Hepatitis B virus particles in serum contain minus strand DNA and degraded pregenomic RNA of variable and inverse lengths.

This study utilized digital PCR to quantify HBV RNA and HBV DNA within three regions of the HBV genome. Analysis of 75 serum samples from patients with chronic infection showed that HBV RNA levels were higher in core than in S and X regions (median 7.20 vs. 6.80 and 6.58 log copies/mL; p < .0001), whereas HBV DNA levels showed an inverse gradient (7.71 vs. 7.73 and 7.77 log copies/mL, p < .001). On average 80% of the nucleic acid was DNA by quantification in core. The core DNA/RNA ratio was associated with viral load and genotype. In individual patients, the relations between RNA levels in core, S and X were stable over time (n = 29; p = .006). The results suggest that pregenomic RNA is completely reverse transcribed to minus DNA in ≈75% of the virus particles, whereas the remaining 25% contain both RNA and DNA of lengths that reflect variable progress of the polymerase.

Enrichment reveals extensive integration of hepatitis B virus DNA in hepatitis delta-infected patients.

INTRODUCTION: Hepatitis B virus (HBV) DNA may become integrated into the human genome of infected human hepatocytes. Expression of integrations can produce the surface antigen (HBsAg) that is required for synthesis of hepatitis D virus (HDV) particles and the abundant subviral particles in the blood of HBV- and HDV-infected subjects. Knowledge about the extent and variation of HBV integrations and impact on chronic HDV is still limited. METHODS: We investigated 50 pieces of liver explant tissue from five patients with hepatitis D-induced cirrhosis, using a deep sequencing strategy targeting HBV RNA. RESULTS: We found that integrations were abundant and highly expressed, however with large variation in number of integration derived (HBV/human chimeric) reads, both between and within patients. The median number of unique integrations for each patient correlated with serum levels of both HBsAg. Still, most of the HBV reads represented a few predominant integrations. CONCLUSIONS: The results suggest that HBV DNA integrates in a large proportion of hepatocytes, and that the HBsAg output from these integrations vary >100-fold depending on clone size and expression rate. A small part of the integrations seems to determine the serum levels of HBsAg and HDV RNA in HBV/HDV co-infected patients with liver cirrhosis.

Detection of drugs and hepatitis C virus in used syringes from a needle exchange in Gothenburg, Sweden.

People who inject drugs (PWID) are exposed to serious health risks such as lethal overdoses, addiction and infections. The patterns of drug use and the prevalence of hepatitis C virus (HCV) infection vary greatly between and even within countries. Data on drugs used for injection are important to inform PWID of risks and adapt healthcare. This study aimed to determine which substances are injected in Gothenburg, Sweden, and estimate the risk of HCV transmission. A total of 150 syringes handed in at the needle and syringe exchange program (NEP) in Gothenburg over a week in November 2021 were analysed for drug content using liquid chromatography coupled with high-resolution mass spectrometry. Using a dose-adjusted comparison, the main drug(s) injected was distinguished from the impurities in the syringes containing several drugs. HCV RNA was quantified by real-time PCR in an additional set of 150 syringes. Drugs were detected in >99% of analysed syringes, and the most common drugs were amphetamine (81%), followed by buprenorphine (8.0%), heroin (6.7%) and alprazolam (4.6%). Less common findings were testosterone (2.7%), methylphenidate (2.0%), MDMA (0.7%), trenbolone (0.7%) and zopiclone (0.7%). Eleven syringes (7.3%) contained more than one drug. HCV RNA was detected in 13% of the syringes, and one in 10 contained enough to potentially transmit an infection. This study underlines the importance of access to NEPs for PWID to reduce the risks associated with drug injection.

GaMF1.39's antibiotic efficacy and its enhanced antitubercular activity in combination with clofazimine, Telacebec, ND-011992, or TBAJ-876.

IMPORTANCE: New drugs are needed to combat multidrug-resistant tuberculosis. The electron transport chain (ETC) maintains the electrochemical potential across the cytoplasmic membrane and allows the production of ATP, the energy currency of any living cell. The mycobacterial engine F-ATP synthase catalyzes the formation of ATP and has come into focus as an attractive and rich drug target. Recent deep insights into these mycobacterial F1FO-ATP synthase elements opened the door for a renaissance of structure-based target identification and inhibitor design. In this study, we present the GaMF1.39 antimycobacterial compound, targeting the rotary subunit γ of the biological engine. The compound is bactericidal, inhibits infection ex vivo, and displays enhanced anti-tuberculosis activity in combination with ETC inhibitors, which promises new strategies to shorten tuberculosis chemotherapy.

Rifaximin potentiates clarithromycin against Mycobacterium abscessus in vitro and in zebrafish.

Background: Mycobacterium abscessus is a non-tuberculous mycobacterium (NTM) that causes chronic pulmonary infections. Because of its extensive innate resistance to numerous antibiotics, treatment options are limited, often resulting in poor clinical outcomes. Current treatment regimens usually involve a combination of antibiotics, with clarithromycin being the cornerstone of NTM treatments. Objectives: To identify drug candidates that exhibit synergistic activity with clarithromycin against M. abscessus. Methods: We performed cell-based phenotypic screening of a compound library against M. abscessus induced to become resistant to clarithromycin. Furthermore, we evaluated the toxicity and efficacy of the top compound in a zebrafish embryo infection model. Results: The screen revealed rifaximin as a clarithromycin potentiator. The combination of rifaximin and clarithromycin was synergistic and bactericidal in vitro and potent in the zebrafish model. Conclusions: The data indicate that the rifaximin/clarithromycin combination is promising to effectively treat pulmonary NTM infections.

Thermomechanical Simulation of Orthogonal Metal Cutting with PFEM and SPH Using a Temperature-Dependent Friction Coefficient: A Comparative Study.

In this work, we apply the Particle Finite Element Method (PFEM) and Smoothed Particle Hydrodynamics (SPH) to simulate the orthogonal cutting chip formation of two workpiece materials, i.e., AISI 1045 steel and Ti6Al4V titanium alloy. A modified Johnson-Cook constitutive model is used to model the plastic behavior of the two workpiece materials. No damage or strain softening is included in the model. The friction between the workpiece and the tool is modeled following Coulomb's law with a temperature-dependent coefficient. The accuracy of PFEM and SPH in predicting thermomechanical loads at various cutting speeds and depths against the experimental data are compared. The results show that both numerical methods can predict the rake face temperature of AISI 1045 with errors less than 34%. For Ti6Al4V, however, the temperature prediction errors are significantly higher than those of the steel alloy. Errors in force prediction were in the range of 10% to 76% for both methods, which compare very well with those reported in the literature. This investigation infers that the Ti6Al4V behavior under machining conditions is difficult to model on the cutting scale irrespective of the choice of numerical method.

Linkage to hepatitis C treatment in two opioid substitution treatment units in Gothenburg, Sweden: a retrospective cohort study.

BACKGROUND: Chronic infection with the hepatitis C virus (HCV) is common in people with former or current injection drug use. Among the patients in the opioid substitution treatment (OST) program in Gothenburg, Sweden, more than 50% had been infected with HCV. However, many patients did not have any follow-up for their infection and the linkage to treatment could be improved. METHODS: A model of care for HCV was introduced at an OST unit in Gothenburg, Sweden, in 2017. The aim was to increase testing and linkage to HCV treatment. A nurse and a medical doctor, both specialized in infectious diseases, performed on-site testing at the OST unit with transient liver elastography (Fibroscan) to evaluate the fibrosis stage and initiated HCV treatment. This study retrospectively reviewed the patients' medical records to assess information regarding participation in the model of care, hepatitis C status, linkage to treatment and treatment outcome. RESULTS: Among the 225 patients enrolled in OST at baseline, 181 were still in the OST program at the end of study (December 31st, 2018). In total, 29 patients, most of whom did not attend the Clinic of Infectious Diseases, were referred to the model of care. By the end of study, 17 patients (100% of those treated) reached sustained virologic response. In parallel, an additional 19 patients got treatment directly at the Clinic of Infectious Diseases. CONCLUSION: Integrating HCV screening and examination in an OST unit successfully linked patients to treatment. However, not all patients received treatment. To reach the goal of eliminating HCV, different models of care are needed.

Long-term self-reported health and disability after COVID-19 in public employees.

BACKGROUND: Although the symptomatology has been assessed in multiple studies among persons recovering from coronavirus disease 2019 (COVID-19), less is known regarding long-term general health and disability. We aimed to assess long-term self-reported disability in public employees after predominantly mild COVID-19 in comparison with individuals who had negative COVID-19 polymerase chain reaction (PCR) test results. METHODS: Public employees within Region Västra Götaland were offered tests to identify SARS-CoV-2 infection (n = 56,221) and were invited to complete an online survey that included the World Health Organization Disability Assessment Schedule. Questionnaires were sent out between January 26 and March 5, 2021. A total of 14,222 (25.3%) employees responded, of which 10,194 (18%) were included (women n = 8749, 85.8%). Of these, 7185 (70.5%) participants had a negative PCR test result (controls). A total of 1425 (14%) had a positive PCR result and were categorized in the sub-acute phase (4-12 weeks post COVID-19), and 1584 (15.5%) had a positive PCR test and were categorized in the post COVID-19 phase (> 12 weeks). RESULTS: Fifty-two percent of controls rated disability of varying degrees, versus 73% and 64% of participants in the sub-acute and post COVID-19 phase, respectively. Being "emotionally affected" was the most frequently reported disability in the sub-acute phase, the post COVID-19 phase, as well as in controls. The proportion of participants reporting difficulties for 20-30 days was higher in the sub-acute phase than in the post COVID-19 phase (27.9% vs. 21.8%, p < 0.001) as well as in a comparison between participants in the post COVID-19 phase and controls (21.8% vs 14.2%, p < 0.001). Compared with controls, severe disability was more common in the post COVID-19 phase among both women (15.8% vs. 10.7%,), and men (9.8% vs. 6.8%). CONCLUSIONS: Disability was present in all groups; however, reported disability was greater in the sub-acute phase than in the post COVID-19 phase. The higher levels of disability reported in the COVID-19 patient population may indicate a persisting need for rehabilitation and recovery. In general, women reported a greater degree of disability than men in the sub-acute and post COVID-19 phases.

Self-reported symptom severity, general health, and impairment in post-acute phases of COVID-19: retrospective cohort study of Swedish public employees.

This study aimed to examine current symptom severity and general health in a sample of primarily non-hospitalized persons with polymerase chain reaction (PCR) confirmed COVID-19 in comparison to PCR negative controls. During the first quarter of 2021, we conducted an online survey among public employees in West Sweden, with a valid COVID-19 test result. The survey assessed past-month severity of 28 symptoms and signs, self-rated health, the WHO Disability Assessment Schedule (WHODAS) 2.0 and illness severity at the time of test. We linked participants' responses to their SARS-CoV-2 PCR tests results. We compared COVID-19 positive and negative participants using univariable and multivariable regression analyses. Out of 56,221 invited, 14,222 (25.3%) responded, with a response rate of 50% among SARS-CoV-2 positive individuals. Analysis included 10,194 participants (86.4% women, mean age 45 years) who tested positive 4-12 weeks (N = 1425; subacute) and > 12 weeks (N = 1584; postcovid) prior to the survey, and 7185 PCR negative participants who did not believe that they had had COVID-19. Symptoms were highly prevalent in all groups, with worst symptoms in subacute phase participants, followed by postcovid phase and PCR negative participants. The most specific symptom for COVID-19 was loss of smell or taste. Both WHODAS 2.0 score and self-rated health were worst in subacute participants, and modestly worse in postcovid participants than in negative controls. Female gender, older age and acute illness severity had larger effects on self-rated health and WHODAS 2.0 score in PCR positive participants than in PCR negative. Studies with longer follow-up are needed to determine the long-term improvement after COVID-19.

Epidemiology and clinical manifestations of different enterovirus and rhinovirus types show that EV-D68 may still have an impact on severity of respiratory infections.

Respiratory infections are often caused by enteroviruses (EVs). The aim of this study was to identify whether certain types of EV were more likely to cause severe illness in 2016, when an increasing spread of upper respiratory infections was observed in Gothenburg, Sweden. The EV strain in 137 of 1341 nasopharyngeal samples reactive for EV by polymerase chain reaction could be typed by sequencing the viral 5'-untranslated region and VP1 regions. Phylogenetic trees were constructed. Patient records were reviewed. Hospital care was needed for 46 of 74 patients with available medical records. The majority of the patients (83) were infected with the rhinovirus (RV). The remaining 54 were infected with EV A, B, C, and D strains of 13 different types, with EV-D68 and CV-A10 being the most common (17 vs. 14). Significantly more patients with EV-D68 presented with dyspnea, both when compared with other EV types (p = 0.003) and compared to all other EV and RV infections (p = 0.04). Phylogenetic analysis of the sequences revealed the spread of both Asian and European CV-A10 strains and 12 different RV C types. This study showed an abundance of different EV types spreading during a year with increased upper respiratory increased infections. EV-D68 infections were associated with more severe disease manifestation. Other EV and RV types were more evenly distributed between hospitalized and nonhospitalized patients. The EV type CV-A10 was also found in infected patients, which warrants further studies and surveillance, as this pathogen could cause more severe disease and outbreaks of hand, foot, and mouth disease.

Abundance of Noncircular Intrahepatic Hepatitis B Virus DNA May Reflect Frequent Integration Into Human DNA in Chronically Infected Patients.

BACKGROUND: Hepatitis B virus (HBV) integration has implications for cancer development and surface antigen (HBsAg) production, but methods to quantify integrations are lacking. The aim of this study was to develop a droplet digital PCR (ddPCR) assay discriminating between circular and integrated HBV DNA, and to relate the distribution between the two forms to other HBV markers. METHODS: ddPCR with primers spanning the typical linearization breakpoint in the HBV genome allowed for quantification of the absolute copy numbers of total and circular HBV DNA, and calculation of linear HBV DNA. RESULTS: Analysis of 70 liver biopsies from patients with chronic HBV infection revealed that the fraction of linear HBV DNA, which includes integrations, was higher in HBeAg-negative patients than HBeAg-positive. The ratio between HBsAg and HBV DNA levels in serum correlated with the intrahepatic proportion of linear HBV DNA. Furthermore, ddPCR experiments on serum samples and experiments with nuclease indicated the contribution of encapsidated double-stranded linear DNA and replication intermediates to be limited. CONCLUSIONS: The degree of integration of intrahepatic HBV DNA in the HBeAg-negative stage may be higher than previously anticipated, and integrated DNA may explain the persistence of high HBsAg serum levels in patients with low HBV DNA levels.

Deep sequencing of hepatitis B virus using Ion Torrent fusion primer method.

BACKGROUND: Hepatitis B virus (HBV) infection is worldwide a major cause of liver cirrhosis and hepatocellular carcinoma. Thousands of years ago, several HBV genotypes (A-I) evolved and have, as a result of human migration, become globally disseminated. Sequencing of HBV is used for genotyping, and investigation of outbreaks or of antiviral resistance. The present study describes a simplified deep sequencing of the whole HBV genome. METHODS: Sequencing by Ion Torrent was evaluated and its performance compared with Sanger sequencing on clinical samples. RESULTS: Amplification of overlapping segments spanning the entire HBV genome was successful at HBV DNA levels in serum as low as 100 IU/mL. The use of primers carrying adapter tags generated libraries without the need for fragmentation and ligation steps, and inclusion of barcode sequences allowed parallel analysis of multiple samples. A streamlined bioinformatic platform generated consensus sequences and superior mutation assessment as compared with Sanger sequencing, with which there was a 99.8 % average agreement. CONCLUSION: Deep sequencing of the whole HBV genome by using PCR primers tagged with adapters that prepare overlapping amplicons for Ion Torrent analysis was efficient and accurate.

Quantification of Viral RNA in Multiple Pieces of Explant Liver Tissue Shows Distinct Focal Differences in Hepatitis B Infection.

Hepatitis B virus (HBV) DNA and RNA were quantified by digital PCR assays in 20-30 tissue pieces from each of 4 liver explants with cirrhosis caused by HBV. The within-patient variability of HBV RNA levels between pieces was up to a 1000-fold. Core RNA and S RNA levels were similar and correlated strongly when replication was high, supporting that transcription was from covalently closed circular DNA (cccDNA). By contrast, enhanced expression of S RNA relative to cccDNA and core RNA in patients with medium-high or low replication supports that HBV surface antigen (HBsAg) can be expressed mainly from integrated HBV DNA in such patients.

Novel Methodology for Experimental Characterization of Micro-Sandwich Materials.

Lightweight components are in demand from the automotive industry, due to legislation regulating greenhouse gas emissions, e.g., CO2. Traditionally, lightweighting has been done by replacing mild steels with ultra-high strength steel. The development of micro-sandwich materials has received increasing attention due to their formability and potential for replacing steel sheets in automotive bodies. A fundamental requirement for micro-sandwich materials to gain significant market share within the automotive industry is the possibility to simulate manufacturing of components, e.g., cold forming. Thus, reliable methods for characterizing the mechanical properties of the micro-sandwich materials, and in particular their cores, are necessary. In the present work, a novel method for obtaining the out-of-plane properties of micro-sandwich cores is presented. In particular, the out-of-plane properties, i.e., transverse tension/compression and out-of-plane shear are characterized. Test tools are designed and developed for subjecting micro-sandwich specimens to the desired loading conditions and digital image correlation is used to qualitatively analyze displacement fields and fracture of the core. A variation of the response from the material tests is observed, analyzed using statistical methods, i.e., the Weibull distribution. It is found that the suggested method produces reliable and repeatable results, providing a better understanding of micro-sandwich materials. The results produced in the present work may be used as input data for constitutive models, but also for validation of numerical models.

Hepatitis B Virus RNA Profiles in Liver Biopsies by Digital Polymerase Chain Reaction.

Replication of hepatitis B virus (HBV) originates from covalently closed circular DNA (cccDNA) and involves reverse transcription of pregenomic RNA (pgRNA), which is also called core RNA and encodes the capsid protein. The RNA coding for hepatitis B surface antigen (HBsAg) in the envelope of viral or subviral particles is produced from cccDNA or from HBV DNA integrated into the host genome. Because only cccDNA can generate the core and the 3' redundancy regions of HBV RNA, we aimed to clarify to what extent such HBV integrations are expressed by quantifying the different HBV RNA species in liver tissue. Digital droplet polymerase chain reaction (ddPCR) was employed to quantify six HBV RNA targets in 76 liver biopsies from patients with chronic infection, comprising 14 who were hepatitis B e antigen (HBeAg) positive and 62 who were HBeAg negative. In patients who were HBeAg negative, HBV RNA from the S RNA region was >1.6 log10 units higher than in the core and 3' redundancy regions (P < 0.0001), indicating that >90% of S RNA was integration derived. HBeAg-negative samples showed 10 times lower levels of pgRNA (5' core) compared with core RNA (3' part of core; P < 0.0001), suggesting that a large proportion of core RNA might have a downstream shift of the transcription starting point. In multiple regression analysis, HBV DNA levels in serum were most strongly dependent on pgRNA. Conclusion: In patients who were HBeAg negative, integration-derived S RNA seemed to predominate and a large proportion of the core RNA lacked the 5' part. Because this part comprises the down-regulator of transcription 1 sequences, which are necessary for virus production (plus strand translocation), the finding might help to explain the low level of HBV DNA in serum that frequently is observed in patients with chronic HBV infection who are HBeAg negative.