor lesions: a retrospective study in 314 cases.

OBJECTIVE: To assess the usefulness of human papilloma virus (HPV) typing for predicting pre-malignant and malignant cervical lesions. STUDY DESIGN: 314 women, who underwent colposcopy, biopsies and high and low-risk HPV typing after a confirmed abnormal routine Pap test were studied. HPV-DNAs were typed by using PCR technique. RESULTS: We found a significant increasing rate of high-risk-HPV by the increasing severity of histology, ranging from 40% in negative cases to 86.9% in those with CIN3 lesions. The positive predictive value of high-risk-HPV ranged from 13.3% in patients with atypical squamous cells of undetermined significance (ASCUS) to 29.4% in those with HSIL. By contrast, negative predictive value was 96% in patients with ASCUS, 97.2% in low-grade squamous intraepithelial lesions (LSIL), and 71.4% in high-grade squamous intraepithelial lesions (HSIL). Sensitivity and specificity for detecting CIN2 or CIN3 was 86.0% and 41.3%, respectively. CONCLUSIONS: The high negative predictive value of high-risk HPV testing suggests that HPV negativity could be used for predicting the absence of important cervical lesions, and therefore avoiding unnecessary colposcopy in ASCUS and LSIL cases.

TTV infection in patients with acute hepatitis of defined aetiology and in acute non-A-E hepatitis.

BACKGROUND/AIMS: Recently, the presence of a novel nonenveloped single-stranded DNA virus (TTV) has been associated with either acute or chronic hepatitis of unknown aetiology, suggesting a possible aetiological role. The aim of this study was to evaluate the prevalence, the significance and the clinical impact of TTV infection in patients with acute viral hepatitis of defined aetiology and in patients with non-A-E acute hepatitis. METHODS: TTV-DNA was tested by hemi-nested PCR in serum samples collected from 121 patients during and after acute hepatitis (103 with acute viral hepatitis of defined aetiology and 18 with acute non-A-E hepatitis) and in 30 healthy controls. RESULTS: Overall, the rate of TTV infection was 12.6% (13/103) in patients with acute hepatitis of defined aetiology, 16.6% (3/18) in patients with non-A-E acute hepatitis and 6.6% (2/30) in the healthy control group, (p=n.s). TTV-DNA was detected in the following proportions: hepatitis B, 13.2% (7/53); hepatitis C, 16.6% (4/24); hepatitis A, 4.7% (1/21); hepatitis E 20% (1/5). Moreover, acute hepatitis with and without TTV infection/coinfection were comparable in terms of both liver biochemistry and chronicity rate. The results of TTV re-testing after serial dilutions of six TTV-DNA positive serum samples during and after the peak of liver transaminases failed to demonstrate a correlation between liver damage and viral titre. CONCLUSIONS: The prevalence of TTV infection appeared to be comparable in patients with non-A-E hepatitis, in acute hepatitis of defined aetiology and in the control group. Hence, an aetiological role of TTV for acute hepatitis of unknown aetiology seems questionable. Moreover, TTV infection does not modify the natural history of acute hepatitis of defined aetiology.

Fibrogenesis serum markers in patients with chronic hepatitis C treated with alpha-IFN.

The correlation between therapeutic response and liver fibrogenesis was studied in serum and liver specimens taken from 31 patients treated with alpha-interferon (IFN) (14 sustained responders and 17 non-responders) for chronic hepatitis C. Serum samples, collected before therapy, and at further 6-month intervals over 2 years, were tested for markers of liver neofibrogenesis. Serum N-terminal procollagen III peptide (PIIINP) displayed a significant and persistent decrease (P < 0.05) in sustained responders but not in non-responders; significantly lowered (P < 0.05) mean levels of C-terminal procollagen I peptide (PICP) were transiently observed in both patient groups, apparently as a result of IFN administration. Serum laminin (Lam) levels remained unchanged. One year after the cessation of treatment, liver biopsy re-testing showed an improvement in necro-inflammatory scores only in sustained responders, with the histological fibrosis scores remaining unaltered in both groups. IFN treatment seemed to exert an influence on serum levels of markers of hepatic connective tissue turnover even in patients that did not respond to therapy, while no effect was observed on preexistent liver fibrosis.

High prevalence of HCV-RNA in the saliva cell fraction of patients with chronic hepatitis C but no evidence of HCV transmission among sexual partners.

The aims of this study were to evaluate the prevalence of HCV-RNA in different fractions of saliva taken from patients with chronic hepatitis C, to establish whether virologic parameters or disease severity exert any influence on the detectability of HCV-RNA in saliva, and to evaluate the prevalence of HCV infection in partners of HCV-infected subjects with respect to the presence of HCV-RNA in saliva. Sera samples and different fractions of saliva (whole saliva, surnatant, and cell fraction) from 48 subjects (45 with chronic hepatitis C and three healthy anti-HCV+ carriers) were examined for HCV-RNA by RT nested PCR and DEIA hybridization. HCV-RNA-positive sera were also tested for genotype and viral titer (bDNA2 method). Twenty-seven stable sexual partners (25 females and 2 males) were screened for anti-HCV antibodies at least twice over a minimum of 12 months. HCV-RNA was detected in the sera of 39/45 patients and of 22/39 viremic patients. In all of the latter, the presence of HCV-RNA was restricted to the cell fraction. Viral titer was significantly higher in patients with HCV-RNA in saliva than in those without (12.3 x 10(6) versus 4.6 x 10(6) eq/ml, P < 0.01). HCV-RNA positivity was unrelated to genotype, duration of disease, Hepatitis Activity Index scores or transaminase levels. Anti-HCV was positive in one of 13 sexual partners of patients with HCV-RNA in saliva and in 1/14 of those without (P = NS). In conclusion, HCV-RNA is detectable in the cell fraction of saliva in a high proportion of highly viremic patients with chronic hepatitis C, but its presence does not seem to be associated with an increased risk of HCV transmission among sexual partners.

HGV/GBV-C in liver tissue and in sera from patients with chronic hepatitis C.

Forty-eight persons (M = 45, F = 3; age range = 20-53, mean = 32.2) affected with chronic hepatitis C were tested for HGV/GBV-C RNA and HCV-RNA by nested PCR and DEIA in serum and in liver specimens to evaluate the prevalence and the impact of HGV/GBV-C coinfection in patients with chronic HCV-related hepatitis. Sera were also assayed for antibodies to HGV/GBV-C E2 protein. Serum HGV/GBV-RNA could be detected in nine (19%) patients, and anti-E2 antibodies in 22 (46%) patients. The presence of HGV/GBV-C RNA or anti-E2 antibodies was mutually exclusive. The cumulative prevalence of HGV/GBV-C infection was 65% (31/48); the majority of these patients (26/31, 84%) were intravenous drug users (IVDUs). In eight of nine patients viraemic for HGV/GBV-C, RNA positivity could be revealed even in liver specimens; these eight patients were also positive for HCV-RNA both in serum and the liver and did not exhibit any specific association with HCV genotype. HGV/GBV-C RNA negative strand RT-PCR testing was negative in all of the eight liver specimens, providing little support to the hypothesis that liver represents the primary site of HGV/GBV-C replication. Moreover, patients with HGV/GBV-C and HCV coinfection were comparable to those with HCV infection alone in terms of biochemistry and liver histology.

Insulin-dependent diabetes mellitus during alpha-interferon therapy for chronic viral hepatitis.

A 29-year-old man was observed to develop insulin-dependent diabetes mellitus following a 5-month treatment with recombinant alpha-2b-interferon for chronic hepatitis C. After the onset of the disease, serum samples that had, respectively, been collected before therapy commencement, at month 3, and at the onset of insulin-dependent diabetes mellitus were tested for islet-cell (ICA-IgG), glutamic acid decarboxylase (GAD-Abs), IA2 (IA2-Abs) and insulin (IA-Abs) autoantibodies. The following results were obtained: ICA-IgG, 5, >80, and >80 JDF-U, respectively; GAD-Abs: >100 U/ml in all three measurements; IA2-Abs and IA-Abs: negative. During treatment, thyroid microsomal autoantibodies increased markedly (from 1:100 to 25,600 titer); thyroid-stimulating hormone was persistently normal. HLA class II typing revealed a genetic predisposition to insulin-dependent diabetes mellitus as demonstrated by the presence of DRB1* 04/08, DQ A1 52 Arg+/Arg+, and DQB1 57 N-Asp/Asp alleles. One year after the onset of insulin-dependent diabetes mellitus, the patient is still receiving 30 IU insulin daily; the liver function tests are normal and HCV-RNA is negative. These data support the hypothesis that, in predisposed patients, alpha-interferon therapy can enhance an ongoing autoimmune process against pancreatic beta-cells and induce overt insulin-dependent diabetes mellitus. We therefore suggest that, in patients with a documented predisposition to insulin-dependent diabetes mellitus, alpha-IFN therapy should be administered with caution.

HGV/GBV-C infection in patients with acute hepatitis of different etiology and in patients with chronic hepatitis C.

To investigate the prevalence of hepatitis G virus (HGV/GBV-C) in patients with liver disease and to confirm its hypothesized ability to cause liver damage, we studied 130 subjects; 61 had chronic hepatitis C virus infection and 69 had acute hepatitis of either defined etiology (n = 57) or of unknown origin (n = 12). Positivity for HGV/GBV-C RNA was detected in 10 of the 61 subjects with chronic hepatitis C (16.3%) and in 11 of the 57 subjects with acute hepatitis of defined etiology (19%), whereas we failed to detect HGV/ GBV-C viremia in subjects with hepatitis of nonestablished etiology. Patients exhibiting positivity for HGV/GBV-C RNA were found to be comparable to those exhibiting negativity for HGV/GBV-C RNA in terms of both liver function tests and Knodell's score (in liver biopsies); the affect of HGV/GBV-C infection on the biohumoral and histological activity in patients with chronic hepatitis C therefore appears to be minimal or absent. Similar clinical features were observed in patients with acute hepatitis of known etiology whether they were positive or negative for HGV/GBV-C RNA. However, long-term clinical studies are still required to clarify the actual impact of HGV/GBV-C co-infection. In our geographic, i.e., a region or north-east Italy, HGV/GBV-C infection appears to be strictly related to intravenous drug use, and this agent does not seem to be responsible for acute hepatitis of unknown etiology; other etiological agents are probably involved.

A DNA hybridization method for typing hepatitis C virus genotype 2c.

A high prevalence of hepatitis C virus (HCV) genotype 2c (22%) was detected in sera from 459 italian patients by core-region amplification and hybridization with specific probes by DNA enzyme immunoassay. Amplified fragments failed to hybridize with 1a, 1b, 2a, 2b and 3a subtype-specific and 4, 5, 6 type-specific oligonucleotides in 105 patients. Hybridization of these samples with type 2 probe, which recognized all the subtypes sequences, showed evidence for genotype 2 distinct from 2a and 2b. Fourteen out of these 105 isolates were cloned and sequenced. The results were consistent with genotyping assay. Nucleotide sequences were partially related to types 2a, 2b, 2d, 2e and 2f (87.0-93.5% of identity). The average nucleotide identity was highest for genotype 2c (95.87%). On the basis of sequence analysis, subtype 2c specific probe was derived. Hybridization efficiency with the newly designed probe was very high and more than 95% (100/105) of type 2 cases were classified as 2c. Evidence of different outcome of therapy inside the same HCV major type account for the need of accurate subtyping. In this study, amplification of the core region followed by hybridization with highly specific probes enabled distinction between HCV subtypes.

Comparison of competitive and non-competitive reverse transcription-polymerase chain reaction (RT-PCR) for the quantification of hepatitis C virus (HCV) RNA.

A competitive reverse transcription (RT)-nested polymerase chain reaction (PCR) assay for HCV RNA was compared with the Roche Amplicor HCV Monitor assay, based on non-competitive, single step RT-PCR. A total of 83 serum samples were tested in parallel by both assays. All samples could be quantified by competitive RT-PCR (cPCR), whereas seven were negative by the non-competitive assay (ncPCR). The HCV RNA titre of the discordant samples assessed by cPCR was significantly lower than that of the remaining 76 (P < 0.001). Absolute HCV RNA titres were higher by cPCR than by ncPCR (P < 0.001), even if the results of the two methods were statistically correlated (P < 0.001). HCV RNA titre tested by cPCR was not significantly different between samples infected with genotype 1 or 2. However, values obtained by ncPCR were higher in samples with genotype 1 (P < 0.001). Furthermore, all seven discordant samples were infected with genotype 2. When both methods were used to measure serial dilutions of standard HCV RNA, we observed a bias to lower measurements with the ncPCR kit. This study shows a good correlation between the results of two PCR-based methods for the quantification HCV RNA. However, the degree of sensitivity and the absolute HCV RNA titre measured may vary according to the assay used.

Altered lipid peroxidation/glutathione ratio in experimental extrahepatic cholestasis.

1. Lipid peroxidation can occur in the presence of a cellular antioxidant-oxidant imbalance, but the role of lipid peroxides in cholestasis is not well understood. 2. This study was undertaken in order to: (i) evaluate the behaviour of a product of lipid peroxidation (thiobarbituric acid-reactive species), and of an important antioxidant tripeptide, reduced glutathione, in the course of experimental extrahepatic cholestasis; and (ii) ascertain whether there was a link between this aspect and the alterations in liver morphology. 3. Forty-five male Sprague-Dawley rats (250-300 g) were double bile duct ligated and followed from 1 to 28 days. At the end of each experimental period, blood and liver samples were collected for thiobarbituric acid-reactive species and glutathione determinations. 4. Bile duct ligated rats showed a marked increase in liver weight which was related to cholestasis duration and to some anatomical alterations such as bile duct proliferation and dilation and liver fibrosis (periportal, perivenular, perineoductular and parenchymal). 5. An increase in serum lipid peroxidation was also observed but this was not linked to hepatic thiobarbituric acid-reactive species. Erythrocyte and hepatic glutathione decreased in relation to cholestasis duration. Serum lipid peroxides and erythrocyte glutathione were correlated with liver cell necrosis. 6. In conclusion, experimental extrahepatic cholestasis determines bile duct proliferation and fibrosis, the degree of which is directly related to the duration of cholestasis itself and to liver cell necrotic phenomena.(ABSTRACT TRUNCATED AT 250 WORDS)

Deoxynucleotide pools and DNA synthesis in resting and PHA-stimulated human lymphocytes treated with mutagens.

In resting and PHA-stimulated PBL treated with uv light or MMS we measured the sizes of the dTTP and dATP pools and the variation of ATP content taken as an indicator of cytotoxicity. The effects on DNA synthesis were examined by measuring DNA repair (unscheduled DNA synthesis) in resting PBL and the inhibition of DNA replication in stimulated PBL. While both treatments affected DNA synthesis, only MMS perturbed dNTP pools and decreased the intracellular concentration of ATP. All the effects were more evident in cycling than in resting lymphocytes.

Detection by fluorescence analysis of DNA unwinding and unscheduled DNA synthesis, of DNA damage and repair induced in vitro by direct-acting mutagens on human lymphocytes.

The sensitivity and reliability of UDS and FADU in detecting mutagenic effects were compared by measuring DNA damage and repair in PBL treated in vitro with UV light, MMS and BPDE. The results indicate that FADU is more sensitive than UDS, as it is able to detect DNA damage at doses 3-4-fold lower. We also determined the DNA damage and repair induced by the above agents on lymphocyte samples from different donors by FADU and UDS, confirming that the DNA repair process in humans is characterized by interindividually variable efficiency.