Increasing hepatitis B vaccination coverage and decreasing hepatitis B co-infection prevalence among people with HIV-1 in Germany, 1996-2019. Results from a cohort study primarily in men who have sex with men.

OBJECTIVES: Viral hepatitis co-infection among people living with HIV is known to accelerate the progression of liver disease and AIDS. An increased prevalence and incidence of hepatitis B virus (HBV) infection among people living with HIV demands continuous monitoring to adapt targeted prevention strategies to reach the global goals of eliminating viral hepatitis as a public health threat. METHODS: We determined the prevalence and incidence of HBV for the years 1996-2019 from yearly blood sample testing and questionnaire reports among people living with HIV belonging to a nationwide, multicentre observational, prospective cohort study. RESULTS: Among this study population of 3479 participants, the majority (87%) indicated that being men who have sex with men (MSM) was their likely HIV transmission route; 51% were recruited from Berlin. HBV prevalence for acute/chronic and resolved infections decreased from 4.1% and 45% in 1996-1999 to 1.3% and 16% in 2019, respectively. Simultaneously, participants with a serological status indicating HBV vaccination increased from 25% in 1996-1999 to 69% in 2019. Among vaccinated participants with relevant information (n = 1135), 38% received their first HBV vaccination after HIV infection. The HBV incidence rate in 565 eligible participants decreased from 6.9/100 person-years in 2004-2007 to 0.45/100 person-years in 2015. CONCLUSION: Increasing vaccination coverage because of a general HBV vaccination recommendation and catch-up vaccination efforts among risk groups decreased HBV infection prevalence over time among this study population of people living with HIV, primarily MSM and from Berlin. Despite this success, the prevalence and incidence of HBV remains higher than in the general population in Germany. This emphasizes the need for continued HBV prevention by promoting HBV vaccination and HBV screening at regular intervals based on the individual risk behaviour.

HIV pre-exposure prophylaxis during the SARS-CoV-2 pandemic: Results from a prospective observational study in Germany.

Aims: Since 2017, HIV pre-exposure prophylaxis (PrEP) care has been provided through an intersectoral collaboration at WIR (Walk-in-Ruhr, Center for Sexual Health and Medicine, Bochum, Germany). The aim of this study was to establish possible impact of COVID-restrictions on the sexual behavior of PrEP users in North Rhine-Westphalia. Methods: The current PrEP study collected data of individuals using PrEP, their sexual behavior and sexually transmitted infections (STIs) before (each quarter of year 2018) and during the COVID-19 pandemic (each quarter of year 2020). Results: During the first lockdown in Germany from mid-March until May 2020, PrEP-care appointments at WIR were postponed or canceled. Almost a third of PrEP users had discontinued their PrEP intake in the 2nd quarter of 2020 due to alteration of their sexual behavior. The number of sexual partners decreased from a median of 14 partners in the previous 6 months in 1st quarter of 2020, to 7 partners in 4th quarter of 2020. Despite such a significant reduction in partner number during the pandemic in comparison to the pre-pandemic period, a steady rate of STIs was observed among PrEP users in 2020. Conclusion: The SARS-CoV-2-pandemic has impacted PrEP-using MSM in North Rhine-Westphalia with respect to their PrEP intake regimen and sexual behavior in 2020. Our study revealed a steady rate of STI among PrEP users even during the pandemic, thus highlighting the importance of ensuring appropriate HIV/STI prevention services in times of crisis.

Influence of dietary supplementation of short-chain fatty acid sodium propionate in people living with HIV (PLHIV).

BACKGROUND: Non-AIDS-associated chronic diseases in HIV+ patients have been on the rise since the advent of antiretroviral therapy. Especially cardiovascular diseases and disruption in the gastrointestinal tract have limited health-related quality of life (QoL). Several of those complications have been associated with chronic systemic inflammation. Short-chain fatty acids (SCFA), with propionate as one of the major compounds, have been described as an important link between gut microbiota and the immune system, defining the pro- and the anti-inflammatory milieu through direct and indirect regulation of T-cell homeostasis. The effects of dietary supplementation of sodium propionate (SP) in people living with HIV (PLHIV) have not yet been investigated prior to this study. OBJECTIVES: To investigate the impact of SP uptake among PLHIV and its relevance to improve QoL, the study aimed to investigate metabolic, immunological, microbiome and patient-reported QoL-related changes post-SP supplementation with follow-up. METHODS: A prospective, non-randomized, controlled, monocentric interventional study was conducted in WIR, Center for Sexual Health and Medicine, in Bochum, Germany. 32 HIV+ patients with unaltered ART-regimen in the last three months were included. Participants were given SP for a duration of 12 weeks in the form of daily oral supplementation and were additionally followed-up for another 12 weeks. RESULTS: The supplementation of SP was well tolerated. We found an improvement in lipid profiles and long-term blood glucose levels. A decrease in pro-inflammatory cytokines and a depletion of effector T cells was observed. Regulatory T cells and IL-10 decreased. Furthermore, changes in taxonomic composition of the microbiome during follow-up were observed and improvement of items of self-reported life-quality assessment. CONCLUSION: Taken together, the beneficial impact of SP in PLHIV reflects its potential in improving metabolic parameters and modulating pro-inflammatory immune responses. Thus, possibly reducing the risk of cardiovascular disorders and facilitating long-term improvement of the gut flora.

[Local health study : Outreach medical services for female sex workers in Bochum]. / Local Health Study : Aufsuchende medizinische Angebote für Sexarbeiterinnen in Bochum.

BACKGROUND: Inadequate access to prevention and medical treatment for female sex workers (SW) represents a challenge for the German health system. Accessibility and care for SW in Bochum (Germany) through a cooperation between the Interdisciplinary Immunology Outpatient Clinic, Center for Sexual Health and Medicine of St. Josef's Hospital, the Bochum health department and the Madonna e.V. was the focus of this work. PATIENTS AND METHODS: Medical outreach services were provided for the diagnosis of sexually transmitted infections (STI) in SW in brothels in Bochum between August 2013 and January 2014. After clarification and verbal consent from the SW, free HIV, syphilis, chlamydia, gonorrhea and trichomoniasis tests were offered and carried out using pseudonyms for the SW. RESULTS: A total of 112 SW were reached (up to 55.4 % within the framework of the STI Outreach Study). Of the SW, 94.6 % had an immigrant background. The majority (61.3 %) of SW were between 20 und 29 years old. Only 19.0 % of the collective had health insurance. The following STIs were diagnosed: 12.5 % chlamydia, 6.2 % syphilis, 3.6 % gonorrhea, 3.6 % trichomoniasis, and 0.9 % HIV. These results were compared with results from STI studies in SW in Germany. Treatment was performed in accordance with the standards of the German STI Society. CONCLUSION: The offer improved the accessibility and the utilization of medical services by SW in Bochum. A further improvement of services is urgently needed.

Parameter uncertainty in biochemical models described by ordinary differential equations.

Improved mechanistic understanding of biochemical networks is one of the driving ambitions of Systems Biology. Computational modeling allows the integration of various sources of experimental data in order to put this conceptual understanding to the test in a quantitative manner. The aim of computational modeling is to obtain both predictive as well as explanatory models for complex phenomena, hereby providing useful approximations of reality with varying levels of detail. As the complexity required to describe different system increases, so does the need for determining how well such predictions can be made. Despite efforts to make tools for uncertainty analysis available to the field, these methods have not yet found widespread use in the field of Systems Biology. Additionally, the suitability of the different methods strongly depends on the problem and system under investigation. This review provides an introduction to some of the techniques available as well as gives an overview of the state-of-the-art methods for parameter uncertainty analysis.

An integrated strategy for prediction uncertainty analysis.

MOTIVATION: To further our understanding of the mechanisms underlying biochemical pathways mathematical modelling is used. Since many parameter values are unknown they need to be estimated using experimental observations. The complexity of models necessary to describe biological pathways in combination with the limited amount of quantitative data results in large parameter uncertainty which propagates into model predictions. Therefore prediction uncertainty analysis is an important topic that needs to be addressed in Systems Biology modelling. RESULTS: We propose a strategy for model prediction uncertainty analysis by integrating profile likelihood analysis with Bayesian estimation. Our method is illustrated with an application to a model of the JAK-STAT signalling pathway. The analysis identified predictions on unobserved variables that could be made with a high level of confidence, despite that some parameters were non-identifiable. AVAILABILITY AND IMPLEMENTATION: Source code is available at: http://bmi.bmt.tue.nl/sysbio/software/pua.html.

A Bayesian approach to targeted experiment design.

MOTIVATION: Systems biology employs mathematical modelling to further our understanding of biochemical pathways. Since the amount of experimental data on which the models are parameterized is often limited, these models exhibit large uncertainty in both parameters and predictions. Statistical methods can be used to select experiments that will reduce such uncertainty in an optimal manner. However, existing methods for optimal experiment design (OED) rely on assumptions that are inappropriate when data are scarce considering model complexity. RESULTS: We have developed a novel method to perform OED for models that cope with large parameter uncertainty. We employ a Bayesian approach involving importance sampling of the posterior predictive distribution to predict the efficacy of a new measurement at reducing the uncertainty of a selected prediction. We demonstrate the method by applying it to a case where we show that specific combinations of experiments result in more precise predictions. AVAILABILITY AND IMPLEMENTATION: Source code is available at: http://bmi.bmt.tue.nl/sysbio/software/pua.html.

Frequency of very low HCV viremia detected by a highly sensitive HCV-RNA assay.

BACKGROUND: Only limited data is available on the frequency and clinical significance of very low hepatitis C viremia (<600 IU/ml) determined by novel sensitive methods for HCV quantification. STUDY DESIGN: We evaluated the new Abbott m2000 RealTime PCR assay in 3213 consecutive anti-HCV-positive sera as well as in 50 HCV-recovered patients with sustained virological response to standard antiviral therapy. RESULTS: The assay showed a linear range between 10(1) IU/ml and 10(7) IU/ml for HCV genotypes 1-6. An HCV viremia below 600 IU/ml was detected more often with the m2000 RealTime PCR assay than with the Cobas Amplicor assay in viremic sera (7.1% versus 1.8%). Seventy-seven cases with HCV levels below 100 IU/ml not related to ongoing antiviral therapy were identified. An HCV-RNA of less than 12 IU/ml was found in nine of the 50 SVR patients. Two patients had a viral load of 34 IU/ml and 84 IU/ml, respectively, one of those showed persistently elevated ALT levels over a period of 5 years after the end of antiviral treatment. CONCLUSION: An HCV viremia below 600 IU/ml can be detected in almost every 40th anti-HCV-positive sera using real-time PCR based assays. Low persisting HCV-RNA in patients after antiviral therapy may be associated with mild liver inflammation in single cases.

Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA.

The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (UNG) contamination control option in the HCV test for previous Roche COBAS Amplicor users was evaluated. It proved to decrease amplicon carryover by 100-fold independent of the amplicon input concentration. The protocol including UNG proved to overcome problems with false-positive negative controls. Comparison with other assays revealed only minor differences. The largest difference was observed between the Abbott HCV RealTime assay and the Roche COBAS Amplicor HCV Monitor version 2.0 assay.

Detection of Treponema pallidum in the vitreous by PCR.

BACKGROUND: Ocular involvement of syphilis still poses a clinical challenge due to the chameleonic behaviour of the disease. As the serodiagnosis has significant limitations, the direct detection of Treponema pallidum (TP) in the vitreous represents a desirable diagnostic tool. METHODS: Real-time polymerase chain reaction (PCR) for the detection of TP was applied in diagnostic vitrectomies of two patients with acute chorioretinitis. Qualitative verification of TP by real-time PCR and melting point analysis according to a modified protocol was ruled out. Patients underwent complete ophthalmological examination with fundus photographs, fluorescein angiography, serological examination, antibiotic treatment and follow-up. RESULTS: In two cases of acute chorioretinitis of unknown origin, real-time PCR of vitreous specimens of both patients provided evidence of TP and was 100% specific. Initial diagnosis of presumed viral retinitis was ruled out by PCR of vitreous specimen. Patients were treated with systemic antibiotics and showed prompt improvement in visual function and resolution of fundus lesions. CONCLUSIONS: With real-time PCR, detection of TP in the vitreous was possible and delivered a sensitive, quick and inexpensive answer to a disease rather difficult to assess. In cases of acute chorioretinitis, the use of PCR-based assays of vitreous specimens in the diagnostic evaluation of patients is advisable. Although syphilitic chorioretinitis is a rare disease, PCR should include search for TP, as diagnostic dilemmas prolong definitive treatment in a sight-threatening disease.

Rapid DNA typing of HLA-B27 allele by real-time PCR using lightcycler technology.

For clinical diagnostic routine we developed a fast DNA typing of HLA-B27 by PCR and real-time detection using LightCycler technology. The method combines the sensitivity and specificity of PCR with the swiftness of the LightCycler system. The amplification step was performed with a primer set coding for a region in the third exon common to B*2701 to B*2705. The PCR cycles were monitored continuously using the SYBR Green I dye. Beta-globin was used as an internal control. An analysis of 32 samples with one PCR run was completed within 40 minutes. After amplification a melting curve analysis permitted the accurate identification of the PCR amplicons. The mean melting temperatures (Tm) were 90.5 degrees C and 87.3 degrees C, which are characteristic for HLA-B27 and beta-globin, respectively. A comparison of 300 samples which were typed for HLA-B27 with a conventional sequence-specific polymerase chain reaction (SSP-PCR) and with the new method demonstrated a perfect correlation (specificity 100%). In summary, the method described is fast, reliable, cost-effective and well adapted for routine laboratory testing.

Mutations in ribosomal protein S19 gene and diamond blackfan anemia: wide variations in phenotypic expression.

Mutations of the ribosomal protein S19 (RPS19) gene were recently identified in 10 patients with Diamond Blackfan anemia (DBA). To determine the prevalence of mutations in this gene in DBA and to begin to define the molecular basis for the observed variable clinical phenotype of this disorder, the genomic sequence of the 6 exons and the 5' untranslated region of the RPS19 gene was directly assessed in DBA index cases from 172 new families. Mutations affecting the coding sequence of RPS19 or splice sites were found in 34 cases (19.7%), whereas mutations in noncoding regions were found in 8 patients (4.6%). Mutations included nonsense, missense, splice sites, and frameshift mutations. A hot spot for missense mutations was identified between codons 52 and 62 of the RPS19 gene in a new sequence consensus motif W-[YFW]-[YF]-x-R-[AT]-A-[SA]-x-[AL]-R-[HRK]-[ILV]-Y. No correlation between the nature of mutations and the different patterns of clinical expression, including age at presentation, presence of malformations, and therapeutic outcome, could be documented. Moreover, RPS19 mutations were also found in some first-degree relatives presenting only with isolated high erythrocyte adenosine deaminase activity and/or macrocytosis. The lack of a consistent relationship between the nature of the mutations and the clinical phenotype implies that yet unidentified factors modulate the phenotypic expression of the primary genetic defect in families with RPS19 mutations.

Identification of new prognosis factors from the clinical and epidemiologic analysis of a registry of 229 Diamond-Blackfan anemia patients. DBA group of Société d'Hématologie et d'Immunologie Pédiatrique (SHIP), Gesellshaft für Pädiatrische Onkologie und Hämatologie (GPOH), and the European Society for Pediatric Hematology and Immunology (ESPHI).

Diamond-Blackfan anemia (DBA) is a constitutional disease characterized by a specific maturation defect in cells of erythroid lineage. We have assembled a registry of 229 DBA patients, which includes 151 patients from France, 70 from Germany, and eight from other countries. Presence of malformations was significantly and independently associated with familial history of DBA, short stature at presentation (before any steroid therapy), and absence of hypotrophy at birth. Two hundred twenty-two patients were available for long-term follow-up analysis (median, 111.5 mo). Of these individuals, 62.6% initially responded to steroid therapy. Initial steroid responsiveness was found significantly and independently associated with older age at presentation, familial history of DBA, and a normal platelet count at the time of diagnosis. Severe evolution of the disease (transfusion dependence or death) was significantly and independently associated with a younger age at presentation and with a history of premature birth. In contrast, patients with a familial history of the disease experienced a better outcome. Outcome analysis revealed the benefit of reassessing steroid responsiveness during the course of the disease for initially nonresponsive patients. Bone marrow transplantation was successful in 11/13 cases; HLA typing of probands and siblings should be performed early if patients are transfusion dependent, and cord blood should be preserved. Incidence of DBA (assessed for France over a 13-y period) is 7.3 cases per million live births without effect of seasonality on incidence of the disease or on malformative status. Similarly, no parental imprinting effect or anticipation phenomenon could be documented in families with dominant inheritance.

Assessment of iron stores in children with transfusion siderosis by biomagnetic liver susceptometry.

To investigate the applicability of noninvasive Superconducting Quantum Interference Device (SQUID) biomagnetic liver susceptometry and its limitations in thalassemic children, 23 patients with beta-thalassemia major and other iron loading anemias (age: 4-16 years) and 16 age-related normal children were studied. Liver iron concentrations ranged from 600 to 11,000 microg/g(liver) for thalassemic patients and from 60 to 340 microg/g(liver) for normal patients. Measuring the respective organ volumes by sonography, liver and spleen iron stores, accounting for 80% of total body iron stores, were estimated. Nonliver contributions from the lung or intestine to the measured SQUID signals in the small-sized patients were not observed. Moreover, livers in thalassemia were found to be enlarged by 18% per 1,000 microg/g (r = 0.75, P < 10(-3)). Serum ferritin values correlate significantly with iron stores (r = 0.64, P < 10(-3)), but predict iron stores only within large error intervals of 4,000 microg/g(liver). Analyzing the experimental data from biomagnetometry and from related transfusion and chelation treatment data within the framework of a two-compartment model, we were able to derive additional information on total body iron elimination and chelation therapy efficacy. The exponential decline of iron stores allows forecast of steady-state conditions of the final iron load for a particular transfusion and chelation therapy regimen.

Effect of cyclosporine A on the release of tissue factor pathway inhibitor from endothelial cells in heart transplant patients and cell culture.

We investigated the influence of cyclosporine A on the concentration of tissue factor pathway inhibitor and von Willebrand factor antigen in plasma of heart transplant outpatients. Tissue factor pathway inhibitor was quantified in plasma of blood donors (n = 50) and heart transplant outpatients (n = 50) by a chromogenic substrate assay with a mean of 32.4 micrograms/l and 98.2 micrograms/l, respectively. Von Willebrand factor antigen was determined with an enzyme-linked immunoassay with a mean of 90.9% for blood donors and 184.5% in plasma of heart transplant recipients. In addition, we investigated the effect of cyclosporine A on endothelial cell cultures over an incubation period of four days. A dose-dependent effect of cyclosporine A on the release of endothelial tissue factor pathway inhibitor and von Willebrand factor antigen was determined in a concentration range from 100 to 200 micrograms/l cyclosporine A. The tissue factor pathway inhibitor and von Willebrand factor antigen concentrations in the cell culture supernatant increased during the incubation time according to the cyclosporine A concentration 2-3 fold and 2 fold, respectively. For a further elucidation of the cyclosporine A effect we investigated the influence of cremophor EL, the vehicle of cyclosporine A. Cremophor EL alone did not increase the tissue factor pathway inhibitor release. However, the release was enhanced 2-4 fold after co-stimulation with the calcium ionophore A 23187 (10(-4) mol/l) in a concentration-dependent mode. We conclude that a generalized endothelial damage or activation is most probably caused by cyclosporine A and its vehicle cremophor EL. This process probably depends upon the increase of cytosolic free calcium, as described for the liberation of von Willebrand factor by endothelial cells.

Detection of the three Kunitz-type single domains of membrane-bound tissue factor pathway inhibitor (TFPI) by flow cytometry.

Tissue factor pathway inhibitor, a natural anticoagulant in the extrinsic pathway of blood coagulation, is associated with the endothelial membrane and presumed to be released by heparin. For flow cytometric detection of membrane-bound tissue factor pathway inhibitor we synthesized polyclonal monospecific antibodies directed against each of the three Kunitz-type domains. Antisera were obtained by immunisation of rabbits with synthetic oligopeptides representing the reactive site of each domain. Kunitz-domain delta 1: 26CAFKDDGPCKAIMKR41, domain delta 2: 101EDPGICRGYITR112 and domain delta 3: 192PADRGLCRANENR204. Different cell lines (chondrosarcoma, synovial sarcoma, synovial cells, leukaemic monocytes) and endothelial cells were investigated by flow cytometric analysis using these antibodies. The three tissue factor pathway inhibitor domains were detected on the surface of all cells by the corresponding antisera. Similar results were obtained by immuno-histochemical staining. Since domain delta 3 was recognised by the appropriate antibody, it would seem that this third domain is not the membrane binding site. To investigate the cellular tissue factor pathway inhibitor release, endothelial cells were cultivated with heparin. Protein resynthesis and translocation were inhibited by puromycin and monensin, respectively. After heparin incubation an increased tissue factor pathway inhibitor concentration was determined in the cell culture medium by a chromogenic substrate assay. However, the tissue factor pathway inhibitor density on the cell surface was not influenced by heparin, as shown by flow cytometry using the three tissue factor pathway inhibitor antisera. Our results suggest that functionally active tissue factor pathway inhibitor is not released from the cell surface. Therefore, the effect of heparin appears to be mediated by secretion of tissue factor pathway inhibitor from intracellular stores.