Estimating the potential to prevent locally acquired HIV infections in a UNAIDS Fast-Track City, Amsterdam.

Background: More than 300 cities including the city of Amsterdam in the Netherlands have joined the UNAIDS Fast-Track Cities initiative, committing to accelerate their HIV response and end the AIDS epidemic in cities by 2030. To support this commitment, we aimed to estimate the number and proportion of Amsterdam HIV infections that originated within the city, from Amsterdam residents. We also aimed to estimate the proportion of recent HIV infections during the 5-year period 2014-2018 in Amsterdam that remained undiagnosed. Methods: We located diagnosed HIV infections in Amsterdam using postcode data (PC4) at time of registration in the ATHENA observational HIV cohort, and used HIV sequence data to reconstruct phylogeographically distinct, partially observed Amsterdam transmission chains. Individual-level infection times were estimated from biomarker data, and used to date the phylogenetically observed transmission chains as well as to estimate undiagnosed proportions among recent infections. A Bayesian Negative Binomial branching process model was used to estimate the number, size, and growth of the unobserved Amsterdam transmission chains from the partially observed phylogenetic data. Results: Between 1 January 2014 and 1 May 2019, there were 846 HIV diagnoses in Amsterdam residents, of whom 516 (61%) were estimated to have been infected in 2014-2018. The rate of new Amsterdam diagnoses since 2014 (104 per 100,000) remained higher than the national rates excluding Amsterdam (24 per 100,000), and in this sense Amsterdam remained a HIV hotspot in the Netherlands. An estimated 14% [12-16%] of infections in Amsterdan MSM in 2014-2018 remained undiagnosed by 1 May 2019, and 41% [35-48%] in Amsterdam heterosexuals, with variation by region of birth. An estimated 67% [60-74%] of Amsterdam MSM infections in 2014-2018 had an Amsterdam resident as source, and 56% [41-70%] in Amsterdam heterosexuals, with heterogeneity by region of birth. Of the locally acquired infections, an estimated 43% [37-49%] were in foreign-born MSM, 41% [35-47%] in Dutch-born MSM, 10% [6-18%] in foreign-born heterosexuals, and 5% [2-9%] in Dutch-born heterosexuals. We estimate the majority of Amsterdam MSM infections in 2014-2018 originated in transmission chains that pre-existed by 2014. Conclusions: This combined phylogenetic, epidemiologic, and modelling analysis in the UNAIDS Fast-Track City Amsterdam indicates that there remains considerable potential to prevent HIV infections among Amsterdam residents through city-level interventions. The burden of locally acquired infection remains concentrated in MSM, and both Dutch-born and foreign-born MSM would likely benefit most from intensified city-level interventions. Funding: This study received funding as part of the H-TEAM initiative from Aidsfonds (project number P29701). The H-TEAM initiative is being supported by Aidsfonds (grant number: 2013169, P29701, P60803), Stichting Amsterdam Dinner Foundation, Bristol-Myers Squibb International Corp. (study number: AI424-541), Gilead Sciences Europe Ltd (grant number: PA-HIV-PREP-16-0024), Gilead Sciences (protocol numbers: CO-NL-276-4222, CO-US-276-1712, CO-NL-985-6195), and M.A.C AIDS Fund.

Phylogenetic estimation of the viral fitness landscape of HIV-1 set-point viral load.

Set-point viral load (SPVL), a common measure of human immunodeficiency virus (HIV)-1 virulence, is partially determined by viral genotype. Epidemiological evidence suggests that this viral property has been under stabilising selection, with a typical optimum for the virus between 104 and 105 copies of viral RNA per ml. Here we aimed to detect transmission fitness differences between viruses from individuals with different SPVLs directly from phylogenetic trees inferred from whole-genome sequences. We used the local branching index (LBI) as a proxy for transmission fitness. We found that LBI is more sensitive to differences in infectiousness than to differences in the duration of the infectious state. By analysing subtype-B samples from the Bridging the Evolution and Epidemiology of HIV in Europe project, we inferred a significant positive relationship between SPVL and LBI up to approximately 105 copies/ml, with some evidence for a peak around this value of SPVL. This is evidence of selection against low values of SPVL in HIV-1 subtype-B strains, likely related to lower infectiousness, and perhaps a peak in the transmission fitness in the expected range of SPVL. The less prominent signatures of selection against higher SPVL could be explained by an inherent limit of the method or the deployment of antiretroviral therapy.

A highly virulent variant of HIV-1 circulating in the Netherlands.

We discovered a highly virulent variant of subtype-B HIV-1 in the Netherlands. One hundred nine individuals with this variant had a 0.54 to 0.74 log10 increase (i.e., a ~3.5-fold to 5.5-fold increase) in viral load compared with, and exhibited CD4 cell decline twice as fast as, 6604 individuals with other subtype-B strains. Without treatment, advanced HIV-CD4 cell counts below 350 cells per cubic millimeter, with long-term clinical consequences-is expected to be reached, on average, 9 months after diagnosis for individuals in their thirties with this variant. Age, sex, suspected mode of transmission, and place of birth for the aforementioned 109 individuals were typical for HIV-positive people in the Netherlands, which suggests that the increased virulence is attributable to the viral strain. Genetic sequence analysis suggests that this variant arose in the 1990s from de novo mutation, not recombination, with increased transmissibility and an unfamiliar molecular mechanism of virulence.

Many but small HIV-1 non-B transmission chains in the Netherlands.

OBJECTIVE: The aim of this study was to investigate introductions and spread of different HIV-1 subtypes in the Netherlands. DESIGN: We identified distinct HIV-1 transmission chains in the Netherlands within the global epidemic context through viral phylogenetic analysis of partial HIV-1 polymerase sequences from individuals enrolled in the ATHENA national HIV cohort of all persons in care since 1996, and publicly available international background sequences. METHODS: Viral lineages circulating in the Netherlands were identified through maximum parsimony phylogeographic analysis. The proportion of HIV-1 infections acquired in-country among heterosexuals and MSM was estimated from phylogenetically observed, national transmission chains using a branching process model that accounts for incomplete sampling. RESULTS: As of 1 January 2019, 2589 (24%) of 10 971 (41%) HIV-1 sequenced individuals in ATHENA had non-B subtypes (A1, C, D, F, G) or circulating recombinant forms (CRF01AE, CRF02AG, CRF06-cpx). The 1588 heterosexuals were in 1224, and 536 MSM in 270 phylogenetically observed transmission chains. After adjustments for incomplete sampling, most heterosexual (75%) and MSM (76%) transmission chains were estimated to include only the individual introducing the virus (size = 1). Onward transmission occurred mostly in chains size 2-5 amongst heterosexuals (62%) and in chains size at least 10 amongst MSM (64%). Considering some chains originated in-country from other risk-groups, 40% (95% confidence interval: 36-44) of non-B-infected heterosexuals and 62% (95% confidence interval: 49-73) of MSM-acquired infection in-country. CONCLUSION: Although most HIV-1 non-B introductions showed no or very little onward transmission, a considerable proportion of non-B infections amongst both heterosexuals and MSM in the Netherlands have been acquired in-country.

Impact of frequent testing on the transmission of HIV and N. gonorrhoeae among men who have sex with men: a mathematical modelling study.

OBJECTIVES: To investigate the impact and efficiency of combined testing for HIV and other STIs on HIV and STI transmission among men who have sex with men (MSM) and to assess what subgroups of MSM should be targeted for frequent testing. METHODS: We developed an agent-based transmission model that simulates infection with HIV or Neisseria gonorrhoeae (NG) among MSM. We examined scenarios with increased percentages of MSM getting tested six monthly, among all MSM or only specific subgroups of MSM (defined according to recent gonorrhoea, number of partners and engagement in condomless anal intercourse (CAI)) and scenarios with reduced intervals between HIV/STI tests. RESULTS: The most efficient strategies were those with increased percentage of MSM getting tested every 6 months among MSM with a recent gonorrhoea diagnosis; or among MSM who had CAI and ≥10 partners; or MSM who had ≥10 partners. Over 10 years, these strategies resulted in 387-718 averted HIV infections and required 29-164 additional HIV tests per averted HIV infection or one to seven additional gonorrhoea tests per averted NG infection. The most effective strategy in reducing HIV transmission was the one where the intervals between tests were reduced by half, followed by the strategy with increased percentage of MSM getting tested every 6 months among all MSM. Over 10 years, these strategies resulted in 1362 and 1319 averted HIV infections, but required 663 and 584 additional HIV tests per averted HIV infection, respectively. CONCLUSIONS: Targeting MSM with recent gonorrhoea diagnosis or MSM with many partners is efficient in terms of HIV/STI tests needed to prevent new HIV or NG infections. Major reductions in HIV incidence can be achieved with consistent HIV/STI testing every 6 months among larger groups, including low-risk MSM. To impede HIV transmission, frequent testing should be combined with other prevention measures.

Phylogenies from dynamic networks.

The relationship between the underlying contact network over which a pathogen spreads and the pathogen phylogenetic trees that are obtained presents an opportunity to use sequence data to learn about contact networks that are difficult to study empirically. However, this relationship is not explicitly known and is usually studied in simulations, often with the simplifying assumption that the contact network is static in time, though human contact networks are dynamic. We simulate pathogen phylogenetic trees on dynamic Erdos-Renyi random networks and on two dynamic networks with skewed degree distribution, of which one is additionally clustered. We use tree shape features to explore how adding dynamics changes the relationships between the overall network structure and phylogenies. Our tree features include the number of small substructures (cherries, pitchforks) in the trees, measures of tree imbalance (Sackin index, Colless index), features derived from network science (diameter, closeness), as well as features using the internal branch lengths from the tip to the root. Using principal component analysis we find that the network dynamics influence the shapes of phylogenies, as does the network type. We also compare dynamic and time-integrated static networks. We find, in particular, that static network models like the widely used Barabasi-Albert model can be poor approximations for dynamic networks. We explore the effects of mis-specifying the network on the performance of classifiers trained identify the transmission rate (using supervised learning methods). We find that both mis-specification of the underlying network and its parameters (mean degree, turnover rate) have a strong adverse effect on the ability to estimate the transmission parameter. We illustrate these results by classifying HIV trees with a classifier that we trained on simulated trees from different networks, infection rates and turnover rates. Our results point to the importance of correctly estimating and modelling contact networks with dynamics when using phylodynamic tools to estimate epidemiological parameters.

Elimination prospects of the Dutch HIV epidemic among men who have sex with men in the era of preexposure prophylaxis.

OBJECTIVE: Preexposure prophylaxis (PrEP) is a promising intervention to help end the HIV epidemic among men who have sex with men (MSM) in the Netherlands. We aimed to assess the impact of PrEP on HIV prevalence in this population and to determine the levels of PrEP coverage necessary for HIV elimination. DESIGN AND METHODS: We developed a mathematical model of HIV transmission in a population stratified by sexual risk behavior with universal antiretroviral treatment (ART) and daily PrEP use depending on an individual's risk behavior. We computed the effective reproduction number, HIV prevalence, ART and PrEP coverage for increasing ART and PrEP uptake levels, and examined how these were affected by PrEP effectiveness and duration of PrEP use. RESULTS: At current levels of ART coverage of 80%, PrEP effectiveness of 86% and PrEP duration of 5 years, HIV elimination required 82% PrEP coverage in the highest risk group (12 000 MSM with more than 18 partners per year). If ART coverage increased by 9%, the elimination threshold was at 70% PrEP coverage. For shorter PrEP duration and lower effectiveness elimination prospects were less favorable. For the same number of PrEP users distributed among two groups with highest risk behavior, prevalence dropped from the current 8 to 4.6%. CONCLUSION: PrEP for HIV prevention among MSM could, in principle, eliminate HIV from this population in the Netherlands. The highest impact of PrEP on prevalence was predicted when ART and PrEP coverage increased simultaneously and PrEP was used by the highest risk individuals.

AIDS Therapy Evaluation in the Netherlands (ATHENA) national observational HIV cohort: cohort profile.

PURPOSE: In 1998, the AIDS Therapy Evaluation in the Netherlands (ATHENA) national observational HIV cohort was established to demonstrate the lifesaving effectiveness of triple combination antiretroviral therapy, including HIV-protease inhibitors, that had recently been made available for clinical use. Subsequently, the HIV Monitoring Foundation was established by the Dutch Ministry of Health, Welfare and Sport to continue ATHENA as an open cohort in order to continue the registration and monitoring of all HIV-positive people as an integral part of HIV care in all 26 HIV treatment centres in the Netherlands. PARTICIPANTS: To date, a total of 25 036 participants have been enrolled in the cohort, with 263 600 person-years of follow-up. As of 1 January 2017, 19 035 HIV-1-positive participants were known to be in care: 18 824 adults (81% men and 19% women) and 211 children (47% boys and 53% girls). The remaining 6001 participants had either died (46%), were lost to care (29%) or had moved abroad (25%). FINDINGS TO DATE: Today, with over 20 years of follow-up, the ATHENA cohort has provided extensive knowledge on HIV treatment, comorbidities and coinfections and created insight into the transmission dynamics of the HIV epidemic. FUTURE PLANS: ATHENA continues to enrol and monitor HIV positive people entering HIV care in the Netherlands. Future research will continue to provide tangible input into HIV care and prevention policies in the Netherlands and internationally.

Easy and accurate reconstruction of whole HIV genomes from short-read sequence data with shiver.

Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of large between- and within-host diversity, including frequent indels, may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions. De novo assembly avoids this bias by aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the tool shiver to pre-process reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with the user's choice of existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We used shiver to reconstruct the consensus sequence and minority variant information from paired-end short-read whole-genome data produced with the Illumina platform, for sixty-five existing publicly available samples and fifty new samples. We show the systematic superiority of mapping to shiver's constructed reference compared with mapping the same reads to the closest of 3,249 real references: median values of 13 bases called differently and more accurately, 0 bases called differently and less accurately, and 205 bases of missing sequence recovered. We also successfully applied shiver to whole-genome samples of Hepatitis C Virus and Respiratory Syncytial Virus. shiver is publicly available from https://github.com/ChrisHIV/shiver.

Correction: Viral genetic variation accounts for a third of variability in HIV-1 set-point viral load in Europe.

[This corrects the article DOI: 10.1371/journal.pbio.2001855.].

Limited overlap between phylogenetic HIV and hepatitis C virus clusters illustrates the dynamic sexual network structure of Dutch HIV-infected MSM.

OBJECTIVE: MSM are at increased risk for infection with HIV-1 and hepatitis C virus (HCV). Is HIV/HCV coinfection confined to specific HIV transmission networks? DESIGN AND METHODS: A HIV phylogenetic tree was constructed for 5038 HIV-1 subtype B polymerase (pol) sequences obtained from MSM in the AIDS therapy evaluation in the Netherlands cohort. We investigated the existence of HIV clusters with increased HCV prevalence, the HIV phylogenetic density (i.e. the number of potential HIV transmission partners) of HIV/HCV-coinfected MSM compared with HIV-infected MSM without HCV, and the overlap in HIV and HCV phylogenies using HCV nonstructural protein 5B sequences from 183 HIV-infected MSM with acute HCV infection. RESULTS: Five hundred and sixty-three of 5038 (11.2%) HIV-infected MSM tested HCV positive. Phylogenetic analysis revealed 93 large HIV clusters (≥10 MSM), 370 small HIV clusters (2-9 MSM), and 867 singletons with a median HCV prevalence of 11.5, 11.6, and 9.3%, respectively. We identified six large HIV clusters with elevated HCV prevalence (range 23.5-46.2%). Median HIV phylogenetic densities for MSM with HCV (3, interquartile range 1-7) and without HCV (3, interquartile range 1-8) were similar. HCV phylogeny showed 12 MSM-specific HCV clusters (clustersize: 2-39 HCV sequences); 12.7% of HCV infections were part of the same HIV and HCV cluster. CONCLUSION: We observed few HIV clusters with elevated HCV prevalence, no increase in the HIV phylogenetic density of HIV/HCV-coinfected MSM compared to HIV-infected MSM without HCV, and limited overlap between HIV and HCV phylogenies among HIV/HCV-coinfected MSM. Our data do not support the existence of MSM-specific sexual networks that fuel both the HIV and HCV epidemic.

Viral genetic variation accounts for a third of variability in HIV-1 set-point viral load in Europe.

HIV-1 set-point viral load-the approximately stable value of viraemia in the first years of chronic infection-is a strong predictor of clinical outcome and is highly variable across infected individuals. To better understand HIV-1 pathogenesis and the evolution of the viral population, we must quantify the heritability of set-point viral load, which is the fraction of variation in this phenotype attributable to viral genetic variation. However, current estimates of heritability vary widely, from 6% to 59%. Here we used a dataset of 2,028 seroconverters infected between 1985 and 2013 from 5 European countries (Belgium, Switzerland, France, the Netherlands and the United Kingdom) and estimated the heritability of set-point viral load at 31% (CI 15%-43%). Specifically, heritability was measured using models of character evolution describing how viral load evolves on the phylogeny of whole-genome viral sequences. In contrast to previous studies, (i) we measured viral loads using standardized assays on a sample collected in a strict time window of 6 to 24 months after infection, from which the viral genome was also sequenced; (ii) we compared 2 models of character evolution, the classical "Brownian motion" model and another model ("Ornstein-Uhlenbeck") that includes stabilising selection on viral load; (iii) we controlled for covariates, including age and sex, which may inflate estimates of heritability; and (iv) we developed a goodness of fit test based on the correlation of viral loads in cherries of the phylogenetic tree, showing that both models of character evolution fit the data well. An overall heritability of 31% (CI 15%-43%) is consistent with other studies based on regression of viral load in donor-recipient pairs. Thus, about a third of variation in HIV-1 virulence is attributable to viral genetic variation.

Phylogenetic evidence for underreporting of male-to-male sex among human immunodeficiency virus-infected donors in the Netherlands and Flanders.

BACKGROUND: Separate transmission networks for human immunodeficiency virus (HIV) coexist. Molecular typing of viral genomes can provide insight in HIV transmission routes in donors for whom risk behavior-based donor selection failed. STUDY DESIGN AND METHODS: This study includes all HIV-infected Dutch and Flemish donors in the period 2005 to 2014 (n = 55). Part of the HIV polymerase (pol) gene was amplified, sequenced, and compared with more than 10,000 HIV strains obtained from HIV-infected Dutch and Flemish patients. The most likely transmission route was determined based on HIV phylogeny and the donor's self-reported risk behavior during the exit interview. RESULTS: HIV-infected donors were predominantly male (69%), were repeat donors (73%), were born in the Netherlands or Belgium (95%), and harbored HIV Subtype B (68%). Seventy-five percent of HIV-infected male donors were part of robust phylogenetic clusters linked to male-to-male sex, while only 24% of HIV-infected male donors reported male-to-male sex during posttest counseling. Sex between men and women accounted for 13% of HIV infections in male donors and 93% of HIV infections in female donors based on phylogenetic analysis. Only 40% of HIV-infected female donors had HIV Subtype B; 65% of female donors reported a foreign partner and indeed HIV sequences interspersed with sequences from HIV-endemic areas abroad, in particular sub-Saharan Africa. CONCLUSION: HIV typing helps to understand HIV transmission routes in donor populations. We found substantial underreporting of male-to-male sex among HIV-infected male donors. Donor education on HIV risk factors and the danger of window-period donations and a donor environment that encourages frank disclosure of sexual behavior will contribute to a decrease of HIV-infected donors.

From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing.

The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5kb, and subsequent recovery of near-complete viral genomes. Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml.

Sources of HIV infection among men having sex with men and implications for prevention.

New HIV diagnoses among men having sex with men (MSM) have not decreased appreciably in most countries, even though care and prevention services have been scaled up substantially in the past 20 years. To maximize the impact of prevention strategies, it is crucial to quantify the sources of transmission at the population level. We used viral sequence and clinical patient data from one of Europe's nationwide cohort studies to estimate probable sources of transmission for 617 recently infected MSM. Seventy-one percent of transmissions were from undiagnosed men, 6% from men who had initiated antiretroviral therapy (ART), 1% from men with no contact to care for at least 18 months, and 43% from those in their first year of infection. The lack of substantial reductions in incidence among Dutch MSM is not a result of ineffective ART provision or inadequate retention in care. In counterfactual modeling scenarios, 19% of these past cases could have been averted with current annual testing coverage and immediate ART to those testing positive. Sixty-six percent of these cases could have been averted with available antiretrovirals (immediate ART provided to all MSM testing positive, and preexposure antiretroviral prophylaxis taken by half of all who test negative for HIV), but only if half of all men at risk of transmission had tested annually. With increasing sequence coverage, molecular epidemiological analyses can be a key tool to direct HIV prevention strategies to the predominant sources of infection, and help send HIV epidemics among MSM into a decisive decline.

A Method to Estimate the Size and Characteristics of HIV-positive Populations Using an Individual-based Stochastic Simulation Model.

It is important not only to collect epidemiologic data on HIV but to also fully utilize such information to understand the epidemic over time and to help inform and monitor the impact of policies and interventions. We describe and apply a novel method to estimate the size and characteristics of HIV-positive populations. The method was applied to data on men who have sex with men living in the UK and to a pseudo dataset to assess performance for different data availability. The individual-based simulation model was calibrated using an approximate Bayesian computation-based approach. In 2013, 48,310 (90% plausibility range: 39,900-45,560) men who have sex with men were estimated to be living with HIV in the UK, of whom 10,400 (6,160-17,350) were undiagnosed. There were an estimated 3,210 (1,730-5,350) infections per year on average between 2010 and 2013. Sixty-two percent of the total HIV-positive population are thought to have viral load <500 copies/ml. In the pseudo-epidemic example, HIV estimates have narrower plausibility ranges and are closer to the true number, the greater the data availability to calibrate the model. We demonstrate that our method can be applied to settings with less data, however plausibility ranges for estimates will be wider to reflect greater uncertainty of the data used to fit the model.

CD4+ cell dynamics in untreated HIV-1 infection: overall rates, and effects of age, viral load, sex and calendar time.

BACKGROUND: CD4 cell count is a key measure of HIV disease progression, and the basis of successive international guidelines for treatment initiation. CD4 cell dynamics are used in mathematical and econometric models for evaluating public health need and interventions. Here, we estimate rates of CD4 decline, stratified by relevant covariates, in a form that is clinically transparent and can be directly used in such models. METHODS: We analyse the AIDS Therapy Evaluation in the Netherlands cohort, including individuals with date of seroconversion estimated to be within 1 year and with intensive clinical follow-up prior to treatment initiation. Owing to the fact that CD4 cell counts are intrinsically noisy, we separate the analysis into long-term trends of smoothed CD4 cell counts and an observation model relating actual CD4 measurements to the underlying smoothed counts. We use a monotonic spline smoothing model to describe the decline of smoothed CD4 cell counts through categories CD4 above 500, 350-500, 200-350 and 200 cells/µl or less. We estimate the proportion of individuals starting in each category after seroconversion and the average time spent in each category. We examine individual-level cofactors which influence these parameters. RESULTS: Among untreated individuals, the time spent in each compartment was 3.32, 2.70, 5.50 and 5.06 years. Only 76% started in the CD4 cell count above 500 cells/µl compartment after seroconversion. Set-point viral load (SPVL) was an important factor: individuals with at least 5 log10 copies/ml took 5.37 years to reach CD4 cell count less than 200 cells/µl compared with 15.76 years for SPVL less than 4 log10 copies/ml. CONCLUSION: Many individuals already have CD4 cell count below 500 cells/µl after seroconversion. SPVL strongly influences the rate of CD4 decline. Treatment guidelines should consider measuring SPVL, whereas mathematical models should incorporate SPVL stratification.

Dispersion of the HIV-1 Epidemic in Men Who Have Sex with Men in the Netherlands: A Combined Mathematical Model and Phylogenetic Analysis.

BACKGROUND: The HIV-1 subtype B epidemic amongst men who have sex with men (MSM) is resurgent in many countries despite the widespread use of effective combination antiretroviral therapy (cART). In this combined mathematical and phylogenetic study of observational data, we aimed to find out the extent to which the resurgent epidemic is the result of newly introduced strains or of growth of already circulating strains. METHODS AND FINDINGS: As of November 2011, the ATHENA observational HIV cohort of all patients in care in the Netherlands since 1996 included HIV-1 subtype B polymerase sequences from 5,852 patients. Patients who were diagnosed between 1981 and 1995 were included in the cohort if they were still alive in 1996. The ten most similar sequences to each ATHENA sequence were selected from the Los Alamos HIV Sequence Database, and a phylogenetic tree was created of a total of 8,320 sequences. Large transmission clusters that included ≥10 ATHENA sequences were selected, with a local support value ≥ 0.9 and median pairwise patristic distance below the fifth percentile of distances in the whole tree. Time-varying reproduction numbers of the large MSM-majority clusters were estimated through mathematical modeling. We identified 106 large transmission clusters, including 3,061 (52%) ATHENA and 652 Los Alamos sequences. Half of the HIV sequences from MSM registered in the cohort in the Netherlands (2,128 of 4,288) were included in 91 large MSM-majority clusters. Strikingly, at least 54 (59%) of these 91 MSM-majority clusters were already circulating before 1996, when cART was introduced, and have persisted to the present. Overall, 1,226 (35%) of the 3,460 diagnoses among MSM since 1996 were found in these 54 long-standing clusters. The reproduction numbers of all large MSM-majority clusters were around the epidemic threshold value of one over the whole study period. A tendency towards higher numbers was visible in recent years, especially in the more recently introduced clusters. The mean age of MSM at diagnosis increased by 0.45 years/year within clusters, but new clusters appeared with lower mean age. Major strengths of this study are the high proportion of HIV-positive MSM with a sequence in this study and the combined application of phylogenetic and modeling approaches. Main limitations are the assumption that the sampled population is representative of the overall HIV-positive population and the assumption that the diagnosis interval distribution is similar between clusters. CONCLUSIONS: The resurgent HIV epidemic amongst MSM in the Netherlands is driven by several large, persistent, self-sustaining, and, in many cases, growing sub-epidemics shifting towards new generations of MSM. Many of the sub-epidemics have been present since the early epidemic, to which new sub-epidemics are being added.

Estimating HIV Incidence, Time to Diagnosis, and the Undiagnosed HIV Epidemic Using Routine Surveillance Data.

BACKGROUND: Estimates of the size of the undiagnosed HIV-infected population are important to understand the HIV epidemic and to plan interventions, including "test-and-treat" strategies. METHODS: We developed a multi-state back-calculation model to estimate HIV incidence, time between infection and diagnosis, and the undiagnosed population by CD4 count strata, using surveillance data on new HIV and AIDS diagnoses. The HIV incidence curve was modelled using cubic splines. The model was tested on simulated data and applied to surveillance data on men who have sex with men in The Netherlands. RESULTS: The number of HIV infections could be estimated accurately using simulated data, with most values within the 95% confidence intervals of model predictions. When applying the model to Dutch surveillance data, 15,400 (95% confidence interval [CI] = 15,000, 16,000) men who have sex with men were estimated to have been infected between 1980 and 2011. HIV incidence showed a bimodal distribution, with peaks around 1985 and 2005 and a decline in recent years. Mean time to diagnosis was 6.1 (95% CI = 5.8, 6.4) years between 1984 and 1995 and decreased to 2.6 (2.3, 3.0) years in 2011. By the end of 2011, 11,500 (11,000, 12,000) men who have sex with men in The Netherlands were estimated to be living with HIV, of whom 1,750 (1,450, 2,200) were still undiagnosed. Of the undiagnosed men who have sex with men, 29% (22, 37) were infected for less than 1 year, and 16% (13, 20) for more than 5 years. CONCLUSIONS: This multi-state back-calculation model will be useful to estimate HIV incidence, time to diagnosis, and the undiagnosed HIV epidemic based on routine surveillance data.

HIV Type 1 transmission networks among men having sex with men and heterosexuals in Kenya.

We performed a molecular phylogenetic study on HIV-1 polymerase sequences of men who have sex with men (MSM) and heterosexual patient samples in Kenya to characterize any observed HIV-1 transmission networks. HIV-1 polymerase sequences were obtained from samples in Nairobi and coastal Kenya from 84 MSM, 226 other men, and 364 women from 2005 to 2010. Using Bayesian phylogenetics, we tested whether sequences clustered by sexual orientation and geographic location. In addition, we used trait diffusion analyses to identify significant epidemiological links and to quantify the number of transmissions between risk groups. Finally, we compared 84 MSM sequences with all HIV-1 sequences available online at GenBank. Significant clustering of sequences from MSM at both coastal Kenya and Nairobi was found, with evidence of HIV-1 transmission between both locations. Although a transmission pair between a coastal MSM and woman was confirmed, no significant HIV-1 transmission was evident between MSM and the comparison population for the predominant subtype A (60%). However, a weak but significant link was evident when studying all subtypes together. GenBank comparison did not reveal other important transmission links. Our data suggest infrequent intermingling of MSM and heterosexual HIV-1 epidemics in Kenya.