L. M. Jarvis

Detection of a novel DNA virus (TT virus) in blood donors and blood products

Summary Background A newly discovered DNA virus, transfusion- transmitted virus (TTV), has been implicated as a cause of post-transfusion hepatitis. We investigated the frequency of TTV viraemia in UK blood donors, and the extent to which TTV contaminates blood products such as factor VIII and IX clotting factors. We also investigated the possible aetiological role of TTV in cryptogenic fulminant hepatic failure (FHF). Methods We extracted DNA from plasma of blood donors and patients with FHF, and from blood products (factor VIII and IX clotting-factor concentrates, immunoglobulin preparations). We detected TTV by PCR using primers from a conserved region in the TTV genome. Findings TTV viraemia was detected in 19 (1·9%) of 1000 non-remunerated regular blood donors. Infection occurred more frequently in older donors (mean age 53 years), compared with the age prolife of donors infected with hepatitis C virus and other parenterally-transmitted viruses. TTV contamination was found in ten (56%) of 18 batches of factor VIII and IX concentrate manufactured from such non- remunerated donors, and in seven (44%) of 16 batches of commercially available products. Whereas solvent or detergent treatment had little effect on the detection of TTV in factor VIII and IX by PCR, this virucidal step seemed to inactivate TTV infectivity. TTV infection was detected in four (19%) of 21 patients with FHF; in three cases, infection was detected at the onset of disease and could thus not be excluded from its aetiology. Interpretation TTV viraemia is frequent in the blood-donor population, and transmission of TTV through transfusion of blood components may have occurred extensively. Clinical assessment of infected donors and recipients of blood and blood products, and assessment of TTVs aetiological role in hepatic and extra-hepatic disease, are urgently needed.

Clinical significance of intrahepatic hepatitis C virus levels in patients with chronic HCV infection

Background—The clinical significance of a single assessment of circulating hepatitis C virus (HCV) RNA and its relation to the level of intrahepatic HCV RNA remains unclear. Aims—To investigate the relation between intrahepatic HCV levels and clinicopathological characteristics of chronic HCV infection. Patients—Ninety eight consecutive patients with chronic HCV infection were studied; none had received α interferon therapy. Of these, 12 patients were repeatedly negative for HCV RNA in serum by reverse transcriptase polymerase chain reaction (RT-PCR). Methods—After diagnostic laparoscopy and liver biopsy, semiquantitative analysis of intrahepatic HCV RNA levels was carried out by limiting dilution of HCV cDNA. HCV genotypes were assessed in 96 patients by restriction fragment length polymorphism analysis of HCV cDNA. Results—Ten out of 12 patients who were RT-PCR negative for HCV RNA in serum were RT-PCR positive in liver; however, this group had a significantly lower intrahepatic HCV level and serum aminotransferase level than the remaining 86 patients. Histological severity (cirrhosis: n=10); histological activity index; HCV genotype (genotype 1: n=41; genotype 2: n=12; genotype 3: n=36; genotype 4: n=7); mode of infection (intravenous drug abuse: n=58; post-transfusion: n=10; haemophiliac: n=4; sporadic: n=26) and alcohol abuse did not affect the intrahepatic virus level. There was no correlation between patient age, duration of infection, and intrahepatic HCV level. Conclusions—Intrahepatic virus levels were not determined by host factors (age of patient, mode or duration of infection) or by virus factors (HCV genotype). Repeatedly negative RT-PCR for HCV RNA in serum does not indicate absence of HCV from the liver.

Sexual transmission of GB virus C/hepatitis G virus

Although it is established that infection with GB virus C (GBV‐C) or hepatitis G virus (HGV) can be transmitted parenterally, the prevalence of GBV‐C/HGV viremia in the general population (2–5%) is relatively high compared with other parenterally borne viruses such as hepatitis C virus. To investigate the possibility of sexual transmission of GBV‐C/HGV, we determined the frequency of viremia by the polymerase chain reaction and serological reactivity to the E2 protein by ELISA in samples collected from individuals at risk for sexually transmitted diseases attending a city genitourinary medicine clinic. GBV‐C/HGV viremia was detected in 27 of 87 male homosexuals (31%) and 9 of 50 prostitutes (18%), frequencies significantly greater than those in matched controls (2/63) and local blood donors (2.3%). Among nonviremic individuals, a high frequency of serological reactivity to the E2 protein of GBV‐C/HGV was also observed in the risk groups (male homosexuals: 14/60; prostitutes: 11/41), although these figures are likely to be underestimates of the frequency of past infection as detectable anti‐E2 reactivity may attenuate rapidly over time following resolution of infection. Infection with GBV‐C/HGV was more frequent among those coinfected with human immunodeficiency virus type 1. Among male homosexuals from whom retrospective samples were available, evidence for de novo infection was found in 9 of 22 individuals over a mean sampling time of 2.9 years, predicting an annualized incidence of GBV‐C/HGV infection of approximately 11% in this group. The high prevalence and incidence of GBV‐C/HGV infection in these individuals and prostitutes provides strong evidence for its spread by sexual contact. Further studies are required to investigate the mechanism of its transmission and the clinical significance of acute and persistent infection in these risk groups. J. Med. Virol. 55:203–208, 1998.

Investigation of chronic hepatitis C infection in individuals with haemophilia : assessment of invasive and non-invasive methods

Hepatitis C virus (HCV) infection is the major cause of chronic liver disease in individuals with haemophilia. A wide spectrum of disease severity is found in this group, ranging from mild hepatitis to cirrhosis. We have studied a cohort of 87 anti‐HCV positive haemophiliacs who have been infected with HCV for 10–25 years and assessed the relative value of invasive and non‐invasive methods of evaluating liver disease. The severity of liver disease was assessed using ultrasound scan (n = 77), upper GI endoscopy (n = 50), laparoscopic liver inspection (n = 33) and liver biopsy (n = 22). Invasive investigations were performed without any significant bleeding complications.  Evidence of severe liver disease was found in approximately 25% of patients. There was agreement between the severity of liver histology and the information derived from the laparoscopic liver inspection, endoscopy and ultrasound in 86%. Co‐infection with HIV was significantly associated with more severe liver disease (P = 0.006).  This study provides further evidence that liver disease is emerging as a major complication in haemophiliacs and severe liver disease is more common in those co‐infected with HIV. We have shown the potential value of laparoscopic liver inspection, in combination with endoscopy and ultrasound, in staging the extent of liver disease, and suggest that most patients may be managed without resorting to liver biopsy.

Association between chronic hepatitis C infection and hepatocellular carcinoma in a Scottish population.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. The geographical prevalence varies considerably in different countries and Scotland is regarded as an area of low risk for the disease. AIMS: To assess the association between chronic hepatitis C infection (HCV) and HCC in a population of patients presenting to a single hospital. PATIENTS: One hundred and fourteen cases of histologically confirmed liver cancer presenting to the Royal Infirmary of Edinburgh between 1985 and 1994 were examined. METHODS: Of 114 cases of HCC, 80 samples of stored sera were available. Samples positive for HCV Ab were genotyped by restriction fragment length polymorphism analysis of HCV c-DNA. A population of 29 cirrhotic patients (diagnosed between 1985 and 1994) with chronic HCV infection was also genotyped. RESULTS: Chronic HCV infection was a major risk factor (30% of tested HCC patients) identified. HCV genotype 1b was predominant (16 of 20 patients). The time from HCV transmission to development of cancer ranged from 10 to 50 years (median 30). In the cirrhotic patient population, a broader distribution of genotypes was present (genotype 1a: 7; genotype 1b: 8; genotype 2b: 3; genotype 3a: 8 and genotype 4: 2). However, this population was significantly younger. (Mean (SD) 52 (14.5) years) (p = 0.0002) and demonstrated a significantly shorter duration of infection: range 10-40 years (median: 19). CONCLUSION: There is a strong association between chronic HCV infection, cirrhosis, and hepatocarcinogenesis in this Scottish population. The study was unable to distinguish whether the high prevalence of genotype 1b in the HCC population reflected increased oncogenicity in itself, or whether 1b was simply the most prevalent genotype in Scotland when these patients were infected.

A combined management protocol for patients with coagulation disorders infected with hepatitis C virus

The case notes of 394 adults with bleeding disorders registered at our centre together with those of the 72 patients who had died since 1971 were reviewed. 36/72 deceased patients had evidence of HCV infection. Liver decompensation was present at time of death in six. 274 (70%) of the currently registered patients had received factor concentrate or cryoprecipitate and 174 of these were screened for HCV infection. 76% of tested patients were RIBA positive. 87% of RIBA‐positive patients were RT‐PCR positive. 50 RIBA‐positive patients, including nine who were HIV infected, have undergone percutaneous liver biopsy following appropriate factor infusion with no complication. The biopsy was assessed using a Histological Activity Index (HAI) ranging from 0 to 13. Patients with HAI ≥6 were offered treatment with interferon. Patients with HAI<6 were followed up with a view to re‐biopsy in 2–3 years to assess progression. The median HAI was 4.5 (range 0–10) with HAI ≥6 in 13 cases (27%). HAI was not correlated with duration of infection, haemophilia severity, RT‐PCR status, HIV status or HCV genotype. Liver biopsy, a safe procedure in our hands, is an important investigation in HCV‐infected patients to assess suitability for interferon therapy.

Prediction of cirrhosis in patients with chronic hepatitis C infection by artificial neural network analysis of virus and clinical factors

The diagnosis of cirrhosis in patients with hepatitis C virus (HCV) infection is currently made using a liver biopsy. In this study we have trained and validated artificial neural networks (ANN) with routine clinical host and viral parameters to predict the presence or absence of cirrhosis in patients with chronic HCV infection and assessed and interpreted the role of the different inputs on the ANN classification. Fifteen routine clinical and virological factors were collated from 112 patients who were HCV RNA positive by reverse transcriptase–polymerase chain reaction (RT–PCR). Standard and Ward‐type feed‐forward fully‐connected ANN analyses were carried out both by training the networks with data from 82 patients and subsequently testing with data from 30 patients plus performing leave‐one‐out tests for the whole patient data set. The ANN results were also compared with those from multiple logistic regression. The performance of both ANN methods was superior compared with the logistic regression. The best performance was obtained with the Ward‐type ANNs resulting in a sensitivity of 92% and a specificity of 98.9% together with a predictive value of a positive test of 95% and a predictive value of a negative test of 97% in the leave‐one‐out test. Hence, further validation of the ANN analysis is likely to provide a non‐invasive test for diagnosing cirrhosis in HCV‐infected patients.

Development of anti-interferon antibodies and breakthrough hepatitis during treatment for HCV infection in haemophiliacs.

The development of anti‐interferon antibodies may lead to treatment failure during interferon therapy. We have studied the development of such antibodies in a group of 39 haemophiliacs receiving interferon‐α2a for chronic hepatitis C virus (HCV) infection.  Anti‐interferon antibodies developed in five (13%) patients and were associated with ‘breakthrough hepatitis’ in three cases. There was an association between the development of anti‐interferon antibodies and infection with HCV genotype 3a (P= 0 .01). This study suggests that the development of anti‐interferon antibodies may lead to treatment failure in a proportion of haemophiliacs with HCV infection. The association with genotype 3a has not previously been reported. Monitoring for the development of breakthrough hepatitis due to anti‐interferon antibodies may provide the opportunity to develop strategies to overcome their effects.

Hepatitis C virus genotypes in multi‐transfused individuals

Non-A non-B hepatitis-associated chronic liver disease is a major complication in multi-transfused individuals with haemophilia [l-31 and is attributed to the use of coagulation factor concentrates which were introduced in the early 1970s [4]. Almost all haemophiliacs who have been treated with non-virus-inactivated factor VIlI of IX concentrates have evidence of past or current non-A nonB hepatitis [5, 61 and it is apparent that the hepatitis C virus (HCV) is the primary cause of the persistent and progressive hepatitis chat occurs in such individuals [7lo]. Several distinct genotypes of HCV, whose distribution shows regional and ethnic variation, have been identified with different types predominant in different parts of the world [ll-151. I t has been hypothesized that biological variations between genotypes may explain some of the differences in the severity and clinical course of HCV infection [IZ], and that some genotypes may be more or less responsive to interferon therapy [12, 16-18]. This may lead to the adoption of different treatment regimens for patient management.

The effect of treatment with alpha-interferon on hepatitis G/GBV-C viraemia. The CONSTRUCT Group.

BACKGROUND Hepatitis G virus (HGV) or GBV-C is frequently detected in patients co-infected with hepatitis C virus (HCV). This study investigated host and virologic factors influencing the response to HGV/GBV-C to alpha-interferon treatment. METHODS HGV/GBV-C was detected and quantified by nested polymerase chain reaction. The influence of variables such as liver biopsy appearance, liver function abnormalities, and response of HCV to interferon treatment was monitored. RESULTS Fourteen of the 25 HGV/GBV-C-infected patients treated with interferon (3-6 MIU three times a week for 6 months) became non-viraemic during treatment, although all relapsed after treatment withdrawal at 6 months, with no net change in virus load between 0 and 12 months. CONCLUSIONS Predictive factors for clearance of HGV/GBV-C viraemia by interferon were pre-treatment severity of liver disease (median Knodell score of 4, compared with 7 for non-responders; P = 0.030) and alanine aminotransferase levels (median, 114, 182 for non-responders; P = 0.039). Clearance was associated with the treatment response of HCV. Nine of 13 who cleared HGV/GBV-C also cleared HCV, compared with 3 of 11 HGV/GBV-C non-responders; P = 0.05). The shared susceptibility of HGV/GBV-C and HCV to interferon treatment suggests a link between the mechanism of clearance of the two viruses.