HCV-RNA detection in liver bioptates--a comparison of automatic and 'home-made' protocols combined with a new procedure of HCV-RNA extraction.

BACKGROUND: For HCV-RNA detection in liver tissue a generally accepted reference method has not been established yet. Therefore, we have developed a procedure of HCV-RNA extraction from liver tissue and compared two methods of HCV-RNA detection. MATERIALS AND METHODS: A set of 32 liver biopsy specimens, obtained from chronically HCV infected patients, has been examined. At the time of biopsy, serum HCV-RNA was detectable in 20 patients in RT-PCR automatic Cobas AmplicorTM Hepatitis C assay, ver. 2.0 (Roche Molecular Systems, Inc, Pleasanton, CA, USA) 2 serum samples were negative for HCV-RNA and 10 patients has not been tested. Liver tissue has been homogenized in the presence of CRSR-Green (Fast RNA Kit-Green, Bio101, Inc, Vista, CA, USA) and a mixture of phenol/chloroform/isoamylic alcohol, using a FastPrep homogenizator (Bio101, Inc, Vista, CA, USA). Then RNA has been isolated from the material obtained using the Total RNA Prep Plus procedure (A&A Biotechnology, Gdansk, Poland). Presence of HCV-RNA was next tested by means either of 'home-made' nested RT-PCR procedure or the RT-PCR automatic Cobas AmplicorTM Hepatitis C assay, ver. 2.0. In case of an inhibition of PCR detected in the first run of automatic assay, both PCR procedures have been repeated. RESULTS: In 5 cases of automatic assay an inhibition of PCR reaction has been detected in the first run, the RT-PCR procedures has been then successfully repeated in the second run. The presence of HCV-RNA in the liver tissues was detected in a total of 22 cases (69%) by mean of automatic assay and in a total of 20 cases (63%) by means of 'home-made' RT-PCR procedure. Except for two cases of HCV-RNA positivity in a biopsy tissue, detected by means of automatic but not 'home-made' procedure, the results obtained employing the methods have been identical. In one serum HCV-RNA positive case, the presence of HCV RNA has not been detected in a liver tissue using both methods. CONCLUSIONS: Our procedure of RNA isolation combined either with automatic Cobas AmplicorTM Hepatitis C assay, ver. 2.0 or 'home-made' RT-PCR procedure may be useful for establishing presence of HCV-RNA in liver tissue. In this combination, the automatic assay seems to be more sensitive than the 'home-made' procedure.

HCV infection in Poland.

BACKGROUND: Hepatitis C virus (HCV) infection is one of the most prevalent infectious diseases in the world. In 1997, hepatitis C became a statutorily noticeable disease in Poland. Nevertheless, to date, only a few notable studies on the prevalence of HCV infection have been carried out in Poland. Therefore, a study to determine the prevalence of HCV infection markers in an unselected population of Polish subjects was performed. METHODS: After several advertisements (in the print media and on television, radio, and other public media) concerning free testing for all volunteers in a hospital laboratory, serum samples of 2,561 subjects (765 men and 1,796 women), with a mean age of 43 years (range 1-88 years), were collected and assessed. In the samples, we first tested for the presence of IgG anti-HCV antibodies using the third generation enzyme immunoassay Anti-HCV EIA Cobas(R) Core Test (Hoffmann La Roche, Basel, Switzerland). The determination of HCV-RNA was then performed on anti-HCV IgG-positive samples by qualitative reverse transcription-polymerase chain reaction (RT-PCR) by automatic Cobas Amplicor (Roche Molecular Systems Hepatitis C Virus Test 2.0, Roche Molecular Systems, Nutley, NJ, USA). RESULTS: The presence of anti-HCV IgG was detected in a total of 48 cases (1.9%). Prevalence was significantly higher in men (2.3%) than in women (1.7%) (p = 0.0057), but was not significantly related to the subject's age (p = 0.51) or domicile (p = 0.35). The presence of HCV-RNA was detected in 31 (65%) anti-HCV-positive cases tested, with no significant relationship to either the age (p = 0.15), domicile (p = 0.24), or gender (p = 0.79) of the subjects. CONCLUSIONS: To the best of our knowledge, this is the largest study on the prevalence of HCV infection in the general population in Poland. The study has several limitations, such as, the use of a nonrandomized population. Nevertheless, the results obtained may be more realistic and applicable to the general population in Poland than those obtained previously (i.e., in voluntary blood donors).

[Hepatitis B (HBV) and C (HCV) virus infections as an eventual cause of chronic hepatic damage in patients undergoing maintenance hemodialysis ]. / Zakazenie wirusami zapalenia watroby typu B (HBV) i C (HCV) jako ewentualna przyczyna przewleklego uszkodzenia watroby u pacjentów leczonych powtarzanymi hemodializami.

In 67 hemodialysis patients with high prevalence of HBV infection (64/67-95.5%) the occurrence of anti-HCV antibodies in second generation enzyme immunoassay was 61.2% (41/67). Among 40/67 (59.7%) patients with actually or previously detected biochemical signs of chronic hepatic injury the H Bs antigen or/and/anti-HCV antibodies were positive in 38 (95.0%) cases. High prevalence of HBV and HCV infections in studied population and their connection with biochemical signs of chronic hepatic injury shows the necessity of introduction the intensive prophylaxis of those infections in polish hemodialysis centers.

Treatment of six patients with chronic active HCV hepatitis, with low dose natural human interferon alpha administered orally.

Following the widely accepted therapeutic standard of treatment of HCV infection with parenteral interferon alpha, and encouraged by the author's won good experience with orally administered natural human interferon alpha in low doses (leuHuIFN alpha (ldou)), applied to chronic active HBV hepatitis patients, this form of interferon was given to six randomly selected HCV infected patients (2 women, 4 men) aged 34-62 years. The diagnosis was made based on a clinical and histological evaluation and confirmed by anti-HCV antibodies detection. In 2 out of 6 patients, leuHuIFN alpha (ldou) was employed immediately after steroid discontinuation. Patients were instructed to take one lozenge daily, in the morning, on an empty stomach, and keep it in the mouth until fully dissolved. Observation period varies from 19 to 69 weeks. In 3 patients the therapy concluded, after 19, 61 and 62 weeks, respectively. One patient after 4 weeks of treatment reported increasingly troublesome small joints pain and swelling, which forced leuHuIFN alpha (ldou) discontinuation after 19 weeks. In no patient transaminases normalization was seen during treatment; biochemical and clinical remission after the drug discontinuation was observed in only one patient, in whom the treatment was interrupted due to articular adverse symptoms. With HCV RNA levels assessment being unavailable at the moment, the treatment impact on the virus replication remains difficult to evaluate objectively. The treatment was well tolerated. All patients stressed significant increase of drive and appetite as well as improvement of the exercise tolerance.

Treatment of fourteen chronic active HBsAg+, HBeAg+ hepatitis patients with low dose natural human interferon alpha administered orally.

The therapy concept is based on a theory of the immunocorrective effect of orally administered natural human interferon alpha in low doses (leuHuIFN alpha (ldou)), manifolded by the logistic amplification system seated in the oral cavity. Fourteen randomly selected patients with chronic active type B hepatitis, aged 7-59 years, were assigned to treatment. All the patients had been treated for several months to several years with steroids, with no beneficial effect--clinical and biochemical symptoms of active liver disease, with histopathological progression (up to liver cirrhosis) had been permanently present. Treatment with leuHuIFN alpha (ldou) (doses: 50-100 U daily) was introduced immediately after the immunosuppression (steroids, steroids+azathioprine) discontinuation, and its influence on the course of the disease was monitored by means of hematological and biochemical tests, humoral and cellular immune response parameters, serological markers of HBV infection, HBV DNA concentration and immunohistochemical evaluation of liver biopsy specimens. The observation period ranges from 15 to 32 months. In all patients, within the first 3-6 weeks of treatment, transient deterioration of biochemical liver function tests was noted (e.g. 2-3 fold increase of ALAT), with no clinical symptoms of the disease exacerbation. The phenomenon lasted for 4-16 weeks. In all the treated patients, intensive immune system activation was seen, which lasted beyond the therapy period. Seven patients eliminated serum HBV DNA; all of them also eliminated HBeAg and seroconverted to HBeAb. Up to date, one person have lost serum HBsAg, in nine others its titre decreased significantly.

[Detection of asymptomatic HBV infection among pregnant women based on personal studies]. / Bezobjawowe zakazenie HBV w populacji kobiet ciezarnych na podstawie wlasnych badan.

The total seroprevalence of HBsAg among pregnant women in Gdansk region is 1.3%. Our examination was based on observation of 226 HBsAg chronic carrier pregnant women. The risk of infection on the way of inoculation in childhood took place in 68%, in 10.8% it was connected with occupation. The presence of HBsAg and HBeAg was revealed in 8.8%. During period of observation none of the women eliminated HBsAg. The clinical course of infection in our group was stable. Liver biopsy was performed in 47 cases. Hepatitis minimalis was found in 35 cases, fibrosis portalis in 2, hepatitis chronica persistens in 5 and normal histological liver structure in 2 cases. Asymptomatic carrier state didn't influence on the course of pregnancy and delivery.

[Preliminary results of the treatment of patients with chronic active hepatitis (CAH) HBsAg+,HBeAg+ with low oral doses of human interferon-alpha]. / Wstepne wyniki leczenia chorych z przewleklym czynnym zapaleniem watroby (p. c. z. w.) HBsAg+, HBeAg+ niskimi doustnymi dawkami ludzkiego interferonu alfa.

The treatment conception was based on the theory of immunocorrective activity function of low doses of human interferon alpha (HuIFN alpha) multi-played by logistic amplifier system included in oral cavity. Twelve patients with chronic hepatitis, HBV infected, aged 7-59, were randomly chosen for treatment. Previously they underwent immunosuppression without success. All of them revealed clinical and biochemical symptoms of steel active disease with morphological progression including active cirrhosis (3 cases). HuIFN alpha therapy was started just after withdrawal of immunosuppression (steroids, steroids and azathioprine). Effects of 50-100 U/d dose of HUIFN alpha were monitored by examination of hematological, biochemical parametres and indicators of humoral and cellular immune response and HBV markers in control liver biopsy specimens too. The HBVDNA serum level was measured besides, using the method of molecular hybridisation. Observation period is from 5 to 17 month, now. Among all patients during 1-2 months from the treatment beginning, transient increase of biochemical parametres of liver function (f. ex: 2-3 times ALAT increase) were observed without any clinical signs of disease exacerbation. All patients revealed immune system activation that lasted longer than incipiens intensive stimulation. Four from twelve patients that finished therapy, eliminated HBVDNA from blood serum. One of them eliminated HBsAg too. Three further confirmed decrease of HBsAg with e system seroconversion. Three from 8 patients treated 6-9 months reveal decrease of HBVDNA concentration in blood serum.

[HBV, HCV and HIV infections among drug addicts-residents of rehabilitation and social adjustment centers in the Gdansk district]. / Zakazenie HBV, HCV i HIV wsród narkomanów, mieszkanców osrodków rehabilitacji i resocjalizacji w województwie gdanskim.

Among 46 i.v. drug abusers we revealed HBV infection in 36, presence of anti-HCV antibodies in 33, and HIV infection in 21. Prevalence of HBV markers and anti-HCV antibodies increased with a period of time of i.v. drug abuse. The course of HBV and HCV infection was often subclinical. IgG levels turned out to be higher in HBV infected persons, with or without HIV infection.