Hepatitis E virus infection of transplanted kidneys.

Immunocompromised patients are at risk of chronic hepatitis E (HEV) infection. Recurrent T cell and borderline rejections in a pediatric patient with high HEV copy numbers led us to study HEV infection within renal transplants. To investigate the frequency of renal HEV infection in transplanted patients, 15 samples from patients with contemporaneous diagnoses of HEV infection were identified at our center. Ten samples had sufficient residual paraffin tissue for immunofluorescence (IF) and RNA-fluorescence-in-situ-hybridization (RNA-FISH). The biopsy of the pediatric index patient was additionally sufficient for tissue polymerase chain reaction and electron microscopy. HEV RNA was detected in paraffin tissue of the index patient by tissue polymerase chain reaction. Subsequently, HEV infection was localized in tubular epithelial cells by IF, RNA-FISH, and electron microscopy. One additional biopsy from an adult was positive for HEV by RNA-FISH and IF. Focal IF positivity for HEV peptide was observed in 7 additional allografts. Ribavirin therapy was not successful in the pediatric index patient; after relapse, ribavirin is still administered. In the second patient, successful elimination of HEV was achieved after short-course ribavirin therapy. HEV infection is an important differential diagnosis for T cell rejection within transplanted kidneys. Immunostaining of HEV peptide does not necessarily prove acute infection. RNA-FISH seems to be a reliable method to localize HEV.

Hepatitis A outbreak in a refugee shelter in Kiel, northern Germany.

In the spring of 2023, three Ukrainian war refugees from a municipal community shelter and a volunteer caregiver at an affiliated daycare center in Kiel, Germany, were diagnosed with infectious jaundice attributable to a single hepatitis A virus (HAV) subgenotype IA strain. Similar HAV sequences have been observed in Germany and other European countries for several years. One refugee and the volunteer required hospitalization. Four children were asymptomatically infected but excreted high levels of HAV ribonucleic acid in the stool. The infections were probably acquired in Germany, but a source could not be determined. The outbreak was contained through vaccination, increased hygiene, and education. The existing HAV vaccination recommendation for refugee shelter staff and volunteers should be consistently implemented.

Immunomodulation of Natural Killer Cell Function by Ribavirin Involves TYK-2 Activation and Subsequent Increased IFN-γ Secretion in the Context of In Vitro Hepatitis E Virus Infection.

Hepatitis E virus (HEV) is a major cause of acute hepatitis globally. Chronic and fulminant courses are observed especially in immunocompromised transplant recipients since administration of ribavirin (RBV) does not always lead to a sustained virologic response. By in vitro stimulation of NK cells through hepatoma cell lines inoculated with a full-length HEV and treatment with RBV, we analyzed the viral replication and cell response to further elucidate the mechanism of action of RBV on immune cells, especially NK cells, in the context of HEV infection. Co-culture of HEV-infected hepatoma cells with PBMCs and treatment with RBV both resulted in a decrease in viral replication, which in combination showed an additive effect. An analysis of NK cell functions after stimulation revealed evidence of reduced cytotoxicity by decreased TRAIL and CD107a degranulation. Simultaneously, IFN-É£ production was significantly increased through the IL-12R pathway. Although there was no direct effect on the IL-12R subunits, downstream events starting with TYK-2 and subsequently pSTAT4 were upregulated. In conclusion, we showed that RBV has an immunomodulatory effect on the IL-12R pathway of NK cells via TYK-2. This subsequently leads to an enhanced IFN-É£ response and thus, to an additive antiviral effect in the context of an in vitro HEV infection.

Molecular epidemiology and genotype-specific disease severity of hepatitis E virus infections in Germany, 2010-2019.

Zoonotic hepatitis E virus (HEV) is endemic in Europe. Genotype 3 (HEV-3) is predominant but information on subtype distribution, trends and clinical implications in Germany is scarce. We analysed 936 HEV RNA positive samples of human origin and corresponding national surveillance data from 2010 to 2019. Samples were referred to the National Consultant Laboratory and sequenced in at least one of four genomic regions. Sequences were analysed using bioinformatics methods and compared to the latest HEV reference set. 1,656 sequences were obtained from 300 female, 611 male and 25 of unknown sex aged 3-92 years (median 55 years). HEV-3c was predominant (67.3%) followed by HEV-3f, HEV-3e and HEV-3i(-like) with 14.3%, 9.7% and 4.0% (other subtypes ≤1.1%). The proportion of HEV-3 group 2 (3abchijklm) strains increased over time. Jaundice, upper abdominal pain, fever, hospitalization, and death due to HEV were significantly more often reported for patients infected with HEV-3 group 1 (3efg) compared to group 2. Larger spatio-temporal clusters of identical sequences were not observed. HEV-3 group 1 infections are more severe as compared to the predominant group 2. Detection of group 2 strains increased over the last years, possibly due to more frequent diagnosis of asymptomatic and mild courses. The diversity of strains and the space-time distribution is compatible with a foodborne zoonosis with supra-regional distribution of the infection vehicle (pork products).

HuH-7-Lunet BLR Cells Propagate Rat Hepatitis E Virus (HEV) in a Cell Culture System Optimized for HEV.

The family Hepeviridae comprises the species Orthohepevirus A-D (HEV-A to -D). HEV-C genotype 1 (HEV-C1, rat HEV) is able to infect humans. This study investigated whether an optimized HEV-A cell culture system is able to propagate the cell culture-derived rat HEV, and if de novo isolation of the virus from rat liver is possible. We tested the liver carcinoma cell lines PLC/PRF/5, HuH-7, and HuH-7-Lunet BLR for their susceptibility to HEV-C1 strains. Cells were infected with the cell culture-derived HEV-C1 strain R63 and rat liver-derived strain R68. Cells were maintained in MEMM medium, which was refreshed every 3-4 days. The viral load of HEV-C1 was determined by RT-qPCR in the supernatant and expressed as genome copies per mL (c/mL). Rat HEV replication was most efficient in the newly introduced HuH-7-Lunet BLR cell line. Even if the rat HEV isolate had been pre-adapted to PLC/PRF/5 by multiple passages, replication in HuH-7-Lunet BLR was still at least equally effective. Only HuH-7-Lunet BLR cells were susceptible to the isolation of HEV-C1 from the liver homogenate. These results suggest HuH-7-Lunet BLR as the most permissive cell line for rat HEV. Our HEV-C1 cell culture system may be useful for basic research, the animal-free generation of large amounts of the virus as well as for the testing of antiviral compounds and drugs.

No Evidence for Orthohepevirus C in Archived Human Samples in Germany, 2000-2020.

Orthohepevirus C1, also known as rat hepatitis E virus (HEV), has been shown to sporadically cause disease in immunocompromised and immunocompetent adults. While routine serological assays vary in reactivity, rat HEV is not detected in routine HEV RT-PCR. Thus, such infections could be either missed or misclassified as conventional HEV (Orthohepevirus A) infections. We conducted a retrospective screening study among serum and plasma samples from patients suspected of having HEV infection, which were archived at the national consultant laboratory for HAV and HEV between 2000 and 2020. We randomly selected n = 200 samples, which were initially tested reactive (positive or borderline) for HEV-IgM and negative for HEV RNA and re-examined them using a highly sensitive Orthohepevirus C genotype 1-specific in-house RT-qPCR (LoD 95: 6.73 copies per reaction) and a nested RT-PCR broadly reactive for Orthohepevirus A and C. Conventional sanger sequencing was conducted for resulting PCR products. No atypical HEV strains were detected (0 of 200 [0.0%; 95% confidence interval: 0.0%-1.89%], indicating that Orthohepevirus C infections in the investigated population (persons with clinical suspicion of hepatitis E and positive HEV-IgM) are very rare.

Omicron's binding to sotrovimab, casirivimab, imdevimab, CR3022, and sera from previously infected or vaccinated individuals.

SARS-CoV-2 Omicron is the first pandemic variant of concern exhibiting an abrupt accumulation of mutations particularly in the receptor-binding domain that is a critical target of vaccination induced and therapeutic antibodies. Omicron's mutations did only marginally affect the binding of ACE2, and the two antibodies Sotrovimab and CR3022 but strongly impaired the binding of Casirivimab and Imdevimab. Moreover, as compared with Wuhan, there is reduced serum reactivity and a pronounced loss of competitive surrogate virus neutralization (sVN) against Omicron in naïve vaccinees and in COVID-19 convalescents after infection and subsequent vaccination. Finally, although the booster vaccination response conferred higher titers and better sVN, the effect was nonetheless significantly lower compared with responses against Wuhan. Overall, our data suggest that the antigenicity of Omicrons receptor binding motive has largely changed but antibodies such as Sotrovimab targeting other conserved sites maintain binding and therefore hold potential in prophylaxis and treatment of Omicron-induced COVID-19.

Phylogeny and spatiotemporal dynamics of hepatitis E virus infections in wild boar and deer from six areas of Germany during 2013-2017.

The hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. Infections with the zoonotic HEV genotype 3, which can be transmitted from infected wild boar and deer to humans, are increasingly detected in Europe. To investigate the spatiotemporal HEV infection dynamics in wild animal populations, a study involving 3572 samples of wild boar and three deer species from six different geographic areas in Germany over a 4-year period was conducted. The HEV-specific antibody detection rates increased between 2013-2014 and 2016-2017 in wild boar from 9.5% to 22.8%, and decreased in deer from 1.1% to 0.2%. At the same time, HEV-RNA detection rates increased in wild boar from 2.8% to 13.3% and in deer from 0.7% to 4.2%. Marked differences were recorded between the investigated areas, with constantly high detection rates in one area and new HEV introductions followed by increasing detection rates in others. Molecular typing identified HEV subtypes 3c, 3f, 3i and a putative new subtype related to Italian wild boar strains. In areas, where sufficient numbers of positive samples were available for further analysis, a specific subtype dominated over the whole observation period. Phylogenetic analysis confirmed the close relationship between strains from the same area and identified closely related human strains from Germany. The results suggest that the HEV infection dynamics in wild animals is dependent on the particular geographical area where area-specific dominant strains circulate over a long period. The virus can spread from wild boar, which represent the main wild animal reservoir, to deer, and generally from wild animals to humans.

Persisting olfactory dysfunction in post-COVID-19 is associated with gustatory impairment: Results from chemosensitive testing eight months after the acute infection.

Olfactory and gustatory disorders are prominent symptoms of acute COVID-19. Although both senses recover in many patients within weeks to months, persistency has been described in up to 60%. However up to now most reports on the course of chemosensitive disorders after COVID-19 are not based on psychophysical testing but only on subjective patients' ratings. In this study we assessed both olfaction and gustation using psychophysical tests eight months after COVID-19. Validated psychophysical testing revealed hyposmia in 18% and hypogeusia in even 32% of 303 included patients. This shows that olfactory and especially gustatory disorders have to be seen as important chronic symptoms post-COVID-19. The high prevalence of gustatory dysfunction indicates that gustatory function does not recover or might even deteriorate in the months following the acute infection.

Gustatory Function in Acute COVID-19 - Results From Home-Based Psychophysical Testing.

OBJECTIVE: Gustatory function during COVID-19 is self-reported by around 50% of patients. However, only a few studies assessed gustation using psychophysical testing during acute infection. The objective of this study is to test gustatory function on threshold tests in the very first days of COVID-19. METHODS: Psychophysical testing consisted of validated and blinded tests for olfaction (NHANES Pocket Smell Test) and gustation (Taste Strips Test). These test kits were sent to home-quarantined patients and self-administered using a detailed instruction sheet. RESULTS: A total of 51 patients were included in this study. Testing was performed 6.5 ± 2.7 days after sampling of respiratory swabs. At this time 37% of patients stated to currently experience a gustatory impairment. The mean Taste Strips score was 10.0 ± 3.4 with 28% scoring in the range of hypogeusia. Interestingly, no significant difference in the results of gustatory testing could be observed between the group with subjectively preserved gustation and the group with self-rated taste impairment. CONCLUSION: During the very first days of COVID-19, psychophysical gustatory testing revealed hypogeusia in 28%. This is far lower than patients' self-reports. Different from previous studies, we did not find clear evidence for an impairment of only certain taste qualities. LEVEL OF EVIDENCE: 3 Laryngoscope, 132:1082-1087, 2022.

Risk of transfusion-transmitted hepatitis E virus infection from pool-tested platelets and plasma.

BACKGROUND AND AIMS: Immunocompromised patients are at risk of chronic hepatitis E which can be acquired by blood transfusions. Currently, screening of blood donors (BDs) for HEV RNA with a limit of detection (LOD) of 2,000 IU/ml is required in Germany. However, this may result in up to 440,000 IU of HEV RNA in blood products depending on their plasma volume. We studied the residual risk of transfusion-transmitted (tt) HEV infection when an LOD of 2,000 IU/ml is applied. METHODS: Highly sensitive individual donor testing for HEV RNA on the Grifols Procleix Panther system (LOD 7.89 IU/ml) was performed. HEV loads were quantified by real-time PCR. RESULTS: Of 16,236 donors, 31 (0.19%) were HEV RNA positive. Three BDs had viral loads between 710 and 2,000 IU/ml, which pose a significant risk of tt hepatitis E with any type of blood product. Eight BDs had viral loads of >32 to 710 IU/ml, which pose a risk of tt hepatitis E with platelet or plasma transfusions because of their higher plasma volume compared to red blood cell concentrates. Eight of these 11 potentially infectious BDs were seronegative for HEV, indicating a recent infection. Only 8 of 31 donors had viral loads >2,000 IU/ml that would also have been detected by the required screening procedure and 12 had very low HEV loads (<32 IU/ml). CONCLUSIONS: Screening of BDs with an LOD of 2,000 IU/ml reduced the risk of tt HEV infection by about 73% for red blood cell concentrates but by just 42% for platelet and fresh frozen plasma transfusions. Single donor screening (LOD <32 IU/ml) should lead to an almost 100% risk reduction. LAY SUMMARY: Immunocompromised patients, such as solid organ or hematopoietic stem cell recipients, are at risk of chronic hepatitis E, which can be acquired via blood transfusions. The risk of transfusion-transmitted hepatitis E in these patients may not be sufficiently controlled by (mini-)pool hepatitis E virus RNA screening of blood donors. Single donor screening should be considered to improve the safety of blood products.

Contribution of High Viral Loads, Detection of Viral Antigen and Seroconversion to Severe Acute Respiratory Syndrome Coronavirus 2 Infectivity.

BACKGROUND: From a public health perspective, effective containment strategies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) should be balanced with individual liberties. METHODS: We collected 79 respiratory samples from 59 patients monitored in an outpatient center or in the intensive care unit of the University Hospital Regensburg. We analyzed viral load by quantitative real-time polymerase chain reaction, viral antigen by point-of-care assay, time since onset of symptoms, and the presence of SARS-CoV-2 immunoglobulin G (IgG) antibodies in the context of virus isolation from respiratory specimens. RESULTS: The odds ratio for virus isolation increased 1.9-fold for each log10 level of SARS-CoV-2 RNA and 7.4-fold with detection of viral antigen, while it decreased 6.3-fold beyond 10 days of symptoms and 20.0-fold with the presence of SARS-CoV-2 antibodies. The latter was confirmed for B.1.1.7 strains. The positive predictive value for virus isolation was 60.0% for viral loads >107 RNA copies/mL and 50.0% for the presence of viral antigen. Symptom onset before 10 days and seroconversion predicted lack of infectivity with negative predictive values of 93.8% and 96.0%. CONCLUSIONS: Our data support quarantining patients with high viral load and detection of viral antigen and lifting restrictive measures with increasing time to symptom onset and seroconversion. Delay of antibody formation may prolong infectivity.

Multicenter Performance Evaluation of Elecsys Anti-HBc II, Anti-HCV II, HIV combi PT, HBsAg II, and Syphilis Immunoassays.

BACKGROUND: The WHO recommends mandatory serological testing of blood donors for hepatitis B virus, hepatitis C virus (HCV), human immunodeficiency virus (HIV), and syphilis. We evaluated the performance of Elecsys® infectious disease immunoassays against commercially available comparator assays. METHODS: Prospective, routine, anonymized patient or donor samples (n = 8,821) were analyzed at three German sites using Elecsys antihepatitis B core antigen (Anti-HBc II), Anti-HCV II, HIV combi PT, hepatitis B surface antigen (HBsAg II), and Syphilis immunoassays (cobas e 411 analyzer) versus ARCHITECT comparator assays. RESULTS: The Elecsys immunoassays demonstrated comparable sensitivity (≤ 1.54% difference) and equivalent specificity (≤ 0.63% difference) to the respective ARCHITECT comparator assays. Overall sensitivity for the Elecsys and ARCHITECT infectious disease panels was 99.78% vs. 99.40%, respectively, and overall specificity was 99.74% vs. 99.80%, respectively. CONCLUSIONS: The Elecsys infectious disease immunoassays demonstrated high sensitivity and specificity, which were similar to comparator assays, supporting their suitability for routine laboratory practice.

Hepatitis A Virus Incidence Rates and Biomarker Dynamics for Plasma Donors, United States.

The United States is currently affected by widespread hepatitis A virus (HAV) outbreaks. We investigated HAV incidence rates among source plasma donors in the United States since 2016. Serial donations from HAV-positive frequent donors were analyzed for common biologic markers to obtain a detailed picture of the course of infection. We found a considerable increase in incidence rates with shifting outbreak hotspots over time. Although individual biomarker profiles were highly variable, HAV RNA typically had a high peak and a biphasic decrease and often remained detectable for several months. One donor had a biomarker pattern indicative of previous exposure. Our findings show that current HAV outbreaks have been spilling over into the plasma donor population. The detailed results presented improve our comprehension of HAV infection and related public health aspects. In addition, the capture of full RNA curves enables estimation of HAV doubling time.

Liver Lipids of Patients with Hepatitis B and C and Associated Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) still remains a difficult to cure malignancy. In recent years, the focus has shifted to lipid metabolism for the treatment of HCC. Very little is known about hepatitis B virus (HBV) and C virus (HCV)-related hepatic lipid disturbances in non-malignant and cancer tissues. The present study showed that triacylglycerol and cholesterol concentrations were similar in tumor adjacent HBV and HCV liver, and were not induced in the HCC tissues. Higher levels of free cholesterol, polyunsaturated phospholipids and diacylglycerol species were noted in non-tumorous HBV compared to HCV liver. Moreover, polyunsaturated phospholipids and diacylglycerols, and ceramides declined in tumors of HBV infected patients. All of these lipids remained unchanged in HCV-related HCC. In HCV tumors, polyunsaturated phosphatidylinositol levels were even induced. There were no associations of these lipid classes in non-tumor tissues with hepatic inflammation and fibrosis scores. Moreover, these lipids did not correlate with tumor grade or T-stage in HCC tissues. Lipid reprogramming of the three analysed HBV/HCV related tumors mostly resembled HBV-HCC. Indeed, lipid composition of non-tumorous HCV tissue, HCV tumors, HBV tumors and HBV/HCV tumors was highly similar. The tumor suppressor protein p53 regulates lipid metabolism. The p53 and p53S392 protein levels were induced in the tumors of HBV, HCV and double infected patients, and this was significant in HBV infection. Negative correlation of tumor p53 protein with free cholesterol indicates a role of p53 in cholesterol metabolism. In summary, the current study suggests that therapeutic strategies to target lipid metabolism in chronic viral hepatitis and associated cancers have to consider disease etiology.

Hepatitis E: An update on One Health and clinical medicine.

The hepatitis E virus (HEV) is one of the main causes of acute hepatitis and the de facto global burden is underestimated. HEV-related clinical complications are often undetected and are not considered in the differential diagnosis. Convincing findings from studies suggest that HEV is clinically relevant not only in developing countries but also in industrialized countries. Eight HEV genotypes (HEV-1 to HEV-8) with different human and animal hosts and other HEV-related viruses are in circulation. Transmission routes vary by genotype and location, with large waterborne outbreaks in developing countries and zoonotic food-borne infections in developed countries. An acute infection can be aggravated in pregnant women, organ transplant recipients, patients with pre-existing liver disease and immunosuppressed patients. HEV during pregnancy affects the fetus and newborn with an increased risk of vertical transmission, preterm and stillbirth, neonatal jaundice and miscarriage. Hepatitis E is associated with extrahepatic manifestations that include neurological disorders such as neuralgic amyotrophy, Guillain-Barré syndrome and encephalitis, renal injury and haematological disorders. The risk of transfusion-transmitted HEV is increasingly recognized in Western countries where the risk may be because of a zoonosis. RNA testing of blood components is essential to determine the risk of transfusion-transmitted HEV. There are currently no approved drugs or vaccines for HEV infections. This review focuses on updating the latest developments in zoonoses, screening and diagnostics, drugs in use and under development, and vaccines.

Comparison of Throat Washings, Nasopharyngeal Swabs and Oropharyngeal Swabs for Detection of SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA is detected by reverse-transcription quantitative real-time PCR (RT-qPCR) from respiratory specimens. This study compares throat washings (TW), nasopharyngeal swabs (NS) and oropharyngeal swabs (OS). A total of 102 samples from 34 adult patients with confirmed SARS-CoV-2 infection were analysed by RT-qPCR with absolute quantification. The median concentrations and diagnostic sensitivities were 5.8×104 copies/mL, 85% (NS), 1.4×104, 79% (OS) and 4.3×103, 85% (TW). Concentration differences were significant between NS and TW (P = 0.019). Saliva (SA) was available from 21 patients (median 3.4×103). OS and TW can be considered for SARS-CoV-2 diagnostics, although with slightly lower concentrations.

Evaluation of a Broad Panel of SARS-CoV-2 Serological Tests for Diagnostic Use.

Serological testing is crucial in detection of previous infection and in monitoring convalescent and vaccine-induced immunity. During the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) pandemic, numerous assay platforms have been developed and marketed for clinical use. Several studies recently compared clinical performance of a limited number of serological tests, but broad comparative evaluation is currently missing. Within this study, a panel of 161 sera from SARS-CoV-2 infected, seasonal CoV-infected and SARS-CoV-2 naïve subjects was enrolled to evaluate 16 ELISA/ECLIA-based and 16 LFA-based tests. Specificities of all ELISA/ECLIA-based assays were acceptable and generally in agreement with the providers' specifications, but sensitivities were lower as specified. Results of the LFAs were less accurate as compared to the ELISAs, albeit with some exceptions. We found a sporadic unequal immune response for different antigens and thus recommend the use of a nucleocapsid protein (N)- and spike protein (S)-based test combination when maximal sensitivity is necessary. Finally, the quality of the immune response in terms of neutralization should be tested using S-based IgG tests.

Virus-specific memory T cell responses unmasked by immune checkpoint blockade cause hepatitis.

Treatment of advanced melanoma with combined PD-1/CTLA-4 blockade commonly causes serious immune-mediated complications. Here, we identify a subset of patients predisposed to immune checkpoint blockade-related hepatitis who are distinguished by chronic expansion of effector memory CD4+ T cells (TEM cells). Pre-therapy CD4+ TEM cell expansion occurs primarily during autumn or winter in patients with metastatic disease and high cytomegalovirus (CMV)-specific serum antibody titres. These clinical features implicate metastasis-dependent, compartmentalised CMV reactivation as the cause of CD4+ TEM expansion. Pre-therapy CD4+ TEM expansion predicts hepatitis in CMV-seropositive patients, opening possibilities for avoidance or prevention. 3 of 4 patients with pre-treatment CD4+ TEM expansion who received αPD-1 monotherapy instead of αPD-1/αCTLA-4 therapy remained hepatitis-free. 4 of 4 patients with baseline CD4+ TEM expansion given prophylactic valganciclovir and αPD-1/αCTLA-4 therapy remained hepatitis-free. Our findings exemplify how pathogen exposure can shape clinical reactions after cancer therapy and how this insight leads to therapeutic innovations.