HIV-1-infection in a man who has sex with men despite self-reported excellent adherence to pre-exposure prophylaxis, the Netherlands, August 2021: be alert to emtricitabine/tenofovir-resistant strain transmission.

In August 2021, a man who has sex with men was diagnosed with HIV-1 infection despite using event-driven pre-exposure prophylaxis for over 2 years with self-reported excellent adherence. Sequencing identified resistance-associated mutations (RAM) M184V and K65R, conferring resistance to emtricitabine and tenofovir, and RAM V108I and E138A. Background RAM prevalence was two of 164 (1.2%) new HIV diagnoses in Amsterdam (2017-19). We reiterate the need for frequent HIV testing among PrEP users and additional testing in case of symptoms.

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.

Human immunodeficiency virus type 1 gp120 envelope characteristics associated with disease progression differ in family members infected with genetically similar viruses.

The human immunodeficiency virus type 1 (HIV-1) envelope protein provides the primary contact between the virus and host, and is the main target of the adaptive humoral immune response. The length of gp120 variable loops and the number of N-linked glycosylation events are key determinants for virus infectivity and immune escape, while the V3 loop overall positive charge is known to affect co-receptor tropism. We selected two families in which both parents and two children had been infected with HIV-1 for nearly 10 years, but who demonstrated variable parameters of disease progression. We analysed the gp120 envelope sequence and compared individuals that progressed to those that did not in order to decipher evolutionary alterations that are associated with disease progression when individuals are infected with genetically related virus strains. The analysis of the V3-positive charge demonstrated an association between higher V3-positive charges with disease progression. The ratio between the amino acid length and the number of potential N-linked glycosylation sites was also shown to be associated with disease progression with the healthier family members having a lower ratio. In conclusion in individuals initially infected with genetically linked virus strains the V3-positive charges and N-linked glycosylation are associated with HIV-1 disease progression and follow varied evolutionary paths for individuals with varied disease progression.

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.

Modest nonadherence to antiretroviral therapy promotes residual HIV-1 replication in the absence of virological rebound in plasma.

BACKGROUND: Modern antiretroviral therapy (ART) regimens are widely assumed to forgive modest nonadherence, because virological suppression in plasma is common at adherence levels of >70%. Yet, it is unknown whether human immunodeficiency virus type 1 (HIV-1) replication is completely suppressed at these levels of adherence. METHODS: We longitudinally quantified levels of cell-associated HIV-1 RNA and DNA in 40 patients (median duration of successful ART before study initiation, 46 months), whose 1-week adherence to therapy prior to the sampling moments was measured electronically. RESULTS: Patients were constantly 100% adherent (the optimal-adherence group), demonstrated improving adherence over time (the improving-adherence group), or neither of the above (the poor-adherence group). Adherence never decreased to <70% in any patient, and no rebound in plasma virological levels was observed. Nevertheless, poor adherence but not optimal or improving adherence caused a significant longitudinal increase in cell-associated HIV RNA levels (P = .006). Time-weighted changes and regression slopes of viral RNA load for the poor-adherence group were significantly higher than those for the optimal-adherence group (P < .01). CONCLUSIONS: Because ART only blocks infection of new cells but not viral RNA transcription in cells infected before therapy initiation, the observed effects strongly suggest that modest nonadherence can cause new cycles of HIV-1 replication that are undetectable by commercial plasma viral load assays.

Intent to vaccinate against SARS-CoV-2 and its determinants across six ethnic groups living in Amsterdam, the Netherlands: A cross-sectional analysis of the HELIUS study.

BACKGROUND: Ethnic minority groups experience a disproportionately high burden of infections, hospitalizations and mortality due to COVID-19, and therefore should be especially encouraged to receive SARS-CoV-2 vaccination. This study aimed to investigate the intent to vaccinate against SARS-CoV-2, along with its determinants, in six ethnic groups residing in Amsterdam, the Netherlands. METHODS: We analyzed data of participants enrolled in the population-based multi-ethnic HELIUS cohort, aged 24 to 79 years, who were tested for SARS-CoV-2 antibodies and answered questions on vaccination intent from November 23, 2020 to March 31, 2021. During the study period, SARS-CoV-2 vaccination in the Netherlands became available to individuals working in healthcare or > 75 years old. Vaccination intent was measured by two statements on a 7-point Likert scale and categorized into low, medium, and high. Using ordinal logistic regression, we examined the association between ethnicity and lower vaccination intent. We also assessed determinants of lower vaccination intent per ethnic group. RESULTS: A total of 2,068 participants were included (median age 56 years, interquartile range 46-63). High intent to vaccinate was most common in the Dutch ethnic origin group (369/466, 79.2%), followed by the Ghanaian (111/213, 52.1%), South-Asian Surinamese (186/391, 47.6%), Turkish (153/325, 47.1%), African Surinamese (156/362, 43.1%), and Moroccan ethnic groups (92/311, 29.6%). Lower intent to vaccinate was more common in all groups other than the Dutch group (P < 0.001). Being female, believing that COVID-19 is exaggerated in the media, and being < 45 years of age were common determinants of lower SARS-CoV-2 vaccination intent across most ethnic groups. Other identified determinants were specific to certain ethnic groups. CONCLUSIONS: Lower intent to vaccinate against SARS-CoV-2 in the largest ethnic minority groups of Amsterdam is a major public health concern. The ethnic-specific and general determinants of lower vaccination intent observed in this study could help shape vaccination interventions and campaigns.

Virological and immunological correlates of HIV posttreatment control after temporal antiretroviral therapy during acute HIV infection.

OBJECTIVE: People with HIV rarely control viral replication after cessation of antiretroviral therapy (ART). We present a person with HIV with extraordinary posttreatment control (PTC) for over 23 years after temporary ART during acute HIV infection (AHI) leading to a new insight in factors contributing to PTC. DESIGN/METHODS: Viral reservoir was determined by HIV qPCR, Intact Proviral DNA Assay, and quantitative viral outgrowth assay. Viral replication kinetics were determined in autologous and donor PBMC. IgG levels directed against HIV envelope and neutralizing antibodies were measured. Immune phenotyping of T cells and HIV-specific T-cell responses were analyzed by flow cytometry. RESULTS: The case presented with AHI and a plasma viral load of 2.7 million copies/ml. ART was initiated 2 weeks after diagnosis and interrupted after 26 months. Replicating virus was isolated shortly after start ART. At 18 years after treatment interruption, HIV-DNA in CD4 + T cells and low levels of HIV-RNA in plasma (<5 copies/ml) were detectable. Stable HIV envelope glycoprotein-directed IgG was present during follow-up, but lacked neutralizing activity. Strong antiviral CD8 + T-cell responses, in particular targeting HIV-gag, were detected during 25 years follow-up. Moreover, we found a P255A mutation in an HLA-B∗44 : 02 restricted gag-epitope, which was associated with decreased replication. CONCLUSION: We describe an exceptional case of PTC, which is likely associated with sustained potent gag-specific CD8 + T-cell responses in combination with a replication attenuating escape mutation in gag. Understanding the initiation and preservation of the HIV-specific T-cell responses could guide the development of strategies to induce HIV control.

HIV-1 dual infection is associated with faster CD4+ T-cell decline in a cohort of men with primary HIV infection.

BACKGROUND: In vitro, animal, and mathematical models suggest that human immunodeficiency virus (HIV) co- or superinfection would result in increased fitness of the pathogen and, possibly, increased virulence. However, in patients, the impact of dual HIV type 1 (HIV-1) infection on disease progression is unclear, because parameters relevant for disease progression have not been strictly analyzed. The objective of the present study is to analyze the effect of dual HIV-1 infections on disease progression in a well-defined cohort of men who have sex with men. METHODS: Between 2000 and 2009, 37 men who had primary infection with HIV-1 subtype B, no indication for immediate need of combination antiretroviral therapy (cART), and sufficient follow-up were characterized with regard to dual infection or single infection and to coreceptor use. Patients were followed to estimate the effect of these parameters on clinical disease progression, as defined by the rate of CD4(+) T-cell decline and the time to initiation of cART. RESULTS: Four patients presented with HIV-1 coinfection; 6 patients acquired HIV-1 superinfection, on average 8.5 months from their primary infection; and 27 patients remained infected with a single strain. Slopes of longitudinal CD4(+) T-cell counts and time-weighted changes from baseline were significantly steeper for patients with dual infection compared with patients with single infection. Multivariate analysis showed that the most important parameter associated with CD4(+) T-cell decline over time was dual infection (P = .001). Additionally, patients with HIV-1 coinfection had a significantly earlier start of cART (P < .0001). CONCLUSIONS: Dual HIV-1 infection is the main factor associated with CD4(+) T-cell decline in men who have untreated primary infection with HIV-1 subtype B.

No treatment versus 24 or 60 weeks of antiretroviral treatment during primary HIV infection: the randomized Primo-SHM trial.

BACKGROUND: The objective of this study was to assess the benefit of temporary combination antiretroviral therapy (cART) during primary HIV infection (PHI). METHODS AND FINDINGS: Adult patients with laboratory evidence of PHI were recruited in 13 HIV treatment centers in the Netherlands and randomly assigned to receive no treatment or 24 or 60 wk of cART (allocation in a 1∶1∶1 ratio); if therapy was clinically indicated, participants were randomized over the two treatment arms (allocation in a 1∶1 ratio). Primary end points were (1) viral set point, defined as the plasma viral load 36 wk after randomization in the no treatment arm and 36 wk after treatment interruption in the treatment arms, and (2) the total time that patients were off therapy, defined as the time between randomization and start of cART in the no treatment arm, and the time between treatment interruption and restart of cART in the treatment arms. cART was (re)started in case of confirmed CD4 cell count < 350 cells/mm(3) or symptomatic HIV disease. In total, 173 participants were randomized. The modified intention-to-treat analysis comprised 168 patients: 115 were randomized over the three study arms, and 53 randomized over the two treatment arms. Of the 115 patients randomized over the three study arms, mean viral set point was 4.8 (standard deviation 0.6) log(10) copies/ml in the no treatment arm, and 4.0 (1.0) and 4.3 (0.9) log(10) copies/ml in the 24- and 60-wk treatment arms (between groups: p < 0.001). The median total time off therapy in the no treatment arm was 0.7 (95% CI 0.0-1.8) y compared to 3.0 (1.9-4.2) and 1.8 (0.5-3.0) y in the 24- and 60-wk treatment arms (log rank test, p < 0.001). In the adjusted Cox analysis, both 24 wk (hazard ratio 0.42 [95% CI 0.25-0.73]) and 60 wk of early treatment (hazard ratio 0.55 [0.32-0.95]) were associated with time to (re)start of cART. CONCLUSIONS: In this trial, temporary cART during PHI was found to transiently lower the viral set point and defer the restart of cART during chronic HIV infection.

Clinical evaluation of single-swab sampling for rapid COVID-19 detection in outbreak settings in Dutch nursing homes.

PURPOSE: To assess whether one swab can be used to perform both the antigen-detection rapid diagnostic test (Ag-RDT) and reverse transcriptase polymerase chain reaction (RT-PCR) for COVID-19 detection during an outbreak in the nursing home (NH) setting. METHODS: The single-swab method (SSM), where the Ag-RDT is performed with the transport medium used for RT-PCR, was evaluated in three Dutch NHs and compared to the laboratory setting. We collected Ag-RDT and RT-PCR results, NH resident characteristics and symptomatology. In addition, two focus groups were held with the involved care professionals to gain insight into the feasibility of the SMM in the NH setting. RESULTS: In the NH setting, the SSM had a sensitivity of 51% and a specificity of 89% compared to RT-PCR. These were lower than in the laboratory setting (69% and 100% respectively). Yet, when stratified for cycle threshold values, the sensitivity became comparable between the settings. Symptoms occurred more frequent in the Ag-RDT+ group than Ag-RDT- group. Resident characteristics did not differ between these groups. Based on the focus groups, the SSM was feasible to perform if certain requirements, such as availability of staff, equipment and proper training, were met. However, the rapid availability of the test results were perceived as a dilemma. CONCLUSION: The advantages and disadvantages need to be considered before implementation of the SSM can be recommended in the NH setting. For the vulnerable NH residents, it is important to find the right balance between effective testing policy and the burden this imposes.

Differences in SARS-CoV-2 infections during the first and second wave of SARS-CoV-2 between six ethnic groups in Amsterdam, the Netherlands: A population-based longitudinal serological study.

BACKGROUND: Surveillance data in high-income countries have reported more frequent SARS-CoV-2 diagnoses in ethnic minority groups. We examined the cumulative incidence of SARS-CoV-2 and its determinants in six ethnic groups in Amsterdam, the Netherlands. METHODS: We analysed participants enrolled in the population-based HELIUS cohort, who were tested for SARS-CoV-2-specific antibodies and answered COVID-19-related questions between June 24-October 9, 2020 (after the first wave) and November 23, 2020-March 31, 2021 (during the second wave). We modelled SARS-CoV-2 incidence from January 1, 2020-March 31, 2021 using Markov models adjusted for age and sex. We compared incidence between ethnic groups over time and identified determinants of incident infection within ethnic groups. FINDINGS: 2,497 participants were tested after the first wave; 2,083 (83·4%) were tested during the second wave. Median age at first visit was 54 years (interquartile range=44-61); 56·6% were female. Compared to Dutch-origin participants (15·9%), cumulative SARS-CoV-2 incidence was higher in participants of South-Asian Surinamese (25·0%; adjusted hazard ratio [aHR]=1·66; 95%CI=1·16-2·40), African Surinamese (28·9%, aHR=1·97; 95%CI=1·37-2·83), Turkish (37·0%; aHR=2·67; 95%CI=1·89-3·78), Moroccan (41·9%; aHR=3·13; 95%CI=2·22-4·42), and Ghanaian (64·6%; aHR=6·00; 95%CI=4·33-8·30) origin. Compared to those of Dutch origin, differences in incidence became wider during the second versus first wave for all ethnic minority groups (all p-values for interaction<0·05), except Ghanaians. Having household members with suspected SARS-CoV-2 infection, larger household size, and low health literacy were common determinants of SARS-CoV-2 incidence across groups. INTERPRETATION: SARS-CoV-2 incidence was higher in the largest ethnic minority groups of Amsterdam, particularly during the second wave. Prevention measures, including vaccination, should be encouraged in these groups. FUNDING: ZonMw, Public Health Service of Amsterdam, Dutch Heart Foundation, European Union, European Fund for the Integration of non-EU immigrants.

SARS-CoV-2 antibody prevalence and correlates of six ethnic groups living in Amsterdam, the Netherlands: a population-based cross-sectional study, June-October 2020.

OBJECTIVES: It has been suggested that ethnic minorities have been disproportionally affected by the COVID-19. We aimed to determine whether prevalence and correlates of past SARS-CoV-2 exposure varied between six ethnic groups in Amsterdam, the Netherlands. DESIGN, SETTING, PARTICIPANTS: Participants aged 25-79 years enrolled in the Healthy Life in an Urban Setting population-based prospective cohort (n=16 889) were randomly selected within ethnic groups and invited to participate in a cross-sectional COVID-19 seroprevalence substudy. OUTCOME MEASURES: We tested participants for SARS-CoV-2-specific antibodies and collected information on SARS-CoV-2 exposures. We estimated prevalence and correlates of SARS-CoV-2 exposure within ethnic groups using survey-weighted logistic regression adjusting for age, sex and calendar time. RESULTS: Between 24 June and 9 October 2020, we included 2497 participants. Adjusted SARS-CoV-2 seroprevalence was comparable between ethnic Dutch (24/498; 5.1%, 95% CI 2.8% to 7.4%), South-Asian Surinamese (22/451; 4.9%, 95% CI 2.2% to 7.7%), African Surinamese (22/400; 8.3%, 95% CI 3.1% to 13.6%), Turkish (30/408; 7.9%, 95% CI 4.4% to 11.4%) and Moroccan (32/391; 7.2%, 95% CI 4.2% to 10.1%) participants, but higher among Ghanaians (95/327; 26.3%, 95% CI 18.5% to 34.0%). 57.1% of SARS-CoV-2-positive participants did not suspect or were unsure of being infected, which was lowest in African Surinamese (18.2%) and highest in Ghanaians (90.5%). Correlates of SARS-CoV-2 exposure varied across ethnic groups, while the most common correlate was having a household member suspected of infection. In Ghanaians, seropositivity was associated with older age, larger household sizes, living with small children, leaving home to work and attending religious services. CONCLUSIONS: No remarkable differences in SARS-CoV-2 seroprevalence were observed between the largest ethnic groups in Amsterdam after the first wave of infections. The higher infection seroprevalence observed among Ghanaians, which passed mostly unnoticed, warrants wider prevention efforts and opportunities for non-symptom-based testing.

Head-to-head validation of six immunoassays for SARS-CoV-2 in hospitalized patients.

BACKGROUND: Detecting SARS-CoV-2 antibodies may help to diagnose COVID-19. Head-to-head validation of different types of immunoassays in well-characterized cohorts of hospitalized patients remains needed. METHODS: We validated three chemiluminescence immunoassays (CLIAs) (Liaison, Elecsys, and Abbott) and one single molecule array assay (SIMOA) (Quanterix) for automated analyzers, one rapid immunoassay RIA (AllTest), and one ELISA (Wantai) in parallel in first samples from 126 PCR confirmed COVID-19 hospitalized patients and 158 pre-COVID-19 patients. Specificity of the AllTest was also tested in 106 patients with confirmed parasitic and dengue virus infections. Specificity of the Wantai assay was not tested due to limitations in sample volumes. RESULTS: Overall sensitivity in first samples was 70.6 % for the Liaison, 71.4 % for the Elecsys, 75.4 % for the Abbott, 70.6 % for the Quanterix, 77.8 % for the AllTest, and 88.9 % for the Wantai assay, respectively. Sensitivity was between 77.4 % (Liaison) and 94.0 % (Wantai) after 10 dpso. No false positive results were observed for the Elecsys and Abbott assays. Specificity was 91.1 % for the Quanterix, 96.2 % for the Liaison, and 98.1 % for the AllTest assay, respectively. CONCLUSION: We conclude that low sensitivity of all immunoassays limits their use early after onset of illness in diagnosing COVID-19 in hospitalized patients. After 10 dpso, the Wantai ELISA has a relatively high sensitivity, followed by the point-of-care AllTest RIA that compares favorably with automated analyzer immunoassays.

Serologic Surveillance and Phylogenetic Analysis of SARS-CoV-2 Infection Among Hospital Health Care Workers.

Importance: It is unclear when, where, and by whom health care workers (HCWs) working in hospitals are infected with SARS-CoV-2. Objective: To determine how often and in what manner nosocomial SARS-CoV-2 infection occurs in HCW groups with varying exposure to patients with COVID-19. Design, Setting, and Participants: This cohort study comprised 4 weekly measurements of SARS-CoV-2-specific antibodies and collection of questionnaires from March 23 to June 25, 2020, combined with phylogenetic and epidemiologic transmission analyses at 2 university hospitals in the Netherlands. Included individuals were HCWs working in patient care for those with COVID-19, HCWs working in patient care for those without COVID-19, and HCWs not working in patient care. Data were analyzed from August through December 2020. Exposures: Varying work-related exposure to patients infected with SARS-CoV-2. Main Outcomes and Measures: The cumulative incidence of and time to SARS-CoV-2 infection, defined as the presence of SARS-CoV-2-specific antibodies in blood samples, were measured. Results: Among 801 HCWs, there were 439 HCWs working in patient care for those with COVID-19, 164 HCWs working in patient care for those without COVID-19, and 198 HCWs not working in patient care. There were 580 (72.4%) women, and the median (interquartile range) age was 36 (29-50) years. The incidence of SARS-CoV-2 was increased among HCWs working in patient care for those with COVID-19 (54 HCWs [13.2%; 95% CI, 9.9%-16.4%]) compared with HCWs working in patient care for those without COVID-19 (11 HCWs [6.7%; 95% CI, 2.8%-10.5%]; hazard ratio [HR], 2.25; 95% CI, 1.17-4.30) and HCWs not working in patient care (7 HCWs [3.6%; 95% CI, 0.9%-6.1%]; HR, 3.92; 95% CI, 1.79-8.62). Among HCWs caring for patients with COVID-19, SARS-CoV-2 cumulative incidence was increased among HCWs working on COVID-19 wards (32 of 134 HCWs [25.7%; 95% CI, 17.6%-33.1%]) compared with HCWs working on intensive care units (13 of 186 HCWs [7.1%; 95% CI, 3.3%-10.7%]; HR, 3.64; 95% CI, 1.91-6.94), and HCWs working in emergency departments (7 of 102 HCWs [8.0%; 95% CI, 2.5%-13.1%]; HR, 3.29; 95% CI, 1.52-7.14). Epidemiologic data combined with phylogenetic analyses on COVID-19 wards identified 3 potential HCW-to-HCW transmission clusters. No patient-to-HCW transmission clusters could be identified in transmission analyses. Conclusions and Relevance: This study found that HCWs working on COVID-19 wards were at increased risk for nosocomial SARS-CoV-2 infection with an important role for HCW-to-HCW transmission. These findings suggest that infection among HCWs deserves more consideration in infection prevention practice.

Cell-associated HIV-1 RNA predicts viral rebound and disease progression after discontinuation of temporary early ART.

Plasma viral load (VL) and CD4+ T cell count are widely used as biomarkers of HIV type 1 (HIV-1) replication, pathogenesis, and response to antiretroviral therapy (ART). However, the clinical potential of cell-associated (CA) HIV-1 molecular markers is much less understood. Here, we measured CA HIV-1 RNA and DNA in HIV-infected individuals treated with temporary ART initiated during primary HIV-1 infection. We demonstrate substantial predictive value of CA RNA for (a) the virological and immunological response to early ART, (b) the magnitude and time to viral rebound after discontinuation of early ART, and (c) disease progression in the absence of treatment. Remarkably, when adjusted for CA RNA, plasma VL no longer appeared as an independent predictor of any clinical endpoint in this cohort. The potential of CA RNA as an HIV-1 clinical marker, in particular as a predictive biomarker of virological control after stopping ART, should be explored in the context of HIV-1 curative interventions.

CD32+CD4+ T Cells Are Highly Enriched for HIV DNA and Can Support Transcriptional Latency.

The HIV latent reservoir forms the major hurdle to an HIV cure. The discovery of CD32 as marker of this reservoir has aroused much interest, but subsequent reports have challenged this finding. Here, we observe a positive correlation between the percentages of CD32+ cells among CD4+ T cells of aviremic cART-treated, HIV-infected individuals and their HIV DNA loads in peripheral blood. Moreover, optimization of the CD32+CD4+ T cell purification protocol reveals prominent enrichment for HIV DNA (mean, 292-fold) in these cells. However, no enrichment for HIV RNA is observed in CD32+CD4+ cells, yielding significantly reduced HIV RNA/DNA ratios. Furthermore, HIV proviruses in CD32+CD4+ cells can be reactivated ex vivo to produce virus, strongly suggesting that these cells support HIV transcriptional latency. Our results underscore the importance of isolating pure, bona fide CD32+CD4+ T cells for future studies and indicate that CD32 remains a promising candidate marker of the HIV reservoir.

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.

High incidence of asymptomatic syphilis in HIV-infected MSM justifies routine screening.

BACKGROUND: Recently, the incidence of syphilis has risen, mainly among men having sex with men (MSM), many of whom are coinfected with HIV. Current guidelines recommend at least yearly syphilis testing in this group. In this study, we assessed the yield of routine syphilis screening in outpatient HIV-patients. METHODS: From March through June 2003, syphilis serology was routinely performed in HIV-infected patients visiting our outpatient clinic. Serology was repeated six months later. Positive test results of the first episode were compared to historical test results (retrospective analysis). Test results of the second episode were compared to test results from the first episode (prospective analysis). Case records of all patients with a new infection were reviewed for symptomatic disease or testing because of syphilis contacts, versus asymptomatic disease. RESULTS: In the retrospective analysis, 1,105 patients were included. In 68 patients, 81 syphilis infections were identified, of which 33% asymptomatic. 77/81 infections were acquired between 2000 and 2003, resulting in a total incidence of 2.7/100 person years (PY) of follow-up, and 0.9/100 PY for asymptomatic disease. In MSM, the incidence rate was 4.6/100 and 1.5/100 PY respectively. In the prospective analysis, 1,010 patients were included. Seventeen patients, all MSM, had a new or recurrent syphilis infection, of whom 4 asymptomatic, accounting for a total event rate of 3.5/100 PY. In MSM, the event rate was 6.2/100 PY, with an incidence of asymptomatic disease of 1.4/100 PY. CONCLUSION: Routine screening for syphilis identifies significant numbers of asymptomatic syphilis infection in HIV-infected MSM.

Dendritic cells potently purge latent HIV-1 beyond TCR-stimulation, activating the PI3K-Akt-mTOR pathway.

BACKGROUND: The latent HIV-1 reservoir in treated patients primarily consists of resting memory CD4+ T cells. Stimulating the T-cell receptor (TCR), which facilitates transition of resting into effector T cells, is the most effective strategy to purge these latently infected cells. Here we supply evidence that TCR-stimulated effector T cells still frequently harbor latent HIV-1. METHODS: Primary HIV-1 infected cells were used in a latency assay with or without dendritic cells (DCs) and reversion of HIV-1 latency was determined, in the presence or absence of specific pathway inhibitors. FINDINGS: Renewed TCR-stimulation or subsequent activation with latency reversing agents (LRAs) did not overcome latency. However, interaction of infected effector cells with DCs triggered further activation of latent HIV-1. When compared to TCR-stimulation only, CD4+ T cells from aviremic patients receiving TCR + DC-stimulation reversed latency more frequently. Such a "one-two punch" strategy seems ideal for purging the reservoir. We determined that DC contact activates the PI3K-Akt-mTOR pathway in CD4+ T cells. INTERPRETATION: This insight could facilitate the development of a novel class of potent LRAs that purge latent HIV beyond levels reached by T-cell activation.

Highly sensitive methods based on seminested real-time reverse transcription-PCR for quantitation of human immunodeficiency virus type 1 unspliced and multiply spliced RNA and proviral DNA.

The effectiveness of highly active antiretroviral therapy (HAART), the standard of care for the treatment of human immunodeficiency virus type 1 (HIV-1) infection, is assessed by measuring the viral RNA load in plasma. A patient is considered to be successfully treated when the HIV-1 load in plasma stays below the detection limit of commercial assays. However, virus replication and evolution do continue in patients under HAART, which may eventually result in the development of drug-resistant HIV-1 strains and therapy failure. To monitor this low-level virus replication in peripheral blood mononuclear cells (PBMC), sensitive methods are required to measure HIV-1 molecular markers. We report the development of highly sensitive methods for the quantitation of unspliced and multiply spliced HIV-1 RNA and proviral DNA in PBMC. The methods are based on innovative seminested real-time reverse transcription-PCR (RT-PCR) that combines the accuracy and precision of real-time PCR and the sensitivity of nested PCR. We show that the newly developed methods are superior to the conventional single-step real-time RT-PCR in their sensitivity, accuracy, dynamic range, and the power of quantitative detection of HIV-1 RNA and DNA in clinical samples. These easy-to-perform methods can be widely used in research, including clinical studies, to monitor intracellular processes of virus replication.