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The variety and limitations of current laboratory methods for estimating HIV-incidence has driven attempts to improve and standardize the performance of serological 'Tests for Recent HIV-Infections' (TRI). Primary and follow-up HIV-1 positive plasma samples from individuals with well-defined dates of infection collected as part of the German Seroconverter Cohort provided specimens highly suitable for use in comparing the performance of three TRIs: the AWARE™ BED™ EIA HIV-1 Incidence test (BED-CEIA), Genetic systems HIV-1/HIV-2 Plus O EIA antibody avidity-based assay (BioRad Avidity) and Sedia™ HIV-1 LAg Avidity EIA (LAg Avidity). The evaluation panel included 180 specimens: 44 from antiretroviral (ARV)-naïve individuals with recently acquired HIV-infection (≤ 130 days; 25 B and 19 non-B subtypes) and 136 from long-term (>12 months) infected individuals [101 ARV-naïve subtype B, 16 non-B subtypes, 14 ARV-treated individuals, 5 slow progressors (SLP)]. For long-term infected, ARV-naïve individuals the false recent rates (FRR) of both the BioRad and LAg Avidity assays were 2% (2/101 for subtype B) and 6% (1/16 for subtype 'non-B'), while the FRR of the BED-CEIA was 7% (7/101 for subtype B) and 25% (4/16 for subtype 'non-B') (all p>0.05). Misclassification of ARV-treated individuals and SLP was rare by LAg (1/14, 0/5) and BioRad Avidity assays (2/14, 1/5) but more frequent by BED-CEIA (5/14, 3/5). Among recently-infected individuals (subtype B), 60% (15/25) were correctly classified by BED-CEIA, 88% (22/25) by BioRad Avidity and significantly fewer by LAg (48%, 12/25) compared to BioRad Avidity (p = 0.005) with a higher true-recency rate among non-B infections for all assays. This study using well-characterized specimens demonstrated lower FRRs for both avidity methods than with the BED-CEIA. For recently infected individuals the BioRad Avidity assay was shown to give the most accurate results.

The aim of this study is to analyse the prevalence of transmitted drug resistance, TDR, and the impact of TDR on treatment success in the German HIV-1 Seroconverter Cohort. Genotypic resistance analysis was performed in treatment-naïve study patients whose sample was available 1,312/1,564 (83.9% October 2008). A genotypic resistance result was obtained for 1,276/1,312 (97.3%). The resistance associated mutations were identified according to the surveillance drug resistance mutations list recommended for drug-naïve patients. Treatment success was determined as viral suppression below 500 copies/ml. Prevalence of TDR was stable at a high level between 1996 and 2007 in the German HIV-1 Seroconverter Cohort (N = 158/1,276; 12.4%; CI(wilson) 10.7-14.3; p(for trend) = 0.25). NRTI resistance was predominant (7.5%) but decreased significantly over time (CI(Wilson): 6.2-9.1, p(for trend) = 0.02). NNRTI resistance tended to increase over time (NNRTI: 3.5%; CI(Wilson): 2.6-4.6; p(for trend)= 0.07), whereas PI resistance remained stable (PI: 3.0%; CI(Wilson): 2.1-4.0; p(for trend) = 0.24). Resistance to all drug classes was frequently caused by singleton resistance mutations (NRTI 55.6%, PI 68.4%, NNRTI 99.1%). The majority of NRTI-resistant strains (79.8%) carried resistance-associated mutations selected by the thymidine analogues zidovudine and stavudine. Preferably 2NRTI/1PIr combinations were prescribed as first line regimen in patients with resistant HIV as well as in patients with susceptible strains (susceptible 45.3%; 173/382 vs. resistant 65.5%; 40/61). The majority of patients in both groups were treated successfully within the first year after ART-initiation (susceptible: 89.9%; 62/69; resistant: 7/9; 77.8%). Overall prevalence of TDR remained stable at a high level but trends of resistance against drug classes differed over time. The significant decrease of NRTI-resistance in patients newly infected with HIV might be related to the introduction of novel antiretroviral drugs and a wider use of genotypic resistance analysis prior to treatment initiation.

Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT). Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System. Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage. Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.

WHO-guidelines for prevention of mother-to-child transmission of HIV-1 in resource-limited settings recommend complex maternal antiretroviral prophylaxis comprising antenatal zidovudine (AZT), nevirapine single-dose (NVP-SD) at labor onset and AZT/lamivudine (3TC) during labor and one week postpartum. Data on resistance development selected by this regimen is not available. We therefore analyzed the emergence of minor drug-resistant HIV-1 variants in Tanzanian women following complex prophylaxis. 1395 pregnant women were tested for HIV-1 at Kyela District Hospital, Tanzania. 87/202 HIV-positive women started complex prophylaxis. Blood samples were collected before start of prophylaxis, at birth and 1-2, 4-6 and 12-16 weeks postpartum. Allele-specific real-time PCR assays specific for HIV-1 subtypes A, C and D were developed and applied on samples of mothers and their vertically infected infants to quantify key resistance mutations of AZT (K70R/T215Y/T215F), NVP (K103N/Y181C) and 3TC (M184V) at detection limits of <1%. 50/87 HIV-infected women having started complex prophylaxis were eligible for the study. All women took AZT with a median duration of 53 days (IQR 39-64); all women ingested NVP-SD, 86% took 3TC. HIV-1 resistance mutations were detected in 20/50 (40%) women, of which 70% displayed minority species. Variants with AZT-resistance mutations were found in 11/50 (22%), NVP-resistant variants in 9/50 (18%) and 3TC-resistant variants in 4/50 women (8%). Three women harbored resistant HIV-1 against more than one drug. 49/50 infants, including the seven vertically HIV-infected were breastfed, 3/7 infants exhibited drug-resistant virus. Complex prophylaxis resulted in lower levels of NVP-selected resistance as compared to NVP-SD, but AZT-resistant HIV-1 emerged in a substantial proportion of women. Starting AZT in pregnancy week 14 instead of 28 as recommended by the current WHO-guidelines may further increase the frequency of AZT-resistance mutations. Given its impact on HIV-transmission rate and drug-resistance development, HAART for all HIV-positive pregnant women should be considered.

Nevirapine (NVP) single-dose is still a widely used antiretroviral prophylaxis for the prevention of vertical HIV-1 transmission in resource-limited settings. However, the main disadvantage of the Non-nucleoside Reverse Transcriptase Inhibitor (NNRTI) NVP is the rapid selection of NVP-resistant virus with negative implications for subsequent NNRTI-based long-term antiretroviral therapy (ART). Here, we analysed the emergence of drug-resistant HIV-1 including minor variants in the early phase after NVP single-dose prophylaxis and the persistence of drug-resistant virus over time. NVP-resistant HIV-1 harbouring the K103N and/or Y181C resistance mutations in the HIV-1 reverse transcriptase gene was measured from 1 week up to 18 months after NVP single-dose prophylaxis in 29 Ugandan women using allele-specific PCR assays capable of detecting drug-resistant variants representing less than 1% of the whole viral population. In total, drug-resistant HIV-1 was identified in 18/29 (62%) women; rates increased from 18% to 38% and 44% at week 1, 2, 6, respectively, and decreased to 18%, 25%, 13% and 4% at month 3, 6, 12 and 18, respectively. The proportion of NVP-resistant virus of the total viral population was significantly higher in women infected with subtype D (median 40.5%) as compared to subtype A (median 1.3%; p = 0.032, Mann-Whitney U test). 33% of resistant virus was not detectable at week 2 but was for the first time measurable 6-12 weeks after NVP single-dose prophylaxis. Three (10%) women harboured resistant virus in proportions >10% still at month 6. Current WHO guidelines recommend an additional postnatal intake of AZT and 3TC for one week to avoid NVP resistance formation. Our findings indicate that a 1-week medication might be too short to impede the emergence of NVP resistance in a substantial proportion of women. Furthermore, subsequent NNRTI-based ART should not be started earlier than 12 months after NVP single-dose prophylaxis.

Introduction The Robert Koch Institute (RKI) aimed to assess a molecular surveillance strategy based on filter‐dried serum spots (DSS) of all newly diagnosed HIV infections in Germany. In 2013, diagnostic laboratories sent DSS to the RKI representing 55% of the newly diagnosed HIV infections reported to the RKI (protection against infection act). DSS were first tested serologically to identify recently acquired infections (<140 days duration of infection); those classified as “recent infection” were processed for HIV‐1 genotyping. The aim of this study was to assess the level of TDR and the current HIV‐1 subtypes in the main HIV transmission group categories (TrGrpC) in 2013: men who have sex with men (MSM), women/men with heterosexual contacts (HET) and injecting drug users (IDUs). Materials and Methods DSS were tested for recency of infection using the BED capture EIA. Viral RNA from “recent infections” was amplified by HIV‐1 group M generic pol‐RT‐PCR covering all resistance‐associated positions in the HIV‐1 protease (AS1‐99) and reverse transcriptase (AS1‐252) if viral loads were ≥6,500 copies/mL. PCR amplicons were sequenced (Sanger) to analyze genotypic resistance and the HIV‐1 subtype. Results were merged to data from the HIV report, i.e. the TrGrpC. Results In 2013, 1027 DSS were classified as recent HIV infections (506 MSM, 118 HET, 31 IDUs, 6 others, 366 unknown). RNA was extracted from 703 recent cases and 389/503 samples with sufficient viral load were PCR‐positive. By June 2014, 276/389 samples were sequenced: TDR was identified in 13% (35/276) of the recent infections including single (PI, NRTI, NNRTI) and dual drug class resistant strains (NRTI/NNRTI; NNRTI/PI). 18% (51/276) of recent HIV‐1 infections were caused by non‐B subtypes (A1, C, CRF01_AE, CRF02_AG, D, F, G, URFs). TDR was observed at comparable levels in all TrGrpC. Proportions of non‐B infections were significantly higher in HET (78%; 14/18) and IDUs (60%; 3/5) compared to MSM (8%; 14/169) (p<0.01). Conclusions The proportion of TDR was similar but the proportion of HIV‐1 subtype non‐B infections was higher as previously described for Germany based on results from the German HIV‐1 Seroconverter Cohort [1,2]. This difference could be the result of a broadened inclusion of HET and IDUs due to the sampling method used making this study representative for molecular surveillance of HIV‐1 in Germany.

Men who have sex with men (MSM) are at higher risk for coinfection with hepatitis B virus (HBV), hepatitis C virus (HCV), and syphilis than the general population. HIV infection and these coinfections accelerate disease progression reciprocally. This study evaluated the prevalence and incidence of these coinfections in HIV1-positive MSM in Germany. As part of a nationwide, multicenter, prospective cohort study of HIV-infected MSM, plasma samples collected yearly were screened for HBsAg and antibodies to HBc, HBs, HCV, and syphilis. Samples with indications of active HBV or HCV infection were confirmed by polymerase chain reaction. Prevalence and incidence of each infection and incidence rates per study participant were calculated, and incidences over 4-year time intervals compared. This study screened 5,445 samples from 1,843 MSM. Median age at HIV seroconversion was 33 years. Prevalences of active, cleared, and occult HBV, and of active/cleared HCV were 1.7%, 27.1%, 0.2%, and 8.2%, respectively, and 47.5% had been effectively vaccinated against HBV. Prevalence of antibodies to Treponema pallidum and of triple or quadruple sexually transmitted infections (STIs) were 39.6% and 18.9%, respectively. Prevalence of STI, cleared HBV, HBV vaccination, and history of syphilis differed significantly among age groups. Incidences of HBV, HCV, and syphilis were 2.51, 1.54, and 4.06 per 100 person-years, respectively. Incidences of HCV and syphilis increased over time. HCV incidence was significantly higher in MSM coinfected with syphilis and living in Berlin, and syphilis incidence was significantly higher for MSM living in Berlin. Despite extensive HBV vaccination campaigns, fewer than 50% of screened MSM were effectively vaccinated, with a high proportion of HIV-positive MSM coinfected with HBV. High rates of STI coinfections in HIV-positive MSM and increasing incidences emphasize the need for better tailored campaigns for HBV vaccination and STI prevention.

Background The HIV surveillance system in Germany is based on mandatory, anonymous notification of newly diagnosed HIV cases by laboratories. Because the time between HIV infection and the diagnosis of HIV varies widely between persons, it is difficult to determine the number of cases of recent HIV infection among newly diagnosed cases of HIV. In Germany, the BED-capture-enzyme immunoassay (BED-CEIA) has been used to distinguish between recent and long-standing HIV infection. The aim of this analysis is to report the proportion of cases of recent HIV infection among newly diagnosed cases in Germany between 2008 and 2014 and to identify factors associated with recent infections. Methods A sample of voluntary laboratories among all HIV diagnostic laboratories was recruited. Residual blood from HIV diagnostic tests was spotted on filter paper as dried serum or dried plasma spots and was sent along with the notification form of the HIV cases. The BED-CEIA test was performed. A case was defined as recent HIV infection with a BED-CEIA test result of less than 0.8 normalized optical density, with the exclusion of CDC stage C. The proportion of recent newly diagnosed HIV infections among different groups (such as transmission groups, gender or age groups) was calculated. We used logistic regression to identify factors associated with recent HIV infection and to identify subpopulations with high proportions of recent HIV infections. Results Approximately 10,257 newly diagnosed cases were tested for recency using the BED-CEIA. In total, 3084 (30.4%) of those were recently infected with HIV. The highest proportion of recent HIV infections was found among men who had sex with men (MSM) (35%) and persons between 18 and 25 years of age (43.0%). Logistic regression revealed that female German intravenous drug users with a recent HIV infection had a higher chance of being detected than German MSM (OR 2.27). Conclusions Surveillance of recent HIV infection is a useful additional tool to monitor the HIV epidemic in Germany. We could observe ongoing HIV transmission in Germany in general and in different subgroups, and we could identify factors associated with recent HIV infection in Germany.

Background People who inject drugs (PWID) are at increased risk of acquiring and transmitting HIV and Hepatitis C (HCV) due to sharing injection paraphernalia and unprotected sex. To generate seroprevalence data on HIV and HCV among PWID and related data on risk behaviour, a multicentre sero- and behavioural survey using respondent driven sampling (RDS) was conducted in eight German cities between 2011 and 2014. We also evaluated the feasibility and effectiveness of RDS for recruiting PWID in the study cities. Methods Eligible for participation were people who had injected drugs within the last 12 months, were 16 years or older, and who consumed in one of the study cities. Participants were recruited, using low-threshold drop-in facilities as study sites. Initial seeds were selected to represent various sub-groups of people who inject drugs (PWID). Participants completed a face-to-face interview with a structured questionnaire about socio-demographics, sexual and injecting risk behaviours, as well as the utilisation of health services. Capillary blood samples were collected as dried blood spots and were anonymously tested for serological and molecular markers of HIV and HCV. The results are shown as range of proportions (min. and max. values (%)) in the respective study cities. For evaluation of the sampling method we applied criteria from the STROBE guidelines. Results Overall, 2,077 PWID were recruited. The range of age medians was 29–41 years, 18.5–35.3 % of participants were female, and 9.2–30.6 % were foreign born. Median time span since first injection were 10–18 years. Injecting during the last 30 days was reported by 76.0–88.4 % of participants. Sharing needle/syringes (last 30 days) ranged between 4.7 and 22.3 %, while sharing unsterile paraphernalia (spoon, filter, water, last 30 days) was reported by 33.0–43.8 %. A majority of participants (72.8–85.8 %) reported incarceration at least once, and 17.8–39.8 % had injected while incarcerated. Between 30.8 and 66.2 % were currently in opioid substitution therapy. Unweighted HIV seroprevalence ranged from 0–9.1 %, HCV from 42.3–75.0 %, and HCV-RNA from 23.1–54.0 %. The implementation of RDS as a recruiting method in cooperation with low-threshold drop in facilities was well accepted by both staff and PWID. We reached our targeted sample size in seven of eight cities. Conclusions In the recruited sample of mostly current injectors with a long duration of injecting drug use, seroprevalence for HIV and HCV varied greatly between the city samples. HCV was endemic among participants in all city samples. Our results demonstrate the necessity of intensified prevention strategies for blood-borne infections among PWID in Germany.

We aimed to study epidemic trends and predictors for transmitted drug resistance (TDR) in our region, its clinical impact and its association with transmission clusters. We included 778 patients from the AIDS Reference Center in Leuven (Belgium) diagnosed from 1998 to 2012. Resistance testing was performed using population-based sequencing and TDR was estimated using the WHO-2009 surveillance list. Phylogenetic analysis was performed using maximum likelihood and Bayesian techniques. The cohort was predominantly Belgian (58.4%), men who have sex with men (MSM) (42.8%), and chronically infected (86.5%). The overall TDR prevalence was 9.6% (95% confidence interval (CI): 7.7-11.9), 6.5% (CI: 5.0-8.5) for nucleoside reverse transcriptase inhibitors (NRTI), 2.2% (CI: 1.4-3.5) for non-NRTI (NNRTI), and 2.2% (CI: 1.4-3.5) for protease inhibitors. A significant parabolic trend of NNRTI-TDR was found (p = 0.019). Factors significantly associated with TDR in univariate analysis were male gender, Belgian origin, MSM, recent infection, transmission clusters and subtype B, while multivariate and Bayesian network analysis singled out subtype B as the most predictive factor of TDR. Subtype B was related with transmission clusters with TDR that included 42.6% of the TDR patients. Thanks to resistance testing, 83% of the patients with TDR who started therapy had undetectable viral load whereas half of the patients would likely have received a suboptimal therapy without this test. In conclusion, TDR remained stable and a NNRTI up-and-down trend was observed. While the presence of clusters with TDR is worrying, we could not identify an independent, non-sequence based predictor for TDR or transmission clusters with TDR that could help with guidelines or public health measures.