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We analyzed 1,397 HIV-1 pol sequences of antiretroviral therapy-naive patients in a total of 7 university hospitals in Bonn, Cologne, Frankfurt, Hamburg, Hannover, and Munich, Germany. Phylogenetic and network analysis elucidated numerous cases of shared drug resistance mutations among genetically linked patients; K103N was the most frequently shared mutation.

In resource-limited or point-of-care settings, rapid diagnostic tests (RDTs), that aim to simultaneously detect HIV antibodies and p24 capsid (p24CA) antigen with high sensitivity, can pose important alternatives to screen for early infections. We evaluated the performance of the antibody and antigen components of the old and novel version of the Determine™ HIV-1/2 Ag/Ab Combo RDTs in parallel to quantifications in a fourth-generation antigen/antibody immunoassay (4G-EIA), p24CA antigen immunoassay (p24CA-EIA), immunoblots, and nucleic acid quantification. We included plasma samples of acute, treatment-naïve HIV-1 infections (Fiebig stages I–VI, subtypes A1, B, C, F, CRF02_AG, CRF02_AE, URF) or chronic HIV-1 and HIV-2 infections. The tests’ antigen component was evaluated also for a panel of subtype B HIV-1 transmitted/founder (T/F) viruses, HIV-2 strains and HIV-2 primary isolates. Furthermore, we assessed the analytical sensitivity of the RDTs to detect p24CA using a highly purified HIV-1NL4-3 p24CA standard. We found that 77% of plasma samples from acutely infected, immunoblot-negative HIV-1 patients in Fiebig stages II–III were identified by the new RDT, while only 25% scored positive in the old RDT. Both RDTs reacted to all samples from chronically HIV-1-infected and acutely HIV-1-infected patients with positive immunoblots. All specimens from chronically infected HIV-2 patients scored positive in the new RDT. Of note, the sensitivity of the RDTs to detect recombinant p24CA from a subtype B virus ranged between 50 and 200 pg/mL, mirrored also by the detection of HIV-1 T/F viruses only at antigen concentrations tenfold higher than suggested by the manufacturer. The RTD failed to recognize any of the HIV-2 viruses tested. Our results indicate that the new version of the Determine™ HIV-1/2 Ag/Ab Combo displays an increased sensitivity to detect HIV-1 p24CA-positive, immunoblot-negative plasma samples compared to the precursor version. The sensitivity of 4G-EIA and p24CA-EIA to detect the major structural HIV antigen, and thus to diagnose acute infections prior to seroconversion, is still superior.

Exclusive breastfeeding is recommended for women living with HIV (WLWH) in low-income-but not in high-income-countries, where milk substitutes are preferred. Some guidelines for high-income countries opted for a shared decision-making process regarding breastfeeding in optimal scenarios with adherence to antiretroviral therapy (cART), suppressed maternal viral load (mVL), and clinical monitoring. Although vertical transmission (VT) risk under cART is estimated below 1% in low-income settings, data from high-income countries are rare.We retrospectively analyzed all 181 live births from WLWH at the LMU Munich university hospital perinatal center in Germany between January 2016 and December 2020. We focused on VT, suppressed mVL and optimal scenario rates, breastfeeding frequency, cART regimens, and infant prophylaxis. All women were counseled according to current guidelines, foremost recommending avoidance of breastfeeding.In the 5-year cohort, no VT was observed. One hundred fifty-one WLWH (83.4%) decided not to breastfeed, even in optimal scenarios. Thrity infants (16.6%) were nursed, of which 25 were within an optimal scenario, whereas in 5 cases, breastfeeding was performed with a detectable VL in pregnancy or the postpartum period. All WLWH were treated with cART at delivery, and 91.7% sustained suppressed mVL. Zidovudine infant prophylaxis was given between 2 and 8 weeks but not necessarily over the whole breastfeeding duration and was declined from 5 breastfeeding WLWH.Although the cohort is too small to assess VT risk through breastfeeding with cART-suppressed mVL, breastfeeding might be an alternative even in high-income countries, but further studies are needed.

Sequestering and reprogramming of cellular metabolism represents one of the principal hallmarks of several cells. Antimicrobial peptides have been shown to exhibit selective anticancer activities. In this study, the secreted metabolome of HeLa cells under action of the antimicrobial peptide Crotamine from the venom of the South American rattlesnake Crotalus durissus terrificus was evaluated. Crotamine has been shown to be selective for highly proliferating cells and is able to extend the in vivo lifespan. The present study using a cell line of cervical cancer, HeLa cells, provide insights into how Crotamine acts in cell metabolism. NMR spectroscopy was used to identify and quantify relative metabolite levels, which are associated with Crotamine uptake. Statistical analysis reveals that Crotamine dramatically affects metabolites related to glycolysis, metabolism and biosynthesis of amino acids and pyruvate metabolism. The developed machine learning model is found to be robust by ROC curve analysis, suggesting that the metabolic state of HeLa cells treated with Crotamine is different from the control samples. To account for metabolite levels, it is suggested that Crotamine would have to act on glycolysis, which, in turn, affects several other metabolic pathways, such as, glutathione metabolism, TCA cycle and pyruvate metabolism. The observed metabolic changes shed light into the mode of Crotamine function.

The C30 Endopeptidase (3C-like protease; 3CLpro) is essential for the life cycle of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) since it plays a pivotal role in viral replication and transcription and is hence a promising drug target. Molecules isolated from animals, insects, plants or microorganisms can serve as a scaffold for the design of novel biopharmaceutical products. Crotamine, a small cationic peptide from the venom of the rattlesnake Crotalus durissus terrificus has been the focus of many studies since it exhibits activities such as analgesic, in vitro antibacterial and hemolytic activities. The crotamine derivative L-peptides (L-CDP) that inhibit the 3CL protease in the low µM range were examined since they are susceptible to proteolytic degradation; we explored the utility of their D-enantiomers form. Comparative uptake inhibition analysis showed D-CDP as a promising prototype for a D-peptide-based drug. We also found that the D-peptides can impair SARS-CoV-2 replication in vivo, probably targeting the viral protease 3CLpro.

Ubiquitous in citrus plants, Hesperidin and Hesperetin flavanones possess several biological functions, including antiviral activity. Arbovirus infections pose an ever-increasing threat to global healthcare systems. Among the severe arboviral infections currently known are those caused by members of the Flavivirus genus, for example, Dengue Virus—DENV, Yellow Fever Virus—YFV, and West Nile Virus—WNV. In this study, we characterize the inhibitory effect of Hesperidin and Hesperetin against DENV2, YFV, and WNV NS2B/NS3 proteases. We report the noncompetitive inhibition of the NS2B/NS3pro by the two bioflavonoids with half maximal inhibitory concentration (IC50) values &lt, 5 µM for HST and &lt, 70 µM for HSD. The determined dissociation constants (KD) of both flavonoids is significantly below the threshold value of 30 µM. Our findings demonstrate that a new generation of anti-flavivirus drugs could be developed based on selective optimization of both molecules.

Human immunodeficiency virus–2 (HIV-2) is endemic in some countries in West Africa. Due to the lower prevalence in industrialized countries, there is limited experience and knowledge on the management of individuals living with HIV-2 in Europe. Compared to HIV-1, there are differential characteristics of HIV-2 regarding diagnostic procedures, the clinical course, and, most importantly, antiretroviral therapy. We integrated the published literature on HIV-2 (studies and reports on epidemiology, diagnostics, the clinical course, and treatment), as well as expert experience in diagnosing and clinical care, to provide recommendations for a present standard of medical care of those living with HIV-2 in Western European countries, including an overview of strategies for diagnosis, monitoring, and treatment, with suggestions for effective drug combinations for first- and second-line treatments, post-exposure prophylaxis, and the prevention of mother-to-child transmission, as well as listings of mutations related to HIV-2 drug resistance and C-C motif chemokine receptor type 5 and C-X-C motif chemokine receptor type 4 coreceptor tropism.

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.

Arboviruses are transmitted by arthropods (arthropod-borne virus) which can be mosquitoes or other hematophagous arthropods, in which their life cycle occurs before transmission to other hosts. Arboviruses such as Dengue, Zika, Saint Louis Encephalitis, West Nile, Yellow Fever, Japanese Encephalitis, Rocio and Murray Valley Encephalitis viruses are some of the arboviruses transmitted biologically among vertebrate hosts by blood-taking vectors, mainly Aedes and Culex sp., and are associated with neurological, viscerotropic, and hemorrhagic reemerging diseases, posing as significant health and socioeconomic concern, as they become more and more adaptive to new environments, to arthropods vectors and human hosts. One of the main families that include mosquito-borne viruses is Flaviviridae, and here, we review the case of the Flavivirus genus, which comprises the viruses cited above, using a variety of research approaches published in literature, including genomics, transcriptomics, proteomics, metabolomics, etc., to better understand their structures as well as virus-host interactions, which are essential for development of future antiviral therapies.

Background RNA viruses are associated with a high frequency of mutations because of the missing proofreading function of polymerases, such as reverse transcriptase. Between 2007 and 2010, six blood donations with false-negative nucleic acid technology (NAT) results were reported in Germany. Therefore, NAT screening in two viral genome regions was introduced by our blood donation service in 2010 on a voluntary basis and became mandatory in Germany since the beginning of 2015. Study design and methods Blood donor screening was done using, in parallel, the German Red Cross (GRC) HIV-1 CE long terminate repeats (LTR) PCR kit and the GRC HIV-1 gag CE PCR kit. In total, 7 million blood donations were screened during the study period from 2010 to 2014 with the GRC dual-target human immunodeficiency virus 1 (HIV-1) NAT system. Additionally, three suspicious specimens were analyzed by four monotargeted NAT assays and by five dual-target NAT assays. Results Three of 7 million donations tested negative using the 5'LTR-polymerase chain reaction, but they were positive if amplification was performed in the gag region. HIV antibodies were detected in all three donations. Nucleic acid sequence analysis identified a deletion of 22 bases within the 5'LTR probe binding region. Three different ltr-based monotargeted assays missed two donations, except for a low-reactive result obtained by one of the assays. In total, the detection rates for HIV-1-positive donations were 37.5% (3/8) for monotargeted assays and 100% (10/10) for dual-target assays. Conclusion The current data demonstrate that dual-target NAT systems reduce the risk of false-negative HIV-1 NAT screening results.

Phospholipase-B-like (SVPLB-like) enzymes are present in relatively small amounts in a number of venoms, however, their biological function and mechanisms of action are un-clear. A three-dimensional model of the SVPLB-like enzyme from Crotalus adamanteus was generated by homology modeling based on the crystal structures of bovine Ntn-hydrolyases and the modeled protein possesses conserved domains characteristic of Ntn-hydrolases. Molecular dynamics simulations indicate that activation by autocatalytic cleavage results in the removal of 25 amino acids which increases accessibility to the active site. SVPLB-like enzymes possess a highly reactive cysteine and are hence amidases that to belong to the N-terminal nucleophile (Ntn) hydrolase family. The Ntn-hydrolases (N-terminal nucleophile) form a superfamily of diverse enzymes that are activated autocatalytically; wherein the N-terminal catalytic nucleophile is implicated in the cleavage of the amide bond.

Transmission of infectious agents might be associated with iatrogenic actions of charitable help in health care. An example is the vaccination against yellow fever in USA that transmitted hepatitis B virus. Another example is injections of praziquantel for treatment and cure of schistosomiasis in Central and Northern Africa, with a focus in Egypt that has spread hepatitis C virus. There is no indication that human T-lymphotropic virus type 1 was spread by injection treatment for African trypanosomiasis, syphilis and treponematosis, but these treatments might have contributed to the early spread of human immunodeficiency virus type 1 (HIV-1) in Central Africa. Slave trade contributed as well to the spread of viruses from Africa to the Americas; it was stopped in 1850. Until that date HIV-1 was not transported to the Americas. By analysis of nucleic acid sequence data it can be concluded that the continental spread of HCV and HIV-1 might have started around 1920 with an exponential phase from 1940 to 1970. Further iatrogenic actions that promoted the spread of HCV and HIV-1 might be vaccinations to prevent deadly diseases. The successful vaccination was followed by diminution of the infectious agent in the population such as small pox, yellow fever and measles. Measurements to reduce the spread of plague and cholera were further benefits increasing survival of diseased subjects in a population. Thus, the reduction of exposure to deadly infectious agents might have given a chance to HIV-1 infected subjects to survive and for HIV-1 to be distributed around the world starting from Central Africa in the 1950s.

HIV-1 diversity poses major challenges to viral load assays because genetic polymorphisms can impede nucleic acid detection. In addition to the on-going viral diversification within the HIV-1 group M pandemic, HIV-1 genetic diversity is further increased by non-group M infections, such as HIV-1 groups O (HIV-1-O), N and P. We here conducted a systematic evaluation of commercially available PCR assays to detect HIV-1-O isolates. We collected 25 primary HIV-1-O isolates covering all genetic clusters within HIV-1-O. Subsequently, this panel of isolates was tested on eight commercially available quantitative and five qualitative HIV-1 PCR-based assays in serial dilutions. Sequence analyses were performed for severe cases of underquantification or lack of detection. We observed differences between the assays in quantification that depended on the HIV-1-O isolate's subgroup. All three tested HIV-1-O subgroup IV isolates were underquantified by the Roche CAP/CTM >800-fold compared to the Abbott RealTime assay. In contrast, the latter assay underquantified several subgroup I isolates >200-fold. Notably, the Xpert HIV-1 Viral Load test from Cepheid failed to detect two of the HIV-1-O isolates, whereas the Roche Cobas 8800 assay readily detected all isolates. Comparative sequence analyses identified polymorphisms in the HIV-1-O long-terminal repeat and integrase genes that likely underlie inadequate nucleic acid amplification. Potential viral load underquantification should be considered in therapeutic monitoring of HIV-1-O-infected patients. Pre-clinical assessments of HIV-1 diagnostic assays could be harmonized by establishing improved and internationally standardized panels of HIV-1 isolates that cover the dynamic diversity of circulating HIV-1 strains.

Due to early antenatal screening and treatment, HIV mother-to-child transmission (MTCT) rarely occurs in Germany. The study aimed to investigate the impact on prevalence of HIV infection in the antenatal population and the incidence of late-presenting HIV-infected mothers attributable to increased numbers of refugees. Retrospective analysis and comparison were performed for all deliveries in HIV-infected pregnant women presenting to medical care in Munich (southern Germany) and Hamburg (northern Germany) covering two time periods, A (2010–2012) and B (2013–2015). In Munich, deliveries in HIV-infected pregnant women increased 1.6-fold from period A (n = 50) to B (n = 79) with late-presenting cases rising significantly from 2% (1/50) in period A to 13% (10/79) in B. In contrast, late-presenting cases in Hamburg decreased from 14% (14/100) in period A to 7% (7/107) in B, while the total number of HIV-infected women giving birth remained stable. From 2010 to 2015, one late-presenting pregnant woman transmitted HIV in Munich by presumed in utero mode of infection (case reviewed here), while no MTCT occurred in Hamburg. HIV infections diagnosed late in pregnancy and leading to delayed ART initiation are rising in Munich compared to Hamburg. Antenatal care of HIV-infected pregnant women in Munich appears to have been more affected by the recent refugee influx than Hamburg. Our study highlights the importance of screening all pregnant women for HIV early in pregnancy and providing timely health care access for pregnant refugees and asylum seekers to effectively prevent MTCT in Germany.

Die diagnostischen Moglichkeiten bei einer Infektion mit dem Hepatitis-B-Virus (HBV) sind vielfaltig. Daher ist die rationale Anwendung der unterschiedlichen Verfahren nicht ganz einfach. Sie wird im Folgenden diskutiert.

Background While Germany has a long tradition in HIV research with many well-established regional cohorts, there was a lack of collaborative efforts toward harmonized data collection and biobanking, both key strategies for efficient translational research projects. Key challenges are heterogeneity of data systems and privacy concepts, of existing study and data collection protocols, and sample collection, storage, and sharing. Methods In 2013, we established the Translational Platform HIV (TP-HIV) with support of the German Centre for Infection Research (DZIF) as a collaboration between university hospitals and specialized HIV care centers throughout Germany. After assessing the individual needs of all partner sites, we have taken comprehensive action to create a common platform for collaboration in all research stages. We developed protocols, rules of operation, biobanking strategies, and privacy concepts for all collaborating partner sites. Patients infected with HIV (PLWH) who sign the informed consent for the TP-HIV are pro- and retrospectively included in the cohort. Results To date, the TP-HIV infrastructure is implemented at 27 member sites from 11 cities, potentially extending to more than 20,000 patients currently treated for HIV across Germany. Facing the special needs in the German research environment, the TP-HIV established a unique data- and biomaterial collection allowing expedited translational research and reduce project overheads, regulatory burden, and data security regulations for investigators. By active surveillance, rapid access to individual patient groups such as patients with acute HIV infection, TP-HIV is an ideal platform for early phase clinical trials with new drug candidates. Researchers with clinical, biological, epidemiological, and statistical expertise have been brought together within the TP-HIV, which enables an effective translational chain from bench to bedside and back. New collaborations have been established with currently 23 active study protocols. Conclusion The TP-HIV has demonstrated to be a powerful tool for generating and testing research hypotheses in PLWH. In the future, we will work to further expand our network and address the pressing needs in the German research environment. Disclosures All authors: No reported disclosures.

Using HIV sequence data to characterize clusters of HIV transmission may provide insight into the epidemic. Phylogenetic and network analyses were performed to infer putative relationships between HIV-1 partial pol sequences from 2,774 individuals receiving care in three German regions between 1999–2016. The regions have in common that they host some of the largest annual festivals in Europe (Carnival and Oktoberfest). Putative links with sequences (n = 150,396) from the Los Alamos HIV Sequence database were evaluated. A total of 595/2,774 (21.4%) sequences linked with at least one other sequence, forming 184 transmission clusters. Clustering individuals were significantly more likely to be younger, male, and report sex with men as their main risk factor (p

RNA viruses are associated with a high frequency of mutations because of the missing proofreading function of polymerases, such as reverse transcriptase. Between 2007 and 2010, six blood donations with false-negative nucleic acid technology (NAT) results were reported in Germany. Therefore, NAT screening in two viral genome regions was introduced by our blood donation service in 2010 on a voluntary basis and became mandatory in Germany since the beginning of 2015.Blood donor screening was done using, in parallel, the German Red Cross (GRC) HIV-1 CE long terminate repeats (LTR) PCR kit and the GRC HIV-1 gag CE PCR kit. In total, 7 million blood donations were screened during the study period from 2010 to 2014 with the GRC dual-target human immunodeficiency virus 1 (HIV-1) NAT system. Additionally, three suspicious specimens were analyzed by four monotargeted NAT assays and by five dual-target NAT assays.Three of 7 million donations tested negative using the 5'LTR-polymerase chain reaction, but they were positive if amplification was performed in the gag region. HIV antibodies were detected in all three donations. Nucleic acid sequence analysis identified a deletion of 22 bases within the 5'LTR probe binding region. Three different ltr-based monotargeted assays missed two donations, except for a low-reactive result obtained by one of the assays. In total, the detection rates for HIV-1-positive donations were 37.5% (3/8) for monotargeted assays and 100% (10/10) for dual-target assays.The current data demonstrate that dual-target NAT systems reduce the risk of false-negative HIV-1 NAT screening results.

Zusammenfassung: Zur Diagnostik der Hepatitis E Virus (HEV) Infektion stehen heutzutage verschiedene virologische Methoden zur Verfügung. Die vermehrte Wahrnehmung sporadischer Fälle akuter Hepatitis E in Deutschland lenkt die Aufmerksamkeit zunehmend auf zoonotische Übertragungen des Virus. Die Kenntnis über unterschiedlich virulente HEV-Genotypen ist sowohl in Hinblick auf Epidemiologie und Krankheitsverlauf, als auch bei der Entwicklung und Auswahl diagnostischer Werkzeuge von Bedeutung. Es existieren eine Vielzahl enzymatischer und proteinbasierter Tests (ELISA, LIA, Western Blot), die anti-HEV IgG oder IgM Antikörper verschiedener HEV-Genotypen detektieren, jedoch große Unterschiede in Bezug auf Sensitivität und Spezifität aufweisen. Die heutzutage gebräuchlichste und am schnellsten auszuwertende Methode zur Diagnosesicherung einer Hepatitis E ist die PCR. Moderne, auch kommerziell erhältliche PCR-Kits können alle vier humanpathogenen Genotypen nachweisen. Zur Differenzierung der Genotypen wird in der Regel eine Sequenzierung durchgeführt, die bisher jedoch nur bei spezieller epidemiologischer Fragestellung von Relevanz ist. Methoden wie Antigennachweis, Virusanzucht oder T-Zell Assays haben bislang keine Bedeutung in der Routinediagnostik. Auch in Zukunft werden neue Erkenntnisse über die Pathogenese des Virus, seine klinische Relevanz bei bestimmten Patientengruppen (z.B. Immunsupprimierten) und die Anwendung antiviraler und prophylaktischer Therapien (Impfung) Leistungsmerkmale existierender Testformate herausfordern und die Anforderungen an durchführende diagnostische Labore erhöhen.

The past decade has witnessed a dramatic increase of anti-retroviral treatment of human immunodeficiency virus (HIV) infected patients in many African countries. Due to costs and lack of currently available commercial viral load assays, insufficient attention has been paid to therapy monitoring through measurement of plasma viral load. This challenge of patient monitoring by tests as viral load, CD4 cell count, and finally HIV drug resistance could reverse achievements already made against HIV/AIDS infection. Loop-mediated isothermal amplification (LAMP) has been shown to be simple, rapid and cost-effective, characteristics which make this assay suitable for viral load monitoring in resource limited settings. This paper describes a revised LAMP assay using primers in the HIV-1 integrase region. The assay can be used for semi-quantitative measurement of HIV-1 group M viral load. The lower limit of detection (LLOD) was determined as 1200copies/mL and lower limit of quantitation (LLOQ) at 9800copies/mL. Sensitivities of 82 and 86% (in 135 and 99 plasma samples respectively from Kenya) and 93% (in 112 plasma samples from Germany) and specificities of 99 and 100% were realized. HIV-1 group O and HIV-2 virus samples were not detected. This LAMP assay has the potential for semi-quantitation of HIV-1 group M viral load in resource limited countries. There is still a need for further improvement by refinement of primers in respect to detection of HIV-1 group M non-B virus.

Background: Scale-up of antiretroviral therapy (ART) and the growing number of longterm treated patients may favor multi-HIV drug resistance (HIVDR) in resource-limited settings. Understanding the burden of HIVDR with ART-exposure may provide new insights for an effective long-term management of infected patients. Methods: Sixty-six HIV-infected individuals (18 ART-naive, 24 failing first-line, 24 failing secondline ART) living in Yaounde-Cameroon were evaluated by sequencing protease-reverse transcriptase (PR-RT, n=62), envelope-V3 loop (V3, n=58) and integrase (IN, n=30) regions. Drug resistance mutations (DRMs) were interpreted using Stanford University HIV drug resistance database and geno2pheno, while viral tropism prediction was done using geno2pheno, position-specific scoring matrices (PSSM) and Net charge rule. Results: Participants, from naive, first- to second-line, had respectively 5.30, 4.85 and 4.66 log HIV RNA, and 532, 203 and 146 CD4 cells/mm 3 ), and infected with diverse HIV-1 non-B clades (58.1% CRF02_AG). Among ART-naive patients, 6.7% harbored K103N, 28.6% had IN accessory-mutations (L74I, E157Q) and 26.7% carried CXCR4-tropic viruses. At first-line failure, 79.2% harbored DRMs to nucleoside and non-nucleoside RT inhibitors, 33.3% had IN accessory-mutations (L68I, L74I, T97A, E157Q), and 47.4% carried CXCR4-tropic viruses. At second-line failure, 91.3% harbored multi-DRMs to PR-RT inhibitors (with 52.2% and 4.3% DRMs to second-generation NNRTIs and darunavir/ r, respectively), 27.3% had IN accessory-mutations (L74I, T97A, E157EQ), and 37.5% carried CXCR4-tropic viruses. Conclusion: Levels of PR-RT resistance increases with ART-exposure, with needs for new ART-options following second-line failure. IN inhibitors and darunavir/r are potentially suitable for a third-line regimen, while the use of maraviroc, etravirine or rilpivirine, requires individual genotypic testing.

Various items are complicating the treatment of HIV-2 infected patients. Compared to HIV-1 there is much less treatment experience, no evidence from randomized control trials, a reduced number of effective drugs and no broadly available test for viral load monitoring. In case of treatment failure there is only limited guidance and presently no easy accessible tool for nucleic acid sequence interpretation available. To solve this problem, we initiated an expert workshop to address some of these problems. A panel of experts from four different European countries voted on a rule set for interpretation of mutations in the HIV-2 protease, reverse transcriptase and integrase. Rules were proposed by each member and were then modified during discussion by considering data gained from HIV-1 and accumulated experience of the follow up of HIV-2-infected patients. Based on the HIV-GRADE internet-tool an online tool was developed to make the rule set easily accessible and usable. Rules were laid down for the interpretation of HIV-2 drug resistance to NRTIs, PIs and INIs (integrase inhibitors). Due to natural resistance of HIV-2, usage of NNRTIs and T-20 was not recommended as part of an antiretroviral regimen for HIV-2. These rules were then translated in a machine interpretable format (algorithm specification interface, ASI) and the HIV-GRADE tool was extended for usage of HIV-2 sequences. Further consensus sequences were generated from the reference sequence data set provided by Los Alamos National Laboratories. In contrast to HIV-1, mutations were compared to a group specific consensus sequence (Group A or Group B) and not to a consensus sequence from the most predominant HIV-2 Group A. This change was necessary due to significant differences between the various HIV-2 strains. We developed a rule set and an automated tool for HIV-2 drug resistance analyses. This tool and the rules will be freely available on the internet. Access to the pre-publication versions can be granted by each of the group members. To keep the algorithm rules up-to-date it will be actualized on a yearly basis. (Published: 11 November 2012) Citation: Abstracts of the Eleventh International Congress on Drug Therapy in HIV Infection Obermeier M et al. Journal of the International AIDS Society 2012, 15 (Suppl 4):18180 http://www.jiasociety.org/index.php/jias/article/view/18180 | http://dx.doi.org/10.7448/IAS.15.6.18180

Before elective operations, particularly orthopaedic surgery, national guidelines in Germany recommend testing for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) to reduce the risk of transmission of the virus through a needlestick or cutting injury. Such testing is expensive. The number of new and unknown diagnoses of viral infections that can be detected by routine screening has not yet been evaluated.The aim of our department of orthopaedic surgery is to screen every adult patient listed for an operation for HBV, HCV and HIV. We retrospectively analysed the number of operations in this single centre from 2001 to 2010, correlated this number with the total number of screens and calculated the number of newly diagnosed infections. An additional cost:benefit ratio was calculated.A total of 20,869 operations were performed by the department between 2001 and 2010. After exclusion of all interventions in children and all patients who had multiple operations, 15,482 patients remained. Test results were found for 10,011 of these patients during this period (screening rate 65 %). Of those screened, in only four cases (0.4 ‰) was a previously unknown infection detected.Two-thirds of the patients included in our study actually underwent screening; this rate was lower than expected. The incidence of newly detected infections was low, putting the benefit of a routine preoperative screening for HBV, HCV and HIV into question. From an economic point of view the low detection rate is a strong argument in favour of omitting routine preoperative screening. Screening only those patients with risk factors may be as safe as screening every patient and would help reduce costs.

Background CCR5-coreceptor antagonists can be used for treating HIV-2 infected individuals. Before initiating treatment with coreceptor antagonists, viral coreceptor usage should be determined to ensure that the virus can use only the CCR5 coreceptor (R5) and cannot evade the drug by using the CXCR4 coreceptor (X4-capable). However, until now, no online tool for the genotypic identification of HIV-2 coreceptor usage had been available. Furthermore, there is a lack of knowledge on the determinants of HIV-2 coreceptor usage. Therefore, we developed a data-driven web service for the prediction of HIV-2 coreceptor usage from the V3 loop of the HIV-2 glycoprotein and used the tool to identify novel discriminatory features of X4-capable variants. Results Using 10 runs of tenfold cross validation, we selected a linear support vector machine (SVM) as the model for geno2pheno[coreceptor-hiv2], because it outperformed the other SVMs with an area under the ROC curve (AUC) of 0.95. We found that SVMs were highly accurate in identifying HIV-2 coreceptor usage, attaining sensitivities of 73.5% and specificities of 96% during tenfold nested cross validation. The predictive performance of SVMs was not significantly different (p value 0.37) from an existing rules-based approach. Moreover, geno2pheno[coreceptor-hiv2] achieved a predictive accuracy of 100% and outperformed the existing approach on an independent data set containing nine new isolates with corresponding phenotypic measurements of coreceptor usage. geno2pheno[coreceptor-hiv2] could not only reproduce the established markers of CXCR4-usage, but also revealed novel markers: the substitutions 27K, 15G, and 8S were significantly predictive of CXCR4 usage. Furthermore, SVMs trained on the amino-acid sequences of the V1 and V2 loops were also quite accurate in predicting coreceptor usage (AUCs of 0.84 and 0.65, respectively). Conclusions In this study, we developed geno2pheno[coreceptor-hiv2], the first online tool for the prediction of HIV-2 coreceptor usage from the V3 loop. Using our method, we identified novel amino-acid markers of X4-capable variants in the V3 loop and found that HIV-2 coreceptor usage is also influenced by the V1/V2 region. The tool can aid clinicians in deciding whether coreceptor antagonists such as maraviroc are a treatment option and enables epidemiological studies investigating HIV-2 coreceptor usage. geno2pheno[coreceptor-hiv2] is freely available at http://coreceptor-hiv2.geno2pheno.org. Electronic supplementary material The online version of this article (doi:10.1186/s12977-016-0320-7) contains supplementary material, which is available to authorized users.

Novel therapeutic options are urgently needed to improve global treatment of virus infections. Herbal products with confirmed clinical safety features are attractive starting material for the identification of new antiviral activities. Here we demonstrate that Cistus incanus (Ci) herbal products inhibit human immunodeficiency virus (HIV) infections in vitro. Ci extract inhibited clinical HIV-1 and HIV-2 isolates, and, importantly, a virus isolate with multiple drug resistances, confirming broad anti-HIV activity. Antiviral activity was highly selective for virus particles, preventing primary attachment of the virus to the cell surface and viral envelope proteins from binding to heparin. Bioassay-guided fractionation indicated that Ci extract contains numerous antiviral compounds and therefore has favorably low propensity to induce virus resistance. Indeed, no resistant viruses emerged during 24 weeks of continuous propagation of the virus in the presence of Ci extracts. Finally, Ci extracts also inhibited infection by virus particles pseudotyped with Ebola and Marburg virus envelope proteins, indicating that antiviral activity of Ci extract extends to emerging viral pathogens. These results demonstrate that Ci extracts show potent and broad in vitro antiviral activity against viruses that cause life-threatening diseases in humans and are promising sources of agents that target virus particles.

Human herpesvirus-8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS), which primarily affects human immunodeficiency virus (HIV)-infected adults with advanced immunodeficiency. Currently, only limited prevalence data for HHV-8 infection in HIV-infected children living in non-endemic areas are available. This multicenter cross-sectional study was conducted in four university hospitals in Germany specializing in pediatric HIV care. Stored serum specimens obtained from 207 vertically HIV-1-infected children and adolescents were tested for antibodies against lytic and latent HHV-8 antigens. Logistic regression was used to assess independent risk factors associated with HHV-8 seropositivity. The overall HHV-8 seroprevalence was 24.6 % (n = 51/207) without significant differences related to sex, age, or ethnicity. In univariate analysis, HHV-8 seropositivity was significantly associated with a child having being born outside Germany, maternal origin from sub-Saharan Africa, a history of breastfeeding, CDC immunologic category 3, and deferred initiation of antiretroviral therapy (>24 months of age). In multivariate analysis, a child's birth outside Germany was the only significant risk factor for HHV-8 seropositivity (odds ratio 3.98; 95 % confidence interval 1.27-12.42). HHV-8-associated malignancies were uncommon; only one patient had a history of KS. Serum specimen of vertically HIV-infected children and adolescents living in Germany showed a high HHV-8 seroprevalence. These findings suggest that primary HHV-8 infection-a risk factor for KS and other HHV-8-associated malignancies-occurs early in life. Thus, management of perinatally HIV-infected children should include testing for HHV-8 coinfection and should consider future risks of HHV-8-associated malignancies.

HIV-1 evolved by zoonotic transmission of chimpanzee lentivirus to man around 1920. HIV-1 is now divided in four groups that had been transmitted by at least four independent events. HIV-2 originates from sooty mangabey monkeys in West Africa and was transmitted to man around 1940. For HIV-2 at least eight transmission events, forming the groups A to H, led to the ongoing epidemic. Regarding HIV and the AIDS epidemic it should be prevented that further animal viruses will be introduced to the human population.

HIV-1 is a chimpanzee virus which was transmitted to humans by several zoonotic events resulting in infection with HIV-1 groups M–P, and in parallel transmission events from sooty mangabey monkey viruses leading to infections with HIV-2 groups A–H. Both viruses have circulated in the human population for about 80 years. In the infected patient, HIV mutates, and by elimination of some of the viruses by the action of the immune system individual quasispecies are formed. Along with the selection of the fittest viruses, mutation and recombination after superinfection with HIV from different groups or subtypes have resulted in the diversity of their patterns of geographic distribution. Despite the high variability observed, some essential parts of the HIV genome are highly conserved. Viral diversity is further facilitated in some parts of the HIV genome by drug selection pressure and may also be enhanced by different genetic factors, including HLA in patients from different regions of the world. Viral and human genetic factors influence pathogenesis. Viral genetic factors are proteins such as Tat, Vif and Rev. Human genetic factors associated with a better clinical outcome are proteins such as APOBEC, langerin, tetherin and chemokine receptor 5 (CCR5) and HLA B27, B57, DRB1*1303, KIR and PARD3B.

Antiretroviral drug resistance is mostly linked to a complex interaction of several amino acids with variable importance or a single amino acid. To facilitate the interpretation of observed mutation patterns, hospital university centers have developed several interpretation systems. All the currently available interpretation algorithms evolved, are being continuously updated and have been improved during the last decade. Some discrepancies are still evident that are partially smoothened by link of the individual programs with other systems. After the interpretation of HIV-1 group M subtype B mutations, a refined algorithm for the other group M subtypes was developed followed by the interpretation of HIV-1 group O and HIV-2 mutations. The process of improvement is ongoing, due to the better understanding and interpretation of single and cluster mutations and the availability of new antiretroviral substances. The knowledge gained from the experience of HIV drug resistance testing has been used to establish the interpretation of HBV polymerase mutations and will be extended for the treatment of HCV infected with protease inhibitors.

The diagnosis of hepatitis E virus (HEV) infections has been recently substantially facilitated by the introduction of a whole range of new different virological assays. The increasing appearance of sporadic cases of acute hepatitis E in Germany directed the focus toward the zoonotic transmission route of the virus. The recognition of HEV genotypes differing in virulence and in pathogenic potential is not only relevant for epidemiology and the course of the disease, but also for the development and choice of diagnostic tools. A broad variety of enzymatic and protein-based assay formats detecting anti-HEV IgG or IgM antibodies directed against the different genotype variants of HEV is available (ELISA, LIA, Western blot); however, sensitivity and specificity of these assays differ notably. Today’s state-of-the art technology that permits fast and reliable assay-based confirmation of HEV infections is PCR. The newly developed commercially available PCR kits will detect all four human pathogenic HEV genotypes. Further subdivision and discrimination can be achieved by sequencing, although this approach is only reasonable in the setting of specific epidemiological demands. Detection of viral antigens, cell culture, and T-cell assays are of no practical importance in a routine diagnostic setting. New insight into the pathogenesis and its clinical relevance for defined groups of patients (immunosuppressed) as well as the implementation of specific antiviral and prophylactic therapies (vaccination) will further challenge the performance of existing assay formats and increase the technical demands for the diagnostic laboratory.

Antiretroviral treatment directed against HIV is highly effective, yet limited by drug resistance mutations. We hypothesized that CD8 T cells targeting drug-resistant HIV mutants are able to inhibit viral replication in the setting of a failing therapeutic regimen. We evaluated CD8 T-cell responses and mapped epitopes in HIV-infected patients by interferon-gamma Elispot and intracellular cytokine staining. Autologous virus was sequenced by RT-PCR. Viral replication inhibition assays were performed using M184V mutant virus and CD8 T cell lines. CD8 T-cell responses toward the regions of viral drug resistance mutations in Pol are frequent. Focusing on the M184V mutation, A*02:01-YQYVDDLYV and A*02:01-VIYQYVDDLYV were identified as optimal epitopes for the majority of study subjects. Viral replication of M184V HIV mutants was inhibited by CD8 T cell lines in vitro. In case of a failing lamivudine/emtricitabine containing regimen, individuals with a CD8 T-cell response toward M184V had a significant lower viral load than those without a CD8 response (p = 0.005). Two study subjects even achieved an undetectable viral load. Our data suggest that control of M184V mutant virus by CD8 T-cell responses is possible in vitro and in vivo. This control has important implications for therapeutic vaccination strategies.

Its stable particle structure combined with its high immunogenicity makes the hepatitis A virus (HAV) a perfect carrier to expose foreign epitopes to the host immune system. In an earlier report [Beneduce, F., Kusov, Y., Klinger, M., Gauss-Müller, V., Morace, G., 2002. Chimeric hepatitis A virus particles presenting a foreign epitope (HIV gp41) at their surface. Antiviral Res. 55, 369-377] chimeric virus-like particles (HAV-gp41) were described that carried at their surface the dominant gp41 epitope 2F5 (2F5e) of the human immunodeficiency virus HIV-1. Extending this work, we now report that chimeric virus HAV-gp41 replicates in HAV-susceptible cells as well as in non-human primates. Infected marmosets developed both an anti-HAV and anti-2F5 epitope immune response. Furthermore, an HIV-neutralizing antibody response was elicited in guinea pigs immunized with HAV-gp41 chimeric particles. The results demonstrate that the replication-competent chimeric HAV-gp41 can serve as either a live or a subunit vaccine for eliciting of antibodies directed against a foreign antigenic epitope.

Objective: We assessed trends in the proportion of transmitted (TDR) and acquired (ADR) HIV drug resistance and associated mutations between 2001 and 2011 in the German ClinSurv-HIV Drug Resistance Study. Method: The German ClinSurv-HIV Drug Resistance Study is a subset of the German ClinSurv-HIV Cohort. For the ClinSurv-HIV Drug Resistance Study all available sequences isolated from patients in five study centres of the long term observational ClinSurv-HIV Cohort were included. TDR was estimated using the first viral sequence of antiretroviral treatment (ART) naive patients. One HIV sequence/patient/year of ART experienced patients was considered to estimate the proportion of ADR. Trends in the proportion of HIV drug resistance were calculated by logistic regression. Results: 9,528 patients were included into the analysis. HIV-sequences of antiretroviral naive and treatment experienced patients were available from 34% (3,267/9,528) of patients. The proportion of TDR over time was stable at 10.4% (95% CI 9.1-11.8; p (for trend)=0.6; 2001-2011). The proportion of ADR among all treated patients was 16%, whereas it was high among those with available HIV genotypic resistance test (64%; 1,310/2,049 sequences; 95% CI 62-66) but declined significantly over time (OR 0.8; 95% CI 0.77-0.83; p (for trend)

1IAME, UMR 1137, Univ Paris Diderot, Sorbonne Paris Cite, F-75018 Paris, France 2IAME, UMR 1137, INSERM, F-75018 Paris, France 3AP-HP, Hopital Bichat-Claude Bernard, Laboratoire de Virologie, F-75018 Paris, France 4Clinical and Epidemiological Virology, Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium 5Universite catholique de Louvain, AIDS Reference Laboratory, Brussels, Belgium 6Max von Pettenkofer Institute, Ludwig Maximilian University Munich, Munich, Germany 7Max Planck Institute for Informatics, Saarbrucken, Germany 8Johann Wolfgang Goethe-University Hospital, Institute for Medical Virology, German National Reference Centre for Retroviruses, Frankfurt, Germany 9Institute of Virology, University of Cologne, Germany 10Medizinisches Labor Dr. Berg, Berlin, Germany

Objective. The effects of two antiretroviral triple combinations including the protease inhibitor indinavir on the surrogate markers, viral load and CD4 cells were evaluated. Methods. Fifteen patients with high viral load or disease progression under their prior antiretroviral therapy were switched to zidovudine/ lamivudine/indinavir (Group A, n = 10) or stavudine/lamivudine/indinavir (Group B, n = 5). Serial determinations of viral load and CD4 cells were performed. Results. The median reduction of the viral load was 0.6 log after 3 months and 0.8 log after 6 months in Group A and 2.5 and 2.4 log after 3 and 6 months in Group B, respectively. After 3 and 6 months 3 of 10 patients in Group A and 3 of 5 patients in Group B had viral load reductions below the detection limit of the assay. Patients with an additional switch of nucleoside analogues at start of indinavir therapy (regardless of the specific reverse transcriptase inhibitor used) had significantly better reductions of the viral load than patients without such a switch (median 2.3 log vs. 0.2 log after 6 months, P < 0.05). In Group A the median of the relative increase of CD4 cells was 37% after 3 months and 57% after 6 months (P = 0.002); in Group B the medians of the relative increase of CD4 cells were 145 and 163% (not significant), respectively. Two patients from Group A and 1 from Group B developed renal calculi, which resolved after adequate hydration. One patient was withdrawn because of intractable vomiting attributed to indinavir. Conclusion. In a small cohort of HIV-infected pediatric patients with extensive prior antiretroviral treatment, triple therapy including indinavir had a sustained effect on the decrease of the viral load and the increase of CD4 cells similar to results obtained in antiretrovirally experienced adults. This effect was significantly better in patients with an additional switch of a nucleoside analogue at start of triple therapy with indinavir than in patients without such a change.

Objective: To assess human immunodeficiency virus (HIV) ribonucleic acid load in children and adolescents with HIV infection who are being treated with antiretroviral combination therapy. Study design: Five patients whose disease progressed with their prior antiretroviral therapy had treatment regimens changed to zidovudine (ZDV)/didanosine (DDI) (group A), and the regimens of six patients were changed to ZDV/lamivudine (3TC) (group B). Patients were followed every 4 to 8 weeks for an average period of 8.6 months. Serial determinations of viral copy numbers and CD4 cells were performed. Results: In group A patients' mean relative changes in CD4 cells showed a 20% increase after 4 months (difference not significant [NS]) and a return to baseline after 8 months; in group B patients' mean relative increases of CD4 cells were 72% ( p = 0.046) and 50% (NS), respectively. In group A mean relative viral load increased 21% (0.08 log 10 , NS) and 71% (0.23 10 log, NS), whereas in group B viral load decreased 22% (0.1 log 10 , NS) and 74% (0.58 log 10 , p = 0.03) after 4 and 8 months, respectively. After starting antiretroviral combination therapy in group A, there was a slight trend of a decreasing ratio of viral load per number of CD4 cells, whereas in group B this ratio significantly decreased, indicating a marked suppression of viral turnover with ZDV/3TC treatment. Conclusion: In a small cohort of pediatric patients, combination therapy with ZDV/3TC was well tolerated and had a strong and sustained effect on the decrease of viral load similar to results obtained in adults. In patients with ZDV/DDI therapy the reduction of viral load was less pronounced, but treatment groups A and B were not comparable for statistic evaluation. (J Pediatr 1997;130:293-9)

The first case of human immunodeficiency virus type 2 (HIV-2) seropositivity in Austria was confirmed in 1993 in a dually human immunodeficiency virus type 1 (HIV-1)- and HIV-2-infected patient from Ghana, who died in 2001. Before this investigation, no further HIV-2 infection was published.The aim of this study was to describe HIV-2 epidemiology in Austria, using serological and molecular techniques, and to perform a sequence analysis of the circulating viral strains.Six additional cases of HIV-2 were identified from 2000 to 2009. All patients originated from high-prevalence areas. In one patient, the HIV-2 infection was revealed 11 years after initial HIV-1 diagnosis, and further analysis confirmed a dual infection.The HIV-2 epidemic has its epicentre in West Africa, but sociocultural issues, especially migration, are contributing to the low but continuous worldwide spread of HIV-2. Diagnosis of HIV-2 implies a different therapeutical management to avoid treatment failure and clinical progression. Differential diagnosis of HIV-1 and HIV-2 is complicated due to antibody cross-reactivity, and paradoxical findings (e.g. declining CD4 cell count despite HIV-1 suppression) may require careful reassessment, especially in patients from endemic countries.

Background: Human herpes virus 8 (HHV-8), a gamma herpes virus, is the etiological agent for Kaposi sarcoma (KS). HIV-infected adults with advanced immunodeficiency are at risk. Prevalence data of HHV-8 infection in HIV-infected children living in non-endemic areas are limited. Serologic studies indicate low seroprevalence rates of 3 - 4% for healthy children living in United States and Germany [1]. Purpose of the study: The aim of the study was to determine the seroprevalence of HHV-8 antibodies among vertically HIV-infected pediatric patients in Germany and to evaluate their association with age, gender, ethnicity, and other demographic factors. Methods: In 2012, a multi-center cross-sectional study was conducted in four University Hospitals in Germany. Stored frozen serum specimens obtained from vertically HIV-infected children and adolescents were tested for antibodies against lytic and latent HHV-8 antigens. Data on patients' demographic characteristics and medical history were recorded. Results: A total of 214 HIV-infected children and adolescents (105 males, 109 females) were included. The median age was 10.2 years (range 1 months - 22.6 years). A high proportion of these children (62%) was born in Western Europe, whereas 65% (139/214) of their mothers were born in countries outside Western Europe. The majoritiy (91%) of the children had been treated with highly active antiretroviral therapy and 55.2% (116/210) had a HIV-viral load < 50 copies/mL. The median CD4 cell count was 1000/μL (range 3 - 4400). The overall seroprevalence of HHV-8 antibodies was 23.8% (51/214). Seroprevalence rates did not show significant differences between age or gender. In the group of young children aged 1 month to 35 months, 19.4% (46/31) had HHV-8 antibodies, compared to 25% (25/100) in children aged 36 months to 11 years, and 24.1% (20/83) children 12 years and older. In the study group, seroprevalence rates were significantly lower in children who were born in Western Europe (p