Developing a next level integrated genomic surveillance: Advances in the molecular epidemiology of HIV in Germany.

Advances in the molecular epidemiological studies of the Human Immunodeficiency Virus (HIV) at the Robert Koch Institute (RKI) by laboratory and bioinformatic automation should allow the processing of larger numbers of samples and more comprehensive and faster data analysis in order to provide a higher resolution of the current HIV infection situation in near real-time and a better understanding of the dynamic of the German HIV epidemic. The early detection of the emergence and transmission of new HIV variants is important for the adaption of diagnostics and treatment guidelines. Likewise, the molecular epidemiological detection and characterization of spatially limited HIV outbreaks or rapidly growing sub-epidemics is of great importance in order to interrupt the transmission pathways by regionally adapting prevention strategies. These aims are becoming even more important in the context of the SARS-CoV2 pandemic and the Ukrainian refugee movement, which both have effects on the German HIV epidemic that should be monitored to identify starting points for targeted public health measures in a timely manner. To this end, a next level integrated genomic surveillance of HIV is to be established.

Decreasing prevalence and stagnating incidence of Hepatitis C-co-infection among a cohort of HIV-1-positive patients, with a majority of men who have sex with men, in Germany, 1996-2019.

Co-infection with Hepatitis C virus (HCV) among HIV-positive patients leads to accelerated progression of liver disease and AIDS. Due to increased HCV prevalence and incidence, co-infection requires monitoring trends among HIV-positive individuals. This will help target prevention strategies and support to reach the global goals of eliminating viral hepatitis as a public health threat. In this analysis HCV prevalence and incidence were determined for the years 1996-2019 from yearly blood samples and questionnaire details among HIV-1-positive patients, with a majority of men who have sex with men, belonging to a nationwide, multicentre observational, prospective cohort study. The results show that HCV prevalence for acute/chronic and resolved infection increased until 2014 to 12%. Since then, prevalence of acute/chronic HCV infection rapidly decreased and prevalence of resolved infections showed a steady increase. HCV incidence was highest in 2010 and lowest in 2017; however, no significant change in HCV incidence could be seen over the years. Therefore, the introduction of directly-acting antiviral agents for HCV treatment notably decreased prevalence and potentially incidence of acute/chronic HCV infection. Nevertheless, prevalence and incidence of HCV among these HIV-1-positive study participants remain high compared with the general population and justify the need for continuous HCV prevention and treatment efforts among HIV-positive individuals.

Reconstruction of the Genetic History and the Current Spread of HIV-1 Subtype A in Germany.

HIV-1 non-B infections have been increasing in Europe for several years. In Germany, subtype A belongs to the most abundant non-B subtypes showing an increasing prevalence of 8.3% among new infections in 2016. Here we trace the origin and examine the current spread of the German HIV-1 subtype A epidemic. Bayesian coalescence and birth-death analyses were performed with 180 German HIV-1 pol sequences and 528 related and publicly available sequences to reconstruct the population dynamics and fluctuations for each of the transmission groups. Our reconstructions indicate two distinct sources of the German subtype A epidemic, with an Eastern European and an Eastern African lineage both cocirculating in the country. A total of 13 German-origin clusters were identified; among these, 6 clusters showed recent activity. Introductions leading to further countrywide spread originated predominantly from Eastern Africa when introduced before 2005. Since 2005, however, spreading introductions have occurred exclusively within the Eastern European clade. Moreover, we observed changes in the main route of subtype A transmission. The beginning of the German epidemic (1985 to 1995) was dominated by heterosexual transmission of the Eastern African lineage. Since 2005, transmissions among German men who have sex with men (MSM) have been increasing and have been associated with the Eastern European lineage. Infections among people who inject drugs dominated between 1998 and 2005. Our findings on HIV-1 subtype A infections provide new insights into the spread of this virus and extend the understanding of the HIV epidemic in Germany.IMPORTANCE HIV-1 subtype A is the second most prevalent subtype worldwide, with a high prevalence in Eastern Africa and Eastern Europe. However, an increase of non-B infections, including subtype A infections, has been observed in Germany and other European countries. There has simultaneously been an increased flow of refugees into Europe and especially into Germany, raising the question of whether the surge in non-B infections resulted from this increased immigration or whether German transmission chains are mainly involved. This study is the first comprehensive subtype A study from a western European country analyzing in detail its phylogenetic origin, the impact of various transmission routes, and its current spread. The results and conclusions presented provide new and substantial insights for virologists, epidemiologists, and the general public health sector. In this regard, they should be useful to those authorities responsible for developing public health intervention strategies to combat the further spread of HIV/AIDS.

Comparative multi-assay evaluation of Determine™ HIV-1/2 Ag/Ab Combo rapid diagnostic tests in acute and chronic HIV infection.

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.

CD4 T cell decline following HIV seroconversion in individuals with and without CXCR4-tropic virus.

Background: The natural clinical and immunological courses following HIV seroconversion with CXCR4-tropic or dual-mixed (X4/DM) viruses are controversial. We compared spontaneous immunological outcome in patients harbouring an X4/DM virus at the time of seroconversion with those harbouring a CCR5-tropic (R5) virus. Methods: Data were included from patients participating in CASCADE, a large cohort collaboration of HIV seroconverters, with ≥2 years of follow-up since seroconversion. The HIV envelope gene was sequenced from frozen plasma samples collected at enrolment, and HIV tropism was determined using Geno2Pheno (false-positive rate 10%). The spontaneous CD4 T cell evolution was compared by modelling CD4 kinetics using linear mixed-effects models with random intercept and random slope. Results: A total of 1387 patients were eligible. Median time between seroconversion and enrolment was 1 month (range 0-3). At enrolment, 202 of 1387 (15%) harboured an X4/DM-tropic virus. CD4 decrease slopes were not significantly different according to HIV-1 tropism during the first 30 months after seroconversion. No marked change in these results was found after adjusting for age, year of seroconversion and baseline HIV viral load. Time to antiretroviral treatment initiation was not statistically different between patients harbouring an R5 (20.76 months) and those harbouring an X4/DM-tropic virus (22.86 months, logrank test P = 0.32). Conclusions: In this large cohort collaboration, 15% of the patients harboured an X4/DM virus close to HIV seroconversion. Patients harbouring X4/DM-tropic viruses close to seroconversion did not have an increased risk of disease progression, estimated by the decline in CD4 T cell count or time to combined ART initiation.

Molecular evolution of HIV-1 integrase during the 20 years prior to the first approval of integrase inhibitors.

BACKGROUND: Detailed knowledge of the evolutionary potential of polymorphic sites in a viral protein is important for understanding the development of drug resistance in the presence of an inhibitor. We therefore set out to analyse the molecular evolution of the HIV-1 subtype B integrase at the inter-patient level in Germany during a 20-year period prior to the first introduction of integrase strand inhibitors (INSTIs). METHODS: We determined 337 HIV-1 integrase subtype B sequences (amino acids 1-278) from stored plasma samples of antiretroviral treatment-naïve individuals newly diagnosed with HIV-1 between 1986 and 2006. Shannon entropy was calculated to determine the variability at each amino acid position. Time trends in the frequency of amino acid variants were identified by linear regression. Direct coupling analysis was applied to detect covarying sites. RESULTS: Twenty-two time trends in the frequency of amino acid variants demonstrated either single amino acid exchanges or variation in the degree of polymorphy. Covariation was observed for 17 amino acid variants with a temporal trend. Some minor INSTI resistance mutations (T124A, V151I, K156 N, T206S, S230 N) and some INSTI-selected mutations (M50I, L101I, T122I, T124 N, T125A, M154I, G193E, V201I) were identified at overall frequencies >5%. Among these, the frequencies of L101I, T122I, and V201I increased over time, whereas the frequency of M154I decreased. Moreover, L101I, T122I, T124A, T125A, M154I, and V201I covaried with non-resistance-associated variants. CONCLUSIONS: Time-trending, covarying polymorphisms indicate that long-term evolutionary changes of the HIV-1 integrase involve defined clusters of possibly structurally or functionally associated sites independent of selective pressure through INSTIs at the inter-patient level. Linkage between polymorphic resistance- and non-resistance-associated sites can impact the selection of INSTI resistance mutations in complex ways. Identification of these sites can help in improving genotypic resistance assays, resistance prediction algorithms, and the development of new integrase inhibitors.

Assessment of ambiguous base calls in HIV-1 pol population sequences as a biomarker for identification of recent infections in HIV-1 incidence studies.

An increase in the proportion of ambiguous base calls in HIV-1 pol population sequences during the course of infection has been demonstrated in different study populations, and sequence ambiguity thresholds to classify infections as recent or nonrecent have been suggested. The aim of our study was to evaluate sequence ambiguities as a candidate biomarker for use in an HIV-1 incidence assay using samples from antiretroviral treatment-naive seroconverters with known durations of infection (German HIV-1 Seroconverter Study). We used 2,203 HIV-1 pol population sequences derived from 1,334 seroconverters to assess the sequence ambiguity method (SAM). We then compared the serological incidence BED capture enzyme immunoassay (BED-CEIA) with the SAM for a subset of 723 samples from 495 seroconverters and evaluated a multianalyte algorithm that includes BED-CEIA results, SAM results, viral loads, and CD4 cell counts for 453 samples from 325 seroconverters. We observed a significant increase in the proportion of sequence ambiguities with the duration of infection. A sequence ambiguity threshold of 0.5% best identified recent infections with 76.7% accuracy. The mean duration of recency was determined to be 208 (95% confidence interval, 196 to 221) days. In the subset analysis, BED-CEIA achieved a significantly higher accuracy than the SAM (84.6 versus 75.5%, P < 0.001) and results were concordant for 64.2% (464/723) of the samples. Also, the multianalyte algorithm did not show better accuracy than the BED-CEIA (83.4 versus 84.3%, P = 0.786). In conclusion, the SAM and the multianalyte algorithm including SAM were inferior to the BED-CEIA, and the proportion of sequence ambiguities is therefore not a preferable biomarker for HIV-1 incidence testing.

The NLRP3 inflammasome is differentially activated by pneumolysin variants and contributes to host defense in pneumococcal pneumonia.

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1ß. This IL-1ß production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1ß production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.

Listeria monocytogenes-infected human peripheral blood mononuclear cells produce IL-1beta, depending on listeriolysin O and NLRP3.

Different NOD-like receptors, including NLRP1, NLRP3, and NLRC4, as well as the recently identified HIN-200 protein, AIM2, form multiprotein complexes called inflammasomes, which mediate caspase-1-dependent processing of pro-IL-1beta. Listeria monocytogenes is an intracellular pathogen that is actively phagocytosed by monocytes/macrophages and subsequently escapes from the phagosome into the host cell cytosol, depending on its pore-forming toxin listeriolysin O (LLO). In this study, we demonstrate that human PBMCs produced mature IL-1beta when infected with wild-type L. monocytogenes or when treated with purified LLO. L. monocytogenes mutants lacking LLO or expressing a noncytolytic LLO as well as the avirulent Listeria innocua induced strongly impaired IL-1beta production. RNA interference and inhibitor experiments in human PBMCs as well as experiments in Nlrp3 and Rip2 knockout bone marrow-derived macrophages demonstrated that the Listeria-induced IL-1beta release was dependent on ASC, caspase-1, and NLRP3, whereas NOD2, Rip2, NLRP1, NLRP6, NLRP12, NLRC4, and AIM2 appeared to be dispensable. We found that L. monocytogenes-induced IL-1beta production was largely dependent on phagosomal acidification and cathepsin B release, whereas purified LLO activated an IL-1beta production independently of these mechanisms. Our results indicate that L. monocytogenes-infected human PBMCs produced IL-1beta, largely depending on an LLO-mediated phagosomal rupture and cathepsin B release, which is sensed by Nlrp3. In addition, an LLO-dependent but cathepsin B-independent NLRP3 activation might contribute to some extent to the IL-1beta production in L. monocytogenes-infected cells.

A Recent Human Immunodeficiency Virus Outbreak Among People Who Inject Drugs in Munich, Germany, Is Associated With Consumption of Synthetic Cathinones.

BACKGROUND: Needle and syringe sharing among people who inject drugs (PWID) can result in a rapid regional spread of a human immunodeficiency virus (HIV) variant. Such outbreaks have been identified recently in several countries and have raised public health attention because of an association with new psychoactive substances (NPS). METHODS: Dried serum spots from approximately 60% of newly diagnosed HIV cases in Germany in 2013-2018 were received together with statutory notification data. Samples were sequenced in the pol-region, genotyped, and viral phylogenies were analyzed. For selected samples, the hepatitis C virus (HCV) status and the presence of NPS were determined. RESULTS: An outbreak of closely related 27 subtype C infections with a core of 11 cases with almost identical sequences was identified using phylogenetic analyses. The first case of the outbreak was diagnosed in 2015, and the last one was in 2018. With exception of 3 infections, all were reported from Munich, the capital of the federal state of Bavaria. Of 26 analyzed outbreak members, 24 (92.3%) had a resolved or viremic HCV coinfection. In 8 of 18 (44%) cases, α-pyrrolidinopentiothiophenone and/or the related substance α-pyrrolidinoheptiophenone was identified. CONCLUSIONS: Despite harm reduction services in place, HIV outbreaks of considerable size can occur in PWID. The establishment of a real-time molecular surveillance is advised to rapidly identify outbreaks and target prevention measures.

Establishment of an anti-hepatitis C virus IgG avidity test for dried serum/plasma spots.

Monitoring recency of infection helps to identify current transmission in vulnerable populations for effective disease control. We have established an in-house avidity based hepatitis C virus (HCV) recency assay based on the Monolisa Anti-HCV PLUS Version 3 ELISA kit for use of dried serum/plasma spots (DS/PS) in order to distinguish recent and long-term infections. A first panel of DS/PS (n = 218; genotype 1 n = 170 and non-genotype 1 n = 48) consisting of primary and at least one follow up sample was used to analyze the temporal changes of the Avidity Index (AI) over time. Sub-panels of longitudinal DS/PS (n = 66) and acute cases (<26 weeks; n = 34) were taken to calculate the Mean Duration of Recent Infection (MDRI) and the False Long-term Rate (FLTR), respectively. A second panel of DS/PS >104 weeks (n = 132) and a third panel of DS/PS prepared from resolved infections (≥180 days since last positive; n = 32) were used to calculate the False Recent Rate (FRR). For all genotypes, the optimal AI cut-off was determined to be 40% resulting in an MDRI of 364 days (95% CI: 223-485). FLTR was 5.9% (95% CI: 0.7-19.7), 8.3% (95% CI: 1-27), and 0% (-) and FRR was 13.6% (95% CI: 8.3-20.7), 11.7% (95% CI: 6.6-19), and 30.6% (95% CI: 9.1-61.4) for all genotypes, genotype 1, and non-genotype 1 infections, respectively. For resolved infections, the FRR was 53.1% (95% CI: 35.8-70.4). Thus, this assay performs particularly well for genotype 1 reaching a high rate of correct discriminations between infections acquired less than a year before diagnosis and those acquired earlier by applying an AI cut-off of 40%. Due to a rapid decline in avidity post resolution of an HCV infection this assay is not recommended to be used in HCV RNA negative patients.

IFNbeta responses induced by intracellular bacteria or cytosolic DNA in different human cells do not require ZBP1 (DLM-1/DAI).

Intracellular bacteria and cytosolic stimulation with DNA activate type I IFN responses independently of Toll-like receptors, most Nod-like receptors and RIG-like receptors. A recent study suggested that ZBP1 (DLM-1/DAI) represents the long anticipated pattern recognition receptor which mediates IFNalpha/beta responses to cytosolic DNA in mice. Here we show that Legionella pneumophila infection, and intracellular challenge with poly(dA-dT), but not with poly(dG-dC), induced expression of IFNbeta, full-length hZBP1 and a prominent splice variant lacking the first Zalpha domain (hZBP1DeltaZalpha) in human cells. Overexpression of hZBP1 but not hZBP1DeltaZalpha slightly amplified poly(dA-dT)-stimulated IFNbeta reporter activation in HEK293 cells, but had no effect on IFNbeta and IL-8 production induced by bacteria or poly(dA-dT) in A549 cells. We found that mZBP1 siRNA impaired poly(dA-dT)-induced IFNbeta responses in mouse L929 fibroblasts at a later time point, while multiple hZBP1 siRNAs did not suppress IFNbeta or IL-8 expression induced by poly(dA-dT) or bacterial infection in human cells. In contrast, IRF3 siRNA strongly impaired the IFNbeta responses to poly(dA-dT) or bacterial infection. In conclusion, intracellular bacteria and cytosolic poly(dA-dT) activate IFNbeta responses in different human cells without requiring human ZBP1.

Prevalence and persistence of transmitted drug resistance mutations in the German HIV-1 Seroconverter Study Cohort.

The prevalence of transmitted drug resistance (TDR) in antiretroviral therapy (ART)-naïve individuals remains stable in most developed countries despite a decrease in the prevalence of acquired drug resistance. This suggests that persistence and further transmission of HIV-1 that encodes transmitted drug resistance mutations (TDRMs) is occurring in ART-naïve individuals. In this study, we analysed the prevalence and persistence of TDRMs in the protease and reverse transcriptase-sequences of ART-naïve patients within the German HIV-1 Seroconverter Study Cohort who were infected between 1996 and 2017. The prevalence of TDRMs and baseline susceptibility to antiretroviral drugs were assessed using the Stanford HIVdb list and algorithm. Mean survival times of TDRMs were calculated by Kaplan-Meier analysis. The overall prevalence of TDR was 17.2% (95% CI 15.7-18.6, N = 466/2715). Transmitted NNRTI resistance was observed most frequently with 7.8% (95% CI 6.8-8.8), followed by NRTI resistance (5.0%, 95% CI 4.2-5.9) and PI resistance (2.8%, 95% CI 2.2-3.4). Total TDR (OR = 0.89, p = 0.034) and transmitted NRTI resistance (OR = 0.65, p = 0.000) decreased between 1996 and 2017 but has remained stable during the last decade. Viral susceptibility to NNRTIs (6.5%-6.9% for individual drugs) was mainly reduced, while <3% of the recommended NRTIs and PIs were affected. The longest mean survival times were calculated for the NNRTI mutations K103N (5.3 years, 95% CI 4.2-5.6) and E138A/G/K (8.0 years, 95% CI 5.8-10.2 / 7.9 years, 95% CI 5.4-10.3 / 6.7 years, 95% CI 6.7-6.7) and for the NRTI mutation M41L (6.4 years, 95% CI 6.0-6.7).The long persistence of single TDRMs indicates that onward transmission from ART-naïve individuals is the main cause for TDR in Germany. Transmitted NNRTI resistance was the most frequent TDR, showing simultaneously the highest impact on baseline ART susceptibility and on TDRMs with prolonged persistence. These results give cause for concern regarding the use of NNRTI in first-line regimens.

Evaluation of a BioRad Avidity assay for identification of recent HIV-1 infections using dried serum or plasma spots.

Serological methods to differentiate between recently acquired and established HIV-1 infections are a useful tool in the HIV-surveillance to characterize the epidemic, identify groups at risk and assess HIV-preventive interventions. Therefore, an avidity-based, modified BioRad Genscreen™ HIV-1/2 assay (BRAEUR) was evaluated according to the avidity-based, modified BioRad HIV-1/2 Plus O protocol (BRAUSA). Overall, 692 well defined samples (82.5% B and 17.5% non-B subtypes) from recent (<180 days, n = 239), intermediate (181-364 days, n = 35) or long term infections (≥365 days, n = 419) were used to determine a 'mean duration of recent infection' (MDRI), a 'median DRI' (MdDRI), the false recent rate (FRR), and concordance between the BRAs and the Sedia BED HIV-1 Capture enzyme immunoassay (BED). The optimal avidity index cut-off was determined to be 70% resulting in an MDRI of 233 days (95% IQR: 174-351) and an MdDRI of 171 days (95% IQR: 142-212). Concordance with the BRAUSA was high with 96.4%. The FRR of 6.0% as well as the MdDRI are similar to the BED (8.4%; 170 (139-214) days). Therefore, the BRAEUR is a suitable alternative to replace the BED and trend analysis will be feasible after minimal adjustments for the MdDRI and the MDRI.

Increasing proportions of HIV-1 non-B subtypes and of NNRTI resistance between 2013 and 2016 in Germany: Results from the national molecular surveillance of new HIV-diagnoses.

BACKGROUND: Molecular surveillance of newly diagnosed HIV-infections is important for tracking trends in circulating HIV-variants, including those with transmitted drug resistances (TDR) to sustain ART efficacy. METHODS: Dried serum spots (DSS) are received together with the statutory notification of a new diagnosis. 'Recent infections' (<155 days) classified by a 'recent infection test algorithm' (BED-CEIA and clinical data) are genotyped in HIV-protease (PR), reverse transcriptase (RT) and integrase (INT) to determine the HIV-1 subtype, to calculate prevalence and trends of TDR, to predict baseline susceptibility and to identify potential transmission clusters for resistant variants. RESULTS: Between January 2013 and December 2016, 1,885 recent infections were analysed regarding the PR/RT genomic region, with 43.5% of these also being subjected to the analysis of INT. The proportion of HIV-1 non-B viruses (31.3%; 591/1,885) increased from 21.6% to 36.0%, particularly the subtypes A (5.0% to 8.3%) and C (3.2% to 7.7%; all ptrends < 0.01). The subtype A increment is mainly due to transmissions within men who have sex with men (MSM) while subtype C transmissions are associated with heterosexuals and people who inject drugs. The prevalence of TDR was stable at 11.0% (208/1,885) over the study period. Resistances to nucleotide RT inhibitors (NRTI) and PR inhibitors (PI) were 4.5% and 3.2%, respectively, without identifiable trends. In contrast, resistances to non-NRTIs (NNRTI, 4.7%) doubled between 2014 and 2016 from 3.2% to 6.4% (ptrend = 0.02) mainly due to the K103N mutation (from 1.7% to 4.1%; ptrend = 0.03) predominantly detected in recently infected German MSM not linked to transmission clusters. Transmitted INSTI mutations were present in only one case (T66I) and resistance to dolutegravir was not identified at all. Reduced susceptibility to recommended first-line therapies was low with 1.0% for PIs, 1.3% for NRTIs and 0.7% for INSTIs, but high for the NNRTIs efavirence (4.9%) and rilpivirine (6.0%) due to the K103N mutation and the polymorphic mutation E138A. These trends in therapy-naïve individuals impact current first-line regimens and require awareness and vigilant surveillance.

Robust and sensitive subtype-generic HIV-1 pol genotyping for use with dried serum spots in epidemiological studies.

HIV-1 genotyping of larger pol-fragments from dried serum/plasma spots (DSS/DPS) is often hindered by RNA-degradation during transportation at ambient temperature. We evaluated an in-house HIV-1 DSS/DPS-genotyping assay designed in two shorter overlapping fragments covering all resistance mutations in protease and reverse transcriptase. Validation criteria such as specificity, detection limit, accuracy, reproducibility and storage conditions were assessed using reference plasma samples prepared as DPS and clinical DSS from the German molecular HIV-1 surveillance processed under real-life transportation conditions. The specificity was 100% for both samples types, and the experimental DPS detection limit of 1000 copies/ml yielded a 98.7% (3,329/3373) success rate for DSS (including all subtypes) above this detection limit. Accuracy for DPS compared to the gold standard was 99.1% and the reproducibility was 100% for DPS replicates and 99.9% for DSS pairs. Storage of DPS at room temperature was possible for 90 or 30 days and at -20 °C for at least 180 or 90 days at viral loads of 10,000 or 1000 copies/ml, respectively. The HIV-1 pol-genotyping assay presented here is a sensitive, robust and subtype generic tool for a large-scale population-based HIV-1 drug resistance surveillance for the use of DSS/DPS.

Decreased emergence of HIV-1 drug resistance mutations in a cohort of Ugandan women initiating option B+ for PMTCT.

BACKGROUND: Since 2012, WHO guidelines for the prevention of mother-to-child transmission (PMTCT) of HIV-1 in resource-limited settings recommend the initiation of lifelong antiretroviral combination therapy (cART) for all pregnant HIV-1 positive women independent of CD4 count and WHO clinical stage (Option B+). However, long-term outcomes regarding development of drug resistance are lacking until now. Therefore, we analysed the emergence of drug resistance mutations (DRMs) in women initiating Option B+ in Fort Portal, Uganda, at 12 and 18 months postpartum (ppm). METHODS AND FINDINGS: 124 HIV-1 positive pregnant women were enrolled within antenatal care services in Fort Portal, Uganda. Blood samples were collected at the first visit prior starting Option B+ and postpartum at week six, month six, 12 and 18. Viral load was determined by real-time RT-PCR. An RT-PCR covering resistance associated positions in the protease and reverse transcriptase HIV-1 genomic region was performed. PCR-positive samples at 12/18 ppm and respective baseline samples were analysed by next generation sequencing regarding HIV-1 drug resistant variants including low-frequency variants. Furthermore, vertical transmission of HIV-1 was analysed. 49/124 (39.5%) women were included into the DRM analysis. Virological failure, defined as >1000 copies HIV-1 RNA/ml, was observed in three and seven women at 12 and 18 ppm, respectively. Sequences were obtained for three and six of these. In total, DRMs were detected in 3/49 (6.1%) women. Two women displayed dual-class resistance against all recommended first-line regimen drugs. Of 49 mother-infant-pairs no infant was HIV-1 positive at 12 or 18 ppm. CONCLUSION: Our findings suggest that the WHO-recommended Option B+ for PMTCT is effective in a cohort of Ugandan HIV-1 positive pregnant women with regard to the low selection rate of DRMs and vertical transmission. Therefore, these results are encouraging for other countries considering the implementation of lifelong cART for all pregnant HIV-1 positive women.

Listeria monocytogenes induced Rac1-dependent signal transduction in endothelial cells.

Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. Small GTPases of the Rho family act as molecular switches in signal transduction. We tested the hypothesis that Rho GTPases contribute to the regulation of cytokine expression following L. monocytogenes infection. L. monocytogenes induced release of distinct CC and CXC, as well as Th1 and Th2 cytokines and growth factors by endothelial cells and activated RhoA and Rac1. Inhibition of Rac1 by inhibitor Nsc23766 reduced cytokine expression, and slightly yet significantly the uptake of bacteria. Blocking of Rho proteins by Clostridium difficile toxin B-10463 (TcdB) reduced Listeria-dependent cytokine expression, whereas activating Rho proteins by Escherichia coli CNF1 increased it. We analyzed regulation of IL-8 expression in more detail: Listeria-induced IL-8 release was reduced by inhibition of RhoA, Rac1 and Cdc42 (TcdB) or Rac1 while blocking of RhoA/B/C by Clostridium limosum C3 fusion toxin (C3FT) or Rho kinase by Y27632 reduced cytokine expression only slightly. Activation of RhoA, Rac1 and Cdc42 (CNF1), but not of RhoA alone (CNF(Y)), enhanced Listeria-dependent IL-8 release significantly. Furthermore, inhibition of RhoA, Rac1 and Cdc42 (TcdB) and Rac1 (Nsc23766), but not of RhoA (C3FT) reduced Listeria-related recruitment of NF-kappaB/p65 and RNA polymerase II to the il8 promoter, as well as acetylation of histone H4 and Ser10/Lys14-phosphorylation/acetylation of histone H3 at the il8 gene promoter in HUVEC. In conclusion, Rac1 contributed to L. monocytogenes-induced cytokine expression by human endothelial cells.

Inferring HIV-1 Transmission Dynamics in Germany From Recently Transmitted Viruses.

BACKGROUND: Although HIV continues to spread globally, novel intervention strategies such as treatment as prevention (TasP) may bring the epidemic to a halt. However, their effective implementation requires a profound understanding of the underlying transmission dynamics. METHODS: We analyzed parameters of the German HIV epidemic based on phylogenetic clustering of viral sequences from recently infected seroconverters with known infection dates. Viral baseline and follow-up pol sequences (n = 1943) from 1159 drug-naïve individuals were selected from a nationwide long-term observational study initiated in 1997. Putative transmission clusters were computed based on a maximum likelihood phylogeny. Using individual follow-up sequences, we optimized our clustering threshold to maximize the likelihood of co-clustering individuals connected by direct transmission. RESULTS: The sizes of putative transmission clusters scaled inversely with their abundance and their distribution exhibited a heavy tail. Clusters based on the optimal clustering threshold were significantly more likely to contain members of the same or bordering German federal states. Interinfection times between co-clustered individuals were significantly shorter (26 weeks; interquartile range: 13-83) than in a null model. CONCLUSIONS: Viral intraindividual evolution may be used to select criteria that maximize co-clustering of transmission pairs in the absence of strong adaptive selection pressure. Interinfection times of co-clustered individuals may then be an indicator of the typical time to onward transmission. Our analysis suggests that onward transmission may have occurred early after infection, when individuals are typically unaware of their serological status. The latter argues that TasP should be combined with HIV testing campaigns to reduce the possibility of transmission before TasP initiation.

A Follow-Up of the Multicenter Collaborative Study on HIV-1 Drug Resistance and Tropism Testing Using 454 Ultra Deep Pyrosequencing.

BACKGROUND: Ultra deep sequencing is of increasing use not only in research but also in diagnostics. For implementation of ultra deep sequencing assays in clinical laboratories for routine diagnostics, intra- and inter-laboratory testing are of the utmost importance. METHODS: A multicenter study was conducted to validate an updated assay design for 454 Life Sciences' GS FLX Titanium system targeting protease/reverse transcriptase (RTP) and env (V3) regions to identify HIV-1 drug-resistance mutations and determine co-receptor use with high sensitivity. The study included 30 HIV-1 subtype B and 6 subtype non-B samples with viral titers (VT) of 3,940-447,400 copies/mL, two dilution series (52,129-1,340 and 25,130-734 copies/mL), and triplicate samples. Amplicons spanning PR codons 10-99, RT codons 1-251 and the entire V3 region were generated using barcoded primers. Analysis was performed using the GS Amplicon Variant Analyzer and geno2pheno for tropism. For comparison, population sequencing was performed using the ViroSeq HIV-1 genotyping system. RESULTS: The median sequencing depth across the 11 sites was 1,829 reads per position for RTP (IQR 592-3,488) and 2,410 for V3 (IQR 786-3,695). 10 preselected drug resistant variants were measured across sites and showed high inter-laboratory correlation across all sites with data (P<0.001). The triplicate samples of a plasmid mixture confirmed the high inter-laboratory consistency (mean% ± stdev: 4.6 ±0.5, 4.8 ±0.4, 4.9 ±0.3) and revealed good intra-laboratory consistency (mean% range ± stdev range: 4.2-5.2 ± 0.04-0.65). In the two dilutions series, no variants >20% were missed, variants 2-10% were detected at most sites (even at low VT), and variants 1-2% were detected by some sites. All mutations detected by population sequencing were also detected by UDS. CONCLUSIONS: This assay design results in an accurate and reproducible approach to analyze HIV-1 mutant spectra, even at variant frequencies well below those routinely detectable by population sequencing.