A Novel Insertion in the Hepatitis B Virus Surface Protein Leading to Hyperglycosylation Causes Diagnostic and Immune Escape.

Chronic hepatitis B virus (HBV) infection is a global health threat. Mutations in the surface antigen of HBV (HBsAg) may alter its antigenicity, infectivity, and transmissibility. A patient positive for HBV DNA and detectable but low-level HBsAg in parallel with anti-HBs suggested the presence of immune and/or diagnostic escape variants. To support this hypothesis, serum-derived HBs gene sequences were amplified and cloned for sequencing, which revealed infection with exclusively non-wildtype HBV subgenotype (sgt) D3. Three distinct mutations in the antigenic loop of HBsAg that caused additional N-glycosylation were found in the variant sequences, including a previously undescribed six-nucleotide insertion. Cellular and secreted HBsAg was analyzed for N-glycosylation in Western blot after expression in human hepatoma cells. Secreted HBsAg was also subjected to four widely used, state-of-the-art diagnostic assays, which all failed to detect the hyperglycosylated insertion variant. Additionally, the recognition of mutant HBsAg by vaccine- and natural infection-induced anti-HBs antibodies was severely impaired. Taken together, these data suggest that the novel six-nucleotide insertion as well as two other previously described mutations causing hyperglycosylation in combination with immune escape mutations have a critical impact on in vitro diagnostics and likely increase the risk of breakthrough infection by evasion of vaccine-induced immunity.

Data on immunogenicity and reactogenicity to COVID-19 vaccination among patients receiving maintenance dialysis.

Compared with the general population, patients receiving maintenance dialysis are at increased risk for morbidity and mortality associated with coronavirus disease 2019 (COVID-19). Currently, data on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-specific immunity post-vaccination in patients on maintenance dialysis are scarce given that the effectiveness of the vaccines has not been explicitly tested in this population due to their common exclusion from SARS-CoV-2 vaccination trials. We herein present data of the specific cellular (interferon-γ and interleukin-2 ELISpot assays) and humoral immune responses (dot plot array and chemiluminescent microparticle immunoassay) at 4 weeks and 6 weeks following a single dose or a complete homologous dual dose SARS-CoV-2 vaccine regimen in 60 adult patients on maintenance dialysis (six with a history of COVID-19). The data was produced in a framework of a project focused on a) quantifying the immune response after full vaccination, b) evaluating the short-term durability of immune response, and c) examining the reactogenicity of SARS-CoV-2 vaccine regimens in patients on maintenance dialysis.

Immunogenicity and reactogenicity of homologous mRNA-based and vector-based SARS-CoV-2 vaccine regimens in patients receiving maintenance dialysis.

Patients receiving maintenance dialysis (MD) are vulnerable to COVID-19-related morbidity and mortality. Currently, data on SARS-CoV-2-specific cellular and humoral immunity post-vaccination in this population are scarce. We conducted a prospective single-center study exploring the specific cellular (interferon-γ and interleukin-2 ELISpot assays) and humoral immune responses (dot plot array and chemiluminescent microparticle immunoassay [CMIA]) at 4 weeks and 6 weeks following a single dose or a complete homologous dual dose SARS-CoV-2 vaccine regimen in 60 MD patients (six with a history of COVID-19). Our results show that MD patients exhibit a high seroconversion rate (91.7%) but the anti-spike IgG antibodies (CMIA) tend to wane rapidly after full immunization. Only 51.7% of the patients developed T cell immune response. High anti-spike IgG antibodies may predict a better cellular immunity. While patients with prior COVID-19 showed the best response after one, SARS-CoV-2-naïve patients may benefit from a third vaccine injection.

[Ten years of the National Reference Center for hepatitis B viruses and hepatitis D viruses in Giessen, Germany: activities and experiences]. / 10 Jahre Nationales Referenzzentrum für Hepatitis-B-Viren und Hepatitis-D-Viren in Gießen: Tätigkeiten und Erfahrungen.

The National Reference Center (NRC) for hepatitis B viruses (HBV) and hepatitis D viruses (HDV) has been located at the Institute of Medical Virology of the Justus Liebig University (JLU) in Giessen, Germany, since its establishment in 2011. This paper describes the NRC's areas of activity and related experience.The NRC offers comprehensive consulting services on all diagnostic and clinical aspects of acute and chronic HBV and HDV infections for the Public Health Service (ÖGD), diagnostic laboratories, clinics, research institutes, and physicians in private practice. Uncertain diagnostic findings can be analyzed and interpreted and epidemiological correlations clarified with the HBV/HDV special diagnostics established at the NRC using state-of-the-art molecular, biochemical, and genetic laboratory tools. The NRC has access to a strain collection of many well-characterized and cloned HBV/HDV isolates, allowing comparative analysis and evaluation of antiviral resistance mutations and immune escape variants. Together with its national and international partner institutions, the NRC initiates and supervises, among other things, interlaboratory studies for the diagnosis of HBV resistance and immune escape for the establishment and validation of international World Health Organization (WHO) standards and for the improvement of quantitative HDV genome determination. The NRC actively participates in current recommendations and guidelines on HBV and HDV and the recommendations of medical societies. It also highlights current HBV/HDV-relevant aspects with contributions in the form of national and international lectures as well as original articles and comments in national and international journals.

Sexual Health in HIV-Positive Men Under Stable Antiretroviral Therapy During a 12-Month Period.

BACKGROUND: Sexual health is becoming increasingly important for many HIV-positive men undergoing highly effective antiretroviral therapy (ART) but remains frequently unaddressed in routine clinical consultation. AIM: To comprehensively evaluate sexual health in male patients with HIV on stable ART over a 12-month period. METHODS: The prospectively registered cohort study comprising 87 HIV-positive men on stable ART (median age: 43 years) was conducted between 2011 and 2015 at a university hospital. Patients were enrolled from the outpatient infectious disease unit and underwent an extensive andrological workup to assess parameters of sexual health (questionnaires, sex hormones, ultrasound, 2-glass urine test including semen analysis with microbiological and viral diagnostics). The study period per patient lasted 12 months. OUTCOME: The primary endpoint was the impact of chronic HIV infection on sexual health. RESULTS: Although, on average, sexual health was fine at baseline, 56% of the patients reported erectile dysfunction, 28% experienced reduced libido, 5% had hypogonadism, 36% showed at least 1 atrophic testicle with a volume of <10 ml, 8% suffered bacterial sexually transmitted infections, 35% had seminal inflammation, and up to 47% showed reduced sperm quality. Sexual satisfaction was linked to mental health (12-Item Short Form Health Survey questionnaire) and International Index of Erectile Function scores. During the study period, the collected parameters on sexual health were generally stable. However, 35% of patients had new sex partners (median: 5 partners), 7% had fathered a child or were planning procreation, 47% reported changed libido, 17% suffered bacterial sexually transmitted infections in the urogenital tract, 16% revealed a positive HIV viral load in blood, 11% had a positive HIV viral load in semen, and 28% were treated for andrological disorders. CLINICAL IMPLICATIONS: Sexual ill-health exists in about one third of patients. This manifests itself in sexual dysfunction, sexually transmitted infections, urogenital tract inflammation, and abnormal sperm parameters, all of which require adequate counseling and therapy. STRENGTH AND LIMITATIONS: The strength of this study is its comprehensive analysis of male sexual health over a 12-month period of stable ART treatment. Limitations are a heterogeneous patient cohort and a rather small percentage of patients with a positive HIV viral load in blood or semen, which prevented multivariate risk analysis. CONCLUSION: Our study provides evidence that sexual health should be actively taken into account in the routine consultation by infectious disease specialists, and an interdisciplinary approach is desirable in the case of symptoms or signs of sexual ill-health. Pilatz A, Maresch CC, Discher T, et al. Sexual Health in HIV-Positive Men Under Stable Antiretroviral Therapy During a 12-Month Period. J Sex Med 2021;18:284-294.

Immunoglobulin deficiency as an indicator of disease severity in patients with COVID-19.

Despite the pandemic status of COVID-19, there is limited information about host risk factors and treatment beyond supportive care. Immunoglobulin G (IgG) could be a potential treatment target. Our aim was to determine the incidence of IgG deficiency and associated risk factors in a cohort of 62 critically ill patients with COVID-19 admitted to two German ICUs (72.6% male, median age: 61 yr). Thirteen (21.0%) of the patients displayed IgG deficiency (IgG < 7 g/L) at baseline (predominant for the IgG1, IgG2, and IgG4 subclasses). Patients who were IgG-deficient had worse measures of clinical disease severity than those with normal IgG levels (shorter duration from disease onset to ICU admission, lower ratio of [Formula: see text] to [Formula: see text], higher Sequential Organ Failure Assessment score, and higher levels of ferritin, neutrophil-to-lymphocyte ratio, and serum creatinine). Patients who were IgG-deficient were also more likely to have sustained lower levels of lymphocyte counts and higher levels of ferritin throughout the hospital stay. Furthermore, patients who were IgG-deficient compared with those with normal IgG levels displayed higher rates of acute kidney injury (76.9% vs. 26.5%; P = 0.001) and death (46.2% vs. 14.3%; P = 0.012), longer ICU [28 (6-48) vs. 12 (3-18) days; P = 0.012] and hospital length of stay [30 (22-50) vs. 18 (9-24) days; P = 0.004]. Univariable logistic regression showed increasing odds of 90-day overall mortality associated with IgG-deficiency (odds ratio 5.14, 95% confidence interval 1.3-19.9; P = 0.018). IgG deficiency might be common in patients with COVID-19 who are critically ill, and warrants investigation as both a marker of disease severity as well as a potential therapeutic target.

[Rapid diagnosis of sexually transmitted infections : Joint statement of DSTIG, RKI, and PEI, as well as the reference centers for HIV, HBV, and HCV and consulting laboratories for Chlamydia, gonococci, and Treponema pallidum]. / Schnelltestdiagnostik sexuell übertragbarer Infektionen : Gemeinsame Stellungnahme von DSTIG, RKI, PEI sowie den Referenzzentren für HIV, HBV und HCV und Konsiliarlaboren für Chlamydien, Gonokokken und Treponema pallidum.

In February 2019, the fourth expert meeting on rapid diagnostic tests (RDTs) for sexually transmitted infections (STI) was held at the Robert Koch Institute (RKI) in Berlin. Novel technical developments and new aspects of RDT applications were discussed by representatives from the German STI Society (DSTIG); RKI; the Paul Ehrlich Institute; national reference centers for HIV, HBV, and HCV; and reference laboratories for Chlamydia, gonococci, and Treponema pallidum.As a result of this meeting, we present a revision of the joint statement on STI diagnostics with RDTs from 2017. The Regulation (EU) 2017/746 of the European Parliament and of the Council on in vitro diagnostic medical devices became effective in May 2017 and includes more stringent regulatory requirements for RDTs, mainly concerning conformity of manufacturing processes and performance characteristics of class D in vitro diagnostics (detection of HIV, HBV, HCV, and T. pallidum). Some RDTs for HIV, HCV, and T. pallidum have been evaluated in clinical studies and/or were WHO prequalified and may be used in low-threshold services. Among them are some HIV RDTs available and approved for self-testing. In addition, some HBV RDTs based on detection of HBs antigen (HBsAg) received WHO prequalification. However, false negative results may occur in samples with low HBsAg levels, as for instance in HIV-coinfected patients receiving antiretroviral therapy. For Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), antigen-based RDTs still do not allow reliable detection of infection. Only PCR-based CT/NG RDTs possess sufficient diagnostic accuracy to be used as point-of-care tests. Rapid PCR tests for NG, however, do not provide any information about antimicrobial resistance.

High prevalence of urogenital infection/inflammation in patients with azoospermia does not impede surgical sperm retrieval.

Considering infection/inflammation to be an important risk factor in male infertility, the aim of this study was to make a comprehensive evaluation of the prevalence of urogenital tract infection/inflammation and its potential impact on sperm retrieval in azoospermic patients. In this prospective study, 71 patients with azoospermia were subjected to an extensive andrological workup including comprehensive microbiological diagnostics (2-glass test, semen, testicular swab and testicular tissue analysis) and testicular biopsy/testicular sperm extraction (TESE). Medical history suggested urogenital tract infection/inflammation in 7% of patients, 11% harboured STIs, 14% showed significant bacteriospermia, 15% had seminal inflammation, 17% fulfilled the MAGI definition, and 27% had relevant pathogens. At the testicular level, 1 patient had a swab positive for bacteria, no viruses were detected, tissue specimens never indicated pathogens, whereas histopathology revealed focal immune cell infiltrates in 23% of samples. Testicular sperm retrieval rate was 100% in obstructive and 46% in nonobstructive azoospermia. None of the infection/inflammation-related variables was associated with the success of sperm retrieval or inflammatory lesions in the testis. The high prevalence of urogenital infection/inflammation among azoospermic men underpins their role as significant aetiologic factors in male infertility. However, this observation does not refer to the chances of sperm retrieval at the time of surgery/TESE.

Indefinite cytomegalovirus prophylaxis with valganciclovir after lung transplantation.

Human cytomegalovirus (HCMV) infections and reactivations are common after lung transplantation and are associated with the development of bronchiolitis obliterans syndrome. Against this background, temporary HCMV prophylaxis is an established standard regimen after lung transplantation in most centers. However, the optimal duration of prophylaxis is unclear. We conducted a retrospective two-center study to determine the efficacy of indefinite lifelong HCMV prophylaxis with oral valganciclovir in a cohort of 133 lung transplant recipients with a mean follow-up time of approximately 5 years. During the follow-up period, HCMV DNA was detected in 22 recipients (16.5%). In one case, HCMV pneumonitis developed after prophylaxis had been terminated. We observed a beneficial safety profile and tolerability in our cohort, as the majority of patients still received valganciclovir after a 1- and 3-year observation period, respectively. Compared to the literature, these data indicate a beneficial effect of extended valganciclovir prophylaxis with an acceptable safety profile.

Schnelltest-Diagnostik sexuell übertragbarer Infektionen in niedrigschwelligen Einrichtungen : Gemeinsame Stellungnahme des RKI, PEI und der DSTIG.

On February 5th, 2016 an expert meeting on rapid diagnostic tests (RDT) for sexually transmitted infections (STI) was held in Berlin at the Robert-Koch-Institute. The aim of the conference was to update a former evaluation of RDTs for diagnosis of HIV, HBV, HCV, T. pallidum, C. trachomatis and N. gonorrhoeae in low-threshold counseling services for STI that had been published after the previous meeting in 2012. According to the strategy to control HIV, hepatitis B and C and other STI, recently adopted by the German Government, there is a lack of test capabilities and a demand for more testing services as well as improved access to testing. Using RDTs as low-threshold test services in counseling centers or even for testing at home may provide an important option to lower the barrier of testing. Based on performance data evaluated in clinical trials some RDTs for HIV, HCV and syphilis are quite well suited as a point-of-care Test (POCT). In contrast, sufficient diagnostic accuracy for detection of C. trachomatis and N. gonorrhoeae can only be achieved by PCR-based POCTs. In Germany the use of POCTs is subjected to legal stipulations of IfSG and MPG. Of importance, it is not allowed to deliver HIV tests to private persons for home testing (§ 11, MPG). Furthermore, both assessment and communication of infectious diseases are reserved to the physician and must not happen as remote diagnostics (§ 24, IfSG). In addition, like all laboratory tests, RDTs are subject to quality assessment according to guidelines of the German Medical Association.

Characterization of the 3rd International Standard for hepatitis B virus surface antigen (HBsAg).

BACKGROUND: HBsAg is the most important marker for laboratory diagnosis of HBV infection. Validation and quality control of HBsAg tests requires International Standards (IS). Recently the 2nd IS was replaced by the 3rd IS. Both IS are made from plasma-derived hepatitis B vaccines, but production and geographical origin are different. OBJECTIVE: Characterization of the HBsAg in the source material (SM) for the 3rd IS and comparison with the 2nd IS and native HBsAg. STUDY DESIGN: The SM was analyzed using solid-phase immunoassays, quantitative immune electrophoresis, ultracentrifugation, immunoblotting and HBV DNA sequencing. RESULTS: The plasma-derived HBsAg of the SM originated from at least two different HBV strains, both of subgenotype (sgt) B4, typical for Vietnam. The HBsAg subtype was heterogeneous with ayw1 and adw2. The HBsAg concentration was 23,700 IU/ml as determined by solid-phase immunoassay; immune electrophoresis calibrated with sgt B2 revealed a concentration of 24,500 IU/ml while calibration with sgt D1 provided lower values. Proteins in the SM are heterogeneous in size containing only traces of preS. The protein subunits are partially cross-linked. CONCLUSIONS: The antigenicity of the 3rd IS is suitable for HBsAg calibration in laboratory tests. In contrast to the 2nd IS, the 3rd IS is representative for a highly endemic region. Similar to the 2nd IS and different from native HBsAg, preS domains are depleted, protein subunits are partially cross-linked and the HBsAg particles are partially aggregated in the 3rd IS. The HBV subgenotype differences between the two IS may lead to variations in different quantitative assays.

International collaborative study on the 3rd WHO International Standard for hepatitis B surface antigen.

BACKGROUND: The WHO International Standard (IS) for hepatitis B surface antigen (HBsAg) is used to standardize HBsAg assays. Stocks of the 2nd IS for HBsAg are depleted. The proposal to establish its replacement was endorsed by WHO in 2012. OBJECTIVE: Preparation of a freeze-dried candidate 3rd IS (NIBSC 12/226); evaluation of its suitability in a WHO international collaborative study; calibration of its potency in International Units (IU). STUDY DESIGN: The 3rd IS is based on plasma-derived, purified, inactivated HBsAg from Vietnam. Qualitative and quantitative HBsAg assays were used to evaluate 12/226 alongside the 2nd IS and 1st IS. Blinded study samples included a duplicate of 12/226, a negative control and two diluted plasma samples representing hepatitis B virus (HBV) genotypes A and B. RESULTS: Twelve laboratories from 9 countries returned 22 data sets from 15 methods. The overall geometric mean potency of 12/226 is 47.3IU/mL (±13% CV) when compared to the 2nd IS with HBV subgenotype A2. The 3rd IS has HBV subgenotype B4 with a heterogeneous HBsAg subtype population of ayw1 and adw2. Some genotype-dependent effects on the inter-laboratory variability were observed but overall mean potencies were virtually identical irrespective of the IS used for calibration. Stability studies indicate that the candidate is stable for long-term use. CONCLUSIONS: 12/226 was established in October 2014 by the WHO Expert Committee on Biological Standardization as the 3rd IS for HBsAg with a potency of 47.3IU per ampoule maintaining the continuity in the standardization of HBsAg assays.

Phylogenetic analysis of human influenza A/H3N2 viruses isolated in 2015 in Germany indicates significant genetic divergence from vaccine strains.

Infections by H3N2-type influenza A viruses (IAV) resulted in significant numbers of hospitalization in several countries in 2014-2015, causing disease also in vaccinated individuals and, in some cases, fatal outcomes. In this study, sequence analysis of H3N2 viruses isolated in Germany from 1998 to 2015, including eleven H3N2 isolates collected early in 2015, was performed. Compared to the vaccine strain A/Texas/50/2012 (H3N2), the 2015 strains from Germany showed up to 4.5 % sequence diversity in their HA1 protein, indicating substantial genetic drift. The data further suggest that two distinct phylogroups, 3C.2 and 3C.3, with 1.6-2.3 % and 0.3-2.4 % HA1 nucleotide and amino acid sequence diversity, respectively, co-circulated in Germany in the 2014/2015 season. Distinct glycosylation patterns and amino acid substitutions in the hemagglutinin and neuraminidase proteins were identified, possibly contributing to the unusually high number of H3N2 infections in this season and providing important information for developing vaccines that are effective against both genotypes.

Studies of nosocomial outbreaks of hepatitis B in nursing homes in Germany suggest a major role of hepatitis B e antigen expression in disease severity and progression.

Hepatitis B virus (HBV) causes acute or chronic hepatitis B. Local outbreaks of HBV infections in skilled nursing facilities is a matter of growing concern in developed countries. Here, we investigated two outbreaks of hepatitis B that recently occurred in nursing homes in Germany. The outbreak at location A was associated with acute fulminant hepatitis with fatal outcome in several cases, while individuals infected at location B developed asymptomatic or mild hepatitis B. Sequence analysis of viruses involved in these outbreaks revealed different, but unique HBV strains for each location. Each of the strains produced high viremia of more than 10(9) virions/mL serum. We found that the mild course of hepatitis B at location B was caused by a circulating wild-type HBV genotype A2 strain, which is commonly found in Central Europe. Complete genome sequences of isolates obtained from infected patients revealed nearly 100% sequence identity at the nucleotide level as well as expression of HBV e protein (HBeAg), a known T cell tolerogen in the incubation or chronic phases of HBV infection. By contrast, the outbreak at location A was associated with an HBV genotype D2 variant that lacked HBeAg expression, suggesting that immunopathology and selection of specific HBV variants played a major role in the severe (or even fulminant) acute hepatitis observed at location A. Importantly, all patients were diagnosed with type 2 diabetes mellitus, a known risk factor for healthcare-associated transmission of HBV. The study leads us to suggest that, besides strict adherence to hygiene standards, additional efforts are required to reduce the risk of HBV transmission and fulminant disease progression in healthcare settings and nursing homes. In this context, a general screening for HBsAg and active hepatitis B vaccination should be considered for people living in nursing homes, especially for those with diagnosed diabetes or other predisposing factors for HBV transmission.

Hepatitis B outbreak in a nursing home associated with reusable lancet devices for blood glucose monitoring, Northern Germany 2010.

In September 2010, an outbreak of acute hepatitis B virus (HBV) infections in a nursing home was notified to public health authorities in Northern Germany. To identify the route of transmission and prevent further cases a retrospective cohort study was conducted. Blood samples of residents were tested for serologic markers of HBV infection and HBV subgenotypes and sequences were analyzed. Outbreak-related cases were defined as residents of the nursing home with detection of hepatitis B surface antigen (HBsAg) and the HBV DNA sequence of the outbreak strain in 2010. Information on possible risk factors as patient care, invasive diagnostic, and therapeutical procedures was collected using a standardized questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were estimated with exact Poisson regression and binomial regression. Sixty-four residents were included in the study, 5 of them were outbreak-related cases, 12 had a past HBV infection. The outbreak strain belonged to HBV genotype D2 (HBsAg subtype ayw3, Ala118) which is not prevalent in Germany but in Eastern Europe. All cases (median age 81) were female, had diabetes, blood glucose monitoring, and chiropody. In multivariable analysis only blood glucose monitoring was associated with HBV infection (RR = 22, 95%CI 3.0-∞). Blood glucose monitoring was reported to be done by nursing home staff with patient-based reusable lancet devices. In nursing home settings the use of single use lancets for blood glucose monitoring is recommended strongly to prevent transmission. National guidelines on the handling of point-of-care devices and reusable equipment in long-term care facilities should be developed.