Epidemiology and Social
Transmission networks of HIV-1 among men having sex with men in the Netherlands.
Bezemer, Danielaa; van Sighem, Arda; Lukashov, Vladimir Vb; van der Hoek, Liab; Back, Nicoleb; Schuurman, Robc; Boucher, Charles ABc,d; Claas, Eric CJe; Boerlijst, Maarten Cf; Coutinho, Roel Ag,h; de Wolf, Franka,i; for the ATHENA observational cohort
aHIV Monitoring Foundation, The Netherlands
bDepartment of Medical Microbiology, Academic Medical Centre, Amsterdam, The Netherlands
cDepartment of Virology, University Hospital Utrecht, Utrecht, The Netherlands
dDepartment of Virology, Erasmus Medical Center, Rotterdam, The Netherlands
eDepartment of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
fInstitute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, The Netherlands
gDepartment of Internal Medicine, Academic Medical Center, Amsterdam, The Netherlands
hCenter for Infectious Disease Control, National Institute of Public Health and the Environment, Bilthoven, The Netherlands
iDepartment of Infectious Disease Epidemiology, Imperial College London, London, UK.
Received 16 June, 2009
Revised 17 September, 2009
Accepted 5 October, 2009
Correspondence to Daniela Bezemer, HIV Monitoring Foundation, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel: +31 20 5667180; fax: +31 205669189; e-mail: D.O.Bezemer@amc.uva.nl
Objective: To obtain insight in the HIV-1 transmission networks among men having sex with men (MSM) in the Netherlands.
Design: A phylogenetic tree was constructed from polymerase sequences isolated from 2877 HIV-1 subtype B-infected patients monitored as part of the AIDS Therapy Evaluation in the Netherlands (ATHENA) nationwide observational cohort.
Methods: For MSM with a known date of infection, the most similar sequences were selected as potential transmission pairs when they clustered with bootstrap value of at least 99%. Time from infection to onward transmission was estimated as the median time between dates of infection for each transmission pair. The source of infections with a resistant strain was traced using the entire phylogenetic tree.
Results: Of sequences from 403 MSM with a known date of infection between 1987 and 2007, 175 (43%) formed 63 clusters. Median time to onward transmission was 1.4 years (interquartile range 0.6–2.7). Twenty-four (6%) MSM carried a virus with resistance-related mutations, 13 of these were in eight clusters together with sequences from 28 other patients in the entire phylogenetic tree. Six clusters contained sequences obtained from 29 men all presenting the same resistance-related mutations.
Conclusion: From our selection of likely transmission pairs, we conclude that onward transmission of HIV-1 from infected MSM in the Netherlands happens both during and after primary infection. Transmission of resistant strains from the antiretroviral therapy-treated population is limited, but strains with resistance-related mutations have formed subepidemics.
Despite the success of combination antiretroviral treatment (cART) in reducing viral load, HIV-1 transmission continues among men having sex with men (MSM) in industrialized countries, including the Netherlands [1,2]. Using a mathematical model to describe trends in the transmission dynamics of HIV-1 among MSM in the Netherlands, we recently estimated that 90% of onward transmission in this risk group takes place from the undiagnosed group. The average time for a recently infected MSM to become diagnosed was estimated to be 2.7 years . However, the impact of primary infection on onward transmission remains unclear. As usually, no diagnostic tests are performed during this initial phase of the infection when the viral load peaks and infectiousness is high [3,4], the rate of partner change may be crucial for epidemic spread .
Despite discrepancies, HIV sequence analysis can reveal information on contact networks . On the basis of the phylogenetic clustering of HIV-1 polymerase (pol) sequences obtained from primary infections, previous studies [7–9] suggested that 25–50% of transmissions among MSM take place during primary infection. However, clustering of sequences obtained from primary infections does not necessarily represent transmission during the period of primary infection . Lewis et al.  applied a Bayesian Monte Carlo Markov Chain method on sequences obtained from 402 patients without a known date of infection and estimated that 25% of transmissions took place within the first 6 months of infection. But in this study, assumptions needed to be made to estimate the dates of infection. We estimated the median time between infection and onward transmission for potential transmission pairs selected from a phylogenetic tree of HIV-1 subtype B pol sequences obtained from 403 MSM shortly after their known date of infection.
Further insight in transmission dynamics of HIV-1 can be obtained by investigating the transmission networks of strains with resistance-related mutations . Transmission soon after infection facilitates transmission of resistant HIV-1 strains, which would revert to the original wild type within a few months in absence of ART [8,13–15]. Certain resistance-related mutations revert to a new wild type, a process that is well documented for amino acid position 215 in reverse transcriptase [13,16]. In previous studies [17,18], we found that 6% of new infections presented resistance-associated mutations, and that 23% of ART-naive patients failing cART were infected with a resistant strain. Several studies [9,15,19] reported phylogenetic clustering of resistant strains obtained from ART-naive patients. We used the set of HIV-1 subtype B pol sequences obtained from 403 MSM with a known date of infection to monitor the transmission of resistant stains over calendar time, and linked these resistant strains to the source of infection in a phylogenetic tree of all 4090 HIV-1 subtype B pol gene sequences obtained from 2877 infected patients, both before and after starting ART. All patients were monitored in one of the 24 HIV treatment centers in the Netherlands as part of the AIDS Therapy Evaluation in the Netherlands (ATHENA) national observational cohort.
The ATHENA cohort encompasses all patients infected with HIV-1 followed longitudinally in one of the 24 HIV treatment centers in the Netherlands since 1996 . Demographic data were collected at entry in the cohort. At each follow-up visit, clinical, virological, and immunological data were collected, as well as data on the use of cART [1,18,21]. HIV-1 pol gene sequences were obtained as part of the screening for resistance to antiretroviral drugs, both before and during treatment with cART [22–24].
Patients were eligible for this study if at least one pol sequence was available containing at least the first 251 amino acids of the reverse transcriptase gene. Population-based nucleotide sequencing of the HIV-1 pol gene was performed as described in detail previously . Sample contamination was checked for at the respective sequencing sites. Multiple sequence alignment was done by hand and using the default parameters of the ClustalX 1.83 program. Subtype B was identified by phylogenetic analysis of combined reverse transcriptase and protease sequences, using reference sequences from the Los Alamos database  and our own database. The percentage of ambiguous sites was estimated for all sequences. Sequences were screened for major resistance-conferring mutations at the amino acid positions described by the International AIDS Society-USA, including alternative substitutions at position 215 .
New HIV-1 infections
For this study, new HIV-1 infections were defined as those infections with either a seroconversion interval of 18 months or less between the last negative and the first positive HIV-1 serology test, or documented evidence of a primary infection. A diagnosis of primary HIV-1 infection was defined as detectable HIV-1 RNA, detectable serum p24 antigen, or both in plasma combined with either a negative HIV-1 antibody test or a positive HIV-1 antibody test with a negative, incomplete, or indeterminate HIV-1 western blot. The estimated date of infection was defined as the midpoint between the last seronegative and the first seropositive sample, or the date of the last seronegative but RNA-positive sample, or the date of an indeterminate result on the western blot . Sequences corresponding to new infections were obtained from a sample taken 18 months or less after the estimated date of seroconversion.
The percentage pairwise sequence distances between all available entire protease sequences and reverse transcriptase sequences cut to equal lengths of 251 amino acids were calculated taking into account ambiguous sites according to a mixed weighted distance method . To study the level of mixing of MSM HIV-1 transmission networks with transmission via other routes, we used a pairwise sequence distance of 1.5% or less between reverse transcriptase sequences as a selection criterion for potential transmission pairs [18,28]. Transmission clusters were defined as groups of linked potential transmission pairs.
Phylogenetic trees were constructed of pol gene RNA sequences that included at least the first 251 amino acids of the reverse transcriptase gene. Trees were constructed with the neighbor-joining method [29,30] within the Molecular Evolutionary Genetics Analysis program , and ambiguous sites were ignored. To prevent false clustering due to convergent evolution, 36 amino acid sites associated with major drug resistance were excluded . When available, multiple sequences per person were included. All trees were rooted against an HIV-1 subtype K sequence (Los Alamos Database accession number AJ249235).
Potential transmission pairs
The first phylogenetic tree included all HIV-1 subtype B pol sequences obtained from therapy-naive MSM within 18 months after their estimated date of infection. The Maximum Composite Likelihood method was used to compute evolutionary distances, and a bootstrap analysis with 1000 replications was performed. From this tree, a selection was made of patients in clusters with a bootstrap value of at least 99% [18,28,32]. Each patient in this selection was combined with the other patient in the same cluster with the smallest pairwise sequence distance to form the most likely transmission pair. The time between infection and onward transmission was estimated as the median time between dates of infection of all these potential transmission pairs. The correlation between the pairwise sequence distance and time between the dates of infection of transmission pairs was estimated from linear regressions.
Tracing the source of resistant strains
In order to trace the infecting source of the transmitted resistant strains with a known date of infection, a second phylogenetic tree was made that contained all subtype B sequences available in the ATHENA database. For the construction of this tree, the Kimura 2-parameter model was used , and a bootstrap analysis with 100 replications was performed. The clusters observed were confirmed in a smaller tree of all sequences that clustered with a resistant strain with a known date of infection. Evolutionary distances in this smaller tree were computed using the maximum composite likelihood method with a gamma distribution of the substitution rate with shape parameter 1.0 , and 1000 bootstrap replications were performed. The clusters were studied with the aim to identify whether patients on treatment transmitted the resistant strain.
By June 2007, 12 951 persons infected with HIV-1 were included in the ATHENA national observational cohort; 6845 (53%) were reported to have been infected by MSM contact, of whom 49% were monitored in an HIV treatment center in Amsterdam. In total, 4090 HIV-1 subtype B pol gene sequences were obtained from 2877 persons, of whom 2022 (70%) were MSM, 486 (17%) were infected via heterosexual contact, 167 (6%) via drug injection, and 202 (7%) via other or unknown transmission routes. The first sequence of each patient was obtained between 1987 and 1996 for 101 (3.5%) patients, between 1996 and 2000 for 503 (17.5%), between 2001 and 2004 for 1114 (38.7%), and after 2004 for 1159 (40.3%) patients. Of 832 patients who had a non-B HIV-1 infection and a sequence available, only 68 (8%) were MSM.
Transmission of HIV-1 between men having sex with men and other risk groups
HIV-1 subtype B reverse transcriptase sequences from 817 (28%) out of 2877 persons clustered within a sequence distance of 1.5% from another person's reverse transcriptase sequence. Of these 817 patients, 603 (74%) were MSM, 91 (11%) were heterosexuals, 51 (6%) were infected via injection drug use, and 72 (9%) were infected via other or unknown transmission routes. Only 8% of MSM had an infection with HIV-1 that was most similar to that found in patients from other risk groups.
From 403 (20%) of the 2022 MSM with a subtype B pol sequence, the approximate date of HIV-1 antibody seroconversion was known (Table 1). The number of new infections by year of seroconversion is shown in Fig. 1(a). For 393 MSM, the pol gene was sequenced, including both protease and reverse transcriptase, whereas for 10 MSM only reverse transcriptase was available. The median percentage of ambiguous sites among the 403 sequences was low [0.08%, interquartile range (IQR) 0.0–0.4], which allowed for calculation of pairwise sequence distances either including or excluding these sites. Of the 403 MSM, 292 (72%) were monitored in an HIV treatment center in Amsterdam, and 294 (73%) reported that they were most likely infected in the Netherlands. Of the 42 MSM infected before 1996, 64% were prospectively identified within the Amsterdam Cohort Studies .
Clustering among new HIV-1 subtype B infections
We constructed a phylogenetic tree of 499 available pol sequences from 403 MSM with at least one reverse transcriptase sequence within 18 months of their estimated date of infection. The tree showed 63 transmission clusters with a bootstrap value of at least 99%, including 175 (43%) patients (Fig. 2a). Both HIV-1 protease and reverse transcriptase sequences were obtained for all 175 patients. The clusters were confirmed in a separate phylogenetic tree containing sequences of the 393 MSM with both protease and reverse transcriptase sequenced. The size of the clusters varied from two to eight patients (median 3, IQR 2–4). The median minimum number of nucleotide substitutions per site per person within each cluster was low (0.0035, IQR 0.0014–0.0065, range 0–0.0158), indicating clustering of potential transmission pairs . The time between the two most distant dates of infection in each cluster ranged from 0.05 to 9.7 years (median 2.0, IQR 0.9–4.2). The median difference in time between the dates of infection of the most likely transmission pair for all patients in a cluster was 1.4 years (IQR 0.6–2.7, range 0.03–9.05). The corresponding median pairwise sequence distance was 0.9% (IQR 0.4–1.5) and increased by 0.33% [95% confidence interval (CI) 0.28–0.38, P < 0.0001] per year of separation in time. The distribution of the median difference in time between the dates of infection of the most likely transmission pair for all patients in a cluster is shown in Fig. 2(b). Constraining the analysis to transmission pairs with a pairwise sequence distance of 1.5% or less or a synonymous sequence distance of 4.5% or less showed that the median difference in time varied between 1.1 and 1.4 years (Table 2, #2–7). When transmissions related to individuals with unknown date of infection were included (Table 2, #9), the time between dates of diagnosis was similar to that found for time between estimated dates of infection, and the median sequence distance was similar to that found for the patient selection in analysis #3–4.
Transmission of resistant HIV-1 subtype B strains
Figure 1(a) shows the annual percentage of infections with a resistant virus strain among all 403 MSM with a new HIV-1 subtype B infection. In total, 24 patients (6.0%, 95% CI 3.8–8.7) were infected with an antiretroviral drug-resistant strain. Of these 24 patients, 18 (4.5%) had one or more mutations associated with resistance to nucleoside reverse transcriptase inhibitors, two (0.5%) patients were resistant to nonnucleoside reverse transcriptase inhibitors, two (0.5%) to a protease inhibitor, and two (0.5%) to two or three drug classes. Fourteen of 24 resistant strains (58%) had a mutation at position 215 in reverse transcriptase. Figure 1(b) shows the annual percentage of infections with HIV-1 strains with a resistance-conferring mutation at reverse transcriptase position 215, separated into 215 resistant mutations (215 Y or F) and 215 revertant mutations (215 C, D, E, or S). After 1996, only revertant mutations were found at position 215.
Networks of transmitted resistant HIV-1 subtype B strains
In order to track the source of infections with a resistant strain, we performed a phylogenetic analysis of all 4090 HIV-1 subtype B pol sequences in the ATHENA database. Herein, we selected sequences from 88 persons that clustered with the sequences obtained from the 24 MSM who were newly infected with a resistant HIV-1 strain. Phylogenetic analysis of this subset of 152 sequences of 112 patients showed eight significant clusters (Fig. 3). The eight clusters contained sequences of 13 (54%) of the 24 newly infected patients who had a resistant strain, including seven with a bootstrap value of at least 99, and one with a bootstrap value of 95 but a pairwise sequence distance of the closest sequence pair of only 1.0%. The same clusters were also identified when clusters were based on connections between potential transmission pairs with a mixed weighted distance of 1.5% or less at reverse transcriptase, except for three patients (M24, M31, and M54). Sequences from the other 11 patients infected with a resistant strain did not cluster significantly with other sequences in the database. One of them (M30) appeared to be infected with an HIV-1 strain resistant to three drug classes and reported the possibility of having been infected by someone from outside the Netherlands. Both M33 (infected in 1994) and M34 (infected in 2002) might have been infected by someone on treatment as their HIV strains contain the reverse transcriptase mutations 70R and 184V, respectively, known for mutating back to wild type in the absence of antiviral therapy .
Twenty-one persons in three clusters had sequences with a revertant mutation at position 215 in reverse transcriptase, specifically 215C in cluster 1 and 8, and 215S in cluster 5. The mutant strain with 215C and 219E at reverse transcriptase in cluster 1 might have been circulating for a period of 15 years, that is, the period between the earliest and the latest date of diagnosis in the cluster. No pretreatment HIV-1 reverse transcriptase sequence was available from the earliest diagnosed patient (M9) in this cluster. However, the sequence obtained from M9 at the time of therapy failure harbored the same 215 revertant mutant as the other sequences in the cluster. A pretreatment reverse transcriptase sequence from patient M11 diagnosed in 1989 that was linked to cluster 1 with a bootstrap value of 80 did not show any resistant mutation. Cluster 2 was linked to one other antiretroviral-naive patient (M16) infected with a strain that harbored a 219Q mutation. In cluster 3, the resistance-associated mutation 33F in HIV-1 protease obtained from patient M22 was not present in any of the other eight persons in the same cluster and was possibly a natural polymorphism . Cluster 4 was neither linked to other persons with a sequence with a 210W mutation in reverse transcriptase nor to persons on cART at the time of infection. Clusters 6 and 7 possibly show a direct transmission from someone failing treatment with a 215Y mutation in cluster 6 and a 215F mutation in cluster 7. In cluster 6, the selection of a resistant strain in the potential initial source failing therapy (M52) was observed.
Our study on transmission networks of HIV-1 subtype B among MSM in the Netherlands indicates that 25% of onward transmissions occur within 7 months after infection, half of transmissions within 17 months, and 75% within 2.7 years. This finding is compatible with our previous analysis of the dynamics of the HIV-1 epidemic among MSM in the Netherlands, in which we estimated that individuals who were unaware of their infection were the source of 90% of new infections, with an average of 2.7 years between infection and diagnosis for those infected after 2000 . Our estimate of the median time between transmissions, for which we used only sequences corresponding to infections with an approximate known date of infection, is in agreement with a study by Lewis et al.  who estimated the time between the nodes in a tree of sequences with an unknown date of infection. They reported an estimated 25% of transmissions taking place within the first 6 months of infection and 50% within 14 months after infection. This might indicate that the HIV-1 transmission dynamics among MSM in the Netherlands and UK are similar.
However, phylogenetic studies on transmission dynamics have shortcomings. Discrepancies have been demonstrated to arise when comparing the viral phylogeny with known sexual contact networks . Our study contained a subset of infections with an estimated date of infection that clustered and formed likely transmission pairs, although it cannot be excluded that there were intermediate transmissions. In addition, distinguishing people who infected more than one person is not always feasible, and individuals that were infected by a common source within a short time period might cluster as a transmission pair. Furthermore, transmission dynamics are known to vary over calendar time , yet we estimated an average transmission rate. The search for new infections was intensified in the later years of our study, resulting in more available sequences, and thus the identification of more transmission pairs in these years. In addition, a significant proportion of patients identified as newly infected were familiar with their HIV-positive status after their first positive test, and thus soon after infection. Consequently, their behavior may differ from those diagnosed at a later stage of infection.
In several countries, the overall transmission of resistant HIV-1 strains is reported to have decreased since the introduction of cART in 1996 [17,36–38]. This has been explained by the efficacy of cART and the lower transmission potential of resistant HIV-1 strains [8,9,13,39–41]. We found that 6% of HIV-1 subtype B strains in new infections among MSM had resistance-related mutations. Tracing the source of these resistant strains showed clusters with mainly transmission of HIV-1 with a revertant mutation at position 215 of reverse transcriptase. These observations are compatible with the estimate that the 215 reverse transcriptase revertant mutations have no significant fitness effect on the fitness of the virus [13,16]. In contrast to an incidence of 20% among new transmissions in 1994, the 215 mutants were not found after the introduction of cART in 1996 until 2003, except for one case in 1999 . However, from 2003 onwards, revertant mutants were present among new infections in all subsequent years. This could be due to the introduction of baseline sequencing around that time, but it might also be a result of changing transmission dynamics. Previously, we estimated that transmission decreased in the early cART era, but a resurgence of the epidemic occurred in later years . Thus, the reappearance of the revertant 215 reverse transcriptase mutation among recent transmissions may be a sentinel for changing transmission dynamics [42,43]. Additionally, the HIV-1 incidence is increasing among older MSM, suggesting the possibility of a re-opened reservoir . However, the initial resistant mutations at position 215 were only observed before the introduction of cART, which reflects the contribution of patients failing monothearpy.
Sampling methods might influence the monitoring of transmitted resistance as viruses from failing patients are often sequenced retrospectively. Contact tracing on an individual basis might also have an impact as recent partners of a person are identified. The cluster of sequences obtained that way could have an impact on the percentage of resistance found in the respective year of infection (whether or not with a resistant strain) that was larger than expected when sampling was random. We reduced these effects by only selecting infections with a known date of infection.
Our study indicates that onward HIV-1 transmission from infected MSM takes place both during and after primary infection. Strains with resistance-related mutations have formed subepidemics, and transmission of resistant strains from the antiretroviral-treated population is limited. However, with the current changes in risk behavior among people using cART [45–47], transmission from the treated population might increase. Intensifying contact tracing and facilitating frequent testing could help to identify people earlier in their infection and prevent onward transmission.
D.B. was supported by grant #7014 from AIDS Fund Netherlands. The authors thank Maus Sabelis for useful discussions, Maria Prins for helping in identifying new infections, Luuk Gras for statistical assistance, and Sally Ebeling for English revision.
D.B. designed the study, performed all analyses, and wrote the first draft of the paper. A.v.S. managed the sequence database, performed the distance matrix calculation, and was involved in the design of the study and critical reading of the manuscript. V.V.L., L.v.d.H., M.C.B., and R.A.C. were involved in the design of the study and critical reading of the manuscript. N.B., R.S., C.A.B.B., and E.C.J.C. were responsible for the data used in the study and critical reading of the manuscript. F.d.W. was involved in the design of the study, critical reading of the manuscript, and was overall responsible for the study and the data monitoring and quality control. All authors gave final approval for submission.
The ATHENA national observational cohort is a collaboration between 24 HIV treatment centers in the Netherlands: Treating physicians (*Site coordinating physicians) Academisch Medisch Centrum bij de Universiteit van Amsterdam, Amsterdam – Dr J.M. Prins*, Dr J.C. Bos, Dr J.K.M. Eeftinck-Schattenkerk, Dr S.E. Geerlings, Dr M.H. Godfried, Professor Dr J.M.A. Lange, Dr J.T.M. van der Meer, Dr F.J.B. Nellen, Dr D.P. Olszyna, Dr T. van der Poll, Professor Dr P. Reiss, Dr S.U.C. Sankatsing, Dr R. Steingrover, Dr M. van der Valk, Dr J.N. Vermeulen, Dr S.M.E. Vrouenraets, Dr M. van Vugt, Dr F.W.M.N. Wit. Academisch Ziekenhuis Maastricht, Maastricht – Dr G. Schreij*, Dr S. van der Geest, Dr A. Oude Lashof, Dr S. Lowe, Dr A. Verbon. Catharina Ziekenhuis, Eindhoven – Dr B. Bravenboer*, Dr M.J.H. Pronk. Emma Kinderziekenhuis, AMC Amsterdam – Professor Dr T.W. Kuijpers, Dr D. Pajkrt, Dr H.J. Scherpbier. Erasmus MC, Rotterdam – Dr M.E. van der Ende*, Dr H. Bax, Dr M. van der Feltz, Dr L.B.S. Gelinck, Dr Mendoca de Melo (until 1 September 2008), Dr J.L. Nouwen, Dr B.J.A. Rijnders, Dr E.D. de Ruiter, Dr L. Slobbe, Dr C.A.M. Schurink, Dr T.E.M.S. de Vries. Erasmus MC, Sophia, Rotterdam – Dr G. Driessen, Dr M. van der Flier, Dr N.G. Hartwig. Flevoziekenhuis, Almere – Dr J. Branger. Haga Ziekenhuis, locatie Leyenburg, Den Haag – Dr R.H. Kauffmann*, Dr K. Pogány (until 1 August 2008), Dr E.F. Schippers (from 1 May 2008). Isala Klinieken, Zwolle – Dr P.H.P. Groeneveld*, Dr M.A. Alleman. Kennemer Gasthuis, Haarlem – Professor Dr R.W. ten Kate*, Dr R. Soetekouw. Leids Universitair Medisch Centrum, Leiden – Dr F.P. Kroon*, Dr S.M. Arend, Dr M.G.J. de Boer, Professor Dr P.J. van den Broek, Professor Dr J.T. van Dissel, Dr C. van Nieuwkoop. Maasstadziekenhuis, locatie Clara, Rotterdam – Dr J.G. den Hollander*. Medisch Centrum Alkmaar, Alkmaar – Dr W. Bronsveld*. Medisch Centrum Haaglanden, locatie Westeinde, Den Haag – Dr R. Vriesendorp*, Dr F.J.F. Jeurissen, Dr E.M.S. Leyten. Medisch Centrum Leeuwarden, Leeuwarden – Dr D. van Houte*, Dr M.B. Polée. Medisch Spectrum Twent e, Enschede: Dr C.H.H. ten Napel*, Dr G.J. Kootstra. Onze Lieve Vrouwe Gasthuis, Amsterdam – Professor Dr K. Brinkman*, Dr G.E.L. van den Berk, Dr W.L. Blok, Dr P.H.J. Frissen, Dr W.E.M. Schouten. St. Medisch Centrum Jan van Goyen, Amsterdam: Dr A. van Eeden*, Dr D.W.M. Verhagen. Slotervaart Ziekenhuis, Amsterdam – Dr J.W. Mulder*, Dr E.C.M. van Gorp, Dr A.T.A. Mairuhu, Dr R. Steingrover, Dr J. Wagenaar. St. Elisabeth Ziekenhuis, Tilburg – Dr J.R. Juttmann*, Dr M.E.E. van Kasteren. St. Lucas Andreas Ziekenhuis, Amsterdam – Dr J. Veenstra*, Dr W.L.E. Vasmel. Universitair Medisch Centrum St. Radboud, Nijmegen – Dr P.P. Koopmans*, Dr A.M. Brouwer, Dr A.S.M. Dofferhoff, Professor Dr R. de Groot, Dr H.J.M. ter Hofstede, Dr M. Keuter, Dr A.J.A.M. van der Ven. Universitair Medisch Centrum Groningen, Groningen – Dr H.G. Sprenger*, Dr S. van Assen, Dr J.T.M. van Leeuwen, Dr C.J. Stek. Universitair Medisch Centrum Groningen, Beatrix Kliniek, Groningen – Dr R. Doedens, Dr E.H. Scholvinck. Universitair Medisch Centrum Utrecht, Utrecht – Professor Dr I.M. Hoepelman*, Dr M.M.E. Schneider, Professor Dr M.J.M. Bonten, Dr P.M. Ellerbroek, Dr C.A.J.J. Jaspers, Dr L.J. Maarschalk-Ellerbroek, Dr J.J. Oosterheert, Dr E.J.G. Peters, Dr T. Mudrikova, Dr M.W.M. Wassenberg, Dr S. Weijer. WilhelminaKinderziekenhuis, UMC Utrecht – Dr S.P.M. Geelen, Dr T.F.W. Wolfs. VU Medisch Centrum, Amsterdam – Professor Dr S.A. Danner*, Dr M.A. van Agtmael, Dr W.F.W. Bierman, Dr F.A.P. Claessen, Dr M.E. Hillebrand, Dr E.V. de Jong, Dr W. Kortmann, Dr R.M. Perenboom, Dr E.A. bij de Vaate. Ziekenhuis Rijnstate, Arnhem – Dr C. Richter*, Dr J. van der Berg, Dr E.H. Gisolf. Ziekenhuis Walcheren, Vlissingen – Dr A.A. Tanis*. St. Elisabeth Hospitaal/Stichting Rode Kruis Bloedbank, Willemstad, Curaçao – Dr A.J. Duits, Dr K. Winkel. Virologists: Academisch Medisch Centrum bij de Universiteit van Amsterdam, Amsterdam – Dr N.K.T. Back, Dr M.E.G. Bakker, Dr H.L. Zaaijer, Professor Dr B. Berkhout, Dr S. Jurriaans. CLB Stichting Sanquin Bloedvoorziening, Amsterdam – Dr Th. Cuijpers. Onze Lieve Vrouwe Gasthuis, Amsterdam – Dr P.J.G.M. Rietra, Dr K.J. Roozendaal. Slotervaart Ziekenhuis, Amsterdam – Dr W. Pauw, Dr P.H.M. Smits, Dr A.P. van Zanten. VU Medisch Centrum, Amsterdam – Dr B.M.E. von Blomberg, Dr A. Pettersson, Dr P. Savelkoul. Ziekenhuis Rijnstate, Arnhem – Dr C.M.A. Swanink. HAGA, ziekenhuis, locatie Leyenburg, Den Haag – Dr P.F.H. Franck, Dr A.S. Lampe; Medisch Centrum Haaglanden, locatie Westeinde, Den Haag – Dr C.L. Jansen. Streeklaboratorium Twente, Enschede – Dr R. Hendriks. Streeklaboratorium Groningen, Groningen – Dr C.A. Benne. Streeklaboratorium Volksgezondheid Kennemerland, Haarlem – Dr J. Schirm, Dr D. Veenendaal. Laboratorium voor de Volksgezondheid in Friesland, Leeuwarden – Dr H. Storm, Dr J. Weel, Dr J.H. van Zeijl. Leids Universitair Medisch Centrum, Leiden – Dr H.C.J. Claas, Professor Dr A.C.M. Kroes. Academisch Ziekenhuis Maastricht, Maastricht – Professor Dr C.A.M.V.A. Bruggeman, Dr V.J. Goossens. Universitair Medisch Centrum St. Radboud, Nijmegen – Professor Dr J.M.D. Galama, Dr W.J.G. Melchers, Dr Verduyn-Lunel. Erasmus MC, Rotterdam – Dr G.J.J. van Doornum, Dr H.G.M. Niesters, Professor Dr A.D.M.E. Osterhaus, Dr M. Schutten. St. Elisabeth Ziekenhuis, Tilburg – Dr A.G.M. Buiting. Universitair Medisch Centrum Utrecht, Utrecht – Dr C.A.B. Boucher, Dr E. Boel, Dr R. Schuurman. Catharina Ziekenhuis, Eindhoven – Dr A.F. Jansz, Dr M. Wulf. Pharmacologists: Medisch Centrum Alkmaar, Alkmaar – Dr A. Veldkamp. Slotervaart Ziekenhuis, Amsterdam – Professor Dr J.H. Beijnen, Dr A.D.R. Huitema. Universitair Medisch Centrum St. Radboud, Nijmegen – Dr D.M.Burger. Academisch Medisch Centrum bij de Universiteit van Amsterdam, Amsterdam – D H.J.M. van Kan.
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