Magnesium in the treatment of alcohol withdrawal syndrome: a multicenter randomized controlled trial.

OBJECTIVE: Alcohol withdrawal syndrome (AWS) is a frequent and potentially life-threatening condition experienced in alcohol use disorder. Since hypomagnesemia is involved in AWS's severity, we conducted a multicenter double-blind randomized placebo-controlled trial to examine the efficacy of oral magnesium supplementation as an adjuvant therapy of AWS. MATERIAL AND METHODS: Inpatients were recruited in six different centers if they had a baseline score higher than eight on the Revised Clinical Institute Withdrawal Assessment for Alcohol (CIWA-Ar). The experimental treatment was magnesium lactate dehydrate, administrated three times per day providing a total of 426.6 mg per day and up to 15 days. The primary endpoint was the significant between-group difference of the CIWA-Ar total score change from baseline to 3 days later. The treatment group and baseline score were introduced as covariables in an analysis of covariance. RESULTS: A total of 98 inpatients were included {71.4% of men; mean age of 49.1 years [standard deviation (SD): 10.3]}. In the intention-to-treat population, the mean reduction of the CIWA-Ar score in the experimental group between baseline and 3 days later was 10.1 (SD: 5.2), whereas it was 9.2 (SD: 3.9) in the control group. The absolute difference of the adjusted mean in the experimental group compared with the control group was -0.69 (SD: 0.72), which did not correspond to a significant between-group difference (P = 0.34). Per-protocol analysis and sensitivity analyses also supported this result. Supplementary analyses found no significant difference regarding benzodiazepine consumption, magnesium blood concentration, and satisfaction to care. CONCLUSIONS: The present study does not support the rationale of systematic oral magnesium supplementation in patients with AWS.

Management of alcohol-related liver disease: the French Association for the Study of the Liver and the French Alcohol Society clinical guidelines.

Excessive alcohol consumption is the leading cause of liver diseases in Western countries, especially in France. Alcohol-related liver disease (ARLD) is an extremely broad context and there remains much to accomplish in terms of identifying patients, improving prognosis and treatment, and standardising practices. The French Association for the Study of the Liver wished to organise guidelines together with the French Alcohol Society in order to summarise the best evidence available about several key clinical points in ARLD. These guidelines have been elaborated based on the level of evidence available in the literature and each recommendation has been analysed, discussed and voted by the panel of experts. They describe how patients with ARLD should be managed nowadays and discuss the main unsettled issues in the field.

Occurrence and severity of cocaine-induced hallucinations: Two distinct phenotypes with shared clinical factors but specific genetic risk factors.

Cocaine-induced transient hallucinations (CIH) are a frequent complication following cocaine intake that is associated with addiction severity. METHODS: Two hundred and forty-two non-psychotic and Caucasian lifetime cocaine users were included in a French multicentric study. Clinical variables and dopamine pathway genotype data were extracted and tested with CIH scores using a zero-inflated binomial model, which allows for the exploration of factors associated with occurrence and severity separately. RESULTS: Cocaine dependence (poccurrence= 6.18 × 10-5, pseverity= 9.25 × 10-8), number of cocaine dependence DSM IV-Tr criteria (poccurrence= 1.22 × 10-7, pseverity= 5.09 × 10-6), and frequency of intake during the worst period of misuse (poccurrence= 8.51 × 10-04, pseverity= 0.04) were associated with greater occurrence and higher severity of CIH. The genetic associations did not yield significant results after correction for multiple tests. However, some nominal associations of SNPs mapped to the VMAT2, DBH, DRD1, and DRD2 genes were significant. In the multivariate model, the significant variables were the number of cocaine dependence criteria, lifetime alcohol dependence, and the nominally associated SNPs. CONCLUSION: Our study shows that CIH occurrence and severity are two distinct phenotypes, with shared clinical risk factors; however, they likely do not share the same genetic background.

Childhood trauma and the severity of past suicide attempts in outpatients with cocaine use disorders.

Introduction: Suicide attempts have been associated with both cocaine use disorder (CocUD) and childhood trauma. We investigated how childhood trauma is an independent risk factor for serious and recurrent suicide attempts in CocUD. Method: 298 outpatients (23% women) with CocUD underwent standardized assessments of substance dependence (Diagnostic and Statistical Manual-mental disorders, fourth edition, text revised), impulsiveness, resilience, and childhood trauma, using validated tools. Suicide attempts history was categorized as single vs. recurrent or non-serious vs. serious depending on the lifetime number of suicide attempts and the potential or actual lethality of the worst attempt reported, respectively. Bivariate and multinomial regression analyses were used to characterize which childhood trauma patterns were associated with the suicide attempts groups. Results: 58% of CocUD patients reported childhood trauma. Recurrent and serious suicide attempts clustered together and were thus combined into "severe SA." Severe suicide attempt risk increased proportionally to the number of childhood traumas (test for trend, p = 9 × 10-7). Non-severe suicide attempt risk increased with impulsiveness and decreased with resilience. In multinomial regression models, a higher number of traumas and emotional abuse were independently and only associated with severe vs. non-severe suicide attempts (effect size = 0.82, AUC = 0.7). The study was limited by its cross-sectional design. Conclusion: These preferential associations between childhood trauma and severe suicide attempts warrant specific monitoring of suicide attempts risk in CocUD, regardless of the severity of addiction profiles.

Assessing Molecular Point-of-Care Testing and Dried Blood Spot for Hepatitis C Virus Screening in People Who Inject Drugs.

BACKGROUND: Injecting drug use is a major driver of hepatitis C virus (HCV) spread worldwide, and the World Health Organization (WHO) has identified people who inject drugs (PWID) as a key population to target for HCV screening and care. Point-of-care (POC) hepatitis C tests and dried blood spot (DBS) sampling offer benefits for the management of patients with HCV infection by increasing HCV testing and linkage to care in different nonclinical settings. The aims of this prospective study were to evaluate the feasibility and the acceptability of use HCV ribonucleic acid (RNA) POC and fingerstick DBS testing in social-medical risk-reduction centers and to describe the cascade of care among PWID in France. METHODS: Between June 2018 and February 2019, 89 consecutive HCV-seropositive PWID attending 2 drug treatment services and 1 supervised consumption room in inner Paris were invited to participate in further evaluation, undergoing a clinical review with a liver assessment and blood tests including fingerstick capillary whole blood POC HCV RNA testing and fingerstick DBS sampling. RESULTS: Of the 89 participants enrolled, HCV RNA was detected in 34 (38.6%) participants. Fingerstick whole blood POC RNA testing and HCV RNA detection from DBS sample were feasible and acceptable among PWID with no major difference in terms of HCV RNA detection rate. Overall, 16 participants received pan-genotypic antiviral treatment. The proportion of PWID with sustained virologic response at 12 weeks was 81.2%, with data for 3 patients still pending. CONCLUSIONS: One-step screening strategy based on the detection of HCV RNA would engage people in care for treatment scale-up and HCV elimination.

Clinical Usefulness of the Iowa Gambling Task in Severe Alcohol Use Disorders: Link with Relapse and Cognitive-Physiological Deficits.

BACKGROUND: Decision-making impairments have been repeatedly evaluated in severe alcohol use disorders (SAUD) using the Iowa Gambling Task (IGT). The IGT, capitalizing on strong theoretical background and ecological significance, allowed identifying large-scale deficits in this population and is now a standard decision-making assessment in therapeutic settings. However, the clinical usefulness of the IGT, particularly regarding its ability to predict relapse and its link with key cognitive-physiological deficits, remains to be clarified. METHODS: Thirty-eight recently detoxified patients with SAUD and 38 matched healthy controls performed the IGT, a neuropsychological task using monetary rewards to assess decision making under uncertainty and under risk. Disease characteristics (e.g., duration and intensity), cognitive abilities, psychopathological comorbidities, and physiological damage were also measured, as well as relapse rates 6 months later. RESULTS: Compared to controls, patients with SAUD presented a dissociation between preserved decision making under uncertainty and impaired decision making under risk. In the SAUD group, while relapsers (55% of the sample) presented lower global cognitive functioning and stronger liver damage than nonrelapsers at detoxification time, no difference was found between these subgroups for the IGT. IGT results were not related to alcohol-consumption characteristics or cognitive-physiological deficits. CONCLUSIONS: SAUD is not related to a global IGT deficit, as suggested earlier, but rather to a specific impairment for decision making under risk. This deficit is not associated with other disease-related variables and has no relapse prediction power. These results question the clinical usefulness of the IGT as a tool identifying key treatment levers and guiding (neuro)psychological rehabilitation.

Integrated Care for the Use of Direct-acting Antivirals in Patients With Chronic Hepatitis C and Substance Use Disorder.

OBJECTIVES: Since little is currently known about predictors of response to direct-acting antiviral agents (DAAs) in people who inject drugs, we undertook an analysis of patients attending a hepatitis clinic with addiction services (outpatient clinics and inpatient services) to examine the outcomes associated with the treatment of difficult-to-manage patients with substance use. Our experience was based on integrated care. METHOD: A retrospective analysis was undertaken of 50 patients with hepatitis C virus (HCV) and a history of addiction who received treatment with DAAs, according to European guidelines. These regimens were sofosbuvir/ledipasvir for 8 weeks (n = 3), sofosbuvir/ledipasvir ±â€Šribavirin for 12 weeks (n = 19), sofosbuvir/daclatasvir for 12 weeks (n = 20), sofosbuvir/simeprevir (n = 1), or sofosbuvir/daclatasvir for 24 weeks (n = 7). Characteristics of patients who did versus did not achieve a sustained virologic response (SVR) 12 weeks after treatment were compared by univariate analysis. RESULTS: Forty-two patients (84%) were male; mean age was 46.2 ±â€Š7.3 years. Genotypes were 1 (n = 21), 2 (n = 4), 3 (n = 18), 4 (n = 6), or 6 (n = 1). Most patients were treatment-naïve (n = 38). Five patients had coinfection with human immunodeficiency virus (n = 4) or hepatitis B (n = 1), 28 (56%) had evidence of cirrhosis on FibroScan (>12.5 kPa), and 34 (68%) were receiving opioid substitution therapy. Psychiatric disease, illicit drug use, unemployment, and homelessness/precarious housing were common. Forty-five patients (90%) achieved SVR, 2 were lost to follow-up, and 3 had treatment relapse. CONCLUSIONS: SVR was not significantly associated with sociodemographic or virological characteristics, treatment, social environment, alcohol/drug use, and adherence. Although adherence was slightly worse than in "usual" patients, it did not affect the SVR rate. In these difficult-to-manage patients with HCV and substance use disorder, the real-world SVR rate (90%) was similar to that in nonaddicted populations.

Transient elastography alone and in combination with FibroTest® for the diagnosis of hepatic fibrosis in alcoholic liver disease.

BACKGROUND & AIMS: The reliability of transient elastography (TE) to assess liver fibrosis is insufficiently validated in alcoholic liver disease (ALD). We aimed to validate the diagnostic utility of TE for liver fibrosis in patients with excessive alcohol consumption and evaluate whether Fibrotest® adds diagnostic value relative to or in combination with TE. METHODS: We conducted a multicentre prospective study on a total of 217 heavy drinkers with high serum aminotransferase levels. Patients underwent liver biopsy, TE, Fibrotest® , PGAA, APRI, FIB-4 and FORNS. The overall diagnostic performance was evaluated by the area under the receiver operating characteristic (AUROC) curves and Obuchowski measures. RESULTS: TE values correlated with fibrosis stage (r=.73; P<.0001) and steatosis stage (r=.19; P<.01). Patients with alcoholic hepatitis had higher TE values than those without alcoholic hepatitis (P<.0001). In an multivariate analysis, fibrosis stage and the presence of alcoholic hepatitis were the only parameters that correlated with liver stiffness. For the diagnosis of advanced fibrosis (F≥3), the AUROC curves were 0.90, 0.85, 0.83, 0.91 and 0.90 for TE, Fibrotest® , PGAA and associations TE-Fibrotest® , TE-PGAA respectively. For the diagnosis of cirrhosis, the AUROC curves were 0.93, 0.88, 0.89, 0.94 and 0.95 respectively. The Obuchowski measures for the diagnosis of fibrosis were 0.94, 0.92, 0.91, 0.95 and 0.94 respectively. The performance of TE was not significantly different than those of Fibrotest® , PGAA and combinations TE-Fibrotest® , TE-PGAA. CONCLUSIONS: TE has excellent diagnostic value for liver fibrosis in alcoholic liver disease. The combined use of TE-Fibrotest® or TE-PGAA does not improve the performance of TE.

[Prognosis assessment of alcoholic liver disease: how and why?]. / Évaluation pronostique de la maladie alcoolique du foie : comment et pourquoi ?

Alcoholic liver disease (ALD) causes more than 5000 deaths per year in France. Most of those deaths could be prevented by an early diagnosis, which would give the patients the opportunity to modify their alcohol consumption while liver lesions are still reversible. Hepatic histology is the main parameter that predicts morbidity and mortality in patients with ALD. Non-invasive methods such as biomarker tests (e.g. FibroTest(®) or FibroMetre A(®)) or hepatic elastography (FibroScan(®)) may allow diagnosing alcohol-induced liver lesion without systematic biopsy. Despite promising preliminary results, those methods are not validated yet in ALD. A validation of non-invasive methods for ALD could allow a large screening of the severe forms of this pathology.

Management of hepatitis C virus infection in heavy drinkers.

AIM: Optimal management of hepatitis C virus (HCV) infection is controversial in heavy drinkers. We compared the management of HCV infection of heavy drinkers with that of patients without a history of alcohol abuse. METHODS: In a retrospective case-control study, 69 HCV-infected heavy drinkers [daily alcohol consumption at referral above 60 g/day, hereafter 'alcohol group'] were compared with matched HCV-infected patients with low alcohol consumption (<40 g/day, 'control group'). RESULTS: Patients of the 'alcohol group' were younger (42 vs. 45 years, P = 0.05), more often male (69.6 vs. 56.5%, P = 0.11) and had been infected by intravenous drug use (85.5 vs. 45.0%, P < 0.0001). The percentage of patients with a recommendation for treatment according to the French 2002 consensus (bridging fibrosis or genotype 2 or 3) was 52 of 69 (75.4%) in both groups, while the proportion of patients treated was higher in the control group (71.0 vs. 44.9%, P = 0.002). In the 'alcohol group', patients had better access to treatment if they were employed or consumed 170 g/day or less at first referral. Sustained virological response (SVR) was obtained in 10 of 31 patients (32.3%) of the 'alcohol group' vs. 8 of 31 patients (25.8%) of the control group matched for genotype and type of treatment (P = 0.58). CONCLUSION: Heavy drinkers are less often considered for antiviral therapy compared with patients without a history of alcohol abuse. However, once treatment is actually initiated, SVR rates are comparable with those achieved in non-drinkers despite the continuation of alcohol consumption during therapy in some patients.

Rapid decline of liver stiffness following alcohol withdrawal in heavy drinkers.

BACKGROUND: Measurement of liver stiffness (LS) using real-time elastography appears as a promising tool to evaluate the severity of chronic liver diseases. Previous studies in patients with alcoholic liver disease have suggested that fibrosis was the only histological parameter to influence LS. To challenge this hypothesis, we have prospectively tested the short-term impact of alcohol withdrawal on LS value. METHODS: Patients hospitalized for alcohol withdrawal in our Liver and Addiction Unit between 2007 and 2010 had an LS determination at entry (D0) and 7 days after alcohol withdrawal (D7). LS value was given as the median of 10 measurements performed with a FibroScan(®) device. For a given patient, variation of LS was considered as significant when the comparison of the 10 measurements at D0 and at D7 yielded a p-value under 0.05 (Wilcoxon test). RESULTS: One hundred and thirty-seven patients were included in the study (median alcohol consumption: 150 g/d; hepatitis C: n = 21 [15.6%]). Considering all patients, median LS value decreased from 7.2 to 6.1 kPa between D0 and D7 (p = 0.00001, paired Wilcoxon test). LS decreased significantly in 62 patients (45.3%), and there was a reduction in the estimated stage of fibrosis in 32 (23.3%). LS increased significantly in 16 patients (11.7%). Subgroup analyses revealed that the decrease in LS was still significant in patients with or without hepatitis C infection, and aspartate transaminase level below or above 100 UI/l. CONCLUSIONS: LS decreases significantly in nearly half of heavy drinkers after only 7 days of abstinence. This result strongly suggests that nonfibrotic lesions (such as the presence of alcoholic hepatitis) may influence LS. From a practical point of view, it also shows that variation of alcohol consumption must be taken into account for the interpretation of LS value.

The role of adherence in virological suppression in patients receiving anti-HBV analogues.

BACKGROUND: Although adherence is of major importance in long-term treatments, few studies have been published regarding the use of anti-HBV analogues in clinical practice. The aim of this study was to evaluate adherence to anti-HBV analogues and associated virological suppression. METHODS: A cross-sectional study was performed between 1 January 2009 and 15 July 2009 in Cochin Hospital, Paris, France. It included all patients being treated with anti-HBV analogues for at least three months, who were without coinfection (HIV, HCV or HDV) and who had not received organ transplants. At the time of enrolment, HBV viral load, analogue regimen and self-reported adherence were collected prospectively. Patients were classified as non-adherent, or moderately or totally adherent using a score based on analysis of self-reports. Other data were obtained retrospectively. RESULTS: Among the 190 patients meeting the inclusion criteria, 33% were initially hepatitis B e antigen-positive and 50% had extensive fibrosis or cirrhosis. Pretreatment viral load was 6.0 log IU/ml (median). The median duration of treatment was 52 months. At enrolment, 61%, 32% and 7% of patients were classified as totally adherent, moderately adherent and non-adherent, respectively. Complete virological suppression (HBV DNA<12 IU/ml) was observed in 83% of patients at enrolment. In the multivariate analysis, lack of virological suppression was associated with an increased pretreatment viral load, with no change in analogue regimen and is classified as non-adherent. CONCLUSIONS: Adherence seems to be an independent factor associated with virological suppression during anti-HBV analogue treatment. Therapeutic education and a systematic evaluation of adherence using self-reports should be promoted to assure long-term anti-HBV analogue efficacy in clinical practice.

Acute cholestatic hepatitis revealing Mycoplasma pneumoniae infection without lung involvement in an adult patient.

Liver involvement is an unusual manifestation of Mycoplasma pneumoniae infection. Cases of cholestatic hepatitis without pulmonary involvement have been described in children with M. pneumoniae infection but only two cases of cytolytic hepatitis have been reported in adults. We report here the case of an 18-year-old woman who presented with febrile epigastric pain of short duration associated with an elevation of gamma-glutamyl transpeptidase and alkaline phosphatase levels and with a mononuclear syndrome. Serological tests for M. pneumoniae were positive for IgG and IgM. Clinical symptoms and blood test perturbations completely resolved after treatment with macrolide.

Influence of liver biopsy on abstinence in alcohol-dependent patients.

BACKGROUND: Liver biopsy indication for the evaluation of alcoholic liver disease is controversial. Our aim was to investigate the influence of the biopsy on the patients' motivation for abstinence. METHODS: We retrospectively analysed, in a population of 324 patients hospitalized for alcohol withdrawal, the impact of liver biopsy on the following clinical outcomes: rapid loss to follow-up (immediately after hospital discharge), early relapse (< 3 months) and long-lasting abstinence (> 12 months). The biopsy was performed in 136 patients who had liver enzymes perturbations. Hepatic lesions were graded as mild (isolated steatosis and/or non-bridging fibrosis), moderate (bridging fibrosis and/or moderate alcoholic hepatitis) or severe (cirrhosis and/or marked alcoholic hepatitis) in 66 (48%), 41 (30%) and 29 (21%) cases, respectively. RESULTS: In univariate analysis, patients who had a liver biopsy were less likely to be rapidly lost to follow-up (12% versus 27%, P = 0.003) but had a lower rate of long-term abstinence (20% versus 34%, P = 0.025). In multivariate analysis, age was the only factor significantly associated with clinical outcome: older patients had higher rate of long-term abstinence (OR = 1.041; P = 0.010). Among patients who had a biopsy, those with severe hepatic lesions had a lower rate of rapid relapse than those with moderate or mild lesions (32% versus 68% and 56%, P = 0.018) but the rate of long-term abstinence was similar in the three groups. CONCLUSION: This observational study does not support the notion that liver biopsy has a significant influence on the maintenance of alcohol abstinence in patients with alcoholic liver disease.

Unexpected mosaicism of R201H-GNAS1 mutant-bearing cells in the testes underlie macro-orchidism without sexual precocity in McCune-Albright syndrome.

McCune-Albright syndrome (MAS), usually presenting with polyostotic bone dysplasia, café-au-lait skin lesions and sexual precocity, results from a somatic activating mutation of the GNAS1 gene, which encodes the Gs-alpha protein involved in signalling of several G-protein-coupled receptors. The clinical spectrum depends on tissue distribution of mutant-bearing cells. Sexual precocity has been ascribed to the occurrence of a mutant GNAS1 allele in the gonadal anlage, from which all somatic cells of the differentiated gonads arise. In boys, precocious activation of Leydig cell androgen secretion results in pubertal spermatogenesis, leading to testicular enlargement, and in the development of secondary sex characteristics. However, sexual precocity is rare in MAS males while isolated testicular enlargement is frequently observed. We recently reported the case of a boy with macro-orchidism and signs of Sertoli cell hyperactivity but no signs of hyperandrogenism, which was unexpected since Gs-alpha is functional in both Sertoli and Leydig cells. To understand its pathophysiology, we microdissected an available testicular biopsy to separate Sertoli from Leydig cells. The R201H-GNAS1 allele was present only in Sertoli cells, resulting in isolated Sertoli cell hyperfunction, evidenced by increased AMH expression and cell hyperplasia leading to prepubertal macro-orchidism, with no signs of Leydig cell activation. The different early embryologic origin of precursors contributing to Sertoli and Leydig cell lineages may underlie the differential existence of the mutated GNAS1 gene. Lack of occurrence of the mutation in Leydig cells may explain why sexual precocity is rarely observed in boys with MAS.

Non-invasive diagnosis of large oesophageal varices with FibroTest in patients with cirrhosis: a preliminary retrospective study.

BACKGROUND AND AIMS: Primary prevention of variceal bleeding with beta-blockers improves survival in patients with large oesophageal varices (LOV). Therefore, cirrhotic patients frequently undergo screening endoscopy. As portal hypertension is related to liver fibrosis, this study aimed to assess the predictive value of FibroTest, a non-invasive marker of liver fibrosis, for the diagnosis of LOV in cirrhotic patients. METHODS: Ninety-nine cirrhotic patients had clinical examination, blood sample (liver function tests, platelet count, FibroTest) and upper endoscopy. Measurements of endoscopic and biochemical parameters were made blindly. Sensitivity, specificity, predictive values and area under the receiver operating characteristic curves were assessed for FibroTest, platelet count and Child-Pugh score. The main endpoint was the presence of LOV. RESULTS: Platelet count, prothrombin time, ascites, FibroTest and Child-Pugh class were significantly different among patients with or without LOV. FibroTest had the highest discriminative power with an area under receiver operating characteristics curves of 0.77 (SE=0.06), compared with 0.64 (0.08) and 0.68 (0.08) for platelet count and Child-Pugh score, respectively (P=0.08). A cut-off at 0.80 had a 86% negative predictive value for the diagnosis of LOV (Se=92%, Sp=21%). CONCLUSION: FibroTest could aid in the diagnosis of LOV and may therefore reduce the indication of endoscopic screening in cirrhotic patients.

Fat, diabetes, and liver injury in chronic hepatitis C.

It is increasingly recognized that host factors can modulate the fibrogenic response in patients with chronic hepatitis C. Obesity, because of its prevalence, and diabetes, which seems to occur more frequently in patients infected by the hepatitis C virus (HCV), are often present in patients with chronic hepatitis C. Both conditions result in fatty liver, which in turn is associated with more severe liver damage, especially fibrosis or inflammation. Steatosis can either originate from associated metabolic alterations (insulin resistance resulting in metabolic steatosis) or from a direct cytopathic effect of the virus (genotype 3, resulting in viral steatosis). Metabolic steatosis seems to be a factor in resistance to antiviral therapy, whereas viral steatosis is reduced in sustained responders. Whether metabolic steatosis has a direct role in liver fibrosis progression or is only a surrogate marker of an underlying defect triggering fibrogenesis, such as insulin resistance, is a subject of debate. High serum glucose levels and diabetes have a strong and independent profibrogenic impact. Exciting new data are expanding our understanding of the mechanisms of steatogenesis in HCV infection and providing potential links between insulin resistance or hyperglycemic states and liver fibrogenesis.

Characterisation of hepatitis B virus X protein mutants in tumour and non-tumour liver cells using laser capture microdissection.

BACKGROUND/AIMS: The analysis of hepatitis B virus (HBV) X protein genetic variability and is correlation with liver disease severity have only been addressed, so far, on whole liver extracts. We have studied, therefore, the HBV X protein (HBx) gene sequence in morphologically well-characterised tumour and non-tumour liver cells from patients with HBV-related hepatocellular carcinoma. METHODS: Using laser capture microdissection (LCM), we picked up six to eight groups of tumour and non-tumour hepatocytes in serial frozen sections from six patients. After global DNA preamplification followed by HBx-specific polymerase chain reaction, the HBx gene was sequenced in each group of microdissected cells. We also validated the quantification of HBV-DNA in microdissected hepatocytes using HBV Amplicor. RESULTS: Heterogeneous mutations in HBx gene were found in distinct cirrhotic nodules and tumour areas from the same patient. Mutations at aa 127, 130 and 131 were frequently detected but there was no distinct point mutation profile between tumour and non-tumour samples. In contrast, deletions in HBx gene, which were found in five/six patients, were more frequent in tumour-derived sequences (6/18) than in non-tumour-derived sequences (1/20). CONCLUSIONS: We have shown that LCM provides a direct insight of intrahepatic HBV infection. Using this technique, we demonstrated the persistence of distinct HBx encoding sequences in clonally expanding cells, thus supporting the hypothesis that HBx deletions may be implicated in liver carcinogenesis.