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Purpose The Ad26.COV2.S vaccine is a replication-incompetent human adenovirus type 26 vector encoding the SARS-CoV-2 spike protein. In a phase 1-2a trial, a single dose of Ad26.COV2.S induced SARS-CoV-2 spike-specific antibodies in ≥ 96% of healthy adults. To investigate vaccine immunogenicity in HIV-1-infection, we measured SARS-CoV-2 spike-specific antibodies in Ad26.COV2.S vaccinated HIV-1-infected patients and analyzed the presence of pre-existing Ad26 neutralizing antibodies. Methods We included all Ad26.COV2.S vaccinated HIV-1-infected patients of Erlangen HIV cohort fulfilling all inclusion criteria. The study cohort consisted of 15 HIV-1-infected patients and three HIV-1-uninfected subjects who received the Ad26.COV2.S vaccine between April and November 2021. Pre-vaccination sera were collected between October 2014 and June 2021, post-vaccination sera between June and December 2021. Neutralizing antibodies towards Ad26 were determined by a FACS-based inhibition assay measuring the expression of SARS-CoV-2 spike and adenoviral proteins in HEK293T cells after in-vitro transduction with Ad26.COV2.S or the control ChAdOx1-S. Results Six out of 15 HIV-1-infected patients failed to develop SARS-CoV-2-specific antibodies and four patients developed weak antibody responses after vaccination with Ad26.COV2.S. Pre-vaccination sera of four of the six vaccine non-responders showed neutralizing activity towards Ad26.COV2.S but not toward the ChAdOx1-S vaccine at 1:50 dilution. After Ad26.COV2.S vaccination, 17 of the 18 subjects developed strong Ad26-neutralizing activity and only one of the 18 subjects showed neutralizing activity towards the ChAdOx1-S vaccine. Conclusion Ad26.COV2.S vaccination showed a high failure rate in HIV-1-infected patients. Pre-existing immunity against Ad26 could be an important contributor to poor vaccine efficacy in a subgroup of patients.

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Purpose:If routine diagnostics are inconclusive, neurologic deterioration and death of patients with brain cancer are attributed to tumor or therapy. Therefore, diagnosing symptoms of encephalopathy caused by human cytomegalovirus (HCMV) reactivation remains uncommon. We investigated the role of HCMV reactivation in neurologic decline and clinical outcome after the start of radiochemotherapy.Experimental Design:HCMV analyses and extended MRI studies including additional independent retrospective neuroradiologic evaluation were performed at predetermined intervals and in case of sudden neurologic decline for 118 adult patients: 63 histologically proven high-grade gliomas, 55 with brain metastases. Immunophenotyping from simultaneously taken whole-blood samples was carried out to detect immune cells serving as prognostic marker for HCMV-associated complications. Symptomatic viremia and overall survival (OS) were the endpoints.Results:Twenty-four percent (28/118) of all patients (12/44 glioblastoma, 3/13 anaplastic astrocytoma; 8/31 non–small cell lung cancer (NSCLC), 13/24 other brain metastases) developed HCMV-viremia during or within 4 weeks after radiotherapy; 21 of 28 patients experienced concurrent major neurologic decline, reversible by antiviral treatment. Identified by immunophenotyping, pretherapeutically low basophil counts predicted a high-risk for HCMV-associated encephalopathy (glioblastoma: P = 0.002, NSCLC: P = 0.02). Median OS was substantially reduced after HCMV-associated encephalopathy without MRI signs of tumor progression [glioblastoma: 99 vs. 570 days (calculated 1-year OS: 22% vs. 69%; P = 0.01) and NSCLC: 47 vs. 219 days (calculated 1-year OS: 0% vs. 32%; P = 0.02)].Conclusions:For patients with brain cancer, HCMV reactivation after the start of radiochemotherapy is a frequent risk for cognitively detrimental but treatable encephalopathy and premature death. Routinely performed HCMV diagnostics, assessing basophil counts and study-based anti-viral regimens, are necessary to combat this hidden threat.See related commentary by Lawler et al., p. 3077

The analysis of T-cell responses in HIV-1-infected controllers may contribute to a better understanding of the protective components of the immune system. Here, we analyzed the HIV-1-specific T-cell response in a 59-year-old HIV-1-infected controller, infected for at least seven years, who presented with low viral loads ranging from 800 cells/µL. In γ-IFN-ELISpot assays using freshly isolated PBMCs, he displayed a very strong polyclonal T-cell response to eight epitopes in Gag, Nef and Rev; with the dominant responses directed against the HLA-B*57-epitope AISPRTLNAW and against a so-far-unknown epitope within Rev. Further analyses using peptide-stimulated T-cell lines in γ-IFN-ELISpot assays delineated the peptide RQRQIRSI (Rev-RI8) as a newly defined HLA-B*52-restricted epitope located within a functionally important region of Rev. Peptide-stimulation assays in 15 HLA-B*52-positive HIV-1-infected subjects, including the controller, demonstrated recognition of the Rev-RI8 epitope in 6/15 subjects. CD4 counts before the start of antiviral therapy were significantly higher in subjects with recognition of the Rev-RI8 epitope. Targeting of the Rev-RI8 epitope in Rev by CTL could contribute to the positive association of HLA-B*52 with a more favorable course of HIV-1-infection.

Puumala hantavirus (PUUV) infections usually show a mild or moderate clinical course, but may sometimes also lead to life-threatening disease. Here, we report on a 60-year-old female patient with common variable immunodeficiency (CVID) who developed a fatal PUUV infection with persistent renal failure, thrombocytopenia, and CNS infection with impaired consciousness and tetraparesis. Hantavirus-specific antibodies could not be detected due to the humoral immunodeficiency. Diagnosis and virological monitoring were based on the quantitative detection of PUUV RNA in blood, cerebrospinal fluid, bronchial lavage, and urine, where viral RNA was found over an unusually extended period of one month. Due to clinical deterioration and virus persistence, treatment with ribavirin was initiated. Additionally, fresh frozen plasma (FFP) from convalescent donors with a history of PUUV infection was administered. Despite viral clearance, the clinical condition of the patient did not improve and the patient died on day 81 of hospitalization. This case underlines the importance of the humoral immune response for the course of PUUV disease and illustrates the need for PCR-based virus diagnostics in those patients. Due to its potential antiviral activity, convalescent plasma should be considered in the therapy of severe hantavirus diseases.

Neurologic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can present with fever, headache, and meningism.

Purpose: If routine diagnostics are inconclusive, neurologic deterioration and death of patients with brain cancer are attributed to tumor or therapy. Therefore, diagnosing symptoms of encephalopathy caused by human cytomegalovirus (HCMV) reactivation remains uncommon. We investigated the role of HCMV reactivation in neurologic decline and clinical outcome after the start of radiochemotherapy. Experimental Design: HCMV analyses and extended MRI studies including additional independent retrospective neuroradiologic evaluation were performed at predetermined intervals and in case of sudden neurologic decline for 118 adult patients: 63 histologically proven high-grade gliomas, 55 with brain metastases. Immunophenotyping from simultaneously taken whole-blood samples was carried out to detect immune cells serving as prognostic marker for HCMV-associated complications. Symptomatic viremia and overall survival (OS) were the endpoints. Results: Twenty-four percent (28/118) of all patients (12/44 glioblastoma, 3/13 anaplastic astrocytoma; 8/31 non–small cell lung cancer (NSCLC), 13/24 other brain metastases) developed HCMV-viremia during or within 4 weeks after radiotherapy; 21 of 28 patients experienced concurrent major neurologic decline, reversible by antiviral treatment. Identified by immunophenotyping, pretherapeutically low basophil counts predicted a high-risk for HCMV-associated encephalopathy (glioblastoma: P = 0.002, NSCLC: P = 0.02). Median OS was substantially reduced after HCMV-associated encephalopathy without MRI signs of tumor progression [glioblastoma: 99 vs. 570 days (calculated 1-year OS: 22% vs. 69%; P = 0.01) and NSCLC: 47 vs. 219 days (calculated 1-year OS: 0% vs. 32%; P = 0.02)]. Conclusions: For patients with brain cancer, HCMV reactivation after the start of radiochemotherapy is a frequent risk for cognitively detrimental but treatable encephalopathy and premature death. Routinely performed HCMV diagnostics, assessing basophil counts and study-based anti-viral regimens, are necessary to combat this hidden threat. See related commentary by Lawler et al., p. 3077

SARS-CoV-2, the causative agent of COVID-19, has spread around the world with more than 700 million cases and 6.8 million deaths. Various variants of concern (VoC) have emerged due to mutations and recombination and concurrent selection for increased viral fitness and immune evasion. The viral protein that primarily determines the pathogenicity, infectivity, and transmissibility is the Spike protein. To analyze the specific impact of variant Spike proteins on infection dynamics, we constructed SARS-CoV-2 with a uniform B.1 backbone but with alternative Spike proteins. In addition, ORF6 was replaced by EYFP as a biological safety measure, and for use of this well-established reporter. We show that namely the delta variant Spike proteins cause a distinct phenotype from the wild type (B.1, D614G) and other variants of concern. Furthermore, we demonstrate that the omicron BA.1 Spike results in lower viral loads and a less efficient spread in vitro. Finally, we utilized viruses with the two different reporters EYFP and mCherry to establish a competitive growth assay, demonstrating that most but not all Spike variant viruses were able to outcompete wild type SARS-CoV-2 B.1.

In 2018, a cluster of pediatric human parechovirus (HPeV) infections in 2 neighboring German hospitals was detected. Viral protein 1 sequence analysis demonstrated co-circulation of different HPeV-3 sublineages and of HPeV-1 and -5 strains, thereby excluding a nosocomial outbreak. Our findings underline the need for HPeV diagnostics and sequence analysis for outbreak investigations.

Background To identify infants with congenital cytomegalovirus (cCMV) saliva polymerase chain reaction (PCR) is an ideal screening method. However, there are only few data on the influence of pre-analytic factors on the analytical sensitivity of the CMV PCR. Objectives This study aimed to evaluate the performance of different swabbing materials, transport time and initial virus concentration regarding to the efficacy of recovery of CMV-DNA. Study design Two CMV suspensions containing a high or low concentration of the laboratory strain AD 169 were prepared as test samples. Sampling was simulated by immersion of different swabs in these CMV suspensions and storing the swabs dry or in specified transport media. Transport conditions were modeled by storing the samples for defined time periods prior to DNA extraction and quantitative PCR analyses. Parallel analyses in two different laboratories allowed determination of lab to lab consistency. Results The duration of storage under the conditions analysed did not have a major effect on the recovery efficiency for the swabbing materials tested. With exception of flocked dry swabs, all tested swabbing materials demonstrated good recovery of CMV DNA. The flocked swab/eNAT system showed the best overall performance. Conclusions All tested swabbing materials (with exception of the flocked dry swabs) seem to be well suited for recovery of CMV DNA and appropriate for use for the diagnosis of cCMV infection in symptomatic cases and in general cCMV screening programs of newborns.

Background More than 15 million people in sub-Saharan Africa receive ART. Treatment failure is common, but the role of HIV drug resistance in treatment failure is largely unknown because drug resistance testing is not routinely done. This study determined the prevalence and patterns of HIV drug resistance in patients with suspected virological failure. Materials and methods A single high viral load of >1000 viral RNA copies/mL of plasma at any point during ART was considered as suspected virological failure. HIV-1 RNA was extracted from plasma samples of these patients using the QIAamp Viral RNA kit. The protease and part of the RT regions of the HIV pol gene were characterized. Results Viral load was determined in 317 patients; 64 (20.2%) had suspected virological failure. We successfully genotyped 56 samples; 48 (85.7%) had at least one major resistance-associated mutation (RAM). Common mutations in RT were M184V (75%), T215Y (41.1%), K103N (39.3%), M41L (32.1%), D67DN (30.3%), G190A (28.6%) and A98G (26.8%). No RAMs were detected in ART regimens based on a ritonavir-boosted PI. Conclusions The Tanzanian national guidelines define ‘virological failure’ as two consecutive viral load measurement results, at 3 month intervals, above the WHO threshold (1000 copies/mL). Here, we show that a single viral load above the WHO threshold is associated with high rates of RAMs. This suggests that a single high viral load measurement could be used to predict virological failure and avoid delays in switching patients from first-line to higher genetic barrier second-line regimens.

SARS-CoV-2 antibody assays are used for epidemiological studies and for the assessment of vaccine responses in highly vulnerable patients. So far, data on cross-reactivity of SARS-CoV-2 antibody assays is limited. Here, we compared four enzyme-linked immunosorbent assays (ELISAs; Vircell SARS-CoV-2 IgM/IgA and IgG, Euroimmun SARS-CoV-2 IgA and IgG) for detection of anti-SARS-CoV-2 antibodies in 207 patients with COVID-19, 178 patients with serological evidence of different bacterial infections, 107 patients with confirmed viral respiratory disease, and 80 controls from the pre-COVID-19 era. In COVID-19 patients, the assays showed highest sensitivity in week 3 (Vircell-IgM/A and Euroimmun-IgA: 78.9% each) and after week 7 (Vircell-IgG: 97.9%; Euroimmun-IgG: 92.1%). The antibody indices were higher in patients with fatal disease. In general, IgM/IgA assays had only limited or no benefit over IgG assays. In patients with non-SARS-CoV-2 respiratory infections, IgG assays were more specific than IgM/IgA assays, and bacterial infections were associated with more false-positive results than viral infections. The specificities in bacterial and viral infections were 68.0 and 81.3% (Vircell-IgM/IgA), 84.8 and 96.3% (Euroimmun-IgA), 97.8 and 86.0% (Vircell-IgG), and 97.8 and 99.1% (Euroimmun-IgG), respectively. Sera from patients positive for antibodies against Mycoplasma pneumoniae, Chlamydia psittaci, and Legionella pneumophila yielded particularly high rates of unspecific false-positive results in the IgM/IgA assays, which was revealed by applying a highly specific flow-cytometric assay using HEK 293 T cells expressing the SARS-CoV-2 spike protein. Positive results obtained with anti-SARS-CoV-2 IgM/IgA ELISAs require careful interpretation, especially if there is evidence for prior bacterial respiratory infections.

SARS-CoV-2 infection fatality ratios (IFR) remain controversially discussed with implications for political measures, but the number of registered infections depends on testing strategies and deduced case fatality ratios (CFR) are poor proxies for IFR. The German county of Tirschenreuth suffered a severe SARS-CoV-2 outbreak in spring 2020 with particularly high CFR.To estimate seroprevalence, dark figure, and IFR for the Tirschenreuth population aged ≥14 years in June/July 2020 with misclassification error control, we conducted a population-based study, including home visits for elderly, and analyzed 4203 participants for SARS-CoV-2 antibodies via three antibody tests (64% of our random sample). Latent class analysis yielded 8.6% standardized county-wide seroprevalence, dark figure factor 5.0, and 2.5% overall IFR. Seroprevalence was two-fold higher among medical workers and one third among current smokers with similar proportions of registered infections. While seroprevalence did not show an age-trend, the dark figure was 12.2 in the young versus 1.7 for ≥85-year-old. Age-specific IFRs were Our data underscore senior care home infections as key determinant of IFR additionally to age, insufficient targeted testing in the young, and the need for further investigations on behavioral or molecular causes of the fewer infections among current smokers.

SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid–based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections

ObjectivesIncreased importance in detection and surveillance of SARS-CoV-2 has been demonstrated due to the emergence of variants of concern (VOCs). In this study we evaluated if a commercially available real-time SARS-CoV-2 PCR assay can identify B.1.1.7 lineage samples by a specific N gene dropout or Ct value shift compared to the S or RdRP gene.MethodsPatients samples with confirmed B.1.1.7 variant by whole-genome sequencing and variant-specific PCR (n=48) and non-B.1.1.7 samples (n=53) were tested by the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay for presence of S, RdRP and N gene of SARS CoV-2. The N gene coding sequence of SARS-CoV-2 with and without D3L mutation (specific for B.1.1.7) were cloned into pCR®-TOPO vectors and Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay was performed.ResultsAll studied B.1.1.7 patient samples showed significantly higher Ct values (Δ 6-10, N-gene dropout on Ct values >29) in the N gene compared to the respective values of S and RdRP gene. Receiver operating characteristic (ROC) curve analysis resulted in 100% sensitivity and specificity for ΔCt N/RdRP and ΔCt N/S. As a result of the reversed genetic experiments we found also the shift in Ct values for the 3L variant N-gene.ConclusionsN gene dropout or Ct value shift is specific for B.1.1.7 positive samples using the Allplex™ SARS-CoV-2/FluA/FluB/RSV PCR assay. This approach can be used as a rapid tool for B.1.1.7 detection in single assay high throughput diagnostics.

Treatment with convalescent plasma has been shown to be safe in coronavirus disease in 2019 (COVID‐19) infection, although efficacy reported in immunocompetent patients varies. Nevertheless, neutralizing antibodies are a key requisite in the fight against viral infections. Patients depleted of antibody‐producing B cells, such as those treated with rituximab (anti‐CD20) for hematological malignancies, lack a fundamental part of their adaptive immunity. Treatment with convalescent plasma appears to be of general benefit in this particularly vulnerable cohort. We analyzed clinical course and inflammation markers of three B‐cell‐depleted patients suffering from COVID‐19 who were treated with convalescent plasma. In addition, we measured serum antibody levels as well as peripheral blood CD38/HLA‐DR‐positive T‐cells ex vivo and CD137‐positive T‐cells after in vitro stimulation with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)‐derived peptides in these patients. We observed that therapy with convalescent plasma was effective in all three patients and analysis of CD137‐positive T‐cells after stimulation with SARS‐CoV‐2 peptides showed an increase in peptide‐specific T‐cells after application of convalescent plasma. In conclusion, we here demonstrate efficacy of convalescent plasma therapy in three B‐cell‐depleted patients and present data that suggest that while application of convalescent plasma elevates systemic antibody levels only transiently, it may also boost specific T‐cell responses., Treatment of B‐cell depleted patients suffering from COVID‐19 with convalescent plasma led to viral clearance and was followed by an increase in peptide‐specific CD4+ T‐cells suggesting that transient antibody titers promoted specific cellular immunity in these patients.

Objectives Detection and surveillance of SARS-CoV-2 is of eminent importance, particularly due to the rapid emergence of variants of concern (VOCs). In this study we evaluated if a commercially available quantitative real-time PCR (qRT-PCR) assay can identify SARS-CoV-2 B.1.1.7 lineage samples by a specific N gene dropout or Ct value shift compared with the S or RdRp gene. Methods VOC B.1.1.7 and non-B.1.1.7 SARS-CoV-2-positive patient samples were identified via whole-genome sequencing and variant-specific PCR. Confirmed B.1.1.7 (n = 48) and non-B.1.1.7 samples (n = 58) were analysed using the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay for presence of SARS-CoV-2 S, RdRp and N genes. The N gene coding sequence of SARS-CoV-2 with and without the D3L mutation (specific for B.1.1.7) was cloned into pCR™II-TOPO™ vectors to validate polymorphism-dependent N gene dropout with the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay. Results All studied B.1.1.7-positive patient samples showed significantly higher Ct values in qRT-PCR (Δ6–10, N gene dropout on Ct values > 29) of N gene than the corresponding values of S (p ≤ 0.0001) and RdRp (p ≤ 0.0001) genes. The assay reliably discriminated B.1.1.7 and non-B.1.1.7 positive samples (area under the curve = 1) in a receiver operating characteristic curve analysis. Identical Ct value shifts (Δ7–10) were detected in reverse genetic experiments, using isolated plasmids containing N gene coding sequences corresponding to D3 or 3L variants. Discussion An N gene dropout or Ct value shift is shown for B.1.1.7-positive samples in the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay. This approach can be used as a rapid tool for B.1.1.7 detection in single assay high throughput diagnostics.

We found that a single nucleotide polymorphism (SNP) in the nucleoprotein gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a patient interfered with detection in a widely used commercial assay. Some 0.2% of the isolates in the EpiCoV database contain this SNP. Although SARS-CoV-2 was still detected by the other probe in the assay, this underlines the necessity of targeting two independent essential regions of a pathogen for reliable detection.

Fatal Borna Disease Virus 1 Infection This report describes a fatal case of severe encephalitis due to Borna disease virus 1.

Introduction:Influenza infection is a viral disease with significant morbidity and mortality during the cold months. Clinical presentation typically includes cough, fever, and pain. Influenza disease is hardly diagnosed only on the basis of clinical symptoms due to similar clinical presentation of other diseases such as a typical cold or other flu-like diseases. We evaluated patients with proven influenza who presented at an emergency department of internal medicine in a university hospital according to the clinical presentation and different age groups.Materials and Methods:From October 2017 to April 2018, 723 reverse transcription-polymerase chain reaction (RT-PCR) tests for influenza were performed in the emergency department on patients with suspected influenza diagnosed clinically. A total of 240 influenza-positive patients were retrospectively assessed for documented main symptoms, vital parameters, risk factors for an unfavorable course, hospitalization, and death.Results:The mean age of influenza patients was 65 years. Overall, 30 patients were aged 18 to 39 years, 48 patients 40 to 59 years, and 162 patients ⩾60 years. Influenza B in 168 (70%) was predominant to 72 influenza A (mostly H1N1). In only 30% of the patients all three typical symptoms (cough, fever, and headache/myalgia) were documented. Headache or myalgia (with 34%) was rather uncommon in influenza B. Sudden onset was cited in only 5.4%; 57% of all influenza patients were in hospital for a mean of 7.1 days, and 5.8% of all influenza patients died. Patients aged above 60 years had more risk factors, showed typical symptoms less frequently, and were hospitalized longer than younger patients (Conclusions:At an emergency department of internal medicine, influenza-diseased patients are of higher age, show an increased number of comorbidities, and are more likely to have milder symptoms documented. Elderly patients with influenza have a higher hospitalization rate with a longer hospital stay as compared with younger patients.

The majority of congenital cytomegalovirus (cCMV) infections are asymptomatic at birth and therefore not diagnosed. Approximately 10-15% of these infants develop late-onset hearing loss and other developmental disorders. Implementation of a universal screening approach at birth may allow early initiation of symptomatic interventions due to a closer follow-up of infants at risk and offers the opportunity to consider treatment of late-onset disease. Real-time PCR assays for the detection of CMV DNA in buccal swab samples demonstrated feasibility and good clinical sensitivity in comparison to a rapid culture screening assay. Because most cCMV infections remain asymptomatic, a universal screening assay that stratifies CMV infected infants according to low and high risk of late-onset cCMV disease could limit the parental anxiety and reduce follow-up costs. We therefore developed and characterized a screening algorithm based on a highly-sensitive quantitative real-time PCR assay that is compatible with centralized testing of samples from universal screening and allows to determine CMV DNA load of saliva samples either as International Units (IU)/ml saliva or IU/105 cell equivalents. 18 of 34 saliva samples of newborns that tested positively by the screening algorithm were confirmed by detection of CMV DNA in blood and/or urine samples obtained during the first weeks of life. All screening samples that could not be confirmed had viral loads of

For both patients with high-grade gliomas and multiple cerebral metastases, radio(chemo)therapy is the standard therapy. Neurological decline during treatment is rarely attributed to infections of the brain but to tumor progression or side effects of radiotherapy.We present 4 cases of cytomegalovirus (CMV) viremia associated with neurological deterioration, which occurred during or shortly after radiotherapy and/or chemotherapy of the brain (brain metastases 2, high-grade glioma 1, carcinoma infiltrating brain 1). In all cases, neurological decline was sudden and unexpected, and causes such as increased intracranial pressure or tumor progression could be excluded radiologically. Treatment with dexamethasone and mannitol had no or only very short-term effects. General infections were either excluded or receding before the neurological symptoms occurred. All patients presented with decreasing levels of thrombocytes. In all cases, CMV (re)activation could be proven using blood test for CMV-DNA. The anti-CMV-IgG status suggested reactivation rather than a primary infection. One patient died within 72 h of onset of the symptoms (results of CMV tests were received postmortem). Diagnosis of 3 patients allowed successful administration of antiviral treatment, which greatly improved the general and neurological conditions of the patients within 48 h.Neurological deterioration during RT is hardly ever attributed to viral infections. These cases suggest that CMV reactivation and subsequent infection might actually be causative and has to be considered and treated.Further prospective studies verifying and investigating this observation in terms of frequency and clinical relevance seem indicated.

Up to 40% of HIV-1 infected individuals are coinfected with human pegivirus type 1 (HPgV-1). The majority of studies, but not all, have reported a beneficial effect of HPgV-1 coinfection on HIV-1 disease progression. So far, the impact of different HPgV-1 genotypes on different HIV-1 subtypes remains unclear.Peptides derived from HPgV-1 envelope protein E2, and representing different viral genotypes, were synthesized using Fmoc/t-Bu-based solid phase peptide synthesis. The inhibitory effect of these peptides on the infection of reporter cell lines was tested using an HIV-1 subtype panel representing clades A (n = 2), AG (n = 2), B (n = 6), C (n = 2), D (n = 2), F (n = 2), G (n = 1), G/H (n = 1), and group O (n = 2).HIV-1 infection was blocked more efficiently by peptides derived from HPgV-1 GT2 than GT1 (P = 0.05). The HIV-1 subtype did not affect the degree of inhibition by a peptide derived from HPgV-1 GT2. All CXCR4-/dual-tropic isolates (n = 12), but only half (four out of eight) CCR5-tropic viruses were inhibited by this peptide (P = 0.014).Our data indicate that the inhibitory effect of peptides derived from HPgV-1 E2 protein is dependent on the genotype, suggesting that coinfection with HPgV-1 GT1 is less likely to confer a beneficial effect on HIV-1 disease progression than GT2. The preferential suppression of more pathogenic CXCR4-tropic HIV-1 by peptides derived from HPgV-1 GT2 may explain the favorable effect in patients harboring these HIV-1 isolates. Consequently, HPgV-1 genotype and HIV-1 coreceptor tropism are likely determinants for the beneficial effect of HPgV-1 co-infection in HIV-1-infected individuals.

Department of Neurology, Christian-Albrecht University and University Medical Center Schleswig-Holstein, Schittenhelmstr. 10, 24105 Kiel, Germany Institute for Infection Medicine, Christian-Albrecht University and University Medical Center Schleswig-Holstein, Brunswiker Str. 4, 24105 Kiel, Germany Department of Nephrology, Christian-Albrecht University and University Medical Center Schleswig-Holstein, Schittenhelmstr. 12, 24105 Kiel, Germany Virology Institute, Friedrich-Alexander University and University Medical Center, Schlossgarten 4, 91054 Erlangen, Germany Department of Radiology and Neuroradiology, Christian-Albrecht University and University Medical Center Schleswig-Holstein, Schittenhelmstr. 10, 4105 Kiel, Germany

Protein kinases represent central and multifunctional regulators of a balanced virus-host interaction. Cyclin-dependent protein kinase 7 (CDK7) plays crucial regulatory roles in cell cycle and transcription, both connected with the replication of many viruses. Previously, we developed a CDK7 inhibitor, LDC4297, that inhibits CDK7 in vitro in the nano-picomolar range. Novel data from a kinome-wide evaluation (>330 kinases profiled in vitro ) demonstrate a kinase selectivity. Importantly, we provide first evidence for the antiviral potential of the CDK7 inhibitor LDC4297, i.e., in exerting a block of the replication of human cytomegalovirus (HCMV) in primary human fibroblasts at nanomolar concentrations (50% effective concentration, 24.5 ± 1.3 nM). As a unique feature compared to approved antiherpesviral drugs, inhibition occurred already at the immediate-early level of HCMV gene expression. The mode of antiviral action was considered multifaceted since CDK7-regulated cellular factors that are supportive of HCMV replication were substantially affected by the inhibitors. An effect of LDC4297 was identified in the interference with HCMV-driven inactivation of retinoblastoma protein (Rb), a regulatory step generally considered a hallmark of herpesviral replication. In line with this finding, a broad inhibitory activity of the drug could be demonstrated against a selection of human and animal herpesviruses and adenoviruses, whereas other viruses only showed intermediate drug sensitivity. Summarized, the CDK7 inhibitor LDC4297 is a promising candidate for further antiviral drug development, possibly offering new options for a comprehensive approach to antiviral therapy.

As part of the Pr55Gag polyprotein, p6 fulfills an essential role in the late steps of the replication cycle. However, almost nothing is known about the functions of the mature HIV-1 p6 protein. Recently, we showed that p6 is a bona fide substrate of the insulin-degrading enzyme (IDE), a ubiquitously expressed zinc metalloprotease. This phenomenon appears to be specific for HIV-1, since p6 homologs of HIV-2, SIV and EIAV were IDE-insensitive. Furthermore, abrogation of the IDE-mediated degradation of p6 reduces the replication capacity of HIV-1 in an Env-dependent manner. However, it remained unclear to which extent the IDE mediated degradation is phylogenetically conserved among HIV-1. Here, we describe two HIV-1 isolates with IDE resistant p6 proteins. Sequence comparison allowed deducing one single amino acid regulating IDE sensitivity of p6. Exchanging the N-terminal leucine residue of p6 derived from the IDE sensitive isolate HIV-1NL4-3 with proline enhances its stability, while replacing Pro-1 of p6 from the IDE insensitive isolate SG3 with leucine restores susceptibility towards IDE. Phylogenetic analyses of this natural polymorphism revealed that the N-terminal leucine is characteristic for p6 derived from HIV-1 group M except for subtype A, which predominantly expresses p6 with an N-terminal proline. Consequently, p6 peptides derived from subtype A are not degraded by IDE. Thus, IDE mediated degradation of p6 is specific for HIV-1 group M isolates and not occasionally distributed among HIV-1.

Acute lymphoblastic leukemia (ALL) is the most common type of cancer in childhood and its incidence has risen steadily over the past decades. Growing evidence from epidemiological studies strongly suggests that the increased leukemia rate is likely related to an abnormal immune response to

Objective The etiology of focal cortical dysplasia type IIb (FCDIIb) remains enigmatic in patients suffering from drug-resistant epilepsy, and an aberrant activation of the mammalian target of rapamycin complex 1 signaling pathway (mTORC1) was detected in this developmental brain malformation. Recently, the human papillomavirus (HPV) oncoprotein E6 has been identified as a potent activator of mTORC1, and HPV16 E6 has been described to persist in balloon cells obtained from surgical FCDIIb specimens. Although this observation was replicated by an independent second report, it contradicts current knowledge of HPV biology. HPV infects the squamous or mucocutaneous epithelium; hematogenic spread into other tissues has not been observed. In addition, brain carcinogenesis has never been reported in FCDIIb patients. Herein, we have tried to confirm 2 previous reports of HPV16 E6 infection using an independent series of 14 surgical specimens with histopathologically confirmed FCDIIb. Methods Snap-frozen FCDIIb specimens were tested for HPV DNA using the primer set for amplification of the complete E6 reading frame of HPV16 and 3 other sets of primers (2 consensus primer sets detecting multiple HPV genotypes, and another primer set specifically used for HPV16). Furthermore, formalin-fixed and paraffin-embedded histopathological preparations were immunohistochemically analyzed using previously described antibodies directed against the HPV E6 oncoprotein. Results All 14 FCDIIb specimens were negative for HPV DNA with all 4 primer sets. Antibodies directed against the HPV E6 epitope showed weak labeling of cytoplasm in balloon cells, as previously described in FCDIIb, but also in other cell populations. Interpretation Our data did not confirm previously reported evidence for HPV16 detection in FCDIIb. Ann Neurol 2015;77:312–319

Background Human T-cell lymphotropic virus type 1 (HTLV-1) screening of blood and organ donors is not mandatory in Germany because of its low prevalence (about 7/100 000). An HTLV-1 transmission event caused by a multiple organ donor was investigated. Validity of diagnostic procedures and HTLV-1 disease association in immunosuppressed organ recipients were analyzed. Methods Two screening immunoassays and an immunoblot (confirmatory assay) were used for detection of HLTV-1/2 antibodies. Proviral DNA was quantified in blood and biopsies of organ recipients by HTLV-1 real-time polymerase chain reaction (PCR). Results Proviral HTLV-1-DNA was detected in all blood samples of 3 organ recipients (1-100 copies/10(2) cells), but seroconversion was delayed for up to 2 years in screening assays and >6 years in the confirmatory assay. In 2 of 3 organ recipients, a cutaneous T-cell lymphoma was diagnosed 2 and 3 years after infection, respectively. Proviral HTLV-1 DNA concentration was almost 100 copies/10(2) cells in cutaneous lymphoma biopsies whereas in biopsies of other tissues ≤3.0 copies/10(2) cells were found. The third organ recipient did not suffer from lymphoma, but detailed clinical data on this patient were not available to us. Conclusions Biopsy results support an etiological role for HTLV-1 in these cases of primary cutaneous T-cell lymphoma after solid organ transplant. HTLV-1-associated lymphoma can arise quickly in immunocompromised transplant recipients. The diagnosis of potentially HTLV-1-associated disease in organ recipients may require PCR because of delayed seroconversion.

The influenza season 2014/15 was dominated by drift variants of influenza A(H3N2), which resulted in a reduced vaccine effectiveness. It was not clear if the performance of commercial nucleic-acid-based amplification (NAT) assays for the detection of influenza was affected. The purpose of this study was to perform a real-life evaluation of two commercial NAT assays. During January-April 2015, we tested a total of 665 samples from patients with influenza-like illness using the Fast Track Diagnostics Respiratory pathogens 21, a commercial multiplex kit, (cohorts 1 and 2, n = 563 patients) and the Xpert Flu/RSV XC assay (cohort 3, n = 102 patients), a single-use cartridge system. An in-house influenza real-time RT-PCR (cohort 1) and the RealStar Influenza RT-PCR 1.0 Kit (cohort 2 and 3) served as reference tests. Compared to the reference assay, an overall agreement of 95.9 % (cohort 1), 95 % (cohort 2), and 98 % (cohort 3) was achieved. A total of 24 false-negative results were observed using the Fast Track Diagnostics Respiratory pathogens 21 kit. No false-negative results occurred using the Xpert Flu/RSV XC assay. The Fast Track Diagnostics Respiratory pathogens 21 kit and the Xpert Flu/RSV XC assay had sensitivities of 90.7 % and 100 % and specificities of 100 % and 94.1 %, respectively, compared to the RealStar 1.0 kit. Upon modification of the Fast Track Diagnostics Respiratory pathogens 21 kit, the sensitivity increased to 97.3 %. Influenza virus strains circulating during the 2014/15 season reduced the detection sensitivity of a commercial NAT assay, and continuous monitoring of test performance is therefore necessary.

Die Deutsche Vereinigung zur Bekampfung der Viruskrankheiten (DVV) e.V. und die Gesellschaft fur Virologie (GfV) e.V. geben nach unserem Wissen erstmalig in Europa konkrete Empfehlungen zum Einsatz von HIV-positiven Mitarbeiterinnen und Mitarbeitern im Gesundheitswesen. Grundsatzlich gelten fur HIV-positives medizinisches Personal klar definierte Auflagen. Bei einer dauerhaften Viruslast von ≤ 50 Kopien/ml konnen unter der Einhaltung der festgelegten Masnahmen (z. B. Tragen doppelter Handschuhe, regelmasige arbeitsmedizinische Betreuung, mindestens vierteljahrliche Kontrollen der HI-Viruslast, regelmasige Betreuung durch einen in der HIV-Therapie erfahrenen Arzt) von dem HIV-positiven medizinischen Beschaftigten alle operativen und invasiven Tatigkeiten durchgefuhrt werden. Die uneingeschrankte Tatigkeit des HIV-positiven medizinischen Beschaftigten ist jedoch nur dann moglich, wenn Adharenz bei der antiretroviralen Therapie besteht und wenn alle zur Vermeidung einer Infektionsubertragung erforderlichen Masnahmen regelrecht eingehalten werden. Unter Einhaltung der Vorgaben dieser Empfehlungen gehen von dem HIV-positiven medizinischen Beschaftigten mit an Sicherheit grenzender Wahrscheinlichkeit keine Gefahren fur Patienten aus.

Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p

BACKGROUND: In February 2007, a 63-year-old man underwent surgery. Retrospective testing with nucleic acid testing (NAT) showed that the patient was human immunodeficiency virus Type 1 (HIV-1) positive 10 days after transfusion. The transfusion-transmitted infection had been identified by a donor-related lookback started in April 2007 after anti-HIV seroconversion.METHODS: Sequence analysis was performed in the gag-pol region as well as in the V3 loop env region. Archived plasma from the transmitting donation was investigated for the individual-donation NAT with the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test (Roche CAP/CTM HIV-1 test) and for HIV antigen/antibody combination testing (Abbott Architect). Additional testing was done on the donor's follow-up sample and on the recipient's sample. RESULTS: The Roche CAP/CTM HIV-1 test failed to detect viral RNA by minipool NAT in the index donation (April 2007) as well as in the donation that caused the infection (January 2007). Phylogenetic analysis showed a very high genetic similarity among viral sequences from both donor and recipient, proving the HIV-1 transmission by sequence data. CONCLUSION: This case represents the first documented HIV-1 transmission by transfusion of red blood cells after mandatory introduction of HIV-1 NAT for blood screening in Germany. Low viral load and mismatches in the primer/probe region might explain the detection failure of the NAT screening assay. A certain risk remains that new virus variants contain mutations at positions critical for amplification or detection of viral genomes. An option to reduce the risk of a detection failure by NAT is the simultaneous use of several conserved regions as amplification targets.

Background Neurological decline during radio(chemo)therapy of the brain is often attributed to disease progression or side effects of radiotherapy. Diagnosis of opportunistic neurotropic infections such as cytomegalovirus (CMV) infections is uncommon, even though high-grade gliomas and some brain metastases are known to contain CMV particles. We prospectively examined the frequency of CMV encephalopathy during radiotherapy of the brain. Methods Fifty patients requiring whole-brain radiotherapy for brain metastases (n = 27) or local radio(chemo)therapy of the brain for high-grade gliomas (n = 23) were observed in the prospective observational GLIO-CMV-01 study. MRIs and blood samples were obtained before, halfway through, and at the end of radiotherapy. MRIs were screened for disease progression or increased intracranial pressure. Blood was tested for anti-CMV immunoglobulin (Ig)M, anti-CMV IgG, and CMV DNA. Results Thirty-two of 50 (64%) patients were positive for anti-CMV IgG before radio(chemo)therapy. Fifteen of those 32 (48%) developed viremia during or up to 28 days after treatment. Thirteen of those 15 (87%) required treatment for CMV-associated encephalopathy. MRIs were negative for disease progression, edema, or bleeding. None of the patients negative for anti-CMV IgG developed viremia, suggesting a reactivation rather than a primary infection.In the group at risk consisting of anti-CMV IgG+ patients, age >65 (P = .004) and the amount of dexamethasone taken during radio(chemo)therapy (P = .004) were associated with an increased risk for CMV-associated encephalopathy. One hundred and fifty days after the start of radio(chemo)therapy, survival was 74% (14/19) (no encephalopathy) versus 54% (7/13) (encephalopathy) (odds ratio, 0.42; 95% CI, 0.03-1.86; P = .25). Conclusion CMV reactivation frequently causes encephalopathy during radio(chemo)therapy of the brain. The unexpected high incidence of this infection makes it highly clinically relevant for every treating physician.

To determine the influence of human immunodeficiency virus type 1 (HIV-1)-specific CD8+T cells on the development of drug resistance mutations in the HIV-1 protease, we analyzed protease sequences from viruses from a human leukocyte antigen class I (HLA class I)-typed cohort of 94 HIV-1-positive individuals. In univariate statistical analyses (Fisher's exact test), minor and major drug resistance mutations as well as drug-associated polymorphisms showed associations with HLA class I alleles. All correlations withPvalues of 0.05 or less were considered to be relevant without corrections for multiple tests. A subset of these observed correlations was experimentally validated by enzyme-linked immunospot assays, allowing the definition of 10 new epitopes recognized by CD8+T cells from patients with the appropriate HLA class I type. Several drug resistance-associated mutations in the protease acted as escape mutations; however, cells from many patients were still able to generate CD8+T cells targeting the escape mutants. This result presumably indicates the usage of different T-cell receptors by CD8+T cells targeting these epitopes in these patients. Our results support a fundamental role for HLA class I-restricted immune responses in shaping the sequence of the HIV-1 protease in vivo. This role may have important clinical implications both for the understanding of drug resistance pathways and for the design of therapeutic vaccines targeting drug-resistant HIV-1.

Diagnostic virus isolation is still frequently used, particularly from respiratory tract secretions. Testing positive virus cultures for all possible viruses is time-consuming, and unexpected or unknown viruses may escape detection. Therefore, a novel random PCR approach was developed that allows sequence-independent amplification of viral nucleic acids from virus isolation-positive cultures. Selectivity for viral sequences is obtained by preferential isolation of nucleic acids that are particle associated and resistant to nucleases. Using primers with a degenerated 3′ end, the isolated nucleic acids are amplified and the randomly amplified PCR products are cloned and sequenced. As proof of the concept, the PAN-PCR approach was applied to supernatants of coxsackievirus B3 and murine adenovirus type 1-infected cells. Enterovirus and adenovirus sequences were obtained, demonstrating that the random PCR approach allows detection of RNA and DNA viruses. As a first application of this PAN-PCR approach, we characterized a virus isolate from mouth-washing material of a patient with chronic fatigue syndrome and high antibody titers to coxsackievirus B2. The virus isolate had tested negative for enteroviruses and respiratory viruses (influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus, and adenovirus) by immunofluorescence and PCR. Particle-associated, nuclease-resistant RNA and DNA were prepared from the supernatant of infected cells. The DNA and the reverse-transcribed RNA were randomly amplified, and PCR products were cloned and sequenced. Of 25 sequences obtained from the DNA preparation, 24 contained herpes simplex virus type 1 (HSV-1) sequences from 14 different loci spread over the HSV-1 genome. This result was confirmed by using a standard diagnostic HSV-PCR, demonstrating that the PAN-PCR correctly identified the virus isolate. Although the identification of HSV-1 in mouth-washing material is not surprising in retrospect, it clearly demonstrates the applicability of the PAN-PCR approach. This method should be particularly useful for characterizing virus isolates that have tested negative for all expected viruses and for identifying unknown viruses.

ZusammenfassungInfektionen mit einer Vielzahl von Viren sind im Zusammenhang mit akuten oder chronischen Arthritiden beschrieben. Gemeinsam ist den virusassoziierten Arthritiden, dass es sich um nicht eitrige Entzündungen handelt. Es kommen sowohl Mono- und Oligoarthritiden vor allem größerer Gelenke als auch Polyarthritiden vor. Als pathogenetischer Mechanismus wird vor allem die Ablagerung von Immunkomplexen angesehen. Ebenfalls beschrieben ist die direkte Infektion von Zellen der Synovia sowie die Bildung von Autoantikörpern. Systematische Untersuchungen zur Epidemiologie der vi-rusassoziierten Arthritis fehlen fast vollständig. Weltweit haben vermutlich Arthritiden im Rahmen von durch Moskitos übertragenen Alphavirus-Infektionen sowie bei Hepatitis B und Hepatitis C die größte Bedeutung. Wo diese Infektionen, wie bei uns in Deutschland, selten sind, steht als weiterer Erreger Parvovirus B19 im Vordergrund. Daneben spielen auch die durch Impfprogramme selten gewordenen Röteln- und Mumps-Infektionen eine gewisse Rolle. Bei Röteln ist zu beachten, dass es auch durch den attenuierten Lebendimpfstoff zu Arthritiden kommen kann. Möglichkeiten der spezifischen antiviralen Therapie und Prophylaxe sind bisher kaum etabliert.

Human immunodeficiency viruses in 321 samples from tenofovir-naïve patients were retrospectively evaluated for resistance to this nucleotide analogue. All virus strains with insertions between amino acids 67 and 70 of the reverse transcriptase (n= 6) were highly resistant. Virus strains with the Q151M mutation were divided into susceptible (n= 12) and highly resistant (n= 8) viruses. This difference was due to the absence or presence of the K65R mutation, which was confirmed by site-directed mutagenesis. Viral clones with various combinations of the mutations M41L, K70R, L210W, and T215F or T215Y were analyzed for cross-resistance induced by thymidine analogue mutations (TAMs). The levels of increased resistance induced by single, double, and triple mutations at the indicated positions could be ranked as follows: for mutants with single mutations, mutations at positions 41 > 215 > 70; for mutants with double mutations, mutations at positions 41 and 215 > 70 and 215 = 210 and 215 > 41 and 70; for mutants with triple mutations, mutations at positions 41, 210, and 215 > 41, 70, and 215. Viral clones with M184V or M184I exhibited slightly increased susceptibilities to tenofovir (0.7-fold). Almost all clones with TAM-induced resistance were resensitized when M184V was present (P< 0.001). Among the viruses in the clinical samples, the rate of tenofovir resistance significantly increased with the number of TAMs both in the samples with 184M and in those with 184V (P= 0.005 andP= 0.003, respectively). A resensitizing effect of M184V was confirmed for all samples exhibiting at least one TAM (P= 0.03). However, accumulation of at least two TAMs resulted in more than 2.0-fold reduced susceptibility to tenofovir, irrespective of the presence of M184V. Decision tree building, a classical machine learning technique, was used to generate models for the interpretation of mutations with respect to tenofovir resistance. The application of previously proposed cutoffs for a reduced response to therapy and treatment failure demonstrated the central roles of positions 215 and 65 for 1.5- and 4.0-fold reduced susceptibilities, respectively. Thus, clinically relevant resistance may be conferred by the accumulation of TAMs, and the resensitizing effect of M184V should be considered only minor.

Motivation: Despite some progress with antiretroviral combination therapies, therapeutic success in the management of HIV-infected patients is limited. The evolution of drug-resistant genetic variants in response to therapy plays a key role in treatment failure and finding a new potent drug combination after therapy failure is considered challenging. Results: To estimate the activity of a drug combination against a particular viral strain, we develop a scoring function whose independent variables describe a set of antiviral agents and viral DNA sequences coding for the molecular targets of the respective drugs. The construction of this activity score involves (1) predicting phenotypic drug resistance from genotypes for each drug individually, (2) probabilistic modeling of predicted resistance values and integration into a score for drug combinations, and (3) searching through the mutational neighborhood of the considered strain in order to estimate activity on nearby mutants. For a clinical data set, we determine the optimal search depth and show that the scoring scheme is predictive of therapeutic outcome. Properties of the activity score and applications are discussed. Contact: beerenwinkel@mpi-sb.mpg.de *To whom correspondence should be addressed. Keywords: HIV, antiretroviral therapy, drug resistance, SVM regression, therapy optimization, sequence space search.

A panel of 245 clinical samples with known treatment histories was retrospectively evaluated for cross-resistance to new protease inhibitors (PI). Samples with resistance to previously approved PI displayed high cross-resistance to atazanavir, whereas cross-resistance to amprenavir was considerably lower. A similar cross-resistance profile was observed for lopinavir, if a higher cut-off for resistance (9.5-fold) was applied. The enhanced efficacy of boosted PI is discussed with respect to clinically relevant cut-offs for drug resistance.

The focus of the current report lies on recent developments of synthetic methods applied to the synthesis of some high-valent complexes containing the nitrido functionality [N]3— as a link between a group 4, 5 or 6 transition metal and a main group element E (E = B, Si, Ge, P, S). Emphasis is put on results, that have been obtained within the “Schwerpunktprogramm “Nitridobrucken” funded by the Deutsche Forschungsgemeinschaft. The synthetic methods include condensation reactions of reactive chloro and oxo complexes (M = V, Nb, Ta, Cr, Mo, W) with silylamines, sulfonylamides, with N-silyl and N-lithio iminophosphoranes, furthermore methatesis reactions of oxo complexes with N-sulfonyl sulfinyl amides (M = V, Cr, Mo, W), the oxidative addition of element azides to d2 metal centers (M = V, W), and finally transamination reactions of N-H iminophosphoranes with amido complexes (M = Ti, Sm). Jungste Fortschritte in der Synthese von N-Heteroatom-substituierten Imidokomplexen mit Nitridobrucken [M=N E] (M = Metall der Gruppe 4, 5 und 6, E = B, Si, Ge, P, S) Der Fokus des vorliegenden Berichts liegt auf der Entwicklung synthetischer Methoden fur die Synthese hohervalenter Komplexe, die eine Nitridobaugruppe [N]3— als Bruckenfunktion zwischen einem Ubergangsmetall der Gruppe 4, 5 oder 6 und einem Hauptgruppenelement E (E = B, Si, Ge, P, S) enthalten. Besonders gewichtet werden Ergebnisse, die im Rahmen des durch die Deutsche Forschungsgemeinschaft geforderten Schwerpunktprogramms “Nitridobrucken

Drug resistance testing significantly improves response to antiretroviral treatment in HIV-1-infected patients, therefore it has recently been implemented into current guidelines for the management of antiretroviral therapy. Knowledge about technologies for measuring drug resistance is important for several reasons: (a) differences exist between different technologies and also between assays based on the same technology; (b) the results of resistance testing are strongly dependent on the reliability and precision of the technology used; and (c) technical aspects have to be considered for a clinically relevant interpretation of drug resistance. The spectrum of genotypic and phenotypic technologies as well as the technical quality is increasing, which shifts the emphasis to the interpretation of resistance profiles. The interpretation is based on the knowledge of drug resistance-associated mutations as well as correlations between genotype and phenotype and clinical response, which are incorporated into rules-based systems. Bioinformatic techniques are used to generate mathematical models for the prediction of drug resistance from genotype. Both approaches are converging toward the prediction of clinical response. Because therapy response is dependent on many additional variables, further efforts are required for the generation of a large clinical database. This will be the basis of a prediction system that will optimize the antiretroviral therapy for each individual patient.

Abacavir is frequently used in antiretroviral combination therapies as a potent nucleoside reverse transcriptase inhibitor (NRTI). Four mutations are selected for by abacavir in vitro and in vivo: K65R, L74V, Y115F, and M184V. Abacavir resistance has also been observed in NRTI multidrug-resistant samples. Furthermore, abacavir resistance has been described in the context of zidovudine resistance. To evaluate the genetic basis of abacavir resistance, the viral genotype and phenotypic resistance were analyzed for 307 patient samples. Low- and high-level resistances were defined as 2.5- to 5.5-fold- and >5.5-fold-reduced susceptibility, respectively. If all samples with abacavir-selected and NRTI multidrug resistance-associated mutations were scored as resistant, 27.6% of the samples were misclassified, mainly due to samples falsely scored as susceptible. Therefore, the relative frequencies of other mutations were evaluated. Mutations at codons 44 and 118 were rarely detected in abacavir-susceptible samples but were overrepresented in resistant samples. Site-directed mutagenesis of E44D, V118I, and M184V resulted in low-level resistance for the double mutant 44/184 and the triple mutant. Low-level abacavir resistance was also detected for a viral clone carrying zidovudine mutations only. Additional insertion of M184V into the zidovudine background doubled the resistance, whereas 44/118 did not lead to a further increase. Incorporating combinations of zidovudine mutations and M184V into the scoring system markedly reduced the number of misclassified samples, whereas 44/118 did not improve the prediction. In conclusion, the combination of M184V with zidovudine mutations gives rise to high-level abacavir resistance, which may be clinically relevant. Thus, options for useful sequential combinations of NRTI are limited.

2036 Background: As recently demonstrated (Neurooncology, 2016), neurological decline of patients with brain cancer (high grade glioma, brain metastases) during radio(chemo)therapy (RCT) of the brain is oftentimes caused by CMV-encephalopathy but not disease progression or therapeutic complications. We examined the impact of clinical and serological CMV-status on the survival one year after the onset of radio(chemo)therapy of the brain. Methods: 118 patients requiring whole-brain radiotherapy for brain metastases (n = 55) or local RCT of the brain for high-grade gliomas (n = 63) were observed in the prospective GLIO-CMV-01 study. MRIs and blood samples were obtained before, halfway through, and at the end of radiotherapy. MRIs were screened for disease progression or increased intracranial pressure. Blood was tested for anti-CMV immunoglobulin (Ig)M, anti-CMV IgG, and CMV DNA. Results: 68 of 118 (58%) patients were positive for anti-CMV IgG before radio(chemo)therapy. 28 of those 68 (41%) developed CMV-viremia during or up to 28 days after the end of irradiation. 21 of those 28 (75%) required treatment for symptomatic CMV-associated encephalopathy. One year after the start of RCT, survival was 72% (34/47) (no encephalopathy, anti-CMV-IgG+) or 68% (34/50) (no encephalopathy, anti-CMV-IgG-) versus 38% (8/21) (encephalopathy) (p = 0.0034). Conclusions: Symptomatic CMV-encephalopathy all but doubles the mortality of brain cancer patients within one year of RCT, despite antiviral treatment with ganciclovir. These findings heavily underline the importance to identify patients with increased risk profile for developing CMV-encephalopathy before initiating RCT.

The etiology of focal cortical dysplasia type IIb (FCDIIb) remains enigmatic in patients suffering from drug-resistant epilepsy, and an aberrant activation of the mammalian target of rapamycin complex 1 signaling pathway (mTORC1) was detected in this developmental brain malformation. Recently, the human papillomavirus (HPV) oncoprotein E6 has been identified as a potent activator of mTORC1, and HPV16 E6 has been described to persist in balloon cells obtained from surgical FCDIIb specimens. Although this observation was replicated by an independent second report, it contradicts current knowledge of HPV biology. HPV infects the squamous or mucocutaneous epithelium; hematogenic spread into other tissues has not been observed. In addition, brain carcinogenesis has never been reported in FCDIIb patients. Herein, we have tried to confirm 2 previous reports of HPV16 E6 infection using an independent series of 14 surgical specimens with histopathologically confirmed FCDIIb.Snap-frozen FCDIIb specimens were tested for HPV DNA using the primer set for amplification of the complete E6 reading frame of HPV16 and 3 other sets of primers (2 consensus primer sets detecting multiple HPV genotypes, and another primer set specifically used for HPV16). Furthermore, formalin-fixed and paraffin-embedded histopathological preparations were immunohistochemically analyzed using previously described antibodies directed against the HPV E6 oncoprotein.All 14 FCDIIb specimens were negative for HPV DNA with all 4 primer sets. Antibodies directed against the HPV E6 epitope showed weak labeling of cytoplasm in balloon cells, as previously described in FCDIIb, but also in other cell populations.Our data did not confirm previously reported evidence for HPV16 detection in FCDIIb.