Prevalence of Respiratory Infections during the 2018-2020 Period in the Paediatric Population of Primary Care Centres in Central Catalonia.

Following the COVID-19 pandemic, policies such as social distancing, hand washing, and the use of masks were implemented, which could play an important role in the reduction of infectious diseases. An observational, descriptive, cross-sectional study was conducted to observe the prevalence of respiratory infections in children under 15 years of age during the 2018-2020 period in Primary Care centres in Central Catalonia. In 2020, there was a 44.3% decrease in total consultations for respiratory infections compared to 2019. All respiratory infections exhibited a significant decrease except flu-like syndrome; children between the ages of 6 and 12 had the highest prevalence of flu-like syndrome (87.6%), and the SARS-CoV-2-19 infection was most frequent (4%) among those between the ages of 12 and 15. Compared to urban centres, rural centres presented a higher prevalence of all infections except flu-like syndrome and SARS-CoV-2. In conclusion, the COVID-19 pandemic caused a significant decrease in the number of consultations for respiratory infections in the paediatric population, except for flu-like syndrome, which increased in cases in January, February, and March 2020. No differences were found between sexes, although differences were found in the distribution of the different age groups.

Prevalence of SARS-CoV-2 antibodies and risk factors in the pandemic epicentre of Catalonia.

To define the seroprevalence of antibodies against SARS-CoV-2 in the municipality of Vilanova del Camí (in the region of Conca d'Ódena, Barcelona, Spain) and to know the risk factors associated with positive seroprevalence. Cross-sectional descriptive study. The population of Vilanova del Camí had the opportunity to voluntarily attend two screenings (October and December 2020) for antibodies against the nucleocapsid protein of SARS-CoV-2 using a Rapid Diagnostic Test (RDT) (Salocor (Salofa Oy). Participants in the screening signed an informed consent form. From the 3,610 attendees at the screening, 2,170 patients were randomly selected. The relationship between antibody test results and other demographic (sex, age, morbidity index) and clinical (diagnoses, smoking and drugs) variables was analysed. The prevalence of antibodies against SARS-CoV-2 was 9.6% (95% CI of 8.4% to 10.9%) and was similar for men and women but increased with age. Among complex chronic patients, 14.3% had antibodies against SARS-CoV-2, and among patients with advanced chronic disease, 25% had antibodies against SARS-CoV-2. Age, AMG (Adjusted Morbidity Groups) index, COVID-19 diagnosis and contact with a COVID-19 case were risk factors for positive seroprevalence. A higher seroprevalence was detected in the October screening (12.16%) than in the December screening (8.38%). In the December screening, obesity was a risk factor for positive seroprevalence. This study demonstrates the high seroprevalence of antibodies against SARS-CoV-2 in the pandemic epicentre of Catalonia.

Hepatitis B Virus Variants with Multiple Insertions and/or Deletions in the X Open Reading Frame 3' End: Common Members of Viral Quasispecies in Chronic Hepatitis B Patients.

Deletions in the 3' end region of the hepatitis B virus (HBV) X open reading frame (HBX) may affect the core promoter (Cp) and have been frequently associated with hepatocellular carcinoma (HCC). The aim of this study was to investigate the presence of variants with deletions and/or insertions (Indels) in this region in the quasispecies of 50 chronic hepatitis B (CHB) patients without HCC. We identified 103 different Indels in 47 (94%) patients, in a median of 3.4% of their reads (IQR, 1.3-8.4%), and 25% (IQR, 13.1-40.7%) of unique sequences identified in each quasispecies (haplotypes). Of those Indels, 101 (98.1%) caused 44 different altered stop codons, the most commonly observed were at positions 128, 129, 135, and 362 (putative position). Moreover, 39 (37.9%) Indels altered the TATA-like box (TA) sequences of Cp; the most commonly observed caused TA2 + TA3 fusion, creating a new putative canonical TATA box. Four (8%) patients developed negative clinical outcomes after a median follow-up of 9.4 (8.7-12) years. In conclusion, we observed variants with Indels in the HBX 3' end in the vast majority of our CHB patients, some of them encoding alternative versions of HBx with potential functional roles, and/or alterations in the regulation of transcription.

Formulating stable hexosome dispersions with a technical grade diglycerol-based surfactant.

We report on the phase behavior of a technical grade and commercially available diglycerol monoisostearate, C41V, and its use for the preparation of nanostructured liquid crystal dispersions (hexosomes). C41V in water forms a reverse hexagonal liquid crystal at room temperature and in a wide range of concentrations (0.5-95 wt%); this hexagonal liquid crystal is stable up to 70 °C. A simple and effective method has been developed to disperse hexosomes with an encapsulated active molecule (Ketoprofen) that consists of (1) producing a nano-emulsion stabilized by an amphiphilic block copolymer (Pluronic F127) and containing ethyl acetate and C41V by using ultrasounds and (2) evaporating the solvent to produce hexosomes. The size of the hexosomes and ultrasound dispersion time is markedly reduced by using ethyl acetate as an auxiliary solvent with an optimal initial ratio of C41V:ethyl acetate of 50:50. Dynamic light scattering shows that the size of the hexosomes decreases as the concentration of stabilizer F127 or encapsulated Ketoprofen is increased. The lattice parameter in the hexagonal structure is calculated from small angle scattering data to be ca. 5.3  nm and is only slightly dependent on the amount of F127 and/or encapsulated Ketoprofen. Cryo electron microscopy reveals that the samples contain hexosomes and these coexist with spherical, likely F127 micelles. Lastly, hexosomes show a pH responsive release of Ketoprofen which could be useful for target delivery in the gastrointestinal tract.

Amino Acid Substitutions within HLA-B*27-Restricted T Cell Epitopes Prevent Recognition by Hepatitis Delta Virus-Specific CD8+ T Cells.

Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-γ) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection.IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development.

Quantitative characterization of hepatitis delta virus genome edition by next-generation sequencing.

AIM: To determine the capacity of next-generation sequencing (NGS) for quantifying edited and unedited HDV populations, and to confirm if edition is a general phenomenon taking place along the entire HDV region analyzed, as we previously reported (Homs M et al. PLoS One 2016, 11, e0158557). METHODS: Four serum samples from 4 patients with chronic HDV/HBV infection were included in the study. The region selected for analysis covered 360 nucleotides (nt), positions 910-1270 of the HDV genome, which included the HDAg ORF editing site (nt 1014 within codon 196). Quantification of edited and unedited genomes was performed by molecular cloning and Sanger sequencing and by NGS. To evaluate the reliability of the NGS values obtained, we combined a clone with an edited codon and one with an unedited codon in known percentages in a series of artificial mixtures, which were then analyzed by NGS. In addition, we determined the nt changes occurring over the complete amplified region after excluding the editing codon (196) to evaluate edition along it. RESULTS: In total, 11,208 quality-filtered sequences were obtained in the 4 samples. The 95% confidence intervals for the proportions of unedited populations by molecular cloning and NGS were overlapping, and those of cloning were wider, indicating that they are comparable and that NGS is more precise than cloning. Unedited genomes predominated over edited ones in all 4 samples analyzed by NGS and in 3 of the 4 samples analyzed by molecular cloning. In total, 83,276 quality-filtered sequences were obtained from the artificial mixtures. Percentages of the two viral populations detected by NGS in these mixtures were comparable to the expected percentages. Evaluation of edition along the HDV coding region showed that transitions were more frequent than transversions, accounting for 63.09% and 36.91%, respectively. Interestingly, among the 4 possible transition-type changes, G:A and A:G accounted for 73.86% of the total. CONCLUSION: Next-generation sequencing proved useful to quantify edited and unedited HDV genomes, and provided relevant information on the HDV quasispecies.

Both interferon alpha and lambda can reduce all intrahepatic HDV infection markers in HBV/HDV infected humanized mice.

Co-infection with hepatitis B (HBV) and D virus (HDV) is associated with the most severe course of liver disease. Interferon represents the only treatment currently approved. However, knowledge about the impact of interferons on HDV in human hepatocytes is scant. Aim was to assess the effect of pegylated interferon alpha (peg-IFNα) and lambda (peg-IFNλ), compared to the HBV-polymerase inhibitor entecavir (ETV) on all HDV infection markers using human liver chimeric mice and novel HDV strand-specific qRT-PCR and RNA in situ hybridization assays, which enable intrahepatic detection of HDV RNA species. Peg-IFNα and peg-IFNλ reduced HDV viremia (1.4 log and 1.2 log, respectively) and serum HBsAg levels (0.9-log and 0.4-log, respectively). Intrahepatic quantification of genomic and antigenomic HDV RNAs revealed a median ratio of 22:1 in untreated mice, resembling levels determined in HBV/HDV infected patients. Both IFNs greatly reduced intrahepatic levels of genomic and antigenomic HDV RNA, increasing the amounts of HDAg- and antigenomic RNA-negative hepatocytes. ETV-mediated suppression of HBV replication (2.1-log) did not significantly affect HBsAg levels, HDV productivity and/or release. In humanized mice lacking adaptive immunity, IFNs but not ETV suppressed HDV. Viremia decrease reflected the intrahepatic reduction of all HDV markers, including the antigenomic template, suggesting that intracellular HDV clearance is achievable.

Deep sequencing in the management of hepatitis virus infections.

The hepatitis viruses represent a major public health problem worldwide. Procedures for characterization of the genomic composition of their populations, accurate diagnosis, identification of multiple infections, and information on inhibitor-escape mutants for treatment decisions are needed. Deep sequencing methodologies are extremely useful for these viruses since they replicate as complex and dynamic quasispecies swarms whose complexity and mutant composition are biologically relevant traits. Population complexity is a major challenge for disease prevention and control, but also an opportunity to distinguish among related but phenotypically distinct variants that might anticipate disease progression and treatment outcome. Detailed characterization of mutant spectra should permit choosing better treatment options, given the increasing number of new antiviral inhibitors available. In the present review we briefly summarize our experience on the use of deep sequencing for the management of hepatitis virus infections, particularly for hepatitis B and C viruses, and outline some possible new applications of deep sequencing for these important human pathogens.

Evidence of an Exponential Decay Pattern of the Hepatitis Delta Virus Evolution Rate and Fluctuations in Quasispecies Complexity in Long-Term Studies of Chronic Delta Infection.

Chronic HDV infection can cause a severe form of viral hepatitis for which there is no specific treatment. Characterization of the hepatitis B or C viral quasispecies has provided insight into treatment failure and disease recurrence following liver transplantation, has proven useful to understand hepatitis B e antigen seroconversion, and has helped to predict whether hepatitis C infection will resolve or become chronic. It is likely that characterization of the hepatitis delta virus (HDV) quasispecies will ultimately have similar value for the management of this infection. This study sought to determine the RNA evolution rates in serum of chronic hepatitis delta (CHD) treatment-naïve patients, using next-generation sequencing methods. The region selected for study encompassed nucleotide positions 910 to 1270 of the genome and included the amber/W codon. Amber/W is a substrate of the editing process by the ADAR1 host enzyme and is essential for encoding the 2 delta antigens (HDAg). The amber codon encodes the small (unedited) HDAg form and the W codon the large (edited) HDAg form. The evolution rate was analyzed taking into account the time elapsed between samples, the percentage of unedited and edited genomes, and the complexity of the viral population. The longitudinal studies included 29 sequential samples from CHD patients followed up for a mean of 11.5 years. In total, 121,116 sequences were analyzed. The HDV evolution rate ranged from 9.5x10-3 to 1.2x10-3 substitutions/site/year and showed a negative correlation with the time elapsed between samples (p<0.05). An accumulation of transition-type changes was found to be responsible for higher evolution rates. The percentages of unedited and edited genomes and the quasispecies complexity showed no relationships with the evolution rate, but the fluctuations in the percentages of genomes and in complexity suggest continuous adaptation of HDV to the host conditions.

Complex Genotype Mixtures Analyzed by Deep Sequencing in Two Different Regions of Hepatitis B Virus.

This study assesses the presence and outcome of genotype mixtures in the polymerase/surface and X/preCore regions of the HBV genome in patients with chronic hepatitis B virus (HBV) infection. Thirty samples from ten chronic hepatitis B patients were included. The polymerase/surface and X/preCore regions were analyzed by deep sequencing (UDPS) in the first available sample at diagnosis, a pre-treatment sample, and a sample while under treatment. HBV genotype was determined by phylogenesis. Quasispecies complexity was evaluated by mutation frequency and nucleotide diversity. The polymerase/surface and X/preCore regions were validated for genotyping from 113 GenBank reference sequences. UDPS yielded a median of 10,960 sequences per sample (IQR 16,645) in the polymerase/surface region and 11,595 sequences per sample (IQR 14,682) in X/preCore. Genotype mixtures were more common in X/preCore (90%) than in polymerase/surface (30%) (p<0.001). On X/preCore genotyping, all samples were genotype A, whereas polymerase/surface yielded genotypes A (80%), D (16.7%), and F (3.3%) (p = 0.036). Genotype changes in polymerase/surface were observed in four patients during natural quasispecies dynamics and in two patients during treatment. There were no genotype changes in X/preCore. Quasispecies complexity was higher in X/preCore than in polymerase/surface (p = 0.004). The results provide evidence of genotype mixtures and differential genotype proportions in the polymerase/surface and X/preCore regions. The genotype dynamics in HBV infection and the different patterns of quasispecies complexity in the HBV genome suggest a new paradigm for HBV genotype classification.

Tenofovir discontinuation after long-term viral suppression in HBeAg negative chronic hepatitis B. Can HBsAg levels be useful?

BACKGROUND: Recent studies have shown that antiviral treatment discontinuation is safe and associated with virologic remission in HBeAg-negative patients. However, the period of viral suppression and follow-up in these studies was relatively short. OBJECTIVES: To investigate whether continuous viral suppression with tenofovir disoproxil fumarate for more than 7 years is associated with HBsAg loss and sustained response after treatment discontinuation and receiving a full course of hepatitis B vaccination. STUDY DESIGN: Patients with HBeAg-negative chronic HBV infection and more than 7 years of persistent viral suppression with tenofovir therapy were selected for treatment discontinuation and HBV vaccination. Follow-up with monthly ALT, HBV-DNA, and HBsAg determinations lasted 72 weeks. In patients with viral relapse, the viral quasispecies in the overlapping reverse transcriptase and small surface protein regions was analysed by ultra-deep pyrosequencing. RESULTS: Eight of 17 HBeAg-negative patients accepted tenofovir discontinuation: 5 patients achieved sustained response (persistent HBV-DNA levels <2000IU/mL and normal ALT) despite an initial virologic relapse, one lost HBsAg, and two needed re-treatment. All patients with an on-treatment HBsAg level decline >5000IU/mL achieved sustained response. Patients with HBsAg level <100IU/mL during an ALT flare after antiviral discontinuation achieved sustained response. Significant changes were seen in the composition of the HBV quasispecies, and half the patients showed changes in HBV genotype. CONCLUSIONS: Even though the majority of patients presented an initial relapse with selection of HBV variants, most achieved sustained response. Changes in HBsAg levels on and off treatment may be useful for predicting the likelihood of virologic remission.

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

Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens, identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott Real-time HCV Genotype II failed subtype 1 calling in 13 (16%) samples each and were unable to identify the HCV genotype and/or subtype in more than half of the non-genotype 1 samples. We concluded that deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections and may be more helpful with respect to informing treatment strategies with new DAA-containing regimens across all HCV subtypes.

Clinical application of estimating hepatitis B virus quasispecies complexity by massive sequencing: correlation between natural evolution and on-treatment evolution.

AIM: To evaluate HBV quasispecies (QA) complexity in the preCore/Core regions in relation to HBeAg status, and explore QA changes under natural evolution and nucleoside analogue (NUC) treatment. METHODS: Ultra-deep pyrosequencing of HBV preCore/Core regions in 30 sequential samples (baseline [diagnosis], treatment-free, and treatment-nonresponse) from 10 retrospectively selected patients grouped according to HBeAg status over time: HBeAg+ (N = 4), HBeAg- (N = 2), and fluctuating HBeAg (transient seroreversion/seroconversion pattern) (N = 4). QA complexity was defined by Shannon entropy, mutation frequency, nucleotide diversity, and mutation frequency of amino acids (MfAA) in preCore and Core. RESULTS: The QA was less complex in HBeAg+ than in HBeAg- or fluctuating HBeAg. High complexity in preCore was associated with decreased viral replication (preCore MfAA negatively correlated with HBV-DNA, p = 0.005). QA complexity in the treatment-free period negatively correlated with values seen during treatment. Specific variants were mainly selected in the Core region in HBeAg- and fluctuating HBeAg patients, suggesting higher immune pressure than in HBeAg+. CONCLUSIONS: The negative correlation between QA natural evolution and on-treatment evolution indicates the importance of pre-treatment QA study to predict QA changes in NUC nonresponders. Study of QA complexity could be useful for managing HBV infection.

Cirrhosis, liver transplantation and HIV infection are risk factors associated with hepatitis E virus infection.

BACKGROUND: Acute and chronic hepatitis E have been associated with high mortality and development of cirrhosis, particularly in solid-organ recipients and patients infected by human immunodeficiency virus. However, data regarding the epidemiology of hepatitis E in special populations is still limited. AIMS: Investigate seroprevalence and possible factors associated with HEV infection in a large cohort of immunosuppressed patients. METHODS: Cross-sectional study testing IgG anti-HEV in serum samples from 1373 consecutive individuals: 332 liver-transplant, 296 kidney-transplant, 6 dual organ recipients, 301 non-transplanted patients with chronic liver disease, 238 HIV-infected patients and 200 healthy controls. RESULTS: IgG anti-HEV was detected in 3.5% controls, 3.7% kidney recipients, 7.4% liver transplant without cirrhosis and 32.1% patients who developed post-transplant cirrhosis (p<0.01). In patients with chronic liver disease, IgG anti-HEV was also statistically higher in those with liver cirrhosis (2% vs 17.5%, p<0.01). HIV-infected patients showed an IgG anti-HEV rate of 9.2%, higher than those patients without HIV infection (p<0.03). Multivariate analysis showed that the factors independently associated with anti-HEV detection were liver cirrhosis, liver transplantation and HIV infection (OR: 7.6, 3.1 and 2.4). HCV infection was a protective factor for HEV infection (OR: 0.4). CONCLUSIONS: HEV seroprevalence was high in liver transplant recipients, particularly those with liver cirrhosis. The difference in anti-HEV prevalence between Liver and Kidney transplanted cases suggests an association with advanced liver disease. Further research is needed to ascertain whether cirrhosis is a predisposing factor for HEV infection or whether HEV infection may play a role in the pathogeneses of cirrhosis.

Relevance of a full-length genomic RNA standard and a thermal-shock step for optimal hepatitis delta virus quantification.

Hepatitis D virus (HDV) is a defective RNA virus that requires the surface antigens of hepatitis B virus (HBV) (HBsAg) for viral assembly and replication. Several commercial and in-house techniques have been described for HDV RNA quantification, but the methodologies differ widely, making a comparison of the results between studies difficult. In this study, a full-length genomic RNA standard was developed and used for HDV quantification by two different real-time PCR approaches (fluorescence resonance energy transfer [FRET] and TaqMan probes). Three experiments were performed. First, the stability of the standard was determined by analyzing the effect of thawing and freezing. Second, because of the strong internal base pairing of the HDV genome, which leads to a rod-like structure, the effect of intense thermal shock (95°C for 10 min and immediate cooling to -80°C) was tested to confirm the importance of this treatment in the reverse transcription step. Lastly, to investigate the differences between the DNA and RNA standards, the two types were quantified in parallel with the following results: the full-length genomic RNA standard was stable and reliably mimicked the behavior of HDV-RNA-positive samples, thermal shock enhanced the sensitivity of HDV RNA quantification, and the DNA standard underquantified the HDV RNA standard. These findings indicate the importance of using complete full-length genomic RNA and a strong thermal-shock step for optimal HDV RNA quantification.

Quasispecies structure, cornerstone of hepatitis B virus infection: mass sequencing approach.

Hepatitis B virus (HBV) is a DNA virus with complex replication, and high replication and mutation rates, leading to a heterogeneous viral population. The population is comprised of genomes that are closely related, but not identical; hence, HBV is considered a viral quasispecies. Quasispecies variability may be somewhat limited by the high degree of overlapping between the HBV coding regions, which is especially important in the P and S gene overlapping regions, but is less significant in the X and preCore/Core genes. Despite this restriction, several clinically and pathologically relevant variants have been characterized along the viral genome. Next-generation sequencing (NGS) approaches enable high-throughput analysis of thousands of clonally amplified regions and are powerful tools for characterizing genetic diversity in viral strains. In the present review, we update the information regarding HBV variability and present a summary of the various NGS approaches available for research in this virus. In addition, we provide an analysis of the clinical implications of HBV variants and their study by NGS.

Removability of a small aperture intracorneal inlay for presbyopia correction.

PURPOSE: To evaluate the safety of the corneal inlay removal procedure and the reversibility of visual acuities, corneal topography, and corneal biomicroscopy changes in a series of cases. METHODS: Ten cases implanted with one of three versions of the AcuFocus Kamra Inlay (ACI 7000, 7000T, and 7000PDT; AcuFocus, Inc., Irvine, CA) were followed for a minimum of 6 months after corneal inlay removal. RESULTS: The reason for removal was related to subjective dissatisfaction with visual symptoms (8 of 10 patients) such as night glare, photophobia, starburst, blurry vision, and halos. One case of removal was related to inadvertent thin flap and the final case was related to insufficient near vision. Mean uncorrected distance visual acuity (UDVA) and uncorrected near visual acuity (UNVA) was 0 ± 0.1 logMAR (Snellen 20/20) and 0.5 ± 0.2 logMAR (Snellen 20/40), respectively, preoperatively and 0.1 ± 0.1 logMAR (Snellen 20/25) and 0.5 ± 0.1 logMAR (Snellen 20/63), respectively, 6 months after corneal inlay removal. Mean corrected distance visual acuity (CDVA) and corrected near visual acuity (CNVA) was 0 ± 0.1 logMAR (Snellen 20/20) and 0 ± 0.1 logMAR (Snellen 20/20), respectively, preoperatively and 0 ± 0.1 logMAR (Snellen 20/20) and 0.1 ± 0.1 logMAR (Snellen 20/25), respectively, 6 months after corneal inlay removal. Mean root mean square (RMS) higher-order aberration (HOA) was 0.50 ± 0.12 (range: 0.30 to 0.70) preoperatively and 0.69 ± 0.14 (range: 0.48 to 0.95) 6 months after corneal inlay removal (P < .8). Weak positive correlation was found between Δt Implant-Removal (Δt I-R), RMS spherical, coma, and HOA at 6 months (Δt I-R vs RMS spherical was r = 0.2, r(2) = 0.5, P < .7; Δt I-R vs RMS coma was r = 0.8, r(2) = 0.6, P < .3; and Δt I-R vs HOA r = 0.8; r(2) = 0.6, P < .9). CONCLUSION: This study suggests that after removal of the corneal inlay, corneal topography and corneal aberrometry are not permanently affected. In more than 60% of patients, CNVA, CDVA, UNVA, and UDVA were similar to the preoperative value.

A comparative study of ultra-deep pyrosequencing and cloning to quantitatively analyze the viral quasispecies using hepatitis B virus infection as a model.

In this study, the reliability and reproducibility of viral quasispecies quantification by three ultra-deep pyrosequencing (UDPS) methods (FLX+, FLX, and Junior) were investigated and results compared with the conventional cloning technique. Hepatitis B virus (HBV) infection was selected as the model. The preCore/Core region, the least overlapped HBV region, was analyzed in samples from a chronic hepatitis B patient by cloning and by UDPS. After computation filtering of the UDPS results, samples A1 and A2 (FLX+) and sample B (FLX) yielded the same 20 polymorphic positions. Junior yielded 18 polymorphic positions that coincided with the FLX results. In contrast, 50 polymorphic positions were detected by cloning. Quasispecies complexity plotted on graphs showed superimposed patterns and the quantitative parameters were similar between FLX+, FLX, Junior, and the cloning sequences. Twenty-two haplotypes were detected by Junior, and 37, 40, and 39 were detected by FLX A1, A2, and B, respectively. These differences may be attributable to methodological differences between FLX and Junior. By cloning, 47 haplotypes were detected. Eight clones with insertions and deletions that induced de novo stop codons were not observed by UDPS because the UDPS filter discarded them. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of the viral quasispecies. Nonetheless, specific mutations, such as insertions and deletions, were only detected by cloning. A filter should be designed to analyze cloning sequences, and UDPS filters should be improved to include the specific mutations.

Quasispecies dynamics in main core epitopes of hepatitis B virus by ultra-deep-pyrosequencing.

AIM: To investigate the variability of the main immunodominant motifs of hepatitis B virus (HBV) core gene by ultra-deep-pyrosequencing (UDPS). METHODS: Four samples (2 genotype A and 2 genotype D) from 4 treatment-naïve patients were assessed for baseline variability. Two additional samples from one patient (patient 4, genotype D) were selected for analysis: one sample corresponded to a 36-mo treatment-free period from baseline and the other to the time of viral breakthrough after 18 mo of lamivudine treatment. The HBV region analyzed covered amino acids 40 to 95 of the core gene, and included the two main epitopic regions, Th50-69 and B74-84. UDPS was carried out in the Genome Sequencer FLX system (454 Life Sciences, Roche). After computer filtering of UDPS data based on a Poisson statistical model, 122,813 sequences were analyzed. The most conserved position detected by UDPS was analyzed by site-directed mutagenesis and evaluated in cell culture. RESULTS: Positions with highest variability rates were mainly located in the main core epitopes, confirming their role as immune-stimulating regions. In addition, the distribution of variability showed a relationship with HBV genotype. Patient 1 (genotype A) presented the lowest variability rates and patient 2 (genotype A) had 3 codons with variability higher than 1%. Patient 3 and 4 (both genotype D) presented 5 and 8 codons with variability higher than 1%, respectively. The median baseline frequencies showed that genotype A samples had higher variability in epitopic positions than in the other positions analyzed, approaching significance (P = 0.07, sample 1 and P = 0.05, sample 2). In contrast, there were no significant differences in variability between the epitopic and other positions in genotype D cases. Interestingly, patient 1 presented a completely mutated motif from amino acid 64 to 67 (E64LMT67), which is commonly recognized by T helper cells. Additionally, the variability observed in all 4 patients was particularly associated with the E64LMT67 motif. Codons 78 and 79 were highly conserved in all samples, in keeping with their involvement in the interaction between the HBV virion capsid and the surface antigens (HBsAg). Of note, codon 76 was even more conserved than codons 78 and 79, suggesting a possible role in HBsAg interactions or even in hepatitis B e antigen conformation. Sequential analysis of samples from patient 4 (genotype D) illustrated the dynamism of the HBV quasispecies, with strong selection of one minor baseline variant coinciding with a decrease in core variability during the treatment-free and lamivudine-treated period. The drop in variability seemed to result from a "steady state" situation of the HBV quasispecies after selection of the variant with greatest fitness. CONCLUSION: Host immune pressure seems to be the main cause of HBV core evolution. UDPS analysis is a useful technique for studying viral quasispecies.