Daniela Bezemer and colleagues investigate the extent to which the resurgent HIV epidemic in the Netherlands is the result of newly introduced strains, or of growth of already circulating strains.
The HIV-1 epidemic amongst men who have sex with men (MSM) is resurgent in many Western countries [1–5]. This may seem paradoxical, as HIV-positive men are being diagnosed at an increasingly earlier stage of HIV infection, and, since 1996, most diagnosed men take effective combination antiretroviral therapy (cART). Not only does successful therapy halt disease progression , but successful suppression of virus replication also reduces the risk of transmitting HIV [7,8]. With the introduction of cART, however, there has been an increase in unprotected sex in many countries. For the Netherlands, this was first demonstrated using a mathematical model that included disease progression to analyze national data from the ATHENA cohort on HIV and AIDS diagnoses and treatment success and failure [1,2]. These findings were later confirmed by analysis of behavioral data from the Amsterdam Cohort Studies, which found that sexual risk behavior increased in a manner similar to model predictions [9,10]. The resulting reproduction number for the epidemic amongst MSM was estimated to be around the epidemic threshold of one . In the period 2007–2010, 2,928 new HIV diagnoses were registered amongst MSM in the Netherlands, very similar to earlier predictions assuming no reductions in risk behavior .
In the current study, we aimed to gain more insight into the transmission clusters that constitute this mainly HIV-1 subtype B epidemic amongst MSM over time . Similarly to an earlier Swiss study, we used phylogenetic analysis of extensive HIV-1 subtype B polymerase (pol) sequence data to identify large transmission clusters . The authors of the Swiss study subsequently estimated a reproduction number for each sub-epidemic directly from the sequence data [13,14]. Here, we also analyze the viral transmission dynamics over time within the different clusters. However, we applied a mathematical model to the estimated incidence of diagnoses within each cluster, using the distribution of time between diagnoses of index cases and secondary cases derived from a previous study [2,15]. Our analysis estimates the time-varying reproduction numbers of the large MSM-majority clusters that together drive the overall dynamics of the epidemic. This will clarify the extent to which the resurgent epidemic is the result of newly introduced strains or of growth of already circulating strains.
The methods used in this study, along with the underlying assumptions, are summarized in Fig 1 and explained in detail below and in S1 Text.
The ATHENA national observational HIV cohort includes anonymized data from all HIV-infected patients followed longitudinally in the 27 HIV treatment centers, in the Netherlands since 1 January 1996 and in the St. Elisabeth Hospital in Willemstad, Curaçao, since 1 January 2005, except 1.5% who opt out . Patients who were diagnosed between 1981 and 1995 were included in the cohort if they were still alive in 1 January 1996 . Demographic data were collected at entry in the cohort, including self-reported most likely risk group of infection, and most likely country of infection. Population-based nucleotide HIV-1 partial pol gene sequences were obtained as part of the screening for resistance to antiretroviral drugs, either before start of cART or at the time of virological failure, sometimes retrospectively, as described in detail previously [11,17]. Subtype B sequences were identified by phylogenetic analysis using reference sequences from the Los Alamos HIV Sequence Database . As of 1 November 2011, the ATHENA cohort included 19,095 HIV-1-infected patients, of whom 7,589 (40%) had at least one sequence; 5,852 (77%) patients were infected with a subtype B strain.
At initiation, the ATHENA observational cohort was approved by the institutional review boards of all participating centers. It has subsequently become an integral part of HIV care and includes anonymized data and stored plasma samples from HIV-infected patients living in the Netherlands and receiving care in one of the designated HIV treatment centers. Patients can opt out after being informed by their treating physician of the purpose of collection of data and samples. Data from patients who opt out are not included in the ATHENA database. Anonymized data may be used for scientific purposes without further review. Patients are informed that if there are future requests for use of stored plasma samples for scientific research, they will be asked for prior consent by their treating HIV physician. Data are anonymized before being provided to investigators. For the purpose of our analysis, only existing data have been used, and therefore no additional review or consent has been necessary.
Recent HIV-1 Infections
Recent HIV-1 infections were defined as infections with either (i) a seroconversion interval of 18 mo or less between the last negative and the first positive HIV-1 serology test or (ii) a diagnosis of primary HIV-1 infection defined as detectable HIV-1 RNA in plasma, detectable serum p24 antigen in plasma, or both, combined with either a negative HIV-1 antibody test or a positive HIV-1 antibody test with a negative or indeterminate HIV-1 Western blot.
Patient Sequences in a Phylogenetic Context
A phylogenetic context for the HIV sequences in our patient cohort was provided by sequences that were selected from the Los Alamos HIV Sequence Database . As of 15 March 2013, there were 56,643 subtype B sequences, one per patient, of minimal length 500, with a known country of sampling that was not the Netherlands. Sixteen sequences from Suriname were added from a previous study . For each of the 5,852 subtype B sequences in our dataset, the ten most similar sequences were identified by BLAST 2.2.22+ . In total, 2,468 unique sequences were added to the study.
A total of 8,320 unique sequences were aligned using Clustal Omega 1.1.0  and manually checked and adjusted. Gblocks 0.91b was used to define the optimal sequence length of consistently aligned positions at 1,254 nucleotides . Major resistance-conferring sites at the amino acid positions described by the International AIDS Society–USA were excluded for phylogenetic analysis, including alternative substitutions at position 215 and two insertions , resulting in an alignment length of 1,137 nucleotides.
A phylogenetic tree was built in FastTree 2.1.3 using a general time-reversible(GTR) model . PhyloPart 2.0 was used to define transmission clusters as those clusters of sequences with a local support value ≥ 0.9 based on 1,000 resamples and for which the median value of all pairwise patristic distances of the sub-tree was below the fifth percentile threshold of the distribution of all pairwise distances of the whole tree , which corresponded to 0.068 mutations per site. We considered only large national transmission clusters with sequences from at least ten patients from within the ATHENA cohort . The median of the median values of all pairwise distances per large cluster was 0.042 (interquartile range [IQR] 0.030–0.057) mutations per site. The duration of a large cluster was defined as the time between the earliest and latest date of diagnosis within the cluster. As a sensitivity analysis, comparisons for three different cluster definitions were made: a “looser” cluster definition, with a local support value ≥ 0.8 and with a median value of all pairwise distances under the tenth percentile threshold, a “more stringent” cluster definition, with a local support value ≥ 0.95 and with a median value of all pairwise distances under the 2.5th percentile threshold, and a third cluster definition, with a local support value ≥ 0.9 and a single linkage distance threshold of 0.042 mutations/site.
Bayesian Time-Scaled Phylogenetic Analysis
We applied a Bayesian Markov chain Monte Carlo (MCMC) method as implemented in BEAST 1.7.5 for phylogenetic analysis of time-stamped sequence data to calculate the time of the most recent common ancestor (MRCA) of each large cluster. This value would indicate the time point since when each cluster has been circulating in the Netherlands, and simultaneously we confirmed the selected clusters by a different phylogenetic method. This was done by grouping clusters together in batches of approximately 200 sequences. Sequences from the Los Alamos HIV Sequence Database were included when the national cluster included ≥5 time-stamped sequences obtained in another country . We used the uncorrelated log-normal relaxed molecular clock with a discretized gamma-distributed general time-reversible substitution model and the Bayesian skyride coalescent model [27,28]. Analyses of 200 million MCMC generations were performed, and evolutionary parameters were sampled every 20,000 generations to retrieve a distribution of posterior trees. After removing 10% of the initial output for burn-in, convergence of the MCMC output with effective sample size above 200 was inspected and confirmed in Tracer . Maximum clade credibility (MCC) trees keeping the heights of the target tree were obtained using TreeAnnotator 1.8.0 after discarding 10% of the posterior trees as burn-in and were visualized using FigTree 1.4.2 . Node ages and correspondent 95% highest posterior density (HPD) intervals were taken as the time of the MRCA and corresponding uncertainty estimates.
Estimation of the Time-Varying Case Reproduction Number within Transmission Clusters
We analyzed HIV-1 transmission dynamics within each of the large transmission clusters containing a majority of MSM. For each cluster, we estimated the case reproduction number for each year t, Rt, defined as the average number of secondary cases infected by an index case diagnosed in year t. We used an extension of the method introduced by Wallinga and Teunis , which allows estimation of Rt based on the incidence of diagnosis time series and what we call the diagnosis interval distribution, i.e., the distribution of the time from diagnosis of an index case to diagnosis of a secondary case.
We extended the Wallinga and Teunis method to allow the diagnosis interval to vary according to the time of diagnosis of the index case, and to allow for the possibility that the diagnosis interval can be negative, because it is possible that individuals may have been infected by index cases who had not yet been diagnosed. The likelihood that case i has been infected by case j is decomposed into the product of the likelihood that the index case of i has been diagnosed and the relative likelihood that if the index case of i has been diagnosed, it is a particular individual j. The relative likelihood that case i has been infected by case j (rather than by another case diagnosed up to year T, here T = 2010) is given by
where wt(.) denotes the distribution of the diagnosis interval for index cases diagnosed in year t.
The likelihood that case i has been infected by a case observed up to year T can be approximated by
assuming that the incidence of diagnosed cases is constant over time (see S1 Text). Additionally assuming that the diagnosis interval distribution remains unchanged after T, this ratio simplifies to
where S > 0 is the maximum possible negative diagnosis interval, such that for all u < −S and wT(u) = 0, the total likelihood that a diagnosed case i has been infected by case j (rather than by another case, diagnosed or not) is given by
Note that the derivation of qi assumes a constant incidence over time. Analyses presented in S1 Text show how qi is modified when this assumption does not hold, and these analyses indicate that the resulting estimates of the reproduction number show consistent temporal trends regardless of the validity of this assumption, although the actual values may vary (S2 and S3 Figs).
The time-varying diagnosis interval wt(.) was obtained by numerically simulating a compartmental HIV transmission model previously fitted to HIV and AIDS diagnosis data on MSM in the Netherlands, where it is assumed no transmission occurs when individuals are on treatment (see S1 Text) . This mathematical model was used to estimate the time-varying diagnosis interval for the whole HIV epidemic amongst MSM, and not to directly fit to cluster dynamics (see S1 Fig).
We further extended the Wallinga and Teunis method to incorporate uncertainty in the number of individuals diagnosed in a certain year t, Dt. The ATHENA cohort records the number St of individuals diagnosed in year t who survived until 1996 and for whom a pol sequence was available. Therefore, we assumed that Dt = Xt + St, with Xt ∼ Negbin(St, 1−φt×πt), where φt is the probability that an individual diagnosed in year t survives until 1996 (taken from ) and πt is the probability that the virus is sequenced (taken from the ATHENA records) given survival up to 1996.
To estimate Rt for each cluster, we first sampled n = 50 realizations of Dt from this negative binomial distribution. For each of these, we then sampled 50 possible transmission chains (for each transmission chain, the index case of each secondary case i is sampled according to a multinomial distribution with probabilities pij), leading to a total of 2,500 likely transmission chains. For each chain, we counted the number of secondary cases corresponding to each case, and averaged over index cases diagnosed in the same year to get one Rt trajectory over time. We pooled the 2,500 trajectories and used their mean and 2.5–97.5 quantiles as the point estimate and 95% confidence interval of Rt. Before pooling, each Rt trajectory was corrected for right censoring to account for the fact that some of the secondary cases of index cases diagnosed in year t may not have been diagnosed yet, which may bias downwards the estimates of Rt, with a stronger effect in recent years. To correct for this effect, Rt was divided by ∑u=−S2010−twt(u), which represents the expected proportion of secondary cases of index cases diagnosed in year t who should be diagnosed in or before 2010, should overall transmission rates remain constant. Note that although this correction assumes that overall transmission rates will remain constant in the future, it still allows for heterogeneity in the times at which secondary cases appear relative to a given index case. We performed a sensitivity analysis to check that this right censoring correction was not introducing a systematic bias in the recent estimates of Rt (see S4 Fig).
Study Population and Phylogenetic Tree
Characteristics of the ATHENA patients are shown in Table 1. Of the 5,852 patients with a HIV-1 subtype B pol sequence in this study, 219 (4%) were registered in the cohort in Curaçao and 5,644 were registered in the Netherlands, of whom 4,288 (73%) reported being MSM, 207 (4%) reported being persons who inject drugs (PWID), and 849 (15%) reported being infected via heterosexual contact. Of HIV-positive MSM registered in the Netherlands and diagnosed before 1 January 1996, 35% (781/2,218) have a pol sequence included, versus 43% (3,507/8,247) of those diagnosed since 1 January 1996. Overall, 48% of MSM registered in the cohort in the Netherlands are treated in the capital city, Amsterdam, and account for 51% of the sequences in this study; 25% of MSM are treated in the other big cities in the western part of the country and account for 27% of the sequences. S7 Fig shows the phylogenetic tree of a total of 8,320 unique pol sequences, including sequences obtained from the Los Alamos HIV Sequence Database.
From the total phylogenetic tree, 106 large transmission clusters were identified that included HIV pol sequences from ≥10 patients from the ATHENA cohort, illustrated in Fig 2 stratified by majority risk group of infection and cluster duration. The 106 clusters included 52% (3,061) of sequences from 5,852 ATHENA patients and 652 sequences obtained from the Los Alamos HIV Sequence Database. Only 15 of the 106 clusters were not MSM-majority clusters (≥50% MSM). Of these, 11 clusters were dominated by heterosexually infected individuals of Suriname or Antillean origin, one was dominated by PWID of Polish origin, and two were mixed heterosexual and MSM clusters of mostly Dutch origin. The largest cluster included sequences from 136 (42%) PWID—hence, from 66% of all PWID with a sequence in this study—and also included sequences from 122 (37%) heterosexually infected individuals and from 24 (7%) MSM. It also included 200 HIV pol sequences from the Los Alamos sequences. Fig 3 illustrates all clusters by region of origin. See S1 Trees for timed trees of all clusters and S1 Text for more detailed information on the largest PWID cluster and the transmission clusters circulating on Curaçao.
Fifty percent (2,128) of the 4,288 MSM registered in the ATHENA cohort in the Netherlands with a sequence in this study were in 91 large MSM-majority (≥50%) transmission clusters including at least ≥10 patients from the ATHENA cohort. A further 28% (1,192) were in 417 small clusters containing 2–9 ATHENA sequences; 21% (900) were singletons, of whom 132 clustered solely with foreign sequences; and only 68 were in nine of the non-MSM-dominated clusters (Fig 4). The median number of MSM registered in the cohort in the Netherlands per large cluster was 16 (IQR 11–27, range 5–121).
At least 59% (54) of the 91 large MSM-majority clusters were already circulating before 1996 (Fig 4). Most clusters were confirmed in the BEAST analysis. Figs 2, 3, and 5 show the estimated time of the MRCA of all clusters, and the 95% HPD intervals are in S2 Table. These estimations indicate that 23 (95% HPD interval 8–33) of the clusters identified in or after 1996 (57%) might have started off before 1996, but also that 14 (95% HPD interval 4–29) of these clusters started off more recently (Fig 4).
Of 3,460 MSM with a sequence in this study who were registered in the ATHENA cohort in the Netherlands and diagnosed after 1996, 1,226 (35%) were infected within 54 transmission clusters circulating before 1996, and 20% (700) within 37 large clusters identified in or after 1996; of the latter, 429 (95% HPD interval 272–621) were in clusters with a time of the MRCA before 1996. Thus, in total, 48% (95% HPD interval 43%–53%) of these men diagnosed in or after 1996 were infected within a cluster that started circulating in the Netherlands before 1996. The proportion of these men diagnosed in the 54 clusters identified before 1996 increased over time to 42% in 2010; the proportion in either these clusters or clusters identified since 1996 but with a time of the MRCA before 1996 increased to 55% (95% HPD interval 53%–62%). Those diagnosed within any of the large clusters increased to 68% (Fig 4).
Of the 54 pre-1996 clusters, 78% (40) included recent infections after 2000 (Fig 5). Since 1996, a recent infection at diagnosis could be identified amongst 41% of MSM in large clusters, 31% of MSM in smaller clusters, and 22% of MSM singletons. Only three (6%) of the 54 pre-1996 clusters had no new diagnoses observed in the last 5 y of the study period, 2006–2010.
Country of infection
Amongst 3,416 MSM registered in the ATHENA cohort in the Netherlands with a sequence in this study who reported a most likely country of infection, 4.3% of MSM in large clusters reported likely having been infected abroad, and likewise 8.1% (risk ratio [RR] = 1.9; 95% CI 1.4–2.6) in smaller clusters, 19.0% (RR = 4.5; 95% CI 3.4–5.9) of singletons, and 27.8% (RR = 6.5; 95% CI 4.4–9.7) of MSM clustering solely with one or more Los Alamos sequences. This indicates that singletons, besides being part of unidentified clusters of national transmission, often represent new introductions.
Data versus theory
Fig 4 illustrates that only a small minority (7%) of all the observed phylogenetic MSM clusters (sum of singletons and small and large clusters) were established “large” transmission clusters including ten or more cases. This is broadly consistent with results from branching theory, which indicates that this proportion should be between 6% and 35% for a reproduction number between 0.8 and 1.2 (see S1 Text). Theoretical results also show that this observed proportion is a good approximation for the proportion of introductions leading to sub-epidemics of ten cases or more, with little sensitivity to the proportion of cases whose virus was sequenced.
Estimation of the Case Reproduction Number within Large MSM-Majority Transmission Clusters
Fig 6 shows the estimated annual case reproduction number for all 91 large MSM-majority transmission clusters. With a reproduction number around one (the critical value that separates epidemic spread from contraction), clusters were self-sustaining over the whole study period. Reproduction numbers were strikingly consistent across clusters. A tendency towards higher numbers was visible over recent years, especially in the more recently introduced clusters (see S6 Fig). Seventy-five percent (68) of the clusters had a mean reproduction number greater than one over the last 5 y (Fig 2).
With the looser cluster definition, 105 large MSM-majority clusters were selected, containing 69% (2,977) of the HIV pol sequences from MSM registered in the ATHENA cohort in the Netherlands, and only 4% (183) of sequences were singletons. In this sensitivity analysis, 17% of all observed phylogentic MSM clusters (including the singletons) included ten or more cases. With the more stringent cluster definition, 67 clusters were identified, containing 39% (1,670) of the pol sequences from MSM registered in the cohort in the Netherlands. With clusters defined under the single linkage branch length threshold, 63 large MSM-majority clusters were selected, containing 69% (2,974) of the pol sequences from MSM registered in the cohort in the Netherlands. In this analysis also, only a small minority (5%) of all observed phylogentic MSM clusters included ten or more cases. The estimations of the case reproduction number for the large MSM-majority clusters in all three sensitivity analyses were similar to those in our main analysis (Fig 6).
Age of MSM at Diagnosis
Fig 7 shows that the HIV epidemic is shifting from the initially infected generations of MSM towards younger generations. The mean age at diagnosis of MSM in our study has been increasing by 0.31 years per calendar year overall. However, this overall trend hides more complicated dynamics because within each of the 91 large MSM-majority clusters, the mean age at diagnosis has increased more rapidly, at a rate of 0.45 years/year, compared to the epidemic as a whole. On the other hand, new clusters have started with a lower initial mean age, which, across clusters, has increased at rate of only 0.28 years/year.
This study provides insight into the different transmission clusters that have contributed to the ongoing resurgent HIV epidemic amongst MSM in the Netherlands. We found that clusters that started spreading before the introduction of cART in 1996 still constitute the main source for new HIV-1 subtype B infections (~55% in 2010). Calculations of the reproduction number separately for each cluster show that whilst the epidemic diminished at the population level during the 1990s [1,2,10], most of these clusters continued spreading in a self-sustaining manner. There is little indication that any of the clusters will stop in the near future as most clusters contain recent infections and are rejuvenated by the inclusion of younger men, and many have a reproduction number greater than the epidemic threshold. While all clusters appear to be aging, new clusters appear to be of lower mean age; if anything, these “younger” clusters have higher epidemic potential than the “older” clusters. It is also conceptually interesting to see the whole HIV-1 subtype B epidemic disaggregated in a single figure (Figs 2 and 3). This perspective illustrates the tight links between clusters on Curaçao with people from the same region living in the Netherlands [19,30–32]. It also shows that the largest transmission cluster, which is dominated by PWID, shows links to heterosexual transmission but is an almost entirely separate epidemic from that amongst MSM.
The median reproduction number of all large clusters together found in this study is consistent with our previous findings of the HIV epidemic amongst MSM in that it is around the epidemic threshold and increases in later years. However, our previous study reported the mean reproduction number aggregated over the whole epidemic, whilst our findings here are based on disaggregating the epidemic into its constituent sub-epidemics. We found that the resurgent epidemic is the result of existing transmission clusters, many of which have been spreading since the early epidemic, to which new transmission clusters are being added. There seems to be a high and continued rate of case importation, but with only a small minority of cases going on to establish new self-sustaining clusters (Fig 5). This heterogeneity of outcomes is in line with expectations from epidemiological theory, as we also show in S1 Text: self-sustaining sub-epidemics are hard to establish but, once initiated, are difficult to stop . The higher percentage of recent infections in large clusters versus small clusters and singletons could indicate an effect of partner tracing. Several studies indicated that a large part of onward transmission amongst MSM is during the early phase of infection [11,34–37]. And thus it is worrying that even though an increasing number of MSM with HIV are diagnosed early in infection, this does not seem to have stopped clusters from growing.
The clusters and findings were confirmed using different phylogenetic methods and cluster definitions. However, by phylogenetic methods alone and using only pol sequences of only a subset of patients, we were not able to fully identify all transmission chains. The focus in our study was on the large transmission clusters. The observation that sequences from patients diagnosed before 1996 were less often part of a large cluster could indicate that some initial clusters have stopped circulating and have gone unnoticed. It is also possible that, since the sample fraction was lower in early years, fewer individuals appear to cluster (due to missing links), even though they were in fact linked to larger transmission clusters via unknown intermediates.
Our estimates of the reproduction number are based on a number of assumptions, including that the distribution of the diagnosis interval (time from diagnosis of an index case to diagnosis of a secondary case) is similar across clusters and similar across the whole HIV-positive population in the Netherlands. These estimates also assume that the probability of having a sequence obtained and the probability of surviving until 1996 are similar across clusters. It should be noted that the reproduction numbers estimated in this study are effective reproduction numbers. Our findings might thus indicate local or temporal saturation effects in certain sexual networks. However, as prevalence of HIV amongst MSM ranges from 5% to 8% , behavior change, not saturation, is expected to be the dominant driver of epidemic trends in this population. For a future study it would be interesting to compare findings, from real and simulated data, with recent studies that obtained reproduction numbers for large transmission clusters directly from sequence data [13,14].
This study, combining phylogenetic methods with mathematical modeling to analyze extensive HIV-1 subtype B sequence data, provides new insight into how different transmission networks together contribute to the resurgent HIV epidemic in the Netherlands. The analysis suggests that the epidemic amongst MSM is dispersed amongst a large number of self-sustaining or growing transmission clusters, many of which persisted throughout the 1990s, before increases in risk behavior became widespread. In particular, the relative homogeneity and consistency of reproduction number estimates for the different networks was unexpected. Our study highlights that many different sub-epidemics have independently persisted for decades, despite the widespread availability of treatment, steadily increasing rates of diagnosis, and increasing tendency for early treatment initiation. The fastest growing sub-epidemics are the newest ones, which also tend to be amongst the youngest men. Preventing further increases in rates of infection will require further developments in prevention services.
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