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.

Occult hepatitis B infection in children born to HBeAg-positive women confers a low long-term risk for HBsAg-positive infection.

PURPOSE: Mother-to-child transmission (MTCT) has been the main cause of chronic hepatitis B virus (HBV) infection, particularly in East Asia. Hepatitis B immunoglobulin (HBIG) and vaccination given directly after birth effectively prevents hepatitis B surface antigen (HBsAg)-positive (overt) HBV infection, but occult hepatitis B infection (OBI) may develop despite adequate prophylaxis. The aim of this study was to investigate the long-term outcome in children born to mothers with very high HBV DNA levels with special focus on children discovered in early childhood with OBI. METHODS: One-year and long-term outcome regarding overt and occult HBV infection were analysed in 66 children born to hepatitis B e antigen (HBeAg)-positive mothers, and were compared with one-year outcome in 69 children born to HBeAg-negative mothers. The children were born between 1998 and 2018. RESULTS: Six children born to HBeAg-positive mothers developed overt chronic HBV infection, in two cases after normal pregnancies and despite HBIG and vaccination, but never when nucleotide analogue treatment was given during pregnancy. OBI with HBV DNA detected in serum in the absence of surface antigen (HBsAg) was observed in four children at the age of 1 year. One of them was transiently HBsAg-positive at the age of 7 years. At long-term follow-up, six children had overt chronic infection, one had OBI and six had previous OBI or positive anti-HBc suggesting resolved unidentified infections. CONCLUSION: The results indicate that children born to mothers with high HBV DNA levels have approximately 10% risk to develop OBI despite antiviral treatment, vaccination and HBIG, but that such OBI confers a minimal long-term risk for overt infection, at least in immunocompetent children.

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.

Complex norovirus transmission dynamics at hospital wards revealed by deep sequencing.

Detailed knowledge regarding norovirus transmission within hospitals is limited. We investigated a norovirus hospital outbreak affecting 65 patients at five different wards. PCR showed that 61 (94%) of the patients were infected with genotype II.4 strains. Successful Ion Torrent deep sequencing of GII.4 positive samples from 59 patients followed by phylogenetic analysis revealed that all sequences but two clustered into four distinct clades. Two of the clades belonged to GII.4 Sydney 2012, while the other two belonged to GII.4 New Orleans 2009. One of the clades was predominant at two wards, while two clades were predominant at one ward each. The fourth clade was found in sporadic cases at several wards. Thus, at four out of five wards, variants from one clade were predominant. At one ward, a single clade accounted for all cases, while at three wards the predominant clade accounted for 60%-71% of cases. Analysis of quasispecies variation identified positions that could further discriminate between variants from separate wards. The results illustrate a complex transmission of healthcare-associated norovirus infections and show that sequencing can be used to discriminate between related and unrelated cases.

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.

Women´s experiences of preeclampsia as a condition of uncertainty: a qualitative study.

BACKGROUND: Preeclampsia is a severe condition that annually affects about 3-8% of pregnancies worldwide. Preeclampsia is thereby one of the most common pregnancy complications for both mother and child. Despite that, there is limited research exploring the women´s perspective of experiencing preeclampsia. AIM: The aim of this study was to describe women´s experiences of preeclampsia to improve the support and care given during and after pregnancy. METHODS: A qualitative descriptive interview study was undertaken. Nine women, diagnosed with preeclampsia, were recruited from a maternity unit in southern Sweden. The descriptive phenomenological method according to Amadeo Giorgi was used to analyse the data. RESULTS: The women´s experiences of PE were expressed as A condition of uncertainty, meaning that it was an unexpected and unknown situation. This main result consisted of 1) incomprehensible diagnosis message, 2) ambivalent feeling when the unexpected happens, 3) confusing contradictory messages, 4) appreciated support from the midwife, 5) need for continuous information. The nature of preeclampsia can sometimes deteriorate rapidly both for the mother and/or the child, often resulting in conversion from a planned vaginal spontaneous delivery to an emergency Caesarean section. The women narrated diffuse symptoms, and they experienced that they got contradictory information from different health care professionals regarding the severity of their disease. Detailed and continuous information is requested throughout the course of the disease, and the postpartum period. CONCLUSION: This qualitative study reveal a need for improved clinical management. Health care professionals must be aware that women and their partners need detailed, consistent and repeated information about severity and prognosis to diminish the condition of uncertainty, confusion and fearful experience. The clinical implication would be a standardized preeclampsia education for pregnant women early on in the pregnancy, to raise awareness of preeclamptic symptoms. Furthermore, there is a need for harmonized guidelines and individualized support to the woman and her partner both at the antenatal care and the maternity ward and inpatient care at the hospital.

Being in the shadow of the unknown - Swedish women's lived experiences of pregnancy during the COVID-19 pandemic, a phenomenological study.

BACKGROUND: The COVID-19 pandemic has had a profound effect on the emotional well-being of expecting mothers. Sweden's unique strategy for managing COVID-19 involved no national lockdown. Emphasis was instead placed on limiting crowding and asking citizens to practice social distancing measures. AIM: To gain a deeper understanding of how women not infected by SARS-CoV-2 experienced pregnancy during the COVID-19 pandemic in Sweden. METHODS: This was a qualitative study with a reflective lifeworld approach. Fourteen women that had not contracted COVID-19 and who were pregnant during the first and second wave of the pandemic were interviewed. Data were analysed with a phenomenological reflective lifeworld approach. FINDINGS: The essence of the women's experiences of being pregnant during the COVID-19 pandemic was best described as being in the shadow of the unknown, where the COVID-19 pandemic could at times totally overshadow the experience of being pregnant, while at other times, rays of sunlight pierced through the clouds. The experience was characterised by having to deal with the uncertainties caused by the pandemic and feelings of being in an information echo. Women felt socially isolated and had to face maternal check-ups without the support of their partners. There was, however, a strong trust in maternal health-care services despite the lack of information available. CONCLUSION: Being in the shadow of the unknown represents the uncertainties posed by the COVID-19 pandemic on the experience of pregnancy. Sufficient information, a companion of choice and screening for emotional well-being are important factors in maternity care during pandemics.

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.

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.

The experience of provided information and care during pregnancy and postpartum when diagnosed with preeclampsia: A qualitative study.

INTRODUCTION: Despite preeclampsia being one of the most severe obstetrical complications there is only scant research describing women's experiences of preeclampsia. The aim of this study was to explore women's experience during pregnancy and the postpartum period regarding the provided information and care concerning preeclampsia. METHODS: A qualitative study was designed. Semi-structured face-to-face interviews were performed with fifteen women who were diagnosed with preeclampsia and included at two maternity units located in southern Sweden. The material was analyzed using content analysis. RESULTS: Suffering from preeclampsia was understood as being stressful, illustrated in four themes: fragmented information, lack of care planning, separation postpartum, and overall stress and worry. CONCLUSIONS: The women experienced fragmented obstetrical care and information deficits when diagnosed with preeclampsia. Our findings indicate a need for additional support and professional guidance due to increased stress, worry, and despair of being separated from the newborn. Future research investigating specific care-planning and postpartum follow-up are suggested as steps to improve care for women with a pregnancy complicated by preeclampsia.

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.

Deep sequencing of liver explant transcriptomes reveals extensive expression from integrated hepatitis B virus DNA.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). Integration of HBV DNA into the human genome may contribute to oncogenesis and to the production of the hepatitis B surface antigen (HBsAg). Whether integrations contribute to HBsAg levels in the blood is poorly known. Here, we characterize the HBV RNA profile of HBV integrations in liver tissue in patients with chronic HBV infection, with or without concurrent hepatitis D infection, by transcriptome deep sequencing. Transcriptomes were determined in liver tissue by deep sequencing providing 200 million reads per sample. Integration points were identified using a bioinformatic pipeline. Explanted liver tissue from five patients with end-stage liver disease caused by HBV or HBV/HDV was studied along with publicly available transcriptomes from 21 patients. Almost all HBV RNA profiles were devoid of reads in the core and the 3' redundancy (nt 1830-1927) regions, and contained a large number of chimeric viral/human reads. Hence, HBV transcripts from integrated HBV DNA rather than from covalently closed circular HBV DNA (cccDNA) predominated in late-stage HBV infection, in particular in cases with hepatitis D virus co-infection. The findings support the suggestion that integrated HBV DNA can be a significant source of HBsAg in humans.

Cardiovascular effects of severe late-onset preeclampsia are reversed within six months postpartum.

OBJECTIVES: Preeclampsia (PE) is a common pregnancy-related disorder associated with cardiovascular long-term disease. Eighty percent are late-onset PE, occurring after 34 gestational weeks, and can present with severe symptoms. Magnitude and reversibility rate of maternal cardiovascular changes after severe late-onset PE have not been characterized. This study therefore evaluated longitudinal dynamics of maternal cardiovascular changes after severe late-onset PE. STUDY DESIGN: Six previously normotensive women with severe late-onset PE and eight pregnant controls were included. Severe PE was defined as systolic blood pressure (SBP) ≥ 160 mmHg or diastolic blood pressure (DBP) ≥ 110 mmHg and proteinuria with/without evidence of end-organ dysfunction, or SBP ≥ 140 mmHg or DBP ≥ 90 mmHg with/without proteinuria and with evidence of end-organ dysfunction. Cardiovascular function was assessed by magnetic resonance imaging at 1-3 days, one week and six months postpartum. RESULTS: Left ventricular mass at 1-3 days postpartum was higher after severe late-onset PE (57 g/m2) compared to after normal pregnancy (48 g/m2; p = 0.01). Pulse wave velocity (PWV) decreased between 1 and 3 days and six months postpartum after PE (6.1 to 5.0 m/s; p = 0.028). There was no difference in PWV 1-3 days postpartum after severe PE compared after normal pregnancy (6.1 versus 5.6 m/s; p = 0.175). Blood pressure normalized within six months in all but one patient. CONCLUSIONS: Cardiac effects after severe late-onset PE were small and transient. This indicates that left ventricular hypertrophy after severe late-onset PE may be a secondary physiologic response to increased peripheral resistance in PE. Vascular mechanisms rather than persistent cardiac hypertrophy postpartum may be the culprit for increased long-term cardiovascular risk after PE.

IFNL4 Genotypes Predict Clearance of RNA Viruses in Rwandan Children With Upper Respiratory Tract Infections.

Polymorphisms in the interferon lambda gene locus (IFNL) such as the IFNL4 genetic variants rs12979860 and rs368234815 are predictive of resolution of hepatitis C virus infection, but information about the impact of these variants in other infections is scarce. This study aimed at determining the potential impact of IFNL4 variation for the clearance of respiratory tract pathogens in Rwandan children (≤5 years old, n = 480) seeking medical care for acute respiratory infections. Nasopharyngeal swabs were retrieved from all children at the first hospital referral and from 161 children at follow-up visits 2 weeks later. The swabs were analyzed for pathogens by real-time PCR and for host cell IFNL4 genotype at rs12979860 and rs368234815. Approximately 1/3 of the children were homozygous for the rs12979860 T allele and the rs368234815 ΔG allele, which are overrepresented in subjects of African descent. These IFNL4 variants were significantly associated with reduced clearance of RNA viruses. Our results suggest that IFNL4 genotypes that are common among subjects of African descent may determine inefficacious clearance of RNA viruses from the respiratory tract.

Comparison of the FilmArray assay and in-house real-time PCR for detection of respiratory infection.

Recently, molecular methods capable of detecting almost all microbial agents that may cause acute respiratory infection have been introduced. The FilmArray Respiratory Panel assay, which integrates nucleic acid extraction, nested amplification and detection in a reaction pouch preloaded with all reagents required for detection of 17 viruses and 3 bacteria, was compared with an in-house real-time PCR that detects these agents in 8 parallel amplifications. When 128 clinical samples representing 18 of these agents were analysed by both assays the agreement was excellent, with kappa values ranging between 0.54 and 1.0. Discordances were mainly observed for adenovirus, but not when version 1.7 of FilmArray was used. The results show that these assays detect a wide range of pathogens with similar performance. FilmArray provides results after approximately 1 h, including ≈ 5 min hands-on time, and does not require advanced equipment or expertise in molecular diagnostics, making it a useful point-of-care-test for acute respiratory infections.

Real-time PCR identification of agents causing diarrhea in Rwandan children less than 5 years of age.

BACKGROUND: Knowledge about causes of acute diarrhea among children in developing countries is insufficient. Molecular methods might improve diagnostics of infectious gastroenteritis, but due to the high sensitivity, findings may be difficult to interpret. METHODS: Feces samples from Rwandan children 0.5-5.0 years of age, with diarrhea for <96 hours (patients, n = 544) or without diarrhea for 14 days (controls, n = 162), were analyzed by real-time polymerase chain reaction targeting 17 pathogens. RESULTS: At least 1 agent was detected in 94% of patients and in 79% of controls, with higher rates in sick children for rotavirus (42% vs. 2%, P < 0.0001) and enterotoxigenic Escherichia coli (ETEC)-estA (21% vs. 9%, P = 0.0006). Detection rates did not differ significantly for adenovirus (39% vs. 36%), ETEC-eltB (29% vs. 30%), Campylobacter (14% vs. 17%) or Shigella (13% vs. 10%), but for Shigella the threshold cycle (Ct) values were lower (pathogen loads were higher) in sick children than in controls. By multivariate analysis, including gender and age, detection of rotavirus (P < 0.0001), ETEC-estA (P = 0.001), Shigella (P = 0.004) and norovirus genogroup II (P = 0.009) was associated with symptomatic infection, and a Ct value below a cutoff (in the range 28-29) improved identification of ETEC-estA, Shigella and norovirus genogroup II. CONCLUSION: Real-time polymerase chain reaction can detect essentially all diarrheagenic agents, and provides Ct values that improve identification of clinically relevant infections.