Translational HIV-1 research: from routine diagnostics to new virology insights in Amsterdam, the Netherlands during 1983-2013.

An HIV-1 diagnostic laboratory was established in the Academic Medical Center (AMC) of the University of Amsterdam after the discovery of human immunodeficiency virus (HIV) as the cause of the acquired immunodeficiency syndrome (AIDS). The first AIDS patients were diagnosed here in 1981 and since 1983 we have tested the samples of 50992 patients using a variety of assays that greatly improved over the years. We will describe some of the basic results from this diagnostic laboratory and then focus on the spin-off in terms of the development of novel virus assays to detect super-infections and ultra-sensitive assays to measure the intracellular HIV-1 RNA load. We also review several original research findings in the field of HIV-1 virology that stem from initial observations made in the diagnostic unit. This includes the study of genetic defects in the HIV-1 genome and time trends of the replication fitness over 30 years of viral evolution, but also the description of novel HIV-1 variants in difficult-to-diagnose clinical specimen.

High prevalence of hepatitis B virus dual infection with genotypes A and G in HIV-1 infected men in Amsterdam, the Netherlands, during 2000-2011.

BACKGROUND: Hepatitis B virus (HBV) is divided into 8 definite (A-H) and 2 putative (I, J) genotypes that show a geographical distribution. HBV genotype G, however, is an aberrant genotype of unknown origin that demonstrates severe replication deficiencies and very little genetic variation. It is often found in co-infections with another HBV genotype and infection has been associated with certain risk groups such as intravenous drug users and men having sex with men (MSM). We aimed to estimate the prevalence of HBV-G in the Netherlands by analysing samples from HBV-positive patients visiting the Academic Medical Center in Amsterdam. METHODS: Ninety-six HBV-infected patients, genotyped as HBV-A or HBV-G infected, were retrieved from the clinical database. Blood plasma samples were analysed with a newly-developed real-time PCR assay that detects HBV-A and HBV-G. For three patients, the HBV plasma viral load (pVL) of both genotypes was followed longitudinally. In addition, three complete genomes of HBV-G were sequenced to determine their relationship to global HBV-G strains. RESULTS: Ten HBV-G infections were found in the selected Dutch patients. All concerned HIV-1 infected males with HBV-A co-infection. Dutch HBV-G strains were phylogenetically closely related to reference HBV-G strains. CONCLUSIONS: In this study, HBV-G infection in the Netherlands is found exclusively in HIV-1 infected men as co-infection with HBV-A. A considerable percentage (37%) of men infected with HBV and HIV-1 are actually co- infected with two HBV genotypes.

Incidence of human immunodeficiency virus type 1 dual infections in Amsterdam, The Netherlands, during 2003-2007.

BACKGROUND: The occurrence of human immunodeficiency virus type 1 (HIV-1) dual infections in Amsterdam, The Netherlands, was examined during 2003-2007 to investigate whether the number of HIV-1 dual infections increased as the number of HIV-1 infected individuals increased during the same period. METHODS: All first HIV-1 genotyping sequences obtained from 2003 through 2007 were retrieved and examined for the number of degenerate base codes in the reverse-transcriptase fragment. A total of 72 patients had >or=34 degenerate base codes; for these patients, a fragment of the V3-V4 region of the env gene was amplified, cloned, and sequenced to verify the presence of an HIV-1 dual infection. The number of dual infections were counted for each year investigated. RESULTS: No significant change in the incidence of dual infections was observed in our population of patients, who were selected on the basis of the number of degenerate base codes in each patient's first HIV-1 sequence obtained from 2003 through 2007. The frequency of HIV-1 dual infections varied between 1.0% and 2.4% each year, with no significant trend over time (P = .49). Patients with HIV-1 dual infections were similar to patients with single HIV-1 infections in The Netherlands with regard to distribution of risk group, sex, and HIV subtype. CONCLUSION: The proportion of HIV-1 dual infections in The Netherlands did not increase from 2003 through 2007, although the HIV-1-infected population expanded in this period.

Characterization of an HIV-1 group M variant that is distinct from the known subtypes.

We identified an HIV-1 variant that belongs to the M group, with limited similarity of short genetic regions (100-200 nt) to subtype K, but the remainder of the genome is unrelated to any established HIV-1 subtype. The isolate was obtained from an HIV-1-positive male, living in the Netherlands, who encountered the virus before 1989, most probably via heterosexual contact in Africa. We describe the full-length genome sequence of four biological clones that were obtained from two samples collected 5 years apart. At both time points all open reading frames were intact. Within the 5-year interval, the person received antiretroviral therapy with zalcitabine and zidovudine for almost 4 years. Evolution of drug-resistant variants is likely given the increase in viral RNA load to +/-10,000 copies/ml during the last year of treatment. Surprisingly, the only regular RT mutation acquired during this period was K70R, which suggests that the genetic background of this variant is perhaps not suitable for the generation of the standard 41L, 67N, and 215Y/F mutations that typically arise during prolonged, nonsuccessful, zidovudine treatment. Awaiting the discovery of at least two additional, epidemiologically unrelated patients with a phylogenetically related HIV-1 variant, we can designate this variant a new HIV-1 subtype, or a distinct branch of subtype K.

Declining trend in transmission of drug-resistant HIV-1 in Amsterdam.

OBJECTIVE: Symptomatic primary HIV infections are over-represented in the mainly hospital-based studies on transmission of resistant HIV-1. We examined a more general population for the prevalence of resistant HIV-1 strains among primary infections. DESIGN: From 1994 to 2002 primary infections were identified within the Amsterdam Cohort Studies (ACS) among homosexual men and drug users, and at the Academic Medical Center (AMC). Whereas primary HIV-1-infected AMC patients, often presented with symptoms of acute retroviral syndrome, ACS participants largely seroconverted during follow-up and thus brought also asymptomatic primary infections to our study. METHODS: Reverse transcriptase (RT) and protease sequences were obtained by population-based nucleotide sequence analysis of the first HIV RNA-positive sample available. Subtypes were identified by phylogenetic analysis. Mutations were identified based on the IAS-USA resistance table. RESULTS: A total of 100 primary HIV-1 infections were identified (32 AMC and 68 ACS). Transmission of drug-resistant strains decreased over calendar time, with 20% [95% confidence interval (CI), 10-34%] of infections bearing drug-resistant mutations before 1998 versus only 6% (95% CI, 1-17%) after 1998. No multi-drug resistance pattern was observed. The median plasma HIV-1 RNA level of the first RNA positive sample was significantly lower for the individuals infected with a resistant strain versus those infected with wild-type, suggesting a fitness-cost to resistance. Four of seven non-B subtypes corresponded with the prevalent subtype in the presumed country of infection, and none showed resistance mutations. CONCLUSIONS: The transmission of drug-resistant HIV-1 strains in Amsterdam has decreased over time. Monitoring should be continued as this trend might change.

Ten years of HIV testing with fourth generation assays: the Amsterdam experience.

Serological HIV assays combining detection of HIV antigen and antibodies are referred to as fourth generation assays. Fourth generation assays were implemented in Europe for routine patient testing about 10 years ago. The Academic Medical Center is one of the main HIV treatment centers in the Netherlands and has now 10 years experience with fourth generation testing, which is summarized here.

Selection of an M184V mutation in the cerebrospinal fluid of a treatment-naive HIV-infected individual starting darunavir-based therapy.

Here, we describe a newly diagnosed HIV-1-infected patient, in whom shortly after the initiation of a darunavir-based regimen, the HIV-1 virus exclusively mutated in the cerebrospinal fluid (CSF), leading to an increase in CSF HIV-1 RNA load and neurological complaints.

Unusual cluster of HIV type 1 dual infections in Groningen, The Netherlands.

In 2007, 14 Dutch men having sex with men (MSM) filed a criminal case against three other men, accusing them of administering sedative drugs, sexual abuse, and deliberate subcutaneous injections with HIV-1-infected blood. Medical files showed that 9 of 17 men presented with an acute HIV-1 infection syndrome during 2006-2007. Two men were not infected with HIV. Analysis of viral strains in the 12 MSM and the three alleged donors showed that one donor and six recipients were double infected with two distinct HIV-1 subtype B strains, while another five recipients and one donor were single infected with either strain. Two men were infected with unrelated strains. The finding of multiple double infections with very similar HIV-1 strains is without precedent.

Multiple transmissions of a stable human leucocyte antigen-B27 cytotoxic T-cell-escape strain of HIV-1 in The Netherlands.

OBJECTIVE: The evolution of HIV-1 is largely shaped by the cytotoxic T-cell (CTL) response of the host as encoded by the human leucocyte antigen (HLA) genes. Certain HLA-B alleles can delay disease progression, but it is uncertain whether this protection will sustain or whether the virus is in the process of adaptation. In The Netherlands, HLA-B27 is moderately prevalent (approximately 8-16% of HLA-B alleles). If adaptation to HLA-B alleles is in progress, virus strains carrying escape mutations to HLA-B27 should appear in the epidemic by now. DESIGN: A subtype B HIV-1 strain carrying a HLA-B27 CTL-escape mutation in the main Gag-p24 KK10 epitope, R264G, together with a compensatory mutation outside this epitope, E260D, was detected in four patients from Amsterdam, The Netherlands, by sequence analysis of the gag gene. The patients were a drug user and three men who have sex with men, and were infected with HIV-1 between 2002 and 2008. METHODS: Characterization and evolutionary analysis of the HIV-1 CTL-escape strain was done by sequence analysis of serial blood plasma samples. RESULTS: The mutations involved were stable during follow-up and after transmission, also in two individuals lacking HLA-B27. CONCLUSION: The finding that a stable HLA-B27 CTL-escape strain is circulating in The Netherlands has important implications for the understanding of virus-host interactions and vaccine design alike. Vaccines targeted at inducing a CTL response might easily be circumvented by the virus. Also, patients carrying protective HLA alleles might not be protected anymore from disease progression in the future.

Phylogenetic reconstruction of transmission events from individuals with acute HIV infection: toward more-rigorous epidemiological definitions.

Phylogenetic reconstructions of transmission events from individuals with acute human immunodeficiency virus (HIV) infection are conducted to illustrate this group's heightened infectivity. Varied definitions of acute infection and assumptions about observed phylogenetic clusters may produce misleading results. We conducted a phylogenetic analysis of HIV pol sequences from 165 European patients with estimated infection dates and calculated the difference between dates within clusters. Nine phylogenetic clusters were observed. Comparison of dates within clusters revealed that only 2 could have been generated during acute infection. Previous analyses may have incorrectly assigned transmission events to the acutely HIV infected when they were more likely to have occurred during chronic infection.

Survey of the temporal changes in HIV-1 replicative fitness in the Amsterdam Cohort.

Changes in virulence and fitness during an epidemic are common among pathogens. Several studies have shown that HIV fitness increases within a patient during disease progression, while bottlenecks, such as sexual transmission, immune pressure and drug treatment can reduce fitness. In this study, we analyzed how these opposing forces have shaped HIV-1 fitness over time. Therefore, we compared the replicative fitness of HIV-1 isolates from newly infected untreated individuals, diagnosed for HIV-1 infection early in the AIDS epidemic in Amsterdam, the Netherlands, with more recent isolates. Twenty-five early and late HIV-1 isolates, carefully matched for seroconversion time, were competed head-to-head in a dual infection/competition assay, employing peripheral blood mononuclear cells. In contrast with previous studies, we observed a trend of increasing fitness over time in the HIV epidemic of Amsterdam. Apparently, the bottleneck, occurring with each transmission event, does not completely reset the fitness increase acquired during disease progression.