Secondary structure of DNA released from purified capsids of human parvovirus B19 under moderate denaturing conditions.

Parvovirus B19 (B19V) possesses a linear single-stranded DNA genome of either positive or negative polarity. Due to intramolecular sequence homologies, either strand may theoretically be folded in several alternative ways. Viral DNA, when extracted from virions by several procedures, presents as linear single-stranded and/or linear double-stranded molecules, except when one particular commercial kit is used. This protocol yields DNA with an aberrant electrophoretic mobility in addition to linear double-stranded molecules, but never any single-stranded molecules. This peculiar kind of DNA was found in all plasma or serum samples tested and so we decided to analyse its secondary structure. In line with our results for one- and two-dimensional electrophoresis, mobility shift assays, DNA preparation by an in-house extraction method with moderate denaturing conditions, density gradient ultracentrifugation, DNA digestion experiments and competition hybridization assays, we conclude that (i) the unique internal portions of this distinctive single-stranded molecules are folded into tight tangles and (ii) the two terminal redundant regions are associated with each other, yielding non-covalently closed pseudo-circular molecules stabilized by a short (18 nucleotides) intramolecular stem, whereas the extreme 3'- and 5'-ends are folded back on themselves, forming a structure resembling a twin hairpin. The question arises as to whether this fairly unstable structure represents the encapsidated genome structure. The answer to this question remains quite relevant in terms of comprehending the initiation and end of B19V genome replication.

Does hepatitis C viremia or genotype predict the risk of mortality in individuals co-infected with HIV?

BACKGROUND & AIMS: The influence of HCV-RNA levels and genotype on HCV disease progression is not well studied. The prognostic value of these markers was investigated in HIV/HCV co-infected individuals from the EuroSIDA cohort. METHODS: EuroSIDA is a prospective cohort of 18,295 HIV-1 infected patients in 105 centres across Europe, Israel, and Argentina. All subjects with known HCV antibody (HCVAb) status (n=13,025) were enrolled in the present study. RESULTS: 4044 (31.0%) patients had detectable HCVAb. After adjustment, HCVAb+ patients had an increased incidence of liver-related death (LRD) compared to HCVAb- individuals (IRR 8.90; 95% CI 5.60-14.14, p<0.0001). Information on HCV-RNA was available for 2709 (67.0%) HCVAb+ patients and 2010 (74.2%) were HCV-RNA+. Of 1907 patients with measured HCV genotype, 1008 (52.9%), 62 (3.3%), 567 (29.7%), and 270 (14.2%) were infected with genotype 1, 2, 3 and 4, respectively. Patients with detectable HCV-RNA had similar incidence of non-LRD, but higher incidence of LRD compared to HCVAb+ aviremic patients (adjusted IRR 1.18; 95% CI 0.93-1.50, p=0.17) and (adjusted IRR 2.11; 95% CI 1.30-3.42, p=0.0025), respectively. In patients with HCV viremia, HCV-RNA levels and HCV genotype did not influence the risk of non-LRD or LRD. CONCLUSIONS: HCV seropositive HIV patients had a 9-fold increased risk of LRD compared to patients who were HCV seronegative. Risk of death from any cause or LRD was not influenced by level of HCV viremia or HCV genotype.

Interferon-lambda serum levels in hepatitis C.

BACKGROUND & AIMS: Dendritic cells (DCs) trigger adaptive immune responses and are an important source of antiviral cytokines. In hepatitis C virus (HCV) infection DC function is markedly impaired. Thus far, studies have focused on types I and II interferon (IFN). We studied IFN-lambda1 (IL-29) and IFN-lambda2/3 (IL-28A/B) serum levels in patients with different outcomes of HCV infection. METHODS: IFN-lambdas were measured by ELISAs detecting IL-29 or IL-28A and IL-28B, respectively. Results were stratified with respect to the recently discovered rs12979860 T/C polymorphism upstream of the IL-28B gene. RESULTS: In general IL-29 serum levels exceeded IL-28A/B at least twofold, with IL-29 and IL-28A/B levels being significantly higher in carriers of the rs12979860 C allele than in TT homozygous individuals (p<0.02). IL-29 levels were substantially lower in patients with chronic hepatitis C than in healthy controls (p=0.005) and patients with spontaneously resolved hepatitis (p=0.001). Patients with acute hepatitis C showed IL-29 levels intermediate between chronic hepatitis C and normal controls; and IL-29 serum levels were higher in patients who spontaneously resolved hepatitis C than in those who became chronic. In vitro HCV proteins NS3 and E2 directly inhibited IL-29 production in poly I:C-stimulated purified DCs. CONCLUSIONS: Our data suggest that HCV proteins modify IFN-lambda production in DCs. Carriers of the rs12979860 C allele associated with resolution of HCV infection exhibited increased IFN-lambda levels. Moreover, high IFN-lambda levels predisposed to spontaneous resolution of HCV infection. Thus, IFN-lambdas seem to play an important role in the control of hepatitis C.

An international collaboration to standardize HIV-2 viral load assays: results from the 2009 ACHI(E)V(2E) quality control study.

Accurate HIV-2 plasma viral load quantification is crucial for adequate HIV-2 patient management and for the proper conduct of clinical trials and international cohort collaborations. This study compared the homogeneity of HIV-2 RNA quantification when using HIV-2 assays from ACHI(E)V(2E) study sites and either in-house PCR calibration standards or common viral load standards supplied to all collaborators. Each of the 12 participating laboratories quantified blinded HIV-2 samples, using its own HIV-2 viral load assay and standard as well as centrally validated and distributed common HIV-2 group A and B standards (http://www.hiv.lanl.gov/content/sequence/HelpDocs/subtypes-more.html). Aliquots of HIV-2 group A and B strains, each at 2 theoretical concentrations (2.7 and 3.7 log(10) copies/ml), were tested. Intralaboratory, interlaboratory, and overall variances of quantification results obtained with both standards were compared using F tests. For HIV-2 group A quantifications, overall and interlaboratory and/or intralaboratory variances were significantly lower when using the common standard than when using in-house standards at the concentration levels of 2.7 log(10) copies/ml and 3.7 log(10) copies/ml, respectively. For HIV-2 group B, a high heterogeneity was observed and the variances did not differ according to the type of standard used. In this international collaboration, the use of a common standard improved the homogeneity of HIV-2 group A RNA quantification only. The diversity of HIV-2 group B, particularly in PCR primer-binding regions, may explain the heterogeneity in quantification of this strain. Development of a validated HIV-2 viral load assay that accurately quantifies distinct circulating strains is needed.

Characterisation of the epitope for a herpes simplex virus glycoprotein B-specific monoclonal antibody with high protective capacity.

Monoclonal antibody (MAb) 2c, specific for glycoprotein B of herpes simplex virus (HSV), had been shown to mediate clearance of infection from the mucous membranes of mice, thereby completely inhibiting mucocutaneous inflammation and lethality, even in mice depleted of both CD4(+) and CD8(+) cells. Additionally, ganglionic infection was highly restricted. In vitro, MAb 2c exhibits a potent complement-independent neutralising activity against HSV type 1 and 2, completely inhibits the viral cell-to-cell spread as well as the syncytium formation induced by syncytial HSV strains (Eis-Hübinger et al. in Intervirology 32:351-360, 1991; Eis-Hübinger et al. in J Gen Virol 74:379-385, 1993). Here, we describe the mapping of the epitope for MAb 2c. The antibody was found to recognise a discontinuous epitope comprised of the HSV type 1 glycoprotein B residues 299 to 305 and one or more additional discontinuous regions that can be mimicked by the sequence FEDF. Identification of the epitope was confirmed by loss of antibody binding to mutated glycoprotein B with replacement of the epitopic key residues, expressed in COS-1 cells. Similarly, MAb 2c was not able to neutralise HSV mutants with altered key residues, and MAb 2c was ineffective in mice inoculated with such mutants. Interestingly, identification and fine-mapping of the discontinuous epitope was not achieved by binding studies with truncated glycoprotein B variants expressed in COS cells but by peptide scanning with synthetic overlapping peptides and peptide key motif analysis. Reactivity of MAb 2c was immensely increased towards a peptide composed of the glycoprotein B residues 299 to 305, a glycine linker, and a C-terminal FEDF motif. If it could be demonstrated that antibodies of the specificity and bioactivity of MAb 2c can be induced by the epitope or a peptide mimicking the epitope, strategies for active immunisation might be conceivable.

Influence of extraction protocol on physical properties of parvovirus B19 DNA.

While establishing a quantification standard for parvovirus B19 diagnostics, we extracted viral DNA from a high-titered human plasma by using three commercial kits. Despite similar viral DNA yields being obtained, striking differences in electrophoretic mobilities of the extracted nucleic acids were observed.

Detection of all species of the genus Alphavirus by reverse transcription-PCR with diagnostic sensitivity.

Clinical arbovirus screening requires exclusion of a broad range of viruses with as few assays as possible. We present a reverse transcription-PCR (RT-PCR) for the detection of all species of the genus Alphavirus qualified for exclusion screening (limit of detection [LOD], 5 to 100 RNA copies per reaction across all Alphavirus species; detection of viremia down to ca. 10,000 copies per ml).

Phylogenetic analysis of acute hepatitis C virus genotype 4 infections among human immunodeficiency virus-positive men who have sex with men in Germany.

BACKGROUND: An ongoing HCV epidemic currently affects a growing proportion of HIV-positive men who have sex with men (MSM) in Europe. Recently in the North-Rhine region of Germany, we have observed an increase in acute HCV infections of genotype 4 (HCV-4). AIMS: To characterize the current spread of HCV-4 among German MSM using a molecular epidemiological approach. METHODS: Patient characteristics and sera were collected for HIV-positive MSM diagnosed with acute HCV-4 infections in the North-Rhine region (n=14), Hamburg (n=14), Frankfurt (n=4) and Berlin (n=4). Part of the HCV NS5B region (436 bp) was amplified, sequenced and compared with HCV-4 sequences from HIV-positive Dutch, English and French MSM (n=50) as well as unrelated HCV risk groups (n=61). RESULTS: NS5B sequences were obtained from 35/36 (97%) of German cases, all of which were HCV subtype 4d (HCV-4d). The phylogenetic analysis of HCV sequences revealed two MSM-specific HCV-4d clusters of 71 and 12 sequences. All except one of the German MSM belonged to a large MSM-specific HCV cluster containing MSM from all four different European countries. None of the HCV-4 strains circulating among injecting drug users or in HCV-4 endemic areas were part of the MSM-specific clusters. CONCLUSIONS: HCV rapidly spreads among European HIV-positive MSM through a joint international transmission network, separate from that of injecting drug users. In order to contain this epidemic, non-parenteral routes of transmission, such as unsafe sex, must be taken into consideration and prevention measures should be refocused accordingly.

Human bocavirus: passenger or pathogen in acute respiratory tract infections?

Human bocavirus (HBoV) is a newly identified virus tentatively assigned to the family Parvoviridae, subfamily Parvovirinae, genus Bocavirus. HBoV was first described in 2005 and has since been detected in respiratory tract secretions worldwide. Herein we review the literature on HBoV and discuss the biology and potential clinical impact of this virus. Most studies have been PCR based and performed on patients with acute respiratory symptoms, from whom HBoV was detected in 2 to 19% of the samples. HBoV-positive samples have been derived mainly from infants and young children. HBoV DNA has also been detected in the blood of patients with respiratory tract infection and in fecal samples of patients with diarrhea with or without concomitant respiratory symptoms. A characteristic feature of HBoV studies is the high frequency of coinciding detections, or codetections, with other viruses. Available data nevertheless indicate a statistical association between HBoV and acute respiratory tract disease. We present a model incorporating these somewhat contradictory findings and suggest that primary HBoV infection causes respiratory tract symptoms which can be followed by prolonged low-level virus shedding in the respiratory tract. Detection of the virus in this phase will be facilitated by other infections, either simply via increased sample cell count or via reactivation of HBoV, leading to an increased detection frequency of HBoV during other virus infections. We conclude that the majority of available HBoV studies are limited by the sole use of PCR diagnostics on respiratory tract secretions, addressing virus prevalence but not disease association. The ability to detect primary infection through the development of improved diagnostic methods will be of great importance for future studies seeking to assign a role for HBoV in causing respiratory illnesses.

A novel diagnostic target in the hepatitis C virus genome.

BACKGROUND: Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (5'-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays. METHODS AND FINDINGS: In this study we determined by de novo sequencing that the 3'-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1-6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3-24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10(-9) IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1-6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5'-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties. CONCLUSION: This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.

Effects of the CCR5-Delta32 mutation on hepatitis C virus-specific immune responses in patients with haemophilia.

In hepatitis C virus (HCV) infection antiviral T cells express the CC chemokine receptor 5 (CCR5). Their recruitment to the liver is an important step in the immune response. A 32 base pair deletion in the CCR5 gene leads to reduced expression and total loss of CCR5 in CCR5-n32 heterozygous and homozygous subjects, respectively. However, the role of this mutation for antiviral immunity remains unclear. Here, we analysed proliferation, IFN-gamma and IL-4 secretion (ELISpot) induced by the HCV antigens core, NS3, NS4, and NS5a in 21 anti-HCV-positive haemophiliac patients in relationship to their CCR5 genotypes (CCR5 wildtype n = 10, CCR5-n32 heterozygous n = 5 and CCR5-n32 homozygous n = 6). Furthermore, T cell migration in response to the CCR5 ligands CCL3, -4 and -5 was studied. Overall IFN-gamma responses to HCV proteins were only slightly greater in CCR5 wild-type patients than in CCR5-n32 carriers (0.6 versus 0.24 SFC/10(4) PBMC; p = 0.043). This difference was consistently seen with all tested HCV antigens. In contrast, neither T cell migration, nor PBMC proliferation, nor IL-4 production differed between CCR5 genotypes. Interruption of the CCR5 signalling pathway due to CCR5-n32 may potentially result in subtle reduction of HCV specific IFN-gamma responses in anti-HCV-positive haemophiliac patients.

Effects of the CCR5-Delta32 mutation on hepatitis C virus-specific immune responses in patients with haemophilia.

In hepatitis C virus (HCV) infection antiviral T cells express the CC chemokine receptor 5 (CCR5). Their recruitment to the liver is an important step in the immune response. A 32 base pair deletion in the CCR5 gene leads to reduced expression and total loss of CCR5 in CCR5-Delta32 heterozygous and homozygous subjects, respectively. However, the role of this mutation for antiviral immunity remains unclear. Here, we analysed proliferation, IFN-gamma and IL-4 secretion (ELISpot) induced by the HCV antigens core, NS3, NS4, and NS5a in 21 anti-HCV-positive haemophiliac patients in relationship to their CCR5 genotypes (CCR5 wildtype n = 10, CCR5-Delta32 heterozygous n = 5 and CCR5-Delta32 homozygous n = 6). Furthermore, T cell migration in response to the CCR5 ligands CCL3, -4 and -5 was studied. Overall IFN-gamma responses to HCV proteins were only slightly greater in CCR5 wild-type patients than in CCR5-Delta32 carriers (0.6 versus 0.24 SFC/10(4) PBMC; p = 0.043). This difference was consistently seen with all tested HCV antigens. In contrast, neither T cell migration, nor PBMC proliferation, nor IL-4 production differed between CCR5 genotypes. Interruption of the CCR5 signalling pathway due to CCR5-Delta32 may potentially result in subtle reduction of HCV specific IFN-gamma responses in anti-HCV-positive haemophiliac patients.

Quality control assessment of human immunodeficiency virus type 2 (HIV-2) viral load quantification assays: results from an international collaboration on HIV-2 infection in 2006.

Human immunodeficiency virus type 2 (HIV-2) RNA quantification assays used in nine laboratories of the ACHI(E)V(2E) (A Collaboration on HIV-2 Infection) study group were evaluated. In a blinded experimental design, laboratories quantified three series of aliquots of an HIV-2 subtype A strain, each at a different theoretical viral load. Quantification varied between laboratories, and international standardization of quantification assays is strongly needed.

Detection of astrovirus in premature infants with necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is a major cause of mortality and morbidity in very low birth weight infants (<1500 g birth weight). Although the etiology remains unknown, infectious agents could play a key role. The aim of this analysis was to examine the role of human astrovirus (HAstV) in infants with NEC. PATIENTS AND METHODS: All patients admitted during a 5-year period at a tertiary neonatal intensive care unit with NEC (Bell stage I-III) who had examination of stool specimens for bacterial and for viral infections were included. Clinical data were reviewed and compared between infants with NEC and astrovirus detection (NEC + HAstV) and infants with NEC without astrovirus detection (NEC - HAstV) in stool specimens. RESULTS: Forty infants with NEC were identified between 2002 and 2006 and 8 patients were excluded from statistical evaluation because of incomplete viral examinations. HAstV was detected in stool specimens of 6 (19%) of the remaining 32 patients with NEC. Double infection with rotavirus was identified in 1 patient. No other viruses were detected. Significant differences in patients with NEC - HAstV and NEC + HAstV were only shown for age at onset of illness (P < 0.001) but not for severity of illness, need for surgical intervention, or mortality. CONCLUSIONS: This study demonstrates that HAstV may be associated with the development of NEC in a subgroup of patients and provides further evidence for the important role of gastrointestinal viral infections in this most common gastrointestinal emergency in premature infants. HAstV should be included in microbiological examination of stool specimens in patients with NEC.

Prospective study of Human Bocavirus (HBoV) infection in a pediatric university hospital in Germany 2005/2006.

BACKGROUND: Human Bocavirus (HBoV), a new species of the genus parvovirus newly detected in 2005, seems to be a worldwide distributed pathogen among children with respiratory tract infection (prevalence 2%-18%). Recently published retrospective studies and one prospective birth cohort study suggest that HBoV-primary infection occurs in infants. METHODS: Prospective single center study over one winter season (November 2005-May 2006) with hospitalized children without age restriction using PCR-based diagnostic methods. RESULTS: HBoV DNA was detected in 11 (2.8%) of 389 nasopharyngeal aspirates from symptomatic hospitalized children (median age 9.0 months; range: 3-17 months). RSV, HMPV, HCoV, and Influenza B were detected in 13.9% (n=54), 5.1% (n=20), 2.6% (n=10), and 1.8% (n=7), respectively. There was no influenza A DNA detected in any of the specimens. The clinical diagnoses were acute wheezing (bronchitis) in four patients, radiologically confirmed pneumonia in six patients (55%) and croup syndrome in one patient. In five to six patients with pneumonia, HBoV was the only pathogen detected. While no patient had to be mechanically ventilated, 73% needed oxygen supplementation. In four (36.4%) patients at least one other viral pathogen was found (plus RSV n=3; 27.3%; Norovirus n=1; 9.1%). CONCLUSION: HBoV causes severe respiratory tract infections in infants and young children. Its role as a copathogen and many other open questions has to be defined in further prospective studies.

Acute life threatening event (ALTE) in an infant with human coronavirus HCoV-229E infection.

In this short report we discuss the temporal association between an acute life threatening event (ALTE) and a RT-PCR confirmed coronavirus HCoV-229E infection in a 4 months old otherwise healthy infant. More detailed microbiological investigations of affected children even without apparent signs of a respiratory tract infection may help to clarify the etiology in some patients and extend our understanding of the pathogenesis. PCR-based techniques should be utilized to increase the sensitivity of detection for old and new respiratory viral pathogens in comparable cases.

Functional domains of the human immunodeficiency virus type 1 Nef protein are conserved among different clades in Cameroon.

The Nef protein of human immunodeficiency virus type 1 (HIV-1) has multiple functional domains, is immunogenic, and contains several cytotoxic T lymphocyte (CTL)-targeted epitopes. Several defined subfunctions of Nef are important for the pathogenesis of HIV-1 infection. In this study, we present the genetic diversity of the nef gene of 55 newly derived HIV-1 sequences obtained from Cameroonian patients. Four genetic subtypes and three circulating recombinant forms (CRFs) were identified: subtypes A (11%), G (7.3%), D (5.4%), F1 (1.8%), F2 (5.4%), CRF01_AE (5.4%), CRF02_AG (58.2%), and CRF11_cpx (1.8%). Two isolates clustered distinctly from the known HIV-1 genetic subtypes in nef and were designated as unclassified. Interestingly, the majority of all functional domains including the myristoylation signal, CD4 binding motif, beta turn motif, and the phosphorylation sites were well conserved in our cohort. Putative CTL-epitopic domains of the central portion of Nef were also well conserved, whereas those at the C-term were not. Our study demonstrated that despite high genetic diversity observed in the nef gene, most described functional domains and CTL epitopes were well conserved among Cameroonian HIV-1 subtypes. These findings could be used for the development of antiretroviral-acting therapeutics and anti-HIV-1 vaccines.

Identification of cell lines permissive for human coronavirus NL63.

Six cell lines routinely used in laboratories were tested for permissiveness to the infection with the newly identified human coronavirus NL63. Two monkey epithelial cell lines, LLC-MK2 and Vero-B4, showed a cytopathic effect (CPE) and clear viral replication, whereas no CPE or replication was observed in human lung fibroblasts MRC-5s. In Rhabdomyosarcoma cells, Madin-Darby-Canine-kidney cells and in an undefined monkey kidney cell line some replication was observed but massive exponential rise in virus yield lacked The results will lead to an improved routine diagnostic algorithm for the detection of the human coronavirus NL63.

HIV-1 infection leads to increased HLA-E expression resulting in impaired function of natural killer cells.

HIV has evolved several strategies to evade recognition by the host immune system including down-regulation of major histocompatibility complex (MHC) class I molecules. However, reduced expression of MHC class I molecules may stimulate natural killer (NK) cell lysis in cells of haematopoietic lineage. Here, we describe how HIV counteracts stimulation of NK cells by stabilizing surface expression of the non-classical MHC class I molecule, HLA-E. We demonstrate enhanced expression of HLA-E on lymphocytes from HIV-infected patients and show that in vitro infection of lymphocytes with HIV results in up-regulation of HLA-E expression and reduced susceptibility to NK cell cytotoxicity. Using HLA-E transfected K-562 cells, we identified the well-known HIV T-cell epitope p24 aa14-22a as a ligand for HLA-E that stabilizes surface expression of HLA-E, favouring inhibition of NK cell cytotoxicity. These results propose HIV-mediated up-regulation of HLA-E expression as an additional evasion strategy targeting the antiviral activities of NK cells, which may contribute to the capability of the virus in establishing chronic infection.

Effect of antiretroviral therapy on liver-related mortality in patients with HIV and hepatitis C virus coinfection.

BACKGROUND: Highly active antiretroviral therapy (HAART) has improved the prognosis of HIV infection. However, replication of hepatitis C virus (HCV) is not inhibited by HAART, and treatment-related hepatotoxicity is common. To clarify the effect of HAART in HIV/HCV-coinfected patients, we studied liver-related mortality and overall mortality in 285 patients who were regularly treated during the period 1990-2002 at our department. METHODS: Survival was analysed retrospectively by Kaplan-Meier and Cox's regression analyses after patients (81% haemophiliacs) had been stratified into three groups according to their antiretroviral therapy (HAART n=93, available after 1995; treatment exclusively with nucleoside analogues n=55, available after 1992; or no treatment, n=137). FINDINGS: Liver-related mortality rates were 0.45, 0.69, and 1.70 per 100 person-years in the HAART, antiretroviral-treatment, and untreated groups. Kaplan-Meier analysis of liver-related mortality confirmed the significant survival benefit in patients with antiretroviral therapy (p=0.018), and regression analysis identified HAART (odds ratio 0.106 [95% CI 0.020-0.564]), antiretroviral treatment (0.283 [0.103-0.780]), CD4-positive T-cell count (0.746 [0.641-0.868] per 0.05x10(9) cells/L), serum cholinesterase (0.962 [0.938-0.986] per 100 U/L), and age (1.065 [1.027-1.105] per year) as independent predictors of liver-related survival. Severe drug-related hepatotoxicity was seen in five patients treated with nucleoside analogues alone and 13 treated with HAART. No patient died from drug-related hepatotoxicity. INTERPRETATION: In addition to improved overall survival, antiretroviral therapy significantly reduced long-term liver-related mortality in our patients. This survival benefit seems to outweigh by far the associated risks of severe hepatotoxicity.