Background : The hepatitis G virus belongs into the group of Flaviviruses (RNA virus). It is nearly the same as the hepatitis C virus (it has only 26% aminoacides identity).

Prevalence in population is high and it is 1.2 - 3% HGV positive people.

Incidence - 0.3% acute viral hepatitis. About 900 - 2000 mostly asymptomatic cases of infection per year (U.S.A., U.K.). 90 - 100% persons with HGV infection have chronic infection. Chronic disease arise rare and it is possible to be as the chronic G hepatitis. Chronic HGV infection is used to be cryptogenic. The HGV infection can cause a) light acute hepatitis or b) persistent infection (it is 15 - 30% adults). Persistent infection sometimes cause chronic disease (sometimes including chronic G hepatitis). In several studies is described how the HGV infection can persist in more than 9 - 14 years and cause the nonimportant clinically chronic hepatitis (the liver biopsy discovered inflammatory necrotic liver changes). Duration of infection its course show the role of HGV infection as the possible cause of chronic liver disease. The HGV infection is mostly not connected with the hepatitis. Another studies say that chronic HGV infection does not appear to cause important liver diseases mainly as the only one. Importance of the HGV infection along with chronic disease does not suggest yet (U.K., U.S.A.). Clinically cryptogenic liver disease is not associated with HGV infection for example in U.K.. Similarly HGV has not been proven to cause fulminant hepatitis, but another studies from Japan show the opposite.

From point of view of liver biopsy finds - in one of studies (Fiordalisi et al. 1996) were discovered histological finds of chronic active hepatitis at 1/6 of patients HGV positive and by the rest of them was found persistent hepatitis. In another studies were discovered connection between HGV and HCV viremy and between portal and periportal inflamming changes. Portal and periportal changes were higher by patients with combined viremy than alone HCV infection. Recent studies show that HGV virus is really replicated in human liver, the other studies suggest that the virus may not even replicate in liver but updated studies say that 60% of patients with liver transplantation according to cryptogenic cirrhosis with persistent HGV infection have hepatitis. Another studies also supposed that hepatitis G virus has no importance of hepatocarcinogenesis.

Incubation time is supposed to be nearly same as incubation time of hepatitis C virus (it means from 3 to 20 weeks) or it is not introduced (in studies where the HGV infection is eliminated as the possibility of HGV infection of chronic liver disease).

The way of transmission (virus transfer) is by blood, blood derivates and other parenteral ways of infection.Sexual transmission and transmission from infected mother to a child during the birth giving and vertical transmission is also possible. Also exist the possibility of transmission via salivas.

The risk groups are injection drug users, patients of hemodialysis, blood donors and recipients of blood derivates and also patients with hemophilia.

Hepatitis G virus is probably transmitted simultaneously with the HCV virus. Studies of blood donors and patients with hemophilia in Scotland discovered low occurrence of HGV infected people with many blood transfusions while occurrence of HGV infection in population was higher. The HCV infection by patients with hemophilia was high while HCV infection is low in population of blood donors. In high occurrence of HGV positive blood donors had not recipients of blood derivates persistent HGV infection.

The HGV infection presented by blood donors is about 1.5 - 1.7%, the HCV infection in blood donors is about 1% and 3% RNA HGV positive patients of hemodialysis.

Clinically is the G hepatitis very slight without icterus. In studies from Japan (Yoshiba et al. 1995 and Tameda et al. 1996) is supposed that the HGV infection is caused by fulminant hepatitis. The fresh HGV infection is not ever intercepted that is why the role of HGV infection in this case of fulminant hepatitis. 10% of patients with chronic liver disease nonA-E have positive RNA HGV virus. 10 - 15% patients with chronic hepatitis C is HGV RNA positive.

More than 20% patients with cryptogenic cirrhosis or with other forms of hepatitis are infected by HGV infection. Mostly is HGV infection detected by patients with HBV infection or HCV infection or both. Injection drug users and homosexual patients have coinfection HGV with HBV in 50% and HGV with HCV in 67%.

Biochemical tests - most of patients (about 59%) without dual infection (with HCV or HBV infection) have higher activity of transaminasis. The other patients have normal values of transaminasis. Dual infections can have frequently higher activity of ALT approximately 79%. The average activity of transaminasis by HGV infection is half than by HCV infection. Patients with dual infection can have chronic liver disease caused by HGV with HBV or HGV with HCV infection. Some of relapsies of chronic active hepatitis B and hepatitis C can be caused by dual infection with HGV virus. In another studies is introduced that the chronic HGV infection should not change course of chronic HCV infection.

Therapy - is mainly connected with dual infection HGV with HBV, HGV with HCV or both but therapy is not the subject of this work.

Patients : We have made blood consumptions for examination PCR RNA HGV by patients with the suspicion of diagnose hepatitis in our Department of infectious diseases from 1^st of January 1998 to 1 ^st of November 1998 (for 10 months). From group of 35 tested patients (all males) was confirmed the diagnose hepatitis of infection etiology by 34 of them and in remaining 1 case was also the diagnose hepatitis but noninfection etiology. 11 patients (31%) so it means 1/3 of patients have positive results of PCR RNA HGV. (graph No.1)

Graph No.1

From group of patients which contained 35 tested patients were 21 injection drug users where 8 of them were HGV positive (that is 38%, graph No.2) and in the group of remaining 14 patients non injection drug users were only 3 RNA HGV positive (21%, graph No.3). In the group of injection drug users infected patients were twice higher occurrence of HGV infection positivity than in other cases.

                        Graph No.2                                                                Graph No.3

In group of 11 HGV positive patients were 8 injection drug users (73%). It means that 3/4 positive patients were injection drug users. In remaining 3 cases (according to anamnesa datas without known risk factors) were the way of transmission tattooing in 1 case, 1 stomatologic intervention and 1 case of sexual transmission (graph No.4).

Graph No.4

In cases of dual infections HGV with HCV, HGV with HBV or both the system of our HGV positive patients was: in 4 cases (way of transmission in 2 cases was i.v. taking drugs, 1 sexual transmission and 1 case tattooing) have occurred dual infection with HCV, 1 case of dual infection with HBV ( the way of transmission was stomatologic intervention ). The diagnose were anti-HBe positive hepatitis with pre-core mutant (HBeAg negative). In 4 cases it was HGV infection along with both HCV and HBV infections (where the way of transmission in all cases were i.v. drug taking) and in 2 cases HGV infection with HCV infection. These patients had at the same time acute viral hepatitis A (the way of transmission of dual infection HGV with HCV was again i.v. drug taking).

These 2 last cases can belong to the group of dual infection with HCV and 1 case of this group can to be likely HGV monoinfection - will see it in future text (VHA infection have passed along with dual infection HGV with HCV but independently on it - according to present time experiences). It all shows tab. No.1.

Tab. No.1

The occurrence of HGV positive patients according to age and along with consistence of dual infections in these age groups shows the next tab. No.2. (The number on the line after the “slash” determining all count of tested patients in respective age group).

Tab. No.2

From these dates follows that the most frequent occurrence of HGV positive patients were in 20 - 24 years age group (together 6 cases). Five of them had as the way of transmission i.v. drug taking. One patient of this group had as the way of transmission sexual transmission. There was also 1 case of dual infection HGV with HCV and along with passing acute VHA. The same case with VHA was in first group in our tablet also. Very interesting is just the case of first age group.

We can classified it as the dual infection with HCV but in this case it can be also only monoinfection HGV with passed acute viral hepatitis A and acute viral hepatitis C.

The patient was brought to our department for diagnosis acute viral hepatitis A in the beginning of March 1998. We have according to laboratory results discovered (information about new elevations of liver transaminasis) the acute viral hepatitis C in a half of April 1998. PCR RNA HCV were repeatly positive while anti HCV was negative. We have received PCR RNA HGV positive results on the beginning of March 1998. The patient had simultaneously acute viral hepatitis A. In the end of May 1998 we have made according to dual infection HGV with HCV liver biopsy where the very suspect chronic active hepatitis was described, ethiology G and C with starting fibrotisation and with hard inflammatorynecrotic changes. From that were followed two explanations: either it was dual infection HGV with HCV (patient caught both of them from January to February 1998 and acute viral hepatitis C as a matter of case was showed in results from laboratory lately according to the time of incubation but the hard bioptic discovery was not acceptable) or during the treatment of patient where the acute viral hepatitis A have passed and in 1,5 of month later acute viral hepatitis C but in conditions of persistent HGV infection.

It was in the view of the fact of bioptic find and anamnesis datas more possible (on primary school apendectomia, since childhood therapy of athopic eczema, in January 1998 the patient took only for the first time drugs i.v. but according to this short time was this bioptical find impossible). After that in this case we could spoken about persistent monoinfection HGV when in this conditions passed acute VHA and than acute VHC (after i.v. taking drug from end of January 1998).

This our consideration had appeared from actual datas of HGV infection (background and as example of case in American College of Physician, University of Palermo).

We have made liver biopsy by selected patients which were HGV positive and had diagnosis of chronic active VHC and also these were suitable for therapy by interferon alpha-2b (4 patients). Their bioptic discoveries were always hard in sense of necroinflammatory changes and starting fibrotisation. They all were not injection drug users. In 3 cases of them we have made therapy by interferon alpha-2b in sense of inductive phase of therapy chronic active VHC. And in 1 remaining case the standard therapy by interferon alpha-2b three times a week. The own therapy and results of it will be the subject of another work which will be written in the future.

Changes in biochemical parameters datas - mostly the elevation of ALT in all cases of dual infections, are always passing given types of hepatitis so the type of passing in dual infection with HGV. In the group of dual infection of HGV with HCV was elevation ALT 10.5 times higher than the top of the norm, in HGV with HBV was 11 times higher and HGV with both was again 11 times higher.In last group where the dual infection HGV with HCV passing along with acute viral hepatitis A the ALT was 28 times higher than norm. So it was the highest value than in other groups of dual infections. These values are same as individual course of VHA.

Clinical symptoms of HGV disease at the same time (HCV, HBV, HAV) are following: in the group of dual infection HGV with HCV was icterus in 2 cases, 1 case of dyspeptic syndrome, 1 case of lymfadenophatia and 2 hepatomegalia. In the group of HGV with HBV were no clinical symptoms of disease, in the group of HGV with HBV and HCV was icterus 1 time and 1 case of dyspeptic syndrome, 1x lymfadenopathia, 1x hepatomegalia and finally 1 case of splenomegalia. The group of HGV with HCV along with passing VHA was icterus presented in 1 case, dyspeptic syndrome in 1 case, 1x lymfadenopathia and 1x hepatomegalia. Another clinical symptoms of these diseases did not occurre. So the summary is 4x icterus, 3x dyspeptic syndrome, 3x lymfadenopathia, 4 cases of hepatomegalia and 1 case of splenomegalia. Seven patients of 11 HGV positive did not have any clinical symptoms of disease. It was 2/3 HGV positive patients (tab. No.3).

Tab. No.3

The table shows that the biggest occurrence of clinical symptoms of disease were in the group of HGV with HBV and HCV infection and in dual infection of HGV with HCV. As a matter of cause of already written facts were these clinical symptoms only about 1/3 HGV positive patients (along with dual infection).

Methods : a) biochemical methods, b) histologic methods - liver biopsy, both these methods are already described in background, c) serologic methods - are described in next articles.

Viral RNA was extracted from serum samples by using QIAamp RNA Spin Column isolation kit (QIAGEN)., according to the manufacturers instruction’s with the following modifications, briefly: 90 microlitres of sera was mixed with 360 microlitres of AVL buffer for lysis and, after 10 minutes incubation (37° C), 360 microlitres of 96% ethanol were added. After being washed, viral RNA was extracted by 40 microlitres of water (Rnasa free) preheated at 80° C. Five microliters of RT Master Mix for reverse transcription (RT) were mixed with 5 microliters of viral RNA and was reverse transcripted (RT). RT master mix included HGV primers 2^nd generation set deduced from 5-non-translated region (5-NCR) and with M-MuLV reverse transcriptase. After RT incubation, 40 microlitres PCR master mix were added. PCR master mix includes DIG-11-duTP and Expanded™ High Fidelity PCR system (Boehringer Mannheim) together with other reagents.

Visualization of PCR product was done by agarose gel (3%) by UV fluorescenceafter staining with ethidium bromide under ultraviolet illumination with with ethidium bromide. Identity of PCR products was also confirmed by hydridization using a commercial ELISA kit with a specific HGV capture probe (Boehringer Mannheim). Absorbance was read in an ELISA reader at 414 nm (reference filter 492nm) after 30 min incubation at 37° C.

The RT-PCR process has been done on Perkin-Elmer thermal cycler. The controls in each case were water, and the HGV-RNA positive serum form acute hepatitis G patient. They were done along with the tests specimens. Positivity and sensitivity were confirmed by a HGV commercial control set (Boehringer Mannheim).

Positivity of the HGV samples was only evaluated for clinical interpretation after both reactivity in electrophoresis and in ELISA detection. Sensitivity of detection of HGV-RNA was minimal 1x10⁴ genom equivalents/ml.

HCV RNA was extracted and detected by a commercial method Hofman - La Roche (Switzerland), system Amplicor - Manual.

HBV DNA was determined by commercial kit for hydridization of system Digene (USA).

Detection of another marks was made by using ELISA test of 3^rd generation.

Results : 1. We performed examination of PCR RNA HGV by our patients (35 males) with diagnosis hepatitis and 11 patients were HGV positive, it is about 31%, so 1/3 of all. Injection drug users formed 73% of HGV positive patients, it is about 3/4 of all HGV positive patients. Injection drug users formed 60% (21 patients) of total number tested patients and from them were 8 HGV positive, it is 38%. At remaining patient (14 patients) were 3 HGV positive, it means patients with other ways of transmission, it is about 21%.

In the group of tested injection drug users were the occurrence of HGV positive patients 2 times higher than in the group of patients with other ways of transmission.

2. In our collection of tested patients is unambiguously confirmed the highest occurrence of HGV positive patients in the group of injection drug users, it was 1/5 (20%) patients of total number. Each other ways of transmission - sexual transmission, tattooing, stomatologic intervention - included 1 patient of each group, it was totally about 9% of all tested patients (always dual infection HGV with HCV, HBV or both).

3. It was certified that HGV infection is detected in all cases of patients with HCV, HBV or both infections. We could suppose persistent monoinfection HGV in 1 case. HGV positive injection drug users we have detected coinfection HGV and monoinfection HCV in 50% (including cases with simultaneously passing HAV infection and also 1 case of possible monoinfection of HGV) and infection HGV with HBV and HCV in 50%. We found no occurrence of case of HGV with HBV between HGV positive injection drug users.

4. Also we have supposed that way of transmission is possible of sexual intercourse (HGV and HCV) in 1 case (in anamnesis at patient was no risk factors or parenteral intervention, HCV positive was his girlfriend). It was discovered in studies from Sweden and Honduras that at individuals without known risk factors.

5. Two cases of dual infections including chronic active hepatitis (as a matter of fact in 1 case is acted about HGV monoinfection with acute VHC, where the VHC was later) were complicated passing acute viral hepatitis A. These patients were accepted just for this VHA into the our department.

6. Patients with dual infections without injection drug addiction with liver biopsy had always max. severe find in the sense of necroinflammatory changes.

7. Biochemical changes at dual infection corresponded with other types of hepatitis. Clinical symptoms of disease in dual infections were not given in present time at 2/3 HGV positive patients. Clinical course of disease was same at injection drug users and other patients.

Conclusions : We have followed occurrence of HGV infection in our patients with diagnose hepatitis, there was clinical symptoms of this disease, biochemical datas, serologic finds, ultrasonic waves finds, liver biopsy at some patients (although always acted about dual infection) always meant max. severe bioptic find. In 1 case where the patient had max. severe bioptic find we took him for persistent monoinfection HGV (anamnesis negative). The high percentage of HGV positive patients was created by injection drug users.

This study was performed because individual studies are full of contradictions. The role of HGV infection in acute and chronic hepatitis remains to be fully defined and it is also very important to do another studies of patients without coinfection HBV and HCV but also studies about dual infections mainly with HCV as we found out from our histologic finds. In our experiences and results we suppose that HGV infection can share with liver disease at our some patients with diagnose hepatitis especially in combination HGV with HCV infection. This is the first clinical study of HGV infection at patients in hospital in Czech Republic with the suspicion of diagnose hepatitis.

Keywords : Hepatitis G virus, HGV infection, acute and chronic G hepatitis, persistent HGV infection, injection drug users, liver biopsy

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Vladimír Strakrle M.D.

Department of infectious diseases

Custody Hospital Brno and

Division of infectious diseases

University Hospital Brno

Jihlavská 20

639 00 Brno, Czech Republic

Tel.: 00-420-5-43515424

E-mail: strakrle@email.cz

Web site: http://come.to/strakrle

Jaroslav König RNDr.

National referency laboratory of hepatitis viruses

State Health Institute, Prague

Šrobárova 48

100 42 Praha 10, Czech Republic

Tel.: 00-420-2-67082455

15^th of December 1998, Brno Czech Republic Vladimír Strakrle M.D.

© Martin Plchút 1998 - 2001
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