Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection

Hepatitis B X gene; Hepatitis B virus; Hepatitis delta virusHepatitis B gen X; Virus d'hepatitis B; Virus d'hepatitis deltaHepatitis B gen X; Virus de hepatitis B; Virus de hepatitis deltaBACKGROUND: Hepatitis delta virus (HDV) seems to strongly suppress hepatitis B virus (HBV) replication, although little is known about the mechanism of this interaction. Both these viruses show a dynamic distribution of mutants, resulting in viral quasispecies. Next-generation sequencing is a viable approach for analyzing the composition of these mutant spectra. As the regulatory hepatitis B X protein (HBx) is essential for HBV replication, determination of HBV X gene (HBX) quasispecies complexity in HBV/HDV infection compared to HBV mono-infection may provide information on the interactions between these two viruses. AIM: To compare HBV quasispecies complexity in the HBX 5' region between chronic hepatitis delta (CHD) and chronic HBV mono-infected patients. METHODS: Twenty-four untreated patients were included: 7/24 (29.2%) with HBeAg-negative chronic HBV infection (CI, previously termed inactive carriers), 8/24 (33.3%) with HBeAg-negative chronic hepatitis B (CHB) and 9/24 (37.5%) with CHD. A serum sample from each patient was first tested for HBV DNA levels. The HBX 5' region [nucleotides (nt) 1255-1611] was then PCR-amplified for subsequent next-generation sequencing (MiSeq, Illumina, United States). HBV quasispecies complexity in the region analyzed was evaluated using incidence-based indices (number of haplotypes and number of mutations), abundance-based indices (Hill numbers of order 1 and 2), and functional indices (mutation frequency and nucleotide diversity). We also evaluated the pattern of nucleotide changes to investigate which of them could be the cause of the quasispecies complexity. RESULTS: CHB patients showed higher median HBV-DNA levels [5.4 logIU/mL, interquartile range (IQR) 3.5-7.9] than CHD (3.4 logIU/mL, IQR 3-7.6) (P = n.s.) or CI (3.2 logIU/mL, IQR 2.3-3.5) (P < 0.01) patients. The incidence and abundance indices indicated that HBV quasispecies complexity was significantly greater in CI than CHB. A similar trend was observed in CHD patients, although only Hill numbers of order 2 showed statistically significant differences (CHB 2.81, IQR 1.11-4.57 vs CHD 8.87, 6.56-11.18, P = 0.038). There were no significant differences in the functional indices, but CI and CHD patients also showed a trend towards greater complexity than CHB. No differences were found for any HBV quasispecies complexity indices between CHD and CI patients. G-to-A and C-to-T nucleotide changes, characteristic of APOBEC3G, were higher in CHD and CI than in CHB in genotype A haplotypes, but not in genotype D. The proportion of nt G-to-A vs A-to-G changes and C-to-T vs T-to-C changes in genotype A and D haplotypes in CHD patients showed no significant differences. In CHB and CI the results of these comparisons were dependent on HBV genotype. CONCLUSION: The lower-replication CHD and CI groups show a trend to higher quasispecies complexity than the higher-replication CHB group. The mechanisms associated with this greater complexity require elucidation.Supported by the Instituto de Salud Carlos III, grants PI15/00856 and PI17/02233; co-financed by the European Regional Development Fund (ERDF

ACE Score Identifies HBeAg-negative Inactive Carriers at a Single-point Evaluation, Regardless of HBV Genotype

HBV DNA; Hepatitis B virus; Inactive carrierADN del VHB; Virus de la hepatitis B; Portador inactivoADN del VHB; Virus de l'hepatitis B; Portador inactiuBackground and Aims Hepatitis B virus (HBV) biomarkers have been used for a better categorization of patients, even though the lack of simple algorithms and the impact of genotypes limit their application. Our aim was to assess the usefulness of noninvasive markers for the identification of HBV inactive carriers (ICs) in a single-point evaluation and to design a predictive model for their identification. Methods This retrospective-prospective study included 343 consecutive HBeAg-negative individuals. Clinical, analytical, and virological data were collected, and a liver biopsy was performed if needed. Subjects were classified at the end of follow-up as ICs, chronic hepatitis B and gray zone.A predictive model was constructed, and validated by 1000-bootstrap samples. Results After 39 months of follow-up, 298 subjects were ICs, 36 were chronic hepatitis B CHB, and nine were gray zone. Eighty-nine (25.9%) individuals required a liver biopsy. Baseline HBV DNA hazard ratio (HR) 6.0, p<0.001), HBV core-related antigen (HBcrAg) (HR 6.5, p<0.001), and elastography (HR 4.6, p<0.001) were independently associated with the IC stage. The ACE score (HBV DNA, HBcrAg, elastography), obtained by bootstrapping, yielded an area under the receiver operating characteristics (AUROC) of 0.925 (95% CI: 0.880–0.970, p<0.001) for identification of ICs. The AUROC for genotype D was 0.95, 0.96 for A, 0.90 for E, and 0.88 for H/F. An ACE score of <1 had a positive predictive value of 99.5%, and a score ≤12 points had a diagnostic accuracy of 93.8%. Conclusions Low baseline HBV DNA, HBcrAg, and liver stiffness were independently associated with the IC phase. A score including those variables identified ICs at a single-point evaluation, and might be applied to implement less intensive follow-up strategies.This study received partial financial support from Instituto de Salud Carlos III (PI17/02233 and PI20/01692)

High-resolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods

HepatitisCvirus(HCV)is classified into seven major genotypesand67 subtypes. Recent studies haveshownthat inHCVgenotype 1-infected patients, response rates to regimens containingdirect-acting antivirals(DAAs)are subtype dependent. Currently available genotypingmethods have limited subtyping accuracy.Wehave evaluated theperformanceof adeep-sequencing-basedHCVsubtyping assay, developed for the 454/GS-Junior platform, in comparisonwith thoseof two commercial assays (VersantHCVgenotype 2.0andAbbott Real-timeHCVGenotype II)andusingdirectNS5Bsequencing as a gold standard (direct sequencing), in 114 clinical specimenspreviously tested by first-generation hybridization assay (82 genotype 1and32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 callingbypopulation Sanger sequencing(69%1b,31%1a) in 81 specimensandidentified amixed-subtype infection (1b/3a/1a) in one sample. Similarly,amongthe 32previously indeterminate specimens, identical genotypeandsubtype results were obtained by directanddeep sequencing in all but four samples with dual infection. In contrast, both VersantHCVGenotype 2.0andAbbott Real-timeHCVGenotype II failed subtype 1 calling in 13 (16%) samples eachandwere unable to identify theHCVgenotype and/or subtype inmore than half of the nongenotype 1 samples.Weconcluded that deep sequencing ismore efficient forHCVsubtyping than currently available methodsandallows qualitative identificationofmixed infectionsandmay bemorehelpfulwith respect to informing treatment strategies withnewDAA-containing regimens across allHCVsubtypesThis study has been supported by CDTI (Centro para el Desarrollo Tecnológico Industrial), Spanish Ministry of Economics and Competitiveness (MINECO), IDI-20110115; MINECO projects SAF 2009-10403; and also by the Spanish Ministry of Health, Instituto de Salud Carlos III (FIS) projects PI10/01505, PI12/01893, and PI13/00456. CIBERehd is funded by the Instituto de Salud Carlos III, Madrid, Spain. Work at CBMSO was supported by grant MINECO-BFU2011-23604, FIPSE, and Fundación Ramón Areces. X. Forns received unrestricted grant support from Roche and has acted as advisor for MSD, Gilead, and Abbvie. M. Alvarez-Tejado, J. Gregori, and J. M. Muñoz work in Roche Diagnostic

Evaluation of factors leading to poor outcomes for pediatric acute lymphoblastic leukemia in Mexico: a multi-institutional report of 2,116 patients

Background and aimsPediatric acute lymphoblastic leukemia (ALL) survival rates in low- and middle-income countries are lower due to deficiencies in multilevel factors, including access to timely diagnosis, risk-stratified therapy, and comprehensive supportive care. This retrospective study aimed to analyze outcomes for pediatric ALL at 16 centers in Mexico.MethodsPatients &lt;18 years of age with newly diagnosed B- and T-cell ALL treated between January 2011 and December 2019 were included. Clinical and biological characteristics and their association with outcomes were examined.ResultsOverall, 2,116 patients with a median age of 6.3 years were included. B-cell immunophenotype was identified in 1,889 (89.3%) patients. The median white blood cells at diagnosis were 11.2.5 × 103/mm3. CNS-1 status was reported in 1,810 (85.5%), CNS-2 in 67 (3.2%), and CNS-3 in 61 (2.9%). A total of 1,488 patients (70.4%) were classified as high-risk at diagnosis. However, in 52.5% (991/1,889) of patients with B-cell ALL, the reported risk group did not match the calculated risk group allocation based on National Cancer Institute (NCI) criteria. Fluorescence in situ hybridization (FISH) and PCR tests were performed for 407 (19.2%) and 736 (34.8%) patients, respectively. Minimal residual disease (MRD) during induction was performed in 1,158 patients (54.7%). The median follow-up was 3.7 years. During induction, 191 patients died (9.1%), and 45 patients (2.1%) experienced induction failure. A total of 365 deaths (17.3%) occurred, including 174 deaths after remission. Six percent (176) of patients abandoned treatment. The 5-year event-free survival (EFS) was 58.9% ± 1.7% for B-cell ALL and 47.4% ± 5.9% for T-cell ALL, while the 5-year overall survival (OS) was 67.5% ± 1.6% for B-cell ALL and 54.3% ± 0.6% for T-cell ALL. The 5-year cumulative incidence of central nervous system (CNS) relapse was 5.5% ± 0.6%. For the whole cohort, significantly higher outcomes were seen for patients aged 1–10 years, with DNA index &gt;0.9, with hyperdiploid ALL, and without substantial treatment modifications. In multivariable analyses, age and Day 15 MRD continued to have a significant effect on EFS.ConclusionOutcomes in this multi-institutional cohort describe poor outcomes, influenced by incomplete and inconsistent risk stratification, early toxic death, high on-treatment mortality, and high CNS relapse rate. Adopting comprehensive risk-stratification strategies, evidence-informed de-intensification for favorable-risk patients and optimized supportive care could improve outcomes