Changing Trends in International Versus Domestic HCV Transmission in HIV-Positive Men Who Have Sex With Men: A Perspective for the Direct-Acting Antiviral Scale-Up Era.

BACKGROUND: Scale-up of direct-acting antiviral therapy is expected to abate hepatitis C virus (HCV) incidence among human immunodeficiency virus (HIV)-positive men who have sex with men (MSM). International transmission could influence this process. We classified HCV infections in HIV-positive MSM as either domestically or internationally acquired, and estimated how this classification changed over time. METHODS: HCV subtype 1a (the most frequent subtype among MSM) genomes from 99 persons enrolled in the Swiss HIV Cohort Study and diagnosed with replicating HCV infections, were sequenced. Sixty-six of these sequences were from MSM. We inferred maximum-likelihood phylogenetic trees and time trees containing a fragment of the NS5B region of these and 374 circulating strains. We inferred transmission clusters from these trees and used the country composition of such clusters to attribute infections to domestic or international transmission. RESULTS: Of HCV transmissions, 50% to 80% were classified as domestic depending on the classification criterion. Between 2000 and 2007, the fraction attributable to domestic transmission was 54% (range 0-75%). It increased to 85% (range 67%-100%) between 2008 and 2016. CONCLUSIONS: International and domestic transmission have played major roles in this epidemic. While international transmission persists, local transmission has established as the main source of infections.

Expression of programmed cell death protein 1 and T-cell immunoglobulin- and mucin-domain-containing molecule-3 on peripheral blood CD4+CD8+ double positive T cells in patients with chronic hepatitis C virus infection and in subjects who spontaneously cleared the virus.

Chronic hepatitis C virus (HCV) infection is characterized by increased proportion of CD4+CD8+ double positive (DP) T cells, but their role in this infection is unclear. In chronic hepatitis C, immune responses to HCV become functionally exhausted, which manifests itself by increased expression of programmed cell death protein 1 (PD-1) and T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) on T cells. The aim of our study was to determine PD-1 and Tim-3 phenotype of DP T cells in subjects with naturally resolved and chronic HCV infection. Peripheral blood mononuclear cells from 16 patients with chronic infection and 14 subjects who cleared HCV in the past were stained with anti-CD3, anti-CD4, anti-CD8, anti-PD-1 and anti-Tim-3 antibodies and, in 12 HLA-A*02-positive subjects, MHC class I pentamer with HCV NS31406 epitope. In chronic and past HCV infection, proportions of total DP T cells and PD-1+ DP T cells were similar but significantly higher than in healthy controls. DP T cells were more likely to be PD-1+ than either CD4+ or CD8+ single positive (SP) T cells. HCV-specific cells were present in higher proportions among DP T cells than among CD8+ SP T cells in both patient groups. Furthermore, while the majority of HCV-specific DP T cells were PD-1+, the proportion of HCV-specific CD8+ T cells which were PD-1+ was 4.9 and 1.9 times lower (chronic and past infection, respectively). PD-1 and Tim-3 were predominantly expressed on CD4high CD8low and CD4low CD8high cells, respectively, and co-expression of both markers was uncommon.

Spontaneous Elimination of Hepatitis C Virus Infection.

Hepatitis C virus (HCV) is the etiological agent of chronic hepatitis C and a major cause of liver cirrhosis and hepatocellular carcinoma. Only a minority of infected individuals can clear the virus spontaneously. The knowledge of the determinants of virus clearance would allow the development of effective methods preventing its further spread and optimizing treatment regimens. Viral factors associated with spontaneous virus clearance in the acute phase of infection, such as HCV genotype, virus heterogeneity, and the impact of viral proteins on the immune system have been characterized. Likewise, host genetic markers, such as the interleukin genotypes, HLA alleles, and factors affecting the T lymphocyte response appear to play an important role. Studies have revealed that natural clearance of HCV infection in the chronic phase is rare and its mechanisms are not well understood. In this review, we present the state-of-the art knowledge on the viral and host factors affecting the spontaneous elimination of HCV infection.

Next-Generation Sequencing of Hepatitis C Virus (HCV) Mixed-Genotype Infections in Anti-HCV-Negative Blood Donors.

The infection with more than one hepatitis C virus (HCV) genotype especially in subjects with a high risk of multiple HCV exposures has been demonstrated. The role of HCV mixed-genotype infection in viral persistence and treatment effect is not fully understood. The prevalence of such infection varies greatly depending on the technique used for genotype determination and studied population. Next-generation sequencing (NGS) which is suitable for extensive analysis of complex viral populations is a method of choice for studying mixed infections. The aim of the present study was to determine the prevalence of mixed-genotype HCV infections in the Polish seronegative, HCV-RNA-positive blood donors (n = 76). Two-step PCR was used for amplification of 5'-UTR of HCV. Using pyrosequencing altogether, 381,063 reads were obtained. The raw reads were trimmed and subjected to similarity analysis against the entire unfiltered NCBI nt database. Results obtained from NGS were compared with the standard genotyping. One (1.3%) mixed-genotype [3a, 2989 reads (94.8%); 1b, 164 reads (5.2%)] infection was found in a sample diagnosed as genotype 3a only by routine testing. Two samples were identified with different genotypes, compared to routine testing. In conclusion, NGS is a sensitive method for HCV genotyping. The prevalence of mixed-genotype HCV infections in blood donors is low.

Next-Generation Sequencing of 5' Untranslated Region of Hepatitis C Virus in Search of Minor Viral Variant in a Patient Who Revealed New Genotype While on Antiviral Treatment.

The role of mixed infections with different hepatitis C virus (HCV) genotypes in viral persistence, treatment effects, and tissue tropism is unclear. Next-generation sequencing (NGS), which is suitable for analysis of large, genetically diverse populations offers unparalleled advantages for the study of mixed infections. The aim of the study was to determine, using two different deep sequencing strategies (pyrosequencing - 454 Life Sciences/Roche and reversible terminator sequencing-by-synthesis by Illumina), the origin of a novel HCV genotype transiently detectable during antiviral therapy (pre-existing minor population vs. de novo superinfection). Secondly, we compared 5' untranslated region (5'-UTR) variants obtained by the two NGS approaches. 5' UTR amplification products from 9 samples collected from genotype 1b infected patient before, during, and after treatment (4 serum and 5 peripheral blood mononuclear cell - PBMC - samples) were subjected to the next-generation sequencing. The sequencing revealed the presence of two (454/Roche) and one (Illumina) genotype 4 variants in PBMC at Week 16. None of these variants were present either in the preceding or following samples as revealed by both platforms. 454/Roche sequencing detected 24 different 5'-UTR variants: 8 were present in serum and PBMC, 4 only in serum and 12 only in PBMC. Illumina sequencing detected 11 different 5'-UTR variants: 5 in serum and PBMC, 4 only in serum and 2 only in PBMC. Six variants were identical for both sequencing platforms. The difference in variants number was primarily due to variability in two 5'-UTR homopolymeric regions. In conclusion, longitudinal analysis of HCV variants, employing two independent deep sequencing methods, suggests that the transient presence of a different genotype strain in PBMC was a result of superinfection and not a selection of pre-existing minor variant.

Sensitivity of Next-Generation Sequencing Metagenomic Analysis for Detection of RNA and DNA Viruses in Cerebrospinal Fluid: The Confounding Effect of Background Contamination.

Next-generation sequencing (NGS) followed by metagenomic enables the detection and identification of known as well as novel pathogens. It could be potentially useful in the diagnosis of encephalitis, caused by a variety of microorganisms. The aim of the present study was to evaluate the sensitivity of isothermal RNA amplification (Ribo-SPIA) followed by NGS metagenomic analysis in the detection of human immunodeficiency virus (HIV) and herpes simplex virus (HSV) in cerebrospinal fluid (CSF). Moreover, we analyzed the contamination background. We detected 102 HIV copies and 103 HSV copies. The analysis of control samples (two water samples and one CSF sample from an uninfected patient) revealed the presence of human DNA in the CSF sample (91 % of all reads), while the dominating sequences in water were qualified as 'other', related to plants, plant viruses, and synthetic constructs, and constituted 31 % and 60 % of all reads. Bacterial sequences represented 5.9 % and 21.4 % of all reads in water samples and 2.3 % in the control CSF sample. The bacterial sequences corresponded mainly to Psychrobacter, Acinetobacter, and Corynebacterium genera. In conclusion, Ribo-SPIA amplification followed by NGS metagenomic analysis is sensitive for detection of RNA and DNA viruses. Contamination seems common and thus the results should be confirmed by other independent methods such as RT-PCR and PCR. Despite these reservations, NGS seems to be a promising method for the diagnosis of viral infections.

Metagenomic Analysis of Cerebrospinal Fluid from Patients with Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of central nervous system of unknown etiology. However, some infectious agents have been suggested to play a significant role in its pathogenesis. Next-generation sequencing (NGS) and metagenomics can be employed to characterize microbiome of MS patients and to identify potential causative pathogens. In this study, 12 patients with idiopathic inflammatory demyelinating disorders (IIDD) of the central nervous system were studied: one patient had clinically isolated syndrome, one patient had recurrent optic neuritis, and ten patients had multiple sclerosis (MS). In addition, there was one patient with other non-inflammatory neurological disease. Cerebrospinal fluid (CSF) was sampled from all patients. RNA was extracted from CSF and subjected to a single-primer isothermal amplification followed by NGS and comprehensive data analysis. Altogether 441,608,474 reads were obtained and mapped using blastn. In a CSF sample from the patient with clinically isolated syndrome, 11 varicella-zoster virus reads were found. Other than that similar bacterial, fungal, parasitic, and protozoan reads were identified in all samples, indicating a common presence of contamination in metagenomics. In conclusion, we identified varicella zoster virus sequences in one out of the 12 patients with IIDD, which suggests that this virus could be occasionally related to the MS pathogenesis. A widespread bacterial contamination seems inherent to NGS and complicates the interpretation of results.

Double Positive CD4+CD8+ (DP) T-Cells Display Distinct Exhaustion Phenotype in Chronic Hepatitis C.

In chronic hepatitis C (CHC), characterized by exhaustion of T-cell function, increased frequencies of double-positive (DP) (CD4+CD8+) cells are present in peripheral blood. We compared the exhaustion phenotype between DP and single positive (SP) T-cells, including HCV-specific cells, and assessed the effect of successful HCV treatment on inhibitory receptors expression. Blood samples from 97 CHC patients were collected before and six months post-treatment. PD-1 (programmed cell death protein 1) and Tim-3 (T-cell immunoglobulin and mucin domain-containing molecule-3) expression was assessed by flow cytometry. DP T-cells displayed significantly higher PD-1 expression, lower Tim-3 expression than CD8+ SP T-cells and lower percentages of PD-1-Tim-3- cells than CD4+ SP T-cells, both before and after treatment. PD-1+Tim-3+ DP T-cells decreased following treatment. HCV-specific cells were more frequent among DP than SP T-cells, both before and after treatment. HCV-specific DP T-cells were characterized by lower PD-1 expression, higher PD-1 and Tim-3 co-expression, and lower percentages of PD-1-Tim-3- cells (both before and after treatment) and higher post-treatment Tim-3 than HCV-specific SP T-cells. Their percentages decreased following treatment, but the exhaustion phenotype remained unchanged. DP T-cells in CHC exhibit a distinct exhaustion phenotype from SP T-cells, and these changes mostly persist following successful treatment.

Cytokine profile and viral diversity in the early seronegative stage of community-acquired hepatitis C virus (HCV) infection.

Most Hepatitis C virus (HCV)-infected subjects develop chronic infection, whereas a minority clear the virus in the early phase of infection. We analyzed factors associated with outcome (chronicity vs clearance) during the preclinical seronegative phase of community-acquired HCV infection. Among 17.5 million blood donations in the years 2000-2016, 124 blood donors were found to be HCV RNA-positive/anti-HCV-negative. All were contacted after 0.5-12.7 years and 40 responded and provided blood sample. Hypervariable region 1 was analyzed by ultradeep pyrosequencing and cytokines in serum were quantified by Luminex (R&D Systems) multiplex immunoassay. Twenty-one (52.5%) donors were found to be HCV-RNA-positive, while 19 (47.5%) were HCV RNA negative (none received antiviral treatment). All but one seroconverted to anti-HCV. Donors with resolving hepatitis did not differ significantly from donors with chronic infection with respect to age, genotypes, IL28B polymorphisms, number of viral variants, nucleotide diversity per site or the overall number of nucleotide substitutions. However, the former group had significantly higher levels of IL-1beta, IL-1RA, IL-6, IFN-gamma and FGF-2 in serum. In our study of community-acquired acute hepatitis C approximately half of all subjects eliminated the virus spontaneously, and this clearance was associated with marked cytokine response in the early seronegative stage of infection.

T-Cell Exhaustion in HIV-1/Hepatitis C Virus Coinfection Is Reduced After Successful Treatment of Chronic Hepatitis C.

Background: T-cell responses during chronic viral infections become exhausted, which is reflected by upregulation of inhibitory receptors (iRs) and increased interleukin 10 (IL-10). We assessed 2 iRs-PD-1 (programmed cell death protein 1) and Tim-3 (T-cell immunoglobulin and mucin domain-containing protein 3)-and IL-10 mRNAs in peripheral blood mononuclear cells (PBMCs) and their soluble analogs (sPD-1, sTim-3, and IL-10) in plasma in chronic HIV-1/hepatitis C virus (HCV) coinfection and explored the effect of HCV treatment on these markers. We also aimed to establish whether iR expression may be determined by the HCV CD8+ T-cell immunodominant epitope sequence. Methods: Plasma and PBMCs from 31 persons with chronic HIV-1/HCV coinfection from the Swiss HIV Cohort Study were collected before and after HCV treatment. As controls, 45 persons who were HIV-1 negative with chronic HCV infection were recruited. Exhaustion markers were assessed by enzyme-linked immunosorbent assay in plasma and by quantitative reverse transcription polymerase chain reaction in PBMCs. Analysis of an HCV epitope sequence was conducted by next-generation sequencing: HLA-A*02-restricted NS31073-1081 and NS31406-1415 and HLA-A*01-restricted NS31436-1444. Results: The study revealed higher plasma sPD-1 (P = .0235) and IL-10 (P = .002) levels and higher IL-10 mRNA in PBMCs (P = .0149) in HIV-1/HCV coinfection. A decrease in plasma sPD-1 (P = .0006), sTim-3 (P = .0136), and IL-10 (P = .0003) and Tim-3 mRNA in PBMCs (P = .0210) was observed following successful HCV treatment. Infection with the HLA-A*01-restricted NS31436-1444 ATDALMTGY prototype variant was related to higher sTim-3 levels than infection with the ATDALMTGF escape variant (P = .0326). Conclusions: The results underscore the synergistic effect of coinfection on expression of exhaustion markers, their reduction following successful HCV treatment and imply that iR levels may operate on an epitope-specific manner.

Seronegative Infection with Toxoplasma gondii in Asymptomatic Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Patients and in Blood Donors.

BACKGROUND: Toxoplasmosis is a common opportunistic infection in AIDS patients. The routine diagnostics is based on serologic testing and IgG avidity index, but it may have limited utility in immunodeficient patients; thus, it is recommendable to detect T. gondii DNA in subjects with advanced HIV disease. The results of the studies published so far focused on patients with clinical symptoms of toxoplasmosis. Our study encompassed a group of HIV-infected subjects on cART therapy, without immunological disturbances and clinical symptoms of T. gondii infection. METHODS: The study was retrospective, and samples were collected between 2013 and 2016. We evaluate the prevalence of serological (IgM, IgG, and avidity IgG) and molecular (DNA) T. gondii infection markers in asymptomatic HIV-infected patients and the control group using serologic (ELISA) and quantitative (real-time PCR) molecular testing. RESULTS: Of 152 HIV-infected in routine follow-up tested for T. gondii IgM and IgG, 6 (3.9%) and 50 (32.9%) were positive, respectively. Of 168 serum samples from blood donors, 1 (0.6%) and 49 (29.2%) were IgM+ and IgG+ positive, respectively. IgM seroprevalence in HIV-infected patients was significantly higher than in blood donors. T. gondii DNA (genotype II) was identified in 47 (30.9%) HIV-infected patients, with 13 (8.6%) IgM-IgG- samples. In blood donors, T. gondii DNA was present in 15 (8.9%) IgM-IgG-. CONCLUSIONS: In both groups, T. gondii DNA was detectable in seronegative subjects, implying the need to supplement the routine serological testing via the molecular method. It can help the accurate monitoring of the reactivation of infection in asymptomatic HIV-infected persons, and the quick introduction of specific therapy, in blood donors, would be of high importance for safe blood donations.

CD8+ T-Cell Exhaustion Phenotype in Chronic Hepatitis C Virus Infection Is Associated With Epitope Sequence Variation.

Background and Aims: During chronic hepatitis C virus (HCV) infection, CD8+ T-cells become functionally exhausted, undergoing progressive phenotypic changes, i.e., overexpression of "inhibitory" molecules such as PD-1 (programmed cell death protein 1) and/or Tim-3 (T-cell immunoglobulin and mucin domain-containing molecule-3). The extreme intrahost genetic diversity of HCV is a major mechanism of immune system evasion, facilitating epitope escape. The aim of the present study was to determine whether T-cell exhaustion phenotype in chronic HCV infection is related to the sequence repertoire of NS3 viral immunodominant epitopes. Methods: The study population was ninety prospective patients with chronic HCV genotype 1b infection. Populations of peripheral blood CD8+ T-cells expressing PD-1/Tim-3 were assessed by multiparametric flow cytometry, including HCV-specific T-cells after magnetic-based enrichment using MHC-pentamer. Autologous epitope sequences were inferred from next-generation sequencing. The correction of sequencing errors and genetic variants reconstruction was performed using Quasirecomb. Results: There was an interplay between the analyzed epitopes sequences and exhaustion phenotype of CD8+ T-cells. A predominance of NS31406 epitope sequence, representing neither prototype KLSGLGLNAV nor cross-reactive variants (KLSSLGLNAV, KLSGLGINAV or KLSALGLNAV), was associated with higher percentage of HCV-specific CD8+PD-1+Tim-3+ T-cells, P=0.0102. Variability (at least two variants) of NS31406 epitope sequence was associated with increased frequencies of global CD8+PD-1+Tim-3+ T-cells (P=0.0197) and lower frequencies of CD8+PD-1-Tim-3- T-cells (P=0.0079). In contrast, infection with NS31073 dominant variant epitope (other than prototype CVNGVCWTV) was associated with lower frequency of global CD8+PD-1+Tim-3+ T-cells (P=0.0054). Conclusions: Our results indicate that PD-1/Tim-3 receptor expression is largely determined by viral epitope sequence and is evident for both HCV-specific and global CD8+ T-cells, pointing to the importance of evaluating autologous viral epitope sequences in the investigation of CD8+ T-cell exhaustion in HCV infection.

Reversal of T Cell Exhaustion in Chronic HCV Infection.

The long-term consequences of T cell responses' impairment in chronic HCV infection are not entirely characterized, although they may be essential in the context of the clinical course of infection, re-infection, treatment-mediated viral clearance and vaccine design. Furthermore, it is unclear whether a complete reinvigoration of HCV-specific T cell response may be feasible. In most studies, attempting to reverse the effects of compromised immune response quality by specific blockades of negative immune regulators, a restoration of functional competence of HCV-specific T cells was shown. This implies that HCV-induced immune dysfunction may be reversible. The advent of highly successful, direct-acting antiviral treatment (DAA) for chronic HCV infection instigated investigation whether the treatment-driven elimination of viral antigens restores T cell function. Most of studies demonstrated that DAA treatment may result in at least partial restoration of T cell immune function. They also suggest that a complete restoration comparable to that seen after spontaneous viral clearance may not be attained, pointing out that long-term antigenic stimulation imprints an irreversible change on the T cell compartment. Understanding the mechanisms of HCV-induced immune dysfunction and barriers to immune restoration following viral clearance is of utmost importance to diminish the possible long-term consequences of chronic HCV infection.

Decrease of T-cells exhaustion markers programmed cell death-1 and T-cell immunoglobulin and mucin domain-containing protein 3 and plasma IL-10 levels after successful treatment of chronic hepatitis C.

During chronic hepatitis C virus (HCV) infection, both CD4+ and CD8+ T-cells become functionally exhausted, which is reflected by increased expression of programmed cell death-1 (PD-1) and T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), and elevated anti-inflammatory interleukin 10 (IL-10) plasma levels. We studied 76 DAA-treated HCV-positive patients and 18 non-infected controls. Flow cytometry measured pretreatment frequencies of CD4+PD-1+, CD4+PD-1+Tim-3+ and CD8+PD-1+Tim-3+ T-cells and IL-10 levels measured by ELISA were significantly higher and CD4+PD-1-Tim-3- and CD8+PD-1-Tim-3- T-cells were significantly lower in patients than in controls. Treatment resulted in significant decrease of CD4+Tim-3+, CD8+Tim-3+, CD4+PD-1+Tim-3+ and CD8+PD-1+Tim-3+ T-cell frequencies as well as IL-10 levels and increase in CD4+PD-1-Tim-3- and CD8+PD-1-Tim-3- T-cells. There were no significant changes in the frequencies of CD4+PD-1+ T-cells, while CD8+PD-1+ T-cells increased. Patients with advanced liver fibrosis had higher PD-1 and lower Tim-3 expression on CD4+T-cells and treatment had little or no effect on the exhaustion markers. HCV-specific CD8+T-cells frequency has declined significantly after treatment, but their PD-1 and Tim-3 expression did not change. Successful treatment of chronic hepatitis C with DAA is associated with reversal of immune exhaustion phenotype, but this effect is absent in patients with advanced liver fibrosis.

Search for Viral Infections in Cerebrospinal Fluid From Patients With Autoimmune Encephalitis.

BACKGROUND: It has been reported that virus-mediated brain tissue damage can lead to autoimmune encephalitis (AE) characterized by the presence of antibodies against neuronal surface antigens. In the study, we investigate the presence of viruses in cerebrospinal fluid (CSF) from patients with AE using reverse transcription polymerase chain reaction (RT-PCR)/PCR and shotgun metagenomics. METHODS: CSF samples collected from 200 patients with encephalitis were tested for the presence of antibodies against antiglutamate receptor (NMDAR), contactin-associated protein 2 (CASPR2), glutamate receptors (type AMPA1/2), leucine-rich glioma-inactivated protein 1 (LGI1), dipeptidyl aminopeptidase-like protein 6 (DPPX), and GABA B receptor, and those found positive were further analyzed with real-time RT-PCR/PCR for common viral neuroinfections and shotgun DNA- and RNA-based metagenomics. RESULTS: Autoantibodies against neuronal cells were detected in CSF from 8 individuals (4% of all encephalitis patients): 7 (3.5%) had anti-NMDAR and 1 (0.5%) had anti-GABA B. RT-PCR/PCR identified human herpes virus type 1 (HSV-1; 300 copies/mL) and the representative of Enterovirus genus (550 copies/mL) in 1 patient each. Torque teno virus (TTV) was found in another patient using metagenomic analysis, and its presence was confirmed by specific PCR. CONCLUSIONS: We detected the presence of HSV, TTV, and Enterovirus genus in CSF samples from 3 out of 8 AE patients. These findings support the concept of viral involvement in the pathogenesis of this disease.

Search for viral agents in cerebrospinal fluid in patients with multiple sclerosis using real-time PCR and metagenomics.

Multiple sclerosis (MS) is a chronic, immune-mediated demyelinating disease of the central nervous system of unclear etiology, but there is some evidence that viral infections could be responsible for triggering autoimmune mechanisms against myelin. We searched for viral RNA and DNA in cerebrospinal fluid (CSF) of 34 MS patients and 13 controls using RT-PCR/PCR against common neurotropic viruses. In addition, shotgun DNA- and RNA-based metagenomics were done in 13 MS patients and 4 controls. Specific quantitative real-time RT-PCR/PCR testing revealed the presence of viral nucleic acid in seven (20.59%) MS patients and in one (7.69%) control patient. In MS patients the most frequently detected was human herpesvirus type 6 (HHV-6; 3 cases; 8.82%); followed by Epstein-Barr virus (EBV; 2 cases; 5.88%), varicella zoster virus (VZV; 1 case; 2.94%) and Enterovirus (EV; 1 case; 2.94%). The single identified virus among controls was EBV (7.69%). DNA and RNA metagenomic assays did not identify any known eukaryotic viruses even though three of the analyzed samples were low-level positive by specific quantitative real-time PCR. In conclusion, we detected the presence of Herpesviridae and occasionally Enteroviridae in CSF from patients with MS but their prevalence was not significantly higher than among controls. Metagenomic analysis seems to be less sensitive than real-time RT-PCR/PCR and it did not detect any potential viral pathogens.

Hepatitis C virus (HCV) genotype 1b displays higher genetic variability of hypervariable region 1 (HVR1) than genotype 3.

Hepatitis C virus (HCV) is characterized by high genetic variability, which is manifested both at the inter-host and intra-host levels. However, its role in the clinical course of infection is less obvious. The aim of the present study was to determine the genetic variability of HCV HVR1 (hypervariable region 1) of genotype 1b and 3 in plasma of blood donors in the early seronegative stage of infection (HCV-RNA+, anti-HCV-) and in samples from chronically infected patients using next-generation sequencing. Sequencing errors were corrected, and haplotypes inferred using the ShoRAH software. Genetic diversity parameters (intra-host number of variants, number of nucleotide substitutions and diversity per site) were assessed by DNA SP and MEGA. During the early infection, the number of variants were significantly lower in subjects infected with genotype 3 than with genotype 1b (p < 0.02). Similarly, intra-host number of variants, number of nucleotide substitutions and diversity per site were lower in genotype 3 chronic infection (p < 0.0005). In addition, early infection was characterized by significantly lower HVR1 variability values (p < 0.04) when compared to chronic infection for both genotypes. It seems that the observed differences in HVR1 variability represent an inherent property of particular viral genotypes.

Next-generation sequencing analysis of a cluster of hepatitis C virus infections in a haematology and oncology center.

Molecular characterization of early hepatitis C virus (HCV) infection remains rare. Ten out of 78 patients of a hematology/oncology center were found to be HCV RNA positive two to four months after hospitalization. Only two of the ten patients were anti-HCV positive. HCV hypervariable region 1 (HVR1) was amplified in seven patients (including one anti-HCV positive) and analyzed by next generation sequencing (NGS). Genetic variants were reconstructed by Shorah and an empirically established 0.5% variant frequency cut-off was implemented. These sequences were compared by phylogenetic and diversity analyses. Ten unrelated blood donors with newly acquired HCV infection detected at the time of donation (HCV RNA positive and anti-HCV negative) served as controls. One to seven HVR1 variants were found in each patient. Sequences intermixed phylogenetically with no evidence of clustering in individual patients. These sequences were more similar to each other (similarity 95.4% to 100.0%) than to those of controls (similarity 64.8% to 82.6%). An identical predominant variant was present in four patients, whereas other closely related variants dominated in the remaining three patients. In five patients the HCV population was limited to a single variant or one predominant variant and minor variants of less than 10% frequency. In conclusion, NGS analysis of a cluster of HCV infections acquired in the hospital setting revealed the presence of low diversity, very closely related variants in all patients, suggesting an early-stage infection with the same virus. NGS combined with phylogenetic analysis and classical epidemiological analysis could help in tracking of HCV outbreaks.

Viral etiologies in adult patients with encephalitis in Poland: A prospective single center study.

Encephalitis is a severe neurological syndrome associated with high morbidity and mortality as well as long-term neurological sequelae. Despite being an important public health problem, very few extensive population-based studies were conducted so far in the world and none in Central Europe. Altogether 114 consecutive patients meeting the initial criteria for encephalitis were enrolled at the Warsaw Hospital for Infectious Diseases between June 2012 and July 2015. Eighteen patients were secondarily excluded from the analysis due to incomplete data or noinfectious cause. Potential pathogen sequences were searched for by molecular methods in the cerebrospinal fluid (CSF) and specific antibodies were detected in CSF and sera. An infectious agent was identified in 41 patients (42.7%). The most frequently diagnosed infections were Human herpesvirus 1 (HHV-1) (22 cases, 24%) followed by Enterovirus (6 cases, 6.3%), Varicella zoster virus (VZV) (5 cases, 5.2%), Tick-borne encephalitis virus (TBEV) (6 cases, 6.3%) and Cytomegalovirus (CMV) (2 cases, 2.1%). There were no cases of human adenovirus, Human herpesvirus 6 (HHV-6) or West Nile virus (WNV) infection identified. In 55 cases (57.3%) the cause of encephalitis remained unknown. Compared to patients in whom the diagnosis was determined the latter group contained more women, was less likely to manifest fever and had lower CSF pleocytosis (p < 0.05) In summary, we identified HHV-1 followed by Enterovirus, VZV and TBEV as the most common causes of encephalitis among adult patients in Poland. In a large proportion of patients the cause of encephalitis remained unknown.