Apostolos Beloukas

Liver Fibrosis by Transient Elastography and Virologic Outcomes After Introduction of Tenofovir in Lamivudine-Experienced Adults With HIV and Hepatitis B Virus Coinfection in Ghana

BACKGROUND Antiretroviral treatment (ART) programs in sub-Saharan Africa have for many years included lamivudine as the sole hepatitis B virus (HBV) inhibitor. Long-term outcomes and the effects of introducing tenofovir as part of ART in these populations have not been characterized. METHODS The study comprised a cross-sectional analysis of 106 human immunodeficiency virus (HIV)/HBV-coinfected subjects maintained on lamivudine, as well as a prospective analysis of 76 lamivudine-experienced subjects who introduced tenofovir. Patients underwent assessment of liver fibrosis by transient elastography (TE) and testing to characterize HIV type 1 (HIV-1) and HBV replication. RESULTS After a median of 45 months of lamivudine treatment, HIV-1 RNA and HBV DNA were detectable in 35 of 106 (33.0%) and 54 of 106 (50.9%) subjects, respectively, with corresponding drug resistance rates of 17 of 106 (16.0%) and 31 of 106 (29.2%), respectively. Median TE values were 5.7 kPa (interquartile range, 4.7-7.2 kPa) and independently associated with HBV DNA load, aspartate aminotransferase levels, and platelet counts; 13 of 106 (12.3%) subjects had TE measurements >9.4 kPa. Twelve months after the first assessment, and a median of 7.8 months after introducing tenofovir, HBV DNA levels declined by a mean of 1.5 log10 IU/mL (P < .001). TE values changed by a mean of -0.2 kPa (P = .097), and declined significantly in subjects who had pretenofovir HBV DNA levels >2000 IU/mL (mean, -0.8 kPa; P = .048) or TE values >7.6 kPa (mean, -1.2 kPa; P = .021). HIV-1 RNA detection rates remained unchanged. CONCLUSIONS A proportion of HIV/HBV-coinfected patients on long-term lamivudine-containing ART had poor HIV and HBV suppression, drug resistance, and TE values indicative of advanced liver fibrosis. Tenofovir improved HBV control and reduced liver stiffness in subjects with high HBV DNA load and TE values.

Hepatitis B Virus Sub-genotype A1 Infection Is Characterized by High Replication Levels and Rapid Emergence of Drug Resistance in HIV-Positive Adults Receiving First-line Antiretroviral Therapy in Malawi

BACKGROUND It has been proposed that hepatitis B virus (HBV) sub-genotype A1 infections have mild outcomes and a low risk of drug-resistance among patients infected with human immunodeficiency virus (HIV) receiving lamivudine-containing antiretroviral therapy (ART) without tenofovir in Africa. METHODS The virologic expression of HBV sub-genotype A1 coinfection was studied over 12 months in HIV-positive adults starting stavudine/lamivudine/nevirapine in Malawi, using Sanger, deep, clonal, and single full-genome sequencing for the sensitive characterization of HBV resistance-associated mutations (RAMs). RESULTS Among 1117 subjects, 133 (12%) tested HBsAg-positive. After starting ART, retention rates were 96/133 (72%) at 6 months and 54/133 (41%) at 12 months. Based upon the last available follow-up, 92/96 (96%) subjects achieved HIV-1 RNA <40 copies/mL, 48/96 (50%) showed HBV DNA <14 IU/mL, and 24/96 (25%) acquired HBV RAMs. At 6 months, M204I was detected in 8/46 (17%) and 16/17 (94%) subjects using Sanger and deep sequencing, respectively. At 12 months, all viremic patients had multiple resistance and compensatory mutations coexisting on the same HBV genomes. Comparing HBeA-positive (67/133, 50%) with HBeAg-negative subjects, 64/67 (96%) vs 35/66 (55%) showed baseline HBV DNA >2000 IU/mL (P = .0006), 39/47 (17%) vs 9/49 (82%) had persistent HBV DNA detection during follow-up (P < .0001), and 23/47 (49%) vs 2/49 (4%) acquired HBV RAMs (P < .0001). Baseline HBV DNA levels were median 8.1 vs 5.3 log10 IU/mL in subjects with vs those without treatment-emergent RAMs (P < .0001). CONCLUSIONS HBV sub-genotype A1 infections showed a severe virologic expression in HIV-positive Malawians. The findings strengthen the urgency of interventions to improve ascertainment and management of chronic hepatitis B in the region.

Development of a new ultra sensitive real-time PCR assay (ultra sensitive RTQ-PCR) for the quantification of HBV-DNA

BackgroundImproved sensitivity of HBV-DNA tests is of critical importance for the management of HBV infection. Our aim was to develop and assess a new ultra sensitive in-house real-time PCR assay for HBV-DNA quantification (ultra sensitive RTQ-PCR).ResultsPreviously used HBV-DNA standards were calibrated against the WHO 1st International Standard for HBV-DNA (OptiQuant® HBV-DNA Quantification Panel, Accrometrix Europe B.V.). The 95% and 50% HBV-DNA detection end-point of the assay were 22.2 and 8.4 IU/mL. According to the calibration results, 1 IU/mL equals 2.8 copies/mL. Importantly the clinical performance of the ultra sensitive real-time PCR was tested similar (67%) to the Procleix Ultrio discriminatory HBV test (dHBV) (70%) in low-titer samples from patients with occult Hepatitis B. Finally, in the comparison of ultra sensitive RTQ-PCR with the commercially available COBAS TaqMan HBV Test, the in-house assay identified 94.7% of the 94 specimens as positive versus 90.4% identified by TaqMan, while the quantitative results that were positive by both assay were strongly correlated (r = 0.979).ConclusionsWe report a new ultra sensitive real time PCR molecular beacon based assay with remarkable analytical and clinical sensitivity, calibrated against the WHO 1st International standard.

Early hCG addition to rFSH for ovarian stimulation in IVF provides better results and the cDNA copies of the hCG receptor may be an indicator of successful stimulation

A simple, safe and cost-effective treatment protocol in ovarian stimulation is of great importance in IVF practice, especially in the case of previous unsuccessful attempts. hCG has been used as a substitute of LH because of the degree of homology between the two hormones. The main aim of this prospective randomized study was to determine, for the first time, whether low dose hCG added to rFSH for ovarian stimulation could produce better results compared to the addition of rLH in women entering IVF-ET, especially in those women that had previous IVF failures. An additional aim was to find an indicator that would allow us to follow-up ovarian stimulation and, possibly, modify it in order to achieve a better IVF outcome; and that indicator may be the cDNA copies of the LH/hCG receptor. Group A patients (n = 58) were administered hCG and Group B rLH (n = 56) in addition to rFSH in the first days of ovarian stimulation. The number of follicles and oocytes and, most importantly, implantation and pregnancy rates were shown to be statistically significantly higher in the hCG group. This study has also determined, for the first time to our best knowledge, m-RNA for LH/hCG receptors in the lymphocytes of peripheral blood 40 h before ovum pick-up. cDNA levels of the hCG receptor after ovarian stimulation were significantly higher among women receiving hCG compared to those receiving LH. In addition, higher levels were encountered among women with pregnancy compared to those without, although this was not statistically significant due to the small number of pregnancies. It seems that hCG permits a highly effective and more stable occupancy of rLH/hCG receptors and gives more follicles and more oocytes. The determination of cDNA copies could be, in the future, a marker during ovulation induction protocols and of course a predictor for the outcome of ART in the special subgroup of patients with previous failures.

Prevalence and Characteristics of Hepatitis B Virus (HBV) Coinfection among HIV-Positive Women in South Africa and Botswana

There is progressive concern about the evolving burden of morbidity and mortality caused by coinfection with HIV-1 and hepatitis B virus (HBV) in sub-Saharan Africa, but the epidemiology and impact of this problem are not well defined. We therefore set out to assimilate more information about the nature of HBV/HIV coinfection in this region by undertaking a retrospective observational study of southern African adult women. We used samples from previously recruited HIV-1 positive women attending antenatal clinics in three settings in South Africa and Botswana (n = 950) and added a small cohort of HIV-negative antenatal South African women for comparison (n = 72). We tested for HBsAg and followed up HBsAg-positive samples by testing for HBeAg, HBV DNA, HBV genotype, presence of drug-resistance associated mutations (RAMs) and HDV. We identified HBsAg in 72 individuals (7% of the whole cohort), of whom 27% were HBeAg-positive, and the majority HBV genotypes A1 and A2. We did not detect any HDV coinfection. HBV prevalence was significantly different between geographically distinct cohorts, but did not differ according to HIV status. Among adults from South Africa, HBV/HIV coinfected patients had lower CD4+ T cell counts compared to those with HIV-monoinfection (p = 0.02), but this finding was not replicated in the cohort from Botswana. Overall, these data provide a snapshot of the coinfection problem at the heart of the HIV/HBV co-epidemic, and are important to inform public health policy, resource allocation, education, surveillance and clinical care.

Quantitative Detection of the M204V Hepatitis B Virus Minor Variants by Amplification Refractory Mutation System Real-Time PCR Combined with Molecular Beacon Technology

ABSTRACT Mutations in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif are frequently associated with resistance to antivirals and represent a major concern in the treatment of hepatitis B virus (HBV) infection. Conventional methods fail to detect minority populations of drug-resistant viral quasispecies if they represent less than 25% of the total sample virus population. The amplification refractory mutation system real-time PCR (ARMS RT-PCR) was combined with molecular beacon technology using the LightCycler system. The samples from HBV patients selected for assay evaluation included (i) 57 samples from treatment-naïve patients for biological discriminatory ability (cutoff) estimation, (ii) 12 samples from patients with treatment failure that were M204V positive by sequencing, and (iii) 13 samples from patients with treatment failure that were negative for mutation at codon 204 by sequencing. The discriminatory ability of the assay was 0.25% when tested with laboratory-synthesized DNA target sequences. The median mutant-to-wild-type ratio for samples from naive patients tested positive for the wild type and for mutant variants was 0.01% (5th and 95th percentiles = 0.0001 and 0.04%, respectively). A value of 0.04% was selected as the biological cutoff of the assay of clinical samples. In all samples M204V positive by sequencing (12/12), the mutant variant was detected as the predominant population (range, 82.76 to 99.43%). Interestingly, in 5 (38%) of 13 samples negative by sequencing, the M204V variant was detected at a ratio above the biological cutoff (0.05 to 28%). The assay represents an efficient technique for the early detection and quantification of M204V variants before mutant strains emerge to dominate the population.

Detection of the NS3 Q80K polymorphism by Sanger and deep sequencing in hepatitis C virus genotype 1a strains in the UK

The Q80K polymorphism in the hepatitis C virus (HCV) NS3 enzyme reduces susceptibility to simeprevir and other novel protease inhibitors. The aims of this study were to determine the prevalence of Q80K in treatment-naïve HCV-1a carriers in the North West region (NW) and South East region (SE) of England, investigate the occurrence of Q80K as a minority variant, and characterize viral phylogeny. Plasma samples from subjects who were naïve to anti-HCV therapy were subjected to conventional (Sanger) and deep (Illumina-Miseq, 1% interpretative cut-off) sequencing of NS3. Q80K occurred in 44 of 238 subjects (18.5%, 95% CI 13.6-23.4%), including 19 of 70 (27.1%) in the NW and 25 of 168 (14.9%) in the SE (p 0.0425), with no difference in HCV RNA load or human immunodeficiency virus (HIV) status. Q80K frequencies in reads of samples subjected to Illumina sequencing were >40% in all cases. Among subjects with Q80K, five of 44 (11.4%) showed one additional major resistance-associated mutation in NS3, detected at frequencies of >10% (V36L and V55A) or <10% (V36M). Phylogenetic analyses identified the two recognized HCV-1a lineages with (clade I) and without (clade II) Q80K. Overall, 148 of 238 (62.2%) sequences occurred within regional or inter-regional clusters, each comprising 3-20 sequences. There was no unique clustering of English sequences relative to strains from continental Europe and North America. In conclusion, Q80K was found at a high prevalence among treatment-naïve HCV-1a carriers in England, and was reliably detected by conventional sequencing, with no increased detection by deep sequencing. English sequences were highly interspersed with sequences from elsewhere in Europe (clade II) and North America (clade I), and their phylogeny was consistent with multiple introductions from different areas.

Prevalence of hepatitis D virus infection in sub-Saharan Africa: a systematic review and meta-analysis

Summary Background Hepatitis D virus (also known as hepatitis delta virus) can establish a persistent infection in people with chronic hepatitis B, leading to accelerated progression of liver disease. In sub-Saharan Africa, where HBsAg prevalence is higher than 8%, hepatitis D virus might represent an important additive cause of chronic liver disease. We aimed to establish the prevalence of hepatitis D virus among HBsAg-positive populations in sub-Saharan Africa. Methods We systematically reviewed studies of hepatitis D virus prevalence among HBsAg-positive populations in sub-Saharan Africa. We searched PubMed, Embase, and Scopus for papers published between Jan 1, 1995, and Aug 30, 2016, in which patient selection criteria and geographical setting were described. Search strings included sub-Saharan Africa, the countries therein, and permutations of hepatitis D virus. Cohort data were also added from HIV-positive populations in Malawi and Ghana. Populations undergoing assessment in liver disease clinics and those sampled from other populations (defined as general populations) were analysed. We did a meta-analysis with a DerSimonian-Laird random-effects model to calculate a pooled estimate of hepatitis D virus seroprevalence. Findings Of 374 studies identified by our search, 30 were included in our study, only eight of which included detection of hepatitis D virus RNA among anti-hepatitis D virus seropositive participants. In west Africa, the pooled seroprevalence of hepatitis D virus was 7·33% (95% CI 3·55–12·20) in general populations and 9·57% (2·31–20·43) in liver-disease populations. In central Africa, seroprevalence was 25·64% (12·09–42·00) in general populations and 37·77% (12·13–67·54) in liver-disease populations. In east and southern Africa, seroprevalence was 0·05% (0·00–1·78) in general populations. The odds ratio for anti-hepatitis D virus detection among HBsAg-positive patients with liver fibrosis or hepatocellular carcinoma was 5·24 (95% CI 2·74–10·01; p<0·0001) relative to asymptomatic controls. Interpretation Findings suggest localised clusters of hepatitis D virus endemicity across sub-Saharan Africa. Epidemiological data are needed from southern and east Africa, and from patients with established liver disease. Further studies should aim to define the reliability of hepatitis D virus testing methods, identify risk factors for transmission, and characterise the natural history of the infection in the region. Funding Wellcome Trust, Royal Society.

Molecular epidemiology of HIV-1 infection in Europe: An overview.

Human Immunodeficiency Virus type 1 (HIV-1) is characterised by vast genetic diversity. Globally circulating HIV-1 viruses are classified into distinct phylogenetic strains (subtypes, sub-subtypes) and several recombinant forms. Here we describe the characteristics and evolution of European HIV-1 epidemic over time through a review of published literature and updated queries of existing HIV-1 sequence databases. HIV-1 in Western and Central Europe was introduced in the early-1980s in the form of subtype B, which is still the predominant clade. However, in Eastern Europe (Former Soviet Union (FSU) countries and Russia) the predominant strain, introduced into Ukraine in the mid-1990s, is subtype A (AFSU) with transmission mostly occurring in People Who Inject Drugs (PWID). In recent years, the epidemic is evolving towards a complex tapestry with an increase in the prevalence of non-B subtypes and recombinants in Western and Central Europe. Non-B epidemics are mainly associated with immigrants, heterosexuals and females but more recently, non-B clades have also spread amongst groups where non-B strains were previously absent - non-immigrant European populations and amongst men having sex with men (MSM). In some countries, non-B clades have spread amongst the native population, for example subtype G in Portugal and subtype A in Greece, Albania and Cyprus. Romania provides a unique case where sub-subtype F1 has predominated throughout the epidemic. In contrast, HIV-1 epidemic in FSU countries remains more homogeneous with AFSU clade predominating in all countries. The differences between the evolution of the Western epidemic and the Eastern epidemic may be attributable to differences in transmission risk behaviours, lifestyle and the patterns of human mobility. The study of HIV-1 epidemic diversity provides a useful tool by which we can understand the history of the pandemic in addition to allowing us to monitor the spread and growth of the epidemic over time.

During Stably Suppressive Antiretroviral Therapy Integrated HIV-1 DNA Load in Peripheral Blood is Associated with the Frequency of CD8 Cells Expressing HLA-DR/DP/DQ

Background Characterising the correlates of HIV persistence improves understanding of disease pathogenesis and guides the design of curative strategies. This study investigated factors associated with integrated HIV-1 DNA load during consistently suppressive first-line antiretroviral therapy (ART). Method Total, integrated, and 2-long terminal repeats (LTR) circular HIV-1 DNA, residual plasma HIV-1 RNA, T-cell activation markers, and soluble CD14 (sCD14) were measured in peripheral blood of 50 patients that had received 1–14 years of efavirenz-based or nevirapine-based therapy. Results Integrated HIV-1 DNA load (per 106 peripheral blood mononuclear cells) was median 1.9 log10 copies (interquartile range 1.7–2.2) and showed a mean difference of 0.2 log10 copies per 10 years of suppressive ART (95% confidence interval − 0.2, 0.6; p = 0.28). It was positively correlated with total HIV-1 DNA load and frequency of CD8+HLA-DR/DP/DQ+ cells, and was also higher in subjects with higher sCD14 levels, but showed no correlation with levels of 2-LTR circular HIV-1 DNA and residual plasma HIV-1 RNA, or the frequency of CD4+CD38+ and CD8+CD38+ cells. Adjusting for pre-ART viral load, duration of suppressive ART, CD4 cell counts, residual plasma HIV-1 RNA levels, and sCD14 levels, integrated HIV-1 DNA load was mean 0.5 log10 copies higher for each 50% higher frequency of CD8+HLA-DR/DP/DQ+ cells (95% confidence interval 0.2, 0.9; p = 0.01). Conclusions The observed positive association between integrated HIV-1 DNA load and frequency of CD8+DR/DP/DQ+ cells indicates that a close correlation between HIV persistence and immune activation continues during consistently suppressive therapy. The inducers of the distinct activation profile warrant further investigation.